CN109824584B - Tertiary leucine-containing peptoid compound and preparation method and application thereof - Google Patents

Abstract

A peptide-like compound containing tert-leucine, its preparation method and application, utilize acylation, Suzuki coupling, condensation, etc. to synthesize the target compound, and construct the compound library, this kind of compound is Bcr-Abl micromolecule tyrosine kinase inhibitor with new molecular structure, the invention adopts the medicament design strategy based on fragment, regard biphenyl pyridine as hinge region binding fragment, introduce L-tert-leucine as flexible Linker, in order to construct the peptide-like micromolecule compound library with kinase inhibitory activity, and through ADP-Glo's kinase activity screening discover tyrosine kinase inhibitor with Bcr-Abl kinase inhibitory activity. Kinase screening tests show that the compounds have certain inhibitory activity on both Abl kinase and T315I mutant Abl kinase, and cell proliferation tests show that most of the compounds have certain inhibitory activity on K562 cells.

Description

A kind of peptoid compound containing tertiary leucine and its preparation method and application

technical field

The present invention relates to a tertiary leucine-containing peptoid compound and its preparation method and application.

Background technique

Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease that occurs in bone marrow hematopoietic stem cells, accounting for 15% to 20% of adult leukemia patients. It is characterized by the detection of Ph chromosome in CML patients. Ph chromosome is a breakpoint cluster-Alberson (BCR-ABL) fusion gene formed by the mutual translocation of human normal chromosome 22 and chromosome 9. This fusion gene encodes Bcr that produces continuous activation of tyrosine kinase activity -Abl fusion protein. At present, there are small molecule tyrosine kinase inhibitors targeting Bcr-Abl on the market, but they all have problems such as drug resistance and other clinical adverse reactions. Subsequently, research and development of novel Bcr-Abl tyrosine kinase inhibitors has become one of the hot spots in the field of pharmacy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a tertiary leucine-containing peptoid compound and its preparation method and application.

For achieving the above object, the present invention adopts the following technical scheme:

A peptoid compound containing tertiary leucine, the structural formula of the compound is as follows:

where R is

A method for preparing a peptoid compound containing tertiary leucine, comprising the following steps:

1) 5-bromo-2-aminopyridine and acid chloride compound generate acylation reaction to prepare acylated 5-bromo-2-aminopyridine;

₂ ) under N protection, 5-bromonicotinic acid, thionyl chloride and amine compound react to prepare the 5-bromonicotinic acid of ammoniation;

3) under the catalysis of tetrakistriphenylphosphine palladium, acylated 5-bromo-2-aminopyridine or aminated 5-bromonicotinic acid and p/m-carboxyphenylboronic acid generate Suzuki coupling reaction to obtain biphenyl compound;

4) Fmoc-L-tert-leucine is condensed with 4-chloro-3-trifluoromethylaniline to form (9fluoren-9-yl)methyl (S)-(1-((4-chloro-3-( Trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate tert-butyl ester;

5) (9Fluoren-9-yl)methyl(S)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxo tert-Butyl-2-yl)carbamate removes the Fmoc protecting group to give (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethyl Butanamide;

6) Condensation of biphenyl compounds with (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide to form tertiary leucine-containing peptoids class compounds.

A further improvement of the present invention is that the specific process of the step 1) is: dissolving 5-bromo-2-aminopyridine in anhydrous dichloromethane, adding triethylamine, and adding dropwise an acid chloride compound under ice bath conditions After the dropwise addition was completed, the ice bath was removed and the reaction was raised to room temperature for 12 h, followed by post-treatment to obtain acylated 5-bromo-2-aminopyridine.

A further improvement of the present invention is that the specific process of step 2) is as follows: under the protection of N ₂ , thionyl chloride is added dropwise to 5-bromonicotinic acid, and after the dropwise addition, the solution is heated under reflux for 2-3h to reach the solution. Clarify, spin off thionyl chloride under reduced pressure to obtain a pale yellow solid, which is dissolved in anhydrous dichloromethane, and then added dropwise to the dichloromethane solution of the amine compound under ice bath conditions; After the dropwise addition, the reaction was raised to room temperature for 12 h, and after the reaction was completed, post-treatment was performed to obtain ammoniated 5-bromo-nicotinic acid.

