WO2016078397A1 - New type of cytidine derivative and application thereof - Google Patents

Abstract

Disclosed are a new type of cytidine derivative, having the general formula (I), and an application thereof. The new type of cytidine derivative of the present invention has a growth inhibitory effect, confirmed by testing, on transplanted colon cancer HCT-116 tumors borne by nude mice; the antitumor activity of the compound of the present invention is high, while the impact on the body weight of the colon cancer HCT-116 tumor-bearing mice, as well as mortality rate data, prove the low toxicity of the compound.

Description

Novel cytidine derivatives and their applications Technical field

The present invention relates to the use of a novel cytidine derivative and a derivative for the preparation of an antitumor drug.

Background technique

Malignant tumors are one of the common diseases that threaten human health, and tumor mortality ranks first among various diseases. At present, the anti-tumor drugs used in clinical practice are a prominent problem that plagues tumor chemotherapy. Improving the therapeutic effect of tumors while reducing the toxicity of drugs is an important research topic in the current treatment of oncology drugs.

Cytosine derivatives having antitumor effects are cytarabine and gemcitabine. Cytarabine is converted into active cytarabine cytarabine in vivo to exert an anticancer effect. Cytarabine triphosphate inhibits the synthesis of DNA and inhibits the growth of cells by inhibiting NDA polymerase and a small amount of DNA, and is mainly used for the treatment of acute myeloid leukemia. However, the cytotoxic side effects of cytarabine are also large. The hematopoietic system is mainly myelosuppression, white blood cells and thrombocytopenia, severe aplastic anemia or megaloblastic anemia can occur; it can occur in the early stage of treatment for leukemia and lymphoma patients. Hyperuricemia, severe cases can occur uric acid nephropathy.

Gemcitabine is a derivative of deoxycytidine that is similar in structure and metabolism to cytarabine. Gemcitabine is catalyzed by the action of nucleotide kinases in cells to form active difluorocytidine diphosphate (dFdCDP) and difluorocytidine triphosphate (dFdCTP), which inhibit DNA polymerase and impede DNA synthesis. Due to incorporation into the DNA, the continued extension of the DNA strand is terminated, thereby inhibiting the growth of tumor cells.

Gemcitabine is indicated for pancreatic cancer (first- and second-line treatment), non-small cell lung cancer, breast cancer, ovarian cancer, and head and neck squamous cell carcinoma. However, gemcitabine is also more toxic. Adverse reactions include myelosuppression, leukopenia, thrombocytopenia, anemia; digestive tract reactions such as mild nausea, vomiting, and abnormal liver function; fever, flu-like symptoms, fatigue, mucositis, and the like.

When the above cytidine derivative enters the human body, the tumor cell will produce a multi-drug resistance gene, and the amino group on the ring is easily acetylated to cause the compound to lose anticancer activity, and other drug resistance factors, the cytosine derivative has a large side effect and It is easy to produce drug resistance.

In order to reduce the toxicity of cytarabine and gemcitabine, and to improve or maintain anti-tumor efficacy, the researchers modified the chemical structure of cytidine derivatives.

For example, "Synthesis and Biological Activity of a Gemcitabine Phosphoramidate Prodrug" (J. Med. Chem 2007, 50, 3743-3746; Weidong Wu) reports a gemcitabine phosphate prodrug.

A gemcitabine prodrug, a pharmaceutical composition and use thereof are disclosed in U. Substituted, the hydrogen atom of the methylol group on the ribofuranose is substituted by H, an acyl group, a substituted acyl group, an acyloxycarbonyl group, a substituted acyloxycarbonyl group, an oxycarbonyl group, a substituted oxycarbonyl group or the like; the hydrogen atom of the hydroxyl group on the ribofuranose is a substituent of H, an acyl group, a substituted acyl group, an acyloxycarbonyl group, a substituted acyloxycarbonyl group, an oxycarbonyl group, a substituted oxycarbonyl group or the like; the amino group is substituted by -N=C(R ¹⁰ )(R ¹¹ ) or -NHR ¹² wherein R ¹² It is a C ₅ -C ₉ acyl group or a C ₅ -C ₉ substituted acyl group. The compound prepared by the patent is a prodrug, and has antitumor activity after being transformed into the body; in addition, clinical studies have found that the gemcitabine prodrug is highly toxic and the antitumor activity is not strong enough, and no drug has been developed yet.

Summary of the invention

The technical problem to be solved by the present invention is to provide a novel cytidine derivative and the use of the above derivative in the preparation of an antitumor drug.

A technical solution for achieving the object of the present invention is: a novel cytidine derivative having the following general formula (I):

Wherein R1 is C ₁ to C ₁₀ alkyl group, substituted alkyl group of C ₁ to C _{10, - (CH 2) n-} Ph, or substituted _{- (CH 2) n-Ph} ; said - (CH ₂₎ n-Ph, wherein n=0, 1, 2, 3-10, Ph is benzene; the substituted alkyl group has a carbon chain independently of one or two or three halogens, a cyano group, a nitro group, Substituted with amino, hydroxy or carboxy; said substituent -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10, independently on the carbon chain or on the phenyl ring by one or two or three Halogen, cyano, nitro, amino, hydroxy or carboxy substituted.

X1是C₁至C₁₀的烷基、C₁至C₁₀的取代烷基、C₁至C₁₀的烷氧基、C₁至C₁₀的取代烷氧基、C₁至C₆的烷基磺酰基、C₁至C₆的烷硫基、-(CH₂)n-Ph、或者是取代-(CH₂)n-Ph；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代烷氧基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的-(CH₂)n-Ph，其中n＝0、1、2、3～10；所述的取代-(CH₂)n-Ph，其中n＝0、1、2、 3～10，其碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。R2 is H, halogen or

X1 is a C ₁ to C ₁₀ alkyl group, a C ₁ to C ₁₀ substituted alkyl group, a C ₁ to C ₁₀ alkoxy group, a C ₁ to C ₁₀ substituted alkoxy group, a C ₁ to C ₆ alkyl group. a sulfonyl group, a C ₁ to C ₆ alkylthio group, -(CH ₂ )n-Ph, or a substituted -(CH ₂ )n-Ph; said substituted alkyl group having a carbon chain independently of one or Substituted by two or three halogens, cyano, nitro, amino, hydroxy or carboxy; said substituted alkoxy, independently of one or two or three halogens, cyano, nitro, on the carbon chain Substituting an amino group, a hydroxyl group or a carboxyl group; said -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10; said substitution -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10, substituted on the carbon chain or on the benzene ring by one or two or three H, halogen, cyano, nitro, amino, hydroxy or carboxy groups.

其中X3是苯环，杂环，稠杂环，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代杂环，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代稠杂环；所述杂环是咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪或哌啶；所述稠杂环是喹啉或吲哚；X2是-(CH₂)n-，其中n＝1、2、3，或者X2是-O-(CH₂)n-，其中n＝0、1、2、3。R3 is H or

Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three a substituted heterocyclic ring substituted with a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group, or a substituted heterocyclic ring independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups a ring; the heterocyclic ring is imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine or piperidine; the fused heterocyclic ring is quinoline or hydrazine; X2 is -(CH ₂ ) n-, wherein n= 1, 2, 3, or X2 is -O-(CH ₂ )n-, where n = 0, 1, 2, 3.

Alternatively, R2 is H.

Further preferably, R2 is not H; and R3 is not H.

X1是-(CH₂)n-Ph或者是取代-(CH₂)n-Ph。When R2 is not H, R2 is halogen or

X1 is -(CH ₂ )n-Ph or is substituted by -(CH ₂ )n-Ph.

Preferable, Rl is a C ₁ to C ₄ alkyl group, a substituted a C ₁ to C ₄ alkyl group, a benzyl group, or a substituted benzyl group; R3 X3, independently by one or two or three halogen, a substituted cyano, nitro, amino, hydroxy or carboxy substituted imidazole, substituted pyridine independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, or independently Or a substituted benzene ring substituted with two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups.

The use of the above compound or a salt thereof for the preparation of a medicament for treating tumors.

The tumor is a hematological tumor or a malignant solid tumor.

The salts are hydrochloride, phosphate, sulfate, carbonate, nitrate, citrate, tartrate, maleate, succinate, sulfonate, p-toluenesulfonate, methanesulfonate. An acid salt, a benzoate or a fumarate.

A pharmaceutical composition comprising, as an active ingredient, a cytidine derivative represented by the formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

The dosage form of the above composition is an injection preparation or an oral dosage form, wherein the injection preparation is a solution injection, a suspension injection, an emulsion injection, or a sterile powder for injection, and the oral dosage form is a tablet, a powder, a granule, a capsule, a micro. Pill preparations, solutions, suspensions, emulsions, syrups or elixirs.

The present invention has a positive effect: the growth inhibition test of the compound of the present invention on colon cancer HCT-116 tumor-bearing nude mouse xenografts confirms that the compound of the present invention has high antitumor activity and simultaneously colonizes human colon cancer HCT-116. The effect of mouse body weight was small, demonstrating that the toxicity of the compound is low.

detailed description

The structural formula of the novel cytidine derivative of the present invention is as shown in formula (I):

Wherein R1 is C ₁ to C ₁₀ alkyl group, substituted alkyl group of C ₁ to C _{10, - (CH 2) n-} Ph, or substituted _{- (CH 2) n-Ph} ; said - (CH ₂₎ n-Ph, wherein n=0, 1, 2, 3-10, Ph is benzene; the substituted alkyl group has a carbon chain independently of one or two or three halogens, a cyano group, a nitro group, Substituted with amino, hydroxy or carboxy; said substituent -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10, independently on the carbon chain or on the phenyl ring by one or two or three Halogen, cyano, nitro, amino, hydroxy or carboxy substituted.

