CN112409431B - Cytarabine structural analogue, and preparation method and application thereof - Google Patents
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- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 title claims abstract description 194
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
The invention discloses a cytarabine structural analogue, a preparation method and application thereof. The cytarabine structural analogues P1-P4 can inhibit proliferation of leukemia cells HL-60 for the first time, and particularly, compared with Ara-C, the cytarabine structural analogue P4 has stronger inhibiting capability on proliferation of leukemia cells, stronger cell penetrating capability, higher drug concentration entering cells and higher concentration of an active metabolite cytarabine triphosphate generated in the cells, and higher bioavailability; can be converted into cytarabine in cells, remarkably improves the blood concentration of target cells in the body, and can avoid toxic and side effects and drug resistance caused by large-dose medication; the half-life period of cytarabine is obviously prolonged, the effective blood concentration is maintained for a long time, the cytarabine can be orally taken, and the inconvenience caused by injection can be avoided. The preparation method is simple and easy to operate, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the field of organic synthesis, and particularly relates to an cytarabine structural analogue, a preparation method and application thereof.
Background
Cytarabine (cytosine arabinoside, ara-C) chemical name: 1-beta-D-arabinofuranosyl-4-amino-2 (1H) -cytosine-none with CAS registry number [147-94-4 ]]The molecular formula: c (C) 9 H 13 N 3 O 5 The structural formula is as follows:
cytarabine belongs to pyrimidine antimetabolites and is mainly used for treating acute myeloid leukemia (acute myeloid leukemia, AML). Ara-C is converted to cytarabine triphosphate (Ara-CTp) in vivo by three steps to exert anticancer effect. Firstly, catalyzing and forming cytarabine monophosphate (Ara-CMp) by intracellular deoxycytidine kinase, and then respectively converting the cytarabine monophosphate (Ara-CDp) and active cytarabine triphosphate (Ara-CTp) by the actions of monophosphate and biphosphite nucleoside kinase, wherein the cytarabine triphosphate influences DNA synthesis by inhibiting DNA polymerase, inhibits the growth of cells and interferes the proliferation of the cells; can also penetrate into DNA in small amounts, interfere with DNA replication, and cause 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 the effect on G1/S and S/G2 conversion phases.
Cytarabine was originally developed by Upjohn in the united states, marketed in the united states 10 months in 1969, and was first registered in the country for 1992. The cytarabine molecule has very high polarity, so that the membrane permeability of the small intestine is poor, the cytarabine has very low oral bioavailability, and only 20 percent of the medicine enters the blood circulation, so that the cytarabine is not orally taken but is used for intravenous administration. Therefore, cytarabine is mainly injection (powder injection) and is produced by a plurality of manufacturers in China at present. In addition, since the medicine is rapidly deaminated by cytosine deaminase in the liver after entering the body, inactive uracil cytarabine is generated, t 1/2 The preparation is short, and only takes 3 to 15 minutes, so that intravenous continuous instillation administration is needed to maintain the effective blood concentration, and a better effect is obtained. For example, for the treatment of acute myeloid leukemia, intravenous injection or intravenous drip is commonly used, 100-200 mg/m per day 2 Or 2-6 mg/Kg for 5-7 days, and the dosage is increased from small to bone marrow suppression. The medicine is repeatedly used after stopping taking the medicine for 5 to 7 days or after recovering bone marrow. Clinically, the high-dose cytarabine treatment effect is better than that of the ordinary long-term treatment, but has toxic and side effects on blood, nervous system and organs, is easy to generate drug resistance, and the survival rate within five years is not obviously improved. The toxic and side effects of cytarabine and the dosage of cytarabine are positively correlated, and the clinical use of cytarabine is limited.
Therefore, there is a need to develop more effective therapeutic agents that can be administered orally, facilitating off-site administration for convalescence patients.
