Preparation of 2'-deoxy-2',2'-difluorocytidine ProTides and their use

Abstract

This invention relates to a series of 2'-deoxy-2',2'-difluorocytidine monophosphate amide derivatives (compounds of formula I) and methods for preparing their pharmaceutically acceptable salts, as well as their applications in the therapeutic field. Furthermore, this invention provides a series of novel, easily prepared, and highly active 2'-deoxy-2',2'-difluorocytidine-5'-monophosphate amide derivatives, namely ProTide, whose structure is mainly derived from the -5' hydroxyl group in the 2'-deoxy-2',2'-difluorocytidine structure to obtain the corresponding phosphate acyl amino acid ester prodrugs. These compounds exhibit good solubility and blood circulation stability, and can be used as single agents or in combination for the treatment of cancer diseases.

Description

Preparation and application of 2'-deoxy-2',2'-difluorocytidine ProTide Technical Field

[0001] This invention relates to the pharmaceutical field. Specifically, this invention provides the preparation and application of a series of novel 2'-deoxy-2',2'-difluorocytidine monophosphate amides. Background Technology

[0002] 2'-Deoxy-2',2'-Difluorocytidine (gemcitabine) is a cytosine nucleoside derivative with potent antitumor activity, primarily acting in the G1 / S phase and effective against various solid tumors. 2'-Deoxy-2',2'-Difluorocytidine enters the cell via nucleoside transporters (NTs) and is activated by deoxycytidine kinase (dCK) to form 2'-Deoxy-2',2'-Difluorocytidine monophosphate (dFdCMP), which is then converted to 2'-Deoxy-2',2'-Difluorocytidine diphosphate (dFdCDP) and 2'-Deoxy-2',2'-Difluorocytidine triphosphate (dFdCTP). Deoxycytidine kinase (dCK) is a key step in the activation of 2'-Deoxy-2',2'-Difluorocytidine. 2'-Deoxy-2',2'-Difluorocytidine triphosphate (2'-DETP) inhibits DNA polymerase and competitively incorporates into the DNA strand, extending it by one nucleotide and causing DNA strand extension to terminate and preventing recognition by DNA repair enzymes. 2'-Deoxy-2',2'-Difluorocytidine diphosphate (2'-DETP) inhibits ribonucleic acid reductase, reducing the production of deoxyribonucleoside triphosphate (DRPT). 2'-Deoxy-2',2'-Difluorocytidine itself inhibits Top1 inhibitors and thymidine nucleotide synthase. 2'-Deoxy-2',2'-Difluorocytidine triphosphate (2'-DETP) can also be incorporated into RNA synthesis, leading to ineffective RNA synthesis. All these effects of 2'-Deoxy-2',2'-Difluorocytidine contribute to apoptosis.

[0003] However, drug resistance often develops during the use of 2'-deoxy-2',2'-difluorocytidine. The main resistance mechanisms include: the lack of nucleoside transporters (NTs) prevents 2'-deoxy-2',2'-difluorocytidine from entering cells; the lack of deoxycytidine kinase (dCK) prevents 2'-deoxy-2',2'-difluorocytidine from forming the key dFdCMP; and deoxycytidine deaminase (dCDA) deaminates 2'-deoxy-2',2'-difluorocytidine to form ineffective dFdU.

[0004] This invention involves derivatization at the 5'-OH of 2'-deoxy-2',2'-difluorocytidine to form a series of 2'-deoxy-2',2'-difluorocytidine-5-phosphate amide derivatives. These compounds can circumvent deoxycytosine kinase resistance and evade the action of deoxycytidine deaminase. These derivatives possess high antitumor activity and low toxicity, and can be used as monotherapy or in combination therapy for cancer treatment. Summary of the Invention

[0005] In a first aspect of the invention, a compound of formula (I), a tautomer, a stereoisomer, a hydrate, a solvate, a prodrug, or a pharmaceutically acceptable salt thereof is provided:

[0006]

[0007] in,

[0008] R1 is a hydrogen, halogen, cyano, amino, carboxyl, or a substituted or unsubstituted group selected from the group consisting of: C 1-6 Alkyl, C 1-6 Ester group, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 3-8 Saturated or partially unsaturated carbocyclic groups, saturated or partially unsaturated 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-12 heteroaryl, -C 1-6 Alkyl-NH2, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C 1-6 Alkyl-NH-C(=O)-NH2, -C 1-6 Alkyl-C 6-10 Aryl and -C 1-6 Alkyl-5-12-membered heteroaryl;

[0009] R2 is H or a substituted or unsubstituted group selected from the group consisting of: C 1-6 Alkyl, C 1-6 Ester group, carboxyl group C 1-6 Alkyl, carboxyl C 3-8 carbonyl group, C 1-6 alkylamine group, C 6-10 Aryl, -C 1-6 Alkyl-C 6-10 Aryl, -C 1-6 Alkyl-5-12-membered heteroaryl, C 3-8 Saturated or partially unsaturated carbocyclic groups, 3-10 membered saturated or partially unsaturated heterocyclic groups, 5-12 membered heteroaryl groups;

[0010] R3 is selected from the following groups: H, C1-6 Ester group, carboxyl group C 1-6 Alkyl, C 1-6 Alkylamine group, α-aminocarboxyl group C 1-6 Alkyl, α-aminocarboxylic acid C 2-6 Acyl-α-aminocarboxyl C 1-6 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-6 Alkyl, C 3-8 Saturated or partially unsaturated carbocyclic groups, 3-10 membered saturated or partially unsaturated heterocyclic groups, 5-12 membered heteroaryl groups;

[0011] X is selected from the following group: O or NH;

[0012] R4 is selected from the following group: C 1-6 Alkyl, C 2-6 Ester group, C 1-6 alkylamine group, C 6-10 Aryl, C 1-6 Alkyl-C 6-10 Aryl, C 3-8 Saturated or partially unsaturated carbon cyclogroups, C 3-8 Saturated or partially unsaturated carbocyclic amino groups, 3-10 membered saturated or partially unsaturated heterocyclic groups, 5-12 membered heteroaryl groups;

[0013] Furthermore, the substitution in the context of substituted or unsubstituted means that the substituent is replaced by one or more substituents selected from the group consisting of: halogen, hydroxyl, amino, carboxyl, C. 1-4 Alkyl, C 6-10 Aryl, C 3-8 Saturated or partially unsaturated carbocyclic groups, 3-10 membered heterocyclic groups, and 5-12 membered heteroaryl groups.

