A piperazine indole amide compound, its preparation method and application

Abstract

The present invention provides a piperazine indole amide compound, a preparation method and an application thereof. The bioactive piperazine indole amide parent nucleus group contained therein is further chemically modified to produce many compounds with higher biological activities or pharmaceutically acceptable salts, hydrates, solvates, metabolites, prodrugs or pharmaceutical compositions thereof, expanding the wide application of such compounds in biomedicine and the prospect of pharmaceutical formulation development. Such compounds can target the STAT3 protein, significantly inhibit the proliferation of various tumor cells such as lung cancer, breast cancer, liver cancer, pancreatic cancer and gastric cancer at low doses (nanomolar), and can effectively inhibit the growth of transplanted tumors in mice, indicating that such compounds have the prospect of being developed into drugs for preventing and / or treating tumors and other diseases related to abnormal STAT3 signaling.

Description

Technical Field

[0001] The present invention belongs to the field of tumor targeted therapy, and particularly relates to a piperazine indole amide compound, a preparation method thereof and an application thereof. Background Art

[0002] Cancer has become an important cause leading to premature death and shortened lifespan of the global population. In 2020, there were approximately 19.3 million newly diagnosed cancer cases globally, and nearly 10 million people died from cancer. Experts predict that by 2035, the number of cancer patients globally will increase by half. Moreover, the incidence and mortality rates of cancer in China account for a high proportion globally. Although there have been great improvements in the current clinical treatment methods for cancer, due to the heterogeneity and continuous evolution of tumors, the response rates of most drugs are low and drug resistance is prone to occur, resulting in a still severe lack of effective therapeutic drugs. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin and capecitabine have the advantages of broad anti-cancer spectrum and good efficacy, but they have large toxic and side effects and are prone to early drug resistance. Therefore, actively exploring and developing new anti-cancer drugs has important clinical significance.

[0003] Signal transducer and activator of transcription 3 (STAT3) is one of the core regulatory factors of cell signal transduction. Abnormal activation of STAT3 signaling exists in approximately 70% of human malignant tumor cells. Activated STAT3 can regulate the transcriptional expression of almost the entire genomic target genes and participate in various pro-cancer events. For example, by upregulating the expression of cyclin D1 or proto-oncogene c-MYC, it promotes the proliferation of tumor cells; by upregulating the expression of anti-apoptotic genes such as BCL-2, BCL-XL, and Survivin, it promotes the survival of tumor cells; by upregulating the expression of vascular endothelial growth factor (VEGF), it promotes tumor angiogenesis; by upregulating the expression of IL-6, IL-10, matrix metalloproteinases (MMPs), and transforming growth factor-β (TGF-β), it regulates the epithelial-mesenchymal transition (EMT) and tumor stem cell (CSC)-like transformation of tumor cells. A large number of studies have shown that STAT3 is significantly associated with poor prognosis of tumors such as lung cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, and leukemia. Knockdown or inhibition of STAT3 expression can significantly inhibit tumor proliferation, survival, angiogenesis, drug resistance, immune escape, metastasis, and recurrence. Therefore, the treatment method targeting STAT3 has the advantages of a broad anti-cancer spectrum and good efficacy. In this application, a class of piperazine indole amide compounds with a completely new structural formula was synthesized. Through some biological technology analyses, it was found that this class of compounds can significantly inhibit the protein expression and phosphorylation of STAT3 at extremely low doses and have good tumor inhibitory activity in various tumor cells such as lung cancer, breast cancer, liver cancer, pancreatic cancer, and gastric cancer. Therefore, further development of such compounds will be of great significance in the application of tumor treatment. Summary of the Invention

[0004] The object of the present invention is to provide a piperazine indole amide compound, a preparation method thereof, and an application.

[0005] Based on the above object, the present invention adopts the following technical solutions:

[0006] A piperazine indole amide compound, the structural formula of which is shown in general formula I:

[0007]

[0008] Wherein, R1 is selected from H, F, Cl, Br, I, -CN, -CH3, -CF3, -OCH3, -OCF3, -SO2NH2;

[0009] R2 is selected from H, F, Cl, Br, I, -CN, -CH3, -CF3, -OCH3, -OCF3, -COOH, OH,

[0010] R3 is selected from H, F, Cl, Br, I, -CN, -CH3, -CF3, -OCH3, -OCF3, -COOH, OH;

[0011] R4 is selected from H, F, Cl, Br, I, -CN, -CH3, -CF3, -OCH3, -OCF3, -COOH, OH;

[0012] R5 is selected from H, F, Cl, Br, I, -CN, -CH3, -CF3, -OCH3, -OCF3, -COOH, OH;

[0013] W, X, G are each independently selected from C, N;

[0014] K is selected from O, NH.

[0015] The above-mentioned piperazine indole amide compounds are specifically compounds with the following structures:

[0016]

[0017] Biologically acceptable salts formed by the above-mentioned piperazine indole amide compounds with at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediaminetetraacetic acid.

