Lysosome-targeted Eudistomin Y-type compounds, their preparation methods and applications

Abstract

This invention discloses Eudistomin Y-class compounds represented by formula (I) or (II) and their medically acceptable salts, as well as methods for their preparation. The Eudistomin Y-class covalent derivatives and their medically acceptable salts provided by this invention exhibit better solubility and lysosomal targeting properties. Therefore, the compounds prepared by this invention can specifically target the lysosomes of cancer cells, affecting the homeostasis of cancer cells and thus enabling targeted killing of cancer cells. The Eudistomin Y-class compounds represented by formula (I) or (II) and their medically acceptable salts provided by this invention can be used to prepare lysosomal-targeted druggable carriers and can be used in combination with other clinical antitumor drugs to improve drug targeting and better exert the antitumor activity of the drugs.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis and medicinal chemistry, specifically to a class of novel Eudistomin Y derivatives with lysosomal targeting, their preparation methods, and their application in the field of anti-triple-negative breast cancer. Background Technology

[0002] According to the latest estimates of the global cancer burden in 2020, the number of malignant tumors and deaths worldwide is rapidly increasing. Triple-negative breast cancer, in particular, lacks known drug targets and has the worst prognosis among breast cancers. It is prone to metastasis, has a high recurrence rate, and is easily resistant to drug resistance due to repeated use, leading to chemotherapy failure. Therefore, the design and research of innovative anti-tumor drugs for triple-negative breast cancer still faces significant challenges, and the development of innovative anti-tumor drugs remains crucial.

[0003] Lysosomes are the most important organelles in cells responsible for degradation. The selective release of drugs into lysosomes is a key mechanism by which tumor cells develop drug resistance. However, lysosomes are also highly involved in various fundamental cellular physiological activities, such as apoptosis, autophagy, and signal transduction, playing a unique role in tumor development and progression. Therefore, drugs that exert their anti-tumor effects via the lysosomal pathway may benefit from lysosomal targeting properties, which could enhance their anti-cancer activity.

[0004] Marine natural products, with their diverse and unique structures, are an important source of innovative drugs. Eudistomin Y compounds are a class of marine β-carboline alkaloids, isolated and identified from Eudistoma sea squirts in 2008, representing a novel type of marine alkaloid. Previous studies have found that Eudistomin Y derivatives possess good antitumor activity (see patent application publication number CN 111423438A), and some Eudistomin Y compounds have also been found to have good activity in reversing multidrug resistance in tumors (see patent application publication number CN 113751815A). However, the Eudistomin Y derivatives disclosed in the prior art have poor water solubility due to the lack of hydrophilic atoms and groups, which limits their subsequent pharmaceutical applications and in vivo drug utilization efficiency, requiring further improvement. Summary of the Invention

[0005] To obtain novel Eudistomin Y-class compounds with novel structures, lysosomal targeting, and good antitumor activity, and further to provide a medicament and composition thereof for treating the corresponding disease or condition, this invention provides two series of derivatives having the structural features of general formula (I) and general formula (II), respectively, and their pharmaceutically acceptable salts. The antitumor activity of these compounds is discovered for the first time, and the compounds are synthesized for the first time.

[0006] The first objective of this invention is to provide a novel Eudistomin Y compound that has lysosomal targeting and good antitumor activity.

[0007] To achieve the objective of this invention, the technical solution adopted is as follows:

[0008] Eudistomin Y-class compounds and their medically acceptable salts, as shown in formula (I) or (II),

[0009]

[0010] In formula (I), R1 represents hydrogen, methoxy or benzyloxy, and R2 represents 2-(4-morpholino)ethyl or N,N-dimethyl-3-aminopropyl.

[0011] In formula (II), R3 represents hydrogen, methoxy or benzyloxy, R4 represents 2-(4-morpholino)ethyl or N,N-dimethyl-3-aminopropyl, and R5 represents methyl or 2-(4-morpholino)ethyl;

[0012] Furthermore, when R3 is preferably methoxy or benzyloxy, R4 is 2-(4-morpholino)ethyl or N,N-dimethyl-3-aminopropyl, and R5 is methyl;

[0013] When R3 is hydrogen, R4 is 2-(4-morpholino)ethyl, and R5 is N,N-dimethyl-3-aminopropyl.

[0014] Furthermore, in preferred formula (I), R1 is benzyloxy and R2 is N,N-dimethyl-3-aminopropyl; as shown in formula 6 below.

[0015]

[0016] In preferred formula (II), R3 is hydrogen, R4 is 2-(4-morpholino)ethyl, and R5 is N,N-dimethyl-3-aminopropyl; or R3 is methoxy or benzyloxy, R4 is N,N-dimethyl-3-aminopropyl, and R5 is methyl. Preferred formula (II) is one of the following compounds:

[0017]

[0018] A second objective of this invention is to provide a method for preparing Eudistomin Y-type compounds with lysosomal targeting, wherein the method for preparing Eudistomin Y-type compounds represented by formula (I) includes the following steps:

[0019] (1) Using tryptophan as a raw material, it is reacted with acetophenone derivatives, I2, and H2O2 as shown in formula (i) in a molar ratio of 1:1~3:0.8~2.5:1.2~2.5 to obtain the compound shown in formula (ii);

[0020] The reaction solvent in step (1) is an aprotic solvent; the reaction temperature is 80℃-150℃; preferably, the reaction solvent is DMSO; the reaction temperature is 110℃-130℃.

