A thiazole-containing cyclic hexapeptide compound and its application

Abstract

The present invention relates to a thiazole-containing cyclic hexapeptide compound and its application. The compound has a structure shown in the following formula I. The application is the application of the thiazole-containing cyclic hexapeptide compound in the preparation of a drug for drug resistance reversal. The thiazole-containing cyclic hexapeptide compound shows safety when used alone and better inhibitory effect than the positive drug verapamil when used in combination, that is, the thiazole-containing cyclic hexapeptide compound has a significant effect as a drug for drug resistance reversal.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal chemistry, and in particular to a thiazole-containing cyclic hexapeptide compound and application thereof. Background Art

[0002] Many cancer cells acquire resistance to multiple structurally and mechanistically distinct anticancer drugs through a phenomenon known as multidrug resistance (MDR). The development of MDR poses a major challenge to cancer chemotherapy, especially in the management of patients with metastatic cancer that is resistant to conventional therapies. MDR occurs intrinsically in certain cancers due to genetic and epigenetic alterations that influence drug sensitivity without prior exposure to chemotherapeutic drugs. MDR can also be acquired during chemotherapeutic treatment of cancers that were initially drug-sensitive but later relapsed in a resistant form. It is also recognized that cancers often consist of a heterogeneous population of drug-sensitive and drug-resistant cells. During treatment, drug-sensitive cells are selectively removed and resistant cells begin to dominate the cancer cell population. The dramatic impact of chemotherapeutic drug resistance has led to extensive research into mechanisms and strategies to understand, modulate, or evade MDR.

[0003] P-gp is the first discovered and most characterized MDR transporter, and has long been considered a viable target for overcoming cancer MDR. P-gp is the most studied efflux transporter. Almost all tumors overexpress P-gp, and its expression level is related to the degree of drug resistance. The expression of P-gp can be induced by drugs. When anti-tumor drugs enter tumor cells, the mRNA level of P-gp will increase as a protective mechanism for cells after receiving this stimulation. When this stimulation continues for a period of time, the expression of P-gp will increase. This shows that this MDR cannot be avoided by reducing the dosage or changing the drug.

[0004] In 1981, it was first discovered that the calcium channel blocker verapamil could effectively reverse the P-gp-mediated resistance to vincristine and vinblastine in the drug-resistant mouse cell line P288 / VCRP, which stimulated everyone's enthusiasm for research on the new target P-gp, and many drugs that reverse MDR by inhibiting the function of P-gp, that is, P-gp inhibitors, were developed. However, there are few reports on achieving a good resistance reversal effect. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a thiazole-containing cyclic hexapeptide compound and its application. The compound not only has good safety, but also has excellent drug resistance reversal activity.

[0006] The present invention provides a thiazole-containing cyclic hexapeptide compound, characterized in that the compound has a structure shown in the following formula I:

[0007] .

[0008] The present invention also provides an application of a thiazole-containing cyclic hexapeptide compound, which is an application of the thiazole-containing cyclic hexapeptide compound in the preparation of a drug resistance reversal drug.

[0009] Furthermore, the application is the application of the thiazole cyclic hexapeptide compound to reverse the drug resistance of vincristine-resistant human oral epidermoid carcinoma cell lines.

[0010] Furthermore, the cell line is a vincristine-resistant human oral epidermoid carcinoma cell line KBV cell.

[0011] Beneficial effects: The thiazole-containing cyclic hexapeptide compound involved in the present invention uses a commercially available compound as a substrate and can be completed by a simple chemical reaction, and has the advantages of high yield and easy separation. The survival rate of the thiazole-containing cyclic hexapeptide compound of the present invention as a single drug for vincristine-resistant human oral epidermoid carcinoma cell line KBV cells is 95.46%, while when used in combination with PTX, the survival rate is only 55.64%. It shows safety when used as a single drug and better inhibitory effect than the positive drug verapamil when used in combination, that is, the use effect of the thiazole-containing cyclic hexapeptide compound as a drug for drug resistance reversal is significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 16 of the present invention.

[0013] Figure 2 This is the carbon NMR spectrum of the thiazole ring-containing hexapeptide compound 16 of the present invention.

[0014] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 17 of the present invention.

[0015] Figure 4 This is the carbon nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 17 of the present invention.

