阅读说明：本技术 阿糖胞苷结构类似物及其制备方法和用途 (Cytarabine structure analogue and preparation method and application thereof ) 是由 付瑞新 黎维勇 周媛 于 2020-12-07 设计创作，主要内容包括：本发明公开了阿糖胞苷结构类似物及其制备方法和用途。本发明首次合成阿糖胞苷结构类似物P1～P4,均能抑制白血病细胞HL-60增殖,尤其,与Ara-C相比,阿糖胞苷结构类似物P4对白血病细胞增殖抑制能力更强,细胞穿透能力更强,进入细胞内的药物浓度和在细胞内产生的活性代谢产物三磷酸阿糖胞苷的浓度更高,有更高的生物利用度；能在细胞内转化成母药阿糖胞苷,显著提高体内靶标细胞的血药浓度,可避免大剂量用药带来的毒副作用和耐药性；显著延长阿糖胞苷的半衰期,在较长时间内维持有效血药浓度,可口服给药,可避免注射用药带来的不便。本发明的制备方法简单易操作,适合工业化生产。(The invention discloses a cytarabine structure analogue and a preparation method and application thereof. The cytarabine structure analogue P1-P4 synthesized for the first time can inhibit the proliferation of the leukemia cell HL-60, and particularly, compared with Ara-C, the cytarabine structure analogue P4 has stronger inhibition capability on the proliferation of the leukemia cell and stronger cell penetrating capability, the concentration of a medicament entering the cell and the concentration of an active metabolite cytarabine triphosphate generated in the cell are higher, and the bioavailability is higher; can be converted into the parent drug cytarabine in cells, obviously improves the blood concentration of target cells in vivo, and can avoid toxic and side effects and drug resistance caused by large-dose medication; obviously prolongs the half-life period of the cytarabine, maintains effective blood concentration for a longer time, can be orally taken, and can avoid the inconvenience caused by injection. The preparation method is simple and easy to operate, and is suitable for industrial production.)

1. An cytarabine structural analogue is characterized in that the structural general formula is as shown in the following formula (I):

wherein R1 ═ Ph or C₁₈H₃₇，

R₂H or

2. The cytarabine structural analog of claim 1, which is any one of the following cytarabine structural analogs P1-P4:

cytarabine structural analogue P1 represented by the following formula (ii):

cytarabine structural analogue P2 represented by the following formula (iii):

cytarabine structural analogue P3 represented by the following formula (iv):

cytarabine structural analog P4 represented by the following formula (V):

。

3. the process for producing a cytarabine structural analogue according to claim 1 or 2, comprising the steps of:

dissolving cytarabine and phosphoryl chloride derivatives in anhydrous tetrahydrofuran, N₂Cooling to-70-78 ℃ under protection, slowly dripping 1-methylimidazole, stirring at-70-78 ℃, heating to room temperature for reaction, and after the reaction is finished, carrying out post-treatment and purification on reaction liquid to obtain a product; wherein the content of the first and second substances,

when the phosphoryl chloride derivative is a compound 2 shown as the following formula (VI), the obtained product is a cytarabine structure analogue P1;

when the phosphoryl chloride derivative is a compound 4 shown as the following formula (VII), the obtained product is a cytarabine structure analogue P3;

。

4. the method for producing a cytarabine structural analog of claim 3, wherein cytarabine: phosphorus oxychloride derivatives: the molar ratio of 1-methylimidazole is 1: (1.5-2.0): (2.0-3.0).

5. The method for preparing a cytarabine structural analog of claim 3, wherein the post-treatment purification comprises: concentrating the reaction solution, adding dichloromethane for dilution, washing with dilute hydrochloric acid, water and saturated sodium chloride solution successively, combining organic phases, drying, concentrating, and purifying by column chromatography to obtain the product.

6. The process for producing a cytarabine structural analogue according to claim 1 or 2, comprising the steps of:

dissolving the selected compound and aspartic acid derivative in anhydrous tetrahydrofuran, N₂Cooling to-5-0 deg.C under protection, slowly dripping 1-propyl phosphoric anhydride, heating to room temperature for reaction, and reacting to obtain the first reaction solution₃Quenching the solution, extracting the water phase by using dichloromethane, combining the organic phases, drying, concentrating, and purifying by column chromatography to obtain an intermediate; dissolving the intermediate in methanol, adding Pd/C, introducing hydrogen for reaction, monitoring by thin-layer chromatography, and performing aftertreatment on the obtained second reaction liquid to obtain a product; wherein the content of the first and second substances,

the aspartic acid derivative is a compound 3 shown as the following formula (VIII)

When the selected compound is cytarabine structural analogue P1, the obtained product is cytarabine structural analogue P2;

when the selected compound is cytarabine structural analogue P3, the resulting product is cytarabine structural analogue P4.

7. The method of making a cytarabine structural analog of claim 6, wherein the compound selected is: aspartic acid derivatives: the molar ratio of 1-propyl phosphoric anhydride is 1: (1.2-1.5): (2.0-2.5).

8. The method of preparing a cytarabine structural analog of claim 6, wherein the post-treatment comprises: and (3) allowing the second reaction solution to pass through a kieselguhr pad to collect first filtrate, eluting the kieselguhr pad with methanol to collect second filtrate, combining the first filtrate and the second filtrate to obtain filtrate, concentrating the filtrate, and stirring and filtering with methyl tert-butyl ether to obtain a product.