[0014] In some embodiments, R1 is H or a substituted or unsubstituted group selected from the group consisting of: C 1-6 Alkyl, -C 1-6 Alkyl-NH2, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C 1-6 Alkyl groups -NH-C(=O)-NH2 and -C 1-6 Alkyl-C 6-10 Aryl.

[0015] In another preferred embodiment, R1 is selected from the following group: H,

[0016] In some implementations, R2 and R3 are each independently selected from the following groups: H, C 1-6 Ester group.

[0017] In some implementations, R4 is selected from the group C 1-6 Alkyl, C 1-6 alkylamine group, C 1-6 Alkyl-C 6-10 Aryl, C 3-8 Saturated or partially unsaturated carbon cyclogroups, C 3-8 Saturated or partially unsaturated carbocyclic amino groups, 3-10 membered saturated or partially unsaturated heterocyclic groups.

[0018] In some embodiments, the compounds are selected from the group consisting of:

[0019]

[0020]

[0021] In a second aspect of the invention, a method for preparing a compound as shown in formula (I) is provided, comprising the steps of:

[0022] 1) Reacting 2'-deoxy-2',2'-difluorocytidine with tert-butyloxycarbonic anhydride yields the corresponding 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside;

[0023] 2) React 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside with phenyl dichlorophosphate, and then react with L-amino acid esters to obtain the corresponding 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside-5'-phosphate (phenyl ester) acyl amino acid esters;

[0024] 3) The 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside-5'-phosphate monophenyl ester amino acid ester was reacted with trifluoroacetic acid to obtain the corresponding 4-amino-1-[2',2'-difluoro-2'-deoxy]cytosine nucleoside-5'-phosphate (phenyl ester) amino acid ester.

[0025] In some embodiments, the amount of tert-butyloxycarbonic anhydride used in step 1) is 2.1-2.2 molar equivalents;

[0026] In another preferred embodiment, in step 2), the amount of phenyl dichlorophosphate used is 1-1.1 molar equivalents, and the amount of amino acid ester used is 1.0-1.2 molar equivalents;

[0027] In another preferred embodiment, the amount of amino acid ester used in step 2) is 1.0-1.2 molar equivalents.

[0028] In another preferred embodiment, the amino acid ester described in step 2) is an amino acid cycloalkyl alcohol ester.

[0029] In another preferred embodiment, the amino acid ester described in step 2) is a cycloalcohol containing 6 atoms, such as piperidine-4-ol, 1,4-cyclohexanediol, or 4-aminocyclohexanol.

[0030] In another preferred embodiment, the trifluoroacetic acid in step 3) is a 10-30% trifluoroacetic acid solution.

[0031] In a third aspect of the invention, a pharmaceutical composition is provided, characterized in that it comprises: (1) a compound, tautomer, stereoisomer, hydrate, solvate, prodrug, or pharmaceutically acceptable salt thereof, or a combination thereof, as described in the first aspect of the invention; and (2) one or more pharmaceutically acceptable carriers, excipients, adjuvants, excipients, and / or diluents.

[0032] In a fourth aspect of the invention, there is provided the use of a compound, a tautomer, a stereoisomer, a hydrate, a solvate, a prodrug, or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention, or a pharmaceutical composition as described in the third aspect of the invention, characterized in that it is used for treating tumors.

[0033] In another preferred embodiment, the tumor is selected from the group consisting of: breast adenocarcinoma and gastric cancer.

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0035] Through extensive and in-depth research, the inventors unexpectedly discovered a series of phosphate ester amide derivatives of 2'-deoxy-2',2'-difluorocytidine. Bioactivity tests revealed that these derivatives exhibit high antitumor activity and low toxicity. Based on this, the present invention was completed.

[0036] definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] As used herein, the term "alkyl" includes straight-chain or branched alkyl groups. For example, C1-C6 alkyl groups refer to straight-chain or branched alkyl groups having 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.

[0039] As used herein, the term "alkenyl" includes straight-chain or branched alkenyl groups. For example, C2-C6 alkenyl refers to straight-chain or branched alkenyl groups having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.

[0040] As used herein, the term "alkynyl" includes straight-chain or branched alkynyl groups. For example, C2-C6 alkynyl refers to straight-chain or branched alkynyl groups having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or similar groups.

[0041] As used herein, the term "cycloalkyl" refers to a cyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, C3-C 10 Alkenyl groups refer to cyclic saturated aliphatic hydrocarbon groups having 3-10 carbon atoms. They can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. They can also be bicyclic, such as bridged or spirocyclic forms.

[0042] As used herein, the term "alkylamino" refers to an amino group substituted with an alkyl group. For example, "C1-C6 alkylamino" refers to an amino group substituted with a C1-C6 alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, ditert-butylamino, etc.

[0043] As used herein, the term "alkoxy" refers to a group having an alkyl-oxy group structure. For example, "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1-6 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.

[0044] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen, wherein the definition of alkyl is as described above.

[0045] As used herein, the term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by a halogen, wherein the definition of alkoxy is as described above.

[0046] As used herein, the term "heterocyclic group" or "heterocyclic alkyl group" refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (e.g., 3-10 ring atoms), wherein 1-3 of these atoms are heteroatoms selected from N, S, and O. It can be monocyclic, bicyclic, or polycyclic, such as bridged or spirocyclic forms. Specific examples include oxobutyranyl, azabutyranyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl, etc.

[0047] As used in this article, the term "C6-C" 10 "Aryl" refers to an aryl group having 6-10 carbon atoms, such as phenyl or naphthyl groups.