[0016] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 18 of the present invention.

[0017] Figure 6 This is the carbon nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 18 of the present invention.

[0018] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 19 of the present invention.

[0019] Figure 8 This is the carbon nuclear magnetic resonance spectrum of the thiazole ring-containing hexapeptide compound 19 of the present invention.

[0020] Fig. 9Rhodamine123 accumulation experiment diagram. DETAILED DESCRIPTION Example 1

[0021] The present invention discloses a thiazole-containing cyclic hexapeptide compound having a structure shown in the following formula I:

[0022] .

[0023] The preparation method of the above compound comprises: starting from cheap and readily available Boc-L-valine, preparing a thiazole-containing cyclic hexapeptide compound through thiazole synthesis and condensation reaction. The specific preparation process is as follows:

[0024]

[0025] The preparation method of the above-mentioned thiazole-containing cyclic hexapeptide compound comprises the following specific steps:

[0026] Step 1. Add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (448 mg, 1.18 mmol), 1-hydroxybenzotriazole (HOBt) (2.4 mg, 0.018 mmol), and N,N-diisopropylethylamine (DIPEA) (228 mg, 1.77 mmol) to a dichloromethane solution of Boc-L-valine (192 mg, 0.88 mmol) in a 50 mL round-bottom flask, stir at room temperature for 2-3 min, add L-threonine methyl ester hydrochloride (100 mg, 0.59 mmol), and react at room temperature for 8 hours. After the reaction is completed by TLC monitoring, it is concentrated under reduced pressure and separated and purified by chromatography column (dichloromethane: methanol = 20:1) to obtain 241 mg of a light yellow oil, namely compound 3, with a yield of 82%.

[0027] Step 2. Compound 3 (3.32 g, 10 mmol) was dissolved in 70 mL of dichloromethane. Stirring was performed at 0 °C, 30 mL of trifluoroacetic acid was added to the reaction system, and the reaction was allowed to react for 1 h. After the reaction was completed by TLC spot plate monitoring, the excess trifluoroacetic acid was removed by rotary evaporation at low temperature, and toluene was added to the reaction mixture for azeotropy, and rotary evaporation was performed for 3-4 times. Then, dichloromethane was added and the reaction mixture was rotary evaporated for 2-3 times to obtain the crude product 4 with exposed amino group, which was used directly in the next step without further purification.

[0028] Step 3. The crude product 4 obtained in the above step was dissolved in 1,4-dioxane, triethylamine (Et3N) (2.62 g, 26 mmol) was added, and then 2-(trimethylsilyl)ethoxycarbonyloxysuccinimide (Teoc-OSu) (2.85 g, 11 mmol) was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, EA and water were used for extraction three times. The organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and separated using a chromatography column (petroleum ether: ethyl acetate = 1:1) to obtain 3.85 g of a light yellow oil, namely compound 5, with a yield of 95%.

[0029] Step 4. Compound 5 (2 g, 5 mmol) was dissolved in 1,2-dichloroethane, 2,2-dimethoxypropane (2.56 g, 25 mmol) was added at room temperature, and the mixture was stirred and refluxed overnight under the catalysis of pyridine p-toluenesulfonate (PPTS) (0.31 g, 1.25 mmol). After the reaction was completed by raw material spot plate monitoring, it was concentrated under reduced pressure. After separation and purification by chromatography column (petroleum ether: ethyl acetate = 2:1), 1.78 g of light yellow oily substance, namely compound 6, was obtained with a yield of 82%.

[0030] Step 5. In a 500 mL round-bottom flask, dissolve Boc-L-valine 7 (2.17 g, 10 mmol) in 200 mL dry dichloromethane, add pentafluorophenyl diphenyl phosphate (FDPP) (3.6 g, 10 mmol) at room temperature, and slowly add triethylamine (2 g, 20 mmol) dropwise, and stir at room temperature for 15 minutes. Then add dimethyl azidocysteine ​​(800 mg, 2.5 mmol) and triphenylphosphine (PPh3) (5.2 g, 10 mmol), and reflux at 50 °C for 5 hours in the dark. After the reaction is completed, cool to room temperature to obtain a thiazoline compound. The obtained thiazoline was dissolved in dry dichloromethane (50 mL), and 1,8-diazabicycloundec-7-ene (DBU) (5.4 g, 30 mmol) and bromotrichloromethane (BrCCl3) (2.0 mg, 20 mmol) were added dropwise at 0 °C, and reacted at room temperature for 30 minutes. After the reaction was completed by TLC monitoring, an equal volume of saturated ammonium chloride solution was added to quench, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 1.12 g of white solid compound 8, with a reaction yield of 72%.

