WO2017041720A1 - 作为高效的疫苗佐剂的甲羟戊酸通路的抑制剂 - Google Patents

作为高效的疫苗佐剂的甲羟戊酸通路的抑制剂 Download PDF

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WO2017041720A1
WO2017041720A1 PCT/CN2016/098371 CN2016098371W WO2017041720A1 WO 2017041720 A1 WO2017041720 A1 WO 2017041720A1 CN 2016098371 W CN2016098371 W CN 2016098371W WO 2017041720 A1 WO2017041720 A1 WO 2017041720A1
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group
alkyl
hydrogen
alkoxy
aryl
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PCT/CN2016/098371
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French (fr)
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张永辉
夏赟
谢永华
于正森
周晓英
李鑫
李丽平
杨云云
高看照
王科
刘万里
赵萌
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清华大学
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Priority to CN201680051521.7A priority Critical patent/CN108289902B/zh
Priority to KR1020207014743A priority patent/KR20200060540A/ko
Priority to JP2018512602A priority patent/JP7076798B2/ja
Priority to US15/757,893 priority patent/US11382971B2/en
Priority to KR1020187009924A priority patent/KR102116528B1/ko
Priority to EP16843658.2A priority patent/EP3348269B1/en
Priority to CN202210417066.5A priority patent/CN114848656A/zh
Publication of WO2017041720A1 publication Critical patent/WO2017041720A1/zh
Priority to JP2022076745A priority patent/JP2022105179A/ja
Priority to US17/664,134 priority patent/US20220387584A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/02—Immunomodulators
    • 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
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3882—Arylalkanephosphonic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/58—Pyridine rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C07F9/65068—Five-membered rings having the nitrogen atoms in positions 1 and 3 condensed with carbocyclic rings or carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to inhibitors of the mevalonate pathway as a highly effective vaccine adjuvant.
  • the invention also relates to an immunogenic composition comprising an inhibitor of the mevalonate pathway as an adjuvant.
  • Adjuvants play an important role in the development and use of vaccines.
  • Adjuvants are also known as non-specific immunopotentiators. It is not antigenic by itself, but it can enhance the immunogenicity of the antigen or change the type of immune response together with the antigen or pre-injection into the body.
  • Live attenuated vaccines and inactivated vaccines may inherently comprise natural adjuvant components, which may include proteins, lipids and oligonucleotides in particulate form. In fact, many attenuated or inactivated vaccines have a very strong protective effect on the body after immunization.
  • a subunit vaccine is a vaccine made up of components that present the major protective immunogens of pathogenic microorganisms. Due to the rise of modern molecular biology, subunit vaccines have become the main trend in the development and application of modern vaccines because of their convenience in quality control, mass production, safety and reliability. However, subunit vaccines also have short-term protective effects and slow onset of action. Adjuvants that make up for these shortcomings of subunit vaccines are an important part of the development and use of modern vaccines.
  • the most widely used adjuvant in vaccine production is aluminum adjuvant.
  • the aluminum salt was first discovered to have an adjuvant effect in 1926 and was first used in diphtheria vaccine in 1936, but due to some limitations of aluminum adjuvant, such as adjuvant effects. Weak, need to cooperate with antigens with strong immunogenicity to play a good role, especially not able to promote the Th1 response that mediates cellular immunity, resulting in aluminum adjuvant not well able to prevent influenza, HIV, tumors and Diseases such as malaria, so these vaccines urgently require new and effective adjuvants.
  • Adjuvants approved clinically in the United States and Europe to date include the use of aluminum salts, oil-in-water emulsions (MF59 AS03 and AF03) and AS04 (MPL aluminum salts).
  • MF59 AS03 and AF03 oil-in-water emulsions
  • AS04 MPL aluminum salts
  • TLRs Toll-like receptors
  • the mevalonate pathway is a metabolic pathway for the synthesis of isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from acetyl-CoA, which is present in all higher eukaryotes and many viruses.
  • the product of this pathway can be regarded as an activated isoprene unit, which is a synthetic precursor of biomolecules such as steroids and terpenoids.
  • acetoacetyl-CoA is first formed from two molecules of acetyl-CoA, and the resulting acetoacetyl-K-A and acetyl-CoA form 3-hydroxy-3-.
  • HMG-CoA 3-hydroxy-3-methylglutary CoA
  • Mevalonate catalyzes the formation of isoprene pyrophosphate (IPP) by mevalonate under the action of two kinases and one decarboxylase.
  • IPP forms farnesyl pyrophosphate (FPP) catalyzed by FPP synthase (FPPS).
  • FPP branches at downstream pathways, such as cholesterol, ubiquinone, Heme A, Sterol, dolichol, and prenylation (prenylated) protein.
  • FPP can synthesize squalene under the action of squalene synthase (SQS), which synthesizes cholesterol under a series of enzyme catalysis.
  • SQS squalene synthase
  • FPP is capable of undergoing farnesylation of some proteins under the action of farnesyl transferase.
  • FPP synthesizes geranyl geranyl pyrophosphate (GGPP) under the catalysis of GGPP synthase (GGPPS); while GGPP can geraniyl geranylation of some proteins under the action of geranyl leaf acyltransferase Modification to form a prenylated protein.
  • GGPP geranyl geranyl pyrophosphate
  • GGPPS GGPP synthase
  • vaccine adjuvants for the following targets can be developed. 1) thiolase (acetoacetyl-CoA transferase); 2) HMG-CoA synthase; 3) HMG-CoA reductase; 4) mevalonate kinase; 5) mevalonate phosphate (Phosphonomevalonate kinase); 6) mevalonate-5-pyrophosphate decarboxylase; 7) isopentenyl-diphosphate isomerase; 8) farnesyl Farnesyl diphosphate synthase (FPPS); 9) Geranylgeranyl diphosphate synthase (GGPPS); 10) Geranylgeranyl transferase I (II) ).
  • the invention relates to an immunogenic composition
  • a substance that affects the geranylation of protein geranium as an adjuvant may include, but are not limited to, 1) thiolase (acetoacetyl-CoA transferase) inhibitors, 2) HMG-CoA synthase inhibitors, 3) HMG-CoA reductase inhibitors, 4) hydroxyl Valerate kinase inhibitor, 5) phosphomevalonate kinase inhibitor, 6) mevalonate-5-pyrophosphate decarboxylase inhibitor, 7) isopentenyl pyrophosphate isomerase inhibitor, 8) farney a pyrophosphate synthase inhibitor, 9) a geranyl-sacrose pyrophosphate synthase inhibitor, and 10) a geranyl-sacyltransferase (I, II) inhibitor.
  • thiolase acetoacetyl-CoA transferase
  • the invention encompasses the above inhibitors for use as an adjuvant.
  • the invention contemplates the use of the above inhibitors as adjuvants in the preparation of immunogenic compositions.
  • the present invention is also directed to a novel compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, which is an inhibitor of farnesyl pyrophosphate synthase (FPPS), which has the formula:
  • the molecular weight is less than 1000, and Ar is a benzimidazole group or an azabenzimidazolyl group;
  • the present invention is also directed to a novel compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, which is an inhibitor of farnesyl pyrophosphate synthase (FPPS), which has the formula:
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 20, more preferably n is an integer from 1 to 15, more preferably n is an integer from 1 to 12.
  • the present invention is also directed to a novel compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, which is an inhibitor of farnesyl pyrophosphate synthase (FPPS), which has the formula:
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl,
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the compound is selected from the group consisting of
  • the present invention is also directed to a novel compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, which is an inhibitor of farnesyl pyrophosphate synthase (FPPS), which has the formula:
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the compound is selected from the group consisting of
  • the present invention is also directed to a novel compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, as a geranylfolate pyrophosphate synthase inhibitor, said compound having the formula:
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the compound is selected from the group consisting of
  • the invention also relates to the use of the novel compounds, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, as adjuvants in the preparation of an immunogenic composition for the prevention or treatment of a disease.
  • a further aspect of the invention relates to a method of immunizing a subject or host comprising administering to the subject or host an immunogenic composition as defined herein.
  • the invention relates to the following specific embodiments:
  • An immunogenic composition comprising an adjuvant selected from the group consisting of:
  • statin compound is selected from the group consisting of pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, sim Ruvastatin, cerivastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • statin compound is selected from the group consisting of simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • the molecular weight is less than 1000, and Ar is a benzimidazole group or an azabenzimidazolyl group;
  • X is any one of the following: hydrogen, hydroxy, thiol, halogen, alkoxy or alkyl;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 8 are each independently selected from any one of the following: hydrogen, hydroxy, aliphatic, mercapto, halogen, amino, alkyl, -O-(CH).
  • Z is hydrogen, a hydroxyl group, a fatty group, an alkoxy group, an amino group or an alkylamine group.
  • n is an integer of 0 or 1-12.
  • immunogenic composition according to any one of 7 to 10, wherein the compound is any one of the following:
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; Selected from hydrogen, hydroxyl, sulfhydryl, halogen, etc.;
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • n is an integer from 1 to 20.
  • R 3 and R 4 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; It is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl and halogen.
  • R 3 and R 4 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, said alkane
  • the alkyl group in the oxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 1 is selected from hydrogen, C 1-10 alkyl, C 1-10 alkynyl, C 1-10 alkylamino, C 1-10 alkylthio, halogen, And a hydroxy group, a carbazolyl group, a C 1-10 alkoxy group, a C 1-10 alkoxy group substituted by a phenyl group or a pyridyl group, which is optionally substituted with a carbamoyl group.
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the immunogenic composition according to any one of 1 to 40, which comprises one or more antigens.
  • Mers Middle Eastern Respiratory Syndrome
  • Hepatitis B virus Hepatitis B virus
  • Melanoma Melanoma
  • An immunogenic composition according to 46 said another adjuvant selected from the group consisting of aluminum adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, AS01, AS02, AS03, AS04, AS15, CAF01 , ISCOMs (immune stimulating complex), Virosomes (virion particles), GLA-SE, liposomes, edible oils, saponins, AF03, TLR agonists.
  • said another adjuvant selected from the group consisting of aluminum adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, AS01, AS02, AS03, AS04, AS15, CAF01 , ISCOMs (immune stimulating complex), Virosomes (virion particles), GLA-SE, liposomes, edible oils, saponins, AF03, TLR agonists.
  • TLR agonist being selected from (eg, triacyl lipoprotein), a TLR2 stimulating agent (eg, peptidoglycan, zymosan, HMGB1 (high-speed kinesin 1), lipid-phosphorus wall Acid), TLR3 stimulator (double-stranded RNA such as PolyI: C), TLR4 stimulator (such as LPS, MPL, RC529, GLA, E6020), TLR5 stimulator (flagellin), TLR6 stimulator (such as triacoprotein, LPS7/8 stimulant (single-stranded RNA, imiquimod), TLR9 stimulator (DNA, such as CPG ODN), C-lectin ligand (such as kelp polysaccharide), CD1d ligand (eg Alpha-galactosylceramide).
  • TLR2 stimulating agent eg, peptidoglycan, zymosan, HMGB1 (high-speed kinesin 1), lipid-phosphorus wall Acid
  • An immunogenic composition according to 49 adapted for immunization by an injection route.
  • An immunogenic composition according to 49 which is adapted to be immunized by injection into the sole, subcutaneous, intramuscular, abdominal and nasal mucosa of the subject.
  • the immunogenic composition according to any one of the preceding 1 to 51 which is used for the preparation of the following vaccines: BCG, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis D vaccine, hepatitis E vaccine, influenza vaccine, polio vaccine , white vaccination vaccine, measles vaccine, Japanese encephalitis vaccine, rabies vaccine, hemorrhagic fever vaccine, pneumonia vaccine, epidemic vaccine, hepatitis A vaccine, mumps vaccine, influenza vaccine, rubella vaccine, varicella vaccine, AIDS vaccine, malaria vaccine; Cancer treatment and prevention vaccines, including but not limited to melanoma therapeutic vaccines, melanoma preventive vaccines, lung cancer therapeutic vaccines, lung cancer preventive vaccines, bladder cancer preventive vaccines, bladder cancer preventive vaccine therapeutic vaccines, cervical cancer treatments Vaccine, cervical cancer preventive vaccine, bladder cancer therapeutic vaccine, bladder cancer preventive vaccine, breast cancer therapeutic vaccine, breast cancer preventive vaccine, liver cancer therapeutic vaccine, liver cancer preventive
  • a thiolase inhibitor for use as an adjuvant for use as an adjuvant.
  • a HMG-CoA synthase inhibitor for use as an adjuvant for use as an adjuvant.
  • a HMG-CoA reductase inhibitor for use as an adjuvant for use as an adjuvant.
  • a mevalonate kinase inhibitor for use as an adjuvant.
  • a phosphomevalonate kinase inhibitor for use as an adjuvant for use as an adjuvant.
  • a mevalonate-5-pyrophosphate decarboxylase inhibitor for use as an adjuvant.
  • Isopentenyl pyrophosphate isomerase inhibitor for use as an adjuvant.
  • a farnesyl pyrophosphate synthase inhibitor for use as an adjuvant.
  • HMG-CoA reductase inhibitor according to 55 wherein the HMG-CoA reductase inhibitor is a statin.
  • statin is selected from the group consisting of pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin , simvastatin, cerivastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • statin compound is selected from the group consisting of simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • the molecular weight is less than 1000, and Ar is a benzimidazole group or an azabenzimidazolyl group;
  • X is any one of the following: hydrogen, hydroxy, thiol, halogen, alkoxy or alkyl;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 8 are each independently selected from any one of the following: hydrogen, hydroxy, aliphatic, mercapto, halogen, amino, alkyl, -O-(CH).
  • Z is hydrogen, a hydroxyl group, a fatty group, an alkoxy group, an amino group or an alkylamine group.
  • n is an integer of 0 or 1-12.
  • the farnesyl pyrophosphate synthase inhibitor according to any one of 68-71, wherein the compound is any one of the following:
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; Selected from hydrogen, hydroxyl, sulfhydryl, halogen, etc.;
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • farnesyl pyrophosphate synthase inhibitor according to 60 wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the formula: or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof:
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • n is an integer from 1 to 20.
  • n is an integer from 1-12.
  • farnesyl pyrophosphate synthase inhibitor according to 60 wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the formula: or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof:
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl,
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 1 is selected from hydrogen, C 1-10 alkyl, C 1-10 alkynyl, C 1-10 alkylamino, C 1-10 alkyl A thio group, a halogen, a hydroxy group, a carbazolyl group, a C 1-10 alkoxy group, a C 1-10 alkoxy group substituted by a phenyl group or a pyridyl group, which is optionally substituted with a carbamoyl group.
  • R 1 is selected from hydrogen, ethoxy-4-methylphenyl, 4,5,6,7-tetrahydro -2H- indazol-2-yl , (2-carbamoylpyridin-4-yl)methoxy, benzyloxy, hexyloxy, methylthio, octylamino, hexyl, octyl, decyl, oct-1-yn-1- Base, hydroxyl, bromine.
  • R 3 is selected from hydrogen, C 1-10 alkyl, C 1-10 alkoxy.
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • statin is selected from the group consisting of pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, west Rivarstatin, or their pharmaceutically acceptable salts, esters, prodrugs, solvates.
  • statin compound is selected from the group consisting of simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • the bisphosphonate compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of zoledronic acid, pamidronic acid, alendronate, ibandronic acid, neridronic acid, risedronate Acid, opadonic acid, minoxidoic acid.
  • the molecular weight is less than 1000, and Ar is a benzimidazole group or an azabenzimidazolyl group;
  • X is any one of the following: hydrogen, hydroxy, thiol, halogen, alkoxy or alkyl;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 8 are each independently selected from any one of the following: hydrogen, hydroxy, aliphatic, mercapto, halogen, amino, alkyl, -O-(CH).
  • Z is hydrogen, a hydroxyl group, a fatty group, an alkoxy group, an amino group or an alkylamine group.
  • n is an integer of 0 or 1-12.
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; It is selected from the group consisting of hydrogen, hydroxyl, thiol, halogen, and the like.
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • n is an integer from 1-20.
  • n is an integer from 1 to 15.
  • n is an integer from 1-12.
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl,
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 1 is selected hydrogen, C 1-10 alkyl, C 1-10 alkynyl, C 1-10 alkylamino, C 1-10 alkylthio, halo, hydroxy, indol thiazolyl, C 1-10 alkoxy, phenyl or pyridyl substituted C 1-10 alkoxy group, a pyridyl group optionally substituted with carbamoyl.
  • R 1 is selected from hydrogen, ethoxy-4-methylphenyl, 4,5,6,7-tetrahydro -2H- indazol-2-yl, (2-carbamoyl pyridine 4-yl)methoxy, benzyloxy, hexyloxy, methylthio, octylamino, hexyl, octyl, decyl, oct-1-yn-1-yl, hydroxy, bromo.
  • R 2 is selected from hydrogen, C 1-10 alkoxy, halogen.
  • R 2 is selected from hydrogen, octyl group, bromo.
  • R 3 is selected from hydrogen, C 1-10 alkyl, C 1-10 alkoxy.
  • R 3 is selected from hydrogen, methyl, hexyl group.
  • R 4 is selected from hydrogen, C 1-10 alkoxy.
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 5 is selected from C 1-10 alkoxy.
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the molecular weight is less than 1000, and Ar is a benzimidazole group or an azabenzimidazolyl group;
  • X is any one of the following: hydrogen, hydroxy, thiol, halogen, alkoxy or alkyl;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 8 are each independently selected from any one of the following: hydrogen, hydroxy, aliphatic, mercapto, halogen, amino, alkyl, -O-(CH).
  • Z is hydrogen, a hydroxyl group, a fatty group, an alkoxy group, an amino group or an alkylamine group.
  • n is an integer of 0 or 1-12.
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; Selected from hydrogen, hydroxyl, sulfhydryl, halogen, etc.;
  • R 1 and R 2 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • R 3 and R 4 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group; It is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl and halogen.
  • R 3 and R 4 are each selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl.
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, and the aryl group, heteroaryl group or heterocyclic group is optionally substituted with an alkyl group or a carbamoyl group.
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl,
  • the alkyl group in the alkoxy group is optionally substituted by an aryl group, a heteroaryl group or a heterocyclic group, which is optionally substituted with an alkyl group or a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 1 is selected from hydrogen, C 1-10 alkyl, C 1-10 alkynyl, C 1-10 alkyl group, C 1-10 alkylthio, halogen, hydroxy, indazolyl, C 1-10 alkoxy, phenyl or pyridyl substituted C 1-10 alkoxy group, a pyridyl group optionally substituted with amino Formyl.
  • R 1 is selected from hydrogen, ethoxy-4-methylphenyl, 4,5,6,7-tetrahydro -2H- Oxazol-2-yl, (2-carbamoylpyridin-4-yl)methoxy, benzyloxy, hexyloxy, methylthio, octylamino, hexyl, octyl, decyl, octyl- 1-alkyn-1-yl, hydroxy, bromo.
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • a method of preparing a compound of formula IXX comprising:
  • n is an integer of 0 or 1-12;
  • n is an integer of 0 or 1-12;
  • n is an integer of 0 or 1-12;
  • step 1) the molar ratio of the compound of the formula XXI to the inorganic base or Lewis base, tert-butyl bromoacetate is 1:1-20:0.2-15;
  • step 1) the reaction temperature is 80-150 ° C, the time is 1-96h;
  • step 2) the temperature of the reduction reaction is 0-100 ° C, the time is 0.5-24h;
  • step 2) the molar ratio of the compound of the formula XXII to formamidine acetate is 1:0.2-10;
  • the temperature of the ring closure reaction is 50-150 ° C, and the time is 1-24 h;
  • the ring closure reaction is carried out in an organic solvent, preferably ethylene glycol monomethyl ether;
  • step 3 the molar ratio of the compound of the formula XXIII to hydrochloric acid or TFA is 1:1 to 100;
  • step 3 the heating and refluxing time is 0.5-96h;
  • step 4 the molar ratio of the compound represented by the formula XIXV to phosphorous acid and phosphorus trichloride is 1:0.2-20:0.2-20;
  • step 4 the reaction time is from 0.5 to 96 h.
  • the eukaryotic organism is a mammal; the tumor cell is a cancer cell; the tumor is a cancer, specifically, the cancer cell is a breast cancer cell; the cancer is a breast cancer, preferably, the breast cancer cell Specifically, it can be human breast cancer cell MDA-MB-231.
  • Figure 1 is a schematic representation of the mevalonate pathway.
  • 2A-D show the adjuvant activity of eight statins in the OVA antibody titer assay, expressed as antibody titers of IgM and IgG at 7 and 14 days post immunization.
  • Figure 3 shows the adjuvant activity of the TH-Z80 series of compounds in the OVA antibody titer assay, expressed as antibody titers of IgM and IgG at 7 and 14 days after immunization.
  • Figure 4 shows the adjuvant activity of the FPPS inhibitor TH-Z97 in the OVA antibody titer assay, expressed as antibody titers of IgG at 7 and 14 days after immunization.
  • Figures 5A-D show the results of comparison of the adjuvant activities of the biphosphate compounds TH-Z80 and TH-Z93 of the present invention with the eight biphosphate drugs on the market in the OVA antibody titer assay.
  • 6A-D show the adjuvant activity of the GGPPS inhibitors TH-Z144 and TH-Z145 in the OVA antibody titer assay, expressed as antibody titers of IgM and IgG at 7 and 14 days after immunization.
  • 7A-D show the adjuvant activity of the dual inhibitors FPPS and GGPPS, BPH-716 and BPH-1222, in the OVA antibody titer assay, expressed as antibody titers of IgM and IgG at 7 and 14 days after immunization, Where Ctrl represents control mice treated with PBS.
  • Figure 8 shows the comparison of the adjuvant activities of the three types of inhibitors of the present invention, namely HMG-CoA reductase inhibitors, FPPS inhibitors, GGPPS inhibitors, with existing adjuvants.
  • the top panel shows the antibody titer of IgG after boost, and the lower panel shows antibody affinity. index.
  • Figures 9A-D show the inhibitory effect of GGOH and GGPP on the adjuvant activity of simvastatin (HMG-CoA reductase inhibitor).
  • Figures 10A-D show the inhibition of the adjuvant activity of TH-Z93 (FPPS inhibitor) by GGOH and GGPP.
  • Figures 11A-D show the inhibitory effect of GGOH and GGPP on the adjuvant activity of TH-Z145 (GGPPS inhibitor).
  • Figure 12A-D shows the results of adjuvant activity studies of selective squalene synthase inhibitor TH-Z66 in OVA antibody titer assay, antibody titers of IgM and IgG at 7 and 14 days after immunization Said.
  • Figure 13 shows the total number of lymph node cells after 24 h immunization of mice with simvastatin, TH-Z80, TH-Z145 as described in the present application, wherein Ctrl represents mice without any treatment.
  • Figure 14 shows the proportion and number of B lymphocytes in lymph nodes after immunization of mice with simvastatin, TH-Z80, and TH-Z145 as described in the present application, wherein Ctrl represents no treatment. Mouse.
  • Figure 15 shows the proportion and number of dendritic cells in lymph nodes after immunization of mice with simvastatin, TH-Z80, and TH-Z145 as described in the present application, wherein Ctrl represents no treatment. Mouse.
  • Figure 16A-B shows the proportion and number of macrophages in lymph nodes after immunization of mice with simvastatin, TH-Z80, and TH-Z145 as described in the present application, wherein Ctrl represents no preparation. Treated mice.
  • Figure 17 shows the proportion and number of T lymphocytes in lymph nodes after immunization of mice with simvastatin, TH-Z80, and TH-Z145 as described in the present application, wherein Ctrl represents no treatment. Mouse.
  • Figures 18-21 show the treatment of mouse bone marrow cells containing GM-CSF and IL-4 with 1 ⁇ M of simvastatin, TH-Z80, TH-Z145 and TH-Z66, respectively, followed by the addition of 100 ng/ml of LPS. Concentrations of IL-6, TNF- ⁇ , IL-1 ⁇ and IL-12p70 after stimulation.
  • 22A-D show the ratio of cell markers B220, F4/80, CD11c in lymph nodes after immunization with simvastatin, TH-Z80, and TH-Z145 as described in the present application, wherein Ctrl represents no preparation Treated mice.
  • Figure 23A-D shows titers of IgM and IgG antibodies in the Middle East Respiratory Syndrome Virus Mers protein after immunization of mice with simvastatin, TH-Z80, and TH-Z144, 7 days after immunization And antibody titers of 14 days of IgM and IgG are expressed.
  • Figures 24A-D show that simvastatin, TH-Z80, and TH-Z144 as adjuvants can promote the production of more antibodies by the hepatitis B surface antigen HbsAg.
  • Figures 25A-D show the adjuvant effect of simvastatin, TH-Z80, TH-Z93, TH-Z145 in a prophylactic vaccine against melanoma.
  • Figures 26A-D show the adjuvant effect of simvastatin, TH-Z80, TH-Z93, TH-Z145 in a therapeutic vaccine for melanoma.
  • Figure 27 shows the adjuvant activity of immunizing different parts of the mouse (foot, subcutaneous, muscle, abdominal cavity and nasal mucosa) with TH-Z80 as an adjuvant antigen, and antibodies against IgG at 7 and 14 days after immunization. The titer is indicated.
  • Figure 28A-D shows the combined adjuvant activity of the FPPS inhibitor TH-Z93 in combination with the TLR agonist imiquimod in the OVA antibody titer assay, with antibody drops of IgM and IgG at 7 and 14 days post immunization Degree representation.
  • Figure 29 is the effect of injection of 100 ⁇ g of different bisphosphonic acids on mouse antibody production.
  • PBS is a phosphate buffer
  • CLD is clodronate
  • ZOL is zoledronic acid
  • PMD pamidronic acid
  • Figure 30 is a diagram showing the effect of the bisphosphonic acid side chain carbon chain substitution length as an adjuvant
  • Figure 31 is a graph showing the effect of TH-Z80 on increasing antibody affinity.
  • PBS is a phosphate buffer
  • Figure 32 is a graph showing the amplification effect of different carbon chain length benzimidazole bisphosphonic acids on ⁇ T cells;
  • Figure 33 is a schematic view showing the crystal structure of TH-Z80 and HsFPPS (pymol plot).
  • Figure 34 is a schematic view showing the crystal structure of the compounds TH-Z82 and PvGGPPS (pymol plot).
  • Figure 35 shows the effects of simvastatin, TH-Z80, TH-Z93 and TH-Z145 on PR8 influenza virus.
  • Figure 36 is a graph showing the inhibitory effect of anti-PD1 antibody in combination with four adjuvants and chicken ovalbumin on B16-OVA tumors.
  • Figure 37 is the adjuvant activity of the HMG-CoA synthase inhibitor in the OVA antibody titer assay.
  • Figure 38 shows the concentrations of IL-6, TNF- ⁇ , IL-1 ⁇ and IL-12p70 after treatment with simvastatin, TH-Z80, TH-Z93, TH-Z145 or these adjuvants mixed with GGOH.
  • an adaptive element of an immune response ie, a B cell or T cell or both
  • an adaptive element of an immune response ie, a B cell or T cell or both
  • the antigen used in the present invention is an immunogen, i.e., an antigen that activates immune cells to produce an immune response against itself.
  • an "immunogenic composition” is a composition suitable for administration to a human or animal patient (e.g., in a test scenario), a substance capable of eliciting a specific immune response (e.g., a pathogen against hepatitis B virus).
  • an immunogenic composition comprises one or more antigens (eg, a complete purified virus or antigenic subunit, eg, a polypeptide thereof) or an epitope.
  • the immunogenic composition may also comprise one or more additional ingredients capable of eliciting or enhancing an immune response, such as excipients, carriers and/or adjuvants.
  • the immunogenic composition is administered to elicit an immune response that aids the patient against the symptoms or conditions caused by the pathogen.
  • the symptoms or diseases caused by the pathogen are prevented (or treated, for example, alleviated or alleviated) by inhibiting replication of the pathogen (e.g., hepatitis B virus) after the patient has been exposed to the pathogen.
  • the term immunogenic composition is understood to encompass a composition (ie, a vaccine composition or vaccine) that is administered to a patient or a population of patients in order to elicit, for example, a protective or palliative immune response against hepatitis B.
  • adjuvant is an agent that enhances an antigen-specific immune response compared to administration of an antigen without it.
  • Common adjuvants include adjuvants containing aluminum, which contain a mineral suspension (or mineral salt such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) to which the antigen can adsorb.
  • the adjuvant is an aluminum-free adjuvant formulated without any of the aluminum salts.
  • Aluminum-free adjuvants include oil and water emulsions such as water-in-oil and oil-in-water types (and variants thereof, including double emulsions and reversible emulsions), glycolipids, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides) , liposomes, Toll-like receptor antagonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 antagonists) and various combinations of these components.
  • oil and water emulsions such as water-in-oil and oil-in-water types (and variants thereof, including double emulsions and reversible emulsions), glycolipids, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides) , liposomes, Toll-like receptor antagonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 antagonists) and various combinations
  • an “immune response” is the response of a cell of the immune system, such as a B cell, T cell, or monocyte to a stimulus.
  • the immune response can be a B cell response that results in the production of specific antibodies, such as antigen-specific neutralizing antibodies.
  • the immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (ie, "antigen-specific response").
  • the antigen-specific response is a "pathogen-specific response.”
  • Protective immune response is the inhibition of pathogens An immune response that is functional or active, reduces pathogen infection, or reduces symptoms (including death) caused by infection with a pathogen.
  • the protective immune response can be measured, for example, by inhibition of viral replication or plaque formation in plaque reduction assays or ELISA-neutralization assays, or by in vivo measurement of resistance to pathogen challenge.
  • antibody describes an immunoglobulin, whether it is naturally occurring or partially or completely synthetically produced.
  • the term also encompasses any polypeptide or protein having a binding domain that is either antigen-binding domain or homologous to an antigen-binding domain.
  • a tumor antigen is an antigenic substance produced in a tumor cell, that is, it triggers an immune response in the host.
  • Tumor antigens are tumor markers useful in diagnostic tests for identifying tumor cells and are potential candidates for use in cancer therapy.
  • MERS Middle East Respiratory Syndrome
  • camel flu MERS-Coronavirus
  • Symptoms range from mild to severe, including fever, cough, diarrhea, and shortness of breath.
  • substituted means that any one or more hydrogens on a given atom or ring are replaced by a group selected from the indicated group, provided that the normal valence of the specified atom or ring atom is not exceeded. And the substitution results in a stable compound.
  • two hydrogens on the atom are replaced.
  • C 1-6 alkyl is intended to include C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.
  • fat based or “aliphatic group” includes alkyl, alkenyl and alkynyl groups as defined below.
  • alkyl is intended to include from 1 to 20 carbons, preferably from 1 to 12 carbons, preferably from 1 to 11 carbons, preferably from 1 to 10 carbons, preferably from 1 to 9 carbons, more in the chain.
  • Preferred are branched and linear saturated aliphatic hydrocarbon groups of 1 to 8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl , heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, decyl, decyl, undecyl, dodecyl, various branches thereof chain isomers thereof, and the group may optionally include 1 to 4 substituents such as halo, for example F, Br, Cl or I or CF 3, alkyl, alkoxy, aryl, aryloxy
  • alkenyl as used herein, alone or as part of another group, unless otherwise indicated, means that the chain contains from 2 to 20 carbons, preferably from 2 to 12 carbons, preferably from 2 to 11 carbons. a linear or branched group of preferably 2 to 10 carbons, preferably 2 to 9 carbons, more preferably 1 to 8 carbons, said group comprising 1 to 6 double bonds in the positive chain, such as a vinyl group , 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3- Heptenyl, 4-heptenyl, 3-octenyl, 3-decenyl, 4-nonenyl, 3-undecenyl, 4-dodecenyl, 4,8,12- Tetradecanoyl and the like, and the group may be optionally substituted by 1 to 4 substituents, ie, halogen,
  • alkynyl as used herein, alone or as part of another group, unless otherwise indicated, means that the chain contains from 2 to 20 carbons, preferably from 2 to 12 carbons, preferably from 2 to 11 carbons. a linear or branched group of 2 to 10 carbons, preferably 2 to 9 carbons, more preferably 2 to 8 carbons, which group contains a hydrazone bond in the normal chain, such as 2-propynyl group , 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4 a heptynyl, 3-octynyl, 3-decynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl, and the like, and the group may be optionally Substituted to 4 substituents,
  • cycloalkyl as used herein, alone or as part of another group, unless otherwise indicated, includes saturated or partially unsaturated (containing 1 or 2 double bonds) rings containing from 1 to 3 rings.
  • a hydrocarbon group comprising a monocyclic alkyl group, a dicyclic alkyl group (or a bicycloalkyl group) and a tricycloalkyl group, which contains a total of 3 to 20 ring-forming carbon atoms, preferably a total of 3 to 10 A ring-forming carbon atom, and the group may be fused to 1 or 2 of the aromatic rings described for the aryl group.
  • the "cycloalkyl group" having 1 ring preferably contains 3 to 8 ring carbon atoms, preferably 3 to 7 ring carbon atoms, more preferably 3 to 6 ring carbon atoms.
  • the cycloalkyl group includes a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclodecyl group, a cyclododecyl group, a cyclohexenyl group,
  • any of these groups may be optionally substituted by 1 to 4 substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, Cycloalkyl, alkylamino, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, fluorenyl and/or alkylthio groups and/or any substitution in the case of alkyl groups base.
  • substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, Cycloalkyl, alkylamino, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, fluorenyl and/or alkylthio groups and/or any substitution in the case of alkyl groups base.
  • alkylene When an alkyl group as defined above has a single bond to another group at two different carbon atoms, the alkyl group is referred to as "alkylene” and may optionally be as defined above for “alkyl” That was replaced.
  • alkenylene When an alkenyl group as defined above and an alkynyl group as defined above have a single bond for attachment at two different carbon atoms, respectively, they are referred to as “alkenylene” and “alkynylene”, respectively, and are optionally It is substituted as defined above for “alkenyl” and “alkynyl”.
  • aryl or “aryl” as used herein, alone or as part of another group, unless otherwise indicated, refers to monocyclic and bicyclic aromatics containing from 6 to 10 carbons in the ring portion. a group such as phenyl or naphthyl (including naphthalen-1-yl and naphthalen-2-yl), and may optionally contain from 1 to 3 with a carbocyclic or heterocyclic ring (such as an aryl ring, a cycloalkyl ring) Other rings fused to a heteroaryl ring or a heterocycloalkyl ring, for example
  • aryl group may be optionally substituted by 1, 2 or 3 substituents through a usable carbon, such as hydrogen, halogen, haloalkyl, alkyl, haloalkyl, alkoxy, haloalkoxy, alkene.
  • alkoxy Unless otherwise indicated, the term "alkoxy”, “aryloxy” or “aralkyloxy” as used herein, alone or as part of another group, includes any of the above alkyl groups attached to an oxygen atom. , aralkyl or aryl.
  • amino refers to an amino group that is unsubstituted or may be substituted with one or two substituents, which may be the same or different, And such as alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, hydroxy Alkyl, alkoxyalkyl or alkylthio. These substituents may be further substituted by a carboxylic acid group and/or an arbitrary R 1 group or a substituent as described above for R 1 .
  • the substituents on the amino group may form a pyrrolidin-1-yl, piperidin-1-yl, aza together with the nitrogen atom to which they are attached.
  • alkylthio Unless otherwise indicated, the terms "alkylthio", “arylthio” or “aralkylthio” as used herein, alone or as part of another group, include any of the foregoing attached to a sulfur atom. Alkyl, aralkyl or aryl.
  • alkylamino as used herein, alone or as part of another group, unless otherwise indicated, includes any alkyl group as defined above attached to a nitrogen atom. , aryl or arylalkyl.
  • heterocyclyl or “heterocyclic system” as used herein, means a stable 5, 6 or 7 membered monocyclic or bicyclic heterocyclic ring or a 7, 8, 9 or 10 membered bicyclic heterocyclic ring.
  • the heterocyclic ring is saturated, partially unsaturated or unsaturated (aromatic) and consists of a carbon atom and 1, 2, 3 or 4 heteroatoms independently selected from N, NH, O and S, and includes Any of the above bicyclic groups in which the above heterocyclic ring is fused to a benzene ring.
  • the nitrogen hetero atom and the sulfur hetero atom may be optionally oxidized.
  • the heterocycle can be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure.
  • the heterocycles described herein can be substituted on carbon or nitrogen, provided that the resulting compound is stable. If specifically indicated, the nitrogen in the heterocycle can optionally be quaternized. Preferably, when the total number of S atoms and O atoms in the heterocycle exceeds 1, Then these heteroatoms are not adjacent to each other.
  • aromatic heterocyclic ring system or “heteroaryl” or “heteroaryl” as used herein, means a stable 5 to 7 membered monocyclic or bicyclic or 7 to 10 membered bicyclic heterocyclic aromatic ring, It consists of a carbon atom and 1 to 4 heteroatoms independently selected from N, O and S, and is aromatic in nature.
  • heterocyclic groups include, but are not limited to, 1H-carbazolyl, 2-pyrrolidone, 2H, 6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 1H-indenyl, 4-piperidine.
  • Keto group 4aH-carbazolyl, 4H-quinazinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azacyclotetradecenyl, benzimidazolyl, benzofuranyl, Benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazole Benzimidazalonyl, carbazolyl, 4aH-carbazolyl, ⁇ -carboline, chromanyl, chromenyl, porphyrin, decahydroquinolinyl, 2H, 6H-1 ,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuranyl, furyl, furazyl, imidazolidinyl, imidazolinyl, imid
  • heterocyclyl includes, but is not limited to, pyridyl, thienyl, furyl, oxazolyl, benzothiazolyl, benzimidazolyl, benzothienyl, benzofuranyl, benzoxan Azyl, benzoisoxazolyl, quinolyl, isoquinolyl, imidazolyl, indolyl, isodecyl, piperidinyl, piperidinone, 4-piperidinyl, piperonyl, Pyrazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl, thiazolyl, oxazolyl, pyrazinyl and pyrimidinyl.
  • the heterocyclic group also includes a fused ring and a spiro compound containing, for example, the above heterocyclic ring.
  • heteroaryl groups are 1H-carbazolyl, 2H, 6H-1,5,2-dithiazinyl, fluorenyl, 4aH-carbazolyl, 4H-quinolizinyl, 6H-1, 2,5 -thiadiazinyl, acridinyl, azacyclotetradecenyl, benzimidazolyl, benzofuranyl, benzothienyl, benzothienyl, benzoxazolyl, benzothiazolyl, benzene And triazolyl, benzotetrazolyl, benzoisoxazolyl, benzisothiazolyl, benzimidazolone, carbazolyl, 4aH-carbazolyl, ⁇ -carboline, chromanyl, Alkenyl, porphyrin, decahydroquinolyl, 2H, 6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetra
  • heteroaryl examples are fluorenyl, benzimidazolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzene And tetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolone, porphyrin, furyl, imidazolyl, oxazolyl, fluorenyl, isoquinolyl, isothiazolyl , isoxazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyrazolotriazinyl, pyridazinyl, pyridyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, Thiazolyl, thienyl and tetrazolyl.
  • cyano refers to a -CN group.
  • nitro refers to a -NO 2 group.
  • hydroxy refers to an -OH group.
  • mercapto refers to a -SH group.
  • phrases "pharmaceutically acceptable” as used herein refers to those compounds, substances, compositions that are suitable for use in contact with humans and animal tissues without reasonable toxicity, irritation, allergies, or other problems or complications within the scope of sound medical judgment. And/or dosage form, which is commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effectiveness and properties of the compounds of the invention, and which are not biologically or otherwise undesirable.
  • the compounds of the invention are capable of forming acid and/or base salts due to the presence of an amino group and/or a carboxyl group or a similar group thereof (e.g., phenol or hydroxyamic acid).
  • a pharmaceutically acceptable acid addition salt can be formed using a mineral acid and an organic acid.
  • the inorganic acid from which the salt can be derived may include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived may include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamon. Acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • a pharmaceutically acceptable base addition salt can be formed using an inorganic base and an organic base.
  • Inorganic bases from which salts can be derived may include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; Ammonium, potassium, sodium, calcium and magnesium salts.
  • Organic bases from which salts can be derived may include, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, alkali ion exchange resins, and the like, particularly such as isopropylamine, trimethyl Amine, diethylamine, triethylamine, tripropylamine and ethanolamine.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, the base moiety or the acid moiety by conventional chemical methods.
  • the salt can be obtained by making the acid form of these compounds with a suitable amount of a suitable base such as sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, magnesium carbonate, potassium carbonate.
  • a suitable base such as sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, magnesium carbonate, potassium carbonate.
  • a suitable base such as sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, magnesium carbonate, potassium carbonate.
  • a suitable base such as sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, magnesium carbonate, potassium carbonate.
  • the reaction is typically carried out in water or in an organic solvent or a mixture of the two.
  • the preferred non-aqueous medium is ether, ethyl acetate, ethanol, isopropanol or acetonitrile. Additional suitable salts listed can be found in, for example, Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.
  • Exemplary acid addition salts include acetates such as those formed with acetic acid or trihaloacetic acid (e.g., trifluoroacetic acid), adipate, alginate, ascorbate, aspartate, Benzoate, besylate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate , lauryl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride Formed with hydrochloric acid), hydrobromide (formed with hydrobromic acid), hydroiodide, maleate (formed with maleic acid), 2-hydroxyethanesulfonate, lactate, methanesulfonate (formed with methanesulfonic acid), naphthalen
  • Exemplary base addition salts include ammonium salts; alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; phosphonium, zinc and aluminum salts; Organic amines) (such as trialkylamines (such as triethylamine), procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1-diphenamine (1-ephenamine) a salt formed by N,N'-dibenzylethylenediamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or a similar pharmaceutically acceptable amine; and amino acid A salt such as arginine, lysine or the like.
  • Organic amines such as trialkylamines (such as triethylamine), procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1-diphenamine (1-ephenamine)
  • Basic nitrogen-containing groups can be quaternized with the following reagents: such as lower alkyl halides (eg methyl chloride, methyl bromide and methyl iodide, ethyl chloride, ethyl bromide and ethyl iodide, propyl chloride, c Bromo and propyl iodide, and butyl chloride, butyl bromide and butyl iodide), dialkyl sulfate (such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate), long Chain halides (eg, mercapto chloride, mercapto bromide and mercapto iodide, lauryl chloride, lauryl bromide and lauryl iodide, myristyl chloride, myristyl bromide and myristyl iodide
  • the invention also includes prodrugs and solvates of the compounds of the invention.
  • prodrug means a compound which, upon administration of the compound to a subject, is chemically converted by metabolic or chemical processes to provide a compound described herein and/or a salt thereof and/or Solvate. Any compound that can be converted in vivo to provide a biologically active substance is a prodrug within the scope and spirit of the invention.