9. Use of a cytarabine structural analogue according to claim 1 or 2 in the manufacture of a medicament for the treatment of leukemia.

10. Use of a cytarabine structural analogue according to claim 1 or 2 in the manufacture of an oral medicament for the treatment of leukemia.

Technical Field

The invention belongs to the field of organic synthesis, and particularly relates to a cytarabine structure analogue, and a preparation method and application thereof.

Background

Cytosine arabinoside (Ara-C) chemical name: 1-beta-D-arabinofuranosyl-4-amino-2 (1H) -cytosine ketone with CAS registry number [147-94-4]The molecular formula is as follows: c₉H₁₃N₃O₅The structural formula is as follows:

cytarabine belongs to pyrimidine antimetabolites and is mainly used for treating Acute Myelogenous Leukemia (AML). Ara-C is converted into arabinoside triphosphate (Ara-CTp) in vivo through three steps to exert an anticancer effect. Firstly, cytarabine monophosphate (Ara-CMp) is formed under the catalysis of intracellular deoxycytidine kinase, and then the cytarabine monophosphate and the cytarabine diphosphate are converted into cytarabine diphosphate (Ara-CDp) and active cytarabine triphosphate (Ara-CTp) through the action of nucleoside diphosphate kinase, wherein the cytarabine triphosphate influences DNA synthesis, inhibits the growth of cells and interferes the proliferation of the cells by inhibiting DNA polymerase; it may also penetrate into the DNA in small amounts, interfering with the replication of the DNA and causing cell death. But has no obvious effect on RNA and protein synthesis, belongs to cell cycle specific drugs acting on S phase, is most sensitive to the effect of cells in S proliferation phase, and also has effect on G1/S and S/G2 conversion phase.

Cytarabine was originally developed by Upjohn in the united states and was marketed in the united states 10 months in 1969, and the time of first registration in the country was 1992. Cytarabine has very high molecular polarity, so that the membrane permeability of small intestine is poor, the oral bioavailability of cytarabine is very low, only 20 percent of the medicine enters blood circulation, and the cytarabine is not taken orally but is taken intravenously. Therefore, the cytarabine sold in the market is mainly an injection (powder injection), and at present, a plurality of manufacturers in China produce cytarabine hydrochloride injection. In addition, the medicine can be deaminated by liver cytosine deaminase to form inactive uracil cytosine arabinoside, t_1/2The time is short, only 3-15 minutes, so that the effective blood concentration can be maintained only by adopting continuous intravenous drip administration, and a better effect is obtained. For example, when the composition is used for treating acute myelocytic leukemia, the composition is usually injected or instilled intravenously, and the daily dose is 100-200 mg/m²Or 2-6 mg/Kg, and the dosage is increased from small to large until bone marrow suppression appears after being used for 5-7 days. Stop the medicine for 5 to 7 daysCan be used repeatedly after bone marrow recovery. Clinically, the large-dose cytarabine has better long-term treatment effect than the common dose, but has toxic and side effects on blood, nervous systems and organs, is easy to generate drug resistance, and the survival rate in five years is not obviously improved. The toxic and side effects of the cytarabine are positively correlated with the dosage, and the clinical use of the cytarabine is also limited.

Therefore, there is a need to develop more effective therapeutic agents that can be administered orally and conveniently to the convalescent patient out of hospital.

Disclosure of Invention

In view of the above, the invention provides cytarabine structural analogues and a preparation method and application thereof. The cytarabine structural analogue can inhibit the proliferation of human leukemia cells, and particularly, compared with cytarabine, some cytarabine structural analogues have better permeability, higher bioavailability and longer half-life period for leukemia cells, can be orally administered, and are expected to be used as novel anti-leukemia drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, cytarabine structural analogs are provided, having the general structural formula (I):

wherein R1 ═ Ph or C₁₈H₃₇，

R₂H or。

In particular, the present invention relates to a method for producing,

the cytarabine structural analogue can be a cytarabine structural analogue P1 shown in the following formula (II):

alternatively, the cytarabine structural analog may be a cytarabine structural analog P2 represented by the following formula (iii):

alternatively, the cytarabine structural analog may be a cytarabine structural analog P3 represented by the following formula (iv):

alternatively, the cytarabine structural analog may be a cytarabine structural analog P4 represented by the following formula (V):

in a second aspect of the invention, a method for preparing a cytarabine structural analog is provided.

A preparation method of cytarabine structural analogues comprises the following steps:

dissolving cytarabine and phosphoryl chloride derivatives in anhydrous tetrahydrofuran, N₂Cooling to-70-78 ℃ under protection, slowly dripping 1-methylimidazole, stirring at-70-78 ℃, heating to room temperature for reaction, and after the reaction is finished, carrying out post-treatment and purification on reaction liquid to obtain a product; wherein the content of the first and second substances,

when the phosphoryl chloride derivative is a compound 2 shown as the following formula (VI), the obtained product is a cytarabine structure analogue P1;

when the phosphoryl chloride derivative is a compound 4 shown as the following formula (VII), the obtained product is a cytarabine structure analogue P3;

。

in a preferred embodiment, cytarabine: phosphorus oxychloride derivatives: the molar ratio of 1-methylimidazole is 1: (1.5-2.0): (2.0-3.0).

