Application of indole small molecules targeting KRAS G-tetrachain in the preparation of drugs for treating triple-negative breast cancer

Abstract

The application provides application of an indole small molecule targeting KRAS G-quadruplex in preparation of a drug for treating triple-negative breast cancer. The ligand provided by the application can specifically bind to a stable KRAS G-quadruplex structure, inhibit expression of KRAS and related signal pathways, and finally inhibit growth of triple-negative breast cancer. Compared with traditional chemotherapy drugs, the ligand has lower toxic side effects and is more effective.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drugs, specifically relating to the application of a novel indole small molecule targeting the KRAS G-tetrachain in the preparation of a drug for treating triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a type of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC accounts for approximately 10–20% of all breast cancers. This type of breast cancer is characterized by high histological grade, poor prognosis, rapid progression, and high invasiveness. Currently, treatment methods for TNBC mainly include chemotherapy, surgery, and radiotherapy. However, patients receiving chemotherapy still face a high risk of recurrence and metastasis, poor prognosis, low recurrence-free survival and overall survival rates, and significant toxic side effects. Furthermore, high-dose chemotherapy drugs often lead to tumor resistance. Therefore, the search for novel and effective treatments for TNBC is of great significance.

[0003] KRAS is the most common mutated oncogene family in human cancers, with approximately 25% of malignant tumors associated with KRAS gene mutations, primarily including lung cancer, pancreatic cancer, and breast cancer. The KRAS gene encodes a guanine nucleotide-binding protein with GTP hydrolase activity. In normal cells, the KRAS protein maintains a homeostatic balance between inactivated and activated states. However, mutations in the KRAS protein lock it into an active tyrosine kinase state, continuously activating cell proliferation signaling pathways (such as RAF-MEK-ERK and PI3K-AKT-mTOR), stimulating sustained cell growth and tumor angiogenesis, ultimately promoting tumor development and progression. Furthermore, KRAS mutations have a significant impact on the tumor microenvironment, promoting the formation of an immunosuppressive microenvironment and mediating immune escape from tumor cells.

[0004] Currently developed inhibitors (Sotorasib and Adagrasib) targeting KRAS mutant proteins are only effective against a specific mutant (G12C). However, KRAS mutations are diverse, limiting the patient population for which these costly drugs are applicable, hindering the development of next-generation inhibitors. Furthermore, acquired resistance inevitably develops with continued administration, posing the greatest challenge to KRAS-targeted therapy. It is worth noting that neither Sotorasib nor Adagrasib is suitable for patients with triple-negative breast cancer. Summary of the Invention

[0005] Therefore, developing small molecule inhibitors with novel mechanisms of action that can block the KRAS pathway at its source and studying their application in the treatment of triple-negative breast cancer is of great scientific significance and clinical research value.

[0006] The KRAS gene promoter contains a nuclease hypersensitive element (NHPPE) crucial for transcription. This element forms an unusual DNA structure called the G-quadruplex and participates in the transcriptional regulation of KRAS, acting as a "molecular switch" for KRAS transcriptional regulation. Given the presence of KRAS gene amplification and oncogene addiction in tumor cells, compounds targeting the G-quadruplex to intervene in KRAS gene expression at its source may have broader application prospects than traditional KRAS protein inhibitors.

[0007] To address the above technical problems, this invention provides the application of a G-quadruplex ligand in the preparation of a drug for treating triple-negative breast cancer, the structural formula of which is: Or a stereoisomer, tautomer, nitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of the compound shown in the structural formula; in the structural formula, Ar is selected from at least one of imidazole, thiazole, oxazole, thiophene, furan, and pyridine.

[0008] This invention also provides a method for preparing G-tetrachain ligands, comprising the following steps:

[0009] Step A: Use The compound was reacted with iodomethane under reflux in acetonitrile solvent to give the compound.

[0010] Step B: In ethanol solvent, The product is reacted with the corresponding aromatic aldehydes under reflux to give the final product.

