Prodrug Compounds

Abstract

To provide nucleoside analog prodrug compounds being less cytotoxic and / or having controllable drug effect expression in cells.SOLUTION: A compound of formula (1), salt or solvate thereof are disclosed. In the formula, R1 is H, a substituted / unsubstituted hydrocarbon group, or -OR11 where R11 is a substituted / unsubstituted hydrocarbon group; R2 is H, a substituted / unsubstituted hydrocarbon group, or -OR12 where R12 is a substituted / unsubstituted hydrocarbon group; R3 is a monovalent group formed by removing one H from the OH group of the sugar moiety of a nucleoside analog; R4 is O or S; where part of C atoms of the hydrocarbon groups may be replaced by a hetero atom, or a ligand molecule and / or enzyme targeting structure may be added thereto; R1 and R2 may be linked together to form a N-containing ring with the adjacent N.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds that can be used as prodrugs. [Background technology]

[0002] Various nucleoside analogs are used as antimetabolites, such as anticancer drugs and antiviral drugs against hepatitis B virus, HIV virus, etc. Examples of such nucleoside analogs include anticancer drugs such as gemcitabine and cytarabine, and antiviral drugs against lamivudine, etc. J Known antiviral drugs include rifampin and zidovudine. These are monophosphorylated within cells and then converted to triphosphates (active forms), which exert their efficacy by inhibiting the synthesis of DNA and RNA necessary for cell and viral proliferation.

[0003] However, because nucleoside analogs such as gemcitabine are transported into cells via transporters, there are concerns about the emergence of resistant cells. Furthermore, the rate of monophosphorylation within cells is slow and uncontrollable. For this reason, various phosphate prodrugs have been developed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Med. Chem. 2008, 51, 2328-2345. [Non-patent document 2] Journal of Medicinal Chemistry 2018, 61, 2211-2226. [Non-patent document 3] Journal of Medicinal Chemistry 2014, 57, 1531-1542. Summary of the Invention [Problem to be solved by the invention]

[0005] To date, various prodrugs, such as BisPOM-type and ProTide-type, have been developed (Non-Patent Documents 1 to 3), but they are limited in terms of the release of toxic substances during deprotection and the variety of deprotection conditions.

[0006] [ka]

[0007] An object of the present invention is to provide novel prodrug compounds of nucleoside analogs.

[0008] An object of the present invention is to provide a prodrug compound of a nucleoside analogue, preferably having lower cytotoxicity, and preferably capable of controlling the expression of a pharmacological effect in cells. [Means for solving the problem]

[0009] Based on this finding, the present inventors have conducted extensive research and have found that a phosphoric acid fluoride-type prodrug compound represented by general formula (1) can solve the above-mentioned problems. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0010] Section 1. General formula (1):

[0011] [ka]

[0012] [In the formula: R 1 represents a hydrogen atom, an optionally substituted hydrocarbon group, or -OR 11 (R 11 represents an optionally substituted hydrocarbon group. 2 represents a hydrogen atom, an optionally substituted hydrocarbon group, or -OR 12 (R 12represents an optionally substituted hydrocarbon group. 3 represents a monovalent group formed by removing one hydrogen atom from the hydroxy group of the sugar moiety of a nucleoside analogue. 4 represents an oxygen atom or a sulfur atom. However, the hydrocarbon group may have some of its carbon atoms replaced with heteroatoms, and may have a ligand molecule and / or an enzyme target structure attached thereto. However, R 1 and R 2 may be linked to each other to form a nitrogen-containing ring together with the adjacent nitrogen atom. A compound represented by the formula (I), a salt thereof, or a solvate thereof.

[0013] Item 2. The compound, salt thereof, or solvate thereof according to Item 1, wherein the hydrocarbon group is an alkyl group, an aryl group, or an aralkyl group.

[0014] Item 3. The compound, salt, or solvate thereof according to Item 1 or 2, wherein the hydrocarbon group is an alkyl group.

[0015] Item 4. The compound, salt thereof, or solvate thereof according to any one of Items 1 to 3, wherein the hydrocarbon group is a branched alkyl group.

[0016] Section 5. Said R 1 is a group other than a hydrogen atom, and the R 2 5. The compound, salt thereof, or solvate thereof according to any one of items 1 to 4, wherein is a hydrogen atom.

[0017] Section 6. Said R 1 is a branched alkyl group, and the R 2 6. The compound, salt thereof, or solvate thereof according to any one of items 1 to 5, wherein is a hydrogen atom.

[0018] Section 7. Said R 1 and the above R 2 and n are each an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group, or a salt thereof, according to Item 2, or a solvate thereof.

[0019] Section 8. Said R 1 is an alkyl group, and the R 2 Item 8. The compound, salt thereof, or solvate thereof according to Item 2 or 7, wherein is an optionally substituted aryl group or an optionally substituted aralkyl group.

[0020] Item 9. The compound, salt, or solvate thereof according to Item 2, 7, or 8, wherein the substituent that the hydrocarbon group may have is at least one selected from the group consisting of an alkoxy group, a halogen atom, a nitro group, an amino group, a thiol group, a sulfone group, a sulfonic acid group, a hydroxyamino group, an amide group, a nitrile group, a phosphate group, a carbonyl group, and a carboxy group.

[0021] Item 10. The compound, salt thereof, or solvate thereof according to any one of Items 1 to 9, wherein the nucleoside analog is an anticancer agent or an antiviral agent.

[0022] Section 11. Said R 3 is represented by the general formula (2):

[0023] [ka]

[0024] [wherein Base represents a monovalent group obtained by removing one hydrogen atom from a nucleic acid base] 。R 31 is -O-, -C(=CH-R 311 )-(R 311 represents a hydrogen atom or an alkyl group, or -NR 312 -(R 312 represents a hydrogen atom or an alkyl group. 32 -CR 321 R 322 -(R 321 and R 322 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R represents -S- or -S-. 33 and R34 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 37 and R 38 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. A double line consisting of a solid line and a dotted line represents a single bond or a double bond (however, in the case of a double bond, R 321 and R 322 Either one of the above and R 33 and R 34 Neither of these exists.) 11. The compound according to any one of items 1 to 10, a salt thereof, or a solvate thereof, wherein the compound is a group represented by the following formula:

[0025] Item 12. A medicine containing at least one compound selected from the group consisting of the compound according to any one of items 1 to 11, a salt thereof, and a solvate thereof.

[0026] Item 13. The pharmaceutical agent according to Item 12, which is for anticancer or antiviral use.

[0027] Item 14. A reagent containing at least one selected from the group consisting of the compound according to any one of items 1 to 11, a salt thereof, and a solvate thereof. [Effects of the Invention]

[0028] According to the present invention, novel prodrug compounds of nucleoside analogs can be provided. Also, according to the present invention, prodrug compounds of nucleoside analogs with lower cytotoxicity can be provided. Furthermore, according to the present invention, it is also possible to provide prodrug compounds of nucleoside analogs that can control the expression of pharmacological effects in cells. [Brief explanation of the drawings]

[0029] [Figure 1] Two variants of compound 3a are shown. [Figure 2] The results of the stability evaluation test for compounds 3a and 3e are shown. The type of test solution (PBS or Milli-Q water (MQ)) is indicated above the graph. The vertical axis indicates the abundance ratio of the test compound and its variants, and the horizontal axis indicates the elapsed time from the time of preparation of the test solution (0 minutes). [Figure 3] The results of the stability evaluation test for compounds 3f and 3h are shown below. The graph notation is the same as in Figure 2. [Figure 4] The results of the stability evaluation test for compounds 4 to 6 are shown below. The notation in the graph is the same as in Figure 2. [Figure 5] The results of the toxicity evaluation test are shown. The vertical axis shows absorbance at 490 nm (higher values ​​indicate lower toxicity). The horizontal axis shows the test compound. The cytidine prodrug form is Compound 4. The concentration in the legend indicates the concentration of the test compound in the medium. [Figure 6] 1 shows the results of LC-MS analysis of cell extracts after administration of compound 3a to cells. The upper row shows the results of extracted ion chromatography of compound 3a, and the lower row shows the results of total ion chromatography. [Figure 7] The upper panel shows the results of extraction ion chromatography of the hydrolysate (monophosphate) of compound 3a, and the lower panel shows the results of LC-MS analysis (total ion chromatography) of the hydrolysate of compound 3a. [Figure 8] It shows that compound 3a is converted into a monophosphate via a hydrolysis product. [Figure 9] This shows that a secondary amine is converted to a monophosphate via a primary amine and a hydrolyzed product. [Figure 10] It shows that Compound 3C and Compound 3E are converted into the monophosphate via hydrolysis. [Figure 11] This shows the results of anticancer activity evaluation test 2. The cells used are shown on the left side of the graph, and the structure of the test compound is shown below the graph. The horizontal axis shows the concentration of the test compound used. DETAILED DESCRIPTION OF THE INVENTION

[0030] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0031] 1.Compound In one aspect, the present invention provides a compound represented by general formula (1):

[0032] [ka]

[0033] The present invention relates to a compound represented by the formula (I), a salt thereof, or a solvate thereof (these may be collectively referred to as "the compound of the present invention" in this specification). This will be explained below.

[0034] <1-1.R 1 , R 2 > R 1 represents a hydrogen atom, an optionally substituted hydrocarbon group, or -OR 11 (R 11 represents an optionally substituted hydrocarbon group. 2 represents a hydrogen atom, an optionally substituted hydrocarbon group, or -OR 12 (R 12 represents an optionally substituted hydrocarbon group.

[0035] The hydrocarbon group is not particularly limited, and examples thereof include alkyl groups, aryl groups, and groups formed by any combination thereof (e.g., aralkyl groups, alkylaryl groups, alkylaralkyl groups). Adjusting the bulkiness of the hydrocarbon group can control the stability of the compound of the present invention (and thus the timing of the intracellular manifestation of its medicinal effect). Specifically, employing a bulky hydrocarbon group can increase stability, while employing a structure with low steric hindrance (e.g., a planar structure) can decrease stability, thereby adjusting the timing of the manifestation of the medicinal effect. In one embodiment of the present invention, preferred examples of the hydrocarbon group include alkyl groups, aryl groups, and aralkyl groups. In one embodiment of the present invention, more preferred examples include alkyl groups. In one embodiment of the present invention, more preferred examples include aryl groups and aralkyl groups, with aralkyl groups being even more preferred.