  • a compound containing a carboxyl group can form a physiologically hydrolyzable ester which is prepared by hydrolysis in vivo to obtain the compound itself.
  • Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reaction of a parent acid with a suitable alcohol, or amides prepared by reaction of a parent acid compound with a substituted or unsubstituted amine, or An acid anhydride, or a mixed acid anhydride. Simple aliphatic or aromatic esters, amides and anhydrides resulting from the acidic groups pendant from the compounds of the invention are specific prodrugs. In some cases, it is desirable to prepare a diester type prodrug such as an (acyloxy)alkyl ester or an ((alkoxycarbonyl)oxy)alkyl ester.
  • a diester type prodrug such as an (acyloxy)alkyl ester or an ((alkoxycarbonyl)oxy)alkyl ester.
  • a C 1 -C 8 alkyl group, a C 2 -C 8 alkenyl group, a C 2 -C 8 alkynyl group, an aryl group, a C 7 -C 12 substituted aryl group and a C 7 -C 12 compound of the present invention Aralkyl ester.
  • the prodrug is preferably administered orally because hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration can be used when the ester itself is active or hydrolysis occurs in the blood.
  • prodrug derivatives are well known in the art.
  • prodrug derivatives see:
  • the ester is typically a compound derived from an acid (organic or inorganic) in which at least one -OH (hydroxy) group is replaced by an -O-alkyl (alkoxy) group.
  • Esters are usually derived from carboxylic acids and alcohols.
  • the ester of the compound of the invention is preferably an in vivo hydrolysable ester.
  • in vivo hydrolysable ester as used herein is understood to mean an in vivo hydrolysable ester of a compound of the invention comprising a carboxy or hydroxyl group, for example pharmaceutically acceptable in a human or animal body to produce a parent acid or alcohol. ester.
  • esters suitable for the carboxy group include, for example, alkyl esters, cycloalkyl esters and optionally substituted phenylalkyl esters (especially benzyl esters), C 1 -C 6 alkoxymethyl esters (for example Methoxymethyl ester), C 1 -C 6 alkanoyloxymethyl ester (eg pivaloyloxymethyl ester), decyl ester, C 3 -C 8 cycloalkoxycarbonyloxy-C 1- C 6 alkyl ester (for example 1-cyclohexylcarbonyloxyethyl ester); 1,3-dioxol-2-carbonylmethyl ester, for example 5-methyl-1,3-di Olecyclo-2-ene methyl ester; and C 1 -C 6 -alkoxycarbonyloxyethyl ester, such as 1-methoxycarbonyloxyethyl ester, and the ester can be used in Formed on any of the carboxyl
  • In vivo hydrolysable esters of the compounds of the invention comprising a hydroxy group include inorganic acid esters (e.g., phosphate esters), [?]-acyloxyalkyl ethers, and related compounds which are cleaved by in vivo hydrolysis of the ester.
  • the parent hydroxyl group examples include acetoxymethyl ether and 2,2-dimethylpropionyloxymethyl ether.
  • the choice of a group which forms an in vivo hydrolysable ester with a hydroxyl group includes an alkanoyl group, a benzoyl group, a phenylacetyl group and a substituted benzoyl group and a phenylacetyl group, an alkoxycarbonyl group (to form an alkyl carbonate), An alkylcarbamoyl group and an N-(dialkylaminoethyl)-N-alkylcarbamoyl group (to form a carbamate), a dialkylaminoacetyl group, and a carboxyacetyl group.
  • the invention includes all such esters.
  • Solvate means a form of a compound that is associated with a solvent or water (also referred to as a "hydrate"), usually by solvent decomposition. This physical association includes hydrogen bonding.
  • solvents include water, ethanol, acetic acid, and the like.
  • the compounds of the invention may be prepared, for example, in crystalline form and may be solvated or hydrated.
  • Suitable solvates include pharmaceutically acceptable solvates such as hydrates, and also include both stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate can be separated, such as when one or more solvent molecules are introduced into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution phases and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.
  • a “subject” or “subject” or “host” for which administration is intended includes, but is not limited to, a human (ie, a male or female of any age group, eg, a pediatric subject (eg, infant, child, Adolescents or adult subjects (eg, young adults, middle-aged adults, or older adults) and/or non-human animals, such as mammals such as primates (eg, cynomolgus monkeys) , rhesus monkeys, cattle, pigs, horses, sheep, goats, rodents such as mice and rats, cats and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human,” “patient,” and “subject” are used interchangeably herein.
  • Effective amount means the amount of a compound that is sufficient to effect such treatment or prevention when administered to a subject for the treatment or prevention of a disease.
  • An “effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.
  • Therapeutically effective amount means an effective amount for therapeutic treatment.
  • Prophylactically effective amount means an effective amount for prophylactic treatment.
  • “Prophylaxis” or “prophylactic treatment” refers to a reduction in the risk of acquiring or developing a disease or condition (ie, at least one of the clinical symptoms of the disease is susceptible to infection, or is not exposed to the pathogen prior to the onset of the disease) The subject did not develop.
  • treating means: (i) preventing the disease, disorder, and/or condition in a patient who may be predisposed to the disease, disorder, and/or condition but has not been diagnosed with the disease, disorder, and/or condition; Ii) inhibiting the disease, disorder and/or condition, ie preventing its development; or (iii) mitigating the disease, disorder and/or condition, ie causing regression of the disease, disorder and/or condition.
  • inhibitor refers to reducing or inhibiting a particular condition, symptom or disease, or significantly reducing the biological activity or baseline activity of the lesion.
  • inhibitor refers to a molecule that is capable of inhibiting (including partial inhibition or allosteric inhibition) one or more biological activities of a target molecule, such as farnesyl pyrophosphate synthase (FPPS).
  • FPPS farnesyl pyrophosphate synthase
  • an inhibitor acts by reducing or suppressing the activity of a target molecule and/or reducing or suppressing signal transduction.
  • the compounds described herein may exist in stereoisomeric forms (eg, they contain one or more asymmetric carbon atoms). Individual stereoisomers (enantiomers and diastereomers) and mixtures of such isomers are included within the scope of the invention. Likewise, it is to be understood that the compounds or salts described herein may exist in tautomeric forms other than those shown in the formula, and such are also included within the scope of the invention. It will be understood that the invention includes all combinations and subclasses of the specific groups defined above. The scope of the invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerically enriched mixtures. It will be understood that the invention includes all combinations and subclasses of the specific groups defined above.
  • One enantiomer of a compound described herein may exhibit superior activity compared to another enantiomer. Therefore, all stereochemistry is considered part of the invention.
  • the separation of the racemic material can be accomplished by using a chiral column for HPLC or by using a resolving reagent such as camphonic chloride for resolution (eg, Young, SDet a1., Antimicrobial). Agents and Chemotherapy 1995, 2602-2605).
  • the invention also includes isotopically-labeled compounds which are identical to those described in formula (I) except that one or more atoms are replaced by atoms of atomic mass or mass number different from the atomic mass or mass number of the natural ones.
  • isotopes which may be incorporated into the compounds of the invention and their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I.
  • Isotopically labeled compounds of the invention such as those incorporating radioisotopes such as 3 H or 14 C, are useful in drug and/or substrate tissue distribution assays.
  • the deuterated 3 H and carbon-14, 14 C isotope are particularly preferred due to their ease of preparation and detectability.
  • the 11 C and 18 F isotopes are particularly useful for PET (positron emission tomography), and the 125 I isotope is particularly useful for SPECT (single photon emission computed tomography), all of which are used for brain imaging.
  • Isotopically labeled compounds of formula (I) and the following compounds of the invention can generally be prepared by carrying out the procedures disclosed in the schemes below and/or in the examples, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • the invention relates to the following embodiments.
  • the invention relates to an immunogenic composition
  • an adjuvant which may include, but is not limited to, a thiolase (acetoacetyl-CoA transferase) inhibitor, 2) HMG-CoA synthase inhibitor, 3) HMG-CoA reductase inhibitor, 4) mevalonate kinase inhibitor, 5) phosphomevalonate kinase inhibitor, 6) mevalonate-5- Pyrophosphate decarboxylase inhibitor, 7) Isopentenyl pyrophosphate isomerase inhibitor, 8) farnesyl pyrophosphate synthase inhibitor, 9) geranyl succinyl pyrophosphate synthase inhibitor and 10) geranyl leaf acyltransferase (I , II) inhibitors.
  • a thiolase acetoacetyl-CoA transferase
  • HMG-CoA synthase inhibitor acetoacetyl-CoA transferase
  • the thiolase (acetoacetyl-CoA transferase) inhibitor includes, but is not limited to, L-660631 as described in biochemical and biophysical research communications, 1989, 163, 548-553.
  • the HMG-CoA synthase inhibitor includes, but is not limited to, L-659699, Agric. Biol. Chem., 55(12), 3129-3131, 1991, described in Biochem. J (1993) 289, 889-895. Dihydroxerulin as described in 1233A/F-244, and Tetrahedron, 2000, 56(3), 479-487, and those disclosed in the following documents: US5064856; EP0411703A1; Agric.Biol. Chern., 1991 , 55(12): 3129-3131; Bioorg. Med. Chem., 1998, 6: 1255-1272; Biochem. Biophys. Res. Commun., 1999, 265: 536-540. All of these patents, as well as other disclosures, are hereby incorporated by reference.
  • HMG-CoA reductase inhibitors include, but are not limited to, those disclosed in the following documents: US 5,102,911-A; EP476493-A1; US5091378-A; EP465970-A; EP465265-A; EP464845-A; EP463456- A; EP456214-A1; EP591165-A; US5049577-A; EP445827-A2; EP442495-A; US5025000-A; EP435322-A2; US5023250-A; JP3112967-A; US5017716-A; US5010105-A; US5011947-A; EP 424 929-A1; EP 422 895-A1; EP 420 266-A2; EP 419 856-A2; EP 418 648-A1; EP 416 383-A2; US 4 496 623 A; US 4,944, 494 - A; EP 415 488-A; US 4,992, 4
  • the HMG-CoA reductase inhibitor is a statin.
  • statins are selected from the group consisting of: pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, cerivastatin, or Pharmaceutically acceptable salts, esters, prodrugs, solvates.
  • Preferred HMG-CoA reductase inhibitors are those already marketed, most preferably simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.
  • HMG-CoA reductase inhibitors are well known to those skilled in the art and these include those commercially available.
  • the HMG-CoA reductase inhibitor can be used in the form of its free acid form, its ester form, or a pharmaceutically acceptable salt thereof.
  • These pharmaceutically acceptable salts include, for example, sodium salts, calcium salts, aluminum salts and ester salts.
  • the HMG-CoA reductase inhibitor can be used in the form of a racemic mixture, or a more active, suitable stereoisomer.
  • the farnesyl pyrophosphate synthase inhibitors include, but are not limited to, those disclosed in the following documents: US Pat. No. 7,462,733; US.20080200679; WO.2006039721; US.7358361; US.7745422; US.20100316676; WO.2007109585; US.7687482; WO.2008128056; US.20080255070; WO.2010033980; WO.2010033981; WO.2008076417; 7781418; WO.2010033978; WO.2009068567; WO.2010043584; WO.2009128918; ACS Med. Chem. Lett. 2013, 4: 423-427; J. Am.
  • the farnesyl pyrophosphate synthase inhibitor is a bisphosphonate compound or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof.
  • bisphosphonic acid (salt) refers to a compound characterized by the binding of two phosphonic acid groups to a central (terminated) carbon atom through a phosphoether linkage. This P-C-P structure is shown in Formula I below. It should be noted that the term "bisphosphonic acid” as used herein in reference to a therapeutic agent of the present invention is also intended to include bisphosphonic acids (salts), bisphosphonic acids, and salts and derivatives thereof. The specific nomenclature used in reference to the bisphosphonic acid is not intended to limit the scope of the invention unless otherwise stated.
  • the bisphosphonic acid (salt) as a drug is described, for example, in EP-A-170,228, EP-A-197,478, EP-A-22,751, EP-A-252,504, EP-A-252,505, EP-A-258,618, EP- A-350, 002, EP-A-273, 190, and WO-A-90/00798, the entire disclosure of which is incorporated herein by reference.
  • Preferred bisphosphonic acids or pharmaceutically acceptable salts thereof are selected from the group consisting of alendronate, cemaronic acid, clodronate, EB-1053, tiludronic acid, etidronic acid, ibandronic acid, incaphosphine Acid, minodronic acid, nalidronic acid, opiateic acid, risedronate, pridronic acid, pamidronic acid, zoledronic acid or an acceptable salt thereof, such as ibandronate monosodium Salt monohydrate.
  • the geranyl-co-pyrophosphate synthase inhibitors include, but are not limited to, those disclosed in the literature: J. Med. Chem. 2009, 52: 8025-37; Biochem. Biophys. Res. Commun. 2007, 353: 921-925; J. Med. Chem. 2002, 45: 2185-2196; Bioorg. Med. Chem. 2008, 16: 390-399; J. Med. Chem., 2008, 51: 5954-5607 ACS Med. Chem. Lett. 2015, 6: 1195-1198; Proc. Natl. Acad. Sci. USA, 2012, 109 (11): 40584063, all of which are incorporated herein by reference.
  • the geranyl leaf acyltransferase (I, II) inhibitors include, but are not limited to, those disclosed in the literature: EP1165084A1; EP1165084A4; EP2014291A2; EP2014291B1; US6103487; US6284910; US6355643B1; US6586461B1; US6638962B2; US7763620B2; US8093274B2; US8815935B2; US9040563B2; US20030219847A1; US20040121985A1; US20060030624A1; US20070249010A1; US20100063114A1; US20110178138A1; US20120035184A1; US20130102639A1; WO1999006376A1; WO2000033826A1; WO2000051614A1; WO2007111948A2; WO2007118009A1; WO2010014054A1; WO201008
  • the present invention also relates to other substances which affect the geranylation of protein geranium, which may also be included as an adjuvant in the immunogenic composition.
  • the farnesyl pyrophosphate synthase inhibitor is a compound of the formula (ie, a compound of the TH-Z80 series) or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof:
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • n is an integer from 1-20. In a further embodiment, n is an integer from 1-15.
  • the compound is selected from the group consisting of
  • the present invention relates to an immunogenic composition
  • an adjuvant selected from the group consisting of the above-mentioned TH-Z80 series of compounds or a pharmaceutically acceptable salt, ester thereof, and the like Medicine, solvate.
  • the present invention also relates to the use of the above compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an adjuvant for the preparation of an immunogenic composition for preventing or treating a disease.
  • the present invention is also directed to a novel bisphosphonate compound (i.e., a compound of the TH-Z97 series) as an inhibitor of farnesyl pyrophosphate synthase (FPPS) or a pharmaceutically acceptable salt, ester or prodrug thereof a solvate having the formula:
  • FPPS farnesyl pyrophosphate synthase
  • n is an integer from 1 to 24, preferably n is an integer from 1 to 12.
  • n is an integer from 1-20. In a further embodiment, n is an integer from 1-15.
  • the compound is selected from the group consisting of
  • the present invention relates to an immunogenic composition
  • an adjuvant selected from the group consisting of the above-mentioned TH-Z97 series of compounds or a pharmaceutically acceptable salt, ester thereof, or the like Medicine, solvate.
  • the present invention also relates to the use of the above compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an adjuvant for the preparation of an immunogenic composition for preventing or treating a disease.
  • the present invention relates to a novel bisphosphonate compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an inhibitor of farnesyl pyrophosphate synthase (FPPS), said compound having Type:
  • R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl, the alkyl of said alkoxy Optionally substituted with an aryl, heteroaryl or heterocyclic group, the aryl, heteroaryl or heterocyclic group being optionally substituted with an alkyl group, a carbamoyl group;
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 2 and R 3 together with the carbon atom to which they are attached form an aromatic or heteroaryl ring;
  • R 4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl.
  • R 1 is selected from the group consisting of hydrogen, C 1-10 alkyl, C 1-10 alkynyl, C 1-10 alkylamino, C 1-10 alkylthio, halogen, hydroxy, oxazolyl , C 1-10 alkoxy, phenyl or pyridyl substituted C 1-10 alkoxy group, a pyridyl group optionally substituted with carbamoyl.
  • R 1 is selected from the group consisting of hydrogen, 4-methylphenylethoxy, 4,5,6,7-tetrahydro-2H-indazol-2-yl, (2-carbamoylpyridine- 4-yl)methoxy, benzyloxy, hexyloxy, methylthio, octylamino, hexyl, octyl, decyl, oct-1-yn-1-yl, hydroxy, bromo.
  • R 2 is selected from the group consisting of hydrogen, C 1-10 alkoxy, halogen. In a further embodiment, R 2 is selected from the group consisting of hydrogen, octyloxy, and bromine.
  • R 3 is selected from the group consisting of hydrogen, C 1-10 alkyl, C 1-10 alkoxy. In a further embodiment, R 3 is selected from hydrogen, methyl, hexyl group.
  • R 2 and R 3 together with the carbon atom to which they are attached form a benzene ring.
  • R 4 is selected from the group consisting of hydrogen, C 1-10 alkoxy. In a further embodiment, R 4 is selected from hydrogen, octyloxy.
  • the compound is selected from the group consisting of
  • the present invention relates to an immunogenic composition
  • an adjuvant selected from the group consisting of the above-mentioned ortho-aminopyridine compounds or pharmaceutically acceptable salts, esters, and prodrugs thereof Solvent.
  • the present invention also relates to the use of the above compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an adjuvant for the preparation of an immunogenic composition for preventing or treating a disease.
  • the present invention relates to a novel bisphosphonate compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an inhibitor of farnesyl pyrophosphate synthase (FPPS), said compound having formula:
  • R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl.
  • R 5 is selected from C 1-10 alkoxy.
  • the compound is selected from the group consisting of
  • the invention relates to an immunogenic composition
  • an adjuvant comprising an adjuvant, said adjuvant being selected From the above-mentioned m-aminopyridine compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof.
  • the present invention also relates to the use of the above compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an adjuvant for the preparation of an immunogenic composition for preventing or treating a disease.
  • the present invention is also directed to a novel bisphosphonate compound, or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, as a geranyl-folate pyrophosphate synthase inhibitor, said compound having the formula:
  • R 9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;
  • R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl.
  • R 9 is selected from C 1-10 alkoxy.
  • the compound is selected from the group consisting of
  • the present invention relates to an immunogenic composition
  • an adjuvant selected from the above benzyl diphosphate compounds or pharmaceutically acceptable salts, esters, prodrugs thereof Solvent.
  • the present invention also relates to the use of the above compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof as an adjuvant for the preparation of an immunogenic composition for preventing or treating a disease.
  • pyrophosphate inhibited ectopic calcification more than 40 years ago.
  • pyrophosphate is unstable and is easily inactivated by enzymatic hydrolysis.
  • the POP group which is easily hydrolyzed by pyrophosphate structure was transformed into a PCP group which is stable to the enzyme, and then a series of bisphosphonate compounds were developed. These compounds have played a good role in the treatment of osteoporosis.
  • Representative drugs include the first generation of sodium etidronate, the second generation of sodium chlorophosphonate, pamidronate and sodium laudronate, and the third generation of alendronate, which has evolved to the present.
  • the second-generation bisphosphonate causes apoptosis of osteoclasts mainly through the protonation of N to the key target enzyme FPPS (Farnesyl pyrophosphate synthase) in the terpene biosynthesis channel.
  • FPPS Fearnesyl pyrophosphate synthase
  • the third-generation representative drug zoledronic acid is used to treat cancer caused by abnormal bone metabolism caused by bone metastasis of malignant tumors such as multiple myeloma, breast cancer, prostate cancer and lung cancer. There are also certain effects in bone metastasis But not significant.
  • One embodiment of the present invention relates to a novel biphosphate compound as described above, which can be used for the preparation of a metabolic bone disease drug, a treatment for a malaria drug, a eukaryotic tumor cell proliferation inhibitor, a drug for preventing and/or treating a tumor , immunotherapeutics and adjuvants for vaccines.
  • the compound of the formula I has a mass content of 0.001-90. %.
  • the compound of the formula I is present in an amount of from 0.001% to 90% by mass.
  • the medicament for treating metabolic bone disease, treating malaria drugs, eukaryotic tumor cell proliferation inhibitors, drugs for preventing and/or treating tumors, immunotherapy drugs and vaccines can be injected, sprayed, nose-dropped, instilled, infiltrated, Absorption, physical or chemical-mediated methods are introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped by other substances and introduced into the body.
  • the bisphosphoric acid compound of the present invention synthesizes the 4,5,6,7 asymmetric bisphosphonic acid of benzimidazole for the first time by modifying the terminal carboxyl group modification, which maintains the activity of the imidazolium acid to FPPS, which is also for GGPPS, malaria And tumor cell proliferation has a good inhibitory effect, especially as a vaccine adjuvant and immunotherapy drugs have good effects, can be used for the preparation of drugs for the treatment of metabolic bone diseases, drugs for the treatment of malaria, eukaryotic tumor cell proliferation inhibitors, A drug for preventing and/or treating a tumor, an immunotherapeutic drug, and a vaccine adjuvant for preparing a vaccine.
  • an immunogenic composition of the invention comprises an antigen derived from a bacterium, a virus, a parasite or a tumor.
  • the one or more antigens are each independently a microbial antigen, an autoantigen, a tumor antigen, an allergen or an addictive substance.
  • Antigens of the invention also include those described in International Patent Application WO 2011/148356.
  • Antigens can be obtained by recombinant means or peptide synthesis, or obtained from natural sources or extracts, and can be derived from any living or non-living organism.
  • Antigens may be derived from bacteria such as, for example, anthrax, campylobacter, cholera, diphtheria, enterotoxin-producing E. coli, giardia. , gonococcus, Helicobacter pylori, Hemophilus influenza B, undeterminable Haemophilus influenzae, meningococcus, pertussis, pneumococcus Pneumococcus), salmonella, shigella, streptococcus B, group A streptococci, tetanus, Vibrio cholerae, yersinia, staphylococci (Staphylococcus), Pseudomonas species, and Clostridia species.
  • bacteria such as, for example, anthrax, campylobacter, cholera, diphtheria, enterotoxin-producing E. coli, giardia. , gonococcus, Helicobacter pylori,
  • the antigen may be derived from a virus such as, for example, adenovirus, dengue 1 to 4 serotype, ebola (Pling et al, Arch Virol Suppl, 11: 135-140, 1996), enterovirus Hepatitis A to E serotype (Blum, Digestion 56: 85-95, 1995; Katkov, Med Clin North Am 80: 189-200, 1996; Lieberman and Greenberg, Adv Pediatr Infect Dis 11: 333-363, 1996; Mast Etc., Annu Rev Med 47: 257-266, 1996), herpes simplex virus 1 or 2, human immunodeficiency virus (Deprez et al, Vaccine 14: 375-382, 1996), influenza, Japanese equine encephalitis, measles, Norwalk (Norwalk), papillomavirus, parvovirus B19, polio, rabies, rotavirus, rubella, rubeola, vaccinia,
  • the antigen can be self-sent Biologically derived
  • the parasites include, for example, Entamoeba histolytica (Zhang et al, Infect Immun 63: 1349-1355); Plasmodium (Bathurst et al, Vaccine 11: 449-456, 1993). ), Toxoplasmosis, and Helminth. (The above 3 paragraphs are from PWH95845A)
  • the antigen may be a tumor specific antigen (TSA) or a tumor associated-antigen (TAA).
  • TSA tumor specific antigen
  • TAA tumor associated-antigen
  • a tumor-specific antigen refers to a new antigen that is expressed only on the surface of a certain tumor cell and not on a normal cell, and is also called a unique tumor antigen.
  • tumor-associated antigens are known in the art, and common tumor-specific antigens include (1) fetal protein globulin (AFP); (2) carcinoembryonic antigen (CEA); (3) CA-125; (4) MUC-1; (5) epithelial tumor antigen (ETA); (6) tyrosinase; (7) melanoma-associated antigen (MAGE); (8) tumor testis antigen; (9) prostate specific antigen (PSA) (10) gp100; (11) Melan A; (12) GAGE, G antigen 12B/C/D/E; (13) BAGE, B melanoma antigen; (14) GM2, ganglioside.
  • Tumor-associated antigens are antigens that are highly associated with certain tumor cells.
  • tumor-associated antigens are known in the art, and common tumor-associated antigens include those listed in International Patent Application WO 2010/009124, such as (1) BMPR1B; (2) E16; (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hlg; (9) ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRIPTO; 14) CD21; (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R ⁇ ; (21) Brevican; (22) EphB2R; (23) ASLG659; 24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29)
  • the antigen is derived from Middle East Respiratory Syndrome (Mers) virus, hepatitis B virus, melanoma.
  • Mers Middle East Respiratory Syndrome
  • the immunogenic compositions of the invention further comprise another adjuvant.
  • the other vaccine adjuvant includes, but is not limited to, aluminum adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, AS01, AS02, AS03, AS04, AS15, CAF01, ISCOMs (immune stimulating complex) ), Virosomes (virions), GLA-SE, liposomes, edible oils, saponins, AF03, TLR agonists.
  • TLR agonists are: TLR1 stimulators (such as triaacyl lipoprotein), TLR2 stimulators (such as peptidoglycan, zymosan, HMGB1 (high-speed kinesin 1), lipoteichoic acid ), TLR3 stimulator (double-stranded RNA such as PolyI: C), TLR4 stimulator (such as LPS, MPL, RC529, GLA, E6020), TLR5 stimulator (flagellin), TLR6 stimulator (such as triacylate) Protein, lipoteichoic acid), TLR7/8 stimulator (single-stranded RNA, imiquimod), TLR9 stimulator (DNA, such as CPG ODN), C-lectin ligand (such as kelp polysaccharide), CD1d ligand (eg alpha-galactosylceramide)
  • TLR1 stimulators such as triaacyl lipoprotein
  • TLR2 stimulators such as peptidoglycan
  • the adjuvants and immunogenic compositions described herein can be used in a variety of vaccines including, but not limited to, BCG, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis D vaccine, hepatitis E vaccine, influenza vaccine, spinal gray matter Inflammation vaccine, DTP vaccine, measles vaccine, JE vaccine, rabies vaccine, hemorrhagic fever vaccine, pneumonia vaccine, epidemic vaccine, hepatitis A vaccine, mumps vaccine, influenza vaccine, rubella vaccine, chickenpox vaccine, AIDS vaccine, malaria vaccine And cancer treatment and prevention vaccines, including but not limited to melanoma treatment Vaccine, melanoma preventive vaccine, lung cancer therapeutic vaccine, lung cancer preventive vaccine, bladder cancer preventive vaccine, bladder cancer preventive vaccine therapeutic vaccine, cervical cancer therapeutic vaccine, cervical cancer preventive vaccine, bladder cancer therapeutic vaccine , bladder cancer preventive vaccine, breast cancer therapeutic vaccine, breast cancer preventive vaccine, prostate cancer prevent
  • the immunogenic compositions of the invention are useful in the treatment of a variety of diseases or conditions, including diseases caused by bacteria, viruses, fungi, parasites.
  • the bacteria include, but are not limited to, the following species and combinations thereof: Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus actinomycetemcomitans, porcine pleura Actinobacillus pleuropneumoniae, Actinomyces israelli, Actinomyces viscosus, Aeromonas hydrophila, Alcaliges eutrophus, sour heat Thermocyclobacillus acidocaldarius, Arhaeglobusfulgidus, Bacillus species, Bacillus antracis, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bacteroides, Bordetella, Bordetella bronchiseptica, Borscht Borrelia (Borrelia burgdorferi ), Brucella (Brucella), Burkholderia cepacia, Burkholderi
  • gingivalis Brachyspirayodysenteria Brachyspira pilosicoli, Camphylobacter species, Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter Jejuni), Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila, Chromobacterium viscosum, Clostridium species, Clostridium botulinum , Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Corynebacterium, Corynebacterium diphtheriae, Canine Ehrlichi a canis), Enterobacter species, Enterobacter aerogenes, Enterococcus species, Erysipelothrix rhusiopathieae, Escherichia species, large intestine Escherichia coli, Fusobacterium nucleatum, Haemophil
  • the virus is a virus that infects an animal, including but not limited to the following species and combinations thereof: avian herpes virus, avian influenza virus, avian leukosis virus, avian paramyxovirus, border disease virus, bovine coronavirus, bovine fever Virus, bovine herpes virus, bovine immunodeficiency virus, bovine leukemia virus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine viral diarrhea virus (BVDV), type I BVDV, type II BVDV, canine adenovirus, canine coronary Virus (CCV), canine distemper virus, canine herpes virus, equine herpes virus, canine influenza virus, canine parainfluenza virus, canine parvovirus, canine respiratory coronavirus, classical swine fever virus, oriental equine encephalitis virus (EEE), Equine infectious anemia virus, equine influenza virus, West Nile virus, cat calici
  • the virus is a human infected virus, including, but not limited to, the adenoviridae (most adenovirus); the arenavirus family (hemorrhagic fever virus); the astrovirus family; the bunia virus family ( Bungaviridae) (eg, hantavirus, bunga virus, vitiligo and endovirus); calciviridae (eg, a strain causing gastroenteritis); coronavirus (eg , coronavirus); fibroviridae (eg, Ebola virus); Flavividae (eg, hepatitis C virus, dengue virus, encephalitis virus, yellow fever virus); hepadnaviridae ( Hepadnaviridae) (HBV); herpesviridae (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus; iridescent virus family (eg, African swine fever virus) Norwalk, Bung
  • fungi include, but are not limited to, spores, molds, and yeasts (eg, Candida species).
  • the parasites include, but are not limited to, the following species and combinations thereof: from the genus Anaplasma, Fasciola hepatica (liver fluke), coccidia, Eimeria, canine neospores Insect, Toxoplasma gondii, Giardia, Filaria (Hymenoptera), Hookworm (Hookworm), Trypanosoma, Leishmania, Trichomonas, Micro Cryptosporidium Insect, Babesia, Schistosoma, Aphis, A. elegans, Aphis, Trichinella, Sarcocyst, Hammond, Isopsora.
  • the parasite is an ectoparasite.
  • ectoparasites include, but are not limited to, mites, including hard genus, genus, genus, burdock, genus, genus, and their combination.
  • the cancer can be a malignant or non-malignant cancer.
  • Cancer or tumors include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer; glioblastoma, Epithelial neoplastic tumors, lymphomas (eg, follicular lymphoma), liver cancer, lung cancer (eg, small cell and non-small cell lung cancer), leukemia (eg, hair cell leukemia, chronic myeloid leukemia, skin T cells) Leukemia), melanoma (eg, malignant melanoma), multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid Cancer, kidney cancer, and other malignancies and s
  • an effective amount of one or more immunogenic compositions can be administered to a subject.
  • Administration of the pharmaceutical compositions of the invention can be accomplished by any means known to those skilled in the art.
  • Preferred routes of administration include, but are not limited to, parenteral (eg, intramuscular, subcutaneous, intradermal, intravenous), topical (eg, transdermal) to the skin, or mucosal (eg, oral, intranasal, intravaginal, In the rectum, through the cheeks, in the eyes or under the tongue.
  • parenteral eg, intramuscular, subcutaneous, intradermal, intravenous
  • topical eg, transdermal
  • mucosal eg, oral, intranasal, intravaginal, In the rectum, through the cheeks, in the eyes or under the tongue.
  • intratumoral administration In the case of cancer treatment, it may include intratumoral administration.
  • an "effective amount" of an immunogenic composition refers to an amount that is necessary or sufficient to achieve the desired biological effect.
  • an effective amount of an immunogenic composition for treating a condition can be an amount necessary to eliminate a microbial infection or tumor.
  • An effective amount for use as a vaccine adjuvant can be an amount that can be used to boost the subject's immune response to the vaccine.
  • An effective amount may vary depending on the disease or condition being treated, the particular immunogenic composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular immunogenic composition without undue experimentation.
  • the immunogenic composition can be administered in a single dosage regimen, or preferably in multiple dosage regimens, i.e., the primary process of vaccination of the immunogenic composition is 1-10 separate doses, followed by maintenance and/or boosting of the immune response. Other doses need to be administered at subsequent intervals, for example, a second dose is administered 1-4 months, and if necessary, subsequent doses are administered after months or years.
  • the medication regimen is also at least partially determined by the individual's needs and depends on the judgment of the medical staff.
  • suitable immunization protocols include: a first dose followed by a second dose between days 7 and 6 months, and optionally a third dose between 1 month and 2 years after the first vaccination; or Other protocols are sufficient to elicit the desired neutralizing antibody titer that confers protective immunity, such as corresponding to established pediatric immunogenic composition vaccination protocols.
  • a booster dose administered at specific intervals can be supplemented to maintain satisfactory protective immunity.
  • the immunogenic composition of the present invention can be prepared into various forms such as an injection solution, a tablet, a powder, a granule, a capsule, an oral solution, a cream, a cream, and the like.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • One or more pharmaceutically acceptable carriers may also be added to the above formulations.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • immunogenic compositions When it is desired to systemically deliver one or more immunogenic compositions, it can be formulated for parenteral administration by injection (e.g., bolus injection or drip).
  • parenteral administration e.g., bolus injection or drip
  • the subject's sole, subcutaneous, intramuscular, abdominal, and nasal mucosa can be immunized.
  • Formulations for injection may be presented in unit dosage form, for example, in an ampule with the added preservative or in a multi-dose container.
  • the composition may take the form of a suspension, solution or emulsion as in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, Stabilizer and / or dispersant.
  • mice were all mice, and the strains of the mice were all C57B/6, purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., and raised in Tsinghua University Biomedicine. Test Center SPF Animal Room.
  • HMG-CoA reductase catalyzes the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate, while the HMG-CoA reductase inhibitor (ie statin) The compound) inhibits the action of HMG-CoA reductase.
  • statins we demonstrated the role of HMG-CoA reductase inhibitors as adjuvants.
  • statins we demonstrated the role of HMG-CoA reductase inhibitors as adjuvants.
  • statins as adjuvants in immunized mice was investigated.
  • statins that are more commonly used clinically are used in this assay.
  • the eight statins are: pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, and simvastatin.
  • Simvastatin was purchased from Tianjin Heathstein, mevastatin and lovastatin purchased from Aladdin, pravastatin and atorvastatin purchased from Anheji, fluvastatin, pitavastatin and Rosuvastatin was purchased from Huazhong Weihai Company.
  • OVA Optalbumin antigen was purchased from Sigma.
  • Chicken ovalbumin also known as chicken egg albumin, consists of 386 amino acids with a molecular weight of approximately 43 kD and is commonly used as a tool protein to study antibody titers.
  • the blood taken out was placed at 4 ° C overnight, centrifuged at 6000 rpm for 8 min, and the supernatant serum was aspirated, and the titer of anti-OVA IgM and IgG antibodies in the serum was measured.
  • OVA protein was diluted with a carbonate solution of pH 9.6 to a concentration of 2 ⁇ g/ml, coated in a 96-well microtiter plate, 50 ⁇ l per well, and coated at 4 ° C overnight.
  • the plate was washed 5 times with PBS containing 0.05% Tween 20, blocked with 1% BSA solution, 200 ⁇ l per well, and blocked at room temperature for 2 h.
  • the plate was washed 5 times with PBST (Tween phosphate buffer), and the mouse serum after immunization with OVA was added, 50 ⁇ l per well, and incubated for 2 h at room temperature.
  • PBST Teween phosphate buffer
  • statins such as fluvastatin, pits
  • IgM and IgG antibodies with significantly higher titers were produced in mice treated with statins, rosuvastatin, simvastatin, lovastatin and mevastatin, especially simvastatin, lovastatin and melamine
  • statins such as fluvastatin, pits
  • IgG antibodies with significantly higher titers were produced in mice treated with statins, rosuvastatin, simvastatin, lovastatin and mevastatin, especially simvastatin, lovastatin and melamine
  • the adjuvant effects of the three statins of statins are most pronounced.
  • statin compounds may not be significantly due to poor bioavailability in vivo such as solubility and poor absorbency, by adjusting the pharmacokinetic properties of these compounds (eg forming salts, esters or prodrugs, or forming aluminum salts) ) to achieve better results.
  • the HMG-CoA reductase inhibitor can be used as an adjuvant to enhance the specific immune response caused by the antigen.
  • FPPS farnesyl pyrophosphate synthase
  • FPP farnesyl pyrophosphate
  • DPP dimethylallyl pyrophosphate
  • FPPS inhibitors inhibit the action of FPPS.
  • bisphosphonic acid compounds are potent inhibitors of FPPS.
  • the adjuvant action of various bisphosphonate compounds was investigated in the following assays.
  • the TH-Z80 series of compounds are newly synthesized bisphosphonic acid compounds whose structures are shown below.
  • the reference source (Sanders, JM, et al., Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption. Journal of medicinal chemistry, 2005.48(8): p. 2957-2963.)
  • Purification method and literature of farnesyl pyrophosphate synthase (HsFPPS) (Zhang, Y., et al., Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation. Journal of the American Chemical Society , 2009.131(14): p.5153-5162.)
  • the enzyme activity test method we determined the inhibitory activity of TH-Z80 series compounds on FPPS targets.
  • HsFPPS with 6 consecutive His at the N-terminus was induced to express in vitro, and collected by Ni column purification.
  • the in vitro HsFPPS enzyme assay was performed in 96-well plates at 200 ⁇ l per well.
  • the buffer of the system was 25 mM HEPES, 2.5 mM MgCl 2 , pH 7.4.
  • DMAPP and IPP as the reaction substrate, the change of UV value at the wavelength of 360 nm was monitored in real time in the phosphate lyase system.
  • Drawing with ORIGIN 8.0 software The IC 50 values of the TH-Z80 series compounds for inhibiting FPPS are shown in the following table, and the unit is micromoles per liter ( ⁇ M).
  • TH-Z80 series compounds can effectively inhibit the activity of FPPS and are potent inhibitors of FPPS.
  • Example 2 we used the OVA evaluation system as described in Example 1 to determine the adjuvant effect of the TH-Z80 series of compounds and its side chain-substituted parent compound BPH-266 in mice.
  • Each test compound was formulated to a concentration of 2 mg/ml, and the concentration of the OVA antigen was also 2 mg/ml, and each compound and OVA were mixed at a ratio of 1:1.
  • Each experimental group of C57B/6 mice was injected with 100 ⁇ l of a mixture (containing 50 ⁇ l of antigen and 50 ⁇ l of the compound), and the right thigh muscle was injected.
  • the BPH-266 compound is used (refer to the literature Ling, Y., et al., Bisphosphonate Inhibitors of Toxoplasma gondi Growth: In Vitro, QSAR, and In Vivo Investigations. Journal of Medicinal Chemistry, 2005.48(9): p.3130 -3140.
  • Compound 8 the structure is as follows:
  • IgM and IgG antibodies with significantly higher titers were produced in mice treated with the TH-Z80 series of compounds, especially TH-Z80, TH-Z81, TH-Z152, TH-Z153.
  • the adjuvant effect is most pronounced.
  • the above assay confirmed that the potent inhibitor of FPPS, that is, the TH-Z80 series compound of the present application can act as an adjuvant, and can enhance the specific immune response caused by the antigen.
  • the TH-Z97 series of compounds are newly synthesized bisphosphonic acid compounds whose structures are shown below.
  • HsFPPS humanized farnesyl pyrophosphate synthase
  • TH-Z97 series compounds can effectively inhibit the activity of FPPS and are potent inhibitors of FPPS.
  • IgG antibodies with significantly higher titers were generated in mice treated with TH-Z97 compared to control mice treated with PBS.
  • the above assay confirmed that the potent inhibitor of FPPS, that is, the TH-Z97 series compound of the present application can act as an adjuvant, and can enhance the specific immune response caused by the antigen.
  • HsFPPS humanized farnesyl pyrophosphate synthase
  • the ortho-aminopyridine bisphosphonic acid compound can effectively inhibit the activity of FPPS and is a potent inhibitor of FPPS.
  • the OVA evaluation system as described in Example 1 was used to determine the adjuvants of the eight bisphosphonate compounds used in clinical practice and the TH-Z80 and TH-Z93 compounds of the present invention in mice.
  • the difference in the activity of the novel bisphosphonate compound synthesized in the present invention and the commercially available bisphosphonate drug as an adjuvant was compared.
  • the eight bisphosphonic acid compounds are respectively zoledronate, pamidronate, alendronate, ibandronate, neridronate, Risedronate, olpadronate, minodronate.
  • mice were intramuscularly injected with a mixture of various test compounds and OVA antigens, 20 ⁇ l each, in which the amount of the OVA antigen and various test compounds were 100 ⁇ g.
  • the control mice were injected with 10 ⁇ l of PBS and 10 ⁇ l of OVA antigen in a 1:1 mixture. 7 days and 14 days after immunization, the mice were subjected to eyelid blood sampling, each The mice took 100 ⁇ l of blood.
  • the blood taken out was placed at 4 ° C overnight, centrifuged at 6000 rpm for 8 min, and the supernatant serum was aspirated, and the titer of anti-OVA IgM and IgG antibodies in the serum was measured.
  • IgM and IgG antibodies having higher titers were produced in the mice treated with the test compound relative to the PBS-treated control mice.
  • these bisphosphonic acid compounds especially the TH-Z80 and TH-Z93 compounds of the present invention have the most remarkable adjuvant effect.
  • the titers of IgM and IgG antibodies in mice treated with the TH-Z80 and TH-Z93 compounds of the present invention were compared with the control group treated with PBS, respectively, 7 days and 14 days after immunization.
  • the antibody titer in mice is more than 5 times higher.
  • the adjuvant effects of the TH-Z80 and TH-Z93 compounds of the present invention are also much higher than those of the eight bisphosphonate compounds which have been clinically used.
  • the above measurements (1) to (4) indicate that the FPPS inhibitor can act as an adjuvant and enhance the specific immune response caused by the antigen.
  • GGPPS geranyl-co-pyrophosphate synthase
  • the compounds TH-Z144 and TH-Z145 are our new bisphosphonate compounds which have the structure shown above.
  • HsGGPPS humanized geranyl-co-pyrophosphate synthase expression purification method and literature (Zhang, Y., et al., Lipophilic Bisphosphonates as Dual Famesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation.Journal The enzyme activity test method of the American Chemical Society, 2009.131(14): p.5153-5162.), we tested the inhibition of GGPPS (geranyl-folate pyrophosphate synthase) by compounds TH-Z144 and TH-Z145. active. The activity test data is as follows:
  • Two compounds of TH-Z144 and TH-Z145 were each formulated to a concentration of 10 mg/ml, and the concentration of OVA antigen was also 10 mg/ml, and the compound and antigen were mixed at a ratio of 1:1.
