CN109180621B - Preparation of Schiff base compounds and application of Schiff base compounds in cancer treatment - Google Patents

Abstract

The invention belongs to the field of medical biology, and particularly provides a design and a preparation method of Schiff base compounds, and an application of the Schiff base compounds in clinical treatment of breast cancer, lung cancer and the like. The biological activity test shows that the compound has obvious inhibiting effect on the growth and reproduction of various cancer cells at low concentration. Through the research of the biological mechanism, the compound related to the invention has obvious inhibition effect on STAT3 cell signaling pathway and the expression of downstream gene (Survivin). The compound has potential significance for clinical treatment and research of related cancers. The preparation method has simple process and easy implementation, and is suitable for the requirement of large-scale industrial production.

Description

Preparation of Schiff base compounds and application of Schiff base compounds in cancer treatment

Technical Field

The invention relates to the field of medicines, in particular to preparation of a novel Schiff base compound and application of the novel Schiff base compound in clinical application of treating cancer.

Background

The latest cancer data in China issued by the national cancer center in 2017 show that in China, every year, new cancer cases reach 429 ten thousand, account for 20 percent of the new cases worldwide, and 281 ten thousand deaths occur. Lung cancer is the first cancer with both morbidity and mortality, and breast cancer is the cancer with the highest tumor incidence in women. Cancer has become one of the factors that affect the health of the Chinese nation and cause significant economic loss. Traditional cancer treatment methods, surgery, radiation, chemotherapy and biotherapy, have limited control of the disease state of patients with tumors (1). In 2001, the emergence of the first generation of targeted drug, gleevec, brought new opportunities and hopes for clinical treatment of cancer patients. The market share of the anti-cancer targeted drugs newly listed in the world in 2017 in China is only about 1%. In 2017, 5 new anti-cancer drugs which are listed on the market by the China Food and Drug Administration (CFDA) have no innovative target, and few new targeted drugs which are originally developed in China reflect that the research and development level of the targeted original drugs in China is behind European and American. How to achieve 'double creation' in the aspect of drug development (biological targets are new targets related to diseases and drugs for attacking the targets are brand new compounds) is a gold test stone for testing the research and development strength of Chinese medicine. The search for new targets and potential drug lead compounds has breakthrough in specific tumor indications, and is a urgent need for Chinese medicine research and development researchers. The JAKs-STAT3 signal transduction promotes the growth and reproduction of various tumor cells, the tumors related to STAT3 comprise head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, chronic granulocytic leukemia, acute granulocytic leukemia, multiple myeloma and the like, more than 30 kinds of STAT3 protein target-based anti-cancer drugs are already approved by the United states Food and Drug Administration (FDA) in clinical testing stages, and recent clinical testing results show that the drugs have huge potential and wide market space in the future tumor clinical treatment (2). The Schiff base compound discovered by the applicant belongs to a STAT3 inhibitor, and the compound has clear mechanism for inhibiting STAT3 cell signaling activation and obvious effect of inducing related tumor cell apoptosis. The STAT3 inhibitor anticancer targeted drug has the characteristics of novel target, wide anticancer spectrum and the like, and the compound is developed by Ruida pharmaceutical technology limited company in Henan province.

The Schiff base compound has good pairing ability due to the unique structural characteristics, namely, a core group contains an N atom with a lone pair electron, and both sides of the group can react with various groups to obtain derivatives with different properties, so that the Schiff base compound has very special practical application, and is particularly widely applied in the aspects of chemistry and biology. O, S, N-containing Schiff base and its metal complex have been paid attention to their excellent biological activities of bacteriostasis, anticancer and antivirus. In particular, nifuroxazide has been studied for cancer therapy (3).

The inventor aims to solve the problem of drug resistance in the targeted therapy of clinical cancer. The method refers to the advanced medicine evaluation system and preparation process in the world, and simultaneously, develops the targeted new medicine with independent intellectual property rights in China by using the high-efficiency medicine research management standard and the clinical transformation method. The compound belongs to Schiff bases, has low synthesis cost, is expected to be 1/3 of the Icotinib price and 1/20 of Gleevec, is expected to be developed successfully to form the first new target targeted drug independently developed in China, and has the characteristics of obvious curative effect, broad-spectrum anticancer, low price and the like.

