JP2022117326A - Prodrug compounds - Google Patents

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

Applied for application of Article 30, Paragraph 2 of the Patent Act (No. 1) Date of publication on website March 5, 2020 Website address https://confit. atlas. jp/guide/event/pharm140/tophttps://conf. atlas. jp/guide/event/pharm140/subject/2L25-34-07/advanced (Part 2) Publication date September 1, 2020 Publication The 14th Bio-Related Chemistry Symposium Abstract (Part 3) Date September 7, 2020 Date to September 8, 2020 Meeting Name, Venue 14th Bio-Related Chemistry Symposium Online

The present invention relates to compounds and the like that can be used as prodrugs.

Various nucleoside analogues are used as antimetabolites, anticancer agents, and antiviral agents against hepatitis B virus, HIV virus, and the like. Examples of such nucleoside analogues include anticancer agents such as gemcitabine and cytarabine, and antiviral agents such as lamivucine and zidovudine. After being monophosphorylated in cells, they are converted to triphosphates (active forms), and exert their efficacy by inhibiting the synthesis of DNA and RNA, which are necessary for cell and virus proliferation.

However, since nucleoside analogues such as gemcitabine are translocated into cells via transporters, there is concern about the emergence of resistant cells. In addition, the rate of intracellular monophosphorylation is slow and cannot be controlled. Therefore, various phosphate prodrugs have been developed.

J. Med. Chem. 2008, 51, 2328-2345. Journal of Medicinal Chemistry 2018, 61, 2211-2226. Journal of Medicinal Chemistry 2014, 57, 1531-1542.

Various prodrugs such as BisPOM type and ProTide type have been developed so far (Non-Patent Documents 1 to 3), but there are limitations in terms of release of toxic substances during deprotection and variety of deprotection conditions. be.

An object of the present invention is to provide novel prodrug compounds of nucleoside analogues.

An object of the present invention is to provide prodrug compounds of nucleoside analogues, preferably with lower cytotoxicity. Another object of the present invention is to provide a prodrug compound of a nucleoside analogue, preferably capable of controlling the expression of drug efficacy in cells.

As a result of intensive studies based on this knowledge, the present inventors have found that the above problems can be solved by the phosphoric acid fluoride-type prodrug compound represented by the general formula (1). The present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.

Section 1. General formula (1):

[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). R ² represents a hydrogen atom, an optionally substituted hydrocarbon group, or —OR ¹² (R ¹² represents an optionally substituted hydrocarbon group). R ³ represents a monovalent group formed by removing one hydrogen atom from the hydroxy group of the sugar moiety of the nucleoside analogue. ^R4 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 added thereto. However, R ¹ and R ² may be linked together to form a nitrogen-containing ring together with the adjacent nitrogen atoms. ]
A compound represented by, a salt thereof, or a solvate thereof.

Section 2. 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.

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

Section 4. 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 chain alkyl group.

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

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

Item 7. Item 3. The compound according to Item 2, or a salt thereof, wherein said R ¹ and said R ² are each an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group. , or a solvate thereof.

Item 8. Item 8. The compound, a salt thereof, or a mixture thereof according to item 2 or 7, wherein said R ¹ is an alkyl group and said R ² is an optionally substituted aryl group or an optionally substituted aralkyl group. Solvate.

Item 9. The substituents that the hydrocarbon group may have are 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 phosphoric acid group, Item 9. The compound, salt thereof, or solvate thereof according to Item 2, 7, or 8, which is at least one selected from the group consisting of a carbonyl group and a carboxy group.

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

Item 11. The R ³ is represented by the general formula (2):

[In the formula: Base represents a monovalent group obtained by removing one hydrogen atom from a nucleic acid base _{. R} ³¹ represents -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); show. R ³² represents -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) or -S- . 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. ^R35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. ^R36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R 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 indicates a single bond or a double bond (however, in the case of a double bond, either one of R ³²¹ and R ³²² and either one of R ³³ and R ³⁴ are absent). .). ]
The compound according to any one of Items 1 to 10, a salt thereof, or a solvate thereof, which is a group represented by

Item 12. A medicament containing at least one selected from the group consisting of the compound according to any one of Items 1 to 11, salts thereof, and solvates thereof.

Item 13. Item 13. The medicament according to item 12, which is for anticancer or antiviral use.

Item 14. Item 12. A reagent containing at least one selected from the group consisting of the compound according to any one of Items 1 to 11, salts thereof, and solvates thereof.

According to the present invention, novel prodrug compounds of nucleoside analogues can be provided. In addition, according to the present invention, prodrug compounds of nucleoside analogues with lower cytotoxicity can be provided. Furthermore, according to the present invention, it is also possible to provide prodrug compounds of nucleoside analogues that are capable of controlling the expression of drug efficacy in cells.

Two variants of compound 3a are shown. 3 shows the results of stability evaluation tests for compounds 3a and 3e. 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 variant, and the horizontal axis indicates the elapsed time from the preparation of the test solution (0 minutes). Figure 2 shows the results of stability evaluation tests for compounds 3f and 3h. The notation of the graph is the same as in FIG. 4 shows the results of stability evaluation tests for compounds 4-6. The notation of the graph is the same as in FIG. The results of the toxicity evaluation test are shown. The vertical axis indicates the absorbance at 490 nm (the higher the value, the lower the toxicity). The horizontal axis indicates test compounds. The cytidine prodrug form is compound 4. Concentrations in the legend indicate test compound concentrations in the medium. 4 shows the results of LC-MS analysis of cell extracts after administration of compound 3a to cells. The top row shows the extracted ion chromatography results for compound 3a, and the bottom row shows the total ion chromatography results. The upper row shows the results of extraction ion chromatography of the hydrolyzate (monophosphate) of compound 3a, and the lower row shows the results of LC-MS analysis (total ion chromatography) of the hydrolyzate of compound 3a. It shows that compound 3a is converted to a monophosphate form via a hydrolyzate. It shows that a secondary amine is converted to a monophosphate via a primary amine and a hydrolyzate. It shows that compounds 3C and 3E are converted to monophosphates via hydrolysates. Shown are the results of Anticancer Activity Evaluation Test 2. The cells used are shown on the left side of the graph, and the structures of test compounds are shown below the graph. The horizontal axis indicates the concentration of test compound used.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

1. In one aspect of the present invention, a compound represented by general formula (1):

A compound represented by, a salt thereof, or a solvate thereof (in the present specification, these may be collectively referred to as the "compound of the present invention"). This will be explained below.

<1-1. ^R1 , ^R2 >
R ¹ represents a hydrogen atom, an optionally substituted hydrocarbon group, or —OR ¹¹ (R ¹¹ represents an optionally substituted hydrocarbon group). R ² represents a hydrogen atom, an optionally substituted hydrocarbon group, or —OR ¹² (R ¹² represents an optionally substituted hydrocarbon group).

