Application of medicinal compound in treatment of solid tumors
Patent Information
- Application Number
- CN202480017373.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-03-10
- Filing Date
- 2024-03-06
- Publication Date
- 2025-10-24
Smart Images
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Abstract
Description
Use of a medicinal compound in treating solid tumors
[0001] Cross-references
[0002] This application claims priority to Chinese patent application No. 202310231050.X, filed on March 10, 2023, entitled “Use of a pharmaceutical compound in the treatment of solid tumors,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field
[0003] The present invention belongs to the field of pharmaceutical technology and relates to the use of a pharmaceutical compound in treating solid tumors. Background Art
[0004] Lung cancer is one of the most aggressive malignancies, with the highest mortality rate of all cancer types, accounting for 19% of cancer-related deaths and 3% of all deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and is the most common type of lung cancer. With the advancement of genetic testing technology, an increasing number of lung cancer driver genes have been discovered, and with the increasing availability of targeted therapeutic drugs, lung cancer has entered an era of precision medicine.
[0005] However, in addition to familiar targets like EGFR and ALK, further breakthroughs have been made for rare or less common targets such as KRAS, RET, MET, and ROS1. New drugs targeting these rare targets have been approved for marketing, and the treatment of these rare targets in lung cancer is gradually seeing the light of day. However, in 10-20% of lung adenocarcinoma patients, no tumor driver genes have been identified.
[0006] Hiroli Izumi et al. (Nature, 2021.11) discovered a new therapeutic target for NSCLC, CLIP1-LTK, which has a 0.4% probability of fusion mutation in NSCLC. Fusion-positive tumors are negative for other known oncogenes, indicating that CLIP1-LTK fusion is mutually exclusive with other identified oncogenes. The above research indicates that CLIP1-LTK fusion is very likely to be an oncogenic driver gene in NSCLC, but there are few reports on small molecule inhibitors of this target. Moreover, more research results are still needed to confirm its true recognition as the next precision treatment target for lung cancer.
[0007] CN108276410A discloses a series of compounds targeting ALK targets. In vitro enzymatic activity experiments and cell proliferation inhibition experiments show that they can significantly inhibit the kinase activity of ALK, including N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4-(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine and other compounds.
[0008] Summary of the Invention
[0009] The inventor discovered in an accidental experiment that N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(Isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine has a significant inhibitory effect on LTK tyrosine kinase and has a significant inhibitory effect on the growth of tumors caused by CLIP1-LTK fusion protein in animals.
[0010] N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 The structure of -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine is shown in the following formula (I):
[0011] The present invention relates to the following.
[0012] One of the objects of the present invention is to provide a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable carrier.
[0013] One of the objects of the present invention is to provide a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or the above composition in the preparation of a drug for treating LTK-mediated diseases.
[0014] One of the objects of the present invention is to provide a method for treating LTK-mediated diseases, which comprises administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.
[0015] One of the objects of the present invention is to provide a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating CLIP1-LTK fusion-positive diseases.
[0016] One of the objects of the present invention is to provide a method for treating CLIP1-LTK fusion-positive diseases, which comprises administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition to a subject.
[0017] As a preferred embodiment, the LTK-mediated disease or CLIP1-LTK fusion-positive disease is a solid tumor.
[0018] As a more preferred embodiment, the solid tumor is non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, systemic histiocytosis and neuroblastoma.
[0019] As the most preferred embodiment, the non-small cell lung cancer is LTK-mediated or CLIP1-LTK fusion-positive non-small cell lung cancer.
[0020] In a specific embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition can also be administered in combination with a second agent, which is a chemotherapeutic drug, a chemo-targeted drug or an antibody drug.
[0021] As used herein, the term "LTK" refers to leukocyte tyrosine kinase (English name: Leukocyte Receptor Tyrosine Kinase). The term "CLIP1" refers to cytoplasmic linker protein 1 (English name: CAP-Gly Domain Containing Linker Protein 1).
[0022] In this article, NIH3T3 cells refer to a mouse embryonic fibroblast cell line established by the National Institutes of Health (NIH). Baf3 cells refer to a mouse primary B lymphocyte cell line. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1: Inhibitory effect of the compound of formula (I) on LTK kinase.
[0024] FIG2A shows the inhibitory activity of the compound of formula (I) on the proliferation of Baf3 cells expressing CLIP1-LTK fusion.
[0025] FIG2B shows the inhibitory activity of the compound of formula (I) on the proliferation of NIH3T3 cells expressing CLIP1-LTK fusion.
[0026] FIG3A shows the effect of the compound of formula (I) on tumor growth in CLIP1-LTK / Baf3 tumor-bearing mice.
