CN109535101B - Preparation method of salicylic acid derivatives and application of salicylic acid derivatives in tumor treatment - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly provides a preparation method of a novel salicylic acid derivative and application of the novel salicylic acid derivative in tumor treatment. A series of novel salicylic acid derivative compounds are obtained by a chemical synthesis method. The biological activity identification finds that the compounds have obvious antagonism effect on various cell lines such as breast cancer, lung cancer, leukemia and the like, and the anti-tumor mechanism research finds that the compounds have obvious inhibition effect on STAT3 cell signaling, which shows that the compounds have great significance in treating STAT3 signaling abnormality type cancers.

Description

Preparation method of salicylic acid derivatives and application of salicylic acid derivatives in tumor treatment

Technical Field

The invention belongs to the field of biological medicine, and relates to a preparation method and a derivative of a novel salicylic acid compound, pharmaceutically acceptable salts thereof, solvates of the derivative or application of solvates of the salts in tumor treatment.

Background

Malignant tumors are "first-hand killers" threatening human health, and the incidence of global tumors is on a rapid trend. The world health organization predicts that the number of new cases worldwide will increase by 70% for the next 20 years, from 1400 tens of thousands in 2012, up to 1900 tens of thousands in 2025, 2400 tens of thousands in 2035 (Freddie Bray, et al, CA CANCER J CLIN 2018; 0:1-31). Drug therapy has been of great importance for cancer treatment. The global anti-tumor field of drug administration structure has shifted from hormone to targeted therapy for the last 10 years, according to the drug category: the 70-year metal platinum and antibiotic anticancer drugs make the clinical chemotherapy technology a step forward towards radical cure; in the 90 s, plant extracts such as paclitaxel and camptothecins are clinically applied, so that the research of tumor cell immunity and cancer suppressor genes enters a stage of white thermalization; until the 21 st century, the era of tumor targeted therapy was truly opened. One of the characteristics of the targeting drugs is that the targeting drugs act on specific targets, the conditions of each patient are different, and the targeting drugs which can be selected are different, so that the individual treatment of tumors is realized to a certain extent. From the trend of medicine demand, obvious curative effect and small side effect are main demand directions of future product development, and under the drive of the market demand, the research and development and clinical application of targeted anti-tumor drugs are one of the main development directions of the future of the anti-tumor drug industry (Lippman S M, et al Journal of Clinical Oncology, 2005, 23 (2): 346-356).

The cestode is a drug which is marketed and is mainly used for treating parasites clinically. According to a large number of literature reports, the compound is found to be a potential drug for treating tumors, a large number of related researches are carried out around the compound at home and abroad, and the compound obtained by carrying out related structural modification on the compound has good anti-tumor biological activity. According to literature reports (Li Y, et al, cancer Letters, 2014, 349 (1): 8-14), possible signal paths are: wnt/beta-catenin, mTORC1, STAT3, NF-kappa B, notch. The numerous research reports provide abundant data for developing the medicaments, and also provide references for developing the targeted medicaments.

The main structure of the cestolin is the structure of ortho-hydroxy benzanilide, according to the structural characteristics, the substituent on the benzene ring is structurally modified to obtain a plurality of compounds, and according to the activity of the compounds, the related structure-activity relationship is obtained. We tried to make some changes in the hydroxyl position and obtain some compounds by chemical synthesis. These have good anti-tumor biological activity, and show great activity difference to different tumor cells, and have certain targeting property.

STAT3-JAK signaling pathways have positive regulatory effects on tumor cell growth, STAT3 protein has been favored as a biological target for the treatment of cancer for nearly ten years, and by 2017, the us FDA approved more than 30 STAT3 signaling pathway-inhibiting anticancer drugs in clinical tests (Johnson D E, et al Nature Reviews Clinical Oncology, 2018, 15 (4): 234). The STAT3 inhibitor anticancer targeting medicine has the characteristics of novel target point, wide anticancer spectrum and the like, and recent clinical test results show that the medicine has great development potential and wide market space in the aspect of future clinical treatment of tumors. The applicant finds that the ortho-hydroxybenzoylaniline compound belongs to a STAT3 inhibitor, and the compound has clear mechanism for inhibiting STAT3 activation and obvious effect for antagonizing tumor cell growth. The compound series were developed by Ruida pharmaceutical technology Co.Ltd.

Disclosure of Invention

The invention mainly solves the technical problem of providing a preparation method of a novel salicylic acid derivative and application of the novel salicylic acid derivative in tumor treatment.

To achieve the above object, the present invention provides a process for preparing a compound of the following general formula.