The hydrocarbon group is not particularly limited and includes, for example, an alkyl group, an aryl group, and a group formed by any combination thereof (eg, an aralkyl group, an alkylaryl group, an alkylaralkyl group). By adjusting the bulkiness of the hydrocarbon group, it is possible to control the stability of the compound of the present invention (and thus the timing of expression of efficacy in cells). That is, by adopting a bulky hydrocarbon group, stability can be increased, and by adopting a structure with low steric hindrance (planar structure, etc.), stability can be reduced, thereby exerting efficacy. timing can be adjusted. In one aspect of the present invention, the hydrocarbon group preferably includes an alkyl group, an aryl group, an aralkyl group, and the like. In one aspect of the present invention, an alkyl group is more preferred. In one aspect of the present invention, an aryl group, an aralkyl group, and the like are more preferred, and an aralkyl group is even more preferred.

The above alkyl groups include any of straight-chain, branched-chain and cyclic ones. From the viewpoint of 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 (linear or branched) is not particularly limited, and is, for example, 1-12. In one aspect of the present invention, the number of carbon atoms is preferably 1-8, more preferably 2-8, even more preferably 3-6, and even more preferably 3-4. The number of carbon atoms is preferably 3-12, more preferably 4-8, even more preferably 5-7 in one embodiment of the present invention. The number of carbon atoms in the alkyl group (in the case of cyclic) is not particularly limited, and is, for example, 3-7, preferably 4-6. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n -hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group and the like. Among these, an isopropyl group is particularly preferred.

The aryl group is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 8 carbon atoms. The aryl group can be either monocyclic or polycyclic (eg, bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include 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 preferably a phenyl group.

The above aralkyl group is not particularly limited, but for example, a linear or branched alkyl group having 1 to 6 (preferably 1 to 3) carbon atoms (for example, 1 to 3, preferably 1 hydrogen atom ) is substituted by the above aryl group. Specific examples of the aralkyl group include benzyl group and phenethyl group.

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

The above alkylaralkyl group is not particularly limited, but for example, hydrogen atoms (eg, 1 to 3, preferably 1 hydrogen atom) on the aromatic ring of the above aralkyl group are linear or branched and have 1 to 1 carbon atoms. Examples thereof include an alkylaralkyl group substituted with 6 (preferably 1 to 2) alkyl groups.

The above hydrocarbon groups may be substituted. Examples of substituents include, but are not limited to, alkoxy groups, halogen atoms, nitro groups, amino groups, thiol groups, sulfone groups, sulfonic acid groups, hydroxyamino groups, amide groups, nitrile groups, phosphoric acid groups, carbonyl groups, A carboxy group and the like can be mentioned. Among these, alkoxy groups, halogen atoms, nitro groups, amino groups and the like are preferred. The alkoxy group includes, for example, a linear or branched alkoxy group having 1 to 8 carbon atoms (preferably 1 to 4, more preferably 1 to 2, still more preferably 1). The halogen atom includes, for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like, preferably a fluorine atom. The number of substituents is not particularly limited, and is, for example, 1-3, preferably 1-2, 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-position and meta-position on the aryl group are preferred.

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.

Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. The carbon atom to be replaced with a hetero atom is not particularly limited, and may be a carbon atom on the main chain of a chain hydrocarbon group or a carbon atom on a side chain of a cyclic hydrocarbon group. It may be a ring-constituting carbon atom.

When some carbon atoms of the above hydrocarbon group are replaced with heteroatoms, the number of heteroatoms is not particularly limited, but is, for example, 1-6, 1-3, and 1.

The hydrocarbon group to which the ligand molecule and/or enzyme target structure is attached is, in other words, a group derived from the ligand molecule and/or enzyme target structure (e.g., one hydrogen atom or A hydrocarbon group substituted with a group from which a functional group has been removed).

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. A ligand is determined in relation to a target molecule (eg, protein). For example, when the protein is a receptor, it may be a substance that binds to the receptor, when the protein is an enzyme, it is a substrate for the enzyme, and when the protein is an antigen, it may be an antibody or an antibody fragment. Ligands include, for example, carbohydrates, cholesterol, lipids, phospholipids, antibodies, lipoproteins, hormones, peptides, vitamins, steroids, cationic lipids and the like. More specifically, for example, by using N-acetylgalactosamine, retinoic acid, galactose, glycyrrhizic acid, etc. as ligands, the compounds of the present invention can act specifically on the liver.

Proteins to be labeled are not particularly limited, and examples thereof include receptors, enzymes, antigens and the like.

Antibodies or fragments thereof include monoclonal antibodies, single chain antibodies such as Fv, scFv, Fab, F(ab')2, Fab', Fd, dAb, CDRs, scFv-Fc fragments, nanobodies, affibodies, diabodies. , Avimer, and Versabodies.

Enzymes include, for example, oxidoreductases, hydrolases, transferases, isomerases, and the like. 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, and 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, cytochrome P450 and the like. Examples of hydrolases include protease, lipase, amylase, invertase, maltase, β-galactosidase, lysozyme, urease, esterase, nuclease, phosphatase and the like. Transferases include, for example, various transferases, kinases, aminotransferases, GSTs and the like. Isomerases include, for example, racemase, phosphoglycerate phosphomutase, glucose 6-phosphate isomerase and the like.

Receptors include muscarinic acetylcholine receptors, adenosine receptors, adrenergic 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 , 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), etc.

By employing a carbohydrate group attached with an enzyme target structure, the compounds of the present invention can be specifically degraded at the site where the enzyme is present. For example, in the compound of the present invention, which is a secondary amine, by adopting a hydrocarbon group to which an enzyme target structure is added, it is specifically degraded at the site where the enzyme exists and is converted to a primary amine, Its site-specific conversion to a hydrolyzate and a monophosphate can lead to manifestation of efficacy.

Enzymes include, for example, the enzymes described above. The enzyme target structure is not particularly limited, and known structures can be adopted. Examples of enzyme target structures, corresponding enzymes, and target sites are shown below.

The number of ligand molecules and/or enzyme target structures attached to the hydrocarbon group is not particularly limited, but is, for example, 1-3, 1-2, and 1.

R ¹ and R ² may be linked together to form a nitrogen-containing ring together with adjacent nitrogen atoms. The nitrogen-containing ring is not particularly limited. For example, R ¹ and R ² are linked together to form a dimethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, etc., and form a nitrogen-containing ring (e.g., piperidine ring, etc.) together with the adjacent nitrogen atom. can be formed.

In one embodiment of the present invention, from the viewpoint of imparting appropriate stability to the compound and appropriately exhibiting efficacy, preferably 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 bound to the phosphorus atom is replaced with an oxygen atom by hydrolysis in vivo, and then it is converted to a monophosphate form by decomposition by an enzyme such as HINT-1, thereby exerting its efficacy. can be done. In this case, from the viewpoint of efficacy, it is particularly preferred that R ¹ is a branched alkyl group and R ² is a hydrogen atom.

On the other hand, when both R ¹ and R ² are groups other than hydrogen atoms, or when R ¹ and R ² are linked together to form a nitrogen-containing ring, the stability of the compound of the present invention is relatively high. Thus, by adding an enzyme target structure to the hydrocarbon group as necessary, the timing, site, etc. of expression of efficacy can be adjusted.