  • Mouse strains were selected for C57B/6. Each experimental group of mice was injected with 20 ⁇ l of a mixture containing the test compound and OVA antigen, that is, 100 ⁇ g (10 ⁇ l) of adjuvant and 100 ⁇ g (10 ⁇ l) of antigen OVA.
  • the control mice were injected with 10 ⁇ l of PBS and 10 ⁇ l of OVA antigen in a 1:1 mixture.
  • IgM and IgG antibodies having significantly higher titers were produced in the mice treated with the test compounds TH-Z144 and TH-Z145, compared with the PBS-treated control mice, confirming GGPPS.
  • the inhibitor acts as an adjuvant and enhances the specific immune response elicited by the antigen.
  • mice treated with BPH-716 and BPH-1222 produced significantly higher titers compared to PBS-treated control mice.
  • IgG and IgM antibodies The antigen used for the detection of BPH-1222 and BPH-716 is hapten, 4-hydroxy-3-nitrophenylacetyl (abbreviated as NP), and NP33-KLH (nitrobenzene attached to hemocyanin (KLH) Mice immunized with BPH-1222 and BPH-716, and NP33-BSA (nitrobenzene linked to bovine serum albumin (BSA)) was used to detect titers against NP-specific antibodies in serum at 7 and 14 days after immunization.
  • the specific implementation and detection methods are the same as the OVA immunization and detection methods
  • this assay confirmed that both the FPPS and GGPPS double inhibitors increase the titer of immunoglobulin, and can exert a good effect as an adjuvant, and can enhance the specific immune response caused by the antigen.
  • HMG-CoA reductase inhibitors HMG-CoA reductase inhibitors
  • Inhibitors FPPS inhibitors (TH-Z80 and TH-Z93), GGPPS inhibitors (TH-Z145) and known common adjuvants (PEI, imiquimod, aluminum adjuvant, MF59, IFA and CFA)
  • PEI common adjuvants
  • IgG titers and antibody affinities after boost were tested in mice treated with these adjuvants.
  • the affinity of an antibody refers to the strength of binding of an antibody to an epitope, and is a very important indicator for assessing the pros and cons of an antibody. It is due to gene mutations in the antibody-forming cells themselves and selective activation of B-cell clones by the antigen. This functional state of the body is the result of long-term evolution and constant adaptation to the external environment, and is of great significance for the body's defense and maintaining its own immune surveillance. In vitro tests were performed to determine the affinity of the antibody by an assay that disrupts antigen-antibody binding by sodium thiocyanate.
  • Antibody affinity assays are based on the fact that sodium thiocyanate is capable of disrupting the binding of the antigen to the antibody; the stronger the affinity of the antibody, the higher the concentration of sodium thiocyanate that dissociates the antigen and the antibody. On the other hand, if the affinity of the antibody is stronger, the effect of the adjuvant is reflected on the other side.
  • PEI polyethyleneimine
  • Lebost Beijing
  • imiquimod purchased from Shanghai Shengsheng Biotechnology Co., Ltd.
  • aluminum adjuvant purchased from Thermo Fisher Company
  • IFA non- Complete Freund's adjuvant
  • CFA complete Freund's adjuvant
  • mice were immunized by intramuscular injection with a mixture of 100 ⁇ l of various test adjuvants and OVA antigen.
  • the control mice were also intramuscularly injected with 50 ⁇ l of PBS and 50 ⁇ l of OVA antigen in a ratio of 1:1.
  • Boosting with 50 ⁇ g of OVA protein was performed 7 days and 14 days after immunization, respectively.
  • the mice were sacrificed on the 7th day after the third immunization, and blood was separated to separate the serum, and the titer of IgG in the serum was measured.
  • the OVA protein was diluted with a pH 9.6 carbonate solution to a concentration of 2 ⁇ g/ml, coated in a 96-well microtiter plate, 50 ⁇ l per well, coated at 4 ° C overnight, and washed with PBS containing 0.05% Tween 20
  • the plate was incubated 5 times with 1% BSA solution, 200 ⁇ l per well, and blocked at room temperature for 2 h.
  • the plate was washed 5 times with PBST, and serum of a certain dilution was added. Specifically, the dilution was not diluted in the PBS group, the simvastatin group was diluted 16 times, and the TH-Z80 group was diluted 16 times.
  • the TH-Z93 group was used.
  • the plate was washed 5 times with PBST and then added at a concentration of 10 mM, 9.5 mM, 9 mM, 8.5 mM, 8 mM, 7.5 mM, 7 mM, 6.5 mM, 6 mM, 5.5 mM, 5 mM, 4.5 mM, 4 mM, 3.5 mM, 3 mM.
  • HRP-labeled goat anti-mouse antibody was added, 50 ⁇ l per well, and incubated for 45 min at room temperature.
  • the plate was washed 5 times with PBST, and sodium citrate OPD coloring solution was added, and the color was blocked for 10 minutes in the dark, and then sulfuric acid was added to terminate.
  • the IC50 of sodium thiocyanate was calculated using Prism software based on the reading of the microplate reader. The higher the IC50 value, the stronger the affinity of the antibody produced by immunization.
  • the three types of inhibitors of the present invention were used with respect to PBS-treated control mice.
  • TH-Z93, TH-Z145 IgG antibodies and antibody affinities with significantly higher titers were produced in mice treated as adjuvants.
  • the inhibitors of the present invention are superior to known common adjuvants such as PEI, imiquimod, aluminum adjuvant, MF59, both in terms of IgG antibody titer and antibody affinity. We believe this result will drive the development of more active adjuvants in the field.
  • Examples 1-4 fully demonstrate that compounds capable of inhibiting the activity of HMG-CoA reductase, FPPS, GGPPS in the mevalonate pathway can serve as adjuvants in immunogenic compositions.
  • HMG-CoA reductase inhibitors, FPPS inhibitors, GGPPS inhibitors can act as adjuvants, thereby enhancing the specific immune response elicited by the antigen.
  • inhibitors of other enzymes in the mevalonate pathway can also act as adjuvants, including but not limited to thiolase (acetoacetyl-CoA transferase), HMG-CoA synthetase, and methotrexate.
  • HMG-CoA reductase inhibitors such as statins, farnesyl pyrophosphate synthase inhibitors such as bisphosphonates, geranium in the mevalonate pathway relative to the negative control group
  • An acyl geranyl pyrophosphate synthase inhibitor such as a bisphosphonate compound significantly increases the titer and antibody affinity of the IgG antibody and can serve as an adjuvant in the immunogenic composition.
  • the geranyl geranyl pyrophosphate synthase is an enzyme downstream of the mevalonate pathway, and our experiments revealed that inhibition of geranyl geranyl pyrophosphate synthetase can exert a good adjuvant effect.
  • GGOH and GGPP were separately formulated to a concentration of 200 mg/ml and 100 mg, respectively.
  • the titer of the antibody when immunized with only 10 ⁇ l of the test compound and 5 ⁇ l of the OVA antigen was mixed without adding GGOH or GGPP.
  • the titer of the antibody when immunized with GGOH or GGPP was measured; specifically, 10 ⁇ l of the test compound, 5 ⁇ l of the OVA antigen, and 5 ⁇ l of each concentration of GGOH were mixed.
  • GGPP total volume 20 ⁇ l
  • Control mice were treated with PBS and antigen alone without the addition of test compounds and GGOH or GGPP.
  • 100 ⁇ l of blood was taken from the eyelids of the mice, and after standing overnight at 4 ° C, serum was separated, and the titer of anti-OVA IgM and IgG antibodies in the serum was measured.
  • the IgG and IgM antibody titers produced in mice treated with simvastatin, TH-Z93, and TH-Z145 of the present invention were significantly increased when GGPP or GGOH was not added.
  • the titer in the control mice above the PBS treatment confirmed its strong adjuvant effect.
  • the titer of IgG and IgM antibodies produced in mice treated with the test compound as an adjuvant is greatly reduced, and the larger the amount of GGPP or GGOH added, the greater the decrease in titer. Big.
  • the titer of the antibody even dropped to the level of titer in the control group treated with PBS alone.
  • the geranyl-glycosylated substrates GGPP and GGOH are very effective in inhibiting the adjuvant action of simvastatin, TH-Z93 and TH-Z145, reflecting the geranyl leaf of the compound by inhibiting the protein.
  • the resulting mechanism acts as an adjuvant. That is, once the geranyl-glycosylated substrates GGPP and GGOH are additionally added from the outside, the compound of the present application does not prevent the formation of the prenylated protein, and thus does not function as an adjuvant.
  • the pathway begins to branch downstream from the formation of FPP, for example, to form cholesterol or the like.
  • HMG-CoA reductase inhibitors act by blocking the intracellular mevalonate pathway and thereby blocking the synthesis of cholesterol.
  • the various enzyme inhibitors of the mevalonate pathway of the present invention exert an adjuvant effect, whether it is because the geranylation of the geranium downstream of the mevalonate pathway is inhibited, or because of other branches. If cholesterol synthesis is inhibited?
  • the selective squalene synthase (SQS) inhibitor TH-Z66 is BPH-652 as described in the following references: Liu CI et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulenc, Science, 2008 Mar 7; 319 ( 5868): 1391-4, which has demonstrated that BPH-652 is a selective squalene synthase (SQS) inhibitor that inhibits the in vivo biosynthesis of cholesterol, which is incorporated herein by reference in its entirety.
  • TH-Z66 was synthesized according to the method described in this document.
  • TH-Z66 was formulated to a concentration of 10 mg/ml, and the concentration of OVA antigen was also 10 mg/ml, mixed in a ratio of 1:1, and each mouse was injected with 20 ⁇ l, ie, containing 100 ⁇ g of TH-Z66 and 100 ⁇ g of antigen. .
  • the mouse is a C57B/6 strain.
  • the mice in the experimental group were injected with the right foot. Control mice were treated similarly except that the same volume of PBS was used instead of TH-Z66.
  • Lymph nodes are important sites for immune response.
  • the lymph nodes are rich in various types of immune cells, which are good for capturing antigens, transmitting antigen information and cell activation and proliferation.
  • B lymphocytes, T lymphocytes, macrophages, and dendritic cells are recruited into the lymph nodes after immunization.
  • T lymphocytes, macrophages, and dendritic cells are recruited into the lymph nodes after immunization.
  • OVA OVA was used as an antigen
  • three inhibitor compounds namely simvastatin, TH-Z80 and TH-Z145, were added as adjuvants, wherein the OVA antigen and the adjuvant were mixed at a ratio of 1:1.
  • Mice were immunized by subcutaneous injection. 24 hours after immunization, the ipsilateral inguinal lymph nodes were removed and separated into single cell suspensions through a 100-mesh sieve.
  • B lymphocytes, T lymphocytes, macrophages, and dendritic cells were measured in lymph nodes by flow cytometry. The proportion and the change in quantity.
  • the marker of B lymphocytes is B220
  • the T lymphocytes are CD3
  • the macrophages are CD11b and F4/80
  • the dendritic cells are CD11c.
  • the three compounds of the present invention were used in comparison with control mice (Ctrl) without any treatment and control mice treated with PBS. , TH-Z80, TH-Z145)
  • the proportion and number of these four cells in the lymph nodes of the mice treated as adjuvants were significantly increased.
  • These adjuvants of the present application were shown to promote the migration of these cells into the lymph nodes to a large extent 24 hours after immunization, reflecting that the compounds of the present application enhance the immune response as an adjuvant.
  • Mevalonate pathway inhibitor enhances DC response to LPS
  • Dendritic cells are one of the most powerful antigen-presenting cells in the body and are a bridge between innate and adaptive immunity. Dendritic cells have highly expressed antigen presenting molecules (MHC-I and MHC-II) and costimulatory molecules (such as CD80, CD86), and thus become powerful antigen presenting cells. Dendritic cells can activate naive T cells and play an important role in the immune response.
  • MHC-I and MHC-II highly expressed antigen presenting molecules
  • costimulatory molecules such as CD80, CD86
  • the adjuvant of the present invention can enhance the stimulation of LPS by dendritic cells in vitro, and can also be applied to dendritic cell vaccines.
  • Dendritic cells are pretreated with these adjuvants and then added to tumor antigens, inactivated tumor cells, tumor cell lysates, plasmids containing tumor antigen DNA, tumor antigen RNA; or these antigens and adjuvants stimulate dendritic Dendritic cell After being treated with these antigens and then added with an adjuvant, it can be used as a novel DC vaccine.
  • Z66 was used to treat the differentiated mouse bone marrow-derived dendritic cells (BMDC) for 24 h, and then stimulated with 100 ng/ml LPS. After 24 h stimulation, the supernatant was collected and the TNF- ⁇ , IL-6 and IL- were measured. 12p70 and IL-1 ⁇ .
  • BMDC was added to 1 ⁇ M of simvastatin, TH-Z80, TH-Z93, TH-Z145 or these adjuvants in a 96-well plate and mixed with 2 ⁇ M of GGOH for 24 hours, with 50,000 cells per well. After 24 h, 50,000 OT-I CD8+ T cells or OT-IICD4+ T cells, and 100 ⁇ g/ml of OVA protein were added and cultured for 72 hours. After 72 h, the culture supernatant of the cells was collected, and the cytokines IL-6, IFN- ⁇ , and TNF- ⁇ secreted in the supernatant were measured.
  • the cytokine assay was purchased from ebioscience with reference to the kit. The assay method was carried out with a 96-well microtiter plate at 4 ° C with a capture antibody of these cytokines, and coated overnight, 100 ⁇ l per well. After 5 aliquots with PBST, they were blocked with blocking solution for 2 h at room temperature. After 5 washes with PBST, the 2-fold diluted cell supernatant was added and incubated for 2 h at room temperature. After 5 washes with PBST, the detection antibody was added and incubated for 1 h at room temperature. After 5 washes with PBST, avidin-conjugated horseradish peroxidase was added and incubated for 45 min at room temperature.
  • APC antigen presenting Cells
  • APC can uptake and process antigen by phagocytosis or pinocytosis, and the treated antigenic determinant-containing polypeptide fragment is bound to MHC class II molecule and then expressed on the cell surface and presented to CD4+TH cells.
  • the cells with antigen presenting mainly include mononuclear/macrophage, dendritic cells and B cells.
  • FITC-labeled OVC was used to label FITC-labeled OVA with the three compounds of the present invention (simvastatin, TH-Z80, TH-Z145) in a similar manner as in Example 1.
  • the mice were subcutaneously immunized, and the ipsilateral lymph nodes were taken out 24 h after immunization, and separated into single cell suspensions.
  • the ratio of FITC-OVA cells contained in the three antigen-presenting cells in the lymph nodes was determined by flow cytometry.
  • the B lymphocyte marker is B220
  • the T lymphocytes are CD3
  • the macrophages are CD11b and F4/80
  • the dendritic cells are CD11c.
  • the experimental group mice treated with the three compounds of the present invention as adjuvants in the lymph nodes the proportion of FITC-positive cells (B220, CD11c, F4/80) was increased, confirming that these inhibitors of the present invention can promote antigen-presenting cells to take up antigens or migrate into lymph nodes, reflecting this The claimed compound enhances the immune response as an adjuvant.
  • Examples 6-8 demonstrate that the adjuvant action of the inhibitors of the present invention is manifested not only in the increase in antibody titer but also in various aspects of the immune response, such as B lymphocytes, T lymphocytes, macrophages, and The recruitment of dendritic cells in lymph nodes is enhanced, assisting LPS to stimulate DC production of IL-1 ⁇ , and promoting antigen-presenting cells to take up antigen.
  • Three inhibitors can act as mucosal adjuvants to promote the production of more antibodies to Mers protein in vivo
  • the titer of the three adjuvant antibodies in the experimental group treated with the three compounds of the present invention as an adjuvant was significantly increased as compared with the control mice treated with PBS, confirming The inhibitor of the present invention is capable of assisting the Mers protein to produce more antibodies, suggesting that it can be used as an adjuvant in the vaccine of the Middle East Respiratory Syndrome virus.
  • Three inhibitors can promote hepatitis B surface antigen HbsAg to produce more antibodies
  • hepatitis B surface antigen (HBsAg, gifted by Beijing Tiantan Biological Products Co., Ltd.) was used as an antigen, and 100 ⁇ g of each of the three compounds of the present invention (simvastatin, TH-Z80, TH-Z144) was added as an adjuvant, according to Mice were immunized by intramuscular injection as in Example 1 except for the procedure.
  • the titer of IgM and IgG of anti-HBsAg protein in mouse serum was measured 7 days and 14 days after immunization.
  • the titer of the three adjuvant antibodies in the experimental group treated with the three compounds of the present invention as an adjuvant was significantly increased as compared with the control mice treated with PBS, confirming The inhibitor of the present invention is capable of assisting hepatitis B surface antigen to produce more antibodies, suggesting that it can be used as an adjuvant in hepatitis B vaccine.
  • mice were immunized with a similar procedure as in Example 1 using the four substances of the present invention, simvastatin, TH-Z80, TH-Z93, and TH-Z145, respectively, as an adjuvant.
  • the mixture used for the primary immunization contained 100 ⁇ g of OVA protein and 100 ⁇ g of adjuvant (ie, the above four substances), and the root of the tail was injected subcutaneously, and it was strengthened two weeks after the immunization.
  • the boost was not used, the root of the tail was subcutaneously injected with 50 ⁇ g of the antigen. Two weeks after the second immunization, it was boosted again and subcutaneously injected with 50 ⁇ g of the base of the antigen.
  • the cells were stably transfected to express luciferase prior to inoculation.
  • the specific method is as follows: 293T cells were transfected with a luciferase-expressing viral plasmid, and 48 h after transfection, the virus-containing 293T cell culture supernatant was collected. After culturing B16-OVA cells with the virus-containing supernatant, the virus in the supernatant infects B16-OVA cells and expresses luciferase, and the cells expressing luciferase are sorted by flow sorting and expanded. to cultivate.
  • the fluorescein substrate was injected, 3 mg/mouse, and 8 min after the injection, the mice were anesthetized, and the volume of the tumor was observed with a living imager.
  • the tumor cells of 3*10 5 (ie, 300,000) B16-OVA (also donated by the Institute of Biomedical Sciences, Soochow University) were inoculated subcutaneously into the right groin of C57B/6 mice. On the 5th day after inoculation, 100 ⁇ g of each was used. A mixture of OVA protein and 100 ⁇ g of adjuvant (ie, the above four substances, simvastatin, TH-Z80, TH-Z93, and TH-Z145) was injected subcutaneously into the tail of the mouse, and was strengthened 7 days after the initial immunization.
  • adjuvant ie, the above four substances, simvastatin, TH-Z80, TH-Z93, and TH-Z145
  • the agent was injected subcutaneously with 50 ⁇ g of antigen at the base of the tail, and re-enhanced 7 days after the second immunization to enhance 50 ⁇ g of antigen.
  • the body weight of the mice and the change in tumor volume were measured every two days.
  • the volume of the tumor is calculated using the formula length * width * width / 2.
  • the cells were stably transfected with luciferase prior to inoculation using the same method as described in Example 11.
  • mice Three mice were randomly taken from each group 7 days, 14 days and 21 days after inoculation.
  • the fluorescein substrate was injected, 3 mg/mouse, and 8 min after the injection, the mice were anesthetized, and the volume of the tumor was observed with a living imager.
  • Fig. 26 The results are shown in Fig. 26. Compared with the control mice treated with PBS, the volume of melanoma was significantly reduced in the experimental group mice treated with the four compounds of the present invention as an adjuvant, suggesting that it can be used as an adjuvant in melanoma. In a therapeutic vaccine.
  • Examples 9-12 demonstrate that the inhibitors of the present invention can be used as adjuvants in a variety of clinical vaccines, such as the Middle East Respiratory Syndrome virus vaccine, hepatitis B vaccine, melanoma therapeutic and prophylactic vaccines, suggesting that Broad prospects for clinical applications.
  • the OVA antigen was mixed with the TH-Z80 of the present invention as an adjuvant to immunize different parts of the mouse, and the parts to be immunized were the sole, subcutaneous, muscle, abdominal cavity and nasal mucosa of the mouse, respectively.
  • the foot and nasal mucosa were immunized with a 20 ⁇ l system, the concentration of TH-Z80 was 10 mg/ml, the concentration of OVA protein was also 10 mg/ml, and the adjuvant and antigen were mixed at a ratio of 1:1, that is, 10 ⁇ l of adjuvant and 10 ⁇ l of antigen.
  • mice were immunized with the same volume of PBS mixed with OVA antigen, and titers of anti-OVA IgM and IgG antibodies in serum at 7 days and 14 days after immunization were determined according to a procedure similar to that in Example 1.
  • concentration of the OVA antigen was 10 mg/ml.
  • 5 ⁇ l of TH-Z93 or imiquimod was added as an adjuvant, and 10 ⁇ l of antigen and 5 ⁇ l of PBS were mixed.
  • 10 ⁇ l of antigen and 5 ⁇ l of PBS were mixed.
  • the strain of the mouse was selected from C57B/6 mice. Control mice used an equal volume of PBS without any adjuvant. The right foot of the mouse was injected.
  • Example 15 Activity experiment of a compound of formula IXX
  • HsFPPS humanized farnesyl pyrophosphate synthase
  • HsFPPs with 6 consecutive Hiss at the N-terminus were induced to express in vitro, and collected by Ni column purification.
  • In vitro HsFPPs enzyme activity assay was performed in 96-well plates at 200 ⁇ l per well.
  • the buffer of the system was 25 mM HEPES, 2.5 mM MgCl 2 , pH 7.4.
  • DMAPP and IPP as the reaction substrate, the change of UV value at the wavelength of 360 nm was monitored in real time in the phosphate lyase system. Drawing with ORIGIN 8.0 software.
  • PvGGPPs with 6 consecutive Hiss at the N-terminus were induced to express in vitro, and collected by Ni column purification.
  • the in vitro PvGGPPs enzyme activity assay was performed in 96-well plates at 200 ⁇ L per well.
  • the buffer of the system was 25 mM HEPES, 2.5 mM MgCl 2 , pH 7.4.
  • a continuous spectrophotometric reading of 360 nm was performed in the presence of a phosphatase system in the presence of GPP and IPP as substrates. Drawing with ORIGIN 8.0 software. The results are as follows:
  • Plasmodium falciparum 3D7 was cultured with IPEM1640 medium supplemented with 10% human type O blood serum and 25 mM HEPES. The culture process was maintained in a 5% carbon dioxide environment in a carbon dioxide incubator. In vitro drug testing experiments were performed using 96-well plates. The drug tested was dissolved in PBS and pre-diluted with complete medium. Infected red blood cells were cultured in triplicate in continuous 3-fold dilutions of the drug for 72 hours. Then add the same amount of SYBR-GREEN1 to each well. Then, it was detected at 485 nm of excitation light and 538 nm of emitted light. The contrast was observed with artemisinin and no drug control group. The graph was fitted with ORIGIN 8.0 software, and the results are shown in Table 1.
  • DMEM medium was purchased from Gibco; fetal bovine serum (FBS) was purchased from BI company; double antibody was purchased from Biyuntian; 0.25% trypsin-EDTA was purchased from Gibco; MTT was purchased from Ameresco; centrifuge was purchased from Anhui Jia Technology Co., Ltd., 4 ° C refrigerator purchased from Haier company, -80 ° C purchased from Thermo company;
  • FBS fetal bovine serum
  • double antibody was purchased from Biyuntian
  • 0.25% trypsin-EDTA was purchased from Gibco
  • MTT was purchased from Ameresco
  • centrifuge was purchased from Anhui Jia Technology Co., Ltd., 4 ° C refrigerator purchased from Haier company, -80 ° C purchased from Thermo company;
  • Cell resuscitation Quickly remove the cryotube from the liquid nitrogen tank, immediately immerse it in water at 37 °C with a forceps, melt it, completely dissolve it, transfer it to a clean bench, aspirate the cell suspension, and add 3 ml of double-antibody.
  • 10% FBS DMEM after mixing, centrifuge at 1000r/min for 4min at 4°C, discard the supernatant, dilute with appropriate amount of DMEM medium containing 10% FBS, and inoculate the culture flask at a density of 0.5 ⁇ 10 6 cells/cm 2 . Mix gently and place in a 37 ° C, 5% CO 2 incubator.
  • Cell passage The cells were first discarded when the cells were passaged, then washed once with pre-warmed PBS at room temperature, and then digested with 0.01% trypsin-EDTA at 37 ° C, 5% CO 2 incubator for 1-3 min, gently The side wall of the culture flask was tapped, the cells were detached from the bottom wall of the culture flask, and the digestion was terminated by adding 2 ml of serum-containing DMEM medium.
  • the cell suspension was transferred to a 15 ml glass centrifuge bottle, and gently blisted to form a single cell suspension, 1000 r/ Centrifuge at 4 ° C for 3 min, discard the supernatant, resuspend the cells with an appropriate amount of medium, mix 50 ⁇ l of the cell suspension with 50 ⁇ l of phenol blue, and count the viable cells on the cell counting plate to 0.5 ⁇ 10 6 cells /
  • the density of cm 2 was introduced into the culture flask, and the medium was gently mixed by adding an appropriate amount of medium, and placed in a 37 ° C, 5% CO 2 incubator for static culture.
  • Cryopreservation of cells Collect the cells in a glass centrifuge tube and count, discard the supernatant, prepare a cell cryopreservation solution according to the ratio of DMSO to serum 1:9, and use a cell cryopreservation solution to treat 1 to 2 ⁇ 10 6 cells. Resuspend the cells at a concentration of /ml, then dispense 1ml to each cryotube, place in a -80 °C refrigerator overnight, and transfer to liquid nitrogen for several years.
  • Drug dissolution Weigh a certain amount of bisphosphonic acid drug, add a small amount of NaOH or NaHCO 3 to dissolve the drug, the storage concentration is 10 ⁇ M;
  • Drug dilution Dilute the prepared bisphosphonate stock solution in DMEM complete medium, the highest concentration is 1 mM, and the ratio is diluted 1:3.2, a total of 11 concentration gradients;
  • MDA-MB-231 cells were digested one day before, centrifuged at 1000 rpm for 3 min, cell count, inoculated with 3000 cells/well of cells into 96 empty plates, 100 ⁇ l per well, cultured for 14-16 h, pay attention to accurate cell counts,
  • the amount of cells inoculated in the well should be the same.
  • the cell suspension can be lightly shaken intermittently, or a row of cells can be lightly blown with a pipette.
  • the speed of the gun into the 96-well plate should not be too fast or too slow to prevent the cells from being
  • the uneven distribution in the orifice plate affects the experiment; the evaporation velocity of the pores around the 96-well plate is slightly faster, and the concentration of the solution changes slightly faster. If necessary, the 60 holes in the middle are selected as the stimulation, and the injected liquid avoids the generation of bubbles.
  • the liquid in the 96-well plate is discarded, and the prepared liquid is added to the cell culture plate laid in the previous day (the total volume of the final treated cells is 100 ⁇ l), Set a blank control group (only medium added), 6 replicates per group;
  • OVA ovalbumin
  • the mice were c57bl/6 of 8 weeks female.
  • OVA was prepared at a concentration of 1 mg/ml, and the concentration of bisphosphonic acid was 1 mg/ml.
  • OVA and bisphosphonic acid were mixed at a ratio of 1:1.
  • Mice were intramuscularly injected with 100 ⁇ l, which was 100 ⁇ g of OVA and 100 ⁇ g per mouse. Bisphosphonic acid.
  • the eyes of the mice were bled, and 100 ⁇ l of each blood was taken.
  • the removed whole blood was placed in a refrigerator at 4 ° C overnight. On the next day, the whole blood at 4 ° C was taken out and centrifuged, the speed of the centrifuge was 6000 rpm, the temperature was 4 ° C, and the mixture was centrifuged for 5 min. After the end of the centrifugation, the supernatant serum was carefully aspirated, centrifuged again, at a speed of 6000 rpm, at a temperature of 4 ° C, and centrifuged for 5 min. The upper serum is then aspirated. The serum was stored frozen at -20 ° C and taken out and thawed at the time of detection.
  • Coat The antigen OVA was diluted to a protein content of 2 ⁇ g/ml with 0.05 M pH 9.6 carbonate coating buffer.
  • the reaction wells of each polystyrene plate were coated with 50 ⁇ l, 4 ° C overnight or 37 ° C for 2 h. The next day, the solution in the well was discarded and washed 5 times with wash buffer PBST.
  • Blocking 2% BSA 150 ⁇ l, blocked at 37 ° for 2 h, washed 5 times with wash buffer PBST.
  • Figure 29 is a graph showing the effect of injection of 100 ⁇ g of different bisphosphonic acids on mouse antibody production.
  • TH-Z80 is able to produce higher titers of antibodies than conventional bisphosphonic acids compared to conventional bisphosphonic acids.
  • Fig. 30 is a view showing the effect of replacing the length of the bisphosphonic acid side chain carbon chain as an adjuvant.
  • the affinity of an antibody refers to the strength of binding of an antibody to an epitope, and is a very important indicator for assessing the pros and cons of an antibody. It This is due to gene mutations in the antibody-forming cells themselves and selective activation of B-cell clones by antigens. This functional state of the body is the result of long-term evolution and constant adaptation to the external environment, and is of great significance for the body's defense and maintaining its own immune surveillance.
  • Figure 31 is a graph showing the effect of TH-Z80 on increasing antibody affinity.
  • TH-Z80 significantly enhanced the affinity of the adjuvant and its antigen compared to PBS.
  • PBMC Human peripheral blood mononuclear cells
  • Figure 32 shows the effect of different carbon chain length benzimidazole bisphosphonic acids on the amplification of ⁇ T cells.
  • Figure 33 is a schematic diagram showing the crystal structure of the compounds TH-Z80 and HsFPPS (pymol plot).
  • Figure 34 is a schematic view showing the crystal structure of the compounds TH-Z82 and PvGGPPS (pymol plot).
  • the main driving force for the combination of TH-Z82 bisphosphonate with PvGGPPS is the chelation of bisphosphonic acid with metal ions and its long carbon.
  • the hydrophobic action of the chain, the benzene ring and the phenol ether in the benzimidazole were not significantly affected by PvGGPPS.
  • This result indicates that the introduction of the N hetero atom in the benzimidazole has no significant effect on the binding activity of the compound to PvGGPPS.
  • the introduction of a long carbon chain in the side chain of the N-heterobenzimidazole compound enhances its hydrophobic interaction with GGPPS and enhances the binding of the compound to GGPPS.
  • Example 16 Effect of simvastatin, TH-Z80, TH-Z93 and TH-Z145 on PR8 influenza virus.
  • hemagglutinin protein (HA1) in PR8 influenza virus was added to phosphate buffer, 20 micrograms of simvastatin, 20 micrograms of TH-Z80, 20 micrograms of TH-Z93 and 20 micrograms of TH. -Z145. And on day 0, day 14 and Mice were immunized intramuscularly on day 21. On day 28, the mouse nasal mucosa was inoculated with PR8 virus. The body weight of the mice was then weighed daily and the death of the mice was observed.
  • HA1 hemagglutinin protein
  • Example 17 Inhibition of B16-OVA tumors by anti-PD1 antibody in combination with four adjuvants and chicken ovalbumin.
  • RESULDS 300,000 tumor cells were inoculated subcutaneously into the right inguinal region of C57B/6 mice.
  • 100 ⁇ g of OVA protein and 100 ⁇ g of adjuvant were used respectively (ie, the above four substances, simvastatin, TH-Z80).
  • the mixture of TH-Z93 and TH-Z145 was injected subcutaneously into the tail of the mouse. It was strengthened 7 days after the initial immunization. When the boost was used, no adjuvant was used.
  • 50 ⁇ g of antigen was injected subcutaneously at the base of the tail, and strengthened again 7 days after the second immunization. Enhance 50 ⁇ g of antigen.
  • Example 18 Adjuvant activity of HMG-CoA synthase inhibitor Hymeglusin in OVA antibody titer assay.
  • the HMG-CoA synthase inhibitor Hymeglusin was formulated to a concentration of 10 mg/ml, the concentration of the OVA antigen was also 10 mg/ml, and the compound and the antigen were mixed at a ratio of 1:1.
  • Mouse strains were selected for C57B/6. Each experimental group of mice was intramuscularly injected with 20 ⁇ l of a mixture containing the test compound and OVA antigen, that is, 100 ⁇ g of Hymeglusin (10 ⁇ l) and 100 ⁇ g (10 ⁇ l) of antigen OVA.
  • the control mice were intramuscularly injected with 10 ⁇ l of PBS and 10 ⁇ l of OVA antigen in a 1:1 mixture. After 14 days from the immunization, 100 ⁇ l of blood was taken from the eye of the mouse, and after standing at 4 ° C overnight, the serum was separated, and the titer of the anti-OVA IgG antibody in the serum was measured.
  • IgG antibodies having a significantly high titer were produced in mice treated with the HMG-CoA synthase inhibitor Hymeglusin as an adjuvant, compared with the PBS-treated control mice.
  • a compound capable of inhibiting the activity of HMG-CoA reductase in the mevalonate pathway can serve as an adjuvant in an immunogenic composition.
  • Step 1 10 mmol (1.11 g) of 2-amino-4-hydroxypyridine was dissolved in 50 mL of acetone, and 30 mmol of anhydrous potassium carbonate was added thereto, and the mixture was heated under reflux with N 2 to give 12 mmol (1.67 mL) of bromohexane. After the reaction was over night, the insoluble material was filtered, and the organic phase was evaporated to dryness, and then purified and purified by petroleum ether / ethyl acetate to give 7.8 mmol (1.51 g) of 2- hexyloxy-2-nitroaniline :78%).
  • Step 2 5 mmol (0.97 g) of 4-hexyloxy-2-amino-pyridine and 5.5 mmol (0.71 g) of trans-4-oxo-2-butenoic acid ethyl ester in 20 mL of acetonitrile at 80 ° After overnight, it was dried, taken-up, and purified by petroleum ether / ethyl acetate to give 4.1 mmol (1.13 g) of 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl. Ethyl acetate, yield 82%.
  • Step 3 3 mmol of ethyl 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl)acetate was heated to reflux in 6N HCl for 6 h then dried and dried. Constant weight, the obtained crude product was directly used for the next reaction.
  • Step 4 After the obtained 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl)acetic acid was dissolved at 9 degrees with 9 mmol (0.74 g) of phosphorous acid and 6 mL of sulfolane After adding 10.2 mmol (1068 ⁇ L) of PCl 3 dropwise, the reaction was carried out for 3.5 hours, 1 mL of water was added, and the mixture was heated under reflux for 2 hours, cooled, and the precipitated solid was filtered, and the methanol was ultrafiltered 3 times, and the obtained pale yellow solid was dried, constant weight After weighing, it was 228 mg of the target product (yield 18%).
  • TH-Z156 was directly prepared from 2-amino-4-methoxypyridine as a starting material according to the synthetic steps 2-4 of TH-Z97.
  • the third step adding 5 mL of 6N hydrochloric acid to the product of the previous step, reacting at 100 ° C for 10 hours, distilling off hydrochloric acid under reduced pressure, and the crude product is firstly washed by acetone for 1 time, and then ultrasonically washed with methanol for 3 times to obtain a pure product.
  • the yield was 95%.
  • the second step 6 mmol of the product of the previous step is dissolved in 50 mL of a solvent, 12 mmol of potassium carbonate and 7.2 mmol of 4-hydroxy-2-aminopyridine are added, and the reaction is carried out at room temperature overnight, the potassium carbonate solid is filtered off, and the acetonitrile solvent is distilled off under reduced pressure.
  • the petroleum ether: ethyl acetate 1:1 silica gel (200-300 mesh) column was isolated to give a pure product, yield 43%.
  • Step 4 Dissolve 2.0 mmol of the product of the third step in 10 mL of DMSO, add 20 mL of 30% hydrogen peroxide and 3.0 mmol of potassium carbonate, react at room temperature for 4 hours, extract with ethyl acetate, dry the organic layer with magnesium sulfate, filter and decompress. Ethyl acetate was distilled off and the next reaction was carried out without isolation.
  • the fifth step was the same as the third step in Example 16.
  • the total reaction yield of the two steps was 78%.
  • Example 19 was prepared in a similar manner to Example 16 except that benzyl bromide was used instead of hexyl bromide as a starting material and allowed to react at room temperature.