Disclosure of Invention

The invention mainly solves the technical problem of providing the preparation of a novel Schiff base compound and the application of the novel Schiff base compound in the aspect of clinical application of treating cancer.

In order to achieve the above purpose, the invention provides the application of the compounds with the following general formula in the preparation and clinical treatment of cancer.

Formula I

Wherein the content of the first and second substances,

r1, R2, R3 and R4 are respectively selected from hydrogen, hydroxyl, amino and nitro;

x is selected from carbon, oxygen, sulfur or nitrogen atom;

y is selected from hydrogen, hydroxyl, nitro, halogen, amino, cyano or carboxyl.

The term "halogen" as used herein refers to fluorine, chlorine, bromine or iodine, the preferred halogen groups being fluorine, chlorine or bromine.

Another aspect of the present invention relates to a process for the preparation of the above compound comprising the steps of:

1) substituted 4-methyl acetylbenzoate is taken as a raw material to react with hydrazine hydrate for 1 to 12 hours under the heating condition to obtain an intermediate (II):

2) reacting the intermediate (II) with a substituted aldehyde for 1 to 12 hours to obtain the compound of claim 1:

wherein X, Y, R1, R2, R3 and R4 are as previously described; the heating temperature is 50-100 ℃.

Another aspect of the present invention relates to the use of a derivative of the above-mentioned compound, a pharmaceutically acceptable salt thereof, a solvate of the derivative, or a solvate of the salt, for the manufacture of a medicament for the treatment and/or prevention of a disease associated with cancer in a mammal. In particular, the mammal is a human.

According to the present invention, it is expected that the compounds of the present invention can be used for the treatment of various cancers, such as head and neck tumors, breast cancer, acute myelogenous leukemia, chronic lymphatic leukemia, etc.

The invention is further described below.

All documents cited herein are incorporated herein by reference in their entirety and to the extent such documents are not inconsistent with this invention, the express disclosure of such documents shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the process of the present invention for the synthesis of compounds of formula I, the various starting materials for the reaction are either prepared by methods known in the literature or are commercially available, as known to the person skilled in the art. The intermediates, starting materials, reagents, reaction conditions, etc. used in the above reaction schemes may be appropriately modified according to the knowledge of those skilled in the art.

The compounds of formula I according to the invention can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions.

The active compound shown in the formula I can be used as an anticancer drug independently or can be used together with one or more other antitumor drugs. Combination therapy is achieved by administering the individual therapeutic components simultaneously, sequentially or separately.

Drawings

FIG. 1 is a schematic diagram of a STAT3(Signal Transducer and Activator of Transcription Type-3) signaling system; binding of cytokines (cytokines) to the receptor triggers dimerization (dimerization) or multimerization (oligomerization) of the receptor and phosphorylation (phosphorylation) of JAKs to activate STAT3 signaling, which is attached to the receptor via phosphorylation of specific tyrosines in the C-terminal region of the receptor cell membrane; after autophosphorylation, STATs form a bi-directional cross-interacting, dimeric parallel conformation; the cell membrane is transported into cell nucleus and combined with specific DNA promoter to induce the expression of relevant anti-cell death gene Bcl-2, Survivin, Bcl-XL, Jun, etc.

FIG. 2 shows the results of experiments on the virtual molecular docking of HN2-020 and STAT3 protein SH2 domain in example 1, wherein HN2-020 is present in the domain interface, none of the nonpolar hydrogen atoms (H) are labeled, and the key amino acids interacting with HN2-020 molecule are in the STAT3 protein SH2 interval: lysine 591 and arginine 609 and serine 611, 613, 636 and glutamic acid 612, 638 are labeled with LYS591, ARG609, SER611, GLU612, SER613, SER636 and GLU638, respectively; the beta sheet, alpha helix and random coil of the STAT3 protein SH2 interval are represented by a flat band, a helical band and a tubule, respectively. The virtual docking result shows that the compound HN2-020 has strong interaction with the phosphorylation site ARG609 of the SH2 functional domain of STAT3 protein, specific amino acid GLU638 and a drug water delivery selective cavity, and the compound is inferred to be a STAT3 signal transduction inhibitor acting on the STAT3 phosphorylation site.

