CN101849934A

CN101849934A - Phosphatidylinositol-3-kinase inhibitor and application thereof

Info

Publication number: CN101849934A
Application number: CN 201010181572
Authority: CN
Inventors: 毛新良; 艾伦·什莫
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2010-05-25
Filing date: 2010-05-25
Publication date: 2010-10-06
Anticipated expiration: 2030-05-25
Also published as: CN101849934B

Abstract

The invention discloses new application of a 2-(4-fluorophenyl)-3-nitro-8-O-ethyl-2-hydro-benzopyran compound, in particular application of the compound serving as phosphatidylinositol-3-kinase (PI3K) inhibitor. The PI3K inhibitor can inhibit PI3K and disturb a PI3K/AKT signal channel, has good treatment effect on multiple tumors, particularly malignant hematological diseases, can effectively control the propagation of tumor cells and induce the apoptosis thereof, and has the effect of inhibiting and treating the tumors; and meanwhile, the PI3K has low toxicity.

Description

A kind of phosphatidylinositol--3-kinase inhibitor and application thereof

Technical field

The invention belongs to medicine for treating tumor thing field, be specifically related to the application of a kind of phosphatidylinositol--3-kinase inhibitor and conduct preparation antitumor drug thereof.

Background technology

Owing to multiple reason, tumor become ascendant trend year by year.And statistics shows, tumor has become the cause of the death of the position that ranks first.Aspect tumour medicine, global cancer therapy drug gross sales amount was 28,200,000,000 dollars in 2005, and China China antitumor drug sales volume is also with annual 15% to 17% speed increment, and domestic only hospital clinical in 2005 medication just surpasses 25,000,000,000 yuan, and annual rate of growth surpasses 30%.Regrettably antitumor drug in the market is many by the control of developed countries such as Great Britain and America, so the new drug that active development has independent intellectual property right effectively has boundless prospect.

Phosphatidylinositol-3-kinase (phosphatidylinositol 3-kinase, PI3K) signal path is the main signal hinge in the cell activities, it also is the signal path of modal unusual sudden change among the human tumor cell, the generation of its excessive activation and tumor, development, prognosis are closely related, be the antitumor drug research and development of target spot with PI3K thereby receive much concern, domestic and international many pharmaceuticals and scientific research institution are developing new PI3K inhibitor, and some candidate's new drug has entered the II clinical trial phase stage.Can be expected in the near future, targeting PI3K anti-cancer agent will bring glad tidings for vast tumor patient.Therefore, actively seeking new PI3K inhibitor is a challenge.

Chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran is a kind of existing chemical compound, Maybridge chemical drugs company limited (Maybridge Chemicals Co.Ltd.) by Britain provides, but does not see the report of any its function and application.

Summary of the invention

The object of the invention provides the new purposes of chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran, specifically is the application as phosphatidylinositol--3-kinase inhibitor.

For achieving the above object, the technical solution used in the present invention is: chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran is as the application of phosphatidylinositol-3-kinase (PI3K) inhibitor.

In the technique scheme, the molecular structural formula of chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran is:

Molecular formula is C ₁₇H ₁₄FNO ₄, molecular weight is 315.3.

In the technique scheme, chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran is provided by the Maybridge chemical drugs company limited (Maybridge Chemicals Co.Ltd.) of Britain.

To discovering of above-mentioned PI3K inhibitor:

(1) suppress the PI3K/Akt signal path: described PI3K inhibitor can significantly suppress the phosphorylation level of Akt, and the enzymatic activity of all I type PI3K is shown the obvious suppression effect;

(2) activity of the whole 4 class PI3K enzymes of inhibition Class I: described PI3K inhibitor all shows inhibitory action to the enzymatic activity of 4 each hypotype of PI3K;

(3) suppress the expression of cyclin D: described PI3K inhibitor has suppressed cyclin D1, D2, D3 in multiple myeloma and leukemia cell line mRNA transcribes and protein expression, thereby makes cell cycle arrest in the G0/G1 phase;

(4) propagation of inhibition blood tumor cell: described PI3K inhibitor can suppress the growth of multiple myeloma cells, leukaemia, solid tumor (for example ovarian cancer and head and neck cancer etc.);

(5) toxicity is little simultaneously to suppress tumor growth: described PI3K inhibitor continuous use 10 days, significantly suppressed the tumor growth of tumor-bearing mice; Simultaneously, described PI3K inhibitor does not have obvious inhibitory action to the growth of normal hematopoietic cell; In the performance antitumor action, to the also not obviously influence of body weight of tumor-bearing mice;

(6) induce the blood tumor cell apoptosis: cell is after Annexin V 488 and two the dying of PI (propidium iodide), and flow cytometer detects the shared ratio of viable apoptotic cell and significantly raises; In addition, the expression of activatory caspase-3, PARP also obviously raises.

Therefore, the present invention's application of claimed described PI3K inhibitor in the preparation antitumor drug simultaneously, in the optimized technical scheme, described tumor is a neoplastic hematologic disorder.