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Abstract

公开了一种作为疫苗佐剂的甲羟戊酸通路的抑制剂及其用途。具体而言，所述抑制剂为乙酰乙酰基-CoA转移酶抑制剂、HMG-CoA合酶抑制剂、HMG-CoA还原酶抑制剂、甲羟戊酸激酶抑制剂、磷酸甲羟戊酸激酶抑制剂、甲羟戊酸-5-焦磷酸脱羧酶抑制剂、异戊烯焦磷酸异构酶抑制剂、法尼基焦磷酸合酶抑制剂、香叶香叶酰基焦磷酸合酶抑制剂和香叶香叶酰基转移（(I，II）抑制剂。还公开了一种免疫原性组合物，所述组合物包含甲羟戊酸通路的抑制剂作为佐剂。

Description

作为高效的疫苗佐剂的甲羟戊酸通路的抑制剂 技术领域
本发明涉及作为高效的疫苗佐剂的甲羟戊酸通路的抑制剂。本发明还涉及免疫原性组合物，所述组合物包含甲羟戊酸通路的抑制剂作为佐剂。
背景技术
佐剂在疫苗的开发和使用中起着重要作用。佐剂又称非特异性免疫增强剂。本身不具抗原性，但同抗原一起或预先注射到机体内能增强抗原的免疫原性或改变免疫反应类型。减毒活疫苗和灭活疫苗本质上可能包含自然佐剂成分，其中可能包括颗粒形式的蛋白质、脂质和寡核苷酸。事实上许多减毒或者灭活的疫苗，免疫后对机体具有非常强的保护作用。但是由于这些减毒和灭活疫苗本身的一些局限性，如减毒的病原微生物突变成强致病的微生物，灭活疫苗在制备中没有灭活完全，当这些疫苗作用于机体，可能会直接导致机体致病。亚单位疫苗是将致病微生物主要的保护性免疫原存在的组分制成的疫苗。由于近现代分子生物学的兴起，亚单位疫苗以其质控方便，可批量生产，安全可靠等优点，成为现代疫苗开发和应用的主要趋势。但是亚单位疫苗也有保护作用短，起效慢等缺点，以弥补亚单位疫苗这些缺点的佐剂是现代疫苗研发和使用中间的一个重要的组成部分。
疫苗生产当中使用最广泛的佐剂为铝佐剂，1926年首次发现铝盐具有佐剂作用，并于1936年首次使用在白喉疫苗当中，但是由于铝佐剂的一些局限性，如佐剂效果弱，需要配合免疫原性较强的抗原才能起到很好的作用，特别是不能够很好的促进介导细胞免疫的Th1反应，导致铝佐剂不能很好的防治流感，HIV，肿瘤以及疟疾等疾病，所以这些疫苗迫切的需要新型有效的佐剂。到目前为止，在美国和欧洲临床批准使用的佐剂包括使用铝盐、水包油乳剂(MF59 AS03和AF03)和AS04(MPL铝盐)。佐剂的开发处于“原始”状态，目前已知的分子靶标仅有TLR(Toll样受体)。本领域迫切需要发现用于佐剂的新的分子靶标。
发明内容
在本发明中，我们首次发现并证实与甲羟戊酸通路(mevalonate pathway)相关的酶可以作为佐剂理性设计的靶标。
甲羟戊酸通路是以乙酰辅酶A为原料合成异戊二烯焦磷酸(IPP)和二甲烯丙基焦磷酸(DMAPP)的一条代谢途径，存在于所有高等真核生物和很多病毒中。该途径的产物可以看作是活化的异戊二烯单位，是类固醇、类萜等生物分子的合成前体。在这条通路中，首先由两分子的乙酰辅酶A(acetyl-CoA)生成乙酰乙酰基辅酶A(acetoacetyl-CoA)，生成的乙酰乙酰基辅酶A再与乙酰辅酶A生成3-羟基-3-甲基戊二酰辅酶A(3-hydroxy-3-methylglutary CoA，即HMG-CoA)，HMG-CoA在HMG-CoA还原酶的作用下，还原成甲羟戊酸。甲羟戊酸在两种激酶和一种脱羧酶的作用下，将甲羟戊酸催化形成异戊二烯焦磷酸(IPP)。IPP在FPP合酶(FPPS)的催化下形成法尼基焦磷酸(FPP)。FPP在下游通路开始分支，例如形成胆固醇、泛醌(ubiquinone)、血红素(Heme A)、甾醇(Sterol)、多萜醇(dolichol)和异戊烯化的 (prenylated)蛋白。例如，FPP可以在角鲨烯合酶(SQS)的作用下合成角鲨烯，角鲨烯在一系列酶的催化下合成胆固醇。FPP能够在法尼基转移酶作用下，对一些蛋白进行法尼基化修饰。另外一方面，FPP在GGPP合酶(GGPPS)的催化下，合成香叶酰香叶酰基焦磷酸(GGPP)；而GGPP能够在香叶香叶酰基转移酶作用下，对一些蛋白进行香叶酰香叶酰化修饰从而形成异戊烯化的(prenylated)蛋白。
我们发现，凡是影响蛋白香叶香叶酰化(geranylgeranylation)的物质都可以用于疫苗佐剂的开发。具体而言，可以开发针对以下靶标的疫苗佐剂。1)硫解酶(乙酰乙酰基-CoA转移酶)；2)HMG-CoA合酶；3)HMG-CoA还原酶；4)甲羟戊酸激酶(Mevalonate Kinase)；5)磷酸甲羟戊酸(Phosphonomevalonate kinase)；6)甲羟戊酸-5-焦磷酸脱羧酶(Mevalonate-5-pyrophosphate decarboxylase)；7)异戊烯焦磷酸异构酶(Isopentenyl-diphosphate isomerase)；8)法尼基焦磷酸合酶(Farnesyl diphosphate synthase，FPPS)；9)香叶香叶酰基焦磷酸合酶(Geranylgeranyl diphosphate synthase，GGPPS)；10)香叶香叶酰基转移酶(I，II)(Geranylgeranyl transferase I，II)。
我们还发现，其它不直接作用于甲羟戊酸通路但间接影响香叶香叶酰化的物质也可以用于疫苗佐剂的开发。
因此，在一个方面，本发明涉及包含影响蛋白香叶香叶酰化的物质作为佐剂的免疫原性组合物。所述物质可包括，但不限于1)硫解酶(乙酰乙酰基-CoA转移酶)抑制剂、2)HMG-CoA合酶抑制剂、3)HMG-CoA还原酶抑制剂、4)甲羟戊酸激酶抑制剂、5)磷酸甲羟戊酸激酶抑制剂、6)甲羟戊酸-5-焦磷酸脱羧酶抑制剂、7)异戊烯焦磷酸异构酶抑制剂、8)法尼基焦磷酸合酶抑制剂、9)香叶香叶酰基焦磷酸合酶抑制剂和10)香叶香叶酰基转移酶(I，II)抑制剂。
在另一方面，本发明涵盖了上述抑制剂，用作佐剂。
在另一方面，本发明涵盖了上述抑制剂作为佐剂在制备免疫原性组合物中的用途。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000001
上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；m＝1-6的整数。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000002
其中n为1-24的整数，优选n为1-20的整数，更优选n为1-15的整数，更优选n为1-12的整数。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000003
其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
在该方面一个优选的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000004
Figure PCTCN2016098371-appb-000005
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000006
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
在该方面一个优选的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000007
在另一方面，本发明还涉及作为香叶香叶酰基焦磷酸合酶抑制剂的新的化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000008
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
在该方面一个优选的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000009
在另一方面，本发明还涉及所述这些新的化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
本发明另外一方面涉及一种免疫受试对象或宿主的方法，包括向所述受试对象或宿主施用如本发明定义的免疫原性组合物。
发明详述
具体地，本发明涉及以下具体实施方案：
1.一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自：
1)硫解酶抑制剂；
2)HMG-CoA合酶抑制剂；
3)HMG-CoA还原酶抑制剂；
4)甲羟戊酸激酶抑制剂；
5)磷酸甲羟戊酸激酶抑制剂；
6)甲羟戊酸-5-焦磷酸脱羧酶抑制剂；
7)异戊烯焦磷酸异构酶抑制剂；
8)法尼基焦磷酸合酶抑制剂；
9)香叶香叶酰基焦磷酸合酶抑制剂；和
10)香叶香叶酰基转移酶(I，II)抑制剂。
2.按照1的免疫原性组合物，其中所述HMG-CoA还原酶抑制剂为他汀类化合物。
3.按照2的免疫原性组合物，其中所述他汀类化合物选自普伐他汀、阿托伐他汀、罗苏伐他汀、氟伐他汀、匹伐他汀、美伐他汀、洛伐他汀、辛伐他汀、西立伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
4.按照2的免疫原性组合物，其中所述他汀类化合物选自辛伐他汀，洛伐他汀和美伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
5.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为双膦酸化合物或其可药用盐、酯、前药、溶剂化物。
6.按照5的免疫原性组合物，其中所述双膦酸化合物或其可药用盐选自唑来膦酸、帕米膦酸、阿仑膦酸，伊班膦酸，奈立膦酸，利塞膦酸，奥帕膦酸，米诺磷酸。
7.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000010
上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
m＝1-6的整数。
8.根据7所述的免疫原性组合物，其中所述式I所示化合物为下述式II-X所示化合物：
Figure PCTCN2016098371-appb-000011
Figure PCTCN2016098371-appb-000012
上述式II-X中，X为下述任意一种：氢、羟基、巯基、卤素、烷氧基或烷基；
每一个M独立地为下述任意一种：负电荷、氢、烷基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；
R1、R2、R3、R4、、R5、R8分别独立地选自下述任意一种：氢、羟基、脂肪基、巯基、卤素、氨基、烷基、-O-(CH2)qCH3、-NH-(CH2)qCH3、-N[(CH2)qCH3]2、-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-O-(CH2)qCH3，其中，p＝1-6，q＝0-6；m＝1-6的整数。
9.根据7或8所述的免疫原性组合物，其中所述化合物为式XI-XVIII所示化合物：
Figure PCTCN2016098371-appb-000013
式XI-XVIII中，Z为氢、羟基、脂肪基、烷氧基、氨基或烷胺基。
10.根据7-9中任一项所述的免疫原性组合物，其中所述化合物为式IXX或XX所示化合物：
Figure PCTCN2016098371-appb-000014
式IXX、式XX中，n为0或1-12的整数。
11.根据7-10中任一项所述的免疫原性组合物，其中所述化合物为下述任意一种：
Figure PCTCN2016098371-appb-000015
12.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000016
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等；
13.按照12的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000017
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
14.按照13的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000018
其中n为1-24的整数，优选n为1-12的整数。
15.按照14的免疫原性组合物，其中n为1-20的整数。
16.按照14的免疫原性组合物，其中n为1-15的整数。
17.按照14的免疫原性组合物，其中所述化合物选自：
Figure PCTCN2016098371-appb-000019
Figure PCTCN2016098371-appb-000020
18.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000021
其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素。
19.按照18的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000022
其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
20.按照19的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000023
其中n为1-24的整数，优选n为1-12的整数。
21.按照20的免疫原性组合物，其中n为1-20的整数。
22.按照20的免疫原性组合物，其中n为1-15的整数。
23.按照20的免疫原性组合物，其中所述化合物选自：
Figure PCTCN2016098371-appb-000024
Figure PCTCN2016098371-appb-000025
24.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000026
其中
R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
25.按照24的免疫原性组合物，其中R1选自氢、C1-10烷基、C1-10炔基、C1-10烷基氨基、C1-10烷基硫基、卤素、羟基、吲唑基、C1-10烷氧基、被苯基或吡啶基取代的C1-10烷氧基，所述吡啶基任选取代有氨基甲酰基。
26.按照25的免疫原性组合物，其中R1选自氢、4-甲基苯乙氧基、4，5，6，7-四氢-2H-吲唑-2-基、(2-氨基甲酰基吡啶-4-基)甲氧基、苄基氧基、己基氧基、甲硫基、辛基氨基、己基、辛基、癸基、辛-1-炔-1-基、羟基、溴。
27.按照24的免疫原性组合物，其中R2选自氢、C1-10烷氧基、卤素。
28.按照27的免疫原性组合物，其中R2选自氢、辛基氧基、溴。
29.按照24的免疫原性组合物，其中R3选自氢、C1-10烷基、C1-10烷氧基。
30.按照29的免疫原性组合物，其中R3选自氢、甲基、己基氧基。
31.按照24的免疫原性组合物，其中R2与R3与它们所连接的碳原子一起形成苯环。
32.按照24的免疫原性组合物，其中R4选自氢、C1-10烷氧基。
33.按照32的免疫原性组合物，其中R4选自氢、辛基氧基。
34.按照24的免疫原性组合物，其中所述化合物选自：
Figure PCTCN2016098371-appb-000027
35.按照1的免疫原性组合物，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000028
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、 芳基和杂芳基。
36.按照35的免疫原性组合物，其中R5选自C1-10烷氧基。
37.按照35的免疫原性组合物，其中所述化合物为
Figure PCTCN2016098371-appb-000029
38.按照1的免疫原性组合物，其中所述香叶香叶酰基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000030
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
39.按照38的免疫原性组合物，其中R9选自C1-10烷氧基。
40.按照38的免疫原性组合物，其中所述化合物为
Figure PCTCN2016098371-appb-000031
41.按照1-40中任一项所述的免疫原性组合物，其包含一种或多种抗原。
42.按照41的免疫原性组合物，其中所述抗原衍生自细菌、病毒、寄生物或肿瘤。
43.按照41的免疫原性组合物，其中所述抗原衍生自炭疽、弯曲杆菌、霍乱、白喉、产肠毒素性大肠杆菌、贾第虫、淋球菌、幽门螺杆菌、乙型流感嗜血菌、不可定型的流感嗜血菌、脑膜炎球菌、百日咳、肺炎球菌、沙门氏菌、志贺氏菌、链球菌B、A组链球菌、破伤风、霍乱弧菌、耶尔森氏菌、葡萄球菌、假单胞菌属物种和梭菌属物种，或者抗原衍生自腺病毒、登革热1至4血清型、埃博拉病毒、肠道病毒、肝炎A至E血清型、单纯疱疹病毒1或2、人免疫缺陷病毒、流感、日本马脑炎、麻疹、诺 沃克、乳头瘤病毒、细小病毒B19、脊髓灰质炎、狂犬病、轮状病毒、风疹、麻疹、痘苗、含有编码其它抗原诸如疟疾抗原的基因的痘苗构建体、水痘、和黄热病，或者抗原衍生自溶组织内阿米巴、疟原虫、弓形体病、和蠕虫，或者抗原衍生自肿瘤。
44.按照41的免疫原性组合物，其中所述抗原衍生自中东呼吸综合征(Mers)病毒、乙型肝炎病毒、黑色素瘤。
45.按照前述1-44中任一项的免疫原性组合物，用于治疗或预防中东呼吸综合征、乙型肝炎病毒、黑色素瘤。
46.按照前述1-45中任一项的免疫原性组合物，还包含另一种佐剂。
47.按照46的免疫原性组合物，所述另一种佐剂选自铝佐剂，完全弗氏佐剂，非完全弗氏佐剂，MF59，AS01，AS02，AS03，AS04，AS15，CAF01，ISCOMs(免疫刺激复合物)，Virosomes(病毒颗粒)，GLA-SE，脂质体，食用油，皂角苷，AF03，TLR激动剂。
48.按照47的免疫原性组合物，所述TLR激动剂选自(如三酰脂蛋白)，TLR2刺激剂(如肽聚糖，酵母多糖，HMGB1(高速泳动蛋白1)，脂磷壁酸)，TLR3刺激剂(双链RNA如PolyI：C)，TLR4刺激剂(如LPS，MPL，RC529，GLA，E6020)，TLR5刺激剂(鞭毛蛋白)，TLR6刺激剂(如三酰脂蛋白，脂磷壁酸)，TLR7/8刺激剂(单链RNA，咪喹莫特)，TLR9刺激剂(DNA，如CPG ODN)，C-凝集素配体(如海带多糖)，CD1d配体(如α-半乳糖基神经酰胺)。
49.按照前述1-48中任一项的免疫原性组合物，适于通过口服、局部或肠胃外途径进行免疫。
50.按照49的免疫原性组合物，适于通过注射途径进行免疫。
51.按照49的免疫原性组合物，适于通过对受试者的脚掌，皮下，肌肉，腹腔以及鼻粘膜注射进行免疫。
52.按照前述1-51中任一项的免疫原性组合物，用于制备以下疫苗：卡介苗，甲肝疫苗，乙肝疫苗，丙肝疫苗，丁肝疫苗，戊肝疫苗，流感疫苗，脊髓灰质炎疫苗，百白破疫苗，麻疹疫苗，乙脑疫苗，狂犬疫苗，出血热疫苗，肺炎疫苗，流脑疫苗，甲肝疫苗，腮腺炎疫苗，流感疫苗，风疹疫苗，水痘疫苗，艾滋病疫苗，疟疾疫苗；以及癌症治疗和预防疫苗，包括但不限于黑色素瘤治疗型疫苗，黑色素瘤预防型疫苗，肺癌治疗型疫苗，肺癌预防型疫苗，膀胱癌预防型疫苗，膀胱癌预防型疫苗治疗型疫苗，宫颈癌治疗型疫苗，宫颈癌预防型疫苗，膀胱癌治疗型疫苗，膀胱癌预防型疫苗，乳腺癌治疗型疫苗，乳腺癌预防型疫苗，肝癌治疗型疫苗，肝癌预防型疫苗，前列腺癌治疗性疫苗，前列腺癌预防性疫苗。
53.硫解酶抑制剂，用作佐剂。
54.HMG-CoA合酶抑制剂，用作佐剂。
55.HMG-CoA还原酶抑制剂，用作佐剂。
56.甲羟戊酸激酶抑制剂，用作佐剂。
57.磷酸甲羟戊酸激酶抑制剂，用作佐剂。
58.甲羟戊酸-5-焦磷酸脱羧酶抑制剂，用作佐剂。
59.异戊烯焦磷酸异构酶抑制剂，用作佐剂。
60.法尼基焦磷酸合酶抑制剂，用作佐剂。
61.香叶香叶酰基焦磷酸合酶抑制剂，用作佐剂。
62.香叶香叶酰基转移酶(I，II)抑制剂，用作佐剂。
63.按照55的HMG-CoA还原酶抑制剂，其中所述HMG-CoA还原酶抑制剂为他汀类化合物。
64.按照63的HMG-CoA还原酶抑制剂，其中所述他汀类化合物选自普伐他汀、阿托伐他汀、罗苏伐他汀、氟伐他汀、匹伐他汀、美伐他汀、洛伐他汀、辛伐他汀、西立伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
65.按照63的HMG-CoA还原酶抑制剂，其中所述他汀类化合物选自辛伐他汀，洛伐他汀和美伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
66.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为双膦酸化合物或其可药用盐、酯、前药、溶剂化物。
67.按照66的法尼基焦磷酸合酶抑制剂，其中所述双膦酸化合物或其可药用盐选自唑来膦酸，帕米膦酸，阿仑膦酸，伊班膦酸，奈立膦酸，利塞膦酸，奥帕膦酸，米诺磷酸。
68.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000032
上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
m＝1-6的整数。
69.根据68所述的法尼基焦磷酸合酶抑制剂，其中所述式I所示化合物为下述式II-X所示化合物：
Figure PCTCN2016098371-appb-000033
Figure PCTCN2016098371-appb-000034
上述式II-X中，X为下述任意一种：氢、羟基、巯基、卤素、烷氧基或烷基；
每一个M独立地为下述任意一种：负电荷、氢、烷基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；
R1、R2、R3、R4、、R5、R8分别独立地选自下述任意一种：氢、羟基、脂肪基、巯基、卤素、氨基、烷基、-O-(CH2)qCH3、-NH-(CH2)qCH3、-N[(CH2)qCH3]2、-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-O-(CH2)qCH3，其中，p＝1-6，q＝0-6；m＝1-6的整数。
70.根据68或69所述的法尼基焦磷酸合酶抑制剂，其中所述化合物为式XI-XVIII所示化合物：
Figure PCTCN2016098371-appb-000035
式XI-XVIII中，Z为氢、羟基、脂肪基、烷氧基、氨基或烷胺基。
71.根据68-70中任一项所述的法尼基焦磷酸合酶抑制剂，其中所述化合物为式IXX或XX所示化合物：
Figure PCTCN2016098371-appb-000036
式IXX、式XX中，n为0或1-12的整数。
72.根据68-71中任一项所述的法尼基焦磷酸合酶抑制剂，其中所述化合物为下述任意一种：
Figure PCTCN2016098371-appb-000037
73.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000038
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等；
74.按照73的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000039
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
75.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000040
其中n为1-24的整数，优选n为1-12的整数。
76.按照75的法尼基焦磷酸合酶抑制剂，其中n为1-20的整数。
77.按照75的法尼基焦磷酸合酶抑制剂，其中n为1-15的整数。
78.按照75的法尼基焦磷酸合酶抑制剂，其中所述化合物选自：
Figure PCTCN2016098371-appb-000041
Figure PCTCN2016098371-appb-000042
79.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000043
其中n为1-12的整数。
80.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000044
其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
81.按照80的法尼基焦磷酸合酶抑制剂，其中R1选自氢、C1-10烷基、C1-10炔基、C1-10烷基氨基、C1-10烷基硫基、卤素、羟基、吲唑基、C1-10烷氧基、被苯基或吡啶基取代的C1-10烷氧基，所述吡啶基任选取代有氨基甲酰基。
82.按照81的法尼基焦磷酸合酶抑制剂，其中R1选自氢、4-甲基苯乙氧基、4，5，6，7-四氢-2H-吲唑-2-基、(2-氨基甲酰基吡啶-4-基)甲氧基、苄基氧基、己基氧基、甲硫基、辛基氨基、己基、辛基、癸基、辛-1-炔-1-基、羟基、溴。
83.按照80的法尼基焦磷酸合酶抑制剂，其中R2选自氢、C1-10烷氧基、卤素。
84.按照83的法尼基焦磷酸合酶抑制剂，其中R2选自氢、辛基氧基、溴。
85.按照80的法尼基焦磷酸合酶抑制剂，其中R3选自氢、C1-10烷基、C1-10烷氧基。
86.按照85的法尼基焦磷酸合酶抑制剂，其中R3选自氢、甲基、己基氧基。
87.按照80的法尼基焦磷酸合酶抑制剂，其中R2与R3与它们所连接的碳原子一起形成苯环。
88.按照80的法尼基焦磷酸合酶抑制剂，其中R4选自氢、C1-10烷氧基。
89.按照88的法尼基焦磷酸合酶抑制剂，其中R4选自氢、辛基氧基。
90.按照80的法尼基焦磷酸合酶抑制剂，其中所述化合物选自：
Figure PCTCN2016098371-appb-000045
91.按照60的法尼基焦磷酸合酶抑制剂，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000046
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、 芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
92.按照91的法尼基焦磷酸合酶抑制剂，其中R5选自C1-10烷氧基。
93.按照91的法尼基焦磷酸合酶抑制剂，其中所述化合物为
Figure PCTCN2016098371-appb-000047
94.按照61的香叶香叶酰基焦磷酸合酶抑制剂，其中所述香叶香叶酰基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000048
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
95.按照94的香叶香叶酰基焦磷酸合酶抑制剂，其中R9选自C1-10烷氧基。
96.按照94的香叶香叶酰基焦磷酸合酶抑制剂，其中所述化合物为
Figure PCTCN2016098371-appb-000049
97.硫解酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
98.HMG-CoA合酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
99.HMG-CoA还原酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
100.甲羟戊酸激酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
101.磷酸甲羟戊酸激酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
102.甲羟戊酸-5-焦磷酸脱羧酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
103.异戊烯焦磷酸异构酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
104.法尼基焦磷酸合酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
105.香叶香叶酰基焦磷酸合酶抑制剂作为佐剂在制备免疫原性组合物中的用途。
106.香叶香叶酰基转移酶(I，II)抑制剂作为佐剂在制备免疫原性组合物中的用途。
107.按照99的用途，其中所述HMG-CoA还原酶抑制剂为他汀类化合物。
108.按照107的用途，其中所述他汀类化合物选自普伐他汀、阿托伐他汀、罗苏伐他汀、氟伐他汀、匹伐他汀、美伐他汀、洛伐他汀、辛伐他汀、西立伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
109.按照107的用途，其中所述他汀类化合物选自辛伐他汀，洛伐他汀和美伐他汀，或它们的可药用盐、酯、前药、溶剂化物。
110.按照109的用途，其中所述法尼基焦磷酸合酶抑制剂为双膦酸化合物或其可药用盐、酯、前药、溶剂化物。
111.按照110的用途，其中所述双膦酸化合物或其可药用盐选自唑来膦酸，帕米膦酸，阿仑膦酸，伊班膦酸，奈立膦酸，利塞膦酸，奥帕膦酸，米诺磷酸。
112.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000050
上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
m＝1-6的整数。
113.根据112所述的用途，其中所述式I所示化合物为下述式II-X所示化合物：
Figure PCTCN2016098371-appb-000051
Figure PCTCN2016098371-appb-000052
上述式II-X中，X为下述任意一种：氢、羟基、巯基、卤素、烷氧基或烷基；
每一个M独立地为下述任意一种：负电荷、氢、烷基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；
R1、R2、R3、R4、、R5、R8分别独立地选自下述任意一种：氢、羟基、脂肪基、巯基、卤素、氨基、烷基、-O-(CH2)qCH3、-NH-(CH2)qCH3、-N[(CH2)qCH3]2、-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-O-(CH2)qCH3，其中，p＝1-6，q＝0-6；m＝1-6的整数。
114.根据112或113所述的用途，其中所述化合物为式XI-XVIII所示化合物：
Figure PCTCN2016098371-appb-000053
式XI-XVIII中，Z为氢、羟基、脂肪基、烷氧基、氨基或烷胺基。
115.根据112-114中任一项所述的用途，其中所述化合物为式IXX或XX所示化合物：
Figure PCTCN2016098371-appb-000054
式IXX、式XX中，n为0或1-12的整数。
116.根据112-115中任一项所述的用途，其中所述化合物为下述任意一种：
Figure PCTCN2016098371-appb-000055
117.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000056
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等。
118.按照117的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000057
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
119.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000058
其中n为1-24的整数，优选n为1-12的整数。
120.按照119的用途，其中n为1-20的整数。
121.按照119的用途，其中n为1-15的整数。
122.按照119的用途，其中所述化合物选自：
Figure PCTCN2016098371-appb-000059
Figure PCTCN2016098371-appb-000060
123.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000061
其中n为1-12的整数。
124.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000062
其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
125.按照124的用途，其中R1选自氢、C1-10烷基、C1-10炔基、C1-10烷基氨基、C1-10烷基硫基、卤素、羟基、吲唑基、C1-10烷氧基、被苯基或吡啶基取代的C1-10烷氧基，所述吡啶基任选取代有氨基甲酰基。
126.按照125的用途，其中R1选自氢、4-甲基苯乙氧基、4，5，6，7-四氢-2H-吲唑-2-基、(2-氨基甲酰基吡啶-4-基)甲氧基、苄基氧基、己基氧基、甲硫基、辛基氨基、己基、辛基、癸基、辛-1-炔-1-基、羟基、溴。
127.按照124的用途，其中R2选自氢、C1-10烷氧基、卤素。
128.按照127的用途，其中R2选自氢、辛基氧基、溴。
129.按照124的用途，其中R3选自氢、C1-10烷基、C1-10烷氧基。
130.按照129的用途，其中R3选自氢、甲基、己基氧基。
131.按照124的用途，其中R2与R3与它们所连接的碳原子一起形成苯环。
132.按照124的用途，其中R4选自氢、C1-10烷氧基。
133.按照132的用途，其中R4选自氢、辛基氧基。
134.按照124的用途，其中所述化合物选自：
Figure PCTCN2016098371-appb-000063
135.按照104的用途，其中所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000064
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
136.按照135的用途，其中R5选自C1-10烷氧基。
137.按照136的用途，其中所述化合物为
Figure PCTCN2016098371-appb-000065
138.按照105的用途，其中所述香叶香叶酰基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000066
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
139.按照138的用途，其中R9选自C1-10烷氧基。
140.按照139的用途，其中所述化合物为
Figure PCTCN2016098371-appb-000067
141.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000068
上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
m＝1-6的整数。
142.根据141所述的化合物，其中所述式I所示化合物为下述式II-X所示化合物：
Figure PCTCN2016098371-appb-000069
Figure PCTCN2016098371-appb-000070
上述式II-X中，X为下述任意一种：氢、羟基、巯基、卤素、烷氧基或烷基；
每一个M独立地为下述任意一种：负电荷、氢、烷基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；
R1、R2、R3、R4、、R5、R8分别独立地选自下述任意一种：氢、羟基、脂肪基、巯基、卤素、氨基、烷基、-O-(CH2)qCH3、-NH-(CH2)qCH3、-N[(CH2)qCH3]2、-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-S-(CH2)qCH3、-O-(CH2)P-O-(CH2)qCH3，其中，p＝1-6，q＝0-6；m＝1-6的整数，优选地，m＝1。
143.根据141或142所述的化合物，其中所述化合物为式XI-XVIII所示化合物：
Figure PCTCN2016098371-appb-000071
式XI-XVIII中，Z为氢、羟基、脂肪基、烷氧基、氨基或烷胺基。
144.根据141-143中任一项所述的化合物，其中所述化合物为式IXX或XX所示化合物：
Figure PCTCN2016098371-appb-000072
式IXX、式XX中，n为0或1-12的整数。
145.根据141-144中任一项所述化合物，其中所述化合物为下述任意一种：
Figure PCTCN2016098371-appb-000073
146.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000074
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等；
147.按照146的化合物或其可药用盐、酯、前药、溶剂化物，其为下式的化合物：
Figure PCTCN2016098371-appb-000075
其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
148.下式的化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000076
其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素。
149.按照148的化合物或其可药用盐、酯、前药、溶剂化物，其为下式的化合物：
Figure PCTCN2016098371-appb-000077
其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基。
150.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000078
其中n为1-24的整数，优选n为1-12的整数。
151.按照150的化合物或其可药用盐、酯、前药、溶剂化物，其中n为1-20的整数。
152.按照150的化合物或其可药用盐、酯、前药、溶剂化物，其中n为1-15的整数。
153.按照150的化合物或其可药用盐、酯、前药、溶剂化物，其中所述化合物选自：
Figure PCTCN2016098371-appb-000079
154.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000080
其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
155.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中R1选自氢、C1-10烷基、C1-10炔基、C1-10烷基氨基、C1-10烷基硫基、卤素、羟基、吲唑基、C1-10烷氧基、被苯基或吡啶基取代的C1-10烷氧基，所述吡啶基任选取代有氨基甲酰基。
156.按照155的化合物或其可药用盐、酯、前药、溶剂化物，其中R1选自氢、4-甲基苯乙氧基、4，5，6，7-四氢-2H-吲唑-2-基、(2-氨基甲酰基吡啶-4-基)甲氧基、苄基氧基、己基氧基、甲硫基、辛基氨基、己基、辛基、癸基、辛-1-炔-1-基、羟基、溴。
157.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中R2选自氢、C1-10烷氧基、卤素。
158.按照157的化合物或其可药用盐、酯、前药、溶剂化物，其中R2选自氢、辛基氧基、溴。
159.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中R3选自氢、C1-10烷基、C1-10烷氧基。
160.按照159的化合物或其可药用盐、酯、前药、溶剂化物，其中R3选自氢、甲基、己基氧基。
161.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中R2与R3与它们所连接的碳原子一起形成苯环。
162.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中R4选自氢、C1-10烷氧基。
163.按照162的化合物或其可药用盐、酯、前药、溶剂化物，其中R4选自氢、辛基氧基。
164.按照154的化合物或其可药用盐、酯、前药、溶剂化物，其中所述化合物选自：
Figure PCTCN2016098371-appb-000081
165.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000082
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、 芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
166.按照165的化合物或其可药用盐、酯、前药、溶剂化物，其中R5选自C1-10烷氧基。
167.按照165的化合物或其可药用盐、酯、前药、溶剂化物，其中所述化合物为
Figure PCTCN2016098371-appb-000083
168.下式化合物或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000084
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
169.按照168的化合物或其可药用盐、酯、前药、溶剂化物，其中R9选自C1-10烷氧基。
170.按照168的化合物或其可药用盐、酯、前药、溶剂化物，其中所述化合物为
Figure PCTCN2016098371-appb-000085
171.一种制备式IXX所示化合物的方法，包括：
Figure PCTCN2016098371-appb-000086
1)在无机碱或路易斯碱作用下，将式XXI所示化合物与溴乙酸叔丁酯进行反应，得到式XXII所示化合物；
Figure PCTCN2016098371-appb-000087
式IV、式V中，n为0或1-12的整数；
2)在钯碳催化下，将式XXII所示化合物在氢气中进行还原反应，所得产物再与醋酸甲脒进行关环反应，得到式XXIII所示化合物；
Figure PCTCN2016098371-appb-000088
式XXIII中，n为0或1-12的整数；
3)将所述式XXIII所示化合物在盐酸或TFA中加热回流，得到式IXXV所示化合物；
Figure PCTCN2016098371-appb-000089
式IXXV中，n为0或1-12的整数；
4)在亚磷酸、环丁砜介质中，将所述式IXXV所示化合物与三氯化磷在回流下反应，得到式IXX所示化合物。
172.根据171所述的制备方法，其特征在于：
步骤1)中，所述式XXI所示化合物与无机碱或路易斯碱、溴乙酸叔丁酯的摩尔比依次为1∶1-20∶0.2-15；
步骤1)中，所述反应的温度为80-150℃，时间为1-96h；
步骤2)中，所述还原反应的温度为0-100℃，时间为0.5-24h；
步骤2)中，式XXII所示化合物与醋酸甲脒的摩尔比为1∶0.2-10；
所述关环反应的温度为50-150℃，时间为1-24h；
所述关环反应在有机溶剂中进行，优选为乙二醇单甲醚；
步骤3)中，所述式XXIII所示化合物与盐酸或TFA的摩尔比为1∶1-100；
步骤3)中，所述加热回流的时间为0.5-96h；
步骤4)中，式IXXV所示化合物与亚磷酸、三氯化磷的摩尔比依次为1∶0.2-20∶0.2-20；
步骤4)中，所述反应的时间为0.5-96h。
174.根据141-170中任一项所述化合物在制备下述产品中的应用：
1)治疗代谢性骨病药物；
2)治疗疟疾药物；
3)真核生物肿瘤细胞增殖抑制剂；
4)预防和/或治疗肿瘤药物；
5)免疫治疗药物；
6)疫苗佐剂；
7)疫苗；
8)抑制HsFPPs酶活性的产品；
9)抑制PvGGPPs酶活性的产品。
175.根据174所述的应用，其特征在于：
所述真核生物为哺乳动物；所述肿瘤细胞为癌细胞；所述肿瘤为癌，具体地，所述癌细胞为乳腺癌细胞；所述癌为乳腺癌，优选地，所述乳腺癌细胞具体可为人乳腺癌细胞MDA-MB-231。
176.根据1的免疫原性组合物、54的HMG-CoA合酶抑制剂或98的用途，所述的HMG-CoA合酶为Hymeglusin(11-[3R-(羟基甲基)-4-氧代-2R-氧杂环丁烷基]-3，5，7R-三甲基-2E，4E-十一二烯酸)。
附图说明
图1是甲羟戊酸通路的示意图。
图2A-D示出了8种他汀类药物在OVA抗体滴度检测中的佐剂活性，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图3示出了TH-Z80系列化合物在OVA抗体滴度检测中的佐剂活性，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图4示出FPPS抑制剂TH-Z97在OVA抗体滴度检测中的佐剂活性，以免疫后7天和14天的IgG的抗体滴度表示。
图5A-D示出了本发明的双磷酸化合物TH-Z80和TH-Z93与市场上的8种双磷酸药物在OVA抗体滴度检测中的佐剂活性的比较结果。
图6A-D示出了GGPPS抑制剂TH-Z144和TH-Z145在OVA抗体滴度检测中的佐剂活性，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图7A-D示出FPPS和GGPPS的双抑制剂BPH-716和BPH-1222在OVA抗体滴度检测中的佐剂活性，以免疫后7天和14天的IgM和IgG的抗体滴度表示，其中Ctrl代表用PBS处理的对照组小鼠。
图8示出了本发明的3类抑制剂即HMG-CoA还原酶抑制剂、FPPS抑制剂、GGPPS抑制剂的佐剂活性与现有佐剂的活性比较。上图为加强免疫后(after boost)的IgG的抗体滴度，下图为抗体亲和力 指数。
图9A-D示出了GGOH和GGPP对辛伐他汀(HMG-CoA还原酶抑制剂)的佐剂活性的抑制作用。
图10A-D示出了GGOH和GGPP对TH-Z93(FPPS抑制剂)的佐剂活性的抑制作用。
图11A-D示出了GGOH和GGPP对TH-Z145(GGPPS抑制剂)的佐剂活性的抑制作用。
图12A-D示出了选择性的角鲨烯合酶抑制剂TH-Z66在OVA抗体滴度检测中的佐剂活性研究结果，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图13示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫小鼠24h后的淋巴结细胞总数量，其中Ctrl代表没有做任何处理的小鼠。
图14示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫小鼠24h后，B淋巴细胞在淋巴结中的比例以及数量，其中Ctrl代表没有做任何处理的小鼠。
图15示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫小鼠24h后，树突状细胞在淋巴结中的比例以及数量，其中Ctrl代表没有做任何处理的小鼠。
图16A-B示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫小鼠24h后，巨噬细胞在淋巴结中的比例以及数量，其中Ctrl代表没有做任何处理的小鼠。
图17示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫小鼠24h后，T淋巴细胞在淋巴结中的比例以及数量，其中Ctrl代表没有做任何处理的小鼠。
