Inhibitors of mevalonate pathway as potent vaccine adjuvants

Abstract

The invention discloses a mevalonic acid pathway inhibitor used as a vaccine adjuvant and application of the mevalonic acid pathway inhibitor. Specifically, the invention provides a method. The inhibitor is an acetoacetyl-CoA transferase inhibitor, an HMG-CoA synthase inhibitor, an HMG-CoA reductase inhibitor, a mevalonate kinase inhibitor, a phosphomevalonate kinase inhibitor, a mevalonic acid-5-pyrophosphate decarboxylase inhibitor, an isopentenyl pyrophosphate isomerase inhibitor and a farnesyl pyrophosphate synthase inhibitor, and the inhibitor can be used for preparing a medicine for preventing and treating diseases caused by the mevalonate kinase inhibitor, the phosphomevalonate kinase inhibitor, the phosphomevalonate kinase inhibitor and the phosphomevalonate decarboxylase inhibitor. A geranyl pyrophosphate synthase inhibitor and a geranyl transfer (I, II) inhibitor. Also disclosed is an immunogenic composition comprising an inhibitor of the mevalonate pathway as an adjuvant.

Description

[0001] This application is a divisional application of an invention patent application with an application date of September 8, 2016, application number 202210417066.5, and invention name “Inhibitors of the mevalonate pathway as highly effective vaccine adjuvants”. Technical Field

[0002] The present invention relates to inhibitors of the mevalonate pathway as highly effective vaccine adjuvants. The present invention also relates to immunogenic compositions comprising inhibitors of the mevalonate pathway as adjuvants. Background Art

[0003] Adjuvants play an important role in the development and use of vaccines. Adjuvants are also called non-specific immunopotentiators. They are not antigenic in themselves, but can enhance the immunogenicity of antigens or change the type of immune response when injected into the body together with antigens or in advance. Live attenuated vaccines and inactivated vaccines may contain natural adjuvant components in nature, which may include proteins, lipids and oligonucleotides in the form of particles. In fact, many attenuated or inactivated vaccines have very strong protective effects on the body after immunization. However, due to some limitations of these attenuated and inactivated vaccines themselves, such as attenuated pathogenic microorganisms mutating into highly pathogenic microorganisms, inactivated vaccines are not completely inactivated during preparation, and when these vaccines act on the body, they may directly cause the body to become diseased. Subunit vaccines are vaccines made from components of the main 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 with their advantages such as convenient quality control, mass production, safety and reliability. However, subunit vaccines also have shortcomings such as short protection and slow onset of action. Adjuvants that compensate for these shortcomings of subunit vaccines are an important component in the development and use of modern vaccines.

[0004] The most widely used adjuvant in vaccine production is aluminum adjuvant. In 1926, aluminum salts were first found to have adjuvant effects and were first used in diphtheria vaccines in 1936. However, due to some limitations of aluminum adjuvants, such as weak adjuvant effects, they need to be combined with antigens with strong immunogenicity to play a good role, especially they cannot promote Th1 responses that mediate cellular immunity, resulting in aluminum adjuvants not being able to prevent and treat influenza, HIV, tumors, malaria and other diseases well. Therefore, these vaccines are in urgent need of new and effective adjuvants. So far, adjuvants approved for clinical use in the United States and Europe include aluminum salts, oil-in-water emulsions (MF59 AS03 and AF03) and AS04 (MPL aluminum salt). The development of adjuvants is in a "primitive" state, and the only known molecular target is TLR (Toll-like receptor). There is an urgent need to discover new molecular targets for adjuvants in this field. Summary of the invention

[0005] In the present invention, we discovered and confirmed for the first time that enzymes related to the mevalonate pathway can serve as targets for rational adjuvant design.

[0006] The mevalonate pathway is a metabolic pathway that uses acetyl-CoA as a raw material to synthesize isoprenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). It exists in all higher eukaryotes and many viruses. The products of this pathway can be regarded as activated isoprene units, which are precursors for the synthesis of biological molecules such as steroids and terpenoids. In this pathway, acetoacetyl-CoA (acetoacetyl-CoA) is first generated by two molecules of acetyl-CoA, and the generated acetoacetyl-CoA then reacts with acetyl-CoA to generate 3-hydroxy-3-methylglutaryl-CoA (3-hydroxy-3-methylglutary CoA, i.e. HMG-CoA). HMG-CoA is reduced to mevalonate under the action of HMG-CoA reductase. Mevalonate catalyzes mevalonate to form isoprenyl pyrophosphate (IPP) under the action of two kinases and a decarboxylase. IPP forms farnesyl pyrophosphate (FPP) under the catalysis of FPP synthase (FPPS). FPP begins to branch in the downstream pathway, such as the formation of cholesterol, ubiquinone, heme (Heme A), sterols (Sterol), dolichol and prenylated proteins. For example, FPP can synthesize squalene under the action of squalene synthase (SQS), and squalene is synthesized into cholesterol under the catalysis of a series of enzymes. FPP can farnesylate some proteins under the action of farnesyl transferase. On the other hand, FPP synthesizes geranylgeranyl pyrophosphate (GGPP) under the catalysis of GGPP synthase (GGPPS); and GGPP can geranylgeranylate some proteins under the action of geranylgeranyltransferase to form prenylated proteins.

[0007] We found that any substance that affects geranylgeranylation of proteins can be used to develop vaccine adjuvants. Specifically, vaccine adjuvants targeting the following targets can be developed. 1) Thiolase (acetoacetyl-CoA transferase); 2) HMG-CoA synthase; 3) HMG-CoA reductase; 4) Mevalonate kinase; 5) Phosphonomevalonate kinase; 6) Mevalonate-5-pyrophosphate decarboxylase; 7) Isopentenyl-diphosphate isomerase; 8) Farnesyl diphosphate synthase (FPPS); 9) Geranylgeranyl diphosphate synthase (GGPPS); 10) Geranylgeranyl transferase (I, II).

[0008] We also found that other substances that do not directly act on the mevalonate pathway but indirectly affect geranylgeranylation can also be used in the development of vaccine adjuvants.

[0009] Thus, in one aspect, the present invention relates to an immunogenic composition comprising a substance that affects geranylgeranylation of a protein as an adjuvant. The substance may include, but is not limited to, 1) thiolase (acetoacetyl-CoA transferase) inhibitors, 2) HMG-CoA synthase inhibitors, 3) HMG-CoA reductase inhibitors, 4) mevalonate kinase inhibitors, 5) phosphomevalonate kinase inhibitors, 6) mevalonate-5-pyrophosphate decarboxylase inhibitors, 7) isopentenyl pyrophosphate isomerase inhibitors, 8) farnesyl pyrophosphate synthase inhibitors, 9) geranylgeranyl pyrophosphate synthase inhibitors and 10) geranylgeranyltransferase (I, II) inhibitors.

[0010] In another aspect, the present invention encompasses the above-described inhibitors for use as adjuvants.

[0011] In another aspect, the present invention encompasses the use of the above-mentioned inhibitors as adjuvants in the preparation of immunogenic compositions.

[0012] In another aspect, the present invention also relates to novel compounds as inhibitors of farnesyl pyrophosphate synthase (FPPS) or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0013]

[0014] In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group;

[0015] X is any of the following: hydrogen, hydroxyl, aliphatic, mercapto, halogen, alkoxy or alkyl; each M can independently be any of the following: negative charge, hydrogen, alkyl, aliphatic, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, mercapto, halogen, amino, aliphatic or alkyl; m=an integer of 1-6.

[0016] In another aspect, the present invention also relates to novel compounds as inhibitors of farnesyl pyrophosphate synthase (FPPS) or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0017]

[0018] wherein n is an integer of 1-24, preferably n is an integer of 1-20, more preferably n is an integer of 1-15, and more preferably n is an integer of 1-12.

[0019] In another aspect, the present invention also relates to novel compounds as inhibitors of farnesyl pyrophosphate synthase (FPPS) or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0020]

[0021] wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0022] R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0023] R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0024] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0025] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0026] In a preferred embodiment of this aspect, the compound is selected from:

[0027]

[0028] In another aspect, the present invention also relates to novel compounds as inhibitors of farnesyl pyrophosphate synthase (FPPS) or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0029]

[0030] in

[0031] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0032] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0033] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0034] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0035] In a preferred embodiment of this aspect, the compound is selected from:

[0036]

[0037] In another aspect, the present invention also relates to novel compounds as geranylgeranyl pyrophosphate synthase inhibitors or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0038]

[0039] in

[0040] R9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0041] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0042] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0043] R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0044] In a preferred embodiment of this aspect, the compound is selected from:

[0045]

[0046] In another aspect, the present invention also relates to the use of the novel compounds or their pharmaceutically acceptable salts, esters, prodrugs, solvates as adjuvants in the preparation of immunogenic compositions for preventing or treating diseases.

[0047] Another aspect of the present invention relates to a method for immunizing a subject or a host, comprising administering to the subject or the host an immunogenic composition as defined in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0049] Specifically, the present invention relates to the following embodiments:

[0050] 1. An immunogenic composition comprising an adjuvant selected from:

[0051] 1) Thiolase inhibitors;

[0052] 2) HMG-CoA synthase inhibitors;

[0053] 3) HMG-CoA reductase inhibitors;

[0054] 4) mevalonate kinase inhibitors;

[0055] 5) Phosphomevalonate kinase inhibitors;

[0056] 6) mevalonate-5-pyrophosphate decarboxylase inhibitors;

[0057] 7) Isopentenyl pyrophosphate isomerase inhibitors;

[0058] 8) Farnesyl pyrophosphate synthase inhibitors;

[0059] 9) Geranylgeranyl pyrophosphate synthase inhibitors; and

[0060] 10) Geranylgeranyl transferase (I, II) inhibitors.

[0061] 2. The immunogenic composition according to 1, wherein the HMG-CoA reductase inhibitor is a statin compound.

[0062] 3. The immunogenic composition according to 2, wherein the statin compound is selected from pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, cerivastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0063] 4. The immunogenic composition according to 2, wherein the statin compound is selected from simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0064] 5. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a bisphosphonic acid compound or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof.

[0065] 6. The immunogenic composition according to 5, wherein the bisphosphonate compound is selected from zoledronic acid, pamidronate, alendronate, ibandronic acid, neridronic acid, risedronate, olpadronate, and minodronic acid.

[0066] 7. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0067]

[0068] In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group;

[0069] X is any of the following: hydrogen, hydroxyl, aliphatic, mercapto, halogen, alkoxy or alkyl; each M can independently be any of the following: negative charge, hydrogen, alkyl, aliphatic, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4+ , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl;

[0070] m=an integer of 1-6.

[0071] 8. The immunogenic composition according to 7, wherein the compound represented by formula I is a compound represented by the following formula II-X:

[0072]

[0073]

[0074] In the above formula II-X, X is any one of the following: hydrogen, hydroxyl, mercapto, halogen, alkoxy or alkyl;

[0075] Each M is independently any of the following: negative charge, hydrogen, alkyl, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group;

[0076] R1, R2, R3, R4, R5, and R8 are independently selected from any one of the following: hydrogen, hydroxyl, aliphatic, thiol, halogen, amino, alkyl, -O-(CH2) q CH3, -NH-(CH2) q CH3, -N[(CH2) q CH3]2, -(CH2) P -S-(CH2) q CH3, -O-(CH2) P -S-(CH2) q CH3, -O-(CH2) P -O-(CH2) q CH3, wherein p=1-6, q=0-6; m=an integer of 1-6.

[0077] 9. The immunogenic composition according to 7 or 8, wherein the compound is a compound represented by formula XI-XVIII:

[0078]

[0079] In formulae XI-XVIII, Z is hydrogen, hydroxy, aliphatic, alkoxy, amino or alkylamino.

[0080] 10. The immunogenic composition according to any one of 7-9, wherein the compound is a compound represented by formula IXX or XX:

[0081]

[0082] In formula IXX and formula XX, n is 0 or an integer of 1-12.

[0083] 11. The immunogenic composition according to any one of 7 to 10, wherein the compound is any one of the following:

[0084]

[0085] 12. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0086]

[0087] wherein R1 and R2 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen, etc.;

[0088] 13. The immunogenic composition according to 12, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0089]

[0090] Wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0091] 14. The immunogenic composition according to 13, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0092]

[0093] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0094] 15. The immunogenic composition according to 14, wherein n is an integer from 1 to 20.

[0095] 16. The immunogenic composition according to 14, wherein n is an integer from 1 to 15.

[0096] 17. The immunogenic composition according to 14, wherein the compound is selected from:

[0097]

[0098]

[0099] 18. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0100]

[0101] Wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxyl, thiol and halogen.

[0102] 19. The immunogenic composition according to 18, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0103]

[0104] Wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0105] 20. The immunogenic composition according to 19, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0106]

[0107] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0108] 21. The immunogenic composition according to 20, wherein n is an integer from 1 to 20.

[0109] 22. The immunogenic composition according to 20, wherein n is an integer from 1 to 15.

[0110] 23. The immunogenic composition according to 20, wherein the compound is selected from:

[0111] n＝ Compound No. 1 TH-Z156 2 TH-Z157 3 TH-Z158 4 TH-Z159 5 TH-Z160 6 TH-Z97 7 TH-Z161 8 TH-Z98 9 TH-Z162 10 TH-Z99 11 TH-Z198 12 TH-Z163.

[0112] 24. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0113]

[0114] in

[0115] R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0116] R2 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R3 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0117] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0118] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0119] 25. The immunogenic composition according to 24, wherein R1 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkynyl, C 1-10 Alkylamino, C 1-10 Alkylthio, halogen, hydroxy, indazolyl, C 1-10 Alkoxy, C substituted by phenyl or pyridyl 1-10 The alkoxy group, the pyridyl group is optionally substituted with a carbamoyl group.

[0120] 26. An immunogenic composition according to 25, wherein R1 is selected from hydrogen, 4-methylphenethoxy, 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-yl, hydroxyl, and bromine.

[0121] 27. The immunogenic composition according to 24, wherein R2 is selected from hydrogen, C 1-10 Alkoxy, halogen.

[0122] 28. An immunogenic composition according to 27, wherein R2 is selected from hydrogen, octyloxy, bromine.

[0123] 29. The immunogenic composition according to 24, wherein R3 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy.

[0124] 30. An immunogenic composition according to 29, wherein R3 is selected from hydrogen, methyl, hexyloxy.

[0125] 31. The immunogenic composition according to 24, wherein R2 and R3 together with the carbon atoms to which they are attached form a benzene ring.

[0126] 32. The immunogenic composition according to 24, wherein R4 is selected from hydrogen, C 1-10 Alkoxy.

[0127] 33. An immunogenic composition according to 32, wherein R4 is selected from hydrogen, octyloxy.

[0128] 34. The immunogenic composition according to 24, wherein the compound is selected from:

[0129]

[0130] 35. The immunogenic composition according to 1, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0131]

[0132] in

[0133] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0134] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0135] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0136] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0137] 36. The immunogenic composition according to 35, wherein R5 is selected from C 1-10 Alkoxy.

[0138] 37. The immunogenic composition according to 35, wherein the compound is

[0139]

[0140] 38. The immunogenic composition according to 1, wherein the geranylgeranyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0141]

[0142] in

[0143] R9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0144] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0145] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0146] R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0147] 39. The immunogenic composition according to 38, wherein R9 is selected from C 1-10 Alkoxy.

[0148] 40. The immunogenic composition according to 38, wherein the compound is

[0149]

[0150] 41. An immunogenic composition according to any one of 1-40, comprising one or more antigens.

[0151] 42. An immunogenic composition according to 41, wherein the antigen is derived from a bacterium, a virus, a parasite or a tumor.

[0152] 43. An immunogenic composition according to 41, wherein the antigen is derived from anthrax, Campylobacter, cholera, diphtheria, enterotoxigenic Escherichia coli, Giardia, Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae type B, non-typeable Haemophilus influenzae, meningococci, pertussis, pneumococci, Salmonella, Shigella, Streptococcus B, Group A Streptococcus, tetanus, Vibrio cholerae, Yersinia, Staphylococcus, Pseudomonas species and Clostridium species, or the antigen is derived from adenovirus, dengue fever 1 to 4 serotypes, Ebola virus, enterovirus, hepatitis A to E serotypes, herpes simplex virus 1 or 2, human immunodeficiency virus, influenza, Japanese equine encephalitis, measles, Norwalk, papillomavirus, parvovirus B19, polio, rabies, rotavirus, rubella, measles, vaccinia, vaccinia constructs containing genes encoding other antigens such as malarial antigens, varicella, and yellow fever, or antigens derived from Entamoeba histolytica, Plasmodium, toxoplasmosis, and helminths, or antigens derived from tumors.

[0153] 44. An immunogenic composition according to 41, wherein the antigen is derived from Middle East Respiratory Syndrome (Mers) virus, Hepatitis B virus, melanoma.

[0154] 45. An immunogenic composition according to any one of 1-44 above, for use in treating or preventing Middle East Respiratory Syndrome, Hepatitis B virus, or melanoma.

[0155] 46. ​​The immunogenic composition according to any one of the above 1-45, further comprising another adjuvant.

[0156] 47. According to the immunogenic composition of 46, the other adjuvant is selected from aluminum adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, AS01, AS02, AS03, AS04, AS15, CAF01, ISCOMs (immunostimulatory complexes), Virosomes (viral particles), GLA-SE, liposomes, edible oils, saponins, AF03, TLR agonists.

[0157] 48. According to the immunogenic composition of 47, the TLR agonist is selected from (such as triacyl lipoprotein), TLR2 stimulator (such as peptidoglycan, yeast polysaccharide, HMGB1 (high-speed swimming protein 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 triacyl lipoprotein, lipoteichoic acid), TLR7 / 8 stimulator (single-stranded RNA, imiquimod), TLR9 stimulator (DNA, such as CPG ODN), C-lectin ligand (such as laminarin), CD1d ligand (such as α-galactosylceramide).

[0158] 49. The immunogenic composition according to any one of 1 to 48 above, suitable for immunization by oral, topical or parenteral route.

[0159] 50. The immunogenic composition according to 49, suitable for immunization by injection.

[0160] 51. The immunogenic composition according to 49, suitable for immunization by injection into the sole, subcutaneous, intramuscular, intraperitoneal and nasal mucosa of a subject.

[0161] 52. The immunogenic composition according to any one of 1-51 above is used to prepare the following vaccines: BCG, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis D vaccine, hepatitis E vaccine, influenza vaccine, polio vaccine, diphtheria and pertussis vaccine, measles vaccine, Japanese encephalitis vaccine, rabies vaccine, hemorrhagic fever vaccine, pneumonia vaccine, meningococcal vaccine, hepatitis A vaccine, mumps vaccine, influenza vaccine, rubella vaccine, varicella vaccine, AIDS vaccine, malaria vaccine; and cancer treatment and prevention vaccines, including but not limited to melanoma therapeutic 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, liver cancer therapeutic vaccine, liver cancer preventive vaccine, prostate cancer therapeutic vaccine, prostate cancer preventive vaccine.

[0162] 53. Thiolase inhibitors, used as adjuvants.

[0163] 54. HMG-CoA synthase inhibitors, used as adjuvants.

[0164] 55. HMG-CoA reductase inhibitors, used as adjuvants.

[0165] 56. Mevalonate kinase inhibitors, used as adjuvants.

[0166] 57. Phosphomevalonate kinase inhibitors, used as adjuvants.

[0167] 58. Mevalonate-5-pyrophosphate decarboxylase inhibitors for use as adjuvants.

[0168] 59. Isopentenyl pyrophosphate isomerase inhibitors, used as adjuvants.

[0169] 60. Farnesyl pyrophosphate synthase inhibitors, used as adjuvants.

[0170] 61. Geranylgeranyl pyrophosphate synthase inhibitors for use as adjuvants.

[0171] 62. Geranylgeranyltransferase (I, II) inhibitors for use as adjuvants.

[0172] 63. The HMG-CoA reductase inhibitor according to 55, wherein the HMG-CoA reductase inhibitor is a statin compound.

[0173] 64. The HMG-CoA reductase inhibitor according to 63, wherein the statin compound is selected from pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, cerivastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0174] 65. The HMG-CoA reductase inhibitor according to 63, wherein the statin compound is selected from simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0175] 66. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a bisphosphonic acid compound or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof.

[0176] 67. A farnesyl pyrophosphate synthase inhibitor according to 66, wherein the bisphosphonate compound is selected from the group consisting of zoledronic acid, pamidronate, alendronate, ibandronic acid, neridronic acid, risedronate, olpadronate, minodronic acid.

[0177] 68. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0178]

[0179] In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group;

[0180] X is any of the following: hydrogen, hydroxyl, aliphatic, mercapto, halogen, alkoxy or alkyl; each M can independently be any of the following: negative charge, hydrogen, alkyl, aliphatic, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl;

[0181] m=an integer of 1-6.

[0182] 69. The farnesyl pyrophosphate synthase inhibitor according to 68, wherein the compound represented by formula I is a compound represented by the following formula II-X:

[0183]

[0184]

[0185] In the above formula II-X, X is any one of the following: hydrogen, hydroxyl, mercapto, halogen, alkoxy or alkyl;

[0186] Each M is independently any of the following: negative charge, hydrogen, alkyl, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group;

[0187] R1, R2, R3, R4, R5, and R8 are independently selected from any one of the following: hydrogen, hydroxyl, aliphatic, thiol, halogen, amino, alkyl, -O-(CH2) q CH3, -NH-(CH2) q CH3, -N[(CH2) q CH3]2, -(CH2) P -S-(CH2) q CH3, -O-(CH2) P -S-(CH2) q CH3, -O-(CH2) P -O-(CH2) q CH3, wherein p=1-6, q=0-6; m=an integer of 1-6.

[0188] 70. The farnesyl pyrophosphate synthase inhibitor according to 68 or 69, wherein the compound is a compound represented by formula XI-XVIII:

[0189]

[0190] In formulae XI-XVIII, Z is hydrogen, hydroxy, aliphatic, alkoxy, amino or alkylamino.

[0191] 71. The farnesyl pyrophosphate synthase inhibitor according to any one of 68-70, wherein the compound is a compound represented by formula IXX or XX:

[0192]

[0193] In formula IXX and formula XX, n is 0 or an integer of 1-12.

[0194] 72. The farnesyl pyrophosphate synthase inhibitor according to any one of 68-71, wherein the compound is any one of the following:

[0195]

[0196] 73. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0197]

[0198] wherein R1 and R2 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen, etc.;

[0199] 74. The farnesyl pyrophosphate synthase inhibitor according to 73, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0200]

[0201] Wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0202] 75. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0203]

[0204] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0205] 76. The farnesyl pyrophosphate synthase inhibitor according to 75, wherein n is an integer from 1 to 20.

[0206] 77. The farnesyl pyrophosphate synthase inhibitor according to 75, wherein n is an integer from 1 to 15.

[0207] 78. The farnesyl pyrophosphate synthase inhibitor according to 75, wherein the compound is selected from:

[0208]

[0209]

[0210] 79. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0211]

[0212] Wherein n is an integer from 1 to 12.

[0213] 80. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0214]

[0215] wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0216] R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0217] R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0218] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0219] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0220] 81. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein R1 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkynyl, C1-10 Alkylamino, C 1-10 Alkylthio, halogen, hydroxy, indazolyl, C 1-10 Alkoxy, C substituted by phenyl or pyridyl 1-10 The alkoxy group, the pyridyl group is optionally substituted with a carbamoyl group.

[0221] 82. A farnesyl pyrophosphate synthase inhibitor according to 81, wherein R1 is selected from hydrogen, 4-methylphenethoxy, 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-yl, hydroxyl, and bromine.

[0222] 83. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein R2 is selected from hydrogen, C 1-10 Alkoxy, halogen.

[0223] 84. The farnesyl pyrophosphate synthase inhibitor according to 83, wherein R2 is selected from hydrogen, octyloxy, bromine.

[0224] 85. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein R3 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy.

[0225] 86. The farnesyl pyrophosphate synthase inhibitor according to 85, wherein R3 is selected from hydrogen, methyl, hexyloxy.

[0226] 87. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein R2 and R3 together with the carbon atoms to which they are attached form a benzene ring.

[0227] 88. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein R4 is selected from hydrogen, C 1-10 Alkoxy.

[0228] 89. The farnesyl pyrophosphate synthase inhibitor according to 88, wherein R4 is selected from hydrogen, octyloxy.

