Compound with antitumor activity and application thereof

Abstract

The invention discloses a compound with antitumor activity and application thereof. The structural formula of the compound is as shown in formula I: # imgabs0. The compound provided by the invention has anti-tumor activity, and an in-vitro anti-tumor activity test result shows that the novel compound has an inhibiting effect on liver cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells and breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a compound with anti-tumor activity and application thereof. Background Art

[0002] Tumors are major diseases that seriously endanger human health. At present, there are various clinical treatments for tumors, mainly including surgical treatment, radiotherapy and drug therapy. In the field of drug therapy, treatment options are becoming more and more extensive, covering traditional chemotherapy drugs, immunotherapy drugs, targeted therapy drugs, endocrine therapy drugs and traditional Chinese medicine. With the rapid development of oncology treatment technology, especially driven by the concept of precision tumor treatment, in-depth research on tumor-related targets has become the key. In addition, the research and development and exploration of new anti-tumor drugs are also crucial. There are many new drug development pathways, including random screening, extraction of active ingredients from natural compounds, mechanism-based drug design, use of existing drugs to obtain new lead compounds, and discovery of new drugs from drug metabolites or degradation products. Therefore, the development and identification of new compounds with anti-tumor activity can not only expand existing treatment methods, but also may bring revolutionary breakthroughs in tumor treatment. Summary of the invention

[0003] One of the purposes of the present invention is to provide a compound represented by formula I or a pharmaceutically acceptable salt, solvent compound, prodrug, polymorph thereof,

[0004]

[0005] The molecular formula of the compound represented by formula I is C 28 H 29 N7O3 is slightly soluble in DMSO, very slightly soluble in methanol, and almost insoluble in water.

[0006] The second object of the present invention is to provide a method for preparing the compound represented by formula I, wherein septinib is dissolved in a solvent, subjected to degradation reaction under acidic conditions, and purified to obtain the compound.

[0007] Further, the solvent is a mixture of solvent A and solvent B, the volume ratio of solvent A to solvent B is 90:10, solvent A is a 2m mol / L potassium dihydrogen phosphate solution containing 0.4% triethylamine, and solvent B is acetonitrile. Further, solvent A is adjusted to pH 2.4 with phosphoric acid.

[0008] Furthermore, the dosage ratio of septinib to the solvent is 600 mg:300 mL.

[0009] Furthermore, the acidic condition is adjusted by adding 150 mL of 4M hydrochloric acid solution into 300 mL of solvent.

[0010] Furthermore, the degradation reaction is carried out at 80° C. for 40 hours.

[0011] Furthermore, after the degradation reaction, the method further comprises a step of adjusting the pH to neutral, preferably neutralizing the reaction solution to neutrality with a sodium hydroxide solution, and more preferably neutralizing the reaction solution to neutrality with a 4M sodium hydroxide solution.

[0012] Furthermore, the purification is performed using a semi-preparative liquid chromatograph, and the chromatographic conditions are: Agilent Eclipse XDB-C 18 (250×9.4mm,5μm); flow rate was 5mL / min; detection wavelength: 235nm; mobile phase A was water, mobile phase B was methanol, gradient elution, elution program was: 0→1min, 40%B→40%B; 1min→8min, 40%B→90%B; 8min→11min, 90%B→90%B; 11min→12min, 90%B→40%B; 12min→15min, 40%B→40%B.

[0013] The third object of the present invention is to provide a pharmaceutical composition comprising the compound represented by formula I or its pharmaceutically acceptable salt, solvent compound, prodrug, polymorph.

[0014] A fourth object of the present invention is to provide a preparation comprising the compound represented by formula I or its pharmaceutically acceptable salt, solvent compound, prodrug, polymorph, and a pharmaceutically acceptable carrier.

[0015] The fifth object of the present invention is to provide a compound represented by formula I or a pharmaceutically acceptable salt, solvent compound, prodrug, polymorph thereof, and the use of the above-mentioned pharmaceutical composition or preparation in the preparation of tumor therapeutic drugs.