[0021] The reaction time for step (1) is 4-10 hours; preferably 5-8 hours. The reaction is typically monitored by TLC until completion.

[0022] After the reaction in step (1) is completed, the reaction solution is post-treated to obtain the compound shown in formula (ii). The post-treatment method is generally as follows: the reaction solution is cooled to room temperature, and ethyl acetate, saturated saline, 10% Na2S2O3 solution and saturated ammonium chloride solution are added sequentially for extraction. The organic phase is dried, filtered, concentrated, and the crude product is separated and purified by column chromatography to obtain the compound shown in formula (ii).

[0023] Step (2): The compound shown in formula (ii) is reacted with R2-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 10 to obtain Eudistomin Y-type compounds shown in formula (I);

[0024] In R2-X, the X group is a halogen element;

[0025] Preferably, the X group is Cl, Br, or I.

[0026] The reaction solvent in step (2) is an aprotic solvent; the reaction temperature is -20℃ to 100℃;

[0027] Preferably, the reaction solvent is anhydrous DMF; the reaction temperature is 65℃-80℃;

[0028] The reaction time for step (2) is 5 to 10 hours; preferably 5 to 8 hours. The reaction is typically monitored by TLC until completion.

[0029] After the reaction in step (2) is completed, the reaction solution is post-treated to obtain Eudistomin Y-type compounds as shown in formula (I). The post-treatment method is generally as follows: after the reaction is completed, water is added to the reaction solution to quench the reaction, and the solution is extracted with ethyl acetate and saturated brine. The organic phase is dried, filtered, concentrated, and the crude product is purified by column chromatography to obtain Eudistomin Y-type compounds as shown in formula (I).

[0030] The reaction formula for the preparation method of Eudistomin Y-type compounds shown in formula (I) is as follows:

[0031]

[0032] The definitions of R1 and R2 are as described above.

[0033] The preparation method of Eudistomin Y-type compounds shown in formula (II) includes the following steps:

[0034] (a) Using the tryptophan derivative shown in formula (iii) as a raw material, reacting it with the acetophenone derivative shown in formula (iv), I2, and H2O2 in a molar ratio of 1:1 to 3:0.8 to 2.5:1.2 to 2.5 to obtain the compound shown in formula (v);

[0035] The reaction solvent in step (a) is an aprotic solvent; the reaction temperature is 80℃-150℃.

[0036] Preferably, the reaction solvent is DMSO; the reaction temperature is 110℃-130℃;

[0037] The reaction time for step (a) is 4-10 hours; preferably 5-8 hours. The reaction is typically monitored by TLC until completion.

[0038] (b) The compound shown in formula (v) was reacted with R4-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 7 to obtain the compound shown in formula (vi);

[0039] In R4-X, the X group is a halogen element;

[0040] Preferably, the X group is Cl, Br, or I.

[0041] The reaction solvent in step (b) is an aprotic solvent; the reaction temperature is -20℃ to 100℃.

[0042] Preferably, the reaction solvent is anhydrous DMF; the reaction temperature is 65℃-80℃;

[0043] The reaction time for step (b) is 5 to 10 hours; preferably 5 to 8 hours. The reaction is typically monitored by TLC until completion.

[0044] (c) The compound shown in formula (vi) is dissolved and hydrolyzed under alkaline conditions to obtain the compound shown in formula (vii);

[0045] The reaction environment in step (c) is alkaline. NaOH is generally added to adjust the reaction environment to be alkaline, and the pH value is generally adjusted to 8-13, preferably 9-12.

[0046] The reaction solvent in step (c) is methanol.

[0047] The reaction temperature in step (c) is -20℃ to 100℃; preferably, the reaction temperature is 65℃ to 80℃.

[0048] TLC is typically used to monitor the reaction until it is complete.

[0049] (d) The compound shown in formula (vii) was reacted with R5-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 7 to prepare Eudistomin Y-type compounds shown in formula (II);

[0050] In R5-X, the X group is a halogen element;

[0051] Preferably, the X group is Cl, Br, or I.

[0052] The reaction solvent in step (d) is an aprotic solvent; the reaction temperature is -20℃ to 100℃.

[0053] Preferably, the reaction solvent is anhydrous DMF; the reaction temperature is 65℃-80℃.

[0054] TLC is typically used to monitor the reaction until it is complete.

[0055] The reaction formula for the preparation method of Eudistomin Y-type compounds shown in formula (II) is as follows:

[0056]

[0057] The definitions of R3, R4, and R5 are as described above.

[0058] Furthermore, when R5 in formula (II) is methyl, the compound shown in formula (vi) obtained in step (b) is the target product.

[0059] A third object of the present invention is to provide the use of Eudistomin Y-class compounds of formula (I) or (II) and their medically acceptable salts in the preparation of lysosomal targeted antitumor drugs.

[0060] Furthermore, the anti-tumor drug mentioned is for the treatment of triple-negative breast cancer, and is not limited to the treatment and prevention of triple-negative breast cancer.

