A compound with antitumor activity and its application

Abstract

This invention discloses a compound with antitumor activity and its applications. The structural formula of the compound is shown in Formula I. The compound provided by this invention has antitumor activity, and in vitro antitumor activity tests show that this new compound has inhibitory effects on liver cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, and breast cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a compound with anti-tumor activity and its applications. Background Technology

[0002] Cancer is a major disease that seriously threatens human health. Currently, there are various clinical treatments for cancer, mainly including surgery, radiotherapy, and drug therapy. In the field of drug therapy, treatment options are becoming increasingly broad, encompassing traditional chemotherapy drugs, immunotherapy drugs, targeted therapy drugs, endocrine therapy drugs, and traditional Chinese medicine. With the rapid development of oncology treatment technologies, especially driven by the concept of precision oncology, in-depth research into tumor-related targets has become crucial. Furthermore, the research and development of novel anti-tumor drugs is also vital. There are numerous pathways for new drug development, including random screening, extraction of active ingredients from natural compounds, mechanism-based drug design, obtaining new lead compounds from existing drugs, and discovering new drugs from drug metabolites or degradation products. Therefore, developing and identifying new compounds with anti-tumor activity can not only expand existing treatment methods but also potentially bring revolutionary breakthroughs in cancer treatment. Summary of the Invention

[0003] One object of this invention is to provide compounds of Formula I or pharmaceutically acceptable salts, solvent compounds, prodrugs, or polymorphs thereof.

[0004]

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

[0006] A second objective of this invention is to provide a method for preparing the compound shown in Formula I, wherein sectinib is dissolved in a solvent, subjected to a degradation reaction under acidic conditions, and purified to obtain the compound.

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

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

[0009] Furthermore, the acidic condition is adjusted by adding 150 mL of 4M hydrochloric acid solution to 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 step of adjusting the pH to neutral is included, preferably by neutralizing the reaction solution to neutral with a sodium hydroxide solution, and more preferably by neutralizing the reaction solution to neutral with a 4M sodium hydroxide solution.

[0012] Furthermore, the purification was performed using a semi-preparative liquid chromatograph under the following chromatographic conditions: Agilent Eclipse XDB-C. 18 (250×9.4mm, 5μm); flow rate: 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.

[0013] A third objective of this invention is to provide a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, a solvent compound, a prodrug, or a polymorph thereof.

[0014] A fourth objective of this invention is to provide a formulation comprising a compound of formula I or a pharmaceutically acceptable salt thereof, a solvent compound, a prodrug, a polymorph thereof, and a pharmaceutically acceptable carrier.

[0015] A fifth objective of this invention is to provide the use of the compound of Formula I or a pharmaceutically acceptable salt, solvent compound, prodrug, or polymorph thereof, in the preparation of a tumor therapeutic drug.

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

[0017] This invention yielded a compound of Formula I from the degradation products of secretinib. In vitro antitumor activity tests showed that this compound inhibited liver cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, and breast cancer cells. In particular, the antitumor activity of this compound against liver cancer cells and gastric cancer cells was superior to that of the active pharmaceutical ingredient secretinib, which is of great significance for the further development of secretinib. Attached Figure Description

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

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

[0020] Figure 3 This is a high-resolution mass spectrum of the compound of the present invention.

[0021] Figure 4 This is a one-dimensional proton NMR spectrum of the compound of this invention.

[0022] Figure 5 This is a one-dimensional carbon NMR spectrum of the compound of this invention.

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

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

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

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

[0027] Figure 10 This is the result of the inhibitory effect of the compound of the present invention on MDA-MB-231 cells. Detailed Implementation

[0028] This invention provides compounds of Formula I or pharmaceutically acceptable salts, solvent compounds, prodrugs, and polymorphs thereof.

[0029]

[0030] The molecular formula of this compound is C2. 28 H 29 N7O3 has a molecular weight of 511.23.

[0031] Specifically, the above-mentioned compound is a white powder, slightly soluble in DMSO, very slightly soluble in methanol, and almost insoluble in water. Its maximum absorption peak in the ultraviolet spectrum is at 235 nm, and its molecular weight is 511.23. This compound is a novel drug produced by the degradation of cepretinib. Its structure has not been previously reported, and it exhibits inhibitory activity against 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, making it a candidate drug with significant potential. In particular, the antitumor activity of the new compound against HepaRG and MKN-1 cells is superior to that of the active pharmaceutical ingredient cepretinib.

[0032] It should be noted that the compounds shown in Formula I, and their pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs, have the ability to inhibit tumor cell proliferation. Furthermore, the terms "solvate," "prodrug," or "polymorph" are conventional technical terms in the art and have no other specific meaning.