In the preferred technical scheme, the stirring time is 1-2 hours.

In a preferred technical scheme, the temperature rise is natural temperature rise.

In a preferred technical scheme, the room temperature reaction time is 12-16 hours.

In a preferred technical scheme, the post-treatment purification comprises the following steps: concentrating the reaction solution, adding dichloromethane for dilution, washing with dilute hydrochloric acid, water and saturated sodium chloride solution successively, combining organic phases, drying, concentrating, and purifying by column chromatography to obtain the product.

In a preferred embodiment, the concentration is carried out under reduced pressure, most preferably under reduced pressure at 40 ℃.

In the preferable technical scheme, the column chromatography adopts dichloromethane/methanol with the volume ratio of 20/1-10/1.

Alternatively, the first and second electrodes may be,

a preparation method of cytarabine structural analogues comprises the following steps:

dissolving the selected compound and aspartic acid derivative in anhydrous tetrahydrofuran, N₂Cooling to-5-0 deg.C under protection, slowly dripping 1-propyl phosphoric anhydride, heating to room temperature for reaction, and reacting to obtain the first reaction solution₃Quenching the solution, extracting the water phase by using dichloromethane, combining the organic phases, drying, concentrating, and purifying by column chromatography to obtain an intermediate; dissolving the intermediate in methanol, adding Pd/C, introducing hydrogen for reaction, monitoring by thin-layer chromatography, and performing aftertreatment on the obtained second reaction liquid to obtain a product; wherein the content of the first and second substances,

the aspartic acid derivative is a compound 3 shown as the following formula (VIII)

When the selected compound is cytarabine structural analogue P1, the obtained product is cytarabine structural analogue P2;

when the selected compound is cytarabine structural analogue P3, the resulting product is cytarabine structural analogue P4.

In a preferred embodiment, the compound is selected from: aspartic acid derivatives: the molar ratio of 1-propyl phosphoric anhydride is 1: (1.2-1.5): (2.0-2.5).

In a preferred technical scheme, the temperature rise is natural temperature rise.

In the preferred technical scheme, the room temperature reaction time is 1-2 hours.

In the preferable technical scheme, petroleum ether/ethyl acetate with the volume ratio of 20/1-1/1 is adopted for column chromatography.

In the preferred technical scheme, the dosage of Pd/C is 2-10% of the mass of the selected compound.

In the preferred technical scheme, the hydrogen is 0.05-0.1 Mpa.

In a preferred technical scheme, the post-treatment comprises the following steps: and (3) allowing the second reaction solution to pass through a kieselguhr pad to collect first filtrate, eluting the kieselguhr pad with methanol to collect second filtrate, combining the first filtrate and the second filtrate to obtain filtrate, concentrating the filtrate, and stirring and filtering with methyl tert-butyl ether to obtain a product.

In a preferred embodiment, the concentration is carried out under reduced pressure, most preferably under reduced pressure at 40 ℃.

In the invention, the room temperature is 20-30 ℃.

In a third aspect of the invention, there is provided the use of a cytarabine structural analogue in the manufacture of a medicament for the treatment of leukaemia.

Furthermore, the invention also provides application of the cytarabine structural analogue in preparing an oral medicament for treating leukemia.

Compared with the prior art, the invention has the following beneficial technical effects:

the cytarabine structural analogues P1-P4 synthesized for the first time can inhibit human leukemia HL-60 cells, have better inhibition effect than cytarabine hydrochloride under low concentration, and are expected to be used as novel anti-leukemia drugs.

Particularly, compared with cytarabine, the cytarabine structural analogue P4 has stronger inhibition capacity on the proliferation of leukemia cells and stronger cell penetrating capacity, the concentration of the medicament entering the cells and the concentration of an active metabolite cytarabine triphosphate generated in the cells are higher, and the bioavailability is higher; the cytarabine is transformed into a parent drug cytarabine in cells, so that the blood concentration of target cells in vivo is remarkably improved, and toxic and side effects and drug resistance caused by large-dose medication can be avoided; obviously prolongs the half-life period of the cytarabine, maintains effective blood concentration for a longer time, and can be orally taken, thereby avoiding the inconvenience caused by injection medication. Therefore, the cytarabine structural analogue P4 completely overcomes the defects of high polarity, poor membrane permeability, easy metabolic inactivation in vivo, short half-life period and the like of cytarabine, provides possibility for oral administration, and fills the technical blank that no new medicine is available in the market at home.

The preparation method of the cytarabine structural analogue is simple and easy to operate, and is expected to be used for industrial production.

Drawings

FIG. 1 is the HNMR spectrum of the compound obtained in example 1, which shows that the structure is shown as formula (II) and is marked as cytarabine structural analogue P1.

FIG. 2 is a graph showing the comparison of the concentrations of intracellular drug and metabolite obtained by incubating different drugs to be tested with human leukemia cell HL60 for different periods of time in example 8.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Those skilled in the art can use conventional methods, for example, according to the techniques or conditions described in the literature in the field or according to the conditions suggested by the suppliers in the product specifications.