[0036] The alkyl group may be linear, branched, or cyclic. From the viewpoint of the efficacy of the compound of the present invention, the alkyl group is preferably a branched alkyl group. The number of carbon atoms in the alkyl group (when linear or branched) is not particularly limited and is, for example, 1 to 12. In one embodiment of the present invention, the number of carbon atoms is preferably 1 to 8, more preferably 2 to 8, even more preferably 3 to 6, and even more preferably 3 to 4. In one embodiment of the present invention, the number of carbon atoms is preferably 3 to 12, more preferably 4 to 8, and even more preferably 5 to 7. The number of carbon atoms in the alkyl group (when cyclic) is not particularly limited and is, for example, 3 to 7, preferably 4 to 6. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, 3-methylpentyl, n-heptyl, and n-octyl. Among these, an isopropyl group is particularly preferred.

[0037] The aryl group is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 8. The aryl group may be either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, naphthyl group, biphenyl group, pentalenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perylenyl group, fluorenyl group, and phenanthryl group, and a preferred example is a phenyl group.

[0038] The aralkyl group is not particularly limited, and examples thereof include aralkyl groups in which the hydrogen atoms (for example, 1 to 3, preferably 1 hydrogen atom) of a linear or branched alkyl group having 1 to 6 carbon atoms (preferably 1 to 3) are substituted with the aryl group. Specific examples of the aralkyl group include a benzyl group and a phenethyl group.

[0039] The alkylaryl group is not particularly limited, and examples thereof include alkylaryl groups in which hydrogen atoms (for example, 1 to 3, preferably 1 hydrogen atom) of the aryl group are substituted with linear or branched alkyl groups having 1 to 6 carbon atoms (preferably 1 to 2). Specific examples of the alkylaryl group include tolyl and xylyl groups.

[0040] The alkylaralkyl group is not particularly limited, and examples thereof include alkylaralkyl groups in which a hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom) on the aromatic ring of the aralkyl group is substituted with a linear or branched alkyl group having 1 to 6 carbon atoms (preferably 1 to 2).

[0041] The hydrocarbon group may be substituted. The substituent is not particularly limited, but examples thereof include an alkoxy group, a halogen atom, a nitro group, an amino group, a thiol group, a sulfone group, a sulfonic acid group, a hydroxyamino group, an amide group, a nitrile group, a phosphate group, a carbonyl group, and a carboxy group. Among these, preferred are an alkoxy group, a halogen atom, a nitro group, and an amino group. Examples of the alkoxy group include a linear or branched alkoxy group having 1 to 8 carbon atoms (preferably 1 to 4, more preferably 1 to 2, and even more preferably 1). Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, with a fluorine atom being preferred. The number of substituents is not particularly limited, but may be, for example, 1 to 3, preferably 1 to 2, and more preferably 2. The position of the substituent is not particularly limited, but when the hydrocarbon group is an aryl group or an aralkyl group, the para- and meta-positions on the aryl group are preferred.

[0042] Some of the carbon atoms in the hydrocarbon group may be replaced with heteroatoms, and a ligand molecule and / or an enzyme target structure may be added.

[0043] The heteroatom is not particularly limited, and examples thereof include a nitrogen atom, an oxygen atom, a sulfur atom, etc. The carbon atom substituted with the heteroatom is not particularly limited, and may be a carbon atom on the main chain of a chain hydrocarbon group, a carbon atom on a side chain, or a ring-constituting carbon atom of a cyclic hydrocarbon group.

[0044] When some of the carbon atoms in the hydrocarbon group are replaced with heteroatoms, the number of heteroatoms is not particularly limited, and may be, for example, 1 to 6, 1 to 3, or 1.

[0045] The hydrocarbon group to which the ligand molecule and / or enzyme target structure is attached is, in other words, a hydrocarbon group substituted with a group derived from the ligand molecule and / or enzyme target structure (e.g., a group obtained by removing one hydrogen atom or functional group from the ligand molecule and / or enzyme target structure).

[0046] By employing a hydrocarbon group to which a ligand molecule is attached, the compound of the present invention can act specifically on the target site of the ligand. The ligand is determined based on its relationship with the target molecule (e.g., a protein). For example, if the protein is a receptor, it can be a substance that binds to the receptor; if the protein is an enzyme, it can be an enzyme substrate; and if the protein is an antigen, it can be an antibody or an antibody fragment. Examples of ligands include carbohydrates, cholesterol, lipids, phospholipids, antibodies, lipoproteins, hormones, peptides, vitamins, steroids, cationic lipids, etc. More specifically, by using, for example, N-acetylgalactosamine, retinoic acid, galactose, glycyrrhizic acid, etc. as the ligand, the compound of the present invention can act specifically on the liver.

[0047] The protein to be labeled is not particularly limited, and examples thereof include receptors, enzymes, and antigens.

[0048] Antibodies or fragments thereof include monoclonal antibodies, single-chain antibodies such as Fv, scFv, Fab, F(ab')2, Fab', Fd, dAb, CDR, scFv-Fc fragments, nanobodies, affibodies, diabodies, avimers, and versabodies.

[0049] Examples of the enzyme include oxidoreductases, hydrolases, transferases, and isomerases. Examples of oxidoreductases include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, formaldehyde oxidase, sorbitol oxidase, fructose oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, xanthine oxidase, ascorbate oxidase, sarcosine oxidase, choline oxidase, amine oxidase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, alcohol dehydrogenase, formaldehyde dehydrogenase, sorbitol dehydrogenase, fructose dehydrogenase, hydroxybutyrate dehydrogenase, glycerol dehydrogenase, glutamate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, catalase, peroxidase, uricase, nitroreductase, and cytochrome P450. Examples of hydrolases include proteases, lipases, amylases, invertases, maltase, β-galactosidase, lysozyme, urease, esterases, nucleases, and phosphatases. Examples of transferases include various transferases, kinases, aminotransferases, and GSTs. Examples of isomerases include racemases, phosphoglycerate phosphomutases, and glucose 6-phosphate isomerases.

[0050] Receptors include muscarinic acetylcholine receptors, adenosine receptors, adrenaline receptors, GABA receptors, angiotensin receptors, cannabinoid receptors, cholecystokinin receptors, dopamine receptors, glucagon receptors, histamine receptors, olfactory receptors, opioid (enkephalin, endorphin, etc.) receptors, rhodopsin, secretin receptors, serotonin receptors, somatostatin receptors, gastrin receptors, P2Y receptors, HER2 receptors, EGF receptors, erythropoietin receptors, insulin receptors, and steroid receptors. These include thrombopoietin receptors, growth factor receptors, cytokine receptors, nicotinic acetylcholine receptors, glycine receptors, glutamate receptors (NMDA receptors, AMPA receptors, kainate receptors), inositol triphosphate (IP3) receptors, P2X receptors, sex hormone (androgen, estrogen, progesterone) receptors, vitamin D receptors, glucocorticoid receptors, mineralocorticoid receptors, thyroid hormone receptors, retinoid receptors, peroxisome proliferator receptors (PPARs), and more.

[0051] By employing a hydrocarbon group having an enzyme target structure attached thereto, the compound of the present invention can be specifically decomposed at the site where the enzyme is present. For example, by employing a hydrocarbon group having an enzyme target structure attached thereto in the compound of the present invention, which is a secondary amine, the compound can be specifically decomposed at the site where the enzyme is present and converted to a primary amine, which can then be site-specifically converted to a hydrolyzed form and a monophosphate, thereby exerting a medicinal effect.

[0052] Examples of the enzyme include the enzymes described above. The enzyme target structure is not particularly limited, and any known structure can be used. Examples of enzyme target structures, corresponding enzymes, and target sites are shown below.

[0053] [ka]

[0054] The number of ligand molecules and / or enzyme target structures added to the hydrocarbon group is not particularly limited, and may be, for example, 1 to 3, 1 to 2, or 1.

[0055] R 1 and R 2 may be linked to each other to form a nitrogen-containing ring together with the adjacent nitrogen atom. The nitrogen-containing ring is not particularly limited. For example, R 1 and R 2 may be linked to each other to form a dimethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, or the like, and may form a nitrogen-containing ring (for example, a piperidine ring) together with the adjacent nitrogen atom.

[0056] In one aspect of the present invention, from the viewpoint of imparting appropriate stability to the compound and allowing it to exhibit appropriate medicinal effects, it is preferable to use R 2 is a hydrogen atom, and more preferably R 1 is a group other than a hydrogen atom and R 2 is a hydrogen atom. In this case, the fluorine atom bonded to the phosphorus atom is replaced by an oxygen atom by hydrolysis in the living body, and then the compound is converted into a monophosphate by degradation with an enzyme such as HINT-1, thereby exerting its medicinal effect. In this case, from the viewpoint of medicinal effect, it is particularly preferable that R 1 is a branched alkyl group, and R 2 is a hydrogen atom.

[0057] On the other hand, R 1 and R 2 are both groups other than hydrogen atoms, or R 1 and R 2 When the groups are linked together to form a nitrogen-containing ring, the stability of the compound of the present invention is relatively high. This allows the timing and site of pharmacological activity to be controlled by adding an enzyme targeting structure to the hydrocarbon group as needed.

[0058] In one aspect of the invention, R 1 and R 2and R are each preferably an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group. 1 is an alkyl group, and the R 2 is preferably an optionally substituted aryl group or an optionally substituted aralkyl group.

[0059] <1-2.R 3 > R 3 represents a monovalent group obtained by removing one hydrogen atom from the hydroxy group of the sugar moiety of a nucleoside analog. The hydroxy group of the sugar moiety of a nucleoside analog is a hydroxy group that is substituted with a phosphate group when the nucleoside analog is converted to a monophosphate, and is not particularly limited thereto.

[0060] Nucleoside analogs are not particularly limited, and examples thereof include those having antimetabolic effects on anticancer drugs, antiviral drugs, etc. Examples of such nucleoside analogs include anticancer drugs such as gemcitabine and cytarabine; J anti-hepatitis B virus agents such as flucloxin and entecavir; anti-HIV agents such as zidovudine, didanosine and stavudine.

