No-s-s-aurb conjugates, preparation method therefor, and use thereof

Abstract

NO-S-S-AurB conjugates, a preparation method therefor, and the use of the NO-S-S-AurB conjugates in drugs having anti-TNBC activity and selectivity, belonging to the technical field of medicines. In particular, the NO-S-S-AurB conjugates are synthesized on the basis of self immolation strategy, and a benzenesulfonyl furazan nitric oxide NO donor is combined with AurB to prepare a twin drug, so as to synthesize a plurality of NO-S-S-AurB conjugates, structures of the NO-S-S-AurB conjugates being confirmed by means of spectrum characterization. Also provided is the preparation method for the NO-S-S-AurB conjugates, which involves a reasonable route, achieves high product yield, facilitates separation and achieves high purity. Since tumor tissue has a characteristic of high glutathione (GSH) expression, a GSH-sensitive disulfide bond linking chain in the series of NO-S-S-AurB conjugates can be self-immolated, thereby remarkably improving the anti-TNBC activity and selectivity of the parent drug AurB.

Description

A NO-SS-AurB coupling compound and its preparation method and application Technical Field

[0001] The present invention relates to a NO-SS-AurB coupler, a preparation method of the NO-SS-AurB coupler, and application of the NO-SS-AurB coupler in active and selective drugs against TNBC, belonging to the technical field of medicine. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women and the second leading cause of death among women worldwide. The results of the 2021 Cancer Annual Statistics Report released by the American Cancer Society indicate that the cancer mortality rate has dropped from a peak of 215.1 / 100,000 in 1991 to 149.0 / 100,000 in 2018. Although the cancer mortality rate has been declining year by year, the incidence of breast cancer has surpassed lung cancer to become the most common cancer in the world, and its incidence ranks first among female malignant tumors, greatly endangering women's life safety. Triple-negative breast cancer (TNBC) is a type of breast cancer that refers to breast cancer in which the immunohistochemical examination results of cancer tissue are negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene (Her-2), accounting for approximately 15% to 20% of all breast cancer types. Currently, there is limited research on the molecular mechanism of TNBC pathogenesis, and there is a lack of clear therapeutic targets. In addition, due to its high risk of recurrence, great potential for metastasis, and extremely poor prognosis, the 5-year survival rate of patients with advanced TNBC is extremely low. Conventional breast cancer treatments, such as hormone therapy and HER2-targeted therapies, have little efficacy for TNBC, leaving clinical treatment options very limited. Traditional chemotherapy remains the mainstay of treatment for TNBC. Currently, the primary first-line chemotherapy agents used in clinical practice include anthracyclines, taxanes, cyclophosphamides, and platinums. However, traditional chemotherapy has significant drawbacks, such as drug resistance and various toxic side effects. Currently, the only drug that has significantly prolonged overall survival in TNBC patients is Trodelvy, an ADC that targets the Trop-2 protein and is linked to the topoisomerase I inhibitor SN-38, which received accelerated approval from the FDA in 2020. Trodelvy is used to treat patients with advanced TNBC. While the launch of Trodelvy marks a significant advancement in TNBC treatment, the molecular mechanisms underlying TNBC are still underrepresented, and clinical treatment options remain limited. Therefore, there is an urgent need to identify potential targeted drugs for the treatment of TNBC.

[0003] Aurovertins are a class of polyene-pyrone derivatives isolated from the fermentation broth of the macrofungus Calcarisporium arbuscular. They are highly reduced polyketides consisting of a 2,6-dioxabicyclo-[3,2,1]-octane ring core linked to a methylated pyrone via a triene linker. Since the identification of aurovertins AD in 1964, 21 aurovertins have been discovered. Among them, AurB selectively inhibits the growth of TNBC cells and exhibits significant anti-metastatic effects against multiple TNBC cell lines in vitro at non-cytotoxic doses. Although aurovertins possess diverse biological activities, they are also highly toxic. Researchers injected these compounds into rabbits and dogs, resulting in death within 50 minutes at a dose of 1 mg / kg. The results indicate that these compounds are highly toxic, making them unsuitable for direct drug development and requiring structural optimization. However, current research on the structure-activity relationship of AurB is insufficient, and structural modifications lack specificity.