I is a kind of

Wherein, the liquid crystal display device comprises a liquid crystal display device,

r1 is selected from hydrogen, halogen, hydroxy, aldehyde, cyano, nitro, aryl, C1-6 alkyl, C1-6 alkoxy;

r2 is selected from 1-piperidyl, 1-pyrrolyl and 4-morpholinyl;

n is Arabic numerals 1-10.

The term "alkyl" as used herein refers to a saturated straight or branched chain monovalent hydrocarbon radical having 1 to 6 carbon atoms (i.e., C1-6 alkyl), 1 to 4 carbon atoms (i.e., C1-4 alkyl), or 1 to 3 carbon atoms (i.e., C1-3 alkyl). Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, neopentyl, n-hexyl, 2-methylpentyl, 2-dimethylbutyl, 3-dimethylbutyl, and the like.

The term "halogen" as used herein means fluorine, chlorine, bromine or iodine, with preferred halogen groups being fluorine, chlorine or bromine.

The preparation method of the compound comprises the following steps:

the preparation method of the compound comprises the following steps: the compounds A and B are reacted by Misunobu to obtain an intermediate C. And (3) hydrolyzing the intermediate C through lithium hydroxide and then acidifying to obtain a compound D. And (3) treating the compound D by using thionyl chloride to obtain acyl chloride E. Intermediate E and F react to obtain target compound G.

Wherein R1, R2 and n are as described above.

The biological significance of the invention is to find out a novel compound with strong STAT3 cell signal transduction inhibition effect and lower toxicity, and the compound has remarkable antagonism effect on the growth of tumor cells and has wide development space in the aspect of clinical treatment of cancers.

The invention also relates to the use of the salicylic acid derivative, the pharmaceutically acceptable salt thereof, the solvate of the derivative or the solvate of the salt in the preparation of medicaments for the treatment or the auxiliary treatment and/or prevention of lung cancer, breast cancer and leukemia of mammals. In particular, the mammal is a human.

The invention also relates to the use of the salicylic acid derivative, the pharmaceutically acceptable salt thereof, the solvate of the derivative or the solvate of the salt in the preparation of a medicament for treating or assisting in the treatment and/or prevention of a tumor mediated by STAT3 or proliferation and migration of tumor cells driven by STAT3 in a mammal. In particular, the mammal is a human.

One aspect of the present invention relates to the use of a salicylic acid derivative, a pharmaceutically acceptable salt thereof, a solvate of said derivative, or a solvate of said salt as described above for the manufacture of a medicament for the treatment and/or prophylaxis of a disease associated with STAT3 cell signalling in a mammal. In particular, the mammal is a human.

The present invention is further described below.

All documents cited herein are incorporated by reference in their entirety and for all purposes to the extent they are inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.

In the method for synthesizing a compound of the present invention, various raw materials used for the reaction are available to those skilled in the art according to the known knowledge, or can be prepared by methods known in the literature, or can be commercially available. The intermediates, raw materials, reagents, reaction conditions and the like used in the above reaction schemes may be appropriately changed according to the knowledge already known to those skilled in the art.

Drawings

FIG. 1 is a drawing of the general formula I.

FIG. 2 is an experimental result of the docking of the virtual molecule of the SH2 domain of the STAT3 protein with 4-chloro-2- (4-nitrobenzoyl) -phenol-4- [2- (1-piperidinyl) ethoxy ] benzoyl ester of the compound presented at the SH2 domain interaction interface of the STAT3 protein, with no tag on the nonpolar hydrogen atom (H), and with key amino acids that interact with the compound molecule above the SH2 domain of the STAT3 protein: lysine 591 and arginine 609, arginine 595 and serine 611, 613, 636 and glutamic acid 612, 638 are labeled with LYS591, ARG609, ARG595, SER611, GLU612, SER613, SER636 and GLU638, respectively; the β -sheet, α -helix and random coil of the SH2 domain of STAT3 protein are represented by the slow straight band, helical band and tubule, respectively. The results of virtual docking show that the compound has strong polar interactions with all of the key amino acids ARG609, LYS591 and ARG595 of the phosphorylation site of the SH2 domain of STAT3 protein, and the compound is deduced to be a STAT3 signaling inhibitor acting on the phosphorylation site of STAT 3.