[0016] Furthermore, the tumor is liver cancer, gastric cancer, pancreatic cancer, lung cancer or breast cancer.

[0017] The present invention obtains a compound of formula I from the degradation product of sepretinib. The in vitro anti-tumor activity test shows that the compound has an inhibitory effect on liver cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells and breast cancer cells; in particular, the anti-tumor activity of the compound on liver cancer cells and gastric cancer cells is better than that of the raw material sepretinib, which is of great significance for the further research and development of sepretinib. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the ultraviolet spectrum of the compound of the present invention.

[0019] Figure 2 It is the liquid chromatogram of the compound of the present invention.

[0020] Figure 3 The high-resolution mass spectrum of the compound of the present invention is shown in FIG.

[0021] Figure 4 The one-dimensional hydrogen nuclear magnetic resonance spectrum of the compound of the present invention is shown in FIG.

[0022] Figure 5 It is a one-dimensional carbon nuclear magnetic resonance spectrum of the compound of the present invention.

[0023] Figure 6 This is the result of the inhibitory effect of the compounds of the present invention on HepaRG cells.

[0024] Figure 7 This is the result of the inhibitory effect of the compounds of the present invention on MKN-1 cells.

[0025] Figure 8 This is the result of the inhibitory effect of the compounds of the present invention on PANC-1 cells.

[0026] Fig. 9 This is the result of the inhibitory effect of the compounds of the present invention on A549 cells.

[0027] Fig.10 This is the result of the inhibitory effect of the compounds of the present invention on MDA-MB-231 cells. DETAILED DESCRIPTION

[0028] The present invention provides a compound represented by formula I or a pharmaceutically acceptable salt, solvent compound, prodrug, polymorph thereof,

[0029]

[0030] The molecular formula of this compound is C 28 H 29 N7O3, molecular weight is 511.23.

[0031] Specifically, the compound is a white powder, slightly soluble in DMSO, very slightly soluble in methanol, almost insoluble in water, with a maximum absorption peak of 235nm in the ultraviolet spectrum and a molecular weight of 511.23. The compound is a new compound produced by the degradation of septinib. The structure of the compound has not been reported before, and it has an inhibitory effect on human lung cancer cells A549, human breast cancer cells MDA-MB-231, human liver cancer cells HepaRG, human pancreatic cancer cells PANC-1, and human gastric cancer cells MKN-1. It is a candidate drug with great potential value. In particular, the anti-tumor activity of the new compound is better than that of the raw drug septinib for HepaRG cells and MKN-1 cells.

[0032] It should be noted that the compound represented by Formula I and its pharmaceutically acceptable salts, solvates, prodrugs or polymorphs have the ability to inhibit tumor cell proliferation. In addition, the solvates, prodrugs or polymorphs are conventional technical terms in the art and have no other specific meanings.

[0033] In one embodiment of the present invention, the preparation method of the compound represented by Formula I is to dissolve septinib in a solvent, perform a degradation reaction under acidic conditions, and purify to obtain the compound.

[0034] Further, the solvent is a mixture of solvent A and solvent B, the volume ratio of solvent A to solvent B is 90:10, solvent A is a 2m mol / L potassium dihydrogen phosphate solution containing 0.4% triethylamine, and solvent B is acetonitrile. Further, solvent A is adjusted to pH 2.4 with phosphoric acid.

[0035] Furthermore, the dosage ratio of septinib to the solvent is 600 mg:300 mL.

[0036] Furthermore, the acidic condition is adjusted by adding 150 mL of 4M hydrochloric acid solution into 300 mL of solvent.

[0037] Furthermore, the degradation reaction is carried out at 80° C. for 40 hours.

[0038] Furthermore, after the degradation reaction, the method further comprises a step of adjusting the pH to neutral, preferably neutralizing the reaction solution to neutrality with a sodium hydroxide solution, and more preferably neutralizing the reaction solution to neutrality with a 4M sodium hydroxide solution.

