Use of a tetrahydro-2-(2-phenylethyl) chromone derivative for the preparation of a medicament for the treatment of mutant lung cancer

Abstract

The application belongs to the field of medicine and relates to application of tetrahydro-2-(2-phenylethyl) chromone derivatives in preparation of drugs for treating mutant lung cancer, in particular, to application of a linaloe-derived tetrahydro-2-(2-phenylethyl) chromone derivative in preparation of drugs for treating KRAS G12C mutant lung cancer. Through CCK8 cell viability experiments and zebrafish CDX model experiments, it is found that THPECs-8# can inhibit cell viability of human KRAS G12C mutant lung cancer cells H23 and H358 and proliferation of tumors in a zebrafish CDX tumor model, verifying that THPECs-8# has an anti-KRAS G12C mutant lung cancer effect and indicating that THPECs-8# has application prospects in preparation of drugs for treating KRAS G12C mutant lung cancer.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the application of a tetrahydro-2-(2-phenylethyl)chromone derivative in the preparation of a drug for treating mutant lung cancer, and more particularly to the application of an agarwood-derived tetrahydro-2-(2-phenylethyl)chromone derivative in the preparation of a drug for treating KRAS G12C mutant lung cancer. Background Technology

[0002] Agarwood is the resinous wood of plants belonging to the genus *Aquilaria* Lam. or *Gyrinops Gaertn.* of the family Thymelaeaceae. It is a natural fragrance and a precious traditional medicinal material. Agarwood is a pungent, bitter, and slightly warm medicinal material, and it enters the spleen, stomach, and kidney meridians. Modern pharmacological studies have shown that the chemical components in agarwood have sedative, analgesic, hypoglycemic, antitumor, antibacterial, and anti-inflammatory effects. Chromones are one of the most important chemical components in agarwood, among which tetrahydro-2-(2-phenylethyl)chromones (THPECs) are a class of chromone compounds found only in agarwood; currently, only more than 60 types of THPECs have been discovered. (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromone (hereinafter referred to as THPECs-8#) is a THPEC isolated from Chinese agarwood by researchers in 2008. It has the activity of inhibiting the proliferation of gastric cancer cells SGC7901 [Jun Liu, Jiao Wu, You Xing Zhao, Yuan Yuan Deng, Wen LiMei, Hao Fu Dai. A new cytotoxic 2-(2-phenylethyl)chromone from Chineseeaglewood. Chinese Chemical Letters 19(2008)934–936.]. However, whether THPECs-8# has an inhibitory effect on KRAS G12C mutant lung cancer has not been reported.

[0003] One-third of human tumors have RAS mutations, with 85% of these mutations being KRAS mutations. Among KRAS mutations, the G12C mutation accounts for 44%, primarily occurring in lung cancer (14%). In 2021, two drugs targeting KRAS G12C-mutant lung cancer, sotorasirb and adagraxirb, were launched for follow-up treatment after progression of first-line therapy. However, the overall response rate of these two drugs for lung cancer is only around 45%. This invention provides a novel candidate compound for the treatment of KRAS G12C-mutant lung cancer. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of a tetrahydro-2-(2-phenylethyl)chromone derivative derived from agarwood in the preparation of a drug for treating KRAS G12C-mutant lung cancer.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] This invention discloses the use of a compound represented by Formula I and its pharmaceutically acceptable salt in the preparation of a drug for treating mutant lung cancer. The structural formula of the compound represented by Formula I is shown below:

[0007]

[0008] The compound represented by Formula I is named (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene, denoted as THEPCs-8#.

[0009] In some embodiments, the mutated lung cancer is KRAS G12C mutation-mediated lung cancer.

[0010] In some embodiments, preferably, the KRAS G12C mutation-mediated lung cancer is KRAS G12C mutation-positive mediated lung cancer.

[0011] In some embodiments, the KRAS G12C mutation is a KRAS G12C mutant H23.

[0012] In some embodiments, the KRAS G12C mutation is a KRAS G12C mutant H358.

[0013] In some embodiments, the KRAS G12C mutation is KRAS G12C mutant H23 and KRAS G12C mutant H358.

[0014] In some embodiments, the pharmaceutically acceptable salt includes, but is not limited to, hydrochloride and sulfate.

[0015] The experiments of this invention demonstrate that THPECs-8# has an inhibitory effect on the cell viability of KRAS G12C mutant lung cancer cells H23 and H358.

