Methotrexate derivative and application thereof

Abstract

The invention provides a methotrexate derivative shown in a formula (I) or pharmaceutically acceptable salt thereof, a preparation method thereof and application of the methotrexate derivative to treatment and prevention of ophthalmic diseases such as retinopathy. # imgabs0 #

Description

Field of the Invention

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to methotrexate derivatives of formula (I) and their use in the treatment and prevention of ophthalmic diseases such as proliferative vitreoretinopathy.

[0002] Background Art

[0003] Proliferative vitreoretinopathy (PVR) refers to recurrent retinal detachment caused by the contraction and traction of extensive fibrotic membranes on the retinal surface and behind the vitreous after the reattachment of rhegmatogenous retinal detachment, and it is one of the complications after the reattachment of rhegmatogenous retinal detachment. The fibrotic membranes are mainly composed of pigment epithelial cells, glial cells, fibrocytes, fibroblasts and macrophages. Pigment epithelial cells play an important role in the occurrence and development of PVR. It is not only the main cell for the formation and contraction of the proliferative membrane, but also can produce chemotactic factors to attract fibroglial cells and fibroblasts to participate in the formation of the proliferative membrane.

[0004] Since PVR is a disease caused by cell proliferation and contraction, inhibiting cell proliferation is the key to drug treatment. Some drugs have been used in laboratories and clinics. For example, colchicine is a drug that inhibits mitosis and has the effect of inhibiting cell proliferation. Some antimetabolic drugs may also be used, such as 5-fluorouridine and daunomycin, which have achieved certain results in clinical applications. In China, homoharringtonine has also shown certain efficacy in experimental traction retinal detachment, but its toxicity to the eye is a major problem. In recent years, many studies have focused on related cytokines and their regulation. However, since the growth and death of cells are controlled by a complex signal network, targeting a single factor is unlikely to be effective.

[0005] The use of methotrexate for intravitreal injection in the treatment of PVR stems from its mechanism of action. Methotrexate can inhibit inflammation and cell replication, both of which are key in the pathogenesis of PVR. PVR usually appears several weeks to several months after surgical repair, so treatment needs to be repeated throughout the risk period rather than a single injection during surgery. In December 2019, the GUARD trial began recruiting subjects. This trial is a multicenter, randomized, controlled, adaptive phase 3 clinical trial aiming to study the efficacy of ADX-2191 (0.8% methotrexate for intravitreal injection, Aldeyra Therapeutics) in preventing recurrent retinal detachment caused by PVR. ADX-2191 has received orphan drug and fast track certifications from the US FDA. However, due to the good water solubility of methotrexate, its half-life in the eye is extremely short, and more than 13 intravitreal injections are required within 16 weeks, resulting in poor patient compliance.

[0006] Due to the poor liposolubility of methotrexate itself, it is impossible to develop sustained-release implants or other sustained-release drug delivery methods through formulation means. Therefore, there is a great demand for sustained-release drugs for the treatment and prevention of PVR in this field. Summary of the Invention

[0007] The inventors have successfully developed a methotrexate derivative with sustained-release properties, which can slowly release methotrexate, greatly increasing the effective concentration of methotrexate after a single intravitreal injection.

[0008] In one aspect, the present disclosure provides a methotrexate derivative that can be used for the treatment and prevention of proliferative vitreoretinopathy. The derivative is prepared by making a prodrug of methotrexate with a vitamin, an amino acid, an ester of an amino acid, or an oligopeptide to increase the liposolubility of methotrexate, increase its half-life in the vitreous body, and ensure that methotrexate can be effectively dissociated to exert its pharmacological effect, and the other part dissociated is not harmful to the eye. The derivative is specifically a compound of formula (I) or a pharmaceutically acceptable salt thereof, and can be administered to the eye of a subject in need of such treatment in an effective amount:

[0009]

[0010] Wherein,

[0011] R is selected from OH, OR1 or NHR1, and at most one R is OH;

[0012] R1 is a vitamin, an amino acid, an amino acid ester or an amino acid oligopeptide.

[0013] In a specific aspect, the derivative is specifically used for the treatment and prevention of the onset of proliferative vitreoretinopathy after the reduction surgery of rhegmatogenous retinal detachment.

[0014] In a specific aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in the form of a topical ophthalmic formulation, which is topically administered to the affected eye. In a more specific aspect, the concentration of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the formulation is 0.001% - 10% of the total amount of the composition, by weight or volume. For example, the aqueous composition contains 0.001%, 0.01%, 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 5.0% or up to 10% of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In another more specific aspect, the topical ophthalmic formulation is a solution, suspension, gel, emulsion, eye drops, suspension, ointment, lotion, injection, intraocular implant, sustained release formulation or cream for use on the periorbital skin. In another more specific aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in the form of an implant or a semi-solid sustained release formulation injected into the affected eye. In another more specific aspect, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the implant is from 1 μg to 100 mg.

[0015] In another aspect, the present disclosure provides a method for preparing the compound of formula (I) described herein, comprising the following reaction:

[0016]

[0017] wherein X is halogen, amino or hydroxy, and R is as defined herein.

[0018] In another aspect, the present disclosure provides a method for refining the crude product of the compound of formula (I) prepared as described above, comprising adding the crude product of the compound of formula (I) to absolute ethanol, pulping, filtering by suction, and drying under vacuum.

