Conjugate of cholesterol and TLR7 agonist, and drug composition containing same

Abstract

The present invention relates to a conjugate of cholesterol and a TLR7 agonist that is useful as a drug, a pharmaceutically acceptable salt thereof, and a drug composition or cancer treatment vaccine containing the same as an active ingredient.

Description

Cholesterol-TLR7 agonist conjugate and pharmaceutical composition containing same

[0001] The present invention relates to a conjugate of cholesterol and a TLR7 agonist that is useful as a medicine, and a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition or a cancer therapeutic vaccine containing the same as an active ingredient.

[0002] An mRNA vaccine is a vaccine composed of lipid nanoparticles (hereinafter referred to as "LNPs") and mRNA, and is known to induce humoral and cellular immunity against proteins produced by in vivo transcription of mRNA (Non-Patent Document 1).

[0003] The role of ionized lipids, one of the components of LNPs, is important when mRNA contained in mRNA vaccines is transcribed. It is known that basic ionized lipids interact with mRNA to form stable LNPs. In endosomes with a weakly acidic pH, the ionized lipids become electrically charged, inducing the induction of LNPs and endosomes. As a result, mRNA escapes from the endosome into the cytoplasm, and the target protein is transcribed. In addition to ionized lipids, LNPs contain cholesterol derivatives, helper lipids, and PEGylated lipids for the purpose of providing formulation stability (Non-Patent Document 1). mRNA vaccines using LNPs are being studied as preventive vaccines for infectious diseases and as therapeutic vaccines for cancer (Non-Patent Document 2).

[0004] Research is being conducted into the addition of adjuvants to enhance the efficacy of LNP-based mRNA (Non-Patent Document 3). These adjuvants include compounds with TLR7 agonist activity, and a conjugate compound (C12-TLR7a) of an ionized lipid and a TLR7 agonist, which combines the properties of an ionized lipid with TLR7 agonist activity, has been reported. However, while C12-TLR7a possesses the properties of an ionized lipid, its transfection activity has been reported to be weaker than that of conventional products. Therefore, the amount added must be optimized to match the transfection activity, making it difficult to optimize the amount of adjuvant (Non-Patent Document 4). Additionally, C16-R848, a conjugate of palmitic acid and a TLR7 agonist, has been reported to be used as an adjuvant for mRNA vaccines using formulations other than LNP (Non-Patent Document 5).

[0005] Among the lipids that make up LNPs, 1V209-Chol, a conjugate of cholesterol and a TLR7 agonist, has been reported. 1V209-Chol-Lip, a liposomal formulation of 1V209-Chol, has been reported to exhibit antitumor effects, but this agent is not an mRNA vaccine (Non-Patent Document 6). Conjugates of cholesterol and the TLR7 agonist imidazoquinoline have also been reported, and there are examples of their use as adjuvants in liposomal and emulsion formulations (Patent Document 1). Despite these reports, it has been reported that conjugation of a TLR7 agonist with cholesterol results in a loss of agonist activity, and there have been cases where prodrug formation is required to maintain TLR7 agonist activity (Patent Document 2).

[0006] Splenic hyperplasia has been reported as one of the side effects of TLR7 agonists (Non-Patent Document 7).

[0007] WO2010 / 048520US2022 / 0008411

[0008] Nat Rev Drug Discov. 2021 Nov; 20(11): 817-838.Nat Med. 2024 Jan 25. doi: 10.1038 / d41591-024-00006-4NPJ Vaccines. 2023 Oct 26; 8(1): 162Nat. Nanotechnol. 18, 1105-1114 (2023).Biomaterials. 2021 Jan: 266: 120431Nano Lett. 2021 Oct 13; 21(19): 7960-7969Blood. 2009 Jan 8; 113(2): 377-88

[0009] An object of the present invention is to provide a TLR7 agonist that is optimized for an mRNA vaccine using LNP.

[0010] As a result of extensive research, the present inventors have found that a conjugate of a TLR7 agonist represented by the following formula (1) and a cholesterol retains TLR7 agonist activity without losing activity as in the case of prodrugs. Additionally, they have found that a conjugate of a TLR7 agonist represented by the following formula (1) and a cholesterol forms a stable formulation with an mRNA vaccine using LNP. Furthermore, they have found that the compound represented by the following formula (1) has a strong immune-inducing effect as an adjuvant for mRNA vaccines. Furthermore, they have found that it is a safe adjuvant that does not cause splenic hyperplasia, a side effect of TLR7 agonists. As a result of these effects, they have obtained a vaccine adjuvant with TLR7 agonist activity optimized for an mRNA vaccine using LNP, thereby completing the invention.

[0011] That is, the present invention is as follows.

[0012] [Term 1] Formula (1): [In formula (1), R １ is C １－６ Alkyl, or C １－６ represents alkoxy, R ２ is halogen, hydroxy, or C １－６C optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy １－６ represents alkyl, R ３ is hydrogen, halogen, cyano, hydroxy, C １－６ Alkyl, or C １－６ L represents *-(CH ２ ) ｐ C(O)O(CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -C(O)-**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, *-NR ４ (CH ２ ) ｐ -**, *-O(CH ２ ) ｐ -** or *-(CH ２ ) ｐ C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６ represents alkyl, p and q each independently represent an integer of 0 to 6, bonded to the benzene ring at *, and bonded to X at **, and X represents a cholesterol, or a pharmaceutically acceptable salt thereof.

[0013] [Section 2] R １ But C １－３ alkyl, R ２ C optionally substituted with 1 to 2 hydroxy groups １－６ alkyl, R ３ is hydrogen or C １－３ Alkoxy, and L is *-(CH ２ )ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, or *-(CH ２ ) ｐ Item 2. The compound according to Item 1, wherein the compound is C(O)-**, or a pharmaceutically acceptable salt thereof.

[0014] [Term 3] Formula (2a): [In formula (2a), R ３ is hydrogen or C １－３ L represents *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ 2. The compound according to claim 1, wherein the compound is represented by the formula: 2.) C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X), and X is a cholesterol.

[0015] [Term 4] Formula (2): [In the formula (2), L is *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ Item 4. The compound according to any one of Items 1 to 3, wherein the compound is represented by the formula: 2(O)-C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X), and X represents a cholesterol, or a pharmaceutically acceptable salt thereof.

[0016] [Item 5] Formula (3a) or (4a): [In formula (3a) and formula (4a), R ３ is hydrogen or C １－３ and X represents a cholesterol or a similar group. Item 4. The compound according to any one of Items 1 to 3, wherein X represents an alkoxy group, or a pharmaceutically acceptable salt thereof.

[0017] [Term 6] Equation (3) or (4): Item 6. The compound according to any one of Items 1 to 5, which is represented by the formula (3) and the formula (4), wherein X represents a cholesterol, or a pharmaceutically acceptable salt thereof.

[0018] [Item 7] Formula (3a) or (4a): [In formula (3a) or formula (4a), R ３ is hydrogen or C １－３ X represents an alkoxy group represented by formula (5): Formula (6): Or formula (7): wherein formula (5), formula (6) or formula (7) is bonded to formula (3a) or formula (4a) at ***.

