Application of (4-oxopentan-1-ynyl)(diphenylcyclohexylphosphine) gold (I) in the preparation of drugs for treating melanoma

Abstract

This invention discloses a (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) and its application in the preparation of drugs for treating melanoma, belonging to the field of biomedicine. This invention provides a novel (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) that effectively combats melanoma by inhibiting the proliferation and growth of melanoma cells, with low side effects. It can serve as a candidate drug for treating melanoma-related diseases caused by various factors, showing promising application prospects in the development of innovative drugs for melanoma treatment. It provides a new drug option for the treatment of melanoma and can alleviate the problems of relatively limited current treatment options and drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a compound, specifically (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I), and its application as a drug for treating melanoma. Background Technology

[0002] Melanoma mainly includes cutaneous melanoma, acral melanoma, and mucosal melanoma, with cutaneous melanoma being the predominant type, accounting for over 90% of all melanoma cases. For patients with early-stage localized melanoma, wide surgical excision is the radical treatment. However, because early-stage melanoma may be asymptomatic or only present as atypical skin lesions, and some patients have already developed regional lymph node metastasis or distant metastasis at the time of diagnosis, treatment becomes significantly more difficult.

[0003] Currently, first-line therapies approved by the U.S. Food and Drug Administration (FDA) for the treatment of advanced or metastatic melanoma include immune checkpoint inhibitors (such as the PD-1 inhibitors pembrolizumab and nivolumab, and the CTLA-4 inhibitor ipilimumab), as well as targeted therapies for patients with BRAF V600 mutations (such as the BRAF inhibitors dabrafenib and vemurafenib). For patients who cannot tolerate immunotherapy or lack indications for targeted therapy (such as BRAF wild-type), the traditional chemotherapy drug dacarbazine can be used as an alternative.

[0004] Although these therapies have significantly improved the prognosis of patients with advanced melanoma, multiple challenges remain in clinical practice. First, targeted therapy is prone to developing resistance due to secondary gene mutations (such as NRAS mutations and abnormal MEK activation), leading to disease progression; while the response rate of immunotherapy is affected by the heterogeneity of the tumor microenvironment, with some patients exhibiting primary resistance. Second, melanoma is highly invasive and prone to metastasis, with common metastatic sites including the lungs, liver, brain, and bones, and treatment responses to metastatic lesions are generally worse than those to primary lesions.

[0005] Overall, while immune checkpoint inhibitors and targeted therapies have provided new treatment options for patients with advanced melanoma, issues such as drug resistance, disease metastasis and recurrence, and the side effects of long-term drug use significantly limit their applicability. Meanwhile, with the promotion of precision oncology, patients with different conditions urgently need more personalized treatment plans to improve treatment outcomes and quality of life for melanoma patients. Therefore, exploring and developing new melanoma drugs is a crucial and increasingly important research direction. Summary of the Invention

[0006] To address the aforementioned problems and shortcomings, and to resolve the issues of relatively limited effective drugs for melanoma treatment, drug resistance, and long-term side effects, this invention provides a (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I), which can be used as a therapeutic drug for melanoma.

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

[0008] A (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) has the following chemical structural formula:

[0009] .

[0010] The preparation method of the (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) includes the following steps:

[0011] 1) Under stirring conditions, dimethyl sulfide was slowly added dropwise to the chloroauric acid solution, causing the solution to change from yellow to colorless and produce a white solid. The reaction was then continued with stirring for 2 hours.

[0012] 2) After filtering the suspension obtained in step 1), collect the white solid, and then wash and vacuum dry it;

[0013] 3) Dissolve the white solid obtained in step 2) in dichloromethane, then add diphenylcyclohexylphosphine for a water bath reaction; after the reaction is complete, filter to remove impurities, and wash the filter cake with dichloromethane. Then combine the filtrate and the washing liquid, let it crystallize at room temperature, and then vacuum dry to obtain a pure crystalline compound.