Disclosure of Invention
In view of this, the present invention provides cytarabine structural analogs, methods of making and uses thereof. Compared with cytarabine, some cytarabine analogues have better permeability, higher bioavailability and longer half-life for leukemia cells, can be orally administered, and are expected to be used as novel anti-leukemia drugs.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a cytarabine structural analogue of the general structural formula (i):
wherein r1=ph or C 18 H 37 ，
R 2 =h or
In particular the number of the elements,
the cytarabine structural analogue can be a cytarabine structural analogue P1 shown in the following formula (II):
alternatively, the cytarabine structural analog may be cytarabine structural analog P2 shown in the following formula (iii):
alternatively, the cytarabine structural analog may be cytarabine structural analog P3 shown in the following formula (iv):
alternatively, the cytarabine structural analog may be 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 method for preparing cytarabine structural analogues, comprising the following steps:
dissolving cytarabine and phosphoryl chloride derivative in anhydrous tetrahydrofuran, N 2 Cooling to-70 to-78 ℃ under protection, slowly dropwise adding 1-methylimidazole, stirring at-70 to-78 ℃, heating to room temperature for reaction, and after the reaction is completed, carrying out post-treatment and purification on the reaction solution to obtain a product; wherein,,
when the phosphorus oxychloride derivative is a compound 2 shown in the following formula (VI), the obtained product is an cytarabine structural analogue P1;
when the phosphorus oxychloride derivative is a compound 4 shown as the following formula (VII), the obtained product is cytarabine structural analogue P3;
in a preferred embodiment, cytarabine: phosphorus oxychloride derivative: the molar ratio of the 1-methylimidazole is 1: (1.5-2.0): (2.0-3.0).
In a 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 embodiment, the post-treatment purification comprises: the reaction solution is concentrated, diluted by adding dichloromethane, washed by dilute hydrochloric acid, water and saturated sodium chloride solution, and the organic phases are combined, dried and concentrated, and purified by column chromatography to obtain the product.
In a preferred embodiment, the concentration is reduced pressure concentration, most preferably 40 ℃.
In the preferred technical scheme, the column chromatography adopts methylene dichloride/methanol with the volume ratio of 20/1-10/1.
Or,
a method for preparing cytarabine structural analogues, comprising the following steps:
dissolving selected compounds and aspartic acid derivatives in anhydrous tetrahydrofuran, N 2 Cooling to-5-0 deg.c under protection, slowly dropping 1-propyl phosphoric anhydride, heating to room temperature to react to obtain the first reaction liquid, and using saturated NaHCO 3 Quenching the solution, extracting the water phase with dichloromethane, merging the organic phases, drying and concentrating, and purifying by column chromatography to obtain an intermediate; dissolving the intermediate in methanol, adding Pd/C, introducing hydrogen for reaction, and performing after-treatment on the obtained second reaction solution after the completion of the reaction monitored by thin-layer chromatography to obtain a product; wherein,,
the aspartic acid derivative is compound 3 shown in the following formula (VIII)
When the selected compound is cytarabine structural analogue P1, the resulting product is cytarabine structural analogue P2;
when the selected compound is cytarabine structural analog P3, the resulting product is cytarabine structural analog P4.
In a preferred embodiment, the compound is selected from: aspartic acid derivatives: the molar ratio of the 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 reaction time at room temperature is 1-2 hours.
In the preferred technical scheme, the column chromatography adopts petroleum ether/ethyl acetate with the volume ratio of 20/1-1/1.
In a preferred technical scheme, the Pd/C dosage 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 embodiment, the post-treatment includes: and (3) collecting the first filtrate from the second reaction solution through a diatomite pad, eluting the diatomite pad by using methanol, collecting the second filtrate, combining the first filtrate and the second filtrate to obtain filtrate, concentrating the filtrate, and stirring and filtering the filtrate by using methyl tertiary butyl ether to obtain a product.
In a preferred embodiment, the concentration is reduced pressure concentration, most preferably 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 leukemia.
Furthermore, the invention also provides application of the cytarabine structural analogue in preparing oral medicines 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, and the inhibition effect of the cytarabine structural analogues is superior to cytarabine hydrochloride at low concentration, so that the cytarabine structural analogues are expected to be used as novel anti-leukemia drugs.
In particular, compared with cytarabine, the cytarabine structural analogue P4 has stronger inhibiting capability to leukemia cell proliferation, stronger cell penetrating capability, higher drug concentration entering cells and higher concentration of an active metabolite cytarabine triphosphate generated in the cells, and higher bioavailability; the cytarabine is converted into the parent drug cytarabine in cells, so that the blood concentration of target cells in the body is obviously improved, and toxic and side effects and drug resistance caused by large-dose medication can be avoided; the half-life period of cytarabine is obviously prolonged, the effective blood concentration is maintained for a long time, and the cytarabine can be orally taken, so that inconvenience caused by injection is avoided. Therefore, the cytarabine structural analogue P4 completely overcomes the defects of large polarity, poor film permeability, easy metabolic inactivation in vivo, short half-life and the like of cytarabine, provides possibility for oral administration, and fills the technical blank that no new medicine of this type is marketed in China.
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 a HNMR spectrum of the compound obtained in example 1, wherein the spectrum shows that the structure is shown as a formula (II), and is marked as an cytarabine structural analogue P1.