[0019] [Term 8] Equation (3) or (4): [In the formula (3) or (4), X represents a group represented by the formula (5): Formula (6): Or formula (7): wherein formula (5), formula (6) or formula (7) is bonded to formula (3) or formula (4) at ***.] The compound according to any one of items 1 to 7, or a pharmaceutically acceptable salt thereof.

[0020] [Term 9] Equation (8), Equation (9), or Equation (10): Item 8. The compound according to any one of Items 1 to 3, 5 and 7, which is represented by the following formula: or a pharmaceutically acceptable salt thereof.

[0021] [Term 10] Equation (8) or Equation (9): Item 10. The compound according to any one of items 1 to 9, wherein the compound is represented by the formula: or a pharmaceutically acceptable salt thereof.

[0022] [Term 11] Formula (8): Item 11. The compound according to any one of Items 1 to 10, which is represented by the following formula: or a pharmaceutically acceptable salt thereof.

[0023] [Term 12] Formula (9): Item 11. The compound according to any one of Items 1 to 10, which is represented by the following formula: or a pharmaceutically acceptable salt thereof.

[0024] [Term 13] Formula (10): Item 10. The compound according to any one of Items 1 to 3, 5, 7, and 9, represented by the formula: or a pharmaceutically acceptable salt thereof.

[0025] [Item 14] A pharmaceutical composition comprising the compound according to any one of items 1 to 13 or a pharmaceutically acceptable salt thereof.

[0026] [Item 15] The pharmaceutical composition according to Item 14, which is a lipid nanoparticle (LNP) formulation.

[0027] [Item 16] The pharmaceutical composition according to Item 15, which is an LNP composed of four types of components: an ionizable lipid, a helper lipid, a PEGylated lipid, and cholesterols.

[0028] [Item 17] The pharmaceutical composition according to Item 16, wherein the ionizable lipid in the LNP is DLin-MC3-DMA (CAS Registry Number 1224606-06-7), SM-102 (CAS Registry Number 2089251-47-6), ALC-0315 (CAS Registry Number 2036272-55-4), A18-iso5-2DC18m (CAS Registry Number 2412492-09-0), Lipid A6 (di(dec-3-yn-1-yl) 9-((4-(dimethylamino) butanoyl)oxy) heptadecanedioate), or 306Oi10 (CAS Registry Number 2322290-93-5).

[0029] [Item 18] The PEGylated lipid of the LNP is PEG2000-C-DMG ((R)-142-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137,143-heptatetracontaoxa-141-azahexatetracontahectane-145,146-diyl Item 18. The pharmaceutical composition according to Item 16 or 17, wherein the PEG-1000 is selected from the group consisting of PEG-1000 ditetradecanoate, ALC-0159 (CAS Registry Number 1849616-42-7), DMG-PEG2000 (CAS Registry Number 160743-62-4), and PEG-1000 ditetradecanoate.

[0030] [Item 19] The pharmaceutical composition according to any one of Items 16 to 18, wherein the helper lipid of the LNP is DSPC (Distearoylphosphatidylcholine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0031] [Item 20] The pharmaceutical composition according to any one of Items 16 to 19, wherein the cholesterol in the LNP is cholesterol, β-sitosterol, or 20α-hydroxycholesterol.

[0032] [Item 21] The pharmaceutical composition according to any one of Items 16 to 20, wherein the ionized lipid of the LNP is DLin-MC3-DMA, the PEGylated lipid is DMG-PEG2000, the helper lipid is DSPC, and the cholesterol is cholesterol.

[0033] [Item 22] The pharmaceutical composition according to any one of Items 14 to 21, which comprises mRNA as an antigen.

[0034] [Item 23] The pharmaceutical composition of Item 22, comprising mRNA encoding a cancer antigen protein.

[0035] [Item 24] A cancer treatment and / or prevention agent comprising the compound according to any one of items 1 to 23, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as an active ingredient.

[0036] [Item 25] The therapeutic and / or prophylactic agent according to Item 24, wherein the cancer is non-small cell lung cancer, head and neck cancer, pancreatic cancer, malignant melanoma, renal cell carcinoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, glioblastoma multiforme, sarcoma, brain tumor, leukemia, myelodysplastic syndrome, multiple myeloma, or malignant lymphoma.

[0037] [Item 26] A method for treating and / or preventing cancer, comprising administering to a patient in need of treatment a therapeutically effective amount of the compound according to any one of items 1 to 23, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

[0038] [Item 27] ​​Use of the compound according to any one of Items 1 to 23, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the manufacture of an agent for treating and / or preventing cancer.

[0039] [Item 28] The compound according to any one of Items 1 to 23, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the treatment and / or prevention of cancer.

[0040] [Item 29] A pharmaceutical comprising a compound according to any one of items 1 to 23, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in combination with at least one or more drugs selected from anticancer agents.

[0041] [Item 30] The compound according to any one of Items 1 to 23, its pharmaceutically acceptable salt, or pharmaceutical composition for use in combination with at least one or more drugs selected from drugs classified as anticancer drugs to treat cancer.

[0042] [Item 31] An mRNA vaccine comprising the compound according to any one of Items 1 to 13 or a pharmaceutically acceptable salt thereof, the LNP according to any one of Items 17 to 21, and mRNA as an antigen.

[0043] The compounds of the present invention exhibit strong immune induction ability when used as adjuvants for mRNA vaccines using LNPs. Furthermore, through strong immune induction, they exhibit strong antitumor activity, making them useful as adjuvants for mRNA vaccines using LNPs administered for the purpose of treating or preventing cancer.

[0044] Figure 1 shows the results of ELISPOT in Test Example 3, in which the adjuvant activity of Examples 3 to 6, in which the compound synthesized in Example 1 was prepared into LNP, was evaluated in the presence and absence of the peptide represented by SEQ ID NO: 1. Figure 2 shows the results of ELISPOT in Test Example 3, in which the adjuvant activity of Examples 3 to 6, in which the compound synthesized in Example 1 was prepared into LNP, was evaluated in the presence and absence of the peptide represented by SEQ ID NO: 2. Figure 3 shows the results of Test Example 4, in which the adjuvant activity of Examples 3 to 6, in which the compound synthesized in Example 1 was prepared into LNP, was evaluated by antitumor effect.

[0045] The terms used in this specification are explained below.

[0046] The number of substituents in a group defined as "optionally substituted" or "substituted" is not particularly limited as long as substitution is possible. In addition, unless otherwise specified, the description of each group also applies when that group is a part or substituent of another group.

[0047] Examples of "halogen" include fluorine, chlorine, bromine, and iodine. Preferred are fluorine and chlorine. More preferred is fluorine.

[0048] "C １－６ "Alkyl" means a straight or branched chain saturated hydrocarbon group having from 1 to 6 carbon atoms. ６ "Alkyl" means alkyl having 6 carbon atoms, and the same applies to other numbers. １－６ As "alkyl", preferably "C １－３ alkyl", and more preferably "C １－２ "C alkyl" is an example. １－２ Specific examples of "alkyl" include methyl and ethyl. １－３Specific examples of "alkyl" include methyl, ethyl, propyl, 1-methylethyl, etc. １－６ Specific examples of "alkyl" include the above-mentioned "C １－３ In addition to the specific examples of "alkyl", examples include butyl, 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, pentyl, 3-methylbutyl, 2-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, and 3-ethylbutyl.