[0011] Its reaction formula is:

[0012]

[0013] Preferably, the triple-negative breast cancer is associated with KRAS gene mutations.

[0014] The present invention also provides the use of the ligand in the preparation of a medicament for inhibiting the growth of triple-negative breast cancer cells.

[0015] The present invention also provides the use of the ligands described herein in the preparation of drugs that enhance the body's antitumor immune activity.

[0016] The present invention also provides the use of the ligands described herein in the preparation of drugs that bind to and stabilize KRAS G-tetrachains.

[0017] The present invention also provides the use of the ligands described herein in the preparation of drugs that inhibit the transcriptional level of KRAS in tumor cells.

[0018] The present invention also provides a composition comprising the said compound, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium, or combination thereof.

[0019] The effective concentration of the G-quadruplex ligand is 5 to 10 mg / kg.

[0020] The preferred administration methods for the above applications are oral, injection, or inhalation.

[0021] Compared with the prior art, the present invention brings the following beneficial effects:

[0022] (1) This type of ligand is simple to prepare, has a stable structure, and is easy to store, providing a simple and effective way to discover anti-tumor drugs targeting KRAS G-tetrachain.

[0023] (2) The ligand provided by the present invention can specifically bind to the stable KRAS G-quadruplex structure, inhibit the expression of KRAS and related signaling pathways, and ultimately inhibit the growth of triple-negative breast cancer.

[0024] (3) Compared with traditional chemotherapy drugs, this type of ligand has lower toxicity and is more effective. Attached Figure Description

[0025] Figure 1 The UV titration curves show the interaction between ligand BN1 and the KRAS G-tetrachain.

[0026] Figure 2 This describes the inhibitory effect of ligand BN1 on KRAS and its related signaling pathways.

[0027] Figure 3 The ligand BN1 inhibits the proliferation and migration of triple-negative breast cancer cells.

[0028] Figure 4 The therapeutic effect of ligand BN1 on a triple-negative breast cancer animal model.

[0029] Figure 5 To investigate the antitumor immunomodulatory activity of ligand BN1 in a triple-negative breast cancer animal model. Detailed Implementation

[0030] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings:

[0031] Example 1: Synthesis of Compound 2

[0032] 2,3,3-trimethyl-4,5-benzoindole (2.0 g) was dissolved in 20 mL of acetonitrile, and 5 molar equivalents of iodomethane were added. The mixture was heated in an oil bath at 60 °C for 5 h. The reaction solution was then poured into 200 mL of ethyl acetate, resulting in the precipitation of a large amount of solid. The solid was filtered under reduced pressure and dried under vacuum to obtain a grayish-white solid with a yield of 85%. The structure was confirmed by mass spectrometry: MS (ESI) m / z 224.1 [MI]+.

[0033] Example 2: Synthesis of BN1 (Ar is imidazole)

[0034] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and 2 molar equivalents of imidazole-2-carboxaldehyde were added. The mixture was refluxed for 20 h. After the reaction was completed, the mixture was allowed to cool naturally, and a solid precipitated out. The solid was filtered under reduced pressure, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-yellow solid with a yield of approximately 55%. MS (ESI) m / z 302.1 [MI]+. 1 H NMR (600MHz, CDCl3) δ8.45(d,J=16.4Hz,1H),8.20(d,J=8.5Hz,1H),8.08(d,J=8.9Hz,1H),8.03(d,J=2.6Hz,1H),8.01(d, J=5.4Hz,1H),7.72(t,J=7.6Hz,1H),7.67(d,J=8.9Hz,1H),7.63(t,J=7.5Hz,1H),7.43(s,2H),4.30(s,3H),2.04(s,6H). 13 C NMR (151MHz, CDCl3) δ182.98,143.24,138.90,138.61,138.54,133.83,131.82, 130.28,128.85,127.80,127.28,122.85,111.57,111.38,54.06,35.81,26.23.