[0061] A fourth object of the present invention is to provide the use of Eudistomin Y-type compounds of formula (I) or (II) and their medically acceptable salts in the preparation of lysosome-targeted druggable carriers, which are not limited to the treatment and prevention of tumor-related diseases.

[0062] A fifth object of the present invention is to provide the use of Eudistomin Y-class compounds of formula (I) or (II) and their medically acceptable salts in the preparation of combination antitumor drugs, wherein the combination antitumor drugs are lysosomal-targeted pharmaceutically acceptable carriers prepared from Eudistomin Y-class compounds of formula (I) or (II) and their medically acceptable salts, used in combination with clinical antitumor drugs, wherein the clinical antitumor drugs may be drugs for treating diseases or conditions such as breast cancer, colon cancer, cervical cancer, liver cancer, gastric cancer or lung cancer.

[0063] The pharmaceutically acceptable salt of the compound of the present invention refers to a conventional acid addition salt that has the same pharmaceutical efficacy as the compound and is formed with a suitable nontoxic organic or inorganic acid.

[0064] The present invention also discloses a pharmaceutical composition comprising Eudistomin Y class compounds of formula (I) or (II) of the present invention or pharmaceutically acceptable salts thereof, which can be added with pharmaceutically acceptable carriers to form common pharmaceutical preparations such as tablets, capsules, powders, syrups, liquids, suspensions, and injections, and can be supplemented with commonly used pharmaceutical excipients such as flavorings, sweeteners, liquid or solid fillers or diluents.

[0065] The pharmaceutical composition described in this invention can be administered clinically via oral administration, injection, or other methods.

[0066] The clinical dosage of the compounds of this invention is 0.01 mg to 1000 mg / day, but may deviate from this range depending on the severity of the condition or the dosage form.

[0067] The present invention also provides the application of Eudistomin Y class compounds of formula (I) or (II) or their pharmaceutically acceptable salts in the preparation of antitumor tracer molecular tool drugs. Eudistomin Y class compounds of formula (I) or (II) are fluorescent compounds that can effectively trace their distribution in cells and tissues through fluorescence characteristics.

[0068] The advantages of this invention compared to the prior art are as follows:

[0069] The Eudistomin Y-class compounds and their medically acceptable salts, represented by formula (I) or (II) of this invention, are synthesized for the first time and exhibit good antitumor activity. This invention, through the introduction of nitrogen-containing groups, for the first time endows them with lysosomal targeting properties. The advantage of lysosomal targeting is that the drug can specifically locate on the lysosomes of cancer cells, affecting the homeostasis of cancer cells and thus enabling targeted killing of cancer cells. Therefore, the Eudistomin Y-class compounds and their medically acceptable salts, represented by formula (I) or (II) of this invention, can be used to prepare pharmaceutically viable lysosomal targeting carriers and can be used in combination with other clinical antitumor drugs to improve drug targeting and better exert antitumor activity. Furthermore, the water solubility of the Eudistomin Y-class compounds represented by formula (I) or (II) of this invention is significantly improved compared to existing technologies, which is beneficial for increasing the effective drug concentration and improving the bioavailability of the drug in vivo. Attached Figure Description

[0070] Figure 1 A bar chart showing the inhibition of triple-negative breast cancer MDA-MB-231 cell proliferation by Eudistomin Y-type compounds.

[0071] Figure 2 Photographs of compounds 6, 7, 10, and 13 targeting the lysosomes of MDA-MB-231 cells. Detailed Implementation

[0072] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0073] Example 1

[0074] Preparation of Compound 1

[0075]

[0076] Tryptophan (481 mg, 3 mmol), acetophenone (350 μL, 3 mmol), and I2 (610 mg, 2.4 mmol) were dissolved in DMSO (6 mL). H2O2 (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na2S2O3 solution, and saturated ammonium chloride solution were added sequentially. Extraction was performed, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain a yellow solid 15 (330 mg, 1.2 mmol, 40%).

[0077]

[0078] Under argon protection, yellow solid 15 (50 mg, 0.2 mmol) and K2CO3 (127 mg, 0.9 mmol) were dissolved in anhydrous DMF (4 mL). 4-(2-chloroethyl)morpholine (125.1 μL, 0.9 mmol) was slowly added at room temperature. After stirring at room temperature, the mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to obtain a pale yellow solid 1 (35 mg, 0.09 mmol, 50%).

[0079] Compound 1, ¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 5.0 Hz, ¹H), 8.18 (d, J = 7.8 Hz, ¹H), 8.12 (d, J = 5.1 Hz, ¹H), 8.03 (d, J = 7.1 Hz, 2H), 7.67–7.60 (m, 2H), 7.55 (d, J = 8.4 Hz, ¹H), 7.50 (ddd, J = 7.3, 5.6, 1.2 Hz, 2H), 7.34 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.49 (t, J = 7.7 Hz, 2H), 3.53 (t, J = 4.6 Hz, 4H), 2.57 (t, J = 7.8 Hz, 2H), 2.28 (t, J = 4.6 Hz, 4H).

[0080] Example 2

[0081] Preparation of compound 2

[0082]

[0083] Under argon protection, yellow solid 15 (60 mg, 0.2 mmol) and K2CO3 (213 mg, 1.5 mmol) were dissolved in anhydrous DMF (4 mL). N,N-dimethylaminochloropropane hydrochloride (104 mg, 0.7 mmol) was slowly added at room temperature. After stirring at room temperature, the mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 2 (51 mg, 0.1 mmol, 65%).