[0018] The preparation method of the above-mentioned piperazine indole amide compounds has the following synthetic route:

[0019]

[0020] The specific synthesis steps are as follows:

[0021] (1) Dissolve compound 1, compound 2, HBTU and DIEA in DMF, stir and react completely at 45 - 55 °C, then dilute the reaction solution with ethyl acetate, wash it with saturated brine, dry the organic phase by evaporation, and slurry it with ethyl acetate to obtain compound 3;

[0022] (2) Dissolve compound 3, compound 4 and potassium carbonate in DMF, stir and react completely at 75 - 85 °C, then dilute the reaction solution with ethyl acetate, wash it with saturated brine, dry the organic phase by evaporation, and slurry it with ethyl acetate to obtain the target compound;

[0023] Further, in step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:1.2:3; in step (2), the molar ratio of compound 3, compound 4 and potassium carbonate is 1:1.1:1.2.

[0024] Use of the piperazine indole amide compound and its biologically acceptable salt as described above in the preparation of anti-tumor drugs.

[0025] Preferably, the anti-tumor drug refers to a drug for treating lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, renal cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome and melanoma cells.

[0026] Specifically, the present invention synthesized a class of piperazine indole amide compounds with a completely new structure, such as IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1, etc. The proliferation inhibitory effect of such compounds on various cancer cells was detected by the CCK-8 method.

[0027] The results showed that the compounds IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 of the present invention can effectively inhibit the proliferation of lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, renal cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome and melanoma cells.

[0028] In summary, the present invention provides the use and potential molecular mechanism of a new piperazine indole amide compound and its derivatives in tumor treatment. Description of the Drawings

[0029] Figure 1 It is the inhibitory effect of IG230309C-1 on the expression and phosphorylation of STAT3 protein in MGC803 cells detected by Western blot. Detailed Embodiments

[0030] In order to make the technical objectives, technical solutions and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below with reference to the drawings and specific embodiments.

[0031] In the method for synthesizing the compound of formula I in the present invention, various raw materials used in the reaction can be prepared by those skilled in the art according to the existing knowledge, or can be prepared by methods known in the literature, or can be commercially purchased. The intermediates, raw materials, reagents, reaction conditions, etc. used in the above reaction scheme can be appropriately changed according to the existing knowledge of those skilled in the art.

[0032] In the present invention, unless otherwise specified, (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20 - 30 °C; (ii) the organic solvents are dried by common drying methods, the evaporation of the solvent is carried out under reduced pressure using a rotary evaporator, and the bath temperature is not higher than 50 °C; the developing agent and eluent are both volume ratios; (iii) the reaction process is tracked by thin layer chromatography (TLC); (iv) the final product has satisfactory proton nuclear magnetic resonance (1H-NMR).

[0033] Example 1: The synthesis of all compounds refers to the following route

[0034] Specific synthesis method, taking the compound IG230309C-1 as an example, the structural formula is as follows:

[0035]

[0036] The name of the compound IG230309C-1 is (1-methyl-6-((5-(1-methyl-1H-indol-2-yl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone,

[0037] The synthesis route is as follows:

[0038]

[0039] Step 1.(6-hydroxy-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)

[0040] benzyl)piperazin-1-yl)methanone (Compound 3)

[0041] Compound 1 (2.0 g, 10.46 mmol, 1.0 eq), compound 2 (2.87 g, 10.46 mmol, 1.0 eq), HBTU (4.77 g, 12.55 mmol, 1.2 eq), and DIEA (4.06 g, 31.38 mmol, 3.0 eq) were dissolved in 30 mL of DMF and reacted at 50°C for 3 hours. Completion of the reaction was monitored by TLC. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL x 3). The organic phase was dried and spin-dried, purified by slurrying with 15 mL of ethyl acetate, and filtered to obtain 3.75 g of compound 3 as a white solid in an 80.1% yield.

[0042] 1 H NMR(CDCl3,300MHz)δ:9.05(d,J=1.1Hz,1H),8.70(d,J=1.1Hz,1H),8.38(d,J=8.2Hz,2H ),7.84(d,J=8.4Hz,2H),7.35(s,1H),6.95(d,J=10.0Hz,2H),4.83(s,4H),1.59(s,9H).

[0043] Step 2.(1-methyl-6-((5-(1-methyl-1H-indol-2-yl)pyrazin-2-yl)oxy)-1H

[0044] -indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone(IG230309C-1)

[0045] Compound 3 (2.0 g, 4.47 mmol, 1.0 eq), compound 4 (1.20 g, 4.92 mmol, 1.1 eq) and potassium carbonate (741 mg, 5.36 mmol, 1.2 eq) were dissolved in 30 mL of DMF and reacted at 80 °C for 12 h. TLC monitoring showed that the raw materials had reacted completely and new spots had appeared. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL * 3). The organic phase was dried and concentrated by rotary evaporation, and then triturated and purified with 25 mL of ethyl acetate and filtered to obtain 1.80 g of white solid compound IG230309C-1, with a yield of 61.4%.

