CN103880707B - STAT3 micromolecular inhibitor and application thereof - Google Patents

Abstract

The invention discloses a STAT3 small molecule inhibitor and its application, and provides a Nˊ-(1-(2,4 dihydroxyphenyl)ethylidene)benzene represented by formula (1) or formula (1a) Formichydrazide derivatives or pharmacologically acceptable salts thereof are STAT3 inhibitors as active ingredients. In the formula, R ¹ and R ² on ring A can be hydroxyl, methoxy, carboxyl and aliphatic, and B can be cyclic or chain. The small molecule inhibitor of STAT3 provided by the present invention has selectivity and high activity. According to the tertiary structure design of STAT3, it is biosynthesized and optimized, and then the activity is detected by methods such as fluorescence polarization and CCK-8, and the inhibitory effect of this type of inhibitor is studied. The mechanism of tumor cell growth, promotion of differentiation and apoptosis, so as to clarify the role of STAT3 small molecule inhibitors in preventing tumorigenesis and treating tumors.

Description

STAT3 small molecule inhibitor and its application

technical field

The invention relates to the field of tumor drugs, in particular to an active small molecule inhibitor designed according to the tertiary structure of signal transduction and transcription factor 3 (STAT3).

Background technique

Signal transducer and transcription factor 3 (STAT3) belongs to the STATs family. There are seven members of this family, STAT1-STAT7, which are encoded by different genes, among which STAT1, STAT3, and STAT5 are highly homologous. The STATs family is stimulated by cell growth factors such as EGFR, IL-6, PDGF, etc. to undergo tyrosine phosphorylation to mediate physiological functions such as cell growth, differentiation, migration, and apoptosis. Studies have shown that excess phosphorylated STAT3 can be detected in prostate cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, and myeloma, while the level of phosphorylated STAT3 in normal epithelial cells is significantly low.

STAT3 consists of four domains: an auxiliary DNA binding domain, a DNA-specific recognition domain, a dimerization domain (SH2), and a transcriptional activity domain, in which there is a tyrosine phosphorylation site in the dimerization domain, which The site plays a key role in STAT3 dimerization. When extracellular growth factors or cytokines (EGF, IL-6, TNF) stimulate the corresponding receptors on the membrane to dimerize, activate JAK, and recruit STAT3 monomers to phosphorylate tyrosine 705, phosphorylation CheSTAT3 monomers form dimers through mutual recognition of each other's SH2 domains and cross the nuclear membrane into the nucleus to bind to the corresponding DNA initiation elements, and then initiate the downstream key proteins (Bcl)-Xl, (Mcl-1) that regulate signal transduction , the transcriptional expression of cyclinD1/D2andc-Myc, etc., thereby regulating cell growth, proliferation, differentiation, and apoptosis. However, STAT3 dimerization plays a nodal role in the JAK/STAT3 signaling pathway, and STAT3 has been used as an anti-tumor drug target in 1997, so inhibiting STAT3 dimerization can be used to promote the apoptosis of tumor cells containing excessively activated STAT3 potential approach as a strategy for the treatment of tumors containing excessively activated STAT3.

At present, the reported inhibitors against STAT3 can be divided into the following categories: phosphorylated peptides, phosphorylated peptide analogs (pTyr-Xxx-Xxx-Gln), non-phosphorylated small molecules (Stattic, STA-21, S31-201, BP-1-102), oligonucleotides (5'-CATTTCCCGTAAATC-3'and3'-GTAAAGGGCATTTAC-3'), kinase inhibitors. However, peptide inhibitors have poor cell permeability, easy metabolism, and poor bioavailability. Kinase inhibitors cannot completely inhibit the activity of the downstream STAT3 pathway, while non-peptide small molecule inhibitors have limited inhibitory activity and there is no known STAT3 inhibitor. Therefore, the development of new selective and highly active STAT3 inhibitors is a huge challenge on the one hand and a major opportunity to overcome tumors on the other hand.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a new small molecule inhibitor of STAT3 with selectivity and high activity.