The various instruments, raw materials and reagents not specifically described in the following examples, those not noted in the manufacturers, are all conventional products commercially available. The particular materials used and their sources set forth in the following examples are illustrative only and not intended to be limiting of the invention, as materials identical or similar in type, quality, nature or function to the tissues, cells, reagents and instruments described below may be used in the practice of the invention.

Description of materials and sources:

cytarabine, CAS: 147-94-4, available from Haohnhong biomedical science and technology, Inc., Shanghai.

Cytarabine hydrochloride, CAS: 69-74-9, available from Haohnhong biomedical science and technology, Inc., Shanghai.

Compound 2, having the structure shown in formula (VI), CAS: 261909-49-3, available from Wuhanxi rui medicine science and technology, Inc.

Compound 3, having the structure shown in formula (VIII), CAS: 4779-31-1, available from Shanghai Haohnhong biomedical science and technology, Inc.

Compound 4, having the structure shown in formula (VII), is available from Wuhanxi Rui pharmaceutical science and technology, Inc.

1-Propylphosphoric anhydride (50% ethyl acetate solution) available from Shanghai Bidi pharmaceutical science, Inc.

Human leukemia cell HL-60, purchased from Wuhan Pronoch Life technologies, Inc.

PBMC cells, peripheral blood mononuclear cells, were isolated from rat blood by Ficoll lymph isolate (sigma).

Sprague-Dawley rats, purchased from Spibefu (Beijing) Biotechnology Ltd, and assigned quality certificate number SYXK (Ee) 2016-0057 for experimental animals.

Example 1 Synthesis of Cytarabine structural analog P1

The synthetic route is shown as the following formula:

the method comprises the following steps: cytarabine (Compound 1, 5.0g, 20.56mmol) was neutralizedCompound 2(10.72g, 30.84mmol, 1.5eq.) is dissolved in anhydrous THF (tetrahydrofuran, 50mL), N₂The temperature is reduced to-78 ℃ under protection, 1-methylimidazole (4.39g, 53.46mmol, 2.6eq.) is slowly dropped, and then the mixture is stirred at-78 ℃ for 2 hours and then naturally heated to room temperature (20 ℃) for reaction for 12 hours. Concentrating the reaction solution at 40 ℃ under reduced pressure, adding DCM (dichloromethane) for dilution, washing with dilute hydrochloric acid (0.5mol/L), water and saturated sodium chloride solution in sequence, combining organic phases, drying over anhydrous sodium sulfate, concentrating at 40 ℃ under reduced pressure, and purifying by column chromatography (DCM/MeOH is 20/1-10/1, V/V, MeOH is methanol) to obtain a white powdery compound.¹H-NMR(400MHz,CD₃OD): δ 7.76(d, J ═ 4.4Hz, 1H), 7.37 to 7.33(m, 2H), 6.23(d, J ═ 4.0Hz, 1H), 5.78(d, J ═ 7.6Hz, 1H), 4.40 to 4.23(m, 2H), 4.07 to 4.06(m, 1H), 4.05 to 3.96(m, 4H), 1.52 to 1.40(m, 1H), 1.38 to 1.33(m, 7H), 0.91 to 0.87(m, 6H). The HNMR spectrum is shown in FIG. 1. The structure of the cytosine arabinoside analogue is analyzed and determined to be shown as a formula (II) and is marked as a cytarabine structural analogue P1.

Example 2 Synthesis of Cytarabine structural analog P2

The synthetic route is shown as the following formula:

the method comprises the following steps: compound P1(3.0g, 5.41mmol) and compound 3(2.32g, 6.49mmol, 1.2eq.) were dissolved in anhydrous DCM (tetrahydrofuran, 50mL), N₂Cooling to 0 deg.C under protection, and slowly adding 1-propyl phosphoric anhydride (50% ethyl acetate solution) (6.88g, wherein, 1-propyl phosphoric anhydride T₃P10.82 mmol, 2.0eq.), and then naturally warmed up to room temperature (20 ℃) for reaction for 1 hour. Saturated NaHCO is used for reaction liquid₃The solution is quenched, the aqueous phase is extracted with DCM (dichloromethane, 50mL × 2), the organic phases are combined and dried over anhydrous sodium sulfate, concentrated under reduced pressure at 40 ℃, and purified by column chromatography (PE/EA ═ 20/1-1/1, PE is petroleum ether, EA is ethyl acetate) to obtain intermediate P1 a. Intermediate P1a was dissolved in methanol (50mL), Pd/C (0.3g) was added, nitrogen was substituted 3 times, and hydrogen (0.1MPa) was introduced for reaction. After completion of the reaction monitored by TLC (thin layer chromatography), the resulting reaction solution was passed through celiteCollecting the first filtrate with a soil pad, rinsing the diatomaceous earth pad with a small amount of methanol to collect the second filtrate, mixing the filtrates, concentrating at 40 deg.C under reduced pressure, and vacuum filtering with MTBE (methyl tert-butyl ether) under stirring to obtain white powder. LCMS: [ M +1 ]]⁺And (4) theoretical calculation: 670.24, actually measuring: 670.20.¹H-NMR (400MHz, DMSO-d): δ 10.48(s,1H), 8.84(d, J ═ 4.0Hz, 1H), 7.92(s, 1H), 7.37 to 7.23(m, 5H), 6.12(d, J ═ 4.0Hz, 1H), 5.87(d, J ═ 7.6Hz, 1H), 5.11(brs, 2H), 4.40 to 4.23(m, 2H), 4.18(s, 1H), 4.07 to 3.93(m, 6H), 3.81 to 3.66(m, 4H), 2.79(d, J ═ 7.6Hz,2H), 1.40 to 1.33(m, 7H), 0.94 to 0.88(m, 6H). Therefore, the structure is determined as shown in the formula (III) and is marked as a cytarabine structural analogue P2.