[0033] In one embodiment of the present invention, the compound represented by Formula I is prepared by dissolving secretinib in a solvent, carrying out a degradation reaction under acidic conditions, and purifying the compound to obtain the compound.

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

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

[0036] Furthermore, the acidic condition is adjusted by adding 150 mL of 4M hydrochloric acid solution to 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 step of adjusting the pH to neutral is included, preferably by neutralizing the reaction solution to neutral with a sodium hydroxide solution, and more preferably by neutralizing the reaction solution to neutral with a 4M sodium hydroxide solution.

[0039] Furthermore, the purification was performed using a semi-preparative liquid chromatograph under the following chromatographic conditions: Agilent Eclipse XDB-C. 18 (250×9.4mm, 5μm); flow rate: 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.

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

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

[0042] In another aspect, the present invention provides the use of the above-mentioned compound in the preparation of a medicament for treating antitumor diseases.

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

[0044] It should be noted that the drug includes the aforementioned compounds and pharmaceutically acceptable carriers.

[0045] It should be noted that the compounds shown in Formula I, as well as their pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs, can be combined with other active pharmaceutical ingredients to enhance their ability to kill cancer cells or to supplement other therapeutic effects and achieve synergistic effects.

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

[0047] It should be noted that pharmaceutically acceptable carriers in the above-mentioned drug formulations 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 polymerization media, surfactants used in drug formulations, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acid partial glycerides, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, etc. Polyvinylpyrrolidone, cellulose substances, 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, hydroxyalkyl cyclodextrins or other soluble derivatives can all be used to promote drug delivery of compounds of Formula I and their pharmaceutically acceptable salts, solvates, prodrugs or polymorphs.

[0048] It should also be noted that, in the above-mentioned pharmaceutical formulations, pharmaceutically acceptable carriers also include pharmaceutically acceptable excipients, such as fillers (e.g., anhydrous lactose, starch, lactose beads, and glucose), binders (e.g., microcrystalline cellulose), disintegrants (e.g., croscarmellose sodium, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and croscarmellose PVP), lubricants (e.g., magnesium stearate), absorption enhancers, flavorings, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers, and colorants, which can also be used as pharmaceutically acceptable carriers to prepare formulations from the above-mentioned compounds and their pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs.

[0049] In some specific embodiments, the pharmaceutical carrier is suitable for liquid dosage forms, solid dosage forms, or ointment dosage forms.

[0050] It should be noted that, based on the administration routes of the compounds shown in Formula I and their pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs, suitable pharmaceutical carriers for different dosage forms can be selected. It should also be noted that liquid dosage forms, solid dosage forms, ointment dosage forms, or emulsion dosage forms are known in the art. For example, liquid dosage forms include, but are not limited to, injections, sprays, or oral solutions; solid dosage forms include, but are not limited to, tablets, powders, granules, or capsules; and ointment dosage forms include, but are not limited to, creams or ointments. In some specific implementations, the compound represented by Formula I and its pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs may be administered via the enteral or non-enteric routes; non-enteric administration preparations include injections, creams, ointments, patches, sprays, etc.; routes of administration include subcutaneous, intradermal, intra-articular, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, or oral, local, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, or urethral administration, or administration via inhalation aerosol, implantation, or acupuncture.

[0051] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

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

[0053] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] Example 1

[0055] 1. Preparation of the compound shown in Formula I

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

[0057] Crude product purification: 600 mg of the crude compound was added to 2 mL of water and dissolved by sonication. The solution was then centrifuged (12000 rpm, 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: Agilent Eclipse XDB-C 18 (250×9.4mm, 5μm); flow rate: 5mL / min; detection wavelength: 235nm; mobile phase A: water, mobile phase B: methanol, gradient elution, elution program: 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 chromatographically purified solution was collected and further processed using a rotary evaporator to remove methanol. The remaining solution was collected and freeze-dried to obtain the pure compound with antitumor activity. Finally, the chromatographic purity of the new compound was determined by high-performance liquid chromatography (HPLC), showing a purity of 99.1%.

[0058] 2. Compound structure characterization

[0059] Ultraviolet (UV) spectroscopy detection: 10 mg of the novel antitumor compound was dissolved and diluted with dimethyl sulfoxide (DMSO) to a concentration of 10 μg / mL, and a UV spectral scan was performed in the range of 200–400 nm. The UV spectrum is shown below. Figure 1 As shown.