X1是C₁至C₁₀的烷基、C₁至C₁₀的取代烷基、C₁至C₁₀的烷氧基、C₁至C₁₀的取代烷氧基、C₁至C₆的烷基磺酰基、C₁至C₆的烷硫基、-(CH₂)n-Ph或者是取代-(CH₂)n-Ph；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代烷氧基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的-(CH₂)n-Ph，其中n＝0、1、2、3～10；所述的取代-(CH₂)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。R2 is H, halogen or

X1 is a C ₁ to C ₁₀ alkyl group, a C ₁ to C ₁₀ substituted alkyl group, a C ₁ to C ₁₀ alkoxy group, a C ₁ to C ₁₀ substituted alkoxy group, a C ₁ to C ₆ alkyl group. a sulfonyl group, a C ₁ to C ₆ alkylthio group, -(CH ₂ )n-Ph or a substituted -(CH ₂ )n-Ph; said substituted alkyl group having independently one or two carbon chains Substituted by three or three halogen, cyano, nitro, amino, hydroxy or carboxy; said substituted alkoxy, independently on the carbon chain from one or two or three halogens, cyano, nitro, amino Substituted by a hydroxyl group or a carboxyl group; said -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10; said substitution -(CH ₂ )n-Ph, wherein n = 0, 1 2, 3 to 10, substituted on the carbon chain or on the benzene ring by one or two or three H, halogen, cyano, nitro, amino, hydroxy or carboxy groups.

其中X3是苯环，杂环，稠杂环，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代杂环，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代稠杂环；所述杂环是咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪或哌啶；所述稠杂环是喹啉或吲哚；X2是-(CH₂)n-，其中n＝1、2、3，或者X2是-O-(CH₂)n-，其中n＝0、1、2、3。R3 is H or

Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three a substituted heterocyclic ring substituted with a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group, or a substituted heterocyclic ring independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups a ring; the heterocyclic ring is imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine or piperidine; the fused heterocyclic ring is quinoline or hydrazine; X2 is -(CH ₂ ) n-, wherein n= 1, 2, 3, or X2 is -O-(CH ₂ )n-, where n = 0, 1, 2, 3.

For the cytidine derivative of the present invention, the following compounds are given in Table 1, but the cytidine derivatives of the present invention are not limited to these compounds.

Table 1

The compound in the above table was prepared, and the solid reagent used in the synthesis was directly used without further treatment, and the liquid reagent was used after being re-distilled and dried.

(Example 1)

The cytidine derivative of the present example is 4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 4, code G2), which is synthesized by a three-step reaction. The reaction formula is as follows (in the reaction formula, HMDS is hexamethyldisilazane, reflux is reflux, chloridate is chloride, rt is room temperature, TEA is triethylamine, the same applies hereinafter).

300 mg (1 mmol) of 2'-deoxy-2',2'-difluorocytidine hydrochloride (Structure 1, code G1), 5 mL (0.023 mmol) of hexamethyldisilazane HMDS, catalytic amount of ammonium sulfate 5 mg was dissolved in 5 mL of 1,4-dioxane and heated under reflux for 2 h. The chemical structure of the reaction product was 2. After completion of the refluxing reaction, the reaction mixture was concentrated, toluene was added thereto, and the mixture was concentrated to dryness twice.

0.24 mL (3 mmol) of N-methylimidazole and 0.32 mL (3 mmol) of butyl chloroformate were added to the above dichloromethane solution, and the reaction was stirred at room temperature for 4 hours, and the reaction product was chemically structured to give a viscous oil.

The above viscous oil was dissolved in a mixed solution of 3 mL of triethylamine and 20 mL of methanol, and stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure.yield eluted eluted eluted eluted eluted eluted eluted eluted eluted elution

G2 NMR characterization:

¹ H-NMR (MeOD-d ₄ , 400 MHz) δ: 8.30 (d, 1H, J = 7.68 Hz, H6), 7.34 (d, 1H, J = 7.68 Hz, H5), 6.28 (t, 1H, J = 7.08 Hz, H1'), 4.33 (m, 1H, H5a'), 4.0 (m, 2H, O-CH ₂ -CH ₂ -), 3.81 (m, 1H, H5b'), 3.79 (m, 1H, H4) '), 1.68 (m, 2H, O-CH ₂ -CH ₂ -), 1.45 (m, 2H, O-CH ₂ -CH ₂ -CH ₂ ), 0.98 (t, 3H, J = 7.4 Hz, -CH ₂ -CH ₃ ).

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 164.28, 156.27, 153.50, 144.39, 128.33, 122.72, 95.81, 84.90, 81.71, 74.87, 68.88, 63.69, 59.15, 30.66, 32.40, 18.81, 11.23, 8.06.

According to the above preparation method, the reaction raw material is changed, and R1 may be other groups other than n-butyl group, such as: C ₁ to C ₁₀ alkyl group, C ₁ to C ₁₀ substituted alkyl group, -(CH ₂ )n- Ph, n = 0, 1, 2, 3 to 10 or substituted -(CH ₂ ) n-Ph, n = 0, 1, 2, 3 to 10, and Ph is benzene; the carbon chain of the substituted alkyl group is independently One or two or three halogen, cyano, nitro, amino, hydroxy or carboxy substituted; substituted -(CH ₂ ) n-Ph on the carbon chain or on the phenyl ring independently from one or two or three halogens Substituted with cyano, nitro, amino, hydroxy or carboxy.

(Example 2)

The cytidine derivative of the present example is 4-N-(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 6, code G3), which is synthesized by a three-step reaction. The reaction formula is as follows.

300mg (1mmol) 2'-deoxy-2',2'-difluorocytidine hydrochloride, 5mL (0.023mmol) hexamethyldisilazane, catalytic amount of ammonium sulfate 5mg dissolved in 5mL 1,4- The dioxane was heated under reflux for 2 hours; after the refluxing reaction was completed, the reaction liquid was concentrated, toluene was added thereto, and the mixture was concentrated to dryness twice.

To the above dichloromethane solution, 0.24 mL (3 mmol) of N-methylimidazole and 416 mg (3 mmol) of di-tert-butyl dicarbonate were added, and the reaction was stirred at room temperature for 4 hours, and the reaction product was chemically structured to give a viscous oil.

The above viscous oil was dissolved in a mixed solution of 3 mL of triethylamine and 20 mL of methanol, and stirred at room temperature overnight. The solvent was then evaporated under reduced pressure. the crude material was purified mjjjjjjj

G3 NMR characterization:

¹ H-NMR (MeOD-d ₄ , 400 MHz) δ: 8.27 (d, 1H, J = 7.68 Hz, H6), 7.32 (d, 1H, J = 7.68 Hz, H5), 6.28 (t, 1H, J = 7.08 Hz, H1'), 4.25 (m, 1H, H5a'), 3.93 (m, 2H, H5b', H4'), 3.78 (m, 1H, H3'), 1.52 (s, 9H, t-Bu) .

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 164.36, 152.17, 144.24, 125.29, 122.71, 120.13, 109.98, 95.78, 82.17, 81.60, 70.45, 69.30, 68.90, 65.57, 55.90, 27.12, 23.57.

(Example 3)

The cytidine derivative of the present example is 4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 8, code G4), which is synthesized by a three-step reaction. The formula is as follows.

300mg (1mmol) 2'-deoxy-2',2'-difluorocytidine hydrochloride, 5mL (0.023mmol) hexamethyldisilazane, catalytic amount of ammonium sulfate 5mg dissolved in 5mL 1,4- In the dioxane, the reaction was heated under reflux for 2 h; after the refluxing reaction was completed, the reaction solution was concentrated, toluene was added thereto, and the mixture was concentrated to dryness twice, and the obtained product was dissolved in 10 mL. In dichloromethane.

0.24 mL (3 mmol) of N-methylimidazole and 340 mg (3 mmol) of decyl chloroformate were added to the above dichloromethane solution, and the reaction was stirred at room temperature for 4 hours, and the reaction product was chemically structured to give a viscous oil.

The above viscous oil was dissolved in a mixed solution of 3 mL of triethylamine and 20 mL of methanol, and stirred at room temperature overnight. The solvent was then evaporated under reduced pressure. the crude material was purified mjjjjjjj

G4 NMR characterization:

¹ H-NMR (MeOD-d ₄ , 400 MHz) δ: 8.31 (d, 1H, J = 7.64 Hz, H6), 7.39 (m, 5H, J = 7.68 Hz, Ph), 6.25 (t, 1H, J = 7.12 Hz, H1'), 5.21 (s, 2H.CH ₂ -Ph), 4.31 (m, 1H, H5a'), 3.82 (m, 2H, H5b, H4'), 3.79 (m, 1H, H3') .

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 164.22, 156.22, 153.27, 144.48, 135.87, 128.42, 128.10, 125.31, 122.74, 120.16, 95.89, 85.35, 84.91, 81.7, 81.66, 68.87, 67.54, 58.31.

(Example 4)

The cytidine derivative of the present example is 4-N-(4-nitrobenzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 10, code G5), after three steps The reaction was synthesized, and the reaction formula was as follows.

300mg (1mmol) 2'-deoxy-2',2'-difluorocytidine hydrochloride, 5mL (0.023mmol) hexamethyldisilazane, catalytic amount of ammonium sulfate 5mg dissolved in 5mL 1,4- The dioxane was heated under reflux for 2 hours; after the refluxing reaction was completed, the reaction liquid was concentrated, toluene was added thereto, and the mixture was concentrated to dryness twice.

To the above dichloromethane solution, 0.24 mL (3 mmol) of N-methylimidazole and 430 mg (3 mmol) of p-nitrobenzyl chloroformate were added, and the reaction was stirred at room temperature for 4 hours, and the reaction product was chemically structured, and the reaction liquid was concentrated to obtain a viscous oil. Shape.

The above viscous oil was dissolved in a mixed solution of 3 mL of triethylamine and 20 mL of methanol, and stirred at room temperature overnight. The solvent was then evaporated under reduced pressure. the crude material was purified eluting with silica gel eluting eluting eluting with with with

G5 NMR characterization:

^{1 H-NMR (DMSO-d} 6, 400MHz) δ: 11.11 (s, 1H), 8.25 (m, 3H, Ph), 7.68 (d, 2H, J = 8.64Hz, Ph), 7.08 (d, 1H, J = 7.44 Hz, H6), 6.29 (d, 1H, J = 7.44 Hz, H5), 6.17 (t, 1H, J = 7.4 Hz, H1 '), 5.33 (s, 2H, CH ₂ -Ph), 5.29 (t, 1H, J = 5.44 Hz), 4.19 (m, 1H, H5a'), 3.66 (m, 1H, H5b'), 3.61 (m, 1H, H4'), 3.29 (m, 1H, H3') .