[0016] The experiments of this invention demonstrate that THPECs-8# has an inhibitory effect on the proliferation of zebrafish CDX model (human lung cancer H23 cell xenograft model).

[0017] Beneficial effects:

[0018] Compared with existing technologies, this invention has the following advantages: This study, through CCK8 cell viability experiments and zebrafish CDX model experiments, found that (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene can inhibit the cell viability of human KRAS G12C mutant lung cancer cells H23 and H358 and tumor proliferation in a zebrafish CDX tumor model, verifying that (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene has anti-KRAS activity. The study suggests that (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene may be a promising candidate for developing drugs to treat KRAS G12C-mutant lung cancer. Attached Figure Description

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0020] Figure 1 The effects of different concentrations of THEPCs-8# on the viability of H23 cells at 24h, 48h, and 72h were investigated.

[0021] Figure 2 The effects of different concentrations of THEPCs-8# on the viability of H358 cells at 24h, 48h, and 72h were investigated.

[0022] Figure 3 Fluorescence imaging images of representative zebrafish from each group in the zebrafish lung cancer H23 cell xenograft experiment.

[0023] Figure 4 The graph shows the antitumor activity data of different concentrations of THPECs-8# in a zebrafish lung cancer H23 cell xenograft model. Detailed Implementation

[0024] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0026] The compounds used in the embodiments of this invention: Its Chinese name is (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene, denoted as THEPCs-8#.

[0027] Example 1: Effect of THEPCs-8# on the viability of human KRAS G12C mutant lung cancer cell lines

[0028] Experimental materials: (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene (THPECs-8#) was provided by Nanjing Shangyuantang Agarwood Biotechnology Co., Ltd. It was a white amorphous powder with the molecular formula C5. 18 H 19 ClO7, with a molecular weight of 382.8 g / mol. Human KRAS G12C mutant lung cancer cells H23 and H358 were donated by Dr. Rafael. RPMI-1640 medium, fetal bovine serum, antibiotics, and trypsin-EDTA solution used for cell culture were all purchased from Thermo Fisher Scientific. CCK8 solution was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0029] Experimental instruments: Hera 150 CO2 incubator (ThermoFisher Scientific, USA), biosafety cabinet (Labconco, USA), Varioskan full-wavelength microplate reader (ThermoFisher Scientific, USA).

[0030] Experimental Methods: Cells (mutant lung cancer cells H23 or H358) were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Once the cells reached the logarithmic growth phase, a single-cell suspension was prepared by digestion with 0.25% trypsin-EDTA solution. 100 μL of the cell suspension (5000 cells / well) was uniformly cultured in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Then, different concentrations of THEPCs-8# were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h, 48 h, and 72 h, respectively (final concentrations of THEPCs-8# were 0 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM). After the intervention period, 10 μL of CCK8 solution was added to each well. After incubation at 37°C for 2 hours, the OD value at 450 nm was measured using a microplate reader, and the inhibition rate of THEPCs-8# against human KRASG12C mutant lung cancer cells H23 and H358 was calculated. The half-maximal inhibitory concentration (IC50) was calculated using Graphpad Prism 8.0 software. 50 The formula for calculating the inhibition rate is shown below:

[0031] Cell inhibition rate % = (1 - average absorbance of cells in the treatment group / average absorbance of cells in the control group) × 100%.

[0032] The control group consisted of dimethyl sulfoxide (DMSO) as the solvent control.

[0033] Experimental Results: The inhibitory effect of the natural compound THEPCes-8# extracted from the traditional Chinese medicine agarwood at different concentrations on the cell viability of human KRAS G12C mutant lung cancer cells H23 and H358 was detected by the CCK8 assay. The results are as follows: Figure 1 , Figure 2 As shown, the results indicated that 0–100 μM THPECs-8# significantly inhibited the viability of KRAS G12C mutant lung cancer cells H23 and H358 after 24 h, 48 h, and 72 h in a time- and concentration-dependent manner. The IC50 of THPECs-8# at 72 h was [not specified in the original text]. 50 The concentrations were 34.22 μM and 50.59 μM, respectively. These results indicate that THPECs-8# significantly inhibits the viability of human KRAS G12C mutant lung cancer cells H23 and H358.