[0031] Step 6. Take compound 8 (3.14 g, 10 mmol), add 70 mL of dichloromethane to dissolve. Stir at 0 °C, add 30 mL of trifluoroacetic acid to the reaction system, react for 1 h, monitor the reaction on a TLC plate, and use a rotary evaporator to remove excess trifluoroacetic acid at low temperature. Add toluene to the reaction mixture for azeotropy, and evaporate 3-4 times. Then add dichloromethane and evaporate 2-3 times to obtain an oily crude product 9, which can be used directly in the next step without further purification.

[0032] Step 7. Take compound 8 (3.14 g, 10 mmol), dissolve it in a mixture of tetrahydrofuran (THF) (30 mL), methanol (30 mL) and water (10 mL), and slowly add 0.5 N lithium hydroxide aqueous solution (6 mL) at 0 °C. Stir at room temperature for 3 hours. After the reaction is completed by TLC spot plate monitoring, dilute with an equal volume of water, adjust the pH value of the reaction system to 3-4 with 10% dilute hydrochloric acid aqueous solution, extract with ethyl acetate three times, dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound 10 with exposed carboxyl group, without further purification.

[0033] Step 8. In a 500 mL round-bottom flask, the hydrolysis product compound 10 was dissolved in 200 mL of dichloromethane, and HATU (5.7 g, 15 mmol), HOBt (40 mg, 0.3 mmol), and DIPEA (5.12 g, 20 mmol) were added in sequence. After stirring at room temperature for 2-3 min, the oily crude product 9 of the de-Boc product was added and reacted at room temperature for 8 hours. After the reaction was completed by TLC monitoring, it was concentrated under reduced pressure and purified by chromatography column separation (petroleum ether: ethyl acetate = 5:1) to obtain 4.2 g of white solid compound 11 with a yield of 85%.

[0034] Step 9. Compound 11 (2.23 g, 5 mmol) was reacted according to the synthesis method of step 7 to obtain carboxylic acid compound 12.

[0035] Step 10. Compound 6 (2.48 g, 5 mmol) was reacted according to the synthesis method of step 6 to obtain amino compound 13.

[0036] Step 11. Compound 12 and compound 13 were reacted according to the synthesis method of step 8. The crude product was separated and purified by chromatography column (petroleum ether: ethyl acetate = 1:1) to obtain 3.32 g of white solid compound 14 with a yield of 82%.

[0037] Step 12. Take compound 14 (810 mg, 1.0 mmol), dissolve it in a mixture of THF (20 mL), methanol (20 mL) and water (10 mL), and slowly add 0.5 N lithium hydroxide aqueous solution (12 mL) at 0 °C. Continue stirring at this temperature for 3 hours. After the reaction is completed by TLC plate monitoring, dilute with an equal volume of water, adjust the pH value of the reaction system to 3-4 with 10% dilute hydrochloric acid aqueous solution, extract with ethyl acetate three times, dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound 15 with naked carboxyl group, without further purification.

[0038] The crude carboxylic acid product obtained in the previous step was dissolved in 100 mL of tetrahydrofuran. Tetrabutylammonium fluoride (TBAF) (522 mg, 2 mmol) was added to the reaction system at room temperature, and the mixture was stirred and refluxed overnight. After the reaction was completed, water / ethyl acetate was used for extraction, and the organic phases were combined and concentrated under reduced pressure to obtain the crude product 15.