In one aspect of the present invention, each of R ¹ and R ² is preferably an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group. Further, in one aspect of the present invention, it is preferable that R ¹ is an alkyl group and R ² is an optionally substituted aryl group or an optionally substituted aralkyl group.

<1-2. ^R3 >
R ³ represents a monovalent group formed by removing one hydrogen atom from the hydroxy group of the sugar moiety of the nucleoside analogue. The hydroxy group of the sugar portion of the nucleoside analogue is the hydroxy group substituted with the phosphate group when the nucleoside analogue is converted to the monophosphate form, and is not particularly limited in this respect.

Nucleoside analogues are not particularly limited, and include, for example, those having antimetabolite action such as anticancer agents and antiviral agents. Examples of such nucleoside analogues include anticancer agents such as gemcitabine and cytarabine; anti-hepatitis B virus agents such as lamivusine and entecavir; anti-HIV agents such as zidovudine, didanosine and stavudine.

A preferred embodiment of R ³ is, for example, general formula (2):

The group represented by is mentioned.

Base represents a monovalent group obtained by removing one hydrogen atom from a nucleic acid base. As the nucleic acid base, any base that constitutes a nucleic acid can be used without any particular limitation. Bases that make up nucleic acids include typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.). However, other bases such as hypoxanthine (I), modified bases and the like are also included. Modified bases include, for example, 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-methylhypoxanthine, 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-iso pentenyl adenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine and the like. These nucleobases may further have one or more substituents.

R ³¹ represents -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); show.

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

R ³² represents -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) or -S- . 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. ^R35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. ^R36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R 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.

Examples of alkynyl groups represented by R ³²¹ , R ³²² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ or R ³⁸ include ethynyl, 1-propynyl, 2-propynyl, butynyl, pentynyl, octenyl and the like. and an alkynyl group having 2 to 8 carbon atoms.

A double line consisting of a solid line and a dotted line indicates a single bond or a double bond. However, in the case of a double bond, either one of R ³²¹ and R ³²² and either one of R ³³ and R ³⁴ do not exist. Further, when R ³² is -S-, the doublet is a single bond.

<1-3. ^R4 >
^R4 represents an oxygen atom or a sulfur atom. ^R4 is preferably an oxygen atom.

<1-4. Others>
The compound represented by the general formula (1) has various isomers such as geometric isomers, configurational isomers, tautomers, optical isomers, stereoisomers, positional isomers, rotational isomers, etc. is included. When having any isomers, mixtures of each isomer are also included in the compound of the present invention. Furthermore, when the compound of the present invention has optical isomers, the optical isomers resolved from the racemate are also included in the compounds of the present invention. When the compound represented by formula (1) has geometrical isomers, configurational isomers, stereoisomers, conformational isomers, etc., each of them can be isolated by known means. When the compound represented by the general formula (1) is an optically active form, it can be separated into the (+) form or (−) form [D form or L form] from the corresponding racemic form by conventional optical resolution means. can.

The salt of the compound represented by formula (1) is not particularly limited as long as it is a pharmaceutically acceptable salt. As the salt, both acid salts and basic salts can be employed. Examples of acid salts include mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid. Salts, organic acid salts such as citrate, methanesulfonate, p-toluenesulfonate, etc. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as salts; salts with ammonia; morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl) Examples thereof include salts with organic amines such as amines.

The solvate of the compound represented by general formula (1) is not particularly limited. Solvents constituting the solvent field include, for example, water, pharmaceutically acceptable organic solvents (eg, ethanol, glycerol, acetic acid, etc.), and the like.

2. Methods of Preparation The compounds of this invention can be prepared in a variety of ways.

The compounds of the present invention can be produced, for example, according to or analogous to the schemes below.

As the compound represented by the general formula (1a), a commercially available compound can be used as it is, and if necessary, it is synthesized according to or according to a known method and/or according to the method described in the Examples. can also be used.

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

The amount of the compound represented by the general formula (1a) to be used is usually preferably 0.5 to 3 mol per 1 mol of the compound represented by the general formula (1b) from the viewpoint of yield, ease of synthesis, etc. , 1 to 2 mol is more preferred.

This reaction is usually carried out in the presence of a reaction solvent. Examples of the reaction solvent include, but are not limited to, dimethylformamide, dichloromethane, acetonitrile, tetrahydrofuran, acetone, toluene and the like, preferably dimethylformamide and the like. A single solvent may be used, or a plurality of solvents may be used in combination.

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

In this reaction, in addition to the above components, additives can be used as appropriate within a range that does not significantly impair the progress of the reaction.

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

The progress of the reaction can be followed by conventional methods such as chromatography. After completion of the reaction, the solvent is distilled off, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. Also, the structure of the product can be identified by elemental analysis, MS (ESI-MS) analysis, IR analysis, ¹ H-NMR, ¹³ C-NMR and the like.

3. Use The compound of the present invention can exhibit various pharmacological effects (eg, anticancer action, antiviral action, etc.) depending on the type of nucleoside analogue moiety. Therefore, the compound of the present invention can be used as an active ingredient of medicines, reagents, etc. (herein sometimes referred to as "the drug of the present invention"), more specifically, anticancer agents, antiviral agents, etc. It can be used as an active ingredient such as a drug.

The drug of the present invention is not particularly limited as long as it contains the compound of the present invention, and may further contain other ingredients as necessary. Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Other components include additives as well as components having pharmacological action. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, A chelating agent and the like are included.

The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted according to its type. 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 an animal), depending on its use. can also

Subjects to which the drug of the present invention is applied are not particularly limited, but examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Moreover, animal cells etc. are mentioned as a cell. The types of cells are not particularly limited, such as blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, ovum), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells. , epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.

When the agent of the present invention is used as an anticancer agent and when used for cancer cells, the target cancer is not particularly limited, and examples include hepatocellular carcinoma, pancreatic cancer, renal cancer, leukemia, and esophagus. cancer, stomach cancer, colon cancer, lung cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, and the like. Among these, solid cancer is preferred, and hepatocellular carcinoma is more preferred.

When using the agent of the present invention as an antiviral agent, the target virus is not particularly limited. Target viruses include, for example, 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, and SARS. enveloped viruses such as viruses, hepatitis viruses (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, lyssa virus; adenoviruses , norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, poliovirus, rhinovirus, and other non-enveloped viruses (viruses without envelopes). Among these, AIDS virus, hepatitis B virus and the like are preferable.

The agent of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, Hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), oral dosage forms such as jelly, injection preparations (e.g., drip injections (e.g., intravenous drip preparations etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external agents (e.g., ointments, poultices, lotions), suppository inhalants, eye agents, eye ointments, nasal drops Parenteral formulations such as pharmaceuticals, ear drops, and liposomes can be used.

The administration route of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and includes enteral administration such as oral administration, tube feeding, and enema administration; intravenous administration, transarterial administration, intramuscular administration, Examples include parenteral administration such as intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

The content of the active ingredient in the drug of the present invention depends on the mode of use, the target of application, the condition of the target of application, etc., and is not limited, but is for example 0.0001 to 100% by weight, preferably 0.001 to 50% by weight. %.