FIG. 3 is MTT assay for inducing apoptosis of breast cancer cells by HN2-020, wherein the result of MTT cell assay is characterized by IC50(mmol/L) value. The experimental result shows that the compound HN2-020 can effectively induce the apoptosis of the breast cancer MBA-MD-231 cell line.

FIG. 4 is MTT assay for inducing apoptosis of lung cancer cells, wherein the results of MTT cell assay are characterized by IC50(mmol/L) values. The experimental result shows that the compound HN2-020 can effectively induce the apoptosis of a lung cancer PC9 cell strain, and can effectively induce the apoptosis of a lung cancer PC9-AR (Afatinib-resistant cell strain) and a lung cancer PC9-GR (gefitinib-resistant cell strain). Shows the potential of the compound HN2-020 in overcoming the drug resistance problem of lung cancer treatment.

FIG. 5 shows the results of western blotting of Compound HN 2-020. And transferring the total cell protein after electrophoretic separation from the gel to a solid support membrane according to the result of the Western blotting experiment, and detecting the expression quantity of the corresponding protein by STAT3, P-STAT3 and beta-Actin antibodies respectively according to the antigen-antibody specific principle. The results are shown in the figure, and it can be seen that under the action of different drugs, with the increase of the drug depth, the amounts of STAT3 and beta-Actin protein expressed by MDA-MB-231 are unchanged, while the expression amount of P-STAT3 is in a descending trend, and the expression of P-STAT3 is obviously inhibited by HN 2-020.

FIG. 6 shows the results of the reverse transcription polymerase chain reaction assay (PCR) for compound HN 2-020. Determining the expression condition of related anti-apoptosis genes by utilizing semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction), and verifying the physiological condition of MDA-MB-231 under the action of different drug concentrations; respectively extracting the MDA-MB-231 under the action of different concentrations through an RNA sample, determining the RNA content, reversely transcribing cDNA by the sample, amplifying beta-Actin and Survivin expression through PCR; comparing the expression of anti-apoptosis gene under different drugs, it can be seen that compound HN2-020 has significant inhibitory effect on Survivin expression.

Detailed Description

The present invention is further described below by way of specific preparation examples and simulations and biological test examples, but it should be understood that these examples and test examples are for illustrative purposes only in more detail and are not to be construed as limiting the invention in any way.

The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well known in the art, unless otherwise specified.

In the present invention, unless otherwise specified, wherein: (i) the temperature is expressed in degrees centigrade (DEG C), and the operation is carried out in a room temperature environment; more specifically, the room temperature is 20-30 ℃; (ii) drying the organic solvent by a common drying method, evaporating the solvent by using a rotary evaporator for reduced pressure evaporation, and keeping the bath temperature not higher than 60 ℃; (iii) the reaction process was followed by Thin Layer Chromatography (TLC); (iv) the final product had satisfactory proton nuclear magnetic resonance (1H-NMR) and Mass Spectral (MS) data.

Example 1: synthesis of Compound HN2-020

Compound HN2-020

1) Synthesis of (Z) -4 (1-hydrazinoethyl) benzoylhydrazine:

4-methyl acetylbenzoate (1.78g, 10mmol) solid, adding 10mL hydrazine hydrate solution, heating to 80 ℃ and stirring for 12 hours after reflux reaction; TLC detection reaction is complete, and recrystallization is carried out to obtain white solid powder which is (Z) -4 (1-hydrazinoethyl) benzoyl hydrazine.