[0229] 90. The farnesyl pyrophosphate synthase inhibitor according to 80, wherein the compound is selected from:

[0230]

[0231] 91. The farnesyl pyrophosphate synthase inhibitor according to 60, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0232]

[0233] in

[0234] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0235] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0236] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0237] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0238] 92. The farnesyl pyrophosphate synthase inhibitor according to 91, wherein R5 is selected from C 1-10 Alkoxy.

[0239] 93. The farnesyl pyrophosphate synthase inhibitor according to 91, wherein the compound is

[0240]

[0241] 94. The geranylgeranyl pyrophosphate synthase inhibitor according to 61, wherein the geranylgeranyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0242]

[0243] in

[0244] R9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0245] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0246] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0247] R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0248] 95. The geranylgeranyl pyrophosphate synthase inhibitor according to 94, wherein R9 is selected from C 1-10 Alkoxy.

[0249] 96. The geranylgeranyl pyrophosphate synthase inhibitor according to 94, wherein the compound is

[0250]

[0251] 97. Use of a thiolase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0252] 98. Use of an HMG-CoA synthase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0253] 99. Use of an HMG-CoA reductase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0254] 100. Use of a mevalonate kinase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0255] 101. Use of a phosphomevalonate kinase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0256] 102. Use of a mevalonate 5-pyrophosphate decarboxylase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0257] 103. Use of an isopentenyl pyrophosphate isomerase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0258] 104. Use of a farnesyl pyrophosphate synthase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0259] 105. Use of a geranylgeranyl pyrophosphate synthase inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0260] 106. Use of a geranylgeranyltransferase (I, II) inhibitor as an adjuvant in the preparation of an immunogenic composition.

[0261] 107. The use according to 99, wherein the HMG-CoA reductase inhibitor is a statin compound.

[0262] 108. The use according to 107, wherein the statin compound is selected from pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, cerivastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0263] 109. The use according to 107, wherein the statin compound is selected from simvastatin, lovastatin and mevastatin, or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof.

[0264] 110. The use according to 109, wherein the farnesyl pyrophosphate synthase inhibitor is a bisphosphonic acid compound or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof.

[0265] 111. The use according to 110, wherein the bisphosphonic acid compound or a pharmaceutically acceptable salt thereof is selected from zoledronic acid, pamidronic acid, alendronic acid, ibandronic acid, neridronic acid, risedronic acid, olpadronic acid, and minodronic acid.

[0266] 112. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0267]

[0268] In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group;

[0269] X is any of the following: hydrogen, hydroxyl, aliphatic, mercapto, halogen, alkoxy or alkyl; each M can independently be any of the following: negative charge, hydrogen, alkyl, aliphatic, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl;

[0270] m=an integer of 1-6.

[0271] 113. The use according to 112, wherein the compound represented by formula I is a compound represented by the following formula II-X:

[0272]

[0273] In the above formula II-X, X is any one of the following: hydrogen, hydroxyl, mercapto, halogen, alkoxy or alkyl;

[0274] Each M is independently any of the following: negative charge, hydrogen, alkyl, -(CH2)p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group;

[0275] R1, R2, R3, R4, R5, and R8 are independently selected from any one of the following: hydrogen, hydroxyl, aliphatic, thiol, halogen, amino, alkyl, -O-(CH2) q CH3, -NH-(CH2) q CH3, -N[(CH2) q CH3]2, -(CH2) P -S-(CH2) q CH3, -O-(CH2) P -S-(CH2) q CH3, -O-(CH2) P -O-(CH2) q CH3, wherein p=1-6, q=0-6; m=an integer of 1-6.

[0276] 114. The use according to 112 or 113, wherein the compound is a compound represented by formula XI-XVIII:

[0277]

[0278] In formulae XI-XVIII, Z is hydrogen, hydroxy, aliphatic, alkoxy, amino or alkylamino.

[0279] 115. The use according to any one of 112-114, wherein the compound is a compound represented by formula IXX or XX:

[0280]

[0281] In formula IXX and formula XX, n is 0 or an integer of 1-12.

[0282] 116. The use according to any one of 112-115, wherein the compound is any one of the following:

[0283]

[0284] 117. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0285]

[0286] Wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxyl, thiol, halogen and the like.

[0287] 118. The use according to 117, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0288]

[0289] Wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0290] 119. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0291]

[0292] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0293] 120. The use according to 119, wherein n is an integer from 1 to 20.

[0294] 121. The use according to 119, wherein n is an integer from 1 to 15.

[0295] 122. The method according to 119, wherein the compound is selected from:

[0296] n＝ Compound No. 1 TH-Z79 2 TH-Z148 3 TH-Z149 4 TH-Z150 5 TH-Z151 6 TH-Z80 7 TH-Z152 8 TH-Z81 9 TH-Z153 10 TH-Z82 11 TH-Z154 12 TH-Z155.

[0297] 123. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0298]

[0299] Wherein n is an integer from 1 to 12.

[0300] 124. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0301]

[0302] wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0303] R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0304] R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0305] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0306] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0307] 125. The method according to 124, wherein R1 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkynyl, C 1-10 Alkylamino, C 1-10 Alkylthio, halogen, hydroxy, indazolyl, C 1-10 Alkoxy, C substituted by phenyl or pyridyl 1-10 The alkoxy group, the pyridyl group is optionally substituted with a carbamoyl group.

[0308] 126. The use according to 125, wherein R1 is selected from hydrogen, 4-methylphenethoxy, 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-yl, hydroxyl, and bromine.

[0309] 127. According to the use of 124, wherein R2 is selected from hydrogen, C 1-10 Alkoxy, halogen.

[0310] 128. The use according to 127, wherein R2 is selected from hydrogen, octyloxy, and bromine.

[0311] 129. According to the use of 124, wherein R3 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy.

[0312] 130. The use according to 129, wherein R3 is selected from hydrogen, methyl, hexyloxy.

[0313] 131. The use according to 124, wherein R2 and R3 together with the carbon atoms to which they are attached form a benzene ring.

[0314] 132. The method according to 124, wherein R4 is selected from hydrogen, C 1-10 Alkoxy.

[0315] 133. The use according to 132, wherein R4 is selected from hydrogen and octyloxy.

[0316] 134. The method according to 124, wherein the compound is selected from:

[0317]

[0318] 135. The use according to 104, wherein the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0319]

[0320] in

[0321] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0322] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0323] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0324] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0325] 136. According to the use of 135, wherein R5 is selected from C 1-10 Alkoxy.

[0326] 137. The method according to 136, wherein the compound is

[0327]

[0328] 138. The use according to 105, wherein the geranylgeranyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0329]

[0330] in

[0331] R9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0332] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0333] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0334] R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0335] 139. According to the use of 138, wherein R9 is selected from C 1-10 Alkoxy.

[0336] 140. The method according to 139, wherein the compound is

[0337]

[0338] 141. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0339]

[0340] In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group;

[0341] X is any of the following: hydrogen, hydroxyl, aliphatic, mercapto, halogen, alkoxy or alkyl; each M can independently be any of the following: negative charge, hydrogen, alkyl, aliphatic, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl;

[0342] m=an integer of 1-6.

[0343] 142. The compound according to 141, wherein the compound represented by formula I is a compound represented by the following formula II-X:

[0344]

[0345]

[0346] In the above formula II-X, X is any one of the following: hydrogen, hydroxyl, mercapto, halogen, alkoxy or alkyl;

[0347] Each M is independently any of the following: negative charge, hydrogen, alkyl, -(CH2) p -O-CO-R, -(CH2) p -CO-R or cation; wherein p=1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group;

[0348] R1, R2, R3, R4, R5, and R8 are independently selected from any one of the following: hydrogen, hydroxyl, aliphatic, thiol, halogen, amino, alkyl, -O-(CH2) q CH3, -NH-(CH2) q CH3, -N[(CH2) q CH3]2, -(CH2) P -S-(CH2) q CH3, -O-(CH2) P -S-(CH2) q CH3, -O-(CH2) P -O-(CH2) q CH3, wherein p=1-6, q=0-6; m=an integer of 1-6, preferably, m=1.

[0349] 143. The compound according to 141 or 142, wherein the compound is a compound represented by formula XI-XVIII:

[0350]

[0351]

[0352] In formulae XI-XVIII, Z is hydrogen, hydroxy, aliphatic, alkoxy, amino or alkylamino.

[0353] 144. A compound according to any one of 141-143, wherein the compound is a compound represented by formula IXX or XX:

[0354]

[0355] In formula IXX and formula XX, n is 0 or an integer of 1-12.

[0356] 145. The compound according to any one of 141-144, wherein the compound is any one of the following:

[0357]

[0358]

[0359] 146. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0360]

[0361] wherein R1 and R2 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen, etc.;

[0362] 147. The compound according to 146 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, which is a compound of the following formula:

[0363]

[0364] Wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0365] 148. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0366]

[0367] Wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxyl, thiol and halogen.

[0368] 149. The compound according to 148 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, which is a compound of the following formula:

[0369]

[0370] Wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl or carbamoyl.

[0371] 150. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0372]

[0373] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0374] 151. A compound according to 150, or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein n is an integer from 1 to 20.

[0375] 152. A compound according to 150, or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein n is an integer from 1 to 15.

[0376] 153. The compound according to 150 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein the compound is selected from:

[0377]

[0378]

[0379] 154. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0380]

[0381] wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0382] R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0383] R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0384] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0385] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0386] 155. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R1 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkynyl, C 1-10 Alkylamino, C 1-10 Alkylthio, halogen, hydroxy, indazolyl, C 1-10 Alkoxy, C substituted by phenyl or pyridyl 1-10 The alkoxy group, the pyridyl group is optionally substituted with a carbamoyl group.

[0387] 156. A compound according to 155 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R1 is selected from hydrogen, 4-methylphenethoxy, 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-yl, hydroxyl, and bromine.

[0388] 157. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R2 is selected from hydrogen, C 1-10 Alkoxy, halogen.

[0389] 158. The compound according to 157 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R2 is selected from hydrogen, octyloxy, bromine.

[0390] 159. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R3 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy.

[0391] 160. The compound according to 159 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R3 is selected from hydrogen, methyl, hexyloxy.

[0392] 161. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R2 and R3 together with the carbon atoms to which they are attached form a benzene ring.

[0393] 162. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof, wherein R4 is selected from hydrogen, C 1-10 Alkoxy.

[0394] 163. A compound according to 162 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R4 is selected from hydrogen, octyloxy.

[0395] 164. The compound according to 154 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein the compound is selected from:

[0396]

[0397] 165. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0398]

[0399] in

[0400] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0401] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0402] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0403] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0404] 166. The compound according to 165 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R5 is selected from C 1-10 Alkoxy.

[0405] 167. The compound according to 165 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein the compound is

[0406]

[0407] 168. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0408]

[0409] in

[0410] R9 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0411] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0412] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0413] R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0414] 169. The compound according to 168 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein R9 is selected from C 1-10 Alkoxy.

[0415] 170. The compound according to 168 or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof, wherein the compound is

[0416]

[0417] 171. A method for preparing a compound of formula IXX, comprising:

[0418]

[0419] Formula IXX, n is 0 or an integer of 1-12,

[0420] 1) reacting the compound represented by formula XXI with tert-butyl bromoacetate in the presence of an inorganic base or a Lewis base to obtain a compound represented by formula XXII;

[0421]

[0422] In Formula XXI and Formula XXII, n is 0 or an integer of 1-12;

[0423] 2) Under palladium-carbon catalysis, the compound represented by formula XXII is subjected to a reduction reaction in hydrogen, and the obtained product is subjected to a ring-closing reaction with formamidine acetate to obtain a compound represented by formula XXIII;

[0424]

[0425] In Formula XXIII, n is 0 or an integer from 1 to 12;

[0426] 3) heating the compound represented by formula XXIII in hydrochloric acid or TFA to reflux to obtain the compound represented by formula IXXV;

[0427]

[0428] In formula IXXV, n is 0 or an integer from 1 to 12;

[0429] 4) reacting the compound represented by formula IXXV with phosphorus trichloride under reflux in a phosphorous acid and sulfolane medium to obtain a compound represented by formula IXX.

[0430] 172. The preparation method according to 171, characterized in that:

[0431] In step 1), the molar ratio of the compound represented by formula XXI to the inorganic base or Lewis base and tert-butyl bromoacetate is 1:1-20:0.2-15 respectively;

[0432] In step 1), the reaction temperature is 80-150°C and the reaction time is 1-96h;

[0433] In step 2), the reduction reaction temperature is 0-100°C and the time is 0.5-24h;

[0434] In step 2), the molar ratio of the compound represented by formula XXII to formamidine acetate is 1:0.2-10;

[0435] The ring-closing reaction temperature is 50-150°C and the time is 1-24h;

[0436] The ring-closing reaction is carried out in an organic solvent, preferably ethylene glycol monomethyl ether;

[0437] In step 3), the molar ratio of the compound represented by formula XXIII to hydrochloric acid or TFA is 1:1-100;

[0438] In step 3), the heating reflux time is 0.5-96h;

[0439] In step 4), the molar ratio of the compound represented by formula IXXV to phosphorous acid and phosphorus trichloride is 1:0.2-20:0.2-20 respectively;

[0440] In step 4), the reaction time is 0.5-96h.

[0441] 174. Use of the compound according to any one of 141-170 in the preparation of the following products:

[0442] 1) Drugs for the treatment of metabolic bone diseases;

[0443] 2) Medicines for the treatment of malaria;

[0444] 3) Eukaryotic tumor cell proliferation inhibitors;

[0445] 4) Drugs for the prevention and / or treatment of tumors;

[0446] 5) Immunotherapy drugs;

[0447] 6) Vaccine adjuvants;

[0448] 7) Vaccines;

[0449] 8) Products that inhibit the activity of HsFPPs enzymes;

[0450] 9) Products that inhibit the enzymatic activity of PvGGPPs.

[0451] 175. The use according to 174, characterized in that:

[0452] 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 breast cancer, preferably, the breast cancer cell can specifically be a human breast cancer cell MDA-MB-231.

[0453] 176. According to the immunogenic composition of 1, the HMG-CoA synthase inhibitor of 54 or the use of 98, the HMG-CoA synthase is Hymeglusin (11-[3R-(hydroxymethyl)-4-oxo-2R-oxetane-1]-3,5,7R-trimethyl-2E,4E-undecadienoic acid). BRIEF DESCRIPTION OF THE DRAWINGS

[0454] Figure 1 is a schematic diagram of the mevalonate pathway.

[0455] Figure 2A-D shows the adjuvant activity of eight statins in an OVA antibody titer assay, expressed as IgM and IgG antibody titers 7 and 14 days after immunization.

[0456] Figure 3 The adjuvant activity of TH-Z80 series compounds in the OVA antibody titer assay is shown, expressed as IgM and IgG antibody titers 7 and 14 days after immunization.

[0457] Figure 4 The adjuvant activity of the FPPS inhibitor TH-Z97 in the OVA antibody titer assay is shown, expressed as IgG antibody titers 7 and 14 days after immunization.

[0458] 5A-D show the results of comparing the adjuvant activity of the bisphosphonic acid compounds TH-Z80 and TH-Z93 of the present invention with eight bisphosphonic acid drugs on the market in the OVA antibody titer test.

[0459] 6A-D show the adjuvant activity of GGPPS inhibitors TH-Z144 and TH-Z145 in an OVA antibody titer assay, expressed as IgM and IgG antibody titers at 7 and 14 days after immunization.

[0460] 7A-D show the adjuvant activity of the dual inhibitors of FPPS and GGPPS, BPH-716 and BPH-1222, in the OVA antibody titer assay, expressed as IgM and IgG antibody titers at 7 and 14 days after immunization, where Ctrl represents the control group of mice treated with PBS.

[0461] Figure 8 The figure shows the comparison of the adjuvant activity of the three types of inhibitors of the present invention, namely HMG-CoA reductase inhibitors, FPPS inhibitors, and GGPPS inhibitors, with the activity of existing adjuvants. The upper figure shows the antibody titer of IgG after boost, and the lower figure shows the antibody affinity index.

[0462] 9A-D show the inhibitory effects of GGOH and GGPP on the adjuvant activity of simvastatin (HMG-CoA reductase inhibitor).

[0463] 10A-D show the inhibitory effects of GGOH and GGPP on the adjuvant activity of TH-Z93 (FPPS inhibitor).

[0464] 11A-D show the inhibitory effects of GGOH and GGPP on the adjuvant activity of TH-Z145 (GGPPS inhibitor).

[0465] 12A-D show the results of the study of the adjuvant activity of the selective squalene synthase inhibitor TH-Z66 in the OVA antibody titer assay, expressed as IgM and IgG antibody titers 7 and 14 days after immunization.

[0466] Fig.13 The total number of lymph node cells in mice 24 hours after immunization with simvastatin, TH-Z80, and TH-Z145 as described in the present application as adjuvants is shown, wherein Ctrl represents mice without any treatment.

[0467] Fig.14The graph shows the proportion and number of B lymphocytes in the lymph nodes 24 hours after mice were immunized with simvastatin, TH-Z80, and TH-Z145 as adjuvants described in the present application, wherein Ctrl represents mice that were not treated in any way.

[0468] Fig.15 The figure shows the proportion and number of dendritic cells in the lymph nodes of mice immunized with simvastatin, TH-Z80, and TH-Z145 as described in the present application as adjuvants 24 hours after the mice were immunized, wherein Ctrl represents mice without any treatment.

[0469] Figure 16A-B The figure shows the proportion and number of macrophages in the lymph nodes 24 hours after mice were immunized with simvastatin, TH-Z80, and TH-Z145 as described in the present application as adjuvants, wherein Ctrl represents mice without any treatment.

[0470] Fig.17 The figure shows the proportion and number of T lymphocytes in the lymph nodes 24 hours after mice were immunized with simvastatin, TH-Z80, and TH-Z145 as adjuvants described in the present application, wherein Ctrl represents mice without any treatment.

[0471] Figure 18-21 The concentrations of IL-6, TNF-α, IL-1β and IL-12p70 after mouse bone marrow cells were treated with 1 μM simvastatin, TH-Z80, TH-Z145 and TH-Z66 in the presence of GM-CSF and IL-4 and then stimulated with 100 ng / ml LPS are shown, respectively.

[0472] Fig.22A -D shows the proportions of cell markers B220, F4 / 80, and CD11c in lymph nodes after immunization with simvastatin, TH-Z80, and TH-Z145 as described in the present application as adjuvants, wherein Ctrl represents mice without any treatment.

[0473] Figures 23A-D show the titers of IgM and IgG antibodies in the Mers protein of Middle East Respiratory Syndrome virus after mice were immunized with simvastatin, TH-Z80, and TH-Z144 as adjuvants, expressed as the antibody titers of IgM and IgG 7 and 14 days after immunization.

[0474] 24A-D show that simvastatin, TH-Z80, and TH-Z144 as adjuvants can promote the production of more antibodies against hepatitis B surface antigen HbsAg.

[0475] Fig.25A-D shows the adjuvant effect of simvastatin, TH-Z80, TH-Z93, and TH-Z145 in preventive vaccines against melanoma.

[0476] Fig.26A -D shows the adjuvant effect of simvastatin, TH-Z80, TH-Z93, and TH-Z145 in therapeutic vaccines for melanoma.

[0477] Fig. 27 The adjuvant activity of mice immunized with TH-Z80 as an adjuvant mixed antigen at different sites (foot sole, subcutaneous, muscle, abdominal cavity and nasal mucosa) is shown, and is expressed as the IgG antibody titer 7 and 14 days after immunization.

[0478] 28A-D show the combined adjuvant activity of the FPPS inhibitor TH-Z93 in combination with the TLR agonist imiquimod in an OVA antibody titer assay, expressed as IgM and IgG antibody titers at 7 and 14 days after immunization.

[0479] Fig.29 The effect of injection of 100 μg of different bisphosphonates on antibody production in mice. PBS is phosphate buffered saline, CLD is clodronic acid, ZOL is zoledronic acid, and PMD is pamidronic acid.

[0480] Fig.30 This is a diagram showing the effect of the length of the carbon chain substitution of the bisphosphonic acid side chain as an adjuvant;

[0481] Fig.31 The effect diagram of increasing antibody affinity for TH-Z80. PBS is a phosphate buffered saline solution;

[0482] Fig.32 The expansion effect of benzimidazole bisphosphonates with different carbon chain lengths on δγT cells;

[0483] Fig.33 Schematic diagram of the crystal structure of TH-Z80 and HsFPPS (pymol drawing).

[0484] Fig.34 Schematic diagram of the crystal structure of compound TH-Z82 and PvGGPPS (pymol drawing).

[0485] FIG. 35 shows the effects of simvastatin, TH-Z80, TH-Z93 and TH-Z145 on PR8 influenza virus.

[0486] Fig.36 The inhibitory effect of anti-PD1 antibody combined with four adjuvants and chicken ovalbumin on B16-OVA tumors.

[0487] Fig.37It is the adjuvant activity of HMG-CoA synthase inhibitors in OVA antibody titer detection.

[0488] Fig.38 The concentrations of IL-6, TNF-α, IL-1β and IL-12p70 after treatment with simvastatin, TH-Z80, TH-Z93, TH-Z145 or these adjuvants mixed with GGOH. DETAILED DESCRIPTION

[0489] the term

[0490] To facilitate review of the various embodiments in the present disclosure, the following explanations of terms are provided. Other terms and explanations are provided in the context of the present disclosure.

[0491] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0492] The term "antigen" is used to refer to any molecule that can be specifically recognized by the adaptive elements of the immune response (ie, B cells or T cells or both).

[0493] Preferably, the antigen used in the present invention is an immunogen, ie an antigen that activates immune cells to produce an immune response against oneself.

[0494] An "immunogenic composition" is a composition of matter suitable for administration to a human or animal patient (e.g., in an experimental setting) that is capable of eliciting a specific immune response (e.g., against a pathogen of hepatitis B virus). For example, an immunogenic composition comprises one or more antigens (e.g., a complete purified virus or an antigenic subunit, such as a polypeptide thereof) or antigenic epitopes. An immunogenic composition may also comprise one or more other ingredients capable of inducing or enhancing an immune response, such as an excipient, a carrier and / or an adjuvant. In some cases, an immunogenic composition is administered to induce an immune response that helps a patient fight symptoms or conditions caused by a pathogen. In some cases, after a patient is exposed to a pathogen, symptoms or diseases caused by a pathogen (e.g., hepatitis B virus) are prevented (or treated, e.g., alleviated or relieved) by inhibiting the replication of the pathogen. In the context of this article, the term immunogenic composition should be understood to encompass a composition (i.e., a vaccine composition or vaccine) administered to a patient or patient population in order to induce, for example, a protective or palliative immune response against hepatitis B.

[0495] "Adjuvant" is a reagent that can enhance antigen-specific immune response compared to the antigen given without it. Common adjuvants include adjuvants containing aluminum, which contain mineral suspensions (or mineral salts, such as aluminum hydroxide, aluminum phosphate, hydroxyaluminum phosphate) on which antigens can be adsorbed. In one case, an adjuvant is an aluminum-free adjuvant prepared in the absence of 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 ingredients.

[0496] "Immune response" is the response of cells of the immune system, such as B cells, T cells or monocytes to stimulation. The immune response can be a B cell response, which leads to 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 to a particular antigen (i.e., an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" is an immune response that inhibits the harmful functions or activities of a pathogen, reduces pathogen infection, or reduces the symptoms (including death) caused by pathogen infection. The protective immune response can be measured, for example, by inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen attack in vivo.

[0497] The term "antibody" describes an immunoglobulin, whether natural or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is or is homologous to an antigen-binding domain.

[0498] Tumor antigens are antigenic substances produced in tumor cells, i.e., they trigger 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 treatment.

[0499] The term "Middle East Respiratory Syndrome (MERS)", also known as camel flu, is a viral respiratory infection caused by the MERS-coronavirus (MERS-CoV). Its symptoms can range from mild to severe and include fever, cough, diarrhea, and shortness of breath.

[0500] As used herein, the term "optionally" means that one or more events described subsequently may or may not occur, and includes both the one or more events that occur and the one or more events that do not occur.

[0501] The term "substituted" as used herein refers to the replacement of any one or more hydrogens on a designated atom or ring by a group selected from the group shown, provided that the normal valence of the designated atom or ring atom is not exceeded and the replacement results in a stable compound. When the substituent is a keto group (i.e., =O), 2 hydrogens on the atom are replaced.

[0502] When a range of values ​​is listed, it is intended to include every value and sub-range within the range. For example, "C 1–6 "Alkyl" is intended to include C1, C2, C3, C4, C5, C6, 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.

[0503]

[0046] As used herein, "aliphatic group" or "aliphatic group" includes alkyl, alkenyl and alkynyl groups as defined below.

