CN105037399A - Bcr-Abl amphiploid inhibitor, preparation method and application thereof - Google Patents

Abstract

The invention discloses a Bcr-Abl amphiploid inhibitor, a preparation method and an application thereof. A compound or pharmaceutically acceptable salts thereof provided in the invention can be used as the Bcr-Abl amphiploid inhibitor which can effectively inhibit the activity of tyrosine kinase. The Bcr-Abl amphiploid inhibitor can be effectively used in treatment of diseases related to abnormal activation of the tyrosine kinase and has excellent treatment effect on malignant tumors. The inhibitor is easy to prepare, is low in cost and is excellent in application prospect.

Description

A kind of Bcr-Abl diploid inhibitor and its preparation method and use

technical field

The invention relates to a Bcr-Abl diploid inhibitor, a preparation method and application thereof.

Background technique

Chronic myelogenous leukemia is a chromosomal gene mutation [t(9:22)(q34;q11)] reciprocal translocation, the ABL gene at chromosome 9 and the BCR gene at chromosome 22 are fused to produce BCR-ABL, the fused gene Encodes a 210kDa tyrosine kinase Bcr-Abl, which is the main cause of chronic myelogenous leukemia. c-Abl is a haploid tyrosine kinase that exists in the cytoplasm in normal cells. After fusion with Bcr, its morphology changes from haploid to tetramer, so the kinase is always activated. lead to the occurrence of tumors. The presence of amino acid sequences that can cause multimerization in the Bcr protein sequence leads to Bcr-Abl tetramerization. Since c-Abl plays an important role in normal cells such as cardiac muscle, if inhibitors that selectively inhibit oncogenic Bcr-Abl but not Abl are developed, it is expected to greatly reduce the extensive side effects of such inhibitors such as cardiotoxicity.

In 2001, the FDA approved the marketing of the first tyrosine kinase inhibitor (TKI), imatinib (Imatinib), for the treatment of chronic myeloid leukemia (CML). As the first-generation Bcr-Abl inhibitor, imatinib has become the first-line drug for the treatment of CML. But most patients eventually develop resistance to imatinib. Subsequent studies have shown that the occurrence of IM drug resistance may be related to gene mutations in Abl kinase active regions such as G250E, Q252H, Y253F, Y253H, E255K, E355G, E255V, T315A, T315I, F317L, F317V, M351T, F359V, H396P, and M244V , gene mutations lead to a decrease in the affinity of imatinib to Abl kinase. Most gene mutations reduce the affinity of imatinib by 5-30 times, which is the main reason for drug resistance. Among them, T315I is special, and the decrease is the most obvious, and the IC ₅₀ drops to about 6400nM. The recently developed ponatinib is not only effective for T315I, but also effective for wild type and most of the mutant strains.

From the x-Ray co-crystal structure of imatinib and ponatinib and the kinase part of the Abl protein, the nitrogen methyl part of the alpha-piperazine part in the two structures is exposed to the solvent, and the two inhibitors The linear distance between the nitrogen atoms is about 24 amps. Since Bcr-Abl is a tetramer, it can combine with four inhibitors at the same time. If the two inhibitors are connected by a chain, it is expected to greatly improve the affinity for tetramerized Bcr-Abl, thereby playing a role Selectively inhibit the action of Bcr-Abl, and because Abl is haploid, it can only be combined with one inhibitor, and the affinity of the double inhibitor to the enzyme will not be greatly affected.

Contents of the invention

The object of the present invention is to provide a Bcr-Abl diploid inhibitor and its preparation method and use.

The compound represented by formula I provided by the present invention or a pharmaceutically acceptable salt thereof has the following structural formula:

Further, Linker is (Z ₁ ) _a –(Z ₂ ) _b –(Z ₃ ) _c ;

Wherein, Z ₁ , Z ₂ , and Z ₃ are independently selected from

C(O)(CH ₂ ) _d NH, CO(CH ₂ ) _e C(O),

(CH ₂ CH ₂ ) _f NHC(O), (CH ₂ CH ₂ ) _g -C(O)NH,

(CH ₂ ) _h C(O)(OCH ₂ CH ₂ ) _i NHC(O)(CH ₂ ) _j C(O),

NH(CH ₂ ) _K (OCH ₂ CH ₂ ) _l -O(CH ₂ ) _m NH,

(CH ₂ ) _n C(O)(OCH ₂ CH ₂ ) _O NH(C(O)(CH ₂ ) _p NH) _q C(O)-alkyl,

C(O)NH(CH ₂ ) _r NHC(O)-(CH ₂ ) _s C(O)(NHCH ₂ CH ₂ ) _t NH(C(O)(CH ₂ ) _u NH) _v -

C(O)-alkyl;

a to v are each independently selected from any value in 1 to 20.