2) Synthesis of Compound HN 2-020:

dissolving (Z) -4 (1-hydrazinoethyl) benzoyl hydrazine (1.92g, 10mmol) in 15mL of absolute ethyl alcohol, adding 5-nitrofurfural (2.1g, 15mmol) into the solution, heating to 80 ℃, refluxing for reaction for 10 hours, pouring the solution into 200mL of distilled water after TLC detection reaction is completed, separating out a light yellow solid, filtering, and recrystallizing the solid by using methanol to obtain light yellow solid powder 1.76g, namely a compound HN 2-020. 1H NMR (300MHz, DMSO) δ 12.35(s, 1H), 8.46(s, 2H), 8.11(d, J ═ 8.2Hz, 2H), 8.03(d, J ═ 8.2Hz, 2H), 7.82(dd, J ═ 6.5, 4.0Hz, 2H), 7.41(d, J ═ 3.8Hz, 1H), 7.30(s, 1H), 2.49(s, 3H), ms (esi)439.1for [ M + H ] (cad 439.1for C19H14N6O7)

Test example 1: molecular docking experiments

The method comprises the following steps: to provide a reasonable set of chemical probes for computer predicted selective target attack STAT3, the inventors used the phosphorylated tyrosine (pY-705) binding region of the STAT3SH2 domain as a computer coordination mimicking (docking) site, consisting essentially of the phosphorylated tyrosine interaction site ARG609 and the hydrophobic interaction site GLU 638. The structural coordinates of STAT3SH2 were taken from the protein structure database (PDB data bank, ID: 1BG 1). Molecular docking (docking) method: all computer coordination simulation (docking) experiments were performed on the operating platform of sybyl X2.1.1, and the tool used for computer coordination simulation (docking) was the SUEFLEX DOCK. Experiments (4, 5) were performed to determine potential profiles (potential gradients) and to perform computer coordination simulations (docking) based on calculations of selected sites (mainly including the phosphotyrosine interaction site ARG609 and the hydrophobic interaction site GLU 638). Scores (Score) and conformational and interaction analysis by simulation (docking).

Test example 2: experiment for inducing apoptosis of breast cancer, lung cancer and cancer cells MTT

The method comprises the following steps: collecting MDA-MB-231 or PC9 cells in logarithmic growth phase, counting, adjusting cell suspension concentration to 50000 cells/ml, and adding 100ul cell suspension into each hole, namely 5000 cells in each hole; MDA-MB-231 or PC9 cells are treated by adding compound drugs, the drugs are added to several gradients with the final concentration of 0.1, 0.3, 1, 3, 10, 30, 100 and 300(um/L), and the culture is continued for 48 h; after the drug treatment, 50ul (1mg/ml) of thiazole blue reagent is added into each hole, incubation is carried out for 4 hours at 37 ℃, liquid in the holes is discarded by throwing a plate, water is drained, residual liquid is completely absorbed by filter paper, then 100ul of dimethyl sulfoxide is added, reaction is carried out for 7-8 minutes on a horizontal oscillator until blue-purple crystals are completely dissolved, reading is carried out by an enzyme labeling instrument, the absorption wavelength is 570nm, and the result is recorded.

Test example 3: western blot (Westernblot) assay

1. Cell culture and dosing:

(1) taking MDA-MB-231 cells in logarithmic growth phase, digesting the cells by pancreatin, preparing single cell suspension with the density of 300000/mL by using RPMI-1640 medium containing 10% fetal bovine serum, and adding 2mL of cell suspension into each hole to inoculate the cells into a 6-hole flat bottom plate.

(2)37℃、5％CO₂Incubator hatchAfter the cells are attached to the wall, drugs with different concentrations are added into the experimental group, the interleukin 6(IL-6) with the concentration of 1mg/mL and 30uL are added after 1 hour to stimulate the cells, and the final concentration of the interleukin 6(IL-6) is 30 ng/mL.

(3) After further culturing for 0.5 hour, the cells were lysed with RIPA lysate to collect the protein.