[0061] R 3 A preferred embodiment of the compound is, for example, a compound represented by the general formula (2):

[0062] [ka]

[0063] Examples of the group include a group represented by the following formula:

[0064] Base represents a monovalent group formed by removing one hydrogen atom from a nucleic acid base. As the nucleic acid base, any base constituting a nucleic acid can be used without particular limitation. The base constituting a nucleic acid includes not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I), modified bases, etc. Examples of modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methyl Examples of nucleic acid bases include 5-hydroxybenzoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc. These nucleic acid bases may further have one or more substituents.

[0065] R 31 is -O-, -C(=CH-R 311 )-(R 311 represents a hydrogen atom or an alkyl group, or -NR 312 -(R 312 represents a hydrogen atom or an alkyl group.

[0066] R 311 or R 312The alkyl group represented by the formula (I) includes any of linear, branched, and cyclic groups (preferably linear or branched, more preferably linear). The number of carbon atoms in the alkyl group (when linear or branched) is not particularly limited and is, for example, 1 to 8. From the viewpoint of detection sensitivity, the number of carbon atoms is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and still more preferably 1. The number of carbon atoms in the alkyl group (when cyclic) is not particularly limited and is, for example, 3 to 7, preferably 4 to 6. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, a 3-methylpentyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, and an n-dodecyl group.

[0067] R 32 -CR 321 R 322 -(R 321 and R 322 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R represents -S- or -S-. 33 and R 34 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 37 and R 38 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group.

[0068] R 321 , R 322 , R 33 , R 34 , R 35 , R 36 , R 37 , or R 38Examples of the alkynyl group represented by the formula (I) include alkynyl groups having 2 to 8 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, butynyl, pentynyl, and octenyl.

[0069] A double line consisting of a solid line and a dotted line indicates a single bond or a double bond. In the case of a double bond, R 321 and R 322 Either one of the above and R 33 and R 34 Neither of these exists. 32 When is -S-, the doublet is a single bond.

[0070] <1-3.R 4 > R 4 represents an oxygen atom or a sulfur atom. 4 is preferably an oxygen atom.

[0071] <1-4.Other> The compound represented by general formula (1) includes various isomers, such as geometric isomers, configurational isomers, tautomers, optical isomers, stereoisomers, positional isomers, and rotational isomers. When the compound of the present invention contains any isomer, a mixture of the isomers is also included in the compound of the present invention. Furthermore, when the compound of the present invention contains optical isomers, the optical isomers resolved from the racemate are also included in the compound of the present invention. When the compound of general formula (1) contains geometric isomers, configurational isomers, stereoisomers, conformational isomers, etc., each can be isolated by known means. When the compound of general formula (1) is optically active, it can be separated from the corresponding racemate into the (+) or (-) isomer [D or L isomer] by conventional optical resolution means.

[0072] The salt of the compound represented by general formula (1) is not particularly limited as long as it is a pharmaceutically acceptable salt. Both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0073] The solvate of the compound represented by general formula (1) is not particularly limited. Examples of the solvent that constitutes the solvate include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0074] 2. Manufacturing method The compounds of the present invention can be prepared in a variety of ways.

[0075] The compounds of the present invention can be produced, for example, according to or in accordance with the following schemes.

[0076] [ka]

[0077] As the compound represented by general formula (1a), commercially available compounds can be used as they are, or compounds synthesized according to or in accordance with known methods and / or according to or in accordance with the methods described in the Examples can also be used as needed.

[0078] As the compound represented by general formula (1b), commercially available nucleoside analogues can be used as they are, or, if necessary, those synthesized according to or in accordance with known methods can also be used.

[0079] From the viewpoints of yield, ease of synthesis, etc., the amount of the compound represented by general formula (1a) used is usually preferably 0.5 to 3 moles, more preferably 1 to 2 moles, per mole of the compound represented by general formula (1b).

[0080] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include dimethylformamide, dichloromethane, acetonitrile, tetrahydrofuran, acetone, toluene, etc., and preferably dimethylformamide, etc. The solvent may be used alone or in combination.

[0081] This reaction is usually carried out in the presence of a base. The base is not particularly limited, and for example, a wide variety of known bases can be used. Specific examples of the base include calcium hydroxide and diisopropylethylamine (DIPEA). The base may be used alone or in combination of two or more.

[0082] In addition to the above components, additives may also be used in this reaction as appropriate, provided that they do not significantly impair the progress of the reaction.

[0083] The reaction can be carried out under heating, at room temperature, or under cooling, and is usually preferably carried out at 10 to 80° C. The reaction time is not particularly limited, and can usually be 30 minutes to 30 hours.

[0084] The progress of the reaction can be monitored by conventional methods such as chromatography. After the reaction is complete, the solvent is removed by distillation, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. The structure of the product can also be determined by elemental analysis, MS (ESI-MS), IR analysis, and the like. 1 H-NMR, 13It can be identified by C-NMR or the like.

[0085] 3.Applications The compounds of the present invention can exhibit various medicinal effects (e.g., anticancer activity, antiviral activity, etc.) depending on the type of nucleoside analog moiety. Therefore, the compounds of the present invention can be used as active ingredients of medicines, reagents, etc. (sometimes referred to as "drugs of the present invention" in this specification), more specifically, as active ingredients of anticancer agents, antiviral agents, etc.

[0086] The pharmaceutical preparation of the present invention is not particularly limited as long as it contains the compound of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharmaceutically acceptable. The other components include not only components having pharmacological effects but also additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0087] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. Depending on the intended use, the agent of the present invention can be used, for example, in vitro (e.g., added to the medium of cultured cells) or in vivo (e.g., administered to animals).

[0088] The agents of the present invention may be applied to, but are not limited to, mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0089] When the agent of the present invention is used as an anticancer agent or when used on cancer cells, the target cancer is not particularly limited and includes, for example, hepatocellular carcinoma, pancreatic cancer, kidney cancer, leukemia, esophageal cancer, stomach cancer, colon cancer, lung cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, etc. Among these, solid cancers are preferred, and hepatocellular carcinoma is more preferred.

[0090] When the agent of the present invention is used as an antiviral agent, the target virus is not particularly limited. Examples of target viruses include influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, arbovirus, respiratory syncytial virus, SARS virus, hepatitis virus (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, and lyssavirus; and non-enveloped viruses (viruses without an envelope) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, poliovirus, and rhinovirus. Among these, AIDS virus and hepatitis B virus are preferred.

[0091] The agents of the present invention may take any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparations such as injectable preparations (for example, drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0092] The route of administration of the drug of the present invention is not particularly limited as long as the desired effect can be obtained, and examples include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0093] The content of the active ingredient in the drug of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0094] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is usually 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in terms of the weight of the active ingredient, in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc. [Example]

[0095] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0096] (1) Compound synthesis 1

[0097] [ka]

[0098] compound 1a Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and isopropylamine (822 μL, 9.65 mmol) diluted in dichloromethane (12.0 mL) was added dropwise with stirring at -65°C. After the addition was complete, the mixture was returned to room temperature and stirred for 3 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated. The resulting white solid was washed with hexane. Yield: 0.840 g, 4.77 mmol, 89%. 1 H NMR (400 MHz, CD3CN): δ 3.66 (m, 1H), 1.27 (d, J = 6 Hz, 6H) 13 C NMR (100 MHz, CDCl3): δ 46.1, 24.1 31 P NMR (162 MHz, CD3CN): δ 13.74.

[0099] compound 1b Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of dodecylamine (1.29 g, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (12.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 3 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered, dehydrated over anhydrous sodium sulfate, and the solvent was evaporated to give a white solid. Yield: 1.14 g, 3.64 mmol, 68%. 1 H NMR (400 MHz, CDCl3): δ 3.14-3.09 (m, 2H), 1.64-1.57 (m, 2H), 1.41-1.21 (m, 26H), 0.872 (t, J = 7.2 Hz, 3H) 13C NMR (100 MHz, CDCl3): δ 46.0, 42.6, 31.9, 30.3, 30.2, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 26.4, 22.7, 14.1, 8.62 31 P NMR (162 MHz, CDCl3): δ 16.2.

[0100] compound 1c Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of hexadecylamine (0.99 g, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (10.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 3 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give a pink oily substance. Yield: 0.795 g, 2.78 mmol, 52%. 1 H NMR (400 MHz, CDCl3): δ 3.11-3.04 (m, 2H), 1.63-1.54 (m, 2H), 1.40-1.21 (m, 18H), 0.872 (t, J = 6.8 Hz, 3H) 13 C NMR (100 MHz, CDCl3): δ 42.7, 32.0, 30.3, 30.2, 29.7, 29.6, 29.5, 29.4, 29.2, 26.5, 22.7, 14.2 31 P NMR (162 MHz, CDCl3): δ 16.9.

[0101] compound 1d Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (12.0 mL), and a solution of hexaneamine (642 μL, 4.82 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (12.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 4 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give a colorless oily substance. Yield: 1.07 g, 4.93 mmol, 92%. 1 H NMR (400 MHz, CDCl3): δ 3.11-3.04 (m, 2H), 1.63-1.55 (m, 2H), 1.37-1.24 (m, 6H), 0.877 (t, J = 6.8 Hz, 3H) 13 C NMR (100 MHz, CDCl3): δ 42.6, 31.2, 30.1, 30.0, 26.1, 22.4 31 P NMR (162 MHz, CDCl3): δ 17.0.

[0102] compound 1e Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of aniline (489 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (10.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 5 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give a white solid. Yield: 775 mg, 3.70 mmol, 69%. 1 H NMR (400 MHz, CDCl3): δ 7.72 (d, J = 11.6 Hz, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.28-7.24 (m, 2H), 7.21-7.17 (m, 1H) 13 C NMR (100 MHz, CDCl3): δ 136.5, 129.6, 125.0, 121.1, 121.0 31 P NMR (162 MHz, CDCl3): δ 9.74.

[0103] compound 1f Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of diethylamine (554 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (10.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 2.5 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give an orange oily substance. Yield: 1.05 g, quant. 1 H NMR (400 MHz, CDCl3): δ 3.28-3.19 (m, 4H), 1.18-1.12 (m, 6H) 13 C NMR (100 MHz, CDCl3): δ 40.7, 40.6, 13.2, 13.1 31 P NMR (162 MHz, CDCl3): δ 17.1.