When the drug of the present invention is administered to animals, the dosage is not particularly limited as long as it is an effective amount that exhibits efficacy. 1000 mg/kg body weight, preferably 0.5-500 mg/kg body weight per day, and 0.01-100 mg/kg body weight per day, preferably 0.05-50 mg/kg body weight for parenteral administration . The above dosage can be adjusted appropriately depending on age, disease state, symptoms and the like.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

(1) Compound synthesis 1

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 completion of the dropwise addition, the mixture was returned to room temperature and stirred for 3 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and water. The organic phase was collected, dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off. The resulting white solid was washed with hexane. Yield 0.840 g, 4.77 mmol, 89% yield.
¹ H NMR (400 MHz, CD ₃ CN): δ 3.66 (m, 1H), 1.27 (d, J = 6 Hz, 6H)
^13C NMR (100 MHz, _CDCl3 ): δ 46.1, 24.1
^31P NMR (162 MHz, _CD3CN ): δ 13.74.

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 heated to 0°C. was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 3 hours, the solvent was distilled off, and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a white solid. Yield 1.14 g, 3.64 mmol, 68% yield.
¹ H NMR (400 MHz, CDCl ₃ ): 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)
¹³ C NMR (100 MHz, CDCl ₃ ): 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, CDCl ₃ ): δ 16.2.

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 diluted to 0 It was added dropwise with stirring at °C. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 3 hours, the solvent was distilled off, and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a pink oily substance. Yield 0.795 g, 2.78 mmol, 52% yield.
¹ H NMR (400 MHz, CDCl ₃ ): 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, CDCl ₃ ): 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, CDCl ₃ ): δ 16.9.

Compound 1d
Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (12.0 mL), and a solution of hexanamine (642 μL, 4.82 mmol) and triethylamine (747 μL, 5.36 mmol) in dichloromethane (12.0 mL) was heated to 0°C. was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 4 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a colorless oily substance. Yield 1.07 g, 4.93 mmol, 92% yield.
¹ H NMR (400 MHz, CDCl ₃ ): 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)
^13C NMR (100 MHz, _CDCl3 ): δ 42.6, 31.2, 30.1, 30.0, 26.1, 22.4
^31P NMR (162 MHz, _CDCl3 ): δ 17.0.

Compound 1e
Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and aniline (489 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) were dissolved in dichloromethane (10.0 mL) at 0°C. It was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 5 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected, dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a white solid. Yield 775 mg, 3.70 mmol, 69% yield.
¹ H NMR (400 MHz, CDCl ₃ ): 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)
^13C NMR (100 MHz, _CDCl3 ): δ 136.5, 129.6, 125.0, 121.1, 121.0
³¹ P NMR (162 MHz, CDCl ₃ ): δ 9.74.

Compound 1f
Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and diethylamine (554 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) were dissolved in dichloromethane (10.0 mL) at 0°C. It was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 2.5 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain an orange oily substance. Yield 1.05 g, yield quant.
¹ H NMR (400 MHz, CDCl ₃ ): δ 3.28-3.19 (m, 4H), 1.18-1.12 (m, 6H)
^13C NMR (100 MHz, _CDCl3 ): δ 40.7, 40.6, 13.2, 13.1
³¹ P NMR (162 MHz, CDCl ₃ ): δ 17.1.

1 g of compound
Phosphoryl chloride (500 μL, 5.36 mmol) was added to dichloromethane (10.0 mL), and piperidine (529 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) were dissolved in dichloromethane (10.0 mL) at 0°C. It was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 2.5 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a colorless oily substance. Yield 1.02 g, yield quant.
¹ H NMR (400 MHz, CDCl ₃ ): δ 3.20-3.18 (m, 4H), 1.54 (bs, 6H)
^13C NMR (100 MHz, _CDCl3 ): δ 45.8, 25.0, 24.9, 23.6
³¹ P NMR (162 MHz, CDCl ₃ ): δ 16.1.

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 heated to 0°C. was added dropwise with stirring. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 9 hours, then the solvent was distilled off and liquid separation was performed with ethyl acetate and cold water. The organic phase was collected and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off to obtain a colorless oily substance. Yield 823 mg, 4.56 mmol, 85% yield.
¹ H NMR (400 MHz, CDCl ₃ ): δ 4.99-4.90 (m, 1H), 1.42-1.40 (m, 6H)
^13C NMR (100 MHz, _CDCl3 ) δ 79.4, 22.9
^31P NMR (162 MHz, _CDCl3 ): δ 5.88.

Compound 2 (common experimental term)
Silver fluoride (2.1 eq.) was added to dichlorophosphoramide (1 eq.) dissolved in dehydrated acetonitrile (1.0 mL), and the mixture was 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.

Compound 3a
Gemcitabine (100 mg, 0.38 mmol) was dissolved in DMF (1 mL) and stirred, then DIPEA (140 μL, 1.15 mmol) and compound 2a (81 mg, 0.57 mmol) in dehydrated acetonitrile solution (1 mL) were added. After that, calcium hydroxide (70 mg, 0.95 mmol) was added and stirred for 1.5 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 25 mg, 0.06 mmol, 17% yield.
¹ H NMR (400 MHz, DMF-d ₆ ): 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)
^13C 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-d ₆ ): δ 5.1 (d, J _PF = 978 Hz), 5.3 (d, J _PF = 974 Hz)
HRMS ₍ ESI) m/z calculated for _{C12H18F3N4O5P} [M+H] ⁺ : _{387.0967 ,} found for: _387.1074 .

Compound 3e
Gemcitabine (61 mg, 0.23 mmol) was dissolved in DMF (1 mL) and stirred, then DIPEA (100 μL, 0.58 mmol) and compound 2e (61.6 mg, 0.35 mmol) in dehydrated acetonitrile solution (1.5 mL) were added. After that, calcium hydroxide (34.4 mg, 0.46 mmol) was added and stirred for 1 hour. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 10 mg, 0.02 mmol, 10% yield.
¹ H NMR (400 MHz, DMSO-d ₆ ): 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-d ₆ ): 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-d ₆ ): δ -70.4 (d, J _PF = 981 Hz), -70.3 (d, J _PF = 981 Hz), -116
³¹ P NMR (162 MHz, DMSO-d ₆ ): δ −1.00 (d, J _PF = 983 Hz), −1.18 (d, J _PF = 987 Hz)
_{HRMS (} ESI) m/z calculated for _{C15H16F3N4O5P} [M+H] ⁺ _421.0889 , found _421.0875 .

compound 3f
Gemcitabine (101.4 mg, 0.39 mmol) was dissolved in DMF (1 mL) and stirred, then DIPEA (166 μL, 0.96 mmol) and compound 2f (90.7 mg, 0.58 mmol) in dehydrated acetonitrile solution (1.5 mL) were added. After that, calcium hydroxide (57.0 mg, 0.77 mmol) was added and stirred for 2.5 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 3.3 mg, 0.003 mmol, 2% yield.
¹ H NMR (400 MHz, DMSO-d ₆ ): 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-d ₆ ): not obtained due to small amount.
¹⁹ F NMR (376 MHz, DMSO-d ₆ ): δ −72.6 (d, J _PF = 999 Hz), −72.7 (d, J _PF = 987 Hz), −114
³¹ P NMR (162 MHz, DMSO-d ₆ ): δ 5.12 (d, J _PF = 991 Hz), 4.73 (d, J _PF = 996 Hz)
_{HRMS (} ESI) m/z calculated for _{C13H20F3N4O5P} [M+H] ⁺ _401.1202 , found _401.1194 .