FIG. 2 is a graph showing the comparison of the concentration of the intracellular drug substance and the metabolite after incubation of the different drugs to be tested with human leukemia cells HL60 for different times in example 8.
Detailed Description
Embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It is to be understood that these examples are for illustration of the present invention only and should not be construed as limiting the scope of the present invention.
The following examples are not to be construed as particular techniques or conditions, and may be carried out in accordance with methods conventional in the art, for example, in accordance with techniques or conditions described in the literature in the field or as suggested by the supplier in the product specification.
The various instruments, materials and reagents not specifically described in the following examples, were not identified to the manufacturer and were commercially available conventional products. The particular materials and sources thereof used, as set forth in the following examples, are illustrative only and are not intended to limit the invention, as materials identical or similar to the type, model, quality, nature, or function of the tissues, cells, reagents, and instruments described below may be used in the practice of the invention.
Material and source description:
cytarabine, CAS:147-94-4 from Shanghai Haohong biological medicine Co., ltd.
Cytarabine hydrochloride, CAS:69-74-9 from Shanghai Haohong biological medicine technologies Co.
Compound 3, having the structure shown in formula (viii), CAS:4779-31-1 from Shanghai Haohong Biotechnology Co., ltd.
The structure of the compound 4 is shown as a formula (VII), and the compound is purchased from the GmbH of pharmaceutical technology of Kadsura.
1-propylphosphoric anhydride (50% ethyl acetate solution) was purchased from Shanghai Bi, medical technologies Co., ltd.
Human leukemia cells HL-60 were purchased from the Living technologies Co., ltd.
PBMC, peripheral blood mononuclear cells, were isolated from rat blood by Ficoll lymph separation (sigma).
Sprague-Dawley rats purchased from St Bei Fu (Beijing) biotechnology Co., ltd., and the laboratory animal quality certificate number SYXK 2016-0057.
EXAMPLE 1 Synthesis of Cytarabine structural analog P1
The synthetic route is shown in the following formula:
the method comprises the following steps: cytarabine (compound 1,5.0g,20.56 mmol) and compound 2 (10.72 g,30.84mmol,1.5 eq.) were dissolved in anhydrous THF (tetrahydrofuran, 50 mL), N 2 1-methylimidazole (4.39 g,53.46mmol,2.6 eq.) was slowly added dropwise under protection, and the mixture was stirred at-78℃for 2 hours, then naturally warmed to room temperature (20 ℃) and reacted for 12 hours. The reaction mixture was concentrated under reduced pressure at 40℃and diluted with DCM (dichloromethane), washed with dilute hydrochloric acid (0.5 mol/L), water and saturated sodium chloride solution, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure at 40℃and purified by column chromatography (DCM/MeOH=20/1 to 10/1, V/V, meOH: methanol) to give the compound as a white powder. 1 H-NMR(400MHz,CD 3 OD)：δ7.76(d，J＝4.4Hz，1H)，7.37～7.33(m，2H)，6.23(d，J＝4.0Hz，1H)，5.78(d，J＝7.6Hz，1H)，4.40～4.23(m，2H)，4.07～4.06(m，1H)，4.05～3.96(m，4H)，1.52～1.40(m，1H)，1.38～1.33(m，7H)，0.91 to 0.87 (m, 6H). The HNMR spectra are shown in FIG. 1. The analysis confirms that the structure is shown as a formula (II) and is marked as cytarabine structural analogue P1.
EXAMPLE 2 Synthesis of Cytarabine structural analog P2
The synthetic route is shown in the following formula:
the method comprises the following steps: compound P1 (3.0 g,5.41 mmol) and compound 3 (2.32 g,6.49mmol,1.2 eq.) were dissolved in anhydrous DCM (tetrahydrofuran, 50 mL), N 2 Cooling to 0deg.C under protection, slowly dropwise adding 1-propylphosphoric anhydride (50% ethyl acetate solution) (6.88 g, wherein 1-propylphosphoric anhydride T 3 P10.82 mmol,2.0 eq.) and then allowed to react for 1 hour at room temperature (20 ℃ C.). Saturated NaHCO was used as the reaction solution 3 The solution was quenched, the aqueous phase was extracted with DCM (dichloromethane, 50ml×2), the organic phases combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure at 40 ℃ and purified by column chromatography (PE/ea=20/1 to 1/1, PE is petroleum ether and EA is ethyl acetate) to give intermediate P1a. Intermediate P1a was dissolved in methanol (50 mL), pd/C (0.3 g) was added, and after 3 times nitrogen substitution, hydrogen (0.1 MPa) was introduced for reaction. After the completion of the reaction, the reaction liquid obtained was collected by passing through a pad of celite, and the second filtrate was collected by eluting the pad of celite with a small amount of methanol, and the filtrates obtained by combining the first filtrate and the second filtrate were concentrated under reduced pressure at 40 c, and then stirred and suction-filtered with MTBE (methyl tert-butyl ether) to obtain a white powdery compound. LCMS: [ M+1 ]] + Theoretical calculation: 670.24, found: 670.20. 1 H-NMR (400 MHz, DMSO-d): δ10.48 (s, 1H), 8.84 (d, j=4.0 hz, 1H), 7.92 (s, 1H), 7.37 to 7.23 (m, 5H), 6.12 (d, j=4.0 hz, 1H), 5.87 (d, j=7.6 hz, 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=7.6 hz, 2H), 1.40 to 1.33 (m, 7H), 0.94 to 0.88 (m, 6H). The structure is shown as a formula (III) and is marked as cytarabine structural analogue P2.