¹³ C-NMR (DMSO-d ₆ , 100 MHz) δ: 163.93, 153.47, 147.86, 144.38, 128.97, 126.72, 126.19, 124.26, 123.62, 95.57, 84.82, 83.99, 81.77, 71.91, 69.13, 68.42, 66.06, 62.34, 59.52.

According to the above synthesis method, various derivatives having different substituent groups such as G5-1 and G5-2 can be synthesized.

(Example 5)

The cytidine derivative of the present example is 5-bromo-4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 11, code G6), reaction formula as follows.

After G2 was obtained by the method of Example 1, 1 g (2.75 mmol) of G2 was dissolved in 150 mL of dimethylformamide, and 500 mg (1.75 mmol) of dibromohydantoin was added thereto with stirring, and the obtained pale yellow solution was stirred at room temperature. After 1 h, the reaction was complete by LCMS and G2 was completely converted to G6. The solvent was evaporated under reduced pressure. EtOAc was evaporated,jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj The total reaction yield was 51%.

G6 NMR characterization:

¹ H-NMR (MeOD-d ₄ , 400 MHz) δ: 8.62 (s, 1H, H5), 6.18 (t, 1H, J = 6.52 Hz, H1 '), 4.19 (m, 1H, H5a'), 4.15 ( m, 2H, H5b', H4'), 3.95 (m, 2H, O-CH2-CH3), 3.17 (m, 1H, H3'), 1.36 (m, 2H, -O-CH2-CH2-CH2-CH3 ), 1.31 (m, 2H, -O-CH2-CH2-CH2-CH3), 0.98 (m, 2H, -O-CH2-CH2-CH2-CH3).

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 143.05, 124.56, 84.59, 81.91, 75.45, 66.17, 58.74, 58.42, 32.96, 30.64, 28.32, 18.84, 12.79, 8.17.

ESIMS: calcd for C ₁₄ H ₁₈ BrF ₂ N ₃ O ₆ m/z 442.03 (M+H) ⁺ , found 442.02.

(Example 6)

The cytidine derivative of the present example is 5-bromo-4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 12, code G7), and the reaction formula is as follows (DMF in the reaction formula is N,N-dimethylformamide, the same applies hereinafter).

After G4 was obtained according to the method of Example 3, 1 g (2.51 mmol) of G4 was dissolved in 150 mL of N,N-dimethylformamide, and 500 mg (1.75 mmol) of dibromohydantoin was added thereto under stirring to obtain a pale yellow solution at room temperature. The reaction was stirred for 1 h, and the reaction was completely confirmed by LCMS, and G4 was completely converted to G7. The solvent was evaporated under reduced pressure. EtOAc was evaporated,jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj The total reaction yield was 32%.

G7 NMR characterization:

¹ H-NMR (MeOD-d ₄ , 400MHz) δ: 8.60 (s, 1H, H5), 7.45 (m, 2H, Ph), 7.36 (m, 3H, Ph), 6.18 (t, 1H, J = 6.52) Hz, H1'), 5.24 (s, 2H, CH ₂ -Ph), 4.33 (m, 1H, H5a'), 4.0 (m, 2H, H5b', H4'), 3.81 (m, 1H, H3') .

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 143.23, 135.93, 128.37, 128.27, 125.22, 122.63, 120.05, 85.41, 85.08, 84.76, 81.85, 68.77, 68.53, 68.31, 67.93, 58.76.

ESIMS: calcd for C ₁₇ H ₁₆ BrF ₂ N ₃ O ₆ m/z 476.02 (M+H) ⁺ , found 477.09.

Similarly, after the substitution on the amino group according to the synthetic route of Example 1, the method according to Example 6 was carried out at 5 G7-1 and G7-2 can be obtained by implementing Br substitution at the position.

According to the above preparation method, a derivative in which R2 is another group can be obtained.

(Example 7)

The cytidine derivative of this example is 5'-O-[3,5-dinitrosalicylate]-4-N-(n-butoxycarbonyl)-2'-deoxy-2', 2'- Difluorocytidine (Structure 14, code G8).

The reaction formula is as follows (in the reaction formula, (Boc) ₂ O is di-tert-butyl dicarbonate, dioxane is 1,4-dioxane, DMAP is 4-dimethylaminopyridine, and EDCL is 1-(3-dimethylamino). Propyl)-3-ethylcarbodiimide hydrochloride, DCM is dichloromethane, TFA is trifluoroacetic acid, the same below):

Compound 13 was first prepared. 60 mg (0.16 mmol) of G2 and 106 mg (1 mmol) of sodium carbonate prepared in Example 1 were mixed, and added to 5 mL of a mixed solution of 1,4-dioxane and water (4:1 by volume). 44 mg (0.2 mmol) of di-tert-butyl dicarbonate (Boc) ₂ O was added to the solution, and then the reaction was stirred at 24 ° C, and TLC was used to detect whether G 2 was completely reacted during the reaction. After the reaction was completed, 2 mL of water was added to the system after the reaction, and then extracted twice with ethyl acetate for 30 mL each time. The extracted organic phase was washed successively with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure, concentrated, and purified by silica gel chromatography using dichloromethane/acetone/methanol (1) :1:0.02) Elution gave 51 mg of Compound 13 in a yield of 76%.

51 mg (0.11 mmol) of the compound 13 prepared above, and 98 mg (0.42 mmol) of 3,5-dinitrogen Salicylic acid, 60 mg (0.31 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCL) was mixed and added to 15 mL of dichloromethane, followed by dichloro 2 mg of 4-dimethylaminopyridine (DMAP) was added to methane, and the reaction was stirred at 24 ° C for 24 hours. During the reaction, thin layer chromatography TLC was used to detect whether or not compound 13 was completely reacted.

After the completion of the reaction, 50 mL of dichloromethane was added to the reaction mixture, followed by washing with 10 mL of water and 20 mL of brine, and dried over anhydrous sodium sulfate. To the material obtained after concentration, 5 mL of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 12 hr. After concentration, it was purified by silica gel chromatography eluting with dichloromethane/methanol (20:1) to afford 18 g of product G8.

其中X3为苯环、杂环、稠杂环、独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯、取代杂环、取代稠杂环；所述杂环包括咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪和哌啶；稠杂环包括喹啉和吲哚；X2为C₁至C₃的-(CH₂)n-或C₀至C₃的-O-(CH₂)n-的化合物。By changing 3,5-dinitrosalicylic acid to another acid, R3 can be prepared.

Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene, a substituted heterocyclic ring or a substituted fused heterocyclic ring independently substituted by one or two or three halogens, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group; The heterocyclic ring includes imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine and piperidine; fused heterocyclic ring includes quinoline and hydrazine; and X2 is C ₁ to C ₃ -(CH ₂ )n- or C a compound of ₀ to C ₃ -O-(CH ₂ )n-.

(Example 8)

The cytidine derivative of this example is 5'-O-[2-(4-nitro-1H-imidazolium) acetate]-4-N-(tert-butoxycarbonyl)-2'-deoxy-2' , 2'-difluorocytidine (Structure 16, code G9).

The reaction formula is as follows:

Compound 15 was first prepared. 60 mg (0.16 mmol) of the compound 6 (G3) prepared in Example 2 and 106 mg (1 mmol) of sodium carbonate were mixed, and then added to a mixed solution of 5 mL of 1,4-dioxane and water (4:1 by volume). 44 mg (0.2 mmol) of di-tert-butyl dicarbonate (Boc) ₂ O was added to the solution, and then the reaction was stirred at 24 ° C, and TLC was used to detect whether G 2 was completely reacted during the reaction. After the reaction was completed, 2 mL of water was added to the system after the reaction, and then extracted twice with ethyl acetate for 30 mL each time. The organic phase extracted was washed successively with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate and then evaporated to dryness. : 1:0.02) Elution gave 48 mg of Compound 15 in a yield of 64%.

51 mg (0.11 mmol) of the compound 15 prepared above was mixed with 98 mg (0.57 mmol) of 2-(4-nitro-1H-imidazole)acetic acid, 60 mg (0.31 mmol) of EDCL, and then added to 15 mL of dichloromethane. Further, 2 mg of DMAP was added to dichloromethane, and the reaction was stirred at 24 ° C for 24 hours. During the reaction, TLC was used to detect whether the compound 15 was completely reacted.

After the completion of the reaction, 50 mL of dichloromethane was added to the reaction mixture, followed by washing with 10 mL of water and 20 mL of brine, and dried over anhydrous sodium sulfate. To the material obtained after concentration, 5 mL of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 12 hr. After concentration, it was purified by silica gel chromatography eluting with methylene chloride/methanol (20:1) to give 18 g of product G9.

(Example 9)

The cytidine derivative of this example has the code G10, and the reaction formula is as follows (DCC in the reaction formula is N,N'-dicyclohexylcarbodiimide):

Compound 22 is first prepared, and the reaction formula is as follows (DMF is N,N-dimethylformamide in the reaction formula):

To the reaction flask was added compound 20, 3,5,6-trichloro-2-pyridinol (5 g, 25.2 mmol), potassium carbonate (7 g, 50.6 mmol) and N,N-dimethylformamide DMF (30 ml) Stir for 15 min. After cooling to 0 ° C, ethyl bromoacetate (2.78 ml, 25.2 mmol) was added dropwise. The reaction was carried out for 4 h at room temperature. Water (15 ml) was added and extracted with dichloromethane (15 ml x 3). The organic phase was dried to give Compound 21 directly to the next reaction.

Compound 21 (5.4 g, 18.8 mmol) was added to 54 mL of water and sodium hydroxide (0.864 g, 21.6 mmol) was added and stirred at 80 ° C for 4 h. Then, concentrated hydrochloric acid was added dropwise to adjust the pH to 1, and the product compound 22 was obtained by filtration, and was applied, 3.3 g (70%).