FIG. 3 shows the result of Western blotting of the compound 4-chloro-2- (4-nitrobenzoyl) -phenol-4- [2- (1-piperidinyl) ethoxy ] benzoyl ester protein in example 4. And (3) transferring the total cell proteins separated by electrophoresis from the gel to a solid support membrane according to the result of a western blotting experiment, and detecting the corresponding protein expression levels through STAT3, p-STAT3 and beta-Actin antibodies according to the antigen-antibody specificity principle. The results are shown in the graph, and under the action of the medicine, the MCF-7 expresses STAT3 and the beta-action protein amount is unchanged along with the increase of the medicine concentration, the p-STAT3 expression amount is in a decreasing trend, and the compound obviously inhibits the expression of the p-STAT 3.

Detailed Description

Example 1: compound synthesis experiment

The compound synthesis method is exemplified by the compound 4-chloro-2- (4-nitroanilide formyl) -phenol-4- [2- (1-piperidinyl) ethoxy ] benzoyl ester:

preparation of 4-chloro-2- (4-nitrobenzoyl) -phenol-4- [2- (1-piperidinyl) ethoxy ] benzoyl ester:

1.3g of N-hydroxyethyl piperidine and 1.67g of methyl 4-hydroxybenzoate were added to a 100mL round bottom flask and dissolved in 30mL of tetrahydrofuran. 3.9g of triphenylphosphine was added to the reaction system, and the mixture was stirred at 0℃for 10 minutes. Then 3.0g of diisopropyl azodicarboxylate was slowly added dropwise to the reaction system. After the completion of the dropwise addition, the reaction was carried out at room temperature overnight. After the reaction, the solvent was removed under reduced pressure, 150mL of a 2mol/L hydrochloric acid solution was added thereto, and the mixture was stirred for 10 minutes. 100mL of ethyl acetate was added to the mixture, the pH of the resulting aqueous phase was adjusted to about 9, the aqueous phase was extracted with ethyl acetate (80 mL. Times.2), the organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 2.1g of the product.

0.65g of the above-obtained product was dissolved in 10mL of ethanol and 10mL of water. To the reaction system was added 0.25g of lithium hydroxide, and the reaction was carried out overnight. After the reaction, the ethanol is removed under reduced pressure, 10mL of water is added, the pH is adjusted to 4 by hydrochloric acid, stirring is continued for 30min, the carboxylic acid is obtained by filtration, and the carboxylic acid is directly used in the next step after being dried.

The obtained carboxylic acid (0.5 g) was taken, 10mL of thionyl chloride was added thereto, and the reaction was carried out at 80℃for 6 hours, after which the solvent was removed under reduced pressure and used directly in the next step.

0.53g of acyl chloride was taken, 20mL of methylene chloride was added for dissolution, 0.55g of 5-chloro-2-hydroxy-N- (4-nitro) -benzanilide and 0.5mL of triethylamine were added for reaction at room temperature for 4 hours. After the completion of the reaction, the solvent was removed under reduced pressure, ethyl acetate and saturated aqueous sodium chloride solution were added, and the organic phase was concentrated and then passed through a column (DCM/meoh=15/1) to give 0.35g of the objective product. 1H-NMR (6 d-DMSO): δ11.11 (s, 1H), 8.22-8.19 (d, 2H), 8.03-8.00 (d, 2H), 7.97-7.94 (d, 1H), 7.89-7.86 (m, 3H), 7.52-7.49 (d, 1H), 7.11-7.08 (d, 2H), 4.26-4.22 (t, 2H), 2.92 (s, 2H), 2.67 (s, 4H), 1.57-1.55 (d, 4H), 1.43-1.42 (d, 2H).

TABLE 1 information relating to compounds

Example 2: molecular docking (docking) experiment

The method comprises the following steps: to verify the interaction mechanism of the compound of formula 1 with STAT3 protein, the inventors used the phosphorylated tyrosine (pY-705) binding region of STAT3 SH2 region as a protein template for computer virtual modeling (docking), with the virtual docking region being concentrated mainly in the vicinity of the phosphorylated tyrosine sites ARG609 and LYS 591. The structure coordinates of STAT3 SH2 were taken from the protein structure database (PDB data bank, ID:1BG 1). Method of molecular docking (docking): all computer coordination modeling (docking) experiments were performed on the sybyl X2.1.1 operating platform using the computer coordination modeling (docking) tool SUEFLEX DOCK. Potential energy plane (potential gradient) was determined and tested in silico (dock) by calculation based on the selected sites, including mainly the phosphotyrosine sites ARG609, LYS591 and ARG 595. Analysis was performed based on the Score (Score) and conformation and interactions of the simulation (dock).