2. Cell collection and lysis

(1) The upper medium was removed and the cells in the six-well plates were washed twice with Phosphate Buffered Saline (PBS). 160. mu.L of pre-cooled RIPA cell lysate (protease inhibitor and phenylmethylsulfonyl fluoride were added to the lysate in advance at a ratio of 1: 100 and mixed) was added. The cell lysate was scraped off with a previously washed cell scraper and collected in a clean 1.5mL centrifuge tube.

(2) Placed on ice, lysed for 30 minutes, and vortexed at regular intervals (6 minutes).

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

(4) The cell supernatant was transferred to a clean centrifuge tube. The cell supernatant is divided into two parts: adding 5 mu L of the mixture into a 1.5mL centrifuge tube for BCA protein content determination, adding 45 mu L of 1 x Phosphate Buffer Solution (PBS) and mixing uniformly for later use; the remaining cell supernatant was quantitatively sampled at 140. mu.L, added with 35. mu.L of 5 XSDS Loading Buffer (Loading Buffer), mixed well, boiled in boiling water for 8 minutes, centrifuged, and stored in a-20 ℃ refrigerator.

(5) Protein concentration determination step:

A. 1 × Phosphate Buffered Saline (PBS) dilution of protein standards:

B. preparing a BCA working solution: and calculating the total required amount of the mixed working solution A and B according to the number of the standard substances and the samples to be detected. Preparing a working solution according to the volume ratio of 50: 1 of the BCA reagent A to the B, and performing vortex oscillation and uniform mixing for later use.

C. The protein standard and the sample supernatant diluted with Phosphate Buffered Saline (PBS) (10-fold dilution) were each added to a new 96-well plate in 25 μ L. Then respectively adding 200 mu L of the BCA working solution prepared in advance and fully mixing. The reaction mixture was subjected to air-blowing, and then the 96-well plate lid was closed, followed by reaction at 37 ℃ for 30 minutes.

D. The 96-well plate was taken out and returned to room temperature for 3 to 5 minutes, and the absorbance value at a wavelength of 562nm was measured on a microplate reader, and a standard curve was made and the 1. mu.L/Protein content of each sample was calculated to prepare a Protein loading.

3. Sodium dodecyl sulfate-Polyacrylamide gel (SDS-PAGE)

(1) The gel plate was fixed and a 10% SDS-PAGE separation gel was prepared.

The separation gel was prepared according to the following table: 10mL

(2) And respectively adding the mixed separation gel into 2 rubber plates, adding the mixed separation gel to a position 1.0 cm away from the top, filling the rubber plates with absolute ethyl alcohol, and standing for 30-45 minutes.

(3) After the separation and gelation are finished, the residual absolute ethyl alcohol is poured out and is completely absorbed by filter paper.

(4) 5mL of 5% concentrated gum was prepared according to the following table

(5) Slowly adding the prepared concentrated glue into a rubber plate to avoid generating bubbles, inserting a sample comb, and standing for 30-45 minutes.

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

(7) Fixing the rubber plate in an electrophoresis tank, adding SDS-PAGE electrophoresis buffer solution, pulling out a sample comb, and adding the processed protein samples into the sample tank in sequence.

(8)80V electrophoresis for 40 min.

(9) Changing the voltage to 120V electrophoresis for about 1.5 hours until the bromophenol blue comes out of the colloid;

4. western-blot membrane conversion

(1) And (3) putting the SDS-PAGE gel after electrophoresis into a TBST buffer solution for rinsing once, and putting the albumin gel into a membrane transfer buffer solution for soaking.

(2) Soaking a layer of spongy cushion in a membrane transfer buffer solution, clamping the spongy cushion on a membrane transfer instrument by using tweezers, sequentially placing and aligning three layers of filter paper, protein glue, a polyvinylidene fluoride (pvdf) membrane, three layers of filter paper and the spongy cushion, and clamping the spongy cushion on the membrane transfer instrument, wherein the filter paper and the spongy cushion are soaked in the membrane transfer buffer solution during operation. If there are air bubbles between each layer, the air bubbles are expelled by gently rolling the glass test tube.