3 g of compound
Gemcitabine (64.1 mg, 0.24 mmol) was dissolved in DMF (1 mL) and stirred. DIPEA (105 μL, 0.61 mmol) and dehydrated acetonitrile solution (1.5 mL) of compound 2g (61.9 mg, 0.37 mmol) were added. After that, calcium hydroxide (36.2 mg, 0.49 mmol) was added and stirred for 1 hour. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 22.9 mg, 0.06 mmol, 23% yield.
¹ H NMR (400 MHz, CD ₃ CN): 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, CD ₃ CN): 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, CD ₃ CN): δ −77.0 (d, J _PF = 982 Hz), −77.5 (d, J _PF = 982 Hz), −118
³¹ P NMR (162 MHz, CD ₃ CN): δ 3.58 (d, J _PF = 977 Hz), 3.04 (d, J _PF = 986 Hz)
_{HRMS (} ESI) m/z calculated for _{C14H20F3N4O5P} [M+H] ⁺ _413.1202 , found _413.1192 .

(2) Synthesis of compound 2

compound 3h
Gemcitabine (56.2 mg, 0.21 mmol) was dissolved in DMF (1 mL) and stirred, then DIPEA (92.2 μL, 0.54 mmol) and compound 2h (46.2 mg, 0.32 mmol) in dehydrated acetonitrile solution (1.5 mL) were added. After that, calcium hydroxide (31.7 mg, 0.43 mmol) was added and stirred for 6 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 36.7 mg, 0.09 mmol, 44% yield.
¹ H NMR (400 MHz, CD ₃ CN): 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)
^13C 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, CD ₃ CN): δ −79.7 (d, J _PF = 993 Hz), −80.0 (d, J _PF = 993 Hz), −118
³¹ P NMR (162 MHz, CD ₃ CN): δ −9.82 (d, J _PF = 993 Hz), −9.89 (d, J _PF = 995 Hz)
_HRMS (ESI) m/z calculated for _{C12H17F3N3O6P} [M+H] ⁺ _{388.0885 ,} found _388.0823 .

compound 4
Deoxycytidine (100 mg, 0.41 mmol) was dissolved in DMF (2 mL) and stirred, then DIPEA (142 μL, 0.82 mmol) and compound 2a (117 mg, 0.82 mmol) in dehydrated acetonitrile solution (1 mL) were added. After the addition, calcium hydroxide (60 mg, 0.82 mmol) was added and stirred for 5 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 35.0 mg, 0.10 mmol, 25% yield.
¹ H NMR (400 MHz, CD ₃ CN): 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 = 6.0Hz, 6H)
^13C 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, CD ₃ CN): δ −75.2 (d, J _PF = 976 Hz), −76.1 (d, J _PF = 981 Hz)
³¹ P NMR (162 MHz, CD ₃ CN): δ 3.2 (d, J _PF = 979 Hz), 3.4 (d, J _PF = 976 Hz)
HRMS ₍ ESI) m/z calculated for _{C12H18F3N4O5P} [M+H] ⁺ : _{351.1234 ,} found for: _351.1356 .

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 dehydrated acetonitrile solution (1 mL) were added. After that, calcium hydroxide (60 mg, 0.82 mmol) was added and stirred for 2 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 35.6 mg, 0.10 mmol, 23% yield.
¹ H NMR (400 MHz, CD ₃ CN): 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)
^13C 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, CD ₃ CN): δ −72.4 (d, J _PF = 970 Hz), −72.8 (d, J _PF = 981 Hz)
³¹ P NMR (162 MHz, CD ₃ CN): δ 4.9 (d, J _PF = 974 Hz), 4.7 (d, J _PF = 978 Hz)
HRMS ₍ ESI) m/z calculated for _{C11H18FN4O4PS} [M+H] ⁺ : _353.0770 , found for: _353.0380 .

compound 6
Entecavir monohydrate (85 mg, 0.29 mmol) was azeotroped with pyridine three times. After adding DIPEA (97 μL, 0.79 mmol) and compound 2a (45.7 mg, 0.32 mmol) in dehydrated acetonitrile solution (1 mL), calcium hydroxide ( 60 mg, 0.82 mmol) was added and stirred for 5 hours. After the reaction, it was filtered and 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) and obtained as a white solid. Yield 45.2 mg, 0.11 mmol, 39% yield.
¹ H NMR (400 MHz, CD ₃ CN): 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, CD ₃ CN): 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
¹⁹ F NMR (376 MHz, CD ₃ CN): δ −72.6 (d, J _PF = 970 Hz), −72.9 (d, J _PF = 975 Hz)
³¹ P NMR (162 MHz, CD ₃ CN): δ 45.4 (d, J _PF = 974 Hz)
HRMS ₍ ESI) m/z calculated for _C15H22FN6O4P [M+H] ⁺ : _401.1502 , found for: _401.1604 .

(3) Stability evaluation test Prodrug compounds (compounds 3a, 3e, 3f, 3h, 4, 5, 6) used for stability evaluation were dispensed 0.2-0.5 mg with 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 minutes, incubated at 37°C, and the test solution at each time was analyzed by RP-HPLC. As a result of the analysis, the prodrug changed into a cyclized form in which the 3' hydroxyl group reacted with the phosphorus atom, in addition to the hydrolyzate in which fluorine was eliminated by water molecules (Fig. 1).

The results are shown in Figures 2-4. The prodrug compound was found to be relatively stable in Milli-Q water. On the other hand, in PBS buffer, it was found to rapidly change to a hydrolyzate (Fig. 1). This difference in stability was attributed to the increased reactivity due to the interaction of ions such as sodium contained in the buffer with the double bond of phosphorus and oxygen.

Among the prodrug compounds tested, the aniline form (3e) was found to be the most susceptible to degradation. In addition, it was found that the diethylamine derivative (3f) and piperidine derivative (3g), which are secondary amines, did not react at all and were stable even in the PBS buffer. This difference in stability is thought to be due to the bulkiness of the phosphorus center, which is subject to nucleophilic attack. In other words, the diethylamine and piperidine derivatives, which are secondary amines, are bulkier than primary amines, and it is thought that water molecules cannot nucleophilically attack phosphorus atoms. On the other hand, since the aniline body has a planar structure, it is considered to be less sterically hindered and easily hydrolyzed.

In vivo, the hydrolyzate is considered to be converted to a monophosphate by removing the amine portion through enzymatic decomposition by HINT-1 and the like (Fig. 8).