[0104] 1g of compound Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of piperidine (529 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (10.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 2.5 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give a colorless oily substance. Yield: 1.02 g, quant. 1 H NMR (400 MHz, CDCl3): δ 3.20-3.18 (m, 4H), 1.54 (bs, 6H) 13 C NMR (100 MHz, CDCl3): δ 45.8, 25.0, 24.9, 23.6 31 P NMR (162 MHz, CDCl3): δ 16.1.

[0105] compound 1h Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and a solution of isopropyl alcohol (423 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (10.0 mL) was added dropwise with stirring at 0°C. After the addition was complete, the mixture was returned to room temperature and stirred for 9 hours, after which the solvent was evaporated and the mixture was separated into ethyl acetate and cold water. The organic phase was recovered and dehydrated over anhydrous sodium sulfate, after which the solvent was evaporated to give a colorless oily substance. Yield: 823 mg, 4.56 mmol, 85%. 1 H NMR (400 MHz, CDCl3): δ 4.99-4.90 (m, 1H), 1.42-1.40 (m, 6H) 13 C NMR (100 MHz, CDCl3) δ 79.4, 22.9 31 P NMR (162 MHz, CDCl3): δ 5.88.

[0106] Compound 2 (Common Experimental Section) Silver fluoride (2.1 eq.) was added to dichlorophosphoramide (1 eq.) dissolved in dehydrated acetonitrile (1.0 mL) and stirred at room temperature for 10 minutes. The solution was filtered through a PTFE syringe filter (13 mm, 0.22 μm), and the filtrate was added to the next reaction solution.

[0107] compound 3a Gemcitabine (100 mg, 0.38 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (140 μL, 1.15 mmol) and compound 2a (81 mg, 0.57 mmol) in anhydrous acetonitrile (1 mL) were added, followed by calcium hydroxide (70 mg, 0.95 mmol) and stirring for 1.5 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-25 min 0-70% (B), 25-30 min 100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 25 mg, 0.06 mmol, 17% yield. 1 H NMR (400 MHz, DMF-d6): δ 7.68 (m, 2H), 7.47 (m, 1H), 6.67 (t, J = 7.2 Hz, 1H), 6.32 (t, J = 16 Hz, 1H), 5.94 (d, J = 6 Hz, 1H), 5.78 (m. 1H), 4.40 (m, 3H), 4.17 (m, 1H), 1.15 (d, J = 6 Hz, 6H) 13 C NMR (100 MHz, DMF-d6): δ 166.4, 155.1, 141.4, 123.0, 95.0, 94.8, 78.6, 70.4, 65.5, 44.2, 25.0, 24.4 19 F NMR (376 MHz, DMF-d6): δ -71.8 (d, J PF = 976 Hz), -71.6 (d, J PF = 970 Hz), -116 31 P NMR (162 MHz, DMF-d6): δ 5.1 (d, J PF = 978 Hz), 5.3 (d, J PF = 974 Hz) HRMS (ESI) m / z calculated for C12 H 18 F3N4O5P [M+H] + :387.0967, found for:387.1074.

[0108] compound 3e Gemcitabine (61 mg, 0.23 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (100 μL, 0.58 mmol) and compound 2e (61.6 mg, 0.35 mmol) in anhydrous acetonitrile (1.5 mL) were added, followed by calcium hydroxide (34.4 mg, 0.46 mmol) and stirring for 1 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-5 min 0-10% (B), 5-35 min 10-60% (B), 35-40 min 60-100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 10 mg, 0.02 mmol, 10% yield. 1 H NMR (400 MHz, DMSO-d6): δ 9.00-8.96 (m, 1H), 7.43 (d, J = 7.6 Hz, 3H), 7.29-7.25 (m, 2H), 7.05 (d, J = 8.0 Hz, 2H), 7.02-6.98 (m, 1H), 6.49 (t, J = 7.6 Hz, 1H), 6.17 (t, J = 7.2 Hz, 1H), 5.71 (dd, J = 7.6, 2.4 Hz, 1H), 4.50-4.41 (m, 2H), 4.19 (bs, 1H), 4.08-4.02 (m, 1H) 13 C NMR (100 MHz, DMSO-d6): δ 165.6, 154.4, 141.1, 138.7, 129.4, 122.4, 118.2, 118.1, 118.0, 94.9, 94.8, 77.9, 69.7, 66.1, 66.0 19 F NMR (376 MHz, DMSO-d6): δ -70.4 (d, J PF = 981 Hz), -70.3 (d, J PF = 981 Hz), -116 31 P NMR (162 MHz, DMSO-d6): δ -1.00 (d, J PF = 983 Hz), -1.18 (d, J PF = 987 Hz) HRMS (ESI) m / z calculated for C 15 H16F3N4O5P [M+H] + 421.0889, found 421.0875.

[0109] compound 3f Gemcitabine (101.4 mg, 0.39 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (166 μL, 0.96 mmol) and compound 2f (90.7 mg, 0.58 mmol) in anhydrous acetonitrile (1.5 mL) were added, followed by calcium hydroxide (57.0 mg, 0.77 mmol) and stirring for 2.5 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-5 min 0-10% (B), 5-35 min 10-60% (B), 35-40 min 60-100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 3.3 mg, 0.003 mmol, yield 2%. 1H NMR (400 MHz, DMSO-d6): δ 7.69-7.66 (m, 1H), 7.45 (d, J= 6.0 Hz, 2H), 6.21 (bs, 1H),5.80 (d, J= 6.8 Hz, 1H), 5.31 (bs, 1H), 5.00 (bs, 1H), 4.14-4.11 (m, 1H), 3.80 (d, J = 12.8 Hz, 1H), 3.71-3.65 (m, 1H), 3.20-3.11 (m, 4H), 1.10-0.96 (m, 6H) 13 C NMR (100 MHz, DMSO-d6): Not obtained due to small amount. 19 F NMR (376 MHz, DMSO-d6): δ -72.6 (d, J PF = 999 Hz), -72.7 (d, J PF = 987 Hz), -114 31 P NMR (162 MHz, DMSO-d6): δ 5.12 (d, J PF = 991 Hz), 4.73 (d, J PF = 996 Hz) HRMS (ESI) m / z calculated for C 13 H 20 F3N4O5P [M+H] + 401.1202, found 401.1194.

[0110] 3g of compound Gemcitabine (64.1 mg, 0.24 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (105 μL, 0.61 mmol) and compound 2g (61.9 mg, 0.37 mmol) in anhydrous acetonitrile (1.5 mL) were added, followed by calcium hydroxide (36.2 mg, 0.49 mmol) and stirring for 1 h. The mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-5 min 0-10% (B), 5-35 min 10-60% (B), 35-40 min 60-100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 22.9 mg, 0.06 mmol, 23% yield. 1 H NMR (400 MHz, CD3CN): δ 7.60 (dd, J= 9.4, 7.6 Hz, 1H), 6.53 (bs, 1H), 6.26 (bs, 1H), 6.18 (bs, 1H), 5.86 (dd, J = 8.0, 2.0 Hz, 1H), 5.02 (bs, 1H), 4.14-4.11 (m, 1H), 3.93 (d, J = 12.8 Hz, 1H), 3.83-3.76 (m, 1H), 3.19 (bs, 4H), 1.62-1.50 (m, 6H) 13 C NMR (100 MHz, CD3CN): δ 166.4, 155.3, 117.4, 94.9, 79.2, 72.5, 72.3, 58.9, 58.8, 45.2, 25.5, 25.4, 23.7, 23.6 19 F NMR (376 MHz, CD3CN): δ -77.0 (d, J PF = 982 Hz), -77.5 (d, J PF = 982 Hz), -118 31 P NMR (162 MHz, CD3CN): δ 3.58 (d, J PF= 977 Hz), 3.04 (d, J PF = 986 Hz) HRMS (ESI) m / z calculated for C 14 H 20 F3N4O5P [M+H] + 413.1202, found 413.1192.

[0111] (2) Compound synthesis 2

[0112] [ka]

[0113] [ka]

[0114] compound 3h Gemcitabine (56.2 mg, 0.21 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (92.2 μL, 0.54 mmol) and compound 2h (46.2 mg, 0.32 mmol) in anhydrous acetonitrile (1.5 mL) were added, followed by calcium hydroxide (31.7 mg, 0.43 mmol) and stirring for 6 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-5 min 0-10% (B), 5-35 min 10-60% (B), 35-40 min 60-100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 36.7 mg, 0.09 mmol, 44% yield. 1H NMR (400 MHz, CD3CN): δ 7.47 (d, J = 7.6 Hz, 1H), 6.18 (t, J = 8.0 Hz, 1H), 5.91 (d, J = 7.6 Hz, 1H), 4.85-4.76 (m, 1H), 4.53-4.48 (m, 1H), 4.45-4.38 (m, 1H), 4.23-4.15 (m, 1H), 4.12-4.11 (m, 1H), 1.35-1.33 (m, 6H) 13 C NMR (100 MHz, CD3CN): δ 167.0, 156.9, 142.2, 96.5, 79.3, 78.3, 70.7, 70.4, 68.1, 68.0, 23.6 19 F NMR (376 MHz, CD3CN): δ -79.7 (d, J PF = 993 Hz), -80.0 (d, J PF = 993 Hz), -118 31 P NMR (162 MHz, CD3CN): δ -9.82 (d, J PF = 993 Hz), -9.89 (d, J PF = 995 Hz) HRMS (ESI) m / z calculated for C 12 H 17 F3N3O6P [M+H] + 388.0885, found 388.0823。

[0115] compound 4 Deoxycytidine (100 mg, 0.41 mmol) was dissolved in DMF (2 mL) and stirred. DIPEA (142 μL, 0.82 mmol) and compound 2a (117 mg, 0.82 mmol) in anhydrous acetonitrile (1 mL) were added, followed by calcium hydroxide (60 mg, 0.82 mmol) and stirring for 5 hours. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-25 min 0-70% (B), 25-30 min 100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 35.0 mg, 0.10 mmol, 25% yield. 1 H NMR (400 MHz, CD3CN): δ 7.67 (m, 1H), 6.11 (t, J = 7.1 Hz, 2H), 5.92 (m, 1H), 4.33 (s, 1H), 4.00 (m, 1H), 3.31 (m, 1H), 2.32 (m, 2H), 1.94 (s, 1H), 1.09 (d, J = 6.0 Hz, 6H) 13 C NMR (100 MHz, CD3CN): δ 166.7, 155.1, 141.4, 95.1, 86.0, 84.2, 70.1, 66.4, 44.0, 39.5, 24.3, 23.8 19 F NMR (376 MHz, CD3CN): δ -75.2 (d, J PF = 976 Hz), -76.1 (d, J PF = 981 Hz) 31 P NMR (162 MHz, CD3CN): δ 3.2 (d, J PF = 979 Hz), 3.4 (d, J PF = 976 Hz) HRMS (ESI) m / z calculated for C 12 H 18F3N4O5P [M+H] + :351.1234, found for:351.1356.