[0504] As used herein, "alkyl" is intended to include branched and straight chain saturated aliphatic hydrocarbon groups containing 1 to 20 carbons, preferably 1 to 12 carbons, preferably 1 to 11 carbons, preferably 1 to 10 carbons, preferably 1 to 9 carbons, and more preferably 1 to 8 carbons in the chain, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, their various branched chain isomers, and the like, and the groups may optionally include 1 to 4 substituents, such as halogens such as F, B R, Cl or I or CF3, alkyl, alkoxy, aryl, aryloxy, aryl (aryl) or biaryl (diaryl), arylalkyl, arylalkyloxy, alkenyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, amino, hydroxy, hydroxyalkyl, acyl, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, alkylthio, arylalkylthio, aryloxyaryl, alkylaminoacyl (alkylamido), alkanoylamino, arylcarbonylamino, nitro, cyano, mercapto, haloalkyl, trihaloalkyl and / or alkylthio.

[0505] Unless otherwise indicated, the term "alkenyl" as used herein alone or as part of another group refers to a straight or branched chain group containing 2 to 20 carbons, preferably 2 to 12 carbons, preferably 2 to 11 carbons, preferably 2 to 10 carbons, preferably 2 to 9 carbons, more preferably 1 to 8 carbons in the chain, the group containing 1 to 6 double bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4 ... -heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, etc., and the group may be optionally substituted by 1 to 4 substituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino, hydroxyl, heteroaryl, heterocycloalkyl, alkanoylamino, alkylaminoacyl, arylcarbonylamino, nitro, cyano, sulfhydryl, alkylthio and / or any one of the substituents on the alkyl described in the present application.

[0506] Unless otherwise indicated, the term "alkynyl" as used herein alone or as part of another group refers to a straight or branched chain group containing 2 to 20 carbons, preferably 2 to 12 carbons, preferably 2 to 11 carbons, preferably 2 to 10 carbons, preferably 2 to 9 carbons, more preferably 2 to 8 carbons in the chain, the group containing a triple bond in the normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-hept ... Alkynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl, etc., and the group may be optionally substituted by 1 to 4 substituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino, heteroaryl, heterocycloalkyl, hydroxyl, alkanoylamino, alkylaminoacyl, arylcarbonylamino, nitro, cyano, sulfhydryl, alkylthio and / or any one of the substituents on the alkyl described in the present application.

[0507] Unless otherwise specified, the term "cycloalkyl" used herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or 2 double bonds) cyclic hydrocarbon groups containing 1 to 3 rings, including monocyclic alkyl, bicyclic alkyl (or bicyclic alkyl) and tricyclic alkyl, which contain a total of 3 to 20 ring carbon atoms, preferably a total of 3 to 10 ring carbon atoms, and the group may be fused with 1 or 2 aromatic rings as described for aryl. "Cycloalkyl" containing 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 includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, cyclohexenyl,

[0508]

[0509] Any of these groups may be optionally substituted by 1 to 4 substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylaminoacyl, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, mercapto and / or alkylthio and / or any substituent in the case of the alkyl group.

[0510] When an alkyl group as defined above has single bonds to other groups at two different carbon atoms, the alkyl group is referred to as an "alkylene group" and may be optionally substituted as defined above for "alkyl".

[0511] When the alkenyl group as defined above and the alkynyl group as defined above have single bonds for attachment at two different carbon atoms, respectively, they are referred to as "alkenylene" and "alkynylene", respectively, and may be optionally substituted as defined above for "alkenyl" and "alkynyl".

[0512] As used herein, "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine; and "haloalkyl" is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups, such as CF3, which have a specific number of carbon atoms and are substituted with one or more halogens (e.g., -C v F w , where v = 1 to 3, and w = 1 to (2v+1)).

[0513] Unless otherwise indicated, the term "aryl" or "aromatic group" as used herein alone or as part of another group refers to monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl or naphthyl (including naphth-1-yl and naphth-2-yl)), and may optionally contain 1 to 3 other rings fused to a carbocyclic or heterocyclic ring (such as an aryl ring, a cycloalkyl ring, a heteroaryl ring or a heterocycloalkyl ring), for example

[0514]

[0515] and aryl groups may be optionally substituted, through available carbon atoms, with 1, 2 or 3 substituents such as hydrogen, halogen, haloalkyl, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylthio, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino, substituted amino (wherein the amino group includes 1 or 2 substituents (the substituents are alkyl, aryl or any other aromatic compound mentioned in the definition), thiol, alkylthio, arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl, arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino or arylsulfonylaminocarbonyl and / or any of the substituents on the alkyl group described herein.

[0516] Unless otherwise stated, the terms "alkoxy," "aryloxy," or "aralkyloxy" as used herein alone or as part of another group include any of the above alkyl, aralkyl, or aryl groups attached to an oxygen atom.

[0517] Unless otherwise indicated, "amino" as used herein alone or as part of another group refers to an amino group that is unsubstituted or substituted by one or two substituents, which may be the same or different and are, for example, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, or alkylthio. These substituents may be further substituted by carboxylic acid groups and / or any R 1 Group or above is R 1 In addition, the substituents on the amino group can form pyrrolidin-1-yl, piperidin-1-yl, aza -1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, 4-alkyl-piperazin-1-yl, 4-arylalkyl-piperazin-1-yl or 4-biarylalkyl-piperazin-1-yl, said group being optionally substituted by alkyl, alkoxy, alkylthio, halogen, trifluoromethyl or hydroxy.

[0518] Unless otherwise stated, the terms "alkylthio", "arylthio" or "aralkylthio" as used herein alone or as part of another group include any of the above alkyl, aralkyl or aryl groups attached to a sulfur atom.

[0519] Unless otherwise stated, the terms "alkylamino", "arylamino" or "arylalkylamino" as used herein alone or as part of another group include any of the above alkyl, aryl or arylalkyl groups attached to a nitrogen atom.

[0520] The term "heterocyclyl" or "heterocyclic ring system" as used herein means a stable 5-, 6- or 7-membered monocyclic heterocycle or a bicyclic heterocycle or a 7-, 8-, 9- or 10-membered bicyclic heterocycle, which is saturated, partially unsaturated or unsaturated (aromatic), and is composed of carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from N, NH, O and S, and includes any bicyclic group in which any of the above heterocycles is fused to a benzene ring. The nitrogen heteroatom and sulfur heteroatom may be optionally oxidized. The heterocycle may be attached to its side group at any heteroatom or carbon atom that results in a stable structure. The heterocycles described herein may be substituted on carbon or nitrogen, provided that the resulting compound is stable. If specifically indicated, the nitrogen in the heterocycle may be optionally quaternized. Preferably, when the total number of S atoms and O atoms in the heterocycle exceeds 1, the heteroatoms are not adjacent to each other. The term "aromatic heterocyclic ring system" or "heteroaryl" or "heteroaromatic" as used herein refers to a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic aromatic ring composed of carbon atoms and 1 to 4 heteroatoms independently selected from N, O and S, and is aromatic in nature.

[0521] Examples of heterocyclic groups include, but are not limited to, 1H-indazolyl, 2-pyrrolidinone, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 1H-indolyl, 4-piperidone, 4aH-carbazolyl, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azoctatetraenyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolonyl (b enzimidazalonyl), carbazolyl, 4aH-carbazolyl, β-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofurano[2,3-b]tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, 3H-indolyl (indolenyl), indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, benzimidazolyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, perylene, phenanthridinyl, phenanthrolinyl, phenpyrazinyl, phenazinyl, phenothiazinyl, phenothioxazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazolyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyridinyl, pyrimidine 1,2,4-thiazolyl, 1,2,5-thiadiazinyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thienyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, and xanthenyl.In another aspect of the present invention, heterocyclic groups include, but are not limited to, pyridyl, thienyl, furanyl, indazolyl, benzothiazolyl, benzimidazolyl, benzothienyl, benzofuranyl, benzoxazolyl, benzisoxazolyl, quinolyl, isoquinolyl, imidazolyl, indolyl, isoindolyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pyrazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl, thiazolyl, oxazolyl, pyrazinyl and pyrimidinyl. Heterocyclic groups also include fused rings and spiro compounds containing, for example, the above-mentioned heterocycles.

[0522] Examples of heteroaryl are 1H-indazolyl, 2H,6H-1,5,2-dithiazinyl, indolyl, 4aH-carbazolyl, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azoctatetraenyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolonyl, carbazolyl, 4aH-carbazolyl, β-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H- 1,5,2-dithiazinyl, dihydrofurano[2,3-b]tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, 3H-indolyl, dihydroindolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4 -oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, perylene-1,1-diazaphenyl, phenanthridinyl, phenanthrolinyl, phenpyrazinyl, phenazinyl, phenothiazinyl, phenothioxazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyrazolotriazinyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, and xanthenyl. In another aspect of the invention, examples of heteroaryl groups are indolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolonyl, cinnolinyl, furanyl, imidazolyl, indazolyl, indolyl, isoquinolyl, isothiazolyl, isoxazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyrazolotriazinyl, pyridazinyl, pyridinyl, pyridinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, thiazolyl, thienyl and tetrazolyl.

[0523]

[0046] The term "cyano," as used herein, refers to a -CN group.

[0524] The term "nitro" as used herein refers to a -NO2 group.

[0525] As used herein, the term "hydroxyl" refers to an -OH group.

[0526] As used herein, the term "thiol" refers to a -SH group.

[0527] The term "carbamoyl" as used herein refers to -C(=O)-amino, wherein the amino group is optionally substituted.

[0528] "Alkanoyl" means a RC(=O)- group where R is alkyl as defined herein.

[0529] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0530] The term "pharmaceutically acceptable salt" used in this application refers to a salt that retains the biological effectiveness and characteristics of the compounds of the present invention, and it is not biologically or otherwise undesirable. In many instances, the compounds of the present invention can form acid salts and / or base salts due to the presence of amino and / or carboxyl groups or their similar groups (e.g., phenol or hydroxyamic acid). Medicinal acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived can include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived can 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, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Medicinal base addition salts can be formed with inorganic bases and organic bases. Inorganic bases from which salts can be derived include, for example, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc.; ammonium salts, potassium salts, sodium salts, calcium salts and magnesium salts are particularly preferred. Organic bases from which salts can be derived include, for example, primary amines, secondary amines and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc., particularly such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, the base part or the acid part by conventional chemical methods. Generally, the salts can be prepared by reacting the acid form of these compounds with a chemical amount of an appropriate base (such as sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, magnesium carbonate, potassium carbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate or potassium bicarbonate, etc.), or by reacting the free base form of these compounds with a chemical amount of an appropriate acid. The reaction is typically carried out in water or in an organic solvent or in a mixture of the two. In general, preferred non-aqueous media are ether, ethyl acetate, ethanol, isopropanol or acetonitrile, if applicable. Additional suitable salts listed can be found in the following literature: for example, Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.

[0531] Exemplary acid addition salts include acetates (such as those formed with acetic acid or a trihaloacetic acid (e.g., trifluoroacetic acid)), adipates, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, 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), naphthalene-2-sulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (such as those formed with sulfuric acid), sulfonate (such as those mentioned in this application), tartrate, thiocyanate, toluenesulfonate (such as toluenesulfonate), undecanoate, and the like.

[0532] 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; barium, zinc and aluminum salts; salts formed with organic bases (e.g., organic amines) (such as trialkylamines (e.g., triethylamine), procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines); and salts formed with amino acids such as arginine, lysine, and the like. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl chloride, methyl bromide, and methyl iodide, ethyl chloride, ethyl bromide, and ethyl iodide, propyl chloride, propyl bromide, and propyl iodide, and butyl chloride, butyl bromide, and butyl iodide), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate), long chain halides (e.g., decyl chloride, decyl bromide, and decyl iodide, lauryl chloride, lauryl bromide, and lauryl iodide, myristyl chloride, myristyl bromide, and myristyl iodide, and stearyl chloride, stearyl bromide, and stearyl iodide), aralkyl halides (e.g., benzyl bromide and phenethyl bromide), and other agents. Preferred salts include monohydrochloride, hydrogen sulfate, methanesulfonate, phosphate, or nitrate.

[0533] The present invention also includes prodrugs and solvates of the compounds of the present invention. The term "prodrug" means the following compound, once the compound is given to a subject, the compound is chemically transformed by a metabolic process or a chemical process to obtain the compound and / or its salt and / or solvate described herein. Any compound that can be transformed in vivo to provide a bioactive substance is a prodrug within the scope and spirit of the present invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester, which acts as a prodrug by hydrolyzing in vivo to obtain the compound itself. Prodrugs include acid derivatives known to practitioners in the art, such as esters prepared by the reaction of a parent acid with a suitable alcohol or amides, or anhydrides, or mixed anhydrides prepared by the reaction of a parent acid compound with a substituted or unsubstituted amine. Simple aliphatic or aromatic esters, amides and anhydrides produced by acidic groups hanging on the compounds of the present invention are specific prodrugs. In some cases, it is desirable to prepare double ester type prodrugs such as (acyloxy) alkyl esters or ((alkoxycarbonyl) oxygen base) alkyl esters. Specifically, the C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C 12 Substituted aryl and C7-C 12 Arylalkyl esters.

[0534] The prodrug is preferably administered orally, since hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.

[0535] Various forms of prodrugs are well known in the art. Examples of such prodrug derivatives are shown in:

[0536] a) Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985), and Methods in Enzymology, 112: 309-396, K. Widder et al., eds., Academic Press (1985);

[0537] b) Bundgaard, H., Chapter 5, "Design and Application of Prodrugs," ATextbook of Drug Design and Development, pp.113-191, P.Krosgaard-Larsen etal., eds., Harwood Academic Publishers (1991); and

[0538] c) Bundgaard, H., Adv.Drug Deliv.Rev., 8:1-38 (1992),

[0539] Each of the above references is incorporated into this application by reference.

[0540] Esters are generally compounds derived from acids (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced by an -O-alkyl (alkoxy) group. Esters are generally derived from carboxylic acids and alcohols.

[0541] Esters of the compounds of the invention are preferably esters which are hydrolysable in vivo.

[0542] The term "in vivo hydrolyzable ester" as used herein is understood to mean an in vivo hydrolyzable ester of a compound of the invention containing a carboxyl or hydroxyl group, for example a pharmaceutically acceptable ester that is hydrolyzed in the human or animal body to produce the parent acid or alcohol. Pharmaceutically acceptable esters suitable for carboxyl groups include, for example, alkyl esters, cycloalkyl esters and optionally substituted phenylalkyl esters (particularly benzyl esters), C1-C6 alkoxymethyl esters (e.g. methoxymethyl esters), C1-C6 alkanoyloxymethyl esters (e.g. pivaloyloxymethyl esters), phthalyl esters, C3-C8 cycloalkyloxycarbonyloxy-C1-C6 alkyl esters (e.g. 1-cyclohexylcarbonyloxyethyl esters); 1,3-dioxole-2-carbonylmethyl esters, for example 5-methyl-1,3-dioxole-2-carbonylmethyl esters; and C1-C6-alkoxycarbonyloxyethyl esters, for example 1-methoxycarbonyloxyethyl esters, and the esters may be formed on any carboxyl group of the compound of the invention.

[0543] In vivo hydrolyzable esters of compounds of the invention containing hydroxyl groups include inorganic acid esters (e.g., phosphates), [α]-acyloxyalkyl ethers, and related compounds that break off to form the parent hydroxyl group due to in vivo hydrolysis of the esters. Examples of [α]-acyloxyalkyl ethers include acetoxymethyl ether and 2,2-dimethylpropionyloxymethyl ether. Selection of groups that form in vivo hydrolyzable esters with hydroxyl groups include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to form alkyl carbonates), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to form carbamates), dialkylaminoacetyl and carboxyacetyl. The present invention includes all such esters.

[0544] "Solvate" refers to a form of a compound that is associated with a solvent or water (also referred to as a "hydrate"), usually by solvolysis. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid, etc. The compounds of the present invention can be prepared, for example, in a crystalline form and can 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 is capable of separation, for example when one or more solvent molecules are introduced into the crystal lattice of the crystalline solid. "Solvate" includes both solution phase and separable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0545] "Subjects" or "subjects" or "hosts" intended for administration include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents such as mice and rats, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0546] "Effective amount" means the amount of a compound that, when administered to a subject for treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. "Effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. "Therapeutically effective amount" refers to an effective amount for therapeutic treatment. "Prophylactically effective amount" refers to an effective amount for preventive treatment.

[0547] "Prevention" or "prophylactic treatment" refers to the reduction of the risk of acquiring or developing a disease or condition (i.e., preventing the development of at least one clinical symptom of a disease in a subject susceptible to the disease, or not exposed to the pathogen prior to the onset of the disease).

[0548] The term "treating," ...

[0549] As used herein, the term "inhibition" or "inhibiting" refers to reducing or suppressing a particular condition, symptom or disease, or significantly reducing the baseline activity of a biological activity or pathology.

[0550] The term "inhibitor" used in this application 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). For example, an inhibitor exerts its effect by reducing or suppressing the activity of a target molecule and / or reducing or suppressing signal transduction.

[0551] The compounds described herein may exist in stereoisomeric forms (e.g., they contain one or more asymmetric carbon atoms). Individual stereoisomers (enantiomers and diastereomers) and mixtures of these isomers are included within the scope of the present invention. Similarly, it should be understood that the compounds or salts described herein may exist in tautomeric forms different from those shown in the formula, and these are also included within the scope of the present invention. It should be understood that the present invention includes all combinations and subclasses of the specific groups defined above. The scope of the present invention includes mixtures of stereoisomers and purified enantiomers or mixtures enriched in enantiomers / diastereomers. It should be understood that the present invention includes all combinations and subclasses of the specific groups defined above.

[0552] One enantiomer of the compounds described in the present application may show superior activity compared to the other enantiomer. Therefore, all stereochemistry is considered as part of the present invention. When necessary, the separation of racemic substances can be achieved as follows: using a chiral column to perform HPLC, or using a resolving agent such as camphane acyl chloride (camphonic chloride) to perform resolution (as described in Young, SD et al., Antimicrobial Agents and Chemotherapy 1995, 2602-2605).

[0553] The present invention also includes isotopically labeled compounds which are identical to those described by formula (I) except for the fact that one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present 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. 18O. 31 P. 32 P. 35 S. 18 F. 36 Cl, 123 I and 125 I.

[0554] Compounds of the invention and pharmaceutically acceptable salts of said compounds containing the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the invention. Isotopically labeled compounds of the invention, for example, compounds into which a radioactive isotope such as 3 H or 14 C, for use in drug and / or substrate tissue distribution assays. 3 H and carbon-14 14 C isotopes are particularly preferred due to their ease of preparation and detectability. 11 C and 18 F isotopes are particularly useful in PET (positron emission tomography), and 125 I isotopes are particularly useful for SPECT (single photon emission computed tomography), all of which are used for brain imaging. In addition, heavier isotopes such as deuterium, 2 H substitution may provide certain therapeutic benefits resulting in greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and is therefore preferred in some circumstances. 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 and / or examples below, by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0555] Specifically, the present invention relates to the following embodiments.

[0556] In one embodiment, the present invention relates to an immunogenic composition comprising an adjuvant, which may include, but is not limited to, thiolase (acetoacetyl-CoA transferase) inhibitors, 2) HMG-CoA synthase inhibitors, 3) HMG-CoA reductase inhibitors, 4) mevalonate kinase inhibitors, 5) phosphomevalonate kinase inhibitors, 6) mevalonate-5-pyrophosphate decarboxylase inhibitors, 7) isopentenyl pyrophosphate isomerase inhibitors, 8) farnesyl pyrophosphate synthase inhibitors, 9) geranylgeranyl pyrophosphate synthase inhibitors and 10) geranylgeranyltransferase (I, II) inhibitors.

[0557] The thiolase (acetoacetyl-CoA transferase) inhibitor includes, but is not limited to, L-660631 described in biochemical and biophysical research communications, 1989, 163, 548-553.

[0558] The HMG-CoA synthase inhibitors include, but are not limited to, L-659699 described in Biochem. J (1993) 289, 889-895, 1233A / F-244 described in Agric. Biol. Chem., 55 (12), 3129-3131, 1991, and Dihydroxerulin described in Tetrahedron, 2000, 56 (3), 479-487, as well as those compounds disclosed in the following documents: US5064856; EP0411703A1; Agric. Biol. Chem., 1991, 55 (12): 3129-3131; Bioorg. Med. Chem., 1998, 6: 1255-1272; Biochem. Biophys. Res. Commun., 1999, 265: 536–540. , all of which patents and other publications are incorporated into this application by reference.

[0559] The HMG-CoA reductase inhibitors include, but are not limited to, those compounds disclosed in the following documents: US5102911-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; 4494-A; EP415488-A; US4992429-A; EP411420-A2; EP409399-A1; EP408806-A1; DE3918364-A; 50675-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; US487628 0-A; EP346759-A2; EP422102-A1; EP344602-A1; DE3805884-A; EP330172-A; EP327166-A; 4-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 etal.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, all of these patents and other publications are incorporated into this application by reference. .

[0560] In a preferred embodiment, the HMG-CoA reductase inhibitor is a statin. Exemplary statins are selected from: pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, simvastatin, cerivastatin, or their pharmaceutically acceptable salts, esters, prodrugs, solvates. Preferred HMG-CoA reductase inhibitors are those drugs that have been listed, and most preferably simvastatin, lovastatin and mevastatin, or their pharmaceutically acceptable salts, esters, prodrugs, solvates. Methods for preparing HMG-CoA reductase inhibitors are well known to those skilled in the art and these ingredients include those commercially available. HMG-CoA reductase inhibitors can be used in the form of their free acid form, their ester form, or their pharmaceutically acceptable salts. These pharmaceutically acceptable salts include, for example, sodium salts, calcium salts, aluminum salts, and ester salts. HMG-CoA reductase inhibitors can be used in the form of racemic mixtures or more active appropriate stereoisomers.

[0561] The farnesyl pyrophosphate synthase inhibitors include, but are not limited to, those compounds disclosed in the following documents: 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.Che m.Lett.,2008,18:2878–82; J.Med.Chem.,2008,51:2187-95; Proc.Natl.Acad.Sci.USA,2007,104:10022–7; Tetrahedron Lett.2011,52:2285–87; Chem.Commun.,2010,46:5340–5342; ExpertOpin.Ther.Pat.2011,21(9):1433–1451; J.Pharmacol.Exp.Ther.,2001,296:235–42; J.Med.Chem.2 003,46:5171-5183; J.Med.Chem.2005,48:2957-2963; J.Med.Chem.2006,49:5804-58 14; J.Med.Chem.2013,56:7939-7950; J.Med.Chem.2008,51:2187–2195; ChemMedChem 2015,10:1884-1891; Biochim.Biophys.Acta,2014,1840 1840:1051–1062; J.Med.Chem.2007;50:5967-75, all of which are incorporated herein by reference as well as other publications.

[0562] In a further embodiment, the farnesyl pyrophosphate synthase inhibitor is a bisphosphonic acid compound or a pharmaceutically acceptable salt, ester, prodrug, solvate thereof. The term "bisphosphonic acid (salt)" refers to a compound characterized by two phosphonic acid groups bonded to a central (geminal) carbon atom via a phosphine ether bond. This PCP structure is shown in Formula I below. It should be noted that the term "bisphosphonic acid" used herein in reference to the therapeutic agents of the present invention is also intended to include bisphosphonic acid (salt), bisphosphonic acids and their salts and derivatives. Unless otherwise specified, the specific nomenclature used in reference to bisphosphonic acids is not intended to limit the scope of the present invention.

[0563] Bisphosphonic acid (salts) as drugs are described in, for example, 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, all of which are incorporated herein by reference.

[0564] "Bisphosphonic acids and pharmaceutically acceptable salts thereof" for use as drugs are also described in, for example, U.S. Patents 4,509,612, 4,666,895, 4,719,203, 4,777,163, 5,002,937, 4,971,958 and 4,958,839 and European Patent Applications 252,504 and 252,505, all of which are incorporated herein by reference.

[0565] Preferred bisphosphonic acids or pharmaceutically acceptable salts thereof are selected from alendronic acid, simadronic acid, clodronic acid, EB-1053, tiludronic acid, etidronic acid, ibandronic acid, incadronic acid, minodronic acid, neridronic acid, olpadronic acid, risedronic acid, piridronic acid, pamidronic acid, zolamdronic acid or an acceptable salt thereof, such as ibandronic acid monosodium salt monohydrate.