Example 3 Synthesis of Cytarabine structural analog P3

The synthetic route is shown as the following formula:

the method comprises the following steps: cytarabine (compound 1, 5.0g, 20.56mmol) and compound 4(16.16g, 30.84mmol, 1.5eq.) were dissolved in anhydrous THF (tetrahydrofuran, 50mL), N₂The temperature is reduced to-78 ℃ under protection, 1-methylimidazole (4.39g, 53.46mmol, 2.6eq.) is slowly dropped, and then the mixture is stirred at-78 ℃ for 2 hours and then naturally heated to room temperature (20 ℃) for reaction for 12 hours. Concentrating the reaction solution at 40 ℃ under reduced pressure, adding DCM (dichloromethane) for dilution, washing with dilute hydrochloric acid (0.5mol/L), water and saturated sodium chloride solution in sequence, combining organic phases, drying with anhydrous sodium sulfate, concentrating at 40 ℃ under reduced pressure, and purifying by column chromatography (DCM/MeOH: 20/1-10/1, V/V) to obtain a white powdery compound. LCMS: [ M +1 ]]⁺And (4) theoretical calculation: 731.46, actually measuring: 731.50.¹H-NMR (400MHz, DMSO-d): δ 9.13(d, J ═ 4.0Hz, 1H), 8.79(brs, 1H), 5.89(d, J ═ 7.6Hz, 1H), 5.87(d, J ═ 4.4Hz, 1H), 4.40 to 4.27(m, 2H), 4.23 to 4.06(m, 8H), 3.58(brs, 2H), 1.87 to 1.77(m, 2H), 1.71 to 1.33(m, 39H), 0.98 to 0.88(m, 6H), and the structure thereof is determined as shown in formula (iv)And is marked as cytarabine structural analogue P3.

Example 4 Synthesis of Cytarabine structural analog P4

The synthetic route is shown as the following formula:

compound P3(3.0g, 4.10mmol) and compound 3(1.76g, 4.93mmol, 1.2eq.) were dissolved in anhydrous DCM (tetrahydrofuran, 50mL), N₂The temperature is reduced to 0 ℃ under protection, 1-propyl phosphoric anhydride (50 percent ethyl acetate solution) (5.21g, 8.20mmol and 2.0eq. of 1-propyl phosphoric anhydride) is slowly dropped, and then the temperature is naturally raised to room temperature (20 ℃) for reaction for 1 hour. Saturated NaHCO is used for reaction liquid₃The solution is quenched, the aqueous phase is extracted with DCM (dichloromethane, 50mL × 2), the organic phases are combined and dried over anhydrous sodium sulfate, concentrated under reduced pressure at 40 ℃, and purified by column chromatography (PE/EA ═ 20/1-1/1, PE is petroleum ether, EA is ethyl acetate) to obtain intermediate P3 a. Intermediate P3a was dissolved in methanol (50mL), Pd/C (0.3g) was added, the mixture was purged with nitrogen 3 times, and then reacted with hydrogen (0.1 MPa). After TLC (thin layer chromatography) monitoring reaction is completed, the obtained reaction liquid passes through a kieselguhr pad to collect a first filtrate, a small amount of methanol is used for leaching the kieselguhr pad to collect a second filtrate, the filtrates obtained by combining the first filtrate and the second filtrate are subjected to reduced pressure concentration at 40 ℃, and are stirred and filtered by MTBE (methyl tert-butyl ether) to obtain a white powdery compound. LCMS: [ M +1 ]]⁺And (4) theoretical calculation: 846.49, actually measuring: 846.50.¹H-NMR (400MHz, DMSO-d): δ 10.48(s,1H), 9.13(d, J ═ 4.0Hz, 1H), 7.92(s, 1H), 5.89(d, J ═ 4.4Hz, 1H), 5.78(d, J ═ 4.0Hz, 1H), 5.11(brs, 2H), 4.40 to 4.27(m, 2H), 4.23 to 4.06(m, 8H), 3.81 to 3.66(m, 6H), 2.79(d, J ═ 7.6Hz,2H), 1.87 to 1.77(m, 2H), 1.71 to 1.33(m, 39H), 0.98 to 0.88(m, 6H). The structure of the derivative is determined by analysis as shown in the formula (V) and is marked as cytarabine structural analogue P4.

Example 5 inhibition of proliferation of human leukemia cells HL-60 by Cytarabine analogs P1-P4

Test compounds: cytarabine hydrochloride and cytarabine analogs P1-P4 prepared in examples 1-4.

Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% fetal calf serum, and adding 5% volume fraction of CO at 37 deg.C₂Carrying out subculture in a saturated humidity incubator at regular intervals, and taking cells in a logarithmic growth phase as cells for subsequent experiments.

Preparing a medicine mother solution: the test compound was dissolved in DMSO (dimethyl sulfoxide) to prepare a mother solution having a concentration of 100mmol/L, and the mother solution was stored for use. The experiment was performed by dilution with DMSO to the desired concentration.