[0048] As used herein, the term "5-12-membered heteroaryl" refers to a cyclic aromatic group having 5-12 atoms, of which 1-3 atoms are heteroatoms selected from the group consisting of N, S, and O. It can be monocyclic or fused-ring. Specific examples include pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrroleyl, pyrazolyl, imidazoleyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thiophenyl, isoxazolyl, thiazolyl, oxazolyl, etc.

[0049] Unless otherwise specified as "substituted or unsubstituted", the groups described in this invention may be substituted by substituents selected from the group consisting of: halogen, nitrile, nitro, hydroxyl, amino, C1-C6 alkyl-amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halo-C1-C6 alkoxy, allyl, benzyl, C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, etc.

[0050] As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl, and Br. "Halogenated" means substituted by an atom selected from F, Cl, Br, and I.

[0051] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers with double bonds, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or geometric isomers (or conformational isomers), is within the scope of this invention.

[0052] As used herein, the term "tautomer" refers to structural isomers with different energies that can cross a low energy barrier and thus interconvert. For example, proton tautomers (i.e., proton shifts) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via some bonding electron recombination.

[0053] As used herein, the term "solvent complex" refers to a complex of the compound of the present invention coordinated with solvent molecules in a specific ratio.

[0054] As used herein, the term "hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0055] The term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0056] Active ingredients

[0057] This invention provides an active ingredient with high antitumor activity. The active ingredient is a compound represented by general formula (I), which can effectively treat tumor diseases.

[0058] Experiments have shown that the active ingredients of this invention can effectively inhibit tumor growth, thereby treating tumor diseases.

[0059] It should be understood that the active ingredients of the present invention include compounds represented by general formula (I), pharmaceutically acceptable salts thereof, or prodrugs thereof. It should also be understood that the active ingredients of the present invention include crystalline forms, amorphous compounds, and deuterated compounds of general formula (I).

[0060] Pharmaceutical Compositions and Administration

[0061] Because the compounds of the present invention have excellent antitumor activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat tumor diseases.

[0062] The pharmaceutical compositions of the present invention comprise the compound of the present invention within a safe and effective range and a pharmaceutically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0063] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0064] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous, intramuscular, or subcutaneous).

[0065] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0066] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0067] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0068] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0069] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0070] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0071] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0072] When administered in combination, the pharmaceutical composition further comprises one or more (two, three, four, or more) other pharmaceutically acceptable therapeutic agents. One or more (two, three, four, or more) of these other pharmaceutically acceptable therapeutic agents may be used simultaneously, separately, or sequentially with the compounds of the present invention to treat oncological diseases.

[0073] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0074] The main advantages of this invention include:

[0075] (1) All compounds of the present invention have good antitumor activity.

[0076] (2) The compounds of the present invention have low toxicity.

[0077] (3) The compounds of the present invention have good pharmacokinetic properties and good drug-likeness.

[0078] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0080] Unless otherwise specified, all experimental materials and reagents used in the following examples are available from commercially available sources.

[0081] Example

[0082] Synthesis of 3',4-bis(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine

[0083] 2'-Deoxy-2',2'-Difluorocytidine (20.0 g, 76 mmol) and Na2CO3 (40.3 g, 380 mmol) were added to a reaction flask containing dioxane and water (5:1, 400 mL). The mixture was stirred at room temperature, and tert-butyloxycarbonic anhydride (16.6 g, 76 mmol) was added. The mixture was stirred at room temperature for another 48 hours. The solid was filtered off, the filtrate was concentrated, and the product was recrystallized from ethyl acetate to give a white solid product, 3'-tert-butyloxycarbonyl-2'-deoxy-2',2'-difluorocytidine (25.6 g, 93% yield).

[0084] The above-mentioned 3'-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine (25.5 g, 70.2 mmol) and tert-butoxycarbonic anhydride (153.1 g, 702 mmol) were dissolved in dioxane (250 mL), stirred at 40 °C for 48 hours, the solvent was concentrated, and petroleum ether was added to precipitate the solid product 3',4-bis(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (27.5 g, yield 85%).

[0085] General Method A: Preparation of Amino Acid Esters

[0086] Fmoc-amino acids (1 eq) were dissolved in DCM, and the corresponding alcohols (1 eq), triethylamine (2 eq), DCC (2 eq), and HOBt (2 eq) were added respectively. The mixture was stirred overnight at room temperature, and the resulting solid was filtered. The filtrate was washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the corresponding Fmoc-amino acid esters.

[0087] The above-mentioned Fmoc-amino acid ester (1 eq) was dissolved in DCM, and diethylamine (1 eq) was added. The mixture was stirred overnight at room temperature, the reaction solution was concentrated, and the corresponding amino acid ester was obtained by silica gel column chromatography.

[0088] General Method B: Preparation of Amino Acid Esters

[0089] Under ice bath cooling, thionyl chloride was added dropwise to a solution containing amino acids and corresponding alcohols, and then stirred at room temperature for 12 hours. The solvent was concentrated, DCM was added, the pH was adjusted to 9 with potassium carbonate, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain an amino acid ester that could be used directly in the next reaction.

[0090] General Method C: 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-citrulline benzyl ester (1)

[0091] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (300 mg, 0.65 mmol), DCM (15 mL), and triethylamine (131 mg, 1.3 mmol) were added to a reaction flask and cooled to 0°C. A solution of phenyl dichlorophosphate (137 mg, 0.65 mmol) in DCM (15 mL) was added with stirring. The mixture was stirred at room temperature for 4 hours, then cooled to 0°C. A solution of L-Cit-OBn (172 mg, 0.65 mmol, prepared by general method 1) and triethylamine (131 mg, 1.3 mmol) in DCM (10 mL) was added with stirring. The mixture was stirred overnight at room temperature. The solvent was concentrated, and the solution was purified by silica gel column chromatography to obtain 3',4-di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phenyl phosphate acyl-L-citrulline benzyl ester (150 mg, yield 27%, HPLC). 96%); LCMS:(M+1) + :867.44 (Calculated value:866.31).