[0049] "C １－６ The term "alkoxy" refers to the same as the above "C １～６ "C" means an oxy group substituted by "alkyl." １－６ As the "alkoxy group", preferably "C １－３ "Alkoxy" is an example. １－３ Specific examples of "alkoxy" include methoxy, ethoxy, propoxy, and 1-methylethoxy. １－６ Specific examples of "alkoxy" include the above-mentioned "C １－３ In addition to the specific examples of "alkoxy", examples include butoxy, 1,1-dimethylethoxy, 1-methylpropoxy, 2-methylpropoxy, pentyloxy, 3-methylbutoxy, 2-methylbutoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, 1,1-dimethylpropoxy, hexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 1-methylpentyloxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, and the like.

[0050] The mRNA vaccine used in the present invention is composed of lipids, mRNA, and a TLR7 agonist included in the present invention.

[0051] The LNPs of the present invention are mainly composed of four lipid components: ionizable lipids, helper lipids, PEGylated lipids, and cholesterols.

[0052] Examples of ionizable lipids include, but are not limited to, DLin-MC3-DMA (CAS Registry Number 1224606-06-7), SM-102 (CAS Registry Number 2089251-47-6), ALC-0315 (CAS Registry Number 2036272-55-4), A18-iso5-2DC18m (CAS Registry Number 2412492-09-0), Lipid A6 (di(dec-3-yn-1-yl) 9-((4-(dimethylamino) butanoyl)oxy) heptadecanedioate), or 306Oi10 (CAS Registry Number 2322290-93-5).

[0053] Examples of PEGylated lipids include PEG2000-C-DMG ((R)-142-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137,143-heptatetracontaoxa-141-azahexatetracontahectane-145,146-diyl ditetradecanoate), ALC-0159 (CAS Registry Number 1849616-42-7), or DMG-PEG2000 (CAS Registry Number 160743-62-4).

[0054] Examples of helper lipids include, but are not limited to, DSPC (Distearoylphosphatidylcholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0055] The cholesterols represented by X in the formula refer to a series of compounds having a steroid skeleton. Cholesterols represented by X in the formula (X-H) include, but are not limited to, cholesterol, β-sitosterol, and 20α-hydroxycholesterol (see below).

[0056] Cholesterols as components of LNPs refer to a series of compounds having a steroid skeleton, including, but not limited to, cholesterol, β-sitosterol, and 20α-hydroxycholesterol.

[0057] mRNA may be a single-stranded ribonucleic acid molecule or a circular ribonucleic acid molecule. It may encode an amino acid sequence for antigen presentation, or it may contain a non-coding sequence to aid translation. From the 5' to 3' end, mRNA typically consists of (1) a cap structure, (2) a 5'-end untranslated region (5'UTR), (3) a coding region (ORF) sequence, (4) a 3'-end untranslated region (3'UTR), and (5) a repeated adenine nucleotide sequence (polyA tail). While 7-methylguanylic acid is commonly used as the cap structure, chemically modified 7-methylguanylic acid may also be used. The untranslated region controls mRNA stability and translation, and the type and length of the sequence may be optimized depending on the antigen. Although mRNA contains naturally occurring nucleotides, it may also contain chemically modified nucleotides, including, but not limited to, pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methoxyuridine (5moU), or 5-methylcytosine. The type of cancer antigen encoded by the mRNA may be one or more. The length of the poly(A) tail is generally 0 to 250 nucleotides. Examples of cancer antigens include, but are not limited to, ovalbumin (OVA, Gene ID: 396058).

[0058] R １ , R ２ , R ３ , R ４ , R ５ Preferred examples of L, X, p, and q are as follows, but the technical scope of the present invention is not limited to the range of compounds listed below.

[0059] R １ Preferably, C １－６ Alkyl, or C １－６ Alkoxy is preferred, and C １－３ Examples thereof include alkyl, and more preferably methyl.

[0060] R ２ Preferably, C １－６ alkyl (wherein the alkyl is halogen, hydroxy, or C １－６ and alkoxy), more preferably C １－６ Examples thereof include alkyl (which may be substituted with 1 to 2 hydroxy groups), and more preferably n-pentyl.

[0061] R ３ is preferably hydrogen, halogen, cyano, hydroxy, C １－６ Alkyl, or C １－６ Alkoxy is preferably a hydrogen atom or C １－３ Examples thereof include alkoxy, and more preferably methoxy.

[0062] L is preferably *-(CH ２ ) ｐ C(O)O(CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -C(O)-**, *-(CH２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, *-NR ４ (CH ２ ) ｐ -**, *-O(CH ２ ) ｐ -**, *-(CH ２ ) ｐ C(O)-**, or *-C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６ alkyl, p and q each independently represent an integer of 1 to 6, * is bonded to the benzene ring, and ** is bonded to X), more preferably *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X), and more preferably *-(CH ２ ) C(O)-** (where * is bonded to the benzene ring and ** is bonded to X).

[0063] X is preferably a cholesterol, more preferably cholesterol, β-sitosterol, or 20α-hydroxycholesterol, and even more preferably cholesterol.

[0064] R ４ and R ５ are preferably each independently hydrogen or C １－６ alkyl, more preferably a hydrogen atom or C １－３ An alkyl group is preferable, and a hydrogen atom is more preferable.

[0065] As p, an integer of 1 to 6 is preferred, an integer of 1 to 3 is more preferred, and 1 is even more preferred.

[0066] q is preferably an integer of 1 to 6, more preferably an integer of 3 to 5, and even more preferably 4.

[0067] Among the compounds represented by formula (1), preferred compounds include the following compounds or pharmaceutically acceptable salts thereof:

[0068] The ionizable lipid preferably includes DLin-MC3-DMA, SM-102, ALC-0315, A18-iso5-2DC18m, Lipid A6, or 306Oi10, and more preferably includes DLin-MC3-DMA.

[0069] The PEGylated lipid is preferably PEG2000-C-DMG, ALC-0159, or DMG-PEG2000, and more preferably PEG2000-C-DMG.

[0070] The helper lipid is preferably DSPC or DOPE, more preferably DSPC.

[0071] Preferred examples of cholesterols as components of LNP include cholesterol, β-sitosterol, and 20α-hydroxycholesterol, and more preferred examples include cholesterol.

[0072] The amounts (molar fractions) of the ionized lipid, PEGylated lipid, helper lipid, and cholesterol are not particularly limited, but preferably ranges of 25 to 75 mol%, 0 to 20 mol%, 0 to 30 mol%, and 15 to 55 mol%, respectively, and more preferably ranges of 35 to 65 mol%, 0 to 10 mol%, 0 to 40 mol%, and 25 to 45 mol%, respectively.

[0073] The amount (molar fraction) of the compound of the present invention to be incorporated is not particularly limited, but is preferably 0 mol % to 50 mol %, more preferably 0 mol % to 30 mol %, and even more preferably 0.01 mol % to 10 mol %, based on the total amount of the compound and lipids (the total of the ionized lipid, the PEGylated lipid, the helper lipid, and the cholesterol).

