Compound containing trifluoromethyl naphthyl urea and application of compound in tumor resistance

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a compound containing trifluoromethyl naphthyl urea and application of the compound in tumor resistance. The invention provides a novel compound 3, 3, 3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl) ureido] phenyl} ethyl propionate, and experiments prove that the novel compound 3, 3, 3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl) ureido] phenyl} ethyl propionate can be combined with GPR43 protein, is high in affinity and also has a remarkable anti-cancer effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a compound containing trifluoromethylnaphthyl urea and its application in anti-tumor treatment. Background Art

[0002] Cancer is a major disease that poses a serious threat to human health and life. Despite significant advances in cancer treatment, curing cancer still faces numerous challenges. Activating or enhancing the immune system to inhibit tumor growth and spread is a key breakthrough in cancer treatment. While significant progress has been made in the study of key tumor immune checkpoints, such as G protein-coupled receptors (GPCRs), there remains significant room for improvement in response rates, synergy, and potential as targets for tumor immunotherapy.

[0003] G protein-coupled receptor 43 (GPR43), also known as free fatty acid receptor 2 (FFAR2), belongs to the class A receptor family of GPCRs. In the immune system, GPR43 is highly expressed in immune cells such as neutrophils, macrophages, and lymphocytes. In tumors such as colon cancer, it can inhibit tumor growth by inhibiting the mTORC1 signaling pathway, and it can also affect the function of myeloid cells, enhance immunosuppressive responses, and promote tumor progression. It also interacts with intestinal flora metabolites, regulates the intestinal microenvironment, and affects tumor development. It has value as a potential drug target. Existing studies have shown that knocking out GPR43 can increase CD8 + The ability of T cells to kill tumor cells. Therefore, the development of GPR43 small molecule antagonists has great clinical significance.

[0004] Currently, the development of GPR43 as a therapeutic target in the field of oncology faces problems such as insufficient molecular skeleton diversity of fatty acids and their derivatives, poor targeting specificity, and lack of clear lead compounds. Therefore, no small molecule anti-tumor immune drugs targeting GPR43 have been approved for marketing.

[0005] In summary, there is an urgent need to propose a new GPR43 antagonist to supplement the deficiencies of existing technologies. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel GPR43 antagonist and its preparation method and application, to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0007] A novel GPR43 antagonist, wherein the novel GPR43 antagonist is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the chemical formula is C 22 H 19 F3N2O4; code name 8019-0710; molecular formula is shown in formula (I): Formula (I).

[0008] A small molecule compound or a pharmaceutically acceptable salt thereof is used in the preparation of an anti-tumor drug. The small molecule compound is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I).

[0009] Furthermore, the types of the tumor include solid tumors and / or hematological tumors.

[0010] Furthermore, the pharmaceutically acceptable salt forms include salts formed between the compound of formula (I) and inorganic or organic acids.

[0011] A small molecule compound is used in the preparation of an immunopotentiation agent targeting GPR43. The small molecule compound is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I).

[0012] Furthermore, the immune enhancement reagent also includes immune cells.

[0013] Since GPR43 is highly expressed in immune cells such as neutrophils, macrophages and lymphocytes, the compound (I) enhances the immune efficacy of the immune cells by binding to GPR43.

[0014] A GPR43-targeting immunopotentiator comprising ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula of the compound is shown in formula (I).

[0015] An antitumor drug comprising ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I).

[0016] Furthermore, the tumor drug may also contain other pharmaceutically acceptable carriers and / or adjuvants.

[0017] The term "pharmaceutically acceptable" as used herein refers to compounds, raw materials, compositions and / or preparations that, within the scope of reasonable medical judgment, can be prepared with the compound of formula (I) of the present invention into a pharmaceutically acceptable dosage form, and that have no excessive toxicity, irritation, allergic reaction or side effect upon contact with patients, and are effective for their intended use.

[0018] Furthermore, the dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration and / or an injection dosage form.

[0019] The "gastrointestinal dosage forms" described in the present invention include common tablets, capsules, solutions, suspensions, etc.

[0020] The "injection dosage form" mentioned in the present invention includes common intravenous injections, intramuscular injections or subcutaneous injections, etc.

[0021] The novel GPR43 antagonist provided by the present invention, ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate, can also be used in combination with one or more tumor treating drugs, including but not limited to the following drugs.