[0092] General Method D: 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-citrulline benzyl ester (2)

[0093] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-citrulline benzyl ester (150 mg, 0.17 mmol) was added to a reaction flask, followed by the addition of trifluoroacetic acid (3 mL). The mixture was stirred overnight at room temperature. The solvent was concentrated, ammonia water was added for neutralization, and the mixture was concentrated under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain solid gemcitabine-5'-phosphophenyl ester-L-citrulline benzyl ester (80 mg, yield 72%, HPLC 98%). 1 H NMR(500MHz,DMSO-d6)δ9.01(s,1H),8.68(s,1H),7.71-7.58(m,1H),7.27(m ,J=6.7,5.6Hz,8H),7.09(t,J=8.7Hz,4H),6.09(dt,J=23.2,9.6Hz,2H),5.9 9-5.80(m,2H),5.00(d,J=7.6Hz,2H),4.31-3.94(m,7H),3.72(m,J=15.5,7. 6Hz,2H),2.81(dt,J=15.0,6.8Hz,2H),1.66-1.41(m,3H),1.35-1.19(m,2H); 13CNMR(126MHz,DMSO)δ173.29,161.91,159.28,151.12,150.02,143.21,136.27,130.11,129.84,128.93,128.55,128.45,128.36,128.33, 125.13,122.84,120.77,120.61,120.54,95.48,84.29,79.38,69.76,66.51,64.75,54.70,39.06,31.11,30.89,27.29,26.69; LCMS: (M+1) + :667.10 (Calculated value:666.20).

[0094] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phenylphosphate-L-citrulline isopropyl ester (3)

[0095] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-citrulline isopropyl ester was prepared according to method C (yield 42%, HPLC 95%); LCMS: (M+1) + :819.37 (Calculated value:818.31).

[0096] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-citrulline isopropyl ester (4)

[0097] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-phenylalanine isopropyl ester was synthesized according to method D (yield 57%, HPLC 97%). 1H NMR (500MHz, DMSO-d6) δ7.77(s,1H),7.64(s,1H),7.52(dd,J=13.0,7.6Hz,1H),7.42-7.32(m,2H),7.19(q,J=8.1,7.1 Hz,3H),6.50(s,1H),6.17(q,J=9.1Hz,1H),6.09(t,J=11.4Hz,1H),5.98(dt,J=17.1,5.8Hz,1H),5.82(t,J=8.9Hz,1H ),5.42(s,2H),4.87(dp,J=12.1,6.2Hz,1H),4.42-4.11(m,3H),4.03(dt,J=24.8,6.9Hz,1H),3.72-3.63(m,1H),2.90 (dp,J=13.2,7.2,6.5Hz,2H),1.69-1.45(m,2H),1.35(dp,J=23.6,8.9,7.5Hz,2H),1.16(d,J=6.0Hz,6H); LCMS: (M+1) + :619.02 (Calculated value:618.20).

[0098] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-alanine (1-Boc-piperidin-4-ol) ester (5)

[0099] Fmoc-L-alanine (1-Boc piperidine-4-ol) ester was synthesized according to general method A (yield 39%), followed by deprotection of the Fmoc protecting group with piperidine to give L-alanine (1-Boc piperidine-4-ol) ester (yield 78%). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-alanine (1-Boc-piperidine-4-ol) ester was synthesized according to general method C (yield 55%, HPLC 98%). 1H NMR (500MHz, CDCl3) δ7.59-7.52(m,1H),7.33-7.04(m,7H),6.36(ddd,J=17.2,11.4,5.8Hz, 1H),5.17-5.03(m,1H),4.89(tt,J=8.0,4.1Hz,1H),4.48-4.37(m,1H),4.36-4.20(m,2H),4. 09-3.93(m,1H),3.75-3.54(m,3H),3.23-3.06(m,2H),1.83-1.72(m,3H),1.53(td,J=8.3,3 .8Hz,5H),1.44(dd,J=3.1,2.2Hz,18H),1.39(d,J=1.7Hz,9H),1.33(dd,J=13.1,7.1Hz,3H); 13 C NMR (126MHz, CDCl3) δ172.62,163.06,154.68,151.55,150.94,150.46,144.52,129.85,125.37,120.07,95. 64,84.77,83.10,79.79,78.04,72.26,71.39,64.21,50.38,30.38,28.41,28.01,27.57,20.99; LCMS: (M+H) + :873.97 (Calculated value:873.34).

[0100] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-alanine (piperidin-4-ol) ester (6)

[0101] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-alanine (piperidin-4-ol) ester was synthesized according to method D (yield 77%, HPLC 98%). 1H NMR(500MHz,DMSO-d6)δ8.80(s,1H),7.60-7.46(m,2H),7.42-7.35(m,2H),7.26-7.08(m,5H),6.51(s ,1H),6.20(m,J=16.2,10.1,4.9Hz,2H),5.85-5.74(m,1H),4.92(m,J=7.2,3.5Hz,1H),4.42-4.11(m, 3H),4.04(dd,J=21.9,6.9Hz,1H),3.97-3.80(m,1H),3.18(q,J=10.3,9.1Hz,2H),3.08(q,J=9.3,7.1 Hz,2H),1.95(m,J=13.0,8.3,3.9Hz,2H),1.73(m,J=13.2,9.8,6.4,5.9Hz,2H),1.27(t,J=7.6Hz,3H); 13 CNMR(126MHz,DMSO)δ172.81,165.77,154.65,151.11,141.68,130.18,125.19,12 0.53,95.38,78.71,69.95,67.24,64.92,50.52,50.37,27.23,20.10; LCMS: (M+H) + :574.01 (Calculated value:573.18).