Further, the compound is:

The present invention also provides a method for preparing the above compound BDB.

The preparation method of above-mentioned compound BDB is characterized in that: it comprises the following operation steps:

Further, it includes the following steps:

a. Preparation of compound 9:

Ethyl chloroformate, compound 8, and diisopropylethylamine were stirred and reacted in tetrahydrofuran at room temperature for 16 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 9;

b. Preparation of Compound 11:

Compound 9, diisopropylethylamine, and 4-(chloromethyl)benzoyl chloride were stirred and reacted at room temperature for 16 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 11;

c. Preparation of compound 12:

Compound 11 was stirred in HCl/ethyl acetate for 2 h at room temperature to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 12;

d. Preparation of Compound 14:

Compound 12, Compound 13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, diisopropylethylamine in dichloromethane, The reaction was stirred at room temperature for 16 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 14;

e. Preparation of compound BDB:

Compound 7, compound 14, and potassium carbonate were reacted in N,N-dimethylformamide with stirring at 60° C. for 5 h to obtain a reaction liquid; the reaction liquid was separated and purified to obtain compound BDB.

further,

In step a, the molar ratio of ethyl chloroformate, compound 8, and diisopropylethylamine is 46.5:44.5:264; the molar volume ratio of ethyl chloroformate to tetrahydrofuran is 46.5:350mmol/ml;

In step b, the molar ratio of the compound 9, diisopropylethylamine, and 4-(chloromethyl)benzoyl chloride is 0.67:0.8:0.8; the molar volume ratio of the compound 9 to dichloromethane is 0.67 : 50mmol/ml;

In step c, the molar volume ratio of the compound 11 to HCl/ethyl acetate is 0.445:20mmol/ml; the concentration of the HCl/ethyl acetate is 2M;

In step d, the compound 12, compound 13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, diisopropylethylamine The molar ratio is 0.445:0.445:0.534:0.534:0.534; the molar volume ratio of the compound 12 to dichloromethane is 0.445:25mmol/ml;

In step e, the molar ratio of compound 7, compound 14, and potassium carbonate is 0.4:1:4.8; the molar volume ratio of compound 7 to N,N-dimethylformamide is 0.4:50 mmol/ml.

Further, in step e, the compound 7 is prepared according to the following steps:

Further, the compound 7 was prepared according to the following steps:

i. Preparation of compound 2:

Compound 1 and BOC anhydride reacted completely in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 2;

ii. Preparation of compound 3:

Compound 2, triethylamine, and glutaryl chloride were stirred and reacted at room temperature for 3 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 3;

iii. Preparation of Compound 4

Compound 3 was stirred and reacted at room temperature for 3 h in HCl/ethyl acetate to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 4;

iv. Preparation of Compound 5

Compound 4, diisopropylethylamine, and 3-chloropropionyl chloride were stirred and reacted at room temperature for 3 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 5;

v, preparation of compound 6

Compound 5, tert-butyl piperazine-1-carboxylate, and diisopropylethylamine were refluxed in dioxane for 18 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 6;

vi. Preparation of Compound 7:

Compound 6 was stirred in HCl/ethyl acetate for 2 h at room temperature to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 7.

further,

In step i, the molar ratio of compound 1 to BOC anhydride is 45.5:43.18; the molar volume ratio of compound 1 to dichloromethane is 45.5:600mmol/ml;

In step ii, the molar ratio of the compound 2, triethylamine and glutaryl chloride is 8.42:14.85:4.17; the molar volume ratio of the compound 2 and dichloromethane is 8.42:120mmol/ml;

In step iii, the molar volume ratio of compound 3 to HCl/ethyl acetate is 3.8:100mmol/ml; the concentration of HCl/ethyl acetate is 2M;

In step iv, the molar ratio of the compound 4, diisopropylethylamine, and 3-chloropropionyl chloride is 0.9:6.15:1.81; the molar volume ratio of the compound 4 dichloromethane is 0.9:50mmol/ml;

In step v, the molar ratio of compound 5, tert-butyl piperazine-1-carboxylate, and diisopropylethylamine is 0.14:2.68:0.77; the molar volume ratio of compound 5 to dioxane is 0.14 : 20mmol/ml;

In step vi, the molar volume ratio of compound 3 to HCl/ethyl acetate is 0.1:20mmol/ml; the concentration of HCl/ethyl acetate is 2M.