[0022] Chemotherapy drugs: such as cyclophosphamide, cisplatin, oxaliplatin, carboplatin, busulfan, thiotepa, mitomycin, methotrexate, pemetrexed, fluorouracil, gemcitabine, cytarabine, doxorubicin, epirubicin, irinotecan, topotecan, etoposide, taxanes, vinca alkaloids, eribulin, and asparaginase.

[0023] Small molecule targeted drugs: EGFR inhibitors: gefitinib, erlotinib, icotinib, osimertinib, ametinib, vometinib, etc.; ALK inhibitors: crizotinib, alectinib, lorlatinib, etc.; MEK inhibitors: trametinib, etc.; HER2 inhibitors: lapatinib, cilotinib, neratinib, tucatinib, etc.; PARP inhibitors: olaparib, niraparib, fluzoparib, etc.; mTOR inhibitors: everolimus, etc.; HDAC inhibitors: cedabenb, etc.; BCR-ABL inhibitors: imatinib, dasatinib, nilotinib, ponatinib, etc.; MET inhibitors: Saivotinib, Capmatinib, etc.; RET inhibitors: Pratinib, BRAF inhibitors: Dabrafenib, Vemurafenib, Encorafenib, etc., CDK4 / 6 inhibitors: Guabecib, Abemaciclib, etc., NTRK inhibitors: Larotrectinib, Entrectinib, etc.; BTK inhibitors: Ibrutinib, Zanubrutinib, etc.; JAK inhibitors: Ruxolitinib, etc.; PI3K inhibitors: Alpelisib, etc., Anti-vascular multikinase inhibitors: Anlotinib, Apatinib, Lenvatinib, Axitinib, Sunitinib, Cabozantinib, Regorafenib, Sorafenib, etc.; PDGFR / c-Kit inhibitors: Imatinib, Nilotinib, Afatinib, etc.; Protease inhibitors: Bortezomib, Ixazomib, etc.; FGFR2 inhibitors: Pemigatinib, etc.; IDH1 inhibitors: Ivosidenib, etc.; Nuclear transport protein inhibitors: Selinexor, etc.

[0024] Antibody targeted drugs: Anti-HER2: trastuzumab, pertuzumab, ZW25, KN026, etc.; Anti-EGFR: cetuximab, nimotuzumab, etc.; Anti-VEGF: bevacizumab, ramucirumab, human endostatin, etc.; Anti-CD20: rituximab, etc., Anti-CD38: daratumumab, etc.; Anti-CD19-CD3: blinatumomab, etc.; Anti-EGFR-MET: JNJ-372, etc.; Anti-DLL4-VEGF: navicixizumab, etc.

[0025] Immune checkpoint inhibitors (Anti-PD1: nivolumab, pembrolizumab, toripalimab, sintilimab, tislelizumab, carrelizumab, etc., Anti-PDL1: atezolizumab, durvalumab, sugemalimab, avelumab, etc., Anti-CTLA4: ipilimumab, etc., Anti-LAG3: relalizumab, etc., Anti-TIGIT: MK-7684A, etc., Anti-PD1 / CLTA4: KN046, XmAb207 17. Cadonilimab, etc., Anti-PD-1 / TIM-3: RO-7121661, etc., Anti-PD1 / CD27: CDX-527, etc., Anti-PDL1 / TGFB: JS201, etc., Anti-PD1 / PDL1: Reozalimab, IB1318, etc., Anti-PD-1 / VEGF: Ivonescimab, etc., Anti-PD-1 / HER2: Fidasimtamab, etc.).

[0026] Immunomodulators: such as thalidomide, lenalidomide, thymosin, retinoic acid, etc.

[0027] Antibody-drug conjugates: brentuximab, U3-1402, trastuzumab emtansine, etc.

[0028] Hormones: tamoxifen, toremifene, letrozole, anastrozole, enzalutamide, bicalutamide, abiraterone, etc.

[0029] Cell therapy: CAR-T, CAR-NK, etc.

