WO2020151295A1 - 含双核苷酸结构的化合物 - Google Patents

含双核苷酸结构的化合物 Download PDF

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WO2020151295A1
WO2020151295A1 PCT/CN2019/115304 CN2019115304W WO2020151295A1 WO 2020151295 A1 WO2020151295 A1 WO 2020151295A1 CN 2019115304 W CN2019115304 W CN 2019115304W WO 2020151295 A1 WO2020151295 A1 WO 2020151295A1
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compound
group
aryl
alkyl
independently selected
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PCT/CN2019/115304
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English (en)
French (fr)
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袁建栋
林祥义
孙占莉
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 WO2020151295A1 publication Critical patent/WO2020151295A1/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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a compound containing a dinucleotide structure with a novel structure. It also relates to a preparation method of the compound containing a dinucleotide structure of the present invention, and its application in the preparation of drugs for treating viral infections, especially hepatitis B virus (HBV) infections and HBV-related liver diseases.
  • HBV hepatitis B virus
  • Hepatitis B is one of the most burdened diseases in our society. At present, about 100 million people in my country are carriers of hepatitis B virus, accounting for about 8%-10% of the total population of my country, and about 20 million people with chronic hepatitis B (inflammatory lesions in the liver). It is estimated that there are 350 million chronic HBV carriers worldwide. According to the Centers for Disease Control, nearly 3 to 7 million people die every year from infection-related complications, such as liver cirrhosis and hepatocellular carcinoma. A large number of patients receiving liver transplantation continue to require anti-HBV therapy. 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.
  • Hepatitis B virus is very harmful to patients. After the patient is infected with hepatitis B virus, it will not cause a lot of damage to the health in the short term, and when it is onset, it has often developed into chronic hepatitis B, which is difficult to treat and has a poor prognosis.
  • prodrug strategies can be used to improve compound stability, increase targeting, overcome first-pass effects, and improve bioavailability.
  • prodrugs Although the 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. In fact, little is known about the existence of this specific transport device that can facilitate the transport of these new compounds across the mucosa through an active transport mechanism.
  • drug molecules used for oral absorption through passive diffusion should have a molecular weight of less than 500 Daltons, and More than 5 hydrogen bond donors (OH and NH groups), no more than 10 hydrogen bond acceptors (notably nitrogen and oxygen), molecular weight less than 500, and LogP value less than 5.
  • dinucleotide prodrugs are compounds with a relatively high molecular weight, so they cannot meet the Ribinski standard for oral absorption in many aspects.
  • the present invention provides a compound containing a dinucleotide structure; another aspect of the present invention also provides a method for preparing these compounds containing a dinucleotide structure and their application in the preparation of drugs for treating HBV infection.
  • the present invention provides a compound represented by formula I or a salt thereof and corresponding diastereomers or salts thereof based on a chiral phosphorus atom P represented by the formulas Sp-I and Rp-I:
  • G is a five-membered heterocyclic group
  • R 1 and R 2 are independently selected from H, C 1-6 alkyl, cycloalkyl, aryl, aralkyl, C 2-6 alkenyl or substituted or unsubstituted by one or more substituents Heterocyclic group;
  • the five-membered heterocyclic group G is optionally substituted by one or more substituents; the substituents are independently selected from halogen, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2.
  • the five-membered heterocyclic group contains two heteroatoms, a cyclic carbonyl group exists between the two heteroatoms, and at least one intracyclic double bond or extracyclic double bond, heteroatom, carbonyl and Double bonds form a conjugated system.
  • a and B are independently selected from -NR 6 -, -O- and -CH 2 -, but A and B are not -CH 2 -at the same time; further preferably, A and B are -O- at the same time; or A and B At least one of them is -O-, and the other is -NR 6 -, or -CH 2 -;
  • R 3, R 4, R 5 , R 6 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 halogens, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2 , protecting group-containing amino, alkoxy, alkylamino, C 2 -C 12 alkenyl substituted amino, cycloalkyl substituted amino, arylamino, alkylcarbonyl, arylcarbonyl, Alkyl carboxy, aryl carboxy, and amide.
  • substituents are independently selected from halogens, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2 , protecting group-containing amino, alkoxy, alkylamino, C 2 -C 12 alkenyl substituted amino, cycloalkyl substituted amino, arylamino, alkylcarbonyl, arylcarbonyl, Alkyl
  • 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.
  • a and B are both -O-, or one of A and B is -O- and the other is -NH-;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from H, C 1 ⁇ 6 alkyl group, C 5 ⁇ 12 aryl group, C 1 ⁇ 6 alkyl substituted C 5 ⁇ 12 aryl group, C 1 ⁇ 6 alkoxy-substituted C 5 ⁇ 12 aryl group, a halogen-substituted C 1-6 alkyl groups, and halogen-substituted C 5-12 aryl groups.
  • the C 1-6 alkyl group is preferably methyl, ethyl, isopropyl, tert-butyl, n-butyl; the halogen is F, Cl, Br, I, preferably F, Cl, the C
  • the 5-12 aryl group is preferably phenyl, thienyl, pyrrolyl or furyl.
  • the present invention also provides compounds represented by formula Ia and their corresponding diastereomers based on chiral phosphorus atom P represented by formulas R p -Ia and S p -Ia body;
  • a and B are independently selected from -O- and -NH-; preferably, A and B are both -O-, or one of A and B is -O- and the other is -NH-;
  • R 1 and R 3 are each independently selected from H, C 1-6 alkyl, such as methyl, ethyl, isopropyl, n-butyl, tert-butyl, C 5-12 aryl, C 1-6 alkane Oxy, C 1-6 alkenyl, or C 5-12 aryl substituted with one or more substituents, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkyl.
  • the substituents are independently selected from C 1-6 alkyl, C 1-6 alkoxy, halogen, preferably methyl, ethyl, isopropyl, tert-butyl, F, Cl, Br, methoxy, Ethoxy, trifluoromethoxy, chloroethoxy;
  • the C 5-12 aryl group is phenyl, thienyl, pyrrolyl, furanyl;
  • the ⁇ 12 aryl group is preferably p-methylphenyl, trifluoromethylphenyl, and dimethylphenyl.
  • *P represents a chiral phosphorus atom.
  • the present invention also provides two diastereomers of the above-mentioned compound BG001 to compound BG011 based on the chiral phosphorus atom P.