The present invention is claimed a kind of antitumor drug simultaneously, and the active component of described antitumor drug is 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran; In the optimized technical scheme, described tumor is a neoplastic hematologic disorder.

In the technique scheme, described antitumor drug comprises also and well known to a person skilled in the art auxiliary ingredients that the dosage form of described tumor radio sensitization medicine comprises: multi-pharmaceuticss such as oral capsule type, oral tablet type, intravenous form, local injection dosage form, drop.Described administering mode comprises oral way or injection system or the like.

Because the technique scheme utilization, the present invention compared with prior art has following advantage:

(1) PI3K inhibitor of the present invention not only can suppress PI3K, disturbs the PI3K/AKT signal path, and can be to kinds of tumors, especially malignant hematologic disease has good therapeutical effect, can control the propagation of tumor cell effectively, induce its apoptosis, tumor be had suppress and therapeutical effect; And be related between the effect to the inhibitory action of PI3K and inducing apoptosis of tumour cell;

(2) PI3K inhibitor of the present invention has therapeutical effect to kinds of tumors and toxicity is less.

Description of drawings

Fig. 1 is the chemical structural formula of embodiment one gained PI3K inhibitor;

Fig. 2 a～2d gives survival rate figure after XM002S is hatched respectively for each cell among the embodiment two;

Fig. 3 a～3b is that the tumor-bearing mice gross tumor volume changes and mice body weight change figure among the embodiment three;

Fig. 4 a induces fluidic cell detection figure behind the apoptosis of blood tumor cell for XM002S among the embodiment four;

Fig. 4 b induces the apoptosis rear electrophoresis figure of blood tumor cell for XM002S among the embodiment four;

Fig. 5 a～5b handles electrophoretogram behind the multiple blood tumor cell for XM002S among the embodiment five;

Fig. 6 A～6B is different tumor cells Western Blot figure after treatment among the embodiment six;

Fig. 7 is the activity figure after PI3K α, β, γ, δ subunit and XM002S are hatched among the embodiment seven.

The specific embodiment

Below in conjunction with drawings and Examples the present invention is further described:

Annotate: represent PI3K inhibitor 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran with XM002S in following examples.

Embodiment one: PI3K inhibitor synthetic

At first synthetic to fluoro-beta-nitrostyrolene with fluorobenzaldehyde and Nitrocarbol., then with 3-ethyoxyl salicylide, to fluoro-beta-nitrostyrolene, pyridine Synthetic 2-to fluorophenyl-3-nitro-8-ethyoxyl-2H-.alpha.-5:6-benzopyran.Concrete method is as follows:

Preparation to fluoro-beta-nitrostyrolene:

With 4-Fluorobenzaldehyde 50.0g, 0.4mol) be dissolved in 3 liters of Nitrocarbol .s, the adding ammonium acetate (34.0g, 0.44mol), back flow reaction 24h.Behind the concentrating under reduced pressure, add 1: 1 mixed liquor of dichloromethane and water, water layer dichloromethane extraction three times.Merge organic layer, after the saturated common salt washing, add anhydrous magnesium sulfate drying.Filter, concentrate.With ethyl acetate-petroleum ether recrystallization, get yellow solid to fluoro-beta-nitrostyrolene 37.3g (92%), fusing point: 98～100 ℃;

2-is synthetic to fluorophenyl-3-nitro-8-ethyoxyl-2H-.alpha.-5:6-benzopyran

With 3-ethyoxyl salicylide (332g, 2mol), (367g 2.2mol) joins in 3 liters of pyridines, stirring at room reaction 48h to fluoro-beta-nitrostyrolene.Reaction mixture is poured in the mixed liquor of ethyl acetate and 2M hydrochloric acid.Tell ethyl acetate layer, use 2M hydrochloric acid, saturated aqueous sodium carbonate and saturated common salt water washing successively.Behind anhydrous magnesium sulfate drying, filter, concentrate.Behind the recrystallization, get yellow solid 200g, be described PI3K inhibitor, structural formula as shown in Figure 1.

Embodiment two: the propagation that suppresses pernicious blood tumor cell

Myeloma cell strain, leukemia cell line, acute myeloid leukemia primary cell and peripheral hematopoietic stem cells are cultivated respectively in RPMI-1640 or IMDM cell culture fluid, in add 10% hyclone, 100 μ g/ml ampicillin, 100 units/ml streptomycin.Cell culture environment is 37 degrees centigrade, 5% carbon dioxide; The XM002S that gives variable concentrations is respectively hatched 72h, with MTS/PMS staining analysis of cells survival rate (wavelength that reads optical density is 490nm), obtain accompanying drawing 2a (myeloma cell strain), accompanying drawing 2b (leukemia cell line), accompanying drawing 2c (acute myeloid leukemia primary cell), accompanying drawing 2d (peripheral hematopoietic stem cells) according to routine techniques.

