Use of imidazoquinazoline compounds for the preparation of a medicament for the treatment of gastrointestinal tumors with KRAS mutations
Imidazoloids and quinazoline compounds inhibit the binding of p-ERK2 to p53, restore p53 transcriptional activity, and selectively inhibit KRAS-mutant gastrointestinal tumor cells, thus solving the problems of poor targeting and drug resistance in existing technologies and achieving highly efficient tumor suppression.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- KUNMING MEDICAL UNIVERSITY
- Filing Date
- 2026-05-08
- Publication Date
- 2026-06-05
AI Technical Summary
Current technologies lack small molecule inhibitors that can specifically target the p-ERK2-p53 complex, resulting in poor treatment outcomes for KRAS-mutant gastrointestinal tumors. In particular, the proportion of KRAS G12C mutations is low and they are not sensitive to existing inhibitors, while drug resistance is common in mutation subtypes such as G12D, G12V, and G13D.
To develop imidazoquinaline small molecule compounds or their pharmaceutically acceptable salts, which can selectively inhibit the proliferation, migration and invasion of KRAS-mutant gastrointestinal tumor cells by restoring the transcriptional activity of p53 through inhibiting the binding of p-ERK2 to p53.
Imidazolidine compounds exhibit significant selective inhibitory activity against KRAS-mutant gastrointestinal tumors, with an in vitro IC50 of approximately 0.42 μM and IC50s of 45.92 μM and 41.27 μM for KRAS wild-type cells, respectively, representing a selectivity index of approximately 100-fold. The in vivo tumor inhibition rate reached 87.56%, and the safety profile was good.
Smart Images
Figure CN122140720A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of an imidazoquinazoline small molecule compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating KRAS-mutant gastrointestinal tumors. Background Technology
[0002] Gastrointestinal tumors are among the most common and deadliest malignant tumors worldwide, primarily including gastric cancer and colorectal cancer. Statistics show that the annual number of new cases of gastric and colorectal cancer ranks among the highest of all malignant tumors globally, and my country is a high-incidence area for gastrointestinal tumors, resulting in a particularly heavy disease burden. Despite continuous advancements in comprehensive treatment methods such as surgical resection, chemotherapy, radiotherapy, and targeted therapy, the five-year survival rate for patients with advanced-stage tumors remains unsatisfactory, mainly due to the high rate of tumor metastasis and drug resistance to existing treatments. Therefore, developing novel treatment strategies and drugs for gastrointestinal tumors is of significant clinical importance and urgently needed.
[0003] KRAS is one of the most common mutated oncogenes in gastrointestinal tumors, with a mutation rate of approximately 40%-50% in colorectal cancer and a high mutation frequency in gastric cancer. KRAS mutations lead to the sustained activation of downstream signaling pathways such as RAF-MEK-ERK, driving unlimited tumor cell proliferation, migration, and resistance to apoptosis. Although covalent inhibitors targeting KRAS G12C mutations (such as sotorasib) have made breakthrough progress in the treatment of non-small cell lung cancer, the proportion of G12C mutations is low in gastrointestinal tumors (approximately 3%), while G12D, G12V, and G13D mutation subtypes are dominant, and these mutants are insensitive to existing G12C inhibitors. Furthermore, while MEK inhibitors targeting downstream pathways of KRAS have shown some efficacy in preclinical studies, the objective response rate of monotherapy is limited, and drug resistance is widespread. Therefore, developing novel therapeutic strategies for KRAS-mutant gastrointestinal tumors, especially identifying new key downstream effector nodes, has become an urgent scientific problem to be solved in this field.
[0004] p53, as a crucial tumor suppressor protein, plays a central role in DNA damage repair, cell cycle arrest, and apoptosis regulation. However, in KRAS-mutant gastrointestinal tumors, p53 often exists in a wild-type state but is functionally inactive. Recent studies have shown that in KRAS-mutant gastrointestinal tumor cells, the persistently activated RAF-MEK-ERK signaling pathway leads to persistent phosphorylation of ERK2 (p-ERK2), which then directly binds to p53 to form the p-ERK2-p53 complex. This interaction interferes with the transcriptional activation function of p53. Therefore, by interfering with the formation of the p-ERK2-p53 complex, the tumor-suppressive function of p53 can be restored, thereby achieving effective inhibition of KRAS-mutant gastrointestinal tumors. However, currently, there is a lack of small-molecule inhibitors that specifically target the p-ERK2-p53 complex.
