CN104974222B - Conjugated peptide compounds as protein tyrosine kinase inhibitors - Google Patents

Abstract

The invention relates to a compound of formula (I) as a protein tyrosine kinase inhibitor, which is a coupling peptide compound; the compounds can be used to treat one or more protein tyrosine kinase mediated diseases.

Description

Conjugated peptide compounds as protein tyrosine kinase inhibitors

Technical Field

The invention belongs to the field of chemistry, and particularly relates to an anticancer compound.

Background

Protein Kinases (PKs) are enzymes that catalyze the phosphorylation of specific serine, threonine or tyrosine residues in cellular proteins. Post-translational modification of these substrate proteins serves as a molecular switch that regulates cell proliferation, activation and/or differentiation. Abnormal or excessive PK activity has been observed in a number of disease states including benign and malignant proliferative disorders. In many cases, in vitro treatment of diseases such as proliferative diseases using PK inhibitors has been feasible, and in many cases, in vivo treatment of diseases such as proliferative diseases using PK inhibitors has been feasible. The kinases are largely divided into two groups, those that specifically phosphorylate serine and threonine and those that specifically phosphorylate tyrosine. In addition, some kinases, called "dual specificity" kinases, are capable of phosphorylating tyrosine as well as serine/threonine residues.

WO2006/000420 (pages 1-8 in particular) discloses details regarding PKs, their mode of action and their relationship to the condition or disorder to be treated. The document also discloses heteroaryl aryl ureas useful in the treatment of protein kinase dependent diseases. Furthermore, W003/023004 and W002/102972 disclose disorders arising from FGFR3 mutations. Furthermore, W005/118580 generically discloses quinoline derivatives useful as HIV inhibitors. In these documents and the references cited therein relating to protein kinases, it is reasonable to expect that modulation of aberrant activity, in particular inhibition of the kinase activity, is useful for the diseases mentioned in the literature.

Although the molecules disclosed in the above references are believed to be active, they still have some disadvantages. Thus, there remains an unmet need for improved (e.g., highly affinity and/or selective) molecules that block aberrant constitutive receptor protein tyrosine kinase activity, particularly FGFR activity, and thereby address the clinical manifestations associated with the above mutations and modulate various biological functions. In view of the large number of protein kinase inhibitors, as well as numerous proliferative diseases and other PK related diseases, there is a continuing need to provide novel compounds useful as PK inhibitors and thus for the treatment of said Protein Tyrosine Kinase (PTK) related diseases.

Disclosure of Invention

The invention aims to provide a peptide chain substituted compound with low toxicity and good anticancer effect aiming at the defects of the prior art.

The present invention is based on certain small molecule compounds that interact with protein tyrosine kinases by introducing a peptide (amino acid) chain into the parent backbone to provide peptidyl substituted compounds that interact with protein tyrosine kinases.

The technical purpose of the invention is realized by the following technical scheme:

(formula I)

A peptidyl substituted compound has a structural formula shown in formula I,

r1 is tripeptide or tetrapeptide.

The amino acid sequence of R1 is GTH, GAK, GKR, GKD, GRQ, GAS, GLA, GEKL, GMAW, GSEH, GRKG. Wherein each capital letter represents an amino acid well known in the art.

Still further provided is a method for preparing the above peptidyl substituted compound, comprising the steps of:

(1) reacting [5- [ [ (2, 4, 6-trimethylphenyl) amino group]Carbonyl radical]-4-methyl-2-thiazolyl]Carbamic acid, 1, 1-dimethylethyl ester (10g, 26.63mmol) in trifluoroacetic acid (100mL)The solution was stirred at room temperature for 3 hours. The solution was concentrated under reduced pressure and the residue was diluted with ethyl acetate (700 mL) and with 5% KHCO₃Aqueous solution (400mL, 2 ×), water and brine wash; drying (MgSO)₄) Filtered and concentrated. The residue was washed with diethyl ether (200mL) and acetonitrile (100mL) to give compound S1 as a white solid;

（S1）

(2) performing a alkylation reaction on S1 and chloroacetic acid in an alkaline environment, and performing a chlorination reaction on the obtained product and thionyl chloride to obtain a compound S2 or realize Cl substitution by adopting other methods commonly used in the field;

（S2）

(3) carrying out substitution reaction on S2 and glycine to obtain a compound S3;

（S3）；

(4) and respectively connecting the compound of S3 and different amino acids protected by Fmoc groups to Rink AmideAM resin to perform condensation reaction to obtain the target compound (shown as formula I). The condensation reaction is carried out by a polypeptide solid phase synthesis method, and then trifluoroacetic acid is used for removing resin, so as to finally obtain the peptidyl substituted compound.

Further provides the application of the peptidyl substituted compound in the preparation of anti-cancer drugs.

The dosage form of the anticancer drug is tablets, pills, capsules, injections, suspending agents or emulsions.