[0004] Nitric oxide (NO) is a crucial signaling molecule in the body, regulating numerous physiological functions and influencing tumor cell growth, proliferation, and metastasis, as well as angiogenesis and promoting apoptosis. NO-donating drugs have become a hot topic in anti-tumor drug research. Low concentrations of NO can promote tumor angiogenesis, while high concentrations can exert anti-tumor effects directly or indirectly. NO donors are compounds that release NO in vivo through enzymatic or non-enzymatic action. These compounds overcome the inherent drawbacks of NO, such as its difficulty in transport and quantification, and its short half-life. Existing NO donors can be categorized by chemical structure into organic nitrates, metal NO complexes, furazan N-oxides, and azodiol-enium salts. Furazan N-oxides offer more stable and sustained NO release than other donors, exhibiting stronger NO-releasing activity and capable of releasing NO in response to both free sulfhydryl groups and sulfhydryl anions. Classic nitrate NO donors require free sulfhydryl groups to release NO. Since free sulfhydryl groups are widely distributed in the body, furazan NO can release NO through non-enzymatic pathways in a variety of tissues or organs to produce biological activity.

[0005] Twin drugs are drugs created by covalently conjugating two or more compounds with similar pharmacological activities into a new drug molecule. These molecules release the parent drug through a series of metabolic reactions in the body. These twin drugs are designed to be compatible with the parent drug's properties, improve its existing physicochemical properties, enhance its selectivity and activity, or generate new pharmacological activities. Twin drug design is common in the treatment of neurodegenerative diseases, psychiatric disorders, and malignant tumors.

[0006] The present invention utilizes a cleavable disulfide bond as a connecting link to combine AurB and an NO donor to create a degradable twin drug. This coupled compound undergoes self-immolative elimination in vivo based on a redox response, releasing the original drug. Because tumors typically express high levels of GSH, this invention is expected to enhance the activity and selectivity of AurB against TNBC.

[0007] Summary of the Invention

[0008] To achieve the above objectives, the present invention provides a NO-SS-AurB coupler and a method for preparing the NO-SS-AurB coupler, as well as the use of the NO-SS-AurB coupler in preparing an active and selective drug against TNBC.

[0009] In a first aspect, the present invention provides a series of releasable NO-aurovertin B conjugates (6a-8g):

[0010] In a second aspect, the present invention provides a series of releasable NO-SS-AurB conjugates for use in preparing active and selective drugs against TNBC.

[0011] In a third aspect, the present invention provides a method for preparing a series of releasable NO-SS-AurB conjugates, the method comprising the following steps:

[0012] Step 1, synthesis of nitric oxide donors and their derivatives:

[0013] Steps (i) and (ii): Phenylthioacetic acid was added to a round-bottom flask, glacial acetic acid was added, and an ice bath was placed. 30% hydrogen peroxide was slowly added dropwise. After completion of the dropwise addition, the reaction was allowed to react at room temperature for 3-5 hours and monitored by TLC. After completion of the reaction, the reaction was placed in an ice bath and fuming nitric acid was slowly added dropwise. The reaction solution was then refluxed in an 80°C water bath for 3 hours. After completion of the reaction, the reaction solution was cooled to room temperature and placed in a -20°C explosion-proof refrigerator overnight. After a large amount of white needle-like crystals precipitated, the reaction solution was filtered and washed with a small amount of water. The solid was collected, dried, and weighed to obtain Product 2.

[0014] Step (iii): Product 2 was dissolved in tetrahydrofuran in a round-bottom flask. The reaction was allowed to proceed in an ice bath. NaOH solution was added and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC. Upon completion of the reaction, the product was extracted with water and ethyl acetate in small amounts several times and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:acetone, and concentrated under reduced pressure to obtain compounds 4a-4e.

[0015] Step (iv): Product 2 was placed in a round-bottom flask, and dichloromethane was added. The reaction was allowed to proceed in an ice bath. DBU was added and the reaction was allowed to proceed at room temperature for 3 h. The reaction was monitored by TLC. Upon completion, the product was extracted with water and ethyl acetate in small amounts several times, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography, eluting with dichloromethane:methanol, and concentrated under reduced pressure to obtain compound 4f-4g.