From Fig. 2 a～2d as can be known: XM002S is dose dependent to the toxicity of pernicious blood tumor cell, but the propagation of normal peripheral hematopoietic stem cells is not had obvious influence.

Embodiment three: suppress the tumor-bearing mice growth of tumor

Age in 5-6 week female NOD/SCID mice, the chivalrous subcutaneous injection K562 cell (5 * 10 of left side shoulder ⁵) or U937 cell (1 * 10 ⁶), treating that tumor is long to can touch the time, lumbar injection XM002S (100mg/kg/d) intervenes, and matched group is then injected equivalent XM002S solvent (10%DMSO), continuous 10d.Measure the variation of gross tumor volume every other day, monitor the body weight change of mice simultaneously, obtain Fig. 3 a (cell tumor-bearing mice cell tumor-bearing mice), Fig. 3 b (U937 cell tumor-bearing mice), wherein, left side figure is a tumor size variation curve, and right figure is the body weight change curve.

According to Fig. 3 a, 3b as can be known: XM002S has significantly suppressed the growth of two kinds of tumor-bearing mice in-vivo tumours, finish since the 6th day to experiment, the gross tumor volume of XM002S intervention group compare with matched group difference all have statistical significance ( ^*P＜0.05; ^*P＜0.01), but XM002S does not have obvious influence to the mice body weight.

Embodiment four: the apoptosis of inducing blood tumor cell

After XM002S handles different blood tumor cell (HL60, U937, JJN3, KMS11) 24h, collecting cell.

Described blood tumor cell is handled after Annexin V-FITC and PI are two dyes, and detects Annexin V with flow cytometer ⁺The ratio that cell is shared gets Fig. 4 a, shows according to Fig. 4 a: after XM002S handled, apoptosis rate all significantly raise than the control group.

Described blood tumor cell is handled back RIPA buffer cracking, get 20 μ g albumen and carry out the SDS-PAGE gel electrophoresis, detect Pro-caspase 3, Pro-PARP and activatory PARP with specific antibody, get Fig. 4 b, the result shows: the inductive apoptosis of XM002S is relevant with the activation of caspase 3 and PARP.

Embodiment five: reduce cyclin D protein expression level

XM002S handles multiple blood tumor cell, collecting cell behind the 24h, extract total protein of cell, through the SDS-PAGE gel electrophoresis, detect protein expression levels such as Pro-caspase 3, PARP, Actin, tubulin, cyclin D1, D2, D3 respectively with specific antibody, Fig. 5 a, the result as can be known: XM002S all shows inhibitory action to the protein expression of 3 kinds of cyclin D.

After KMS11 and K562 cell are handled 24h with XM002S respectively, extract total protein of cell, through the SDS-PAGE gel electrophoresis, detect protein expression levels such as Pro-caspase 3, PARP, Actin, tubulin, cyclin D1, D2, D3 respectively with specific antibody, get Fig. 5 b, find that the inhibitory action of D-cycilin is the dose dependent relation.

Embodiment six: the phosphorylation that suppresses Akt

After different tumor cell low serum overnight (0.5%) are cultivated, through XM002S (XM, 100 μ M) or LY294002 (LY, 100 μ M) locate behind the 2hr with IGF1 effect 15min, collecting cell extracts total protein, detects the expression of phosphorylation Akt (p-Akt) and total Akt with Western blot, β-actin gets Fig. 6 a as confidential reference items.

After serum-free medium processing KMS11 cell spends the night, give XM002S (XM) and handle 30min, 1hr or 2hr, LY294002 is as the positive control medicine, IGF-1 induces 15min then, collecting cell extracts total protein, detects the expression of phosphorylation Akt (p-Akt) and total Akt with Western blot, β-actin gets Fig. 6 b as confidential reference items.

The result shows: IGF-1 has significantly induced the phosphorylation of Akt, and LY294002 (100 μ M) has then effectively suppressed the expression of pAkt; XM002S handles 1hr or 2hr has all significantly suppressed the expression of p-Akt, and presents dose-effect and time-effect relationship.

Embodiment seven: the enzymatic activity that suppresses I type PI3K.

Immuno-precipitation extracts intracellular PI3K α, β, γ, δ subunit, by Concentraton gradient XM002S is hatched with it, and the activity change of each enzyme of HotSpot technology for detection is calculated IC ₅₀(for avoiding experimental error, carrying out three parallel laboratory tests) gets Fig. 7 (each figure all is 3 result of experiment), and as shown in the figure: XM002S all shows inhibitory action to the enzymatic activity of 4 each hypotype of PI3K.

Claims

1. chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran is as the application of phosphatidylinositol--3-kinase inhibitor.

2. the application of chemical compound 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran in the preparation antitumor drug.

3. an antitumor drug is characterized in that, the active component of described antitumor drug is 2-(4-fluorophenyl)-3-nitro-8-oxygen-ethyl-2-hydrogen-.alpha.-5:6-benzopyran.