Compound 12, 5-bromo-4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (2 g, 4.21 mmol) and sodium carbonate (3.3 g, 31.1 mmol) After mixing, it was added to a system of 1,4-dioxane and water (volume ratio 4:1, 200 mL). Add (Boc) ₂ O (1.8g, 8.25mmol, Di-t-butyldicarbonate), stir the reaction at 25 ° C for 48h, TLC detection during the reaction, after the reaction is completed, add 20mL water to dilute, use 2 × 100mL ethyl acetate 2 extractions, the organic phase was washed with 50 mL of water and 50 mL of brine, and dried over anhydrous sodium sulfate, and the column chromatography (dichloromethane/acetone/methanol, 1:1: 0.02) gave compound 17 yield 690 mg yield 29%.

ESIMS: calcd for C ₂₂ H ₂₄ BrF ₂ N ₃ O ₈ m/z 576.07 (M+H)+, found 576.16.

Compound 17 (350 mg, 0.61 mmol) was mixed with compound 22 (186 mg, 0.73 mmol), DCC (250 mg, 1.21 mmol, Dicydohexylcarbodiimide, N, N'-dicyclohexylcarbodiimide) and added to 7 mL of dichloromethane. DMAP (15 mg, 0.12 mmol, 4-dimethylaminopyridine, 4-dimethylaminopyridine) was added, and the reaction was stirred at room temperature overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted with 2×20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate and 45:1) Intermediate 18 (260 mg, yield 70%). The intermediate is straight Treatment with a solution of TFA (trifluoroacetic acid) in dichloromethane afforded product G10 ( 175 mg, yield 77%).

G10 NMR characterization:

¹ H-NMR (CDCl ₃ , 400 MHz) δ 7.81 (s, 1H), 7.75 (s, 1H), 7.35 (m, 5H), 6.24 (m, 1H), 5.27 (s, 2H), 5.22 (s) , 2H), 5.03 (m, 2H), 4.67 (m, 1H), 4.47 (m, 1H), 4.30 (s, 2H).

¹³ C NMR (CDCl ₃ , 100 MHz) δ 167.80, 162.56, 157.21, 155.57, 143.28, 140.91, 128.90, 128.78, 124.55, 123.44, 118.57, 117.47, 72.61, 68.68, 64.94, 63.83, 63.37, 62.84.

ESIMS: calcd for C ₂₄ H ₁₈ BrC _l3 F ₂ N ₄ O ₈ m/z 712.93 (M+H) ⁺ , found 712.99.

(Embodiment 10)

The code of the cytidine derivative of this example is G11.

Compound 24 is first prepared and has the following reaction formula:

2'-Deoxy-2',2'-difluorocytidine (5 g, 16 mmol) was dissolved in acetic acid (56 mL) and stirred at 35 ° C for 2 h; chloroform (17 mL) was added and stirred at 0 ° C for 15 min. A mixture of chloroform/acetyl chloride (28 ml/33 ml) was added dropwise, and the reaction was stirred at 50 ° C for 24 h. The solvent was evaporated to dryness, then methanol (35 ml) was evaporated and evaporated

The reaction flask was charged with iodine (2.8 g, 11 mmol), iodic acid (0.83 g, 4.7 mmol), acetic acid (37.5 mL), carbon tetrachloride (25.5 mL), water (25.5 ml) and compound 23, and stirred at 40 ° C. Reaction for 24h. The solvent was dried and dichloromethane and water were added. The pH was adjusted to 6-7, and the organic phase was washed with sodium thiosulfate and washed with water. The organic phases were combined and dried over anhydrous sodium sulfate. Filter and the filtrate was dried. The compound 24, i.e., the product 5 g, was obtained (yield 55% in two steps).

ESIMS: calcd for C ₁₃ H ₁₄ F ₂ N ₃ O ₆ m/z 473.99 (M+H) ⁺ , found 474.14.

The reaction formula for preparing compound G11 is as follows:

Compound 24 (2.5 g, 5.3 mmol), pyridine (1.24 g, 15.8 mmol) was dissolved in dichloromethane (35 mL); butyl chloroformate (2.15 g, 15.8 mmol) was added dropwise at 0 ° C and allowed to react overnight at 10 ° C . The solvent was evaporated to dryness, and then purified (yield: methylene chloride:methanol = 60:1) to afford 1.9 g (yield: 63%) Compound 25.

ESIMS: calcd for C ₁₈ H ₂₂ F ₂ N ₃ O ₈ m/z 574.04 (M+H) ⁺ , found 574.14.

Compound 25 (2.5 g, 4.3 mmol) was dissolved in methanol (40 mL), stirred for 5 min, EtOAc (EtOAc, EtOAc, EtOAc. 26.

ESIMS: calcd for C ₁₄ H ₁₈ F ₂ N ₃ O ₆ m/z 490.02 (M+H) ⁺ , found 490.07.

Compound 26 (3.9 g, 8 mmol) and sodium carbonate (4.24 g, 40 mmol) were dissolved in a mixture of 1,4-dioxane (22.5 mL) and water (4.5 mL) and stirred for 10 min. Di-tert-butyl dicarbonate (2.1 g, 9.6 mmol) was added. React at room temperature for at least 24 h. The solvent was evaporated to dryness and dichloromethane (EtOAc) (EtOAc) The organic phase is dried. Column separation (dichloromethane:methanol = 80:1) gave 2.8 g (yield: 60%) of Compound 27.

ESIMS:calcd for C ₁₉ H ₂₆ F ₂ IN ₃ O ₈ m/z 590.07(M+H) ⁺ ,found 590.02.

Compound 27 (50 mg, 0.08 mmol) was mixed with compound 22 (32.4 mg, 0.13 mmol), DCC (35 mg, 0.17 mmol), and then added to 2 mL of dichloromethane, and DMAP (2 mg, 0.016 mmol) was added to react at normal temperature. Stir overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted twice with 2×20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate and Methanol, 150:1) gave intermediate (50 mg, yield 71%). This intermediate was directly treated with a solution of TFA in dichloromethane to give G11 (35 mg, yield 80%).

The representation of G11 is as follows:

ESIMS: calcd for C ₂₁ H ₂₀ Cl ₃ F ₂ IN ₄ O ₈ m/z 726.94 (M+H) ⁺ , found 727.12.

1H-NMR (MeOD-d4, 400MHz) δ 8.06 (s, 1H), 5.49 (s, 1H), 5.07 (m, 3H), 4.51 (s, 1H), 4.48 (m, 1H), 4.21. , 5H), 1.69 (m, 3H), 1.44 (m, 3H), 0.97 (m, 3H).

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ 168.06, 141.08, 140.91, 122.67, 81.73, 66.15, 63.85, 63.54, 59.38, 58.64, 33.27, 30.68, 19.73, 18.89, 15.75, 12.84, 8.78.

(Example 11)

The code of the cytidine derivative of this example is G12, and the reaction formula is as follows:

Compound 27 (300 mg, 0.51 mmol) was mixed with 2-(4-nitro-1H-imidazole)acetic acid (105 mg, 0.61 mmol), DCC (210 mg, 1.02 mmol), and then added to 10 mL of dichloromethane. 9 mg, 0.073 mmol), and the reaction was stirred at room temperature overnight. TLC detection, after completion of the reaction, was diluted with 20 mL of water, extracted with 3×30 mL of dichloromethane three times, and the organic phase was washed with 20 mL of water and 20 mL of brine, dried over anhydrous sodium sulfate and Methanol, 66:1) afforded intermediate 200 mg (yield: 53%).

(Embodiment 12)

The code of the cytidine derivative of this example is G13.

Compound 35 was first prepared.

Compound 24 (5 g, 0.01 mol), (Ph ₃ )PdCl ₂ (1.5 g, 2.14 mmol), CuI (1.0 g, 5.27 mmol) was added to a pre-dried reaction flask, and dry THF (100 ml) was added under N ₂ protection. ), Me3SiC≡CH (5.2 g, 0.053 mol) was injected, and TEA (15 ml) was injected. The reaction was carried out at room temperature overnight. After completion of the reaction, the solvent was distilled off under reduced pressure. Purification using a silica gel column (ethyl acetate / petroleum ether: 1:1) afforded 2.27 g (yield 57%) of Compound 30.

Compound 30 (10 g, 1.1 mmol) was added to MeOH (200 mL). Cool at 15 ° C for 15 minutes. K ₂ CO ₃ (10.92 g, 3.95 mmol) was added and stirred at 0 ° C for 2 h. After completion of the reaction, potassium carbonate was removed by filtration, and the solvent was evaporated under reduced pressure. Purification using a silica gel column (dichloromethane/methanol: 10:1) gave 5.46 g (yield 84.27%) of Compound 31.

N2保护下常温搅拌36小时。反应结束后减压蒸馏除去溶剂。使用硅胶层析柱(二氯甲烷/甲醇：15∶1)纯化得到4.88g(产率61.0％)化合物32。Compound 61 (5.46g, 0.019mol), CuSO 4 .5H 2 O (475mg, 1.9mmol), Vc.Na (1.13g, 5.7mmol), was added to a THF (55mL) and water (49ml) in, N ₂ Stir at room temperature for 10 minutes under protection. Then inject

Stir at room temperature for 36 hours under N2 protection. After completion of the reaction, the solvent was distilled off under reduced pressure. Purification using a silica gel column (dichloromethane / methanol: 15:1) gave 4.88 g (yield 61.0%) of Compound 32.

Compound 32: ESIMS: Calcd for C ₁₈ H ₁₈ F ₂ N ₆ O ₄ m/z 421.18 (M+H)+, found 421.36.

In the above reaction formula, 1,4-dioxane is 1,4-dioxane, and HMDS is hexamethyldisilazane.