The second technical problem to be solved by the present invention is to provide the application of STAT3 small molecule inhibitors in the preparation of tumor drugs.

In order to solve the above-mentioned first technical problem, the present invention provides a small molecule inhibitor of STAT3, which contains N'-(1-(2,4 dihydroxyphenyl)ethylene oxide represented by formula (1) base) benzoyl hydrazide derivatives or pharmacologically acceptable salts thereof;

In formula (1), R ¹ and R ² on ring A are the same or different, representing a hydrogen atom, a substituted or unsubstituted hydroxyl group, a substituted or unsubstituted methoxy group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted carbomethoxy group, Substituted or unsubstituted ethyl carboxylate;

In formula (1), R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ on ring B are the same or different, and represent hydrogen atom, halogen, said halogen refers to fluorine, chlorine, bromine and iodine, substituted or unsubstituted alkanes substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted ester cyclic heterocyclic group, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic heterocyclic Cyclic, substituted or unsubstituted aromatic heterocycloalkyl, or hydroxyl, nitro, amino, sulfonamide, mercapto, methoxy, ethoxy, benzyloxy, methyl, cyano; in ring B, V , W, X, Y, Z are mono-substituted or multi-substituted carbon atoms or nitrogen atoms; B ring itself is a benzene ring, pyridine ring, furan ring, thiophene ring, pyrrole ring, pyrazole ring, imidazole, oxazole, iso Oxazole, indole, triazole, tetrazole, piperidine ring, naphthalene ring or anthracycline;

A hydrazide is used in the middle of ring A and ring B to form substituted or unsubstituted derivatives.

As a preferred version, in formula (1), the B ring part is a furan ring, and its substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, Substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine.

As a preferred embodiment, in formula (1), ring B is a thiophene ring, and its substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, Substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine.

As a preferred version, in formula (1), ring B is a benzene ring, and its substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, Substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine.

As a preferred version, in formula (1), the B ring part is a pyridine ring, and its substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, Substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine.

As a preferred embodiment, in formula (1), the B ring part is a heterocyclic ring, and the heterocyclic group is pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazole Azolyl, piperidinyl, pyranyl, pyrazinyl, pyrimidinyl, isothiazolyl, triazinyl, and substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxy, Substituted or unsubstituted cyano, substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine ,iodine.

As a preferred embodiment, in formula (1), the B ring part is a ring and ring, and the ring and ring group includes naphthyl, quinolinyl, isoquinolyl, indolyl, purinyl, pteridyl, quinolinyl, Azolinyl, benzothienyl, benzofuryl, benzoxazolyl, benzopyrazinyl, benzopyrimidinyl, pyridopyrimidinyl, pyrimidopyrimidinyl, thianthryl, benzindazolyl , benzotriazolyl, and substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, substituted or unsubstituted methoxy, substituted Or unsubstituted benzyloxy, substituted or unsubstituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine.

The present invention also provides a small molecule inhibitor of STAT3, which contains N'-(1-(2,4 dihydroxyphenyl)ethylidene)benzohydrazide derivative represented by formula (1a) or a pharmacologically acceptable salt thereof;

In formula (1a), R ¹ and R ² on ring A are the same or different, representing a hydrogen atom, a substituted or unsubstituted hydroxyl group, a substituted or unsubstituted methoxy group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted carbomethoxy group, Substituted or unsubstituted ethyl carboxylate;

In the formula (1a), R ⁸ of the B part represents an aliphatic chain structure, and the aliphatic chain structure refers to ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl , cyclobutyl, n-pentyl, isopentyl, cyclopentyl, n-hexyl, cyclohexyl, n-heptyl, cycloheptyl, allyl and their derivatives, or substituted or unsubstituted cinnamic acids, including substituted or unsubstituted nitro, substituted or unsubstituted amino, substituted or unsubstituted hydroxyl, substituted or unsubstituted cyano, substituted or unsubstituted methoxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted Substituted methyl, substituted or unsubstituted halogen, said halogen refers to fluorine, chlorine, bromine, iodine;

A hydrazide is used between ring A and part B to form substituted or unsubstituted derivatives.