[0039] Furthermore, the purification is performed using a semi-preparative liquid chromatograph, and the chromatographic conditions are: Agilent Eclipse XDB-C 18 (250×9.4mm,5μm); flow rate was 5mL / min; detection wavelength: 235nm; mobile phase A was water, mobile phase B was methanol, gradient elution, elution program was: 0→1min, 40%B→40%B; 1min→8min, 40%B→90%B; 8min→11min, 90%B→90%B; 11min→12min, 90%B→40%B; 12min→15min, 40%B→40%B.

[0040] It should be noted that the chromatographic purity obtained by the purification method of the above compound can reach more than 99%.

[0041] It should be noted that the purification also includes removing the organic solvent by a rotary evaporator.

[0042] Another aspect of the present invention provides the use of the above compound in the preparation of anti-tumor drugs.

[0043] In some specific embodiments, the tumor includes lung cancer, breast cancer, liver cancer, pancreatic cancer, and gastric cancer.

[0044] It should be noted that the drug includes the above compound and a pharmaceutically acceptable carrier.

[0045] It should be noted that the compound represented by formula I and its pharmaceutically acceptable salts, solvates, prodrugs or polymorphs can be used in combination with other active drugs to enhance the killing ability of cancer cells or supplement other therapeutic effects to achieve synergistic effects.

[0046] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-mentioned compound and a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof; and a pharmaceutically acceptable carrier.

[0047] It should be noted that, in the above-mentioned pharmaceutical preparations, pharmaceutically acceptable carriers include but are not limited to: ion exchange materials, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-vitamin E polyethylene glycol 1000 succinate, Tween or other similar polymer media and other surfactants for pharmaceutical preparations, serum proteins such as human serum albumin, buffer substances such as phosphates, aminoacetic acid, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, etc. Polyvinyl pyrrolidone, cellulosic materials, polyvinyl alcohol, sodium carboxymethyl cellulose, polyacrylates, ethylene-polyoxyethylene-block polymers and lanolin, cyclodextrins such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or their chemically modified derivatives such as 2- and 3-hydroxypropyl-propylcyclodextrin and other hydroxyalkyl cyclodextrins or other soluble derivatives can all be used to promote the drug delivery of the compound represented by formula I and its pharmaceutically acceptable salts, solvates, prodrugs or polymorphs.

[0048] It should also be noted that in the above-mentioned pharmaceutical preparations, pharmaceutically acceptable carriers also include pharmaceutically acceptable excipients, such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption promoters, flavoring agents, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers and colorants, etc., which can also be used as pharmaceutically acceptable carriers for preparing the above-mentioned compounds and their pharmaceutically acceptable salts, solvates, prodrugs or polymorphs into preparations.

[0049] In some embodiments, the pharmaceutically acceptable carrier is suitable for liquid dosage forms, solid dosage forms or paste dosage forms.

[0050] It should be noted that pharmaceutically acceptable carriers suitable for different dosage forms can be selected according to the administration method of the compound represented by Formula I and its pharmaceutically acceptable salt, solvate, prodrug or polymorph. It should also be noted that liquid dosage forms, solid dosage forms, paste dosage forms or emulsion dosage forms are known in the art, such as liquid dosage forms including but not limited to injections, sprays or oral liquids; solid dosage forms including but not limited to tablets, powders, granules or capsules; paste dosage forms including but not limited to creams or ointments. In some specific embodiments, the compound of formula I and its pharmaceutically acceptable salts, solvates, prodrugs or polymorphs can be administered by enteral or parenteral routes; parenteral preparations include injections, creams, ointments, patches, sprays, etc.; administration routes include subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, or oral, topical, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, or administration by inhalation of aerosol or implanted accumulation or acupuncture.

[0051] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0053] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0054] Example 1

[0055] 1. Preparation of the compound represented by formula I

[0056] Preparation of crude product: 600 mg of septinib API was dissolved in 300 mL of 2 m mol / L potassium dihydrogen phosphate solution (containing 0.4% triethylamine, pH adjusted to 2.4 with phosphoric acid)-acetonitrile (90:10), and then 150 mL of 4 M hydrochloric acid solution was added. The mixture was reacted at 80 ° C for 40 h. After the reaction was completed, it was neutralized to neutrality with 4 M sodium hydroxide solution. Subsequently, it was concentrated to a suitable volume using a rotary evaporator and freeze-dried to obtain a crude product.