[0074] One embodiment of the compound represented by formula (1) is the following (Aspect A). Aspect (A) R １ is C １－６ Alkyl, or C １－６ alkoxy; R ２ is C １－６ alkyl (wherein the alkyl is halogen, hydroxy, or C １－６ and R is optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy; ３ is hydrogen, halogen, cyano, hydroxy, C １－６ Alkyl, or C １－６ L is *-(CH ２ ) ｐ C(O)O(CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -C(O)-**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, *-NR ４ (CH ２ ) ｐ -**, *-O(CH ２ ) ｐ -**, *-(CH ２ ) ｐ C(O)-**, or *-C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６ alkyl, p and q each independently represent an integer of 1 to 6, bond to the benzene ring at *, and bond to X at **; and X is a cholesterol or a similar compound, or a pharmaceutically acceptable salt thereof.

[0075] One embodiment of the compound represented by formula (1) is the following (Aspect B). Aspect (B) R １ is C １－３ and R ２ is C １－６ alkyl, which may be substituted with 1 to 2 hydroxy groups; R ３ is hydrogen or C １－３ L is *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, or *-(CH ２ ) ｐ C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６ alkyl, p and q each independently represent an integer of 1 to 6, are bonded to the benzene ring at *, and are bonded to X at **; and X is cholesterol, β-sitosterol, or 20α-hydroxycholesterol, or a pharmaceutically acceptable salt thereof.

[0076] One embodiment of the compound represented by formula (1) is the following (Aspect C). Aspect (C) R １ is methyl; R ２ is n-pentyl; R ３ is hydrogen or methoxy; L is *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X); and X is cholesterol, β-sitosterol, or 20α-hydroxycholesterol, or a pharmaceutically acceptable salt thereof.

[0077] One embodiment of the compound represented by formula (1) is the following (embodiment D). １ is methyl; R２ is n-pentyl; R ３ is methoxy; L is *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X); and X is cholesterol, β-sitosterol, or 20α-hydroxycholesterol, or a pharmaceutically acceptable salt thereof.

[0078] One embodiment of the compound represented by formula (1) is the following (Embodiment E). Embodiment (E) R １ is methyl; R ２ is n-pentyl; R ３ is hydrogen or methoxy; L is *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X); and X is cholesterol, or a pharmaceutically acceptable salt thereof.

[0079] One embodiment of the compound represented by formula (1) is the following (Aspect F). Aspect (F) R １ is methyl; R ２ is n-pentyl; R ３ is methoxy; L is *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X); and X is cholesterol, or a pharmaceutically acceptable salt thereof.

[0080] One embodiment of the compound represented by formula (1) is the following (Aspect G): Aspect (G) A compound of formula (8), formula (9) or formula (10) or a pharmaceutically acceptable salt thereof.

[0081] One embodiment of the compound represented by formula (1) is the following (Embodiment H): Embodiment (H) A compound of formula (8) or formula (9) or a pharmaceutically acceptable salt thereof.

[0082] "Pharmaceutically acceptable salts" include acid addition salts and base addition salts. For example, acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Furthermore, base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, "pharmaceutically acceptable salts" also include amino acid salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid.

[0083] Suitable salts of starting compounds and intermediates, and salts acceptable as pharmaceutical raw materials, are conventional non-toxic salts, including acid addition salts such as organic acid salts (e.g., acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, para-toluenesulfonate, etc.) and inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.); salts with amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.); metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.); ammonium salts; and organic base salts (e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), which can be appropriately selected by those skilled in the art.

[0084] When it is desired to obtain a salt of the compound of the present invention, if the compound of the present invention is obtained in the form of a salt, it may be purified as is, or if it is obtained in the free form, it may be dissolved or suspended in an appropriate organic solvent, and an acid or a base may be added to form a salt by a conventional method.

[0085] In the present invention, any one or more compounds represented by formula (1) １ H ２ Deuterium-converted forms converted to H(D) are also encompassed by the compound represented by formula (1). The present invention includes compounds represented by formula (1) or pharmaceutically acceptable salts thereof. In addition, the compounds of the present invention may exist in the form of hydrates and / or solvates with various solvents (ethanol solvates, etc.), and these hydrates and / or solvates are also included in the compounds of the present invention. Furthermore, the present invention includes all crystalline forms and mixtures thereof.

[0086] Compound (1) of the present invention may include optical isomers based on optically active centers, atropisomers based on axial or planar chirality resulting from restricted intramolecular rotation, other stereoisomers, tautomers, geometric isomers, and the like, and all possible isomers and mixtures thereof, including these, are included in the scope of the present invention.

[0087] In particular, optical isomers and atropisomers can be obtained as racemates, or as optically active isomers when optically active starting materials or intermediates are used. If necessary, at an appropriate stage in the production process described below, the racemates of the corresponding starting materials, intermediates, or final products can be physically or chemically resolved into their optical antipodes by known separation methods such as a method using an optically active column or fractional crystallization. Specifically, for example, in the diastereomeric method, two diastereomers are formed from a racemate by reaction with an optically active resolving agent. These different diastereomers generally have different physical properties and can be resolved by known methods such as fractional crystallization.

[0088] Methods for producing the compound of the present invention are described below, but the methods for producing the compound of the present invention are not limited to these. The compound of the present invention represented by formula (1) can be produced, for example, by the following Production Methods 1 and 2.

[0089] Production Method 1 Among the compounds represented by formula (1), the compound represented by formula a2 can be produced, for example, by the following production method. (In the formula, R １ , R ２ , R ３ and X are the same as those in item 1, and La is *-(CH ２ ) ｐ C(O)-** or *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６alkyl, p and q each independently represent an integer of 1 to 6, * is bonded to the benzene ring, and ** is bonded to X).

[0090] [Step A-1] Compound a2 is produced by condensing compound a1 with X—H in a suitable solvent in the presence or absence of various condensing agents and / or bases. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (including hydrochloride), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU). The base may be appropriately selected from the bases exemplified below, with triethylamine or diisopropylethylamine being preferred. The solvent may be appropriately selected from the solvents exemplified below, with dimethylformamide or dichloromethane being preferred. The reaction time is typically 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually 4°C to 100°C, preferably 4°C to 40°C. Compound a1 is produced according to the method described in WO2009 / 067081. ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ The C(O)-** linker is produced according to the method described in WO2020 / 204172.

[0091] Production Method 2 Among the compounds represented by formula (1), the compound represented by b5 can be produced, for example, by the following production method. (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , and X are as defined in item 1, and Lb is -(CH ２ ) ｑ - (wherein q represents an integer of 0 to 6), PG represents an amino-protecting group, and LG represents a leaving group.

[0092] [Step B-1] Compound b2 is produced by condensing compound b1 with X—H in a suitable solvent in the presence of various condensing agents and bases. Examples of condensing agents include triphosgene and oxalyl chloride. Examples of bases include those listed below, but preferably include triethylamine or diisopropylethylamine. Examples of solvents include those listed below, but preferably include dimethylformamide or dichloromethane. The reaction time is usually 5 minutes to 48 hours, and preferably 1 hour to 24 hours. The reaction temperature is usually 4° C. to 100° C., and preferably 4° C. to 40° C.