Compound 32 (2.0 g, 4.76 mmol), hexamethyldisilazane HMDS (20 mL), (NH ₄ ) ₂ SO ₄ (50.3 mg, 0.38 mmol) was added to 1,4-dioxane (20 mL) The mixture was stirred under reflux at 80 ° C for 4 hours. After the completion of the reaction, the solvent was evaporated under reduced pressure, and toluene (5 ml × 2) was added to the solvent, and the oil pump was drained. Dichloromethane (40 ml) and pyridine (1.13 g, 14.3 mmol) were added, and the mixture was cooled to 0 ° C, and p-nitro chloro chloroformate (3.08 g, 14.3 mmol) was slowly added and stirred at 10 ° C overnight. After completion of the reaction, the solvent was distilled off under reduced pressure. Methanol (40 mL) was added, and the mixture was cooled at 0 ° C for 15 min, and triethylamine (6.7 ml) was added dropwise. Stir at 10 ° C overnight. After the completion of the reaction, the solvent was evaporated under reduced pressure and purified using silica gel chromatography (dichloromethane/methanol: 40:1).

Compound 33: ESIMS: Calcd for C ₂₆ H ₂₃ F ₂ N ₇ O ₈ m/z 600.22 (M+H)+, found 600.25.

Compound 33 (780 mg, 1.3 mmol) and Na ₂ CO ₃ (690 mg, 6.5 mmol) were added to 1,4-dioxane (20 ml) and water (5 ml), and stirred to dissolve. (Boc) ₂ O (340.6 mg, 1.56 mmol) was added at room temperature. After stirring at room temperature overnight, the solvent was evaporated under reduced pressure, and purified using silica gel chromatography (dichloromethane/methanol: 80:1) to afford 366 mg (yield 40.2%) Compound 34.

Compound 34: ESIMS: Calcd for C ₃₁ H ₃₁ F ₂ N ₇ O ₁₀ m/z 700.28 (M+H)+, found 700.08.

Compound 34 (250 mg, 0.36 mmol), Compound 22 (109 mg, 0.43 mmol), DCC (148 mg, 0.72 mmol), DMF (5 mg) was added to dichloromethane (12 ml) and stirred at room temperature overnight. After completion of the reaction, DCC was removed by suction filtration, and the solvent was evaporated under reduced pressure, and purified using silica gel chromatography (dichloromethane/methanol: 100:1) to afford 210 mg (yield 60.0%) of Compound 35.

Compound 35: ESIMS: Calcd for C ₃₈ H ₃₃ C _l3 F ₂ N ₈ O ₁₂ m/z 937.28 (M+H)+, found 937.28.

Compound 35 (210 mg, 0.224 mmol) was added to 10 ml of DCM/TFA=5:1 system, and stirred at room temperature for 4 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, using silica gel chromatography column (dichloromethane/methanol: 60 : 1) Purification gave the product 140 mg (yield 75.0%) of G13.

ESIMS: calcd for C ₃₃ H ₂₅ Cl ₃ F ₂ N ₈ O ₁₀ m/z 837.17 (M+H)+, found 837.27.

¹ H-NMR (CDCl ₃ , 400 MHz) δ 8.34 (s, 1H), 8.23 (m, 3H), 7.57 (m, 3H), 7.33 (m, 3H), 7.23 (m, 2H), 6.38 (m) , 1H), 5.49 (m, 3H), 5.30 (s, 3H), 5.13 (m, 2H), 4.73 (m, 1H), 4.33 (m, 3H).

进行反

烷基、C₁ Replace the azide compound that is currently available on the market or that has been synthesized

Counter

Alkyl, C ₁

To C ₁₀ alkoxy, C ₁ to C ₁₀ substituted alkoxy, C alkylsulfonyl group of ₁ to C _6, C ₁ to C ₆ alkylthio _{of, - (CH 2) n-} Ph or substituted -(CH ₂ )n-Ph; wherein the substituted alkyl, substituted alkoxy carbon chain is independently substituted by one or two or three halogens, cyano, nitro, amino, hydroxy or carboxy; -(CH ₂ ) n-Ph and substituted -(CH ₂ )n-Ph n = 0, 1, 2, 3 - 10; substituted -(CH ₂ ) n-Ph on the carbon chain or on the benzene ring by one or two Or three H, halogen, cyano, nitro, amino, hydroxy or carboxy substituted.

(Example 13)

The code of the cytidine derivative of this example is G14.

Compound 39 was first prepared.

Compound 32 (1.5 g, 3.57 mmol), HMDS (15 mL), (NH ₄ ) ₂ SO ₄ (37.7 mg, 0.285 mmol) was added to 1,4-dioxane (15 mL) and stirred at 80 ° C Reflux for 4 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and toluene (5 ml × 2) was added to the solvent, and the oil pump was drained. Dichloromethane (30 mL), pyridine (0.846 g, 0.01 mol) were added, and the mixture was cooled to 0 ° C, and then ethyl chloroformate (1.83 g, 0.01 mol) was added and stirred at 10 ° C overnight. After completion of the reaction, the solvent was distilled off under reduced pressure. 40 mL of methanol was added, and the mixture was cooled at 0 ° C for 15 minutes, and 6 mL of triethylamine was added dropwise. Stir at 10 ° C overnight. After the completion of the reaction, the solvent was evaporated under reduced pressure and purified using silica gel chromatography (dichloromethane/methanol: 40:1) to afford 570 mg (yield 28.8%) Compound 37.

ESIMS: calcd for C ₂₅ H ₂₄ F ₂ N ₆ O ₆ m/z 555.24 (M+H)+, found 555.04.

Compound 37 (520 mg, 0.939 mmol), Na ₂ CO ₃ (497.5 mg, 4.69 mmol) was added to 12 mL of 1,4-dioxane and 3 mL of water and stirred until dissolved; (Boc) ₂ O (245.5) Mg, 1.126 mmol), stirred at room temperature overnight. After completion of the reaction, the solvent was evaporated under reduced pressure and purified using silica gel chromatography (dichloromethane/methanol: 80:1) to afford 278 mg (yield: 45.

ESIMS: calcd for C ₃₁ H ₃₂ F ₂ N ₆ O ₈ m/z 655.28 (M+H)+, found 655.08.

Compound 38 (200 mg, 0.3 mmol), Compound 22 (93.6 mg, 0.367 mmol), DCC (126 mg, 0.612 mmol), DMF (10 mg) was added to dichloromethane (10 ml) and stirred at room temperature overnight. After completion of the reaction, DCC was removed by suction filtration, and the solvent was evaporated under reduced pressure, and purified using silica gel column (dichloromethane/methanol: 125:1) to yield 170 mg (yield 62.5%) of Compound 39.

ESIMS: calcd for C ₃₈ H ₃₄ Cl ₃ F ₂ N ₇ O ₁₀ m/z 914.14 (M+Na)+, found 914.36.

Compound 39 (140 mg, 0.157 mmol) was added to 10 mL of DCM/TFA=5:1 system, and stirred at room temperature for 4 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, using silica gel chromatography column (dichloromethane/methanol: 50 : 1) Purification gave product G14 95 mg (yield 76.43%).

ESIMS: calcd for C ₃₃ H ₂₆ Cl ₃ F ₂ N ₇ O ₈ m/z 814.26 (M+Na)+, found 814.61.

¹ H-NMR (CDCl ₃ , 400 MHz) δ 8.50 (s, 1H), 8.36 (s, 1H), 7.63 (s, 1H), 7.33 (m, 9H), 7.23 (m, 2H), 6.38 (m) , 1H), 5.49 (m, 2H), 5.30 (s, 4H), 5.13 (m, 1H), 4.80 (m, 1H), 4.50 (m, 2H), 4.30 (m, 1H).

¹³ C-NMR (CDCl ₃ , 100 MHz) δ 168.61157.65155.68146.20143.22140.59128.87128.02123.07117.2468.1964.1154.4012.69.

进行反

C₁至C₁₀的烷氧基、C₁至C₁₀的取代烷氧基、C₁至C₆的烷基磺酰基、C₁至C₆的烷硫基、-(CH₂)n-Ph或取代-(CH₂)n-Ph；其中取代烷基、取代烷氧基的碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；-(CH₂)n-Ph和取代-(CH₂)n-Ph的n＝0、1、2、3～10；取代-(CH₂)n-Ph的碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。Replace the azide compound that is currently available on the market or that has been synthesized

Counter

C ₁ to C ₁₀ alkoxy group, C ₁ to C ₁₀ substituted alkoxy group, C ₁ to C ₆ alkylsulfonyl group, C ₁ to C ₆ alkylthio group, -(CH ₂ )n-Ph Or substituted -(CH ₂ )n-Ph; wherein the substituted alkyl, substituted alkoxy carbon chain is independently substituted by one or two or three halogens, cyano, nitro, amino, hydroxy or carboxy; (CH ₂₎ n-Ph, and substituted - (CH ₂₎ n-Ph of n = 0,1,2,3 ~ 10; substituted - a (CH ₂₎ n-Ph, or a carbon chain on the phenyl ring by one or Two or three H, halogen, cyano, nitro, amino, hydroxy or carboxy groups are substituted.

(Example 14)

The code of the cytidine derivative of this example is G15.

Compound 43 was first prepared.

Compound 32 (1.3 g, 3.0 mmol), HMDS (13 mL), (NH ₄ ) ₂ SO ₄ (32.7 mg, 0.247 mmol) was added to 1,4-dioxane (13 mL) and stirred at 80 ° C Reflux for 4 hours. After the completion of the reaction, the solvent was distilled off under reduced pressure, and toluene (5 ml × 2) was added to the solvent, and the oil pump was drained. Dichloromethane (26 ml), pyridine (0.733 g, 9.3 mmol) was added, and the mixture was cooled to 0 ° C slowly butyl chloroformate (1.268 g, 9.3 mmmol) and stirred at 10 ° C overnight. After completion of the reaction, the solvent was distilled off under reduced pressure. Methanol (30 ml) was added, and the mixture was cooled at 0 ° C for 15 minutes, and triethylamine (4.5 ml) was added dropwise. Stir at 10 ° C overnight. After the completion of the reaction, the solvent was evaporated under reduced pressure and purified using silica gel chromatography (dichloromethane/methanol: 40:1).

