Selective inhibitors of txnrd1 and uses thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to a propynoic amide derivative targeting TXNRD1 and application thereof in the aspect of anti-tumor, and discloses an inhibitor selectively targeting TXNRD1, namely a propynoic amide derivative small-molecule compound, a structure of which is shown as formula (I). The molecule plays an anti-tumor role by targeting TXNRD1, and shows obvious anti-tumor effects in the aspects of molecular level, cell level and mouse models.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a propynoic amide derivative targeting TXNRD1 and application thereof in the aspect of anti-tumor. BACKGROUND

[0002] At present, there are nearly one hundred drugs for treating cancer, and various innovative therapies have made breakthrough progress, but in fact, chemotherapy is still one of the most important systemic anticancer methods at present. The anti-tumor drugs currently used in clinical practice cannot meet the requirements of treatment, and there is still a lack of effective drugs for treating tumors. Therefore, it is of great significance to further develop new anti-tumor drugs.

[0003] The TXN system is an important regulatory system for the intracellular redox homeostasis and signal transduction. TXNRD reduces the physiological substrate TXN. Subsequently, TXN reduces its downstream molecular targets, which play an important role in the growth, proliferation and differentiation of cells. Studies have shown that TXNRD is highly expressed in tumors and is a potential target for cancer treatment. A large number of drugs exhibit anti-tumor activity by inhibiting TXNRD.

[0004] So far, there is no officially approved TXNRD inhibitor drug molecule on the market, most of which are in the preclinical research stage, mainly including natural products and artificially synthesized drugs, such as small white chrysanthemum lactone, costunolide, curcumin, goldin and cisplatin. There are relatively few selective TXNRD inhibitors in clinical research. Therefore, it is of great scientific research significance and clinical medical application value to develop new small molecule inhibitors targeting TXNRD and reveal the interaction mechanism between the inhibitors and TXNRD. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a selective TXNRD1 inhibitor, which is a propynoic amide derivative small molecule compound. The present application found that the propynoic amide derivative small molecule compound has good anti-tumor activity and has a wide range of applications in the preparation of anti-tumor drugs.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A selective TXNRD1 inhibitor, which is a propynoic amide derivative small molecule compound (the molecular formula of which is C 26 H 22 BrFN2O4, the molecular weight of which is 525.37), the structure of the inhibitor is shown in formula (I):

[0008]

[0009] The application also provides the use of the selective TXNRD1 inhibitor in the preparation of a drug targeting TXNRD1.

[0010] The application also provides the use of the selective TXNRD1 inhibitor in the preparation of an anti-tumor drug.

[0011] The application also provides the use of the propynoic amide derivative small molecule compound in the preparation of an anti-tumor drug.

[0012] Preferably, the tumor includes, but is not limited to, breast cancer.

[0013] The application also provides the use of the selective TXNRD1 inhibitor in the preparation of a drug for inhibiting the growth of tumor cells.

[0014] Preferably, the tumor cells include, but are not limited to, breast cancer cells.

[0015] The application also provides a drug for selectively targeting TXNRD1, an anti-tumor drug, or a drug for inhibiting the growth of tumor cells, wherein the drug takes the selective TXNRD1 inhibitor as a main active ingredient.

[0016] Preferably, in the above use and drug regimen, the selective TXNRD1 inhibitor also includes a propynoic amide derivative small molecule compound mixed with a pharmaceutically acceptable carrier and / or excipient to prepare a composition and a clinically acceptable dosage form.

[0017] Preferably, the excipient refers to diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, and other pharmaceutical bases used in the pharmaceutical field.

[0018] Preferably, the carrier refers to functional pharmaceutical excipients acceptable in the pharmaceutical field, including surfactants, suspending agents, emulsifying agents, and some novel pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), hyaluronic acid, etc.