(3) The membrane rotating instrument is opened, and the membrane is rotated for 75 minutes at 300 mA.

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

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

(6) 3mL of antibody incubation with 3. mu.L of primary antibody (Stat3 and p-Stat 31: 1000) was incubated overnight at 4 ℃ with a 60rpm horizontal shaker.

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

(8) The pvdf membrane was incubated with 20m1 antibody incubation solution with 2 μ L secondary antibody and a horizontal shaker at 60rpm at room temperature for 2 hours.

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

(10) The chemiluminescent substrate reagent solution A and solution B were taken 1ml each and developed for 5 minutes at room temperature.

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

Test example 4: reverse transcription polymerase chain reaction assay (RT-PCR)

1. Cell culture and dosing:

(1) taking MDA-MB-231 cells in logarithmic growth phase, digesting the cells by pancreatin, preparing single cell suspension with the density of 100000/mL by using RPMI-1640 medium containing 10% fetal bovine serum, and adding 2mL of cell suspension into each hole to inoculate the cells into a 6-hole flat bottom plate.

(2)37℃、5％CO₂And (3) incubating in an incubator, replacing a fresh culture medium after the cells adhere to the wall, adding medicaments containing different concentrations of 3, 10, 30 and 100(uM) into the experimental group, adding interleukin 6(IL-6) with the concentration of 1mg/mL and 30uL to stimulate the cells after 1 hour, and finally adding interleukin 6(IL-6) with the concentration of 30 ng/mL.

(3) After further incubation for 24 hours, total RNA was extracted from each group of cells as indicated in the RNA extraction kit.

2. RNA sample preparation:

(1) when the cell growth density of the 6-well plate is 90-100%, taking out the 6-well plate from the sterile room, removing the supernatant, adding 1mL of TRIZOL total RNA extraction reagent into each well, shaking uniformly, digesting for 5 minutes on ice, blowing and beating by using a pipette tip, and sucking the liquid of each well into a 1.5mL centrifuge tube.

(2) 0.2ml of chloroform was added thereto, the mixture was shaken gently for 15 seconds, and the mixture was allowed to stand at room temperature for 3 minutes.

(3) Centrifuge at 12000rpm for 15 minutes at 4 ℃. After centrifugation, the liquid is divided into three layers, the upper layer is colorless water sample layer RNA, the middle white layer is DNA, the bottom layer is red protein, and the upper layer colorless liquid is carefully absorbed and transferred into a new centrifuge tube.

(4) About 0.4mL of the supernatant was added to an equal volume of isopropanol and allowed to stand at room temperature for 10 minutes.

(5) Centrifuge at 12000rpm for 10 minutes at 4 ℃.

(6) The liquid in the centrifuge tube was discarded, and 1mL of 75% ethanol was added.

(7) Centrifuging at 7500rpm for 5 min at 4 deg.C, carefully discarding the supernatant, placing in a clean bench, and air drying for 5-10 min.

(8) Adding 20-30uL of diethyl cokenate (DEPC) aqueous solution, subpackaging and storing at-70 ℃ for later use.

(9) 2uL of total RNA is taken and detected by a K5500 ultramicro spectrophotometer to determine the concentration and the purity. Total RNA purity was characterized by the ratio of OD260/OD280 and OD260/OD 230. OD260/OD280 between 1.8 and 2.0 indicate better purity.

(10) 5uL of total RNA was mixed with 1uL of loading buffer, and the RNA quality was observed on a gel imaging system by electrophoresis on a 1% agarose gel (containing GleRed) at 120V for 30 minutes (electrophoresis buffer 1 XTAE). If the RNA is not degraded, the next experiment can be carried out.

3. Synthesis of cDNA

Reverse Transcription-Polymerase Chain Reaction (RT-PCR), the experimental principle is as follows: first, total RNA in each group of cells is extracted, and complementary DNA single-stranded (complementary DNA) cDNA is synthesized under the catalytic action of reverse transcriptase and under the guide of random primer or polythymidine (oligo (dT)) or gene-specific primer by using mRNA in the total RNA as a template. Then, the cDNA is used as a template, and PCR amplification reaction of the target gene is carried out under the guide of a specific primer. Since the cDNA is obtained by reverse transcription using mRNA as a template, the target gene sequence to be amplified is an encodable gene sequence containing no intron.