[0027] FIG3B : Effects of the compound of formula (I) on the body weight of CLIP1-LTK / Baf3 tumor-bearing mice.
[0028] FIG4A shows the effect of the compound of formula (I) on tumor growth in CLIP1-LTK / NIH3T3 tumor-bearing mice.
[0029] FIG4B : Effects of the compound of formula (I) on the body weight of CLIP1-LTK / NIH3T3 tumor-bearing mice.
[0030] Detailed Description of the Invention
[0031] The present invention is further illustrated below by way of examples and drawings, but is not intended to limit the present invention.
[0032] Example 1
[0033] Compound of formula (I) (N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 LTK kinase inhibitory activity assay of 2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine).
[0034] A homogeneous time-resolved fluorescence (HTRF) assay platform was established to determine compound activity. The reaction buffer consisted of 50 mM HEPES, pH 7.5; 2 μM Na₃VO₄; 0.001% Tween-20; 5 mM MgCl₂; 1 mM DTT; 33.3 nM SEB (Cisbio); and 0.01% BSA. Compounds were serially diluted three-fold starting at 10 μM using 10% DMSO in reaction buffer (10 concentrations total). At each concentration, 2 μL of the diluted compound was added to 48 μL of reaction buffer and mixed thoroughly. 2.5 μL of the sample was added to a 384-well plate (OptiPlate-384, PerkinElmer). Then, 5 μL of GST-LTK (498-796aa, final concentration 0.1 nM) was added and centrifuged to mix thoroughly. The reaction was initiated by adding 2.5 μL of ATP (final concentration 10 μM) and a mixture of TK peptide substrates (final concentration 1 μM, Cisbio) to a total reaction volume of 10 μL. The 384-well plate was incubated at 23°C for 1 hour. The reaction was then stopped by adding 5 μL of TK Antibody (Cisbio) and 5 μL of Streptavidin-XL665 (Cisbio). After an additional 1 hour of incubation in the incubator, fluorescence was read on an Envision reader (PerkinElmer) (excitation at 320 nm, emission at 665 nm and detection at 620 nm; the ratio of the two wavelengths represents the LTK kinase activity signal). The signal value of the compound at each concentration was measured, and the IC value of the compound for LTK enzyme inhibition was calculated using GraphPad Prism software. 50 As shown in Figure 1, the compound has a strong inhibitory activity against LTK kinase, and the IC 50 The value is 2.07nM.
[0035] Unexpectedly, the applicant used the above method to test other specific compounds disclosed in the patent document CN108276410A. The specific compounds of Examples 2, 13-14, 16-17, 18, and 20 in the patent had poor inhibitory ability on LTK kinase activity. 50 The values were all greater than 50 nM, which could not effectively inhibit the LTK kinase activity.
[0036] Example 2
[0037] Determination of cell proliferation inhibitory activity of the compound of formula (I).
[0038] LTK fusion cell lines CLIP1-LTK / Baf3 and CLIP1-LTK / NIH3T3 were constructed using lentiviral infection. CLIP1-LTK / Baf3 cells were cultured in suspension using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin (P / S, purchased from Life Technology); CLIP1-LTK / NIH3T3 cells were cultured in adherent culture using DMEM medium supplemented with 10% fetal bovine serum (FBS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin (P / S, purchased from Life Technology). Cells were cultured in a cell culture incubator at 37°C and 5% CO2. The day before compound testing, CLIP1-LTK / Baf3 cells were plated at a density of 2000 cells / 195 μL / well in a 96-well plate (Corning); CLIP1-LTK / NIH3T3 cells were plated at a density of 1000 cells / 195 μL / well in a 96-well plate. After 24 hours, the compound was serially diluted three-fold starting at 10 mM in 100% DMSO (11 concentrations total). Then, 2 μL of each concentration was added to 48 μL of serum-free medium for further dilution. 5 μL of each diluted compound concentration was added to the cell culture plate and cultured in a cell incubator for an additional 3 days. After 3 days, 45 μL of Cell-Titer Glo reagent (Promega) was added directly to the culture medium for CLIP1-LTK / Baf3 cells; for CLIP1-LTK / NIH3T3 cells, the culture medium was aspirated and 25 μL of Cell-Titer Glo reagent was added. Incubate at room temperature for 5-10 minutes. Read the fluorescence signal value on the Envision machine and calculate the IC value of the compound for cell proliferation inhibition using GraphPad Prism software. 50As shown in Figures 2A and 2B, the compounds of the present invention have significant inhibitory activity on the proliferation of CLIP1-LTK fusion-expressing Baf3 and NIH3T3 cells, IC 50 The values were 43.0 nM and 70.7 nM, respectively.