图18-21分别示出了用1μM的辛伐他汀，TH-Z80，TH-Z145和TH-Z66处理含有GM-CSF和IL-4的小鼠骨髓细胞后，再加入100ng/ml的LPS进行刺激之后，IL-6，TNF-α，IL-1β和IL-12p70的浓度。
图22A-D示出了采用本申请所述的辛伐他汀、TH-Z80、TH-Z145作为佐剂免疫后淋巴结中细胞标志物B220、F4/80、CD11c的比例，其中Ctrl代表没有做任何处理的小鼠。
图23A-D示出了采用辛伐他汀、TH-Z80、TH-Z144作为佐剂免疫小鼠后，在中东呼吸综合征病毒Mers蛋白中的IgM和IgG抗体的滴度，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图24A-D示出辛伐他汀、TH-Z80、TH-Z144作为佐剂能够促进乙肝表面抗原HbsAg产生更多的抗体。
图25A-D示出辛伐他汀，TH-Z80，TH-Z93，TH-Z145在黑色素瘤的预防性疫苗中的佐剂作用。
图26A-D示出辛伐他汀，TH-Z80，TH-Z93，TH-Z145在黑色素瘤的治疗性疫苗中的佐剂作用。
图27示出用TH-Z80作为佐剂混合抗原对小鼠不同的部位(脚掌，皮下，肌肉，腹腔以及鼻粘膜)进行免疫的佐剂活性，以免疫后7天和14天的IgG的抗体滴度表示。
图28A-D示出FPPS抑制剂TH-Z93与TLR激动剂咪喹莫特组合使用在OVA抗体滴度检测中的联合佐剂活性，以免疫后7天和14天的IgM和IgG的抗体滴度表示。
图29为注射100μg不同双膦酸对小鼠抗体产生的影响。其中，PBS为磷酸缓冲液，CLD为氯屈膦酸，ZOL为唑来膦酸，PMD为帕米膦酸；
图30为双膦酸侧链碳链取代长短作为佐剂的效果图；
图31为TH-Z80增加抗体亲和力的效果图。其中，PBS为磷酸缓冲液；
图32为不同碳链长度苯并咪唑类双膦酸对δγT细胞的扩增效果；
图33为TH-Z80与HsFPPS的晶体结构示意图(pymol作图)。
图34为化合物TH-Z82与PvGGPPS的晶体结构示意图(pymol作图)。
图35为辛伐他汀，TH-Z80，TH-Z93和TH-Z145对PR8流感病毒的作用。
图36为抗PD1抗体联合四种佐剂和鸡卵清蛋白对B16-OVA肿瘤的抑制作用。
图37为HMG-CoA合酶抑制剂在OVA抗体滴度检测中的佐剂活性。
图38为经辛伐他汀，TH-Z80，TH-Z93，TH-Z145或这些佐剂混合GGOH处理后的IL-6，TNF-α，IL-1β和IL-12p70的浓度。
具体实施方式
术语
为了便于审查本公开中的各种实施方案，特提供对术语的以下解释。其他术语和解释在本公开的上下文中有提供。
除非另作解释，本文使用的所有科技术语与本公开所属领域中普通技术人员通常理解的含义相同。
术语“抗原”用于指免疫应答的适应性(adaptive)元件(即B细胞或T细胞或两者)可以特异性识别的任何分子。
优选地，本发明中使用的抗原是免疫原，即活化免疫细胞以产生针对自身的免疫应答的抗原。
“免疫原性组合物”是适合给予人或动物患者(例如在试验情景中)，能够引发特异免疫反应(例如抗乙肝病毒的病原)的物质的组合物。比如，免疫原性组合物包含一或多种抗原(例如，完整的纯化病毒或抗原亚基，例如其多肽)或抗原表位。免疫原性组合物还可以包含能够引发或增强免疫反应的一或多种其他成分，比如赋形剂、载体和/或佐剂。某些情况中，给予免疫原性组合物以引发帮助患者对抗病原所引起的症状或状况的免疫反应。某些情况中，在患者接触病原后，通过抑制病原(例如乙肝病毒)的复制来预防(或治疗，例如减轻或缓解)病原所导致的症状或疾病。在本文语境中，术语免疫原性组合物应理解为涵盖为了引发例如抗乙肝的保护性或缓解性免疫反应，给予患者或患者群体的组合物(即，疫苗组合物或疫苗)。
“佐剂”是与没有它的情况下给予抗原相比，能够增强抗原特异性免疫反应的试剂。常见佐剂包括含有铝的佐剂，其包含抗原可以吸附其上的矿物质悬液(或矿物盐，比如氢氧化铝、磷酸铝、羟基磷酸铝)。在一种情形中，佐剂是在没有任何所述铝盐的情况下配制的无铝佐剂。无铝佐剂包括油和水乳剂，比如油包水和水包油型(及其变体，包括复乳剂和可逆乳剂)、糖脂、脂多糖、免疫刺激核酸(比如CpG寡核苷酸)、脂质体、Toll样受体拮抗剂(特别是，TLR2、TLR4、TLR7/8和TLR9拮抗剂)和这些成分的各种组合。
“免疫反应”是免疫系统的细胞，比如B细胞、T细胞或单核细胞对刺激的反应。免疫反应可以是B细胞反应，这种反应导致产生特异抗体，比如抗原特异性中和抗体。免疫反应也可以是T细胞反应，比如CD4+反应或CD8+反应。某些情况中，反应是对特定抗原特异的(即“抗原特异性反应”)。如果抗原来源于病原体，抗原特异性反应是“病原体特异性反应”。“保护性免疫反应”是抑制病原体的有害 功能或活性、减轻病原感染或者减少病原体感染所导致的症状(包括死亡)的免疫反应。保护性免疫反应可以通过例如在噬斑减少分析或ELISA-中和检测中病毒复制或噬菌斑形成的抑制，或者通过体内测量对病原攻击的抗性来测量。
术语“抗体”描述了免疫球蛋白，无论其是天然的还是部分或完全合成产生的。所述术语还涵盖具有为抗原-结合域或与抗原-结合域同源的结合域的任意多肽或蛋白质。
肿瘤抗原是在肿瘤细胞中产生的抗原物质，即它会在宿主中触发免疫应答。肿瘤抗原是在识别肿瘤细胞的诊断测试中有用的肿瘤标记物，并且是在癌症治疗中使用的潜在候选物。
术语“中东呼吸综合征(MERS)”，也称为camel流感，是由MERS-冠状病毒(MERS-CoV)引起的病毒性呼吸道感染。其症状可从轻度至重度，包括发热、咳嗽、腹泻和呼吸急促。
本申请使用的术语“任选”表示随后描述的一个或多个事件可能发生或可能不发生，并且同时包括发生的一个或多个事件和不发生的一个或多个事件。
本申请使用的术语“取代”是指在所指定原子或者环上的任意一个或者多个氢被选自所示组群的基团替换，其条件是没有超过所指定原子或者环原子的正常化合价，且所述取代得到稳定的化合物。当取代基是酮基(即＝O)时，原子上的2个氢被替换。
当列举数值的范围时，意图包括在所述范围内的每个数值和子范围。例如“C1-6烷基”意图包括C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5和C5-6烷基。
本申请使用的“脂肪基”或“脂肪族基团”包括如下所定义的烷基，烯基和炔基。
本申请使用的“烷基”意在包括在链中含有1至20个碳，优选1至12个碳，优选1至11个碳，优选1至10个碳，优选1至9个碳，更优选1至8个碳的支链和直链饱和脂族烃基团，诸如甲基、乙基、丙基、异丙基、丁基、叔丁基、异丁基、戊基、己基、异己基、庚基、4，4-二甲基戊基、辛基、2，2，4-三甲基戊基、壬基、癸基、十一烷基、十二烷基、它们的各种支链异构体等，及所述基团可任选包括1至4个取代基，诸如卤素例如F、Br、Cl或者I或者CF3、烷基、烷氧基、芳基、芳基氧基、芳基(芳基)或者联芳基(diaryl)、芳基烷基、芳基烷基氧基、烯基、环烷基、环烷基烷基、环烷基烷基氧基、氨基、羟基、羟基烷基、酰基、杂芳基、杂芳基氧基、杂芳基烷基、杂芳基烷氧基、芳基氧基烷基、烷基硫基、芳基烷基硫基、芳基氧基芳基、烷基氨酰基(alkylamido)、烷酰基氨基、芳基羰基氨基、硝基、氰基、巯基、卤代烷基、三卤代烷基和/或烷基硫基。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“烯基”是指在链中含有2至20个碳，优选2至12个碳，优选2至11个碳，优选2至10个碳，优选2至9个碳，更优选1至8个碳的直链或者支链基团，所述基团在正链中包含1至6个双键，诸如乙烯基、2-丙烯基、3-丁烯基、2-丁烯基、4-戊烯基、3-戊烯基、2-己烯基、3-己烯基、2-庚烯基、3-庚烯基、4-庚烯基、3-辛烯基、3-壬烯基、4-癸烯基、3-十一碳烯基、4-十二碳烯基、4，8，12-十四碳三烯基等，且所述基团可任选被1至4个取代基取代，所述取代基即卤素、卤代烷基、烷基、烷氧基、烯基、炔基、芳基、芳基烷基、环烷基、氨基、羟基、杂芳基、杂环烷基、烷酰基氨基、烷基氨酰基、芳基羰基氨基、硝基、氰基、巯基、烷基硫基和/或本申请所述烷基上的取代基中的任意一种。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“炔基”是指在链中含有2至20个碳，优选2至12个碳，优选2至11个碳，优选2至10个碳，优选2至9个碳，更优选2至8个碳的直链或者支链基团，所述基团在正链中包含一个叁键，诸如2-丙炔基、3-丁炔基、2-丁炔基、4-戊炔基、3-戊炔基、2-己炔基、3-己炔基、2-庚炔基、3-庚炔基、4-庚炔基、3-辛炔基、3-壬炔基、4-癸炔基、3-十一碳炔基、4-十二碳炔基等，且所述基团可任选被1至4个取代基取代，所述取代基即卤素、卤代烷基、烷基、烷氧基、烯基、炔基、芳基、芳基烷基、环烷基、氨基、杂芳基、杂环烷基、羟基、烷酰基氨基、烷基氨酰基、芳基羰基氨基、硝基、氰基、巯基、烷基硫基和/或本申请所述烷基上的取代基中的任意一种。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“环烷基”包括饱和或者部分不饱和(含有1或者2个双键)的含有1至3个环的环状烃基团，包括单环烷基、二环状烷基(或者二环烷基)和三环烷基，其含有总数为3至20个的成环碳原子，优选总数为3至10个的成环碳原子，且所述基团可与1或者2个就芳基所述的芳族环稠合。含有1个环的“环烷基”优选包含3至8个环碳原子，优选包含3至7个环碳原子，更优选包含3至6个环碳原子。所述环烷基包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环癸基和环十二烷基、环己烯基、
Figure PCTCN2016098371-appb-000090
这些基团中的任意一种可任选被1至4个取代基取代，所述取代基为诸如卤素、烷基、烷氧基、羟基、芳基、芳基氧基、芳基烷基、环烷基、烷基氨酰基、烷酰基氨基、氧代、酰基、芳基羰基氨基、氨基、硝基、氰基、巯基和/或烷基硫基和/或就烷基而言的任意取代基。
当如上定义的烷基在两个不同碳原子处具有与其它基团连接的单键时，所述烷基被称为“亚烷基”，且可任选如以上就“烷基”所定义的那样被取代。
当如上定义的烯基和如上定义的炔基分别在两个不同碳原子处具有用于连接的单键时，它们分别被称为“亚烯基”和“亚炔基”，且可任选如以上就“烯基”和“炔基”所定义的那样被取代。
本申请使用的“卤代”或者“卤素”是指氟、氯、溴和碘；且“卤代烷基”意在包括支链和直链饱和脂族烃基团，例如CF3，所述烃基团具有特定的碳原子数目，且被一个或者多个卤素取代(例如-CvFw，其中v＝1至3，且w＝1至(2v+1))。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“芳基”或“芳香基”是指在环部分中含有6至10个碳的单环和二环芳族基团(诸如苯基或者萘基(包括萘-1-基和萘-2-基))，且可任选包含1至3个与碳环或者杂环(诸如芳基环、环烷基环、杂芳基环或者杂环烷基环)稠合的其它环，例如
Figure PCTCN2016098371-appb-000091
且芳基可任选通过可用的碳而被1、2或者3个取代基取代，所述取代基为例如氢、卤素、卤代烷基、烷基、卤代烷基、烷氧基、卤代烷氧基、烯基、三氟甲基、三氟甲氧基、炔基、环烷基烷基、杂环烷基、杂环烷基烷基、芳基、杂芳基、芳基烷基、芳基氧基、芳基氧基烷基、芳基烷氧基、芳基硫基、芳基偶氮基、杂芳基烷基、杂芳基烯基、杂芳基杂芳基、杂芳基氧基、羟基、硝基、氰基、氨基、取代的氨基(其中所述氨基包括1或者2个取代基(所述取代基为烷基、芳基或者定义中所提到的任意其它芳基化合物))、巯基、烷基硫基、芳基硫基、杂芳基硫基、芳基硫基烷基、烷氧基芳基硫基、烷基羰基、芳基羰基、烷基氨基羰基、芳基氨基羰基、烷氧基羰基、氨基羰基、烷基羰基氧基、芳基羰基氧基、烷基羰基氨基、芳基羰基氨基、芳基亚磺酰基、芳基亚磺酰基烷基、芳基磺酰基氨基或者芳基磺酰氨基羰基和/或本申请所述烷基上的取代基中的任意一种。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“烷氧基”、“芳基氧基”或者“芳烷氧基”包括与氧原子相连的任意上述烷基、芳烷基或者芳基。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的“氨基”是指未取代的或可被一个或两个取代基取代的氨基，所述取代基可以相同或不同，且为诸如烷基、芳基、芳基烷基、杂芳基、杂芳基烷基、杂环烷基、杂环烷基烷基、环烷基、环烷基烷基、卤代烷基、羟基烷基、烷氧基烷基或者烷基硫基。这些取代基可进一步被羧酸基团和/或任意R1基团或者以上就R1所述的取代基取代。另外，氨基上的取代基可与它们所连接的氮原子一起形成吡咯烷-1-基、哌啶-1-基、氮杂
Figure PCTCN2016098371-appb-000092
-1-基、吗啉-4-基、硫吗啉-4-基、哌嗪-1-基、4-烷基-哌嗪-1-基、4-芳基烷基-哌嗪-1-基或者4-联芳基烷基-哌嗪-1-基，所述基团任选被烷基、烷氧基、烷基硫基、卤素、三氟甲基或者羟基取代。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“烷基硫基”、“芳基硫基”或者“芳烷基硫基”包括与硫原子相连的任意上述烷基、芳烷基或者芳基。
除非另有说明，本申请单独使用或者作为另一基团的部分而使用的术语“烷基氨基”、“芳基氨基”或者“芳基烷基氨基”包括与氮原子相连的任意上述烷基、芳基或者芳基烷基。
本申请使用的术语“杂环基”或者“杂环系统”意指稳定的5、6或者7元单环杂环或者二环杂环或者7、8、9或者10元二环杂环，所述杂环是饱和、部分不饱和或者不饱和的(芳族的)，且由碳原子和1、2、3或者4个独立选自N、NH、O和S的杂原子构成，且包括其中任意上述杂环与苯环稠合的任意二环基团。所述氮杂原子和硫杂原子可任选被氧化。杂环可在能得到稳定结构的任意杂原子或者碳原子处与其侧基相连。本申请描述的杂环可在碳或者氮上被取代，其条件是所得化合物是稳定的。如果专门指出，则杂环中的氮可任选被季铵化。优选的是，当杂环中S原子和O原子的总数超过1时， 则这些杂原子不彼此相邻。本申请使用的术语“芳族杂环系统”或者“杂芳基”或者“杂芳香基”意指稳定的5至7元单环或者二环或者7至10元二环杂环芳环，其由碳原子和1至4个独立选自N、O和S的杂原子构成，且本质上是芳香性的。
杂环基的实例包括但不限于1H-吲唑基、2-吡咯烷酮基、2H，6H-1，5，2-二噻嗪基、2H-吡咯基、1H-吲哚基、4-哌啶酮基、4aH-咔唑基、4H-喹嗪基、6H-1，2，5-噻二嗪基、吖啶基、氮杂环辛四烯基、苯并咪唑基、苯并呋喃基、苯并噻吩基(benzothiofuranyl)、苯并噻吩基(benzothiophenyl)、苯并噁唑基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异噁唑基、苯并异噻唑基、苯并咪唑酮基(benzimidazalonyl)、咔唑基、4aH-咔唑基、β-咔啉基、色满基、色烯基、噌啉基、十氢喹啉基、2H，6H-1，5，2-二噻嗪基、二氢呋喃并[2，3-b]四氢呋喃基、呋喃基、呋咱基、咪唑烷基、咪唑啉基、咪唑基、吲唑基、3H-吲哚基(indolenyl)、二氢吲哚基(indolinyl)、吲嗪基、吲哚基、异苯并呋喃基、异色满基、异吲唑基、异二氢吲哚基、异吲哚基、异喹啉基、苯并咪唑基、异噻唑基、异噁唑基、吗啉基、二氮杂萘基、八氢异喹啉基、噁二唑基、1，2，3-噁二唑基、1，2，4-噁二唑基、1，2，5-噁二唑基、1，3，4-噁二唑基、噁唑烷基、噁唑基、噁唑烷基、萘嵌间二氮杂苯基、菲啶基、菲咯啉基、吩吡嗪基、吩嗪基、吩噻嗪基、吩噻噁基、吩噁嗪基、酞嗪基、哌嗪基、哌啶基、蝶啶基、哌啶酮基、4-哌啶酮基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑烷基、吡唑啉基、吡唑基、哒嗪基、吡啶并噁唑基、吡啶并咪唑基、吡啶并噻唑基、吡啶基、吡啶基、嘧啶基、吡咯烷基、吡咯啉基、吡咯基、喹唑啉基、喹啉基、4H-喹嗪基、喹喔啉基、奎宁环基、咔啉基、四氢呋喃基、四氢异喹啉基、四氢喹啉基、6H-1，2，5-噻二嗪基、1，2，3-噻二唑基、1，2，4-噻二唑基、1，2，5-噻二唑基、1，3，4-噻二唑基、噻蒽基、噻唑基、噻吩基、噻吩并噻唑基、噻吩并噁唑基、噻吩并咪唑基、噻吩基、三嗪基、1，2，3-三唑基、1，2，4-三唑基、1，2，5-三唑基、1，3，4-三唑基、四唑基和呫吨基。在本发明另一个方面，杂环基包括但不限于吡啶基、噻吩基、呋喃基、吲唑基、苯并噻唑基、苯并咪唑基、苯并噻吩基、苯并呋喃基、苯并噁唑基、苯并异噁唑基、喹啉基、异喹啉基、咪唑基、吲哚基、异吲哚基、哌啶基、哌啶酮基、4-哌啶酮基、胡椒基、吡唑基、1，2，4-三唑基、1，2，3-三唑基、四唑基、噻唑基、噁唑基、吡嗪基和嘧啶基。杂环基还包括含有例如上述杂环的稠环和螺环化合物。
杂芳基的实例为1H-吲唑基、2H，6H-1，5，2-二噻嗪基、吲哚基、4aH-咔唑基、4H-喹嗪基、6H-1，2，5-噻二嗪基、吖啶基、氮杂环辛四烯基、苯并咪唑基、苯并呋喃基、苯并噻吩基、苯并噻吩基、苯并噁唑基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异噁唑基、苯并异噻唑基、苯并咪唑酮基、咔唑基、4aH-咔唑基、β-咔啉基、色满基、色烯基、噌啉基、十氢喹啉基、2H，6H-1，5，2-二噻嗪基、二氢呋喃并[2，3-b]四氢呋喃基、呋喃基、呋咱基、咪唑烷基、咪唑啉基、咪唑基、吲唑基、3H-吲哚基、二氢吲哚基、吲嗪基、吲哚基、异苯并呋喃基、异色满基、异吲唑基、异二氢吲哚基、异吲哚基、异喹啉基(苯并咪唑基)、异噻唑基、异噁唑基、吗啉基、二氮杂萘基、八氢异喹啉基、噁二唑基、1，2，3-噁二唑基、1，2，4-噁二唑基、1，2，5-噁二唑基、1，3，4-噁二唑基、噁唑烷基、噁唑基、噁唑烷基、萘嵌间二氮杂苯基、菲啶基、菲咯啉基、吩吡嗪基、吩嗪基、吩噻嗪基、吩噻噁基、吩噁嗪基、酞嗪基、哌嗪基、哌啶基、蝶啶基、哌啶酮基、4-哌啶酮基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑烷基、吡唑啉基、吡唑基、吡唑并三嗪基、哒嗪基、吡啶并噁唑基、吡啶并咪唑基、吡啶并噻唑基、吡啶基、 吡啶基、嘧啶基、吡咯烷基、吡咯啉基、吡咯基、喹唑啉基、喹啉基、4H-喹嗪基、喹喔啉基、奎宁环基、咔啉基、四氢呋喃基、四氢异喹啉基、四氢喹啉基、6H-1，2，5-噻二嗪基、1，2，3-噻二唑基、1，2，4-噻二唑基、1，2，5-噻二唑基、1，3，4-噻二唑基、噻蒽基、噻唑基、噻吩基、噻吩并噻唑基、噻吩并噁唑基、噻吩并咪唑基、噻吩基、三嗪基、1，2，3-三唑基、1，2，4-三唑基、1，2，5-三唑基、1，3，4-三唑基、四唑基和呫吨基。在本发明另一个方面，杂芳基的实例为吲哚基、苯并咪唑基、苯并呋喃基、苯并噻吩基、苯并噁唑基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异噁唑基、苯并异噻唑基、苯并咪唑酮基、噌啉基、呋喃基、咪唑基、吲唑基、吲哚基、异喹啉基、异噻唑基、异噁唑基、噁唑基、吡嗪基、吡唑基、吡唑并三嗪基、哒嗪基、吡啶基、吡啶基、嘧啶基、吡咯基、喹唑啉基、喹啉基、噻唑基、噻吩基和四唑基。
本申请使用的术语“氰基”是指-CN基团。
本申请使用的术语“硝基”是指-NO2基团。
本申请使用的术语“羟基”是指-OH基团。
本申请使用的术语“巯基”是指-SH基团。
本申请使用的术语“氨基甲酰基”是指-C(＝O)-氨基，其中所述氨基为任选被取代的氨基。
“烷酰基”是指RC(＝O)-基团，其中R为本申请定义的烷基。
本申请使用的短语“可药用”是指在合理医药判断范围内适用于与人类和动物组织接触而没有过度毒性、刺激性、变态反应或者其它问题或者并发症的那些化合物、物质、组合物和/或剂型，这与合理的益处/风险比例相称。
本申请使用的术语“可药用盐”是指保留本发明化合物的生物有效性和特性的盐，且其不是在生物学上或者其它方面不期望的。在许多实例中，本发明的化合物能够由于氨基和/或者羧基或者其类似基团(例如苯酚或者羟基酰胺基酸(hydroxyamic acid))的存在而形成酸盐和/或者碱盐。用无机酸和有机酸可形成药用酸加成盐。可从中衍生为盐的无机酸可包括，例如盐酸、氢溴酸、硫酸、硝酸、磷酸等。可从中衍生为盐的有机酸可包括，例如乙酸、丙酸、羟乙酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、枸橼酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸等。用无机碱和有机碱可形成药用碱加成盐。可从中衍生为盐的无机碱可包括，例如钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等；特别优选的是铵盐、钾盐、钠盐、钙盐和镁盐。可从中衍生为盐的有机碱可包括，例如伯胺、仲胺和叔胺、取代的胺(包括天然存在的取代的胺)、环胺、碱离子交换树脂等，特别的诸如异丙胺、三甲胺、二乙胺、三乙胺、三丙胺和乙醇胺。本发明的药用盐可由母体化合物、碱部分或者酸部分通过常规化学方法来合成。通常，所述的盐可通过使这些化合物的酸形式与化学量的适当的碱(诸如氢氧化钠、氢氧化钙、氢氧化镁、氢氧化钾、碳酸钠、碳酸钙、碳酸镁、碳酸钾、碳酸氢钠、碳酸氢钙、碳酸氢镁或者碳酸氢钾等)反应来制备，或者通过使这些化合物的游离碱的形式与化学量的适当的酸反应来制备。该反应典型地在水中或者在有机溶剂中或者在两者的混合物中进行。通常，如果可行，优选的非水介质为醚、乙酸乙酯、乙醇、异丙醇或者乙腈。所列的额外的适当的盐可见于下述文献：例如Remington′s Pharmaceutical Sciences，20th ed.，Mack Publishing Company，Easton，Pa.，(1985)，将其引入本申请作为参考。
示例性的酸加成盐包括乙酸盐(诸如与乙酸或三卤代乙酸(例如三氟乙酸)形成的那些盐)、己二酸盐、海藻酸盐、抗坏血酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、硼酸盐、丁酸盐、柠檬酸盐、樟脑酸盐、樟脑磺酸盐、环戊烷丙酸盐、二葡糖酸盐(digluconate)、十二烷基硫酸盐、乙磺酸盐、富马酸盐、葡糖庚酸盐(glucoheptanoate)、甘油磷酸盐(glycerophosphate)、半硫酸盐、庚酸盐、己酸盐、盐酸盐(与盐酸形成)、氢溴酸盐(与氢溴酸形成)、氢碘酸盐、马来酸盐(与马来酸形成)、2-羟基乙磺酸盐、乳酸盐、甲磺酸盐(与甲磺酸形成)、萘-2-磺酸盐、烟酸盐(nicotinate)、硝酸盐、草酸盐、果胶酸盐(pectinate)、过硫酸盐、3-苯基丙酸盐、磷酸盐、苦味酸盐、特戊酸盐、丙酸盐、水杨酸盐、琥珀酸盐、硫酸盐(诸如与硫酸形成的那些盐)、磺酸盐(诸如本申请提及的那些磺酸盐)、酒石酸盐、硫氰酸盐、甲苯磺酸盐(诸如甲苯磺酸盐)、十一烷酸盐等。
示例性的碱加成盐包括铵盐；碱金属盐，诸如钠盐、锂盐及钾盐；碱土金属盐，诸如钙盐及镁盐；钡盐、锌盐和铝盐；与有机碱(例如有机胺)(诸如三烷基胺(如三乙胺)、普鲁卡因(procaine)、二苄胺、N-苄基-β-苯乙胺、1-二苯羟甲胺(1-ephenamine)、N，N’-二苄基乙二胺、脱氢枞胺(dehydroabietylamine)、N-乙基哌啶、苄胺、二环己胺或类似的药用胺)形成的盐；及与氨基酸诸如精氨酸、赖氨酸等形成的盐。碱性含氮基团可用以下试剂来季铵化：诸如低级烷基卤化物(例如甲基氯、甲基溴及甲基碘，乙基氯、乙基溴及乙基碘，丙基氯、丙基溴及丙基碘，和丁基氯、丁基溴及丁基碘)、硫酸二烷基酯(例如硫酸二甲酯、硫酸二乙酯、硫酸二丁酯及硫酸二戊酯)、长链卤化物(例如癸基氯、癸基溴及癸基碘，月桂基氯、月桂基溴及月桂基碘，肉豆蔻基氯、肉豆蔻基溴及肉豆蔻基碘，和硬脂酰氯、硬脂酰溴及硬脂酰碘)、芳烷基卤化物(例如苄基溴及苯乙基溴)及其它试剂。优选的盐包括一盐酸盐、硫酸氢盐、甲磺酸盐、磷酸盐或硝酸盐。
本发明也包括本发明化合物的前药和溶剂化物。术语“前药”表示以下化合物，一旦将所述化合物给予受试者，所述化合物就通过代谢过程或化学过程来进行化学转化，从而得到本申请所述的化合物和/或其盐和/或溶剂化物。可在体内转化以提供生物活性物质的任何化合物是在本发明的范围和主旨内的前药。例如，含有羧基的化合物可形成生理上可水解的酯，其通过在体内水解以得到化合物本身而充当前药。前药包括为本领域的实践者所公知的酸衍生物，例如通过母体酸与合适的醇的反应制备的酯、或通过母体酸化合物与取代的或未取代的胺的反应制备的酰胺、或酸酐、或混合酸酐。由悬垂在本发明的化合物上酸性基团产生的简单的脂族或芳族酯、酰胺和酸酐是具体的前药。在一些情况中，期望制备双酯型前药例如(酰氧基)烷基酯或((烷氧基羰基)氧基)烷基酯。具体地，本发明的化合物的C1-C8烷基、C2-C8烯基、C2-C8炔基、芳基、C7-C12取代的芳基和C7-C12芳烷基酯。
所述前药优选口服给药，这是因为水解在许多情况下主要在消化酶的影响下发生。当酯本身具有活性或水解发生在血液中时，可使用肠胃外给药。
各种形式的前药是本领域公知的。所述前药衍生物的实例请参见：
a)Design of Prodrugs，H.Bundgaard，ed.，Elsevier(1985)，and Methods in Enzymology，112：309-396，K.Widder et al.，eds.，Academic Press(1985)；
b)Bundgaard，H.，Chapter 5，“Design and Application of Prodrugs，”A Textbook of Drug Design and Development，pp.113-191，P.Krosgaard-Larsen et al.，eds.，Harwood Academic Publishers(1991)；和
c)Bundgaard，H.，Adv.Drug Deliv.Rev.，8：1-38(1992)，
将上述每篇文献引入本申请作为参考。
酯通常是由酸(有机酸或无机酸)衍生的化合物，其中至少一个-OH(羟基)基团被-O-烷基(烷氧基)基团替代。酯通常衍生自羧酸和醇。
本发明化合物的酯优选为体内可水解的酯。
本文使用的术语“体内可水解的酯”应理解为表示包含羧基或羟基团的本发明化合物的体内可水解的酯，例如在人体或动物体内被水解从而产生母体酸或醇的药学可接受的酯。适合羧基的药学可接受的酯包括例如烷基酯、环烷基酯和任选地取代的苯基烷基酯(特别是苄基酯)、C1-C6烷氧基甲基酯(例如甲氧基甲基酯)、C1-C6烷酰氧基甲基酯(例如特戊酰氧基甲基酯)、酞基酯、C3-C8环烷氧基羰氧基-C1-C6烷基酯(例如1-环己基羰氧基乙基酯)；1，3-二氧杂环戊烯-2-羰基甲基酯，例如5-甲基-1，3-二氧杂环戊烯-2-羰基甲基酯；以及C1-C6-烷氧基羰氧基乙基酯，例如1-甲氧基羰氧基乙基酯，并且所述酯可在本发明化合物的任意羧基上形成。
包含羟基的本发明化合物的体内可水解的酯包括无机酸酯(例如磷酸酯)、[α]-酰氧基烷基醚和相关化合物，所述相关化合物由于所述酯的体内水解而断裂形成母体羟基。[α]-酰氧基烷基醚的实例包括乙酰氧基甲基醚和2，2-二甲基丙酰氧基甲基醚。与羟基形成体内可水解的酯的基团的选择包括烷酰基、苯甲酰基、苯基乙酰基和取代的苯甲酰基和苯基乙酰基、烷氧羰基(以形成碳酸烷基酯)、二烷基氨基甲酰基和N-(二烷基氨基乙基)-N-烷基氨基甲酰基(以形成氨基甲酸酯)、二烷基氨基乙酰基和羧基乙酰基。本发明包括所有此类酯。
“溶剂化物”是指化合物的与溶剂或水缔合的形式(也称作“水合物”)，通常通过溶剂分解。该物理缔合包括氢键合。常规的溶剂包括水、乙醇、乙酸等。本发明的化合物可例如以结晶形式制备且可为溶剂化的或水合的。合适的溶剂化物包括药学上可接受的溶剂化物例如水合物，且还包括化学计量的溶剂化物和非化学计量的溶剂化物两者。在一些情况中，溶剂化物能够分离，例如当一个或多个溶剂分子被引入结晶固体的晶格中时。“溶剂化物”包括溶液相和可分离的溶剂化物两者。代表性的溶剂化物包括水合物、乙醇合物和甲醇合物。
意图进行施用的“受试者”或“受试对象”或“宿主”包括，但不限于，人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人))和/或非人的动物，例如哺乳动物例如灵长类(例如，食蟹猴(cynomolgus monkey)、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物如小鼠和大鼠、猫和/或狗。在一些实施方案中，受试者为人。在一些实施方案中，受试者为非人的动物。术语“人”、“患者”和“受试者”在本文中可互换地使用。
“有效量”意指当施用到受试者用于治疗或预防疾病时足以实现这样的治疗或预防的化合物的量。“有效量”可取决于化合物、疾病及其严重度、以及待治疗的受试者的年龄、体重等改变。“治疗有效量”是指用于治疗性治疗的有效量。“预防有效量”是指用于预防性治疗的有效量。
“预防”或“预防性治疗”是指疾病或病症的获得或发展的风险的降低(即，使疾病的临床症状的至少一种在疾病发作之前易受疾病感染、或未暴露于致病原的受试者中不发展。
术语“治疗”是指：(i)在可能易患疾病、障碍和/或病症但尚未诊断患有所述疾病、障碍和/或病症的患者中预防所述疾病、障碍和/或病症；(ii)抑制所述疾病、障碍和/或病症，即阻止其发展；或(iii)减轻所述疾病、障碍和/或病症，即引起所述疾病、障碍和/或病症的消退。
本申请使用的术语″抑制(inhibition)″或者“抑制(inhibiting)”是指减少或者抑制特定的病症、症状或者疾病，或者显著地减少生物活性或者病变的基线活性(baseline activity)。
本申请使用的术语“抑制剂”是指以下分子，其能够抑制(包括部分抑制或变构抑制)靶标分子例如法尼基焦磷酸合酶(FPPS)的一种或多种生物活性。例如，抑制剂通过降低或遏制靶标分子的活性和/或降低或遏制信号转导来发挥作用。
本申请所描述的化合物可按立体异构形式存在(例如，其含有一个或多个不对称碳原子)。单独的立体异构体(对映异构体和非对映异构体)和这些异构体的混合物包括在本发明范围内。同样地，应理解本申请所描述的化合物或盐可按不同于该式中所示的互变异构形式存在，并且这些也包括在本发明范围内。应理解，本发明包括上文定义的具体组的所有组合和亚类。本发明的范围包括立体异构体的混合物以及纯化的对映异构体或对映异构体/非对映异构体富集的混合物。应理解，本发明包括上文定义的具体组的所有组合和亚类。
本申请所描述的化合物的一种对映异构体与另一种对映异构体相比可能显示出优越的活性。因此，所有立体化学都视为本发明的一部分。当需要时，对外消旋物质进行的分离可如下实现：使用手性柱来进行HPLC，或者使用拆分试剂诸如莰烷酰氯(camphonic chloride)来进行拆分(如Young，S.D.et a1.，Antimicrobial Agents and Chemotherapy 1995，2602-2605中所述)。
本发明还包括同位素标记的化合物，其除以下事实外与式(I)所述的那些化合物相同：一个或多个原子被原子质量或质量数不同于天然常见的原子质量或质量数的原子代替。可引入本发明化合物及其可药用盐中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如2H、3H、11C、13C、14C、15N、17O、18O、31P、32P、35S、18F、36Cl、123I和125I。
含有上述同位素和/或其他原子的其他同位素的本发明的化合物和所述化合物的可药用盐在本发明范围内。同位素标记的本发明的化合物，例如引入了放射性同位素诸如3H或14C的那些化合物，用于药物和/或底物组织分布测定。氚代的即3H和碳-14即14C同位素由于其易于制备及可检测性而是特别优选的。11C和18F同位素特别用于PET(正电子发射断层扫描术)，以及125I同位素特别用于SPECT(单光子发射计算机断层扫描)，所有这些都用于脑成像。此外，用较重的同位素诸如氘即2H取代可提供某些导致更大的代谢稳定性的治疗益处，例如体内半衰期增加或剂量需求减小，由此在一些情况中是优选的。通常可通过进行下文的方案和/或实施例中披露的操作，通过用容易获得的同位素标记的试剂代替非同位素标记的试剂来制备同位素标记的式(I)化合物和本发明的以下化合物。
具体地，本发明涉及如下的实施方案。
在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂可包括，但不限于硫解酶(乙酰乙酰基-CoA转移酶)抑制剂、2)HMG-CoA合酶抑制剂、3)HMG-CoA还原酶抑制剂、4)甲羟戊酸激酶抑制剂、5)磷酸甲羟戊酸激酶抑制剂、6)甲羟戊酸-5-焦磷酸脱羧酶抑制剂、 7)异戊烯焦磷酸异构酶抑制剂、8)法尼基焦磷酸合酶抑制剂、9)香叶香叶酰基焦磷酸合酶抑制剂和10)香叶香叶酰基转移酶(I，II)抑制剂。
所述硫解酶(乙酰乙酰基-CoA转移酶)抑制剂包括，但不限于biochemical and biophysical research communications，1989，163，548-553中所述的L-660631。
所述HMG-CoA合酶抑制剂包括，但不限于Biochem.J(1993)289，889-895中所述的L-659699，Agric.Biol.Chem.，55(12)，3129-3131，1991中所述的1233A/F-244，以及Tetrahedron，2000，56(3)，479-487中所述的Dihydroxerulin以及公开在下述文献中的那些化合物：US5064856；EP0411703A1；Agric.Biol.Chern.，1991，55(12)：3129-3131；Bioorg.Med.Chem.，1998，6：1255-1272；Biochem.Biophys.Res.Commun.，1999，265：536-540。，将所有这些专利以及其他公开引入本申请作为参考。
所述HMG-CoA还原酶抑制剂包括，但不限于公开在下述文献中的那些化合物：US 5102911-A；EP476493-A1；US5091378-A；EP465970-A；EP465265-A；EP464845-A；EP463456-A；EP456214-A1；EP591165-A；US5049577-A；EP445827-A2；EP442495-A；US5025000-A；EP435322-A2；US5023250-A；JP3112967-A；US5017716-A；US5010105-A；US5011947-A；EP424929-A1；EP422895-A1；EP420266-A2；EP419856-A2；EP418648-A1；EP416383-A2；US4996234-A；US4994494-A；EP415488-A；US4992429-A；EP411420-A2；EP409399-A1；EP408806-A1；DE3918364-A；EP401705-A；EP391185-A1；US4957940-A；US4950675-A；US4946860-A；US4940727-A；US4939143-A；US4937264-A；US4937263-A；EP402154-A1；EP375156-A2；US4929620-A；US4927851-A；US4904692-A；EP468974-A1；US4904646-A；WO9113616-A1；US4897402-A；US4892884-A；EP355846-A2；US4885314-A；EP349063-A；US4876280-A；EP346759-A2；EP422102-A1；EP344602-A1；DE3805884-A；EP330172-A；EP327166-A；EP327165-A；WO8905639-A；JP1068367-A；EP306264-A；US4792614-A；DE3632893-A1；US4719229-A；EP251625-A2；EP232997-A1；EP211416-A2；EP183132-A2；EP164049-A；FR2516087-A1；Wang K et al.J.Nat.Prod.，2015，78：1977-1989；Wess G et al.J Med.Chem.，1994，37：3240-3246；Procopiou PA et al.J Med.Chem.，1993，36：3655-3662；Pfefferkorn JA et al.J Med.Chem.，2008，51：31-45；Ahmad S et al.J Med.Chem.，2008，51：2722-2733；Sarver RW et al.J Med.Chem.，2008，51：3804-3813，将所有这些专利以及其他公开引入本申请作为参考。
在优选的实施方案中，所述HMG-CoA还原酶抑制剂为他汀类化合物。示例性他汀类化合物选自：普伐他汀、阿托伐他汀、罗苏伐他汀、氟伐他汀、匹伐他汀、美伐他汀、洛伐他汀、辛伐他汀、西立伐他汀，或它们的可药用盐、酯、前药、溶剂化物。优选的HMG-CoA还原酶抑制剂是那些已上市的药物，最优选的是辛伐他汀，洛伐他汀和美伐他汀，或它们的可药用盐、酯、前药、溶剂化物。制备HMG-CoA还原酶抑制剂的方法是本领域技术人员熟知的且这些成分包括可商购获得的那些。HMG-CoA还原酶抑制剂可以其游离酸形式、其酯形式、或其可药用盐的形式使用。这些可药用盐包括例如，钠盐、钙盐、铝盐和酯盐。HMG-CoA还原酶抑制剂可以以消旋混合物、或更强活性的适当的立体异构体的形式进行使用。
所述的法尼基焦磷酸合酶抑制剂包括，但不限于公开在下述文献中那些化合物：US.7462733； US.20080200679；WO.2006039721；US.7358361；US.7745422；US.20100316676；WO.2007109585；US.7687482；WO.2008128056；US.20080255070；WO.2010033980；WO.2010033981；WO.2008076417；US.7781418；WO.2010033978；WO.2009068567；WO.2010043584；WO.2009128918；ACS Med.Chem.Lett.2013，4：423-427；J.Am.Chem.Soc.，2009，131：5153-62；Nat.Chem.Biol.，2010，6：660-6；Bioorg.Med.Chem.Lett.，2008，18：2878-82；J.Med.Chem.，2008，51：2187-95；Proc.Natl.Acad.Sci.U.S.A.，2007，104：10022-7；Tetrahedron Lett.2011，52：2285-87；Chem.Commun.，2010，46：5340-5342；Expert Opin.Ther.Pat.2011，21(9)：1433-1451；J.Pharmacol.Exp.Ther.，2001，296：235-42；J.Med.Chem.2003，46：5171-5183；J.Med.Chem.2005，48：2957-2963；J.Med.Chem.2006，49：5804-5814；J.Med.Chem.2013，56：7939-7950；J.Med.Chem.2008，51：2187-2195；ChemMed Chem2015，10：1884-1891；Biochim.Biophys.Acta，2014，18401840：1051-1062；J.Med.Chem.2007；50：5967-75，将所有这些专利以及其他公开引入本申请作为参考。
在进一步的实施方案中，所述法尼基焦磷酸合酶抑制剂为双膦酸化合物或其可药用盐、酯、前药、溶剂化物。术语“双膦酸(盐)”是指特征在于两个膦酸基团通过膦醚键结合到一个中心(孪位的)碳原子上的化合物。这种P-C-P结构如下面的式I所示。应该指出的是，这里在提到本发明的治疗剂时所用的术语“双膦酸”也旨在包括双膦酸(盐)、双膦酸以及它们的盐和衍生物。除非特别说明，在提到双膦酸时所用的具体命名无意限制本发明的范围。
作为药物的双膦酸(盐)记载在例如EP-A-170,228、EP-A-197,478、EP-A-22,751、EP-A-252,504、EP-A-252,505、EP-A-258,618、EP-A-350,002、EP-A-273,190和WO-A-90/00798等中，将所有这些专利引入本申请作为参考。
用作药物的“双膦酸及其药学上可接受的盐”还记载在例如美国专利4,509,612、4,666,895、4,719,203、4,777,163、5,002,937、4,971,958和4,958,839以及欧洲专利申请252,504和252,505中，将所有这些专利引入本申请作为参考。
优选的双膦酸或其可药用盐选自阿仑膦酸、西马膦酸、氯屈膦酸、EB-1053、替鲁膦酸、依替膦酸、伊班膦酸、英卡膦酸、米诺膦酸、奈立膦酸、奥帕膦酸、利塞膦酸、匹瑞膦酸、帕米膦酸、唑仑膦酸或其可接受的盐，例如伊班膦酸单钠盐一水合物。
所述香叶香叶酰基焦磷酸合酶抑制剂包括，但不限于公开在如下文献中的那些化合物：J.Med.Chem.2009，52：8025-37；Biochem.Biophys.Res.Commun.，2007，353：921-925；J.Med.Chem.2002，45：2185-2196；Bioorg.Med.Chem.2008，16：390-399；J.Med.Chem.，2008，51：5594-5607；ACS Med.Chem.Lett.2015，6：1195-1198；Proc.Natl.Acad.Sci.U.S.A.，2012，109(11)：40584063，将所有这些专利以及文献引入本申请作为参考。
所述香叶香叶酰基转移酶(I，II)抑制剂包括，但不限于公开在如下文献中的那些化合物：EP1165084A1；EP1165084A4；EP2014291A2；EP2014291B1；US6103487；US6284910；US6355643B1；US6586461B1；US6638962B2；US7763620B2；US8093274B2；US8815935B2；US9040563B2；US20030219847A1；US20040121985A1；US20060030624A1；US20070249010A1；US20100063114A1；US20110178138A1；US20120035184A1；US20130102639A1；WO1999006376A1；WO2000033826A1；WO2000051614A1；WO2007111948A2；WO2007118009A1；WO2010014054A1；WO2010088457A2；WO2012034038A2；WO.2009106586.；Angew.Chem.Int.Ed.2011，50，4957-4961；J.Med.Chem.2010， 53：3454-64；J.Biol.Chem.2001，276：48213-22；Bone.2005，37：349-58；J.Biol.Chem.2009，284：6861-8；Eur.J.Med.Chem.，2011，46(10)：4820-4826；Drug Discov.Today，2015，20：267-276；J.Med.Chem.2012，55，8330-8340；J.Am.Chem.Soc.2007，129：5843-5845；J.Med.Chem.2009，52：8025-8037；J.Med.Chem.1999，42：1333-1340；PLoS ONE，2011，6：e26135；Bioorg.Med.Chem.，2005，13：677-688；IL Farmaco，2004，59：857-861；Org.Biomol.Chem.，2006，4，1768-1784；J.Biol.Chem.，2006，281(18)：12445-12450；J.Biol.Chem.，2008，283(15)：9571-9579；及GGTI-298，其在McGuire et al(1996)Platelet-derived growth factor receptor tyrosine kinase phosphorylation requires protein geranylgeranylation but not farnesylation.J.Biol.Chem.271 27402.PMID：8910319中描述，结构如下：
Figure PCTCN2016098371-appb-000093
GGTI-298的结构
将所有这些专利以及文献引入本申请作为参考。
除上述抑制剂之外，本发明还涉及影响蛋白香叶香叶酰化的其他物质，这些其他物质也可作为佐剂包含在免疫原性组合物中。
在另一方面，所述法尼基焦磷酸合酶抑制剂为下式的化合物(即TH-Z80系列的化合物)或其可药用盐、酯、前药、溶剂化物：
Figure PCTCN2016098371-appb-000094
其中n为1-24的整数，优选n为1-12的整数。
在进一步的实施方案中，n为1-20的整数。在进一步的实施方案中，n为1-15的整数。
在更具体的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000095
Figure PCTCN2016098371-appb-000096
因此，在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自上述TH-Z80系列的化合物或其可药用盐、酯、前药、溶剂化物。在一个实施方案中，本发明还涉及上述化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的双膦酸化合物(即TH-Z97系列的化合物)或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000097
其中n为1-24的整数，优选n为1-12的整数。
在进一步的实施方案中，n为1-20的整数。在进一步的实施方案中，n为1-15的整数。
在更具体的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000098
因此，在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自上述TH-Z97系列的化合物或其可药用盐、酯、前药、溶剂化物。在一个实施方案中，本发明还涉及上述化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的双膦酸化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000099
其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基。
在一个实施方案中，R1选自氢、C1-10烷基、C1-10炔基、C1-10烷基氨基、C1-10烷基硫基、卤素、羟基、吲唑基、C1-10烷氧基、被苯基或吡啶基取代的C1-10烷氧基，所述吡啶基任选取代有氨基甲酰基。
在进一步的实施方案中，R1选自氢、4-甲基苯乙氧基、4，5，6，7-四氢-2H-吲唑-2-基、(2-氨基甲酰基吡啶-4-基)甲氧基、苄基氧基、己基氧基、甲硫基、辛基氨基、己基、辛基、癸基、辛-1-炔-1-基、羟基、溴。
在一个实施方案中，R2选自氢、C1-10烷氧基、卤素。在进一步的实施方案中，R2选自氢、辛基氧基、溴。
在一个实施方案中，R3选自氢、C1-10烷基、C1-10烷氧基。在进一步的实施方案中，R3选自氢、甲基、己基氧基。
在一个实施方案中，R2与R3与它们所连接的碳原子一起形成苯环。
在一个实施方案中，R4选自氢、C1-10烷氧基。在进一步的实施方案中，R4选自氢、辛基氧基。
在进一步的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000100
Figure PCTCN2016098371-appb-000101
因此，在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自上述邻氨基吡啶类化合物或其可药用盐、酯、前药、溶剂化物。在一个实施方案中，本发明还涉及上述化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