The present invention also provides the use of the above-mentioned compound and its pharmaceutically acceptable salt in the preparation of medicine for treating cell proliferation diseases.

Use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cell proliferation diseases.

Further, the drug is a tyrosine kinase inhibitor.

Further, the drug is an ABL inhibitor drug.

Further, the drug is an inhibitor of BCR-ABL or its mutant strain.

Further, the BCR-ABL mutant is a T315I mutant.

Further, the cell proliferative disease is cancer.

Further, the cancer is chronic myeloid leukemia, intestinal stromal tumor, gynecological tumor, dermatofibrosarcoma protuberans, Ph+ALL.

The compound provided by the present invention or a pharmaceutically acceptable salt thereof, as a Bcr-Abl diploid inhibitor, can effectively inhibit the activity of tyrosine kinase, and can be effectively used for the treatment of diseases related to the abnormal activation of such kinases, and can effectively treat malignant tumors. It has good therapeutic effect, and the preparation method of this type of inhibitor is simple and low in cost, and has good application prospect.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed ways

The raw materials and equipment used in the specific embodiment of the present invention are all known products, obtained by purchasing commercially available products.

Abbreviated Chinese name: DCM: dichloromethane; (Boc) ₂ O: BOC anhydride;

The preparation of embodiment 1 compound BDB of the present invention

The synthetic route is as follows:

Concrete synthetic steps are as follows:

Preparation of Compound 2:

Compound 1 (10g, 45.5mmol; manufacturer: Sigma-Aldrich) was dissolved in 500ml of DCM, and (Boc) ₂ O (9.4g, 43.18mmol) was dissolved in 100ml of DCM at room temperature, and slowly added dropwise to the reaction flask , react overnight; add 200ml of water to the reaction solution, adjust the pH to 3-4, extract the impurities in the organic phase, adjust the pH to 10 in the water phase, then add sodium chloride to saturate, extract the product, and then use saturated Aqueous sodium chloride solution (10ml×6) was used to wash away unreacted raw materials, dried over sodium sulfate, and evaporated to dryness to obtain a colorless oily compound 2 (4.7g, yield 34.1%).

¹ HNMR (400MHz, CDCl3): δ5.11 (bs, 1H), 3.63-3.45 (m, 12H), 3.21 (t, J = 6.0Hz, 2H), 2.81 (t, J = 6.4Hz, 2H), 1.77-1.42(m,6H),1.42(s,9H).

Preparation of compound 3:

At room temperature, compound 2 (2.7g, 8.42mmol), triethylamine (1.5g, 14.85mmol) were dissolved in dichloromethane (100ml), glutaryl chloride (0.7g, 4.17mmol) was dissolved in dichloro Add methane (20ml) dropwise to the reaction solution for more than 1h. After the addition, stir and react at room temperature for 3h, then wash the organic phase with saturated aqueous sodium chloride (100ml×2), and wash the organic phase with sodium sulfate After drying, the solvent was concentrated in vacuo and evaporated to dryness. The residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1) to obtain a colorless oily compound 3 (3.1 g, yield 100%).

ESI-MSm/z:737.4[M+H] ⁺ ;

¹ HNMR (400MHz, CDCl3): δ6.55 (bs, 2H), 5.06 (bs, 2H), 3.63-3.49 (m, 24H), 3.34 (m, 4H), 3.20 (m, 4H), 2.22 (t , J=7.2Hz, 4H), 1.93(m, 2H), 1.78-1.70(m, 8H), 1.41(s, 18H).

Preparation of compound 4:

Compound 3 (2.8g, 3.80mmol) was added to HCl/ethyl acetate (2M, 100ml), stirred at room temperature for 3h, concentrated in vacuo and evaporated to dryness to obtain a colorless oily liquid, namely compound 4 (2.6g, Yield: ~ 100%), without further purification, directly used in the next step reaction;

Preparation of Compound 5:

At room temperature, compound 4 (484mg, 0.90mmol), diisopropylethylamine (DIPEA) (800mg, 6.15mmol) were dissolved in dichloromethane (40ml), and 3-chloropropionyl chloride (230mg, 1.81mmol ) was diluted with dichloromethane (10ml), added dropwise to the reaction solution for more than 1h, stirred and reacted at room temperature for 3h after the addition was completed, then washed the organic phase with saturated aqueous sodium chloride solution (50ml×2), The organic phase was dried over sodium sulfate, concentrated under reduced pressure and evaporated to dryness, added n-hexane to the residue, stirred evenly, and filtered to obtain a white solid, which was compound 5 (330 mg; yield 50.9%).