Since the secondary amine was stable even in the PBS buffer, it is considered that the timing of conversion to the hydrolyzate and monophosphate can be delayed by using the secondary amine. In addition, if it is a prodrug compound in which an enzyme target structure is added to a secondary amine, it is specifically degraded at the site where the enzyme exists and converted to a primary amine, and the site-specific hydrolysis pair and monoline It is thought that the conversion to the acid form can lead to the manifestation of efficacy (Fig. 9).

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

The results are shown in FIG. It has been found that the prodrug structures of the present invention are not toxic.

(5) Detection of intracellular conversion to monophosphate form Gemcitabine prodrug (Compound 3a) (DMSO, for negative control experiment) was added to the cell culture medium, and after 6 hours of incubation, the contents of the wells were transferred to an Eppendorf tube. The cells were transferred and disrupted with a homogenizer. Under ice-cooling, the same amount of 20% trichloroacetic acid aqueous solution as the cell lysate was added, allowed to stand for 30 minutes, and centrifuged (4°C, 13000-15000G, 5-10 minutes). Subsequently, the supernatant was transferred to another tube, and the residue (pellet) was washed with a small amount of 50% aqueous acetone solution. After centrifugation (4°C, 13000-15000G, 5-10 minutes), the supernatant was added to the above solution. The combined supernatant was centrifuged again (4°C, 13000-15000 G, 5-10 minutes), the organic solvent was removed, and the remaining aqueous solution was lyophilized. The extract thus obtained was subjected to LC/MS analysis under the following conditions.

The results are shown in Figures 6-7. It has been found that the prodrugs of the present invention are converted intracellularly to their monophosphate form.

(6) Synthesis of compound 3
Compounds 3B and 3C are prodrugs activated by CYP450 (Fig. 10).

Compound 1A
A stirred solution of phosphoryl chloride (500 μL, 5.36 mmol) in dry CH2Cl2 (10.0 mL), isopropylamine (822 μL, 9.65 mmol) and triethylamine (742 μL, 5.36 mmol) diluted with CH2Cl2 (12.0 mL) was added dropwise at −65° C. and the reaction mixture was stirred at rt for 2 hours. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 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, CD ₃ CN): δ 3.66 (m, 1H), 1.27 (d, J = 6 Hz, 6H)
¹³ C-NMR (100 MHz, CDCl ₃ ): δ 46.1, 24.1
^31P -NMR (162 MHz, _CD3CN ): δ 13.74.

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 CH2Cl2 (12.0 mL) diluted with CH2Cl2 (12.0 mL) was added dropwise at 0° C. to the stirred solution and the reaction mixture was stirred at rt for 3 h. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 and evaporated to give 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, CDCl ₃ ): δ 7.40-7.13 (m, 5H), δ 4.42 (d, J = 8 Hz, 2H), δ 2.72 (d, J = 16 Hz, 3H).
¹³ C-NMR (100 MHz, CDCl ₃ ): δ 135.22, 128.96, 128.42, 128.34, 53.40, 33.71
³¹ P-NMR (162 MHz, CDCl ₃ ): δ 19.32.

Compound 1C
To a stirred solution of phosphoryl chloride (205.7 μL, 2.2 mmol) in dry CH2Cl2 (15.0 mL) was added 4-methoxy-N-methylbenzylamine (297 μL, 1.98 mmol) diluted with CH2Cl2 (15.0 mL) at 0 °C. It was added dropwise and the reaction mixture was stirred at rt for 3 hours. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 and evaporated to give a yellow oily crude compound (448 mg, 1.88 mmol, 95%) which was used in the next step without further purification.
¹ H-NMR (400 MHz, CD ₃ CN): 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 = 16.4 Hz, 3H).
¹³ C-NMR (100 MHz, CDCl ₃ ): δ 159.56, 129.76, 126.98, 114.15, 55.12, 52.59, 33.24.
^31P -NMR (162 MHz, _CD3CN ): δ 19.14.

Compound 1D
To a stirred solution of phosphoryl chloride (500 μL, 5.36 mmol) in dry CH2Cl2 (12.0 mL) was added N,N-methylisopropylamine (560 μL, 5.36 mmol) and triethylamine (747 μL, 5.36 mmol) in CH2Cl2 (12.0 mL). ) was added dropwise at 0° C. and the reaction mixture was stirred at rt for 3 h. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 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, CD ₃ CN): δ 4.21 (m, 1H), δ 2.72 (d, J = 16 Hz, 3H), δ 1.20 (d, J = 7.2 Hz, 6H).
^13C -NMR (100 MHz, _CDCl3 ): δ 48.44, 26.43, 19.23
^31P -NMR (162 MHz, _CD3CN ): δ 17.69.

Compound 1E
A stirred solution of phosphoryl chloride (523.6 μL, 5.60 mmol) in dry CH2Cl2 (12.0 mL) was diluted with dimethylbutylamine (505.9 mg, 5 mmol) and triethylamine (776.3 μL, 5.60 mmol) in CH2Cl2 (12.0 mL). was added dropwise at -40°C and the reaction mixture was stirred at rt for 3 hours. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 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.
¹ H-NMR (400 MHz, CDCl ₃ ): 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, CDCl ₃ ): δ 48.32, 47.55, 24.71, 22.56, 22.43
^31P -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 CH2Cl2 (12.0 mL), adamantineamine (756.3 mg, 5 mmol) and triethylamine (776.3 μL, 5.60 mmol) were diluted with CH2Cl2 (12.0 mL). was added dropwise at -40°C and the reaction mixture was stirred at rt for 3 hours. Volatiles were removed under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was dried over Na2SO4 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, CD ₃ CN): δ 1.96 (3H, m), 1.92 (6H, m), 1.64 (6H, t, J = 14 Hz)
^13C -NMR (100 MHz, _CDCl3 ): δ 56.99, 44.13, 44.07, 35.82, 29.86
^31P -NMR (162 MHz, _CD3CN ): δ 8.50.

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

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) was added followed by Ca(OH)2 (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 analyzed by RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQ B) ACN, gradient: 0-25 min. -70% (B), 25-35 min 100% (B), flow rate 8 mL/min, detection: 250 nm) to give compound 3A (25 mg, 0.06 mmol, 17%) as a white solid. Obtained.
¹ H-NMR (400 MHz, DMF-d ₆ ): 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-d ₆ ): 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-d ₆ ): δ 5.1 (d, J = 978 Hz), 5.3 (d, J = 974 Hz)
HRMS ₍ ESI) m/z calculated for _{C12H18F3N4O5P} [M+H] ⁺ : _{387.0967 ,} found for: _387.1074 .

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) was added followed by (CF3SO3)2Ca (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 analyzed by RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQ B) ACN, gradient: 0-5 min. -50% (B), 5-24 min 50-80% (B), 24-25 min 80-100% 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, CD ₃ OD): 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 (100 MHz, CD ₃ CN): 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, 32.67,
¹⁹ F-NMR (376 MHz, CD ₃ CN): δ -77.3 (d, J = 987.0 Hz), -76.9 (d, J = 999.0 Hz), -116
³¹ P-NMR (162 MHz, CD ₃ CN): δ 5.1 (d, J = 1018 Hz), 4.5 (d, J = 1035 Hz),
HRMS ₍ ESI+) _calcd for _{C17H20F3N4O5P} [M+Na] ⁺ : _{471.1123 ,} found for: 471.1195.