[0116] compound 5 Lamivudine (100 mg, 0.44 mmol) was dissolved in DMF (1.5 mL) and stirred. DIPEA (142 μL, 1.48 mmol) and compound 2a (71.3 mg, 0.50 mmol) in anhydrous acetonitrile (1 mL) were added, followed by calcium hydroxide (60 mg, 0.82 mmol) and stirring for 2 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-25 min 0-70% (B), 25-30 min 100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 35.6 mg, 0.10 mmol, 23% yield. 1 H NMR (400 MHz, CD3CN): δ 7.75 (m, 1H), 6.24 (m, 1H), 5.91 (dd, J = 23.7, 7.5 Hz, 1H), 5.35 (m, 1H), 4.34 (m, 2H), 3.48 (m, 1H), 3.29 (m, 1H), 3.11 (m, 1H), 1.20(m, 6H) 13 C NMR (100 MHz, CD3CN): δ 166.3, 156.4, 141.4, 95.3, 87.9, 82.7, 67.8, 44.6, 36.8, 24.3, 24.2 19 F NMR (376 MHz, CD3CN): δ -72.4 (d, J PF = 970 Hz), -72.8 (d, J PF = 981 Hz) 31 P NMR (162 MHz, CD3CN): δ 4.9 (d, J PF= 974 Hz), 4.7 (d, J PF = 978 Hz) HRMS (ESI) m / z calculated for C 11 H 18 FN4O4PS [M+H] + :353.0770, found for:353.0380.

[0117] compound 6 Entecavir monohydrate (85 mg, 0.29 mmol) was azeotroped three times with pyridine. This was dissolved in DMF (5 mL) and stirred. DIPEA (97 μL, 0.79 mmol) and compound 2a (45.7 mg, 0.32 mmol) in anhydrous acetonitrile (1 mL) were added, followed by calcium hydroxide (60 mg, 0.82 mmol) and stirring for 5 h. After the reaction, the mixture was filtered and purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), solvent: A) MQ, B) acetonitrile, gradient: 0-25 min 0-70% (B), 25-30 min 100% (B), flow rate: 8 mL / min, detection: 250 nm) to obtain a white solid. Yield: 45.2 mg, 0.11 mmol, 39% yield. 1 H NMR (400 MHz, CD3CN): δ 7.65 (d, J = 14.1 Hz, 1H), 5.38 (t, J = 8.6 Hz, 1H), 5.26 (d, J = 2.5 Hz, 1H), 4.86 (s, 1H), 4.30 (m, 3H), 3.37 (m, 1H), 2.86 (m, 1H), 2.30 (m, 2H), 1.13 (m, 6H) 13 C NMR (100 MHz, CD3CN): δ 158.4, 153.5, 151.7, 148.0, 138.1, 116.7, 112.3, 71.0, 67.7, 56.0, 51.5, 44.5, 38.5, 24.3, 24.2 19F NMR (376 MHz, CD3CN): δ -72.6 (d, J PF = 970 Hz), -72.9 (d, J PF = 975 Hz) 31 P NMR (162 MHz, CD3CN): δ 45.4 (d, J PF = 974 Hz) HRMS (ESI) m / z calculated for C 15 H 22 FN6O4P [M+H] + : 401.1502, found for:401.1604.

[0118] (3) Stability evaluation test A 0.2-0.5 mg aliquot of the prodrug compounds (compounds 3a, 3e, 3f, 3h, 4, 5, and 6) used for stability evaluation was dispensed onto a spatula and dissolved in acetonitrile (10 μL). PBS buffer (pH 7.3) or Milli-Q water was added to prepare a test solution with a total volume of 200 μL. The test solution was prepared at 0 min, and incubated at 37°C. The test solution was analyzed by RP-HPLC at each time point. The analysis revealed that the prodrugs were converted not only into hydrolyzed products in which the fluorine atom was removed by water molecules, but also into cyclized products in which the 3'-hydroxyl group reacted with the phosphorus atom (Figure 1).

[0119] The results are shown in Figures 2 to 4. The prodrug compound was found to be relatively stable in Milli-Q water. On the other hand, it was found to rapidly convert to a hydrolyzed form (Figure 1) in PBS buffer. This difference in stability is thought to be due to increased reactivity caused by the interaction of ions such as sodium contained in the buffer with the phosphorus-oxygen double bond.

[0120] Of the prodrug compounds tested, the aniline derivative (3e) was found to be the most susceptible to decomposition. Furthermore, the secondary amines, diethylamine derivative (3f) and piperidine derivative (3g), were found to be completely stable in PBS buffer, showing no reaction. This difference in stability is thought to be due to the bulkiness of the phosphorus center that is susceptible to nucleophilic attack. In other words, the secondary amines, diethylamine derivative and piperidine derivative, are bulkier than primary amines, preventing water molecules from nucleophilically attacking the phosphorus atom. On the other hand, the aniline derivative has a planar structure, which means it has less steric hindrance and is therefore more susceptible to hydrolysis.

[0121] In vivo, the hydrolysate is thought to be converted to the monophosphate form by enzymatic degradation by HINT-1 or the like, resulting in the removal of the amine moiety (Figure 8).

[0122] Since secondary amines were stable even in PBS buffer, it is believed that the timing of conversion to hydrolysates and monophosphates can be delayed by using secondary amines. Furthermore, if a prodrug compound has an enzyme targeting structure attached to a secondary amine, it is thought that it will be specifically decomposed and converted to a primary amine at the site where the enzyme is present, and then converted to a hydrolysate and monophosphate in a site-specific manner, leading to the expression of pharmacological effects (Figure 9).

[0123] (4) Toxicity evaluation test The CellTiter 96® AQueous One Solution Cell Proliferation Assay protocol was followed. 48 hours after addition of the prodrug compound (Compound 4), CellTiter 96® AQueous One Solution Reagent (Promega) was added, and the cells were incubated at 37°C and 5% CO2 for 2 hours. Absorbance was measured at 490 nm using a plate reader.

[0124] The results are shown in Figure 5. The prodrug structures of the present invention were found to be non-toxic.

[0125] (5) Detection of intracellular conversion to monophosphate Gemcitabine prodrug (compound 3a) (DMSO, for negative control experiments) was added to the cell culture medium. After 6 hours of incubation, the contents of the wells were transferred to an Eppendorf tube and the cells were disrupted using a homogenizer. An equal volume of 20% trichloroacetic acid solution was added to the cell lysate under ice cooling, and the mixture was left to stand for 30 minutes. The mixture was then centrifuged (4°C, 13,000-15,000G, 5-10 minutes). The supernatant was then transferred to another tube, and the residue (pellet) was washed with a small amount of 50% acetone. After centrifugation (4°C, 13,000-15,000G, 5-10 minutes), the supernatant was added to the above solution. The combined supernatant was then centrifuged again (4°C, 13,000-15,000G, 5-10 minutes), the organic solvent was removed, and the remaining aqueous solution was lyophilized. The resulting extract was analyzed by LC / MS under the following conditions.

[0126] [Table 1]

[0127] The results are shown in Figures 6 and 7. It was found that the prodrug of the present invention was converted into the monophosphate form within the cells.

[0128] (6) Compound synthesis 3 Compounds 3B and 3C are prodrugs activated by CYP450 (Figure 10).

[0129] [ka]

[0130] Compound 1A To a stirred solution of phosphoryl chloride (500 μL, 5.36 mmol) in dry CHCl (10.0 mL) was added dropwise isopropylamine (822 μL, 9.65 mmol) and triethylamine (742 μL, 5.36 mmol) diluted in CHCl (12.0 mL) at −65 °C, and the reaction mixture was stirred at rt for 2 h. The volatiles were removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give the crude compound (0.840 g, 4.77 mmol, 89%) as a white solid without further purification. 1 H-NMR (400 MHz, CD3CN): δ 3.66 (m, 1H), 1.27 (d, J = 6 Hz, 6H) 13 C-NMR (100 MHz, CDCl3): δ 46.1, 24.1 31 P-NMR (162 MHz, CD3CN): δ 13.74.

[0131] Compound 1B Phosphoryl chloride (258 μL, 2.76 mmol), N,N-methylbenzylamine (319 μL, 2.48 mmol), and triethylamine (384.6 μL, 2.76 mmol) in dry CHCl (12.0 mL) were added dropwise to a stirred solution diluted with CHCl (12.0 mL) at 0 °C, and the reaction mixture was stirred at rt for 3 h. The volatiles were removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give the crude compound (512.9 mg, 2.50 mmol, 91%) as a yellow oil, which was used in the next step without further purification. 1 H-NMR (400 MHz, CDCl3): δ 7.40-7.13 (m, 5H), δ 4.42 (d, J = 8 Hz, 2H), δ 2.72 (d, J = 16 Hz, 3H). 13C-NMR (100 MHz, CDCl3): δ 135.22, 128.96, 128.42, 128.34, 53.40, 33.71 31 P-NMR (162 MHz, CDCl3): δ 19.32.

[0132] Compound 1C To a stirred solution of phosphoryl chloride (205.7 μL, 2.2 mmol) in dry CHCl (15.0 mL) was added dropwise 4-methoxy-N-methylbenzylamine (297 μL, 1.98 mmol) diluted with CHCl (15.0 mL) at 0 °C, and the reaction mixture was stirred at rt for 3 h. The volatiles were removed in vacuo, and the residue was partitioned with ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give the crude compound (448 mg, 1.88 mmol, 95%) as a yellow oil, which was used in the next step without further purification. 1 H-NMR (400 MHz, CD3CN): δ 7.25 (d, J = 8 Hz, 2H), δ 6.90 (d, J = 8 Hz, 2H), δ 4.32 (s, 2H), δ 3.81 (s, 3H), δ 3.81 (s, 3H), δ 2.72 (d, J = 16.4Hz, 3H). 13 C-NMR (100 MHz, CDCl3): δ 159.56, 129.76, 126.98, 114.15, 55.12, 52.59, 33.24 31 P-NMR (162 MHz, CD3CN): δ 19.14.