[0016] Furthermore, in step (iii), the concentration of the NaOH solution is 25%, which is 2.5 times the amount of product 2, and the reaction time at room temperature is 2.5-3.5 hours. The concentration of the sodium hydroxide solution in this reaction should not be too high, but should be appropriate, and the reaction time should not be too long, otherwise the phenylsulfonyl group at position 3 will also be replaced, forming a disubstituted byproduct.

[0017] Step 2 Synthesis of NO-SS-AurB conjugate:

[0018] Step (a): Dissolve 2,2-dithiodiacetic acid in dichloromethane, add DMAP and DCC, react at room temperature, then add AurB and react at room temperature. Monitor the reaction by TLC. After the reaction is terminated, dry the mixture, add ether, filter, and concentrate the filtrate under reduced pressure to obtain a crude product. The crude product is chromatographed on a crude silica gel column, eluted with petroleum ether:acetone, and concentrated under reduced pressure to obtain a yellow oil. Dissolve 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid) and DMAP in dichloromethane, react at room temperature, then add AurB and EDCI and react at room temperature. After the reaction is terminated, extract with water and dichloromethane, and concentrate under reduced pressure to obtain a crude product. The crude product is chromatographed on a crude silica gel column, eluted with petroleum ether:acetone, and concentrated under reduced pressure to obtain a yellow oil, 5X1, 5X2, 5X3.

[0019] Step (b): Dissolve 5X1, 5X2, 5X3, and DMAP in an appropriate amount of dichloromethane. After reaction at room temperature, add 4a-4e and EDCI. The reaction was continued at room temperature and monitored by TLC. Upon completion, the mixture was extracted with water and dichloromethane and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:acetone, and concentrated under reduced pressure to obtain a yellow oil, 6a-6e, 7a-7e, and 8a-8e.

[0020] Step (c): Dissolve 5X1, 5X2, 5X3, and HOBT in an appropriate amount of dichloromethane. After reaction at room temperature, add 4f-4g and EDCI. The reaction was continued at room temperature and monitored by TLC. Upon completion, the mixture was extracted with water and dichloromethane and concentrated under reduced pressure to obtain a crude product. The crude product was analyzed by chromatography using a gradient elution method and concentrated under reduced pressure to obtain white solids (6f-6g, 7f-7g, and 8f-8g).

[0021] Furthermore, in step (a), the ratio of the 2,2-dithiodiacetic acid, DMAP, and DCC catalyst is 1.1:0.5:2. During the reaction of 2,2-dithiodiacetic acid and AurB, the amount of DMAP catalyst should be carefully considered. DMAP is highly alkaline, and excessive addition can lead to disulfide bond cleavage and the formation of a byproduct of the coupling of thioglycolic acid and AurB. EDCI cannot be used as a condensing agent; the reaction does not occur when EDCI is used, and the reaction solution changes from a clear yellow liquid to a turbid brown liquid.

[0022] Furthermore, in step (a), the ratio of the amounts of 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid), DMAP, AurB, and EDCI is 1.1:0.5:1:1. During the reaction of 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid) and AurB, attention should be paid to the amounts of DMAP and EDCI, and the reaction time should not be too long, as disulfide bonds are easily broken in an alkaline environment, generating related by-products.

[0023] Furthermore, in step (b), during the separation and purification process, the Rf value must be reduced to below 0.1. During the separation and purification process, the Rf value must be reduced to below 0.1 to separate more pure compounds.

[0024] Furthermore, in step (c), the crude product was subjected to RP-18 reverse-phase column chromatography with a gradient elution of methanol:water = 40:60 → 100:0. Step (c) requires the use of HOBT and DMAP in combination, which results in a higher reaction yield but makes separation more difficult. Compounds in the f and g series are not suitable for silica gel column chromatography due to their high adsorption and low yield. Using reverse-phase column chromatography to separate the product is less prone to adsorption, resulting in higher separation yield and purity.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention utilizes the high expression of glutathione (GSH) in tumor tissue, introduces a GSH-sensitive disulfide self-eliminating linker, synthesizes a NO-AurB coupler based on the self-elimination strategy, and combines a phenylsulfonylfuran nitrogen oxide-type NO donor with AurB to prepare a twin drug. A total of 20 derivatives in three series were synthesized, and their compound structures were confirmed by spectral characterization. The connection between AurB and the NO donor in the synthesized NO-AurB coupler is a disulfide bond. Such a structure can be cleaved by tumor cells that highly express glutathionease, thereby releasing the two parts of the drug in the tumor tissue in a targeted manner.