[0057] Purification of crude product: 600 mg of crude new compound was added to 2 mL of water and dissolved by ultrasonic treatment. Centrifugation was then performed (12000 r / min, 10 min) and the resulting precipitate was collected. 1 mL of 60% methanol aqueous solution was added to the precipitate to dissolve it. Chromatographic conditions were: Agilent Eclipse XDB-C 18 (250×9.4mm,5μm); flow rate is 5mL / min; detection wavelength: 235nm; mobile phase A is water, mobile phase B is methanol, gradient elution, elution program is: 0→1min, 40%B→40%B; 1min→8min, 40%B→90%B; 8min→11min, 90%B→90%B; 11min→12min, 90%B→40%B; 12min→15min, 40%B→40%B. The solution after chromatographic purification was collected and further treated by rotary evaporator to remove the methanol component in the solution. The remaining solution was collected and freeze-dried to obtain a pure compound with anti-tumor activity. Finally, the chromatographic purity of the new compound was detected by high performance liquid chromatography, and the results showed that its purity was 99.1%.

[0058] 2. Compound structure characterization

[0059] UV spectrum detection: Take 10 mg of the new compound with anti-tumor activity, dissolve it in dimethyl sulfoxide (DMSO) and dilute it to a concentration of 10 μg / mL, and perform UV spectrum scanning in the range of 200-400 nm. The UV spectrum is shown in the figure below. Figure 1 shown.

[0060] Liquid chromatography: High performance liquid chromatography was used to detect new compounds with anti-tumor activity. 18 (2) column (250 mm×4.6 mm, 5 μm), flow rate 1.0 mL / min, detection wavelength 235 nm, 2 m mol / L potassium dihydrogen phosphate solution (containing 0.4% triethylamine, pH adjusted to 2.5 with phosphoric acid)-acetonitrile (90:10) as mobile phase A, acetonitrile as mobile phase B; the gradient elution program is: 0→2 min, 5% B→5% B; 2 min→15 min, 5% B→15% B; 15 min→30 min, 15% B→35% B; 30 min→35 min, 35% B→45% B; 35 min→36 min, 45% B→5% B; 36 min→45 min, 5% B→5% B; the chromatogram of the anti-tumor active compound detected is as shown in the figure Figure 2 shown.

[0061] Mass spectrometry detection: Take 10 mg of the new compound with anti-tumor activity, dissolve it in 50% methanol aqueous solution, and dilute it to a solution with a concentration of 100 ng / mL. The molecular weight is determined by a high-resolution mass spectrometer. The high-resolution mass spectrum is shown in Figure 3 shown.

[0062] Nuclear magnetic resonance spectrum detection: Weigh 10 mg and 20 mg of the new compound with anti-tumor activity respectively, dissolve it in deuterated DMSO and then test it on the machine. The one-dimensional nuclear magnetic resonance hydrogen spectrum is as follows Figure 4 As shown, the one-dimensional carbon NMR spectrum is as follows Figure 5 shown.

[0063] 3. Anti-tumor activity

[0064] The new compound was subjected to an in vitro antitumor activity experiment. HepaRG (human liver cancer cell), MKN-1 (human gastric cancer cell), PANC-1 (human pancreatic cancer cell), A549 (human lung cancer cell), and MDA-MB-231 (human breast cancer cell) cell lines were selected. The MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) method was used to study the inhibitory activity of the new compound against five human cancer cell lines, and the drug concentration when the inhibition rate reached 50% was calculated, i.e., IC 50 The specific steps are as follows:

[0065] (1) Cell culture: HepaRG, MKN-1, PANC-1, A549, and MDA-MB-231 cells were cultured in a 37°C, 5% CO2 incubator. The culture medium for A549, PANC-1, MKN-1, and MDA-MB-231 cells was DMEM, and the culture medium for HepaRG cells was RPMI-1640. 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin were added to the culture medium.