[0060] Liquid chromatography detection: New compounds with antitumor activity were detected using high-performance liquid chromatography (HPLC) with Agilent 5HC-C. 18 (2) Column (250 mm × 4.6 mm, 5 μm), flow rate 1.0 mL / min, detection wavelength 235 nm, mobile phase A was 2 mmol / L potassium dihydrogen phosphate solution (containing 0.4% triethylamine, pH adjusted to 2.5 with phosphoric acid) - acetonitrile (90:10), and mobile phase B was acetonitrile; the gradient elution program was: 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 obtained antitumor active compound is shown in the figure. Figure 2 As shown.

[0061] Mass spectrometry detection: 10 mg of the novel antitumor compound was dissolved in 50% methanol aqueous solution and diluted to a concentration of 100 ng / mL. The molecular weight was then determined using a high-resolution mass spectrometer. The high-resolution mass spectrum is shown below. Figure 3 As shown.

[0062] Nuclear magnetic resonance (NMR) spectroscopy detection: 10 mg and 20 mg of the novel antitumor compound were weighed out, dissolved in deuterated DMSO, and then analyzed by the instrument. The one-dimensional proton NMR spectrum is shown below. Figure 4 As shown, the one-dimensional carbon NMR spectrum is as follows: Figure 5 As shown.

[0063] 3. Antitumor activity

[0064] In vitro antitumor activity experiments were conducted on the new compound. HepaRG (human liver cancer cells), MKN-1 (human gastric cancer cells), PANC-1 (human pancreatic cancer cells), A549 (human lung cancer cells), and MDA-MB-231 (human breast cancer cells) cell lines were selected. The MTT assay (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) was used to study the inhibitory activity of the new compound against these five human cancer cell lines, and the drug concentration at which the inhibition rate reached 50%, i.e., IC50, was calculated. 50 Value. 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℃, 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 assay: Five types of tumor cells in the logarithmic growth phase were digested with trypsin, and the culture medium was prepared to a concentration of 1×10⁻⁶. 5 Cell suspension was cultured at 100 cells / mL and seeded into 96-well plates. After 24 hours, different concentrations of ceprtinib and its novel antitumor compounds were added, with 8 replicates for each concentration. After 48 hours, the supernatant was discarded, and the cells were washed twice with PBS. 100 μL of freshly prepared MTT solution (1 mg / mL) was added to each well. The plates were incubated for another 4 hours. Then, the supernatant in the 96-well plates was carefully discarded, and 150 μL of DMSO was added to each well. The plates were incubated at 37°C in the dark for 30 minutes. After the crystals were completely dissolved, the optical density (OD) of each well was measured at 490 nm using a microplate reader.

[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 As shown, the results indicate that ceprtinib and the novel antitumor compound all exhibit varying degrees of growth inhibition against the aforementioned tumor cells. Ceprtinib and the novel antitumor compound also showed effects on the IC50 values ​​of HepaRG cells. 50 The concentrations were 29.21 μg / mL and 21.50 μg / mL, respectively; the IC50 concentrations against MKN-1 cells were... 50 The concentrations were 40.53 μg / mL and 30.96 μg / mL, respectively; the IC50 concentrations against PANC-1 cells were... 50 The concentrations were 9.96 μg / mL and 36.95 μg / mL, respectively; the IC50 concentrations against A549 cells were... 50 The concentrations were 8.56 μg / mL and 41.87 μg / mL, respectively; the IC50 concentrations against MDA-MB-231 cells were... 50 The concentrations were 8.61 μg / mL and 53.11 μg / mL, respectively. MTT assay results showed that the antitumor activity of this new compound against PANC-1, A549, and MDA-MB-231 cells was lower than that of the active pharmaceutical ingredient (API), cepretinib; however, against HepaRG and MKN-1 cells, the antitumor activity of the new compound was superior to that of cepretinib.

Claims

1. Use of a compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor, wherein the tumor is liver cancer or gastric cancer, ； The preparation method of the compound represented by Formula I is that cipatinib is dissolved in a solvent, a degradation reaction is carried out under acidic conditions, and the compound is obtained by purification; the acidic conditions are that 150 mL of a 4 M hydrochloric acid solution is added to 300 mL of the solvent for adjustment.

2. Use according to claim 1, 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 2 m mol / L potassium dihydrogen phosphate solution containing 0.4% triethylamine, and solvent B is acetonitrile.

3. Use according to claim 1, characterized in that, The degradation reaction is carried out at 80 ℃ for 40 h.

4. Use according to claim 1, characterized in that, The purification is carried out by using a semi-preparative liquid chromatograph, and the chromatographic conditions are as follows: the chromatographic column is Agilent Eclipse XDB-C 18 18, the flow rate is 5 mL / min, the detection wavelength is 235 nm, the mobile phase A is water, the mobile phase B is methanol, gradient elution is carried out, and the elution program is as follows: 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.

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