[0019] In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof described herein, and a pharmaceutically acceptable excipient, and optionally, other therapeutic agents.

[0020] In another aspect, the present disclosure provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof described herein in the preparation of a medicament for the treatment or prevention of retinopathy.

[0021] In another aspect, the present disclosure provides the compound of formula (I) or a pharmaceutically acceptable salt thereof described herein for the treatment or prevention of retinopathy.

[0022] In another aspect, the present disclosure provides a method for treating or preventing retinopathy in a subject in need thereof, which comprises administering to the subject the compound of formula (I) or a pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein.

[0023] In a specific aspect, the retinopathy is proliferative vitreoretinopathy.

[0024] Specifically, the present invention relates to the following technical solutions:

[0025] 1. A compound of formula (I), or a pharmaceutically acceptable salt thereof:

[0026]

[0027] Wherein,

[0028] R is selected from OH, OR1 or NHR1, and at most one R is OH;

[0029] R1 is a vitamin, an amino acid, an amino acid ester or an amino acid oligopeptide.

[0030] 2. The compound of formula (I) according to Technical Solution 1, or a pharmaceutically acceptable salt thereof, wherein the vitamin is a fat-soluble vitamin, preferably selected from vitamin A, vitamin D, vitamin E or vitamin K, preferably selected from vitamin A1, vitamin A2, vitamin D2, vitamin D3, a-tocopherol or b-tocopherol.

[0031] 3. The compound of formula (I) according to Technical Solution 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine in L or R configuration and their esters, preferably selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, threonine in L or R configuration and their esters, preferably selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, tyrosine, phenylalanine in L or R configuration and their esters.

[0032] 4. The compound of formula (I) according to Technical Solution 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid ester is selected from the C 1-6 alkyl ester, C 1-6 haloalkyl ester, C 2-6 alkenyl ester, C 2-6 alkynyl ester, C 5-10 cycloalkyl ester, 5-10 membered heterocyclic ester, C 6-10 aryl ester or 5-10 membered heteroaryl ester of the amino acid, preferably selected from the C 1-6 alkyl ester or C 1-6 haloalkyl ester of the amino acid.

[0033] 5. The compound of formula (I) according to Technical Solution 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid oligopeptide is an oligopeptide of 2 to 5 amino acids, preferably an oligopeptide of 2 to 3 amino acids.

[0034] 6. The compound of formula (I) according to any one of Technical Solutions 1-5, or a pharmaceutically acceptable salt thereof, which has the following structure:

[0035] or

[0036] wherein R is selected from OR1 or NHR1, preferably OR1,

[0037] wherein R1 is as defined in any one of Technical Solutions 1-5.

[0038] 7. The compound of formula (I) according to any one of Technical Solutions 1-5, or a pharmaceutically acceptable salt thereof, which has the following structure:

[0039] or

[0040] wherein R1 is as defined in any one of Technical Solutions 1-5.

[0041] 8. The compound of formula (I) according to Technical Solution 1, or a pharmaceutically acceptable salt thereof, which has the following structure:

[0042] 、 、 or 。

[0043] 9. A method for preparing the compound of formula (I) according to Technical Solution 1, comprising the following reaction:

[0044]

[0045] wherein X is halogen, amino or hydroxyl,

[0046] R is as defined in any one of Technical Solutions 1-5.

[0047] 10. The method according to Technical Solution 9, wherein the molar ratio of methotrexate of formula (II) to the compound of formula (III) in the reaction is 1:1 to 1:5, preferably 1:1 to 1:2, preferably 1:1 - 1:1.5, preferably 1:1.

[0048] 11. The method according to Technical Solution 9 or 10, wherein the reaction is carried out in the presence of a condensing agent, preferably the condensing agent is selected from DCC, CDI or EDCI, preferably DCC or EDCI, preferably DCC.

[0049] 12. The method of Technical Solution 11, wherein the molar ratio of methotrexate in Formula (II) to the condensing agent in the reaction is 1:2 to 1:5, preferably 1:3 to 1:5, and more preferably 1:3.

[0050] 13. The method of any one of Technical Solutions 9 - 12, wherein the reaction is carried out in the presence of a base, preferably the base is selected from organic bases such as pyridine, DMAP, DIPEA or triethylamine, preferably DMAP or DIPEA, and more preferably DMAP.

[0051] 14. The method of Technical Solution 13, wherein the molar ratio of methotrexate in Formula (II) to the organic base in the reaction is 1:1 to 1:5, preferably 1:1 to 1:3, and more preferably 1:1.

[0052] 15. The method of any one of Technical Solutions 9 - 14, wherein the reaction is carried out in a solvent, preferably the solvent is an aprotic solvent, preferably an aprotic polar solvent, preferably selected from DMF, DMA or DMSO, and more preferably DMSO.

[0053] 16. The method of any one of Technical Solutions 9 - 15, wherein the reaction is carried out at a temperature of 0 - 50 °C, preferably the temperature is 15 - 25 °C, and more preferably 20 - 25 °C.

[0054] 17. The method of any one of Technical Solutions 9 - 16, wherein the reaction is carried out for at least 8 h, preferably for at least 12 h.