[0102] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phenylphosphate-L-ε-Trt-lysine cyclohexanol ester (7)

[0103] Fmoc-L-ε-Trt-lysine cyclohexanol ester was synthesized according to general method A (yield 39%), followed by deprotection of the Fmoc protecting group with piperidine to give L-ε-Trt-lysine cyclohexanol ester (yield 80%). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-ε-Trt-lysine cyclohexanol ester was synthesized according to general method C (yield 56%, HPLC 97%). 1H NMR (500MHz, CDCl3) δ7.67 (dd, J=7.6, 1.9Hz, 1H), 7.57-7.49 (m, 1H), 7.37 (m, J=7.4, 3.8, 2.2Hz, 6H) ,7.28-6.96(m,16H),6.34(m,J=18.2,11.8,6.1Hz,1H),5.12-4.98(m,1H),4.70(d,J=7.0Hz,1H),4. 37(m,J=12.1,5.7,2.6Hz,1H),4.34-4.15(m,2H),3.97-3.77(m,1H),3.63-3.42(m,1H),1.97(m,J=6 .9,3.2Hz,2H),1.77-1.55(m,8H),1.43(m,J=5.3,2.8Hz,19H),1.38-1.23(m,8H),1.23-1.14(m,3H); 13 C NMR (126MHz, CDCl3) δ172.23,163.07,154.69,151.51,150.89,150.50,146 .22,144.52,129.86,129.81,128.62,127.78,126.22,125.25,120.14,120. 01,95.67,84.74,83.00,77.97,74.16,72.58,70.84,64.15,54.56,43.30, 34.58,31.45,30.41,28.03,27.57,27.00,25.22,23.62,22.60; LCMS: (M+H) + :1072.03 (Calculated value:1071.46).

[0104] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-lysine cyclohexanol ester (8)

[0105] 2'-Deoxy-2',2'-Difluorocytidine-5'-phenylphosphate-L-lysine cyclohexanol ester was synthesized according to method D (yield 52%, HPLC 97%). 1H NMR(600MHz,DMSO-d6)δ7.99-7.85(m,2H),7.74(d,J=11.7Hz,1H),7.62-7.42 (m,5H),6.57(s,1H),6.22-5.99(m,2H),5.88-5.75(m,1H),4.64(qt,J=8.6,4 .1Hz,1H),4.39-4.16(m,3H),4.07-3.98(m,1H),3.73-3.62(m,2H),2.67(dt, J=16.3,7.6Hz,2H),1.78-1.57(m,5H),1.56-1.40(m,4H),1.39-1.15(m,8H); 13 C NMR (151MHz, DMSO) δ172.86,165.91,159.23,159.02,158.82,158.61,130.10,118.58,116.60,95.47,9 5.35,73.08,54.80,54.59,48.36,38.27,33.20,30.83,26.89,26.52,25.24,23.46,22.41; LCMS: (M+H) + :630.13 (Calculated value:629.24).

[0106] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-phenylalanine (1-Boc-piperidin-4-ol) ester (9)

[0107] Fmoc-L-phenylalanine (1-Boc piperidine-4-ol) ester was synthesized according to general method A (yield 69%), followed by deprotection of the Fmoc protecting group with piperidine to give L-phenylalanine (1-Boc piperidine-4-ol) ester (yield 92%). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenylesteryl-L-phenylalanine (1-Boc-piperidine-4-ol)

[0108] The ester was synthesized according to general method C (yield 45%, HPLC 96%). 1H NMR (500MHz, CDCl3) δ7.51-7.45(m,1H),7.30-7.01(m,15H),6.32(m,J=14 .7,5.6,5.0Hz,1H),5.01(m,J=13.9,5.7,2.8Hz,1H),4.83(m,J=13.9,9.2 ,4.8Hz,1H),4.27-4.08(m,3H),3.70-3.41(m,4H),3.15-3.03(m,3H),2.9 9-2.92(m,2H),1.75-1.64(m,3H),1.46-1.43(m,16H),1.40-1.36(m,13H); 13 C NMR (126MHz, CDCl3) δ171.68,163.19,154.65,151.55,151.00,150.41,144.37,135.55,129.89,129.84,129.56,129.47,128.70,127 .27,125.34,120.14,95.73,84.77,82.92,79.78,72.39,71.46,64.00,56.01,55.69,40.36,30.38,28.41,28.02,27.57; LCMS: (M+H) + :950.11 (Calculated value:949.37).

[0109] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-phenylalanine (piperidin-4-ol) ester (10)

[0110] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-phenylalanine (piperidin-4-ol) ester was synthesized according to method D (yield 57%, HPLC 94%). 1H NMR (500MHz, DMSO-d6) δ8.79(d,J=58.8Hz,3H),7.68(m,J=15.6,14.0,7.5Hz,1H),7.26(m,J=43.0,18.4,17.1,8.2H z,9H),7.08-7.00(m,2H),6.39(dt,J=21.2,11.3Hz,1H),6.15(dt,J=15.8,8.0Hz,1H),5.99(m,J=24.9,13.1,12.1,7 .0Hz,1H),4.86(d,J=6.4Hz,1H),4.14-4.11(m,3H),4.06-3.99(m,6H),3.20-3.13(m,1H),3.02(m,J=31.4,13.9,7.5 Hz,5H),2.87(dd,J=13.6,7.9Hz,1H),2.00-1.75(m,3H),1.67(dt,J=13.1,6.6Hz,1H),1.54(dd,J=15.1,8.5Hz,1H); 13 C NMR (126MHz, DMSO) δ171.81,158.90,151.01,142.91,137.22,130.12,129.83,128.78,127.18,125.15,120.46,1 20.34,118.46,116.10,95.52,79.13,69.81,68.23,67.16,64.51,62.99,57.31,36.59,31.74,26.67; LCMS: (M+H) + :650.10 (Calculated value:649.21).

[0111] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-valine (piperidin-4-ol) ester (11)

[0112] Synthesis of Fmoc-Val-(1-N-Boc piperidine-4-ol) ester: Fmoc-Val-(1-N-Boc piperidine-4-ol) ester was synthesized using Fmoc-Val-OH and 1-N-Boc piperidine-4-ol according to method A (7.1 g, yield 77%).