[0039] Step 13. The crude product 15 was dissolved in 300 mL DCM:DMF=(2:1), and benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (1.04 g, 2.0 mmol) and 4-dimethylaminopyridine (244 mg, 2.0 mmol) were slowly added to the solution. After the addition was completed, the mixture was stirred overnight. After the reaction was completed by TLC monitoring, 200 mL of water was added, extracted three times with DCM, concentrated under reduced pressure, and purified by chromatography column (petroleum ether: ethyl acetate = 2:1) to obtain 513 mg of solid, i.e., the thiazole-containing cyclic hexapeptide compound 16 of the present invention, with a yield of 85%. 1 H NMR (500 MHz, CDCl3) δ 9.14 (d, J = 8.0Hz, 1H), 8.78 (d, J = 9.5 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.06 (s, 1H), 8.02 (s, 1H), 5.41 (dd, J = 8.0, 4.5 Hz, 1H), 5.04 (d, J = 10.0 Hz, 1H), 4.89(m, 2H), 4.37 (d, J = 8.0 Hz, 1H), 2.40 – 2.33 (m, 2H), 2.12 (q, J= 7.0 Hz,1H), 1.86 (s, 3H), 1.61 (s, 3H), 1.29 (d, J = 6.0 Hz, 3H), 1.19 (d, J = 6.5Hz, 3H), 1.08 (d, J = 7.0 Hz, 3H), 1.02 – 0.98 (m, 9H), 0.84 (d, J = 7.0 Hz,3H). 13 C NMR (126 MHz, CDCl3) δ 172.8, 168.9, 168.4, 167.0, 162.5, 160.1,150.5, 149.5, 123.4, 122.8, 94.7, 69.7, 66.3, 58.0, 57.2, 56.5, 35.5, 34.6,30.9, 26.7, 25.8, 20.2, 19.5, 19.4, 19.1, 18.6, 18.1, 15.2; ESI-HRMS calcdfor C 28 H 40 N6O5S2([M + Na] + ) 627.2394, found 627.2385.

[0040] Comparative Example 1

[0041] 60 mg of the thiazole cyclic hexapeptide compound 16 obtained in Example 1 was dissolved in 10 mL of dichloromethane solution. Under ice-water conditions, 10 mL of trifluoroacetic acid was added to the system and stirring was continued overnight. After the reaction was completed by TLC monitoring, the system was concentrated under reduced pressure and purified using a chromatography column (petroleum ether: ethyl acetate = 1:1) to obtain 47 mg of a colorless oil 17 with a yield of 85%. 1 H NMR (500MHz, DMSO- d 6) δ 8.49 (d, J = 10.5 Hz, 1H), 8.43 (d, J = 10.0 Hz, 1H), 8.30(s, 1H), 8.24 (s, 1H), 8.14 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 10.0 Hz, 1H),5.33 (dd, J= 10.0, 7.5 Hz, 1H), 5.21 (t, J = 10.0 Hz, 1H), 5.19 (d, J = 9.0Hz, 1H), 4.34 (t, J = 11.0 Hz, 1H), 4.30 (dd, J = 10.0, 2.5 Hz, 1H), 4.14 –4.12 (m, 1H), 2.26 – 2.20 (m, 1H), 2.17 – 2.12 (m, 2H), 1.11 (d, J = 6.0 Hz,3H), 1.05 (d, J = 1.5 Hz, 3H), 1.04 (d, J = 3.0 Hz, 3H), 1.03 (d, J = 1.5 Hz,3H), 1.00 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 7.0 Hz,3H). 13 C NMR (125 MHz, DMSO- d 6) δ 170.1, 170.1, 169.5, 160.0, 159.7, 149.0,148.3, 125.5, 124.2, 67.7, 61.3, 58.9, 55.4, 55.0, 34.7, 34.3, 30.8, 21.2,19.8, 19.5, 19.4, 18.9, 18.8, 18.6; ESI-HRMS calcd for C 25 H 36 N6O5S2([M + Na] + )587.2081, found 587.2097.