[0055] 18. A method for refining the crude product of the compound of Formula (I) prepared by any one of Technical Solutions 9 - 17, comprising adding the crude product of the compound of Formula (I) to absolute ethanol, pulping, filtering by suction, and drying under vacuum.

[0056] 19. The method of Technical Solution 18, wherein the amount of absolute ethanol used is 10 - 30 times the mass of the crude product of the compound of Formula (I), preferably 10 - 20 times, and more preferably 20 times.

[0057] 20. The method of Technical Solution 18 or 19, wherein the pulping is carried out at a temperature of 0 - 40 °C, preferably the temperature is 0 - 30 °C, more preferably 10 - 30 °C, and most preferably 20 - 30 °C.

[0058] 21. The method of any one of Technical Solutions 18 - 20, wherein the pulping is not less than 1 h, preferably not less than 2 h, and more preferably 3 h.

[0059] 22. A method for preparing the following compound, comprising the following reaction:

[0060]

[0061] Among them, the reaction is carried out in DMSO at a temperature of 20 - 25 °C, using DCC as a condensing agent and DMAP as a base for at least 12 h. The molar ratio of the compound of formula (II) to the compound of formula (IIIa) is 1:1, the molar ratio of the compound of formula (II) to the condensing agent is 1:3, and the molar ratio of the compound of formula (II) to the organic base is 1:1. The crude product obtained from the reaction is slurried in 20 times the mass of absolute ethanol at room temperature for about 3 hours.

[0062] 23. A pharmaceutical composition comprising the compound of formula (I) according to any one of Technical Solutions 1 - 8 or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable excipient, and optionally, other therapeutic agents.

[0063] 24. The pharmaceutical composition of Technical Solution 23, which is in a form selected from eye drops, suspensions, ointments, gels, emulsions, injections, intraocular implants, sustained release agents, and creams for use on the skin around the eye.

[0064] 25. The pharmaceutical composition of Technical Solution 23 or 24, wherein the pharmaceutically acceptable excipient is selected from one or more of the following: surfactants, buffers, antioxidants, stabilizers, osmotic pressure regulators, viscosity regulators, pH regulators, preservatives, carriers, excipients, media, and water.

[0065] 26. Use of the compound of formula (I) according to any one of Technical Solutions 1 - 8 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment or prevention of retinopathy.

[0066] 27. The use of Technical Solution 26, wherein the retinopathy is retinal detachment, proliferative vitreoretinopathy, retinopathy of prematurity, preferably proliferative vitreoretinopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Is the linear curve of methotrexate injection. DETAILED DESCRIPTION OF THE INVENTION

[0068] DEFINITIONS

[0069] CHEMICAL DEFINITIONS

[0070] The definitions of specific functional groups and chemical terms are described in more detail below.

[0071] When a numerical range is listed, each value and sub - ranges within the range are included. For example, "C 1-6 alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C2-6 , 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 groups.

[0072] "C 1-6 alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms, also referred to herein as "lower alkyl". In some embodiments, C 1-4 alkyl and C 1-3 alkyl are particularly preferred. Examples of said alkyl groups include, but are not limited to: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6). Whether or not the alkyl group is preceded by "substituted", each of the alkyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined below.

[0073] "C 2-6 alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having 2 to 6 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). One or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). In some embodiments, C 2-4 alkenyl is particularly preferred. Examples of said alkenyl groups include, but are not limited to: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Whether or not the alkenyl group is preceded by "substituted", each of the alkenyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined below.

[0074] "C 2-6 alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having 2 to 6 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2 or 3 carbon-carbon triple bonds) and optionally one or more carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). In some embodiments, C 2-4An alkynyl group is particularly preferred. In some embodiments, the alkynyl group does not contain any double bonds. One or more carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of the alkynyl group include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. Whether or not the alkynyl group is preceded by "substituted", each of the alkynyl groups is independently optionally substituted, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined as follows.

[0075] "Halogenated" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). In some embodiments, the halogen group is F, Cl, or Br. In some embodiments, the halogen group is F or Cl. In some embodiments, the halogen group is F.

[0076] Thus, "C 1-6 haloalkyl" refers to the above-mentioned "C 1-6 alkyl" which is substituted by one or more halogen groups. In some embodiments, C 1-4 haloalkyl is particularly preferred, and more preferably C 1-3 haloalkyl and C 1-2 haloalkyl. Exemplary haloalkyls include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethylethyl, and the like.