[0039] Example 3
[0040] In vivo pharmacodynamic evaluation of compounds
[0041] 6-week-old SPF-grade BALB / c-nude female mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were injected subcutaneously on both sides of the back with 0.1 mL of 2.0×10 6 CLIP1-LTK / Baf3 cells or 7.5×10 6 CLIP1-LTK / NIH3T3 cells and 30% Matrigel (purchased from BD) in PBS were used to establish CLIP1-LTK / Baf3 and CLIP1-LTK / NIH3T3 mouse subcutaneous transplant tumor models, respectively.
[0042] When the average tumor volume reaches 100-200 mm 3 When the experiment was conducted, mice with tumors of regular shape and uniform size were selected for the experiment, and the mice were randomly divided into groups according to the tumor volume and weight. The experimental animals were orally administered with the solvent and different concentrations of the compound, starting from the day of grouping, once a day for 9 consecutive days. During the experiment, the long diameter (cm) and short diameter (cm) of the tumor were measured with a vernier caliper, and the tumor volume was calculated using the formula. Tumor volume (cm 3 )=0.5×long diameter (cm)×short diameter (mm) 2 Tumor diameters were measured three times per week, tumor volumes were calculated, and tumor growth curves were plotted. At the end of the experiment, mice were sacrificed, and tumor tissues were collected, photographed, and weighed.
[0043] Among them, the experimental results for CLIP1-LTK / Baf3 tumor-bearing mice are shown in Figures 3A and 3B. The growth of tumors was inhibited in a dose-dependent manner in CLIP1-LTK / Baf3 tumor-bearing mice. Figure 3A was analyzed and sorted to obtain the effects of the compound on the tumor volume growth inhibition rate (Table 1) and the relative tumor proliferation rate (Table 2) of CLIP1-LTK / Baf3 tumor-bearing mice. In terms of compound safety, no drug-induced death or weight change was observed in the tumor-bearing mice in the compound-treated group, and no obvious abnormalities were observed in the general condition of the tumor-bearing mice, indicating that the compound has good tolerance (Figure 3B).
[0044] Table 1
[0045] Table 2
[0046] Among them, the experimental results for CLIP1-LTK / NIH3T3 tumor-bearing mice are shown in Figures 4A and 4B. The growth of tumors was inhibited in a dose-dependent manner in CLIP1-LTK / NIH3T3 tumor-bearing mice. Figure 4A was analyzed and sorted to obtain the effects of the compound on the tumor volume growth inhibition rate (Table 3) and the relative tumor proliferation rate (Table 4) of CLIP1-LTK / NIH3T3 tumor-bearing mice. In terms of compound safety, no drug-induced death or weight change was observed in the tumor-bearing mice in the compound-treated group, and no obvious abnormalities were observed in the general state of each tumor-bearing mouse, indicating that the compound has good tolerance (Figure 4B).
[0047] Table 3
[0048] Table 4
[0049] Although the above describes the specific implementation methods of the present invention in combination with the embodiments, it does not limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.
Claims
1. N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine or a pharmaceutically acceptable salt thereof or a therapeutic agent comprising any one of the above in the preparation of a drug for treating LTK-mediated diseases.
2. A method for treating a LTK-mediated disease, It is characterized in that The method comprises administering to the subject a therapeutically effective amount of N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine or containing N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine therapeutic agents. 3.N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine or a pharmaceutically acceptable salt thereof, or a therapeutic agent comprising any one of the foregoing, in the preparation of a drug for treating CLIP1-LTK fusion-positive diseases.
4. A method for treating a CLIP1-LTK fusion-positive disease, It is characterized in that The method comprises administering to the subject a therapeutically effective amount of N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine or containing N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine therapeutic agents.
5. A therapeutic agent for LTK-mediated or CLIP1-LTK fusion-positive diseases, It is characterized in that The therapeutic agent comprises N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.
6. The use, method or therapeutic agent according to any one of claims 1 to 5, It is characterized in that The N 2 -(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)-N 4 The structure of -(2-(isopropylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine is shown in Formula I below:
7. The use, method or therapeutic agent according to any one of claims 1 to 5, It is characterized in that The LTK-mediated disease or CLIP1-LTK fusion-positive disease is a solid tumor.
8. The use, method or therapeutic agent according to claim 7, It is characterized in that The solid tumors are non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, systemic histiocytosis and neuroblastoma.
9. The use, method or therapeutic agent according to claim 8, It is characterized in that The non-small cell lung cancer is LTK-mediated or CLIP1-LTK fusion-positive non-small cell lung cancer.
10. The therapeutic agent according to any one of claims 5 to 9, It is characterized in that Administered in combination with a second agent, the second agent being a chemotherapeutic drug, a chemically targeted drug or an antibody drug.