[0030] Beneficial technical effects: The present invention provides a novel compound, ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate, which has a parent ring structure of trifluoromethylnaphthylurea. Experiments have shown that ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate can bind to the GPR43 protein with high affinity, and therefore has the potential to be prepared as a GPR43 antagonist and related drugs targeting GPR43. Furthermore, the present invention also verifies that ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate has immune activation efficacy and anti-tumor efficacy. It can significantly promote the expression of cytokines TNF-α and IFN-γ, and exhibits effective anti-cancer effects in animal experiments. Finally, ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate has the characteristics of skeleton diversity, which provides direction and ideas for subsequent drug design and optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0032] Figure 1 The NMR spectrum of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate provided in one embodiment of the present invention; Figure 2 This is the result of verifying the expression of the tumor-promoting factor of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate in one of the embodiments of the present invention; Figure 3 This is an experiment on subcutaneous transplanted tumors in mice in one of the embodiments of the present invention to verify the anti-tumor effect of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate; Figure 4 This is the analysis result of the binding of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate to GPR43 protein in one of the examples of the present invention; Figure 5 This is the analysis result of the binding of the reference compound to the GPR43 protein in one of the examples of the present invention. DETAILED DESCRIPTION

[0033] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0036] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0037] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0038] Definition of noun: The "antagonist" mentioned in the present invention refers to a class of chemical substances that do not cause biological effects themselves after binding to a receptor, but block the effects mediated by the receptor agonist.

[0039] Currently available antagonists include but are not limited to: (1) Chemokine receptor antagonists: Plerixafor: CXCR4 antagonist, used to mobilize hematopoietic stem cells into the bloodstream in cancer patients. (2) 5-HT1F receptor antagonists: Lasmiditan: selective 5-HT1F receptor antagonist, used to treat migraines. (3) Histamine receptor antagonists: Pitolisant: selective histamine H3 receptor antagonist, used to treat daytime sleepiness associated with narcolepsy. (4) Endopeptidase receptor antagonists: Fosnetupitant: endopeptidase receptor antagonist, used for chemotherapy-induced nausea and vomiting. (5) CGRP receptor antagonists: Erenumab: CGRP receptor antagonist monoclonal antibody, used to prevent migraines.

[0040] Example 1 This example provides a GPR43 antagonist, ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate, code-named 8019-07101, and its structural characterization is shown in the NMR image. Figure 1 shown.

[0041] Example 2 Verification of tumor-promoting factor expression of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate.

[0042] Experimental method: Jurkat cells were co-cultured with the compound of formula I at a concentration of 10 μM, and DMSO was set as the control group. After 24 hours of culture, the culture supernatant was collected and the relative expression levels of cytokines TNF-α and IFN-γ were detected by qRT-PCR (HiScript II One Step gRT-PCR SYBRGreen Kit Q221-01) to evaluate the activation of immune cells by the compound of formula I. The cytokine CD8 + / TNF-α + and CD8 + / IFN-γ + changes.

[0043] Experimental results: see Figure 2 . Figure 2 AB represents the effect of RT-qPCR detection of the expression of TNF-α and IFN-γ in Jurkat cells by the compound of formula I, which shows that the compound of formula I can significantly promote the expression of TNF-α and IFN-γ compared with the control. Figure 2 C is the flow cytometry analysis of CD8 in mouse spleen cells after administration of the compound of formula I + / TNF-α + and CD8 + / IFN-γ + Cell changes showed that the compound of formula I can stimulate CD8 + / TNF-α + and CD8 + / IFN-γ + The increase of cells. Figure 2 DE is the CD8 in mouse spleen cells detected by flow cytometry after administration of the compound of formula I + / TNF-α + and CD8 + / IFN-γ + The percentage expression statistics of cell changes showed that the compound of formula I can promote CD8 + / TNF-α + Cells increased by 13.5%, promoting CD8 + / IFN-γ + This example demonstrates that ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate has an immune-activating effect and has the potential to be combined with immune cells to prepare an immunopotentiator for clinical research.

[0044] Example 3 The mouse subcutaneous transplant tumor experiment verified the killing effect of the compound of formula I on liver cancer cells.

[0045] Experimental method: BALB / C mice were subcutaneously inoculated with H22 hepatoma cells (1×10 5 On the 8th day after inoculation, the mice were intraperitoneally injected with 5 mg / kg of the compound of formula I every other day. The mice were euthanized on the 16th day for pathological analysis.

[0046] Experimental results: see Figure 3 . Figure 3 A is the experimental scheme of subcutaneous tumor transplantation in mice. Figure 3 B shows the changes in tumor volume in the mouse subcutaneous transplant tumor model; Figure 3 C is a picture of the H22 transplanted tumor at the end of the experiment, showing that the tumor volume was significantly reduced after administration of the compound of formula I. Figure 3 D is the weight change of mice during the experiment; Figure 4 E is the weight of the tumor tissue at the end of the experiment. This indicates that the body weight of mice administered with the compound of Formula I increased slightly compared to the control group, but tumor tissue weight was significantly suppressed, demonstrating that the compound of Formula I has a significant anti-tumor effect. Figure 3 FG is a test of blood cells and liver function in experimental mice. The results are close to those of the control group, indicating that the compound of formula I has good safety.