[0005] In the prior art, there are no reports on the use of the imidazoquinoline compounds of the present invention or their pharmaceutically acceptable salts in the preparation of medicaments for treating KRAS-mutant gastrointestinal tumors. Summary of the Invention
[0006] To overcome the shortcomings of the prior art, this invention aims to provide the use of imidazoquinaline small molecule compounds of formula (I) or pharmaceutically acceptable salts thereof in the preparation of anti-gastrointestinal tumor drugs. These compounds selectively inhibit the proliferation, migration, and invasion of KRAS-mutant gastrointestinal tumor cells, and induce cell cycle arrest and apoptosis.
[0007] This invention is achieved through the following technical solution: The present invention provides the use of an imidazoquinaline compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating KRAS-mutant gastrointestinal tumors.
[0008]
[0009] Wherein, R is H, halogen, C1-C6 alkyl, or C1-C6 alkoxy.
[0010] Furthermore, the structures of the imidazoquinoline compounds or their pharmaceutically acceptable salts are as follows:
[0011] Furthermore, the gastrointestinal tumor is gastric cancer or colorectal cancer.
[0012] Furthermore, the gastrointestinal tumor is a KRAS-mutated gastric cancer or colorectal cancer.
[0013] The KRAS mutation is one or more of the following: KRAS G12D, KRAS G12V, and KRAS G13D.
[0014] Preferably, the KRAS mutation is the KRAS G12D mutation.
[0015] The imidazoquinaline compounds or their pharmaceutically acceptable salts exert their antitumor effects by upregulating the expression of downstream target genes of p53.
[0016] The imidazoquinaline compounds or their pharmaceutically acceptable salts exert their antitumor effects by inhibiting the binding of p-ERK2 to p53 protein and restoring the transcriptional activity of p53 protein.
[0017] In the applications described above, the imidazoquinoline compounds or their pharmaceutically acceptable salts can be prepared into pharmaceutical compositions with pharmaceutically acceptable carriers or excipients.
[0018] Beneficial effects
[0019] (1) For the first time, imidazoline compounds or their pharmaceutically acceptable salts were found to have selective inhibitory activity against KRAS-mutant gastrointestinal tumors. These compounds effectively inhibited the proliferation, migration, and invasion of KRAS-mutant gastrointestinal tumor cells, induced cell cycle arrest and apoptosis, and exerted antitumor effects by inhibiting the binding of p-ERK2 to p53 and restoring p53 transcriptional activity. In vitro activity experiments showed that imidazoline compounds or their pharmaceutically acceptable salts had an effect on the IC50 of KRAS-mutant gastric cancer AGS cells. 50 The IC50 concentration was approximately 0.42 μM, while the IC50 concentration against KRAS wild-type human colon cancer HT-29 cells and human gastric cancer SNU-216 cells was [missing value]. 50 The concentrations were 45.92 μM and 41.27 μM, respectively, showing a selectivity index of approximately 100-fold. This selective inhibitory property indicates that imidazoline compounds or their pharmaceutically acceptable salts have no inhibitory activity against unmutated gastric or colon cancer. However, they exhibit significant inhibitory activity against KRAS-mutated gastric cancer, thus demonstrating a specific mechanism of action and suggesting a better therapeutic window and lower toxicity.
[0020] (2) In vivo pharmacodynamic experiments showed that imidazoline compounds or their pharmaceutically acceptable salts inhibited the tumor growth rate of subcutaneous xenografts of KRAS-mutant gastric cancer in nude mice by 87.56%, and the weight of mice did not decrease significantly during the administration period, indicating that imidazoline compounds or their pharmaceutically acceptable salts have good safety at effective doses.