[0042] Comparative Example 2

[0043] 56 mg of compound 17 obtained in Comparative Example 1 was dissolved in 30 mL of pyridine solution, acetic anhydride was added to the system and stirring was continued overnight. After the reaction was completed as monitored by TLC, the mixture was concentrated under reduced pressure and purified using a chromatography column (petroleum ether: ethyl acetate = 2:1) to obtain 54 mg of colorless oil 18 with a yield of 90%. 1H NMR (400 MHz, DMSO- d 6) δ 8.52 (d, J = 9.6Hz, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.36 (d, J = 9.2 Hz, 1H), 8.35 (s, 1H), 8.33 (s, 1H), 5.45 (dd, J = 9.6, 6.4 Hz, 1H), 5.35 (dd, J = 8.4, 5.2 Hz, 1H),5.07 (dd, J = 8.4, 5.2 Hz, 1H), 2.58 (s, 3H), 2.25 - 2.16 (m, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ 168.9, 168.5, 160.5, 159.7, 159.5, 159.0, 153.2, 148.3,147.8, 127.8, 125.3, 124.8, 54.8, 54.6, 52.2, 34.5, 34.4, 32.8, 18.9, 18.5,18.3, 18.1, 18.0, 18.0, 11.3; ESI-HRMS calcd for C 25 H 32 N6O4S2([M + Na] + )567.1819, found 567.1815.

[0044] Comparative Example 3

[0045] 12 mg of compound 17 obtained in comparative example 1 was dissolved in 100 ml of dichloromethane and heated at -78 °C under nitrogen protection. oDiethylaminosulfur trifluoride (DAST) reagent (5 mg, 0.03 mmol) was slowly added dropwise at 40 °C, and the reaction was maintained at low temperature for 2 h. After the reaction was completed by TLC monitoring, solid potassium carbonate (50 mg, 0.36 mmol) was added to quench the reaction. After slowly returning to room temperature, saturated sodium carbonate and dichloromethane were used for extraction three times. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in 10 mL of dichloromethane. DBU (60 mg, 0.4 mmol) and BrCCl3 (80 mg, 0.4 mmol) were added dropwise at 0 °C in sequence. The reaction was allowed to proceed at room temperature for 30 minutes. After the reaction was completed by TLC monitoring, an equal volume of saturated ammonium chloride solution was added to quench the reaction, and dichloromethane was used for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 7 mg of colorless oil 19. The yield of the reaction was 60%. 1 H NMR (400 MHz, CDCl3) δ 8.08(s, 1H), 5.27 (s, 1H), 4.88 (s, 1H), 3.92 (s, 3H), 2.42 (s, 1H), 1.43 (s,9H), 0.96 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz,CDCl3 ) δ 173.7, 162.0, 155.5, 147.1, 127.2, 80.2, 58.2, 52.5, 33.4, 28.4,19.5, 17.4; ESI-HRMS calcd for C 14 H 22 N2O6S ([M + Na] + ) 337.1192, found337.1186.

[0046] Test Example 1: Pharmacological Test

[0047] To evaluate the inhibitory activity of thiazole ring-containing hexapeptides against vincristine-resistant human oral epidermoid carcinoma cell line KBV cells when used alone or in combination.

[0048] Detection principle: MTT can penetrate the cell membrane and enter the cell. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to blue-purple needle-shaped formazan crystals that are insoluble in water and deposited in the cells. The crystals can be dissolved by dimethyl sulfoxide (DMSO). The absorbance measurement can reflect the cell survival rate.

[0049] The determination method is as follows:

[0050] 1. Seed the cells in a 96-well plate at a density of 2000 cells / well and culture them in a CO2 incubator for 24 hours;

[0051] 2. Dilute the test compound (stock solution 10 mM) to 150 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.25 μM, 0.6 μM, and 0 μM; set up three sets of parallel wells for each concentration, add the test compound to the seeded cells, and continue to culture for 72 hours;

[0052] 3. Take out the 96-well plate, add 20 μl of MTT solution to each well, and incubate in the incubator for 2 hours;

[0053] 4. Discard the upper layer of liquid, add 150 μl DMSO to each well, shake for 15 min until the formazan is completely dissolved;

[0054] 5. Measure the absorbance at 570 nm.

[0055] Three parallel tests were performed as shown in Table 1 below, and the experimental results were averaged.

[0056] As shown in the table below, when paclitaxel was administered alone, it had almost no inhibitory effect on drug-resistant KBV cells, and the cell survival rate reached 121.99%.

[0057] When the commercially available resistance-reversing drug verapamil is administered in combination with paclitaxel, its inhibitory activity against drug-resistant KBV cells is significantly increased.

[0058] When compounds 16 and 18 were co-administered with paclitaxel, the survival rates of drug-resistant KBV cells were only 55.64% and 72.65%, respectively. This indicates that the synthetic compounds have good resistance reversal effects, especially compound 16, which is better than the commercially available drug verapamil.