[0035] Example 3: Synthesis of BN2 (Ar is thiazole)

[0036] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and 2 molar equivalents of 2-aldehydethiazole were added. The mixture was refluxed for 20 h. After the reaction was completed, the mixture was allowed to cool naturally, and a solid precipitated out. The solid was filtered under reduced pressure, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-red solid with a yield of approximately 50%. MS (ESI) m / z 319.1 [MI]+. 1H NMR (600MHz, DMSO) δ8.72(d,J=16.1Hz,1H),8.47(d,J=8.2Hz,1H),8.37–8.28(m,3H),8.25(d,J=7.9Hz,1H),8.20 (d,J=8.7Hz,1H),7.88(d,J=16.1Hz,1H),7.84(t,J=7.3Hz,1H),7.77(t,J=7.2Hz,1H),4.30(s,3H),2.02(s,6H). 13 C NMR (151MHz, DMSO) δ182.35,162.45,146.66,141.01,139.95,139.33,133.92,131.53, 130.53,129.04,128.50,128.06,127.07,123.91,115.10,114.02,54.57,35.96,25.13.

[0037] Example 4: Synthesis of BN3 (Ar is oxazole)

[0038] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and 2 molar equivalents of oxazol-2-carboxaldehyde were added. The mixture was refluxed for 20 h. After the reaction was completed, the mixture was allowed to cool naturally, and a solid precipitated out. The solid was filtered under reduced pressure, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-red solid with a yield of approximately 55%. MS (ESI) m / z 303.1 [MI]+. 1 H NMR (600MHz, DMSO) δ8.59(s,1H),8.45(d,J=8.5Hz,1H),8.35(d,J=8.9Hz,1H),8.26(d,J=8.1Hz,1H),8.20 (dd,J=15.0,12.8Hz,2H),7.84(ddd,J=8.3,6.9,1.3Hz,1H),7.82–7.74(m,3H),4.32(s,3H),1.99(s,6H). 13 C NMR(151MHz,DMSO)δ182.37,159.42,143.88,139.92,139.59,134.03,133.10,131.81, 131.61,130.53,129.11,128.24,127.02,123.97,118.22,114.11,54.77,36.34,24.68.

[0039] Example 5: Synthesis of BN4 (Ar is thiophene)

[0040] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and 2 molar equivalents of thiophene-2-carboxaldehyde were added. The mixture was refluxed for 20 h. After the reaction was completed, a solid precipitated. The solid was filtered under reduced pressure while hot, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-yellow solid with a yield of approximately 80%. MS (ESI) m / z 318.1 [MI]+. 1 H NMR (600MHz, DMSO) δ8.77(d,J=16.1Hz,1H),8.43(d,J=8.3Hz,1H),8.30(d,J=8.8Hz,1H),8.24–8.18(m,3H),8.11(d,J=8.8 Hz,1H),7.82(t,J=7.4Hz,1H),7.73(t,J=7.3Hz,1H),7.43–7.39(m,1H),7.36(d,J=16.1Hz,1H),4.23(s,3H),2.01(s,6H). 13 C NMR (151MHz, DMSO) δ182.30,145.03,140.76,139.91,138.31,137.12,136.71,133.58,131. 33,130.51,130.28,128.89,127.57,127.13,123.64,113.72,111.13,54.05,35.25,25.66.

[0041] Example 6: Synthesis of BN5 (Ar is furan)

[0042] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and two molar equivalents of furan-2-carboxaldehyde were added. The mixture was refluxed for 20 h. After the reaction was complete, a solid precipitated. The solid was filtered under reduced pressure while hot, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-yellow solid with a yield of approximately 85%. MS (ESI) m / z 302.1 [MI]+. 1 H NMR (600MHz, DMSO) δ8.47–8.39(m,2H),8.33–8.24(m,2H),8.22(d,J=8.0Hz,1H),8.12(d,J=8.8Hz,1H),7.81(t,J=7 .4Hz,1H),7.76–7.69(m,1H),7.59(d,J=2.3Hz,1H),7.30(d,J=16.2Hz,1H),6.94(s,1H),4.20(s,3H),1.99(s,6H). 13C NMR (151MHz, DMSO) δ182.11,151.83,150.49,139.93,138.29,137.24,133.58,131.35,130. 50,128.89,127.59,127.15,123.78,123.66,115.36,113.71,109.19,53.91,35.11,25.67.