[0021] (3) Mechanistic studies have shown that imidazoline compounds or their pharmaceutically acceptable salts can inhibit the binding of p-ERK2 to p53, restore p53 transcriptional activity, upregulate the expression of the downstream pro-apoptotic gene PUMA, and specifically target the p-ERK2-p53 complex, thereby exerting an anti-tumor effect. This mechanism of action is different from traditional KRAS or MEK inhibitors and is original.
[0022] (4) This invention provides a new treatment option for patients with KRAS-mutant gastrointestinal tumors, and has important clinical value and industrial application prospects. Attached Figure Description
[0023] Figure 1 Inhibitory effect of IQZ-17k on the proliferation of KRAS-mutant gastric cancer AGS cells.
[0024] Figure 2 Inhibitory effect of IQZ-17k on the proliferation of KRAS wild-type colon cancer HT-29 cells and gastric cancer SNU-216 cells.
[0025] Figure 3 Inhibitory effect of IQZ-17k on the binding of p-ERK2 to p53.
[0026] Figure 4 Effects of IQZ-17k on the expression of downstream target genes of p53.
[0027] Figure 5 Flow cytometry results of IQZ-17k-induced apoptosis in AGS cells.
[0028] Figure 6 Effects of IQZ-17k on cell cycle distribution in AGS cells.
[0029] A: Cell cycle flow cytometry data after treatment with different concentrations of IQZ-17k.
[0030] B: Statistical graph of cell cycle distribution after treatment with different concentrations of IQZ-17k.
[0031] C: The proportion of cells in the G2 / M phase after treatment with different concentrations of IQZ-17k.
[0032] Figure 7 Inhibitory effect of IQZ-17k on the migration ability of AGS cells.
[0033] Figure 8 Inhibitory effect of IQZ-17k on the invasive ability of AGS cells.
[0034] Figure 9Inhibitory effect of IQZ-17k on the growth of human gastric cancer AGS cell subcutaneous xenografts in nude mice.
[0035] A: Body weight of mice in each group during drug administration.
[0036] B: Graph showing changes in tumor volume in mice during drug administration.
[0037] C: Photographs of tumors in mice from each group.
[0038] D: Statistical chart of tumor weight in mice of each group. Detailed Implementation
[0039] The present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.
[0040] Example 1: Synthesis of IQZ-17k
[0041] Step 1: Compound A (20 mmol, 1.0 eq.), cyanamide (80 mmol, 4.0 eq.), and 4A molecular sieve (3.34 g) were dissolved in methanol (168 mL). Sodium tert-butoxide (80 mmol, 4.0 eq.) was added in portions under ice bath conditions. The mixture was stirred at 25 °C for 30 minutes, followed by the addition of NBS (80 mmol, 4.0 eq.) under ice bath conditions. The mixture was then heated to 50 °C and reacted for 12 hours. After concentration, dichloromethane (140 mL) and a saturated sodium thiosulfate solution (100 mL) were added, and the mixture was stirred for 10 minutes before filtration. The product was extracted with dichloromethane, dried, concentrated, and purified by petroleum ether-ethyl acetate (5:1) column chromatography to obtain 2.92 g of compound B, named methyl (E)-N-cyano-2-nitrobenzimidate.
[0042] Step 2: Dissolve 2-chloroethylamine hydrochloride (6 mmol, 2.0 eq.) and sodium bicarbonate (6 mmol, 2.0 eq.) in methanol (12 mL). React at room temperature for 30 minutes, then add compound B (3 mmol, 1.0 eq.) and heat to 50 °C for 6 hours. After compound B has reacted completely, add iron powder (12 mmol, 4.0 eq.), 12 M hydrochloric acid (2.4 mL), and methanol (150 mL), and reflux for 3 hours. Add potassium carbonate (30 mmol, 10.0 eq.) and reflux for 6 hours. Filter with diatomaceous earth, concentrate the filtrate, and wash with methanol to obtain 1.21 g of crude product C.
[0043] Step 3: Using 2-furanic acid, the reaction scale was 0.5 mmol. The crude product C was purified by petroleum ether-ethyl acetate (1:1) column chromatography to give 56 mg of pink solid, namely compound IQZ-17k. Its name is N-(2,3-dihydroimidazo[1,2-c]quinazolin-5-yl)furan-2-carboxamide.
[0044] 1 HNMR (600 MHz, CDCl3) δ 12.67 (s, 1H), 8.03 – 7.87 (m, 1H), 7.54 (d,J = 37.2 Hz,2H), 7.21 (s, 2H), 7.06 (d, J = 6.1 Hz, 1H), 6.48 (s, 1H), 4.15(s, 4H). 13 C NMR (151MHz, CDCl3) δ 170.3, 152.1, 151.8, 151.2, 145.9, 136.7,133.8, 126.4, 124.9, 116.7,113.6, 111.9, 53.9, 45.5.
[0045]
[0046] Example 2: Inhibitory effect of IQZ-17k on the proliferation of KRAS-mutant gastric cancer cells Experimental methods: Logarithmic growth phase human gastric cancer AGS cells (KRAS G12D mutation) were harvested, digested, and counted. Cells were seeded at 3 × 10³ cells per well in 96-well plates. After overnight adhesion, different concentrations (0, 0.16, 0.8, 4, 20, 100 μM) of IQZ-17k were added, with three replicates for each concentration. After 72 hours of culture, 10 μL of CCK8 solution was added to each well, and incubation continued for 1–4 hours. Absorbance was measured at 450 nm to calculate cell viability and half-maximal inhibitory concentration (IC50). 50 ).
[0047] Experimental results showed that IQZ-17k inhibited the IC50 of AGS cell survival rate. 50 The concentration was approximately 0.42 μM, and it inhibited cell proliferation in a concentration-dependent manner. Figure 1 ).
[0048] Example 3: Inhibitory effect of IQZ-17k on the proliferation of KRAS wild-type gastrointestinal tumor cells Experimental methods: Human colon cancer HT-29 cells and human gastric cancer SNU-216 cells (KRAS wild-type) in logarithmic growth phase were collected and CCK8 cell viability was detected according to the method in Example 2. Cell viability and half-maximal inhibitory concentration (IC50) were calculated. 50 ).
[0049] Experimental results show that IQZ-17k has an effect on the IC50 of HT-29 and SNU-216 cells. 50 The concentrations were 45.92 μM and 41.27 μM, respectively, indicating that IQZ-17k had extremely weak effects on KRAS wild-type cells, essentially rendering it inactive in antitumor studies and posing no value for further investigation. Figure 2 ).
[0050] It is evident that IQZ-17k exhibits selective inhibitory activity against KRAS mutant cells, particularly KRAS G12D mutant gastric cancer cells, with a selectivity index of approximately 100-fold. Therefore, it possesses specific targeting for KRAS G12D mutant gastric cancer cells.
[0051] Example 4: Inhibitory effect of IQZ-17k on the binding of p-ERK2 and p53
[0052] Experimental methods: AGS cells (KRAS G12D mutant) were seeded in 6-well plates at a density of 5 × 10⁶ cells per well. 5 Cells were treated with IQZ-17k for 24 hours after cell adhesion. Cells were collected, lysed with lysis buffer containing protease inhibitors, and total protein was extracted. An equal volume of protein sample was taken and subjected to immunoprecipitation with p53 antibody. The precipitate complex was separated by SDS-PAGE and then detected by Western blot using p-ERK2 antibody and p53 antibody, respectively.
[0053] Immunoprecipitation assays showed that, compared with the control group, the coprecipitation signal of p-ERK2 and p53 was significantly reduced after IQZ-17k treatment, indicating that IQZ-17k can effectively inhibit the binding of endogenous p-ERK2 and p53. Figure 3 ).
[0054] Example 5: Effects of IQZ-17k on the expression of downstream target genes of p53
[0055] Experimental methods: After AGS cells (KRAS G12D mutant) were treated with different concentrations of IQZ-17k (0, 1, 2, 4 μM) for 24 hours, total protein was extracted, and the expression levels of PUMA and p21 proteins were detected by Western blot.
[0056] Western blot results showed that IQZ-17k treatment significantly upregulated the expression levels of PUMA and p21 proteins. Figure 4 ).
[0057] Example 6: Detection of IQZ-17k-induced apoptosis in AGS cells
[0058] Experimental methods: AGS cells (KRAS G12D mutant) were treated with different concentrations of IQZ-17k (0, 1, 2, 4 μM) for 72 hours. Cells were then collected, stained with Annexin V-FITC / PI double staining, and DMSO was used as a control. The apoptosis rate of cells treated with different concentrations of IQZ-17k was detected by flow cytometry.
[0059] Experimental results: The apoptosis rate in the control group was 8.25%; the apoptosis rates after treatment with 1μM, 2μM, and 4μM IQZ-17k for 72 h were 38.23%, 77.63%, and 81.57%, respectively. Figure 5 Compared to the control group, the apoptosis rate of cells treated with compound IQZ-17k increased in a concentration-dependent manner.
[0060] Example 7: Effect of IQZ-17k on AGS cell cycle distribution
[0061] Experimental methods: AGS cells (KRAS G12D mutant) were treated with different concentrations of IQZ-17k (0, 1, 2, 4 μM) for 24 hours, then fixed, stained with PI, and with DMSO as a negative control. The cell cycle distribution of cells treated with different concentrations of IQZ-17k was detected by flow cytometry.
[0062] Experimental results: The proportions of cells in the negative control group at G1, S, and G2 / M phases were 44.03%, 30.22%, and 25.76%, respectively. Figure 6 -B); After treatment with compound IQZ-17k at 1 μM, 2 μM, and 4 μM for 24 h, the proportion of cells in the G2 / M phase was 42.26±0.91%, 47.12±0.68%, and 48.3±1.38%, respectively, indicating that IQZ-17k can arrest the cell cycle at the G2 / M phase ( Figure 6 -C).
[0063] Example 8: Inhibitory effect of IQZ-17k on the migration ability of AGS cells
[0064] Experimental methods: AGS cells (KRAS G12D mutant) were seeded into 6-well plates. When the cell density reached more than 95%, the cells were scratched with a 200 μL pipette tip, washed away with PBS, and serum-free medium containing different concentrations of IQZ-17k was added. Photos were taken at 0 hours and 24 hours to calculate the healing rate.
[0065] Experimental results: Compared with the control group, administration of compound IQZ-17k effectively inhibited the migration of AGS cells. Administration of the compound at 0.5 μM, 1 μM, 2 μM, and 4 μM reduced the healing rate of AGS cells by 11.56%, 35.15%, 30.06%, and 35.15%, respectively. Figure 7 It was significantly better than the positive control group Nutlin3a.
[0066] Example 9: Inhibitory effect of IQZ-17k on the invasive ability of AGS cells
[0067] Experimental methods: AGS cells (KRAS G12D mutant) in the logarithmic growth phase were collected, resuspended in 1640 basal medium, and seeded in chambers lined with Matrigel. The next day, the upper chamber was replaced with basal medium containing IQZ-17k, and the lower chamber was added with 1640 medium containing 20% FBS. After culturing for 24 hours, the cells were fixed, stained, photographed, and analyzed using ImageJ.
[0068] The results showed that, compared with the control group, the number of AGS cells that crossed the chamber membrane was significantly reduced after treatment with compound IQZ-17k, suggesting that compound IQZ-17k significantly inhibited the invasive ability of AGS. Figure 8 It is superior to the positive control group Nutlin3a.