EXAMPLE 3 Synthesis of Cytarabine structural analog P3
The synthetic route is shown in the following formula:
the method comprises the following steps: cytarabine (compound 1,5.0g,20.56 mmol) and compound 4 (16.16 g,30.84mmol,1.5 eq.) were dissolved in anhydrous THF (tetrahydrofuran, 50 mL), N 2 1-methylimidazole (4.39 g,53.46mmol,2.6 eq.) was slowly added dropwise under protection, and the mixture was stirred at-78℃for 2 hours, then naturally warmed to room temperature (20 ℃) and reacted for 12 hours. The reaction mixture was concentrated under reduced pressure at 40℃and diluted with DCM (dichloromethane), washed with dilute hydrochloric acid (0.5 mol/L), water and saturated sodium chloride solution, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure at 40℃and purified by column chromatography (DCM/MeOH=20/1 to 10/1, V/V) to give the compound as a white powder. LCMS: [ M+1 ]] + Theoretical calculation: 731.46, found: 731.50. 1 H-NMR (400 MHz, DMSO-d): δ9.13 (d, j=4.0 hz, 1H), 8.79 (brs, 1H), 5.89 (d, j=7.6 hz, 1H), 5.87 (d, j=4.4 hz, 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), whereby the structure thereof is determined as shown in formula (iv), and is denoted as cytarabine structural analogue P3.
EXAMPLE 4 Synthesis of Cytarabine structural analog P4
The synthetic route is shown in the following formula:
compound P3 (3.0 g,4.10 mmol) and compound 3 (1.76 g,4.93mmol,1.2 eq.) were dissolved in anhydrous DCM (tetrahydrofuran, 50 mL), N 2 1-propylphosphoric anhydride (50% ethyl acetate solution) (5.21 g, wherein, 8.20mmol,2.0 eq.) was slowly added dropwise under protection, and then the mixture was naturally warmed to room temperature (20 ℃) to react for 1 hour. Saturated NaHCO was used as the reaction solution 3 Quenching the solution, and water phaseExtraction with DCM (dichloromethane, 50ml×2), drying of the combined organic phases over anhydrous sodium sulfate, concentration at 40 ℃ under reduced pressure, and purification by column chromatography (PE/ea=20/1 to 1/1, PE is petroleum ether, EA is ethyl acetate) gives intermediate P3a. Intermediate P3a was dissolved in methanol (50 mL), pd/C (0.3 g) was added, and after 3 times nitrogen substitution, hydrogen (0.1 MPa) was introduced for reaction. After the completion of the reaction, the reaction liquid obtained was collected by passing through a pad of celite, and the second filtrate was collected by eluting the pad of celite with a small amount of methanol, and the filtrates obtained by combining the first filtrate and the second filtrate were concentrated under reduced pressure at 40 c, and then stirred and suction-filtered with MTBE (methyl tert-butyl ether) to obtain a white powdery compound. LCMS: [ M+1 ]] + Theoretical calculation: 846.49, found: 846.50. 1 H-NMR (400 MHz, DMSO-d): δ10.48 (s, 1H), 9.13 (d, j=4.0 hz, 1H), 7.92 (s, 1H), 5.89 (d, j=4.4 hz, 1H), 5.78 (d, j=4.0 hz, 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=7.6 hz, 2H), 1.87 to 1.77 (m, 2H), 1.71 to 1.33 (m, 39H), 0.98 to 0.88 (m, 6H). The analysis determines that the structure is shown as a formula (V) and is marked as cytarabine structural analogue P4.
EXAMPLE 5 inhibition of proliferation of human leukemia cells HL-60 by cytarabine structural analogues P1-P4
Test compounds: cytarabine structural analogues P1-P4 prepared in examples 1-4 and cytarabine hydrochloride.
Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% foetal calf serum at 37deg.C with volume fraction of 5% CO 2 And (3) carrying out periodic subculture in a saturated humidity incubator, and taking the cells in the logarithmic growth phase as the cells for subsequent experiments.
Preparing a medicine mother solution: the test compound is dissolved in DMSO (dimethyl sulfoxide) to prepare mother solution with the concentration of 100mmol/L, and the mother solution is preserved for standby. The experiments were diluted with DMSO to the desired concentration.
Inhibition rate measurement: taking HL-60 cells in logarithmic growth phase, and counting to obtain a density of 5×10 4 Each/mL of the cell suspension was inoculated into a 24-well culture plate, and 2mL (the number of cells was10 5 And/or holes). Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 The culture was carried out in a saturated humidity incubator for 24 hours, the culture broth was discarded, and 100. Mu.L of a culture broth containing the test compound at a concentration of 1.5625. Mu. Mol/L was added to each well. Control wells were additionally provided, and no test compound was added to the control wells, and only 100. Mu.L of culture medium was added. Each group was provided with 3 duplicate wells. Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 Culturing in saturated humidity incubator for 72 hr, taking out 24-well plate, taking out cells from the well, adding trypan blue in equal proportion, and mixing. The number of cells in the control well and the dosing well, respectively, was counted using a blood cell counting plate. The growth inhibition rate was calculated as follows:
growth inhibition (%) = (1-dosing well cell number/control well cell number) ×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 measured cell proliferation inhibition rates were respectively: 18%, 12%, 14%, 56%, 10%. It can be found that: at the lower concentration, the cytarabine analogues P1-P4 have stronger inhibition effect on proliferation and growth of human leukemia cells HL60 than cytarabine hydrochloride.
EXAMPLE 6 inhibition of proliferation of human leukemia cell HL-60 by Cytarabine structural analog P1
Test compounds: cytarabine structural analogue P1 prepared in example 1 and cytarabine hydrochloride.
Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% foetal calf serum at 37deg.C with volume fraction of 5% CO 2 And (3) carrying out periodic subculture in a saturated humidity incubator, and taking the cells in the logarithmic growth phase as the cells for subsequent experiments.
Preparing a medicine mother solution: the test compound is dissolved in DMSO (dimethyl sulfoxide) to prepare mother solution with the concentration of 100mmol/L, and the mother solution is preserved for standby. The experiments were diluted with DMSO to the desired concentration.
Inhibition rate measurement: taking HL-60 cells in logarithmic growth phaseCell count was made to a density of 5X 10 4 Each/mL of the cell suspension was inoculated into a 24-well culture plate, and 2mL (cell number 10 5 And/or holes). Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 The culture was carried out in a saturated humidity incubator for 24 hours, the culture solution was discarded, 100. Mu.L of the corresponding culture solution containing test compounds at different concentrations was added to each well, and 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) were set for each test compound, and 3 multiplex wells were set for each concentration. Control wells were additionally provided, and no test compound was added to the control wells, and only 100. Mu.L of culture medium was added. Control wells were set with 3 duplicate wells. Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 Culturing in saturated humidity incubator for 72 hr, taking out 24-well plate, taking out cells from the well, adding trypan blue in equal proportion, and mixing. The number of cells in the control well and the dosing well, respectively, was counted using a blood cell counting plate. The growth inhibition rate was calculated as follows:
growth inhibition (%) = (1-dosing well cell number/control well cell number) ×100%
The experiment is divided into a group of cytarabine structural analogues P1 and a group of cytarabine hydrochloride. The results of the inhibition of HL-60 cell proliferation assay for each group are shown in Table 1 below:
TABLE 1 results of measurement of HL-60 cell proliferation inhibition by each test compound group
| P1 | Cytarabine hydrochloride | |
| 1.5625μmol/L | 18％ | 10％ |
| 3.125μmol/L | 30％ | 21％ |
| 6.25μmol/L | 50％ | 30％ |
| 12.5μmol/ |
60％ | 40％ |
| 25μmol/L | 64％ | 56％ |
As can be seen from the results in Table 1, the cytarabine structural analog P1 has an inhibitory effect on HL60 cells at various gradient concentrations. This means: like cytarabine hydrochloride, cytarabine structural analog P1 can inhibit proliferation and growth of human leukemia cells HL 60. Furthermore, at each identical concentration, the inhibition rate of cytarabine structural analogue P1 on HL60 cell growth was higher than cytarabine hydrochloride, showing that: the inhibition effect of the cytarabine structural analogue P1 on HL60 cell proliferation is stronger than that of cytarabine hydrochloride.