[0133] Compound 1D To a stirred solution of phosphoryl chloride (500 μL, 5.36 mmol) in dry CHCl (12.0 mL) was added dropwise N,N-methylisopropylamine (560 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) diluted in CHCl (12.0 mL) at 0 °C, and the reaction mixture was stirred at rt for 3 h. The volatiles were removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give a clear, oily crude oil (760.0 mg, 4.02 mmol, 75%), which was used in the next step without further purification. 1 H-NMR (400 MHz, CD3CN): δ 4.21 (m, 1H), δ 2.72 (d, J = 16 Hz, 3H), δ 1.20 (d, J = 7.2 Hz, 6H). 13 C-NMR (100 MHz, CDCl3): δ 48.44, 26.43, 19.23 31 P-NMR (162 MHz, CD3CN): δ 17.69.

[0134] Compound 1E To a stirred solution of phosphoryl chloride (523.6 μL, 5.60 mmol) in dry CHCl (12.0 mL) was added dropwise dimethylbutylamine (505.9 mg, 5 mmol) and triethylamine (776.3 μL, 5.60 mmol) diluted in CHCl (12.0 mL) at −40 °C, and the reaction mixture was stirred at rt for 3 h. The volatiles were removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give a white solid (1015.9 mg, 4.66 mmol, 83.2%), which was used in the next step without further purification. 1H-NMR (400 MHz, CDCl3):δ 3.56 (1H, m,), 1.76 (1H, m,), 1.43(2H, t,), 1.26 (3H, d), 0.94 (6H, d) 13 C-NMR (100 MHz, CDCl3): δ 48.32, 47.55, 24.71, 22.56, 22.43 31 P-NMR (162 MHz, CDCl3): δ 13.65, 13.71 Compound 1F To a stirred solution of phosphoryl chloride (523.6 μL, 5.60 mmol) in dry CHCl (12.0 mL) was added dropwise adamantineamine (756.3 mg, 5 mmol) and triethylamine (776.3 μL, 5.60 mmol) diluted in CHCl (12.0 mL) at −40 °C, and the reaction mixture was stirred at rt for 3 h. The volatiles were removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over NaSO and evaporated to give a white solid (997.6 mg, 3.72 mmol, 66.4%), which was used in the next step without further purification. 1 H-NMR (400 MHz, CD3CN): δ 1.96 (3H, m), 1.92 (6H, m), 1.64 (6H, t, J = 14 Hz) 13 C-NMR (100 MHz, CDCl3): δ 56.99, 44.13, 44.07, 35.82, 29.86 31 P-NMR (162 MHz, CD3CN): δ 8.50.

[0135] Compounds 2A-F (General procedure) To a stirred solution of phosphoramidodichloride (1 eq.) in dry acetonitrile, AgF (2.1 eq.) was added at rt, and the reaction mixture was stirred for 10 min. The reaction mixture was filtered through a 0.22 μm membrane filter, and the filtrate was added to the reaction mixture for the next step.

[0136] Compound 3A To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (100 mg, 0.38 mmol) in dry DMF (1 mL) was added DIPEA (140 μL, 1.15 mmol) and compound 2A (81 mg, 0.57 mmol), followed by Ca(OH) (70 mg, 0.95 mmol). The reaction mixture was stirred at RT for 1.5 h, filtered through a 0.22 μm membrane filter, and the filtrate was purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQB) ACN, gradient: 0-25 min 0-70% (B), 25-35 min 100% (B), flow rate 8 mL / min, detection at 250 nm) to give compound 3A (25 mg, 0.06 mmol, 17%) as a white solid. 1 H-NMR (400 MHz, DMF-d6): δ 7.68 (m, 2H), 7.47 (m, 1H), 6.67 (t, J= 7.2 Hz, 1H), 6.32 (t, J = 16 Hz, 1H), 5.94 (d, J =6 Hz, 1H), 5.78 (m. 1H), 4.40 (m, 3H), 4.17 (m, 1H), 1.15 (d, J = 6 Hz, 6H) 13 C-NMR (100 MHz, DMF-d6): δ 166.4, 155.1, 141.4, 123.0, 95.0, 94.8, 78.6, 70.4, 65.5, 44.2, 25.0, 24.4 19 F-NMR (376 MHz, DMF-d6): δ -71.8 (d, J = 976 Hz), -71.6 (d, J = 970 Hz), -116 31 P-NMR (162 MHz, DMF-d6): δ 5.1 (d, J = 978 Hz), 5.3 (d, J = 974 Hz) HRMS (ESI) m / z calculated for C 12 H 18 F3N4O5P [M+H] + :387.0967, found for:387.1074.

[0137] Compound 3B To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (22.9 mg, 0.087 mmol) in dry DMF (1 mL) was added DIPEA (37 μL, 0.26 mmol) and compound 2B (53.5 mg, 0.26 mmol) in dry acetonitrile (1 mL), followed by the addition of (CFSO)Ca (55 mg, 0.17 mmol). The reaction mixture was stirred at rt for 2 h, filtered through a 0.22 μm membrane filter, and the filtrate was purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQB) ACN, gradient: 0-5 min 0-50% (B), 5-24 min 50-80% (B), 24-25 min 80-100% (B), flow rate 8 mL / min, detection: 250 nm) to give the desired compound 3B (15.4 mg, 0.03 mmol, 40%) as a white solid. 1 H-NMR (400 MHz, CD3OD):δ 7.81 (1H, m), 7.34 (5H, m), 6.27 (1H, t), 5.91 (1H, dd, J = 7.6 Hz, J = 2.4 Hz), 5.07 (1H, m,), 4.27 (2H, d,), 4.18 (1H, t,) , 3.97 (1H, t), 3.82 (1H, m) , 2.63 (3H, m) 13 C-NMR (100MHz, CD3CN): δ 166.4, 156.3, 141.5, 136.7, 129.0, 128.5, 128.4, 128.2, 118.3, 95.9, 79.3, 73.4, 58.7, 58.6, 52.7, 32.7, 32.6 19F-NMR (376MHz, CD3CN): δ -77.3 (d, J = 987.0 Hz), -76.9 (d, J = 999.0 Hz), -116 31 P-NMR (162MHz, CD3CN): δ 5.1(d, J = 1018 Hz), 4.5 (d, J = 1035 Hz), HRMS (ESI+) calculation for C 17 H 20 F3N4O5P [M+Na] + :471.1123, found for: 471.1195.

[0138] Compound 3C To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (45.8 mg, 0.174 mmol) in dry DMF (1 mL), DIPEA (74 μL, 0.52 mmol) and compound 2C (122.3 mg, 0.52 mmol) in dry acetonitrile (1 mL) were added (CFSO)Ca (58.9 mg, 0.17 mmol). The reaction mixture was stirred overnight at RT, filtered through a 0.22 μm membrane filter, and the filtrate was purified by RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQB) ACN, gradient: 0-5 min 0-50% (B), 5-24 min 50-80% (B), 24-25 min 80-100% (B). Purification at a flow rate of 8 mL / min (detection at 250 nm) gave the desired compound 3C (9.2 mg, 0.02 mmol, 11%) as a white solid. 1 H-NMR (400 MHz, CD3CN): δ 7.65 (1H, d, J = 7.6 Hz ), 7.23 (2H, d, J = 6.8 Hz ), 6.91 (2H, m), 6.20 (1H, m), 5.94 (1H, dd, J = 7.6 Hz, J = 2.4 Hz), 4.14 (2H, m,), 3.90 (1H, m,), 3.75 (4H, m,), 2.57 (3H, m), 1.95 (3H, m) 13 C-NMR (100MHz, CD3CN): δ 166.44, 159.50, 156.26, 130.00, 129.94, 128.68, 114.28, 95.94, 79.27, 58.68, 58.57, 55.29, 52.08, 32.54, 32.40 19 F-NMR (376MHz, CD3CN): δ -74.9 (d, J = 988.0 Hz), -77.48 (d, J = 977 Hz), -116 31 P-NMR (162MHz, CD3CN): δ 7.9 (d, J = 977 Hz), δ 1.9 (d, J = 990 Hz), HRMS (ESI + ) calcd for C 18 H 22 F3N4O6P [M+H] + :479.1129, found for: 479.1118.

[0139] compound 3d To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (22.9 mg, 0.087 mmol) in dry DMF (1 mL) was added DIPEA (37 μL, 0.26 mmol) and compound 2D (53.5 mg, 0.26 mmol) in dry acetonitrile (1 mL), followed by the addition of (CFSO)Ca (55 mg, 0.17 mmol). The reaction mixture was stirred at room temperature for 2 h, filtered through a 0.22 μm membrane filter, and the filtrate was purified by column chromatography on amino silica gel (DCM / MeOH = 5 / 1) to give the desired compound 3D (5.5 mg, 0.02 mmol, 23%) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ 7.57 (1H, m), 6.24 (1H, s), 5.90 (1H, d, J = 8.0 Hz) , 5.41 (1H, t,) , 4.07 (1H, s,) , 4.05 (1H, m,) 3.82(1H, m,), 3.81 (1H, m,), 2.63 (1H, m), 1.93 (3H, s) , 1.18 (6H, d) 13 C-NMR (100MHz, DMSO-d6): δ 165.8, 154.5, 141.2, 121.5, 95.0, 78.8, 58.8, 48.6, 47.6, 26.3, 19.8 19 F-NMR (376MHz, DMSO-d6): δ -77.2 (d, J = 999.0 Hz), -76.9 (d, J = 987.0 Hz), -116 31 P-NMR (162MHz, DMSO-d6): δ 7.6(d, J = 991 Hz), δ1.45(d, J = 1000 Hz), HRMS (ESI + ) calcd for C 13 H 20 F3N4O5P [M+H] + :401.1123, found for: 401.1165。