  • the present invention also provides compounds represented by formula Ib and corresponding diastereomers based on chiral phosphorus atom P represented by formulas R p -Ib and S p -Ib ;
  • a and B are independently selected from -O- and -NH-; preferably, A and B are both -O-, or one of A and B is -O- and the other is -NH-;
  • R 1 , R 4 , R 5 is each independently selected from H, C 1-6 alkyl, such as methyl, ethyl, isopropyl, n-butyl, tert-butyl, C 5-12 aryl, C 1-6 alkoxy, C 1-6 alkenyl, or C 5-12 aryl substituted with one or more substituents, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkyl.
  • R 1 , R 4 , and R 5 are all H.
  • a and B are both -O-, R 1 , R 4 , and R 5 are both H, or R 4 and R 5 are H, and R 1 is C 1-6 alkyl.
  • R 1 is C 1-6 alkyl.
  • the substituents are independently selected from C 1-6 alkyl groups, such as methyl, ethyl, isopropyl, tert-butyl, and C 1-6 alkoxy groups, such as methoxy, ethoxy, trifluoromethyl Oxy, chloroethoxy, halogen, preferably F, Cl, Br;
  • the C 5-12 aryl group is preferably phenyl, thienyl, pyrrolyl, furanyl;
  • the substituted C 5-12 aryl groups are preferably p-methylphenyl, trifluoromethylphenyl, and dimethylphenyl, 3,4-methylenedioxyphenyl;
  • the C 1-6 Alkenyl for example, vinyl, propenyl, substituted C 1-6 alkenyl, such as styryl and the like.
  • *P represents a chiral phosphorus atom.
  • the present invention also provides two diastereomers of the aforementioned compound BG012 to compound BG023 based on the chiral phosphorus atom P.
  • more preferred compounds have the following structure:
  • aryl in the present invention refers to a monocyclic or polycyclic carbocyclic ring system having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indan Base, indenyl, etc.
  • the aryl group of the present invention includes any one of unsubstituted aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups.
  • heteroaryl refers to a monocyclic or polycyclic (for example, bi-, or tricyclic, or more cyclic) aryl group.
  • aryl ring atoms one or more ring atoms are selected from S, O and N, and the remaining ring atoms are carbon, where any one of N or S contained in the ring can be optionally oxidized.
  • Heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, furyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazole, oxadiazole Azole, thiophenyl, furyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, etc.
  • aralkyl or "arylalkyl” refers to an alkyl group containing aryl substitution, such as benzyl, benzhydryl, trityl, phenethyl, and diphenylethyl.
  • 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.
  • alkenyl refers to an unsaturated hydrocarbon chain group having one or two double bonds, preferably one double bond, and 2-10 carbon atoms.
  • the substituted aryl group, substituted alkyl group, and substituted cycloalkyl group refer to the aryl group, alkyl group, and ring as defined above in which one or more hydrogen atoms in the aryl group or alkyl group are replaced by a substituent group Alkyl
  • the hydroxy protecting group may be (C 1-10 alkyl or aryl) 3 silyl group, for example: triethylsilyl, triisopropylsilyl, tert-butyldimethyl Silyl, tert-butyl diphenylsilyl, etc.; can be C 1-10 alkyl or substituted alkyl, for example: methyl, tert-butyl, allyl, benzyl, methoxymethyl, ethoxy Group ethyl, 2-tetrahydropyranyl (THP), etc.; can be (C 1-10 alkyl or aryl) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1- 6 alkyl or C 6-10 aryl)sulfonyl; it can also be (C 1-6 alkoxy or C6-10 aryloxy)carbonyl.
  • silyl group for example: triethylsilyl,
  • leaving group is known to those skilled in the art (Advanced Organic Chemistry: reactions, mechanisms and structure 4th edition, edited by Jerry March, John Wiley and Sons; 1992, p. 351-357 pages), when the substrate molecule undergoes a substitution reaction (such as a nucleophilic substitution reaction), the leaving group is subsequently substituted; leaving groups include but are not limited to halogens (F, Cl, Br and I), Preferably Cl, Br or I; tosylate group, mesylate group, triflate group, acetate group and the like.
  • *P means that the phosphorus atom is chiral, and it has the corresponding Cahn-Ingold-Prelog designation of "R” or "S”, which has the conventional meaning recognized in the art.
  • the present invention also provides a preparation method of the dinucleoside precursor compound, including:
  • L is a leaving group, and the leaving group is selected from halogen, such as Cl, Br, I, p-toluenesulfonic acid group, methylsulfonic acid group, or trifluoroacetate group;
  • R 1 and R 2 are each independently selected from H, C 1-6 alkyl, cycloalkyl, aryl, aralkyl, C 1-6 alkenyl or heterocyclic group;
  • the five-membered heterocyclic group G is optionally substituted by one or more substituents; the substituents are independently selected from halogen, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2.
  • the compound NBI (where L is a leaving group, preferably Cl, Br, I) and SM3 in a suitable solvent solvent (preferably N,N-dimethylformamide, N,N-dimethyl ethyl Amide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane) to be converted into a compound of formula (I), preferably an acid binding agent such as DIEA, and a catalyst are added to the reaction system Sodium iodide.
  • a suitable solvent solvent preferably N,N-dimethylformamide, N,N-dimethyl ethyl Amide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane
  • a suitable solvent solvent preferably N,N-dimethylformamide, N,N-dimethyl ethyl Amide, N
  • the purified compound of general formula (I) was obtained by HPLC preparation and purification, and lyophilization, which was characterized by 1 H-NMR and 13 C-NMR.
  • the present invention provides a method for preparing a compound of formula (Ia), which comprises: reacting compound SM3 with compound NBII to convert compound Ia into compound Ia, specifically the following scheme A:
  • compound Ia is further separated to obtain enantiomers Rp-Ia and Sp-Ia.
  • 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 halogens, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2 , protecting group-containing amino, alkoxy, alkylamino, C 2 -C 12 alkenyl substituted amino, cycloalkyl substituted amino, arylamino, alkylcarbonyl, arylcarbonyl, Alkyl carboxy, aryl carboxy, and amide.
  • substituents are independently selected from halogens, -OH, hydroxyl groups containing protective groups, -NO 2 , -CN, -NH 2 , protecting group-containing amino, alkoxy, alkylamino, C 2 -C 12 alkenyl substituted amino, cycloalkyl substituted amino, arylamino, alkylcarbonyl, arylcarbonyl, Alkyl
  • R 3 is H, Me, ethyl, isopropyl, t-Bu, phenyl, p-methylphenyl, p-methoxyphenyl, 2-chloro-4-methoxyphenyl, Or trifluoromethylphenyl, etc., and it is more preferable that R 3 is a methyl group.