And (3) determination of inhibition rate: taking HL-60 cells in logarithmic growth phase, counting the cells to obtain the cell density of 5 multiplied by 10⁴one/mL of the cell suspension was inoculated into a 24-well plate, and 2mL (10 cells) of the cell suspension was added to each well⁵One/hole). Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂The cells were incubated in a saturated humidity incubator for 24 hours, and the culture medium was discarded, and 100. mu.L of the culture medium containing the test compound at a concentration of 1.5625. mu. mol/L was added to each well. Control wells were also provided, and 100. mu.L of the culture medium was added to the control wells without the test compound. Each set was provided with 3 multiple wells. Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Culturing in a saturated humidity incubator for 72 hours, taking out the 24-well plate, taking out the cells from the well, adding trypan blue in equal proportion, and mixing uniformly. The number of cells in the control well and the dosing well was counted separately using a hemocytometer. The growth inhibition rate was calculated as follows:

growth inhibition (%) was (1-number of drug-added wells/number of control wells) × 100%

The experiments were divided into 5 groups: the first group is a cytarabine structural analogue P1 group, the second group is a cytarabine structural analogue P2 group, the third group is a cytarabine structural analogue P3 group, the fourth group is a cytarabine structural analogue P4 group, and the fifth group is a cytarabine hydrochloride group. The cell proliferation inhibition rates were measured as: 18%, 12%, 14%, 56%, 10%. It can be found that: at the lower concentration, the inhibition effect of the cytarabine structural analogues P1-P4 on the proliferation growth of human leukemia cells HL60 is stronger than that of cytarabine hydrochloride.

Example 6 inhibition of proliferation of human leukemia cells HL-60 by Cytarabine structural analog P1

Test compounds: cytarabine hydrochloride, a cytarabine structural analog P1 prepared in example 1.

Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% fetal calf serum, and adding 5% volume fraction of CO at 37 deg.C₂Carrying out subculture in a saturated humidity incubator at regular intervals, and taking cells in a logarithmic growth phase as cells for subsequent experiments.

Preparing a medicine mother solution: the test compound was dissolved in DMSO (dimethyl sulfoxide) to prepare a mother solution having a concentration of 100mmol/L, and the mother solution was stored for use. The experiment was performed by dilution with DMSO to the desired concentration.

And (3) determination of inhibition rate: taking HL-60 cells in logarithmic growth phase, counting the cells to obtain the cell density of 5 multiplied by 10⁴one/mL of the cell suspension was inoculated into a 24-well plate, and 2mL (10 cells) of the cell suspension was added to each well⁵One/hole). Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Incubate in a saturated humidity incubator for 24h, discard the medium, add 100. mu.L of the corresponding medium containing different concentrations of test compound to each well, set 5 concentration gradients (25. mu. mol/L, 12.5. mu. mol/L, 6.25. mu. mol/L, 3.125. mu. mol/L, 1.5625. mu. mol/L) for each test compound, and set 3 multiple wells for each concentration. Control wells were also provided, and 100. mu.L of the culture medium was added to the control wells without the test compound. Control wells were set with 3 replicates. Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Culturing in a saturated humidity incubator for 72 hours, taking out the 24-well plate, taking out the cells from the well, adding trypan blue in equal proportion, and mixing uniformly. The number of cells in the control well and the dosing well was counted separately using a hemocytometer. The growth inhibition rate was calculated as follows:

growth inhibition (%) was (1-number of drug-added wells/number of control wells) × 100%

The experiments were divided into cytarabine structural analogue P1 group and cytarabine hydrochloride group. The results of measurement of the HL-60 cell proliferation inhibition rate of each group are shown in the following table 1:

TABLE 1 measurement of HL-60 cell proliferation inhibition ratio of each test compound group

	P1	Cytarabine hydrochloride
			1.5625μmol/L	18％	10％
3.125μmol/L	30％	21％
			6.25μmol/L	50％	30％
12.5μmol/L	60％	40％
			25μmol/L	64％	56％

As can be seen from the results in Table 1, the cytarabine structural analogue P1 has an inhibitory effect on HL60 cells at different gradient concentrations. This means that: like cytarabine hydrochloride, cytarabine structural analogue P1 can inhibit proliferation of human leukemia cell HL 60. In addition, at each of the same concentrations, cytarabine structural analogue P1 inhibited the growth of HL60 cells more than cytarabine hydrochloride, showing that: the cytarabine structural analogue P1 has stronger inhibiting effect on HL60 cell proliferation than cytarabine hydrochloride.

Example 7 inhibition of proliferation of human leukemia cells HL-60 by Cytarabine structural analog P4

Test compounds: cytarabine hydrochloride, cytarabine structural analog P4 prepared in example 4.

Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% fetal calf serum, and adding 5% volume fraction of CO at 37 deg.C₂Carrying out subculture in a saturated humidity incubator at regular intervals, and taking cells in a logarithmic growth phase as cells for subsequent experiments.

Preparing a medicine mother solution: the test compound was dissolved in DMSO (dimethyl sulfoxide) to prepare a mother solution having a concentration of 100mmol/L, and the mother solution was stored for use. The experiment was performed by dilution with DMSO to the desired concentration.