(1) Synthesis of cDNA

A. Taking a nuclease-free centrifugal tube, and sequentially adding the following solutions in an ice bath:

deoxyribonucleoside triphosphate mixture (dNTP Mix)2.5mM/each 4 uL: primer Mix (Primer Mix)2 uL; RNA Template (RNA Template)1 ug; RNase Free Water (RNase-Free Water) was added to 15uL, reaction conditions: incubate at 70 ℃ for 10 min and ice-wash rapidly for 2 min.

B. The following components were added in sequence in an ice bath and mixed gently with a pipette:

4uL of 5 times of First Strand buffer (5 XFirst-Strand buffer); reverse transcriptase (M-MLV)1uL, reaction conditions: the reaction was terminated by heating at 42 ℃ for 50 minutes and 85 ℃ for 5 minutes.

C. 2-3uL was used directly for PCR reaction or stored at-20 ℃.

(2) Semi-quantitative PCR amplification of target gene

A. The following reaction systems were added to the PCR tube in order on ice:

cDNA 2.5 uL; forward primer (Forward primer) (10mM)1 uL; reverse primer (Reverse primer) (10mM)1 uL; 2 times PCR premixed enzyme (2 × Taq PCR Mix)12.5 uL; double distilled water (ddH)₂O) to 25 uL.

PCR cycling conditions:

cycling at the following temperatures:

1) pre-denaturation at 94 ℃ for 5 min; 2) denaturation at 94 ℃ for 30 seconds; 3) annealing at 61 ℃ for 30 seconds; 4) extension at 72 ℃ for 1 min; 5) repeating the steps 2-4 for 30 cycles; 6) extension at 72 ℃ for 5 minutes; 7) storing at 4 ℃.

C. And (4) detecting a result: and (3) after the reaction is finished, taking 5uL of reaction product, and carrying out agarose gel electrophoresis separation and identification.

Citations

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2.Johnson D E，O’Keefe R A，Grandis J R.Targeting the IL-6/JAK/STAT3 signalling axis in cancer[J].Nature Reviews Clinical Oncology，2018，15(4).

3.Nelson E A，Kepich W A.Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3[J].Blood，2008，112(13)：5095-5102.

4.Becker S，Groner B，Muller CW(1998)Three-dimensional structure of the Stat3[beta]homodimer bound to DNA.Nature 394：145-151.

5.Totrov M，Abagyan R(1997)Proteins 1：215-220.

Claims (6)

1. Schiff base compounds are characterized by having a structure shown in a general formula I:

formula I

Wherein the content of the first and second substances,

R₁、R₂、R₃、R₄are all hydrogen; x is selected from oxygen or sulfur atom; y is nitro.

2. A process for preparing a compound as claimed in claim 1, characterized in that: the method comprises the following steps:

i) substituted 4-methyl acetylbenzoate is taken as a raw material to react with hydrazine hydrate for 1 to 12 hours under the heating condition to obtain an intermediate (II):

II) reacting the intermediate (II) with a substituted aldehyde for 1 to 12 hours to obtain the compound of claim 1:

wherein the content of the first and second substances,

R₁、R₂、R₃、R₄are all hydrogen; x is selected from oxygen or sulfur atom; y is nitro.

3. The use of schiff base compounds according to claim 1 and pharmaceutically acceptable salts thereof for the preparation of anti-cancer drugs.

4. The use of claim 3, wherein the cancer comprises breast cancer.

5. The use of claim 3, wherein the cancer comprises lung cancer.

6. The use according to claim 3, wherein the cancer comprises head and neck, liver, colon and prostate cancer associated with modulation of the JAKs-STAT3 signaling pathway, and chronic and acute myeloid leukemia.

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