[0077] "C 5-10 cycloalkyl" refers to a non-aromatic cycloaliphatic hydrocarbon group having 5 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 5-8 cycloalkyl is preferred, and C 5-6 cycloalkyl is particularly preferred. Cycloalkyl also includes a ring system in which the above cycloalkyl ring is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ), and so on. Whether or not the cycloalkyl group is preceded by "substituted", each of the cycloalkyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0078] "5- to 10-membered heterocyclic group" or refers to a group of a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In a heterocyclic group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom as long as the valence allows. In some embodiments, a 5- to 6-membered heterocyclic group is preferred, which is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. The heterocyclic group also includes a ring system in which the above-mentioned heterocyclic group ring is fused to one or more cycloalkyl, aryl, or heteroaryl groups, wherein the point of attachment is on the heterocyclic group ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclic group ring system. Whether or not the heterocyclic group is preceded by "substituted", each of the heterocyclic groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0079] "C 6-10 aryl" refers to a group of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system having 6 to 10 ring carbon atoms and zero heteroatoms (e.g., having 6 or 10 π electrons arranged in a cyclic arrangement and shared). In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C 10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, phenyl is particularly preferred. The aryl group also includes a ring system in which the above-mentioned aryl group ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aryl group ring. In such a case, the number of carbon atoms continues to represent the number of carbon atoms in the aryl group ring system. Whether or not the aryl group is preceded by "substituted", each of the aryl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0080] "5-10 membered heteroaryl" refers to a group having a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system of ring carbon atoms and 1-4 ring heteroatoms (e.g., having 6 or 10 π electrons shared in a cyclic arrangement), where each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, provided that the valence allows. The bicyclic heteroaryl system can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above heteroaryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5 membered heteroaryl is particularly preferred, which is a 5 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Whether or not the heteroaryl is preceded by "substituted", each of the heteroaryls is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0081] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NRbb ) OR aa 、 - OC(=NR bb ) R aa 、 - OC(=NR bb ) OR aa 、 - C(=NR bb ) N(R bb )2、 - OC(=NR bb ) N(R bb )2、 - NR bb C(=NR bb ) N(R bb )2、 - C(=O) NR bb SO2R aa 、 - NR bb SO2R aa 、 - SO2N(R bb )2、 - SO2R aa 、 - SO2OR aa 、 - OSO2R aa 、 - S(=O) R aa 、 - OS(=O) R aa 、 - Si(R aa )3、 - OSi(R aa )3、 - C(=S) N(R bb )2、 - C(=O) SR aa 、 - C(=S) SR aa 、 - SC(=S) SR aa 、 - SC(=O) SR aa 、 - OC(=O) SR aa 、 - SC(=O) OR aa 、 - SC(=O) R aa 、 - P(=O)2R aa 、 - OP(=O)2R aa 、 - P(=O)(R aa )2、 - OP(=O)(R aa )2、 - OP(=O)(OR cc )2、 - P(=O)2N(R bb )2、 - OP(=O)2N(R bb )2、 - P(=O)(NR bb )2、 - OP(=O)(NR bb )2、 - NR bb P(=O)(OR cc )2、 - NR bb P(=O)(NR bb )2、 - P(R cc )2、 - P(R cc)3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ) alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd groups;

[0082] or two geminal hydrogens on a carbon atom are substituted by the groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc ;

[0083] Each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, or two R aa groups combine to form a heterocyclic group or a heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd groups;

[0084] Each R bb is independently selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SRcc 、 -P(=O)2R aa 、 -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, or two R bb groups combine to form a heterocyclic group or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups;

[0085] R cc each independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, or two R cc groups combine to form a heterocyclic group or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups;

[0086] R dd each independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、 -ON(R ff )2, -N(R ff )2, 、 -N(R ff )3 + X - 、 -N(OR ee )R ff 、 -SH, -SR ee 、 -SSR ee 、 -C(=O)R ee 、 -CO2H, -CO2R ee 、 -OC(=O)R ee 、 -OCO2R ee 、 -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee 、 -NR ff CO2R ee 、 -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee 、 -OC(=NR ff )R ee 、 -OC(=NRff ) OR ee 、 -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee 、 -SO2N(R ff )2, -SO2R ee 、 -SO2OR ee 、 -OSO2R ee 、 -S(=O)R ee 、 -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee 、 -C(=S)SR ee 、 -SC(=S)SR ee 、 -P(=O)2R ee 、 -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups, or two geminal R dd substituents may combine to form =O or =S;

[0087] R ee each of which is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, aryl, heterocyclic group and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups;

[0088] R ff each of which is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, or two R ff groups combine to form a heterocyclic group or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups;

[0089] Rgg Each of which is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6(alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 (alkyl), -SO2N(C 1-6 (alkyl)2, -SO2NH(C 1-6 (alkyl), -SO2NH2, -SO2C 1-6 (alkyl), -SO2OC 1-6 (alkyl), -OSO2C 1-6 (alkyl), -SOC 1-6 (alkyl), -Si(C 1-6 (alkyl)3, -OSi(C 1-6 (alkyl)3, -C(=S)N(C 1-6 (alkyl)2, C(=S)NH(C 1-6 (alkyl), C(=S)NH2, -C(=O)S(C 1-6 (alkyl), -C(=S)SC 1-6 (alkyl), -SC(=S)SC 1-6 (alkyl), -P(=O)2(C 1-6 (alkyl), -P(=O)(C 1-6 (alkyl)2, -OP(=O)(C 1-6 (alkyl)2, -OP(=O)(OC 1-6 (alkyl)2, C 1-6 (alkyl), C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic group, C6-C 10 aryl, C3-C7 heterocyclic group, C5-C 10 heteroaryl; or two geminal R gg substituents may combine to form =O or =S; wherein, X - is a counterion.

[0090] Exemplary substituents on the nitrogen atom include but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SORaa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl, or two R groups attached to the nitrogen atom combine to form a heterocyclic group or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic group, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R groups, and wherein R cc , R dd , R aa , R bb , R cc and R dd are as described above.