[0019] Preferably, the dosage form refers to injection, tablet, capsule, etc. commonly used in the clinic. The pharmaceutical preparation can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally), and if some drugs are unstable under the stomach condition, they can be prepared into enteric-coated tablets.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The propynoic amide derivative small molecule compound in the application can specifically target TXNRD1, thereby achieving killing of tumor cells. The propynoic amide derivative small molecule compound has a strong anti-tumor effect at the molecular level, the cell level and the cancer mouse model, and the anti-tumor activity is better than that of other TXNRD1 inhibitors, and is expected to be prepared into an anti-tumor drug. The propynoic amide derivative small molecule compound has a good inhibitory effect on human breast cancer cells (MDA-MB-231), and the IC 50 value can be as low as 4.72 nM, and can be prepared into an anti-breast cancer drug. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Chemical formula for preparation of the propynoic amide derivative small molecule compound;

[0023] Figure 2 XF-5079-D is the test result of TXNRD1 at the molecular level and the cell level, A is the IC 50 curve of the inhibition of the compound on the recombinant TXNRD1 pure enzyme, B is the IC 50 curve of the inhibition of the compound on the cell TXNRD, and C is the anti-proliferation effect of the compound on various cells in the in-vitro test.

[0024] Figure 3 A is the change of the tumor volume within 27 days of administration, B is the mass diagram of the breast cancer subcutaneous tumor peeled off after 27 days of administration, C is the appearance diagram of the breast cancer subcutaneous tumor peeled off after 27 days of administration, and D is the tumor inhibition rate of the compound and the positive control paclitaxel.

[0025] Figure 4 Molecular structural formula of the inhibitor. DETAILED DESCRIPTION

[0026] The specific embodiments of the application are further described below. It should be noted that the description of these embodiments is used to help understand the application, but does not constitute a limitation on the application. In addition, the technical features involved in each embodiment of the application described below can be combined with each other as long as there is no conflict.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0028] Example 1

[0029] The present embodiment provides a preparation method of a propynoic amide derivative small molecule compound (XF-5079-D).

[0030] The research finds that the propynamide derivative small molecule compound can specifically target TXNRD1, and the preparation method of the propynamide derivative small molecule compound is carried out according to the reaction formula shown in the description Figure 1

[0031] According to Figure 1 The reaction formula is shown in the description, and the compound shown in formula 1 (imine, 133 mg, 0.48 mmol), the compound shown in formula 2 (propynamide, 27 mg, 0.40 mmol), rhodium octanoate (Rh2(oct)4, 3 mg, 0.004 mmol), phosphoric acid (binaphthyl phosphate, 3.5 mg, 0.01 mmol), and water (0.5 mL) are added to a 25 mL single-neck flask, and the mixture is stirred at room temperature for 12 hours. Molecular sieves (50 mg) are added to a 25 mL single-neck flask, treated with nitrogen replacement, and then anhydrous dichloromethane solvent (DCM, 8 mL) is added to prepare a mixed solution a, and the temperature is reduced to 0°C; formula 3 (diazonium compound, 123 mg, 0.60 mmol) is dissolved in anhydrous dichloromethane solvent (DCM, 8 mL) to prepare solution b, and the mixed solution b is slowly injected into solution a by using a peristaltic pump, and the injection is completed in about 2 hours, and after the dripping is completed, the solution is stirred at 0°C for 8 hours. TLC detects that the consumption of diazonium raw materials is complete, the molecular sieves are removed by filtration, the filtrate is concentrated under reduced pressure, and the crude product is separated by column chromatography (PE:EA=15:1) to obtain a white solid, which is recrystallized with petroleum ether and dichloromethane to obtain white crystals of formula (I) (XF-5079-D, 160 mg, 76%). The spectral information of the product is as follows:

[0032] 1 H NMR (500 MHz, CDCl3) δ 7.5-7.4 (m, 2H), 7.2-7.1 (m, 2H), 7.1-6.9 (m, 4H), 6.9-6.8 (m, 2H), 6.4-6.3 (m, 1H), 6.3-6.2 (m, 1H), 6.2-6.1 (m, 1H), 5.7 (d, J=6.8 Hz, 1H), 5.4 (d, J=6.8 Hz, 1H), 3.9 (s, 3H), 3.8 (s, 3H), 2.8 (s, 1H). 13 C NMR (125 MHz, CDCl3) δ 170.59, 164.88, 162.94, 159.62, 151.17, 147.69, 147.61, 136.70, 131.67, 130.42, 130.34, 129.64, 129.08, 127.13, 122.47, 113.68, 109.27, 109.25, 104.74, 104.57, 100.46, 100.25, 73.87, 69.54, 59.06, 55.29, 53.79. 19 F NMR (471 MHz, CDCl3) δ -112.47.​

[0033] Example 2

[0034] As Figure 2 shown in the following, this example provides the determination of the TXNRD1 enzyme activity in vitro by the propynylamide derivative (XF-5079-D).