Compound 41 (800 mg, 1.538 mmol), Na ₂ CO ₃ (815 mg, 7.69 mmol) was added to 25 mL of 1,4-dioxane and 6 mL of water, and stirred until dissolved; (Boc) ₂ O (403 mg, 1.85) was added at normal temperature. Methyl), stirred at room temperature overnight. After the completion of the reaction, the solvent was evaporated under reduced pressure and purified using silica gel chromatography (dichloromethane/methanol: 80:1) to afford 458 mg (yield 48.1%) Compound 42.

ESIMS: calcd for C ₂₈ H ₃₄ F ₂ N ₆ O ₈ m/z 637.76 (M+NH4)+, found 637.46.

Compound 42 (380 mg, 0.613 mmol), Compound 22 (187.5 mg, 0.736 mmol), DCC (252.5 mg, 1.262 mmol), and DMAP (20 mg) were added to dichloromethane (20 ml) and stirred at room temperature overnight. After completion of the reaction, the DCC was removed by suction filtration, and the solvent was evaporated under reduced pressure, and purified using silica gel chromatography (dichloromethane/methanol: 125:1) to afford 333 mg (yield: 63.

ESIMS: calcd for C ₃₅ H ₃₆ Cl ₃ F ₂ N ₇ O ₁₀ m/z 858.18 (M+H)+, found 858.28.

Compound 43 (310 mg, 0.362 mmol) was added to 12 ml of DCM/TFA=5:1 system, and stirred at room temperature for 4 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, using silica gel chromatography column (dichloromethane/methanol: 50 : 1) Purification afforded 235 mg (yield 85.8%) of product G15.

ESIMS: calcd for C ₃₀ H ₂₈ Cl ₃ F ₂ N ₇ O ₈ m/z 758.18 (M+H)+, found 758.18.

¹ H NMR (CDCl ₃ , 400 MHz) δ 8.43 (s, 1H), 8.33 (s, 1H), 8.11 (m, 1H), 7.70 (m, 1H), 7.63 (m, 1H), 7.34 (m, 4H) ), 6.34 (m, 1H), 5.53 (m, 3H), 5.21 (m, 2H), 4.76 (m, 1H), 4.54 (m, 1H), 4.46 (m, 1H), 4.43 (m, 1H) , 4.20 (m, 2H), 1.70 (m, 2H), 1.41 (m, 2H), 0.95 (m, 3H).

进行反

C₁至C₁₀的烷氧基、C₁至C₁₀的取代烷氧基、C₁至C₆的烷基磺酰基、C₁至C₆的烷硫基、-(CH₂)n-Ph或取代-(CH₂)n-Ph；其中取代烷基、取代烷氧基的碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；-(CH₂)n-Ph和取代-(CH₂)n-Ph的n＝0、1、2、3～10；取代-(CH₂)n-Ph的碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。Replace the azide compound that is currently available on the market or that has been synthesized

Counter

C ₁ to C ₁₀ alkoxy group, C ₁ to C ₁₀ substituted alkoxy group, C ₁ to C ₆ alkylsulfonyl group, C ₁ to C ₆ alkylthio group, -(CH ₂ )n-Ph Or substituted -(CH ₂ )n-Ph; wherein the substituted alkyl, substituted alkoxy carbon chain is independently substituted by one or two or three halogens, cyano, nitro, amino, hydroxy or carboxy; (CH ₂₎ n-Ph, and substituted - (CH ₂₎ n-Ph of n = 0,1,2,3 ~ 10; substituted - a (CH ₂₎ n-Ph, or a carbon chain on the phenyl ring by one or Two or three H, halogen, cyano, nitro, amino, hydroxy or carboxy groups are substituted.

(Example 15)

The code of the cytidine derivative of this example is G16.

Compound 11 (2 g, 4.52 mmol), sodium carbonate (3.4 g) was dissolved in a mixture of 133 mL of 1,4-dioxane and 34 mL of water and stirred for 10 min; di-tert-butyl dicarbonate (1.47 g, 6.78 mmol) was added. ), react at room temperature for at least 48 h. The solvent was evaporated, and dichloromethane (EtOAc) (EtOAc) The organic phase is dried. Column separation (dichloromethane:methanol = 80:1) gave 1.2 g (48.9%) of Compound 45.

ESIMS: calcd for C19H26BrF2N3O8m/z 542.09(M+H) ⁺ ,found 542.11

Boc-protected compound 45 (355 mg, 0.65 mmol) was combined with compound 22 (499 mg, 1.95 mmol), DCC (401 mg, 1.95 mmol), and then added to 45 mL of dichloromethane, and DMAP (2 mg, 0.016 mmol) was added. Stir at room temperature overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted with 2×20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate and then added to TFA to give the target compound G16 ( 110 mg, 2 steps yield 24%).

¹ H-NMR (MeOD-d ₄ , 400MHz) δ: 8.05 (s, 2H, Ar), 6.22 (t, 1H, J = 7.6 Hz, H1'), 5.15 (m, 2H), 4.67 (m, 1H) , H5a'), 4.48 (m, 4H, H5b', H4', O-CH2-CH3), 1.70 (m, 2H, -O-CH2-CH2-CH2-CH3), 1.28 (m, 2H, -O) -CH2-CH2-CH2-CH3), 0.97 (m, 2H, -O-CH2-CH2-CH2-CH3).

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 168.12, 155.98, 142.88, 141.03, 124.15, 122.75, 117.32, 82.73, 78.37, 77.97, 63.76, 63.55, 62.67, 41.82, 31.04, 30.63, 29.98, 18.88, 18.84, 12.81, 8.29.

ESIMS: calcd for C21H20BrCl3F2N4O8m/z 681.94 (M+2H) ⁺ , found 681.15.

(Embodiment 16)

The code of the cytidine derivative of this example is G17.

Compound 10 (911 mg, 1.75 mmol) was dissolved in 50 mL of dimethylformamide, and dibromohydantoin (500 mg, 1.75 mmol) was added, and the pale yellow solution was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure. EtOAcjjjjjjjj

¹ H-NMR (CDCl 3 , 400 MHz) δ 8.40 (s, 1H, Ar), 8.23 (d, J = 4 Hz, 2H, Ar), 7.60 (d, J = 4 Hz, 2H, Ar), 6.15 (m, 1H, H1'), 5.30 (s, 2H, Ar-CH ₂ ), 4.50 (m, 1H, H5a'), 4.48 (m, 4H, H5b', H4', O-CH2-CH3).

Compound 47 (2.33 g, 5.72 mmol), sodium carbonate (4.12 g) was dissolved in 133 mL of 1,4-dioxane and 34 mL of water and stirred for 10 min. Di-tert-butyl dicarbonate (1.72 g, 7.9 mmol) was added; the reaction was carried out at room temperature for at least 48 h. The solvent was evaporated to dryness and dichloromethane (EtOAc) (EtOAc) The organic phase is dried. Column separation (dichloromethane:methanol = 80:1) gave 1.4 g (49%) of Compound 48.

The Boc-protected compound 48 (540 mg, 0.869 mmol) was combined with compound 22 (667 mg, 2.60 mmol), DCC (537 mg, 2.60 mmol), then added to 45 mL of dichloromethane and DMAP (2 mg, 0.016 mmol) Stir at room temperature overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted with 2×20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous sodium sulfate and then added to TFA to obtain the target compound G17 ( 115mg, 2 step yield 17%)

¹ H-NMR (MeOD-d ₄ , 400MHz) δ: .05 (s, 2H, Ar), 6.22 (t, 1H, J = 7.6 Hz, H1'), 5.15 (m, 2H), 4.67 (m, 1H, H5a'), 4.48 (m, 4H, H5b', H4', O-CH2-CH3), 1.70 (m, 2H, -O-CH2-CH2-CH2-CH3), 1.28 (m, 2H, - O-CH2-CH2-CH2-CH3), 0.97 (m, 2H, -O-CH2-CH2-CH2-CH3).

¹³ C-NMR (MeOD-d ₄ , 100 MHz) δ: 67.81, 157.47, 155.55, 142.93, 141.00, 128.98, 128.42, 123.99, 117.54, 79.92, 76.89, 76.55, 66.69, 65.80, 63.78, 62.54.

ESIMS: calcd for C24H17BrCl3F2N5O10m/z 760.92 (M+2H) ⁺ , found 760.10.

(Example 17 hydrochloride of cytidine derivative)

In the present example, the hydrochloride salt of 4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine of the compound of Example 1 was prepared.

Take 0.50 g of 4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine dissolved in 60 mL of ethyl acetate, and pass dry hydrochloric acid under ice bath, stir 15 The solvent was removed after a minute to give a white solid product.

The hydrochloride of other cytidine derivatives is prepared as above.

In addition to the above hydrochloride salt, it is also possible to prepare a phosphate, a sulfate, a carbonate, a nitrate, a citrate, a tartrate, a maleate, a succinate, a sulfonate, a p-toluene of a cytidine derivative. Sulfonate, methanesulfonate, benzoate or fumarate.

(Example 18, lyophilized powder injection for injection of cytidine derivative)

This example prepared a lyophilized powder injection of the compound G14 of Example 13.

The lyophilized powder injection of G14 comprises 30 g of compound G14, mannitol (20% w/v) 300 g, buffer buffer 7 g of sodium dihydrogen phosphate dihydrate, and surfactant poloxamer 188 (F68) 4.0 g.

Sodium dihydrogen phosphate dihydrate, poloxamer 188 (F68) (CAS number: 9003-11-6), mannitol (20% w/v) accurately weighed according to the above prescribed amount, 300 g pre-cooled to 10 After dissolving in water for injection below °C, adjust the pH of the solution to 7.3-7.5 with 0.1 mol/L NaOH; add the prescribed amount of G14 to the above solution and mix well, using 0.1 mol/L NaOH solution or 0.1 mol/ The pH of L HCl was adjusted to 7.3±0.2 (7.5 in this example); water was added to 2000g, and the solution was filtered and sterilized by 0.22μm microporous membrane; the filtrate was 2.0g per bottle. Dispense in a controlled bottle, half-plugged and placed in a freeze dryer to freeze-dry, after drying, vacuum plug, capping, labeling, that is, lyophilized powder injection 1000, stored at 2 ~ 8 ° C temperature.