[0027] The present invention provides a preparation method for synthesizing a NO-AurB coupler based on a self-elimination strategy, which has a reasonable route, high product yield, is easy to separate, and has high purity.

[0028] The NO-AurB couplers synthesized in the present invention have a significant inhibitory effect on triple-negative breast cancer MDA-MB-231 cells, and the inhibitory effect is significantly stronger than that of the positive drug paclitaxel and the mother drug AurB. It also shows a good inhibitory effect on mouse triple-negative breast cancer 4T1 cells, indicating that this series of NO-AurB couplers significantly improves the activity and selectivity of the mother drug AurB against TNBC. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the evaluation of the coupling agent on the survival rate of MDA-MB-231 cells

[0030] Figure 2 is the evaluation of the coupling agent on the survival rate of 4T1 cells

[0031] Figure 3 is the evaluation of the viability of A549 cells by the coupling agent

[0032] Figure 4 shows the inhibition rate of MDA-MB-231 and 4T1 cells at each concentration of 6g, 7g, 8g, 2* DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration of the present invention and are not intended to limit the scope of the present invention.

[0034] Experimental materials and equipment

[0035] Experimental instruments

[0036] The main experimental instruments and equipment required for the experiment are shown in Table 1.

[0037] Table 1 Experimental instruments and equipment

[0038] Experimental reagents

[0039] The main chemical reagents required for the experiment are shown in Table 2.

[0040] Table 2 Experimental reagents

[0041] Note: AurB is derived from the secondary metabolite of the fruiting body of the macrofungus Calcarisporium arbuscula.

[0042] Explanation of symbols ATPase—adenosine triphosphatase; ADC—antibody drug conjugate; AurB—Aurovertin B DMSO—dimethyl sulfoxide; DMF—N,N-dimethylformamide; DMAP—4-dimethylaminopyridine; DCC—dicyclohexylcarbodiimide; ER—estrogen receptor; EDCI—carbodiimide; GSH——glutathione; H2O2——hydrogen peroxide; IC 50 ———half maximal inhibitory concentration; MTT—methyl thiazolyl tetrazolium; NK—natural killer cell; NO donor—nitric oxide donor; NaH—sodium hydride; NaOH—sodium hydroxide; NMR—nuclear magnetic resonance; PR—progesterone receptor; PMSF—phenylmethanesulfonyl fluoride; TNBC—triple-negative breast cancer;

[0043] Example 1 Synthesis of Nitric Oxide Donors and Their Derivatives

[0044] The synthetic routes of nitric oxide donors and their derivatives are as follows:

[0045] Steps (i) and (ii): Phenylthioacetic acid was added to a round-bottom flask, glacial acetic acid was added, and an ice bath was placed. 30% hydrogen peroxide was slowly added dropwise. After completion of the dropwise addition, the reaction was allowed to react at room temperature for 3-5 hours and monitored by TLC. After completion of the reaction, the reaction was placed in an ice bath and fuming nitric acid was slowly added dropwise. The reaction solution was then refluxed in an 80°C water bath for 3 hours. After completion of the reaction, the reaction solution was cooled to room temperature and placed in a -20°C explosion-proof refrigerator overnight. After a large amount of white needle-like crystals precipitated, the reaction solution was filtered and washed with a small amount of water. The solid was collected, dried, and weighed to obtain Product 2.