[0093] [Step B-2] Compound b3 can be obtained by deprotecting the protecting group PG of b2. This step can be carried out according to the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter G.M. Wuts, John Wiley & Sons, Inc., 1999).

[0094] [Step B-3] Compound b5 can be obtained by condensing compound b3 with compound b4. This step can be performed in the same manner as in step A-1. Compound b4 can be produced according to the method described in WO2009 / 067081, etc.

[0095] The compounds of the present invention have the effect of enhancing immune induction through TLR7 agonist activity. By enhancing immune induction, they have the effect of enhancing the pharmacological efficacy of mRNA vaccines. An mRNA vaccine comprising a cancer antigen mRNA, LNP, and the compound of the present invention can serve as a novel therapeutic and / or preventive agent for cancer. The mRNA vaccine of the present invention can serve as a novel therapeutic and / or preventive agent for non-small cell lung cancer, head and neck cancer, pancreatic cancer, malignant melanoma, renal cell carcinoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, glioblastoma multiforme, sarcoma, brain tumor, leukemia, myelodysplastic syndrome, multiple myeloma, or malignant lymphoma. In the present invention, "prevention" refers to the administration of the active ingredient of the present invention to a healthy person who has not developed a disease, for example, with the purpose of preventing the onset of a disease. "Treatment" refers to the act of administering the active ingredient of the present invention to a person (patient) who has been diagnosed by a doctor as having developed a disease.

[0096] The compound of the present invention is administered primarily via parenteral administration. The daily dosage varies depending on the type of compound, the administration method, the patient's symptoms, age, and other factors. For example, in the case of oral administration, the compound of the present invention can be administered in a dose of about 0.01 to 1000 mg, more preferably about 0.1 to 500 mg, per kg body weight of a human or mammal, in one or several divided doses. In the case of parenteral administration such as intravenous injection, the compound of the present invention can be administered in a dose of about 0.01 to 300 mg, more preferably about 0.01 to 1 mg, per kg body weight of a human or mammal.

[0097] The compound of the present invention can be administered parenterally, either directly or after being formulated using an appropriate dosage form. Examples of dosage forms include, but are not limited to, solutions, suspensions, injections, patches, and poultices. The formulations are produced by known methods using pharmaceutically acceptable additives. Depending on the purpose, additives that can be used include excipients, solubilizers, solubilizers, thickeners, dispersants, stabilizers, and the like.

[0098] The compounds of the present invention can be used in combination with other anti-cancer agents. Examples of anticancer agents include chemotherapeutic agents (e.g., ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enocitabine, capecitabine, carmofur, gemcitabine, cytarabine, tegafur, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, irinotecan, etoposide, sobuzoxane, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, actinomycin D, aclarubicin, idarubicin, epirubicin, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cisplatin, nedaplatin), kinase inhibitors (e.g., , gefitinib, erlotinib, osimertinib, afatinib, imatinib, dasatinib, bosutinib, vandetanib, sunitinib, axitinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, ibrutinib, crizotinib, ceritinib, alectinib, tofacitinib, baricitinib, ruxolitinib, olaparib, sorafenib, vemurafenib, dabrafenib, Examples of the immune checkpoint inhibitor include cyclosporine-1 (CSF-1), cyclosporine-1 (CSF-2), cyclosporine-1 (CSF-3), cyclosporine-1 (CSF-4), cyclosporine-1 (CSF-5), cyclosporine-1 (CSF-6), cyclosporine-1 (CSF-7), cyclosporine-1 (CSF-8), cyclosporine-1 (CSF-9), cyclosporine-1 (CSF-10), cyclosporine-1 (CSF-11), cyclosporine-1 (CSF-12), cyclosporine-1 (CSF-13), cyclosporine-1 (CSF-14), cyclosporine-1 (CSF-15), cyclosporine-1 (CSF-16), cyclosporine-1 (CSF-17), cyclosporine-1 (CSF-18), cyclosporine-1 (CSF-19 ...Furthermore, preferred immune checkpoint inhibitors are antibodies, such as antibodies against PD-1, PD-L1, CTLA4, LAG-3, TIM-3, VISTA, HVEM, BTLA, CD160, TIGIT, CD47, CCR8, or PVR. Preferred antibodies that are immune checkpoint inhibitors include antibodies against PD-1 (nivolumab, pembrolizumab, AMP-224, AMP-514 (MEDI0680), pidilizumab (CT-011) and the like), antibodies against PD-L1 (durvalumab (MEDI4736), atezolizumab (MPDL3280A), BMS-936559, avelumab (MSB0010718C) and the like), antibodies against LAG-3 (IMP-321, BMS-986016 and the like), antibodies against TIM-3, antibodies against VISTA, antibodies against HVEM, antibodies against BTLA, antibodies against CD160, antibodies against TIGIT, antibodies against CD47, antibodies against CCR8, and antibodies against PVR. More preferred are antibodies against PD-1 (nivolumab, pembrolizumab) or antibodies against PD-L1 (durvalumab, atezolizumab (MPDL3280A), avelumab (MSB0010718C) or BMS-936559).

[0099] The present invention will be explained in more detail below with reference to the following examples and experimental examples, but the present invention is not limited thereto. The compound names shown in the following examples and experimental examples do not necessarily conform to the IUPAC nomenclature.

[0100] To simplify the description in the specification, the following abbreviations may be used in the Reference Examples, Examples, and Tables in the Examples: LNP: lipid nano particle TFA: trifluoroacetic acid Boc: tert-butyloxycarbonyl HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0101] Symbols used in NMR include s for singlet, d for doublet, dd for doublet of doublets, t for triplet, td for doublet of triplets, q for quartet, m for multiplet, br for broad, brs for broad singlet, brs for broad multiplet and J for coupling constant.

[0102] The measurement conditions for the high performance liquid chromatograph mass spectrometer (LCMS) were as follows, and the observed mass spectrometry value [MS (m / z)] was calculated as M+H ＋ The retention time is indicated as Rt (minutes). The measurement conditions used for each measured value are also noted.

[0103] Measurement condition A Detector: Shimadzu LCMS-2020 Column: Phenomenex Kinetex (1.7 μm C18, 50 mm × 2.10 mm) Solvent: Solution A: 0.05% TFA / H2O, Solution B: Acetonitrile Gradient Condition: 0.0-1.7 min (linear gradient from B 10% to 99%) 1.7-1.9 min (B 99%) 1.9-3.0 min (B 10%) Flow rate: 0.5 mL / min UV: 254 nm Column temperature: 40°C

[0104] Reference Example 1 (4-{[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl}-3-methoxyphenyl)acetic acid Reference Example 1 was synthesized by the method described in WO2009 / 067081. LCMS (measurement condition A): 373 [M+H] +

[0105] Reference Examples 2 and 3 Step 1 Reference Example 2 tert-Butyl cholest-5-en-3β-yl butane-1,4-diylbiscarbamate N,N-Diisopropylethylamine (3.5 mL) was added to a chloroform solution (200 mL) of N-Boc-1,4-diaminobutane (2.08 g) and cholesteryl chloroformate (4.5 g), and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to give Reference Example 2 (5 g). 1H-NMR (400 MHz, CDCl3): 4.85 (d, 1H, J = 4.8, Hz), 4.11 (brs, 1H), 4.00-3.97 (m, 2H), 1.86-1.81 (m, 1H), 1.77-1.72 (m, 1H), 1.51-1.42 (m, 2H), 1.35-1.26 (m, 3H), 1.08 (brs, 1H), 1.03-0.97 (m, 3H), 0.97 (s, 9H), 0.84-0.72 (m, 5H), 0.70-0.54 (m, 8H), 0.49 (s, 3H), 0.46-0.42 (m, 4H), 0.40 (d, 3H, J = 6.4 Hz), 0.36 (d, 3H, J = 2.0 Hz), 0.34 (d, 3H, J = 1.2Hz).