Compound 3C
To a stirred solution of 2'-deoxy-2',2'-difluorocytidine (45.8 mg, 0.174 mmol) in dry DMF (1 mL) was added DIPEA (74 µL, 0.52 mmol) and Compound 2C (122.3 mg, 0.52 mmol) was added and (CF3SO3)2Ca (58.9 mg, 0.17 mmol) was added and stirred. The reaction mixture was stirred overnight at rt, filtered through a 0.22 μm membrane filter and the filtrate was RP-HPLC (YMC Triart C18 (250 × 20 mm), Elute A) MQ B) ACN, gradient: 0-5 min. ~50% (B), 5-24 minutes 50-80% (B), 24-25 minutes 80-100%. Purification at flow rate 8 mL/min, detection: 250 nm) gave the desired compound 3C (9.2 mg, 0.02 mmol, 11%) as a white solid.
1H ^- 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 (100 MHz, CD ₃ CN): 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, 32.40,
¹⁹ F-NMR (376 MHz, CD ₃ CN): δ -74.9 (d, J = 988.0 Hz), -77.48 (d, J = 977 Hz), -116
³¹ P-NMR (162 MHz, CD ₃ CN): δ 7.9 (d, J = 977 Hz), δ 1.9 (d, J = 990 Hz),
HRMS ₍ ESI ⁺ ) calcd for _{C18H22F3N4O6P} [M+H] ⁺ : _{479.1129 ,} found for: _479.1118 .

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) was added followed by (CF3SO3)2Ca (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 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.
¹ H-NMR (400 MHz, DMSO-d ₆ ): 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 (100 MHz, DMSO-d ₆ ): 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 (162 MHz, DMSO-d ₆ ): δ 7.6 (d, J = 991 Hz), δ 1.45 (d, J = 1000 Hz),
HRMS ₍ ESI ⁺ ) _calcd for _{C13H20F3N4O5P} [M+H] ⁺ : _{401.1123 ,} found for: 401.1165.

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) was added followed by (CF3SO3)2Ca (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 analyzed by RP-HPLC (YMC Hydrosphere C18 (250 × 20 mm), Elute A) MQ B) ACN, gradient: 0-5 min. ~50% (B), 5-24 minutes 50-80% (B), 24-25 minutes 80-100%. Purification at flow rate 8 mL/min, detection: 250 nm) gave the desired compound 3E (13 mg, 0.03 mmol, 3%) as a white solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.53 (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-d ₆ ): 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 (376 MHz, DMSO-d ₆ ): δ -68.6 (dq, J = 959.6 Hz), -70.6 (dq, J = 963.5 Hz), -116
³¹ P-NMR (162 MHz, DMSO-d ₆ ): δ 8.54 (d, J = 964 Hz), 2.59 (d, J = 964 Hz),
HRMS ₍ ESI ⁺ ) _calcd for _{C15H24F3N4O5P} [M+H] ⁺ : _{429.1436 ,} found for: 429.1581.

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) was added followed by Ca(OH)2 (145.9 mg, 1.87 mmol). The reaction mixture was stirred at rt overnight, filtered using a 0.22 μm membrane filter and the filtrate was purified by column chromatography on silica gel (DCM/MeOH=100/2-100/8) to give the desired compound. 3F (5.66 mg, 0.01 mmol, 2%) was obtained as a white solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.59 (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 = 14Hz)
¹³ C-NMR (100 MHz, DMSO-d ₆ ): 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, 35.54, 18.52, 29.
¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): δ -60.95 (d, J = 987 Hz), -62.38 (d, J = 999 Hz), -113
³¹ P-NMR (162 MHz, DMSO-d ₆ ): δ 6.45 (d, J = 982 Hz), 0.33 (d, J = 1000 Hz)
HRMS ₍ ESI ⁺ ) _calcd for _{C19H26F3N4O5P (} M+H) ⁺ : _479.1593 , found for: 479.1546.

(7) Synthesis of compound 4
Compounds 15 and 25 are nitroreductase-activated prodrugs (Figure 10).

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 hours, then cooled to 0° C., sodium borohydride (5.00 g, 165 mmol) was added and stirred for 27 hours. The reaction mixture was evaporated in vacuo and the residue 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 pore-evaporated 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 obtained as an oil.
¹ H-NMR (400 MHz, Chloroform-d ₁ ): 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 _C10H14N2O2 , [M ₊ H] ₊ : _195.1128 , found for 195.1199 ^.

Compound 13
To a stirred solution of phosphoryl chloride (2.90 mL, 31.0 mmol) in dry CH2Cl2 (20.0 mL) was added 12 (1.00 g, 5.15 mmol) and triethylamine (4.34 mL, 30.9 mmol) in dichloromethane (20.0 mL) at 0°C. was added dropwise and the reaction mixture was stirred at room temperature for 21 hours under an argon atmosphere. The reaction mixture was evaporated in vacuo and the residue partitioned with ethyl acetate and brine. The organic phase was dried over Na2SO4. The filtrate was pit-evaporated without further purification to give the crude desired compound 13 (1.51 g, 4.85 mmol, 94%) as an orange oil.
¹ H-NMR (400 MHz, Chloroform-d ₁ ): 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-d ₁ ): δ 18.7.
HRMS (ESI) m/z calculated for _{C10H13Cl2N2O3P ,} [M ₊ Na] ₊ : _332.9933 , [M ^{+K] + :} 348.9673, found for [M+Na] ⁺ : 332.9938, [M+K] ⁺ : 348.9676.

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. After the reaction mixture was filtered using a 0.22 μm membrane filter and confirmed by ¹⁹ F NMR and ³¹ P NMR, the filtrate was added to the reaction mixture in the next step.
¹⁹ F-NMR (376 MHz, Chloroform-d ₁ ): δ -75.0 (d, JPF = 1033 MHz).
³¹ P-NMR (162 MHz, Chloroform-d ₁ ): δ -2.97 (t, JPF = 1017 MHz).

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.) was added followed by calcium hydroxide (139 mg, 1.86 mmol). The reaction mixture was stirred at room temperature for 22 hours under an argon atmosphere. The mixture was filtered using 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 subjected to RP-HPLC (YMC Triart C18 hydrospheres (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-d ₄ ): 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).
¹³ C-NMR (100 MHz, Methanol-d ₄ ): 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-d ₄ ): δ 4.88 (d, JPF = 1000 MHz), 4.49 (d, JPF = 1010 MHz).
HRMS (ESI) m/z calculated for _{C19H23F3N5O7P ,} [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.

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. After the solution was stirred at room temperature for 19 hours, it was cooled to 0° C., anhydrous sodium borohydride (815 mg, 21.6 mmol) was added, and the mixture was stirred at room temperature for 8 hours. The reaction mixture was evaporated in vacuo and the residue 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 pore-evaporated 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 obtained as an oil.
¹ H-NMR (400 MHz, Chloroform-d ₁ ): 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-d ₁ ): δ162.3, 159.8, 133.1, 132.4, 120.4, 112.3, 48.7, 48.0, 23.0.
¹⁹ F-NMR (376 MHz, Chloroform-d ₁ ): δ -112.1.
HRMS (ESI) m/z calculated for _C10H13FN2O2 , [M ₊ H] ₊ : _213.1034 , found for [M+H] ⁺ : 213.1039 ^.