[0014] 4) Dissolve the crystalline compound obtained in step 3) in methanol by stirring, then add the alkynyl compound, stir and mix well, and then add potassium hydroxide solution dropwise to carry out the reaction at room temperature.

[0015] 5) After the reaction is complete, the solid is filtered, washed with anhydrous methanol, and then dried under vacuum to obtain the target compound.

[0016] Further, the chloroauric acid solution in step 1) is prepared by dissolving 1g of chloroauric acid in a mixed solution of 10ml of anhydrous ethanol and deionized water; wherein the volume ratio of anhydrous ethanol to deionized water is 5:1.

[0017] Furthermore, in step 3), the molar ratio of the white solid to diphenylcyclohexylphosphine is 1:1.05.

[0018] Furthermore, the water bath reaction in step 3) is carried out at a temperature of 37°C for 2 hours.

[0019] Furthermore, the concentration of the potassium hydroxide solution in step 4) is 1 mmol / mL.

[0020] Furthermore, in step 4), the molar ratio of the crystalline compound, the alkynyl compound, and potassium hydroxide is 1:1.2:2.

[0021] Furthermore, the room temperature reaction time described in step 4) is 12 hours.

[0022] The (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) can be used to prepare drugs for treating melanoma.

[0023] Furthermore, it specifically uses the (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I) as the active ingredient, and combines it with pharmaceutical carriers or excipients, additives and other pharmaceutical excipients to prepare a drug for treating melanoma.

[0024] Furthermore, the drug for treating melanoma is a medically acceptable dosage form, such as tablets, capsules, granules, or injections, for enteral or non-enteric administration.

[0025] The significant advantages of this invention are:

[0026] (1) This invention provides a novel (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I), and for the first time it was discovered that it can effectively resist melanoma by inhibiting the proliferation and growth of melanoma cells, thus it can be used as a candidate drug for treating melanoma-related diseases caused by various reasons, and has good application prospects for creating drugs to treat melanoma.

[0027] (2) Through various experiments, the present invention has analyzed and verified that the compound has excellent inhibitory effect on the proliferation and growth of melanoma cells and has low side effects. It can be formulated into an intestinal or non-intestinal dosage form according to the known methods in the art and applied to the treatment of melanoma. It provides a new drug option for the treatment of melanoma and effectively alleviates the problems of relatively single current treatment drugs and drug resistance. Attached Figure Description

[0028] Figure 1 The diagram shows the structure of TRI-01~04 prepared for the example.

[0029] Figure 2 The reaction flow diagram for preparing TRI-03 is shown in the example.

[0030] Figure 3 Infrared spectrum (a) and nuclear magnetic resonance phosphorus spectrum (b) of TRI-03 prepared for the example.

[0031] Figure 4This is a comparison chart showing the survival rates of human melanoma cell line A375 treated with various compounds in Application Example 1.

[0032] Figure 5 This is a comparison of the survival curves of human melanoma cell line A375 treated with TRI-03, dacarbazine, and cisplatin using a solubility gradient in Application Example 2.

[0033] Figure 6 This is a comparison of the growth curves of mouse melanoma xenografts in the blank control group and the TRI-03 treatment group in Application Example 3.

[0034] Figure 7 Comparison of melanoma xenografts excised from the blank control group and the TRI-03 treatment group in Example 3 (a) and comparison of average tumor weight (b).

[0035] Figure 8 The images show a comparison of melanoma lung metastases in different treatment groups of mice in Example 4: (a) comparison of the number of lung metastases, (b) comparison of the average body weight after treatment, and (c) comparison of the lung metastases.

[0036] Figure 9 This is a comparison chart of the average serum levels of aspartate aminotransferase (a), alanine aminotransferase (b), creatinine (c), and blood urea nitrogen (d) in melanoma metastasis mice from different treatment groups in Example 4. Detailed Implementation

[0037] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods. Example

[0039] 1) Solvent A is prepared by mixing anhydrous ethanol and deionized water at a volume ratio of 5:1 at room temperature;

[0040] 2) Dissolve 1g of chloroauric acid in 10ml of solvent A to obtain a yellow chloroauric acid solution;

[0041] 3) At room temperature, slowly add dimethyl sulfide dropwise to the chloroauric acid solution obtained in step 2) while stirring until the solution changes from yellow to colorless and a white solid is produced. Then continue stirring for 2 hours to ensure the reaction is complete.

[0042] 4) At room temperature, the suspension obtained in step 3) was filtered to obtain a white solid. The solid was washed with 2 ml of solvent A at 4℃, and the process was repeated twice. Then, the solid was dried under vacuum to obtain a white solid AuCl(Me2S).

[0043] 5) Take 1 mmol of AuCl(Me2S) obtained in step 4), dissolve it in 15 ml of dichloromethane, and add 1.05 mmol of diphenylcyclohexylphosphine. Stir the reaction in a water bath at about 37°C for 2 hours. After the reaction is complete, filter to remove impurities, and wash the filter paper repeatedly with 10 ml of dichloromethane. Then collect the filtrate and washing liquid in the same container, let it crystallize at room temperature, and then dry it under vacuum to obtain pure crystalline compound A1.

[0044] 6) At room temperature, place 0.5 mmol of crystalline compound A1 in a vial, add 5 ml of methanol and stir for 20 minutes to dissolve it. Then add 0.6 mmol of 2-methyl-3-butyn-2-ol, 3-butyn-2-one, 4-pentyn-2-one and 4-ethynyl anisole, stir for 10 minutes to mix thoroughly, and then add 1 ml of solution containing 1 mmol of potassium hydroxide. React at room temperature for 12 hours.

[0045] 7) After the reaction is complete, filter the solution containing flocculent precipitate generated in step 6), wash with 2 ml of anhydrous methanol at 4℃, repeat the washing twice, and then dry under vacuum to obtain the compounds, which are labeled as TRI-01~04 respectively.

[0046] Application Example 1

[0047] The effects of the compounds obtained in the examples on melanoma cell death were detected by flow cytometry.

[0048] The experimental method is as follows:

[0049] 1. Cell seeding: Take human melanoma cells A375 in the logarithmic growth phase, digest them with trypsin and pipette them into single cells, then suspend the cells in complete culture medium containing 10% fetal bovine serum to prepare a cell suspension, and seed 200,000 cells per well into a 12-well plate with a volume of 1000 µl per well.

[0050] 2. Cell culture: After the cells adhered to the wall, A375 melanoma cells were treated with 1 µM TRI-01, TRI-02, TRI-03 and TRI-04 respectively, and the cells were cultured at 37 ℃ and 5% CO2 for 24 hours.

[0051] 3. Cell Collection: Collect cells and trypsin-digested adherent cells from the culture supernatant by centrifugation at 2000 rpm for 5 min into a 2 ml Eppendorf microcentrifuge tube. Discard the supernatant, wash once with 1 ml PBS buffer, and transfer to a 1.5 ml Eppendorf microcentrifuge tube. Centrifuge at 2000 rpm for 5 min at 4°C, and discard the supernatant. Resuspend the cells in 1 ml PBS, centrifuge again, and discard the supernatant.

[0052] 4. Flow cytometry: Finally, the cells were resuspended in 1 mL of PBS buffer containing 5 µg / mL ethidium bromide. Incubation was performed at 4°C for 10 min in the dark. Cell viability under different compound treatments was determined using flow cytometry. One-way ANOVA was used for statistical analysis between different groups. The significance levels were set as follows: * indicates p < 0.05, which is significant; ** indicates p < 0.01, which is highly significant; *** indicates p < 0.001, which is extremely significant.

[0053] The experimental results are as follows:

[0054] Figure 4 A comparison of the survival rates of human melanoma cell line A375 after treatment with different compounds. Figure 4 As shown, treatment of A375 cell lines with each compound for 24 hours induced cell death to varying degrees. Specifically, the cell viability rates were 42.05% in the TRI-01 treatment group, 43.45% in the TRI-02 treatment group, 23.40% in the TRI-03 treatment group, and 81.50% in the TRI-04 treatment group.

[0055] It is evident that TRI-03 has a significantly stronger ability to induce melanoma cell death than other structural analogs, with the difference reaching a highly significant or extremely significant level.

[0056] Application Example 2

[0057] The effect of TRI-03 on the proliferation and growth of melanoma cells was analyzed using the MTT assay, with dacarbazine and cisplatin used as positive controls.

[0058] The experimental method is as follows:

[0059] 1. Cell seeding: Take human melanoma cells A375 in the logarithmic growth phase, digest them with trypsin and pipette them into single cells, suspend the cells in complete culture medium containing 10% fetal bovine serum to prepare a cell suspension, and seed 6000 cells per well into a 96-well plate with a volume of 200 µl per well.

[0060] 2. Cell culture: After melanoma cells A375 adhered to the wall, they were treated with TRI-03, dacarbazine and cisplatin in a concentration gradient, and the cells were cultured at 37 ℃ and 5% CO2 for 72 hours.

[0061] 3. Color development: After 72 hours of incubation, add 20 µl of MTT solution (5 mg / ml, dissolved in PBS) to each well and continue incubation for 4 hours. Then carefully aspirate the culture supernatant from the wells, add 150 µl of DMSO to each well, and shake for 10 minutes to fully dissolve the crystals.

[0062] 4. Colorimetric assay: Using a wavelength of 490 nm, measure the absorbance of each well on an ELISA reader and record the results. Calculate the cell viability under different concentrations of the compound, and plot the compound concentration (Cell Viability, %) on the x-axis and cell viability (%) on the y-axis to obtain the compound concentration at 50% inhibition, i.e., IC50. 50 .

[0063] The experimental results are as follows:

[0064] Figure 5 Survival curves of A375 cell lines treated with TRI-03, dacarbazine, and cisplatin at varying concentrations for 72 hours are shown. Figure 2 As shown, treatment of A375 cell lines with TRI-03, dacarbazine, or cisplatin for 72 hours all affected the proliferation and growth of A375 cells to varying degrees. Among them, the IC50 value of the TRI-03 treatment group was significantly lower. 50 The concentration of dacarbazine in the treatment group was 0.45 µM. 50 104.9 µM, cisplatin-treated IC 50 It is 27.07 µM.

[0065] It is evident that, compared to dacarbazine, a clinical drug for melanoma, and cisplatin, a classic cancer treatment drug, TRI-03 exhibits 233 times the inhibitory effect on melanoma cells and 60 times the inhibitory effect on cisplatin, demonstrating that it can more significantly inhibit the proliferation and growth of human melanoma cells A375.

[0066] Application Example 3

[0067] The inhibitory effect of TRI-03 on the growth of melanoma xenografts was detected using a nude mouse xenograft experiment, with a blank control group used for comparison.

[0068] The experimental method is as follows:

[0069] First, human melanoma cells A375 were subcutaneously injected into the right back of nude mice. The transplanted tumor was allowed to grow to 100 mm. 3After ensuring the successful establishment of the model, the mice were randomly divided into two groups: (1) blank control group; (2) TRI-03 treatment group, with a dose of 10 mg / kg each time.

[0070] The TRI-03 treatment group received intratumoral injections of TRI-03 every two days, while the blank control group received intratumoral injections of drug-free solvent every two days, both for 2 weeks. Tumor size was recorded daily. After 10 days, mice were weighed, euthanized by cervical dislocation, dissected, photographed (arranged in order of size), and the tumor weight was measured. SPSS 13.0 statistical software was used for data analysis. Results are expressed as mean ± variance (Mean ± SD). The independent samples t-test (unpaired t-test) was used to analyze the differences between the control and experimental groups. Significance levels were set as follows: ** indicates p < 0.01 (highly significant); *** indicates p < 0.001 (extremely significant).