  • Reaction conditions (a) KO 2 CCH2CO 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 two Imidazole, 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 preparation method can refer to the literature: Tetrahedron Letters 43 (2002) 1161-1164.
  • the compounds BG001 to BG011 of the present invention can be prepared according to the method described in the above compound Ia.
  • the compound represented by general formula (Ib) can be combined with SM3 (where L is a leaving group, such as Cl, Br, I, etc.) and SM3 under the protection of nitrogen according to the method described in Scheme B.
  • 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 are added to the reaction system.
  • the purified compound of general formula (Ib) was obtained by HPLC preparation and purification, and lyophilization, which was characterized by 1 H-NMR and 13 C-NMR.
  • compound Ib is further separated to obtain enantiomers Rp-Ib and Sp-Ib.
  • NBIII can be prepared according to the following scheme 1b:
  • the compounds BG012 to BG023 provided by the present invention can be prepared according to the method of Scheme B above.
  • the compound of general formula (Ic) can be prepared according to the method described in Scheme C, for example, the compound NBIV (where L is a leaving group, such as Cl, Br, I, etc.) and SM3 under nitrogen protection, Reaction in a solvent (preferably N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane), Preferably, an acid binding agent such as DIEA and a catalyst sodium iodide are added to the reaction system.
  • 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 are added to the
  • the purified compound of general formula (Ic) was obtained by HPLC preparation and purification, and lyophilization, which was characterized by 1 H-NMR and 13 C-NMR.
  • the compound (I) of the present invention When the compound (I) of the present invention is in a free form, it can be converted into a 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 a solvate (such as a hydrate, etc.), and both solvate and nonsolvate are included in the scope of the present invention.
  • the salt of compound (I) of 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 Salts formed by amines such as triethylamine, trimethylamine, tert-butylamine, pyridine, etc., basic amino acids such as arginine, lysine, ornithine, etc., and 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 Salts formed by amines such as triethylamine, trimethylamine, tert-butylamine, pyridine, etc., basic amino acids such as arginine, lysine, ornithine, etc., and ammonia
  • amines such as triethylamine, trimethylamine, tert-but
  • the inventors studied the pharmacodynamic activity of a large number of compounds, and analyzed the metabolic processes of these compounds in vivo, and finally determined that in the general formula of compound I, the five-membered heterocycle, the carbonyl group between the heteroatoms A and B, and the five-membered ring Or a conjugated system consisting of a double bond outside the ring is an important pharmacodynamic group of compound I, and it is also the key to satisfying that such compounds can absorb, distribute and release the active substance SM3 in the body and exert their pharmacological effects.
  • the inventors have studied the pharmacokinetics of compounds with different structural series and in vitro and in vivo pharmacodynamics, and initially believes that the carbonate esters of compound Ia and compound Ib are first hydrolyzed in the in vivo microenvironment, and then compounds Iaa and Ibb containing double bonds are obtained.
  • Compound Iaa and compound Ibb are isomerized by enol formula or Schiff base, and finally compound Iaa and compound Ibb are isomerized to obtain compound SMP, and SMP is finally decomposed into compound SM3 in the body to exert its efficacy.
  • the decomposition mechanism in the body is as follows:
  • the present invention provides a dinucleotide precursor compound with a novel structure, which exhibits excellent anti-HBV activity and good bioavailability in vivo. Compared with the existing dinucleotide prodrugs:
  • the compound (I) provided by the present invention has a variety of unexpected technical effects such as high pharmacodynamic activity, low dosage, and low toxic and side effects; in the 35 S-labeled dinucleotide precursor compound for mice
  • the administration of BG002 as a representative showed that after absorption, the compound rapidly distributed from the central ventricle to the tissue outside the blood vessel. And a large amount of concentration in the liver, only a small dose was observed in other tissues.
  • the present invention provides a method for preparing compound I, which is easy to operate and has high yield and purity.
  • the present invention also separates two diastereomerically pure compounds of compound I. Preliminary studies have found that compounds with S configuration have significant advantages in terms of solubility, which means that compounds with S configuration may have advantages in preparing preparations.
  • BG002 Add 0.3g of BG002 to the reaction solution, dissolve it with 50mL of 50% CH3CN/H2O mixture, filter through a 0.45um organic filter membrane, and purify the filtrate by preparative HPLC, then preparative HPLC for desalination, concentration to remove most of the CH3CN, and freeze-drying to obtain White solid BG002a 47mg, BG002b 60mg, HPLC ⁇ 99%.
  • Tetrahydrofuran 250ml
  • purified water 120ml
  • BG004-03 (12.7g, 48.7mmol)
  • Rh(OAc) 4 165mg, 0.37mmol
  • the temperature was reduced to room temperature. It was concentrated under reduced pressure, and the remaining solution was extracted 3 times with ethyl acetate.
  • the combined organic layer was extracted and washed with saturated sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate. Filtered and concentrated to obtain BG004-04 (12.0 g) as a yellow oil.
  • BG004-06 (9.98g, 36.1mmol), add absolute ethanol (230ml), stir to dissolve. Then, palladium hydroxide on carbon (20%, 490 mg) was added, the hydrogen balloon was replaced three times, and the reaction was stirred for 70 min. Filter and concentrate the filtrate under reduced pressure. A white solid BG004-07 (6.56 g, 98%) was obtained.
  • hydroxyacetone (21.0g, 280mmol) and dichloromethane (200ml) were added to the reaction flask.
  • the temperature was lowered to 0°C, and triphosgene (30.0 g, 110 mmol) was added. Control the temperature ⁇ -8°C, and add N,N-dimethylaniline (37.0g, 300mmol) dropwise.
  • the temperature was controlled at 0°C, the reaction was stirred for 15 minutes, and then the reaction was conducted at room temperature for more than 2 hours.
  • the temperature of the reaction solution was lowered to 5°C, and then frozen 3M diluted hydrochloric acid (40ml), water (30ml) and saturated sodium chloride (30ml) were used in sequence.
  • the organic layer was dried by adding anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to 1/2 volume, and refluxed for 3 hours. After being concentrated to dryness, an oily substance was obtained. The temperature was raised to 170° C. and stirred for 2.5 hours. At this time, the reaction liquid was dark gray. Then under reduced pressure distillation (130-140°C/30mmHg), a light yellow oily substance BG012-02 (15g) was obtained.
  • 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 were selected for the experiment. 176 mice were divided into 22 groups with 8 mice in each group. The specific groups and the administration of each group of mice are as follows:
  • the day of administration was D1, and the administration was administered every afternoon for the following 8 weeks.