[0566] The geranylgeranyl pyrophosphate synthase inhibitors include, but are not limited to, those compounds disclosed in the following documents: 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. USA, 2012, 109 (11): 4058-4063, all of which are incorporated herein by reference.

[0567] The geranylgeranyltransferase (I, II) inhibitors include, but are not limited to, those compounds disclosed in the following documents: 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; and GGTI-298, which is described in McGuire et al (1996) Platelet-derived growth factor receptor tyrosine kinase phosphorylation requires protein geranylgeranylation but not farnesylation. J. Biol. Chem. 27127402. PMID: 8910319, and has the following structure:.

[0568]

[0569] All of these patents and publications are incorporated into this application by reference.

[0570] In addition to the above-mentioned inhibitors, the present invention also relates to other substances that influence the geranylgeranylation of proteins, which other substances can also be included in the immunogenic composition as adjuvants.

[0571] In another aspect, the farnesyl pyrophosphate synthase inhibitor is a compound of the following formula (i.e., a compound of the TH-Z80 series) or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof:

[0572]

[0573] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0574] In a further embodiment, n is an integer from 1 to 20. In a further embodiment, n is an integer from 1 to 15.

[0575] In a more specific embodiment, the compound is selected from:

[0576] n＝ Compound No. 1 TH-Z79 2 TH-Z148 3 TH-Z149 4 TH-Z150 5 TH-Z151 6 TH-Z80 7 TH-Z152 8 TH-Z81 9 TH-Z153 10 TH-Z82 11 TH-Z154 12 TH-Z155.

[0577] Therefore, in one embodiment, the present invention relates to an immunogenic composition, the composition comprising an adjuvant, the adjuvant being selected from the above-mentioned TH-Z80 series of compounds or their pharmaceutically acceptable salts, esters, prodrugs, solvates. In one embodiment, the present invention also relates to the use of the above-mentioned compounds or their pharmaceutically acceptable salts, esters, prodrugs, solvates as adjuvants in the preparation of immunogenic compositions for the prevention or treatment of diseases.

[0578] In another aspect, the present invention also relates to novel bisphosphonic acid compounds (i.e. compounds of the TH-Z97 series) as inhibitors of farnesyl pyrophosphate synthase (FPPS) or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0579]

[0580] wherein n is an integer of 1-24, preferably n is an integer of 1-12.

[0581] In a further embodiment, n is an integer from 1 to 20. In a further embodiment, n is an integer from 1 to 15.

[0582] In a more specific embodiment, the compound is selected from:

[0583] n＝ Compound No. 1 TH-Z156 2 TH-Z157 3 TH-Z158 4 TH-Z159 5 TH-Z160 6 TH-Z97 7 TH-Z161 8 TH-Z98 9 TH-Z162 10 TH-Z99 11 TH-Z198 12 TH-Z163.

[0584] Therefore, in one embodiment, the present invention relates to an immunogenic composition, the composition comprising an adjuvant, the adjuvant being selected from the above-mentioned TH-Z97 series of compounds or their pharmaceutically acceptable salts, esters, prodrugs, solvates. In one embodiment, the present invention also relates to the use of the above-mentioned compounds or their pharmaceutically acceptable salts, esters, prodrugs, solvates as adjuvants in the preparation of immunogenic compositions for preventing or treating diseases.

[0585] In another aspect, the present invention also relates to novel bisphosphonic acid compounds or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof as inhibitors of farnesyl pyrophosphate synthase (FPPS), wherein the compounds have the following formula:

[0586]

[0587] wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl;

[0588] R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;

[0589] R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;

[0590] or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and

[0591] R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl.

[0592] In one embodiment, R1 is selected from hydrogen, C 1-10 Alkyl, C 1-10 Alkynyl, C 1-10 Alkylamino, C1-10 Alkylthio, halogen, hydroxy, indazolyl, C 1-10 Alkoxy, C substituted by phenyl or pyridyl 1-10 The alkoxy group, the pyridyl group is optionally substituted with a carbamoyl group.

[0593] In a further embodiment, R1 is selected from hydrogen, 4-methylphenethoxy, 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-yl, hydroxy, bromine.

[0594] In one embodiment, R2 is selected from hydrogen, C 1-10 In a further embodiment, R2 is selected from hydrogen, octyloxy, bromine.

[0595] In one embodiment, R3 is selected from hydrogen, C 1-10 Alkyl, C 1-10 In a further embodiment, R3 is selected from hydrogen, methyl, hexyloxy.

[0596] In one embodiment, R2 and R3 together with the carbon atoms to which they are attached form a benzene ring.

[0597] In one embodiment, R4 is selected from hydrogen, C 1-10 In a further embodiment, R4 is selected from hydrogen, octyloxy.

[0598] In a further embodiment, the compound is selected from:

[0599]

[0600]

[0601] Therefore, in one embodiment, the present invention relates to an immunogenic composition, the composition comprising an adjuvant, the adjuvant being selected from the above-mentioned o-aminopyridine compound or its pharmaceutically acceptable salt, ester, prodrug, solvate. In one embodiment, the present invention also relates to the use of the above-mentioned compound or its pharmaceutically acceptable salt, ester, prodrug, solvate as an adjuvant in the preparation of an immunogenic composition for preventing or treating a disease.

[0602] In another aspect, the present invention also relates to novel bisphosphonic acid compounds or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof as inhibitors of farnesyl pyrophosphate synthase (FPPS), wherein the compounds have the following formula:

[0603]

[0604] in

[0605] R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;

[0606] R6 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;

[0607] R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl, and heteroaryl; and

[0608] R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl, and heteroaryl.

[0609] In one embodiment, R5 is selected from C 1-10 Alkoxy.

[0610] In a further embodiment, the compound is selected from:

[0611]

[0612] Therefore, in one embodiment, the present invention relates to an immunogenic composition, the composition comprising an adjuvant, the adjuvant being selected from the above-mentioned meta-aminopyridine compound or its pharmaceutically acceptable salt, ester, prodrug, solvate. In one embodiment, the present invention also relates to the use of the above-mentioned compound or its pharmaceutically acceptable salt, ester, prodrug, solvate as an adjuvant in the preparation of an immunogenic composition for preventing or treating a disease.

[0613] In another aspect, the present invention also relates to novel bisphosphonic acid compounds as geranylgeranyl pyrophosphate synthase inhibitors or pharmaceutically acceptable salts, esters, prodrugs, solvates thereof, wherein the compounds have the following formula:

[0614]

[0615] in

[0616] R9 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl;

[0617] R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl, and heteroaryl;

[0618] R 11 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl, and heteroaryl; and

[0619] R 12is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, aryl and heteroaryl.

[0620] In one embodiment, R9 is selected from C 1-10 Alkoxy.

[0621] In a further embodiment, the compound is selected from:

[0622]

[0623] Therefore, in one embodiment, the present invention relates to an immunogenic composition, the composition comprising an adjuvant, the adjuvant being selected from the above-mentioned benzyl diphosphate compound or its pharmaceutically acceptable salt, ester, prodrug, solvate. In one embodiment, the present invention also relates to the use of the above-mentioned compound or its pharmaceutically acceptable salt, ester, prodrug, solvate as an adjuvant in the preparation of an immunogenic composition for preventing or treating a disease.

[0624] Bisphosphonic acid compounds

[0625] As early as more than 40 years ago, Fleisch et al. found that pyrophosphate has the effect of inhibiting ectopic calcification. However, pyrophosphate is unstable and easily inactivated by enzyme hydrolysis. Later, the POP group in the pyrophosphate structure that is easily hydrolyzed by enzymes was transformed into a PCP group that is stable to enzymes, and then a series of bisphosphonate compounds were developed, which have played a good therapeutic effect in the treatment of osteoporosis. Representative drugs include the first generation of etidronate, the second generation of clodronate, pamidronate and tiludronate, and the third generation of alendronate, neridronate, olpadronate, risedronate, ibandronate, zoledronate, etc. Unlike the first generation of bisphosphonic acids, the second and third generation bisphosphonic acids mainly act on the key target enzyme FPPS (Farnesyl pyrophosphate synthase) in the terpene biosynthesis pathway through the protonation of their N, thereby causing osteoclast apoptosis.

[0626] The third-generation representative drug zoledronic acid has excellent effects in treating osteoporosis. It also has certain effects in treating cancer bone metastasis caused by abnormal bone metabolism caused by bone metastasis of malignant tumors such as multiple myeloma, breast cancer, prostate cancer and lung cancer, but the effect is not significant.

[0627] One embodiment of the present invention relates to the novel bisphosphonic acid compound as described above, which can be used to prepare metabolic bone disease drugs, malaria treatment drugs, eukaryotic tumor cell proliferation inhibitors, drugs for preventing and / or treating tumors, immunotherapy drugs and adjuvants for vaccines.

[0628] In the drug for treating metabolic bone diseases, the drug for treating malaria, the inhibitor of eukaryotic tumor cell proliferation, the drug for preventing and / or treating tumors or the immunotherapy drug, the mass content of the compound represented by formula I is 0.001-90%.

[0629] In the vaccine, the mass content of the compound represented by formula I is 0.001-90%.

[0630] The drug for treating metabolic bone disease, drug for treating malaria, inhibitor of eukaryotic tumor cell proliferation, drug for preventing and / or treating tumor, immunotherapy drug and vaccine can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or wrapped with other substances.

[0631] The bisphosphonic acid compound of the present invention synthesizes asymmetric bisphosphonic acid at positions 4, 5, 6, and 7 of benzimidazole for the first time by improving the modification of the terminal carboxyl group. The bisphosphonic acid compound maintains the activity of imidazole phosphonic acid on FPPS and has a good inhibitory effect on GGPPS, malaria and tumor cell proliferation, especially has a good effect as a vaccine adjuvant and immunotherapy drug, and can be used to prepare drugs for treating metabolic bone diseases, drugs for treating malaria, eukaryotic tumor cell proliferation inhibitors, drugs for preventing and / or treating tumors, immunotherapy drugs, and as a vaccine adjuvant for preparing vaccines.

[0632] antigen

[0633] In one embodiment, the immunogenic composition of the present invention comprises an antigen derived from a bacterium, a virus, a parasite or a tumor. In certain aspects, the one or more antigens are each independently a microbial antigen, a self antigen, a tumor antigen, an allergen or an addictive substance. Antigens of the present invention also include those described in International Patent Application WO2011 / 148356.

[0634] Antigens may be obtained by recombinant means or peptide synthesis, or obtained from natural sources or extracts, and may be derived from any living or non-living organism.

[0635] Antigens can be derived from bacteria such as, for example, anthrax, campylobacter, cholera, diphtheria, enterotoxigenic E. coli, giardia, gonococcus, Helicobacter pylori, Hemophilus influenza B, non-typable Haemophilus influenzae, meningococcus, pertussis, pneumococcus, salmonella, shigella, Streptococcus B, Group A Streptococcus, tetanus, Vibrio cholerae, yersinia, Staphylococcus, Pseudomonas species, and Clostridia species.

[0636] Alternatively, the antigen can be derived from a virus, such as, for example, adenovirus, dengue serotypes 1 to 4, ebola (Jahrling et al., Arch Virol Suppl, 11: 135-140, 1996), enterovirus, hepatitis serotypes A to E (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 et al., 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, papillomavirus, parvovirus B19, polio, rabies, rotavirus, rubella, rubeola, vaccinia, vaccinia constructs containing genes encoding other antigens such as malarial antigens, varicella, and yellow fever. Alternatively, the antigens can be derived from parasites, including, for example: Entamoeba histolytica (Zhang et al., Infect Immun 63:1349-1355); Plasmodium (Bathurst et al., Vaccine 11:449-456, 1993), Toxoplasmosis, and Helminth.

[0637] Alternatively, the antigen may be a tumor specific antigen (TSA) or a tumor associated-antigen (TAA). Tumor specific antigen refers to a novel antigen that is only expressed on the surface of a certain tumor cell and not present on normal cells, and is therefore also called a unique tumor antigen. Such tumor associated antigens are known in the art, and common tumor specific antigens include (1) alpha fetoprotein (AFP); (2) carcinoembryonic antigen (CEA); (3) CA-125; (4) MUC-1; (5) epithelial cell 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 correlated with certain tumor cells. They are not usually found, or are found to a lesser extent, on normal cells. Such tumor-associated antigens are known in the art. 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) CD7 9b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2 R; (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; and (36) TENB2.It is incorporated into the present application by reference.

[0638] In a further embodiment, the antigen is derived from Middle East Respiratory Syndrome (Mers) virus, Hepatitis B virus, melanoma.

[0639] In another aspect, the immunogenic composition of the present invention further comprises 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 complexes), Virosomes (virion), GLA-SE, liposomes, edible oils, saponin, AF03, TLR agonists.

[0640] In a further embodiment, the another adjuvant is selected from a TLR agonist. Exemplary TLR agonists are: TLR1 stimulators (such as triacyl lipoprotein), TLR2 stimulators (such as peptidoglycan, yeast polysaccharide (zymosan), HMGB1 (high-speed swimming protein 1), lipoteichoic acid), TLR3 stimulators (double-stranded RNA such as PolyI: C), TLR4 stimulators (such as LPS, MPL, RC529, GLA, E6020), TLR5 stimulators (flagellin), TLR6 stimulators (such as triacyl lipoprotein, lipoteichoic acid), TLR7 / 8 stimulators (single-stranded RNA, imiquimod), TLR9 stimulators (DNA, such as CPGODN), C-lectin ligands (such as laminarin), CD1d ligands (such as α-galactosylceramide)

[0641] Vaccines used

[0642] The adjuvant and immunogenic composition described in the present application can be applied to various vaccines, including but not limited to: BCG, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis D vaccine, hepatitis E vaccine, influenza vaccine, polio vaccine, diphtheria and pertussis vaccine, measles vaccine, Japanese encephalitis vaccine, rabies vaccine, hemorrhagic fever vaccine, pneumonia vaccine, meningococcal vaccine, hepatitis A vaccine, mumps vaccine, influenza vaccine, rubella vaccine, varicella vaccine, AIDS vaccine, malaria vaccine; and cancer treatment and prevention vaccines, including but not limited to melanoma therapeutic 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, liver cancer therapeutic vaccine, liver cancer preventive vaccine, prostate cancer preventive vaccine, prostate cancer preventive vaccine.

[0643] Indications

[0644] The immunogenic compositions of the present invention can be used to treat a variety of diseases or conditions, including those caused by bacteria, viruses, fungi, and parasites.

[0645] In various aspects of the invention, bacteria include, but are not limited to, the following species and combinations thereof: Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces israelli, Actinomyces viscosus, Aeromonas hydrophila, Alcaliges eutrophus, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus species, Bacillus antracis, Bacillus brevis. pumilus), Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bacteroides species, Bordetella species, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella species, Burkholderia cepacia, Burkholderia glumae, Brachyspira species, Brachyspira hyodysenteria, Brachyspira pilosicoli, Campylobacter species, Campylobacter coli, Campylobacter fetus fetus), Campylobacter hyointestinalis, Campylobacter jejuni, Chlamydia psittaci, Chlamydia trachomatistrachomatis), Chlamydophila species, Chromobacterium viscosum, Clostridium species, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium species, Corynebacterium diphtheriae, Ehrlichia canis, Enterobacter species, Enterobacter aerogenes, Enterococcus species, Erysipelothrix rhusiopathieae, Escherichia species, Escherichia coli coli), Fusobacterium nucleatum, Haemophilus species, Haemophilus influenzae, Haemophilus somnus, Helicobacter species, Helicobacter pylori, Helicobacter suis, Klebsiella species, Klebsiella pneumoniae, Lactobacillus acidophilis, Lawsonia intracellularis, Legionella species, Legionella pneumophilia, Leptospira species (e.g., Leptospira canis), canicola), Leptospira typhimurium, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-pashino,borgpetersenii hardjo-prajitno), Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira, Leptospira bratislava, etc.), Listeria species, Listeria monocytogenes, Meningococcal bacteria, Moraxella species, Mycobacterium species, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, kansaii), Mycobacterium gordonae, Mycoplasma species (such as Mycoplasma hyopneumoniae, Mycoplasma synoviae, Mycoplasma hyorhinis, Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides, Neisseria species, Neisseria gonorrhoeae, Neisseria meningitidis, Odoribacter denticanis, Pasteurella species, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida multocida), Photorhabdus luminescens, Porphyromonas gingivalis, Porphyromonas gulae, Porphyromonas salivarius,salivosa), Propionibacterium acnes, Proteus species, Proteus vulgaris, Pseudomonas species, Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas species B11-1, Psychrobacter immobilis, Rickettsia spp., Rickettsia prowazekii prowazekii), Rickettsia rickettsia, Salmonella species, Salmonella bongori, Salmonella choleraeuis, Salmonella dublin, Salmonella enterica, Salmonella newport, Salmonella typhimurium, Salmonella typhi, Serratia marcescens, Shigella species, Spirlina platensis, Staphylococci species, Staphlyoccocus aureus, Staphylococcus epidermidis epidermidis), Staphylococcus hyicus, Streptococcus species, Streptobacillus moniliformis, beta-hemolytic Streptococcus, Streptococcus pyogenespyogenes) (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Viridans Group), Streptococcus faecalis, Streptococcus bovis, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus (anaerobic species), Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptomyces albus, Streptomyces cinnamoneus, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius acidocaldarius, Syechocystis, Treponena species, Treponema denticola, Treponema minutum, Treponema palladium, Treponema pertenue, Treponema phagedenis, Treponema refractorens, Treponema vincentii, Vibrio species, Vibrio cholerae and Yersinia species.

[0646] In certain aspects, the virus is a virus that infects animals, including but not limited to the following species and combinations thereof: avian herpesvirus, avian influenza virus, avian leukosis virus, avian paramyxovirus, border disease virus, bovine coronavirus, bovine epidemic fever virus, bovine herpesvirus, 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 coronavirus (CCV), canine distemper virus, canine herpesvirus, equine herpesvirus, canine influenza virus, canine parainfluenza virus, canine parvovirus, canine respiratory coronavirus, classical swine fever virus, Eastern equine encephalitis virus (EEE), equine infectious anemia virus, equine influenza virus, West Nile virus, feline calicivirus, feline enteric coronavirus, Feline immunodeficiency virus, feline infectious peritonitis virus, feline herpesvirus, feline influenza virus, feline leukemia virus (FeLV), feline viral rhinotracheitis virus, lentivirus, Marek's disease virus, Newcastle disease virus, ovine herpesvirus, ovine parainfluenza type 3 virus, ovine progressive pneumonia virus, ovine pulmonary adenocarcinoma virus, pantropical CCV, porcine circovirus (PCV) type I, PCV type II, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine herpesvirus, porcine parvovirus, porcine reproductive and respiratory syndrome (PRRS) virus, pseudorabies virus, rabies virus, provirus, rhinovirus, rinderpest virus, swine influenza virus, transmissible gastroenteritis virus, turkey coronavirus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, West Nile virus, and western equine encephalitis virus.

[0647] In certain aspects, the virus is a virus that infects humans, including but not limited to: Adenoviridae (most adenoviruses); Arenaviridae (hemorrhagic fever viruses); Astroviridae; Bunyaviridae (e.g., Hantavirus, Bunyavirus); virus, phlebovirus, and nairovirus); Calciviridae (e.g., strains that cause gastroenteritis); Coronaviridae (e.g., coronaviruses); Filoviridae (e.g., Ebola virus); Flaviridae (e.g., hepatitis C virus, dengue virus, encephalitis virus, yellow fever virus); Hepadnaviridae (hepatitis B virus); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus); Iridoviridae (e.g., African swine fever virus); Norwalk virus and related viruses; Orthomyxoviridae (e.g., influenza virus); Papovaviridae (papillomavirus, polyomavirus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Middle East respiratory syndrome virus; =Mers virus; Parvoviridae (parvoviruses); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Poxviridae (e.g., smallpox virus, vaccinia virus, poxvirus); Reoviridae (reovirus, reovirus, orbivirus, and rotavirus); Retroviruses (e.g., human immunodeficiency viruses such as HIV-1 or HIV-2 (also known as HTLV-III, LAV or HTLV-III / LAV) or HIV-III; and other isolates such as HIV-LP); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Togaviridae (e.g., equine encephalitis virus, rubella virus); and unclassified viruses (e.g., the etiology of spongiform encephalopathy, the etiology of hepatitis D (thought to be a defective satellite of hepatitis B virus).

[0648] In some aspects of the invention, fungi include, but are not limited to, spores, molds, and yeasts (eg, Candida species).

[0649] In some aspects of the invention, parasites include, but are not limited to, the following species and combinations thereof: proteins from the genus Anaplasma, Fasciola hepatica (liver fluke), Coccidia, Eimeria species, Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (dog heartworm), Uncinaria (hookworm), Trypanosoma species, Leishmania species, Trichomonas species, Cryptosporidium parvum, Babesia, Schistosoma, Cestodia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, Isopsora. In some aspects, parasites are ectoparasites. In some aspects, ectoparasites include, but are not limited to, ticks, including species of Ixodes, Rhipicephalus, Dermacentor, Boophilus microplus, Hyalomma, Haemaphysalis and combinations thereof.

[0650] In some aspects of the invention, 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, intraepithelial neoplasia, lymphoma (e.g., follicular lymphoma), liver cancer, lung cancer (e.g., small cell and non-small cell lung cancer), leukemia (e.g., hairy cell leukemia, chronic myeloid leukemia, cutaneous T-cell leukemia), melanoma (e.g., 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 sarcomas (e.g., squamous cell tumor, renal cell tumor, prostate tumor, bladder tumor or colon tumor).

[0651] Administration and dosage

[0652] For treatment, one or more immunogenic compositions of an effective amount can be applied to a subject. The administration of the pharmaceutical composition of the present invention can be achieved by any means known to those skilled in the art. Preferred routes of administration include, but are not limited to, parenteral (e.g., intramuscular, subcutaneous, intradermal, intravenous injection), topical (e.g., transdermal) or mucosal (e.g., oral, intranasal, intravaginal, rectal, transbuccal, intraocular or sublingual) administration to the skin. In the case of cancer treatment, it can include intratumoral administration.

[0653] In some aspects of the invention, an "effective amount" of an immunogenic composition refers to an amount that is necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an immunogenic composition for treating a condition may be an amount necessary to eliminate a microbial infection or a tumor. An effective amount used as a vaccine adjuvant may be an amount that can be used to enhance the immune response of a subject to a vaccine. The effective amount may vary according to the following parameters: the disease or condition to be treated, the specific immunogenic composition administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may determine the effective amount of a specific immunogenic composition empirically without excessive experimentation.

[0654] The immunogenic composition can be administered in a single dose regimen, or preferably in a multiple dose regimen, i.e., the main course of immunogenic composition vaccination is 1-10 independent doses, followed by other doses at subsequent time intervals according to the need to maintain and / or strengthen the immune response, such as 1-4 months to give a second dose, and if necessary, a subsequent dose after a few months or years. The medication regimen is also determined at least in part by the needs of the individual and depends on the judgment of the medical staff. Examples of suitable immunization regimens include: a first dose, followed by a second dose between the 7th day and 6 months, and optionally a third dose between 1 month and 2 years after the first vaccination; or other regimens sufficient to induce the desired virus neutralizing antibody titer conferring protective immunity, such as corresponding established pediatric immunogenic composition vaccination regimens. A booster dose given at a specific interval (e.g., every two years) can be supplemented to maintain satisfactory protective immunity.

[0655] The immunogenic composition of the present invention can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, ointment, cream, etc. The above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field. One or more pharmaceutically acceptable carriers can also be added to the above-mentioned preparations. The carriers include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0656] When it is desired that one or more immunogenic compositions be systemically delivered, it can be formulated for parenteral administration by injection (e.g., push injection or instillation). For example, the sole of the foot, subcutaneous, intramuscular, peritoneal and nasal mucosal injections of the subject can be immunized. Injectable preparations can be presented in unit dosage form, for example, in an ampoule or in a multidose container with an added preservative. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can include a formulating agent, such as a suspending agent, a stabilizer and / or a dispersant.

[0657] Example

[0658] The present invention is further illustrated by reference to the following examples. However, it should be noted that these examples, like the above-described embodiments, are illustrative and should not be construed as limiting the scope of the present invention in any way.