In addition to the above-mentioned lyophilized powder injection, that is, a sterile powder for injection, the cytidine derivative of the present invention can be prepared into other forms of injections such as a solution injection, a suspension injection, and an emulsion injection.

(Example 19, oral pharmaceutical composition of cytidine derivative)

The pharmaceutical composition of the cytidine derivative of the present embodiment is composed of an active ingredient and an adjuvant, wherein the pharmaceutically active component is the cytidine derivative prepared in the above examples or a corresponding salt thereof. The proportion of the pharmaceutically active component in the composition is from 1% to 95% (30% in this embodiment). The excipient consists of water, lactose, corn starch, hydroxypropyl methylcellulose and magnesium stearate. The pharmaceutical composition of the present embodiment is in the form of a tablet.

Appropriate Formulations of Pharmaceutical Compositions In addition to the tablet forms referred to above, the pharmaceutically active component can be formulated into oral powders, granules, capsules, pellets, solutions, suspensions, emulsions, syrups or An expectorant, or a sustained release and controlled release preparation in oral form, or a pharmaceutical composition in other oral form, which contains common corresponding excipients (additives, addenda, etc. depending on the effect), such as additives There are pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin salts, cellulose or magnesium sulfate.

In achieving the above oral dosage form, a pharmaceutically acceptable addenda may be selected as a carrier for the pharmaceutically active ingredient, including materials mature in the prior art, such as inert solid diluents, aqueous solvents, liposomes, microspheres or/and none. Toxic organic solvents, etc.; preferred additions are: moisturizer, emulsifier, pH buffer, human serum albumin, antioxidants, preservatives, bacteriostatic agents, glucose, sucrose, trehalose, maltose, lecithin, glycine, Sorbic acid, propylene alcohol, polyethylene, protamine, boric acid, sodium chloride, or potassium chloride, mineral oil, vegetable oil, etc.; one or several combinations may be selected as a pharmaceutical carrier.

The target tumor of the pharmaceutical composition of the present invention includes a hematological tumor or a malignant solid tumor; specifically, the target tumor includes lung cancer, prostate cancer, breast cancer, colon cancer, gastric cancer, pancreatic cancer, liver cancer, esophageal cancer, brain tumor, ovarian cancer , uterine cancer, kidney cancer, head and neck cancer, skin cancer, bladder cancer, vulvar cancer, testicular tumor, rectal cancer, villus cancer, germ cell tumor, malignant lymphoma, leukemia and multiple myeloma, and even more preferred target tumor Pancreatic cancer (first- and second-line treatment), non-small cell lung cancer, breast cancer, ovarian cancer, and head and neck squamous cell carcinoma, colon cancer may be included, but the present invention is not limited thereto.

(Application Example 1, a single intraperitoneal administration of a series of compounds in the maximum tolerated dose test in ICR mice)

This experiment is to study the toxicity of a single intraperitoneal administration of the test substance to ICR mice, and determine the maximum tolerated dose (MTD) of each test substance. The maximum tolerated dose (MTD) is the dose at which the animal does not die, the animal's weight loss does not exceed 10% (compared to Day 0), or does not produce significant toxic side effects.

1. The test object is configured as follows.

The solvent used to dissolve the test substance is as follows:

Weigh the corresponding test substance in a 5mL glass test tube, dissolve it in ethanol under the stirring of 5mm magnetic stirrer, add all the solution, add Cremophor EL, keep stirring, add the labeled amount of physiological saline before use, stir evenly, configure The volume ratio of ethanol, Cremophor EL, and physiological saline was 5:5:90.

2. Test animals

Varieties and strains: ICR mice; grade: SPF; gender: female.

Source: Shanghai Slack Laboratory Animal Co., Ltd.

Certificate number: 0130749.

The experiment began with animal weight: 18-20 g.

Quantity and gender: 41 females.

Feeding method: six cages.

Adaptation period: 5 to 7 days, the same feeding conditions as in the experiment.

The animal room has an ambient temperature of 18-26 ° C, a relative humidity of 30-70%, and 12 hours of light. The experimental animals were acclimated for 5-7 days before the experiment. The SPF large mouse growth and propagation feed Co60 was sterilized and purchased from Beijing Keao Xieli Co., Ltd. The experimental animals were filtered and sterilized with water, and the animals were free to eat and drink.

3. Experimental methods

Mode of administration: ip. If the animal dies, the dose is reduced until the animal survives, and if there is no animal death, the dose is increased; if the animal is normally alive at a given high dose, the experiment is over. Finally, the MTD of the test subject was determined according to the experimental results; the animals were continuously observed for 7 days after acute administration.

All animals in the experiment were subjected to detailed clinical observations of all the tested animals. Two times a day (10:00, 16:00 each), continuous observation for 14 days, including but not limited to: skin, Hair, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs and feet, respiratory and circulatory systems, autonomic effects (such as salivation), nervous system (such as tremors, convulsions, stress reactions, and abnormal behavior).

The body weight of the animals was weighed before administration, and then the body weight of the animals was weighed and recorded at the same time every day.

The observations, animal weight, and animal survival after one week of dosing were recorded in detail daily.

4 test results

G3 and G4 can tolerate at 350mg/kg, G5 can tolerate at 300mg/kg, G6 can tolerate at 200mg/kg, G7 can tolerate at 200mg/kg, G8 can tolerate at 300mg/kg, G10 at 400mg /kg can be tolerated, G11 can tolerate at 400mg/kg, G12 can tolerate at 400mg/kg, G13 can tolerate at 400mg/kg, G15 can tolerate at 400mg/kg, and G16 can tolerate at 400mg/kg .

(Application Example 2, series of compounds inhibit tumor growth)

In this application example, the growth inhibition effect and toxicity of single intraperitoneal injection of compound G2 to G17 on colon cancer HCT-116 tumor-bearing nude mice were evaluated by observing the formation of tumor at the inoculation site and the change in body weight of the test animals.

1. Purpose of the test

The growth inhibitory effect and toxicity of the cytidine derivative sample of the present invention on transplanted tumor of colon cancer HCT-116 tumor-bearing nude mice were determined.

2. Preparation of test materials

The solvent used to dissolve the test substance is as follows:

Solvent lot number Vendor Absolute ethanol 10009218 Sinopharm Chemical Reagent Co., Ltd. Cremophor EL 27963 Sigma 0.9% saline 13083004 Huayu Pharmaceutical Co., Ltd.

Weigh the corresponding test substance in a 5mL glass test tube, dissolve it in ethanol under the stirring of 5mm magnetic stirrer, add all the solution, add Cremophor EL, keep stirring, add the labeled amount of physiological saline before use, stir evenly, configure The volume ratio of ethanol, Cremophor EL, and physiological saline was 5:5:90.

3. Experimental animals

Variety and strain: Balb/c Nude mice; Level: SPF; Gender: Female.

Source: Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.

Number of animals: Order 100, choose the ones that are in good health for the experiment.

Animal certificate number: 0123627.

Animal age at the start of the experiment: 7-9 weeks old.

Animal weight at the start of the experiment: 18-22 grams.

Adapt to the environment time: 5-7 days.

Animal numbering method: tail number.

The animal room environment maintained a temperature of 23 ± 2 ° C, humidity of 40-70%, alternating 12 hours of light and dark.

Animal feed (SLAC-M01) was purchased from Beijing Keao Xieli Co., Ltd. The experimental animals were filtered and sterilized with water. Animals were free to eat and drink during the experiment.

4. Experimental methods

4.1 Tumor cells: Colon cancer HCT-116 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in a carbon dioxide incubator at 37 ° C, saturated humidity, and containing a volume fraction of 5% CO ₂ and 95% air using F-12 medium (containing 10% FBS). Logarithmic growth phase cells were taken before inoculation, digested with 0.25% trypsin, washed once with PBS, resuspended in PBS, resuspended in serum-free medium, and adjusted to a cell concentration of about 3 x 10^7 cells/mL.

4.2 Animal inoculation and grouping: Each nude mouse was subcutaneously inoculated with 0.1 mL of cell suspension (3x10^6 cells/mouse) under sterile conditions. When the tumor grows to a volume of about 60-150 mm ³ , nude mice with similar tumor volume and good shape are selected (the shape is as single spherical as possible, no irregular shape or multiple tumors are gathered together), grouped, each group 6 Only, the grouping situation is as follows:

IP: intraperitoneal injection; QD × 1: injection once.

The control control group, that is, the model control group, was injected with a mixed solution of 5:5:90 ethanol, Cremophor EL, and physiological saline.

4.3 Animal administration and observation

The formation of tumors at the inoculation site of each group of nude mice was observed. The diameter of the tumor nodules (D) was measured with a round hole rule three times a week, and the volume (V) of the tumor nodules was calculated as follows: V=3/4π ( D/2) ³ .

The evaluation index of antitumor activity is the tumor growth inhibition rate TGI (%), and the relative tumor growth rate T/C (%).

The formula for calculating the tumor growth inhibition rate TGI (%) is: TGI (%) = (V _control - V _Treatment ) / V _control ) × 100%.

The relative tumor volume (RTV) is calculated as: RTV = Vt / V0. Where V0 is the tumor volume at the time of group administration, and Vt is the tumor volume at the time of measurement.

Relative tumor proliferation rate T/C (%), calculated as: T / C (%) = T _RTV / C _RTV × 100%.

T _RTV : treatment group RTV; C _RTV : negative control group RTV.

The body weight of the mice was weighed 3 times a week.

4.4 clinical symptoms

All clinical symptoms of each animal should be recorded at the beginning of the experiment and during the experiment. Observations should be made at the same time every day.

If the weight loss is >20% after administration of the test substance, the sudden death of the animal or the tumor volume exceeds 2800 mm^3, the CO _{2 is} sacrificed, the tumor is isolated and weighed, autopsy is performed, and the diseased organ is visually observed and recorded.

4.5 Data Statistics

The experimental data were expressed by Mean±SEM unless otherwise specified. The data between the two groups were analyzed by unpaired T test. P<0.05 was considered to be significant.