¹ HNMR (400MHz, CDCl3): δ6.85 (bs, 2H), 6.58 (bs, 2H), 3.81 (t, J = 6.4Hz, 4H), 3.66-3.53 (m, 24H), 3.39-3.31 (m , 8H), 2.64 (m, 4H), 2.24 (t, J=7.2Hz, 4H), 1.81 (m, 2H), 1.55-1.47 (m, 8H).

Preparation of compound 6:

Compound 5 (100mg, 0.14mmol), tert-butyl piperazine-1-carboxylate (500mg, 2.68mmol), diisopropylethylamine (DIPEA) (100mg, 0.77mmol), dioxane (20ml) were added Put it into a round bottom flask, heat it under reflux and react for 18h, then cool the reaction liquid to room temperature, evaporate the solvent under reduced pressure, dissolve the residue with dichloromethane, wash the organic phase with saturated sodium chloride aqueous solution, and dry it with sodium sulfate The organic phase was concentrated under reduced pressure and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (dichloromethane/methanol=10/1) to obtain a colorless oily liquid, Compound 6 (50 mg; yield 35.2%).

¹ HNMR (400MHz, CDCl3): δ7.98 (bs, 2H), 7.07 (bs, 2H), 3.66-3.53 (m, 24H), 3.46 (m, 8H), 3.36-3.31 (m, 8H), 2.71 (m, 4H), 2.47 (m, 12H), 2.27 (t, J=6.8Hz, 4H), 1.95 (m, 2H), 1.79-1.75 (m, 8H), 1.45 (s, 18H).

Preparation of compound 7

Compound 6 (100mg, 0.10mmol) was added to HCl/ethyl acetate (2M, 20ml), stirred at room temperature for 2h, then concentrated in vacuo and evaporated to dryness to obtain a colorless oily liquid, compound 7 (100mg; harvested rate ~ 100%).

¹ HNMR (400MHz, D2O) δ3.62 (m, 30H), 3.53 (m, 14H), 3.21 (q, J = 7, 2Hz, 8H), 2.77 (t, J = 6.8Hz, 4H), 2.21 ( t, J=7.6Hz, 4H), 1.82(m, 2H), 1.74(m, 8H).

Preparation of Compound 9:

Ethyl chloroformate (4.5ml, 46.5mmol) was dissolved in tetrahydrofuran (100ml), cooled in an ice bath, compound 8 (10g, 44.5mmol) and diisopropylethylamine (DIPEA) (46ml, 264mmol) were dissolved in Tetrahydrofuran (250ml) was slowly added dropwise to the reaction solution at 0-5°C. After the addition, it was naturally raised to room temperature and stirred for 16 hours. Then the reaction solution was concentrated in vacuo and evaporated to dryness. The residue was dissolved in ethyl acetate, and Wash the organic phase with water, dry the organic phase with sodium sulfate, evaporate the solvent under reduced pressure in vacuo, and purify the residue by silica gel column chromatography (ethyl acetate:petroleum ether=1:3) to obtain a white solid, which is compound 9 (8.0 g; yield 60.7%).

¹ HNMR (400MHz, CD3OD) δ4.72(s, 2H), 4.40(q, J=7.2Hz, 2H), 4.12(d, J=7.2Hz, 2H), 1.53(s, 9H), 1.40(t , J=6.8Hz, 3H).

Preparation of Compound 11:

Compound 9 (200mg, 0.67mmol), diisopropylethylamine (DIPEA) (103mg, 0.8mmol) were dissolved in dichloromethane (25ml), cooled with ice water; 4-(Chloromethyl)benzoyl chloride (Compound 10) (151mg, 0.8mmol) was dissolved in dichloromethane (25ml), and added dropwise to the reaction solution at 0-5°C. After the addition, it was naturally raised to room temperature and stirred for 16h, and the reaction solution was reduced in vacuo. The solvent was evaporated to dryness under reduced pressure, the residue was dissolved in ethyl acetate, and the organic phase was washed with water, the organic phase was dried over sodium sulfate, the solvent was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether= 1:2), a yellow solid was obtained, namely compound 11 (220 mg, yield 73%).