[0659] In this application, unless otherwise indicated, the following abbreviations are used:

[0660] FPPS Farnesyl pyrophosphate synthase

[0661] GGPPS Geranylgeranyl pyrophosphate

[0662] Enzymes

[0663] SQS Squalene synthase

[0664] GGPP Geranylgeranyl pyrophosphate

[0665] GGOH Geranylgeraniol

[0666] OVA Chicken egg albumin

[0667] IgM Immunoglobulin M

[0668] IgG Immunoglobulin G

[0669] PBS Phosphate Buffered Saline

[0670] PBST Phosphate Buffered Tween

[0671] BSA bovine serum albumin

[0672] HRP Horseradish Peroxidase

[0673] OPD o-phenylenediamine

[0674] DMSO Dimethyl sulfoxide

[0675] min

[0676] h hour

[0677] DMAPP Dimethylallyl pyrophosphate

[0678] IPP isoprene pyrophosphate

[0679] PEI Polyethyleneimine

[0680] IFA Incomplete Freund's Adjuvant

[0681] CFA Complete Freund's Adjuvant

[0682] LPS Lipopolysaccharide

[0683] DC Dendritic cells

[0684] BMDC bone marrow-derived dendritic cells

[0685] FITC Fluorescein isothiocyanate

[0686] Mers Middle East Respiratory Syndrome

[0687] KLH Hemocyanin

[0688] TLR Toll-like receptor

[0689] In biological experiments, unless otherwise specified, the experimental animals used were mice of C57B / 6 strain purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and kept in the SPF animal room of the Biomedical Testing Center of Tsinghua University.

[0690] Biological Examples

[0691] We first discovered and confirmed that enzymes related to the mevalonate pathway can be used as targets for rational adjuvant design, and proposed that any substance that affects protein geranylgeranylation, such as inhibitors of all enzymes involved in the mevalonate pathway, can be used as adjuvants for the preparation of vaccines or immunogenic compositions. We demonstrated this view through the following biological experiments.

[0692] Example 1

[0693] Determining the adjuvant effect of HMG-CoA reductase inhibitors (statins) in mice

[0694] In the mevalonate pathway, HMG-CoA reductase catalyzes the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate, while HMG-CoA reductase inhibitors (i.e., statins) inhibit the action of HMG-CoA reductase.

[0695] We used statins as an example to demonstrate the role of HMG-CoA reductase inhibitors as adjuvants. In this assay, the role of statins as adjuvants in immunized mice was studied.

[0696] Eight statins commonly used in clinical practice were used in this assay, including pravastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, mevastatin, lovastatin, and simvastatin.

[0697] Sources of materials: Simvastatin was purchased from Tianjin Xiensi Company, mevastatin and lovastatin were purchased from Aladdin Company, pravastatin and atorvastatin were purchased from Anergy Company, fluvastatin, pitavastatin and rosuvastatin were purchased from Huazhong Weihai Company. OVA (Ovalbumin) antigen was purchased from Sigma Company.

[0698] The OVA evaluation system is used to reflect the adjuvant activity by antibody titer. Chicken ovalbumin, also known as chicken egg white albumin, is composed of 386 amino acids and has a molecular weight of about 43kD. It is usually used as a tool protein to study antibody titer.

[0699] Experimental method: Various statins and OVA antigens were mixed in a ratio of 1:1, and the concentrations of statins and OVA were both 10 mg / ml. The mouse strain used was C57B / 6. The experimental group of mice was injected with the mixture of various statins and OVA antigens, 20 μl per mouse. The control group of mice was injected with PBS or DMSO, 10 μl of each and 10 μl of OVA antigen in a ratio of 1:1 (PBS or DMSO was 10 μl, and antigen was 10 μl) per mouse. The mice were injected with 20 μl per mouse. 7 days and 14 days after immunization, the mice were bled from the eye sockets, and 100 μl of blood was collected from each mouse. The blood was placed at 4°C overnight, centrifuged at 6000 rpm for 8 minutes, the upper serum was aspirated, and the titers of anti-OVA IgM and IgG antibodies in the serum were measured.

[0700] Detection of OVA antibody titer: Dilute OVA protein with a carbonate solution of pH 9.6 to a concentration of 2μg / ml, apply to a 96-well ELISA plate, 50μl per well, and apply overnight at 4°C. Wash the ELISA plate 5 times with PBS containing 0.05% Tween 20, block with 1% BSA solution, 200μl per well, and block at room temperature for 2h. Wash the ELISA plate 5 times with PBST (Tween phosphate buffer), add mouse serum diluted in multiples after immunization with OVA, 50μl per well, and incubate at room temperature for 2h. After washing the ELISA plate 5 times with PBST, add HRP (horseradish peroxidase) labeled sheep anti-mouse IgM or IgG antibodies and incubate at room temperature for 45min. Wash the ELISA plate 5 times with PBST, add sodium citrate OPD (o-phenylenediamine) colorimetric solution, color for 10min in the dark, and then add sulfuric acid to stop. The OD490 reading was read using an enzyme-labeled instrument. The maximum serum dilution factor when the light absorption ratio between the experimental group and the control group was ≥2.0 was the titer of anti-OVA antibodies in the serum.

[0701] As shown in the results of Figure 2, compared with the control group mice treated with PBS or DMSO, mice treated with statins (such as fluvastatin, pitavastatin, rosuvastatin, simvastatin, lovastatin and mevastatin) produced significantly high titers of IgM and IgG antibodies, among which the adjuvant effects of simvastatin, lovastatin and mevastatin were the most significant. The insignificant effect of some statins may be due to poor bioavailability in vivo, such as poor solubility and absorption, and better effects can be achieved by adjusting the pharmacokinetic properties of these compounds (such as forming salts, esters or prodrugs, or forming aluminum salts).

[0702] Therefore, the above studies confirmed that HMG-CoA reductase inhibitors can be used as adjuvants to enhance the specific immune response caused by antigens.

[0703] Example 2

[0704] Determination of the adjuvant effect of FPPS inhibitors in mice

[0705] In the mevalonate pathway, farnesyl pyrophosphate synthase (FPPS) catalyzes the formation of farnesyl pyrophosphate (FPP) from dimethylallyl pyrophosphate (DPP). FPPS inhibitors inhibit the action of FPPS. We investigated the role of FPPS inhibitors as adjuvants in immunized mice. It is well known that bisphosphonates are potent inhibitors of FPPS. In the following assays, the adjuvant effects of various bisphosphonates were investigated.

[0706] (1) Adjuvant effect of TH-Z80 series compounds in mice

[0707] TH-Z80 series compounds are newly synthesized bisphosphonic acid compounds, and their structures are shown below.

[0708] First, referring to the humanized farnesyl diphosphate synthase (HsFPPS) purification method in the literature (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.) and the enzyme activity test method in the literature (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.), we determined the inhibitory activity of TH-Z80 series compounds against the FPPS target.

[0709] The assay method is briefly described as follows: Induce the expression of HsFPPS with 6 consecutive His at the N-terminus in vitro, collect and purify with Ni column. Perform in vitro HsFPPS enzyme activity test in 96-well plates, with 200 μl of solution in each well. The buffer of the system is 25 mM HEPES, 2.5 mM MgCl2, pH 7.4. Use DMAPP and IPP as reaction substrates, and monitor the changes of UV value at 360 nm wavelength in real time in the phosphate lyase system. Use ORIGIN 8.0 software to draw the graph and fit. IC of TH-Z80 series compounds for inhibiting FPPS 50 The values ​​are shown in the table below and are in micromoles per liter (μM).

[0710]

[0711]

[0712] From the above data, it can be seen that the TH-Z80 series compounds can effectively inhibit the activity of FPPS and are potent inhibitors of FPPS.

[0713] Next, we used the OVA evaluation system as described in Example 1 to determine the adjuvant effect of the TH-Z80 series compounds and its non-side chain substituted core compound BPH-266 in mice. Each test compound was formulated into a concentration of 2 mg / ml, 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 μl of the mixture (containing 50 μl of antigen and 50 μl of compound) into the right thigh muscle. 100 μl of blood was collected from the mouse eye sockets 7 and 14 days after immunization. After standing at 4°C overnight, it was centrifuged at 6000 rpm for 8 minutes, and the upper serum was aspirated. The anti-OVA IgM and IgG antibody titers in the serum were measured. The experimental results are shown in Figure 3 .

[0714] The BPH-266 compound used (see the document 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) has the following structure:

[0715]

[0716] like Figure 3As shown, mice treated with the TH-Z80 series of compounds produced significantly high titers of IgM and IgG antibodies, and the adjuvant effects of TH-Z80, TH-Z81, TH-Z152, and TH-Z153 were particularly significant.

[0717] Therefore, the above determinations confirm that the potent inhibitor of FPPS, namely the TH-Z80 series compounds of the present application, can act as adjuvants and can enhance the specific immune response caused by antigens.

[0718] (2) Adjuvant effect of TH-Z97 series compounds in mice

[0719] TH-Z97 series compounds are newly synthesized bisphosphonic acid compounds, and their structures are shown below.

[0720] First, we conducted a HsFPPS (humanized farnesyl pyrophosphate synthase) activity test to determine the inhibitory activity of the TH-Z97 series of compounds against the FPPS target. The same method as the above test of the TH-Z80 series of compounds was used. The IC50 values ​​of the TH-Z97 series of compounds for inhibiting FPPS are shown in the following table, in micromoles / liter.

[0721]

[0722] From the above data, it can be seen that the TH-Z97 series compounds can effectively inhibit the activity of FPPS and are potent inhibitors of FPPS.

[0723] Next, we used the OVA evaluation system described in Example 1 to determine the adjuvant effect of TH-Z97 in mice. The results are shown in Figure 4 middle.

[0724] like Figure 4 As shown, IgG antibodies with significantly higher titers were produced in mice treated with TH-Z97 compared with control mice treated with PBS.

[0725] Therefore, the above determinations confirm that the potent inhibitor of FPPS, namely the TH-Z97 series compounds of the present application, can act as adjuvants and can enhance the specific immune response caused by antigens.

[0726] (3) Data on the activity of o-aminopyridine compounds and their activity in HsFPPS (humanized farnesyl pyrophosphate synthase)

[0727] We tested the HsFPPS (humanized farnesyl pyrophosphate synthase) activity of some newly synthesized o-aminopyridine bisphosphonic acid compounds and determined the inhibitory activity of these compounds against the FPPS target. The same method as the above test of TH-Z80 series compounds was used. The IC values ​​of these compounds for inhibiting FPPS 50 The values ​​are shown in the table below and are in micromoles per liter (μM).

[0728]

[0729] From the above data, it can be seen that the o-aminopyridine bisphosphonic acid compound can effectively inhibit the activity of FPPS and is a potent inhibitor of FPPS.

[0730] The adjuvant effect of this class of o-aminopyridine bisphosphonic acid compounds (eg TH-Z93) in mice was demonstrated in the following test (4).

[0731] (4) Adjuvant effects of other FPPS inhibitor compounds in mice

[0732] In this assay, the OVA evaluation system as described in Example 1 was used to determine the adjuvant effects of 8 commonly used bisphosphonic acid compounds in clinical practice and the TH-Z80 and TH-Z93 compounds of the present invention in mice, and the difference in the activity of the new bisphosphonic acid compound synthesized in the present invention and the bisphosphonic acid drugs on the market as adjuvants was compared. The 8 bisphosphonic acid compounds are zoledronate, pamidronate, alendronate, ibandronate, neridronate, risedronate, olpadronate, and minodronate. These compounds were synthesized with reference to 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, which is incorporated herein by reference in its entirety.

[0733] Methods: Various test compounds (the ten bisphosphonates as described above) and OVA antigen were mixed at a ratio of 1:1, and the concentrations of bisphosphonates and OVA were both 10 mg / ml. The mouse strain used was C57B / 6. The experimental group of mice was injected with a mixture of various test compounds and OVA antigen, 20 μl per mouse, where the amount of OVA antigen and various test compounds was 100 μg. The control group of mice was injected with 10 μl PBS and 10 μl OVA antigen mixed at a ratio of 1:1. 7 days and 14 days after immunization, the mice were bled from the eye sockets, and 100 μl of blood was collected from each mouse. The blood was placed at 4°C overnight, centrifuged at 6000 rpm for 8 minutes, and the upper serum was aspirated to determine the titers of anti-OVA IgM and IgG antibodies in the serum.

[0734] As shown in the results of FIG. 5 , IgM and IgG antibodies with higher titers were produced in mice treated with the test compound relative to the control group mice treated with PBS. Among these bisphosphonic acid compounds, the adjuvant effects of the TH-Z80 and TH-Z93 compounds of the present invention are particularly significant. For example, as can be seen from FIG. 5 , the titers of IgM and IgG antibodies in mice treated with the TH-Z80 and TH-Z93 compounds of the present invention are more than 5 times higher than those in the control group mice treated with PBS at 7 and 14 days after immunization. In addition, the adjuvant effects of the TH-Z80 and TH-Z93 compounds of the present invention are also much higher than the 8 bisphosphonic acid compounds that are already on the market and are used more clinically.

[0735] The above-mentioned determinations (1)-(4) indicate that FPPS inhibitors can act as adjuvants and can enhance the specific immune response induced by antigens.

[0736] Example 3

[0737] (1) Determination of the adjuvant effect of GGPPS inhibitors in mice

[0738] In the mevalonate pathway, geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes the formation of geranylgeranyl pyrophosphate (GGPP) from FPP. We investigated the role of GGPPS inhibitors as adjuvants in immunized mice.

[0739] Compounds TH-Z144 and TH-Z145 are new bisphosphonic acid compounds synthesized by us, and their structures are shown above.

[0740] Reference was made to the expression and purification method of HsGGPPS (humanized geranylgeranyl diphosphate synthase) in the literature (Szabo, CM, 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.) and the literature (Zhang, Y., et al., Lipophilic Bisphosphonates as Dual Farnesyl / Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMRI investigation. Journal of the American Chemical Society Society, 2009.131(14): p.5153-5162.) We tested the inhibitory activity of compounds TH-Z144 and TH-Z145 against GGPPS (geranylgeranyl pyrophosphate synthase). The activity test data are shown below:

[0741]

[0742] From the above data, it can be seen that compounds TH-Z144 and TH-Z145 can effectively inhibit the activity of GGPPS and are potent inhibitors of GGPPS.

[0743] Next, we used the OVA evaluation system described in Example 1 to study the adjuvant effects of TH-Z144 and TH-Z145.

[0744] Methods: The two compounds TH-Z144 and TH-Z145 were each formulated to a concentration of 10 mg / ml, and the concentration of the OVA antigen was also 10 mg / ml, and the compound and antigen were mixed in a ratio of 1:1. The mouse strain used was C57B / 6. Each experimental group of mice was injected with 20 μl of a mixture containing the test compound and the OVA antigen, i.e., 100 μg (10 μl) of adjuvant and 100 μg (10 μl) of the antigen OVA, into the right foot of the mouse. The control group of mice was injected with 10 μl PBS and 10 μl OVA antigen mixed in a ratio of 1:1 into the foot. 100 μl of blood was collected from the eye sockets of the mice 7 days and 14 days after immunization. After standing at 4°C overnight, the serum was separated and the titers of IgM and IgG antibodies against OVA in the serum were determined. The results are shown in Figure 6.

[0745] As shown in the results of FIG6 , mice treated with the test compounds TH-Z144 and TH-Z145 produced significantly higher titers of IgM and IgG antibodies than those of the PBS-treated control group mice, confirming that GGPPS inhibitors can act as adjuvants to enhance the specific immune response induced by antigens.

[0746] (2) Adjuvant effect of dual inhibitors of FPPS and GGPPS in mice

[0747] Compounds BPH-716 and BPH-1222 have been reported to be dual inhibitors of FPPS and GGPPS (references: Zhang, Y., et al., Lipophilic pyridinium bisphosphonates: potent γδT cellstimulators. 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), which have the following structure:

[0748]

[0749] We investigated the adjuvant effects of BPH-716 and BPH-1222 using the OVA evaluation system described in Example 1. The results are shown in Figures 7A-D.

[0750] As shown in Figures 7A-D, at 7 days and 14 days after immunization, mice treated with BPH-716 and BPH-1222 produced significantly higher titers of IgG and IgM antibodies compared to mice in the PBS-treated control group. (The antigen used for the detection of BPH-1222 and BPH-716 is a hapten, 4-hydroxy-3-nitrophenylacetyl (abbreviated as NP). Mice were immunized with NP33-KLH (nitrobenzene linked to hemocyanin (KLH)) mixed with BPH-1222 and BPH-716. The titers of NP-specific antibodies in the serum were detected with NP33-BSA (nitrobenzene linked to bovine serum albumin (BSA)) 7 days and 14 days after immunization. The specific implementation and detection methods are the same as those of OVA immunization and detection methods)

[0751] Therefore, this assay confirmed that the dual inhibitors of FPPS and GGPPS both increased the titers of immunoglobulins and could play a good role as adjuvants, capable of enhancing the specific immune response induced by antigens.

[0752] Example 4

[0753] Comparison of the adjuvant activity of HMG-CoA reductase inhibitors, FPPS inhibitors, and GGPPS inhibitors with the activity of existing adjuvants

[0754] The OVA evaluation system described in Example 1 was used to compare the differences in adjuvant effects of three types of inhibitors, namely HMG-CoA reductase inhibitors (simvastatin), FPPS inhibitors (TH-Z80 and TH-Z93), and GGPPS inhibitors (TH-Z145) with known common adjuvants (PEI, imiquimod, aluminum adjuvant, MF59, IFA, and CFA). Specifically, the IgG titer and antibody affinity after boosting immunization in mice treated with these adjuvants were tested.

[0755] The affinity of an antibody refers to the strength of the binding between the antibody and the antigen epitope, and is a very important indicator for evaluating the quality of an antibody. It is due to the gene mutation of the antibody-forming cells themselves and the selective activation of the B cell clone by the antigen. This functional state of the body is the result of long-term evolution and continuous adaptation to the external environment, and is of great significance to the body's defense and maintenance of autoimmune monitoring. In in vitro testing, the affinity of the antibody is determined by an experiment in which sodium thiocyanate destroys the binding of antigen and antibody. The antibody affinity determination is based on the following facts: sodium thiocyanate can destroy the binding of antigen and antibody; the stronger the affinity of the antibody, the higher the concentration of sodium thiocyanate that dissociates the binding of antigen and antibody. On the other hand, if the affinity of the antibody is stronger, it reflects that the effect of the adjuvant is better.

[0756] Source of materials: PEI (polyethyleneimine) was purchased from Labster (Beijing) Technology Co., Ltd., Imiquimod was purchased from Shanghai Yisheng Biotechnology Co., Ltd., aluminum adjuvant was purchased from Thermo Fisher Scientific, IFA (incomplete Freund's adjuvant) and CFA (complete Freund's adjuvant) were purchased from Sigma. MF59 was prepared in our own laboratory. The preparation method can be found in the literature "The adjuvant 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. 2013 Jul 18; 31 (33): 3363-9, which is incorporated into this application as a reference in its entirety.

[0757] Methods: Various test adjuvants and OVA antigen were mixed in a ratio of 1:1, with the concentration of OVA antigen being 2 mg / ml and the volume being 50 μl. Simvastatin, TH-Z80, TH-Z93, TH-Z145, PEI and Imiquimod were each 100 μg; aluminum adjuvant, MF59, IFA (incomplete Freund's adjuvant) and CFA (complete Freund's adjuvant) were each 50 μl. After the antigen and adjuvant were pre-mixed, they were left to stand at 4°C overnight before immunizing mice. Among them, the mice in the experimental group were immunized by intramuscular injection of 100 μl of a mixture of various test adjuvants and OVA antigen. The mice in the control group were also intramuscularly injected with 50 μl PBS and 50 μl OVA antigen mixed in a ratio of 1:1. 50 μg of OVA protein was used for booster immunization 7 days and 14 days after immunization. The mice were killed on the 7th day after the third immunization, and blood was collected to separate serum and measure the titer of IgG in the serum.

[0758] The OVA protein was diluted with a carbonate solution of pH 9.6 to a concentration of 2 μg / ml, coated in a 96-well ELISA plate, 50 μl per well, coated overnight at 4°C, washed 5 times with PBS containing 0.05% Tween 20, blocked with 1% BSA solution, 200 μl per well, blocked at room temperature for 2 hours. The ELISA plate was washed 5 times with PBST, and serum of a certain dilution was added. Specifically, the PBS group was not diluted, the simvastatin group was diluted 16 times, the TH-Z80 group was diluted 16 times, the TH-Z93 group was diluted 32 times, the TH-Z145 group was diluted 16 times, the imiquimod group was diluted 8 times, the PEI group was diluted 8 times, the aluminum adjuvant group was diluted 8 times, the MF59 group was diluted 16 times, the incomplete Freund's adjuvant group was diluted 32 times, and the complete Freund's adjuvant group was diluted 64 times. When sodium thiocyanate was not added, the amount of anti-OVA IgG antibody in the diluted serum was the same, 50μl per well, and incubated at room temperature for 2h. After washing the ELISA plate with PBST for 5 times, sodium thiocyanate with concentrations of 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 was added, 50μl per well, and incubated at room temperature for 30min. After washing the ELISA plate with PBST for 5 times, HRP-labeled goat anti-mouse antibody was added, 50μl per well, and incubated at room temperature for 45min. Wash the ELISA plate with PBST for 5 times, add sodium citrate OPD colorimetric solution, color for 10min in dark, and then add sulfuric acid to stop. According to the readings of the microplate reader, the IC50 of sodium thiocyanate was calculated using Prism software. The higher the IC50 value, the stronger the affinity of the antibody produced by immunization.

[0759] Depend on Figure 8 It can be seen that, compared with the control group mice treated with PBS, mice treated with the three types of inhibitors of the present invention (simvastatin, TH-Z80, TH-Z93, TH-Z145) as adjuvants produced IgG antibodies with significantly high titers and antibody affinities. In addition, it is particularly noteworthy that, in terms of both IgG antibody titers and antibody affinity, the inhibitors of the present invention are even superior to known common adjuvants such as PEI, imiquimod, aluminum adjuvants, and MF59. We believe that this result will promote the development of more active adjuvants in the art.

[0760] Examples 1-4 fully demonstrate that compounds that can inhibit the activity of HMG-CoA reductase, FPPS, and GGPPS in the mevalonate pathway can act as adjuvants in immunogenic compositions. In other words, HMG-CoA reductase inhibitors, FPPS inhibitors, and GGPPS inhibitors can act as adjuvants, thereby enhancing the specific immune response caused by the antigen. We believe that 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, mevalonate kinase, phosphomevalonate kinase, mevalonate-5-pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, and geranylgeranyl transferase (I, II).

[0761] Example 5

[0762] Verification that various adjuvants of the present invention act by inhibiting geranylgeranylation of proteins

[0763] As described above, we found that HMG-CoA reductase inhibitors in the mevalonate pathway, such as statins, farnesyl pyrophosphate synthase inhibitors, such as bisphosphonates, and geranylgeranyl pyrophosphate synthase inhibitors, such as bisphosphonates, significantly increased the titer and affinity of IgG antibodies compared to the negative control group, and can act as adjuvants in immunogenic compositions. Geranylgeranyl pyrophosphate synthase is a downstream enzyme in the mevalonate pathway, and our experiments revealed that inhibiting geranylgeranyl pyrophosphate synthase can exert a good adjuvant effect. Therefore, we further speculate that these inhibitors exert adjuvant effects by inhibiting the geranylgeranylation of proteins in the mevalonate pathway. It should be understood that in the mevalonate pathway, if signal transduction is carried out through post-translational modification of proteins, namely geranylgeranylation, then the relevant biological behavior can be rescued by the substrates required for geranylgeranylation, namely geranylgeranyl pyrophosphate (GGPP) or geranylgeraniol (GGOH); that is, once the geranylgeranylation substrates GGPP and GGOH are additionally added from the outside, these inhibitors can no longer effectively exert their inhibitory effects, and thus cannot prevent the formation of prenylated proteins.

[0764] To prove our hypothesis, we designed adjuvant + OVA + GGPP (or GGOH) to verify whether the various inhibitors of the present invention play an adjuvant role by inhibiting the mechanism of geranylgeranylation. Specifically, we studied the effects of GGOH and GGPP on the adjuvant activity of simvastatin (HMG-CoA reductase inhibitor), TH-Z93 (FPPS inhibitor), and TH-Z145 (GGPPS inhibitor).