As a preferred solution, R ¹ and R ² on ring A in formula (1) and formula (1a) are the same or different, and represent hydroxyl or carboxyl.

In order to solve the second technical problem above, the present invention provides the application of STAT3 small molecule inhibitors in the preparation of drugs for treating cancer.

The advantage of the present invention is that the present invention provides a new selective and highly active small molecule inhibitor of STAT3, and its active small molecule inhibitor is designed according to the tertiary structure of STAT3, biosynthesized and optimized, and then passed Fluorescence polarization and CCK-8 and other methods are used to detect the activity and study the mechanism of such inhibitors in inhibiting tumor cell growth and promoting differentiation and apoptosis, so as to clarify the role of small molecule inhibitors of STAT3 in preventing tumorigenesis and treating tumors.

Description of drawings

Figure 1 shows the fluorescence polarization values of small molecule compounds. The IC ₅₀ value of each small molecule compound was obtained by robustfit analysis. The IC ₅₀ of 1-177 was 8.972uM, and the IC ₅₀ of 1-078 was 16.05uM.

Figure 2 shows the growth inhibition of small molecule compounds on cancer cell DU145. The IC ₅₀ of 1-078 in the in vitro competition experiment was 16.05uM, but its water solubility and membrane permeability were slightly weaker than those of 1-177. 1-177 and 1- The IC ₅₀ of 078 is 25.42uM and 222.5uM, respectively.

Figure 3 shows the inhibitory effect of 1-177 on phosphorylated STAT3 in MDA-MB-468 cells detected by Western Blot.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer .

Embodiment 1. Preparation of compound general formula (2a)

General formula (2a) is a specific compound type of general formula (1) and general formula (1a). Since part B can be a ring or chain structure, it is represented by R in general formula (2a).

Substituted or unsubstituted benzene rings, heterocycles, cinnamic acids or aliphatic acids are refluxed in anhydrous ethanol-concentrated sulfuric acid (20:1) solution for 9 hours, spin to dry 2/3 of the solvent, and add an appropriate amount of crushed ice to the residue , then adjust the pH to neutral with NaHCO ₃ solution, extract the product with ethyl acetate, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, and spin the solvent to obtain a colorless liquid, which is product 3. Dissolve the ester obtained above in absolute ethanol, add an equivalent amount of hydrazine hydrate, and reflux for 24 hours. Spin off part of the solvent, then add an appropriate amount of crushed ice, and a solid precipitates out, filter, wash with water, dry, and recrystallize with 90% ethanol to obtain product 4. The method for preparing 2 includes the use of methanol or ethanol, or the reaction with sodium azide, or the reaction of methyl iodide with a base, such as sodium hydroxide, potassium carbonate, potassium tert-butoxide and other inorganic bases.

Product 3 and 2,4 dihydroxy-acetophenone were dissolved in absolute ethanol at a ratio of 1:1, then one equivalent of acetic acid was added, refluxed for 4 hours, the reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethanol and dried , the resulting solid is product 5. The acid used in the reaction may be acetic acid or hydrochloric acid.

In the above production method, when the defined group changes under the conditions of the method or is not suitable for the method, it can be introduced and removed by using the protective group commonly used in organic chemistry ("Protective Groups in Organic Synthesis" East China University of Science and Technology Press) and so on to obtain the target compound. In addition, the conversion of the functional group contained in each substitution can be performed by a known method other than the above-mentioned production method, and in the compound general formula (2a), it may be poured into other derivatives as a synthetic intermediate.

The intermediates and target compounds of the above-mentioned production methods can be separated and purified by purification methods commonly used in organic synthetic chemistry, such as neutralization, filtration, extraction, washing, drying, concentration, recrystallization, and various chromatography. In addition, intermediates can be used in the following reaction without special purification.

When it is desired to obtain the salt of the compound general formula (2a), when the compound (2a) is obtained in the form of a salt, it can be purified directly, or, when it is obtained in a free form later, as long as it is dissolved or suspended in an appropriate organic solvent What is necessary is just to add an acid and form a salt by a usual method.