EXAMPLE 7 inhibition of proliferation of human leukemia cell HL-60 by Cytarabine structural analog P4
Test compounds: the cytarabine structural analogues P4, cytarabine and cytarabine hydrochloride prepared in example 4.
Cell culture: placing human leukemia cell line HL-60 in RPMI-1640 culture solution containing 10% foetal calf serum at 37deg.C with volume fraction of 5% CO 2 And (3) carrying out periodic subculture in a saturated humidity incubator, and taking the cells in the logarithmic growth phase as the cells for subsequent experiments.
Preparing a medicine mother solution: the test compound is dissolved in DMSO (dimethyl sulfoxide) to prepare mother solution with the concentration of 100mmol/L, and the mother solution is preserved for standby. The experiments were diluted with DMSO to the desired concentration.
Inhibition rate measurement: taking HL-60 cells in logarithmic growth phase, and counting to obtain a density of 5×10 4 Each/mL of the cell suspension was inoculated into a 24-well culture plate, and 2mL (cell number 10 5 And/or holes). Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 The culture was carried out in a saturated humidity incubator for 24 hours, the culture solution was discarded, 100. Mu.L of the corresponding culture solution containing test compounds at different concentrations was added to each well, and 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) were set for each test compound, and 3 multiplex wells were set for each concentration. Control wells were additionally provided, and no test compound was added to the control wells, and only 100. Mu.L of culture medium was added. Control wells were set with 3 duplicate wells. Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 Culturing in saturated humidity incubator for 72 hr, taking out 24-well plate, taking out cells from the well, adding trypan blue in equal proportion, and mixing. The number of cells in the control well and the dosing well, respectively, was counted using a blood cell counting plate. The growth inhibition rate was calculated as follows:
growth inhibition (%) = (1-dosing well cell number/control well cell number) ×100%
The experiments were divided into 3 groups: cytarabine group, cytarabine structural analogue P4 group, cytarabine hydrochloride group. The results of the inhibition of HL-60 cell proliferation assay for each group are shown in Table 2 below:
TABLE 2 results of measurement of HL-60 cell proliferation inhibition by each test compound group
| Cytarabine | P4 | Cytarabine hydrochloride | |
| 1.5625μmol/ |
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) Each test compound has inhibitory effect on HL60 cells at different gradient concentrations. This means: like cytarabine and cytarabine hydrochloride, cytarabine structural analog P4 can inhibit proliferation and growth of human leukemia cells HL 60.
(2) At each identical concentration, the inhibition rate of cytarabine structural analog P4 on HL60 cell growth was highest and significantly higher than that of cytarabine and cytarabine hydrochloride groups. At a concentration of 1.5625. Mu. Mol/L, the inhibition rate of cytarabine structural analogue P4 to HL60 cell growth was 56%, while the inhibition rates of cytarabine and cytarabine hydrochloride to HL60 cell growth were 20% and 10%, respectively; at a concentration of 25. Mu. Mol/L, the inhibition rate of the cytarabine structural analogue P4 on the growth of HL60 cells is as high as 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 the cytarabine structural analogue P4 has a significantly stronger inhibitory effect on HL60 cells at each concentration compared to cytarabine and cytarabine hydrochloride.
EXAMPLE 8 drug concentration of Cytarabine structural analog incubated in HL-60 cells for various times
Taking HL-60 cells in logarithmic growth phase, and counting to obtain a density of 5×10 4 Each/mL of the cell suspension was inoculated into a 24-well culture plate, and 2mL (cell number 10 5 And/or holes). Placing 24-well plate at 37deg.C with volume fraction of 5% CO 2 Culturing in a saturated humidity incubator for 24 hours, discarding the culture solution, adding 100 mu L of culture solution containing 100 mu mol/L of drug to be detected into each hole, wherein the drug to be detected is cytarabine and the cytarabine structural analogue P4 prepared in example 4 respectively. 3 multiple holes are arranged in each group, and the groups are placed at 37 ℃ and 5% CO 2 The culture was continued in a saturated humidity incubator. After various incubation times (0.5, 1, 3, 6 hours), the concentration of the original drug (cytarabine, designated Ara-C in the figures; or cytarabine structural analogue P4, designated BX20-1-004 in the figures) and the corresponding active metabolite cytarabine triphosphate (designated Ara-CTP (Ara-C) and Ara-CTP (004) in the figures) in HL-60 cells, respectively, was tested. The concentration comparison chart is shown in fig. 2.