¹ HNMR (400MHz, CD3OD) δ7.98(d, J＝8.4Hz, 2H).7.60(d, J＝7.6Hz, 2H).4.74(s, 2H), 4.69(s, 4H), 4.50(q , J=7.2Hz, 2H), 1.55(s, 9H), 1.45(t, J=7.2Hz, 3H).

Preparation of Compound 12:

Compound 11 (200mg, 0.445mmol) was added to HCl/ethyl acetate (2M, 20ml), stirred at room temperature for 2h, the solvent was evaporated to dryness in vacuo, and a white solid was obtained, which was compound 12 (172mg; yield ~ 100 %); directly used in the next step without further purification;

¹ HNMR (400MHz, CD3OD) δ7.98(d, J=8.0Hz, 2H), 7.60(d, J=8.0Hz, 2H), 4.74(s, 2H), 4.71(s, 4H), 4.52(q , J=7.2Hz, 2H), 1.46(t, J=7.2Hz, 3H).

Preparation of Compound 14:

At room temperature, compound 12 (153 mg, 0.445 mmol), compound 13 (74 mg, 0.445 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) ( 91mg, 0.534mmol) and 1-hydroxybenzotriazole (HOBT) (72mg, 0.534mmol) were dissolved in dichloromethane (25ml), then slowly added diisopropylethylamine (DIPEA) dropwise in the reaction solution ( 115mg, 0.89mmol), and stirred for 16h after the addition. Then add water to the reaction solution to dilute, and extract the aqueous phase with dichloromethane (100ml×2), combine the organic phase, and wash the organic phase with saturated aqueous sodium chloride solution, organic The phase was dried over sodium sulfate, the solvent was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:1) to obtain a yellow solid, namely compound 14 (100 mg; yield 45%) .

¹ HNMR (400MHz, CD ₃ OD) δ7.96(d, J＝8.0Hz, 2H), 7.59(d, J＝8.0Hz, 2H), 7.37～7.48(m, 4H), 5.13(s, 1H) , 4.69 ~ 4.79 (m, 6H), 4.50 (q, J = 7.2Hz, 2H), 3.44 (s, 3H), 1.46 (t, J = 7.2Hz, 3H).

Prepare BDB:

Compound 7 (327mg, 0.4mmol), compound 14 (500mg, 1mmol) and K ₂ CO ₃ (664mg, 4.8mmol), N,N-dimethylformamide (50ml) were added to a round bottom flask at 60 Stir the reaction at ℃ for 5 h, after the reaction solution is cooled to room temperature, pour the reaction solution into 200ml of water, extract the water phase with ethyl acetate, combine the organic phase, wash the organic phase with saturated sodium chloride aqueous solution, and dry the organic phase with sodium sulfate , and the solvent was evaporated to dryness under reduced pressure to obtain a light yellow oily liquid, namely compound BDB (522 mg, yield 81%).

¹ HNMR (400MHz, D2O) δ7.77(d, J=7.2Hz, 2H), 7.68(d, J=8Hz, 2H), 7.47(d, J=7.2Hz, 2H), 7.43(d, J= 7.6Hz, 2H), 7.35(bs, J=7.2Hz, 10H), 5.01(s, 1H), 4.98(s, 1H, s), 4.38(s, 4H), 3.54～3.42(m, 44H), 3.27～3.26(bs,6H),3.11(t,J＝6.8Hz,4H),3.04(t,J＝6.8Hz,4H),2.66(bs,4H),2.05(m,4H),1.75～1.54 (m,10H).

The medicinal activity of test example 1 compound of the present invention

1. Tyrosine Kinase Inhibition

Reference: Amy Cardetal., Journal of Biomolecular Screening 14(1) 2009; Jude Dunne et al., Assay & Drug Development Technologies Vol.2, No.2, 2004.

Using the method of MobilityShiftAssay, the in vitro kinase Abl was screened for the compound BDB of the present invention. The concentration of the compound BDB was sequentially diluted to 0.005 μM by 100 μM triple dilution method with 100% DMSO, and a total of 10 concentration points were used for detection (2 duplicate holes). The compound staurosporine was used as a standard control, and a vehicle control (10% DMSO) and a negative control (EDTA) were set.

The inhibition of kinase Abl by the compound BDB at various concentrations was detected by enzyme-linked immunoreaction on a 384-well reaction plate, the conversion rate data was read on the Caliper, and the conversion rate was converted into an inhibition rate.