[0043] Example 7: Synthesis of BN6 (Ar is pyridine)

[0044] Compound 2 was dispersed in 20 mL of ethanol (0.5 g), and 2 molar equivalents of pyridine-2-carboxaldehyde were added. The mixture was refluxed for 20 h. After the reaction was completed, a solid precipitated. The solid was filtered under reduced pressure while hot, and the filter cake was washed three times with a small amount of ethanol to obtain a brownish-yellow solid with a yield of approximately 90%. MS (ESI) m / z 313.1 [MI]+. 1 H NMR (600MHz, DMSO) δ8.85(d,J=4.6Hz,1H),8.53(d,J=16.1Hz,1H),8.48(d,J= 8.5Hz,1H),8.34(d,J=8.9Hz,1H),8.26(d,J=8.1Hz,1H),8.21(d,J=8.9Hz,1H) ,8.19(d,J=7.7Hz,1H),8.11–8.05(m,2H),7.84(ddd,J=8.3,6.9,1.2Hz,1H),7 .80–7.75(m,1H),7.62(ddd,J=7.6,4.6,1.1Hz,1H),4.31(s,3H),2.04(s,6H). 13 C NMR (151MHz, DMSO) δ182.96,152.02,151.07,149.62,139.92,139.20,138.25,133.92,131.50, 130.53,129.03,128.48,128.02,127.09,127.00,123.89,115.65,114.05,54.65,35.95,25.24.

[0045] Example 8: Study on the interaction between BN-1 and KRAS G-quadruplex

[0046] The compound was diluted to a concentration of 5 μM with Tris-HCl buffer and added to a suitable cuvette. The cuvette was then placed in a UV spectrometer, and the absorption spectrum was acquired at room temperature, scanning the range of 300–600 nm. Subsequently, G-quadruplex DNA (KRAS-32R and KRAS-28Rm) was added to the cuvette, increasing the concentration by 1 μM until reaching a cumulative total of 10 μM. Each titration curve was recorded. The results are as follows: Figure 1 As shown.

[0047] from Figure 1 It is evident that after the addition of KRAS G-tetrachain, the absorption spectrum of BN-1 exhibits a significant hypochromic effect and a clear red shift, ultimately revealing a distinct isochromatic point. This indicates that BN1 can undergo strong π-π stacking of KRAS G-tetrachain in vitro.

[0048] Example 9: BN1 inhibits KRAS gene transcription and related signaling pathways in triple-negative breast cancer cells

[0049] MDA-MB-231 cell suspension was collected, counted, and seeded into six-well plates at approximately 250,000 cells per well, and cultured for 24 hours. The old culture medium was removed, and different concentrations of the drug (0, 1, 2, 4 μM) were added, followed by 24 hours of culture. Cell samples were collected, and RNA was extracted. 400 ng of RNA was used to prepare 20 μL of RT reaction solution according to the reverse transcription kit instructions, and then reverse transcribed into cDNA in a PCR instrument. 2 μL of cDNA was used to prepare 20 μL of PCR reaction solution according to the PCR kit instructions. The mixture was amplified in a PCR instrument, with the program set as follows: 95℃ 30s → 55℃ 30s → 72℃ 15min. 10 μL of PCR product was added to loading buffer, mixed well, and loaded onto the plate. Electrophoresis was performed at a constant voltage of 90V. The agarose gel was then placed in a gel scanner to observe and photograph the results, as shown below. Figure 2 As shown in Figure A.