  • the mice in each group were weighed in the morning on D15/29/43/57, and blood was taken from the orbit and the number of HBV DNA copies in the blood was detected. The animals were sacrificed after 8 weeks.
  • D0 is the day before administration.
  • the anti-HBV effects of the 20 tested compounds were better than the control compound SB9200, and the antiviral effects of BG001, BG002, BG003, BG004 and BG005 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.
  • the structurally modified precursor compounds BG001-BG020 especially BG001, BG002, BG004, BGB005, etc. can significantly reduce liver HBV DNA, and these reductions are statistically significant.
  • 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 CTG method was used to test the 50% inhibitory concentration of the cell proliferation of compounds BG001, BG002, BG007, BG009, BG015, BG018, BG021, BG022 and SB9200 in human normal lung fibroblasts (MRC-9) and human umbilical vein endothelial cells (HUVEC) (IC 50 ).
  • the other compounds provided by the present invention are subjected to the same anti-HBV activity study, pharmacokinetic study and in vitro cytotoxicity study, and the experimental results show that compared with SB9200, significant improvement has been achieved.

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Abstract

提供了一种结构新颖的含双核苷酸结构的化合物，还提供了所述含双核苷酸结构的化合物的制备方法，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。另一方面，涉及分离得到的光学异构体纯的含双核苷酸结构化合物，其具有显著的抗HBV DNA活性、使用剂量低、安全性高等优点，并且还具有良好的药代动力学特性。

Description

含双核苷酸结构的化合物 技术领域
本发明涉及一种结构新颖的含双核苷酸结构的化合物。还涉及本发明的含双核苷酸结构的化合物的制备方法，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
乙肝是我国社会负担最大的疾病之一。目前，我国约有1亿左右的人群为乙肝病毒携带者，约占我国总人口数的8％-10％，慢性乙肝患者(肝脏已出现炎性病变)约2000万人。据估计，全世界有3亿5千万慢性HBV携带者。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。
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
本发明提供了一含双核苷酸结构的化合物；本发明另一方面还提供了这些含双核苷酸结构的化 合物的制备方法以及其在制备治疗HBV感染的药物中的应用。
首先，本发明提供了由式I表示的化合物或其盐及其由式Sp-I和Rp-I表示的相应基于手性磷原子P的非对应异构体或其盐：
Figure PCTCN2019115304-appb-000001
其中，G为五元杂环基；
R 1、R 2独立的选自H、被一种或多种取代基取代或未被取代的C 1～6烷基、环烷基、芳基、芳烷基，C 2～6烯基或杂环基；
所述的五元杂环基G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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基、酰胺基；*P表示手性磷原子。
进一步的优选，式I化合物中，五元杂环基中含有两个杂原子，两个杂原子之间存在环羰基，并且至少含有一个环内双键或环外双键，杂原子，羰基以及双键构成共轭体系。
在本发明的另一优选实施方式中，所述式(I)化合物，其中，G为下式所示的基团：
Figure PCTCN2019115304-appb-000002
其中，
A、B独立的选自-NR 6-，-O-和-CH 2-，但A和B不同时为-CH 2-；进一步的优选，A和B同时为-O-；或者A和B中至少一个为-O-，另一个为-NR 6-，或-CH 2-；
R 3、R 4、R 5、R 6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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基和酰胺基。
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。
更进一步的优选，A和B同时为-O-，或者A和B中一个为-O-，另一个为-NH-；R 1、R 2、R 3、R 4、R 5各自独立的选自H、C 1～6烷基、C 5～12芳基，C 1～6烷基取代的C 5～12芳基，C 1～6烷氧基取代的C 5～12芳基，卤素取代的C 1～6烷基，和卤素取代的C 5～12芳基。
其中，所述C 1～6烷基优选为甲基，乙基，异丙基，叔丁基，正丁基；所述卤素为F，Cl，Br，I，优选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例中，本发明还提供了由式Ia表示的化合物及其由式R p-Ia和S p-Ia表示的相应基于手性磷原子P的非对映异构体；
Figure PCTCN2019115304-appb-000003
其中，
A和B独立的选自-O-和-NH-；优选，A和B同时为-O-，或者A和B中一个为-O-，另一个为-NH-；
R 1、R 3各自独立的选自H、C 1～6烷基，如甲基、乙基、异丙基、正丁基、叔丁基，C 5～12芳基，C 1～6烷氧基，C 1～6烯基，或被一种或多种取代基取代的C 5～12芳基，C 1～6烷氧基，C 1～6烯基，C 1～6烷基。
进一步的优选，式(I)化合物中，A和B同时为-O-或-NH-；R 1、R 3同时为H，或者R 1为H，R 3为甲基，乙基，异丙基，叔丁基，C 5～12芳基，或被一种或多种取代基取代的C 5～12芳基；或者，A和B其中一个为-O-，另一个为-NH-，R 1为H，R 3为甲基，乙基，异丙基，叔丁基，C 5～12芳基，或被一种或多种取代基取代的C 5～12芳基；