5 test results

(1) Clinical observation and mortality

One animal died in the G4 350 mg/kg group on the fourth day after administration, and three animals showed clinical symptoms such as decreased activity, decreased body weight, and lower body surface temperature than normal. Animals in the G8 350 mg/kg group died on the fourth day after administration. Five animals died in the G6 300 mg/kg group on the fourth day after administration. There were no significant abnormalities in the clinical symptoms of the model control group and the remaining compound groups (G3, G5, G7, G9, G10, G11, G12, G13, G15, G16). The mortality of each group of animals is shown in Table 2 (QD*1 in the table: single intraperitoneal injection).

Table 2

(2) Effect of test compound on body weight of human colon cancer HCT-116 tumor-bearing mice.

The average body weight of each group of animals is shown in Table 3.

Table 3-1 G3 to G9 mouse weights on different dates

*p<0.05, **p<0.01vs vehicle group

Table 3-2 G10 to G16 mouse weights on different dates

*p<0.05, **p<0.01vs vehicle group

Table 4-1 G3 to G9 weight change rate

*p<0.05, **p<0.01vs vehicle group

The weight change rate of G10 to G16 is shown in Table 4-2.

Table 4-2

*p<0.05, **p<0.01vs vehicle group

From the above chart data, in the growth inhibition effect of the compound on colon cancer HCT-116 tumor-bearing nude mice, the body weight of G4 350mg/kg was significantly decreased on the 4th day of administration (p<0.05), and the average weight loss rate was average. After 10.91±3.45%, the body weight increased steadily, and the body weight was significantly increased on the 18th to 20th day compared with the model control group (p<0.05). The body weight of G5 325mg/kg was significantly decreased (p<0.05), and the body weight loss rate was <10%. After that, the body weight increased steadily, and the body weight increased significantly on the 13th to 20th day compared with the model control group (p< 0.05 to 0.01). G7 250mg/kg showed a significant decrease in body weight (p<0.05) on days 4 and 6 of administration, and the body weight loss rates were 12.28±4.78% and 4.39±3.6%, respectively, and then the body weight increased steadily. There was no significant difference in body weight between the other drug-administered groups and the model control.

(3) Effect of test compound on tumor volume of human colon cancer HCT-116 tumor-bearing mice

Specific data of tumor volume of each group are shown in Table 5.

Table 5-1 G3-G9 tumor volume

*p<0.05, **p<0.01vs vehicle group

Table 5-2 G10-G16 tumor volume

*p<0.05, **p<0.01vs vehicle group

It can be seen from the data of the tumor volumes of the above groups that the cytidine derivative of the present invention has a significant inhibitory effect on tumors.

(4) Growth inhibition rate (TGI%) of test compound against human colon cancer HCT-116 tumor-bearing mice

The growth inhibition rate (TGI) of test compounds against human colon cancer HCT-116 tumor-bearing mice was shown in Table 6 below:

Table 6-1 Growth inhibition rate of G3-G9 on human colon cancer HCT-116 tumor-bearing mice

Table 6-2 Growth inhibition rate of G10-G16 on human colon cancer HCT-116 tumor-bearing mice

The maximum tumor inhibition rate of the compound G3 350 mg/kg group was 58.10% in Day8 and 46.82% in Day22. The tumor inhibition rate of the compound G4 350 mg/kg group was better at Day 11 and reached a maximum of 92.58%, and remained at 70% or more for Day 22 days. The tumor inhibition rate of the compound G5 325mg/kg group was better at Day11, reaching a maximum of 94.46%, and remained above 70% for Day24. The maximum tumor inhibition rate in the G9 325 mg/kg group was 80.77% in Day4 and about 40% in Day22. The maximum tumor inhibition rate in the G7 250 mg/kg group was 82.62% in Day8, and the inhibition rate in Day22 was 44.07%.

(5) Tumor relative volume (RTV) of test compound against human colon cancer HCT-116 tumor-bearing mice

The tumor relative volume of test compound G3-G9 against human colon cancer HCT-116 tumor-bearing mice is shown in Table 7-1 below:

Table 7-1

The relative tumor volume of the compound G4 350 mg/kg group was significantly lower from Day 4 to Day 18 (p<0.05 to 0.01) compared with the model control group. Compared with the model control group, the G5 325 mg/kg group had a significant decrease in tumor relative volume from Day 4 to Day 18 (p<0.05 to 0.01). Compared with the model control group, the G7 250 mg/kg group had a significant decrease in tumor relative volume from Day 4 to Day 15 (p<0.05 to 0.01). Compared with the model control group, the relative volume of tumor in the G9 325 mg/kg group was only significantly decreased in Day 4 (p<0.05). There was no significant difference in tumor relative volume between the other drug-administered groups and the model control group.

The relative tumor volume of G10-G16 in human colon cancer HCT-116 tumor-bearing mice is shown in Table 7-2 below:

Table 7-2

(6) Relative tumor growth rate (T/C%) of test compound against human colon cancer HCT-116 tumor-bearing mice

The relative tumor growth rate data of the test compound on human colon cancer HCT-116 tumor-bearing mice are shown in Table 8 below:

Table 8-1 Relative tumor growth rate of G3-116 in human colon cancer HCT-116 tumor-bearing mice

The relative tumor proliferation rate of the compound G4 350 mg/kg group reached a minimum of 17.62% in Day 13, and the tumor proliferation rate in Day 22 was 75.38%. The relative tumor proliferation rate of the compound G5 325 mg/kg group reached a minimum of 10.01% on Day 11, and the tumor proliferation rate on Day 22 was 68.77%. Compound G7 250mg/kg group relative tumor proliferation The rate reached a minimum of 17.67% in Day 8 and a tumor proliferation rate of 58.62% in Day 22 days.

In the growth inhibition experiment of a series of compounds on human colon cancer HCT-116 tumor-bearing nude mice, the compound G4, G5, G7 had a good tumor inhibition rate on colon cancer HCT-116 tumor-bearing nude mice xenografts. Day 8 to Day 13 had good tumor inhibition after intraperitoneal administration. The relative growth rate of G5 in Day 11 reached a minimum of 10.01%, and the effect on animal body weight was reduced, and the average weight loss rate was less than 10%.

Table 8-2 Relative tumor growth rate of G10-G16 in human colon cancer HCT-116 tumor-bearing mice

Based on the results of the above experiments, it was confirmed that the novel cytidine derivative of the present invention provides an excellent action as an antitumor agent.

The above-described embodiments are illustrative of the present invention, and those skilled in the art will appreciate that various modifications are possible and are within the scope of the present invention.

Claims (10)

A novel cytidine derivative having the following general formula (I):

Wherein, R1 is a C ₁ to C ₁₀ alkyl group is, C ₁ to C ₁₀ alkyl substituted _{a, - (CH 2) n-} Ph, or substituted _{- (CH 2) n-Ph} ; said - (CH ₂ n-Ph, wherein n=0, 1, 2, 3-10, Ph is benzene; the substituted alkyl group has independently one or two or three halogens, cyano groups and nitro groups on the carbon chain Substituted with amino, hydroxy or carboxy; said substituent -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10, independently on the carbon chain or on the phenyl ring by one or two or Substituted by three halogens, cyano, nitro, amino, hydroxy or carboxy; R2 is H, halogen, or

X1 is a C ₁ to C ₁₀ alkyl group, a C ₁ to C ₁₀ substituted alkyl group, a C ₁ to C ₁₀ alkoxy group, a C ₁ to C ₁₀ substituted alkoxy group, a C ₁ to C ₆ alkyl group. a sulfonyl group, a C ₁ to C ₆ alkylthio group, -(CH ₂ )n-Ph, or a substituted -(CH ₂ )n-Ph; said substituted alkyl group having a carbon chain independently of one or Substituted by two or three halogens, cyano, nitro, amino, hydroxy or carboxy; said substituted alkoxy, independently of one or two or three halogens, cyano, nitro, on the carbon chain Substituting an amino group, a hydroxyl group or a carboxyl group; said -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10; said substitution -(CH ₂ )n-Ph, wherein n = 0, 1, 2, 3 to 10, substituted on the carbon chain or on the benzene ring by one or two or three H, halogen, cyano, nitro, amino, hydroxyl or carboxyl groups; R3 is H or

Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three a substituted heterocyclic ring substituted with a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group, or a substituted heterocyclic ring independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups a ring; the heterocyclic ring is imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine or piperidine; the fused heterocyclic ring is quinoline or hydrazine; X2 is -(CH ₂ ) n-, wherein n= 1, 2, 3, or X2 is -O-(CH ₂ ) n-, where n = 0, 1, 2, 3. The novel cytidine derivative according to claim 1, wherein R2 is H. The novel cytidine derivative according to claim 1, wherein R2 is not H and R3 is not H. The novel cytidine derivative according to claim 3, wherein R2 is halogen or

X1 is -(CH ₂ )n-Ph or is substituted by -(CH ₂ )n-Ph. The novel cytidine derivative according to claim 4, wherein R1 is a C ₁ to C ₄ alkyl group, a C ₁ to C ₄ substituted alkyl group, a benzyl group, or a substituted benzyl group; Is a substituted imidazole independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three halogens, cyano, nitro, amino, hydroxy Or a carboxy substituted substituted pyridine, or a substituted phenyl ring independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups. Use of the compound of claim 1 or a salt thereof for the preparation of a medicament for treating a tumor. The use of the compound according to claim 6 for the preparation of a medicament for treating tumors, characterized in that the tumor is a hematological tumor or a malignant solid tumor. A pharmaceutically acceptable salt of a compound according to claim 6, wherein the salt is a hydrochloride, a phosphate, a sulfate, a carbonate, a nitrate, a citrate, a tartrate, a horse. Acidate, succinate, sulfonate, p-toluenesulfonate, methanesulfonate, benzoate or fumarate. A pharmaceutical composition comprising, as an active ingredient, the cytidine derivative of the formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipient. The pharmaceutical composition according to claim 9, wherein the composition is in the form of an injection or an oral preparation, wherein the injection is a solution injection, a suspension injection, an emulsion injection, or a sterile powder for injection, orally. The dosage form is a tablet, a powder, a granule, a capsule, a pellet, a solution, a suspension, an emulsion, a syrup or an elixir.