[0050] from Figure 2 As can be seen from RT-PCR, BN1 has a strong inhibitory effect on KRAS gene transcription with high selectivity, and a smaller inhibitory effect on transcription of other genes except NRAS, HRAS and others.

[0051] MDA-MB-231 cell suspension was collected, counted, and seeded into six-well plates at approximately 250,000 cells per well, and cultured for 24 h. The old culture medium was removed, and different concentrations of the drug were added, followed by culturing for another 24 h. Cell samples were collected, 100-120 μL of lysis buffer was added, and the plates were centrifuged at 4 °C for 15 min. The supernatant was collected, and protein concentration was determined according to the BCA protein kit instructions. The extracted protein supernatant was mixed with protein loading buffer at the appropriate dilution ratio and heated at 95 °C for 10 min to denature the protein. A 12% (v / v) separating gel and a 5% (v / v) stacking gel were prepared, and electrophoresis was performed at a constant voltage of 90 V for 120 min. After electrophoresis, the gel and PVDF membrane were placed in a transfer apparatus and transferred on ice. The transfer conditions were a constant current of 250 mA for 120 min. The PVDF membrane was blocked with TBST containing 5% skim milk powder on a shaker at room temperature for 2 h. After washing twice with TBST, the PVDF membrane was placed in the primary antibody incubation solution and incubated overnight at 4°C. The primary antibody was recovered, the membrane was thoroughly washed with TBST, and the secondary antibody incubation solution was added. The membrane was then shaken at room temperature for 2 hours. The secondary antibody was recovered, the membrane was thoroughly washed with TBST, and ECL developing solution was prepared. Development and exposure were performed using a chemiluminescence analyzer. The results are as follows: Figure 2 As shown in B.

[0052] from Figure 2 As can be seen from the Western Blot results, BN1 has a significant inhibitory effect on KRAS protein expression and related signaling pathways (MEK, ERK).

[0053] Example 10: BN1 inhibits the proliferation and migration of triple-negative breast cancer cells

[0054] MDA-MB-231 cell suspension was collected, counted, and seeded into six-well plates at approximately 250,000 cells per well, and cultured for 24 hours. After confluence of cells at the bottom of the plate, the old culture medium was aspirated, and cells were streaked evenly along the center line of each well using a micropipette tip. The streaked cells were washed away with PBS, and drug solutions with concentration gradients of 0 μM, 1 μM, 2 μM, and 4 μM were prepared using serum-free medium. The results were photographed and recorded. After culturing in an incubator for 48 hours, the changes in cell distribution at the streaked areas were photographed again. The results are as follows: Figure 3 As shown in Figure A.

[0055] Figure 3 In Figure A, without the drug, the cell migration area was significantly reduced after 48 hours; as the drug concentration increased, cell migration decreased and the scratches became more obvious, indicating that BN1 has a relatively significant inhibitory effect on the migration of MDA-MB-231 cells.

[0056] MDA-MB-231 cells were seeded into 6-well plates at 500 cells per well and cultured in a 5% CO2 cell culture incubator for 24 hours to allow cell adhesion. Culture medium containing the compound (0, 0.0625, 0.125, 0.25, 0.5, 1 μM) was added to the culture dishes, and the cells were cultured for another 7 days to allow clonal formation. The culture medium was discarded, and the cells were carefully washed twice with PBS to remove serum-containing medium. Cells were fixed with anhydrous methanol for 15 min, and then stained with 0.1% crystal violet methanol staining solution at room temperature for 30 min. After discarding the staining solution, excess staining solution was gently washed away with water, and the cells were air-dried upside down. The culture dishes were photographed under white light, and the results were stored. Results are as follows: Figure 3 As shown in B.

[0057] Figure 3 In sample B, the number of visible cell clones decreased as the drug concentration increased, indicating that BN1 has an inhibitory effect on the formation of MDA-MB-231 clones.