[0071] The experimental results are as follows:

[0072] Figure 6 This is a comparison of growth curves of melanoma xenografts in mice from the blank control group and the TRI-03 treatment group. Figure 6 As shown, the growth of melanoma xenografts in the TRI-03 treatment group was significantly slower than that in the blank control group.

[0073] Figure 7 Comparative images of melanoma xenografts removed from mice in the blank control group and the TRI-03 treatment group, along with a comparison of average tumor weight. Figure 7 As shown, the melanoma xenografts in the TRI-03 treatment group were significantly smaller than those in the blank control group. The average tumor weight in the blank control group was 0.4803 g, while the average tumor weight in the TRI-03 treatment group was 0.1249 g, and the difference between the two groups was highly significant.

[0074] Experimental results show that TRI-03 has a significant inhibitory effect on human melanoma xenografts in vivo.

[0075] Application Example 4

[0076] The inhibitory effect of TRI-03 on melanoma metastasis in vivo was detected by a mouse B16 melanoma lung metastasis assay, with dacarbazine as a positive control.

[0077] The experimental method is as follows:

[0078] C57BL / 6J mice were used for in vivo tumor metastasis experiments after reaching 8 weeks of age and weighing 18-20 g. Mouse melanoma cells (B16) were resuspended in PBS and then injected via the tail vein into the mice (0.2 mL per mouse, approximately 5 × 10⁶ cells).5 Two days after cell injection, the mice were randomly divided into three groups: a blank control group (injected with PBS containing 2% Tween 80), a dacarbazine group (dacarbazine dissolved in PBS containing 2% Tween 80 for injection, 10 mg / kg body weight / mouse), and a TRI-03 group (TRI-03 dissolved in PBS containing 2% Tween 80 for injection, 10 mg / kg body weight / mouse). Injections were administered every other day. Two weeks later, mice were anesthetized, and blood was collected from the orbital venous plexus. After the blood had settled, serum was separated by centrifugation. Liver function indicators (aspartate aminotransferase, alanine aminotransferase) and kidney function indicators (creatinine, blood urea nitrogen) were measured using a biochemical analyzer. Mice were then euthanized by cervical dislocation, dissected, and the number of metastatic tumors was observed and counted. SPSS 13.0 statistical software was used for statistical analysis, and the results are expressed as mean ± variance (Mean ± SD). Differences between the control and experimental groups were analyzed using one-way ANOVA and two-way ANOVA, supplemented by Fisher's test for further validation.

[0079] The experimental results are as follows:

[0080] Mouse melanoma cells B16 injected via the tail vein can migrate to the lungs through blood circulation and form melanomas. Figure 8 Images of dissected lungs, lung metastases, and average body weight after treatment are shown for mice in the blank control group, dacarbazine treatment group, and TRI-03 treatment group. Figure 8 As shown, the number of melanoma lung metastases in mice treated with TRI-03 and dacarbazine was significantly lower than that in the blank control group, and the number of melanoma lung metastases in the TRI-03 treatment group was significantly lower than that in the dacarbazine treatment group. Meanwhile, there was no significant difference in the average body weight of mice in the TRI-03 and dacarbazine treatment groups compared to the average body weight of mice in the blank control group (mean body weight of mice in the blank control group was 23.53 g, mean body weight of mice in the dacarbazine treatment group was 22.28 g, and mean body weight of mice in the TRI-03 treatment group was 22.45 g).