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

1. the novel peptidyl substituted compound protein tyrosine kinase inhibits and shows good inhibitory activity to cancer cells, thereby having remarkable inhibitory action to various cancer cell strains;

2. the novel peptidyl substituted compound has low toxicity to normal cells and high safety in the application of preparing anticancer drugs;

3. the novel peptidyl substituted compound can be prepared into various dosage forms of anti-cancer drugs, and has high medical value and wide market prospect.

Detailed Description

EXAMPLE 1 preparation of peptidyl substituted Compounds

(1) Reacting [5- [ [ (2, 4, 6-trimethylphenyl) amino group]Carbonyl radical]-4-methyl-2-thiazolyl]A solution of carbamic acid, 1, 1-dimethylethyl ester (10g, 26.63mmol) in trifluoroacetic acid (100mL) was stirred at room temperature for 3 hours. The solution was concentrated under reduced pressure and the residue was diluted with ethyl acetate (700 mL) and with 5% KHCO₃Aqueous solution (400mL, 2 ×), water and brine wash; drying (MgSO)₄) Filtered and concentrated. The residue was washed with diethyl ether (200mL) and acetonitrile (100mL) to give compound S1 as a white solid;

(2) performing a alkylation reaction on S1 and chloroacetic acid in an alkaline environment, and performing a chlorination reaction on the obtained product and thionyl chloride to obtain a compound S2 or realize Cl substitution by adopting other methods commonly used in the field;

(3) carrying out substitution reaction on S2 and glycine to obtain a compound S3;

(4) the compound of S3 was linked to Rink Amide AM resin with different amino acids (GTH, GAK, GKR, GKD, GRQ, GAS, GLA, GEKL, GMAW, GSEH, GRKG) protected with Fmoc group, respectively, and then subjected to condensation reaction to obtain the objective compound. The condensation reaction is carried out by a polypeptide solid phase synthesis method, and then trifluoroacetic acid is used for removing resin, so as to finally obtain the peptidyl substituted compound. Through tandem liquid quality identification, the compound structure is correct.

Example 2 functional validation of peptidyl substituted Compounds

Human lung adenocarcinoma cell line A549 was cultured in RPMI1640 medium (containing 10% inactivated calf serum, 100U/mL penicillin and 100U/mL streptomycin) and grown in A5% CO2 incubator at 37 ℃.

Cell suspension was prepared at 1X 105/mL by digestion, and 100. mu.L of cell suspension was added to each well in a 96-well plate and cultured for 24 h for adherence. Changing the solution, adding culture solution containing drugs with different concentrations, each well is 100 μ L, each concentration is provided with 4 parallel wells, and the final concentration of the compound is 2.0 mg/L. The culture was continued for 72 h. mu.L of MTT (5 mg/mL) was added to each well and returned to the incubator for further incubation for 4 h. Discard the supernatant, add 150. mu.L DMSO into each well, shake slightly for 15 min to dissolve the crystal completely, adjust the blank control well to zero, select 490 nm wavelength within 20 min, and measure the absorbance value (A) of each well on an automatic fluorescence microplate reader. The experiment was repeated 3 times and the average was taken. Calculating the inhibition rate of cell proliferation: inhibition% = (1-experimental group a value/control group a value) × 100%. The inhibition results were as follows:

class of compounds	Human lung adenocarcinoma cell strain A549 inhibition rate%	Inhibition rate of normal human epidermal cells%
			Compound S1	30%	20.8%
Substituted by the formula I-GTH	90.2%	1.2%
			Substituted by the formula I-GAK	91.3%	2.1%
A substitution of the formula I-GKR	92.5%	1.7%
			Substituted by the formula I-GKD	89.7%	1.5%
Substituted of formula I-GRQ	89.1%	1.1%
			Substitution of formula I-GAS	90.2%	1.8%
Substituted by formula I-GLA	91.5%	2.0%
			Substitution of formula I-GEKL	88.3%	2.3%
Substituted by the formula I-GMAW	92.4%	1.4%
			Substitution of formula I-GSEH	93.5%	1.8%
Substituted by the formula I-GRKG	90.1%	1.7%

The compound has obvious inhibition effect on cancer cells when the concentration is 2 mg/L. The function characteristics and the structure-activity relationship of the compounds can be well illustrated. Therefore, the peptidyl substituted compound can be used for preparing an anticancer drug taking kinase as a target.

Meanwhile, MCF-7 cells are adopted for testing, and basically consistent test results are obtained. Indicating that the compound has broad-spectrum anticancer effect.

Claims (3)

1. A compound is characterized in that the structural formula is shown as a formula I,

and R1 is GTH, GAK, GKR, GKD, GRQ, GAS, GLA, GEKL, GMAW, GSEH or GRKG.

2. The use of a compound according to claim 1 in the preparation of an anti-cancer medicament, wherein said anti-cancer medicament is an anti-lung or breast cancer medicament.

3. The use of the compound of claim 1 in the preparation of an anticancer agent in the form of a tablet, pill, capsule, injection, suspension or emulsion, wherein the anticancer agent is an anti-lung cancer or breast cancer agent.