[0084] Compound 2,1H NMR (400MHz, CDCl3) δ8.50(d,J=5.0Hz,1H),8.18(d,J=7.9Hz,1H),8.12(d,J=5.1Hz,1H),8.04(d,J=7.1Hz,2H),7.75–7.45 (m,5H),7.32(dd,J=14.8,1.0Hz,1H),4.39(t,J=7.3Hz,2H),2.10(t,J=7.1Hz,2H),2.02(s,6H),1.76(quin,J=7.1Hz,2H).

[0085] Example 3

[0086] Preparation of compound 3

[0087]

[0088] Tryptophan (481 mg, 3 mmol), p-methoxyacetophenone (417 μl, 3 mmol), and I2 (610 mg, 2.4 mmol) were dissolved in DMSO (6 mL). H2O2 (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na2S2O3, and saturated ammonium chloride were added sequentially. The mixture was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain a yellow solid 16 (543 mg, 1.8 mmol, 60%).

[0089]

[0090] Under argon protection, yellow solid 16 (45 mg, 0.1 mmol) and K2CO3 (144 mg, 1.0395 mmol) were dissolved in anhydrous DMF (4 mL). 4-(2-chloroethyl)morpholine (40 μL, 0.3 mmol) was slowly added at room temperature. After stirring at room temperature, the mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 3 (42 mg, 0.1 mmol, 68%).

[0091] Compound 3, 1H NMR (400MHz, CDCl3) δ8.50(d,J=5.1Hz,1H),8.18(d,J=7.8Hz,1H),8.10(d,J=5.1H z,1H),8.01(d,J=9.0Hz,2H),7.62(ddd,J=8.3,7.0,1.2Hz,1H),7.53(d,J=8.4Hz, 1H),7.33(ddd,J=7.9,7.1,0.9Hz,1H),6.97(d,J=9.0Hz,2H),4.47(t,J=7.7Hz,2H ),3.88(s,3H),3.55(t,J=4.6Hz,4H),2.55(t,J=7.7Hz,2H),2.29(t,J=4.6Hz,4H).

[0092] Example 4

[0093] Preparation of compound 4

[0094]

[0095] Under argon protection, yellow solid 16 (45 mg, 0.1 mmol) and K2CO3 (144 mg, 1.0 mmol) were dissolved in anhydrous DMF (4 mL). N,N-dimethylaminochloropropane hydrochloride (47 mg, 0.3 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 4 (46 mg, 0.1 mmol, 80%).

[0096] Compound 4,1H NMR (400MHz, CDCl3) δ8.50(d,J=5.1Hz,1H),8.17(d,J=7.8Hz,1H),8.10(d,J=5.1 Hz,1H),8.01(d,J=8.9Hz,2H),7.61(ddd,J=8.2,6.9,1.2Hz,1H),7.56(d,J=8.3H z,1H),7.31(ddd,J=7.8,6.8,1.1Hz,1H),6.96(d,J=8.9Hz,2H),4.37(t,J=7.4Hz ,2H),3.87(s,3H),2.09(t,J=7.0Hz,2H),2.04(s,6H),1.75(quin,J=7.2Hz,2H).

[0097] Example 5

[0098] Preparation of compound 5

[0099]

[0100] Tryptophan (384 mg, 2.4 mmol), p-benzyloxyacetophenone (543 mg, 2.4 mmol), and I2 (488 mg, 1.9 mmol) were dissolved in DMSO (3 mL). H2O2 (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na2S2O3, and saturated ammonium chloride were added sequentially. The mixture was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain a yellow solid 17 (499 mg, 1.3 mmol, 55%).

[0101]

[0102] Under argon protection, yellow solid 17 (50 mg, 0.12 mmol) and K2CO3 (83 mg, 0.6 mmol) were dissolved in anhydrous DMF (3 mL). 4-(2-chloroethyl)morpholine (82 μL, 0.6 mmol) was slowly added at room temperature. After stirring at room temperature, the mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 5 (50 mg, 0.1 mmol, 85%).

[0103] Compound 5,1H NMR (400MHz, CDCl3) δ8.49(d,J=5.1Hz,1H),8.16(d,J=7.8Hz,1H),8.08(d,J =5.1Hz,1H),8.00(d,J=8.9Hz,2H),7.61(dd,J=15.4,1.1Hz,1H),7.52(d,J=8 .0Hz,1H),7.45–7.29(m,6H),7.04(d,J=9.0Hz,2H),5.14(s,2H),4.46t,J=7. 7Hz, 2H), 3.54 (t, J = 4.4Hz, 4H), 2.54 (t, J = 7.2Hz, 2H), 2.28 (t, J = 4.0Hz, 4H).

[0104] Example 6

[0105] Preparation of compound 6

[0106]

[0107] Under argon protection, yellow solid 17 (50 mg, 0.12 mmol) and K2CO3 (83 mg, 0.6 mmol) were dissolved in anhydrous DMF (4 mL). N,N-dimethylaminochloropropane hydrochloride (95 mg, 0.6 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 6 (34 mg, 0.07 mmol, 62%).