A further improvement of the present invention is that the step 3) the specific process is: adding the acylated 5-bromo-2-aminopyridine and p/m-carboxyphenylboronic acid into the reaction vessel, or adding the ammoniated 5-bromo pyridine to the reaction vessel. - Nicotinic acid and p/m-carboxyphenylboronic acid were added to the reaction vessel, then cesium carbonate and tetrakistriphenylphosphine palladium _were added in turn; then a mixed solution of acetonitrile and water was added, protected by N, and the temperature was raised to 90°C for 48h; After completion of the reaction, post-treatment is performed to obtain a biphenyl compound.

A further improvement of the present invention is that the specific process of the step 4) is as follows: 4-chloro-3-trifluoromethylaniline, HOBT and HATU are dissolved in anhydrous dichloromethane, and then DIPEA is added dropwise, and the dropwise addition is completed. After that, the dichloromethane solution of Fmoc-L-tert-leucine was added dropwise, and the reaction was carried out at room temperature for 5 h, followed by post-treatment to obtain (9fluoren-9-yl)methyl (S)-(1-((4-chloro- 3-(Trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate tert-butyl ester.

A further improvement of the present invention is that the specific process of the step 5) is: at 0 °C, the (9fluoren-9-yl)methyl (S)-(1-((4-chloro-3-(trifluoro) Methyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamic acid tert-butyl ester was dissolved in anhydrous dichloromethane, and then 20% volume concentration piperidine was added dropwise After 1 h of reaction, post-treatment was performed to obtain (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide.

A further improvement of the present invention is that the specific process of the step 6) is as follows: dissolving the biphenyl compound and PyBOP in anhydrous DMF, adding DIPEA dropwise, reacting at room temperature for 5 min, adding (S)-2-amino-N( 4-Chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide, react at room temperature for 12 h, and perform post-treatment to obtain a tertiary leucine-containing peptoid compound.

A tertiary leucine-containing peptoid compound is used in the preparation of a drug for inhibiting the activity of Abl kinase and T315I mutant Abl kinase.

A peptidomimetic compound containing tertiary leucine, and the application of the peptidomimetic compound in the preparation of antitumor drugs.

时对T315I突变Abl激酶的活性最佳。细胞增殖试验表明大部分化合物对K562细胞具有一定的抑制活性。构效关系分析发现：引入L-叔亮氨酸的衍生物与Abl激酶的ATP位点的空间匹配良好，作用模式和参照小分子伊马替尼一致，说明L-叔亮氨酸的引入对化合物的抑制活性具有重要作用。在吡啶环上引入酰胺侧链以提高小分子与受体的亲和力，可以作为以Bcr-Abl为靶标的酪氨酸激酶抑制的新型药效片段。Compared with the prior art, the present invention has the beneficial effects: the present invention utilizes acylation, Suzuki coupling, condensation and other reactions to synthesize target compounds, and builds a compound library, which is a Bcr-Abl small compound with a novel molecular structure. Molecular tyrosine kinase inhibitor, and the structure of the target compound was characterized by MS, NMR and other means. The invention adopts a fragment-based drug design strategy, uses biphenylpyridine as the hinge region binding fragment, and introduces L-tert-leucine as a flexible Linker to construct a peptoid small molecule compound library with kinase inhibitory activity, and through ADP- Glo's kinase activity screen identified tyrosine kinase inhibitors with Bcr-Abl kinase inhibitory activity. Kinase screening test showed that these compounds have certain inhibitory activity on Abl kinase and T315I mutant Abl kinase, where R is

The best activity against T315I mutant Abl kinase. Cell proliferation assay showed that most of the compounds had certain inhibitory activity on K562 cells. The structure-activity relationship analysis found that the derivatives introduced into L-tert-leucine had a good spatial match with the ATP site of Abl kinase, and the mode of action was consistent with the reference small molecule imatinib, indicating that the introduction of L-tert-leucine had a significant impact on the The inhibitory activity of the compound plays an important role. The amide side chain was introduced on the pyridine ring to improve the affinity of the small molecule to the receptor, which can be used as a novel pharmacodynamic fragment for tyrosine kinase inhibition targeting Bcr-Abl.

Description of drawings

Fig. 1 is the synthetic route diagram of the present invention.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings.

Referring to Figure 1, the structural formula of a tertiary leucine-containing peptoid compound of the present invention is:

Among them, R is as follows, see Table 1:

Table 1 Groups represented by R

The following description will be given through specific embodiments.