[0765] A. Rescue experiments with GGPP and GGOH

[0766] Methods: Simvastatin, TH-Z93, and TH-Z145 were prepared at a concentration of 10 mg / ml, the concentration of OVA antigen was 20 mg / ml, and GGOH and GGPP were prepared into a series of solutions with concentrations of 200 mg / ml, 100 mg / ml, 40 mg / ml, and 20 mg / ml, respectively. For the experimental group, on the one hand, the titer of the antibody was measured when immunization was performed by mixing only 10 μl of the test compound and 5 μl of the OVA antigen without adding GGOH or GGPP. On the other hand, the titer of the antibody was measured when immunization was performed after adding GGOH or GGPP; specifically, 10 μl of the test compound, 5 μl of the OVA antigen, and 5 μl of each concentration of GGOH or GGPP (total volume of 20 μl) were mixed and immunized by injection into the right foot of the mouse. The control group mice were treated with PBS and antigen only, without adding the test compound and GGOH or GGPP. 100 μl of blood was collected from the eye sockets of mice 7 and 14 days after immunization and allowed to stand overnight at 4°C. The serum was separated and the titers of anti-OVA IgM and IgG antibodies in the serum were measured.

[0767] As can be seen from Figures 9-11, when GGPP or GGOH was not added, the titers of IgG and IgM antibodies produced in mice treated with simvastatin, TH-Z93, and TH-Z145 of the present invention as adjuvants were significantly higher than those in the control group mice treated with PBS, confirming their strong adjuvant effect. However, after the addition of GGPP or GGOH, the titers of IgG and IgM antibodies produced in mice treated with the test compound as an adjuvant were greatly reduced, and the greater the amount of GGPP or GGOH added, the greater the magnitude of the titer reduction. After the addition of 1 mg of GGPP, the titer of the antibody even dropped to the titer level of the control group treated with only PBS.

[0768] It can be seen that the geranyl-geranylated substrates GGPP and GGOH can effectively inhibit the adjuvant effect of simvastatin, TH-Z93 and TH-Z145, reflecting that the compounds play an adjuvant role by inhibiting the geranyl-geranylation of proteins. That is, once the geranyl-geranylated substrates GGPP and GGOH are additionally added from the outside, the compounds of the present application cannot prevent the formation of prenylated proteins, and thus cannot play an adjuvant role.

[0769] B. Study on the adjuvant activity of selective squalene synthase inhibitors

[0770] In the mevalonate pathway, the pathway begins to branch downstream from the generation of FPP, such as the formation of cholesterol. It is known in the prior art that HMG-CoA reductase inhibitors work by blocking the intracellular mevalonate pathway and thereby blocking the synthesis of cholesterol. There is such a question, whether the various enzyme inhibitors of the mevalonate pathway of the present invention play an adjuvant role because the geranyl geranylation downstream of the mevalonate pathway is inhibited, or because other branches such as cholesterol synthesis are inhibited? To this end, we studied the adjuvant effect of the selective squalene synthase (SQS) inhibitor TH-Z66, with the aim of determining whether the compounds of the present application work by inhibiting cholesterol formation. It is well known that squalene synthase (SQS) inhibitors can inhibit the biosynthesis of cholesterol in vivo.

[0771] The selective squalene synthase (SQS) inhibitor TH-Z66 is BPH-652 described in the following reference: Liu CI et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureusvirulenc, Science, 2008 Mar 7; 319 (5868): 1391-4, which has confirmed that BPH-652 is a selective squalene synthase (SQS) inhibitor that can inhibit cholesterol biosynthesis in vivo, and is incorporated herein by reference in its entirety. TH-Z66 is synthesized according to the method described in the reference.

[0772]

[0773] Methods: TH-Z66 was prepared to a concentration of 10 mg / ml, and the concentration of OVA antigen was also 10 mg / ml. They were mixed in a 1:1 ratio, and each mouse was injected with 20 μl, which contained 100 μg of TH-Z66 and 100 μg of antigen. The mice were of the C57B / 6 strain. The right paw of the experimental group of mice was injected. The control group of mice was treated similarly, except that the same volume of PBS was used instead of TH-Z66. 100 μl of blood was collected from the eye sockets of the mice 7 days and 14 days after immunization. After standing at 4°C overnight, the serum was separated and the titers of anti-OVA IgM and IgG antibodies in the serum were determined.

[0774] As shown in Figure 12, the study showed that the titers of IgG and IgM antibodies produced in mice treated with the SQS inhibitor TH-Z66 were not significantly different from those in the control group mice treated with PBS, and even the IgG antibody titers 7 days after immunization were lower than those in the control group mice. This shows that TH-Z66 does not have an adjuvant effect, thereby eliminating the influence of the cholesterol-lowering effect of the compounds of the present application on the adjuvant effect. It can be seen that the mevalonate pathway inhibitor of the present invention exerts an adjuvant effect not by inhibiting cholesterol synthesis.

[0775] The above experiments A and B fully demonstrate that the mevalonate pathway inhibitor of the present invention exerts its adjuvant effect not by inhibiting cholesterol synthesis but by inhibiting geranylgeranylation of proteins.

[0776] Through the studies in Examples 1-5, we confirmed that inhibitors of various enzymes in the mevalonate pathway can act as adjuvants, thereby enhancing the specific immune response induced by antigens, and that this adjuvant effect is achieved by inhibiting geranylgeranylation of proteins.

[0777] Example 6

[0778] Recruitment of cells in lymph nodes after immunization using the inhibitor of the present application as an adjuvant

[0779] Lymph nodes are important sites for immune response. Lymph nodes are rich in various types of immune cells, which are conducive to capturing antigens, transmitting antigen information and cell activation and proliferation. Usually, B lymphocytes, T lymphocytes, macrophages and dendritic cells are recruited to lymph nodes after immunization. Here, we studied the recruitment of cells in lymph nodes after immunization using the inhibitor of the present invention as an adjuvant.

[0780] Method: According to the similar operation as in Example 1, OVA was used as an antigen, and 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, and mice were immunized by subcutaneous injection. 24 hours after immunization, the inguinal lymph nodes on the same side of the immunization were removed, separated into single cell suspensions through a 100-mesh sieve, and the proportions and changes in the number of B lymphocytes, T lymphocytes, macrophages, and dendritic cells in the lymph nodes were determined by flow cytometry. The markers of B lymphocytes were B220, T lymphocytes were CD3, macrophages were CD11b and F4 / 80, and dendritic cells were CD11c.

[0781] like Figure 13-17As shown, after adding adjuvant, the proportion and number of these four cells in the lymph nodes of mice treated with the three compounds of the present invention (simvastatin, TH-Z80, TH-Z145) as adjuvants were significantly increased compared with the control group mice (Ctrl) without any treatment and the control group mice treated with PBS. It shows that 24h after immunization, these adjuvants of the present application can greatly promote the migration of these cells to the lymph nodes, reflecting that the compounds of the present application as adjuvants enhance the immune response.

[0782] Example 7

[0783] Mevalonate pathway inhibitors enhance DC responses to LPS

[0784] Dendritic cells (DC) are one of the most powerful antigen presenting cells in the body and are the bridge between innate immunity and adaptive immunity. Dendritic cells have highly expressed antigen presenting molecules (MHC-I and MHC-II) and co-stimulatory molecules (such as CD80, CD86) on their surface, making them powerful antigen presenting cells. Dendritic cells can activate naive T cells and play an important role in immune response. Extracting dendritic cells from the peripheral blood of tumor patients, or separating monocytes from peripheral blood and adding cytokines to induce differentiation into dendritic cells, then adding tumor antigens, inactivated tumor cells, tumor cell lysate, plasmids containing tumor antigen DNA or RNA to dendritic cells, adding some stimulants to stimulate and activate these dendritic cells to express more co-stimulatory molecules, and then adoptively infusing the treated dendritic cells into the body of tumor patients can have a good therapeutic effect on tumors.

[0785] The adjuvant of the present invention can enhance the stimulating effect of LPS on dendritic cells in vitro, and can also be applied to dendritic cell vaccines. Dendritic cells are treated with these adjuvants in advance and then tumor antigens, inactivated tumor cells, tumor cell lysate, plasmids containing tumor antigen DNA, tumor antigen RNA are added; or these antigens and adjuvants are used to stimulate dendritic cells together, or dendritic cells are treated with these antigens and then treated with adjuvants, which can be used as a new type of DC vaccine.

[0786] Methods: Bone marrow cells were taken from mice, and 10 ng / ml recombinant mouse GM-CSF and IL-4 were added. After 7 days of in vitro induction, the differentiated mouse bone marrow derived dendritic cells (BMDC) were treated with 1 μM simvastatin, TH-Z80, TH-Z145 and TH-Z66 for 24 hours, and then stimulated with 100 ng / ml LPS. After 24 hours of stimulation, the supernatant was collected and the TNF-α, IL-6, IL-12p70 and IL-1β were determined. BMDC in 96-well plates were added with 1 μM simvastatin, TH-Z80, TH-Z93, TH-Z145 or a mixture of these adjuvants and 2 μM GGOH for 24 hours, with 50,000 cells per well. After 24 hours, 50,000 OT-ⅠCD8+T cells or OT-ⅡCD4+T cells and 100 μg / ml OVA protein were added and cultured for 72 hours. After 72 hours, the culture supernatant of the cells was collected and the secreted cytokines IL-6, IFN-γ, and TNF-α in the supernatant were measured.

[0787] The cytokine determination kit was purchased from ebioscience. The determination method was based on the product manual. The capture antibodies of these cytokines were coated with a 96-well ELISA plate at 4°C overnight, with 100 μl per well. After elution with PBST 5 times, the plate was blocked with blocking solution at room temperature for 2 hours. After elution with PBST 5 times, a 2-fold diluted cell supernatant was added and incubated at room temperature for 2 hours. After elution with PBST 5 times, the detection antibody was added and incubated at room temperature for 1 hour. After elution with PBST 5 times, avidin-linked horseradish peroxidase was added and incubated at room temperature for 45 minutes. After elution with PBST 5 times, 3,3",5,5"-tetramethylbenzidine solution was added for color development for 15 minutes, 2 molar sulfuric acid was added for termination, and the OD450 reading was read with an ELISA reader. The concentration of the target cytokine in the supernatant was calculated based on the standard.

[0788] The results showed that these adjuvants had no stimulatory effect on mouse bone marrow-derived dendritic cells (BMDC), but could enhance the stimulatory effect of LPS on DC. In particular, these inhibitors could assist LPS in stimulating DC to produce IL-1β ( Figure 18-21 , Fig.38 ), reflecting that the inhibitor of the present application enhances the immune response and can be applied to dendritic cell vaccines.

[0789] Example 8

[0790] The uptake of OVA by antigen-presenting cells in lymph nodes after immunization using the inhibitor of the present application as an adjuvant

[0791] In the immune response process, in addition to T cells and B cells playing a core role, monocytes / macrophages and dendritic cells also participate in the process, mainly processing and presenting antigens, so they are called antigen presenting cells (APC), which can also be called accessory cells (A cells) or A cells. APC can take up and process antigens through phagocytosis or endocytosis, and bind the processed polypeptide fragments containing antigenic determinants to MHC class II molecules, and then express them on the cell surface and present them to CD4+TH cells. Cells with antigen presenting function mainly include monocytes / macrophages, dendritic cells and B cells. Here, we studied the uptake of OVA antigen by antigen presenting cells after immunization using the inhibitor of the present invention as an adjuvant.

[0792] Method: OVA was labeled with FITC (fluorescein isothiocyanate), and the FITC-labeled OVA was mixed with three compounds of the present invention (simvastatin, TH-Z80, and TH-Z145), and mice were subcutaneously immunized according to the similar operation as in Example 1. The lymph nodes on the same side of the immunization were taken out 24 hours after immunization, separated into single cell suspensions, and the proportion of FITC-OVA cells contained in the three antigen-presenting cells in the lymph nodes was determined by flow cytometry. The markers of B lymphocytes are B220, T lymphocytes are CD3, macrophages are CD11b and F4 / 80, and dendritic cells are CD11c.

[0793] As can be seen from Figure 22, compared with the control group mice (Ctrl) that did not undergo any treatment and the control group mice treated with PBS, the proportion of FITC-positive cells (B220, CD11c, F4 / 80) in the lymph nodes of the experimental group mice treated with the three compounds of the present invention as adjuvants increased, confirming that these inhibitors of the present invention can promote the uptake of antigens by antigen-presenting cells or their migration into the lymph nodes, reflecting that the compounds of the present application enhance the immune response as adjuvants.

[0794] Examples 6-8 demonstrate that the adjuvant effect of the inhibitor of the present invention is not only reflected in the increase in antibody titer, but also in the enhancement of various aspects of the immune response, such as the enhanced recruitment of B lymphocytes, T lymphocytes, macrophages and dendritic cells in lymph nodes, assisting LPS in stimulating DC to produce IL-1β, and promoting the uptake of antigens by antigen-presenting cells.

[0795] In the following experiments, we investigated the adjuvant effects of the inhibitors of the present invention on several specific antigens.

[0796] Example 9

[0797] Three inhibitors can act as mucosal adjuvants to promote the production of more antibodies against Mers protein in vivo

[0798] Take 20 μg of Mers protein (the expression and purification of the protein refer to Jiang L et al, Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Sci Transl Med. 2014Apr 30), add 100 μg of the three compounds of the present invention (simvastatin, TH-Z80, TH-Z144) as adjuvants, and immunize mice through the nasal mucosa according to the similar operation as in Example 1. 100 μg of C48 / 80 (a mast cell stimulator, commonly used as a mucosal adjuvant) was used as a comparison. 7 days and 14 days after immunization, the titers of IgM and IgG antibodies against the Mers protein of the Middle East respiratory syndrome virus in the mouse serum were detected.

[0799] As can be seen from Figure 23, compared with the control group mice treated with PBS, the titers of the three adjuvant antibodies in the experimental group mice treated with the three compounds of the present invention as adjuvants were significantly increased, confirming that the inhibitor of the present invention can assist the Mers protein in producing more antibodies, suggesting that it can be used as an adjuvant in the Middle East Respiratory Syndrome virus vaccine.

[0800] Example 10

[0801] Three inhibitors can promote the production of more antibodies against hepatitis B surface antigen HbsAg

[0802] 20 μg of hepatitis B surface antigen (HBsAg, donated by Beijing Tiantan Biological Products Co., Ltd.) was used as an antigen, and 100 μg of each of the three compounds of the present invention (simvastatin, TH-Z80, and TH-Z144) was added as an adjuvant. Mice were immunized by intramuscular injection according to a similar operation as in Example 1. The titers of IgM and IgG against HBsAg protein in the mouse serum were detected 7 days and 14 days after immunization.

[0803] As can be seen from Figure 24, compared with the control group mice treated with PBS, the titers of the three adjuvant antibodies in the experimental group mice treated with the three compounds of the present invention as adjuvants were significantly increased, confirming that the inhibitor of the present invention can assist the hepatitis B surface antigen in producing more antibodies, suggesting that it can be used as an adjuvant in hepatitis B vaccine.

[0804] Embodiment 11

[0805] Preparation of preventive vaccine against melanoma

[0806] The four substances of the present invention, simvastatin, TH-Z80, TH-Z93, and TH-Z145, were used as adjuvants and mixed with OVA, and mice were immunized according to the same operation as in Example 1. The mixture used for the initial immunization contained 100 μg of OVA protein and 100 μg of adjuvant (i.e., the above four substances) respectively, and was subcutaneously injected at the base of the tail. Two weeks after the immunization, the booster was performed without adjuvant, and 50 μg of antigen was subcutaneously injected at the base of the tail. Two weeks after the second immunization, the booster was performed again, and 50 μg of antigen was subcutaneously injected at the base of the tail. Two weeks after the third immunization, 3*10 5 B16-OVA (B16 melanoma cells stably expressing OVA, donated by the Institute of Biomedical Sciences, Soochow University). The weight and tumor volume of mice were measured every two days. The tumor volume was calculated using the formula length*width*width / 2.

[0807] The cells are stably transfected to express luciferase before inoculation. The specific method is as follows: 293T cells are transfected with a viral plasmid expressing luciferase, and 48 hours after transfection, the 293T cell culture supernatant containing the virus is collected. After culturing B16-OVA cells with the supernatant containing the virus, the virus in the supernatant infects the B16-OVA cells and expresses luciferase. The cells expressing luciferase are sorted out by flow cytometry and expanded for culture.

[0808] Three mice were randomly selected from each group at 7, 14, and 21 days after inoculation. 3 mg / mouse of luciferin substrate was injected. 8 minutes after injection, the mice were anesthetized and the tumor volume was observed using a live imaging device.

[0809] The results are shown in Figure 25. Compared with the control group 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 adjuvants, suggesting that they can be used as adjuvants in preventive vaccines for melanoma.

[0810] Example 12

[0811] Preparation of therapeutic vaccines against melanoma cells

[0812] C57B / 6 mice were subcutaneously inoculated with 3*10 5300,000 B16-OVA (also donated by the Institute of Biomedical Sciences, Soochow University) tumor cells were inoculated. On the 5th day after inoculation, the mice were subcutaneously injected with a mixture containing 100 μg of OVA protein and 100 μg of adjuvant (the above four substances, simvastatin, TH-Z80, TH-Z93, and TH-Z145) at the base of the tail. 7 days after the first immunization, the mice were boosted without adjuvant and 50 μg of antigen was subcutaneously injected at the base of the tail. 7 days after the second immunization, the mice were boosted again with 50 μg of antigen. The weight and tumor volume of the mice were detected every two days. The volume of the tumor was calculated using the formula length*width*width / 2.

[0813] The same method as described in Example 11 was used to stably express luciferase in the cells before inoculation.

[0814] Three mice were randomly selected from each group at 7, 14, and 21 days after inoculation. 3 mg / mouse of luciferin substrate was injected. 8 minutes after injection, the mice were anesthetized and the tumor volume was observed using a live imaging device.

[0815] The results are shown in Figure 26. Compared with the control group 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 adjuvants, suggesting that they can be used as adjuvants in therapeutic vaccines for melanoma.

[0816] Examples 9-12 demonstrate that the inhibitor of the present invention can be used as an adjuvant in a variety of clinical vaccines, such as Middle East Respiratory Syndrome virus vaccine, hepatitis B vaccine, and therapeutic and preventive vaccines for melanoma, suggesting its broad prospects in clinical application.

[0817] Example 13

[0818] Adjuvant effects at different immunization sites

[0819] The TH-Z80 of the present invention was used as an adjuvant to mix the OVA antigen to immunize different parts of the mouse, and the immunized parts were the soles of the feet, subcutaneous, muscle, abdominal cavity and nasal mucosa of the mouse. The soles of the feet and nasal mucosa were immunized using a 20μl system, the concentration of TH-Z80 was 10mg / ml, the concentration of OVA protein was also 10mg / ml, and the adjuvant and antigen were mixed according to 1:1, that is, 10μl of adjuvant and 10μl of antigen. The muscle, subcutaneous and abdominal immunizations used a 100μl system, the concentration of TH-Z80 was 2mg / ml, the concentration of OVA protein was also 2mg / ml, and the adjuvant and antigen were mixed according to 1:1, that is, 50μl of adjuvant and 50μl of antigen. The same volume of PBS was mixed with OVA antigen to immunize the control group of mice, and the titers of anti-OVA IgM and IgG antibodies in the serum were determined 7 days and 14 days after immunization according to similar operations as in Example 1.

[0820] Depend on Fig. 27 It can be seen that no matter where the immunization was performed, the antibody titer of TH-Z80 was significantly increased compared with the control group mice treated with PBS, which confirmed that the compound of the present invention can achieve a good adjuvant effect when injected at various sites.

[0821] Embodiment 14

[0822] The combined adjuvant effect of FPPS inhibitor and TLR agonist

[0823] In this assay, we investigated the inhibitors of the invention in combination with other adjuvants known in the prior art, wherein the other adjuvant employed was Imiquimod (a TLR agonist).

[0824] The two compounds TH-Z93 and Imiquimod were formulated into a concentration of 4 mg / ml, and the concentration of OVA antigen was 10 mg / ml. When used alone, 5 μl of TH-Z93 or Imiquimod was added as an adjuvant, and then 10 μl of antigen and 5 μl of PBS were mixed. When the two adjuvants were used in combination, 5 μl of each adjuvant was added, and then 10 μl of antigen was mixed, and the total volume was 20 μl. The mouse strain used was C57B / 6 mice. The control group mice used an equal volume of PBS without any adjuvant. The right foot of the mouse was injected. 100 μl of blood was collected from the eye sockets of the mice 7 days and 14 days after immunization. After standing at 4°C overnight, the serum was separated and the titers of anti-OVA IgM and IgG antibodies in the serum were measured.

[0825] As can be seen from Figure 28, compared with the control group mice treated with PBS, mice treated with TH-Z93 or imiquimod alone produced significantly higher antibody titers, and the antibody titers of mice treated with TH-Z93 alone were significantly better than those of mice treated with imiquimod alone. When the two adjuvants were mixed, a certain synergistic effect was produced in vivo. This study shows that the various inhibitors of the present invention can be used in combination with other adjuvants known in the prior art, so as to exert the adjuvant effect of increasing the immune response to a greater extent.

[0826] Example 15: Activity test of the compound represented by formula IXX

[0827] Target activity test

[0828] a: HsFPPS (humanized farnesyl pyrophosphate synthase) activity test

[0829] HsFPPs with 6 consecutive His at the N-terminus were induced to express in vitro, and the samples were collected and purified by Ni column. In vitro HsFPPs enzyme activity test was performed in a 96-well plate, with 200 μl of solution in each well. The buffer of the system was 25 mM HEPES, 2.5 mM MgCl2, pH 7.4. DMAPP and IPP were used as reaction substrates, and the changes of UV values ​​at a wavelength of 360 nm were monitored in real time in the phosphate lyase system. ORIGIN 8.0 software was used for plotting and fitting.

[0830] b: PvGGPPS (Plasmodium geranylgeranyl pyrophosphate synthase) activity test,

[0831] PvGGPPs with 6 consecutive His at the N-terminus were induced to express in vitro and collected for Ni column purification. In vitro PvGGPPs enzyme activity test was performed in a 96-well plate, with 200 μL of solution in each well. The buffer of the system was 25 mM HEPES, 2.5 mM MgCl2, pH 7.4. GPP and IPP were used as reaction substrates, and continuous spectrophotometric detection readings were performed at 360 nm in the presence of a phosphate lyase system. ORIGIN 8.0 software was used for plotting and fitting. The results are shown in the table below:

[0832] Table: Activity results of synthetic compounds against different targets

[0833] Compound <![CDATA[HsFPPS(IC 50 / μM)]]> <![CDATA[PvGGPPS(IC 50 / μM)]]> <![CDATA[malaria(IC 50 / 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

[0834] Zoledronic acid was used as a positive control compound and its IC 50 The IC values ​​of these compounds synthesized in the present invention for HsFPPS are about 100 nM. 50 Many values ​​are around 100 nM. At the same time, the compounds of the present invention have good inhibitory effects on PvGGPPS and malaria.

[0835] Cell viability test:

[0836] A: Plasmodium activity test,

[0837] Plasmodium falciparum 3D7 was cultured in IPEM1640 medium mixed with 10% human O-type blood serum and 25mM HEPES. The culture process was maintained in a carbon dioxide incubator with 5% carbon dioxide. In vitro drug testing experiments were performed using 96-well plates. The tested drugs were dissolved in PBS and pre-diluted with complete culture medium. Infected red blood cells were cultured in triplicate for 72 hours in drugs diluted 3 times in succession. Then an equal amount of SYBR-GREEN1 was added to each well. Then the excitation light was 485nm and the emission light was 538nm. Artemisinin and a control group without any drug were used for observation and comparison. ORIGIN8.0 software was used for graph fitting, and the results are shown in Table 1.

[0838] B: MDA-MB-231 activity test

[0839] Reagents and instruments

[0840] DMEM medium was purchased from Gibco; fetal bovine serum (FBS) was purchased from BI; double antibody was purchased from Bio-Tech; 0.25% trypsin-EDTA was purchased from Gibco; MTT was purchased from Ameresco; centrifuge was purchased from Anhui Zhongjia Technology Co., Ltd., 4℃ refrigerator was purchased from Haier, and -80℃ refrigerator was purchased from Thermo;

[0841] DMEM complete medium

[0842] Add 10 ml of fetal bovine serum to each 90 ml of DMEM medium and add the double antibody at a ratio of 1:100 to make a complete culture medium and store it at 4°C.