As can be seen from fig. 2:
(1) With the prolonged incubation time, the concentration of the active metabolite in the HL-60 cell is increased, and when the incubation time is 3 hours or more, the concentration of the active metabolite cytarabine triphosphate in the HL-60 cell exceeds the concentration of the active metabolite. This means that both cytarabine and cytarabine structural analogue P4 are able to enter HL-60 cells and be converted into active metabolites in the cells.
(2) The concentration of the cytarabine structural analogue P4 in the cells is significantly higher than that of cytarabine under the same incubation time, which indicates that the cytarabine structural analogue P4 is more capable of penetrating the cell membrane into the cells than cytarabine.
(3) The concentration of the active metabolite cytarabine triphosphate in cells treated with the cytarabine structural analogue P4 was much higher than the concentration of the active metabolite cytarabine triphosphate in cells treated with cytarabine at the same incubation time. This suggests: the cytarabine structural analog P4 prepared in example 4 was able to produce a higher concentration of active metabolite than cytarabine, thereby exerting a stronger anti-leukemia cell proliferation activity. This demonstrates that the structural analogue P4 of cytarabine prepared in example 4 has significantly higher bioavailability than cytarabine.
EXAMPLE 9 in vivo oral single dose pharmacokinetic study in rats
8 Sprague-Dawley rats were divided into two groups: cytarabine group and cytarabine structural analogue group, 4 rats in each group, were orally administered with cytarabine structural analogue P4 solution (physiological saline solution, drug concentration 10 mg/mL) and cytarabine solution (physiological saline solution, drug concentration 10 mg/mL) in a single dose, respectively, with the following oral administration doses: rats were 10mg/Kg calculated as cytarabine.
Determination was performed using liquid chromatography-mass spectrometry (HPLC-MS/MS): (1) Concentration of cytarabine in plasma and PBMC cells following oral administration of cytarabine; (2) Concentration of proto-drug (cytarabine structural analogue P4) in plasma and PBMC cells following oral administration of cytarabine structural analogue P4; (3) Concentration of metabolite cytarabine in plasma and PBMC cells following oral administration of cytarabine structural analog P4. The results are shown below in table 3 for pharmacokinetic parameters (n=4) of the proto-drug and the metabolite cytarabine in plasma and PBMC cells after a single oral administration of rats. The cytarabine structural analogue P4 is designated 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 drug level curve, which means the area surrounded by the blood concentration curve with respect to the time axis, in ng.h/mL. This parameter is an important index for evaluating the degree of drug absorption, reflecting the in vivo exposure characteristics of the drug. C (C) max Peak concentration refers to the highest value of blood concentration occurring after administration. This parameter is an important indicator reflecting the rate and extent of absorption of the drug in the body. t is t max The time to peak refers to the time required to reach peak drug concentration after administration. The parameter reflects the speed of the drug entering the body, and the peak time is short if the absorption speed is high. t is t 1/2 Is the terminal elimination half-life, which refers to the time required for the blood concentration of the terminal phase to drop by half. This parameter intuitively 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 the PBMC cells after administration of cytarabine structural analogue P4 was significantly higher than the AUC of cytarabine entering the PBMC cells after administration of cytarabine, indicating that cytarabine structural analogue P4 has better cell penetration than cytarabine.
(2) The AUC of cytarabine in plasma after administration of cytarabine structural analogue P4 is significantly lower than the AUC of cytarabine in plasma after administration of cytarabine, which means that less uracil cytarabine is subsequently produced in plasma and the bioavailability of cytarabine structural analogue P4 is higher.
(3) The peak concentration of cytarabine in PBMC cells (986 ng/mL) after administration of cytarabine structural analog P4 was more than twice the peak concentration of cytarabine in PBMC cells (436 ng/mL) after administration of cytarabine. The cytarabine structural analogue P4 can be converted into the parent cytarabine in cells, so that the blood concentration of target cells in the body is obviously improved, and the bioavailability is higher.
(4) The peak time of cytarabine in PBMC cells after administration of cytarabine structural analogue P4 (26.1 h) was significantly higher than the peak time of cytarabine in PBMC cells after administration of cytarabine (0.83 h). This means: the cytarabine structural analogue P4 significantly prolongs the time of cytarabine in cells.
(5) The half-life of cytarabine in PBMC cells after administration of cytarabine structural analogue P4 (25.5 h) was significantly higher than the peak time of cytarabine in PBMC cells after administration of cytarabine (1.02 h). This means: the cytarabine structural analogue P4 orally taken has longer half-life period, has good stability of cytarabine in PBMC cells and can maintain effective blood concentration for a longer time.