Percentinhibition=(max-conversion)/(max-min)*100. Wherein max refers to the conversion rate of DMSO control, and min refers to the conversion rate of no enzyme activity control. The half inhibitory concentration IC ₅₀ of the enzymatic activity of the compound BDB of the present invention to c-Abl tyrosine kinase was calculated according to the inhibition rate of each concentration.

The suppression results are as follows:

IC ₅₀ of compound BDB: 0.05 μM;

The test results show that the compound BDB of the present invention has strong inhibitory activity on c-Abl tyrosine kinase.

2. Compound BDB of the present invention inhibits proliferation of KBM5 (Bcr-Abl positive) and KBM5R (T315I mutant) tumor cells in vitro

Use the ATP method to measure the cytotoxicity of the BDB series compounds of the present invention, adjust the appropriate cell density of KBM5 and KBM5R cells, inoculate a 96-well plate with 140ul cell suspension per well, and the inoculation density of each cell is: 2000-6000; The above-mentioned cell culture plate was placed in the incubator for 24 hours to make it completely adhere to the hole wall, and each compound in the compound BDB series of the present invention was prepared into appropriate different concentrations by three-fold dilution method, and added to 96 cells inoculated in advance. In the corresponding wells of the orifice plate, 10 μL per well, so that the final concentrations are: 100 μM, 33.3 μM, 11.1 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM. Three replicate wells were set up for each concentration, and after incubation in a 37°C incubator for 72 hours, the cell proliferation in each well was measured by ATP method, and the inhibitory rate and half lethal concentration ( _IC50 ) of each drug concentration on the proliferation of KBM5 and KBM5R cells were calculated. ).

test results:

The cytotoxicity of compound BDB to KBM5 (human wild-type Bcr-Abl) tumor cells is as follows:

IC ₅₀ of compound BDB: 7 μM;

The cytotoxicity of compound BDB to KBM5R tumor cells is as follows:

IC ₅₀ of compound BDB: 8.9 μM;

The test results show that the compound BDB of the present invention has strong inhibitory activity at the cell level in vitro.

3. In vivo leukemia subcutaneous xenograft proliferation inhibition test of compound BDB of the present invention

According to the results of the in vitro proliferation inhibition test, the inhibitor with the smallest IC ₅₀ was selected among the BDB series compounds of the present invention, and the growth inhibition of BDB on human leukemia KBM5 and KBM5R cells transplanted subcutaneously in C57BL/6 (Es-1 ^c ) nude mice was evaluated. Effect.

The administration dosage of compound BDB of the present invention is respectively: 80mg/Kg, 160mg/Kg, 350mg/Kg, set positive control group (Imatinib Imatinib of 160mg/Kg) and blank control group (normal saline) simultaneously, tumor-bearing The mice were grouped and administered once a day, and after continuous intraperitoneal injection for two weeks, the tumor volume was detected.

To see if tumor growth can be inhibited, delayed or cured. Tumor diameters were measured with vernier calipers twice a week or every other day. The formula for calculating the tumor volume is: V=0.5a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively.

Data analysis: T test was used for comparison between two groups; one-way ANOVA was used for comparison between three or more groups. If there is a significant difference in F values, multiple comparisons should be performed after ANOVA analysis. Two-way ANOVA was used to analyze the potential synergistic effects of the combined drug groups. All data analyzes were performed with SPSS 17.0; p<0.05 was considered significant difference.

Test results: For the human leukemia KBM5 (Bcr-Abl positive) subcutaneous xenograft tumor model, the compound BDB of the present invention has anti-tumor efficacy, and its inhibitory effect is equivalent to that of imatinib 160 mg/kg at a dose of 80 mg/kg. For the human leukemia KBM5R (Bcr-AblT315I mutant) subcutaneous xenograft tumor model, the compound BDB of the present invention has an inhibitory effect equivalent to that of KBM5 subcutaneous xenograft tumor, and imatinib has no obvious inhibitory effect.

4. Preliminary pharmacokinetic study of the compound BDB of the present invention

After female C57BL/6 (Es-1 ^c ) nude mice were given a single dose intravenously of the compound BDB of the present invention, its concentration in main tissues and blood drug concentration were quantitatively determined by liquid chromatography tandem mass spectrometry, and the effect of the test drug on females was investigated. Differences in distribution between blood and major tissues in C57BL/6(Es-1 ^c ) nude mice; compared with imatinib.

The experimental method and process are as follows: female C57BL/6(Es-1 ^c ) nude mice, body weight 18-25g, 6-8 weeks old, in addition, 3 female C57BL/6(Es-1 ^c ) nude mice were used to collect blank samples, and prepare the standard curve required for analysis. The dosage for intravenous injection is 1mg/kg, and the administration volume is 5mL/kg. Vehicle for intravenous injection: DMSO:SolutolHS15:Saline=5:5:90, v/v/v. Establish the LC-MS/MS method, and use the internal standard method to quantitatively determine the blood drug concentration and tissue concentration of the tested drug. The linear range is 1-1000 ng/mL, and the lower limit of quantification is generally 1 ng/mL. Female C57BL/6 (Es-1 ^c ) nude mice were administered intravenously, and about 20 μL of whole blood was collected from the tail vein of the mice at 0.25h, 2h, 8h and 24h, anticoagulated with K ₂ EDTA, and added according to the volume ratio Blood samples were diluted with 3 times redistilled water and stored at -70°C until analysis. At the same time, tissue samples such as heart, liver, spleen, lung, kidney, stomach, small intestine, and pancreas were collected, washed with normal saline, blotted dry with filter paper, weighed and recorded, and then stored at -70°C until analysis. After thawing the weighed organ tissues, add 3 times PBS buffer salt to the heart, liver, spleen, lung, kidney, stomach, small intestine and pancreas and use Beadbeater to homogenize; when collecting bone marrow samples, use 0.3mL PBS buffer salt to wash, Then centrifuge at 12,000 rpm for 5 minutes, remove 0.25 mL of supernatant, and add 150 mL of PBS buffered saline to the remaining cell samples for homogenization.

Data analysis: WinNonlin (version 6.2) software was used to calculate pharmacokinetic parameters according to non-compartmental model.

The experimental results show that the half-life (t _1/2 ) of the compound BDB of the present invention is 3.8h, which is far greater than the half-life (t _1/2 is 1.5h) of imatinib; compared with imatinib, the compound of the present invention BDB has very obvious advantages in pharmacokinetic properties after intravenous injection.

In summary, the compounds provided by the present invention or their pharmaceutically acceptable salts, as Bcr-Abl diploid inhibitors, can effectively inhibit the activity of tyrosine kinases, and can be effectively used for diseases related to the abnormal activation of such kinases It has a good therapeutic effect on malignant tumors, and the preparation method of this type of inhibitor is simple and low in cost, and has good application prospects.

Claims (16)