In addition, the compound of general formula (2a) and its pharmacologically acceptable salts or adducts of various solutions exist, and these adducts can also be used as the STAT3 inhibitor of the present invention.

Preparation scheme one:

Preparation scheme two:

Preparation scheme three:

Preparation scheme four:

Table 1. Specific examples of compounds of general formula (2a) obtained by the above-mentioned production method

Embodiment 2. Preparation of compound general formula (3a)

Similarly, the general formula (3a) is a specific compound type of the general formula (1) and the general formula (1a). Since part B can be a ring or chain structure, it is represented by R in the general formula (3a).

Substituted or unsubstituted benzene rings, heterocycles, cinnamic acids or aliphatic acids are refluxed in anhydrous ethanol-concentrated sulfuric acid (20:1) solution for 9 hours, spin to dry 2/3 of the solvent, and add an appropriate amount of crushed ice to the residue , then adjust the pH to neutral with NaHCO ₃ solution, extract the product with ethyl acetate, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, and spin the solvent to obtain a colorless liquid, which is product 3. Dissolve the ester obtained above in absolute ethanol, add an equivalent amount of hydrazine hydrate, and reflux for 24 hours. Spin off part of the solvent, then add an appropriate amount of crushed ice, and a solid precipitates out, filter, wash with water, dry, and recrystallize with 90% ethanol to obtain product 4. The method for preparing 2 includes the use of methanol or ethanol, or the reaction with sodium azide, or the reaction of methyl iodide with a base, such as sodium hydroxide, potassium carbonate, potassium tert-butoxide and other inorganic bases.

m-Hydroxybenzoic acid and acetic anhydride (1:1.1), concentrated sulfuric acid (1-2 drops), reflux in solvent toluene for 3h. The reaction solution was cooled to room temperature, and a solid precipitated out. The reaction solution was dissolved in ethyl acetate, washed with water, dried over anhydrous NaSO ₄ , and concentrated to dryness. Recrystallization from ethyl acetate gave white crystals. Mix the white solid obtained above with 4 times the amount of anhydrous AlCl ₃ , raise the temperature to 160° C., and react for 3 hours. Pour the reaction solution into ice water, add a certain amount of hydrochloric acid to pickle, extract with ethyl acetate, wash with water until neutral, dry over anhydrous NaSO ₄ , and concentrate to dryness to obtain a dark red syrupy solid. Pass through the column to obtain a dark yellow solid, and then use absolute ethanol or ethyl acetate to recrystallize and purify the product multiple times to obtain compound 8.

Compound 4 and 3-hydroxy-4-acetyl-benzoic acid were dissolved in absolute ethanol at a ratio of 1:1, then one equivalent of acetic acid was added, refluxed for 4 hours, the reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethanol, After drying, the resulting solid is product 9. As the acid used in the reaction, acetic acid or hydrochloric acid may be mentioned.

In the above production method, when the defined group changes under the conditions of the method or is not suitable for the method, it can be introduced and removed by using the protective group commonly used in organic chemistry ("Protective Groups in Organic Synthesis" East China University of Science and Technology Press) and so on to obtain the target compound. In addition, the conversion of the functional group contained in each substitution can be performed by a known method other than the above-mentioned production method, and in the compound general formula (3a), it may be poured into other derivatives as a synthetic intermediate.

The intermediates and target compounds of the above-mentioned production methods can be separated and purified by purification methods commonly used in organic synthetic chemistry, such as neutralization, filtration, extraction, washing, drying, concentration, recrystallization, and various chromatography. In addition, intermediates can be used in the following reaction without special purification.

When it is desired to obtain the salt of the compound general formula (3a), when the compound (3a) is obtained in the form of a salt, it can be purified directly, or, when it is obtained in a free form later, as long as it is dissolved or suspended in an appropriate organic solvent What is necessary is just to add an acid and form a salt by a usual method.

In addition, the compound of general formula (3a) and its pharmacologically acceptable salts or adducts of various solutions exist, and these adducts can also be used as the STAT3 inhibitor of the present invention.