[0108] Compound 6,1H NMR (400MHz, CDCl3) δ8.49(d,J=5.1Hz,1H),8.17(d,J=7.8Hz,1H),8.10(d,J=5.1Hz,1H),8.00(d,J=9.0Hz,2H),7.62(dd,J=15.3,1.2Hz,1H),7.56(d,J =8.3Hz,1H),7.44–7.29(m,6H),7.03(d,J=9.0Hz,2H),5.14(s,2H),4.36(t, J=7.6Hz,2H),2.15(t,J=7.1Hz,2H),2.07(s,6H),1.79(quin,J=7.2Hz,2H).

[0109] Example 7

[0110] Preparation of compound 7

[0111]

[0112] Tryptophan methyl ester (637 mg, 2.5 mmol), acetophenone (291.2 μL, 2.5 mmol), and I₂ (508 mg, 2 mmol) were dissolved in DMSO (6 mL). H₂O₂ (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na₂S₂O₃, and saturated ammonium chloride were added sequentially. The mixture was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain a yellow solid 18 (283.5 mg, 0.92 mmol, 37%).

[0113]

[0114] Under argon protection, yellow solid 18 (60 mg, 0.2 mmol) and K2CO3 (134 mg, 1.0 mmol) were dissolved in anhydrous DMF (6 mL). 4-(2-chloroethyl)morpholine (131.3 μL, 1.0 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 19 (62 mg, 0.1 mmol, 72%).

[0115]

[0116] Yellow solid 19 (62 mg) was dissolved in 3 mL of MeOH, and the pH was adjusted to 10 by adding NaOH. The mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain yellow solid 20 (59 mg, 0.1 mmol, 93%).

[0117]

[0118] Under argon protection, yellow solid 20 (25 mg, 0.1 mmol) and K2CO3 (56 mg, 0.4 mmol) were dissolved in anhydrous DMF (3 mL). N,N-dimethylaminochloropropane hydrochloride (28 mg, 0.2 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 7 (24 mg, 0.05 mmol, 82%).

[0119] Compound 7, 1 H NMR(400MHz, CDCl3)δ9.00(s,1H),8.29(d,J=7.8Hz,1H),8.08(d,J=7.1Hz,2H),7.73–7.62(m,2H),7.61–7.50(m,3H),7.43(ddd,J=7.8,7.0,0.7H z,1H),4.51–4.47(m,4H),3.51(t,J=4.7Hz,4H),2.83(t,J=7.7Hz,2H),2.56(t,2H),2.49(s,6H),2.26(t,J=4.7Hz,4H),2.21(quin,J=6.5Hz,2H).

[0120] Example 8

[0121] Preparation of compound 8

[0122]

[0123] Under argon protection, yellow solid 18 (80 mg, 0.3 mmol) and K2CO3 (241 mg, 1.8 mmol) were dissolved in anhydrous DMF (3 mL). N,N-dimethylaminochloropropane hydrochloride (79 mg, 0.5 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 21 (59 mg, 0.1 mmol, 57%).

[0124]

[0125] Yellow solid 21 (59 mg) was dissolved in 4 mL of MeOH, and the pH was adjusted to 10 with NaOH. The mixture was heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain yellow solid 22 (56 mg, 0.1 mmol, 96%).

[0126]

[0127] Under argon protection, yellow solid 22 (56 mg, 0.1 mmol) and K2CO3 (139 mg, 1.0 mmol) were dissolved in anhydrous DMF (3 mL). 4-(2-chloroethyl)morpholine (60 μL, 0.433 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 8 (50 mg, 0.1 mmol, 67%).

[0128] Compound 8, 1HNMR(400MHz, CDCl3)δ9.00(s,1H),8.26(d,J=7.9Hz,1H),8.08(d,J=7.1Hz,2H),7 .71–7.60(m,3H),7.53–7.47(m,2H),7.41(ddd,J=7.8,6.8,1.1Hz,1H),4.55(t,J= 5.7Hz,2H),4.40(t,J=7.5Hz,2H),3.63(t,J=4.7Hz,4H),2.79(t,J=5.7Hz,2H),2. 56(t,J=4.7Hz,4H),2.17(t,J=7.0Hz,2H),2.08(s,6H),1.83(quin,J=7.2Hz,2H).

[0129] Example 9

[0130] Preparation of compound 9

[0131]

[0132] Tryptophan methyl ester (764 mg, 3 mmol), p-methoxyacetophenone (417 μl, 3 mmol), and I₂ (610 mg, 2.4 mmol) were dissolved in DMSO (5 mL). H₂O₂ (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na₂S₂O₃, and saturated ammonium chloride were added sequentially. The mixture was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain a yellow solid 23 (337 mg, 0.9 mmol, 31%).

[0133]

[0134] Under argon protection, yellow solid 23 (100 mg, 0.3 mmol) and K2CO3 (285 mg, 2.1 mmol) were dissolved in anhydrous DMF (5 ml). 4-(2-chloroethyl)morpholine (120 μl, 0.9 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 9 (85 mg, 0.2 mmol, 61%).

[0135] Compound 9, 1H NMR (400MHz, CDCl3) δ9.00 (s, 1H), 8.25 (d, J = 7.9Hz, 1H), 8.03 (d, J = 8.3Hz, 2H ),7.67(ddd,J=8.2,7.1,1.1Hz,1H),7.57(d,J=8.4Hz,1H),7.41(ddd,J=8.0, 7.1,0.9Hz,1H),6.97(d,J=8.4Hz,2H),4.47(t,J=7.7Hz,2H),4.01(s,3H),3. 89(s,3H),3.55(t,J=4.7Hz,4H),2.58(t,J=6.7Hz,2H),2.30(t,J=4.3Hz,4H).

[0136] Example 10

[0137] Preparation of compound 10

[0138]

[0139] Under argon protection, yellow solid 23 (100 mg, 0.3 mmol) and K2CO3 (285.4 mg, 2.1 mmol) were dissolved in anhydrous DMF (3 mL). N,N-dimethylaminochloropropane hydrochloride (93.2 mg, 0.6 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 10 (114 mg, 0.3 mmol, 93%).

[0140] Compound 10, 1 HNMR (400MHz, CDCl3) δ9.01 (s, 1H), 8.25 (d, J = 7.8Hz, 1H), 8.04 (d, J = 8.9Hz, 2H),7.66(ddd,J=8.0,7.0,1.1Hz,1H),7.60(d,J=8.4Hz,1H),7.39(ddd,J=7 .9,6.8,1.1Hz,1H),6.96(d,J=9.0Hz,2H),4.37(t,J=7.5Hz,1H),4.01(s,3H ),3.88(s,3H),2.10(t,J=6.9Hz,2H),2.05(s,6H),1.78(quin,J=7.1Hz,2H).

[0141] Example 11

[0142] Preparation of compound 11

[0143]

[0144] Yellow solid 10 (94 mg, 0.2 mmol) was dissolved in 3 mL of MeOH, and NaOH was added to adjust the pH to 10. The mixture was stirred at room temperature and then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 24 (90 mg, 0.2 mmol, 95%).

[0145]

[0146] Under argon protection, yellow solid 24 (95 mg, 0.2 mmol) and K2CO3 (214.1 mg, 1.6 mmol) were dissolved in anhydrous DMF (3 mL). 4-(2-chloroethyl)morpholine (90.3 μL, 0.7 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 11 (51 mg, 0.1 mmol, 42%).

[0147] Compound 11, 1 HNMR (400MHz, CDCl3) δ8.98(s,1H),8.24(d,J=7.8Hz,1H),8.06(d,J=8.9Hz,2H),7.66(dd,J= 15.2,1.0Hz,1H),7.61(d,J=8.3Hz,1H),7.39(dd,J=14.8,0.9Hz,1H),6.96(d,J=9.0Hz,2H),4 .57(t,J=5.8Hz,2H),4.39(t,J=7.5Hz,2H),3.89(s,3H),3.75(t,J=4.6Hz,4H),2.81(t,J=5. 8Hz,2H),2.58(t,J=4.5Hz,4H),2.12(t,J=7.0Hz,2H),2.07(s,6H),1.79(quin,J=7.0Hz,2H).

[0148] Example 12

[0149] Preparation of compound 12

[0150]

[0151] Tryptophan methyl ester (764 mg, 3 mmol), p-benzyloxyacetophenone (679 mg, 3 mmol), and I2 (609.6 mg, 5.4 mmol) were dissolved in DMSO (6 mL). H2O2 (30%, 1.5 eq) was added at room temperature, and the mixture was stirred until dissolved. The temperature was then raised to 110 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and ethyl acetate, saturated brine, 10% Na2S2O3, and saturated ammonium chloride were added sequentially. The mixture was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain a yellow solid 25 (746 mg, 1.7 mmol, 57%).

[0152]

[0153] Under argon protection, yellow solid 25 (50 mg, 0.1 mmol) and K2CO3 (83 mg, 0.6 mmol) were dissolved in anhydrous DMF (5 ml). 4-(2-chloroethyl)morpholine (82 μl, 0.6 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 12 (51 mg, 0.09 mmol, 90%).

[0154] Compound 12, 1 HNMR (400MHz, CDCl3) δ8.99(s,1H),8.24(d,J=7.7Hz,1H),8.03(d,J=9.0Hz,2H),7.66(dd,J=15.5,1.1Hz,1H),7.56(d,J=8.4Hz,1H),7.46–7.30( m,6H),7.04(d,J=9.0Hz,2H),5.15(s,2H),4.45(t,J=7.7Hz,2H),4.00(s ,3H),3.54(t,J=4.6Hz,4H),2.55(t,J=7.5Hz,2H),2.28(t,J=4.3Hz,4H).

[0155] Example 13

[0156] Preparation of compound 13

[0157]

[0158] Under argon protection, yellow solid 25 (40 mg, 0.1 mmol) and K2CO3 (93 mg, 0.7 mmol) were dissolved in anhydrous DMF (4 mL). N,N-dimethylaminochloropropane hydrochloride (45.6 mg, 0.3 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain yellow solid 13 (41 mg, 0.1 mmol, 79%).