Example 3: MTT assay to induce apoptosis in breast, lung and leukemia cancer cells

Materials: 96-well cell culture plates, MDA-MB-231, MCF-7, PC9-AR, PC9-GR, MOLT-13, jurkat cell lines, tetramethyl azoazole blue (MTT), fetal bovine serum.

The method comprises the following steps: the cells are cultured according to conventional culture methods. During the test, collecting breast cancer cell strains MDA-MB-231 and MCF-7 in logarithmic growth phase, colon cancer cell strains HCT-116 and HT-29, lung cancer cell strains PC9, PC9-AR, PC9-GR and HCC827, leukemia cell strains Jurkat and MOLT-13, counting, adjusting the cell suspension concentration to 40000/mL, and adding 200 mu L of cell suspension into each hole, namely 8000 cells in each hole; the cell strain is treated by adding a compound, the final concentration is respectively 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 mu M, blank control is arranged, and the culture is continued for 48 hours; after the drug treatment, 20. Mu.L (5 mg/mL) of thiazole blue reagent is added to each well, and the wells are incubated for 4 hours at 37 ℃; removing liquid in the hole after incubation, draining water, sucking the residual liquid with filter paper, adding 100ul dimethyl sulfoxide, and reacting on a horizontal oscillator for 7-8min until the blue-violet crystal is completely dissolved; the OD value at the absorption wavelength of 570nm was measured by an enzyme-labeled instrument, and the result was recorded.

Inhibition (%) = (control well mean OD value-experimental well mean OD value)/control well mean OD value x 100%

The data were counted by GrapHPad Prism 7 and IC50 values were calculated.

IC50 values of compounds of Table 2 for tumor cells

Note that: the numerical values in the table are average values of three experiments; the values after "±" represent standard deviations

Example 4: western immunoblotting (Westernblot) experiment to detect influence of compound on cell p-STAT3 expression

Materials: 6-well cell culture plates, MCF-7 cell lines, fetal bovine serum, RIPA cell lysates, protein assay kits, polyvinylidene fluoride (PVDF) membranes, STAT3/p-STAT 3/beta-Actin primary antibodies, and STAT3/p-STAT 3/beta-Actin secondary antibodies.

The method comprises the following steps:

1. cell culture and drug addition

(1) After taking MDA-MB-231 cells in logarithmic growth phase and digesting the cells with pancreatin, single cell suspension with the density of 300000 cells/mL is prepared by using RPMI-1640 medium containing 10% bovine serum, and 2mL of cell suspension is added to each well to inoculate the cell culture plate with 6 holes.

（2）37℃、5% CO ₂ After incubation in an incubator and cell attachment, the final concentrations of compound were 1, 3, 10 and 30 μm, respectively, and a blank group was set. After 1h, 30. Mu.L of stimulated cells were added to each group except the blank group at a concentration of 1mg/mL interleukin-6 (IL-6) and the final interleukin 6 (IL-6) concentration was 30ng/mL.

2. Cell lysis, protein extraction and protein content determination

(1) The upper medium was removed and the six-well cell culture plate was washed twice with Phosphate Buffered Saline (PBS). 100. Mu.L of precooled RIPA cell lysate (protease inhibitor, phosphatase inhibitor and phenylmethylsulfonyl fluoride, all added in advance at a ratio of 1:100) was added. The cell lysate was scraped off with a pre-washed cell scraper and collected in a clean 1.5mL centrifuge tube.

(2) Placing on ice, cracking for 30min, and swirling once every 6 min.

(3) Centrifuge at 12000rpm for 12 min at 4 ℃.

(4) After centrifugation, the cell supernatant was collected in two parts: adding 5 μl into 1.5mL centrifuge tube for BCA protein content measurement, and adding 45 μl of 1×phosphate buffer (PBS) for mixing; the remaining cell supernatant was quantitatively taken at 60. Mu.L, added with 15. Mu.L of 5 XSDS Loading Buffer (Loading Buffer), mixed well, boiled in boiling water for 8min, centrifuged and stored in a refrigerator at-20 ℃.

(5) Protein concentration determination, specifically the steps:

A. protein standards were diluted with 1 x Phosphate Buffer (PBS) and run as follows:

B. BCA working solution preparation: the total required amount of A and B mixed working fluid is calculated according to the number of the standard substance and the sample to be tested. Volume ratio of BCA reagent a to B50: 1, preparing working solution, and uniformly mixing by vortex oscillation for standby.

C. Protein standards and sample supernatants diluted with Phosphate Buffered Saline (PBS) (10-fold dilution) were added to fresh 96-well plates at 25 μl each. Then 200. Mu.L of BCA working solution prepared in advance was added respectively and mixed well. The bubbles are not generated by blowing, the cover of the 96-well plate is tightly covered, and the reaction is carried out for 30min in a constant temperature box at 37 ℃.