[0113] Synthesis of Val-(1-N-Boc piperidine-4-ol) ester: Fmoc-Val-(1-N-Boc piperidine-4-ol) ester was reacted with diethylamine according to method A to give Val-(1-N-Boc piperidine-4-ol) ester (0.86 g, yield 64%).

[0114] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-valine (1-Boc-piperidin-4-ol) ester was synthesized by general method C (yield 48%, HPLC 97%). 1 H NMR (500MHz, CDCl3) δ7.7-7.68(m,1H),7.55(dt,J=9.9,4.9Hz,1H),7.31-7.05(m,7H),6.36(m,J=17.8,11 .1,6.0Hz,1H),5.18-5.02(m,1H),4.90(m,J=7.2,3.5Hz,1H),4.41(m,J=11.7,6.1,2.5Hz,1H),4.35-4.19( m,2H),3.77(m,J=9.8,5.1Hz,1H),3.61(s,2H),3.55-3.45(m,1H),3.19-3.10(m,2H),2.08-1.98(m,2H),1 .78(d,J=12.2Hz,3H),1.57-1.49(m,3H),1.44(t,J=3.2Hz,19H),1.39(d,J=3.1Hz,9H),0.91-0.78(m,6H); 13 C NMR (126MHz, CDCl3) δ171.83,163.06,154.68,151.50,150.84,150.55,144.82,129.81,125.27,120.11,95.56, 84.75,83.13,79.77,72.42,71.27,64.19,59.99,32.17,30.49,28.41,28.01,27.55,18.95,17.18; LCMS: (M+H) + :902.21 (Calculated value:901.37).

[0115] 2'-Deoxy-2',2'-Difluorocytidine-5'-phenylphosphate yl-L-valine (piperidin-4-ol) ester was synthesized according to method D (yield 93%, HPLC 94%); LCMS: (M+H) + :602.10 (Calculated value: 601.21).

[0116] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-leucine (piperidin-4-ol) ester (12)

[0117] Synthesis of Fmoc-Leu-(1-N-Boc piperidine-4-ol) ester: Fmoc-Leu-(1-N-Boc piperidine-4-ol) ester was synthesized using Fmoc-Leu-OH and 1-N-Boc piperidine-4-ol according to method A (10.8 g, yield 89%).

[0118] Synthesis of Leu-(1-N-Boc piperidine-4-ol) ester: Fmoc-Leu-(1-N-Boc piperidine-4-ol) ester was reacted with diethylamine according to method A to give Leu-(1-N-Boc piperidine-4-ol) ester (1.3 g, yield 51%).

[0119] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-leucine (1-Boc-piperidin-4-ol) ester was synthesized by general method C (yield 43%, HPLC 95%); LCMS: (M+H) + :916.10 (Calculated value:915.38).

[0120] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-leucine (piperidin-4-ol) ester was synthesized according to method D (yield 94%, HPLC 93%). 1 H NMR(500MHz,DMSO-d6)δ8.73(d,J=50.8Hz,2H),7.79(dt,J=14.9,7.0Hz,1H),7.42-7.33( m,2H),7.19(m,J=10.9,7.6,3.6Hz,3H),6.22-5.99(m,3H),4.95(m,J=7.0,3.5Hz,2H),4. 43-4.06(m,7H),3.66-3.49(m,1H),3.19(m,J=9.6,8.8,4.0Hz,2H),3.15-3.02(m,2H),1. 95(m,J=13.6,8.2,7.5Hz,3H),1.70(dt,J=16.9,9.7Hz,2H),0.83(dd,J=30.9,6.6Hz,6H); 13 C NMR (126MHz, DMSO) δ171.83,151.12,143.47,130.14,125.19,122.79,120.44,118.13,115.78,113.43,95. 52,79.45,69.80,67.14,65.07,64.87,60.89,31.84,31.62,27.27,19.34,19.26,18.47,18.38; LCMS: (M+H) +:616.10 (Calculated value:615.23).

[0121] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-ε-Trt-lysine (tetrahydropyran-4-ol) ester (13)

[0122] Fmoc-L-ε-Trt-lysine (tetrahydropyran-4-ol) ester was synthesized according to general method A (4.2 g, 92% yield), followed by deprotection of the Fmoc protecting group with piperidine to give L-ε-Trt-lysine (tetrahydropyran-4-ol) ester (1.7 g, 61% yield). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-ε-Trt-lysine (tetrahydropyran-4-ol) ester was synthesized according to general method C (0.18 g, 27% yield, HPLC: 96%); LCMS: (M+H). + :1074.45 (Calculated value:1073.44).

[0123] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-lysine cyclohexanol ester (14)

[0124] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-lysine (tetrahydropyran-4-ol) ester was synthesized according to method D (0.078 g, yield 78%, HPLC 97%). 1 H NMR (500MHz, DMSO-d6) δ7.89-7.68(m,3H),7.55-7.46(m,1H),7.37(td,J=7.8,2. 5Hz,2H),7.24-7.14(m,3H),6.51(s,1H),6.26-6.03(m,2H),5.86-5.74(m,1H),4 .40-4.08(m,3H),4.08-3.88(m,3H),3.73(qd,J=9.4,5.4Hz,1H),2.69(dt,J=15. 7,7.5Hz,2H),1.70-1.39(m,5H),1.29(pd,J=7.1,3.1Hz,6H),0.90-0.78(m,6H); 13C NMR (126MHz, DMSO) δ173.26,165.63,154.45,151.11,141.60,130.12,125.10,120.66,120.46,9 5.36,78.82,69.99,66.66,54.59,52.19,38.98,33.13,26.92,22.99,22.44,11.22; LCMS: (M+H) + :632.20 (Calculated value:631.22).