[0091] Other definitions

[0092] The term "pharmaceutically acceptable salts" refers to those salts which, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail by Berge et al. in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also included are salts formed using conventional methods in the art, for example, ion exchange methods. Other pharmaceutically acceptable salts include: adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, gluconates, glycerophosphates, glucuronates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 (alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, etc. Other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed with counterions, if appropriate, the counterions such as halide ions, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

[0093] "Subjects" to whom administration is made 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 elderly adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, 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" may be used interchangeably herein.

[0094] The terms "disease", "disorder" and "condition" may be used interchangeably herein.

[0095] Unless otherwise specified, the term "treatment" as used herein includes the effects that occur when a subject has a specific disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows the development of the disease, disorder or condition ("therapeutic treatment"), and also includes the effects that occur before a subject begins to have a specific disease, disorder or condition ("preventive treatment").

[0096] "Vitamin" is a class of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions and plays an important role in the growth, metabolism and development of the human body. Vitamins neither participate in the formation of human cells nor provide energy for the human body. Vitamins participate in the biochemical reactions of the human body and regulate the metabolic functions of the human body. If the intake of vitamins is insufficient, it will lead to an imbalance in human metabolism, a decline in immunity and may lead to malnutrition and susceptibility to various diseases.

[0097] "Fat-soluble vitamins" are a class of vitamins that are insoluble in water but soluble in fat and non-polar organic solvents (such as benzene, ether and chloroform, etc.), including vitamin A, vitamin D, vitamin E, vitamin K, etc. This class of vitamins generally contains only three elements: carbon, hydrogen and oxygen, and mostly coexists with lipids in food. Fat-soluble vitamins are mostly relatively stable.

[0098] "Vitamin A" is a class of active 20-carbon unsaturated hydrocarbons composed of a β-ionone ring and an unsaturated monohydric alcohol. Its hydroxyl group can be esterified or converted into an aldehyde or an acid, and it can also exist in the state of a free alcohol. Vitamin A includes two types: vitamin A1 (retinol) and vitamin A2 (dehydroretinol). The difference between the two is that there is an additional double bond between C3 and C4 in the ionone ring of vitamin A2.

[0099] "Vitamin D" is a class of sterols containing a cyclopentanoperhydrophenanthrene structure. Six types of vitamin D have been identified, namely vitamin D2, vitamin D3, vitamin D4, vitamin D5, vitamin D6 and vitamin D7. Among them, the most important ones are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Their structures are very similar, and vitamin D2 has one more double bond and a methyl group in the side chain than vitamin D3. "Vitamin E" is the general term for tocopherols and tocotrienols with α-tocopherol-like activity. Tocopherols and tocotrienols are both derivatives of 6-hydroxychromane. Tocotrienols have double bonds at the 3', 7' and 11' positions of the side chain, and the other parts have exactly the same structure as tocopherols. Eight types of vitamin E are known, and their differences lie in the number and position of methyl groups on the cyclic structure. Among them, the most important ones are the derivatives of 4 tocopherols, namely α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol.

[0100] "Vitamin K" is a general term for a series of 2-methyl-1,4-naphthoquinone derivatives. Natural vitamin K includes vitamin K1 (phylloquinone) and vitamin K2 (menaquinone). Vitamin K3 (2-methylnaphthoquinone) is synthetic. The difference between these derivatives lies in whether there is a terpene side chain at the 3-position.

[0101] "Amino acid" refers to an organic compound containing a basic amino group and an acidic carboxyl group. It is the basic structural unit of proteins and the basis for post-translational modification of proteins. There are a total of 20 kinds. In addition, based on these basic amino acids, organisms will also synthesize derived amino acid types such as hydroxyproline and hydroxylysine. In fireflies, even D-amino acids will be synthesized. These amino acids synthesized by organisms are collectively called "natural amino acids". Natural amino acids are generally of the L-type. The 20 most common natural amino acids are shown in the following table:

[0102]

[0103] "Amino acid ester" refers to an amino acid in which the acidic carboxyl group of the amino acid exists in the form of an ester.

[0104] "Amino acid oligopeptide" refers to a short polypeptide in which 2 to 10 amino acids are connected to each other by peptide bonds. For example, an amino acid oligopeptide can be an oligopeptide formed by 2 - 5 amino acids, or an oligopeptide formed by 2 - 3 amino acids.

[0105] "Aprotic solvent" is also known as non-proton transfer solvent or non-protic solvent. The proton self-transfer reaction of such solvents is extremely weak or has no self-transfer tendency. Aprotic solvents can be divided into: aprotic non-polar solvents, such as alkanes, for example n-hexane, heptane, benzene, toluene, diethyl ether, carbon tetrachloride, etc.; aprotic polar solvents, such as amides, ketones, nitriles, dimethyl sulfoxide, pyridine, dichloromethane, N,N-dimethylformamide, acetone, etc. Example

[0106] The technical solution of the present invention will be clearly and completely described below in conjunction with the examples. Obviously, the described examples are only used to illustrate the present invention, rather than to limit the present invention. All other examples obtained by those of ordinary skill in the art based on the examples of the present invention without creative work fall within the scope of protection of the present invention.

[0107] DMF: N,N-dimethylformamide, DMA: N,N-dimethylacetamide, DMSO: dimethyl sulfoxide, DMAP: 4-dimethylaminopyridine, DIPEA: N,N-diisopropylethylamine, DCC: dicyclohexylcarbodiimide, EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, CDI: carbonyldiimidazole.

[0108] The method for synthesizing the compound of formula (I) is shown as follows:

[0109]

[0110] React the compound of formula (II) (methotrexate) with the compound of formula (III) (wherein representative X is halogen, amino or hydroxyl) to obtain the compound of formula (I). Preferably, the reaction is carried out in an aprotic polar solvent in the presence of a condensing agent and a base.

[0111] Specifically, the compound (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamide)-5-(((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonane-2,4,6,8-tetraen-1-yl)oxy)-5-oxopentanoic acid (IV) is taken as an example to elaborate in detail on the investigation of the process parameters of this type of compound.

[0112]

[0113] Example 1: Synthesis of Compound (S)-2-(4-(((2,4-Diaminopteridin-6-yl)methyl)(methyl)amino)benzamide)-5-(((2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-Trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl)oxy)-5-oxopentanoic Acid (IV)

[0114] Take a 250 mL single-necked flask, measure 100 mL of DMSO and add it to the flask. Weigh 9.08 g of methotrexate and add it to the flask. Weigh 5.72 g of vitamin A and add it to the flask. Weigh 2.44 g of DMAP and add it to the flask. Weigh 12.40 g of DCC and add it to the flask. Under magnetic stirring, react at room temperature overnight. The solid dissolves to form a yellow solution. The next day, filter to remove the filter cake. Drop the filtrate into 1000 mL of purified water, and a large amount of bright yellow solid precipitates. Filter by suction, wash the filter cake with purified water, and dry it under vacuum to obtain a solid filter cake. Dry the solid, add 200 mL of ethanol, slurry for 3 hours, filter by suction, dry it under vacuum to obtain a solid, grind and crush it to obtain 9.23 g of solid product, with a yield of 63.80%. 1 H NMR (400 MHz, DMSO) δ 8.61 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H),7.73 - 7.59 (m, 1H), 7.49 (d, J = 8.9 Hz, 3H), 6.79 (d, J = 9.0 Hz, 2H), 6.71 - 6.52 (m, 2H), 5.57 (d, J = 8.0 Hz, 1H), 4.79 (s, 3H), 4.04 - 3.73 (m, 1H),3.58 - 3.41 (m, 1H), 3.24 (s, 3H), 2.33 - 1.99 (m, 2H), 1.50 (m, 28H).

[0115] Experiment 1: Influence of Different Feed Ratios on the Preparation of Compound (IV)

[0116] Table 1: Influence of Different Feed Ratios on the Preparation of Compound (IV)

[0117]

[0118] Table 2: Results of the Preparation of Compound (IV) at Different Feed Ratios

[0119]

[0120] When the dosage of vitamin A is increased, the reaction yield changes little. Therefore, the preferred feeding ratio of methotrexate to vitamin A is 1:1 to 1:5, preferably 1:1 to 1:2, more preferably 1:1 - 1:1.5, and most preferably 1:1.

[0121] Experiment 2: Effect of different reaction temperatures on the preparation of the compound of formula (IV)

[0122] Table 3: Effect of different reaction temperatures on the preparation of the compound of formula (IV)

[0123]

[0124] Table 4: Results of the preparation of the compound of formula (IV) at different reaction temperatures

[0125]

[0126] When the temperature is lower than 30 °C, the reaction can proceed smoothly and the yield is relatively stable; when the temperature is too high (exceeding 35 °C), other impurities will be generated, resulting in a decrease in the yield after purification of the product. Therefore, the preferred reaction temperature is 0 - 50 °C, preferably 15 - 25 °C, more preferably 20 - 25 °C.

[0127] Experiment 3: Effect of different organic bases on the preparation of the compound of formula (IV)

[0128] Table 5: Effect of different organic bases on the preparation of the compound of formula (IV)

[0129]

[0130] Table 6: Results of the preparation of the compound of formula (IV) with different organic bases

[0131]

[0132] It can be found from the above experimental results that after adding the organic base, the required reaction time is basically between 12 - 16 h, and the relevant yields are not much different. Comparing the purity of the obtained product and the difficulty of post-treatment, DMAP or DIPEA is preferred, and DMAP is more preferred.

[0133] Experiment 4: Effect of different condensing agents on the preparation of the compound of formula (IV)

[0134] Table 7: Effect of different condensing agents on the preparation of the compound of formula (IV)

[0135]

[0136] Table 8: Results of the preparation of the compound of formula (IV) with different condensing agents

[0137]

[0138] According to the above experimental results, we found that when CDI was used for condensation, the reaction rate was relatively fast, but the obtained products were relatively complex. After purification, the product yield was relatively low. When EDCI was used for condensation, the reaction proceeded slowly and it was difficult to complete the reaction completely. After purification, the product yield was not high either. Therefore, DCC was preferably used as the condensing agent.

[0139] Experiment Five: Influence of Different DCC Dosages on the Preparation of Compound (IV)

[0140] Table 9: Influence of Different DCC Dosages on the Preparation of Compound (IV)

[0141]

[0142] Table 10: Results of the Preparation of Compound (IV) with Different DCC Dosages

[0143]

[0144] When the feeding amount of DCC was 3 times that of methotrexate, the reaction could be completely carried out in 12 hours. When the amount of DCC was reduced, the reaction could not be completely completed in the same time, and the relative yield was relatively low after purification. When the amount of DCC was continuously increased, the yield of the reaction would not continue to increase. Therefore, the optimal multiple was 3 times, that is, the molar ratio of methotrexate to DCC dosage was 1:3 to 1:5, preferably 1:3.