In conclusion, the cytarabine structural analogues P1-P4 can inhibit human leukemia HL-60 cells for the first time, and the inhibition effect of the cytarabine structural analogues is superior to cytarabine hydrochloride at low concentration, so that the cytarabine structural analogues are expected to be used as novel anti-leukemia drugs.
In particular, compared with cytarabine, the cytarabine structural analogue P4 has stronger proliferation inhibition capability and cell penetration capability on leukemia cells HL60, has higher drug concentration entering cells and higher concentration of an active metabolite cytarabine triphosphate generated in the cells, and has higher bioavailability; the cytarabine can be converted into the parent drug cytarabine in cells, the blood concentration of target cells in the body is obviously improved, and toxic and side effects and drug resistance caused by large-dose medication can be avoided; can remarkably prolong the half-life period of cytarabine, maintain effective blood concentration for a long time, can be orally taken, and can avoid inconvenience caused by injection. Therefore, the cytarabine structural analogue P4 completely overcomes the defects of large polarity, poor film permeability, easy metabolic inactivation in vivo, short half-life and the like of cytarabine, provides possibility for oral administration, and fills the technical blank that no new medicine of this type is marketed in China.
The preparation method of the cytarabine structural analogue is simple and easy to operate, and is expected to be used for industrial production.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.
Claims (8)
1. A cytarabine structural analogue, which is characterized by being a cytarabine structural analogue P4 shown in the following formula (V):
2. a process for the preparation of cytarabine structural analogues as claimed in claim 1 comprising the steps of:
dissolving cytarabine analogue P3 and aspartic acid derivative in anhydrous tetrahydrofuran, N 2 Cooling to-5-0 deg.c under protection, slowly dropping 1-propyl phosphoric anhydride, heating to room temperature to react to obtain the first reaction liquid, and using saturated NaHCO 3 Quenching the solution, extracting the water phase with dichloromethane, merging the organic phases, drying and concentrating, and purifying by column chromatography to obtain an intermediate; dissolving the intermediate in methanol, adding Pd/C, introducing hydrogen for reaction, and performing after-treatment on the obtained second reaction solution after the completion of the reaction monitored by thin-layer chromatography to obtain a product; wherein,,
the aspartic acid derivative is compound 3 shown in the following formula (VIII)
Cytarabine structural analog P3 is a compound shown in the following formula (IV):
it is prepared by the following method:
dissolving cytarabine and phosphoryl chloride derivative in anhydrous tetrahydrofuran, N 2 Cooling to-70 to-78 ℃ under protection, slowly dropwise adding 1-methylimidazole, stirring at-70 to-78 ℃, heating to room temperature for reaction, and after the reaction is completed, carrying out post-treatment and purification on the reaction solution to obtain a product; wherein the phosphoryl chloride derivative is a compound 4 shown in the following formula (VII):
3. the method for preparing the cytarabine structural analogue according to claim 2, wherein the cytarabine structural analogue P3: aspartic acid derivatives: the molar ratio of the 1-propyl phosphoric anhydride is 1: (1.2-1.5): (2.0-2.5).
4. The method for producing a cytarabine structural analogue according to claim 2, wherein, in the production of cytarabine structural analogue P3, cytarabine: phosphorus oxychloride derivative: the molar ratio of the 1-methylimidazole is 1: (1.5-2.0): (2.0-3.0).
5. The method for producing a cytarabine structural analog according to claim 2, wherein in the production of cytarabine structural analog P3, the post-treatment purification of the reaction solution comprises: the reaction solution is concentrated, diluted by adding dichloromethane, washed by dilute hydrochloric acid, water and saturated sodium chloride solution, and the organic phases are combined, dried and concentrated, and purified by column chromatography to obtain the product.
6. The method for preparing a cytarabine structural analog according to claim 2, wherein the post-treatment of the second reaction solution comprises: and (3) collecting the first filtrate from the second reaction solution through a diatomite pad, eluting the diatomite pad by using methanol, collecting the second filtrate, combining the first filtrate and the second filtrate to obtain filtrate, concentrating the filtrate, and stirring and filtering the filtrate by using methyl tertiary butyl ether to obtain a product.
7. Use of a cytarabine structural analogue according to claim 1 in the manufacture of a medicament for the treatment of leukemia.
8. Use of a cytarabine structural analogue according to claim 1 in the manufacture of an oral medicament for the treatment of leukemia.
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Denomination of invention: Structural analogues of cytarabine and their preparation methods and applications Granted publication date: 20230421 Pledgee: Bank of China Limited Wuhan Hanyang sub branch Pledgor: Wuhan Bereheng Pharmaceutical Technology Co.,Ltd. Registration number: Y2024980022569 |