[0843] 1.Resuscitation, passaging and cryopreservation of MDA-MB-231 cell lines

[0844] Cell recovery: Take out the cryopreserved tube from the liquid nitrogen tank quickly, hold it with tweezers and immediately immerse it in 37℃ water to accelerate thawing. After it is completely dissolved, transfer it to the clean bench, aspirate the cell suspension, add 3ml of DMEM containing double antibody and 10% FBS, mix well, centrifuge at 1000r / min and 4℃ for 3min, discard the supernatant, add appropriate amount of DMEM medium containing 10% FBS to dilute to 0.5×10 6 cells / cm 2 Density inoculated in a culture bottle, gently mixed, and cultured in a 37°C, 5% CO2 incubator. Two days after cell recovery, observe cell morphology under a microscope, change the culture medium once, and culture for three days. Before subculturing, observe cells under a microscope. If the cells are plump and have strong refractive index, take the cells in the logarithmic growth phase for the test.

[0845] Cell passaging: When passing cells, first discard the culture medium, then wash once with preheated PBS at room temperature, and then digest with 0.01% trypsin-EDTA in a 37°C, 5% CO2 incubator for 1-3 minutes. Gently tap the side wall of the culture flask to separate the cells from the bottom wall of the culture flask, add 2ml of DMEM culture medium containing serum to terminate the digestion, and then transfer the cell suspension to a 15ml glass centrifuge bottle, gently blow and evenly make a single cell suspension, centrifuge at 1000r / min and 4°C for 3min, discard the supernatant, resuspend the cells with an appropriate amount of culture medium, take 50μl of the cell suspension and mix it evenly with 50μl of trypan blue, and count the living cells with a cell counting plate, with 0.5×10 6 cells / cm 2Transfer the cells to the culture flask at a density of 100 μg / cm2, add an appropriate amount of culture medium and mix gently, then place in a 37°C, 5% CO2 incubator for static culture.

[0846] Cell freezing: Collect cells in a glass centrifuge tube and count them, discard the supernatant, prepare cell freezing solution at a ratio of DMSO to serum of 1:9, and use the cell freezing solution at a rate of 1-2×10 6 Resuspend the cells at a concentration of 1 cell / ml, then dispense 1 ml into each cryovial, place in a -80°C freezer overnight, and then transfer to liquid nitrogen for storage for several years.

[0847] 2. Drug screening test steps:

[0848] Drug dissolution: Weigh a certain mass of bisphosphonate drugs, add a small amount of NaOH or NaHCO3 to dissolve the drugs, and the storage concentration is 10μM;

[0849] Drug dilution: dilute the prepared bisphosphonic acid stock solution with DMEM complete medium, the highest concentration is 1mM, and dilute it in a ratio of 1:3.2, with a total of 11 concentration gradients;

[0850] a. Digest MDA-MB-231 cells the day before, centrifuge at 1000 rpm for 3 min, count the cells, inoculate 3000 cells / well into a 96-well plate, with a volume of 100 μl per well, and culture for 14-16 hours. Pay attention to accurate cell counting and the same amount of cells inoculated in each well. Gently shake the cell suspension between tapping, or tap a row of cells and blow the cell suspension gently with a pipette. The speed of the gun into the 96-well plate should not be too fast or too slow, so as to avoid uneven distribution of cells in the well plate and affect the experiment. The wells around the 96-well plate evaporate slightly faster, and the concentration of the solution changes slightly faster. If not necessary, try to choose the 60 wells in the middle for stimulation, and avoid bubbles in the injected liquid.

[0851] b. After the cells adhere to the wall, the liquid in the 96-well plate was discarded, and the prepared drug solution was added to the cell culture plate prepared the day before (the final total volume of treated cells was 100 μl). At the same time, a blank control group (only culture medium was added) was set up, with 6 replicates per group;

[0852] c. After 72 hours of drug action, add the prepared MTT solution (weigh a certain amount of MTT powder and add PBS to dissolve it into 5 mg / ml, try to prepare it before use, protect it from light, and filter it with a 0.22 filter membrane before use) directly to each well, 20 μl per well, and continue to culture for 4 hours;

[0853] d. Lay out the filter paper (absorbent paper can be used instead), gently invert the well plate (be careful to use gentle force to prevent the crystals in the wells from falling), absorb the liquid in the wells and add 150μl DMSO to dissolve, use an oscillator to shake and mix, and the time is just enough to dissolve the crystals. Use an enzyme marker to measure the absorbance (OD value) of each well at 570nm, calculate the mean of the blank control as ODBlank, the OD value of each well = OD measured - ODBlank, calculate the mean of ODNT, and use the mean of ODNT to calculate the relative percentage of cell death = (1-OD / ODNT)*100% (or the relative percentage of cell survival = OD / ODNT*100%), then calculate the mean and standard error, and use GraphPrism software to fit the mean and standard error of each group of drug concentrations (uM) and the corresponding drug inhibition rate to the drug IC 50 The results are shown in the table below:

[0854] Table: Proliferation inhibition activity of compounds with different carbon chain lengths on MDA-MB-231

[0855] Drug Name TH-Z79 TH-Z80 TH-Z81 TH-Z82 TH-Z148 <![CDATA[IC 50 (μM)]]> 50.93 75.46 59.63 63.09 7.302 Drug Name TH-Z149 TH-Z150 TH-Z151 TH-Z152 TH-Z153 <![CDATA[IC 50 (μM)]]> 36.07 127.5 406.4 428.2 234.5

[0856] Biological experiments as adjuvants:

[0857] 1. Mouse immunization and serum isolation

[0858] During the immunization process, the antigen we used was chicken ovalbumin (OVA). The mice were 8-week-old female c57bl / 6. The concentration of OVA was 1 mg / ml, and the concentration of bisphosphonic acid was 1 mg / ml. During immunization, OVA and bisphosphonic acid were mixed at a ratio of 1:1, and 100 μl was injected intramuscularly into the mice, i.e., 100 μg of OVA and 100 μg of bisphosphonic acid per mouse. 14 days after immunization, blood was collected from the eye sockets of the mice, and 100 μl of blood was collected from each mouse.

[0859] Place the whole blood in a 4℃ refrigerator overnight. The next day, take out the whole blood at 4℃ and centrifuge it at 6000rpm and 4℃ for 5min. After centrifugation, carefully aspirate the upper serum and centrifuge it again at 6000rpm and 4℃ for 5min. Aspirate the upper serum again. The serum is frozen at -20℃ and taken out to melt when testing.

[0860] 2. Potency Monitoring

[0861] 1. Coating: Dilute the antigen OVA to a protein content of 2 μg / ml with 0.05M pH 9.6 carbonate coating buffer. Coat 50 μl in each well of the polystyrene plate and incubate at 4°C overnight or at 37°C for 2 hours. The next day, discard the solution in the wells and wash 5 times with PBST washing buffer.

[0862] 2. Blocking: 150 μl of 2% BSA, block at 37° for 2 h, and wash 5 times with washing buffer PBST.

[0863] 3. Incubate the serum: dilute the serum with diluent at 1:200, then dilute it again in multiples, add 50 μl of the diluted serum to each well, and incubate at 37°C for 2 hours. Wash 5 times with PBST (do blank wells and negative control wells at the same time). The diluent is 0.1% BSA.

[0864] 4. Add enzyme-labeled secondary antibody: Add 50 μl of freshly diluted HRP-labeled Goat-anti mouse IgM or Goat-anti mouse IgM (1:5000) to each reaction well. Incubate at 37°C for 1 hour, and wash 5 times with washing buffer PBST.

[0865] 5. Add substrate for color development: Add 50 ml of pre-prepared OPD color development solution to each reaction well.

[0866] 6. Termination of reaction: After 10 min of color development, add 50 μl of 2 M sulfuric acid to each reaction well.

[0867] 7. Read the OD value at 490nm on the E.LISA detector. Calculate the titer of serum antibodies. The serum dilution with an OD value greater than or equal to 1.5 times the reading of the negative control well is the titer of serum antibodies.

[0868] Fig.29 The graph shows the effect of injection of 100 μg of different bisphosphonates on antibody production in mice.

[0869] from Fig.29 It can be seen that compared with traditional bisphosphonates, TH-Z80 can produce higher titer antibodies than traditional bisphosphonates.

[0870] Fig.30 This is a diagram showing the effect of the length of the carbon chain substitution of the bisphosphonic acid side chain as an adjuvant.

[0871] from Fig.30 It can be seen that when the side chain carbon chain length increases from 1 to 5, the adjuvant effect does not change much, but when it increases from 6 to 8, the adjuvant effect is significantly enhanced, and starting from 8, the adjuvant effect weakens. Compound TH-Z81 has the strongest adjuvant effect.

[0872] The affinity of an antibody refers to the strength of the binding between the antibody and the antigen epitope, and is a very important indicator for evaluating the quality of an antibody. It is due to the gene mutation of the antibody-forming cells themselves and the selective activation of the B cell clone by the antigen. This functional state of the body is the result of long-term evolution and continuous adaptation to the external environment, and is of great significance to the body's defense and maintenance of autoimmune monitoring.

[0873] In vitro, we measured the affinity of antibodies by using NaSCN to disrupt antigen-antibody binding. The operation was as follows: OVA 2μg / ml was coated on a 96-well ELISA plate, and after blocking with 2% BSA, the diluted serum of mice 7 days after primary and booster immunization with TH-Z80 as an adjuvant with the same titer was added, incubated at 37°C for 2h, washed with PBS-T, and sodium thiocyanate (NaSCN) of different dilutions was added, 50μl per well, and incubated at 37°C for 0.5h; after washing with PBS-T, 1:5000 diluted HRP-labeled goat anti-mouse IgG was added, incubated at 37°C for 1h, washed with PBS-T 5 times, and developed with OPD-phosphocitrate buffer system (PH9.6), the reaction was terminated with 2M H2SO4 solution, and the absorbance value at OD490 nm was read. The IC of NaSCN inhibition was calculated. 50 Value, IC of NaSCN 50 The higher the value, the stronger the antibody affinity produced by immunization.

[0874] Fig.31 The effect of increasing antibody affinity for TH-Z80.

[0875] Depend on Fig.31 It can be seen that compared with PBS, TH-Z80 significantly enhanced the affinity of the adjuvant and its antigen.

[0876] Human peripheral blood mononuclear cells (PBMCs) were prepared from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. 1 × 10 5 PBMC were placed in 0.2 mL culture medium, and 4 μM of the tested bisphosphonates and 200 U / mL of rIL-2 were added. Zoledronic acid was used as a positive control. Cells were collected on day 12, stained with FITC-anti-CD3 (Miltenyi Biotec) and PE-anti-Vδ2 (Miltenyi Biotec) monoclonal antibodies, detected by flow cytometry, and data were analyzed and collated by Prism 5.0.

[0877] Fig.32 The expansion effect of benzimidazole bisphosphonates with different carbon chain lengths on δγT cells.

[0878] It can be clearly seen from the figure that the carbon chain length significantly affects Vγ9Vδ2T cells, and the compound TH-Z80 has the best effect on the expansion of Vγ9Vδ2T cells.

[0879] Fig.33 Schematic diagram of the crystal structure of compounds TH-Z80 and HsFPPS (pymol drawing).

[0880] It can be clearly seen from the figure that the main driving force for the binding of TH-Z80 bisphosphonic acid compounds to FPPS lies in the chelation of bisphosphonic acid and metal ions. At the same time, its long carbon chain is in a hydrophobic cavity. The benzene ring and phenol ether in benzimidazole have no obvious effect on FPPS. This result shows that the introduction of N heteroatoms to the benzene ring in benzimidazole has no significant effect on the binding activity of the compound with FPPS. The introduction of long carbon chains in the side chains of N-heterobenzimidazole compounds enhances its hydrophobic interaction with FPPS and improves the binding force between the compound and FPPS.

[0881] Fig.34 Schematic diagram of the crystal structure of compound TH-Z82 and PvGGPPS (pymol drawing).

[0882] It can be clearly seen from the figure that, similar to the crystal structures of TH-Z80 and HsFPPS, the main driving force for the binding of TH-Z82-type bisphosphonic acid compounds to PvGGPPS lies in the chelation of bisphosphonic acid with metal ions and the hydrophobic effect of its long carbon chain. The benzene ring and phenol ether in benzimidazole also have no significant effect on PvGGPPS. This result shows that the introduction of N heteroatoms to the benzene ring in benzimidazole has no significant effect on the binding activity of the compound with PvGGPPS. The introduction of long carbon chains in the side chains of N-heterobenzimidazole compounds enhances its hydrophobic interaction with GGPPS and improves the binding force between the compound and GGPPS.

[0883] According to the crystal structure (calculation results), FPPS and GGPPS are both present in the hydrophobic cavity, so the alkoxy groups on the side chains can be replaced by other hydrophobic groups. Therefore, we also claim to protect N-heterobenzimidazole compounds substituted by other hydrophobic groups.

[0884] Example 16: Effects of simvastatin, TH-Z80, TH-Z93 and TH-Z145 on PR8 influenza virus.

[0885] Methods: 5 μg of HA1 protein from PR8 influenza virus was added to phosphate buffer, 20 μg of simvastatin, 20 μg of TH-Z80, 20 μg of TH-Z93 and 20 μg of TH-Z145. Mice were immunized by intramuscular injection on days 0, 14 and 21. PR8 virus was inoculated into the nasal mucosa of mice on day 28. The mice were weighed every day and the death of mice was observed.

[0886] Results: As shown in Figure 35, the mice without simvastatin, TH-Z80, TH-Z93 and TH-Z145 had a continuous weight loss after virus inoculation and died within 5 to 10 days. However, the mice with simvastatin, TH-Z80, TH-Z93 and TH-Z145 did not lose weight and did not die.

[0887] Example 17: Inhibitory effect of anti-PD1 antibody combined with four adjuvants and chicken ovalbumin on B16-OVA tumors.

[0888] Methods: 300,000 tumor cells were inoculated subcutaneously in the right groin of C57B / 6 mice. On the 5th day after inoculation, a mixture containing 100 μg of OVA protein and 100 μg of adjuvant (i.e., the above four substances, simvastatin, TH-Z80, TH-Z93, and TH-Z145) was subcutaneously injected into the base of the tail of the mice. 7 days after the first immunization, no adjuvant was used for the booster, and 50 μg of antigen was subcutaneously injected into the base of the tail. 7 days after the second immunization, the booster was again strengthened with 50 μg of antigen. At the same time, 100 microliters of anti-PD1 antibody were injected on the 8th, 11th, 15th, 18th, 22nd, and 25th days after tumor inoculation. The weight of the mice and the changes in tumor volume were detected every two days. The volume of the tumor was calculated using the formula length*width*width / 2.

[0889] Results: Fig.36 As shown, anti-PD1 antibody combined with four adjuvants and chicken ovalbumin showed inhibitory effect on B16-OVA tumors.

[0890] Example 18: Adjuvant activity of HMG-CoA synthase inhibitor Hymeglusin in OVA antibody titer detection.

[0891] Methods: The HMG-CoA synthase inhibitor Hymeglusin was formulated into 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. The mouse strain used was C57B / 6. Each mouse in the experimental group was intramuscularly injected with 20 μl of a mixture containing the test compound and the OVA antigen, namely 100 μg Hymeglusin (10 μl) and 100 μg (10 μl) of the antigen OVA. The mice in the control group were intramuscularly injected with 10 μl PBS mixed with 10 μl OVA antigen at a ratio of 1:1. 100 μl of blood was collected from the eye sockets of the mice 14 days after immunization. After standing at 4°C overnight, the serum was separated and the titer of anti-OVA IgG antibodies in the serum was determined.

[0892] Results: Fig.37 As shown, compared with the PBS-treated control mice, mice treated with the HMG-CoA synthase inhibitor Hymeglusin as an adjuvant produced IgG antibodies with significantly higher titers, which fully demonstrated that compounds capable of inhibiting the activity of HMG-CoA reductase in the mevalonate pathway can serve as adjuvants in immunogenic compositions.

[0893] Compound Preparation Examples

[0894] All the NMR characterization data of the compounds were performed on a Bruker Avance DRX-400 spectrometer, with chemical shifts (δ, expressed in ppm) using D2O at 4.79ppm, CDCl3 at 7.26ppm, and MeOD at 3.31ppm as references, and the NMR spectrum peak splitting was expressed as d, t, q, and m, i.e., doublet, triplet, quartet, and multiplet, and the coupling constant was in Hertz. High-resolution mass spectrometry was performed on a Waters Xevo G2 QTof using ESI as the ion source.

[0895] Starting materials can be obtained from commercial sources or prepared by established literature methods known to those skilled in the art.

[0896] Example 1 Preparation of TH-Z97 series compounds

[0897] Preparation of TH-Z97(n=6)((1-hydroxy-2-(7-n-hexyloxyimidazo[1,2-a]pyridin-3-yl)ethane-1,1-diyl)bisphosphonic acid)

[0898]

[0899] Step 1: Dissolve 10 mmol (1.11 g) of 2-amino-4-hydroxypyridine in 50 mL of acetone, add 30 mmol of anhydrous potassium carbonate, heat under reflux under N2 protection, and then add 12 mmol (1.67 mL) of bromohexane. After reacting overnight, filter the insoluble matter, spin dry the organic phase, load the sample, and separate and purify with petroleum ether / ethyl acetate to obtain 7.8 mmol (1.51 g) of 4-hexyloxy-2-amino-pyridine (yield: 78%).

[0900] Step 2: 5 mmol (0.97 g) of 4-hexyloxy-2-amino-pyridine and 5.5 mmol (0.71 g) of trans-4-oxy-2-butenoic acid ethyl ester were reacted in 20 mL of acetonitrile at 80 degrees overnight, then spin-dried, loaded, and separated and purified with petroleum ether / ethyl acetate to obtain 4.1 mmol (1.13 g) of ethyl 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl)acetate with a yield of 82%.

[0901] Step 3: Heat 3 mmol of ethyl 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl)acetate in 6N HCl under reflux for 6 h, then spin dry and dry to constant weight. The crude product is directly used in the next reaction.

[0902] Step 4: Dissolve the obtained 2-(7-(n-hexyloxy)imidazo[1,2-a]piperidin-3-yl)acetic acid with 9 mmol (0.74 g) of phosphorous acid and 6 mL of cyclohexane sulfone at 75 degrees, add 10.2 mmol (1068 μL) of PCl3 dropwise, react for 3.5 hours, add 1 mL of water, heat to reflux for 2 h, cool, filter the precipitated solid, ultrafilter with methanol 3 times, dry the obtained light yellow solid, and weigh 228 mg of the target product after constant weight (yield 18%).

[0903] Structural characterization data: 1 H NMR (400MHz, D2O), δ (ppm): 8.49 (d, J1=7.2Hz1H), 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.22

[0904] Compounds TH-Z157, TH-Z158, TH-Z159, TH-Z160, TH-Z97, TH-Z161, TH-Z98, TH-Z162, TH-Z99, TH-Z198 and TH-Z163 were prepared by reacting the corresponding bromoalkanes with 2-amino-4-hydroxypyridine in a manner similar to the synthesis of TH-Z97 described above.

[0905] TH-Z156 is directly prepared from 2-amino-4-methoxypyridine according to the synthetic steps 2-4 of TH-Z97.

[0906] The characterization data of these compounds are as follows:

[0907] TH-Z98: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.92

[0908] TH-Z99: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.70

[0909] TH-Z156: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.97

[0910] TH-Z157: Structural characterization data: 1 H 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, J 1=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), 31 PNMR(162MHz,D2O),δ(ppm):18.00

[0911] TH-Z158: Structural characterization data: 1 H NMR (400MHz, D2O), δ (ppm): 8.61 (d, J = 7.6Hz, 1H), 7.37 (s, 1H), 6.87 (s, 1H), 6.65 (d, J = 7.2 Hz,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), 31 P NMR (162MHz, D2O), δ (ppm): 16.76

[0912] TH-Z159: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 16.56

[0913] TH-Z160: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.18

[0914] TH-Z161: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.52

[0915] TH-Z162: Structural characterization data: 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.74

[0916] TH-Z163: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 18.05

[0917] TH-Z198: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 17.67

[0918] Example 2

[0919] Preparation of (((4-(Hexyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid (TH-Z93)

[0920]

[0921] Step 1: Take 10 mmol of 2-amino-4-hydroxypyridine and dissolve it in 100 mL of acetonitrile, add 20 mmol of potassium carbonate and 12 mmol of 1-bromohexane, protect with nitrogen, react at 60° C. overnight, monitor by TLC until the raw material reacts completely, cool the reaction solution, filter out potassium carbonate, concentrate under reduced pressure, and separate on a silica gel (200-300 mesh) column with petroleum ether / ethyl acetate = 1:1 to obtain a pure product with a yield of 75%.

[0922] Step 2: Take 6 mmol of the product from the first step and dissolve it in 20 mL of toluene. Add 15 mmol of diethyl phosphite and 24 mmol of triethyl orthoformate. React at 120°C for 10 h. Separate on a silica gel (200-300 mesh) column with petroleum ether / ethyl acetate = 1:1 to obtain a pure product. Distill off the toluene under reduced pressure. Separate on a silica gel (200-300 mesh) column with ethyl acetate:methanol = 30:1 to obtain a pure product with a yield of 78%.

[0923] Step 3: Add 50 mL of 6N hydrochloric acid to the product of the previous step, react at 100°C for 10 hours, distill off the hydrochloric acid under reduced pressure, and wash the crude product once with acetone ultrasonically, and then wash it three times with methanol ultrasonically to obtain a pure product with a yield of 95%.

[0924] TH-Z93 Characterization Data:

[0925] 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). 31 P NMR (162MHz, D2O):δ15.12.

[0926] HRMS (ESI): calculated for C12H22N2O7P2: 369.0980; found: 369.0969.

[0927] Example 3

[0928]

[0929] (((4-(4,5,6,7-Tetrahydro-2H-indazol-2-yl)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0930]

[0931] Example 3 was prepared in a manner similar to Example 2, except that the first step was prepared under the following conditions: under argon protection, 10 mmol of 4-bromo-2-aminopyridine was dissolved in 100 mL of anhydrous DMF, 20 mmol of cesium carbonate and 12 mmol of 4,5,6,7-tetrahydroindazole were added, and the mixture was heated to 120° C. for 8 hours. The cesium carbonate was filtered off, the solvent was evaporated under reduced pressure, and the pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 1:1, with a yield of 73%.

[0932] 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). 31 P NMR (162MHz, D2O):δ14.92.

[0933] HRMS (ESI): C13H18N4O6P2 calculated value: 389.0780; found value: 389.0783

[0934] Example 4

[0935]

[0936] (((4-((2-Carbamoylpyridin-4-yl)methoxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0937]

[0938] Step 1: Dissolve 10 mol of 2-cyano-4-methylpyridine in 100 mL of carbon tetrachloride, add 11 mol of benzoyl peroxide and 11 mol of bromosuccinimide respectively, react at 77° C. for 4 hours, evaporate the solvent under reduced pressure, and separate on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 3:1 to obtain a pure product with a yield of 67%.

[0939] Step 2: 6 mmol of the product from the previous step was dissolved in 50 mL of solvent, 12 mmol of potassium carbonate and 7.2 mmol of 4-hydroxy-2-aminopyridine were added, the reaction was carried out at room temperature overnight, the potassium carbonate solid was filtered out, the acetonitrile solvent was evaporated under reduced pressure, and the pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 1:1, with a yield of 43%.

[0940] Step 3: Dissolve 2.5 mmol and 6 mmol of the product of the previous step, diethyl phosphite and 10 mmol of triethyl orthoformate in 20 mL of toluene, react at 120° C. overnight, evaporate the excess solvent under reduced pressure, and separate the pure product on a silica gel (200-300 mesh) column with ethyl acetate:methanol=30:1, with a yield of 82%.

[0941] Step 4: Dissolve 2.0 mmol of the product from step 3 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 evaporate the ethyl acetate under reduced pressure, and proceed to the next step without separation.

[0942] The fifth step is the same as the third step in Example 2, and the total reaction yield of the two steps is 78%.

[0943] 1 H 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). 31 P NMR (162 MHz, D2O): δ 12.19.

[0944] HRMS (ESI): C13H16N4O8P2 calculated value: 419.0522; found value: 419.0518

[0945] Example 5

[0946]

[0947] (((4-Hydroxypyridin-2-yl)amino)methylene)bisphosphonic acid

[0948] Example 5 was prepared in a similar manner to Example 2, except that benzyl bromide was used as a raw material instead of hexyl bromide in the first step and the reaction was carried out at room temperature.

[0949] 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) 31 P NMR (162 MHz, D2O): δ 15.27.

[0950] HRMS (ESI): C6H10N2O7P2 calculated value: 284.0051; found value: 284.0059

[0951] Example 6

[0952]

[0953] (((4-(Benzyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0954] Example 6 was prepared in a manner similar to Example 5, except that trimethylsilyl bromide was used in place of 6N hydrochloric acid in the third step, and hydrolysis was performed in dichloromethane at room temperature.