所述取代基独立的选自C 1～6烷基，C 1～6烷氧基，卤素，优选甲基，乙基，异丙基，叔丁基，F，Cl，Br，甲氧基，乙氧基，三氟甲氧基，氯代乙氧基；所述C 5～12芳基，为苯基，噻吩基，吡咯基，呋喃基；所述C 1～6烷基取代的C 5～12芳基优选为对甲基苯基，三氟甲基苯基，和二甲基苯基。
更进一步的，本发明优选的代表化合物，用式(Ia)表示，其中，R 1为H，A，B，R 3如下表所示：
Figure PCTCN2019115304-appb-000004
*P表示手性磷原子。
相应的，本发明还提供了上述化合物BG001～化合物BG011基于手性磷原子P的两种非对映异构体。
在本发明的另一个优选实施例中，本发明还提供了由式Ib表示的化合物及其由式R p-Ib和S p-Ib表示的相应基于手性磷原子P的非对应异构体；
Figure PCTCN2019115304-appb-000005
其中，
A和B独立的选自-O-和-NH-；优选，A和B同时为-O-，或者A和B中一个为-O-，另一个为-NH-；R 1、R 4、R 5各自独立的选自H、C 1～6烷基，如甲基、乙基、异丙基、正丁基、叔丁基，C 5～12芳基，C 1～6烷氧基，C 1～6烯基，或被一种或多种取代基取代的C 5～12芳基，C 1～6烷氧基，C 1～6烯基，C 1～6烷基。
优选，R 1、R 4、R 5均为H。
进一步的优选，式(Ib)化合物中，A和B同时为-O-，R 1、R 4、R 5同时为H，或者R 4、R 5为H，R 1为C 1～6烷基，如甲基、乙基、异丙基、正丁基、叔丁基，C 5～12芳基，C 1～6烷氧基，C 1～6烯基，或被一种或多种取代基取代的C 5～12芳基，C 1～6烷氧基，C 1～6烯基，C 1～6烷基；或者，优选A和B同时为-NH-，R 1、R 4、R 5同时为H；或者R 4、R 5为H，R 1为C 1～6烷基，如甲基、乙基、异丙基、正丁基、叔丁基，C 5～12芳基，C 1～6烷氧基，C 1～6烯基，或被一种或多种取代基取代的C 5～12芳基，C 1～6烷氧基，C 1～6烯基，C 1～6烷基；
所述取代基独立的选自C 1～6烷基，如甲基、乙基、异丙基、叔丁基，C 1～6烷氧基，如甲氧基、乙氧基、三氟甲氧基、氯代乙氧基，卤素，优选，F、Cl、Br；所述C 5～12芳基，优选为苯基，噻吩基，吡咯基，呋喃基；所述C 1～6烷基取代的C 5～12芳基优选为对甲基苯基，三氟甲基苯基，和二甲基苯基，3,4-亚甲基二氧基苯基；所述的C 1～6烯基，例如，乙烯基，丙烯基，取代的C 1～6烯基，例如苯乙烯基等。
更进一步的，本发明优选的代表化合物，用式(Ib)表示，其中，R 4、R 5同时为H，R 1如下表所示：
Figure PCTCN2019115304-appb-000006
*P表示手性磷原子。
对应的，本发明还提供了上述化合物BG012～化合物BG023基于手性磷原子P的两种非对映异构体。
在本发明的另一方面，更优选的化合物具有如下结构：
Figure PCTCN2019115304-appb-000007
本发明所述的术语“芳基”是指具有一个或两个芳环的单环或多环碳环系统，所述芳环包括但不限于苯基、萘基、四氢萘基、茚满基、茚基等。本发明的芳基包括任何一种未取代的芳基、取代的芳基、杂芳基和取代的杂芳基。
术语“杂芳基”是指一种单环或多环的(例如，二、或三环的，或更多环的)芳基，在芳基环原子中，一个或一个以上环原子选自S、O和N，而剩余的环原子是碳，其中环内包含的任何一种N或S可以任选地被氧化。杂芳基包括，但不仅限于，吡啶基、吡嗪基、嘧啶基、吡咯基、吡唑基、呋喃基、咪唑基、噻唑基、噁唑基、异噁唑基、噻二唑、噁二唑、苯硫基、呋喃基、喹啉基、异喹啉基、苯并咪唑基、苯并噁唑基、喹喔啉基等。
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- 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；C 1- 6烷基基团的例子包括甲基、乙基、丙基、异丙基、正丁基、叔丁基、新戊基和正己基。
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。
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。
术语“烯基”是指具有一个或两个双键，优选具有一个双键，具有2-10个碳原子的不饱和烃链基团。
所述的取代芳基、取代烷基、取代环烷基是指所述的芳基、烷基中的一个或多个氢原子被取代基所置换的如前述定义的芳基、烷基、环烷基，所述取代基包括但不限于卤素(如-F，-Cl，-Br，-I)，-OH，保护的羟基，-NO 3，-CN，-NH 2，保护的氨基，烷氧基(如CH 3O-，C 2H 5O-等)、烷基氨基(如CH 3NH-，C 2H 5NH-，异丙基氨基等)、C 2-C 12烯基取代氨基(例如，包括但不限于CH 2＝CHNH-，CH 3CH＝CHNH-，CH 2＝CHCH 2NH-等)、环烷基取代氨基(例如，包括但不限于环己烷氨基，环戊烷氨基等)、芳基氨基(例如，包括但不限于苯胺基，对甲苯氨基等)、烷基羰基(例如，包括但不限于乙酰基、丙酰基等)、芳基羰基(例如，包括但不限于苯甲酰基)、烷基羧基(例如，包括但不限于CH 3COO-，C 2H 5COO-等)、芳基羧基(例如，包括但不限于苯甲羧基)、酰胺基(例如，包括但不限于乙酰氨基，甲酰胺基等)；其中烷基氨基优选-NH-C 1-12-烷基，如-NH-甲基；环烷基取代氨基优选-NH-C 3-12-环烷基。
术语“含保护基的羟基”是指羟基被保护基保护，所述的羟基保护基为本领域常用的如参见文献(“Protective Groups in Organic Synthesis”，5Th Ed.T.W.Greene&P.G.M.Wuts)中的羟基保护基团。作为示例，优选地，所述的羟基保护基可以是(C 1-10烷基或芳基) 3硅烷基，例如：三乙基硅基，三异丙基硅基，叔丁基二甲基硅基，叔丁基二苯基硅基等；可以是C 1-10烷基或取代烷基，例如：甲基，叔丁基，烯丙基，苄基，甲氧基甲基，乙氧基乙基，2-四氢吡喃基(THP)等；可以是(C 1-10烷基或芳香基)酰基，例如：甲酰基，乙酰基，苯甲酰基等；可以是(C 1-6烷基或C 6-10芳基)磺酰基；也可以是(C 1-6烷氧基或C6-10芳基氧基)羰基。
术语“含保护基的氨基”是指氨基被氨基保护基保护，常用的氨基保护基例如可以是，苄氧羰基，叔丁氧羰基，丙烯氧羰基(Alloc)等。
术语“离去基团”对本领域技术人员而言其功能以及可选择性是已知的(Advanced Organic Chemistry:reactions,mechanisms and structure第4版，由Jerry March,John Wiley和Sons编；1992，第351-357页)，在底物分子进行取代反应时(如亲核取代反应)，离去基团随后被取代；离去基团包括但不限于：卤素(F，Cl,Br和I)，优选Cl，Br或I；甲苯磺酸酯基，甲磺酸酯基，三氟甲基磺酸酯基，乙酸酯基等。
本发明所使用的“任选的”或“任选地”是指后续描述的事件或事项可能发生但未必繁琐，并且这一描述包括事件或事项发生的情况和事件或事项不发生的情况，举例说明，“任选的，进一步将化合物I分离得到非对映异构体Rp-I和Sp-I”是指可以将化合物I进一步分离得到非对映异构体Rp-I和Sp-I，也可以不对化合物I做进一步的分离。
术语“*P”是表示磷原子是手性的，并且其具有对应的“R”或“S”的Cahn-Ingold-Prelog命名，其具 有本领域公认的常规含义。
本发明说明书中提到的“化合物SM3”，“SM3”以及“SB9000”和“化合物SB9000”具有相同的含义，均是指具有如下结构的化合物：
Figure PCTCN2019115304-appb-000008
另一方面，本发明还提供了所述双核苷前体化合物的制备方法，包括：
使化合物SM3与NBI反应，转化成式(I)化合物：
Figure PCTCN2019115304-appb-000009