[0125] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-ε-Trt-lysine-1,4-cyclohexanediol monoester (15)

[0126] Fmoc-L-ε-Trt-lysine 1,4-cyclohexanediol monoester was synthesized according to general method A (2.5 g, 54% yield), followed by deprotection of the Fmoc protecting group with piperidine to give L-ε-Trt-lysine 1,4-cyclohexanediol monoester (0.78 g, 44% yield). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-ε-Trt-lysine 1,4-cyclohexanediol monoester was synthesized according to general method C (0.18 g, 57% yield, HPLC 94%); LCMS: (M+H) + :1088.10 (Calculated value:1087.45).

[0127] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-lysine 1,4-cyclohexanediol monoester (16)

[0128] 2'-Deoxy-2',2'-Difluorocytidine-5'-phenylphosphate-L-lysine 1,4-cyclohexanediol monoester was synthesized according to method D (0.08 g, yield 35%, HPLC 96%); LCMS: (M+H) + :646.20 (Calculated value:645.24).

[0129] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-alanine(4-Bocamino)cyclohexanol ester (17)

[0130] Fmoc-L-alanine (4-Bocamino)cyclohexanol ester was synthesized according to general method A (4.0 g, yield 61%), followed by deprotection of the Fmoc protecting group with piperidine to give L-alanine (4-Bocamino)cyclohexanol ester (1.5 g, yield 67%). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-alanine (4-Bocamino)cyclohexanol ester was synthesized according to general method C (0.40 g, yield 70%, HPLC 95%). 1 H NMR (600MHz, DMSO-d6) δ10.59(s,1H),7.96(dd,J=23.8,7.7Hz,1H),7.37(q,J=8.0Hz,2H),7.22(d,J=8.0Hz,1H),7.19(t, J=7.4Hz,2H),7.08(dd,J=25.3,7.7Hz,1H),6.72(d,J=8.1Hz,1H),6.34(dt,J=25.4,8.0Hz,1H),6.18-6.12(m,1H),5.26(s ,1H),4.57-4.45(m,2H),4.44-4.29(m,2H),3.81(dh,J=14.0,7.1Hz,1H),3.24(d,J=10.6Hz,1H),1.85-1.80(m,2H),1.77( d,J=10.8Hz,2H),1.47(s,9H),1.45(d,J=3.4Hz,9H),1.37(d,J=2.4Hz,9H),1.32(d,J=23.1Hz,3H),1.24(d,J=6.7Hz,3H); 13 C NMR (151MHz, DMSO-d6) δ173.20,173.03,164.21,155.31,154.29,152.35,151.56,151. 10,151.06,151.02,130.12,130.07,125.13,121.63,120.61,120.59,120.56,95.75,9 5.70,84.36,81.86,77.91,77.28,72.91,64.68,64.54,50.40,50.27,48.28,31.61,30 .29,30.15,29.84,29.48,28.72,28.20,27.57,20.20,20.15,20.11,20.07; LCMS: (M+H) + :888.50 (Calculated value:887.35).

[0131] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-alanine (4-amino)cyclohexanol ester (18)

[0132] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-alanine (4-amino)cyclohexanol ester was synthesized according to method D (0.075 g, yield 93%, HPLC 97%). 1 H NMR (500MHz, DMSO-d6) δ8.35 (s, 1H), 7.89 (m, 3H), 7.72-7.54 (m, 1H), 7.38 (dt, J = 7.7, 4. 8Hz,1H),7.19(dt,J=18.2,8.4Hz,2H),6.64-6.37(m,1H),6.15(dt,J=15.1,13.0,9.2Hz, 1H),5.94-5.85(m,1H),4.55(s,1H),4.40-3.99(m,5H),3.81(m,J=15.1,7.6Hz,2H),3.03 (s,1H),1.93(q,J=19.3,15.8Hz,3H),1.51-1.33(m,4H),1.28-1.19(m,2H),1.11(s,1H); 13 C NMR (126MHz, DMSO) δ172.71,170.05,150.81,142.20,130.14,129.78,125.14,12 0.63,95.19,73.09,72.12,50.25,49.20,48.34,28.90,28.24,27.30; LCMS: (M+H) + :588.24 (Calculated value:587.20).

[0133] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-phenylalanine (4-Boc amino)cyclohexanol ester (19)

[0134] Fmoc-L-phenylalanine (4-Bocamino)cyclohexanol ester was synthesized according to general method A (4.5 g, 75% yield), followed by deprotection of the Fmoc protecting group with piperidine to give L-phenylalanine (4-Bocamino)cyclohexanol ester (2.2 g, 78% yield). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl ester-L-phenylalanine (4-Bocamino)cyclohexanol ester was synthesized according to general method C (0.28 g, 46% yield, 94%). 1H NMR(600MHz,DMSO-d6)δ10.59(s,1H),7.96-7.88(m,1H),7.34-7.12(m,7H),7.05(ddd,J=21.4,17.0,7.8Hz,3 H),6.70(t,J=8.1Hz,1H),6.30(dq,J=13.2,9.3,8.1Hz,2H),5.18(s,1H),4.47-4.36(m,2H),4.25-4.14(m,1H) ,4.12-4.06(m,1H),3.91(dq,J=25.4,8.9,8.2Hz,1H),3.19(s,1H),2.96-2.90(m,1H),2.85-2.79(m,1H),1.80 -1.70(m,4H),1.64(s,1H),1.47-1.44(m,18H),1.37(d,J=3.5Hz,9H),1.28(d,J=12Hz,2H),1.23-1.16(m,4H); 13 CNMR(151MHz,DMSO-d6)δ172.24,164.17,155.29,154.25,152.36,151.54,150.98,150.94, 137.32,137.27,130.05,130.01,129.81,129.79,129.73,128.67,128.64,127.02,125.04, 121.56,120.49,120.46,120.41,120.38,120.36,95.72,84.37,81.86,77.90,77.10,72.92 ,64.39,56.74,56.68,48.23,30.09,29.81,29.58,28.71,28.20,27.58,27.56; LCMS: (M+H) + :964.47 (Calculated value:963.38).