Preparation scheme five:

Preparation scheme six:

Preparation scheme seven:

Preparation scheme eight:

Preparation scheme nine:

The specific example of the compound general formula (3a) that table 2 above-mentioned production method obtains

Example 3. Competitive experiments to detect the activity of STAT3 small molecule inhibitors

In this experiment, the affinity of small molecular compounds to STAT3 was detected by the fluorescence polarization (FP) method, and small molecular compounds were used to compete for the binding of fluorescent peptide segment FITC-pYLPQTV-NH2 to STAT3 protein. Ac-pYLPQTV-NH2 was used as a strong positive control, and Ac-YLPQTV- NH2 weak positive control, Ac-pYLKTKF negative control. When the STAT3 protein and the fluorescent peptide are combined with each other, the polarized light generated by the fluorescent group will increase, and the addition of these peptides as a control will compete for the binding of the fluorescent peptide to the STAT3 protein, reducing the polarization value. The concentration of STAT3 used in the experiment was 5uM, and the concentration of FITC-pYLPQTV-NH2 was 1nM. Experimental grouping: Negative control is STAT3 protein and FITC-pYLPQTV-NH2; positive control is STAT3 protein, FITC-pYLPQTV-NH2, and peptides with different activity levels; sample group is STAT3 protein, FITC-pYLPQTV-NH2, and small molecular compound. Make gradient dilutions of small molecular compounds or positive peptides in the 96-well plate of corningnbs3995#, then add the mixture of STAT3 protein and FITC-pYLPQTV-NH2 into each well, keep away from light, and mix on the shaker for 3 hours until the reaction When the balance is reached, the Biotekreader is used to measure, the excitation light wavelength is 485nm, the emission light wavelength is 525nm, and the fluorescence polarization value is read.

Graphpadprimz5 software was used to process the fluorescence polarization value, and the IC ₅₀ value of each small molecular compound was obtained by robustfit analysis. The IC ₅₀ of 1-177 was 8.972uM, and the IC ₅₀ of 1-078 was 16.05uM, as shown in Figure 1.

Embodiment 4. The effect of this series of compounds on the growth and survival of cancer cells

Through FP experiments, this series of compounds has a high affinity for STAT3 at the molecular level in vitro. Next, it is verified whether this series of compounds has growth inhibitory effect on breast cancer cells overexpressing STAT3. Use Cellcountingkit-8 (CCK-8) to detect the effect of K series compounds on the growth of DU145, plant cells in a 96-well plate, 10000Cells/well, add K series compounds at a preset concentration after 24 hours, and then add 10uLcck-8/well for 24 hours well, 37°C, 4h, use BioTekreader to measure the absorbance at 450nm, the growth inhibition of K series compounds on DU145 is shown in Figure 2, although the IC ₅₀ of 1-078 in the in vitro competition experiment is 16.05uM, but its water solubility and membrane permeability Relatively weaker than 1-177, the IC ₅₀ of 1-177 and 1-078 are 25.42uM and 222.5uM, respectively. Therefore, the following functional and mechanism studies mainly focus on 1-177.

Example 5.WesternBlot detection of the inhibitory effect of 1-177 on phosphorylated STAT3 in MDA-MB-468 cells

MDA-MB-468 cells were treated with different concentrations of 1-177 for 2 hours, stimulated with IL-6 (10ng/ul), collected cells after 24 hours, and then used westernblot method and p-STAT3, STAT3, p -AKT, AKT, P-Src, Src antibodies for detection. As shown in Figure 3, the cells were treated with DMSO and 0uM, 10uM, and 30uM of 1-177, and p-STAT3 and p-AKT were significantly reduced after 10uM treatment, and the p-STAT3 and p-AKT bands were very weak after 30uM treatment. Basically 0. Therefore, K116 has an obvious inhibitory effect on STAT3 phosphorylation, and has no effect on the upstream kinase P-Src of STAT3.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims (1)

1. the application of compound 1-078 in preparation STAT3 micromolecular inhibitor, the structural formula of described 1-078 is as follows: