3, 4, 5-trihydroxy phenylacetate compound as well as preparation method and application thereof

Abstract

The invention relates to a 3, 4, 5-trihydroxy phenylacetate compound and a preparation method and application thereof, the 3, 4, 5-trihydroxy phenylacetate compound belongs to a compound shown in a general formula I and a general formula II, R1, R2 and corresponding derivatives thereof can activate a copper death pathway mediated by endogenous copper ions, have an obvious anti-tumor effect, and have a stronger anti-tumor effect in chemotherapy of drug-resistant tumors. The tumors comprise melanoma, liver cancer, gastric cancer, breast cancer, oral squamous cell carcinoma and the like. The compound is a non-copper ion carrier compound with a brand new structure, induced copper death does not depend on exogenous copper ions, and endogenous copper ions are dissociated by reducing the content of glutathione which is a copper ion chelate in cells. The copper ions are further combined with microfilament skeleton related protein to cause disintegration of the microfilament skeleton, so that copper death is induced. The compound disclosed by the invention can be used as a potential lead compound for inducing endogenous copper ion mediated copper death for anti-tumor therapy.

Description

Technical Field

[0001] This invention relates to the fields of medicinal chemistry and tumor treatment, and particularly to a 3,4,5-trihydroxyphenylacetic acid ester compound, its preparation method, and its application. Background Technology

[0002] Copper death, first reported in *Science* in 2022 by Tsvetkov et al., is a novel form of cell death induced by copper overload and is closely related to intracellular copper levels. The mitochondrial protein FDX1 not only reduces copper ions entering the mitochondria to the more toxic cuprous ions but also promotes lipoylation modification of proteins; subsequently, copper ions directly bind to lipoylated proteins, inducing their polymerization. Furthermore, copper-induced degradation of iron-sulfur cluster proteins leads to aberrant protein stress responses, ultimately inducing copper death.

[0003] Copper ion carriers can transport external copper into cells, and once it accumulates to a certain threshold, it can induce copper death. There are four main classes of copper ion carrier drugs: ilisimo, disulfiram, cloiodriquinone, and 8-hydroxyquinone. Ilisimo is a mitochondrial-targeting copper ion carrier; its sodium salt formulation has been developed for clinical trials and can be used in combination with paclitaxel, as well as as monotherapy for various solid tumors and acute myeloid leukemia. Ilisimo has a good safety profile, but has not shown good clinical efficacy. Disulfiram is an FDA-approved aldehyde dehydrogenase (ALDH) inhibitor with multi-target antitumor activity; its anticancer efficacy is significantly enhanced when used in combination with copper. However, the toxicity of disulfiram is closely related to its ability to promote intracellular copper accumulation.

[0004] Copper chelators have demonstrated potential in preclinical animal and clinical trials for cancer treatment, primarily by impairing angiogenesis and inhibiting tumor cell proliferation and metastasis. Representative drugs include ammonium tetrathiomolybdate, choline tetrathiomolybdate, D-penicillamine, and trientine. Ammonium tetrathiomolybdate is an oral copper chelator. Studies have shown that ammonium tetrathiomolybdate helps inhibit the development of papillary thyroid carcinoma, colon cancer, and melanoma by reducing copper levels and inhibiting MEK1 / 2 kinase activity. Choline tetrathiomolybdate is a second-generation ammonium tetrathiomolybdate analogue, exhibiting better stability and a longer half-life. D-penicillamine was the first copper chelator used to treat Wilson's disease and has been preclinically shown to inhibit tumor proliferation and angiogenesis. Trientine is an alternative copper chelator used clinically in Wilson's disease patients who are intolerant to D-penicillamine, showing more significant inhibitory effects.

[0005] Although therapies using copper ion carriers to induce copper death and suppress tumors have proven effective in combating tumors, the uncontrollable nature of exogenous copper ions can lead to an imbalance in copper homeostasis, resulting in serious side effects such as liver damage. Therefore, developing antitumor therapies based on endogenous physiological copper ion-mediated copper death, rather than relying on exogenous copper ion supplementation, may be a potentially effective approach with fewer side effects. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a 3,4,5-trihydroxyphenylacetic acid ester compound, its preparation method and application, as a novel inducer for activating endogenous copper ion-mediated copper death, rather than an exogenous copper ion carrier. This provides a novel lead compound for copper death-based antitumor therapy. This invention has important guiding significance for the research and development of copper death inducers that are not copper ion carriers.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A 3,4,5-trihydroxyphenylacetic acid ester compound comprising at least one of the following structural formulas I and II:

[0009]

[0010] R1 is selected from C4~C12 alkyl chains, cyclopentane, and cyclohexane; R2 is selected from hydrogen, halogen, cyano, methyl, ethyl, trifluoromethyl, and carboxyl.

[0011] The method for preparing the 3,4,5-trihydroxyphenylacetic acid ester compound is as follows:

[0012] .

[0013] The application of the 3,4,5-trihydroxyphenylacetic acid ester compound is to prepare an inducer for copper death in tumor cells, specifically, to prepare an inducer that activates endogenous copper ion-mediated cytoskeleton disintegration, thereby activating the copper death pathway.

[0014] The application of the 3,4,5-trihydroxyphenylacetic acid ester compounds is characterized by their biological activity, particularly their antitumor application based on the regulation of copper death mechanisms, thereby enabling their use in the preparation of drugs for treating cancer, especially in chemotherapy-resistant tumors with higher endogenous copper ion content. The antitumor activity of these compounds covers melanoma, liver cancer, gastric cancer, breast cancer, and oral squamous cell carcinoma, as well as their corresponding chemotherapy-resistant tumors.

[0015] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0016] 1. This invention provides a class of non-copper ion carrier compounds that can activate the endogenous copper ion-mediated copper death pathway without relying on exogenous copper ions, thus avoiding side effects such as copper homeostasis imbalance and liver damage that may be caused by exogenous copper ions.

[0017] 2. The compounds of this invention induce copper death in tumor cells by downregulating the intracellular glutathione (GSH) content, releasing intracellular free copper ions, which then bind to and destroy microfilament cytoskeleton proteins.

[0018] 3. This invention has significant anti-tumor effects on various tumor cells (melanoma, liver cancer, gastric cancer, breast cancer, oral squamous cell carcinoma, etc.), and exhibits stronger inhibitory activity in chemotherapy-resistant tumors.

[0019] 4. The compounds of this invention are novel lead compounds with good drug development potential and structural modifiability, providing an important foundation for the subsequent development of copper death-based antitumor drugs. Attached Figure Description

[0020] Figure 1 To investigate the effect of HThPA on endogenous copper-mediated copper death, A375 cells were pretreated with TTM and D-pen for 2 hours, followed by HThPA treatment for 24 hours. Cell morphology and ATP levels were observed and measured. The results showed that both the copper ion chelators TTM and D-pen could inhibit HThPA-induced copper death.

[0021] Figure 2 The compound HThPA cannot transport exogenous copper ions into cells. A375 cells were pretreated with copper ions (CuSO4) for 2 hours, followed by treatment with HThPA or the copper ion carrier DSF for 4 hours. Intracellular copper ion levels were detected by ICP-MS. The results showed that HThPA did not increase intracellular copper ion levels, proving that HThPA is not a copper ion carrier.

[0022] Figure 3 HThPA was used to induce copper death in various tumor cell lines. Different concentrations of HThPA were used to treat various tumor cell lines, and ATP levels were measured after 24 hours. The results showed that HThPA could induce copper death in various cell types, exhibiting a gradient effect.

[0023] Figure 4 The compound HThPA was used to downregulate intracellular glutathione (GSH) levels. Intracellular GSH levels were measured after treating A375 cells with different concentrations of HThPA for 2 hours. The results showed that HThPA downregulated intracellular GSH levels in a gradient effect.

[0024] Figure 5The compound HThPA was investigated to inhibit breast cancer with a gradient effect. In a PDX model, nude mice were inoculated abdominally with patient-derived breast cancer tissue blocks and intraperitoneally injected with HThPA (20 mg / kg) every two days. Mice were sacrificed when the tumor reached a predetermined size, and tumor weight was photographed and recorded. The results showed that HThPA significantly inhibited breast cancer growth.

[0025] Figure 6 The compound HThPA was used to inhibit the growth of primary gastric cancer. Cldn18 -CreERT2; Apc fl / fl ; Trp53 fl / fl ; Kras G12D (Cldn18-ATK) mice were injected with Tamoxifen (100 mg / kg) for two weeks, followed by intraperitoneal injection of 5-FU (25 mg / kg, once a week), HThPA (20 mg / kg, once every two days), or a combination thereof. One month later, the mice were sacrificed, and the proportion of gastric tumor area to total gastric area was recorded. The results showed that HThPA could inhibit gastric cancer, and 5-FU synergistically enhanced the antitumor effect of HThPA.

[0026] Figure 7 The compound HThPA was used to inhibit primary liver cancer. After inducing liver tumors in mice using the Sleeping Beauty system, HThPA was administered intraperitoneally (once every two days). One month later, the mice were sacrificed, and liver weight, body weight, and tumor number were recorded. The results showed that HThPA significantly inhibited the growth of liver cancer.

[0027] Figure 8 To investigate the effect of compound HThPA on inducing stronger copper death in drug-resistant cell lines, parental cell lines (P) and their corresponding drug-resistant cell lines (R) (A375 for melanoma, A549 for lung cancer, KB for oral squamous cell carcinoma, and AGS for gastric cancer) were treated with different concentrations of HThPA, and ATP levels were measured after 24 hours. The results showed that the drug-resistant cell lines were more sensitive to HThPA than the parental cell lines.

[0028] Figure 9 To demonstrate the superior inhibitory effect of compound HThPA on xenografts induced by drug-resistant strains, xenograft models were established in the left and right wings of the same nude mouse using the parental oral squamous cell carcinoma KB cell line (P) and the corresponding vincristine-resistant KB cell line (R). HThPA (20 mg / kg) was administered intraperitoneally every two days, and tumor volume was measured. Mice were sacrificed when the tumor reached the predetermined size, and tumor weight was photographed and recorded. Results showed that HThPA exhibited better inhibitory effects on xenografts induced by drug-resistant strains.

[0029] Figure 10 Compounds 1-10 can all induce endogenous copper-mediated copper death. Cells were treated with these small molecule compounds for 24 hours, and cell morphology and ATP levels were measured. The results showed that compounds 1-10 can all induce endogenous copper-mediated copper death, demonstrating their activity in inducing copper death. Detailed Implementation

[0030] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0031] Example 1: Synthesis of intermediate compound a (3,4,5-trimethoxyphenylacetic acid hexyl ester).

[0032]

[0033] 3,4,5-Trimethoxyphenylacetic acid (2.2623 g), bromohexane (1.1239 g), and sodium bicarbonate (0.9240 g) were dissolved in N,N-dimethylformamide and stirred at 78 °C for 6 hours. The reaction was analyzed by thin-layer chromatography and monitored under ultraviolet light. After the reaction was completed, the mixture was cooled to room temperature, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography using a hexane:ethyl acetate system to obtain a colorless oily compound a with a yield of 80%.

[0034] 1 H NMR (600 MHz, DMSO- d 6 ): δ 14.52 (s, 1H), 7.94 (dd, J = 1.8, 7.3 Hz, 1H), 7.92 (d, J =12.1 Hz, 1H), 7.36 (dt, J = 1.6, 7.7 Hz, 1H), 6.78 - 6.86 (m,2H), 5.98 (d, J = 12.1 Hz, 1H), 3.21 (s, 3H), 3.00 (s, 3H).

[0035] Example 2: Synthesis of intermediate compound b (3,4,5-trimethoxyphenylacetic acid phenylpropyl ester).

[0036]

[0037] 3,4,5-Trimethoxyphenylacetic acid (1.6967 g), 1-hydroxybenzotriazole (1.3512 g), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.9170 g) were dissolved in dichloromethane and stirred in an ice bath for 1 hour. Then, 3-phenylpropanol (1.6809 g) and triethylamine (1.5 mL) were added to the mixture, and the mixture was stirred at room temperature for 12 hours. The reaction was analyzed by thin-layer chromatography and monitored under UV light. After the reaction was complete, the mixture was cooled to room temperature, extracted three times with water and dichloromethane, and the organic layers were combined. The mixture was then washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography using a hexane:ethyl acetate system to obtain a colorless oily compound b in 85% yield.

[0038] 1 H NMR (600 MHz, DMSO- d6 ): δ 7.25 (t, J = 7.6 Hz, 2H), 7.14 – 7.10 (m,2H), 6.58 (s, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.73 (s, 6H), 3.61 (s, 3H), 3.58(s, 2H), 2.58 (dd, J = 8.6, 6.7 Hz, 2H), 1.89 – 1.81 (m, 2H).

[0039] Example 3: Synthesis of compound 1.

[0040]

[0041] Compound 1

[0042] Under argon protection, intermediate compound a (0.2404 g) was dissolved in dichloromethane solution (15 mL), and boron tribromide solution (0.25 mL) was slowly added dropwise at -5 °C. The reaction was stirred at room temperature for 8 hours. After the reaction was complete, the mixture was directly concentrated, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography using a dichloromethane:methanol system of 1:60 to obtain brown oily compound 1 in 35.7% yield.

[0043] 1 H NMR (600 MHz, DMSO- d6 ): δ8.75 (s, 2 H), 7.95 (s, 1 H), 6.16 (s, 2H), 3.99 (t, J = 6.6 Hz, 2 H), 3.33 (s, 2 H), 1.54 (q, J = 6.9 Hz, 2 H), 1.22 –1.30 (m, 4 H), 0.85 (t, J = 6.9 Hz, 3 H); 13 C NMR (150 MHz, DMSO- d6 ): δ 172.0,146.4, 132.3, 124.8, 108.4, 64.4, 40.7, 28.3, 28.0, 22.2, 14.3; MS (ESI): m / z C 13 H 18 O5 [M+H + Calculated value: 255.1, Measured value: 255.0.

[0044] Example 4: Synthesis of compound 2.

[0045] Similar to the synthesis method of compound 1 in Example 3, brownish-black oily compound 2 was obtained with a yield of 86.2%.

[0046]

[0047] Compound 2

[0048] 1 H NMR (600 MHz, MeOD): δ 6.27 (s, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.37(s, 2H), 1.63 – 1.58 (m, 2H), 1.52 (dp, J = 13.4, 6.7 Hz, 1H), 1.21 – 1.16 (m,2H), 0.87 (d, J = 6.6 Hz, 6H); 13 C NMR (151 MHz, MeOD): δ174.18, 147.01, 133.26, 126.41, 109.21, 66.17, 49.43, 49.28, 49.14, 49.00, 48.86, 48.72, 48.58, 41.90, 36.10, 28.85, 27.61, 22.84.

[0049] Example 5: Synthesis of compound 3.

[0050] The brownish-black oily compound 3 was prepared by a similar synthetic method to that used for compound 1 in Example 3, with a yield of 35.7%.

[0051]

[0052] Compound 3

[0053] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.75 (s, 2 H), 7.95 (s, 1 H), 6.14 (s, 2H), 3.97 (t, J = 6.6 Hz, 2 H), 3.32 (s, 2 H), 1.52 (dq, J = 8.1, 6.5 Hz, 2 H), 1.28 – 1.19 (m, 6 H), 0.87 – 0.80 (m, 3 H); 13 C NMR (150 MHz, DMSO- d6 ): δ 171.50,145.93, 131.83, 124.27, 107.93, 63.98, 40.22, 39.94, 39.80, 39.66, 39.52,39.38, 39.24, 39.10, 30.82, 28.07, 24.95, 21.96, 13.85; HRMS (ESI): m / z C 14 H 20 O5[MH] – Calculated value: 267.1234; Measured value: 267.1237.

[0054] Example 6: Synthesis of compound 4.

[0055] The brownish-black oily compound 4 was prepared by a similar synthetic method to that used for compound 1 in Example 3, with a yield of 42%.

[0056]

[0057] Compound 4

[0058] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.75 (s, 2 H), 7.95 (s, 1 H), 6.15 (s, 2H), 3.98 (t, J = 6.7 Hz, 2 H), 3.33 (s, 2 H), 1.54 (q, J = 6.5 Hz, 2 H), 1.17–1.30 (m, 8 H), 0.82–0.89 (m, 3 H); 13 C NMR (150 MHz, DMSO- d6 ): δ 172.50, 146.4,132.3, 124.7, 108.4, 64.5, 40.7, 31.6, 28.7, 28.6, 25.7, 22.5, 14.4. MS(ESI): m / z C 15 H 25 O5 [M+H + Calculated value: 283.2, Measured value: 283.2.

[0059] Example 7: Synthesis of compound 5.

[0060] Brown oily compound 5 was prepared by a similar synthetic method to compound 1 in Example 3, with a yield of 33.0%.

[0061]

[0062] Compound 5

[0063] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.73 (s, 2H), 7.94 (s, 1H), 6.14 (s, 2H), 3.97 (t, J = 6.7 Hz, 2H), 1.56 – 1.51 (m, 6H), 1.48 – 1.42 (m, 2H), 1.25 (ddq, J = 17.0, 10.3, 6.3, 5.6 Hz, 5H); 13 C NMR (151 MHz, DMSO- d6 ): δ 171.50, 145.92, 131.83, 124.26, 107.92, 64.18, 40.24, 40.06, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.17, 39.10, 32.27, 32.13, 31.69, 27.41, 24.69.

[0064] Example 8: Synthesis of compound 6.

[0065] The brownish-black oily compound 6 was prepared by a similar synthetic method to compound 1 in Example 3, with a yield of 37.9%.

[0066]

[0067] Compound 6

[0068] 1 H NMR (600 MHz, MeOD): δ 6.26 (s, 2H), 4.04 (t, J = 6.6 Hz, 2H), 3.37(s, 2H), 1.72 – 1.66 (m, 4H), 1.66 – 1.57 (m, 2H), 1.24 (dt, J = 12.1, 2.7 Hz, 2H), 1.22 – 1.15 (m, 3H), 0.86 (h, J = 9.4 Hz, 2H); 13 C NMR (151 MHz, MeOD): δ 174.18, 147.01, 133.26, 126.41, 109.21, 66.17, 49.43, 49.28, 49.14, 49.00, 48.86, 48.72, 48.58, 41.90, 36.10, 28.85, 27.61, 22.84.

[0069] Example 9: Synthesis of compound 7.

[0070]

[0071] Compound 7

[0072] Under argon protection, intermediate compound b (1.4764 g) was dissolved in dichloromethane solution, and boron tribromide solution (1.985 mL) was slowly added dropwise at -5°C. The reaction was stirred at room temperature for 8 hours. After the reaction was complete, the mixture was directly concentrated, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography using a dichloromethane:methanol system to obtain a brown oily substance in 40.9% yield.

[0073] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.76 (s, 2H), 7.95 (s, 1H), 7.25 (t, J =7.6 Hz, 2H), 7.19 – 7.11 (m, 3H), 6.17 (s, 2H), 3.97 (t, J = 6.5 Hz, 2H), 3.34(s, 2H), 2.57 (t, J = 7.7 Hz, 2H), 1.87 – 1.79 (m, 2H); 13 C NMR (151 MHz, DMSO- d6 ): δ 171.48, 145.97, 141.10, 131.87, 128.32, 128.31, 125.82, 107.96, 63.22, 40.26, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10, 31.29, 29.80.

[0074] Example 10: Synthesis of compound 8.

[0075] Compound 8, a pale yellow solid, was prepared by a similar synthetic method to compound 7 in Example 9, with a yield of 6.0%.

[0076]

[0077] Compound 8

[0078] 1 H NMR (600 MHz, DMSO- d6 ): δ8.80 (s, 2H), 7.99 (s, 1H), 7.33 – 7.26(m, 2H), 7.16 – 7.07 (m, 2H), 6.17 (s, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.33(s, 2H), 2.60 – 2.51 (m, 2H), 1.85 – 1.77 (m, 2H); 13 C NMR (151 MHz, DMSO- d6 ): δ 171.51, 146.02, 140.16, 131.92, 130.46, 128.24, 128.17, 107.98, 63.06, 40.32, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10, 30.57, 29.62.

[0079] Example 11: Synthesis of compound 9.

[0080] Compound 9, a brownish-black oily substance, was prepared by a similar synthetic method to compound 7 in Example 9, with a yield of 25.1%.

[0081]

[0082] Compound 9

[0083] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.80 (s, 2H), 7.99 (s, 1H), 7.45 – 7.37 (m, 2H), 7.11 – 7.01 (m, 2H), 6.17 (s, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.33 (s,3H), 2.53 (dd, J = 8.6, 6.7 Hz, 2H), 1.84 – 1.78 (m, 2H); 13 C NMR (151 MHz, DMSO- d6 ): δ171.52, 146.03, 140.59, 131.17, 131.10, 130.71, 124.34, 118.90, 107.99, 63.05, 40.33, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10, 30.63, 29.57.

[0084] Example 12: Synthesis of compound 10.

[0085] Compound 10, a grayish-green oily substance, was prepared by a similar synthetic method to compound 7 in Example 9, with a yield of 78.3%.

[0086]

[0087] Compound 10

[0088] 1 H NMR (600 MHz, DMSO- d6 ): δ 8.87 (s, 1H), 8.17 (s, 1H), 7.79 – 7.66 (m, 2H), 7.36 – 7.30 (m, 2H), 6.33 (q, J = 2.0 Hz, 2H), 3.98 (t, J = 6.4 Hz, 2H), 3.70 (s, 2H), 2.65 (dd, J = 8.7, 6.7 Hz, 2H), 1.88 – 1.82 (m, 2H); 13 C NMR (151MHz, DMSO- d6 ): δ 171.40, 148.24, 147.34, 145.71, 132.25, 129.49, 124.30, 119.01, 109.94, 104.64, 63.09, 40.34, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10, 31.40, 29.22.

[0089] Example 13: Compound 3 (HThPA) induces copper death in melanoma cells.

[0090] The experimental method is as follows:

[0091] 1. Take human melanoma A375 cells in the logarithmic growth phase and seed them at a concentration of 1.5 × 10⁻⁶ cells. 5Cells were cultured in 12-well plates in DMEM medium containing 10% serum.

[0092] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation), pretreated with copper ion chelator D-pen and TTM for 2 hours, and then treated with HThPA (15 μM) for 24 hours. Cell morphology was observed and recorded. Cells were digested with trypsin and the digestion was terminated with 1 mL of culture medium. 50 μL of cell suspension was added to a 96-well plate, and an equal amount of ATP assay reagent was added. After equilibration for 15 minutes, the readings were taken using a microplate reader.

[0093] Experimental results are as follows Figure 1 As shown, HThPA induces copper death in melanoma cells, accompanied by a decrease in ATP. Both phenotypes are reversed by the copper ion chelators D-pen and TTM.

[0094] Example 14: Compound 3 (HThPA) is not a copper ion carrier and does not have the ability to transport exogenous copper ions.

[0095] The experimental method is as follows:

[0096] 1. Take human melanoma A375 cells in the logarithmic growth phase and seed them at a concentration of 1.5 × 10⁻⁶ cells. 6 Cells were cultured in 10 cm plates in DMEM medium containing 10% serum.

[0097] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation), pretreated with copper ions for 2 hours, and then treated with HThPA (15 μM) or copper ion carrier DSF for 4 hours. Cells were then harvested, and the intracellular copper ion content was determined by ICP-MS. The specific steps are as follows: Cells were dissolved in 1 mL of nitric acid using microwave, and the temperature was increased to 120℃ within 5 minutes and held for 30 seconds; then increased to 190℃ within 5 minutes and held for 7 minutes. The copper ion content was then determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0098] Experimental results are as follows Figure 2 As shown, there was no difference in intracellular copper ion content between co-treatment with HThPA and copper ions and treatment with copper ions alone. However, the intracellular copper ion content was significantly increased after co-treatment with copper ion carrier DSF and copper ions compared to treatment with copper ions alone. This proves that HThPA does not have the ability to transport exogenous copper and is not a copper ion carrier.

[0099] Example 15: Compound 3 (HThPA) induces copper death in various tumor cells with a gradient effect.

[0100] The experimental method is as follows:

[0101] 1. Take various tumor cells in the logarithmic growth phase and inoculate them at a concentration of 1.5 × 10⁻⁶. 5 Cells were cultured in 12-well plates in DMEM medium containing 10% serum.

[0102] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation) and treated with different concentrations of HThPA for 24 hours. ATP levels were then measured to indicate cell viability. The measurement method was the same as in Example 13.

[0103] Experimental results are as follows Figure 3 As shown, HThPA can induce copper death in various tumor cells and exhibits a gradient effect.

[0104] Example 16: Compound 3 (HThPA) induces a decrease in glutathione (GSH) through a gradient effect.

[0105] The experimental method is as follows:

[0106] 1. Human melanoma A375 cells in logarithmic growth phase were seeded at a density of 3000 cells in a 96-well plate and cultured in DMEM medium containing 10% serum.

[0107] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation). After treatment with different concentrations of HThPA for 2 hours, total glutathione was measured using a glutathione assay kit (Promega, Cat#V6912). Cells were incubated on a shaker with 100 μL of 1× GSH-Glo™ reagent for 30 minutes, lysed, and then incubated with 100 µL of recombinant luciferin assay reagent for 15 minutes. The results were then read using a microplate reader.

[0108] Experimental results are as follows Figure 4 As shown: Glutathione is an intracellular copper ion chelate. HThPA reduces intracellular glutathione levels through a gradient effect, which facilitates the release of intracellular copper, leading to an increase in free copper and inducing copper death.

[0109] Example 17: Compound 3 (HThPA) inhibits the growth of PDX breast cancer tumors.

[0110] The experimental method is as follows:

[0111] A patient-derived xenograft (PDX) model of breast cancer was established using female BALB / c nude mice (7-8 weeks old, weighing 18-22 g). Fresh tumor tissue blocks (0.1-0.2 cubic centimeters per mouse) were subcutaneously implanted into the flank of the mice. After the tumors grew to a suitable size, the mice were randomly divided into a control group and a group receiving intraperitoneal injections of 5 mg / kg or 10 mg / kg HThPA. Administration was performed every other day. When the tumors reached the predetermined size, the mice were euthanized, the tumor tissue was removed, photographed, and the weight of the tumor was measured.

[0112] The results are as follows Figure 5 As shown, HThPA can effectively inhibit tumor growth and size, and exhibits a concentration gradient effect, demonstrating that HThPA has the potential to inhibit breast cancer growth.

[0113] Example 18: Compound 3 (HThPA) synergistically enhances the inhibition of primary gastric cancer growth with 5-Fu.

[0114] The experimental method is as follows:

[0115] Used male 8-week-old Cldn18-CreERT2; Apc fl / fl Trp53 fl / fl Kras G12D The therapeutic effect of HThPA on primary gastric cancer was evaluated in (Cldn18-ATK) mice. Intraperitoneal injection of 100 mg / kg Tamoxifen induced spontaneous gastric cancer development in mice. Two weeks after Tamoxifen induction, mice were intraperitoneally injected with either the clinical antitumor drug 5-FU (25 mg / kg, once a week), HThPA (20 mg / kg, once every two days), or a combination of 5-FU and HThPA. One month later, the mice were euthanized, and their stomachs were photographed and the proportion of the stomach area occupied by the tumor was recorded.

[0116] The results are as follows Figure 6 As shown, both HThPA and 5-Fu can inhibit the growth of gastric cancer. The combined use of 5-Fu and HThPA can significantly enhance the effect of inhibiting gastric cancer, proving that HThPA and 5-Fu have a synergistic effect.

[0117] Example 19: Compound 3 (HThPA) can inhibit the growth of primary liver cancer.

[0118] The experimental method is as follows:

[0119] Hepatocellular carcinoma was induced in 8-week-old male wild-type mice using the "Sleeping Beauty System" transposase vector to evaluate the therapeutic effect of HThPA on primary hepatocellular carcinoma. The specific steps were as follows: Liver tumors were induced by injection of the "Sleeping Beauty" (SB) transposase vector containing c-Myc (5 μg) and Trp53 (7.5 μg). The plasmid was suspended in 1 mL of sterile saline and rapidly injected via the tail vein within 5 seconds. Two weeks after induction, mice were intraperitoneally injected with HThPA (20 mg / kg, every two days). One month later, the mice were euthanized, and their livers were photographed and recorded. The number of hepatocellular carcinomas and liver weight were also assessed.

[0120] The results are as follows Figure 7 As shown, HThPA can effectively inhibit the progression of liver cancer, demonstrating its potential for treating liver cancer.