1. The compound shown in formula I or its pharmaceutically acceptable salt, its structural formula is as follows: 2. The compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Linker is (Z ₁ ) _a -(Z ₂ ) _b -(Z ₃ ) _c ; Wherein, Z ₁ , Z ₂ , and Z ₃ are independently selected from C(O)(CH ₂ ) _d NH, CO(CH ₂ ) _e C(O), (CH ₂ CH ₂ ) _f NHC(O), (CH ₂ CH ₂ ) _g -C(O)NH, (CH ₂ ) _h C(O)(OCH ₂ CH ₂ ) _i NHC(O)(CH ₂ ) _j C(O), NH(CH ₂ ) _k (OCH ₂ CH ₂ ) _l -O(CH ₂ ) _m NH, (CH ₂ ) _n C(O)(OCH ₂ CH ₂ ) _o NH(C(O)(CH ₂ ) _p NH) _q C(O)-alkyl, C(O)NH(CH ₂ ) _r NHC(O )-(CH ₂ ) _s C(O)(NHCH ₂ CH ₂ ) _t NH(C(O)(CH ₂ ) _u NH) _v -C(O)-alkyl; a to v are each independently selected from any value in 1 to 20. 3. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: the compound is: 4. the preparation method of compound BDB described in claim 3 is characterized in that: it comprises the following operating steps: 5. The preparation method according to claim 4, characterized in that: it comprises the following steps: a. Preparation of compound 9: Ethyl chloroformate, compound 8, and diisopropylethylamine were stirred and reacted in tetrahydrofuran at room temperature for 16 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 9; b. Preparation of Compound 11: Compound 9, diisopropylethylamine, and 4-(chloromethyl)benzoyl chloride were stirred and reacted at room temperature for 16 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 11; c. Preparation of compound 12: Compound 11 was stirred in HCl/ethyl acetate for 2 h at room temperature to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 12; d. Preparation of Compound 14: Compound 12, Compound 13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, diisopropylethylamine in dichloromethane, The reaction was stirred at room temperature for 16 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 14; e. Preparation of compound BDB: Compound 7, compound 14, and potassium carbonate were reacted in N,N-dimethylformamide with stirring at 60° C. for 5 h to obtain a reaction solution; the reaction solution was separated to obtain compound BDB. 6. The preparation method according to claim 5, characterized in that: In step a, the molar ratio of ethyl chloroformate, compound 8, and diisopropylethylamine is 46.5:44.5:264; the molar volume ratio of ethyl chloroformate to tetrahydrofuran is 46.5:350mmol/ml; In step b, the molar ratio of the compound 9, diisopropylethylamine, and 4-(chloromethyl)benzoyl chloride is 0.67:0.8:0.8; the molar volume ratio of the compound 9 to dichloromethane is 0.67 : 50mmol/ml; In step c, the molar volume ratio of the compound 11 to HCl/ethyl acetate is 0.445:20mmol/ml; the concentration of the HCl/ethyl acetate is 2M; In step d, the compound 12, compound 13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, diisopropylethylamine The molar ratio is 0.445:0.445:0.534:0.534:0.534; the molar volume ratio of the compound 12 to dichloromethane is 0.445:25mmol/ml; In step e, the molar ratio of compound 7, compound 14, and potassium carbonate is 0.4:1:4.8; the molar volume ratio of compound 7 to N,N-dimethylformamide is 0.4:50 mmol/ml. 7. The preparation method according to claim 5, characterized in that: in step e, the compound 7 is prepared according to the following steps: 8. The preparation method according to claim 7, characterized in that: in step e, the compound 7 is prepared according to the following steps: i. Preparation of compound 2: Compound 1 and BOC anhydride reacted completely in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 2; ii. Preparation of compound 3: Compound 2, triethylamine, and glutaryl chloride were stirred and reacted at room temperature for 3 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 3; iii. Preparation of Compound 4 Compound 3 was stirred and reacted at room temperature for 3 h in HCl/ethyl acetate to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 4; iv. Preparation of Compound 5 Compound 4, diisopropylethylamine, and 3-chloropropionyl chloride were stirred and reacted at room temperature for 3 hours in dichloromethane to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 5; v, preparation of compound 6 Compound 5, tert-butyl piperazine-1-carboxylate, and diisopropylethylamine were refluxed in dioxane for 18 hours to obtain a reaction solution; the reaction solution was separated and purified to obtain compound 6; vi. Preparation of Compound 7: Compound 6 was stirred in HCl/ethyl acetate for 2 h at room temperature to obtain a reaction solution; the solvent was removed from the reaction solution to obtain Compound 7. 9. The preparation method according to claim 7, characterized in that: In step i, the molar ratio of compound 1 to BOC anhydride is 45.5:43.18; the molar volume ratio of compound 1 to dichloromethane is 45.5:600mmol/ml; In step ii, the molar ratio of the compound 2, triethylamine and glutaryl chloride is 8.42:14.85:4.17; the molar volume ratio of the compound 2 and dichloromethane is 8.42:120mmol/ml; In step iii, the molar volume ratio of compound 3 to HCl/ethyl acetate is 3.8:100mmol/ml; the concentration of HCl/ethyl acetate is 2M; In step iv, the molar ratio of compound 4, diisopropylethylamine, and 3-chloropropionyl chloride is 0.9:6.15:1.81; the molar volume ratio of compound 4 to dichloromethane is 0.9:50mmol/ml; In step v, the molar ratio of compound 5, tert-butyl piperazine-1-carboxylate, and diisopropylethylamine is 0.14:2.68:0.77; the molar volume ratio of compound 5 to dioxane is 0.14 : 20mmol/ml; In step vi, the molar volume ratio of compound 6 to HCl/ethyl acetate is 0.1:20mmol/ml; the concentration of HCl/ethyl acetate is 2M. 10. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cell proliferation diseases. 11. The use according to claim 10, characterized in that the drug is a tyrosine kinase inhibitor. 12. The use according to claim 10 or 11, characterized in that: the drug is an ABL inhibitor drug. 13. The use according to claim 10 or 11, characterized in that: the drug is BCR-ABL or its mutant strain inhibitor drugs. 14. The use according to claim 13, characterized in that: the BCR-ABL mutant is a T315I mutant. 15. The use according to any one of claims 10-14, characterized in that the cell proliferative disease is cancer. 16. The use according to claim 14, characterized in that: the cancer is chronic myeloid leukemia, intestinal stromal tumor, gynecological tumor, dermatofibrosarcoma protuberans, Ph+ALL.