任选的，进一步将化合物I分离得到分对映异构体Rp-I和Sp-I；
其中，L为离去基团，所述离去基团选自：卤素，如Cl、Br、I，对甲苯磺酸基、甲基磺酸基、或三氟乙酸基；
R 1、R 2各自独立的选自H、C 1～6烷基、环烷基、芳基、芳烷基，C 1～6烯基或杂环基；
所述的五元杂环基G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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基、酰胺基；
优选的，将化合物NBI(其中L为离去基团，优选为Cl，Br，I)与SM3在合适的溶剂溶剂(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，转化成式(I)化合物，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，得到纯化后的通式(I)化合物，使用 1H-NMR和 13C-NMR对其定性。
举例说明的，本发明提供了一种式(Ia)化合物的制备方法，包括：使化合物SM3与化合物NBII反应，转化成化合物Ia，具体如下方案A：
Figure PCTCN2019115304-appb-000010
任选的，进一步将化合物Ia分离得到分对映异构体Rp-Ia和Sp-Ia。
上述方案中式NBII所示化合物根据A、B、R 1、R 3的具体取代基进行合成。举例说明的，例如当A，B均为O，时，NBII可以按照如下方案1a或方案2a制备：
Figure PCTCN2019115304-appb-000011
R 3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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基和酰胺基。
进一步的，优选R 3为H,Me，乙基，异丙基，t-Bu，苯基，对甲基苯基，对甲氧基苯基，2-氯-4-甲氧基苯基，或三氟甲基苯基等，更进一步的优选R 3为甲基。
Figure PCTCN2019115304-appb-000012
反应条件：(a)KO 2CCH2CO 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。
制备方法可以参考文献：Tetrahedron Letters 43(2002)1161-1164。
本发明所述的化合物BG001～BG011均可按照上述化合物Ia所述的方法制得。
进一步的，举例说明，通式(Ib)所示化合物可按照方案B描述的方法，将化合物NBⅢ(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，得到纯化后的通式(Ib)化合物，使用 1H-NMR和 13C-NMR对其定性。
Figure PCTCN2019115304-appb-000013
任选的，进一步将化合物Ib分离得到分对映异构体Rp-Ib和Sp-Ib。
上述方案中式NBⅢ所示化合物根据A、B，R 1的具体取代基情况进行合成。举例说明的，例如当A，B均为O，时，NBIII可以按照如下方案1b制备：
Figure PCTCN2019115304-appb-000014
以THF和水做溶剂，室温反应条件下，R 1CHO与
Figure PCTCN2019115304-appb-000015
进行反应，然后将反应底物在二氯甲烷作溶剂，三苯基磷存在条件下，进行溴代反应，得到化合物NBIII，文献Tetrahedron Letters 43(2002)1161-1164也公开了此反应具体方法，其公开的内容引入本发明中。
本发明提供的化合物BG012～BG023均可按照上述方案B的方法制得。
进一步的，举例说明，通式(Ic)化合物可按照方案C描述的方法制备，如将化合物NBⅣ(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，得到纯化后的通式(Ic)化合物，使用 1H-NMR和 13C-NMR对其定性。
文献Tetrahedron Letters 43(2002)1161-1164和文献ORGANIC LETTERS 2011 Vol.13，No.17 4720-4723等也都公开了上述制备NBI的类似反应，当A、B，R1，R3，R4，R5为本发明前述其他基团时候，均可参考类似方法制备得到相应的化合物NBI，制备这些不同取代基替换的化合物NBI是本领普通技术人员在本发明公开的方法基础上通过常规调整反应底物可以实现的。
当本发明的化合物(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。
本发明人通过研究大量的化合物药效活性，以及分析检测这些化合物的体内代谢过程，最终确定，化合物I通式中，五元杂环、杂原子A和B之间的羰基以及五元环内或环外含有一个双键构成的共轭体系，是化合物I的重要药效基团，也是满足这类化合物能够在体内吸收，分布以及释放活性物质SM3，发挥药效的关键。
发明人通过对不同结构系列的化合物及体内外药效药代研究，初步认为，化合物Ia和化合物Ib，在体内微环境中，碳酸酯先水解，然后得到含双键的化合物Iaa和化合物Ibb，化合物Iaa和化合物Ibb中通过烯醇式或席夫碱进行异构化，最终化合物Iaa和化合物Ibb异构化均得到化合物SMP，SMP最终在体内分解成化合物SM3发挥药效作。体内分解机制如下所示：
Figure PCTCN2019115304-appb-000016
本发明提供了一种结构新颖的双核苷酸前体化合物，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：
(1)本发明提供的化合物(I)具有药效活性高，使用剂量低，毒副作用小等多方面意料不到的技术效果；在对对小鼠进行 35S标记的双核苷酸前体化合物以BG002为代表给药表明，在吸收之后，化合物迅速地从中央室分布到血管外的组织中。并在肝脏大量的集中，在其他组织中只观察到很少剂量。研究表明，在吸收之后，化合物BG002在肝脏中发生显著的分布。由于肝脏是HBV和HCV的靶器官，这一研究显示本发明提供的双核苷酸前体化合物能够容易的进入肝细胞。这些双核苷酸前体化合物在老鼠模型中的有效抗病毒活性也可以被上述研究支持。
(2)实验表明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。
(3)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。
(4)本发明提供了一种化合物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，经过发明人的初步研究发现其中S构型的化合物在溶解性方面具有显著的优势，这意味着，S构型的化合物在制备制剂方面可能具备优势。
具体实施例
以下结合具体实施例对本发明的内容以及有益效果做进一步说明。应当理解，下面这些实施是本发明的一些优选实施方式的进一步阐释，不应该用于限制本发明。
实施例1 化合物BG002的合成方法：
Figure PCTCN2019115304-appb-000017
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BG002a、BG002b：
Figure PCTCN2019115304-appb-000018
反应液中加入0.3g BG002用50mL的50％CH3CN/H2O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CH3CN，冻干，得到白色固体BG002a 47mg，BG002b 60mg，HPLC≥99％。
制备纯化方法
Figure PCTCN2019115304-appb-000019