And (3) determination of inhibition rate: taking HL-60 cells in logarithmic growth phase, counting the cells to obtain the cell density of 5 multiplied by 10⁴one/mL of the cell suspension was inoculated into a 24-well plate, and 2mL (10 cells) of the cell suspension was added to each well⁵One/hole). Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Incubate in a saturated humidity incubator for 24h, discard the medium, add 100. mu.L of the corresponding medium containing different concentrations of test compound to each well, set 5 concentration gradients (25. mu. mol/L, 12.5. mu. mol/L, 6.25. mu. mol/L, 3.125. mu. mol/L, 1.5625. mu. mol/L) for each test compound, and set 3 multiple wells for each concentration. Control wells were also provided, and 100. mu.L of the culture medium was added to the control wells without the test compound. Control wells were set with 3 replicates. Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Culturing in a saturated humidity incubator for 72 hours, taking out the 24-well plate, taking out the cells from the well, adding trypan blue in equal proportion, and mixing uniformly. The number of cells in the control well and the dosing well was counted separately using a hemocytometer. The growth inhibition rate was calculated as follows:

growth inhibition (%) was (1-number of drug-added wells/number of control wells) × 100%

The experiments were divided into 3 groups: cytarabine hydrochloride, cytarabine analog P4, and cytarabine hydrochloride. The results of measurement of the HL-60 cell proliferation inhibition rate of each group are shown in the following table 2:

TABLE 2 measurement of HL-60 cell proliferation inhibition ratio of each test compound group

	Cytarabine	P4	Cytarabine hydrochloride
				1.5625μmol/L	20％	56％	10％
3.125μmol/L	34％	73％	21％
				6.25μmol/L	52％	86％	30％
12.5μmol/L	66％	90％	40％
				25μmol/L	75％	96％	56％

According to the results of Table 2:

(1) under different gradient concentrations, each test compound has inhibitory effect on HL60 cells. This means that: like cytarabine and cytarabine hydrochloride, cytarabine structural analogue P4 can inhibit the proliferation and growth of human leukemia cell HL 60.

(2) At each same concentration, the inhibition rate of the cytarabine structural analogue P4 on the growth of HL60 cells is the highest and is obviously higher than that of the cytarabine group and the cytarabine hydrochloride group. When the concentration is 1.5625 mu mol/L, the inhibition rate of the cytarabine structural analogue P4 on the growth of HL60 cells is 56 percent, and the inhibition rates of cytarabine and cytarabine hydrochloride on the growth of HL60 cells are 20 percent and 10 percent respectively; when the concentration is 25 mu mol/L, the inhibition rate of the cytarabine structural analogue P4 on the growth of HL60 cells is up to 96%, and the inhibition rates of cytarabine and cytarabine hydrochloride on the growth of HL60 cells are 75% and 56% respectively. It can be seen that cytarabine structural analogue P4 has a much stronger inhibitory effect on HL60 cells at each concentration than cytarabine and cytarabine hydrochloride.

Example 8 drug concentrations of Cytarabine structural analogs incubated in HL-60 cells for various times

Taking HL-60 cells in logarithmic growth phase, counting the cells to obtain the cell density of 5 multiplied by 10⁴one/mL of the cell suspension was inoculated into a 24-well plate, and 2mL (10 cells) of the cell suspension was added to each well⁵One/hole). Placing a 24-pore plate in CO with the volume fraction of 5% at 37 DEG C₂Culturing in a saturated humidity incubator for 24h, discarding the culture solution, adding 100 μ L of culture solution containing 100 μmol/L of the drug to be tested into each well, wherein the drug to be tested is cytarabine and the cytarabine prepared in example 4Cytidine structural analog P4. Each group is provided with 3 multiple holes and is placed at 37 ℃ and 5% CO₂And (5) continuing culturing in a saturated humidity incubator. After incubation for various periods of time (0.5 hours, 1 hour, 3 hours, 6 hours), the concentrations of the parent drug (cytarabine, designated Ara-C in the figure; or, cytarabine structural analogue P4, designated BX20-1-004 in the figure) and the corresponding active metabolite cytarabine triphosphate (designated Ara-CTP (Ara-C) and Ara-CTP (004) in the figure) were tested in HL-60 cells, respectively. The concentration profile is shown in figure 2.

As can be seen from fig. 2:

(1) with the prolonging of the incubation time, the concentrations of the original drug and the corresponding active metabolite in the HL-60 cells are increased, and when the incubation time is 3 hours or more, the concentration of the active metabolite cytarabine triphosphate in the HL-60 cells exceeds the original drug concentration. This means that cytarabine and the cytarabine structural analogue P4 both entered HL-60 cells and were converted into active metabolites inside the cells.

(2) The intracellular concentration of cytarabine structural analogue P4 was significantly higher than that of cytarabine for the same incubation time, indicating that cytarabine structural analogue P4 had a greater ability to penetrate the cell membrane into the cell than cytarabine.

(3) The concentration of cytarabine triphosphate, an active metabolite in cells treated with cytarabine structural analogue P4, was much higher compared to the concentration of cytarabine triphosphate, an active metabolite in cells treated with cytarabine for the same incubation time. This suggests that: the cytarabine structural analog P4 prepared in example 4 was able to produce higher concentrations of active metabolites than cytarabine, thereby exerting stronger anti-leukemic cell proliferative activity. This indicates that the bioavailability of cytarabine structural analog P4 prepared in example 4 was significantly higher than that of cytarabine.