Compound 23
To a stirred solution of phosphoryl chloride (2.63 mL, 28.2 mmol) in dry CH2Cl2 (10.0 mL) was added 22 (1.00 g, 4.71 mmol) and triethylamine (3.93 mL, 28.2 mmol) in dichloromethane (10.0 mL) at 0°C. was added dropwise and the reaction mixture was stirred at room temperature for 23 hours under an argon atmosphere. The mixture was evaporated and the residue partitioned with ethyl acetate and brine. The organic layer was dried over Na2SO4. The filtrate was evaporated empty without further purification to give the crude desired compound 19 (1.54 g, 4.66 mmol, 99%) as an orange solid.
¹ H-NMR (400 MHz, Chloroform-d ₁ ): 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 = 6.8Hz, 6H).
¹⁹ F-NMR (376 MHz, Chloroform-d ₁ ): δ -111.0.
³¹ P-NMR (162 MHz, Chloroform-d ₁ ): δ 19.0.
HRMS (ESI) m/z calculated for _{C10H12FCl2N2O3P ,} [M ₊ Na] ₊ : _350.9839 , [M ^{+K] + :} 366.9579, found for [M+Na] ⁺ : 350.9839, [M+K] ⁺ : 366.9578.

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. After the reaction mixture was filtered using a 0.22 μm membrane filter and confirmed by ¹⁹ F NMR and ³¹ P NMR, the filtrate was added to the reaction mixture in the next step.
¹⁹ F-NMR (376 MHz, Chloroform-d ₁ ): δ -74.8 (d, JPF = 1034 MHz), -111.5.
³¹ P-NMR (162 MHz, Chloroform-d ₁ ): 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.) was added followed by calcium hydroxide (236 mg, 3.18 mmol). The reaction mixture was stirred at room temperature for 22 hours under an argon atmosphere. The mixture was filtered using 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 hydrospheres (250×20.0 mmL.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%, mixture of diastereomers) as a white solid.
¹ H-NMR (400 MHz, Acetonitrile-d ₃ ): 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). (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, 43.4, 50.4
¹⁹ F-NMR (376 MHz, Acetonitrile-d ₃ ): δ -71.0 (d, JPF = 1004 MHz), -71.2 (d, JPF = 1010 MHz), -114.1, -115.8.
³¹ P-NMR (162 MHz, Acetonitrile-d ₃ ): δ 4.89 (d, JPF = 1004 MHz), 4.76 (d, JPF = 1013 MHz).
_HRMS (ESI) m/z calculated for _{C19H22F4N5O7P} , [M+H] ⁺ : _540.1266 , [M+Na] ⁺ : _562.1085 , [M+K] ⁺ : _578.0825 , [M +Et ₃ NH] ⁺ : 641.2470, found for [M+H] ⁺ : 540.1265, [M+Na] ⁺ : 562.1086, [M+K] ⁺ : 578.0824, [M+Et ₃ NH] ⁺ : 641.2467.

(10) Anticancer action evaluation test This experiment was performed by MTT assay according to the protocol of CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation Assay. Each cell was seeded in 96 well plates (3 plates) at 3000 cells/well. The next day, each concentration of test compound solution (1% DMSO) was added and incubated at 37°C, 5% CO ₂ . After 72 hours, 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Reagent (Promega) was added to each well, incubated at 37°C, 5% CO ₂ for 2 hours, and absorbance at 490 nm was measured using a MithrasLB940 plate reader. , to calculate the anticancer activity of the test compound.

As test compounds, Compound 3C, Compound 3D, Compound 3E, Compound 25, gemcitabine, and Protide (gemcitabine prodrugs introduced protides of existing prodrugs, Non-Patent Document 3) were used. As cells, PK1 (normal pancreatic cancer cells) and gemcitabine-resistant PK1 cells (RPK1) were used.

The results are shown in FIG. It has been found that the prodrugs of the present invention exert anticancer effects.

Claims (14)

General formula (1):

[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). R ² represents a hydrogen atom, an optionally substituted hydrocarbon group, or —OR ¹² (R ¹² represents an optionally substituted hydrocarbon group). R ³ represents a monovalent group formed by removing one hydrogen atom from the hydroxy group of the sugar moiety of the nucleoside analogue. ^R4 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 added thereto. However, R ¹ and R ² may be linked together to form a nitrogen-containing ring together with the adjacent nitrogen atoms. ]
A compound represented by, a salt thereof, or a solvate thereof. 2. The compound, its salt, or its solvate according to claim 1, wherein said hydrocarbon group is an alkyl group, an aryl group, or an aralkyl group. 3. The compound, salt or solvate thereof according to claim 1 or 2, wherein said hydrocarbon group is an alkyl group. 4. The compound, salt thereof, or solvate thereof according to any one of claims 1 to 3, wherein said hydrocarbon group is a branched alkyl group. 5. The compound, salt thereof, or solvate thereof according to any one of claims 1 to 4, wherein said R ¹ is a group other than a hydrogen atom and said R ² is a hydrogen atom. 6. The compound, salt thereof, or solvate thereof according to any one of claims 1 to 5, wherein said R ¹ is a branched alkyl group and said R ² is a hydrogen atom. 3. The compound according to claim 2, wherein said R ¹ and said R ² are each an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted aralkyl group; salts or solvates thereof; 8. The compound, salt thereof, or thereof according to claim 2 or 7, wherein said R ¹ is an alkyl group and said R ² is an optionally substituted aryl group or an optionally substituted aralkyl group solvate of The substituents that the hydrocarbon group may have are 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 phosphoric acid group, 9. The compound, salt thereof, or solvate thereof according to claim 2, 7, or 8, which is at least one selected from the group consisting of a carbonyl group and a carboxy group. A compound, a salt thereof, or a solvate thereof according to any one of claims 1 to 9, wherein said nucleoside analogue is an anticancer agent or an antiviral agent. The R ³ is represented by the general formula (2):

[In the formula: Base represents a monovalent group obtained by removing one hydrogen atom from a nucleic acid base. R ³¹ represents -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); show. R ³² represents -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) or -S- . 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. ^R35 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. ^R36 represents a hydrogen atom, a hydroxy group, a halogen atom, an azide group, an alkynyl group, or a cyano group. R 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 indicates a single bond or a double bond (however, in the case of a double bond, either one of R ³²¹ and R ³²² and either one of R ³³ and R ³⁴ are absent). .). ]
The compound according to any one of claims 1 to 10, a salt thereof, or a solvate thereof, which is a group represented by A medicament containing at least one selected from the group consisting of the compound according to any one of claims 1 to 11, salts thereof, and solvates thereof. The pharmaceutical according to claim 12, which is for anticancer or antiviral use. A reagent containing at least one selected from the group consisting of the compound according to any one of claims 1 to 11, salts thereof, and solvates thereof.

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