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BG004的合成方法：
Figure PCTCN2019115304-appb-000020
(1)BG004-03制备：
N 2保护，在0℃，于反应瓶依次加入乙腈(420ml)、BG004-02(12.0g，51.3mmol)、对甲苯磺酰叠氮(12.3g，51.3mmol)，之后加入三乙胺(21.4ml，15.4mmol)，维持0℃，搅拌30min，之后室温反应4h。反应液减压浓缩干，用150ml×2乙醚/石油醚＝2:1混合试剂打浆。抽滤。滤液减压浓缩干，用乙酸乙酯/正己烷混合溶剂柱层析纯化得油状物BG004-03 12.79g，收率96％。
(2)BG004-04制备：
于反应瓶依次加入四氢呋喃(250ml)、纯化水(120ml)、BG004-03(12.7g，48.7mmol)、Rh(OAc) 4(165mg，0.37mmol)，回流反应5h。反应结束，降至室温。减压浓缩，剩余溶液用乙酸乙酯萃取3次。合并有机层饱和氯化钠水溶液萃洗。有机层无水硫酸钠干燥。过滤，浓缩，得黄色油状物BG004-04(12.0g)。
(3)BG004-06制备：
N 2保护下，量取无水四氢呋喃(230ml)，加入BG004-04(11.76g，46.9mmol)，搅拌溶清。降温至0℃加入CDI(15.3g，94.4mmol)，加毕滴加DIEA(19mmol)。维持0℃搅拌反应5h，室温反应过夜。反应液减压浓缩。浓缩结束加入乙酸乙酯和5％KHSO4水溶液，搅拌均匀，静置分层。有机层依次用水、饱和氯化钠萃洗。无水硫酸钠干燥，过滤，浓缩，得油状物。经乙酸乙酯正庚烷柱层析得无色油状物BG004-06(9.99g)。
(4)BG004-07制备：
称量BG004-06(9.98g，36.1mmol)加入无水乙醇(230ml)，搅拌溶解。之后加入氢氧化钯碳(20％，490mg)，氢气球置换三次，搅拌反应70min。过滤，减压浓缩滤液。得白色固体BG004-07(6.56g，98％)。
(5)BG004-08制备：
氮气保护下，量取无水二氯甲烷150ml，加入BG004-07(6.47g，34.7mmol)、无水DMF(350μl)，控温0℃，滴加草酰氯(3.33ml，38.2mmol)。滴加结束，维持0℃，搅拌反应30min，室温反应1h。随后减压浓缩至干。残余物加入无水二氯甲烷(200ml)搅拌溶解。N2保护下，控温-78℃，滴加Bu 4NBH 4(9.86g，38.3mmol)二氯甲烷(60ml)溶液。滴加结束后，维持-78℃搅拌反应1h，滴加0.1N盐酸(100ml)淬灭反应。缓慢升至室温。减压浓缩，剩余物加入乙酸乙酯(200ml)、水(50ml)。摇匀，分液。水层加入氯化钠至饱和，加入乙酸乙酯(100ml)萃取。合并乙酸乙酯层。饱和氯化钠水溶液萃洗。无水硫酸钠干燥，减压浓缩得油状物，经乙酸乙酯/正己烷柱层析纯化得油状物BG004-08(4.25g，收率69％)。
(6)BG004-09制备：
氮气保护下，量取二氯甲烷150ml，加入BG004-08(2.0g，11.6mmol)，降温至0℃。随后依次加入四溴化碳(4.62g，13.9mmol)、三苯基磷(3.35g，12.8mmol)。维持0℃搅拌反应30min，室温搅拌反应1h。反应液减压浓缩。经柱层析纯化得BG004-09(2.15g，收率79％)。
(7)BG004制备：
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)、BG004-09(1.9g，8.4mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG004 4.56g，纯度97％，收率80％。MS Calcd:741；MS Found:742[M+H] +。
参考实施例2方法制备化合物BG001，BG003，BG005～BG011：
化合物编号 MS Calcd MS Found：[M+H] + 化合物编号 MS Calcd MS Found：[M+H] +
BG001 685 686 BG008 829 830
BG003 713 714 BG009 791 792
BG005 727 728 BG010 684 685
BG006 727 728 BG011 683 684
BG007 741 742      
实施例3 化合物BG012的合成方法：
Figure PCTCN2019115304-appb-000021
(1)BG012-02制备：
氮气保护下，于反应瓶加入羟基丙酮(21.0g，280mmol)、二氯甲烷(200ml)。降温至0℃，加入三光气(30.0g，110mmol)。控温≤-8℃，滴加N,N-二甲基苯胺(37.0g，300mmol)。控温0℃，搅拌反应15min，之后室温反应2h以上。反应液降温至5℃，依次用冰冻3M稀盐酸(40ml)、水(30ml)、饱和氯化钠(30ml)。有机层加入无水硫酸镁干燥，过滤，减压浓缩至1/2体积，回流3h。之后浓缩至干，得油状物，升温至170℃搅拌2.5h，此时反应液为暗灰色。随后经减压蒸馏(130-140℃/30mmHg)，得淡黄色油状物BG012-02(15g)。
(2)BG012-03制备：
于反应瓶中，依次加入四氯化碳(200ml)、BG012-02(10g，100mmol)，加毕搅拌溶清。之后加入NBS(23g，130mmol)、AIBN(10mg)。回流反应1.5h。降压浓缩至一半体积，过滤，滤液浓缩至干，经减压蒸馏得BG012-03(14g)。
(3)BG012-04制备：
于四氢呋喃水溶液中，加入铟，苯甲醛，BG012-03，室温搅拌反应15min。反应结束，过滤，浓缩，经纯化得BG012-04(收率96％)。
(4)BG012-05制备：
氮气保护下，量取二氯甲烷150ml，加入BG012-04(10g，49mmol)，降温至0℃。随后依次加入四溴化碳(19.3g，58mmol)、三苯基磷(14.0g，53.4mmol)。维持0℃搅拌反应30min，室温搅拌反应1h。反应液减压浓缩。经柱层析纯化得BG012-05(10.5，收率80％)。
(5)BG012制备：
氮气保护下，反应瓶中，依次加入SM3(2g，3.4mmol)、DMF(20ml)、二异丙基乙胺(0.52g，4.0mmol)、碘化钠(0.52g，3.5mmol)、BG012-05(1.08g，4.0mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG012 1.60g，纯度98.5％，收率61％。MS Calcd:775；MS Found:776[M+H] +
参考实施3类似方法制备化合物BG013～BG023；
Figure PCTCN2019115304-appb-000022
Figure PCTCN2019115304-appb-000023
实施例4：受试化合物抗HBV活性研究
选取约200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。176只小鼠分成22组，每组8只。具体分组及每组小鼠给药情况如下：
Figure PCTCN2019115304-appb-000024
给药当天为D1，随后的8周每天下午给药，各组小鼠在D15/29/43/57上午称重，并眼眶取血并检测血液中HBV DNA拷贝数。8周后处死动物。D0为给药前一天。
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BG001、BG002、BG003、BG004和BG005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 PCTCN2019115304-appb-000025