[0135] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-phenylalanine (4-amino)cyclohexanol ester (20)

[0136] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylacetyl-L-phenylalanine (4-amino)cyclohexanol ester was synthesized according to method D (0.070 g, yield 91%, HPLC 96%). 1H NMR (500MHz, DMSO-d6) δ8.49 (s, 1H), 7.85 (s, 3H), 7.68-7.56 (m, 1H), 7.31 (d, J = 8.1Hz, 1H), 7.27-7.25 (m, 2H), 7.2 4-7.13(m,4H),7.05-7.02(m,1H),6.50(s,1H),6.35-6.25(m,1H),6.15(dt,J=16.6,7.9Hz,1H),5.93(dd,J=16.2, 9.0Hz,1H),4.50-4.43(m,1H),4.19-4.07(m,3H),4.06-4.01(m,1H),3.97(s,1H),3.94-3.87(m,1H),3.03-2.88(m ,2H),2.85-2.79(m,1H),1.87(d,J=22.3Hz,2H),1.68(d,J=10.7Hz,1H),1.40-1.30(m,3H),1.23(d,J=5.0Hz,1H); 13 C NMR (126MHz, DMSO) δ172.27,163.19,151.05,137.23,130.08,129.91,129.79,129.74,129.07,128.75,128.72,127.13,125.07,120.54,1 20.50,120.43,120.39,120.35,116.04,95.60,94.76,78.71,73.22,72.45,56.75,56.65,48.26,28.83,28.63,28.16,27.30; LCMS: (M+H) + :664.20 (Calculated value:663.23).

[0137] 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phenylphosphate-L-alanyl(4-Boc-1,4-)butanediamine (21)

[0138] Fmoc-L-alanyl(4-Boc-1,4-)butanediamine was synthesized by general method A (68% yield), followed by deprotection of the Fmoc protecting group with piperidine to give L-alanyl(4-Boc-1,4-)butanediamine (77% yield). 3',4-Di(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine-5'-phosphophenyl esteryl-L-alanyl(4-Boc-1,4-)butanediamine was synthesized by general method C (42% yield, HPLC 97%); LCMS: (M+H) + :861.28 (Calculated value:860.35).

[0139] 2'-Deoxy-2',2'-Difluorocytidine-5'-Phenylphosphobenzoyl-L-alanine (piperidin-4-ol) ester (22)

[0140] 2'-Deoxy-2',2'-difluorocytidine-5'-phenylphosphate-L-alanyl(4-Boc-1,4-)butanediamine was synthesized according to method D (yield 47%, HPLC 96%); LCMS: (M+H) + :561.02 (Calculated value:560.20).

[0141] Inhibits tumor cell growth activity

[0142] Human esophageal cancer cells OE33 (human breast adenocarcinoma cells SK-BR-3, or human gastric cancer cells NCI-N87) were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax). Tumor cells in the exponential growth phase were diluted with the culture medium to 1×10⁻⁶. 5 Cells / mL, 100 μL was added to each well of a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, the compound was diluted to 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.13 nM using culture medium. 2 μL of the diluted compound was added to each well of the 96-well cell culture plate, with three replicates for each concentration. 2 μL of the diluent was added to each well of the negative control and blank control group (no compound added). After addition, the plates were incubated at 37°C with 5% CO2 for 72 h. After incubation, the cell culture plates were removed, the culture medium was aspirated, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 h. After incubation, remove the culture plate, protect it from light, and place it in a microplate. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance values, calculate the IC50 using four-parameter regression in GraphPad. 50 Values ​​(Table 2).

[0143] Table 2. IC50s (μM) values ​​of some compounds inhibiting tumor cell growth

[0144]

[0145] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: (I) Among them, R1 is selected from the following group: 、 or R2 is H; R3 is H; X is O; R4 is selected from the following group: isopropyl, C 1-6 Alkyl-C6 aryl, cyclohexyl, cyclohexyl-amino, or 3-10 saturated heterocyclic groups; and one atom in the 3-10 saturated heterocyclic group is a heteroatom selected from N or O.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the following group:

3. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the group consisting of:

4. A method for preparing the compound as described in claim 1 or 3, comprising the steps of: 1) reacting 2'-deoxy-2',2'-difluorocytidine with tert-butyloxycarbonic anhydride to obtain the corresponding 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside; 2) reacting 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside with phenyl dichlorophosphate, and then reacting with an L-amino acid ester to obtain the corresponding 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside-5'-phosphate (phenyl ester) acyl amino acid ester; 3). The 4-Bocamino-1-[2',2'-difluoro-2'-deoxy-3'-Bocoxy]cytosine nucleoside-5'-phosphate (phenyl ester) acyl amino acid ester was reacted with trifluoroacetic acid to give the corresponding 4-amino-1-[2',2'-difluoro-2'-deoxy]cytosine nucleoside-5'-phosphate (phenyl ester) acyl amino acid ester.

5. The method as described in claim 4, characterized in that, In step 1), the amount of tert-butyloxycarbonic anhydride used is 2.1-2.2 molar equivalents.

6. The method as described in claim 4, characterized in that, In step 2), the amount of phenyl dichlorophosphate used is 1-1.1 molar equivalents, and the amount of amino acid ester used is 1.0-1.2 molar equivalents.

7. The method as described in claim 4, characterized in that, In step 2), the amount of amino acid ester used is 1.0-1.2 molar equivalents.

8. The method as described in claim 4, characterized in that, The amino acid ester mentioned in step 2) is an amino acid cycloalkyl alcohol ester.

9. The method as described in claim 4, characterized in that, In step 3), the trifluoroacetic acid is a 10-30% trifluoroacetic acid solution.

10. A pharmaceutical composition, characterized in that, It comprises: (1) the compound of claim 1 or 3 or a pharmaceutically acceptable salt thereof, or a combination thereof; and (2) one or more pharmaceutically acceptable carriers.

11. The use of the compound of claim 1 or 3 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 10, characterized in that, Used to prepare drugs for the treatment of esophageal cancer, breast cancer, or stomach cancer.

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