[0046] 1 H NMR (CDCl3, 300 MHz) δ: 9.05 (d, J = 1.1 Hz, 1H), 8.70 (d, J = 1.1 Hz, 1H), 8.38 (d, J = 8.2 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.35 (s, 1H), 6.95 (d, J = 10.0 Hz, 2H), 4.83 (s, 4H)

[0047] Example 2: Proliferation inhibitory effects of IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1 on lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, renal cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome and melanoma cells

[0048] H460, MDA-MB-468, HepG2, BxPC-3, MKN-45, TPC-1, U251, CNE-1, TE-11, HuH28, SW480, NTERA-2cl.D1, Thy0517, GRC-1, PC-3, EJ-1, Hela, SKOV3, WERI-Rb-1, MPP-89, U2OS, Jurkat, MM1.S, U937, SKM-1 and MEL202 cells in the logarithmic growth phase were collected respectively, counted, and the cell suspension concentration was adjusted to 5×10 4cells / mL, add them to a 96-well cell culture plate, with a volume of 100 μL per well. Using DMSO as a solvent control, dilute the compounds IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 of the present invention with DMSO and add them to the culture wells, so that the final concentrations of the compounds in the system are 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 (μmol / L) respectively. After continuing the culture for 48 h, add 10 μL of CCK-8 solvent to each well, incubate at 37 °C for 1 h, read the value with an enzyme-linked immunosorbent assay (ELISA) reader, measure the OD value at an absorption wavelength of 450 nm, record the results, and plot the cell growth curve with the dose of the compound as the abscissa and the absorbance value as the ordinate. The statistical results of the half-maximal inhibitory concentration (IC50 value) of the compounds against tumor cells are shown in Tables 1 to 4 below:

[0049] Table 1. Inhibitory effect of CCK-8 assay on the proliferation of IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 on lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, and thyroid cancer cells

[0050]

[0051] Table 2. CCK-8 assay of the inhibitory effect of IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 on the proliferation of glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer and thymoma cells

[0052]

[0053]

[0054] Table 3. CCK-8 assay of the inhibitory effect of IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 on the proliferation of renal cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma and mesothelioma cells

[0055]

[0056]

[0057] Table 4. Proliferation inhibitory effects of IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 on osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome and melanoma cells

[0058]

[0059]

[0060] Data in Tables 1, 2, 3 and 4 show that IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 have good proliferation inhibitory effects on lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, kidney cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome and melanoma cells. In this application, taking IG230309C-1 as an example, the in vivo anti-tumor mechanism of this kind of compound was further studied.

[0061] Example 3. Western blot detection of the regulatory effect of IG230309C-1 on STAT3 protein expression

[0062] Inoculate MGC803 cells in the logarithmic growth phase into a 6-well cell culture plate, 4×10 per well 5Cells. After the cells adhered to the wall, IG230309C-1 was added to make its final concentrations 0, 30, 100, 300, and 1000 nM respectively. After about 24 h, the cells were lysed with RIPA lysis buffer to collect proteins, and Western blot analysis was performed. The expression levels of the corresponding proteins were detected by anti-STAT3, p-STAT3-705, p-STAT3-727, and GAPDH antibodies respectively.

[0063] The results are as Figure 1 shown that compared with the solvent control wells, after treatment with IG230309C-1, the expression levels of Total-STAT3, p-STAT3-Tyr705, and p-STAT3-Ser727 could be significantly inhibited, and it was dose-dependent. It indicated that the compound IG230309C-1 could target and inhibit the expression and phosphorylation of STAT3 protein.

[0064] In summary, the above results showed that IG230307C-1, IG230308C-1, IG230309C-1, IG230310C-1, IG230311C-1, IG230312C-1, IG230313C-1, IG230314C-1, IG230315C-1, IG230316C-1, IG230317C-1, IG230318C-1, IG230319C-1, IG230320C-1, IG230321C-1, IG230322C-1, IG230323C-1, IG230324C-1 could significantly inhibit the proliferation of lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, kidney cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome, and melanoma cells, and the antitumor mechanism of such compounds was related to targeting and inhibiting the expression and activation of STAT3 signaling. Therefore, such drugs had good anti-cancer effects and development potential.

[0065] According to the general approach of drug development (first conducting conventional in vitro anti-tumor screening, and then conducting targeted research), the compounds of the present invention could be applied to anti-cancer drugs related to abnormal cell proliferation, and anti-tumor drugs could be prepared by salifying with a pharmaceutically acceptable substance or mixing with a pharmaceutical carrier.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Any equivalent replacement of the present invention and modifications or partial replacements without departing from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A piperazine indole amide compound, characterized in that, Specifically, a compound having the following structure:

2. A biologically acceptable salt formed by the piperazine indole amide compound according to claim 1 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.

3. Use of the piperazine indole amide compound according to claim 1 or 2 and its biologically acceptable salt in the preparation of an anti-tumor drug.

4. The application according to claim 3, wherein: The anti-tumor drug refers to a drug for treating diseases related to STAT3 signal transduction.

5. The application according to claim 3 or 4, characterized in that: The tumors include lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, thyroid cancer, glioma, head and neck cancer, esophageal cancer, cholangiocarcinoma, colorectal cancer, testicular cancer, thymoma, kidney cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, retinoblastoma, mesothelioma, osteosarcoma, lymphoma, multiple myeloma, leukemia, chronic myelodysplastic syndrome, and melanoma.

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