[0140] Compound 3E To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (313 mg, 1.19 mmol) in dry DMF (3 mL) was added DIPEA (508 μL, 3.57 mmol) and compound 2E (660.7 mg, 3.57 mmol) in dry acetonitrile (4 mL), followed by (CFSO)Ca (805.0 mg, 2.38 mmol). The reaction mixture was stirred overnight at RT, filtered through a 0.22 μm membrane filter, and the filtrate was purified by RP-HPLC (YMC Hydrosphere C18 (250 × 20 mm), Elute A) MQB) ACN, gradient: 0-5 min 0-50% (B), 5-24 min 50-80% (B), 24-25 min 80-100% (B). Purification at a flow rate of 8 mL / min, detection at 250 nm gave the desired compound 3E (13 mg, 0.03 mmol, 3%) as a white solid. 1 H-NMR (400 MHz, DMSO-d6):δ7.53 (1H, d, J =6.8 Hz), 7.43(1H, s), 6.49 (1H, s), 6.17 (1H, s), 5.79 (2H, s), 4.23 (3H, s,), 3.82(1H, m,), 3.19 (1H, s,), 1.65(1H, m,), 1.30(1H, m,), 1.16 (2H, t), 1.07 (3H, d), 0.839 (6H, d), 13 C-NMR (100MHZ, DMSO-d6): δ 165.64, 154.54, 140.85, 122.71, 94.77, 78.41, 63.83, 52.48, 45.21, 24.29, 23.59, 22.65. 19 F-NMR (376MHz, DMSO-d6): δ -68.6 (dq, J =959.6 Hz), -70.6 (dq, J = 963.5 Hz), -116 31 P-NMR (162MHz, DMSO-d6): δ 8.54 (d, J = 964 Hz), 2.59(d, J = 964 Hz), HRMS (ESI + ) calcd for C 15 H 24 F3N4O5P [M+H] + : 429.1436, found for: 429.1581.

[0141] Compound 3F To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (176.3 mg, 0.67 mmol) in dry DMF (3 mL) was added DIPEA (118 μL, 0.83 mmol) and compound 2F (439.6 mg, 1.87 mmol) in dry acetonitrile (5 mL), followed by Ca(OH) (145.9 mg, 1.87 mmol). The reaction mixture was stirred overnight at room temperature, filtered through a 0.22 μm membrane filter, and the filtrate was purified by column chromatography on silica gel (DCM / MeOH = 100 / 2 to 100 / 8) to give the desired compound 3F (5.66 mg, 0.01 mmol, 2%) as a white solid. 1 H-NMR (400 MHz, DMSO-d6): δ7.59 (1H, d, J = 7.6 Hz), 7.45 (1H, s), 6.17 (2H, s), 5.75 (1H, d, J =7.2 Hz), 5.15 (1H, s,), 4.33 (3H, br,), 4.06 (1H, s,), 3.13 (1H, s,), 1.94 (2H, d), 1.75 (4H, d), 1.72 (3H, m), 1.58 (6H, t, J = 14 Hz) 13 C-NMR (100MHz, DMSO-d6): δ 165.04, 164.86, 141.3, 94.88, 79.18, 79.08, 56.03, 51.13, 50.74, 48.30, 43.46, 43.32, 35.62, 35.52, 29.04, 18.56. 19F-NMR (376MHz, DMSO-d6): δ -60.95 (d, J = 987 Hz), -62.38 (d, J = 999 Hz), -113 31 P-NMR (162MHz, DMSO-d6): δ 6.45 (d, J = 982 Hz), 0.33 (d, J = 1000 Hz) HRMS (ESI + ) calcd for C 19 H 26 F3N4O5P (M+H) + :479.1593, found for:479.1546.

[0142] (7) Compound synthesis 4 Compounds 15 and 25 are nitroreductase-activated prodrugs (Figure 10).

[0143] [ka]

[0144] [ka]

[0145] compound 12 To a solution of p-nitrobenzaldehyde (5.00 g, 33.0 mmol) in dry ethanol (100 mL) was added isopropylamine (11.7 mL, 198 mmol) under an argon atmosphere. The solution was stirred at room temperature for 20 h, then cooled to 0 °C. Sodium borohydride (5.00 g, 165 mmol) was added and stirred for 27 h. The reaction mixture was evaporated in vacuo, and the residue was dissolved in dichloromethane. The organic phase was washed successively with saturated solutions of NaHCO3, water, and brine. The organic phase was dried over Na2SO4. The filtrate was evaporated in vacuo and purified by column chromatography on silica gel (hexane / ethyl acetate / triethylamine = 133 / 66 / 1) to give the desired compound 12 (5.75 g, 29.6 mmol, 89%) as an orange oil. 1 H-NMR (400 MHz, Chloroform-d1): δ 8.18-8.14 (m, 2H), 7.51-7.49 (m, 2H), 3.88 (s, 2H), 2.85 (sep, 1H), 1.09 (d, J = 6.4 Hz, 6H). HRMS (ESI) m / z calculated for C 10 H 14 N2O2, [M+H] + : 195.1128, found for 195.1199.

[0146] compound 13 To a stirred solution of phosphoryl chloride (2.90 mL, 31.0 mmol) in dry CHCl (20.0 mL) was added compound 12 (1.00 g, 5.15 mmol) and triethylamine (4.34 mL, 30.9 mmol) in dichloromethane (20.0 mL) dropwise at 0 °C, and the reaction mixture was stirred at room temperature under an argon atmosphere for 21 h. The reaction mixture was evaporated in vacuo, and the residue was partitioned between ethyl acetate and brine. The organic phase was dried over NaSO. The filtrate was evaporated in vacuo to give the crude desired compound 13 (1.51 g, 4.85 mmol, 94%) as an orange oil without further purification. 1 H-NMR (400 MHz, Chloroform-d1): δ 8.23-8.20 (m, 2H), 7.56-7.53 (m, 2H), 4.52 (s, 2H), 2.85 (m, 1H), 1.19 (d, J = 6.8 Hz, 6H). 31 P-NMR (162 MHz, Chloroform-d1): δ 18.7. HRMS (ESI) m / z calculated for C 10 H 13 Cl2N2O3P, [M+Na] + : 332.9933, [M+K] + : 348.9673, found for [M+Na] + : 332.9938, [M+K] + : 348.9676.

[0147] compound 14 To a stirred solution of compound 13 (1 eq.) in dry acetonitrile (3.00 mL) was added silver fluoride (4 eq.) at room temperature under an argon atmosphere. The reaction mixture was stirred for 60 minutes. The reaction mixture was filtered using a 0.22 μm membrane filter. 19 F NMR and 31 After confirmation by P NMR, the filtrate was added to the reaction mixture for the next step. 19 F-NMR (376 MHz, Chloroform-d1): δ -75.0 (d, JPF = 1033 MHz). 31 P-NMR (162 MHz, Chloroform-d1): δ -2.97 (t, JPF = 1017 MHz).

[0148] compound 15 To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (248 mg, 0.940 mmol) in dry DMF (2.00 mL) was added DIPEA (0.975 mL, 5.59 mmol) and compound 14 (2 eq.) in dry acetonitrile (3.00 mL), followed by calcium hydroxide (139 mg, 1.86 mmol). The reaction mixture was stirred at room temperature under an argon atmosphere for 22 hours. The mixture was filtered through a 0.22 μm membrane filter. The filtrate was evaporated in vacuo and purified by column chromatography on NH2-modified silica gel (dichloromethane / methanol = 10 / 1). The crude product was purified by RP-HPLC (YMC Triart C18 Hydrosphere (250 × 10.0 mmL.D., S-5 μm, 12 nm), Elute A) MQ, B) acetonitrile, Gradient: 0–25 min 20–80% (B), Flow rate: 3 mL / min, Detection: 250 nm). The collected fractions were lyophilized to give the desired compound 15 (49.9 mg, 94.0 μmol, 9.6%, diastereomeric mixture) as a white solid. 1 H-NMR (400 MHz, Methanol-d4): δ 8.21 (d, J = 12 Hz, 2H), 8.20 (d, J = 12 Hz, 2H), 7.78 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 6.26 (br s, 2H), 5.92 (s, 1H), 5.90 (s, 1H), 5.21-5.09 (br, 2H), 4.47 (s, 2H), 4.44 (s, 2H), 4.29-4.11 (m, 2H), 4.03-3.92 (m, 2H), 3.90-3.64 (m, 4H), 1.19-1.16 (m, 12H). 13C-NMR (100 MHz, Methanol-d4): δ 166.4, 156.3, 147.4, 146.7, 141.2, 128.2, 123.4, 121.3, 95.4, 84.8, 79.6, 73.0, 58.5, 47.9, 47.5, 20.3. 19 F-NMR (376 MHz, Methanol-d4): δ -72.1 (d, JPF = 999 MHz), -72.6 (d, JPF = 1010 MHz), -116. 31 P-NMR (162 MHz, Methanol-d4): δ 4.88 (d, JPF = 1000 MHz), 4.49 (d, JPF = 1010 MHz). HRMS (ESI) m / z calculated for C 19 H 23 F3N5O7P, [M+H] + : 522.1360, [M+Na] + : 544.1179, [M+K] + : 560.0919, found for [M+H] + : 522.1345, [M+Na] + : 544.1168, [M+K] + : 560.0904.。

[0149] compound 22 To a solution of 4-fluoro-2-nitrobenzaldehyde (1.00 g, 5.91 mmol) in dry ethanol (20.0 mL) was added isopropylamine (3.04 mL, 35.4 mmol) under an argon atmosphere. The solution was stirred at room temperature for 19 h, then cooled to 0 °C. Anhydrous sodium borate (815 mg, 21.6 mmol) was added and stirred at room temperature for 8 h. The reaction mixture was evaporated in vacuo, and the residue was dissolved in dichloromethane. The organic phase was washed successively with saturated solutions of NaHCO3, water, and brine. The organic phase was dried over Na2SO4. The filtrate was evaporated in vacuo and purified by column chromatography on silica gel (hexane / ethyl acetate / triethylamine = 133 / 66 / 1) to give the desired compound 22 (1.07 g, 5.08 mmol, 86%) as an orange oil. 1 H-NMR (400 MHz, Chloroform-d1): δ 7.66 (d, J = 2.8 Hz, 1H), 7.64 (d, J = 2.8 Hz, 1H), 7.31-7.26 (m, 1H), 3.97 (s, 2H), 2.82 (sep, J = 6.4 Hz, 1H), 1.08 (d, J = 6.4 Hz, 6H). 13 C-NMR (100 MHz, Chloroform-d1): δ162.3, 159.8, 133.1, 132.4, 120.4, 112.3, 48.7, 48.0, 23.0. 19 F-NMR (376 MHz, Chloroform-d1): δ -112.1. HRMS (ESI) m / z calculated for C 10 H 13 FN2O2, [M+H] + : 213.1034, found for [M+H] + : 213.1039.