[0035] (1) Determination of recombinant TXNRD1 enzyme activity by DTNB reduction method

[0036] 1) Preparation of compounds, enzymes and buffers

[0037] ① Prepare phosphate PB buffer (pH 7.4) containing EDTA (0.4 mM).

[0038] ② Dilute the recombinant pure enzyme Thioredoxin reductase 1 (rat, recombinant, purchased from Cayman Company, Item No. 30586) 200 times with the prepared PB buffer.

[0039] ③ Prepare 1 mM NADPH solution and 15 mM DTNB solution with the prepared PB buffer, and dilute the compound XF-5079-D with DMSO to a mother liquor with a target concentration of 60X.

[0040] ④ Determine the residual activity of the enzyme using a 100 uL standard reaction system in a 96-well plate: add 56 uL PB buffer, 3 uL pure enzyme, and 1 uL compound XF-5079-D to each well in turn, and incubate at 37°C for 30 minutes.

[0041] 2) Determination of absorbance

[0042] After incubation, quickly add 20 uL of NADPH and DTNB solution to each well, and track the amount of TNB generated at 412 nm within 10 minutes by kinetic detection, which is used to calculate the enzyme activity. -

[0043] 3) Experimental results

[0044] Process the data obtained by kinetic detection for 1 minute, and the half-inhibitory concentration of the compound on the recombinant TXNRD1 enzyme activity is shown in Table 1

[0045] Table 1 Half-inhibitory concentration of recombinant TXNRD1 enzyme activity

[0046] Compound Half maximal inhibitory concentration of recombinant TXNRD1 enzyme activity XF-5079-D 0.86 ± 0.36 nM

[0047] (2) Determination of TXNRD enzyme activity in cells

[0048] 1) Cell culture

[0049] ​The MDA-MB-231 cells (purchased from ATCC) used in this experiment were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and incubated at 37°C in a 5% CO2 cell culture incubator.

[0050] 2) Determination of cellular TXNRD enzyme activity using the DTNB reduction method

[0051] ① The reagent used in this experiment was a thioredoxin reductase activity assay kit (Solarbio, BC1115). Different concentration gradients of compounds were used to treat 7 x 10⁻⁶ wells in a six-well plate. 5 After incubating the adhered cells for 2 hours, process and measure the samples according to the kit instructions.

[0052] ② Protein concentration was determined using the BCA method. The reagent used in this experiment was the BCA protein concentration assay kit (Beyotime P0010). The protein concentration in the cell lysate supernatant obtained in the enzyme activity experiment was determined according to the kit steps.

[0053] 3) Experimental Results

[0054] TNB at 412nm was tracked by dynamic detection. - The amount produced within 10 minutes was used to reflect enzyme activity. The resulting cellular TXNRD activity was normalized using protein concentrations measured by the BCA method. Data obtained from kinetic assays over 3 minutes were processed, and the half-maximal inhibitory concentrations (IC50) of the compounds on cellular TXNRD enzyme activity are shown in Table 2.

[0055] Table 2. Half-maximal inhibitory concentration of TXNRD enzyme activity

[0056] Compound Half maximal inhibitory concentration of TXNRD enzyme activity XF-5079-D 55.87 ± 29.51 nM

[0057] Example 3

[0058] like Figure 2 As shown in this embodiment, the inhibitory effect of propyneamide derivative (XF-5079-D) on tumor cell growth in an in vitro model was determined.

[0059] (1) CCK-8 assay for cell viability

[0060] 1) Cell culture:

[0061] MDA-MB-231, LX-2 (purchased from ATCC) cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double-antibiotic (penicillin and streptomycin), and SKOV3, Caki-1, Kyse-520 (purchased from ATCC) cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double-antibiotic (penicillin and streptomycin). The above cells were placed in a 37°C, 5% CO2 incubator for culture.

[0062] 2) Compound activity test

[0063] ①Inoculate cell suspension 200uL (5000 cells / well) in a 96-well plate, and place the 96-well plate in a 37°C, 5% CO2 incubator for culture for 24h. Dissolve the compound with DMSO, add different concentrations of the compound to the culture plate 1uL, and continue to incubate for 48h.