Example 9 in vivo oral Single dose pharmacokinetic Studies in rats

8 Sprague-Dawley rats were divided into two groups: the cytarabine and cytarabine structural analogue groups, each group comprising 4 rats, were orally administered with a single dose of a cytarabine structural analogue P4 solution (physiological saline solution, drug concentration 10mg/mL) and a cytarabine solution (physiological saline solution, drug concentration 10mg/mL), respectively, at the following doses: the concentration of cytosine arabinoside is 10mg/Kg rat.

Determination by liquid chromatography-Mass Spectrometry (HPLC-MS/MS): (1) (ii) cytarabine concentration in plasma and PBMC cells following oral administration of cytarabine; (2) the concentration of the proto-drug (cytarabine structural analogue P4) in plasma and PBMC cells following oral administration of cytarabine structural analogue P4; (3) the metabolite cytarabine concentration in plasma and PBMC cells following oral administration of cytarabine structural analogue P4. The results are shown in table 3 below as the pharmacokinetic parameters (n-4) of the proto-drug and the metabolite cytarabine in plasma and PBMC cells after a single oral administration in rats. Cytarabine structural analogue P4 is shown as BX20-1-004 in Table 3.

Table 3: pharmacokinetic parameters of proto-drug and metabolite cytarabine in plasma and PBMC cells after oral single administration in rats

In Table 3, AUC is the area under the curve when drug is administered, and means the area enclosed by the curve of plasma concentration with respect to the time axis, and is expressed in ng.h/mL. The parameter is an important index for evaluating the absorption degree of the drug and reflects the exposure characteristic of the drug in vivo. C_maxThe peak concentration refers to the highest blood concentration after administration. This parameter is an important index reflecting the rate and extent of absorption of the drug in vivo. t is t_maxThe peak reaching time refers to the time required to reach the peak concentration of the drug after administration. The parameter reflects the speed of the drug entering the body, and the peak reaching time is short when the absorption speed is high. t is t_1/2Is the terminal elimination half-life, which refers to the time required for the terminal phase blood concentration to decrease by half. This parameter visually reflects the rate of elimination of the drug from the body.

As can be seen from table 3:

(1) the AUC of the cytarabine structural analogue P4 (BX 20-1-004 in the table) entering PBMC after the cytarabine structural analogue P4 is administered is obviously higher than that of the cytarabine entering PBMC after the cytarabine is administered, which indicates that the cytarabine structural analogue P4 has better cell penetrability than the cytarabine.

(2) The AUC of cytarabine in plasma after administration of the cytarabine structural analogue P4 was significantly lower than the AUC of cytarabine in plasma after administration of cytarabine, which means that less inactive uracil cytarabine was subsequently produced in plasma and the bioavailability of the cytarabine structural analogue P4 was higher.

(3) The peak cytarabine concentration in PBMC cells after administration of cytarabine structural analogue P4 (986ng/mL) was more than twice the peak cytarabine concentration in PBMC cells after administration of cytarabine (436 ng/mL). The cytarabine structural analogue P4 can be converted into the parent cytarabine in cells, the blood concentration of target cells in vivo is obviously improved, and the bioavailability is higher.

(4) The time to peak of cytarabine in PBMC cells after administration of cytarabine structural analogue P4 (26.1h) was significantly higher than the time to peak of cytarabine in PBMC cells after administration of cytarabine (0.83 h). This means that: the cytarabine structural analogue P4 significantly prolonged the intracellular time of cytarabine.

(5) The half-life of cytarabine in PBMC cells after administration of cytarabine structural analogue P4 (25.5h) was significantly higher than the time to peak of cytarabine in PBMC cells after administration of cytarabine (1.02 h). This means that: the cytosine arabinoside structural analogue P4 for oral administration has longer half-life, good stability of cytosine arabinoside in PBMC cells, and can maintain effective blood concentration for a longer time.

In conclusion, the cytarabine structural analogues P1-P4 synthesized for the first time can inhibit human leukemia HL-60 cells, have better inhibition effect than cytarabine hydrochloride under low concentration, and are expected to be used as novel anti-leukemia drugs.

Particularly, compared with cytarabine, the cytarabine structural analogue P4 has stronger proliferation inhibition capacity on leukemia cells HL60 and stronger cell penetrating capacity, the concentration of the medicine entering the cells and the concentration of an active metabolite cytarabine triphosphate generated in the cells are higher, and the bioavailability is higher; can be converted into the parent drug cytarabine in cells, obviously improves the blood concentration of target cells in vivo, and can avoid toxic and side effects and drug resistance caused by large-dose medication; can obviously prolong the half-life period of the cytarabine, maintain the effective blood concentration for a longer time, can be orally taken, and can avoid the inconvenience caused by injection. Therefore, the cytarabine structural analogue P4 completely overcomes the defects of high polarity, poor membrane permeability, easy metabolic inactivation in vivo, short half-life period and the like of cytarabine, provides possibility for oral administration, and fills the technical blank that no new medicine is available in the market at home.

The preparation method of the cytarabine structural analogue is simple and easy to operate, and is expected to be used for industrial production.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.