[0106] Step 2 Reference Example 3 Cholest-5-en-3β-yl (4-aminobutyl)carbamate TFA (10 mL) was added to a chloroform solution (40 mL) of Reference Example 2 (4.7 g) and the mixture was stirred overnight. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting residue was dissolved in chloroform and washed with saturated aqueous sodium bicarbonate, and the organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (elution solvent: hexane:ethyl acetate) to give Reference Example 3 (2.87 g). 1 H-NMR (400 MHz, CDCl3): 7.68 (brs, 1H), 4.85 (d, 1H J = 4.8, Hz), 3.92 (brs, 1H), 2.67 (brs, 2H), 2.54 (brs, 2H), 1.83-1.75 (m, 10H), 1.50-1.42 (m, 2H), 1.35-1.26 (m, 4H), 1.16 (brs, 6H), 1.13-0.97 (m, 10H), 0.97 (s, 9H), 0.84-0.72 (m, 2H), 0.65-0.56 (m, 8H), 0.49 (s, 3H), 0.46-0.43 (m, 2H), 0.40 (d, 3H, J = 6.4 Hz), 0.36 (dd, 6H, J = 6.0, 1.2 Hz), 0.16 (s, 3H).

[0107] Reference Examples 4 and 5 The peptides represented in Reference Examples 4 and 5 are commercially available.

[0108] Reference Example 6 mRNA encoding OVA (CleanCap TM OVA mRNA (5 moU) (Trilink) is commercially available. See Table 2 below for the formulation.

[0109] Reference Example 7 (4-{[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl}phenyl)acetic acid Reference Example 7 was synthesized by the method described in WO2009 / 067081. LCMS (measurement condition A): 343 [M+H] +

[0110] Example 1 Cholest-5-en-3β-yl (4-{[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl}-3-methoxyphenyl) acetate Diisopropylethylamine (0.14 mL) was added to a chloroform solution (10 mL) of Reference Example 1 (100 mg), cholesterol (157 mg), and HATU (205 mg), and the mixture was stirred at room temperature for 10 hours. After the reaction was completed, the mixture was diluted with chloroform and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (elution solvent: hexane / ethyl acetate) to give Example 1 (20 mg). 1 H-NMR (400 MHz, CDCl3): 6.81-6.69 (m, 3H), 5.29 (brs, 1H), 4.83 (s, 1H), 4.73 (s, 2H), 4.51-4.54 (m, 1H), 3.83 (s, 3H), 3.58 (s, 2H), 3.48 (s, 2H), 3.23-3.19 (m, 2H), 2.74-2.73 (m, 1H), 2.25-2.24 (m, 5H), 1.98-1.76 (m, 6H),

[0111] Example 2 Cholest-5-en-3β-yl {4-[2-(4-{[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl}-3-methoxyphenyl)acetamido]butyl}carbamate Diisopropylethylamine (0.16 mL) was added to a chloroform solution (10 mL) of Reference Example 1 (113 mg), Reference Example 3 (152 mg), and HATU (231 mg), and the mixture was stirred at room temperature for 10 hours. After completion of the reaction, the mixture was diluted with chloroform and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (elution solvent: hexane / ethyl acetate) to give Example 2 (157 mg). 1 H-NMR (400 MHz, CDCl3): 6.89-6.79 (m, 3H), 6.22 (brs, 1H), 5.72 (brs, 1H), 5.34 (d, 2H, J = 4.8 Hz), 4.65 (brs, 1H), 4.45 (brs, 1H), 3.90 (s, 2H), 3.62 (brs, 2H), 3,49 (brs, 2H), 3.33 (dd, 2H, J = 8.8, 6.8 Hz), 3.23 (d, 2H, J = 6.0 Hz), 3.11 (d, 2H, J = 6.4 Hz), 2.52 (brs, 2H),2.31-2.24 (m, 1H), 2.00-1.76 (m, 4H), 1.56-1.38 (m, 51H), 1.34-1.23 (m, 8H), 1.19-1.02 (m, 8H), 0.99 (s, 3H), 0.96-0.92 (m, 2H), 0.90 (d, 3H, J = 8.4 Hz), 0.86-0.83 (m, 9H), 0.65 (s, 3H).

[0112] Examples 3 to 6 LNP formulations containing the compound of Example 1 DLin-MC3-DMA (manufactured by Medchemexpress), DSPC (manufactured by NOF Corp.), cholesterol (manufactured by Sigma), DMG-PEG2000 (manufactured by NOF Corp.), and the compound of Example 1 were dissolved in ethanol to the concentrations shown in Table 2. This ethanol solution and modified mRNA (CleanCap) encoding the full-length ovalbumin (hereinafter referred to as OVA) protein were mixed. TM The mixture was mixed with 10 mM citrate buffer (pH 4) containing 0.17 mg / mL OVA mRNA (5 moU, TriLink) at a ratio of 1 / 3 (v / v) in a microchannel (Syrris). The resulting mixture was diluted 5-fold with 10 mM citrate buffer (pH 4), and the external phase was replaced with 10 mM Tris / 9.4% sucrose buffer (pH 7.5) in a dialysis cassette (Slide-A-Lyzer G2 Dialysis Cassettes 20K MWCO, Thermo Scientific).

[0113] The pharmacological test results of representative compounds of the present invention are shown below, and the pharmacological actions of the compounds are explained, but the present invention is not limited to these test examples.

[0114] Example 9 Cholest-5-en-3β-yl {4-[2-(4-{[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl}phenyl)acetamido]butyl}carbamate Diisopropylethylamine (0.16 mL) was added to a chloroform solution (10 mL) of Reference Example 7 (101 mg), Reference Example 3 (222 mg), and HATU (225 mg), and the mixture was stirred at room temperature for 10 hours. After completion of the reaction, the mixture was diluted with chloroform and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (elution solvent: chloroform:methanol) to give Example 9 (187 mg). 1H-NMR (400 MHz, CDCl3): 7.19 (d, 2H, J = 8.0Hz), 6.99 (d, 2H, J = 8.0Hz), 5.59 (brs, 1H), 5.30 (d, 2H, J = 4.8 Hz), 4.60 (brs, 1H), 4.41 (m, 1H), 3.67 (s, 2H), 3.45 (s, 2H), 3.28 (dd, 2H, J = 8.8, 6.8 Hz), 3.17 (dd, 2H, J = 8.8, 6.8 Hz), 3.11 (d, 2H, J = 6.4 Hz), 2.38 (brs, 2H), 2.28-2.17 (m, 1H), 2.00-1.88 (m, 3H), -1.72 (m, 3H), 1.56-1.38 (m, 72H), 0.61 (s, 3H).