[0150] compound 23 To a stirred solution of phosphoryl chloride (2.63 mL, 28.2 mmol) in dry CHCl (10.0 mL) was added compound 22 (1.00 g, 4.71 mmol) and triethylamine (3.93 mL, 28.2 mmol) in dichloromethane (10.0 mL) dropwise at 0 °C, and the reaction mixture was stirred at room temperature under an argon atmosphere for 23 h. The mixture was evaporated, and the residue was partitioned between ethyl acetate and brine. The organic layer was dried over NaSO. The filtrate was evaporated in vacuo to give the crude desired compound 19 (1.54 g, 4.66 mmol, 99%) as an orange solid without further purification. 1 H-NMR (400 MHz, Chloroform-d1): δ 7.78-7.72 (m, 2H), 7.40-7.35 (m, 1H), 4.75 (s, 2H), 4.13-3.99 (m, 1H), 1.19 (d, J = 6.8 Hz, 6H). 19 F-NMR (376 MHz, Chloroform-d1): δ -111.0. 31 P-NMR (162 MHz, Chloroform-d1): δ 19.0. HRMS (ESI) m / z calculated for C 10 H 12 FCl2N2O3P, [M+Na] + : 350.9839, [M+K] + : 366.9579, found for [M+Na] + : 350.9839, [M+K] + : 366.9578.

[0151] compound 24 To a stirred solution of compound 23 (700 mg, 2.13 mmol) in dry acetonitrile (3.00 mL) was added silver fluoride (403 mg, 3.18 mmol) at room temperature under an argon atmosphere. The reaction mixture was stirred for 60 minutes. The reaction mixture was filtered using a 0.22 μm membrane filter. 19 F NMR and31 After confirmation by P NMR, the filtrate was added to the reaction mixture for the next step. 19 F-NMR (376 MHz, Chloroform-d1): δ -74.8 (d, JPF = 1034 MHz), -111.5. 31 P-NMR (162 MHz, Chloroform-d1): δ -3.10 (t, JPF = 1041 MHz). compound 25 To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (279 mg, 1.06 mmol) in dry DMF (2.00 mL) was added DIPEA (0.554 mL, 3.18 mmol) and compound 24 (2 eq.) in dry acetonitrile (3.00 mL), followed by calcium hydroxide (236 mg, 3.18 mmol). The reaction mixture was stirred at room temperature under an argon atmosphere for 22 hours. The mixture was filtered through a 0.22 μm membrane filter. The filtrate was evaporated in vacuo and purified by column chromatography on NH2-modified silica gel (dichloromethane / methanol = 10 / 1). The crude product was purified by RP-HPLC (YMC Triart C18 Hydrosphere (250 × 20.0 mm L.D., S-5 μm, 8 nm), Elute A) MQ, B) acetonitrile, Gradient: 0–25 min 20–80% (B), Flow rate: 10 mL / min, Detection: 250 nm). The collected fractions were lyophilized to give the desired compound 25 (82.1 mg, 0.148 mmol, 14%, diastereomeric mixture) as a white solid. 1H-NMR (400 MHz, Acetonitrile-d3): δ 7.81-7.73 (m, 4H), 7.62-7.52 (m, 2H), 7.50-7.44 (m, 2H), 7.11 (br, 2H), 6.55 (br, 2H), 6.15 (br. 2H), 5.87 (d, J = 8.0, 1H), 5.86 (d, J = 8.0, 1H), 5.14 (br, 2H), 4.66-4.54 (m, 4H), 4.14-4.10 (m, 2H), 3.94-3.87 (m, 2H), 3.79-3.71(m, 2H), 3.69-3.58 (m, 2H), 1.19-1.09 (m, 12H). 13 C-NMR (100 MHz, Acetonitrile-d3): δ 166.4, 162.4, 159.8, 155.4, 148.3, 141.6, 131.0, 124.3, 120.9, 119.0, 112.5, 95.3, 79.3, 72.9, 58.8, 50.4, 43.9, 20.3. 19 F-NMR (376 MHz, Acetonitrile-d3): δ -71.0 (d, JPF = 1004 MHz), -71.2 (d, JPF = 1010 MHz), -114.1, -115.8. 31 P-NMR (162 MHz, Acetonitrile-d3): δ 4.89 (d, JPF = 1004 MHz), 4.76 (d, JPF = 1013 MHz). HRMS (ESI) m / z calculated for C 19 H 22 F4N5O7P, [M+H] + : 540.1266, [M+Na] + : 562.1085, [M+K] + : 578.0825, [M+Et3NH] + : 641.2470, found for [M+H] + : 540.1265, [M+Na] +: 562.1086, [M+K] + : 578.0824, [M+Et3NH] + : 641.2467.

[0152] (10) Anticancer activity evaluation test This experiment was performed using an MTT assay, following the CellTiter 96® AQueous One Solution Cell Proliferation Assay protocol. Each cell type was seeded at 3000 cells / well in triplicate onto 96-well plates. The following day, test compound solutions (1% DMSO) at various concentrations were added and incubated at 37°C and 5% CO2. After 72 hours, 20 μL of CellTiter 96® AQueous One Solution Reagent (Promega) was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, absorbance at 490 nm was measured using a Mithras LB940 plate reader, and the anticancer activity of the test compounds was calculated.

[0153] The test compounds used were Compound 3C, Compound 3D, Compound 3E, Compound 25, gemcitabine, and Protide (a gemcitabine prodrug incorporating protide from an existing prodrug, Non-Patent Document 3). PK1 (normal pancreatic cancer cells) and gemcitabine-resistant PK1 cells (RPK1) were used as cells.

[0154] The results are shown in Figure 11. It was found that the prodrug of the present invention exhibits anticancer activity.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula: R 1 represents a hydrogen atom or a hydrocarbon group having 1 to 12 carbon atoms which may be substituted with 1 to 3 substituents. 2 represents a hydrogen atom or a hydrocarbon group having 1 to 12 carbon atoms which may be substituted with 1 to 3 substituents. R 3 represents a monovalent group obtained by removing one hydrogen atom from the hydroxy group of the sugar moiety of lamivudine, or a group represented by the general formula (2): 【Chemistry 2】 Here, Base represents a monovalent group formed by removing one hydrogen atom from a nucleic acid base. 31 is -O-, -C (=CH-R 311 )-(R 311 represents a hydrogen atom or an alkyl group, or —NR 312 - (R 312 represents a hydrogen atom or an alkyl group. 32 is -CR 321 R 322 - (R 321 and R 322 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R 33 and R 34 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. 37 and R 38 are the same or different and represent a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. A double line consisting of a solid line and a dotted line represents a single bond or a double bond (however, in the case of a double bond, R 321 and R 322 Either one of the above and R 33 and R 34 Either one of the following is not present. R 4 represents an oxygen atom or a sulfur atom. However, some of the carbon atoms in the hydrocarbon group may be replaced by heteroatoms, and examples of the oxidases that can be used include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, formaldehyde oxidase, sorbitol oxidase, fructose oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, xanthine oxidase, ascorbate oxidase, sarcosine oxidase, choline oxidase, amine oxidase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, alcohol dehydrogenase, formaldehyde dehydrogenase, sorbitol dehydrogenase, fructose oxidase, and the like. Alternatively, a target structure for at least one enzyme selected from the group consisting of R dehydrogenase, hydroxybutyrate dehydrogenase, glycerol dehydrogenase, glutamate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, catalase, peroxidase, uricase, nitroreductase, cytochrome P450, protease, lipase, amylase, invertase, maltase, β-galactosidase, lysozyme, urease, esterase, nuclease, phosphatase, kinase, aminotransferase, GST, racemase, phosphoglycerate phosphomutase, and glucose 6-phosphate isomerase may be added. 1 and R 2 may be linked to each other to form a nitrogen-containing ring together with the adjacent nitrogen atom. A compound represented by the formula (I), a salt thereof, or a solvate thereof.

2. The compound, its salt, or solvate thereof according to claim 1 , wherein the hydrocarbon group is an alkyl group, an aryl group, or an aralkyl group.

3. The compound, salt thereof, or solvate thereof according to claim 1 or 2, wherein the hydrocarbon group is an alkyl group.

4. The compound, its salt, or solvate thereof according to any one of claims 1 to 3, wherein the hydrocarbon group is a branched alkyl group.

5. R 1 is a group other than a hydrogen atom, and the R 2 The compound, its salt, or solvate thereof according to any one of claims 1 to 4, wherein is a hydrogen atom.

6. R 1 is a branched alkyl group, and the R 2 The compound, its salt, or solvate thereof according to any one of claims 1 to 5, wherein is a hydrogen atom.

7. R 1 and the above R 2 and n are each an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group, or a salt thereof according to claim 2, or a solvate thereof.

8. R 1 is an alkyl group, and the R 2 The compound, salt thereof, or solvate thereof according to claim 2 or 7, wherein is an optionally substituted aryl group or an optionally substituted aralkyl group.

9. 9. The compound, salt thereof, or solvate thereof according to claim 2, 7, or 8, wherein the substituent that the hydrocarbon group may have is at least one selected from the group consisting of an alkoxy group, a halogen atom, a nitro group, an amino group, a thiol group, a sulfone group, a sulfonic acid group, a hydroxyamino group, an amide group, a nitrile group, a phosphate group, a carbonyl group, and a carboxy group.

10. The compound, salt thereof, or solvate thereof according to any one of claims 1 to 9, wherein the group represented by general formula (2) is a monovalent group obtained by removing one hydrogen atom from a hydroxy group in a sugar moiety of a nucleoside analog that is an anticancer agent or an antiviral agent.

11. A medicine comprising at least one selected from the group consisting of the compound according to any one of claims 1 to 10, a salt thereof, and a solvate thereof.

12. The pharmaceutical composition according to claim 11, which is for anticancer or antiviral use.

13. A reagent comprising at least one selected from the group consisting of the compound according to any one of claims 1 to 10, a salt thereof, and a solvate thereof.

Images