[0046] Step (iii): Dissolve 1 eq. of NO donor in 5 mL of tetrahydrofuran in a round-bottom flask. The mixture was reacted for 5 min in an ice bath. Then, 2.5 eq. of 25% NaOH solution and 5 eq. of 3a-3e were added. The reaction was allowed to react at room temperature for 3 h. The reaction was monitored by TLC (using petroleum ether:acetone in a ratio of 3:1 as the developing solvent; the product spot Rf value was approximately 0.2). Upon completion of the reaction, the mixture was extracted with water and ethyl acetate in small amounts several times, and concentrated under reduced pressure to obtain the crude product. The crude product was chromatographed on a 200-300 mesh silica gel column, eluting with petroleum ether:acetone in a ratio of 3:1. The mixture was concentrated under reduced pressure to obtain compounds 4a-4e as white powdery solids. The yield of 4a-4e, calculated based on the amount of NO donor charged, ranged from 50% to 95%.

[0047] Step (iv): 1 eq. of NO donor was placed in a round-bottom flask, 5 mL of dichloromethane was added, and the mixture was reacted in an ice bath for 5 min. 3 eq. of DBU and 5 eq. of 3f-3g were added, and the mixture was reacted at room temperature for 3 h. The reaction was monitored by TLC (using dichloromethane:methanol = 10:1 as the developing solvent, and the product spot R f The reaction was terminated with extraction with water and ethyl acetate in small amounts several times, followed by concentration under reduced pressure to obtain a crude product. The crude product was chromatographed on a 200-300 mesh silica gel column, eluting with dichloromethane:methanol = 10:1. The product was concentrated under reduced pressure to afford compound 4f-4g as a pale yellow oil. The yield, calculated based on the amount of NO donor added, was 93%-95% for 4f-4g.

[0048] Example 2: Synthesis of NO-SS-AurB Coupled

[0049] The synthetic route of NO-SS-AurB conjugate is as follows:

[0050] Step (a): Dissolve 1.1 eq. of 2,2-dithiodiacetic acid in dichloromethane, add 0.5 eq. of DMAP and 2 eq. of DCC, and react at room temperature for 2 hours. Then, add 1 eq. of AurB and continue to react at room temperature for 24 hours. Monitor the reaction by TLC. Upon completion, the dichloromethane is suspended to dryness, filtered three times with ether, and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product is chromatographed on a 200-300 mesh coarse silica gel column using a 3:2 ratio of petroleum ether to acetone. Concentrate under reduced pressure to obtain a yellow oil. Dissolve 1.1 eq. of 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid) and 0.5 eq. of DMAP in dichloromethane, react at room temperature for 20 minutes, then add 1 eq. of AurB and 1 eq. of EDCI and continue to react at room temperature for 24 hours. Upon completion, extract the product with small amounts of water and dichloromethane several times. The dichloromethane layer is concentrated under reduced pressure to obtain the crude product. The crude product was chromatographed on a 200-300 mesh silica gel column with petroleum ether:acetone = 3:2 as eluent and concentrated under reduced pressure to give a yellow oil. The yields, calculated based on the amount of AurB added, were 60% for compound 5X1, 85% for 5X2, and 88% for 5X3.

[0051] Step (b): Dissolve 1 eq. 5X1, 5X2, 5X3, and 0.5 eq. of DMAP in an appropriate amount of dichloromethane. React at room temperature for 20 minutes. Then, add 1 eq. of 4a4e and 1 eq. of EDCI. Continue reacting at room temperature for 24 hours. Monitor the reaction by TLC. Upon completion, extract the mixture with water and dichloromethane in small amounts several times. The dichloromethane layer is concentrated under reduced pressure to obtain the crude product. The crude product is chromatographed on a 200-300 mesh silica gel column using a 3:1 ratio of petroleum ether to acetone. Concentrate under reduced pressure to obtain a yellow oil. The yields, calculated based on the amount of AurB added, are 60%-88% for compounds 6a-6e, 65%-90% for 7a-7e, and 70%-90% for 8a-8e.

[0052] Step (c): Dissolve 1 eq. 5X1, 5X2, 5X3, and 1 eq. of HOBT in an appropriate amount of dichloromethane. React at room temperature for 20 minutes. Then, add 1 eq. 4f-4g and 1 eq. of EDCI. React at room temperature for 24 hours. Monitor the reaction by TLC (using petroleum ether:acetone = 3:2 as the developing solvent; the product spot Rf value is approximately 0.2). Upon completion, extract the mixture with water and dichloromethane in small amounts several times. The dichloromethane layer is concentrated under reduced pressure to obtain the crude product. The crude product is purified by reverse-phase column chromatography on an RP-18 column using a gradient elution ratio of methanol:water = 40:60 → 100:0. Concentrate under reduced pressure to obtain a white solid. The yields, calculated based on the amount of AurB added, are 79%-83% for compounds 6f-6g, 85%-87% for 7f-7g, and 82%-88% for 8f-8g.