在另一方面，本发明还涉及作为法尼基焦磷酸合酶(FPPS)的抑制剂的新的双膦酸化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000102
其中
R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；以及
R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基。
在一个实施方案中，R5选自C1-10烷氧基。
在进一步的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000103
因此，在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选 自上述间氨基吡啶类化合物或其可药用盐、酯、前药、溶剂化物。在一个实施方案中，本发明还涉及上述化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
在另一方面，本发明还涉及作为香叶香叶酰基焦磷酸合酶抑制剂的新的双膦酸化合物或其可药用盐、酯、前药、溶剂化物，所述化合物具有下式：
Figure PCTCN2016098371-appb-000104
其中
R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；
R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基；以及
R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、芳基和杂芳基。
在一个实施方案中，R9选自C1-10烷氧基。
在进一步的实施方案中，所述化合物选自：
Figure PCTCN2016098371-appb-000105
因此，在一个实施方案中，本发明涉及一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自上述苄基二磷酸化合物或其可药用盐、酯、前药、溶剂化物。在一个实施方案中，本发明还涉及上述化合物或其可药用盐、酯、前药、溶剂化物作为佐剂在制备用于预防或治疗疾病的免疫原性组合物中的用途。
双磷酸化合物
早在40多年前Fleisch等人发现焦磷酸盐(pyrophosphate)有抑制异位钙化的作用。但焦磷酸盐不稳定，易被酶水解失活。后来人们将焦磷酸盐结构中易被酶水解的P-O-P基团改造为对酶稳定的P-C-P基团，而后发展了一系列双膦酸盐类化合物，这类化合物在治疗骨质疏松症发挥了良好的疗效。代表性的药物包括第一代的依替膦酸钠，第二代的氯膦酸钠、帕米膦酸钠和替鲁膦酸钠以及发展到现在的第三代的阿仑膦酸钠、奈立膦酸钠(neridronate)、奥帕膦酸钠(olpadronate)、利塞膦酸钠以及伊本膦酸钠，唑来膦酸钠等。与第一代双膦酸不同，第二三代双膦酸主要通过其N的质子化作用于萜烯生物合成通道中的关键靶酶FPPS(Farnesyl pyrophosphate synthase)，从而导致破骨细胞凋亡。
第三代的代表性药物唑来磷酸除了具有优良的治疗骨质疏松症外，在治疗由多发性骨髓瘤、乳腺癌、前列腺癌及肺癌等恶性肿瘤骨转移引起的骨代谢异常所致的癌症骨转移类疾病方面也有一定的效 果，但是并不显著。
本发明的一个实施方案涉及如前所述的新的双磷酸化合物，其能够用于制备代谢性骨病药物、治疗疟疾药物、真核生物肿瘤细胞增殖抑制剂、预防和/或治疗肿瘤的药物、免疫治疗药物以及用于疫苗的佐剂。
所述治疗代谢性骨病药物、治疗疟疾药物、真核生物肿瘤细胞增殖抑制剂、预防和/或治疗肿瘤的药物或免疫治疗药物中，所述式I所示化合物的质量含量为0.001-90％。
所述疫苗中，所述式I所示化合物的质量含量为0.001-90％。
所述治疗代谢性骨病药物、治疗疟疾药物、真核生物肿瘤细胞增殖抑制剂、预防和/或治疗肿瘤的药物、免疫治疗药物以及疫苗可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
本发明的双磷酸化合物通过改进末端羧基修饰，首次合成了苯并咪唑4，5，6，7位非对称的双膦酸，其保持了咪唑膦酸对FPPS的活性，其还对GGPPS，malaria及肿瘤细胞增殖具有良好的抑制效果，尤其是作为疫苗佐剂和免疫治疗药物具有良好的效果，可用于制备治疗代谢性骨病的药物、治疗疟疾的药物、真核生物肿瘤细胞增殖抑制剂、预防和/或治疗肿瘤的药物，免疫治疗药物以及作为疫苗佐剂用于制备疫苗。
抗原
在一个实施方案中，本发明的免疫原性组合物包含衍生自细菌、病毒、寄生物或肿瘤的抗原。在某些方面，所述一种或多种抗原各自独立地为微生物抗原、自身抗原、肿瘤抗原、过敏原或成瘾性物质。本发明的抗原还包括国际专利申请WO2011/148356中所述的那些。
抗原可以通过重组手段或肽合成获得，或者获自天然来源或提取物，并且可以自任何活的或非活的生物体衍生。
抗原可以自细菌衍生，所述细菌诸如例如炭疽(anthrax)、弯曲杆菌(campylobacter)、霍乱(cholera)、白喉(diphtheria)、产肠毒素性大肠杆菌(E.coli)、贾第虫(giardia)、淋球菌(gonococcus)、幽门螺杆菌(Helicobacter pylori)、乙型流感嗜血菌(Hemophilus influenza B)、不可定型的流感嗜血菌、脑膜炎球菌(meningococcus)、百日咳(pertussis)、肺炎球菌(pneumococcus)、沙门氏菌(salmonella)、志贺氏菌(shigella)、链球菌B、A组链球菌、破伤风(tetanus)、霍乱弧菌(Vibrio cholerae)、耶尔森氏菌(yersinia)、葡萄球菌(Staphylococcus)、假单胞菌属(Pseudomonas)物种和梭菌属(Clostridia)物种。
或者，抗原可以自病毒衍生，所述病毒诸如例如腺病毒、登革热1至4血清型、埃博拉病毒(ebola)(Jahrling等，Arch Virol Suppl，11：135-140，1996)、肠道病毒、肝炎A至E血清型(Blum，Digestion 56：85-95，1995；Katkov，Med Clin North Am 80：189-200，1996；Lieberman and Greenberg，Adv Pediatr Infect Dis 11：333-363，1996；Mast等，Annu Rev Med 47：257-266，1996)、单纯疱疹病毒1或2、人免疫缺陷病毒(Deprez等，Vaccine 14：375-382，1996)、流感、日本马脑炎、麻疹、诺沃克(Norwalk)、乳头瘤病毒、细小病毒B19、脊髓灰质炎、狂犬病、轮状病毒、风疹(rubella)、麻疹(rubeola)、痘苗、含有编码其它抗原诸如疟疾抗原的基因的痘苗构建体、水痘(varicella)、和黄热病(yellow fever)。或者，抗原可以自寄 生物衍生，所述寄生物包括例如：溶组织内阿米巴(Entamoeba histolytica)(Zhang等，Infect Immun 63：1349-1355)；疟原虫(Plasmodium)(Bathurst等，Vaccine 11：449-456，1993)、弓形体病(Toxoplasmosis)、和蠕虫(Helminth)。(以上3段来自PWH95845A)
或者，抗原可以为肿瘤特异性抗原(tumor specific antigen，TSA)或肿瘤相关抗原(tumor associated-antigen，TAA)。肿瘤特异性抗原指仅表达于某种肿瘤细胞表面而不存在于正常细胞上的新抗原，故又称独特肿瘤抗原(unique tumor antigen)。这样的肿瘤相关抗原是本领域已知的，常见的肿瘤特异性抗原包括(1)胎甲球蛋白(AFP)；(2)癌胚抗原(CEA)；(3)CA-125；(4)MUC-1；(5)上皮细胞肿瘤抗原(ETA)；(6)酪氨酸酶；(7)黑色素瘤相关抗原(MAGE)；(8)肿瘤睾丸抗原；(9)前列腺特异抗原(PSA)；(10)gp100；(11)Melan A；(12)GAGE，G antigen 12B/C/D/E；(13)BAGE，B melanoma antigen；(14)GM2，ganglioside。肿瘤相关抗原是与某些肿瘤细胞高度相关的抗原，它们通常未见于正常细胞，或相对于肿瘤细胞较小程度的见于正常细胞(antigens that are highly correlated with certain tumor cells.They are not usually found，or are found to a lesser extent，on normal cells)。这样的肿瘤相关抗原是本领域已知的，常见的肿瘤相关抗原包括国际专利申请WO 2010/009124中列出的那些，例如(1)BMPR1B；(2)E16；(3)STEAP1；(4)0772P；(5)MPF；(6)Napi3b；(7)Sema 5b；(8)PSCA hlg；(9)ETBR；(10)MSG783；(11)STEAP2；(12)TrpM4；(13)CRIPTO；(14)CD21；(15)CD79b；(16)FcRH2；(17)HER2；(18)NCA；(19)MDP；(20)IL20Rα；(21)Brevican；(22)EphB2R；(23)ASLG659；(24)PSCA；(25)GEDA；(26)BAFF-R；(27)CD22；(28)CD79a；(29)CXCR5；(30)HLA-DOB；(31)P2X5；(32)CD72；(33)LY64；(34)FcRH1；(35)IRTA2；以及(36)TENB2。将其引入本申请作为参考。
在进一步的实施方案中，所述抗原衍生自中东呼吸综合征(Mers)病毒、乙型肝炎病毒、黑色素瘤。
在另外一方面，本发明的免疫原性组合物还包含另一种佐剂。所述另一种疫苗佐剂包括，但不限于铝佐剂，完全弗氏佐剂，非完全弗氏佐剂，MF59，AS01，AS02，AS03，AS04，AS15，CAF01，ISCOMs(免疫刺激复合物)，Virosomes(病毒颗粒)，GLA-SE，脂质体，食用油，皂角苷，AF03，TLR激动剂。
在进一步的实施方案中，所述另一种佐剂选自TLR激动剂。示例性TLR激动剂为：TLR1刺激剂(如三酰脂蛋白(triaacyl lipoprotein))，TLR2刺激剂(如肽聚糖，酵母多糖(zymosan)，HMGB1(高速泳动蛋白1)，脂磷壁酸)，TLR3刺激剂(双链RNA如PolyI：C)，TLR4刺激剂(如LPS，MPL，RC529，GLA，E6020)，TLR5刺激剂(鞭毛蛋白(Flagellin))，TLR6刺激剂(如三酰脂蛋白，脂磷壁酸)，TLR7/8刺激剂(单链RNA，咪喹莫特)，TLR9刺激剂(DNA，如CPG ODN)，C-凝集素配体(如海带多糖)，CD1d配体(如α-半乳糖基神经酰胺)
应用的疫苗
本申请所述的佐剂和免疫原性组合物可应用于各种疫苗中，包括但不限于：卡介苗、甲肝疫苗、乙肝疫苗，丙肝疫苗，丁肝疫苗，戊肝疫苗，流感疫苗，脊髓灰质炎疫苗，百白破疫苗，麻疹疫苗，乙脑疫苗，狂犬疫苗，出血热疫苗，肺炎疫苗，流脑疫苗，甲肝疫苗，腮腺炎疫苗，流感疫苗，风疹疫苗，水痘疫苗，艾滋病疫苗，疟疾疫苗；以及癌症治疗和预防疫苗，包括但不限于黑色素瘤治疗型 疫苗，黑色素瘤预防型疫苗，肺癌治疗型疫苗，肺癌预防型疫苗，膀胱癌预防型疫苗，膀胱癌预防型疫苗治疗型疫苗，宫颈癌治疗型疫苗，宫颈癌预防型疫苗，膀胱癌治疗型疫苗，膀胱癌预防型疫苗，乳腺癌治疗型疫苗，乳腺癌预防型疫苗，肝癌治疗型疫苗，肝癌预防型疫苗，前列腺癌预防性疫苗，前列腺癌预防性疫苗。
适应症
本发明的免疫原性组合物可用于治疗各种疾病或病症，包括由细菌、病毒、真菌、寄生物引起的疾病。
在本发明的各方面中，细菌包括但不限于以下物种及其组合：醋酸钙不动杆菌(Aceinetobacter calcoaceticus)、帕氏醋酸菌(Acetobacterpaseruianus)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans)、猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)、以色列放线菌(Actinomyces israelli)、粘性放线菌(Actinomyces viscosus)、嗜水气单胞菌(Aeromonas hydrophila)、富养产碱菌(Alcaliges eutrophus)、酸热耐热杆菌(Alicyclobacillus acidocaldarius)、闪烁古生球菌(Arhaeglobusfulgidus)、芽孢杆菌属(Bacillus)种、炭疽芽孢杆菌(Bacillus antracis)、短小芽孢杆菌(Bacillus pumilus)、嗜热脂肪芽孢杆菌(Bacillus stearothermophillus)、枯草芽孢杆菌(Bacillus subtilis)、热链形芽孢杆菌(Bacillus thermocatenulatus)、拟杆菌属(Bacteroides)种、博德特菌属(Bordetella)种、支气管败血性博德特菌(Bordetella bronchiseptica)、博氏疏螺旋体(Borrelia burgdorferi)、布鲁氏菌属(Brucella)种、洋葱伯克霍尔德氏菌(Burkholderia cepacia)、类鼻疽伯克霍尔德氏菌(Burkholderiaglumae)、短螺旋体属(Brachyspira)种、猪痢疾短螺旋体(Brachyspirahyodysenteria)、肠道短螺旋体(Brachyspira pilosicoli)、弯曲菌属(Camphylobacter)种、大肠弯曲杆菌(Campylobacter coli)、胎儿弯曲杆菌(Campylobacter fetus)、猪肠弯曲杆菌(Campylobacter hyointestinalis)、空肠弯曲杆菌(Campylobacter jejuni)、鹦鹉热衣原体(Chlamydia psittaci)、沙眼衣原体(Chlamydia trachomatis)、嗜衣原体属(Chlamydophila)种、粘稠色杆菌(Chromobacterium viscosum)、(Clostridium species)、肉毒梭状芽孢杆菌(Clostridium botulinum)、艰难梭状芽孢杆菌(Clostridiumdifficile)、产气荚膜梭状芽孢杆菌(Clostridium perfringens)、破伤风梭状芽孢杆菌(Clostridium tetani)、棒状杆菌属(Corynebacterium)种、白喉棒状杆菌(Corynebacterium diphtheriae)、犬艾里西体(Ehrlichia canis)、肠杆菌属(Enterobacter)种、产气肠杆菌(Enterobacter aerogenes)、肠球菌属(Enterococcus)种、猪红斑丹毒丝菌(Erysipelothrix rhusiopathieae)、埃希氏菌属(Escherichia)种、大肠杆菌(Escherichia coli)、具核梭形杆菌(Fusobacterium nucleatum)、嗜血杆菌属(Haemophilus)种、流感嗜血杆菌(Haemophilus infiuenzae)、睡眠嗜血杆菌(Haemophilus somnus)、螺杆菌属(Helicobacter)种、幽门螺杆菌(Helicobacter pylori)、猪螺杆菌(Helicobacter suis)、克雷伯菌属(Klebsiella)种、肺炎克雷伯菌(Klebsiellapneumoniae)、嗜酸乳杆菌(Lactobacillus acidophilis)、胞内劳森氏菌(Lawsonia intracellularis)、军团杆菌属(Legionella)种、肺炎军团杆菌(Legionella pneumophilia)、钩端螺旋体属(Leptospira)种(如犬型钩端螺旋体(Leptospira canicola)、流行性伤寒钩端螺旋体(Leptospiragrippotyposa)、哈德焦钩端螺旋体(Leptospira hardjo)、博氏哈德焦-牛钩端螺旋体(Leptospira borgpetersenii hardjo-bovis)、博氏哈德焦-帕希诺钩端螺旋体(Leptospira borgpetersenii hardjo-prajitno)、问号钩端螺旋体(Leptospira interrogans)、 出血性黄疸钩端螺旋体(Leptospiraicterohaemorrhagiae)、波蒙娜钩端螺旋体(Leptospira pomona)、钩端螺旋体(Leptospira)、伯拉第斯拉瓦钩端螺旋体(Leptospira bratislava)等)、李斯特菌属(Listeria)种、单核细胞增多性李斯特菌(Listeriamonocytogenes)、脑膜炎双球菌类细菌(Meningococcal bacteria)、莫拉氏菌属(Moraxella)种、分支杆菌属(Mycobacterium)种、牛分支杆菌(Mycobacterium bovis)、结核分支杆菌(Mycobacterium tuberculosis)、鸟分支杆菌(Mycobacterium avium)、胞内分枝杆菌(Mycobacteriumintracellulare)、堪萨斯分支杆菌(Mycobacterium kansaii)、戈登氏分枝杆菌(Mycobacterium gordonae)、支原体属(Mycoplasma)种(如猪肺炎支原体(Mycoplasma hyopneumoniae)、关节液支原体(Mycoplasma synoviae)、猪鼻支原体(Mycoplasma hyorhinis)、肺炎支原体(Mycoplasmapneumoniae)、蕈状枝原体(Mycoplasma mycoides)蕈状LC亚种、奈瑟氏菌属(Neisseria)种、淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、(Odoribacter denticanis)、巴斯德氏菌属(Pasteurella)种、溶血性巴斯德氏菌(曼海姆菌)(Pasteurella(Mannheimia)haemolytica)、多杀性巴斯德氏菌(Pasteurella multocida)、发光无色杆菌(Photorhabdus luminescens)、牙龈卟啉单胞菌(Porphyromonasgingivalis)、牙周卟啉单胞菌(Porphyromonas gulae)、唾液卟啉单胞菌(Porphyromonas salivosa)、痤疮丙酸杆菌(Propionibacterium acnes)、变形杆菌属(Proteus)种、普通变形杆菌(Proteus vulgaris)、假单胞菌属(Pseudomonas)种、威斯康辛假单胞菌(Pseudomnas wisconsinensis)、铜绿假单胞菌(Pseudomonas aeruginosa)、荧光C9假单胞菌(Pseudomonasfluorescens C9)、荧光SIKW1假单胞菌(Pseudomonas fluorescensSIKW1)、脆弱假单胞菌(Pseudomonas fragi)、浅黄假单胞菌(Pseudomonas luteola)、解油假单胞菌(Pseudomonas oleovorans)、假单胞菌种B11-1、静止嗜冷杆菌(Psychrobacter immobilis)、立克次体属(Rickettsia spp)、普氏立克氏体(Rickettsia prowazekii)、立克次氏立克次体(Rickettsia rickettsia)、沙门氏菌属(Salmonella)种、邦戈尔沙门氏菌(Salmonella bongori)、猪霍乱沙门氏菌(Salmonella choleraeuis)、都柏林沙门氏菌(Salmonella dublin)、肠道沙门氏菌(Salmonella enterica)、新港沙门氏菌(Salmonella newport)、鼠伤寒沙门氏菌(Salmonellatyphimurium)、伤寒沙门氏菌(Salmonella typhi)、粘质沙雷菌(Serratiamarcescens)、志贺氏菌属(Shigella)种、钝顶螺旋藻(Spirlina platensis)、葡萄球菌属(Staphylococci)种、金黄葡萄球菌(Staphlyoccocus aureus)、表皮葡萄球菌(Staphyloccoccus epidermidis)、葡萄球菌(Staphylococcushyicus)、链球菌属(Streptococcus)种、猪葡萄球菌(Streptobacillusmoniliformis)、β-溶血性链球菌(beta-hemolytic Streptococcus)、化脓链球菌(Streptococcus pyogenes)(A组链球菌)、无乳链球菌(Streptococcusagalactiae)(B组链球菌)、链球菌(vifidans组)、粪链球菌(Streptococcusfaecalis)、牛链球菌(Streptococcus bovis)、乳房链球菌(Streptococcusuberis)、停乳链球菌(Streptococcus dysgalactiae)、链球菌(厌氧种)、肺炎链球菌(Streptococcus pneumoniae)、突变链球菌(Streptococcusmutans)、远缘链球菌(Streptococcus sobrinus)、血液链球菌(Streptococcussanguis)、白色链霉菌(Streptomyces albus)、肉桂色链霉菌(Streptomycescinnamoneus)、脱角质链霉菌(Streptomyces exfoliates)、疥链霉菌(Streptomyces scabies)、嗜酸热硫化叶菌(Sulfolobus acidocaldarius)、集胞藻属(Syechocystis)、密螺旋体属(Treponena)种、齿垢密螺旋体(Treponema denticola)、微小密螺旋体(Treponema minutum)、梅毒密螺旋体(Treponema palladium)、雅司密螺旋体(Treponema pertenue)、蚀疮溃疡密螺旋体(Treponema phagedenis)、屈折密 螺旋体(Treponemarefringens)、文氏密螺旋体(Treponema vincentii)、弧菌属(Vibrio)种、霍乱弧菌(Vibrio cholerae)和耶尔森氏菌属(Yersinia)种。
在某些方面，病毒是感染动物的病毒，包括但不限于以下物种及其组合：禽疱疹病毒、禽流感病毒、禽白血病病毒、禽副粘病毒、边界病病毒、牛冠状病毒、牛流行热病毒、牛疱疹病毒、牛免疫缺陷病毒、牛白血病病毒、牛副流感病毒3、牛呼吸道合胞体病毒、牛病毒性腹泻病毒(BVDV)、I型BVDV、II型BVDV、犬腺病毒、犬冠状病毒(CCV)、犬瘟热病毒、犬疱疹病毒、马疱疹病毒、犬流感病毒、犬副流感病毒、犬细小病毒、犬呼吸道冠状病毒、古典猪瘟病毒、东方马脑炎病毒(EEE)、马传染性贫血病毒、马流感病毒、西尼罗病毒、猫杯状病毒、猫肠冠状病毒、猫免疫缺陷病毒、猫传染性腹膜炎病毒、猫疱疹病毒、猫流感病毒、猫白血病病毒(FeLV)、猫病毒性鼻气管炎病毒、慢病毒、马立克氏病病毒、新城疫病毒、绵羊疱疹病毒、绵羊副流感3型病毒、绵羊进行性肺炎病毒、绵羊肺腺癌病毒、泛热带CCV、I型猪环状病毒(PCV)、II型PCV、猪流行性腹泻病毒、猪血凝性脑脊髓炎病毒、猪疱疹病毒、猪细小病毒、猪繁殖与呼吸综合征(PRRS)病毒、伪狂犬病毒、狂犬病毒、原病毒、鼻病毒、牛瘟病毒、猪流感病毒、传染性肠胃炎病毒、火鸡冠状病毒、委内瑞拉马脑炎病毒、水泡性口炎病毒、西尼罗病毒和西方马脑炎病毒。
在某些方面，所述病毒是感染人的病毒，包括但不限于：腺病毒科(大多数腺病毒)；沙粒病毒科(出血热病毒)；星形病毒科；布尼亚病毒科(Bungaviridae)(例如，汉坦病毒、布尼亚病毒(bunga virus)、白蛉病毒和内罗病毒)；杯状病毒科(Calciviridae)(例如，导致肠胃炎的毒株)；冠状病毒科(例如，冠状病毒)；纤丝病毒科(例如，埃博拉病毒)；黄病毒科(Flaviridae)(例如，丙肝病毒、登革病毒、脑炎病毒、黄热病病毒)；嗜肝DNA病毒科(Hepadnaviridae)(乙肝病毒)；疱疹病毒科(单纯疱疹病毒(HSV)1和2、水痘-带状疱疹病毒、巨细胞病毒(CMV)、疱疹病毒)；虹彩病毒科(例如，非洲猪瘟病毒)；诺瓦克病毒和相关病毒；正粘病毒科(例如，流感病毒)；乳多空病毒科(乳头瘤病毒、多瘤病毒)；副粘病毒科(例如，副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒)；中东呼吸综合征(Mers)病毒；细小病毒科(细小病毒)；微小核糖核酸病毒科(例如，脊髓灰质炎病毒、甲肝病毒、肠病毒、人柯萨奇病毒、鼻病毒、埃可病毒)；痘病毒科(例如，天花病毒、牛痘病毒、痘病毒)；呼肠孤病毒科(呼肠孤病毒、呼肠孤病毒、环状病毒和轮状病毒)；逆转录病毒(例如，人免疫缺陷病毒如HIV-1或HIV-2(也称HTLV-III、LAV或HTLV-III/LAV)或HIV-III；以及其他分离株，如HIV-LP)；弹状病毒科(例如，水泡性口炎病毒、狂犬病毒)；披膜病毒科(例如，马脑炎病毒、风疹病毒)；和未分类的病毒(例如，海绵状脑病的病原、丁型肝炎的病原(据认为是乙型肝炎病毒的缺陷性随体(satellite))。
在本发明的一些方面，真菌包括但不限于孢子、霉菌和酵母(例如，念珠菌物种)。
在本发明的一些方面，寄生物包括但不限于以下物种及其组合：来自无形体属(Anaplasma)的蛋白、肝片吸虫(肝吸虫)、球虫属、艾美虫属种、犬新孢子虫、刚地弓形虫、贾第虫属、恶丝虫属(犬恶丝虫)、钩虫属(钩虫)、锥虫属种、利什曼原虫属种、毛滴虫属种、微小隐孢子虫、巴贝斯虫属、血吸虫属、绦虫属、类圆线虫属、蛔虫属、毛线虫属、肉孢子虫属、哈蒙德虫属、等孢子球虫属(Isopsora)。在某些方面，寄生物是外寄生物。在某些方面，外寄生物包括但不限于蜱类，包括硬蜱属、扇头蜱属、草原革蜱属、微小牛蜱属、璃眼蜱属、嗜群血蜱属的物种以及它们的组合。
在本发明的一些方面，癌症可以是恶性或非恶性的癌症。癌症或肿瘤包括但不限于：胆道癌、膀胱癌、脑癌、乳腺癌、子宫颈癌、绒毛膜癌、结肠癌、大肠癌、子宫内膜癌、食道癌、胃癌；成胶质细胞瘤、上皮内赘生瘤、淋巴瘤(例如、滤泡性淋巴瘤)、肝癌、肺癌(例如、小细胞和非小细胞肺癌)、白血病(例如、毛发细胞白血病、慢性髓细胞性白血病、皮肤T细胞白血病)、黑色素瘤(例如、恶性黑色素瘤)、多发性骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、直肠癌、肾癌、肉瘤、皮肤癌、睾丸癌、甲状腺癌、肾癌、以及其他恶性瘤和肉瘤(例如、鳞状细胞瘤、肾细胞瘤、前列腺瘤、膀胱瘤或结肠瘤)。
施用方式和剂量
为用于治疗，可以将有效量的一种或多种免疫原性组合物施用于受试对象。本发明的药物组合物的施用可以通过本领域技术人员已知的任何方式来实现。优选的施用途径包括但不限于胃肠外(例如，肌内、皮下、皮内、静脉内注射)、对皮肤的局部(例如，透皮)或粘膜(例如，口服、鼻内、阴道内、直肠内、透颊、眼内或舌下)。在癌症治疗的情形中，其可以包括肿瘤内施用。
在本发明的一些方面，免疫原性组合物的“有效量”是指对于实现所需的生物学效果而言必须或足够的量。例如，对于治疗病症的免疫原性组合物的有效量可以是消除微生物感染或肿瘤所必需的量。用作疫苗佐剂的有效量可以是可用于加强受试对象对于疫苗的免疫应答的量。有效量可能根据如下参数而有所不同：待治疗的疾病或病况、所施用的特定免疫原性组合物、受试对象的大小或者疾病或病况的严重性。本领域普通技术人员可以无需过度实验而通过经验确定特定的免疫原性组合物的有效量。
免疫原性组合物可以以单个剂量方案给予，或者优选以多个剂量方案给予，即免疫原性组合物接种的主要过程是1-10个独立的剂量，之后根据维持和/或加强免疫反应的需要在随后的时间间隔给予其他剂量，例如1-4个月给予第二个剂量，并且如果需要，在几个月或几年后给予随后的剂量。用药方案还至少部分由个体的需要决定并取决于医务人员的判断。合适的免疫接种方案的例子包括：第一剂量，之后在第7天到6个月之间给予第二剂量，以及任选在首次接种后1个月到2年之间给予第三剂量；或者其他足够引发赋予保护性免疫力所期望的病毒中和抗体效价的方案，例如对应已建立的儿科免疫原性组合物接种方案。可以补充以特定间隔(例如每两年)给予的加强剂量来维持满意的保护性免疫力。
本发明的免疫原性组合物可以制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。在上述制剂中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
当希望将一种或多种免疫原性组合物系统性输送时，可以将其配制用于通过注射(例如，推注或滴注)的胃肠外施用。例如，可对受试者的脚掌，皮下，肌肉，腹腔以及鼻粘膜注射进行免疫。注射用制剂可以以单元剂型呈现，例如，与添加的防腐剂处于安瓿瓶中或处于多剂量容器中。组合物可以采取如处于油性或水性载具中的悬浮液、溶液剂或乳化液的形式，并且可以包含配方剂，如悬浮剂、 稳定剂和/或分散剂。
实施例
通过参考以下实施例进一步说明本发明。但是，应注意，这些实施例中，正如同上述的实施方案，是示例性的并且不应被解释为以任何方式限制本发明的范围。
在本申请中，除非另有说明，使用以下缩写：
FPPS       法尼基焦磷酸合酶
GGPPS      香叶香叶酰基焦磷酸合酶
SQS        角鲨烯合酶
GGPP       香叶香叶酰基焦磷酸
GGOH       香叶基香叶醇
OVA        鸡卵白蛋白
IgM        免疫球蛋白M
IgG        免疫球蛋白G
PBS        磷酸盐缓冲液
PBST       吐温磷酸缓冲液
BSA        牛血清白蛋白
HRP        辣根过氧化物酶
OPD        邻苯二胺
DMSO       二甲基亚砜
min        分钟
h          小时
DMAPP      二甲烯丙基焦磷酸
IPP        异戊二烯焦磷酸
PEI        聚乙烯亚胺
IFA        非完全弗氏佐剂
CFA        完全弗氏佐剂
LPS        脂多糖
DC         树突状细胞
BMDC       骨髓来源的树突状细胞
FITC       异硫氰酸荧光素
Mers       中东呼吸综合症
KLH        血蓝蛋白
TLR        Toll样受体
在生物学实验中，除非另有说明，所使用的实验动物都为小鼠，小鼠的品系都为C57B/6，购买自北京维通利华实验动物技术有限公司，饲养于清华大学生物医学测试中心SPF级动物房。
生物学实施例
我们首次发现并证实与甲羟戊酸通路相关的酶可以作为佐剂理性设计的靶标，并提出凡是影响蛋白香叶香叶酰化的物质例如甲羟戊酸通路中涉及的所有酶的抑制剂都可以用作佐剂，用于制备疫苗或免疫原性组合物。我们通过以下生物学实验证明了该观点。
实施例1
测定HMG-CoA还原酶抑制剂(他汀类化合物)在小鼠中的佐剂作用
在甲羟戊酸通路中，HMG-CoA还原酶催化3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原成甲羟戊酸，而HMG-CoA还原酶抑制剂(即他汀类化合物)抑制HMG-CoA还原酶的作用。
我们以他汀类化合物为例，证实了HMG-CoA还原酶抑制剂作为佐剂的作用。在该测定中，研究了他汀类化合物在免疫小鼠中作为佐剂的作用。
在该测定中采用了临床较常使用的8种他汀类药物。这8种他汀类药物分别为：普伐他汀、阿托伐他汀、罗苏伐他汀、氟伐他汀、匹伐他汀、美伐他汀、洛伐他汀、辛伐他汀。
材料来源：辛伐他汀购买自天津希恩思公司，美伐他汀和洛伐他汀购买自阿拉丁公司，普伐他汀和阿托伐他汀购买自安耐吉公司，氟伐他汀、匹伐他汀和罗苏伐他汀购买自华中威海公司。OVA(Ovalbumin)抗原购买自Sigma公司。
采用OVA评估体系，以抗体滴度来反映佐剂活性。鸡卵白蛋白也称鸡卵清白蛋白，由386个氨基酸组成，分子量约43kD，通常作为一种工具蛋白来研究抗体的滴度。
实验方法：将各种他汀和OVA抗原按照1∶1混合，他汀和OVA的浓度都为10mg/ml。小鼠品系选用C57B/6。实验组小鼠用所得的各种他汀和OVA抗原的混合物进行脚掌注射，每只20μl。对照组小鼠采用PBS或DMSO，各自10μl与10μl OVA抗原按照1∶1混合(其中PBS或DMSO分别是10μl，抗原是10μl)进行脚掌注射，每只20μl。免疫后7天以及14天，对小鼠进行眼眶取血，每只小鼠取血100μl。将取出的血放4℃过夜，以6000rpm离心8min，将上层血清吸出，测定血清中抗OVA的IgM和IgG抗体的滴度。
OVA抗体滴度的检测：用pH 9.6的碳酸盐溶液稀释OVA蛋白至浓度为2μg/ml，涂覆在96孔酶标板中，每孔50μl，4℃涂覆过夜。用含有0.05％吐温20的PBS清洗酶标板5次，用1％的BSA溶液封闭，每孔200μl，室温封闭2h。用PBST(吐温磷酸缓冲液)清洗酶标板5次，加入倍比稀释免疫OVA后的鼠血清，每孔50μl，室温孵育2h。用PBST清洗酶标板5次后，加入HRP(辣根过氧化物酶)标记的羊抗鼠IgM或者IgG的抗体，室温孵育45min。用PBST清洗酶标板5次，加入柠檬酸钠OPD(邻苯二胺)显色液，避光显色10min后，加入硫酸终止。用酶标仪OD490读数，实验组和对照组光吸收比值≥2.0时最大的血清稀释倍数为血清中抗OVA抗体的滴度。
如图2的结果显示，相对于PBS或DMSO处理的对照组小鼠，用他汀类药物(如氟伐他汀、匹伐 他汀、罗苏伐他汀、辛伐他汀，洛伐他汀和美伐他汀)处理的小鼠中产生了具有显著高的滴度的IgM和IgG抗体，其中尤其是以辛伐他汀，洛伐他汀和美伐他汀三种他汀的佐剂效果最为显著。部分他汀类化合物的效果不显著可能是由于体内生物利用度差如溶解性、吸收性差导致的，可通过调节这些化合物的药代动力学性质(例如形成盐、酯或前药，或者形成铝盐)而实现较好的效果。
因此，上述研究证实，HMG-CoA还原酶抑制剂可以作为佐剂使用，能够增强抗原引起的特异性免疫反应。
实施例2
测定FPPS抑制抑制剂在小鼠中的佐剂作用
在甲羟戊酸通路中，法尼基焦磷酸合酶(FPPS)催化二甲基烯丙基焦磷酸(DPP)形成法尼基焦磷酸(FPP)。而FPPS抑制剂会抑制FPPS的作用。我们研究了FPPS抑制剂在免疫小鼠中作为佐剂的作用。众所周知的是，双膦酸化合物是FPPS的强效抑制剂。在以下测定中，研究了各种双膦酸化合物的佐剂作用。
(1)TH-Z80系列化合物在小鼠中的佐剂作用
TH-Z80系列化合物是新合成的双膦酸化合物，其结构如下文所示。
首先，参照文献(Sanders，J.M.，et al.，Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption.Journal of medicinal chemistry，2005.48(8)：p.2957-2963.)的人源化法尼基焦磷酸合酶(HsFPPS)纯化方法和文献(Zhang，Y.，et al.，Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors：An X-ray and NMR Investigation.Journal of the American Chemical Society，2009.131(14)：p.5153-5162.)的酶活性测试方法，我们测定了TH-Z80系列化合物对FPPS靶标的抑制活性。
测定方法简单介绍如下：将N端带有6个连续His的HsFPPS在体外诱导表达，收集Ni柱纯化。在96孔平板中进行体外HsFPPS酶活测试，每孔溶液200μl。体系的缓冲液为25mM HEPES，2.5mM MgCl2，pH 7.4。以DMAPP与IPP为反应底物，在磷酸裂解酶体系中实时监测360nm波长处紫外值的变化。用ORIGIN 8.0软件作图拟合。TH-Z80系列化合物的抑制FPPS的IC50值如下表所示，单位是微摩尔/升(μM)。
Figure PCTCN2016098371-appb-000106
Figure PCTCN2016098371-appb-000107
从以上数据可以看出，TH-Z80系列化合物能够有效地抑制FPPS的活性，是FPPS的强效抑制剂。
接着，我们采用了如实施例1所述的OVA评估体系，测定了TH-Z80系列化合物以及其无侧链取代的母核化合物BPH-266在小鼠中的佐剂作用。将各测试化合物配制成2mg/ml的浓度，OVA抗原的浓度也为2mg/ml，各化合物和OVA按照1∶1混合。每只实验组C57B/6小鼠注射100μl的混合物(包含50μl抗原和50μl的化合物)，右侧大腿肌肉注射。免疫后7天以及14天小鼠眼眶取血100μl，4℃静置过夜后，6000rpm离心8min，吸取上层的血清。测定血清中抗OVA的IgM和IgG的抗体滴度。实验结果见图3。
其中，采用的BPH-266化合物(参照文献Ling，Y.，et al.，Bisphosphonate Inhibitors of Toxoplasma gondi Growth：In Vitro，QSAR，and In Vivo Investigations.Journal of Medicinal Chemistry，2005.48(9)：p.3130-3140.中化合物8]，其结构如下：
Figure PCTCN2016098371-appb-000108
如图3所示，用TH-Z80系列化合物处理的小鼠中产生了具有显著高的滴度的IgM和IgG抗体，其中尤其是以TH-Z80、TH-Z81、TH-Z152、TH-Z153的佐剂效果最为显著。
因此，上述测定证实，FPPS的强效抑制剂即本申请的TH-Z80系列化合物可以作为佐剂起作用，能够增强抗原引起的特异性免疫反应。
(2)TH-Z97系列化合物在小鼠中的佐剂作用
TH-Z97系列化合物是新合成的双膦酸化合物，其结构如下文所示。
首先，我们进行了HsFPPS(人源化法尼基焦磷酸合酶)活性测试，测定了TH-Z97系列化合物对FPPS靶标的抑制活性。采用了与上文测试TH-Z80系列化合物相同的方法。TH-Z97系列化合物抑制FPPS的IC50值如下表所示，单位是微摩尔/升。
Figure PCTCN2016098371-appb-000109
从以上数据可以看出，TH-Z97系列化合物能够有效地抑制FPPS的活性，是FPPS的强效抑制剂。
接着，我们采用了如实施例1所述的OVA评估体系，测定了TH-Z97在小鼠中的佐剂作用。结果示于图4中。
如图4所示，与用PBS处理的对照小鼠相比，用TH-Z97处理的小鼠中产生了具有显著高的滴度的IgG抗体。
因此，上述测定证实，FPPS的强效抑制剂即本申请的TH-Z97系列化合物可以作为佐剂起作用，能够增强抗原引起的特异性免疫反应。
(3)邻氨基吡啶化合物及其在HsFPPS(人源化法尼基焦磷酸合酶)的活性测试数据
我们对部分新合成的邻氨基吡啶双膦酸化合物进行了HsFPPS(人源化法尼基焦磷酸合酶)活性测试，测定了这些化合物对FPPS靶标的抑制活性。采用了与上文测试TH-Z80系列化合物相同的方法。这些化合物抑制FPPS的IC50值如下表所示，单位是微摩尔/升(μM)。
Figure PCTCN2016098371-appb-000110
Figure PCTCN2016098371-appb-000111
从以上数据可以看出，邻氨基吡啶双膦酸化合物能够有效地抑制FPPS的活性，是FPPS的强效抑制剂。
这类邻氨基吡啶双膦酸化合物(例如TH-Z93)在小鼠中的佐剂作用体现在以下的测试(4)中。
(4)其他FPPS抑制剂化合物在小鼠中的佐剂作用
在该测定中，采用了如实施例1所述的OVA评估体系，测定了临床较常使用的8种双膦酸化合物以及本发明的TH-Z80、TH-Z93化合物在小鼠中的佐剂作用，并比较了本发明中合成的新的双磷酸化合物与市场上双磷酸药物作为佐剂的活性的差异。所述8种双膦酸化合物分别为唑来膦酸(zoledronate)，帕米膦酸(pamidronate)，阿仑膦酸(alendronate)，伊班膦酸(ibandronate)，奈立膦酸(neridronate)，利塞膦酸(risedronate)，奥帕膦酸(olpadronate)，米诺磷酸(minodronate)。这些化合物参照Zhang，Yonghui，Annette Leon，Yongcheng Song，Danielle Studer，Christa Haase，Lukasz A.Koscielski and Eric Oldfield.″Activity of Nitrogen-Containing and Non-Nitrogen-Containing Bisphosphonates on Tumor Cell Lines.″Journal of Medicinal Chemistry，2006：5804-5814合成，将其整体引入本申请作为参考。
方法：将各种测试化合物(如上所述的十种双膦酸)和OVA抗原各按照1∶1混合，双膦酸和OVA的浓度都为10mg/ml。小鼠品系选用C57B/6。实验组小鼠用各种测试化合物和OVA抗原的混合物进行脚掌注射，每只20μl，其中OVA抗原和各种测试化合物的量都为100μg。对照组小鼠采用10μl PBS与10μl OVA抗原按照1∶1混合进行脚掌注射。免疫后7天以及14天，对小鼠进行眼眶取血，每只 小鼠取血100μl。将取出的血置于4℃过夜，6000rpm离心8min，将上层血清吸出，测定血清中抗OVA的IgM和IgG抗体的滴度。
如图5的结果所示，相对于PBS处理的对照组小鼠，用测试化合物处理的小鼠中产生了具有较高滴度的IgM和IgG抗体。而在这些双膦酸化合物中，尤其是本发明的TH-Z80和TH-Z93化合物的佐剂效果最为显著。例如，由图5中可以看出，在免疫后7天以及14天，用本发明的TH-Z80和TH-Z93化合物处理的小鼠中IgM和IgG抗体的滴度分别比PBS处理的对照组小鼠中的抗体滴度高5倍以上。此外，本发明的TH-Z80和TH-Z93化合物的佐剂效果也远高于已经上市的临床使用较多的8种双膦酸化合物。
上述(1)-(4)的测定表明，FPPS抑制剂可以发挥佐剂的作用，能够增强抗原引起的特异性免疫反应。
实施例3
(1)测定GGPPS抑制剂在小鼠中的佐剂作用
在甲羟戊酸通路中，香叶香叶酰基焦磷酸合酶(GGPPS)催化FPP形成香叶酰香叶酰基焦磷酸(GGPP)。我们研究了GGPPS抑制剂在免疫小鼠中作为佐剂的作用。
化合物TH-Z144和TH-Z145是我们合成的新的双膦酸化合物，其结构如上文所示。
参照文献(Szabo，C.M.，et al.，Inhibition of Geranylgeranyl Diphosphate Synthase by Bisphosphonates and Diphosphates：A Potential Route to New Bone Antiresorption and Antiparasitic Agents.Journal of Medicinal Chemistry，2002.45(11)：p.2185-2196.)的HsGGPPS(人源化香叶香叶酰基焦磷酸合酶)表达纯化方法和文献(Zhang，Y.，et al.，Lipophilic Bisphosphonates as Dual Famesyl/Geranylgeranyl Diphosphate Synthase Inhibitors：An X-ray and NMR Investigation.Journal of the American Chemical Society，2009.131(14)：p.5153-5162.)的酶活性测试方法，我们测试了化合物TH-Z144和TH-Z145对GGPPS(香叶香叶酰基焦磷酸合酶)的抑制活性。活性测试数据如下所示：
Figure PCTCN2016098371-appb-000112
从以上数据可以看出，化合物TH-Z144和TH-Z145能够有效地抑制GGPPS的活性，是GGPPS 的强效抑制剂。
接着，我们采用了如实施例1所述的OVA评估体系，研究了TH-Z144和TH-Z145的佐剂作用。
方法：将TH-Z144和TH-Z145两个化合物各配制成10mg/ml的浓度，OVA抗原的浓度也为10mg/ml，化合物和抗原按照1∶1混合。小鼠品系选用C57B/6。每只实验组小鼠右侧脚掌注射20μl含测试化合物和OVA抗原的混合物，即100μg(10μl)的佐剂和100μg(10μl)的抗原OVA。对照组小鼠采用10μl PBS与10ul OVA抗原按照1∶1混合进行脚掌注射。免疫后7天以及14天小鼠眼眶取血100μl，4℃静置过夜后，分离血清，测定血清中抗OVA的IgM和IgG抗体的滴度。结果见图6。
如图6的结果所示，相对于PBS处理的对照组小鼠，用测试化合物TH-Z144和TH-Z145处理的小鼠中产生了具有显著高的滴度的IgM和IgG抗体，证实了GGPPS抑制剂可以作为佐剂起作用，能够增强抗原引起的特异性免疫反应。
(2)FPPS和GGPPS的双抑制剂在小鼠中的佐剂作用
化合物BPH-716和BPH-1222已被报道是FPPS和GGPPS这两种酶的双抑制剂(参考文献：Zhang，Y.，et al.，Lipophilic pyridinium bisphosphonates：potentγδT cell stimulators.Angewandte Chemie，2010.122(6)：p.1154-1156.，Zhang，Y.，et al.，Chemo-Immunotherapeutic Antimalarials Targeting Isoprenoid Biosynthesis.ACS Medicinal Chemistry Letters，2013.4(4)：p.423-427)，其具有如下的结构：
Figure PCTCN2016098371-appb-000113
我们采用了如实施例1所述的OVA评估体系，研究了BPH-716和BPH-1222的佐剂作用。结果示于图7A-D中。
如图7A-D所示，在免疫后7天以及14天，与PBS处理的对照组小鼠相比，用BPH-716和BPH-1222处理的小鼠中产生了具有显著高的滴度的IgG和IgM抗体。(BPH-1222和BPH-716检测使用的抗原为半抗原，4-羟基-3-硝基苯基乙酰基(简称NP)，用NP33-KLH(硝基苯连接在血蓝蛋白上(KLH))混合BPH-1222和BPH-716免疫小鼠，免疫后7天和14天用NP33-BSA(硝基苯连接在牛血清白蛋白上(BSA))检测血清中针对NP特异性抗体的滴度。具体的实施以及检测方法与OVA的免疫以及检测方法相同)
因此，该测定证实，FPPS和GGPPS的双抑制剂都增加了免疫球蛋白的滴度，作为佐剂可以发挥良好的效果，能够增强抗原引起的特异性免疫反应。
实施例4
HMG-CoA还原酶抑制剂、FPPS抑制剂、GGPPS抑制剂的佐剂活性与现有佐剂的活性比较
采用了如实施例1所述的OVA评估体系，比较了3类抑制剂即HMG-CoA还原酶抑制剂(辛伐他 汀)、FPPS抑制剂(TH-Z80和TH-Z93)、GGPPS抑制剂(TH-Z145)与已知的常见佐剂(PEI、咪喹莫特、铝佐剂、MF59、IFA和CFA)的佐剂作用的差异。具体地，测试了经这些佐剂处理的小鼠中加强免疫后(after boost)的IgG滴度和抗体亲和力。
抗体的亲和力是指抗体与抗原表位结合强度的大小，是评定抗体优劣的一个非常重要的指标。它是由于抗体形成细胞本身的基因突变和抗原对B细胞克隆的选择性激活。机体的这种功能状态是长期进化和对外界环境不断适应的结果，对机体防御和维持自身免疫监控有着十分重要的意义。在体外测试，通过硫氰酸钠破坏抗原抗体结合的实验来测定抗体的亲和力大小。抗体亲和力测定是基于以下事实：硫氰酸钠能够破坏抗原和抗体的结合；抗体的亲和力越强，解离抗原和抗体结合的硫氰酸钠浓度就越高。而若抗体的亲和力越强，则从另一方面反映出佐剂的效果越好。