[0159] Compound 13, 1 HNMR(400MHz, CDCl3)δ9.00(s,1H),8.24(d,J=7.8Hz,1H),8.04(d,J=9.0Hz,2H),7.75(dd,J=15.3,1.2Hz,1H),7.60(d,J=8.3Hz,1H),7.45–7.3 0(m,6H),7.03(d,J=9.0Hz,2H),5.15(s,2H),4.36(d,J=7.6Hz,2H),4.01(s,3H),2.07(t,J=6.9Hz,2H),2.03(s,6H),1.75(quin,J=7.0Hz,2H).

[0160] Example 14

[0161] Preparation of compound 14

[0162]

[0163] Yellow solid 13 (55 mg, 0.1 mmol) was dissolved in 3 mL of MeOH, and the pH was adjusted to 10 with NaOH. The mixture was stirred at room temperature and then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain yellow solid 26 (51 mg, 0.1 mmol, 96%).

[0164]

[0165] Under argon protection, yellow solid 26 (30 mg, 0.1 mmol) and K2CO3 (60 mg, 0.4 mmol) were dissolved in anhydrous DMF (5 ml). 4-(2-chloroethyl)morpholine (25.1 μl, 0.2 mmol) was slowly added at room temperature and stirred at room temperature. The mixture was then heated and stirred in a water bath at 75 °C. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain a pale yellow solid 14 (48 mg, 0.1 mmol, 48%).

[0166] Compound 14, 1 HNMR (400MHz, CDCl3) δ8.97 (s, 1H), 8.24 (d, J = 7.6Hz, 1H), 8.04 (d, J = 9.0Hz, 2H), 7. 62(d,J=8.3Hz,1H),7.46–7.31(m,7H),7.03(d,J=9.0Hz,2H),5.16(s,2H),4.56(t, J=5.8Hz,2H),4.45(t,J=5.9Hz,4H),4.38(t,J=7.7Hz,2H),3.91(s,6H),2.81(t,J= 5.8Hz,2H),2.74(t,J=5.9Hz,4H),2.29(t,J=6.8Hz,2H),1.89(quin,J=7.2Hz,2H).

[0167] Example 15

[0168] Comparative experiment on water solubility of Eudistomin Y-type compounds

[0169] 1. Experimental Methods

[0170] Fourteen newly synthesized lysosome-targeted Eudistomin Y compounds were compared with Eudistomin Y compounds 2 and 4 in the authorized patent (see patent application publication number CN 111423438A) and Eudistomin Y compounds H1k and H2a in a published paper (Yang G, Xie H, Wang C, Zhang C, Yu L, Zhang L, Liu X, Xu R, Song Z, Liu R, Ueda M. Design, synthesis, and discovery of Eudistomin Y derivatives as lysosome-targeted antiproliferation agents. Eur J MedChem. 2023 Mar 15; 250:115193. doi:10.1016 / j.ejmech.2023.115193.Epub). 2023Feb8.PMID:36774698.) Prepare 100 μmol PBS solutions containing 1% DMSO, shake thoroughly, let stand for two hours, and observe the state of the solutions under an optical microscope.

[0171] 2. Experimental Results

[0172] Microcrystals were found to precipitate in solutions of Eudistomin Y derivatives 2, 4, H1k, and H2a, while 14 newly synthesized lysosomal-targeted Eudistomin Y derivatives were completely dissolved without microcrystal precipitation.

[0173] The results of Example 15 show that the Eudistomin Y derivatives involved in this invention have significantly improved water solubility compared with the original Eudistomin Y derivatives.

[0174] The following are the pharmacological experimental results of some compounds of this invention.

[0175] Example 16

[0176] In vitro inhibition of MDA-MB-231 tumor cell activity by Eudistomin Y-type compounds

[0177] 1. Experimental Methods

[0178] Evaluation using the MTT method:

[0179] ① Digest and count MDA-MB-231 tumor cells in the logarithmic growth phase, 2.5 × 10⁻⁶ 3 / wells were seeded into 96-well plates and cultured in a cell culture incubator for 24 hours;

[0180] ② Prepare a 100 mM stock solution of Eudistomin Y-type compounds using DMSO;

[0181] ③ Dilute with culture medium again, add to 96-well plate, final concentration 50 μM, and incubate in cell culture incubator for 72 hours;

[0182] ④ Add 20 μL of 5 mg / mL MTT to each well, incubate for 4 h, discard the supernatant, add 150 μL of DMSO, shake in the dark for 10 min, detect OD570, and calculate the inhibition rate.

[0183] Inhibition rate = [1 – experimental group (OD570) / blank (OD570)] x 100%

[0184] 2. Experimental Results

[0185] Figure 1 This study demonstrates the inhibitory effect of Eudistomin Y-type compounds on the proliferation of triple-negative breast cancer MDA-MB-231 cells.

[0186] The results of Example 16 show that the Eudistomin Y derivatives involved in this invention exhibit good inhibitory activity against tumor cell proliferation at a concentration of 50 μM, significantly superior to the antitumor activity of 5-fluorouracil, with an inhibition rate more than twice that of 5-fluorouracil. In particular, compounds 6, 7, 10, and 13 show strong inhibitory activity against tumor cell proliferation, inhibiting the proliferation of MDA-MB-231 tumor cells by more than 80% at a concentration of 50 μM.