[0064] ②Discard the culture medium, add 10% CCK-8 fresh culture medium, and incubate at 37°C, 5% CO2 for 2h, then measure the absorbance at 450nm.

[0065] 3) Experimental results

[0066] As shown in Table 3, compound XF-5079-D showed nanomolar range IC 50 in various cell lines, with certain cell line differences, and the best activity in triple-negative breast cancer cell MDA-MB-231, with a selectivity of 300 times for normal human hepatic stellate cell line LX2.

[0067] Table 3 IC of compound XF-5079-D in various cell lines 50

[0068]

[0069] Example 4

[0070] As Figure 3 shown, this example provides a study on the inhibitory effect of propynylamide derivative (XF-5079-D) on tumor growth in an in vivo model.

[0071] (1) Effect on mouse subcutaneous breast cancer 4T1 cell transplantation tumor model

[0072] 1) Tumor mouse model construction and dosing regimen

[0073] ①Take mouse breast cancer cells (4T1 breast cancer cells) in logarithmic growth phase, count after digestion, mix pre-cooled PBS and Matrigel at a ratio of 1:1, resuspend the cells to obtain a concentration of 5x10 6The cell suspension was placed on ice.

[0074] ② 4-5 weeks old Balb / C mice were injected with 100 uL of cell suspension on both sides of the back, and when the subcutaneous tumor size reached 50 mm, the mice were randomly divided into four groups, and the group and administration were as follows:

[0075] A blank control group;

[0076] A drug administration group: XF-5079-D 5 mg / Kg group; XF-5079-D 10 mg / Kg group;

[0077] A positive control group: Taxol 15 mg / Kg group.

[0078] Administration: The control group was injected with 100 uL of placebo (i.e. the solvent used for dissolving the drug: 15% castor oil + 85% sterile PBS) intraperitoneally every other day; the other drug administration groups were injected with 100 uL of drug solution of different doses intraperitoneally every other day.

[0079] ③ During the 27 days of continuous administration, the body weight and tumor size of the mice were measured every other day, and the growth curve of the body weight of the mice was drawn.

[0080] ④ After 27 days of administration, the mice were sacrificed for dissection, and the subcutaneous tumors were peeled off and weighed.

[0081] 2) Experimental results

[0082] The experimental results are shown in Table 4, which is the tumor inhibition rate after administration in the mouse subcutaneous breast cancer cell transplantation tumor model. The 10 mg / kg compound XF-5079-D has an activity comparable to that of 15 mg / kg paclitaxel, and can inhibit the growth of tumors in mice.

[0083] Table 4 Tumor inhibition rate after administration in the mouse subcutaneous breast cancer cell transplantation tumor model

[0084] Group Tumor inhibition rate TGI (%) XF-5079-D 5 mg / Kg 27.7％ XF-5079-D 10 mg / Kg 66.3％ Taxol 15 mg / Kg 65.5％

[0085] It is found through the subcutaneous cell transplantation tumor model experiment of Balb / c mice that the propynamide derivative small molecule compound can effectively inhibit the growth of breast cancer cells.

[0086] The embodiments of the application are described in detail above, but the application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the application, and still fall within the protection scope of the application.

Claims

1. An inhibitor that selectively targets TXNRD1, characterized in that, The inhibitor is a small molecule compound of the propyne amide derivative, and the structure of the inhibitor is shown in formula (I):

2. The use of the selective TXNRD1 inhibitor of claim 1 in the preparation of a drug targeting TXNRD1.

3. The use of the selective TXNRD1 inhibitor according to claim 1 in the preparation of an anti-breast cancer drug.

4. The use of the selective TXNRD1 inhibitor of claim 1 in the preparation of a drug for inhibiting the growth of breast cancer cells.

5. A drug comprising the selectively targeting TXNRD1 as described in claim 1, characterized in that, The selective TXNRD1 inhibitor is the main active ingredient.

6. The drug selectively targeting TXNRD1 according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

7. The drug selectively targeting TXNRD1 according to claim 6, characterized in that, The carrier includes surfactants, suspending agents, and emulsifiers.

8. The drug selectively targeting TXNRD1 according to claim 6, characterized in that, The excipients include diluents, binders, lubricants, disintegrants, solubilizers, and stabilizers.

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