材料来源：PEI(聚乙烯亚胺)购买自莱博斯特(北京)科技有限公司、咪喹莫特购买自上海翊圣生物科技有限公司、铝佐剂购买自赛默飞公司，IFA(非完全弗氏佐剂)和CFA(完全弗氏佐剂)都购买自Sigma公司。MF59为自己实验室制作完成的，制作方法可参照文献“The adiuvant effect of MF59 is due to the oil-in-water emulsion formulation，none of the individual components induce a comparable adjuvant effect.”，Calabro S1，Tritto E，Pezzotti A，Taccone M，Muzzi A，Bertholet S，De Gregorio E，O′Hagan DT，Baudner B，Seubert A.Vaccine.2013Jul 18；31(33)：3363-9，将其整体引入本申请作为参考。
方法：将各种测试佐剂和OVA抗原按照1∶1混合，其中OVA抗原的浓度各为2mg/ml，体积为50μl。辛伐他汀、TH-Z80、TH-Z93、TH-Z145、PEI以及咪喹莫特(Imiquimod)各采用100μg；铝佐剂、MF59、IFA(非完全弗氏佐剂)和CFA(完全弗氏佐剂)各采用50μl。抗原和佐剂预先混合好后，4℃静置过夜后再免疫小鼠。其中，实验组小鼠通过用100μl的各种测试佐剂和OVA抗原的混合物肌肉注射进行免疫。对照组小鼠采用50μl PBS与50μl OVA抗原按照1∶1混合也进行肌肉注射。免疫后7天以及14天分别用50μg的OVA蛋白进行加强免疫。在第三次免疫后第7天将小鼠处死，取血分离血清，测定血清中IgG的滴度。
用pH 9.6的碳酸盐溶液稀释OVA蛋白至浓度为2μg/ml，涂覆在96孔酶标板中，每孔50μl，4℃涂覆过夜，用含有0.05％吐温20的PBS清洗酶标板5次，用1％的BSA溶液封闭，每孔200μl，室温封闭2h。用PBST清洗酶标板5次，加入一定稀释度的血清，就所述稀释度具体而言，PBS组不稀释，辛伐他汀组稀释16倍，TH-Z80组稀释16倍，TH-Z93组稀释32倍，TH-Z145组稀释16倍，咪喹莫特组稀释8倍，PEI组稀释8倍，铝佐剂组稀释8倍，MF59组稀释16倍，非完全弗氏佐剂组稀释32倍，完全弗氏佐剂组稀释64倍。在没有加入硫氰酸钠时，稀释后的血清中抗OVA的IgG型抗体量相同，每孔50μl，室温孵育2h。用PBST清洗酶标板5次后，分别加入浓度为10mM，9.5mM，9mM，8.5mM，8mM，7.5mM，7mM，6.5mM，6mM，5.5mM，5mM，4.5mM，4mM，3.5mM，3mM，2.5mM，2mM，1.5mM，0.5mM的硫氰酸钠，每孔50μl，室温孵育30min。用PBST清洗酶标板5次后，加入HRP标记的羊抗鼠抗体，每孔50μl，室温孵育45min。用PBST清洗酶标板5次，加入柠檬酸钠OPD显色液，避光显色10min后，加入硫酸终止。根据酶标仪的读数，用Prism软件计算出硫氰酸钠的IC50。IC50值越高，说明免疫所产生的抗体亲和力越强。
由图8可以看出，相对于PBS处理的对照组小鼠，在用本发明的3类抑制剂(辛伐他汀，TH-Z80， TH-Z93，TH-Z145)作为佐剂处理的小鼠中产生了具有显著高的滴度的IgG抗体和抗体亲和力。另外，尤其值得注意的是，无论是IgG抗体滴度还是抗体亲和力而言，本发明的抑制剂甚至都优于已知的常见佐剂如PEI、咪喹莫特、铝佐剂、MF59。我们相信这一结果会推动在本领域中开发出活性更高的佐剂。
实施例1-4充分证明，能够抑制甲羟戊酸通路中的HMG-CoA还原酶、FPPS、GGPPS的活性的化合物可以充当免疫原性组合物中的佐剂。换言之，HMG-CoA还原酶抑制剂、FPPS抑制剂、GGPPS抑制剂能够作为佐剂起作用，从而增强抗原引起的特异性免疫反应。我们相信甲羟戊酸通路中其他酶的抑制剂也能起到佐剂的作用，这些酶包括但不限于硫解酶(乙酰乙酰基-CoA转移酶)、HMG-CoA合成酶、甲羟戊酸激酶、磷酸甲羟戊酸激酶、甲羟戊酸-5-焦磷酸脱羧酶、异戊烯焦磷酸异构酶和香叶香叶酰基转移酶(I，II)。
实施例5
本发明的各种佐剂通过抑制蛋白质的香叶香叶酰化的机制发挥作用的验证
如上所述，我们发现，相对于阴性对照组，甲羟戊酸通路中的HMG-CoA还原酶抑制剂如他汀类化合物、法尼基焦磷酸合成酶抑制剂如双膦酸类化合物、香叶酰基香叶酰焦磷酸合成酶抑制剂如双膦酸类化合物都显著提高了IgG抗体的滴度和抗体亲和力，可以充当免疫原性组合物中的佐剂。香叶酰基香叶酰焦磷酸合成酶是甲羟戊酸通路中下游的酶，而我们的实验揭示抑制香叶酰基香叶酰焦磷酸合成酶则可以发挥良好的佐剂效应。因此，我们进一步推测这些抑制剂通过抑制甲羟戊酸通路中蛋白质的香叶香叶酰化的机制发挥佐剂作用。应该理解，在甲羟戊酸通路中如果信号传导是通过蛋白的翻译后修饰，即香叶香叶酰化进行，那么相关的生物学行为可以通过香叶香叶酰化所需要的底物即香叶香叶酰基焦磷酸(GGPP)或香叶基香叶醇(GGOH)进行挽回；即一旦从外部额外加入香叶香叶酰化的底物GGPP和GGOH，则这些抑制剂便不能有效地发挥其抑制作用，进而不能阻止异戊烯化的蛋白的形成。
为了证明我们的这一推测，我们设计了佐剂+OVA+GGPP(或GGOH)来验证本发明的各种抑制剂是否通过抑制香叶香叶酰化的机制来发挥佐剂的作用。具体而言，我们研究了GGOH和GGPP对辛伐他汀(HMG-CoA还原酶抑制剂)、TH-Z93(FPPS抑制剂)、TH-Z145(GGPPS抑制剂)的佐剂活性的影响。
A.GGPP和GGOH的挽救实验
方法：将辛伐他汀，TH-Z93，TH-Z145三个化合物分别配制成10mg/ml的浓度，OVA抗原的浓度为20mg/ml，将GGOH和GGPP分别配制成浓度各为200mg/ml、100mg/ml、40mg/ml、20mg/ml的一系列溶液。对于实验组而言，一方面，测定不加入GGOH或GGPP，而仅混合10μl的测试化合物、5μl的OVA抗原进行免疫时的抗体的滴度。另一方面，测定加入了GGOH或GGPP后进行免疫时的抗体的滴度；具体而言，混合10μl的测试化合物、5μl的OVA抗原和5μl的各浓度的GGOH 或者GGPP(总体积为20μl)，通过对小鼠右侧脚掌注射进行免疫。对照组小鼠仅采用PBS和抗原处理，而不加入测试化合物和GGOH或GGPP。免疫后7天以及14天小鼠眼眶取血100μl，4℃静置过夜后，分离血清，测定血清中抗OVA的IgM和IgG抗体的滴度。
由图9-11可以看出，在未加入GGPP或GGOH时，在用本发明的辛伐他汀，TH-Z93，TH-Z145作为佐剂处理的小鼠中产生的IgG和IgM抗体滴度显著高于PBS处理的对照组小鼠中的滴度，证实了其很强的佐剂作用。但是，在加入了GGPP或GGOH后，在用测试化合物作为佐剂处理的小鼠中产生的IgG和IgM抗体的滴度大大下降，并且GGPP或GGOH的加入量越大，滴度下降的幅度越大。在加入了1mg GGPP后，抗体的滴度甚至下降到了与仅用PBS处理的对照组中的滴度水平。
可见，香叶香叶酰化的底物GGPP和GGOH都能很有效的抑制辛伐他汀，TH-Z93和TH-Z145的佐剂作用，反映了所述化合物通过抑制蛋白的香叶香叶酰化来的机制发挥佐剂的作用。即，一旦从外部额外加入香叶香叶酰化的底物GGPP和GGOH，则本申请化合物不能阻止异戊烯化的蛋白的形成，进而不能发挥佐剂的作用。
B.选择性的角鲨烯合酶抑制剂的佐剂活性研究
在甲羟戊酸通路中，从FPP的生成开始该通路在下游开始分支，例如形成胆固醇等。现有技术中已知的是HMG-CoA还原酶抑制剂通过阻断细胞内甲羟戊酸通路，进而阻断胆固醇的合成而发挥作用。存在这样的疑问，本发明的甲羟戊酸通路的各种酶抑制剂之所以发挥佐剂作用，是否确实是因为甲羟戊酸通路下游的香叶香叶酰化受到抑制，还是因为其他分支如胆固醇合成受到抑制？为此，我们研究了选择性的角鲨烯合酶(SQS)抑制剂TH-Z66的佐剂作用，目的是确定本申请的化合物是否通过抑制胆固醇形成而发挥作用。众所周知的是，角鲨烯合酶(SQS)抑制剂可以抑制胆固醇的体内生物合成。
选择性的角鲨烯合酶(SQS)抑制剂TH-Z66即下述参考文献中所述的BPH-652：Liu CI et al.A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulenc，Science，2008Mar 7；319(5868)：1391-4，该文献已经证实了BPH-652是一种选择性的角鲨烯合酶(SQS)抑制剂，可以抑制胆固醇的体内生物合成，将其整体引入本申请作为参考。TH-Z66按照该文献所述的方法合成。
Figure PCTCN2016098371-appb-000114
方法：将TH-Z66配制成10mg/ml的浓度，OVA抗原的浓度也为10mg/ml，按照1∶1的比例混合，每只小鼠注射20μl，即含有100μg的TH-Z66和100μg的抗原。小鼠为C57B/6品系。对实验组小鼠右侧脚掌注射。对照组小鼠进行类似的处理，不同的是用相同体积的PBS代替TH-Z66。免疫后7天以及14天小鼠眼眶取血100μl，4℃静置过夜后，分离血清，测定血清中抗OVA的IgM和IgG抗体的滴度。
如图12所示，该研究表明，用SQS抑制剂TH-Z66处理的小鼠中产生的IgG和IgM抗体滴度与PBS处理的对照组小鼠中的滴度没有显著差异，甚至在免疫7天后的IgG抗体滴度还低于对照组小鼠 中的滴度。这说明了TH-Z66不具有佐剂作用，从而排除了本申请的化合物降胆固醇作用对佐剂效应的影响。由此可见，本发明甲羟戊酸通路抑制剂发挥佐剂作用不是通过抑制胆固醇合成进行。
以上实验A和实验B充分证明了本发明甲羟戊酸通路抑制剂发挥佐剂作用不是通过抑制胆固醇合成，而是通过抑制蛋白质的香叶香叶酰化进行。
通过实施例1-5的各项研究，我们确认，甲羟戊酸通路中各种酶的抑制剂可以作为佐剂起作用，从而增强抗原引起的特异性免疫反应，并且这种佐剂作用是通过抑制蛋白质的香叶香叶酰化实现的。
实施例6
采用本申请的抑制剂作为佐剂免疫后淋巴结中细胞的募集
淋巴结是免疫应答的重要场所，淋巴结中富含各种类型的免疫细胞，利于捕捉抗原、传递抗原信息和细胞活化增殖。通常，在免疫后B淋巴细胞，T淋巴细胞，巨噬细胞以及树突状细胞会募集至淋巴结中。在此我们研究了采用本发明的抑制剂作为佐剂免疫后淋巴结中细胞的募集。
方法：按照如实施例1类似的操作，以OVA为抗原，分别加入三种抑制剂化合物即辛伐他汀、TH-Z80、TH-Z145作为佐剂，其中OVA抗原和佐剂按照1∶1混合，通过皮下注射免疫小鼠。免疫后24h，将免疫同侧的腹股沟淋巴结取出，过100目的筛网分离成单个细胞悬液，采用流式细胞技术测定了B淋巴细胞、T淋巴细胞、巨噬细胞以及树突状细胞在淋巴结中的比例以及数量的变化。其中B淋巴细胞的标志物为B220，T淋巴细胞为CD3，巨噬细胞为CD11b和F4/80，树突状细胞为CD11c。
如图13-17所示，加入佐剂后，相比于没有经过任何处理的对照组小鼠(Ctrl)和用PBS处理的对照组小鼠，在用本发明的三种化合物(辛伐他汀，TH-Z80，TH-Z145)作为佐剂处理的小鼠的淋巴结中这四种细胞的比例和数量都显著升高。表明免疫后24h，本申请的这些佐剂都能够很大程度地促进这些细胞向淋巴结中迁移，反映出本申请的化合物作为佐剂增强了免疫应答。
实施例7
甲羟戊酸通路抑制剂增强DC对LPS的反应
树突状细胞(DC)是体内功能最强的抗原呈递细胞之一，是连接固有免疫和适应性免疫的桥梁。树突状细胞表面具有高表达的抗原呈递分子(MHC-I和MHC-II)和共刺激分子(如CD80、CD86)等，因而成为功能强大的抗原呈递细胞。树突状细胞可以活化初始型T细胞，在免疫应答中发挥着重要的作用。将肿瘤病人外周血中的树突状细胞提取出来，或者是将外周血中的单核细胞分离出来后加入细胞因子诱导分化成树突状细胞，再将肿瘤抗原，灭活的肿瘤细胞，肿瘤细胞裂解液，包含有肿瘤抗原DNA的质粒或者是RNA等加入树突状细胞中，加入一些刺激剂刺激活化这些树突状细胞以表达更多的共刺激分子，再将处理好的树突状细胞过继回输到肿瘤患者体内，对肿瘤可以起到很好的治疗效果。
本发明的佐剂可以在体外增强树突状细胞对LPS的刺激作用，也可以应用于树突状细胞疫苗中。将树突状细胞预先用这些佐剂处理再加入肿瘤抗原、灭活的肿瘤细胞、肿瘤细胞裂解液、包含有肿瘤抗原DNA的质粒、肿瘤抗原RNA；或者是这些抗原和佐剂一起刺激树突状细胞，或者是树突状细胞 被这些抗原处理过后再加入佐剂处理，可以作为一种新型的DC疫苗使用。
方法：将小鼠的骨髓细胞取出，加入10ng/ml重组的小鼠GM-CSF和IL-4，体外诱导7天后使用，分别用1μM的辛伐他汀、TH-Z80、TH-Z145和TH-Z66处理诱导分化好的小鼠骨髓来源的树突状细胞(BMDC)24h，再加入100ng/ml的LPS刺激，刺激24h后，收集上清，测定其中的TNF-α、IL-6、IL-12p70和IL-1β。96孔板中BMDC加入1μM的辛伐他汀，TH-Z80，TH-Z93，TH-Z145或这些佐剂混合加入2μM的GGOH培养24h，每孔5万细胞。24h后，加入5万OT-I CD8+T细胞或OT-IICD4+T细胞，以及100μg/ml的OVA蛋白，培养72h。72h后，将细胞的培养上清收集，测定上清中分泌的细胞因子IL-6，IFN-γ，TNF-α。
细胞因子的测定参照试剂盒购买自ebioscience，测定方法参照产品说明书，用96孔酶标板4℃包被这些细胞因子的捕获抗体，包被过夜，每孔100μl。用PBST洗脱5遍后，用封闭液室温封闭2h。用PBST洗脱5遍后，加入2倍稀释的细胞上清，室温孵育2h。用PBST洗脱5遍后，加入检测抗体，室温孵育1h。用PBST洗脱5遍后，加入亲和素连接的辣根过氧化物酶，室温孵育45min。用PBST洗脱5遍后，加入3，3″，5，5″-四甲基联苯胺溶液显色15min，加入2摩尔的硫酸终止，用酶标仪OD450读数，根据标准品计算出上清里面目的细胞因子的浓度。
结果显示，这些佐剂对小鼠骨髓来源的树突状细胞(BMDC)没有刺激作用，但是可以增强LPS对DC的刺激作用，特别是这些抑制剂都可以辅助LPS刺激DC产生IL-1β(图18-21，图38)，反映出本申请的抑制剂增强了免疫应答，可以应用于树突状细胞疫苗中。
实施例8
采用本申请的抑制剂作为佐剂免疫后淋巴结中抗原递呈细胞对OVA的摄取
在免疫应答过程中，除T细胞和B细胞起核心作用外，单核/巨噬细胞和树突状细胞也参加发挥作用，主要是处理和递呈抗原，故称抗原递呈细胞(antigen presenting cells，APC)，亦可称为辅佐细胞(accessory cells，A cells)或A细胞。APC能通过吞噬或胞饮作用摄取和处理抗原，并将经过处理得到的含有抗原决定簇的多肽片段与MHC II类分子结合，然后表达于细胞表面递呈给CD4+TH细胞。具有抗原递呈作用的细胞主要有单核/巨噬细胞、树突状细胞和B细胞三类。在此我们研究了采用本发明的抑制剂作为佐剂免疫后抗原递呈细胞对OVA抗原的摄取。
方法：用OVA标记FITC(异硫氰酸荧光素)，将FITC标记的OVA混合本发明的三种化合物(辛伐他汀、TH-Z80、TH-Z145)，按照如实施例1类似的操作，对小鼠进行皮下免疫，免疫后24h将免疫同侧的淋巴结取出，分离成单个细胞悬液，采用流式细胞技术测定淋巴结中三种抗原递呈细胞中含有的FITC-OVA细胞的比例。B淋巴细胞的标志物为B220，T淋巴细胞为CD3，巨噬细胞为CD11b和F4/80，树突状细胞为CD11c。
由图22可以看出，相比于没有经过任何处理的对照组小鼠(Ctrl)和用PBS处理的对照组小鼠，在用本发明的三种化合物作为佐剂处理的实验组小鼠的淋巴结中，FITC阳性的细胞(B220、CD11c、F4/80)比例都升高了，证实了本发明的这些抑制剂都能够促进抗原递呈细胞对抗原的摄取或向淋巴结中迁移，反映出本申请的化合物作为佐剂增强了免疫应答。
实施例6-8证实，本发明的抑制剂的佐剂作用不仅体现在抗体滴度的增加，还体现在免疫应答的各个方面得到了增强，如B淋巴细胞、T淋巴细胞，巨噬细胞以及树突状细胞在淋巴结中的募集增强，辅助LPS刺激DC产生IL-1β，以及促进抗原递呈细胞对抗原的摄取。
在以下的实验中，我们研究了本发明的抑制剂对几种特定的抗原的佐剂作用。
实施例9
三种抑制剂能够作为黏膜佐剂在体内促进Mers蛋白产生更多的抗体
取20μg的Mers蛋白(该蛋白的表达纯化参考文献Jiang L et al，Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to theviral spike glycoprotein.Sci Transl Med.2014 Apr 30)，里面各加入100μg的本发明的三种化合物(辛伐他汀，TH-Z80，TH-Z144)作为佐剂，按照如实施例1类似的操作，通过鼻黏膜对小鼠进行免疫。100μg的C48/80(一种肥大细胞的刺激剂，常用作于黏膜佐剂)作为比较。免疫后7天以及14天，检测小鼠血清中的抗中东呼吸综合征病毒Mers蛋白的IgM和IgG抗体的滴度。
由图23可以看出，相比于用PBS处理的对照组小鼠，在用本发明的三种化合物作为佐剂处理的实验组小鼠三种佐剂抗体的滴度都显著增加，证实了本发明的抑制剂能够辅助Mers蛋白产生更多的抗体，提示其可以作为佐剂用在中东呼吸综合征病毒的疫苗中。
实施例10
三种抑制剂能够促进乙肝表面抗原HbsAg产生更多的抗体
用20μg的乙肝表面抗原(HBsAg，由北京天坛生物制品有限公司馈赠)作为抗原，里面各加入100μg的本发明的三种化合物(辛伐他汀、TH-Z80、TH-Z144)作为佐剂，按照如实施例1类似的操作，通过肌肉注射对小鼠进行免疫。免疫后7天14天检测小鼠血清中抗HBsAg蛋白的IgM和IgG的滴度。
由图24可以看出，相比于用PBS处理的对照组小鼠，在用本发明的三种化合物作为佐剂处理的实验组小鼠三种佐剂抗体的滴度都显著增加，证实了本发明的抑制剂能够辅助乙肝表面抗原产生更多的抗体，提示其可以作为佐剂用在乙肝疫苗中。
实施例11
黑色素瘤的预防性疫苗的制备
用本发明的四种物质辛伐他汀、TH-Z80、TH-Z93、TH-Z145分别作为佐剂混合OVA，按照如实施例1类似的操作，免疫小鼠。初次免疫使用的混合物中各含有100μg的OVA蛋白和100μg的佐剂(即上述四种物质)，尾根部皮下注射，免疫后两周加强，加强时候不用佐剂，尾根部皮下注射50μg的抗原，第二次免疫后两周，再次加强，用50μg的抗原尾根部皮下注射。第三次免疫后两周，在小鼠右侧腹股沟皮下接种3*105 B16-OVA(稳定表达OVA的B16黑色素瘤细胞，由苏州大学生物医学 研究院馈赠)。每两天检测小鼠的体重以及肿瘤体积的变化。肿瘤的体积使用公式长*宽*宽/2计算。
使细胞在接种前稳转表达荧光素酶。具体方法如下：用表达荧光素酶的病毒质粒转染293T细胞，转染后48h，收集含有病毒的293T细胞培养上清。用含有病毒的上清培养B16-OVA细胞后，上清中的病毒感染B16-OVA细胞，并表达荧光素酶，用流式分选的方法，分选出表达荧光素酶的细胞，并扩大培养。
在接种后7天、14天以及21天，在每一组随机取出三只。注射荧光素底物，3mg/只，注射8min后，将小鼠麻醉，用活体成像仪观测肿瘤的体积。
结果示于图25中。相比于用PBS处理的对照组小鼠，在用本发明的四种化合物作为佐剂处理的实验组小鼠中，黑色素瘤的体积显著减小，提示其可以作为佐剂用在黑色素瘤的预防性疫苗中。
实施例12
黑色素瘤细胞的治疗性疫苗的制备
向C57B/6小鼠右侧腹股沟皮下接种3*105个(即300000个)B16-OVA(同样由苏州大学生物医学研究院馈赠)的肿瘤细胞，接种后第5天，用分别含有100μg的OVA蛋白和100μg的佐剂(即上述四种物质辛伐他汀、TH-Z80、TH-Z93、TH-Z145)的混合物对小鼠尾根部皮下注射，初次免疫后7天加强，加强时候不用佐剂，尾根部皮下注射50μg的抗原，第二次免疫后7天，再次加强，加强50μg的抗原。每两天检测小鼠的体重以及肿瘤体积的变化。肿瘤的体积使用公式长*宽*宽/2计算。
采用如实施例11所述相同的方法，使细胞在接种前稳转表达荧光素酶。
在接种后7天，14天以及21天，在每一组随机取出三只小鼠。注射荧光素底物，3mg/只，注射8min后，将小鼠麻醉，用活体成像仪观测肿瘤的体积。
结果示于图26中。相比于用PBS处理的对照组小鼠，在用本发明的四种化合物作为佐剂处理的实验组小鼠中，黑色素瘤的体积显著减小，提示其可以作为佐剂用在黑色素瘤的治疗性疫苗中。
实施例9-12证明，本发明的抑制剂可以作为佐剂用于临床上的多种疫苗，如中东呼吸综合征病毒的疫苗、乙肝疫苗、黑色素瘤的治疗性和预防性疫苗，提示其在临床应用上的广阔前景。
实施例13
不同免疫部位的佐剂作用
用本发明的TH-Z80作为佐剂分别混合OVA抗原对小鼠不同的部位进行免疫，免疫的部位分别是小鼠的脚掌、皮下、肌肉、腹腔以及鼻粘膜。脚掌和鼻粘膜免疫使用20μl的体系，TH-Z80的浓度为10mg/ml，OVA蛋白的浓度也10mg/ml，佐剂和抗原按照1∶1混合，即10μl的佐剂和10μl的抗原。肌肉、皮下和腹腔免疫使用100μl的体系，TH-Z80的浓度为2mg/ml，OVA蛋白的浓度也2mg/ml，佐剂和抗原按照1∶1混合，即50μl的佐剂和50μl的抗原。采用相同体积的PBS混合OVA抗原免疫对照组小鼠，按照如实施例1类似的操作，测定免疫后7天以及14天血清中抗OVA的IgM和IgG抗体的滴度。
由图27可以看出，无论在什么部位免疫，与PBS处理的对照组小鼠相比，TH-Z80的抗体滴度都显著增加，证实了本发明的化合物在各种部位注射都能达到很好的佐剂作用。
实施例14
FPPS抑制剂与TLR激动剂的联合佐剂作用
在该测定中，我们研究了本发明的抑制剂与现有技术已知的其他佐剂的联合作用，其中所采用的其他佐剂为咪喹莫特(Imiquimod，一种TLR激动剂)。
将TH-Z93和咪喹莫特两个化合物配制成4mg/ml的浓度，OVA抗原的浓度为10mg/ml。单独使用时，加入5μl的TH-Z93或者咪喹莫特作为佐剂，再混合10μl的抗原和5μl的PBS。两种佐剂联用时，分别加入两种佐剂各5μl，再混合10μl抗原，总体积都为20μl。小鼠的品系选用C57B/6小鼠。对照组小鼠采用相等体积的PBS而不加任何佐剂。对小鼠的右侧脚掌注射。免疫后7天以及14天小鼠眼眶取血100μl，4℃静置过夜后，分离血清，测定血清中抗OVA的IgM和IgG抗体的滴度。
由图28可以看出，相对于PBS处理的对照组小鼠，在单独用TH-Z93或咪喹莫特处理的小鼠中产生了具有显著高的抗体滴度，单独用TH-Z93处理的小鼠的抗体滴度显著优于单独用咪喹莫特处理的小鼠的抗体滴度。而将两者佐剂混合使用时，在在体内产生了一定的协同作用。该研究表明本发明的各种抑制剂可以与现有技术已知的其他佐剂的联合使用，从而更大程度的发挥增加免疫应答的佐剂作用。
实施例15：式IXX所示化合物的活性实验
靶点活性测试
a：HsFPPS(人源化法呢基焦磷酸合酶)活性测试
将N端带有6个连续His的HsFPPs在体外诱导表达，收集Ni柱纯化。在96孔平板中进行体外HsFPPs酶活测试，每孔溶液200μl。体系的缓冲液为25mM HEPES，2.5mM MgCl2，pH 7.4。以DMAPP与IPP为反应底物，在磷酸裂解酶体系中实时监测360nm波长处紫外值的变化。用ORIGIN 8.0软件作图拟合。
b：PvGGPPS(疟原虫香叶基香叶基焦磷酸合酶)活性测试，
将N端带有6个连续His的PvGGPPs在体外诱导表达，收集Ni柱纯化。在96孔平板中进行体外PvGGPPs酶活测试，每孔溶液200μL。体系的缓冲液为25mM HEPES，2.5mM MgCl2，pH 7.4。以GPP与IPP为反应底物，在存在磷酸裂解酶体系中进行360nm的连续分光光度检测读数。用ORIGIN8.0软件作图拟合。结果见下表：
表：合成化合物对不同靶点的活性结果
化合物 HsFPPS(IC50/μM) PvGGPPS(IC50/μM) malaria(IC50/μM)
TH-Z79 0.1-0.3 0.61 0.96
TH-Z148 0.068 0.56 1.86
TH-Z149 0.27 0.47 0.92
TH-Z150 0.14 0.28 0.78
TH-Z151 0.12 0.46 1.25
TH-Z80 0.11 0.37 1.76
TH-Z152 0.14 0.95 1.21
TH-Z81 0.48 2.07 1.95
TH-Z153 4.9 1.09 1.28
TH-Z82 9.5 3.05 0.83
TH-Z154 ＞5 5.16 0.75
TH-Z155 ＞5 10.23 3.26
II-7 0.16 0.82 0.69
以唑来膦酸作为阳性对照化合物，其对HsFPPS的IC50值在100nM左右，本发明合成的这些化合物对HsFPPS的IC50值有不少在100nM左右，同时本发明的化合物对PvGGPPS和malaria都有良好的抑制效果。
细胞活性测试：
A：疟原虫活性测试，
将恶性疟原虫3D7，用混合了10％的人类O型血的血清和25mM HEPES的IPEM1640培养基培养。培养过程保持在二氧化碳培养箱5％二氧化碳的环境中。用96孔平板进行体外药物测试实验。所测药物用PBS溶解，用完全培养基预稀释。感染的红细胞一式三份在连续3倍稀释的药物中连续培养72小时。然后在每个孔里加入等量SYBR-GREEN1。然后在激发光485nm，发射光538nm情况检测。用青蒿素和不添加任何药物对照组来观察对比。用ORIGIN 8.0软件作图拟合，结果见表1。
B：MDA-MB-231活性测试
试剂与仪器
DMEM培养基购自Gibco公司；胎牛血清(FBS)购自BI公司；双抗购自碧云天公司；0.25％胰蛋白酶-EDTA购自Gibco公司；MTT购自Ameresco公司；离心机购自安徽中佳科技有限公司，4℃冰箱购自海尔公司，-80℃购自Thermo公司；
DMEM完全培养基
每90ml DMEM培养基中，加入10ml胎牛血清，双抗按照1∶100添加，制成完全培养液，于4℃保存
1.MDA-MB-231传代细胞复苏，传代和冻存
细胞复苏：将冻存管从液氮罐里迅速取出，用镊子夹持立刻浸到37℃的水中加速融化，完全溶化后转移至超净台内，吸出细胞悬液，并加入3ml含有双抗10％FBS的DMEM，混匀后，1000r/min 4℃离心3min，弃去上清，加入适量含有10％FBS的DMEM培养基稀释，以0.5×106个细胞/cm2密度接种于培养瓶中，轻轻混匀，放入37℃，5％CO2培养箱中培养。细胞复苏两天后，在显微镜下观察细胞形态，更换一次培养基，培养至三天，传代前，在镜下观察细胞，细胞饱满，折光性强，取其对数生长期细胞进行试验。
细胞传代：细胞传代时首先弃掉培养基，然后用预热的PBS在室温下清洗一次，再用0.01％胰蛋白酶-EDTA在37℃，5％CO2培养箱内消化1-3min，轻轻拍打培养瓶侧壁，使细胞脱离培养瓶底壁，加入2ml含有血清的DMEM培养基终止消化，然后将细胞悬液转移至15ml玻璃离心瓶中，轻轻吹打均匀成为单细胞悬液，1000r/min 4℃离心3min，弃掉上清，用适量的培养基重悬细胞，取50μl细胞悬液与50μl台酚蓝混合均匀，细胞计数板对活细胞进行计数，以0.5×106个细胞/cm2的密度传入培养瓶，加入适量培养基轻轻混匀，放入37℃，5％CO2培养箱静置培养。
细胞冻存：将细胞收集于玻璃离心管中并计数，弃去上清，按照DMSO与血清为1∶9的比例配制细胞冻存液，并用细胞冻存液以1～2×106个细胞/ml的浓度重悬细胞，然后分装1ml至每支冻存管，放入-80℃冰箱中过夜，再转移至液氮，可保存数年。
2.药物筛选试验步骤：
药物溶解：称取一定质量的双膦酸药物，添加少量NaOH或NaHCO3，将药物溶解，储存浓度为10μM；
药物稀释：用DMEM完全培养基稀释配制好的双膦酸储存液，最高浓度为1mM，按照1∶3.2进行倍比稀释，共11个浓度梯度；
a.前一天将MDA-MB-231细胞进行消化，1000rmp离心3min，细胞计数，接种3000个细胞/孔细胞到96空板中，每孔体积100μl，培养14-16h，注意细胞计数准确，每孔接种的细胞量要一致，点板间歇可以轻晃细胞悬液，或者点一排细胞用吸管轻吹细胞悬液，枪打入96孔板的速度，不宜太快或太慢，以免细胞在孔板中分布不均，影响实验；96孔板周边的孔蒸发速度稍快，溶液的浓度变化稍快，如非必要尽量选择中间的60个孔做刺激，且打入的液体避免产生气泡。
b.待细胞贴壁后，将96孔板中的液体吸弃，将以上配制好的药液以此加入到前一天铺好的细胞培养板中(最终处理细胞的总体积为100μl)，同时设置空白对照组(只添加培养基)，每组6个重复；
c.药物作用72h后，将配制好的MTT溶液(称取一定量的MTT粉末加PBS溶解成5mg/ml，尽量现配现用，避光，用前用0.22的滤膜过滤)直接加到各孔中，每孔20μl，继续培养4小时；
d.铺好滤纸(可用吸水纸代替)，将孔板轻轻倒置放置(注意用力要轻，防止孔中结晶掉落)，吸干孔中液体后加入150μl DMSO溶解，用振荡器将震荡混匀，时间以刚好溶解结晶为宜，用酶标仪于570nm处测定各孔吸光值(OD值)，计算空白对照均值为ODBlank，每孔OD值＝OD实测-ODBlank，计算ODNT均值，以ODNT均值计算细胞死亡相对百分比＝(1-OD/ODNT)*100％(或细胞存活相对百分比＝OD/ODNT*100％)，然后计算均值和标准误，用Graph Prism软件将每组药物浓度(uM)及对应的药物抑制率的均值和标准误拟合出药物的IC50。结果见下表：
表：不同碳链长度化合物对MDA-MB-231的增殖抑制活性
药物名称 TH-Z79 TH-Z80 TH-Z81 TH-Z82 TH-Z148
IC50(μM) 50.93 75.46 59.63 63.09 7.302
药物名称 TH-Z149 TH-Z150 TH-Z151 TH-Z152 TH-Z153
IC50(μM) 36.07 127.5 406.4 428.2 234.5
作为佐剂的生物学实验：
一、小鼠免疫及血清分离
免疫过程中，我们使用的抗原为鸡卵清蛋白(OVA)。小鼠为8周雌性的c57bl/6。OVA配制浓度为1mg/ml，双膦酸的浓度为1mg/ml，免疫时将OVA和双膦酸按1∶1混合，小鼠肌肉注射100μl，即为每只小鼠100μg的OVA和100μg的双膦酸。免疫后14天，小鼠的眼眶采血，每只取血100μl。
将取出的全血放入4℃冰箱中过夜。第二天将4℃的全血取出离心，离心机转速为6000rpm，温度为4℃，离心5min。离心结束后将上层的血清小心吸出，再次离心，转速为6000rpm，温度为4℃，离心5min。再将上层血清吸出。血清冻存于-20℃中，检测时取出融化。
二、效价监测
1.包被：用0.05M pH9.6碳酸盐包被缓冲液将抗原OVA稀释至蛋白质含量为2μg/ml。在每个聚苯乙烯板的反应孔包被50μl，4℃过夜或37℃ 2h。第二天，弃去孔内溶液，用洗涤缓冲液PBST洗5次。
2.封闭：2％的BSA150μl，37°封闭2h，用洗涤缓冲液PBST洗5次。
3.孵育血清：将血清用稀释液按照1∶200稀释，然后再倍比稀释，每孔加入50μl的稀释后的血清，37℃孵育2小时。用洗涤缓冲液PBST洗5次(同时做空白孔，阴性对照孔)。稀释液为0.1％BSA。
4.加酶标二抗：于各反应孔中，加入新鲜稀释的HRP标记的Goat-anti mouse IgM或者Goat-anti mouse IgM(1∶5000)50μl。37℃孵育1h，用洗涤缓冲液PBST洗5次。
5.加底物显色：于各反应孔中加入预先配制的OPD显色液50ml。
6.终止反应：在显色10min后，各反应孔中加入2M硫酸50μl。
7.E.LISA检测仪上，于490nm读OD值。计算血清抗体的滴度。计算按OD值大于或等于阴性对照孔读数1.5倍的血清稀释度为血清抗体的滴度。
图29为注射100μg不同双膦酸对小鼠抗体产生的影响图。
从图29可以看出：与传统的双膦酸相比，TH-Z80能够比传统的双膦酸产生更高滴度的抗体。
图30为双膦酸侧链碳链取代长短作为佐剂的效果图。
从图30可以看出：侧链碳链长度从1增加到5时，佐剂效应变化不大，而从6增加到8时，佐剂效应显著增强，而从8开始，佐剂效应又减弱。化合物TH-Z81具有最强的佐剂效应。
抗体的亲和力是指抗体与抗原表位结合强度的大小，是评定抗体优劣的一个非常重要的指标。它 是由于抗体形成细胞本身的基因突变和抗原对B细胞克隆的选择性激活。机体的这种功能状态是长期进化和对外界环境不断适应的结果，对机体防御和维持自身免疫监控有着十分重要的意义。
在体外，我们通过NaSCN破坏抗原抗体结合的实验来测定抗体的亲和力大小。操作如下：OVA2μg/ml包被96孔酶标板，2％BSA封闭后，加入稀释后相同效价以TH-Z80作为佐剂OVA初次免疫及加强免疫小鼠后7天的血清，37℃孵育2h，PBS-T洗涤后加入不同稀释度的硫氰酸钠(NaSCN)，50μl每孔，37℃孵育0.5h；PBS-T洗涤后加入1∶5000稀释的HRP标记的羊抗小鼠IgG，37℃孵育1h，PBS-T洗涤5次，OPD-磷酸柠檬酸缓冲液系统显色(PH9.6)，2M H2SO4溶液终止反应，读取OD490nm吸光度值。计算出NaSCN抑制的IC50值，NaSCN的IC50值越高，说明免疫所产生的抗体亲和力越强。
图31为TH-Z80增加抗体亲和力的效果图。
由图31可以看出：相比于PBS，TH-Z80明显增强了佐剂及其抗原的亲和力。
从健康志愿者通过Ficoll-Hypaque密度梯度离心法制备人外周血单核细胞(PBMC)。在96孔圆底板中种植1×105个PBMC于0.2mL培养基中，加入4μM测试的双膦酸类化合物和200U/mL的rIL-2，唑来磷酸作为阳性对照。在第12天收集细胞，以FITC-anti-CD3(MiltenyiBiotec)和PE-anti-Vδ2(MiltenyiBiotec)单克隆抗体染色，流式细胞仪检测，Prism 5.0分析整理数据。
图32为不同碳链长度苯并咪唑类双膦酸对δγT细胞的扩增效果。
从图中可以明显看到，碳链长度显著影响Vγ9Vδ2 T细胞，化合物TH-Z80对Vγ9Vδ2 T细胞的扩增效果最好。
图33为化合物TH-Z80与HsFPPS的晶体结构示意图(pymol作图)。
从图中可以明显看到，TH-Z80类双膦酸类化合物与FPPS结合的主要驱动力在于双膦酸与金属离子的螯合，同时其长碳链处于一个疏水腔内，苯并咪唑中苯环及酚醚与FPPS作用不明显，这一结果表明在苯并咪唑中苯环引入N杂原子对化合物与FPPS的结合活性影响不显著。在N-杂苯并咪唑化合物侧链引入长碳链，增强了其与FPPS的疏水作用，提高化合物与FPPS的结合力。
图34为化合物TH-Z82与PvGGPPS的晶体结构示意图(pymol作图)。
从图中可以明显看到，与TH-Z80和HsFPPS的晶体结构相类似，TH-Z82类双膦酸类化合物与PvGGPPS结合的主要驱动力在于双膦酸与金属离子的螯合及其长碳链的疏水作用，苯并咪唑中苯环及酚醚与PvGGPPS作用也不显著，这一结果表明在苯并咪唑中苯环引入N杂原子对化合物与PvGGPPS也的结合活性影响不显著。在N-杂苯并咪唑化合物侧链引入长碳链，增强了其与GGPPS的疏水作用，提高化合物与GGPPS的结合力。
根据晶体结构(计算结果)显示，FPPS、GGPPS都存在在疏水腔，所以侧链上的烷氧基可以被其它疏水基团代替。因而我们同时要求保护其它疏水性基团取代的N杂苯并咪唑类化合物。
实施例16：辛伐他汀，TH-Z80，TH-Z93和TH-Z145对PR8流感病毒的作用。
方法：5微克的PR8流感病毒中的血球凝集素蛋白(HA1)分别加入到磷酸盐缓冲液，20微克的辛伐他汀，20微克的TH-Z80，20微克的TH-Z93和20微克的TH-Z145中。并在第0天，第14天和 第21天给小鼠肌肉注射免疫。第28天给小鼠鼻粘膜接种PR8病毒。之后每天称量小鼠的体重，并观测小鼠的死亡。
结果：如图35所示，没有加入辛伐他汀，TH-Z80，TH-Z93和TH-Z145的小鼠在接种病毒后，体重一直都在下降，并在5到10天内死亡。而加入辛伐他汀，TH-Z80，TH-Z93和TH-Z145的小鼠体重不减轻，也没有死亡的现象。
实施例17：抗PD1抗体联合四种佐剂和鸡卵清蛋白对B16-OVA肿瘤的抑制作用。
方法：向C57B/6小鼠右侧腹股沟皮下接种300000个的肿瘤细胞，接种后第5天，用分别含有100μg的OVA蛋白和100μg的佐剂(即上述四种物质辛伐他汀、TH-Z80、TH-Z93、TH-Z145)的混合物对小鼠尾根部皮下注射，初次免疫后7天加强，加强时候不用佐剂，尾根部皮下注射50μg的抗原，第二次免疫后7天，再次加强，加强50μg的抗原。同时在接种肿瘤后第8天，11天，15天，18天，22天和第25注射100微升抗PD1的抗体。每两天检测小鼠的体重以及肿瘤体积的变化。肿瘤的体积使用公式长*宽*宽/2计算。
结果：如图36所示，抗PD1抗体联合四种佐剂和鸡卵清蛋白对B16-OVA肿瘤显示抑制作用。
实施例18：HMG-CoA合酶抑制剂Hymeglusin在OVA抗体滴度检测中的佐剂活性。
方法：将HMG-CoA合酶抑制剂Hymeglusin配制成10mg/ml的浓度，OVA抗原的浓度也为10mg/ml，化合物和抗原按照1∶1混合。小鼠品系选用C57B/6。每只实验组小鼠肌肉注射20μl含测试化合物和OVA抗原的混合物，即100μg Hymeglusin(10μl)和100μg(10μl)的抗原OVA。对照组小鼠采用10μl PBS与10μl OVA抗原按照1∶1混合进行肌肉注射。免疫后14天小鼠眼眶取血100μl，4℃静置过夜后，分离血清，测定血清中抗OVA的IgG抗体的滴度。
结果：如图37所示，相对于PBS处理的对照组小鼠，在用HMG-CoA合酶抑制剂Hymeglusin作为佐剂处理的小鼠中产生了具有显著高的滴度的IgG抗体，充分证明了能够抑制甲羟戊酸通路中的HMG-CoA还原酶的活性的化合物可以充当免疫原性组合物中的佐剂。
化合物制备实施例
所有的化合物核磁表征数据在Bruker Avance DRX-400光谱仪上进行，化学位移(δ，以ppm表示)以D2O在4.79ppm，CDCl3在7.26ppm，MeOD在3.31ppm作为参照，核磁光谱峰裂分分别以d、t、q、m表示，即双重峰、三重峰、四重峰、多重峰，耦合常数以赫兹为单位。高分辨质谱在Waters Xevo G2 QTof上以ESI为离子源测试完成。
起始物质可自商业来源获得，或用本领域技术人员已知的既定的文献方法制备。
实施例1 TH-Z97系列化合物的制备
TH-Z97(n＝6)((1-羟基-2-(7-正己氧基咪唑并[1，2-a]吡啶-3-基)乙烷-1，1-二基)双膦酸)的制备
Figure PCTCN2016098371-appb-000115
步骤1：将10mmol(1.11g)2-氨基-4-羟基吡啶溶于50mL丙酮，加入30mmol无水碳酸钾，在N2保护下加热回流后，加入12mmol(1.67mL)溴己烷。反应过夜后，将其中的不溶物过滤，有机相旋干，上样，以石油醚/乙酸乙酯分离纯化得到7.8mmol(1.51g)2-己氧基-2-硝基-苯胺(产率：78％)。
步骤2：将5mmol(0.97g)4-己氧基-2-氨基-吡啶与5.5mmol(0.71g)反式-4-氧基-2-丁烯酸乙酯在20mL乙腈中于80度反应过夜后，旋干，上样，以石油醚/乙酸乙酯分离纯化得到4.1mmol(1.13g)2-(7-(正己氧基)咪唑并[1，2-a]哌啶-3-基)乙酸乙酯，产率82％。
步骤3：将3mmol2-(7-(正己氧基)咪唑并[1，2-a]哌啶-3-基)乙酸乙酯在6N HCl中加热回流6h，然后将其旋干再烘干至恒重，得到的粗品直接用于下一步反应。
步骤4：将得到的2-(7-(正己氧基)咪唑并[1，2-a]哌啶-3-基)乙酸与9mmol(0.74g)亚磷酸和6mL的环丁砜在75度溶解后，逐滴加入10.2mmol(1068μL)PCl3后，反应3.5小时，加入1mL水，加热回流2h，冷却，将析出的固体过滤，甲醇超滤3次，将得到的淡黄色固体烘干，恒重后称重为228mg目标产物(产率18％)。
结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.49(d，J1＝7.2Hz 1H)，7.39(s，1H)，6.79-6.75(m，2H)，4.02(t，J＝6.0Hz，2H)，3.47(m，2H)，1.95(m，2H)，1.39-1.25(m，2H)，1.47(m，6H)，0.79(t，J＝6.4Hz，3H)，31p NMR(162MHz，D2O)，δ(ppm)：17.22
化合物TH-Z157、TH-Z158、TH-Z159、TH-Z160、TH-Z97、TH-Z161、TH-Z98、TH-Z162、TH-Z99、TH-Z198和TH-Z163按照类似于上述TH-Z97的合成方法，以相应的溴代烷烃和2-氨基-4-羟基吡啶反应，进行制备。
TH-Z156直接以2-氨基-4-甲氧基吡啶为原料，按照TH-Z97的合成步骤2-4，逐步进行制备。
这些化合物的表征数据如下：
TH-Z98：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.58(d，J＝7.6Hz，1H，7.37(s，1H)，6.86(s，1H)，6.65(d，J＝7.2Hz，1H)，4.12(t，J＝6.4Hz，2H，)，3.57(t，J＝12.0Hz，2H)，1.81(m，2H)，1.48-1.44(m，2H)，1.27(m，8H)，0.79(t，J＝6.4Hz，3H)，31p NMR(162MHz，D2O)，δ(ppm)：17.92
TH-Z99：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.51(d，J＝7.6Hz，1H，7.37(s，1H)，6.87(s，1H)，6.65(d，J＝7.2Hz，1H)，4.12(t，J＝6.4Hz，2H，)，3.55(t，J＝12.0Hz，2H)，1.95(m，2H)，1.46-1.44(m， 2H)，1.27(m，10H)，0.79(t，J＝6.8Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：17.70
TH-Z156：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.60(d，J＝7.6Hz，1H，7.37(s，1H)，6.87(s，1H)，6.65(d，J＝7.6Hz，1H)，3.58(t，J＝12.0Hz，2H)，31P NMR(162MHz，D2O)，δ(ppm)：17.97
TH-Z157：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.59(d，J＝7.2Hz，1H，7.37(s，1H)，6.85(d，J＝1.6Hz，1H)，6.63(dd，J1＝7.6Hz，J2＝1.8Hz，1H)，4.16(q，J＝7.2Hz，2H，)，3.57(t，J＝11.6Hz，2H)，1.41(t，J＝6.8Hz，2H)，31P NMR(162MHz，D2O)，δ(ppm)：18.00
TH-Z158：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.61(d，J＝7.6Hz，1H)，7.37(s，1H)，6.87(s，1H)，6.65(d，J＝7.2Hz，1H)，4.12(t，J＝6.4Hz，2H，)，3.55(t，J＝12.0Hz，2H)，1.95(m，2H)，0.97(t，J＝6.8Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：16.76
TH-Z159：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.64(d，J＝7.6Hz，1H)，7.55(s，1H)，6.99(d，J＝7.6Hz，1H)，6.96(s，1H)，4.13(t，J＝6.4Hz，2H)，3.61(t，J＝12.0Hz，2H)，1.80(m，2H)，1.49(m，2H)，0.96(t，J＝7.2Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：16.56
TH-Z160：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.61(d，J＝7.2Hz，1H)，7.38(s，1H)，6.88(d，J＝2.1Hz，1H)，6.66(dd，J1＝7.6Hz，J2＝2.2Hz，1H)，4.14(t，J＝6.4Hz，2H)，3.59(t，J＝11.7Hz，2H)，1.83(m，2H)，1.50-1.34(m，4H)，0.92(t，J＝7.2Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：17.18
TH-Z161：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.46(d，J＝7.6Hz，1H)，7.38(s，1H)，6.89(s，1H)，6.68(d，J＝8.0Hz，1H)，4.13(t，J＝6.4Hz，2H)，3.54(t，J＝11.7Hz，2H)，1.82(m，2H)，1.48-1.29(m，6H)，0.92(t，J＝7.2Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：17.52
TH-Z162：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.53(d，J＝7.6Hz，1H)，7.40(s，1H)，6.90(s，1H)，6.68(d，J＝7.2Hz，1H)，4.15(t，J＝6.4Hz，2H)，3.58(t，J＝11.6Hz，2H)，1.83(m，2H)，1.48-1.28(m，12H)，0.86(t，J＝6.0Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：17.74
TH-Z163：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.63(d，J＝7.2Hz，1H)，7.40(s，1H)，6.89(s，1H)，6.67(d，J＝6.4Hz，1H)，4.15(t，J＝6.4Hz，2H)，3.60(t，J＝11.6Hz，2H)，1.83(m，2H)，1.48-1.28(m，18H)，0.86(t，J＝6.0Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：18.05
TH-Z198：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.39(d，J＝6.8Hz，1H)，7.27(s，1H)，6.77(s，1H)，6.56(d，J＝6.4Hz，1H)，4.02(t，J＝6.4Hz，2H)，3.45(t，J＝11.0Hz，2H)，1.71(brs，2H)，1.36-1.16(m，16H)，0.73(brs，3H)，31P NMR(162MHz，D2O)，δ(ppm)：17.67
实施例2
(((4-(己基氧基)吡啶-2-基)氨基)亚甲基)双膦酸(TH-Z93)的制备
Figure PCTCN2016098371-appb-000116
第一步：取10mmol的2-氨基4-羟基吡啶溶于100mL的乙腈中，加入20mmol的碳酸钾和12mmol的1-溴正己烷，氮气保护，在60℃下反应过夜，TLC监测至原料完全反应，反应液冷却后，过滤掉碳酸钾，减压浓缩，石油醚/乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率75％。
第二步：取第一步产物6mmol溶于20mL甲苯，加入15mmol亚磷酸二乙酯和24mmol原甲酸三乙酯，120℃反应10h，石油醚/乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，减压蒸掉甲苯，乙酸乙酯∶甲醇＝30∶1硅胶(200-300目)柱分离得纯产品，产率78％。
第三步：在上一步的产物中加入6N的盐酸50mL，在100℃下反应10小时，减压蒸馏掉盐酸，粗产品先经丙酮超声洗涤1次，再甲醇超声洗涤3次得纯产品，产率95％。
TH-Z93表征数据：
1H NMR(400MHz，D2O)：δ7.62-7.63(d，1H，J＝8.0Hz)，6.04-6.1(m，2H)，4.01-4.04(t，2H，J＝6.4Hz)，1.64-1.69(m，2H)，1.24-1.35(m，6H)，0.77-0.80(t，3H，J＝6.4Hz).31P NMR(162MHz，D2O)：δ15.12.