[0121] Example 20: Compound 3 (HThPA) has a stronger ability to induce copper death in chemotherapy-resistant tumor cell lines.

[0122] The experimental method is as follows:

[0123] 1. Take various tumor cells in logarithmic growth phase (P: parental cell line and R: drug-resistant cell line) and inoculate them at 1.5 × 10⁻⁶ m². 5 Cells were cultured in 12-well plates in DMEM medium containing 10% serum.

[0124] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation) and treated with different concentrations of HThPA for 24 hours. ATP levels were then measured to indicate cell viability. The measurement method was the same as in Example 13.

[0125] Experimental results are as follows Figure 8 As shown, HThPA can induce copper death in both parental and drug-resistant tumor cells, but it has a stronger ability to induce copper death in drug-resistant cell lines.

[0126] Example 21: Compound 3 (HThPA) has a better inhibitory effect on xenografts generated by chemotherapy-resistant cell lines.

[0127] The experimental method is as follows:

[0128] 1. Oral squamous cell carcinoma KB-P cell line (parental line) and its corresponding drug-resistant cell line KB-R (vincristine-resistant line) (2×10⁻⁶) 6 The cells were resuspended in 100 μL of DMEM medium. Twenty male BALB / c nude mice (7-8 weeks old) were selected, and the cells were subcutaneously injected into the left and right wings of the same mouse to establish xenograft tumor models derived from the parental strain and the drug-resistant strain, respectively.

[0129] 2. Once the tumors have grown to a suitable size, the mice are divided into two groups. The control group (solvent) and the HThPA group (20 mg / kg) are injected intraperitoneally once every two days. The size of the tumors is measured with calipers.

[0130] 3. Once the tumor reaches the predetermined size, the mouse is euthanized, and the tumor weight is recorded.

[0131] Experimental results are as follows Figure 9 As shown, HThPA has an inhibitory effect on both parental and drug-resistant xenografts, but it has a better inhibitory effect on xenografts from drug-resistant strains, suggesting the specificity of HThPA in treating chemotherapy-resistant tumors.

[0132] Example 22: Compounds 1-10 can all induce endogenous copper-mediated copper death in melanoma cells.

[0133] The experimental method is as follows:

[0134] 1. Take human melanoma A375 cells in the logarithmic growth phase and seed them at a concentration of 1.5 × 10⁻⁶ cells. 5 Cells were cultured in 12-well plates in DMEM medium containing 10% serum.

[0135] 2. After overnight culture, the cells were replaced with 0.5% serum DMEM medium (without exogenous copper ion supplementation) and treated with different compounds (1-10, 15 μM) for 24 hours. Cell morphology was observed and recorded. Cells were digested with trypsin and the digestion was terminated with 1 mL of culture medium. 50 μL of cell suspension was added to a 96-well plate, and an equal amount of ATP assay reagent was added. After equilibration for 15 minutes, the readings were taken using a microplate reader.

[0136] Experimental results are as follows Figure 10 As shown, compounds 1-10 can induce copper death in melanoma cells, accompanied by a decrease in ATP. These results demonstrate that compounds 1-10 possess copper-inducing activity.

Claims

1. A 3,4,5-trihydroxyphenylacetic acid ester compound, characterized in that, Includes at least one of the following structural formulas I and II: R1 is selected from C4~C12 alkyl chains, cyclopentane, and cyclohexane; R2 is selected from hydrogen, halogen, cyano, methyl, ethyl, trifluoromethyl, and carboxyl.

2. The method for preparing a 3,4,5-trihydroxyphenylacetic acid ester compound according to claim 1, characterized in that, The synthesis route is as follows: 。 3. The application of the 3,4,5-trihydroxyphenylacetic acid ester compound according to claim 1, characterized in that: Used to prepare an inducer that induces copper death in tumor cells.

4. The application of the 3,4,5-trihydroxyphenylacetic acid ester compound according to claim 1, characterized in that: Used to prepare drugs for treating cancer, including melanoma, liver cancer, breast cancer, stomach cancer, or oral squamous cell carcinoma.

5. The application as described in claim 4, characterized in that: The cancer in question is one that has developed resistance to chemotherapy drugs.

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