[0081] Figure 9 This is a comparison of the average serum levels of aspartate aminotransferase, alanine aminotransferase, creatinine, and blood urea nitrogen in mice from the blank control group, dacarbazine treatment group, and TRI-03 treatment group. Figure 9 As shown, there were no significant changes in liver function parameters (aspartate aminotransferase, alanine aminotransferase) and kidney function parameters (serum creatinine, blood urea nitrogen) in the TRI-03 treatment group, dacarbazine treatment group, and blank control group.

[0082] Experimental results show that, compared with the clinical chemotherapy drug dacarbazine, TRI-03 can more significantly inhibit the metastasis of melanoma cells in vivo, and TRI-03 does not cause strong changes in body weight and liver and kidney function parameters in mice, indicating that its side effects are mild.

[0083] In summary, this invention discovers a novel pharmaceutical application for (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine) gold (I), which can effectively combat melanoma by inhibiting the proliferation and growth of melanoma cells, with low side effects, thus serving as a candidate drug for the treatment of melanoma-related diseases. This invention provides a new drug option for the treatment of melanoma, alleviating the problems of relatively limited drug options, drug resistance, and long-term side effects in current treatments, and has promising prospects for the development of innovative drugs for the treatment of melanoma.

[0084] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine)gold(I) characterized in that, The chemical structural formula is as follows: 。 2. A process for the preparation of (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine)gold(I) as claimed in claim 1, characterized in that, The method comprises the following steps: 1) slowly adding dimethyl sulfide into the chloroauric acid solution under stirring, so that the solution changes from yellow to colorless and white solid is produced, and then the reaction is continuously stirred for 2 hours; 2) the white solid is collected by suction filtration of the suspension solution obtained in step 1), and then washed and vacuum dried; 3) the white solid obtained in step 2) is dissolved in dichloromethane, and then diphenylcyclohexyl phosphine is added for water bath reaction; after the reaction is completed, impurities are removed by filtration, and the filter cake is washed with dichloromethane; then the filtrate and the washing liquid are combined, and the mixture is crystallized at room temperature and then vacuum dried to obtain pure crystalline compound; 4) the crystalline compound obtained in step 3) is stirred and dissolved in methanol, then an alkynyl compound is added, the mixture is stirred and mixed, and then a potassium hydroxide solution is added dropwise for reaction at room temperature; 5) after the reaction is completed, the mixture is filtered, and the solid is washed with anhydrous methanol and then vacuum dried to obtain the target compound.

3. The production method according to claim 2, characterized by, The chloroauric acid solution in step 1) is prepared by dissolving 1 g of chloroauric acid in a mixed solution of 10 ml of anhydrous ethanol and deionized water, wherein the volume ratio of the anhydrous ethanol to the deionized water is 5:

1.

4. The production method according to claim 2, characterized by, The molar ratio of the white solid to diphenylcyclohexyl phosphine used in step 3) is 1:1.

05.

5. The preparation method according to claim 2, characterized in that, The temperature of the water bath reaction in step 3) is 37℃, and the time is 2 hours.

6. The preparation method according to claim 2, characterized in that, The concentration of the potassium hydroxide solution in step 4) is 1 mmol / mL.

7. The preparation method according to claim 2, characterized in that, The molar ratio of the crystalline compound, the alkynyl compound and potassium hydroxide used in step 4) is 1:1.2:

2.

8. The preparation method according to claim 2, characterized in that, The time of the reaction at room temperature in step 4) is 12 hours.

9. Use of (4-oxopent-1-ynyl)(diphenylcyclohexylphosphine)gold (I) as defined in claim 1 for the manufacture of a medicament for the treatment of melanoma, characterized in that: The (4-oxopent-1-alkynyl) (diphenylcyclohexyl phosphine) gold (I) is used as an active ingredient, which is combined with a pharmaceutical carrier or a pharmaceutical excipient to prepare a medicine for treating melanoma.

10. Use according to claim 9, characterized in that, The medicine for treating melanoma is a tablet, a capsule, a granule or an injection.

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