Referring to Fig. 1, the preparation method of the tertiary leucine-containing peptoid compound of the above structure comprises the following steps:

1) 5-bromo-2-aminopyridine is acylated with corresponding acid chloride compound to prepare acylated 5-bromo-2-aminopyridine;

The specific process of the step 1) is as follows: 5-bromo-2-aminopyridine is dissolved in anhydrous dichloromethane, triethylamine is added, and the corresponding acid chloride compound is slowly added dropwise to the above solution under ice bath conditions. After the dropwise addition was completed, the ice bath was removed and the temperature was raised to room temperature for 12 h. After the reaction was completed, dichloromethane was added to dilute, washed with water, washed with saturated sodium bicarbonate, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated by column chromatography to obtain a white solid, which was acylated 5- Bromo-2-aminopyridine.

₂ ) under N protection, 5-bromonicotinic acid reacts with sulfur oxychloride and corresponding amine compound to prepare the 5-bromonicotinic acid of ammoniation;

The specific process of the step ₂ ) is: under the protection of N , add thionyl chloride dropwise to 5-bromonicotinic acid, after the dropwise addition, heat under reflux for 2-3h until the solution is clarified, and spin under reduced pressure to remove chlorine. sulfoxide to obtain a pale yellow solid, the pale yellow solid was dissolved in anhydrous dichloromethane, and the active intermediate solution was slowly added dropwise to the dichloromethane solution of the corresponding amine compound under ice bath conditions After the dropwise addition, the reaction was raised to room temperature for 12h. After the reaction was completed, K ₂ CO ₃ solution was added to the reaction system, and the dichloromethane phase was separated and the aqueous phase was extracted with dichloromethane. The organic phases were combined and anhydrous Na ₂ SO ₄ drying; column chromatography separation and purification to obtain a white solid, that is, ammoniated 5-bromo-nicotinic acid.

3) under the catalysis of tetrakistriphenylphosphine palladium, acylated 5-bromo-2-aminopyridine or aminated 5-bromonicotinic acid and p/m-carboxyphenylboronic acid generate Suzuki coupling reaction to obtain biphenyl compound;

Described step 3) concrete process is: the 5-bromo-2-aminopyridine of acylation and p/m-carboxyphenylboronic acid are added in the pear-shaped bottle, or the 5-bromo-nicotinic acid of ammoniation and p/m m-carboxyphenylboronic acid was added to the pear-shaped bottle, followed by cesium carbonate and tetrakistriphenylphosphine palladium; to the above mixture, a mixed solution of acetonitrile/water was added, protected by N ₂ , and the oil bath was heated to 90 °C for 48 h; the reaction After the completion, the reaction solution was lowered to room temperature and suction filtered. The filtrate was adjusted to pH 4 with hydrochloric acid, a white solid was precipitated, suction filtered, and the filter cake was vacuum-dried to obtain the product, which was a biphenyl compound.

4) Fmoc-L-tert-leucine is condensed with 4-chloro-3-trifluoromethylaniline to form (9fluoren-9-yl)methyl (S)-(1-((4-chloro-3-( Trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate tert-butyl ester;

The specific process of the step 4) is as follows: 4-chloro-3-trifluoromethylaniline, HOBT and HATU are dissolved in anhydrous dichloromethane, and then DIPEA is added dropwise. Fmoc-L-tert-leucine solution in dichloromethane. The reaction was carried out at room temperature for 6 h; after the reaction, water was added to terminate the reaction; extracted with dichloromethane, the organic phases were combined, washed with saturated NaCl water, dried over anhydrous NaSO ₄ , and separated by column chromatography to obtain (9fluoren-9-yl)methyl (S)- (1-((4-Chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate tert-butyl ester.

5) (9Fluoren-9-yl)methyl(S)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxo tert-Butyl-2-yl)carbamate removes the Fmoc protecting group to give (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethyl Butanamide;

The specific process of the step 5) is as follows: (9fluoren-9-yl)methyl (S)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3 will be obtained, 3-Dimethyl-1-oxobutan-2-yl) tert-butyl carbamate was dissolved in anhydrous dichloromethane, at 0 ℃, 20% piperidine in DMF solution (volume concentration) was added dropwise, and then at room temperature The reaction was carried out for 1 h; after the reaction was completed, the solvent was removed by spinning, and column chromatography was performed to obtain (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide .

6) Condensation of biphenyl compounds with (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide to form tertiary leucine-containing peptoids class compounds.

The specific process of the step 6) is as follows: the biphenyl compound and PyBOP are dissolved in anhydrous DMF, the DIPEA solution is added dropwise, and after the reaction at room temperature for 5 min, (S)-2-amino-N(4-chloro-3(tri) is added. Fluoromethyl)phenyl)-3,3-dimethylbutanamide. The reaction was carried out at room temperature for 12 h, and post-treatment was performed to obtain a peptoid compound containing tertiary leucine.

A tertiary leucine-containing peptoid compound prepared by the above method is used for inhibiting the activity of Abl kinase and T315I mutant Abl kinase.

The compounds have anti-tumor effects and can be used in the preparation of anti-tumor drugs.

Example 1

时，制备方法如下：A peptoid compound containing tertiary leucine, R is

, the preparation method is as follows:

1) Synthesis of N-(5-bromopyridin-2-yl)acetamide: 5-bromo-2-aminopyridine (5.19 g, 30 mmol) was dissolved in 100 ml of anhydrous dichloromethane, and 20 ml of triethylamine was added. Under ice bath conditions, acetyl chloride (2.54ml) was slowly added dropwise to the above solution, after the dropwise addition, the ice bath was removed, and the reaction was carried out at room temperature overnight (ie, the reaction was 12h). After the reaction, dichloromethane was added to dilute, washed with water (30ml×3), washed with saturated NaHCO ₃ solution (30ml×3), washed with saturated NaCl (30ml), and the organic phase was dried over anhydrous Na ₂ SO ₄ . Column chromatography gave 5.65 g of a white solid with a yield of 88%. Mp 78-81°C; EI-MS (m/z): 214 [M] ⁺ .

2) Synthesis of 4-(6-(acetylamino)pyridin-3-yl)benzoic acid: N-(5-bromopyridin-2-yl)acetamide (4.30g, 20mmol), p-carboxyphenylboronic acid (3.66g) , 22 mmol) was added to a 250 ml pear-shaped bottle, followed by cesium carbonate (13.0 g, 40 mmol) and tetrakistriphenylphosphine palladium (1.2 g, 1 mmol). To the above mixture was added 200 ml of acetonitrile/water (V:V=3:2). Protected by _N2 , the oil bath was heated to 90 °C and reacted for 48 h. After the reaction, the reaction solution was lowered to room temperature and filtered with suction. The filtrate was adjusted to pH 4 with 6 mol/L hydrochloric acid, a white solid was precipitated, suction filtered, and the filter cake was vacuum-dried to obtain 3.89 g of the product with a yield of 76%. Mp156-158°C; EI-MS (m/z): 256 [M] ⁺ .

3) Dissolve 4-chloro-3-trifluoromethylaniline (0.49g, 2.5mmol), HOBT (0.69g, 4.2mmol), HATU (1.6g, 4.2mmol) in 30ml of anhydrous dichloromethane, Then DIPEA (1.43 ml) was added dropwise, and after the dropwise addition was completed, a solution of Fmoc-L-tert-leucine (1.0 g, 2.8 mmol) dissolved in anhydrous dichloromethane was added dropwise. The reaction was carried out at room temperature for 6 h; after the reaction was completed, 20 ml of water was added to terminate the reaction; (3) was extracted with dichloromethane, the organic phases were combined, washed with saturated NaCl water, dried over anhydrous NaSO ₄ , and separated by column chromatography to obtain (9fluoren-9-yl)methane tert-butyl (S)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate Ester 0.54g. Light yellow oily solid, 36.2% yield. EI-MS (m/z): 529.0 [M] ⁺ .

4) (9fluoren-9-yl)methyl(S)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1 will be obtained -Oxobutan-2-yl) tert-butyl carbamate (0.54g, 1.0mmol) was dissolved in anhydrous dichloromethane (20ml), at 0°C, 1ml of 20% piperidine in DMF solution (volume concentration) was added dropwise ), and then reacted at room temperature for 1 h; after the reaction was over, the solvent was removed, and column chromatography was performed to obtain (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-di Methylbutanamide 0.34g pale yellow oily solid. Yield 94.6%. EI-MS (m/z): 337[M] ⁺ .