[0145] Experiment Six: Influence of Different Multiples of Absolute Ethanol on the Preparation of Compound (IV)

[0146] Table 11: Influence of Different Multiples of Absolute Ethanol on the Preparation of Compound (IV)

[0147]

[0148] Table 12: Results of the Preparation of Compound (IV) with Different Multiples of Absolute Ethanol

[0149]

[0150] When the amount of ethanol used for pulping was too small, the product yield was relatively high, but the purity was relatively low; when the amount of absolute ethanol was too large, the product purity changed little, but the yield decreased significantly and the product loss was serious. To sum up, the preferred amount of absolute ethanol was 10 - 20 times, preferably 20 times.

[0151] Experiment Seven: Influence of Different Pulping Temperatures on the Preparation of Compound (IV)

[0152] Table 13: Influence of Different Pulping Temperatures on the Preparation of Compound (IV)

[0153]

[0154] Table 14: Results of the preparation of the compound of formula (IV) at different beating temperatures

[0155]

[0156] When the beating temperature is too low, the purification effect of the product is poor and the purity of the pure product is low; when the beating temperature is too high, the product yield is too low. In summary, the preferred beating temperature is 10 - 30 °C, preferably 20 - 30 °C.

[0157] Experiment 8: Effect of different beating times on the preparation of the compound of formula (IV)

[0158] Table 15: Effect of different beating times on the preparation of the compound of formula (IV)

[0159]

[0160] Table 16: Results of the preparation of the compound of formula (IV) at different beating times

[0161]

[0162] Since the obtained wet product is directly beaten without drying, when the beating time is insufficient, some water will remain in the product. To ensure the stability of the product, the drying method is vacuum drying at room temperature. When a large amount of water remains, the drying time will inevitably be extended. In summary, the preferred beating time is 3 hours.

[0163] Example 22: Synthesis of Compound V

[0164]

[0165] Take a 250 mL single-necked flask, measure 100 mL of DMSO and add it to the flask. Weigh 9.08 g of methotrexate and add it to the flask. Weigh 9.02 g of ethyl tyrosine and add it to the flask. Weigh 2.44 g of DMAP and add it to the flask. Weigh 12.40 g of DCC and add it to the flask. Under magnetic stirring, react overnight at room temperature. The solid dissolves to form a yellow solution. Filter the next day, remove the filter cake, drop the filtrate into 1000 mL of purified water, a large amount of bright yellow solid precipitates, filter by suction, wash the filter cake with purified water, and dry it in vacuo to obtain a solid filter cake. The dried solid is slurried with 200 mL of ethanol for 3 hours, filtered by suction, and dried in vacuo to obtain a solid. Grind and crush it to obtain 12.08 g of solid product with a yield of 69.97%. 11H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.57 (d, J = 1.3 Hz, 1H), 8.28 - 7.93 (m, 2H), 7.71 (m, 1H), 7.04 - 6.75 (m, 4H), 6.73 - 6.37 (m, 4H), 4.79 (s, 1H), 4.52 - 4.15 (m, 2H), 4.06 - 3.93 (m, 2H), 3.27 - 3.10 (m, 3H), 3.00 - 2.61 (m, 3H), 2.54 (s, 1H), 2.45 - 1.91 (m, 2H), 1.89 - 1.76 (m, 3H), 1.74 - 1.56 (m, 3H), 1.50 (dd, J = 8.0, 3.5 Hz, 2H), 1.33 - 1.17 (m, 6H), 1.11 – 1.03 (m, 4H).

[0166] Example 23: Synthesis of Compound VI

[0167]

[0168] Take a 250 mL single-necked flask, measure 100 mL of DMSO and add it to the flask. Weigh 9.08 g of methotrexate and add it to the flask. Weigh 4.92 g of ethyl leucinate and add it to the flask. Weigh 2.44 g of DMAP and add it to the flask. Weigh 12.40 g of DCC and add it to the flask. Under magnetic stirring, react at room temperature overnight. The solid dissolves to form a yellow solution. The next day, filter to remove the filter cake. Drop the filtrate into 1000 mL of purified water, and a large amount of bright yellow solid precipitates. Filter by suction, wash the filter cake with purified water, and dry it in vacuo to obtain a solid filter cake. Pulverize the dried solid with 200 mL of ethanol for 3 hours, filter by suction, and dry it in vacuo to obtain a solid. Grind and crush it to obtain 8.85 g of solid product with a yield of 60.10%. 11H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.33 - 7.93 (m, 3H), 7.63 (m, 4H), 6.82 (d, J = 8.5 Hz, 2H), 6.59 (s, 2H), 4.79 (s, 2H), 4.52 - 4.35 (m, 1H), 4.21 (d, J = 25.7 Hz, 2H), 4.15 - 3.92 (m, 4H), 3.21 (s, 3H), 2.39 - 2.07 (m, 2H), 2.06 - 1.77 (m, 2H), 1.78 - 1.33 (m, 6H), 1.31 - 1.05 (m, 6H), 0.99 - 0.65 (m, 12H).