HRMS(ESI)：C12H22N2O7P2计算值：369.0980；实测值：369.0969.
实施例3
Figure PCTCN2016098371-appb-000117
(((4-(4，5，6，7-四氢-2H-吲唑-2-基)吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000118
实施例17以类似于实施例16的方法制备，不同的是第一步采用了如下条件制备：在氩气保护下，将10mmol的4-溴-2-氨基吡啶溶解在100mL无水DMF中，加入20mmol碳酸铯和12mmol的4，5，6，7-四氢吲唑，加热到120℃反应8小时，过滤掉碳酸铯，减压蒸掉溶剂，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率73％。
1H NMR(400MHz，D2O)：δ8.06(s，1H)，7.91-7.92(m，2H)，6.77(s，1H)，3.81(s，1H)，3.65(q，2H，J＝6.8Hz)，2.60-2.69(m，4H)，1.76-1.84(m，4H)，1.18(t，3H，J＝7.2Hz).31p NMR(162MHz，D2O)：δ14.92.
HRMS(ESI)：C13H18N4O6P2计算值：389.0780；实测值：389.0783
实施例4
Figure PCTCN2016098371-appb-000119
(((4-((2-氨基甲酰基吡啶-4-基)甲氧基)吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000120
第一步：将10mol 2-氰基-4-甲基吡啶溶于100mL四氯化碳中，分别加入11mol过氧化苯甲酰和11mol溴代琥珀酰亚胺，77℃反应4小时，减压蒸掉溶剂，石油醚∶乙酸乙酯＝3∶1硅胶(200-300目)柱分离得纯产品，产率67％。
第二步：将上一步产物6mmol溶于50mL溶剂中，加入12mmol的碳酸钾和7.2mmol的4-羟基-2-氨基吡啶，室温反应过夜，过滤掉碳酸钾固体，减压蒸掉乙腈溶剂，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率43％。
第三步：将上一步产物2.5mmol和6mmol的亚磷酸二乙酯和10mmol的原甲酸三乙酯溶于20mL 的甲苯中，120℃反应过夜，减压蒸掉多余的溶剂，乙酸乙酯∶甲醇＝30∶1硅胶(200-300目)柱分离得纯产品，产率82％。
第四步：将第三步的产物2.0mmol溶于10mL DMSO中，加入20mL30％双氧水和3.0mmol的碳酸钾，常温反应4小时，乙酸乙酯萃取，有机层用硫酸镁干燥，过滤并减压蒸掉乙酸乙酯，未经分离直接做下一步反应。
第五步同实施例16中的第三步，两步总反应产率78％。
1H NMR(400MHz，D2O)：δ8.71(bs，1H)，8.09(bs，2H)，7.67(bs，1H)，6.58(bs，2H)，5.39(bs，1H)，3.86-3.95(t，1H，17.2Hz).31P NMR(162MHz，D2O)：δ12.19.
HRMS(ESI)：C13H16N4O8P2计算值：419.0522；实测值：419.0518
实施例5
Figure PCTCN2016098371-appb-000121
(((4-羟基吡啶-2-基)氨基)亚甲基)双膦酸
实施例19以类似于实施例16的方法制备，不同的是第一步采用了苄溴替代了己基溴作为原料并在室温下反应。
1H NMR(400MHz，D2O)：δ7.41(s，1H)，5.88(s，1H)，5.69(s，1H)，3.51-3.60(t，2H，J＝19.2Hz)31P NMR(162MHz，D2O)：δ15.27.
HRMS(ESI)：C6H10N2O7P2计算值：284.0051；实测值：284.0059
实施例6
Figure PCTCN2016098371-appb-000122
(((4-(苄基氧基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例20以类似于实施例19的方法制备，不同的是第三步采用了三甲基溴硅烷替代了6N盐酸，室温下在二氯甲烷中进行水解。
1H NMR(400MHz，D2O)：δ7.70-7.71(d，1H，J＝6.0Hz)，7.39-7.50(m，5H)，6.19-6.22(m，2H)，5.18(s，2H)，3.76(bs，1H).31P NMR(162MHz，D2O)：δ15.33.
HRMS(ESI)：C13H16N2O7P2计算值：375.0511；实测值：375.0519.
实施例7
Figure PCTCN2016098371-appb-000123
(((6-(己基氧基)吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000124
实施例21以类似于实施例16的方法制备，第一步除外。
第一步：在室温条件下，10mmol6-羟基-4-氨基吡啶溶于100mL二氧六环中，加入12mmol的三苯基磷和偶氮二甲酸二乙酯，缓慢加入20mmol的正己醇，反应4小时后，反应液减压蒸馏掉溶剂，石油醚∶乙酸乙酯＝2∶1硅胶(200-300目)柱分离得纯产品，产率85％。
其他反应步骤同实施例16。
1H NMR(400MHz，D2O)：δ7.62(d，1H，J＝2.4Hz)，7.43(dd，1H，J1＝9.2Hz，J2＝2.4Hz)，7.62(d，1H，J＝9.2Hz)，4.03(t，2H，J＝6.4Hz)，3.96(t，1H，J＝19.2Hz)，1.71-1.78(m，2H)，1.43-1.46(m，2H)，1.33-1.34(m，4H)，0.89(t，3H，J＝6.8Hz).31P NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C12H22N2O7P2计算值：369.0980；实测值：369.0973.
实施例8
Figure PCTCN2016098371-appb-000125
(((4-溴吡啶-2-基)氨基)亚甲基)双膦酸
实施例22以类似于实施例16的方法制备，不同的是第一步采用了2-氨基-4-溴吡啶作为原料。
1H NMR(400MHz，D2O)：δ7.67(s，1H)，7.09-7.13(m，1H)，6.84-6.86(m，1H)，4.06(t，1H，J＝19.2Hz).31P NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C6H9BrN2O6P2计算值：347.9143；实测值：347.9145.
实施例9
Figure PCTCN2016098371-appb-000126
(((4-(甲硫基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例23以类似于实施例16的方法制备，不同的是第一步采用了2-氨基-4-硫基吡啶和碘甲烷作为原料。
1H NMR(400MHz，D2O)：δ7.36(s，1H)，6.61(s，1H)，6.51(s，1H)，3.82(t，1H，J＝16.0Hz)，2.46(s，3H).31P NMR(162MHz，D2O)：δ13.0.
HRMS(ESI)：C7H12N2SO6P2计算值：314.9969；实测值：314.9969.
实施例10
Figure PCTCN2016098371-appb-000127
(((4-(己基氧基)喹啉-2-基)氨基)亚甲基)双膦酸
实施例24以类似于实施例16的方法制备，不同的是第一步采用了2-氨基-4-羟基喹啉作为原料。
1H NMR(400MHz，D2O)：δ7.98(d，1H，J＝8.0Hz)，7.58(d，1H，J＝8.0Hz)，7.51(t，1H，J＝8.0Hz)，7.21(t，1H，J＝8.0Hz)，6.0(s，1H)，4.1(t，2H，J＝6.4Hz)，3.80(t，1H，J＝20.0Hz)，1.90-2.04(m，2H)，1.52-1.54(m，2H)，1.37-1.38(m，2H)，1.23-1.25(m，4H)，0.92(s，3H，J＝6.4Hz).31P NMR(162MHz，D2O)：δ13.1.
HRMS(ESI)：C16H24N2O7P2计算值：419.1137；实测值：419.1145.
实施例11
Figure PCTCN2016098371-appb-000128
(((4-(4-甲基苯乙氧基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例25以类似于实施例16的方法制备，不同的是第一步采用了1-(2-溴-乙基)-4-甲基-苯作为原 料。
1H NMR(400MHz，D2O)：δ7.49(d，1H，J＝6.0Hz)，7.20-7.27(m，5H)，6.34(s，1H)，6.23(d，1H，J＝6.0Hz)，4.37(t，1H，J＝6.0Hz)，3.80(t，1H，J＝18.4Hz)，3.07(t，J＝5.6Hz)，2.30(s，3H)，2.21(s，3H).31P NMR(162MHz，D2O)：δ13.1.
HRMS(ESI)：C15H20N2O7P2计算值：403.0824；实测值：403.0829.
实施例12
Figure PCTCN2016098371-appb-000129
(((5-溴-4-(己基氧基)吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000130
实施例26以类似于实施例16的方法制备，不同的是对实施例16中间体4-己氧基-2-氨基吡啶进行了一步溴代反应。操作步骤如下：将10mmol的4-己氧基-2-氨基吡啶溶于30mL冰醋酸中，室温下加入11mmol溴单质，加热到70℃反应4小时，然后用饱和硫代硫酸钠淬灭反应，用乙酸乙酯萃取，有机层用无水硫酸镁干燥，减压浓缩，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率73％。
1H NMR(400MHz，D2O)：δ7.75(s，1H)，6.10(s，1H)，4.06(t，2H，J＝8.0Hz)，3.68(bs，1H)，1.66-1.73(m，2H)，1.34-1.35(m，2H)，1.22(s，4H)，0.76(t，3H，J＝6.8Hz).31P NMR(162MHz，D2O)：δ15.5.
HRMS(ESI)：C12H21BrN2O7P2计算值：448.0032；实测值：448.0033.
实施例13
Figure PCTCN2016098371-appb-000131
(((3-(己基氧基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例27以类似于实施例16的方法制备，不同的是第一步采用了2-氨基-3-羟基吡啶作为原料。
1H NMR(400MHz，D2O)：δ7.39(d，1H，J＝6.4Hz)，7.29(d，1H，J＝8.0Hz)，6.82(t，J＝7.2Hz)，4.18(t，2H，J＝6.8Hz)，4.10(t，1H，J＝20.4Hz)，1.81-1.88(m，2H)，1.44(s，2H)，1.32(s，4H)，0.87(t，3H，J＝6.8Hz).31P NMR(162MHz，D2O)：δ11.1.
HRMS(ESI)：C12H22N2O7P2计算值：369.0980；实测值：369.0978.
实施例14
Figure PCTCN2016098371-appb-000132
(((4-己基吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000133
第一步：将10mmol的2-氨基-4-溴吡啶溶于100mL无水四氢呋喃中，加入10ml三乙胺和15mmol1-己炔，0.5mmol碘化亚铜和1mmol三苯基磷氯化钯，在氩气保护下60℃反应4小时，减压蒸馏掉溶剂，石油醚∶乙酸乙酯＝3∶1硅胶(200-300目)柱分离得纯产品，产率88％。
第二步：将第一步的产物8.8mmol溶解在50mL乙酸中，加入200mg氢氧化钯，氢气还原反应24小时，氢氧化钯用硅藻土过滤掉，减压蒸掉乙酸，石油醚∶乙酸乙酯＝2∶1硅胶(200-300目)柱分离得纯产品，产率95％。
其他反应步骤类似实施例16中的第二步和第三步。
1H NMR(400MHz，D2O)：δ7.63(d，1H，J＝6.0Hz)，6.90(s，1H)，6.73(d，J＝5.6Hz)，4.04(t，1H，J＝19.6Hz)，2.64(s，2H)，1.62(s，2H)，1.28(s，6H)，0.84(s，3H).31P NMR(81MHz，D2O)：δ11.7.
HRMS(ESI)：C12H22N2O6P2计算值：353.1031；实测值：353.1037
实施例15
Figure PCTCN2016098371-appb-000134
(((4-(辛-1-炔-1-基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例29以类似于实施例28的方法制备，不同的是第一步采用了1-辛炔作为原料。不经第二步 氢气还原直接进行第三步反应。
1H NMR(400MHz，D2O)：δ7.64(d，1H，J＝5.6Hz)，7.03(s，1H)，6.63(d，J＝6.0Hz)，4.06(t，2H，J＝19.6Hz)，2.45(s，2H)，1.56-1.58(m，2H)，1.41(s，2H)，1.29(s，4H)，0.86(s，3H).31P NMR(162MHz，D2O)：δ11.6.
HRMS(ESI)：C14H22N2O6P2计算值：377.1031；实测值：377.1033.
实施例16
Figure PCTCN2016098371-appb-000135
(((4-(辛基氨基)吡啶-2-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000136
第一步：将10mmol2，4-二氨基吡啶溶解在100mL吡啶中，冷却到0℃，缓慢在反应液中滴加辛酰氯，反应4小时后，加压蒸掉溶剂，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率37％。
第二步：将第一步的产物3mmol溶于50mL无水四氢呋喃中，冷却到0℃，缓慢加入5mmol的四氢铝锂，继续反应2小时，用饱和氯化铵淬灭反应，150mL乙酸乙酯萃取，有机相用无水硫酸镁干燥，减压浓缩，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率89％。
第三步和第四步反应和实施例16中的第二步和第三步类似。
1H NMR(400MHz，D2O)：δ7.72(s，1H)，7.62(s，1H)，7.05(s，1H)，4.11(t，2H，J＝6.8Hz)，3.55(t，1H，J＝14.4Hz)，1.76-1.79(m，2H)，1.43-1.45(m，2H)，1.29-1.33(m，8H)，0.89(t，3H，J＝6.8Hz).31P NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C14H27N3O6P2计算值：396.1463；实测值：396.1471.
实施例17
Figure PCTCN2016098371-appb-000137
(((5-(辛基氧基)吡啶-2-基)氨基)亚甲基)双膦酸
实施例31以类似于实施例18的方法制备，不同的是第一步采用了5-羟基-2-氨基吡啶和正辛醇作为原料。
1H NMR(400MHz，D2O)：δ7.76-7.71(M，2H)，6.89-6.91(m，1H)，4.02(t，2H，J＝8.0Hz)，3.88(t，1H，J＝20.0Hz)，1.78-1.85(m，2H)，1.45-1.49(m，2H)，1.34-1.36(m，8H)，0.91(t，3H，J＝6.4Hz).31P NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C14H26N2O7P2计算值：397.1293；实测值：397.1298.
实施例18
Figure PCTCN2016098371-appb-000138
(((4-辛基吡啶-2-基)氨基)亚甲基)双膦酸
实施例32以类似于实施例18的方法制备，不同的是第一步采用了1-辛炔作为原料。
1H NMR(400MHz，D2O)：δ7.65(d，1H，J＝6.0Hz)，6.92(s，1H)，6.75(d，J＝5.6Hz)，4.06(t，1H，J＝19.6Hz)，2.65(s，2H)，1.64(s，2H)，1.29(s，10H)，0.85(s，3H).31P NMR(162MHz，D2O)：δ11.8.
HRMS(ESI)：C14H26N2O6P2计算值：381.1316；实测值：381.1320.
实施例19
Figure PCTCN2016098371-appb-000139
(((6-甲基-3-(辛基氧基)吡啶-2-基)氨基)亚甲基)双磷酸
实施例33以类似于实施例17的方法制备，不同的是第一步采用了2-氨基-3-羟基-6-甲基喹啉作为原料。
1H NMR(400MHz，D2O)：δ7.37(d，1H，J＝7.2Hz)，7.21(d，1H，J＝7.2Hz)，4.02(t，2H，J＝8.0Hz)，3.88(t，1H，J＝20.0Hz)，2.43(s，3H)，1.78-1.85(m，2H)，1.45-1.49(m，2H)，1.34-1.36(m，12H)，0.91(t，3H，J＝6.4Hz).31P NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C15H29N2O7P2计算值：411.1421；实测值：411.1417.
实施例20
Figure PCTCN2016098371-appb-000140
(((4-癸基吡啶-2-基)氨基)亚甲基)双膦酸
此化合物合成类似于实施例32的方法制备，不同的是第一步采用了1-癸炔作为原料。
1H NMR(400MHz，D2O)：δ7.63(d，1H，J＝6.0Hz)，6.89(s，1H)，6.72(d，J＝5.6Hz)，4.02(t，1H，J＝19.6Hz)，2.61(s，2H)，1.61(s，2H)，1.27(s，14H)，0.82(s，3H).31P NMR(162MHz，D2O)：δ11.5.
HRMS(ESI)：C16H30N2O6P2计算值：410.1657；实测值：410.1663.
实施例21
Figure PCTCN2016098371-appb-000141
(((5-(辛基氧基)吡啶-3-基)氨基)亚甲基)双膦酸
Figure PCTCN2016098371-appb-000142
第一步：10mmol的5-羟基-3-羧基吡啶溶于100mL甲醇中，缓慢加入5mL氯化亚砜，在60℃反应4小时，减压蒸掉溶剂，不经提纯进行下一步反应。
第二步：将第一步产物溶于150mL乙腈中，加入30mmol的碳酸钾，60℃反应过夜，滤掉固体，减压蒸掉溶剂，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，两步产率73％。
第三步：将7.3mmol产物溶解于100mL二氧六环中，加入4N的NaOH20mL，室温下反应8小时，反应完毕，用稀盐酸调节PH到6，用200mL乙酸乙酯萃取，有机相用无水硫酸镁干燥，减压蒸掉溶剂，粗产品未经提纯直接进行下一步的反应。
第四步：将上一步的粗产物溶于150mL二氯甲烷中，在0℃加入10mL三乙胺，缓慢滴加8mmol叠氮磷酸二苯酯，反应4小时至室温，减压蒸掉溶剂，未经分离直接进行下一步反应。
第五步：在耐压管中，将上一步的粗产物溶于100mL的四氢呋喃中，加入10mL苯甲醇，90℃ 反应8小时，减压蒸掉溶剂，石油醚∶乙酸乙酯＝3∶1硅胶(200-300目)柱分离得纯产品，三步总产率78％。
第六步：将上一步的产物溶于100mL甲醇中，加入500mg的钯碳，室温下用氢气还原24小时，硅藻土滤掉钯碳，滤液减压浓缩，石油醚∶乙酸乙酯＝1∶1硅胶(200-300目)柱分离得纯产品，产率97％。
剩余的两步反应和实施例16中的步骤2和3相同。
1H NMR(400MHz，D2O)：δ7.62(s，1H)，7.42(s，1H)，6.73(s，1H)，4.11(t，2H，J＝6.8Hz)，3.55(t，1H，J＝14.4Hz)，1.76-1.79(m，2H)，1.43-1.45(m，2H)，1.29-1.33(m，8H)，0.89(t，3H，J＝6.8Hz).31p NMR(162MHz，D2O)：δ13.5.
HRMS(ESI)：C14H26N2O7P2计算值：397.1293；实测值：397.1298.
实施例22
TH-Z144制备：
Figure PCTCN2016098371-appb-000143
(2-(3-(己基氧基)苯基)乙烷-1，1二基)双膦酸
Figure PCTCN2016098371-appb-000144
第一步：将原料3-羟基苯甲酸甲酯10mmol溶于100mL乙腈中，加入20mmol碳酸钾和12mmol的1-溴正己烷，氮气保护，在60℃下反应过夜，反应液冷却至室温，过滤掉碳酸钾，减压浓缩，粗产品未经柱分离进行下一步反应。
第二步：将上一步的产物在0℃下溶于200mL无水四氢呋喃，分批加入20mmol的四氢铝锂，继续0℃反应4小时，薄层色谱检测原料几乎完全反应，用饱和氯化铵淬灭反应，用200mL乙酸乙酯萃取，有机相用无水硫酸镁干燥，石油醚∶乙酸乙酯＝8∶1硅胶(200-300目)柱分离得纯产品，两步产率90％。
第三步：将上步的产物溶于100mL无水二氯甲烷中，在0℃下加入10.8mmol的三苯基磷，然后缓慢滴加10.8mmol的四溴化碳的二氯甲烷溶液，继续反应4小时，TLC检测原料完全反应，减压蒸掉二氯甲烷，石油醚∶乙酸乙酯＝30∶1硅胶(200-300目)柱分离得纯产品，产率95％。
第四步：将亚甲基二磷酸四乙酯6mmol溶于100mL无水四氢呋喃中，在0℃下加入7mmol的钠 氢，待反应30分钟后，将上一步的产物8mmol溶于10mL无水四氢呋喃滴加到上述混合液中，反应至室温，波层色谱检测原料大部分反应，加入100mL饱和氯化钠，用200mL乙酸乙酯萃取，有机相用无水硫酸镁干燥，石油醚∶乙酸乙酯＝3∶1硅胶(200-300目)柱分离得纯产品，产率75％。
第五步：将上步产物中加入6N的盐酸50mL，在100℃下反应10小时，减压蒸馏掉盐酸，粗产品经丙酮超声洗涤3次得纯产品，产率93％。
1H NMR(400MHz，MeOD)：δ7.13-7.17(t，1H，J＝8.0Hz)，6.88-6.90(m，2H)，6.71-6.74(dd，J1＝8.0Hz，J2＝2.4Hz)，3.93-3.97(t，2H，J＝6.4Hz)，3.14-3.24(td，2H，J1＝16.8Hz，J2＝6.0Hz)，2.43-2.58(m，1H)，1.72-1.79(m，2H)，1.44-1.51(m，2H)，1.34-1.37(m，4H)，0.91-0.94(t，3H，J＝8.0Hz).31P NMR(162MHz，MeOD)：δ21.78.HRMS(ESI)：C16H28O7P2计算值：367.1080；实测值：367.1081
实施例23
TH-Z145的制备
Figure PCTCN2016098371-appb-000145
(2-(3-(辛基氧基)苯基)乙烷-1，1二基)双膦酸
TH-Z145以类似于H-Z144的方法制备，不同的是第一步采用了1-溴正辛烷替代了1-溴正己烷作为原料并在60℃反应。
1H NMR(400MHz，MeOD)：δ7.13-7.18(t，1H，J＝8.0Hz)，6.86-6.88(m，2H)，6.70-6.73(dd，J1＝8.0Hz，J2＝2.4Hz)，3.90-3.94(t，2H，J＝6.4Hz)，3.12-3.22(td，2H，J1＝16.8Hz，J2＝6.0Hz)，2.40-2.55(m，1H)，1.70-1.77(m，2H)，1.42-1.49(m，2H)，1.33-1.36(m，8H)，0.90-0.93(t，3H，J＝8.0Hz).31P NMR(162MHz，MeOD)：δ21.67.HRMS(ESI)：C14H24O7P2计算值：395.1393；实测值：395.1387.
实施例24
TH-Z80的制备：按照下列合成路线制备TH-Z80。
Figure PCTCN2016098371-appb-000146
步骤1：将10mmol(1.54g)3-硝基-4-氨基苯酚溶于50mL丙酮，加入30mmol无水碳酸钾，在N2保护下加热回流后，加入12mmol(1.67mL)溴己烷。反应过夜后，将其中的不溶物过滤，有机相旋干，上样，以石油醚/乙酸乙酯分离纯化得到7.3mmol(1.74g)4-己氧基-2-硝基-苯胺(产率：73％)。
步骤2：将5mmol(1.19g)4-己氧基-2-硝基-苯胺与60mmol(9.69mL)溴乙酸叔丁酯，7.5mmol(1.04g)无水碳酸钾在N2保护下于110度反应12h，过滤反应液中的不溶物，旋干，上样，以石油醚/乙酸乙酯分离纯化得到2.1mmol(0.74g)(4-(己氧基)-2-硝基苯基)甘氨酸叔丁酯，产率42％。
步骤3：将2mmol(0.70g)(4-(己氧基)-2-硝基苯基)甘氨酸叔丁酯溶于10mL乙醇中，加入0.1g 5％的钯碳，在H2下反应4h。硅藻土过滤，有机相旋干，得到的粗品溶于10mL乙二醇单甲醚，加入7mmol(0.72g)的醋酸甲脒，加热回流4h后冷却，将反应液旋干，上样，过柱得到0.7mmol(0.23g)2-5-(己氧基)-1H-苯并[d]咪唑-1基-)乙酸叔丁酯，两步总产率35％。
步骤4：将0.6mmol(0.2g)2-5-(己氧基)-1H-苯并[d]咪唑-1基-)乙酸叔丁酯在6N HCl中加热回流6h，然后将其旋干再烘干至恒重，得到的粗品直接用于下一步反应。
步骤5：将得到的2-5-(己基氧基)-1H-苯并[d]咪唑-1基-)乙酸与1.8mmol(0.15g)亚磷酸和1mL的环丁砜在75度溶解后，逐滴加入2mmol(178μL)PCl3后，反应3.5小时，加入1mL水，加热回流2h，冷却，将析出的固体过滤，甲醇超滤3次，将得到的淡黄色固体烘干，恒重后称重为38mg目标产物(产率15％)。
结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.19(s，1H)，7.76(d，J＝8.9Hz，1H)，7.26(d，J＝2.2Hz，1H)，7.04(dd，J1＝8.9Hz，J2＝2.3Hz，1H，)，4.77(m，2H)，4.14(t，J＝6.6Hz，2H，)，1.79(m，2H)，1.47(m，2H)，1.33(m，4H)，0.88(t，J＝7.1Hz，3H)，13C NMR(100MHz，D2O)，δ(ppm)：154.18，147.63，142.09，131.10，113.20，112.78，102.72，77.79，76.49，75.19，69.96，50.51，30.80，28.40，24.91，21.94，13.32，31P NMR(162MHz，D2O)，δ(ppm)：15.67
参照上述制备方法，本发明还分别制备得到了n＝1、2、3、4、5、7、8、9、10、11和12时对应的下式化合物
Figure PCTCN2016098371-appb-000147
依次记为TH-Z79、TH-Z148、TH-Z149、TH-Z150、TH-Z151、TH-Z152、TH-Z81、TH-Z153、TH-Z82、TH-Z154和TH-Z 155。
将TH-Z80制备中的步骤2中的4-己氧基-2-硝基-苯胺原料用4-羟基2-硝基苯胺替代，依次进行步骤2、3、4和5，得到BPH-266(R1、R2、R4和R5＝H，m＝1，X＝OH，M＝H，n＝0时对应的式I所示化合物)。
除TH-Z79直接以2-氨基-4-甲氧基苯胺为原料外，其它TH-Z80系列化合物按照TH-Z80的合成步骤合成相应化合物。
TH-Z80系列化合物表征数据如下：
TH-Z79：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.32(s，1H)，7.70(d，J＝7.6Hz，1H)，7.19(s，1H)，6.97(d，J＝7.6Hz，1H)，4.70(s，2H)，3.83(t，J＝7.0Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：15.33
TH-Z81：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.40(s，1H)，7.78(d，J＝9.0Hz，1H)，7.27(s，1H)，7.05(d，J＝9.0Hz，1H)，4.70(s，2H)，4.16(t，J＝6.4Hz，2H)，1.81(m，2H)，1.47(m，2H)，1.29(m，8H)，0.87(t，J＝6.8Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.60
TH-Z82：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.40(s，1H)，7.78(d，J＝9.0Hz，1H)，7.27(s，1H)，7.05(d，J＝9.0Hz，1H)，4.72(s，2H)，4.16(t，J＝6.4Hz，2H)，1.81(m，2H)，1.47(m，2H)，1.28(m，8H)，0.86(t，J＝6.8Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.58
TH-Z148：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.65(s，1H)，7.82(d，J＝9.0Hz，1H)，7.19(s，1H)，7.13(d，J＝9.0Hz，1H)，4.84(s，2H)，4.17(q，J＝6.7Hz，2H)，1.43(t，J＝6.8Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：14.82
TH-Z149：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.90(s，1H)，7.86(d，J＝9.0Hz，1H)，7.27(s，1H)，7.05(d，J＝9.0Hz，1H)，4.70(s，2H)，4.16(t，J＝6.4Hz，2H)，1.81(m，2H)，1.47(m，2H)，1.29(m，8H)，0.87(t，J＝6.8Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：15.60
TH-Z150：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.39(s，1H)，7.77(d，J＝8.9Hz，1H)，7.28(s，1H)，7.05(d，J＝8.4Hz，1H)，4.74(s，2H)，4.15(t，J＝6.4Hz，2H)，1.78(m，2H)，1.48(m，2H)，0.96(t，J＝7.2Hz，3H)，31P NMR(162MHz，D2O)，δ(ppm)：15.60
TH-Z151：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.37(s，1H)，7.76(d，J＝8.9Hz，1H)，7.26(s，1H)，7.05(d，J＝9.0Hz，1H)，4.73(s，2H)，4.14(t，J＝6.5Hz，2H)，1.79(m，2H)，1.46(m，2H)，1.36(m，2H)，0.90(t，J＝7.2Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：14.02
TH-Z152：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.34(s，1H)，7.74(d，J＝9.0Hz，1H)，7.27(s，1H)，7.06(d，J＝9.0Hz，1H)，4.72(s，2H)，4.14(t，J＝6.4Hz，2H)，1.80(m，2H)，1.47(m，2H)，1.31(m，8H)，0.86(t，J＝6.4Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.32
TH-Z153：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.40(s，1H)，7.78(d，J＝9.0Hz，1H)，7.27(s，1H)，7.05(d，J＝9.0Hz，1H)，4.75(s，2H)，4.15(t，J＝5.6Hz，2H)，1.80(m，2H)，1.47(m，2H)，1.29(m，10H)，0.87(t，J＝6.2Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.41
TH-Z154：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.32(s，1H)，7.76(d，J＝8.8Hz，1H)，7.29 (s，1H)，7.07(d，J＝8.9Hz，1H)，4.73(s，2H)，4.16(t，J＝6.4Hz，2H)，1.81(m，2H)，1.48(m，2H)，1.28(m，14H)，0.85(t，J＝6.4Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.33
TH-Z155：结构表征数据：1H NMR(400MHz，D2O)，δ(ppm)：8.37(s，1H)，7.78(d，J＝8.9Hz，1H)，7.28(s，1H)，7.05(d，J＝9.0Hz，1H)，4.72(s，2H)，4.16(t，J＝6.4Hz，2H)，1.81(m，2H)，1.47(m，2H)，1.29(m，16H)，0.86(t，J＝6.8Hz，3H)31P NMR(162MHz，D2O)，δ(ppm)：15.39
本申请参考了各种发行的专利、公开的专利申请、期刊文章和其他出版物，将所有这些引入本申请作为参考。若任何引入的参考文献和本说明书有冲突，则以本说明书为准。此外，落入现有技术范围的本发明的任何具体实施方案可以明确地从任何一个或多个权利要求中排除。因为所述实施方案被认为是本领域技术人员已知的，它们可以被排除，即使所述排除没有在本申请中明确列出。本发明的任何具体实施方案可从任何权利要求中以任何理由排除，不管是否与现有技术的存在有关。
仅使用常规实验，本领域技术人员会认识到或者能够确定对本申请所述的具体实施方案的许多等价形式。本申请所述的本发明实施方案的范围不意在限制为上述说明书，而是如随附的权利要求所列出的那样。本领域技术人员会理解，可实现本说明书的各种变化和修饰，而不背离如权利要求所定义的本发明的主旨或范围。

Claims (10)

  1. 一种免疫原性组合物，所述组合物包含佐剂，所述佐剂选自：
    1)硫解酶抑制剂；
    2)HMG-CoA合酶抑制剂；
    3)HMG-CoA还原酶抑制剂；
    4)甲羟戊酸激酶抑制剂；
    5)磷酸甲羟戊酸激酶抑制剂；
    6)甲羟戊酸-5-焦磷酸脱羧酶抑制剂；
    7)异戊烯焦磷酸异构酶抑制剂；
    8)法尼基焦磷酸合酶抑制剂；
    9)香叶香叶酰基焦磷酸合酶抑制剂；和
    10)香叶香叶酰基转移酶(I，II)抑制剂。
  2. 权利要求1的免疫原性组合物，其中
    所述法尼基焦磷酸合酶抑制剂为双膦酸化合物或其可药用盐、酯、前药、溶剂化物；
    或
    所述法尼基焦磷酸合酶抑制剂为式I所示化合物或其药学上可接受的盐或水合物：
    Figure PCTCN2016098371-appb-100001
    上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
    X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6，R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
    m＝1-6的整数；
    或
    所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100002
    其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环 烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等；
    或
    所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100003
    其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素；
    或
    所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100004
    其中
    R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
    R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
    R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    或
    所述法尼基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100005
    其中
    R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
    R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
  3. 权利要求1的免疫原性组合物，其中所述香叶香叶酰基焦磷酸合酶抑制剂为下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100006
    其中
    R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
    R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
  4. 如前所述的硫解酶抑制剂、HMG-CoA合酶抑制剂、HMG-CoA还原酶抑制剂、甲羟戊酸激酶抑制剂、磷酸甲羟戊酸激酶抑制剂、甲羟戊酸-5-焦磷酸脱羧酶抑制剂、异戊烯焦磷酸异构酶抑制剂、异戊烯焦磷酸异构酶抑制剂、香叶香叶酰基焦磷酸合酶抑制剂和香叶香叶酰基转移酶(I，II)抑制剂，其用作佐剂。
  5. 如前所述的硫解酶抑制剂、HMG-CoA合酶抑制剂、HMG-CoA还原酶抑制剂、甲羟戊酸激酶抑制剂、磷酸甲羟戊酸激酶抑制剂、甲羟戊酸-5-焦磷酸脱羧酶抑制剂、异戊烯焦磷酸异构酶抑制剂、异戊烯焦磷酸异构酶抑制剂、香叶香叶酰基焦磷酸合酶抑制剂和香叶香叶酰基转移酶(I，II)抑制剂作为佐剂在制备免疫原性组合物中的用途。
  6. 式I所示化合物或其药学上可接受的盐或水合物：
    Figure PCTCN2016098371-appb-100007
    上述式I中，其分子量小于1000，Ar为苯并咪唑类基或氮杂苯并咪唑基；
    X为下述任意一种：氢、羟基、脂肪基、巯基、卤素、烷氧基或烷基；每一个M可独立地为下述任意一种：负电荷、氢、烷基、脂肪基、-(CH2)p-O-CO-R、-(CH2)p-CO-R或阳离子；其中，p＝1-6， R为氢、烷基或芳香基；所述阳离子为Li+、Na+、K+、Ca2+、Mg2+、NH4 +或N(R’)4 +，其中R’为烷基；R6、R7分别独立地选自下述任意一种：氢、羟基、巯基、卤素、氨基、脂肪基或烷基；
    m＝1-6的整数。
  7. 下式的化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100008
    其中R1、R2各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素等；
    其中R3、R4各自选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；X选自氢、羟基、巯基、卤素。
  8. 下式化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100009
    其中R1选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基，所述烷氧基中的烷基任选被芳基、杂芳基或杂环基取代，所述芳基、杂芳基或杂环基任选取代有烷基、氨基甲酰基；
    R2选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R3选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    或者R2与R3与它们所连接的碳原子一起形成芳环或杂芳环；以及
    R4选自氢、烷基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
  9. 下式化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100010
    其中
    R5选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R6选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R7选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
    R8选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
  10. 下式化合物或其可药用盐、酯、前药、溶剂化物：
    Figure PCTCN2016098371-appb-100011
    其中
    R9选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R10选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；
    R11选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基；以及
    R12选自氢、烷基、烯基、炔基、烷氧基、烷基氨基、烷基硫基、卤素、羟基、环烷基、杂环基、芳基和杂芳基。
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