[0115] Test Example 1 Human TLR7 reporter gene assay TLR7 / NF-κB / SEAPorter ＴＭ The HEK293 cell line (Imgenex Corporation) is a stable co-transfected cell line expressing full-length human TLR7 and a secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of the NF-κB response element. TLR7 expression in this cell line was examined by flow cytometry. Stably expressing transformants were selected using the antibiotics blasticidin and geneticin. TLR signaling leads to NF-κB translocation, and promoter activation results in SEAP gene expression. TLR7-specific activation was assessed by measuring the level of SEAP produced after overnight incubation of the cells with example compounds at 37°C in the presence of 0.1% (v / v) dimethyl sulfoxide (DMSO). The degree of human TLR7 activation by the compounds of the present invention was assessed using a human TLR7 reporter gene assay, and the concentration of compound that produces half-maximal levels of SEAP (EC ５０ ) are shown in Table 3.

[0116] As shown in Test Example 1, the example compounds of the present invention exhibited TLR7 agonist activity despite being conjugated with cholesterol. This suggests that the position at which the TLR7 agonist is conjugated with cholesterol and adjustment of the linker are important for maintaining TLR7 agonist activity.

[0117] Test Example 2: Evaluation of LNP Formulations DLin-MC3-DMA (Target Mol), DSPC (NOF Corp.), cholesterol (Sigma), DMG-PEG2000 (NOF Corp.), and Example 1 or Example 2 were dissolved in ethanol to the concentrations shown in Table 4 or Table 5, respectively. This ethanol solution was mixed with 10 mM citrate buffer (pH 4) at a ratio of 1 / 3 (v / v) in a microchannel (Syrris Corp.). The external phase of a dialysis cassette (Slide-A-Lyzer G2 Dialysis Cassettes 20K MWCO, Thermo Scientific Corp.) was replaced with 10 mM Tris / 9.4% sucrose buffer (pH 7.5).

[0118] The results of repeated freezing (-80°C) and thawing of the LNPs of Examples 7 and 8 are shown in Table 6. The polydispersity index of merit of the LNPs of Examples 7 and 8 after one freeze-thaw was 0.2. The polydispersity index of the LNP of Example 7 after three freeze-thaws was also 0.2.

[0119] Test Example 3: Evaluation of in vivo antigen-specific T cell induction ability The in vivo adjuvant activity of the compound synthesized in Example 1 was evaluated by the ability to induce antigen-specific cytotoxic T cells (CTLs) and antigen-specific helper T cells when a vaccine prepared by adding the compound synthesized in Example 1 to an LNP vaccine encapsulating modified mRNA encoding the full-length ovalbumin (hereinafter, referred to as OVA) protein (hereinafter, referred to as "adjuvant-containing vaccine") was administered to mice.

[0120] Whether administration of the adjuvant-containing vaccine induces CTLs against the OVA-derived antigen epitope (SEQ ID NO: 1) was determined by measuring whether IFNγ was produced when splenocytes derived from mice administered the adjuvant-containing vaccine were restimulated with peptide (SEQ ID NO: 1). Whether administration of the adjuvant-containing vaccine induces helper T cells against the OVA-derived antigen epitope (SEQ ID NO: 2) was determined by measuring whether IFNγ was produced when splenocytes derived from mice administered the adjuvant-containing vaccine were restimulated with peptide (SEQ ID NO: 2). Furthermore, whether the compound synthesized in Example 1 exerts adjuvant activity in vivo was determined by comparing the number of CTLs or helper T cells induced by administration of an LNP vaccine without adjuvant with the number of CTLs or helper T cells induced by administration of an adjuvant-containing vaccine in which the compound synthesized in Example 1 was added to the LNP vaccine, and determining whether there was an increase in the number of CTLs or helper T cells.

[0121] Specifically, the LNP vaccine of Reference Example 6 and the adjuvant-containing vaccines of Examples 3 to 6 were administered intradermally at the base of the tail to mice implanted intradermally with EE.G7-OVA, at a modified mRNA dose of 0.55 μg / mouse. Administration was performed once or twice at one-week intervals to three mice in each group. One week after the final administration, the mice were then inoculated with CO ２ After euthanasia by gas, the spleens were removed and splenocytes were prepared. IFNγ production was measured using an IFNγ ELISPOT assay kit. The day before splenocyte preparation, the ELISPOT plate was treated with anti-mouse IFNγ antibody, and on the day of preparation, the plate was blocked with RPMI 1640 medium containing 10% fetal bovine serum (FBS). 2 × 10 prepared splenocytes were cultured. ５ The cells were seeded onto a blocked ELISPOT plate at 1000x1000 cells / well. The peptide (SEQ ID NO: 1) or peptide (SEQ ID NO: 2) was added to the splenocytes at a final concentration of 10 μg / mL in the presence of 0.1% (v / v). The peptide-added splenocytes were incubated at 37°C, 5% CO ２After incubation, the supernatant was removed, and the number of spots on the colored ELISPOT plate was counted using an Immuno Spot Analyzer (CTL).

[0122] The results of the IFNγ ELISPOT assay for three mice per group are shown in Figures 1 and 2. In Figure 1, the vertical axis indicates the number of cells that produced IFNγ in response to stimulation in the inoculated cells, and the horizontal axis indicates the vaccine administered to the mice. The black and white bars in Figure 1 represent the results of culturing splenocytes from vaccinated mice in the presence and absence of the peptide represented by SEQ ID NO: 1. That is, the difference between the values ​​of the black and white bars indicates the number of IFNγ-producing cells, i.e., CTLs, specific to the peptide represented by SEQ ID NO: 1, induced in vivo by vaccine administration. The values ​​of the white bars in Figure 1 are almost absent. This indicates that the mouse splenocytes hardly reacted in the absence of the target peptide. As a result of this test, the induction of CTLs reactive to the peptide represented by SEQ ID NO: 1 was confirmed in mice administered the adjuvant-containing vaccine. Furthermore, the number of CTLs reactive to the peptide represented by SEQ ID NO: 1 was greater with the adjuvant-containing vaccine loaded with the compound synthesized in Example 1 at molar fraction concentrations of 0.08, 0.4, and 2 mol% than with the LNP vaccine (Reference Example 6) that did not contain an adjuvant. The black and white bars in Figure 2 show the results of culturing splenocytes from vaccinated mice in the presence and absence of the peptide represented by SEQ ID NO: 2. That is, the difference between the values ​​of the black and white bars indicates the number of IFNγ-producing cells specific to the peptide represented by SEQ ID NO: 2, i.e., helper T cells, induced in vivo by administration of the vaccine. The values ​​of the white bars in Figure 2 are almost absent. This indicates that the mouse splenocytes hardly reacted in the absence of the target peptide. As a result of this test, the induction of helper T cells reactive to the peptide represented by SEQ ID NO: 2 was confirmed in mice administered the adjuvant-containing vaccine. Furthermore, the number of peptide-reactive helper T cells represented by sequence number 2 was found to be greater with the adjuvant-containing vaccine loaded with the compound synthesized in Example 1 at molar fraction concentrations of 0.08, 0.4, and 2 mol% than with the LNP vaccine without adjuvant (Reference Example 6).