[0955] 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). 31 P NMR (162 MHz, D2O): δ 15.33.

[0956] HRMS (ESI): calculated for C13H16N2O7P2: 375.0511; found: 375.0519.

[0957] Example 7

[0958]

[0959] (((6-(Hexyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0960]

[0961] Example 7 was prepared in a similar manner to Example 2, except for the first step.

[0962] Step 1: At room temperature, 10 mmol of 6-hydroxy-4-aminopyridine was dissolved in 100 mL of dioxane, 12 mmol of triphenylphosphine and diethyl azodicarboxylate were added, and 20 mmol of n-hexanol was slowly added. After reacting for 4 hours, the solvent was distilled off under reduced pressure, and the pure product was separated on a silica gel (200-300 mesh) column with petroleum ether: ethyl acetate = 2:1, with a yield of 85%.

[0963] The other reaction steps are the same as in Example 2.

[0964] 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). 31 P NMR (162 MHz, D2O): δ 13.5.

[0965] HRMS (ESI): calculated for C12H22N2O7P2: 369.0980; found: 369.0973.

[0966] Example 8

[0967]

[0968] (((4-Bromopyridin-2-yl)amino)methylene)bisphosphonic acid

[0969] Example 8 was prepared in a manner similar to Example 2, except that 2-amino-4-bromopyridine was used as a raw material in the first step.

[0970] 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). 31 P NMR (162 MHz, D2O): δ 13.5.

[0971] HRMS (ESI): calculated for C6H9BrN2O6P2: 347.9143; found: 347.9145.

[0972] Example 9

[0973]

[0974] (((4-(Methylthio)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0975] Example 9 was prepared in a manner similar to Example 2, except that 2-amino-4-thiopyridine and iodomethane were used as raw materials in the first step.

[0976] 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). 31 P NMR (162 MHz, D2O): δ 13.0.

[0977] HRMS (ESI): calculated for C7H12N2SO6P2: 314.9969; found: 314.9969.

[0978] Example 10

[0979]

[0980] (((4-(Hexyloxy)quinolin-2-yl)amino)methylene)bisphosphonic acid

[0981] Example 10 was prepared in a manner similar to Example 2, except that 2-amino-4-hydroxyquinoline was used as a raw material in the first step.

[0982] 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). 31 P NMR (162 MHz, D2O): δ 13.1.

[0983] HRMS (ESI): calculated for C16H24N2O7P2: 419.1137; found: 419.1145.

[0984] Embodiment 11

[0985]

[0986] (((4-(4-Methylphenethoxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0987] Example 11 was prepared in a similar manner to Example 2, except that 1-(2-bromo-ethyl)-4-methyl-benzene was used as the starting material in the first step.

[0988] 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=6.0H z), 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). 31 P NMR (162 MHz, D2O): δ 13.1.

[0989] HRMS (ESI): calculated for C15H20N2O7P2: 403.0824; found: 403.0829.

[0990] Example 12

[0991]

[0992] (((5-Bromo-4-(hexyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[0993]

[0994] Example 12 was prepared in a similar manner to Example 2, except that the intermediate 4-hexyloxy-2-aminopyridine of Example 2 was subjected to a one-step bromination reaction. The operation steps were as follows: 10 mmol of 4-hexyloxy-2-aminopyridine was dissolved in 30 mL of glacial acetic acid, 11 mmol of bromine was added at room temperature, heated to 70° C. for 4 hours, then the reaction was quenched with saturated sodium thiosulfate, extracted with ethyl acetate, the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 1:1 to obtain a pure product with a yield of 73%.

[0995] 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). 31 P NMR (162 MHz, D2O): δ 15.5.

[0996] HRMS (ESI): calculated for C12H21BrN2O7P2: 448.0032; found: 448.0033.

[0997] Example 13

[0998]

[0999] (((3-(Hexyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[1000] Example 13 was prepared in a manner similar to Example 2, except that 2-amino-3-hydroxypyridine was used as the starting material in the first step.

[1001] 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). 31 P NMR (162 MHz, D2O): δ 11.1.

[1002] HRMS (ESI): calculated for C12H22N2O7P2: 369.0980; found: 369.0978.

[1003] Embodiment 14

[1004]

[1005] (((4-Hexylpyridin-2-yl)amino)methylene)bisphosphonic acid

[1006]

[1007] The first step: 10 mmol of 2-amino-4-bromopyridine was dissolved in 100 mL of anhydrous tetrahydrofuran, 10 ml of triethylamine and 15 mmol of 1-hexyne, 0.5 mmol of cuprous iodide and 1 mmol of triphenylphosphine palladium chloride were added, and the mixture was reacted at 60° C. for 4 hours under argon protection, and the solvent was distilled off under reduced pressure. The pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether to ethyl acetate of 3:1, and the yield was 88%.

[1008] Step 2: 8.8 mmol of the product of the first step was dissolved in 50 mL of acetic acid, 200 mg of palladium hydroxide was added, and hydrogen reduction reaction was carried out for 24 hours. The palladium hydroxide was filtered out with diatomaceous earth, and the acetic acid was evaporated under reduced pressure. The pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 2:1, and the yield was 95%.

[1009] The other reaction steps are similar to the second and third steps in Example 2.

[1010] 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). 31 P NMR (81 MHz, D2O): δ 11.7.

[1011] HRMS (ESI): C12H22N2O6P2 calculated value: 353.1031; found value: 353.1037

[1012] Embodiment 15

[1013]

[1014] (((4-(Oct-1-yn-1-yl)pyridin-2-yl)amino)methylene)bisphosphonic acid

[1015] Example 15 was prepared in a similar manner to Example 14, except that 1-octyne was used as a raw material in the first step, and the third step reaction was directly carried out without the second step hydrogen reduction.

[1016] 1H NMR (400MHz, D2O): δ7.64 (d, 1H, J = 5.6Hz), 7.03 (s, 1H), 6.63 (d, J = 6.0Hz), 4.06 (t, 2 H, 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). 31 P NMR (162 MHz, D2O): δ 11.6.

[1017] HRMS (ESI): calculated for C14H22N2O6P2: 377.1031; found: 377.1033.

[1018] Example 16

[1019]

[1020] (((4-(Octylamino)pyridin-2-yl)amino)methylene)bisphosphonic acid

[1021]

[1022] Step 1: Dissolve 10 mmol 2,4-diaminopyridine in 100 mL pyridine, cool to 0°C, slowly add octanoyl chloride to the reaction solution, react for 4 hours, evaporate the solvent under pressure, and separate the pure product on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 1:1, with a yield of 37%.

[1023] Step 2: 3 mmol of the product of the first step was dissolved in 50 mL of anhydrous tetrahydrofuran, cooled to 0°C, 5 mmol of lithium aluminum tetrahydride was slowly added, the reaction was continued for 2 hours, the reaction was quenched with saturated ammonium chloride, extracted with 150 mL of ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 1:1 to obtain a pure product with a yield of 89%.

[1024] The third and fourth step reactions are similar to the second and third steps in Example 2.

[1025] 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). 31 P NMR (162 MHz, D2O): δ 13.5.

[1026] HRMS (ESI): calculated for C14H27N3O6P2: 396.1463; found: 396.1471.

[1027] Embodiment 17

[1028]

[1029] (((5-(Octyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[1030] Example 17 was prepared in a manner similar to Example 2, except that 5-hydroxy-2-aminopyridine and n-octanol were used as raw materials in the first step.

[1031] 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).31 P NMR (162 MHz, D2O): δ 13.5.

[1032] HRMS (ESI): calculated for C14H26N2O7P2: 397.1293; found: 397.1298.

[1033] Embodiment 18

[1034]

[1035] (((4-Octylpyridin-2-yl)amino)methylene)bisphosphonic acid

[1036] Example 18 was prepared in a manner similar to Example 14, except that 1-octyne was used as the starting material in the first step.

[1037] 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). 31 P NMR (162 MHz, D2O): δ 11.8.

[1038] HRMS (ESI): calculated for C14H26N2O6P2: 381.1316; found: 381.1320.

[1039] Embodiment 19

[1040]

[1041] (((6-methyl-3-(octyloxy)pyridin-2-yl)amino)methylene)bisphosphonic acid

[1042] Example 19 was prepared in a manner similar to Example 2, except that 2-amino-3-hydroxy-6-methylpyridine was used as the starting material in the first step.

[1043] 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.

[1044] HRMS (ESI): calculated for C15H29N2O7P2: 411.1421; found: 411.1417.

[1045] Embodiment 20

[1046]

[1047] (((4-Decypyridin-2-yl)amino)methylene)bisphosphonic acid

[1048] The synthesis of this compound is similar to the method of Example 20, except that 1-decyne is used as the raw material in the first step.

[1049] 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). 31 P NMR (162 MHz, D2O): δ 11.5.

[1050] HRMS (ESI): calculated for C16H30N2O6P2: 410.1657; found: 410.1663.

[1051] Embodiment 21

[1052]

[1053] (((5-(Octyloxy)pyridin-3-yl)amino)methylene)bisphosphonic acid

[1054]

[1055] Step 1: 10 mmol of 5-hydroxy-3-carboxypyridine was dissolved in 100 mL of methanol, 5 mL of thionyl chloride was slowly added, and the mixture was reacted at 60° C. for 4 hours. The solvent was evaporated under reduced pressure, and the next step of reaction was carried out without purification.

[1056] Step 2: The product of the first step was dissolved in 150 mL of acetonitrile, 30 mmol of potassium carbonate was added, the reaction was carried out at 60° C. overnight, the solid was filtered off, the solvent was evaporated under reduced pressure, and the pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 1:1. The two-step yield was 73%.

[1057] Step 3: Dissolve 7.3 mmol of the product in 100 mL of dioxane, add 20 mL of 4N NaOH, and react at room temperature for 8 hours. After the reaction is completed, adjust the pH to 6 with dilute hydrochloric acid, extract with 200 mL of ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, evaporate the solvent under reduced pressure, and directly proceed to the next step without purification.

[1058] Step 4: Dissolve the crude product from the previous step in 150 mL of dichloromethane, add 10 mL of triethylamine at 0°C, slowly dropwise add 8 mmol of diphenylphosphoryl azide, react for 4 hours to room temperature, evaporate the solvent under reduced pressure, and proceed to the next step without separation.

[1059] Step 5: In a pressure tube, the crude product of the previous step was dissolved in 100 mL of tetrahydrofuran, 10 mL of benzyl alcohol was added, and the reaction was carried out at 90° C. for 8 hours. The solvent was evaporated under reduced pressure, and the pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 3:1. The total yield of the three steps was 78%.

[1060] Step 6: Dissolve the product of the previous step in 100 mL of methanol, add 500 mg of palladium carbon, reduce with hydrogen at room temperature for 24 hours, filter out the palladium carbon with diatomaceous earth, concentrate the filtrate under reduced pressure, and separate on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 1:1 to obtain the pure product with a yield of 97%.

[1061] The remaining two steps of the reaction are the same as Steps 2 and 3 in Example 2.

[1062] 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). 31 P NMR (162 MHz, D2O): δ 13.5.

[1063] HRMS (ESI): calculated for C14H26N2O7P2: 397.1293; found: 397.1298.

[1064] Embodiment 22

[1065] TH-Z144 preparation:

[1066]

[1067] (2-(3-(Hexyloxy)phenyl)ethane-1,1-diyl)bisphosphonic acid

[1068]

[1069] Step 1: Dissolve 10 mmol of the raw material methyl 3-hydroxybenzoate in 100 mL of acetonitrile, add 20 mmol of potassium carbonate and 12 mmol of 1-bromohexane, protect with nitrogen, react at 60°C overnight, cool the reaction solution to room temperature, filter out the potassium carbonate, concentrate under reduced pressure, and use the crude product for the next step of reaction without column separation.

[1070] Step 2: The product of the previous step was dissolved in 200 mL of anhydrous tetrahydrofuran at 0°C, 20 mmol of lithium aluminum hydroxide was added in batches, and the reaction was continued at 0°C for 4 hours. The raw material was almost completely reacted according to thin layer chromatography. The reaction was quenched with saturated ammonium chloride, extracted with 200 mL of ethyl acetate, and the organic phase was dried over anhydrous magnesium sulfate. The pure product was separated on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 8:1. The two-step yield was 90%.

[1071] Step 3: The product of the previous step was dissolved in 100 mL of anhydrous dichloromethane, 10.8 mmol of triphenylphosphine was added at 0°C, and then 10.8 mmol of a dichloromethane solution of carbon tetrabromide was slowly added dropwise, and the reaction was continued for 4 hours. TLC detected that the raw material was completely reacted, and the dichloromethane was evaporated under reduced pressure. The pure product was separated on a silica gel (200-300 mesh) column with petroleum ether:ethyl acetate = 30:1, and the yield was 95%.

[1072] Step 4: 6 mmol of tetraethyl methylene diphosphate was dissolved in 100 mL of anhydrous tetrahydrofuran, and 7 mmol of sodium hydrogen was added at 0°C. After the reaction for 30 minutes, 8 mmol of the product of the previous step was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the above mixed solution. The mixture was reacted to room temperature. Most of the raw materials were detected by wave chromatography. 100 mL of saturated sodium chloride was added, and the mixture was extracted with 200 mL of ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, and the pure product was separated on a silica gel (200-300 mesh) column with a ratio of petroleum ether:ethyl acetate = 3:1, with a yield of 75%.

[1073] Step 5: Add 50 mL of 6N hydrochloric acid to the product of the previous step, react at 100°C for 10 hours, distill off the hydrochloric acid under reduced pressure, and wash the crude product with acetone ultrasonically for 3 times to obtain a pure product with a yield of 93%.

[1074] 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). 31 PNMR (162MHz, MeOD): δ21.78. HRMS (ESI): C16H28O7P2 calculated value: 367.1080; found value: 367.1081

[1075] Embodiment 23

[1076] Preparation of TH-Z145

[1077]

[1078] (2-(3-(Octyloxy)phenyl)ethane-1,1-diyl)bisphosphonic acid

[1079] TH-Z145 was prepared in a similar manner to H-Z144, except that 1-bromooctane was used as the raw material instead of 1-bromohexane in the first step and the reaction was carried out at 60 °C.

[1080] 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). 31 PNMR (162MHz, MeOD): δ21.67. HRMS (ESI): C14H24O7P2 calculated value: 395.1393; found value: 395.1387.

[1081] Embodiment 24

[1082] Preparation of TH-Z80: TH-Z80 was prepared according to the following synthetic route.

[1083]

[1084] Step 1: Dissolve 10 mmol (1.54 g) of 3-nitro-4-aminophenol in 50 mL of acetone, add 30 mmol of anhydrous potassium carbonate, heat under reflux under N2 protection, and then add 12 mmol (1.67 mL) of bromohexane. After reacting overnight, filter the insoluble matter, spin-dry the organic phase, load the sample, and separate and purify with petroleum ether / ethyl acetate to obtain 7.3 mmol (1.74 g) of 4-hexyloxy-2-nitro-aniline (yield: 73%).

[1085] Step 2: 5mmol (1.19g) of 4-hexyloxy-2-nitro-aniline was reacted with 60mmol (9.69mL) of tert-butyl bromoacetate and 7.5mmol (1.04g) of anhydrous potassium carbonate at 110 degrees under N2 protection for 12h. The insoluble matter in the reaction solution was filtered out, spin-dried, loaded with sample, and separated and purified with petroleum ether / ethyl acetate to obtain 2.1mmol (0.74g) of (4-(hexyloxy)-2-nitrophenyl)glycine tert-butyl ester with a yield of 42%.

[1086] Step 3: 2mmol (0.70g) (4-(hexyloxy)-2-nitrophenyl) glycine tert-butyl ester was dissolved in 10mL ethanol, 0.1g 5% palladium carbon was added, and the mixture was reacted under H2 for 4h. Celite was filtered, the organic phase was dried by rotation, the crude product was dissolved in 10mL ethylene glycol monomethyl ether, 7mmol (0.72g) formamidine acetate was added, the mixture was heated under reflux for 4h and then cooled, the reaction solution was dried by rotation, the sample was loaded, and column was passed to obtain 0.7mmol (0.23g) 2-5-(hexyloxy)-1H-benzo[d]imidazol-1yl-)acetic acid tert-butyl ester, and the total yield of the two steps was 35%.

[1087] Step 4: Heat 0.6 mmol (0.2 g) of tert-butyl 2-5-(hexyloxy)-1H-benzo[d]imidazol-1-yl-)acetate in 6N HCl under reflux for 6 h, then spin dry and dry to constant weight. The crude product is directly used in the next reaction.

[1088] Step 5: Dissolve the obtained 2-5-(hexyloxy)-1H-benzo[d]imidazole-1-yl-)acetic acid with 1.8mmol (0.15g) of phosphorous acid and 1mL of cyclopentane sulfone at 75 degrees, add 2mmol (178μL) of PCl3 dropwise, react for 3.5 hours, add 1mL of water, heat to reflux for 2h, cool, filter the precipitated solid, ultrafilter with methanol 3 times, dry the obtained light yellow solid, and weigh 38mg of the target product after constant weight (yield 15%).

[1089] Structural characterization data: 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), 13 C NMR(100MHz,D2O),δ(ppm):154.18,147.63,142.09,131.10,113.20,112.78,1 02.72,77.79,76.49,75.19,69.96,50.51,30.80,28.40,24.91,21.94,13.32, 31 P NMR (162MHz, D2O), δ (ppm): 15.67

[1090] Referring to the above preparation method, the present invention also prepares the following compounds corresponding to n=1, 2, 3, 4, 5, 7, 8, 9, 10, 11 and 12 respectively:

[1091]

[1092] They are recorded as TH-Z79, TH-Z148, TH-Z149, TH-Z150, TH-Z151, TH-Z152, TH-Z81, TH-Z153, TH-Z82, TH-Z154 and TH-Z 155 respectively.

[1093] The 4-hexyloxy-2-nitro-aniline raw material in step 2 of the preparation of TH-Z80 is replaced by 4-hydroxy 2-nitroaniline, and steps 2, 3, 4 and 5 are carried out in sequence to obtain BPH-266 (the corresponding compound shown in formula I when R1, R2, R4 and R5 = H, m = 1, X = OH, M = H, and n = 0).

[1094] Except for TH-Z79 which directly uses 2-amino-4-methoxyaniline as raw material, other TH-Z80 series compounds are synthesized according to the synthesis steps of TH-Z80.

[1095] The characterization data of TH-Z80 series compounds are as follows:

[1096] TH-Z79: Structural characterization data: 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), 31 PNMR(162MHz,D2O),δ(ppm):15.33

[1097] TH-Z81: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.60

[1098] TH-Z82: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.58

[1099] TH-Z148: Structural characterization data: 1 H 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), 31 P NMR (162MHz, D2O), δ (ppm): 14.82

[1100] TH-Z149: Structural characterization data: 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), 31 PNMR(162MHz,D2O),δ(ppm):15.60

[1101] TH-Z150: Structural characterization data: 1 H NMR (400MHz, D2O), δ (ppm): 8.39 (s, 1H), 7.77 (d, J = 8.9Hz, 1H), 7.28 (s, 1H), 7.05 (d, J = 8.4H z,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), 31 P NMR (162MHz, D2O), δ (ppm): 15.60

[1102] TH-Z151: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):14.02

[1103] TH-Z152: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.32

[1104] TH-Z153: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.41

[1105] TH-Z154: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.33

[1106] TH-Z155: Structural characterization data: 1 H 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) 31 PNMR(162MHz,D2O),δ(ppm):15.39

[1107] This application refers to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, this specification shall prevail. In addition, any specific embodiment of the present invention that falls within the scope of the prior art may be explicitly excluded from any one or more claims. Because the embodiments are considered to be known to those skilled in the art, they may be excluded, even if the exclusion is not explicitly listed in this application. Any specific embodiment of the present invention may be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[1108] Using only routine experimentation, those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited to the above description, but rather to that set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications of this specification may be achieved without departing from the spirit or scope of the invention as defined in the claims.

Claims

1. An immunogenic composition comprising an adjuvant selected from: 1) Thiolase inhibitors; 2) HMG-CoA synthase inhibitors; 3) HMG-CoA reductase inhibitors; 4) mevalonate kinase inhibitors; 5) Phosphomevalonate kinase inhibitors; 6) mevalonate-5-pyrophosphate decarboxylase inhibitors; 7) Isopentenyl pyrophosphate isomerase inhibitors; 8) Farnesyl pyrophosphate synthase inhibitors; 9) Geranylgeranyl pyrophosphate synthase inhibitors; and 10) Geranylgeranyl transferase (I, II) inhibitors.

2. The immunogenic composition of claim 1, wherein The farnesyl pyrophosphate synthase inhibitor is a bisphosphonic acid compound or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof; or The farnesyl pyrophosphate synthase inhibitor is a compound represented by formula I or a pharmaceutically acceptable salt or hydrate thereof: In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group; X is any one of the following: hydrogen, hydroxyl, aliphatic group, thiol, halogen, alkoxy or alkyl; each M can independently be any one of the following: negative charge, hydrogen, alkyl, aliphatic group, -(CH2)pO-CO-R, -(CH2)p-CO-R or cation; wherein p =1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl; m=an integer from 1 to 6; or The farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: wherein R1 and R2 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen, etc.; or The farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: wherein R3 and R4 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen; or The farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: in R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; or The farnesyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: in R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

3. The immunogenic composition of claim 1, wherein the geranylgeranyl pyrophosphate synthase inhibitor is a compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: in R9 is selected from 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; and R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

4. Thiolase inhibitors, HMG-CoA synthase inhibitors, HMG-CoA reductase inhibitors, mevalonate kinase inhibitors, phosphomevalonate kinase inhibitors, mevalonate-5-pyrophosphate decarboxylase inhibitors, isopentenyl pyrophosphate isomerase inhibitors, isopentenyl pyrophosphate isomerase inhibitors, geranylgeranyl pyrophosphate synthase inhibitors and geranylgeranyl transferase (I, II) inhibitors as described above, which are used as adjuvants.

5. Use of the aforementioned thiolase inhibitors, HMG-CoA synthase inhibitors, HMG-CoA reductase inhibitors, mevalonate kinase inhibitors, phosphomevalonate kinase inhibitors, mevalonate-5-pyrophosphate decarboxylase inhibitors, isopentenyl pyrophosphate isomerase inhibitors, isopentenyl pyrophosphate isomerase inhibitors, geranylgeranyl pyrophosphate synthase inhibitors and geranylgeranyl transferase (I, II) inhibitors as adjuvants in the preparation of immunogenic compositions.

6. The compound represented by formula I or its pharmaceutically acceptable salt or hydrate: In the above formula I, the molecular weight is less than 1000, Ar is a benzimidazole group or an azabenzimidazole group; X is any one of the following: hydrogen, hydroxyl, aliphatic group, thiol, halogen, alkoxy or alkyl; each M can independently be any one of the following: negative charge, hydrogen, alkyl, aliphatic group, -(CH2)pO-CO-R, -(CH2)p-CO-R or cation; wherein p =1-6, R is hydrogen, alkyl or aromatic; the cation is Li + 、Na + , K + , Ca 2+ Mg 2+ NH4 + or N(R')4 + , wherein R' is an alkyl group; R6 and R7 are independently selected from any one of the following: hydrogen, hydroxyl, thiol, halogen, amino, aliphatic or alkyl; m=an integer of 1-6.

7. A compound of the following formula or a pharmaceutically acceptable salt, ester, prodrug, or solvate thereof: wherein R1 and R2 are each selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxy, thiol, halogen, etc.; Wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic group, and the aryl, heteroaryl or heterocyclic group is optionally substituted by alkyl, carbamoyl; X is selected from hydrogen, hydroxyl, thiol and halogen.

8. The compound of the following formula or its pharmaceutically acceptable salt, ester, prodrug, solvate: wherein R1 is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclic, aryl and heteroaryl, the alkyl in the alkoxy is optionally substituted by aryl, heteroaryl or heterocyclic, and the aryl, heteroaryl or heterocyclic is optionally substituted by alkyl, carbamoyl; R2 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R3 is selected from hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R2 and R3 together with the carbon atoms to which they are attached form an aromatic or heteroaromatic ring; and R4 is selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

9. The compound of the following formula or its pharmaceutically acceptable salt, ester, prodrug, solvate: in R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

10. The compound of the following formula or its pharmaceutically acceptable salt, ester, prodrug, solvate: in R9 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; and R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, halogen, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

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