[0066] (2) MTT test: Five tumor cells in the logarithmic growth phase were digested with trypsin and the culture medium was prepared to 1×10 5 / mL cell suspension, then inoculate the cell suspension in a 96-well plate, add different concentrations of septinib and its new anti-tumor active compounds after 24 hours, set 8 replicate wells for each concentration, discard the supernatant after 48 hours, wash twice with PBS, add 100μL of freshly prepared MTT solution (1mg / mL) to each well. Place in an incubator and continue incubation for 4 hours, then carefully discard the supernatant in the 96-well plate, add 150μL DMSO to each well and react at 37℃ in the dark for 30 minutes. After the crystals are completely dissolved, use an enzyme reader to detect the optical density (OD) of each well at 490nm;

[0067] (3) Calculation of inhibition rate: Growth inhibition rate = (1-OD value of experimental group / OD value of control group) × 100%;

[0068] (4) Calculate IC based on concentration and growth inhibition rate 50 .

[0069] The results are as follows Figure 6-10 The results showed that sepretib and the new anti-tumor active compounds had different degrees of growth inhibition on the above tumor cells. IC of sepretib and the new anti-tumor active compounds on HepaRG cells 50 The IC values ​​of MKN-1 cells were 29.21 μg / mL and 21.50 μg / mL respectively. 50 The IC values ​​of PANC-1 cells were 40.53 μg / mL and 30.96 μg / mL respectively. 50 The IC values ​​for A549 cells were 9.96 μg / mL and 36.95 μg / mL, respectively. 50 The IC values ​​of MDA-MB-231 cells were 8.56 μg / mL and 41.87 μg / mL, respectively. 50 The results of MTT experiments showed that the anti-tumor activity of the new compound on PANC-1 cells, A549 cells, and MDA-MB-231 cells was lower than that of the raw material drug sepretib; however, the anti-tumor activity of the new compound on HepaRG cells and MKN-1 cells was better than that of the raw material drug sepretib.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, solvate, prodrug, or polymorph thereof, 2. The method for preparing the compound of formula I according to claim 1, characterized in that: Seputinib is dissolved in a solvent, subjected to degradation reaction under acidic conditions, and purified to obtain the compound.

3. The preparation method according to claim 2, characterized in that: The solvent is a mixture of solvent A and solvent B, the volume ratio of solvent A to solvent B is 90:10, solvent A is a 2m mol / L potassium dihydrogen phosphate solution containing 0.4% triethylamine, and solvent B is acetonitrile.

4. The preparation method according to claim 2, characterized in that: The acidic condition was adjusted by adding 150 mL of 4 M hydrochloric acid solution into 300 mL of solvent.

5. The preparation method according to claim 2, characterized in that: The degradation reaction was carried out at 80° C. for 40 h.

6. The preparation method according to claim 2, characterized in that: The purification was performed using a semi-preparative liquid chromatograph, and the chromatographic conditions were: the chromatographic column was Agilent Eclipse XDB-C 18 The flow rate was 5 mL / min, the detection wavelength was 235 nm, the mobile phase A was water, the mobile phase B was methanol, the gradient elution was: 0→1 min, 40% B→40% B; 1 min→8 min, 40% B→90% B; 8 min→11 min, 90% B→90% B; 11 min→12 min, 90% B→40% B; 12 min→15 min, 40% B→40% B.

7. A pharmaceutical composition, characterized in that It includes the compound represented by formula I as described in claim 1 or its pharmaceutically acceptable salt, solvent compound, prodrug, polymorph.

8. A pharmaceutical preparation, characterized in that It includes the compound represented by formula I as described in claim 1 or its pharmaceutically acceptable salt, solvent compound, prodrug, polymorph, and pharmaceutically acceptable carrier.

9. Use of the compound of formula I according to claim 1 or its pharmaceutically acceptable salt, solvate, prodrug, polymorph, the pharmaceutical composition according to claim 7 or the pharmaceutical preparation according to claim 8 in the preparation of tumor therapeutic drugs.

10. The use according to claim 9, characterized in that: The tumor is liver cancer, stomach cancer, pancreatic cancer, lung cancer or breast cancer.

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