[0123] This demonstrates that the addition of the compound synthesized in Example 1 to an LNP vaccine increases the number of induced CTLs and helper T cells, strongly suggesting that the compound synthesized in Example 1 has adjuvant activity in vivo.

[0124] Test Example 4 Enhancement of the in vivo tumor growth inhibitory effect of a vaccine E.G7-OVA cells were suspended in Hank's balanced salt solution and transplanted intradermally into the ventral skin of C57BL / 6 mice (5 x 10 cells per mouse). ６ One and eight days after tumor cell transplantation, an LNP vaccine containing modified mRNA encoding the full-length OVA protein at a concentration of 5.5 μg / mL was administered intradermally at the base of the tail to achieve a modified mRNA dose of 0.55 μg / mouse. Alternatively, an adjuvant-containing vaccine containing the compound synthesized in Example 1 at molar fractions of 0.08, 0.4, 2, or 10 mol% was administered intradermally at the base of the tail to achieve a modified mRNA dose of 0.55 μg / mouse. Three mice per group were administered the vaccine, and 15 days after tumor transplantation, tumor diameters were measured and tumor volumes were calculated.

[0125] The average tumor volumes of three mice from each group 15 days after tumor inoculation are shown in Figure 3. Addition of the compound synthesized in Example 1 to the LNP vaccine more significantly suppressed tumor cell growth.

[0126] These results demonstrate that the addition of the compound of the present invention to a vaccine enhances tumor growth suppression.

[0127] Test Example 5: Evaluation of in vivo mouse spleen weight In Test Example 3, the spleen weights of the group administered with the LNP vaccine containing the compound of the present invention and the group administered with the LNP vaccine not containing the compound of the present invention were examined, and no significant spleen weight was observed in either group. This suggests that the compound of the present invention can be used as a safe mRNA vaccine adjuvant.

[0128] The compounds of the present invention have TLR7 agonist activity. When used as adjuvants for mRNA vaccines consisting of LNPs and mRNA, the compounds of the present invention can induce strong anti-tumor immunity. As a result, mRNA vaccines containing the compounds of the present invention as adjuvants can be provided that are expected to have therapeutic and preventive effects on cancer.

Claims

1. Formula (1): [In formula (1), R １ is C １－６ Alkyl, or C １－６ represents alkoxy, R ２ is halogen, hydroxy, or C １－６ C optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy １－６ represents alkyl, R ３ is hydrogen, halogen, cyano, hydroxy, C １－６ Alkyl, or C １－６ L represents *-(CH ２ ) ｐ C(O)O(CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -C(O)-**, *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, *-NR ４ (CH ２ ) ｐ -**, *-O(CH ２ ) ｐ -** or *-(CH ２ ) ｐ C(O)-** (where R ４ and R ５ are each independently hydrogen or C １－６ represents alkyl, p and q each independently represent an integer of 0 to 6, bonded to the benzene ring at *, and bonded to X at **, and X represents a cholesterol, or a pharmaceutically acceptable salt thereof.

2. R １ But C １－３ alkyl, R ２ C optionally substituted with 1 to 2 hydroxy groups １－６ alkyl, R ３ is hydrogen or C １－３ Alkoxy, and L is *-(CH ２ ) ｐ C(O)NR ４ (CH ２ ) ｑ -NR ５ C(O)-**, or *-(CH ２ ) ｐ The compound according to claim 1, which is C(O)-**, or a pharmaceutically acceptable salt thereof.

3. Formula (2a): [In formula (2a), R ３ is hydrogen or C １－３ L represents *-(CH ２ )C(O)NH(CH ２ ) ４ -NHC(O)-**, or *-(CH ２ 3. The compound according to claim 1 or 2, wherein the compound is represented by the formula: )C(O)-** (wherein * is bonded to the benzene ring and ** is bonded to X), and X represents a cholesterol, or a pharmaceutically acceptable salt thereof.

4. Formula (3a) or (4a): [In formula (3a) and formula (4a), R ３ is hydrogen or C １－３ and X represents an alkoxy, and X represents a cholesterol or a similar group.

5. Formula (3a) or (4a): [In formula (3a) or formula (4a), R ３ is hydrogen or C １－３ X represents an alkoxy group represented by formula (5): Formula (6): Or formula (7): wherein formula (5), formula (6) or formula (7) is bonded to formula (3a) or formula (4a) at ***.

6. Equation (8), (9), or (10):

6. The compound according to any one of claims 1 to 5, wherein:

7. Formula (8): The compound according to any one of claims 1 to 6, wherein:

8. Formula (9): The compound according to any one of claims 1 to 6, wherein:

9. Formula (10): The compound according to any one of claims 1 to 6, wherein:

10. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition according to claim 10, which is a lipid nanoparticle (LNP) formulation.

12. The pharmaceutical composition according to claim 11, which is an LNP composed of four types of components: an ionizable lipid, a helper lipid, a PEGylated lipid, and cholesterols.

13. The pharmaceutical composition of claim 12, wherein the ionizable lipid of the LNP is DLin-MC3-DMA, SM-102, ALC-0315, A18-iso5-2DC18m, Lipid A6, or 306Oi10.

14. The pharmaceutical composition of claim 12 or 13, wherein the PEGylated lipid of the LNP is PEG2000-C-DMG, ALC-0159, or DMG-PEG2000.

15. The pharmaceutical composition according to any one of claims 12 to 14, wherein the helper lipid of the LNP is DSPC or DOPE.

16. The pharmaceutical composition according to any one of claims 12 to 15, wherein the cholesterol in the LNP is cholesterol, β-sitosterol, or 20α-hydroxycholesterol.

17. The pharmaceutical composition according to any one of claims 12 to 16, wherein the ionizable lipid of the LNP is DLin-MC3-DMA, the PEGylated lipid is DMG-PEG2000, the helper lipid is DSPC, and the cholesterol is cholesterol.

18. The pharmaceutical composition according to any one of claims 10 to 17, comprising mRNA as an antigen.

19. The pharmaceutical composition of claim 18, comprising mRNA encoding a cancer antigen protein.

20. A cancer treatment and / or prevention agent comprising, as an active ingredient, the compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

21. The therapeutic and / or preventive agent according to claim 20, wherein the cancer is non-small cell lung cancer, head and neck cancer, pancreatic cancer, malignant melanoma, renal cell carcinoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, glioblastoma multiforme, sarcoma, brain tumor, leukemia, myelodysplastic syndrome, multiple myeloma, or malignant lymphoma.

22. A method for treating and / or preventing cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

23. Use of a compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the manufacture of an agent for the treatment and / or prevention of cancer.

24. A compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the treatment and / or prevention of cancer.

25. A medicine comprising a compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in combination with at least one drug selected from anticancer agents.

26. A compound according to any one of claims 1 to 19, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for treating cancer in combination with at least one or more drugs selected from drugs classified as anticancer drugs.

27. An mRNA vaccine comprising the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, the LNP according to any one of claims 13 to 17, and mRNA as an antigen.

Images