上述结果表明，与对照组相比，结构修饰的前体化合物BG001～BG020，特别是BG001，BG002，BG004，BGB005等能使肝HBV DNA显著降低，并且这些降低具有统计学意义。
实施例5：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。
动物分组及给药信息详见下表。
Figure PCTCN2019115304-appb-000026
Figure PCTCN2019115304-appb-000027
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 PCTCN2019115304-appb-000028
Figure PCTCN2019115304-appb-000029
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BG001、BG002、BG003、BG004和BG005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6：体外细胞毒性：
用CTG方法测试化合物BG001，BG002，BG007，BG009，BG015，BG018，BG021，BG022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。供试化合物在2个细胞系的细胞增殖IC 50结果如下所示。
化合物在MRC-9和HUVEC细胞系的细胞增殖IC 50结果
Figure PCTCN2019115304-appb-000030
Figure PCTCN2019115304-appb-000031
由以上结果可以看出，本发明提供的化合物具有较低的体细胞毒性。
将本发明提供的其他化合物进行同样的抗HBV活性研究，药代动力学研究及体外细胞毒性研究，实验结果显示与SB9200相比，均取得显著的改善。

Claims (15)

  1. 由式(Ⅰ)表示的化合物或其盐：
    Figure PCTCN2019115304-appb-100001
    其中G为五元杂环基；
    R 1、R 2独立的选自H、被一种或多种取代基取代或未被取代的C 1～6烷基、环烷基、芳基、芳烷基，C 2～6烯基或杂环基；
    所述的五元杂环基G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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基、酰胺基；
    *P表示手性磷原子。
  2. 根据权利要求1所述的化合物，其特征在于，G为下式所示的基团：
    Figure PCTCN2019115304-appb-100002
    其中，
    A、B独立的选自-NR 6-，-O-和-CH 2-，但A和B不同时为-CH 2-；
    R 3、R 4、R 5、R 6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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基和酰胺基。
  3. 根据权利要求2所述的化合物，其特征在于，A和B同时为-O-；或者A和B中至少一个为-O-，另一个为-NR 6-，或-CH 2-；或者A和B同时为-NR 6-；
    其中，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；R 1还可以选自C 1～6烯基；
    所述C 1～6烷基，C 1～6烯基和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，C 5～12芳基。
  4. 根据权利要求2所述的化合物，其特征在于，A和B同时为-O-，或者A和B中一个为-O-，另一个为-NH-；
    R 1、R 2、R 3、R 4、R 5各自独立的选自H、C 1～6烷基、C 5～12芳基，C 1～6烷氧基，C 1～6烷基取代的C 5～12芳基，C 1～6烷氧基取代的C 5～12芳基，C 1～6烷基取代的C 1～6烯基。
  5. 根据权利要求2所述的化合物，其特征在于，G为下式所示的基团：
    Figure PCTCN2019115304-appb-100003
    其中，R 1、R 2、R 3、R 4、R 5各自独立的选自H、和C 1～6烷基，对甲基苯基，三氟甲基苯基，二甲基苯基，丙烯基，N-乙基吡咯基，苯氧甲基，苯乙烯，噻吩基，和苯基。
  6. 根据权利要求2所述化合物，其特征在于，具有如下结构：
    Figure PCTCN2019115304-appb-100004
    其中，R 1为H，A，B，R 3如下所示：
    Figure PCTCN2019115304-appb-100005
    *P表示手性磷原子。
  7. 根据权利要求2所述化合物，其特征在于，具有如下结构：
    Figure PCTCN2019115304-appb-100006
    其中，R 4、R 5同时为H，R 1如下所示：
    Figure PCTCN2019115304-appb-100007
    *P表示手性磷原子。
  8. 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I包含两种非对映异构体，表示为R p-I和S p-I：
    Figure PCTCN2019115304-appb-100008
  9. 根据权利要求2～6任一项所述化合物，其特征在于，所述化合物包含两种非对映异构体，表示为R p-Ia和S p-Ia：
    Figure PCTCN2019115304-appb-100009
  10. 根据权利要求2～7任一项所述化合物，其特征在于，所述化合物包含两种非对映异构体，表示为R p-Ib和S p-Ib：
    Figure PCTCN2019115304-appb-100010
  11. 根据权利要求1所述化合物，其特征在于，具有如下结构：
    Figure PCTCN2019115304-appb-100011
  12. 一种权利要求1所述化合物的制备方法，包括：使化合物SM3与NBI反应，转化成式(I)化合物：
    Figure PCTCN2019115304-appb-100012
    任选的，进一步将化合物I分离得到非对映异构体Rp-I和Sp-I；
    其中，L为离去基团，所述离去基团选自：卤素，对甲苯磺酸基、甲基磺酸基、和三氟乙酸基；
    R 1、R 2各自独立的选自H、C 1～6烷基、环烷基、芳基、芳烷基，C 1～6烯基或杂环基；
    所述的五元杂环基G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，含保护基的羟基、-NO 2、-CN、-NH 2、含保护基的氨基、烷氧基、烷基氨基、C 2-C 12烯基取代氨基、环烷基取代氨基、芳基氨基、烷基羰基、芳基羰基、烷基羧基、芳基羧基、酰胺基；
    *P表示手性磷原子；
    优选，向反应体系中加入缚酸剂DIEA，以及催化剂碘化钠。
  13. 一种权利要求1或2所述的化合物在制备用于治疗病毒感染的药物中的应用；优选，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。
  14. 一种药物组合物，包含权利要求1或2所述化合物和至少一种药学上可接受的载体或赋形剂，优选所述药物组合物制备成片剂，胶囊剂，口服混悬剂或固体分散体。
  15. 权利要求14所述的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。
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