[0047] Example 4 Analysis of the binding affinity of ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalen-2-yl)ureido]phenyl}propionate to GPR43 protein.

[0048] The binding affinity and potential binding mechanism of the compound of formula I to GPR43 protein were revealed by plasma resonance (SPR) experiments and computer molecular docking technology.

[0049] Experimental Methods: Samples of Formula I compound and GPR43 recombinant protein were prepared. A CM5 sensor chip was activated with a mixture (400 mM EDC and 100 mM NHS) for 420 seconds at a flow rate of 10 μL / min. GPR43 recombinant protein was diluted to 20 μg / mL in immobilization buffer and injected into the sample channel at a flow rate of 10 μL / min. The chip was deactivated with 1 M ethanolamine hydrochloride at a flow rate of 10 μL / min for 420 seconds. Formula I compound was diluted to eight concentrations in the same analyte buffer and injected into the channel at a flow rate of 20 μL / min. The association phase was set to 100 seconds, followed by a dissociation phase of 180 seconds. Eight cycles of analysis were repeated in ascending order of compound concentration. Based on the experimental data, binding affinity curves were plotted and statistical analysis of the binding affinities was performed to compare the effects of different compound concentrations on binding affinity.

[0050] Experimental results: see Figure 4. Figure 4 A is the result of the SPR technique determination of the binding affinity of the compound of formula I to GPR43, and its binding affinity is K D =12.5 μM. Figure 4 Figure B shows the GPR43 protein-ligand binding pattern analyzed by computational molecular docking. The results indicate that the compound of Formula I is primarily located in the central binding pocket comprised of the transmembrane regions (TM3, TM4, TM5, and TM6). Residues F87, M136, V144, H140, L183, and L187 primarily form hydrophobic interactions with the ligand, while residue H242 forms hydrogen bonds with residues B019-B0710. This binding pattern suggests that the compound of Formula I may inhibit GPR43 function and promote the expression of anti-tumor cytokines through these key interactions. The binding energy of the compound of Formula I to GPR43 is -12.31 kcal / mol.

[0051] Example 5 This example provides a comparison of the predicted binding abilities of representative naphthyl ureas to GPR43.

[0052] Binding energy prediction of the representative naphthyl urea compound IY210316B-1 on GPR43.

[0053] Reference compound: IY210316B-1; the structural formula is shown in formula (II).

[0054] Formula (II).

[0055] Experimental method: The same method as in Example 4 was used to select a representative naphthyl urea compound (compound of Formula II) for binding energy prediction with GPR43 protein by computer molecular docking technology, and the 3D structure of the compound was obtained using Obabel software.

[0056] Experimental results: see Figure 5 The binding energy of the compound of Formula II to the GPR43 protein was -9.88 kcal / mol, while the binding energy of the compound of Formula I to the GPR43 protein was -12.31 kcal / mol. The results showed that the compound of Formula I had a stronger binding affinity for GPR43 than the compound of Formula II and had a better anti-tumor effect.

[0057] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A novel GPR43 antagonist, characterized in that: The novel GPR43 antagonist is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I): Formula (I).

2. Use of a small molecule compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, characterized in that: The small molecule compound is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I): Formula (I).

3. The use according to claim 2, characterized in that The types of tumors include solid tumors and / or hematological tumors.

4. The use according to claim 2, characterized in that The pharmaceutically acceptable salt forms include salts formed between the compound of formula (I) and inorganic or organic acids.

5. Use of a small molecule compound in the preparation of an immunopotentiation agent targeting GPR43, characterized in that: The small molecule compound is ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I): Formula (I).

6. The use according to claim 5, characterized in that The immune enhancement reagent also includes immune cells.

7. An immunopotentiator targeting GPR43, characterized in that: The immunopotentiator comprises ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I): Formula (I).

8. An anti-tumor drug, characterized in that: The anti-tumor drug comprises ethyl 3,3,3-trifluoro-2-hydroxy-2-{4-[3-(naphthalene-2-yl)ureido]phenyl}propionate; the molecular formula is shown in formula (I): Formula (I).

9. The antitumor drug according to claim 8, characterized in that The tumor drug may further comprise other pharmaceutically acceptable carriers and / or adjuvants.

10. The antitumor drug according to claim 8, characterized in that The dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration and / or an injection dosage form.

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