5) Dissolve 4-(6-(acetylamino)pyridin-3-yl)benzoic acid (0.1g, 3.9mmol) and PyBOP (0.17g, 3.3mmol) in anhydrous DMF (10ml), add DIPEA ( 0.168ml) solution, after 5min reaction at room temperature, (S)-2-amino-N(4-chloro-3(trifluoromethyl)phenyl)-3,3-dimethylbutanamide (0.1g, 3.3 mmol). The reaction was carried out at room temperature for 12 h, 100 ml of water was added to terminate the reaction, extracted with ethyl acetate (3×30 ml), the organic phases were combined, washed with saturated NaCl water, dried over anhydrous NaSO ₄ , and separated by column chromatography to obtain (S)-4-(6-acetamidopyridine) -3-yl)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)benzene formamide (B7). White powdery solid, 75% yield. EI-MS (m/z): 545.165 [M] ⁺ ; 547.165 [M] ⁻ . MP: 57.6～59.9℃; ¹ H NMR (400MHz, DMSO)δ10.68(s,1H),8.67(s,2H),8.27(d,J=1.7Hz,1H),8.21(d,J=8.7 Hz, 1H), 7.99(dd, J＝27.1, 8.2Hz, 2H), 7.89(d, J＝8.7Hz, 1H), 7.74(d, J＝8.2Hz, 2H), 7.69(d, J＝8.8 Hz, 1H), 6.92(s, 2H), 4.65(t, J=6.8Hz, 1H), 2.89(s, 1H), 2.71(d, J=17.8Hz, 1H), 1.08(s, 9H), 0.87(t,J=9.2Hz,1H).

Example 2

时，制备方法如下：A peptoid compound containing tertiary leucine, R is

, the preparation method is as follows:

1) Under the protection of N ₂ , thionyl chloride (36 ml, 494 mmol) was added dropwise to 5-bromonicotinic acid (5.00 g, 24.7 mmol). Swirl to remove thionyl chloride to obtain pale yellow solid. The solid was dissolved in 30 ml of anhydrous dichloromethane, and the active intermediate solution was slowly added dropwise to a solution of cyclopropylamine (3.77 ml) in dichloromethane (30 ml) under ice bath conditions. After the dropwise addition, it was warmed to room temperature and reacted overnight. After the reaction, 20ml of 2mol/LK ₂ CO ₃ solution was added to the reaction system. The dichloromethane phase was separated and the aqueous phase was extracted with dichloromethane (15 ml×3). The organic phases were combined and dried over anhydrous Na ₂ SO ₄ . Column chromatography separation and purification. (Petroleum ether:ethyl acetate=1:1) to obtain 5.27 g of white solid with a yield of 89%. Mp 140-142°C; EI-MS (m/z): 240 [M] ⁺ .

Steps 2) to 6) are the same as in Example 1 to obtain compound B3, white solid, yield 28%; EI-MS (m/z): 571[M] ⁺ ; 573[M] ^- . MP: 149～152.5℃; ¹ H NMR (400MHz, DMSO) δ 10.71(s, 1H), 9.09(d, J=1.9Hz, 1H), 8.99(d, J=1.7Hz, 1H), 8.79( d, J＝3.9Hz, 1H), 8.47(s, 1H), 8.31(dd, J＝27.4, 5.5Hz, 2H), 8.05(d, J＝8.3Hz, 2H), 7.96–7.88(m, 3H ),7.69(d,J＝8.8Hz,1H),4.66(d,J＝8.7Hz,1H),2.90(td,J＝7.3,3.8Hz,1H),1.09(s,9H),0.77–0.71 (m,2H),0.64–0.60(m,2H).

The synthesis steps of B1, B2 and B4 are the same as those of B3.

Compound B1, pale yellow solid, yield 54%. EI-MS (m/z): 587 [M] ⁺ ; 589 [M] ⁻ . MP: 97.6～99.7℃; ¹ H NMR (400MHz, DMSO) δ 10.70(s, 1H), 9.05(s, 1H), 8.62(s, 1H), 8.31(dd, J=30.2, 5.6Hz, 2H ),8.16(s,1H),8.03(d,J＝8.4Hz,2H),7.90(dd,J＝11.8,5.4Hz,3H),7.69(d,J＝8.8Hz,1H),4.66(d ,J=8.8Hz,1H),3.49(d,J=6.9Hz,2H),3.24(d,J=6.8Hz,2H),2.55–2.48(m,2H),1.20(dd,J=13.3, 6.4Hz, 4H), 1.09(s, 9H).