[0169] Example 24: Synthesis of Compound VII

[0170]

[0171] Take a 250 mL single-necked flask, measure 100 mL of DMSO and add it to the flask. Weigh 9.08 g of methotrexate and add it to the flask. Weigh 3.92 g of serine ethyl ester and add it to the flask. Weigh 2.44 g of DMAP and add it to the flask. Weigh 12.40 g of DCC and add it to the flask. Under magnetic stirring, react overnight at room temperature. The solid dissolves to form a yellow solution. The next day, filter to remove the filter cake. Drop the filtrate into 1000 mL of purified water, and a large amount of bright yellow solid precipitates. Filter by suction, wash the filter cake with purified water, and dry it in vacuo to obtain a solid filter cake. Pulverize the dried solid with 200 mL of ethanol for 3 hours, filter by suction, and dry it in vacuo to obtain a solid. Grind and crush it to obtain 8.47 g of solid product with a yield of 61.89%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.23 (m, 3H), 7.65 (m, 4H), 6.82 (d, J = 7.6 Hz, 2H), 6.60 (s, 2H), 4.79 (s, 2H), 4.47 (s, 1H), 4.28 (s, 2H), 4.05 (s, 4H), 3.21 (s, 3H), 2.95 (s, 1H), 2.26 (d, J = 8.5 Hz, 2H), 1.97 (d, J = 8.6 Hz, 2H), 1.42 - 0.93 (m, 8H), 0.85 (s, 1H).

[0172] Test Example: In Vitro Intravitreal Release Test of Methotrexate and Compound of Formula (IV)

[0173] Experimental Procedure:

[0174] 1. Under this liquid phase condition, simultaneously inject the compound of formula (IV), and the compound of formula (IV) is not added to the vitreous body (to exclude the influence of methotrexate impurities contained in the compound of formula (IV) itself).

[0175] 2. Further investigate the release of the compound of formula (IV) in the in vitro vitreous experiment (at different time gradients, in a 37°C water bath).

[0176] Purpose: Through the study of the metabolic situation in the in vitro vitreous body, test whether the compound of formula (IV) can be degraded into methotrexate by vitreous enzymes in the rabbit eye.

[0177] In Vitro Experimental Conditions:

[0178] Temperature: 37°C shaker water bath oscillation

[0179] Release Medium: Rabbit vitreous humor, Medium Volume: Approximately 1 mL

[0180] Disperse approximately 3 mg of the compound of formula (IV) and methotrexate raw drug respectively in the rabbit vitreous body and shake in a water bath.

[0181] Shaking Time: Sample at intervals of 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h, grind the vitreous body, send for analysis and testing, and detect the content of methotrexate in the vitreous body by liquid phase. The experiment is carried out in duplicate in parallel.

[0182] Figure 1 The following table shows the linear curve of the methotrexate injection.

[0183]

[0184]

[0185] Results of This Test:

[0186] The compound of formula (IV) releases a small amount of methotrexate in the vitreous body, and there is a gradually increasing trend with time, and the influence of the impurities in the raw material itself is excluded. This indicates that the compound of formula (IV) has a tendency to be degraded in the vitreous body and can release methotrexate, and methotrexate can exert its efficacy at extremely low drug concentrations. At the same time, due to the greatly improved liposolubility of the compound of formula (IV), it can effectively combine with the development of sustained-release implants at the preparation end or other sustained-release drug delivery methods to develop effective intraocular sustained-release drugs. This is beneficial to effectively reducing the dosing frequency during the subsequent treatment process, increasing patient compliance, and reducing patient pain.

[0187] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: in, R is selected from OH, OR1 or NHR1, and at most one R is OH; R1 is a vitamin, an amino acid, an amino acid ester or an amino acid oligopeptide.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the vitamin is a fat-soluble vitamin, preferably selected from vitamin A, vitamin D, vitamin E or vitamin K, preferably selected from vitamin A1, vitamin A2, vitamin D2, vitamin D3, α-tocopherol or β-tocopherol.

3. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine in L or R configuration and their esters, preferably selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, threonine and their esters in L or R configuration, preferably selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, tyrosine, phenylalanine and their esters in L or R configuration.

4. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid ester is selected from the C 1-6 Alkyl esters, C 1-6 Halogenated alkyl esters, C 2-6 Alkenyl ester, C 2-6 Alkynyl esters, C 5-10 Cycloalkyl esters, 5-10 membered heterocyclic esters, C 6-10 Aryl ester or 5-10 membered heteroaryl ester, preferably selected from the C 1-6 Alkyl ester or C 1-6 Haloalkyl esters.

5. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid oligopeptide is an oligopeptide of 2 to 5 amino acids, preferably an oligopeptide of 2 to 3 amino acids.

6. A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, having the following structure: or wherein R is selected from OR1 or NHR1, preferably OR1, wherein R1 is as defined in any one of claims 1-5.

7. A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, having the following structure: or wherein R1 is as defined in any one of claims 1-5.

8. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, having the following structure: , , or .

9. A method for preparing a compound of formula (I) according to claim 1, comprising the following reaction: Wherein X is halogen, amino or hydroxyl, R is as defined in any one of claims 1-5.

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