[0034] Example 2: Effects of THEPCS-8# on tumor growth in a zebrafish CDK model

[0035] Experimental materials: (5S,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7-trihydroxy-8-chloro-5,6,7,8-tetrahydrochromene (THPECs-8#) was provided by Nanjing Shangyuantang Agarwood Biotechnology Co., Ltd. It was a white amorphous powder with the molecular formula C5. 18 H 19 ClO7, with a molecular weight of 382.8 g / mol. Human KRAS G12C mutant lung cancer cells H23 were donated by Dr. Rafael. RPMI-1640 medium, fetal bovine serum, antibiotics, and trypsin-EDTA solution used for cell culture were all purchased from Thermo Fisher Scientific. CellTracker CM-DiI dye was from Invitrogen, USA. Wild-type AB strain zebrafish were raised in aquaculture water at 28℃ (water quality: 200 mg of readily soluble sea salt per 1 L of reverse osmosis water; conductivity 500–600 μS / cm; pH 7.0–8.0; hardness 50–100 mg / L CaCO3), provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd.

[0036] Experimental instruments: MVX10 fluorescence stereomicroscope (OLYMPUS, Japan), DP73 CCD camera (OLYMPUS, Japan), PICOSPRITZERⅢ microinjection apparatus (Parker, USA), PC-10 needle puller (Narishige, Japan), GHP-400 light incubator (Shanghai Jiangren Experimental Equipment Co., Ltd.).

[0037] Experimental Methods: H23 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Once the cells reached the logarithmic growth phase, human KRAS G12C mutant lung cancer cells (H23) were collected by digestion with 0.25% trypsin-EDTA. The cells were washed once with 1 mL of PBS buffer (pH 7.4, 0.01 M), centrifuged to remove the supernatant, and then 1 mL of PBS (pH 7.4, 0.01 M) was added. 1 μL of CM DiI dye was then added, and the cells were stained at 37°C for 5 min, followed by incubation at 4°C for 15 min. After centrifugation, the supernatant was discarded, and the cells were washed three times with PBS (pH 7.4, 0.01 M). The cells were then resuspended in 5–20 μL of PBS (pH 7.4, 0.01 M) and prepared for injection. Wild-type AB zebrafish embryos at 48 hpf were fixed onto low-melting-point agar plates. The tumor cell suspension stained in the previous step was injected into the perivitelline space of the zebrafish at a dose of 400 cells / embryo. 24 hours post-transplantation, zebrafish with a consistent number of transplanted tumor cells were randomly divided into four groups of 30 each and placed in six-well plates. The control solvent DMSO (DMSO group) and different final concentrations of THPECs-8# were added (15 μM THPECs-8# group, 30 μM THPECs-8# group, and 60 μM THPECs-8# group). The six-well plates were placed in a 34°C light incubator with 14 hours of light followed by 10 hours of darkness for 3 consecutive days. After drug administration, 10 juvenile fish at 4 dpi were randomly selected from each group, fixed with low-melting-point adhesive, and subjected to fluorescence imaging. Image processing was performed using ImageJ software, and statistical analysis was conducted using Graphpad Prism8 software.

[0038] Experimental results: Figure 3 It can be seen that, compared with the solvent control group (DMSO group), the fluorescence intensity of zebrafish in all THEPCs-8# drug-treated groups was reduced; Figure 4Fluorescence intensity scanning analysis revealed that the 15 μM THPECs-8# treatment group showed a 42.8% reduction in tumor cell proliferation compared to the solvent control group (***P=0.0003, n=10), the 30 μM THPECs-8# treatment group showed a 68.6% reduction in tumor cell proliferation compared to the solvent control group (****P<0.0001, n=10), and the 60 μM THPECs-8# treatment group showed a 63.7% reduction in tumor cell proliferation compared to the solvent control group (****P<0.0001, n=10). These results indicate that THPECs-8# can inhibit the proliferation of human KRAS G12C mutant lung cancer cells H23 in zebrafish.

[0039] This invention provides a concept and method for the application of tetrahydro-2-(2-phenylethyl)chromone derivatives in the preparation of drugs for treating mutant lung cancer. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The use of a compound of Formula I and its pharmaceutically acceptable salt in the preparation of a drug for treating mutant lung cancer, wherein the compound of Formula I has the following structural formula: ； The mutated lung cancer is KRAS G12C mutation-mediated lung cancer.

2. The application according to claim 1, characterized in that, The KRAS G12C mutation becomes KRAS G12C mutant H23.

3. The application according to claim 1, characterized in that, The KRAS G12C mutation becomes KRAS G12C mutant H358.

4. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is either a hydrochloride or a sulfate.

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