D. Taking out the 96-well plate, recovering to room temperature for 3-5min, measuring absorbance value at 562nm wavelength on an enzyme label instrument, making a standard curve, and calculating 1 mu L/Protein content of each sample for Protein loading.

3. Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE)

(1) Fixing the gel plate and preparing 12% SDS-PAGE separating gel.

The release gel was prepared according to the following table: 10mL (mL)

(2) Adding the mixed separating glue into 2 glue plates, respectively, adding into the position 1.0cm away from the top, filling the glue plates with absolute ethyl alcohol, and standing for 30-45min.

(3) After the gel is separated, the residual absolute ethyl alcohol is poured out, and the residual absolute ethyl alcohol is sucked clean by filter paper.

(4) 5% concentrate was prepared according to the following table: 5mL

(5) Slowly adding the prepared concentrated gel into a gel plate to avoid bubble generation, inserting a sample comb, and standing for 30-45min.

(6) Taking out the protein sample, heating in water bath at 100deg.C for 5min, rotating at 10000rpm, and centrifuging for 10min.

(7) The gel plate is fixed in an electrophoresis tank, SDS-PAGE electrophoresis buffer is added, a sample comb is pulled out, and the processed protein samples are added into the sample tank in sequence.

(8) 80V electrophoresis for 40min.

(9) The voltage was changed to 120V to electrophoresis of approximately 1.5 h until bromophenol blue had run out of the gel.

4. Transfer film

(1) And (3) placing the SDS-PAGE gel subjected to electrophoresis into TBST buffer solution for rinsing once, and placing the protein gel into transfer buffer solution for soaking.

(2) Soaking a layer of foam cushion in a film transfer buffer solution, clamping the foam cushion onto a film transfer device by using tweezers, and soaking the foam cushion, the three layers of filter paper, the albumin glue, the polyvinylidene fluoride (PVDF) film, the three layers of filter paper and the foam cushion in sequence, aligning the foam cushion, clamping the foam cushion onto the film transfer device, and during operation, soaking the filter paper and the foam cushion in the film transfer buffer solution. If bubbles exist between each two layers, the bubbles are removed by using a glass test tube to gently roll.

(3) The film transfer instrument was turned on, and 300mA was transferred for 75min.

(4) The membrane was removed and placed in TBST buffer and rinsed 3 times with a 60rpm horizontal shaker for 8min each.

(5) Blocking was performed with 20mL of 5% Bovine Serum Albumin (BSA) blocking solution at 60rpm with a horizontal shaker at room temperature for 2h.

(6) Incubation was performed overnight with a 3.mu.L 3-mL antibody incubation with 1:1000 of primary antibodies (STAT 3 and p-STAT 3), 60rpm horizontal shaker at 4deg.C.

(7) The pvdf membrane was washed three times, 10min each, with 10mL TBST, a 60rpm horizontal shaker at ambient temperature.

(8) PVDF membrane 2h was incubated with 20mL antibody incubation with 2. Mu.L secondary antibody at room temperature with a 60rpm horizontal shaker.

(9) The PVDF membrane was washed three times, 10min each, with 10mL TBST, a 60rpm horizontal shaker at ambient temperature.

(10) 1mL each of chemiluminescent substrate reagent solution A and solution B was taken and developed for 5min at room temperature.

(11) The liquid on the membrane was blotted dry with filter paper and developed.

Claims (4)

1. Salicylic acid derivatives or pharmaceutically acceptable salts thereof, wherein the structure of the derivatives is shown as formula I:

i is a kind of

Wherein R is ₁ Selected from halogen or C1-6 alkyl; r is R ₂ Selected from 1-piperidinyl.

2. A process for preparing a compound according to any one of claim 1, characterized in that: the method comprises the following steps:

the preparation method of the compound comprises the following steps: the compound A and the compound B are subjected to Mitsunobu reaction to obtain an intermediate C; the intermediate C is hydrolyzed by lithium hydroxide and then acidized to obtain a compound D; the compound D is treated by thionyl chloride to obtain acyl chloride E; intermediate E and F react to obtain target compound G.

3. Use of a compound as claimed in claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of tumors, wherein the use is in the manufacture of a medicament for the treatment of STAT3 mediated tumors or STAT3 driven tumor cell proliferation and migration.

4. The use according to claim 3, wherein the tumour is breast cancer, lung cancer or leukaemia.