Compound B2, white solid, 50% yield, EI-MS (m/z): 587 [M] ⁺ ; 589 [M] ⁻ . MP: 93.4～94.8℃; ¹ H NMR (400MHz, DMSO) δ 10.74(s, 1H), 9.08(d, J=2.2Hz, 1H), 8.60(d, J=1.9Hz, 1H), 8.56( d,J＝8.9Hz,1H),8.28(d,J＝2.5Hz,1H),8.21(t,J＝2.0Hz,2H),7.92(ddd,J＝12.5,11.6,5.0Hz,3H), 7.69(d,J＝8.8Hz,1H),7.62(t,J＝7.8Hz,1H),4.71(d,J＝8.9Hz,1H),3.49(d,J＝6.8Hz,2H),3.32– 3.18(m, 2H), 1.17(dd, J=21.9, 15.1Hz, 6H), 1.09(s, 9H).

Compound B4, white solid, 54% yield, EI-MS (m/z): 571 [M] ⁺ ; 573 [M] ⁻ . MP: 138.2～140.6℃; ¹ H NMR (400MHz, DMSO) δ 10.74(s, 1H), 9.14(d, J=1.9Hz, 1H), 8.99(d, J=1.7Hz, 1H), 8.78( d, J=4.0Hz, 1H), 8.51 (dd, J=11.2, 5.4Hz, 2H), 8.30–8.25 (m, 2H), 7.96 (dd, J=7.7, 6.1Hz, 2H), 7.90 (dd , J=8.8, 2.2Hz, 1H), 7.71–7.61 (m, 2H), 4.71 (d, J=8.8Hz, 1H), 2.73 (s, 1H), 1.09 (s, 9H), 0.80–0.71 ( m,2H),0.66–0.58(m,2H).

B5, B6, B8～B10 have the same synthesis steps as B7

Compound B5, white solid, 58% yield, EI-MS (m/z): 581 [M] ⁺ ; 583 [M] ⁻ . MP: 92.5～95.1°C; ¹ H NMR (400MHz, DMSO) δ 10.69 (s, 1H), 8.27 (dd, J=5.5, 3.1Hz, 2H), 8.14 (dd, J=8.7, 2.5Hz, 1H) ), 8.01(s, 1H), 7.99(s, 1H), 7.96(s, 1H), 7.90(dd, J=8.8, 2.3Hz, 1H), 7.81(s, 1H), 7.79(s, 1H) ,7.69(d,J＝8.8Hz,1H),7.10(d,J＝8.7Hz,1H),4.64(d,J＝8.8Hz,1H),3.01(td,J＝6.6,3.9Hz,1H) ,2.89(s,3H),1.08(d,J＝4.6Hz,9H).

Compound B6, white solid, 58% yield, EI-MS (m/z): 581 [M] ⁺ ; 583 [M] ⁻ . MP: 67.3～69.4°C; ¹ H NMR (400MHz, DMSO) δ 8.69 (d, J=2.3Hz, 1H), 8.39 (d, J=9.2Hz, 1H), 8.16 (dd, J=8.5, 2.4 Hz, 2H), 7.85(dd, J＝10.0, 8.7Hz, 2H), 7.57(dd, J＝10.2, 5.3Hz, 1H), 7.10(d, J＝8.6Hz, 1H), 5.05(d, J = 9.2Hz, 1H), 2.89(s, 1H), 2.73(s, 1H), 1.60–1.56(m, 2H), 1.40(s, 1H), 1.04(s, 9H), 0.95(d, J= 2.8Hz, 1H), 0.85 (ddd, J=6.6, 5.7, 2.4Hz, 2H).

Compound B8, white solid, 65% yield, EI-MS (m/z): 545 [M] ⁺ ; 547 [M] ⁻ . MP: 130.3～131.6℃; ¹ H NMR(400MHz, DMSO)δ10.73(s,1H),10.65(s,1H),8.74(s,1H),8.48(d,J=8.8Hz,1H), 8.28(d,J＝2.3Hz,1H),8.18(d,J＝12.7Hz,3H),7.89(dd,J＝12.3,5.1Hz,3H),7.69(d,J＝8.8Hz,1H), 7.58(t, J=7.8Hz, 1H), 4.70(d, J=8.8Hz, 1H), 2.13(s, 3H), 1.09(s, 9H).