[0069] Example 10: Evaluation of the in vivo antitumor activity of IQZ-17k
[0070] Experimental methods: Human gastric cancer AGS cells (KRAS G12D mutation) in logarithmic growth phase were harvested, digested, resuspended, and the cell density was adjusted to 1×10⁻⁶. 7Human gastric cancer subcutaneous xenograft model was established in nude mice by subcutaneous inoculation of BALB / c mice at a rate of 0.1 mL / milliliter. When the tumor volume reached approximately 100 mm³, the tumor-bearing mice were randomly divided into a solvent control group and an IQZ-17k treatment group, with 6 mice in each group. The IQZ-17k treatment group received daily intraperitoneal injections (20 mg / kg and 40 mg / kg), the solvent control group received an equal volume of solvent, and the positive control group received trametinib 3 mg / kg. Mouse body weight and tumor volume were measured every two days during the treatment period, for 25 consecutive days. After the last administration, the animals were sacrificed, the tumor tissue was dissected and weighed, and the tumor inhibition rate was calculated. The formula for calculating the tumor inhibition rate is: Tumor inhibition rate (%) = (average tumor weight in the control group - average tumor weight in the treatment group) / average tumor weight in the control group × 100%.
[0071] Experimental results: During the administration period, there was no significant difference in body weight between the IQZ-17k treatment group and the control group. Figure 9 The presence of -A indicates that IQZ-17k is well-tolerated at the administered dose.
[0072] Tumor volume measurements showed that, compared to the solvent control group, tumor volume growth was significantly slowed in the IQZ-17k treatment group. Figure 9 -B) The tumor was removed, weighed, and photographed after the last administration of the drug. Figure 9 -C), the average tumor weight in the solvent control group was 1.11 g, and the average tumor weight in the IQZ-17k 20mg / kg and 40mg / kg treatment groups was 0.38 and 0.14 g, respectively. Figure 9 The tumor inhibition rates calculated were 65.64% and 87.56%, respectively, indicating that IQZ-17k has a good inhibitory effect on KRAS-mutant gastric cancer AGS subcutaneous xenografts in nude mice.
[0073] This invention provides the application of IQZ-17k in the preparation of drugs for treating KRAS-mutant gastrointestinal tumors. This compound exhibits selective inhibitory activity against KRAS-mutant tumors (IC50-90% inhibition on KRAS-mutant cells). 50 The concentration was 0.42 μM, and the IC50 concentration in KRAS wild-type cells was [missing value]. 50 The effective concentrations were 45.92 μM and 41.27 μM (with a selectivity index of approximately 100-fold), respectively. The in vivo tumor inhibition rate reached 87.56%, and the safety profile was good. This discovery provides a new treatment option for patients with KRAS-mutant gastrointestinal tumors, possessing significant clinical value and promising industrial application prospects. Drugs containing IQZ-17k can be formulated into oral or injectable formulations, showing broad clinical application potential.
Claims
1. The use of imidazoquinaline compounds represented by formula (Ⅰ) or pharmaceutically acceptable salts thereof in the preparation of drugs for treating gastrointestinal tumors: ； in, R can be H, halogen, C1-C6 alkyl, or C1-C6 alkoxy.
2. The application according to claim 1, characterized in that, The gastrointestinal tumors mentioned are gastric cancer or colorectal cancer.
3. The application according to claim 1 or 2, characterized in that, The gastrointestinal tumors mentioned are KRAS-mutated gastrointestinal tumors.
4. The application according to claim 3, characterized in that, The KRAS mutation is one or more of the following: KRAS G12D, KRAS G12V, and KRAS G13D.
5. The application according to any one of claims 1-4, characterized in that, The imidazoquinaline compounds or their pharmaceutically acceptable salts have the following structures: 。 6. The application according to any one of claims 1-5, characterized in that, The imidazoquinaline compounds or their pharmaceutically acceptable salts exert their antitumor effects by inhibiting the binding of p-ERK2 to p53 protein.
7. The application according to any one of claims 1-5, characterized in that, The imidazoquinaline compounds or their pharmaceutically acceptable salts exert their antitumor effects by upregulating the expression of the downstream pro-apoptotic gene PUMA of p53.
8. The application according to any one of claims 1-7, characterized in that, The imidazoquinazolinoid compound or its pharmaceutically acceptable salt is prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.