[0058] Example 11: BN1 inhibits the growth of triple-negative breast cancer in vivo.

[0059] MDA-MB-231 cells in the logarithmic growth phase were collected and resuspended in serum-free DMEM medium to a density of 102. 7 100 μL of cell suspension was injected into the axilla of each BALB / c mouse's forelimb. The mice were fed for one week until the tumor grew to approximately 50 mm². 3 At that time, the antitumor experiment was initiated. BALB / c mice bearing subcutaneous tumors were randomly divided into three groups of eight mice each: a solvent group, a compound group (5 mg / kg), and a compound group (10 mg / kg). Mouse body weight and tumor volume were measured every two days, and the appropriate concentration of drug was injected intraperitoneally every three days. After 18 days of continuous administration, the mice were euthanized by cervical dislocation. Results are as follows... Figure 4 As shown.

[0060] from Figure 4 It is evident that BN1 can effectively inhibit tumor growth in a triple-negative breast cancer mouse model. Figure 4 A), with fewer side effects, does not affect the reduction of mouse body weight. Figure 4 B).

[0061] Example 12: Antitumor immunomodulatory activity of BN1 in vivo

[0062] Collect 4T1 cells in the logarithmic growth phase and resuspend them in serum-free DMEM medium to a density of 102. 5 100 μL of cell suspension was injected into the axilla of each BALB / c mouse's forelimb. The mice were fed for one week until the tumor grew to approximately 50 mm².3 At that time, the antitumor experiment was initiated. BALB / c mice bearing subcutaneous tumors were randomly divided into three groups of eight mice each: a solvent group, a compound group (5 mg / kg), and a compound group (10 mg / kg). The drugs were administered every three days. After the drug administration was completed, the mice were sacrificed, and the tumors were collected. Single-cell suspensions were prepared by grinding and enzymatic digestion, stained, and CD45 levels were detected by flow cytometry. + / CD3 + / CD4 + T cells, CD45 + / CD3 + / CD8 + T cells and CD45 + / CD3 + / CD8 + / IFN-γ + The proportion of T cells. Results as follows: Figure 5 As shown.

[0063] The results showed that, to some extent, BN1 can increase CD4 levels in the tumor microenvironment. + T cells, CD8 + T cells and CD8 cells that produce IFN-γ + The proportion of T cells suggests that BN1 plays a role in remodeling the tumor immune microenvironment and inhibiting immune escape.

[0064] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. The application of a G-quadruplex ligand in the preparation of a drug for treating triple-negative breast cancer, characterized in that, Its structural formula is: In this structural formula, Ar is selected from imidazoles substituted at position 2.

2. The application as described in claim 1, characterized in that, The triple-negative breast cancer mentioned above is associated with KRAS gene mutations.

3. The use of the ligand as described in claim 1 in the preparation of a medicament for inhibiting the growth of triple-negative breast cancer cells.

4. The use of the ligand as described in claim 1 in the preparation of a medicament for treating triple-negative breast cancer that enhances the body's antitumor immune activity.

5. The use of the ligand as described in claim 1 in the preparation of a medicament for treating triple-negative breast cancer that binds to and stabilizes the KRAS G-tetrachain.

6. The use of the ligand as described in claim 1 in the preparation of a medicament for treating triple-negative breast cancer by inhibiting the transcriptional level of KRAS in tumor cells.

7. A composition comprising the ligand as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

8. The application as described in claims 1-6, characterized in that, The effective concentration of the G-quadruplex ligand is 5 to 10 mg / kg.

9. The application as described in claims 1-6, characterized in that, The application is administered orally, by injection, or by inhalation.

10. A method for preparing G-quadruplex ligands, comprising the following steps: Step A: Use The compound was reacted with iodomethane under reflux in acetonitrile solvent to give the compound. ; Step B: In ethanol solvent, The product was reacted with imidazole-2-carbaldehyde under reflux to give the final product. ; Its reaction formula is: .

Images