Compound B9, white solid, 65% yield, EI-MS (m/z): 587 [M] ⁺ ; 589 [M] ⁻ . MP: 57.6～59.9℃; ¹ H NMR(400MHz, DMSO)δ10.73(s,1H),10.04-9.96(m,1H),8.78-8.69(m,1H),8.28(dd,J=5.6, 3.1Hz, 2H), 8.18 (d, J=1.4Hz, 2H), 8.01 (d, J=8.4Hz, 2H), 7.90 (dd, J=8.8, 2.4Hz, 1H), 7.84 (d, J= 8.4Hz, 2H), 7.69(d, J=8.8Hz, 1H), 4.66(d, J=8.8Hz, 1H), 1.26(s, 9H), 1.08(s, 9H).

Compound B10, white solid, 90% yield, EI-MS (m/z): 587 [M] ⁺ ; 589 [M] ⁻ . MP: 76.2～78.7℃; ¹ H NMR(400MHz, DMSO)δ10.73(s,1H),9.96(s,1H),8.76-8.74(m,1H),8.48(d,J=8.8Hz,1H) ),8.28(d,J＝2.5Hz,1H),8.20(d,J＝3.2Hz,2H),8.16(s,1H),7.96(s,1H),7.92–7.87(m,3H),7.68 (d, J=8.8Hz, 1H), 7.58 (t, J=7.8Hz, 1H), 4.70 (d, J=8.8Hz, 1H), 1.26 (s, 9H), 1.08 (d, J=7.0Hz) ,9H).

Next, the Bcr-Abl kinase inhibitory activity was screened for the derivatives of tertiary leucine with anti-tumor activity prepared in the present invention.

The measurement method is as follows:

Kinase ABL1, ABL(T315I) and substrate Abltide were purchased from Signal-Chem Company, and the ADP-Glo ^™ Kinase Assays detection kit of Promega Company was used to detect the enzymatic inhibitory activity of the target compound. The operation method was carried out according to the kit instructions.

In the Abl experiment, ATP (1 mM) was diluted 80 times with buffer (2×) (Tris 80 mM, MgCl ₂ 20 mM, BSA 0.2 mg/ml, DTT 2 mM) to prepare a buffer (2×) solution of ATP (125 μM). ; Mix 125 μM ATP solution and Abltide solution volume 1:1 to prepare a mixed solution of ATP (62.5 μM)-Abltide (0.5 μg/μl) for later use; ABL1 kinase solution was prepared with buffer (1×) (Tris 40 mM, MgCl ₂ 10 mM, BSA 0.1mg/ml, DTT 1mM) diluted 100 times to prepare ABL1 (1ng/μl) buffer (1×) solution for use.

The preparation steps of ATP-Abltide and ABL1(T315I) in the Abl(T315I) experiment are the same as above, except that the concentration of ATP is 12.5 μM, and the concentration of ABL1(T315I) is 2ng/μl

The target compound and positive control drug (Imatinib) were prepared into 6×10 ^-6 mol/L sample solutions with buffer (1×) respectively, and 2 μl of ATP-Abltide mixed solution and 1 μl of sample solution were added to each well of the 384-well plate in turn. , 2 μl enzyme solution; blank wells add 3 μl buffer and 2 μl ATP-Abltide mixed solution; control wells add 2 μl ATP-Abltide mixed solution, 1 μl buffer, 2 μl enzyme solution, after adding, incubate at 30°C for 60 min; add ADP-Glo Reagent 5μl, incubate at 25°C for 40min; add Kinase detection reagent, then incubate at 25°C for 30min. The chemiluminescence module of PerkinElmer multi-function microplate reader was used to measure the luminescence value of each well, and the inhibition rate and IC ₅₀ of the compound to Abl were calculated.

The structural formula of a tertiary leucine-containing peptoid compound of the present invention is:

where R is

The kinase inhibitory activities of the tertiary leucine-containing peptoid compounds of the above structural formula are shown in Table 2

Table 2 Inhibition rate of Bcr-Abl/Bcr-Abl ^T315I by peptoid compounds containing tertiary leucine (%)

It can be seen from Table 2 that most of the compounds have inhibitory activity on Bcr-Abl kinase, and most of the compounds have certain inhibitory activity on Bcr-Abl ^T315 kinase, and the inhibition rate is in the range of 53.4% to 98%. The inhibition rate of some compounds (B1, B3) on T315I mutant Abl kinase reached more than 90%, and the activity was good. The activity results showed that the difference of the substituents would directly affect the inhibitory activity of the compound on the kinase.