[0059] Interestingly, although compound 17 has a similar skeleton structure to compound 16, the presence of exposed hydroxyl groups completely causes it to lose its resistance-reversing ability, indicating that protecting the hydroxyl group plays an important role in maintaining its resistance-reversing activity.

[0060] When compound 19 was used in combination with paclitaxel, the inhibitory effect on drug-resistant KBV cells was further enhanced. However, it itself had strong cytotoxicity. When it was used alone, the survival rate of KBV cells was only 62.32%.

[0061] In summary, the thiazole-containing cyclic hexapeptide compound 16 of the present invention not only has good safety, but also has excellent drug resistance reversal activity, and has the potential to be the next generation of drug resistance reversal agent.

[0062] Table 1 Pharmacological tests of examples and comparative examples

[0063]

[0064] Verapamil (VL) was used as a control.

[0065] Pharmacological experiments showed that compound 16 of the present invention had no biological activity against vincristine-resistant human oral epidermoid carcinoma cell line KBV cells when used alone, but when used in combination with PTX, it showed better biological activity than the positive drug verapamil.

[0066] Specific IC 50 As shown in Table 2, the combination of compound 16 and paclitaxel showed a nearly 250-fold improvement in the cytotoxicity of compound 16 against KBV200 cells compared with paclitaxel, demonstrating its excellent drug resistance reversal activity.

[0067] Table 1 IC of compound 16 obtained in Example 1 50 value

[0068]

[0069] Experimental Example 2: Mechanism Experiment - Rhodamine123 Accumulation in Cells

[0070] Rhodamine123 is a substrate of P-gp and is usually used to study the function of P-gp protein. The fluorescence intensity of Rhodamine123 in cells can reflect the changes in the drug pump function of P-gp under the action of compounds.

[0071] Experimental methods:

[0072] After KBV cells were digested, 3×105 cells / well were inoculated in 6-well plates. After incubation for 24 h, different doses (0 μM, 2.5 μM, 5 μM, 10 μM) of compound 16 and 10 μM of taliciquid and verapamil were added. After incubation for 3 h, 10 μM Rhodamine123 was added and incubated for 30 min. The cells were collected and washed 3 times with PBS. The fluorescence intensity of Rhodamine123 in the cells was detected by flow cytometry. 10,000 cells were counted to obtain the relative fluorescence intensity of Rhodamine123.

[0073] like Fig. 9As shown in the figure, after the commercially available drugs Taliquida and Verapamil were incubated with cells, strong Rhodamine123 fluorescence was detected in the cells. However, after compound 16 and Rhodamine123 were co-incubated with KBV cells, there was no fluorescence enhancement in the cells, indicating that the function of P-gp protein was not inhibited, which was completely different from the results of Taliquida and Verapamil. This experiment shows that compound 16 has a different resistance reversal mechanism from Taliquida and Verapamil, and does not target P-gp protein.

[0074] The thiazole-containing cyclic hexapeptide compound of the present invention, when used in combination with paclitaxel PTX, exhibited a better inhibitory effect on vincristine-resistant human oral epidermoid carcinoma cell line KBV than the positive drug verapamil combined with PTX. The thiazole-containing cyclic hexapeptide compound of the present invention has the characteristics of cheap and readily available raw materials and significant combined anti-cancer effect. The present invention can be summarized in other specific forms that do not violate the spirit or main features of the present invention. Therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be regarded as an explanation of the present invention and cannot limit the present invention. The claims indicate the scope of the present invention, while the above description does not indicate the scope of the present invention. Therefore, any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims of the present invention.

Claims

1. A thiazole-containing cyclic hexapeptide compound, characterized in that: The compound has a structure shown in the following formula I: 。 2. A use of the thiazole-containing cyclic hexapeptide compound as claimed in claim 1 in the preparation of a drug for drug resistance reversal, characterized in that: The application is the application of the thiazole cyclic hexapeptide compound in combination with paclitaxel to reverse the drug resistance of vincristine-resistant human oral epidermoid cancer cell lines.

3. The use according to claim 2, characterized in that The cell line is a vincristine-resistant human oral epidermoid carcinoma cell line KBV cell.

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