[0187] Example 17

[0188] Eudistomin Y-type compound lysosomal targeting properties experiment

[0189] 1. Experimental Methods

[0190] MDA-MB-231 tumor cells were seeded in 35 mm laser confocal microscopy culture dishes and then cultured in a cell culture incubator for 24 h. After culture, compounds 6, 7, 10, and 13 from the above examples were added to prepare a 10 μM solution. The cells were then cultured in a cell culture incubator for another 3 h, after which the culture medium was aspirated and discarded. Lysosome localization agent lyso-Tracker was added, and the cells were cultured in an incubator for 0.5 h. The cells were then washed three times with PBS. The samples were placed on the stage of a laser confocal microscope to observe and record the degree of lysosomal aggregation of compounds 6, 7, 10, and 13 in the cells.

[0191] 2. Experimental Results

[0192] Figure 2The results show the effects of compounds 6, 7, 10, and 13 targeting the lysosomes of MDA-MB-231 cells. Scale bar: 10 μm.

[0193] The first column of the figure shows the fluorescence localization of compounds 6, 7, 10, and 13 in MDA-MB-231 tumor cells, with green fluorescence representing the compounds. The second column shows the fluorescence localization of lysosomal localizers in MDA-MB-231 tumor cells, with red fluorescence representing lysosomal targeting localizers. The third column shows the distribution of compounds 6, 7, 10, and 13 and lysosomal localizers in the same MDA-MB-231 tumor cells. The degree of overlap between red and green fluorescence indicates the degree of lysosomal targeting of the compounds. The third column shows actual images of MDA-MB-231 tumor cells.

[0194] The results of Example 17 show that the novel Eudistomin Y derivatives with lysosome targeting designed and synthesized in this invention can effectively target the lysosomes of tumor cells, providing a new approach for exploring the mechanism of action of lysosome-targeting compounds in cells.

[0195] The above description is merely a partial embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the combination of the above-described technical features, but should also cover other technical solutions formed by any combination of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with, but not limited to, technical features disclosed in this application that have similar functions.

Claims

1. Eudistomin Y-class compounds represented by formula (I) or (II) and their medically acceptable salts, In formula (I), R1 is benzyloxy and R2 is N,N-dimethyl-3-aminopropyl; In formula (II), R3 is hydrogen, R4 is 2-(4-morpholino)ethyl, and R5 is N,N-dimethyl-3-aminopropyl; R3 is methoxy or benzyloxy, R4 is N,N-dimethyl-3-aminopropyl, and R5 is methyl. Formula (I) represents the following compounds; Formula (II) is one of the following compounds:

2. The method for preparing Eudistomin Y-type compounds as shown in formula (I) according to claim 1, characterized in that... The method includes the following steps: (1) Using tryptophan as a raw material, it is reacted with acetophenone derivatives, I2, and H2O2 as shown in formula (i) in a molar ratio of 1:1~3:0.8~2.5:1.2~2.5 to obtain the compound shown in formula (ii); Step (2): The compound shown in formula (ii) is reacted with R2-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 10 to obtain Eudistomin Y-type compounds shown in formula (I); In R2-X, the X group is a halogen element; The reaction equation is shown below: The definitions of R1 and R2 are as described in claim 1.

3. The method for preparing Eudistomin Y-type compounds as shown in formula (II) according to claim 1, characterized in that... The method includes the following steps: (a) Using the tryptophan derivative shown in formula (iii) as a raw material, reacting it with the acetophenone derivative shown in formula (iv), I2, and H2O2 in a molar ratio of 1:1 to 3:0.8 to 2.5:1.2 to 2.5 to obtain the compound shown in formula (v); (b) The compound shown in formula (v) was reacted with R4-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 7 to obtain the compound shown in formula (vi); In R4-X, the X group is a halogen element; (c) The compound shown in formula (vi) is dissolved and hydrolyzed under alkaline conditions to obtain the compound shown in formula (vii); (d) The compound shown in formula (vii) was reacted with R5-X and K2CO3 in a molar ratio of 1:1.3 to 6:1.2 to 7 to prepare Eudistomin Y-type compounds shown in formula (II); In R5-X, the X group is a halogen element; The reaction formula for the preparation method of Eudistomin Y-type compounds shown in formula (II) is as follows: The definitions of R3, R4, and R5 are as described in claim 1.

4. The use of Eudistomin Y-type compounds of formula (I) or (II) as described in claim 1 and their medically acceptable salts in the preparation of lysosomal targeted antitumor drugs.

5. The application as described in claim 4, characterized in that... The anti-tumor drug mentioned is a drug for treating triple-negative breast cancer.

6. The use of Eudistomin Y-type compounds of formula (I) or (II) as described in claim 1 and their medically acceptable salts in the preparation of lysosomal targeted pharmaceutically acceptable carriers.

7. The use of Eudistomin Y-class compounds and their medically acceptable salts as described in formula (I) or (II) of claim 1 in the preparation of combination antitumor drugs, wherein the combination antitumor drug is a lysosome-targeted pharmaceutically acceptable carrier prepared from Eudistomin Y-class compounds and their medically acceptable salts as described in formula (I) or (II) and used in combination with a clinical antitumor drug, wherein the clinical antitumor drug is a drug for treating breast cancer, colon cancer, cervical cancer, liver cancer, gastric cancer, or lung cancer.

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