Next, the growth inhibitory activity of tertiary leucine-containing peptoids on tumor cells was determined. MTT assay was used to test the growth inhibitory activity of tertiary leucine-containing peptoids on tumor cells.

The tertiary leucine-containing peptoid compound provided by the present invention has an anti-tumor effect. It has the effect of inhibiting the proliferation of tumor cells in vitro, and has the effect of inhibiting the proliferation of tumor cells in human leukemia cells (K562 cells), and can be used for the treatment of leukemia.

Take human leukemia cells (K562 cells) in logarithmic growth phase, dilute them with RPMI1640 medium to a cell solution of the order of 10 ⁴ cells/ml, and inoculate them in parallel in 96-well culture plates (2000-4000 cells/well), inoculated in each well The volume is 180 μl and cultured in a 37°C, 5% _CO2 incubator for 12h;

Add 20 μl of different concentrations of the compounds to be tested to each well, so that the final concentration of the compounds in the wells is 1.5×10 ^-5 mol/L, 3 duplicate wells are set for each concentration, and 6 negative control cells are added without the compounds. Duplicate wells, nilotinib or imatinib as positive controls, continue to culture for 48h;

Add 20 μl of MTT (5 mg/ml) to each well to make the final concentration of MTT in the wells 0.5 mg/ml, incubate at 37°C for 4 hours, carefully aspirate the supernatant, add 150 μl of DMSO to each well, shake for 15 minutes, use an enzyme-linked immunosorbent assay (ELISA) Measure the ultraviolet absorption value (OD value) at 490 nm of each well, then calculate the cell inhibition rate, and calculate the IC ₅₀ value of the compound according to the inhibition rate by linear regression method;

The formula for calculating the cell inhibition rate is:

Inhibition rate%=(average OD value of control wells-average OD value of drug group)/average OD value of control wells×100%;

The test results showed that compared with the negative control group, the alanine-containing peptoid compounds had different degrees of in vitro inhibitory effects on the above tumor cells, as shown in Table 3.

K562 cell proliferation activity:

Table 3 Inhibitory rate (%) of peptoid compounds containing tertiary leucine on K562 cells

As can be seen from Table 3, the cell activity screening test showed that the compound had a certain cell proliferation inhibitory activity on K562 cells. Among them, the compound with better activity was B5, and the inhibition rate reached 36.3%. For tertiary leucine-containing peptoid compounds, the introduction of different substituents on the pyridine ring has a great difference in the impact on the biological activity, and the different positions of the substituents have different effects on the biological activity. Compounds B1 and B3 had better inhibitory activity on Bcr-Abl ^T315I kinase; compound B5 had better inhibitory activity on K562 cells, which deserves further research.

The invention is based on the research on the previous Bcr-Abl tyrosine kinase inhibitor and the interaction analysis between Bcr-Abl protein and ligands, etc., adopts a fragment-based drug design strategy, takes biphenylpyridine as the hinge region binding fragment, and introduces L - Tert-Leucine is a flexible Linker to construct a library of peptoid compounds with kinase inhibitory activity, and a tyrosine kinase inhibitor with Bcr-Abl kinase inhibitory activity was found through the kinase activity screening of ADP-Glo. The compound can be used in the preparation of anti-tumor (chronic myeloid leukemia) drugs, has the activity of inhibiting Bcr-Abl and Bcr-Abl ^T315I kinase, and has the activity of inhibiting cell proliferation on K562 cells. The introduction of tertiary leucine structure can expand the structural diversity of Bcr-Abl kinase inhibitors. At the same time, the activity test shows that tertiary leucine Linker plays an important role in the inhibitory activity of the compound, and can improve the affinity between the receptor and the compound. Pharmacodynamic fragment as Bcr-Abl tyrosine kinase inhibitor.

Claims (3)

1. The tert-leucine-containing peptoid compound is characterized in that the structural formula of the compound is as follows:

2. use of the tertiary leucine containing peptoid compound of claim 1 in the preparation of a medicament for inhibiting the activity of Abl kinase, T315I mutant Abl kinase.

3. Use of the tert-leucine containing peptoid according to claim 1 for the preparation of an anti-tumor medicament, wherein the tumor is leukemia.

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