[0053] Spectral characterization of some compounds confirmed their structures as follows

[0054] Table 3 NMR spectra and mass spectrometry data of some compounds

[0055] Example 3: Study on the activity of AurB-NO conjugates on MDA-MB-231 cells, 4T1 cells and A549 cells

[0056] 1. Experimental instruments

[0057] The main experimental instruments required for this experiment are shown in Table 4.

[0058] Table 4 Experimental instruments and equipment

[0059] 2. Experimental Reagents

[0060] The main chemical reagents required for this experiment are shown in Table 5.

[0061] Table 5 Experimental materials and reagents

[0062] Preparation of main reagents

[0063] DMEM complete medium: Add 50 mL of FBS and 5 mL of penicillin-streptomycin solution to 445 mL of DMEM, shake well, and store at 4°C until ready to use.

[0064] RPMI-1640 complete medium: Add 50 mL of FBS and 5 mL of penicillin-streptomycin solution to 445 mL of RPMI-1640, shake well, and incubate at 4°C.

[0065] 10% ammonium persulfate (APS) solution: Weigh 1 g of APS into a centrifuge tube, add 10 mL of ultrapure water, and vortex to dissolve.

[0066] 10% separating gel: Add 5.9 mL of ultrapure water, 5 mL of Acrylamide Bis, 3.8 mL of 1.5 M Tris-HCl (pH 8.8), 0.15 mL of 10% SDS, 0.15 mL of 10% AP, and 0.015 mL of TEMED to a beaker and stir to mix.

[0067] 5% stacking gel: Add 4.2 mL of ultrapure water, 1 mL of Acrylamide Bis, 0.76 mL of 0.5 M Tris-HCl (pH 6.8), 0.06 mL of 10% SDS, 0.06 mL of 10% AP, and 0.006 mL of TEMED to a beaker and stir to mix.

[0068] Electrophoresis fluid: Measure 100 mL of electrophoresis fluid (10×) and 900 mL of ultrapure water into a beaker and stir evenly.

[0069] Electrotransfer solution: Weigh 2.42 g Tris-base and 11.5 g glycine into a beaker, add 800 mL of distilled water, stir to dissolve, then add 200 mL of methanol, mix well, and use for electrotransfer. Prepare and use immediately.

[0070] TBS buffer (10×): Weigh 24.2 g Tris-base and 80 g NaCl in a beaker, add 1000 mL distilled water, stir to dissolve, and adjust the pH to 7.6 with HCl.

[0071] TBST buffer: Add 100 mL of TBS buffer (10×) and 1 mL of Tween-20 to 900 mL of ultrapure water and stir to mix.

[0072] 5% blocking solution: Weigh 5 g of skim milk powder into a beaker, add 100 mL of TBST buffer, stir to dissolve, recycle and reuse, and store at -20°C.

[0073] Compound stock solution: Weigh appropriate amount of compound into EP tube, add corresponding volume of DMSO solution, vortex to dissolve, prepare 20mM compound stock solution, and store at -20℃.

[0074] MTT solution: Weigh 1 g of MTT powder into a beaker, add 200 mL of PBS, sonicate in the dark to dissolve, and store in the dark at 4°C.

[0075] 3. Experimental Methods

[0076] 3.1 Cell culture conditions

[0077] MDA-MB-231 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Culture conditions: DMEM complete medium (containing 10% FBS and 1% PS), 5% CO2, and cultured at 37°C.

[0078] 4T1 cells: from ATCC (American Type Culture Collection). Culture conditions: RPMI-1640 complete medium (containing 10% FBS and 1% PS), 5% CO2, constant temperature culture at 37°C.

[0079] A549 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Culture conditions: RPMI-1640 complete medium (containing 10% FBS and 1% PS), 5% CO2, and cultured at 37°C.

[0080] 3.2 Cell recovery, passaging, and cryopreservation

[0081] (1) Cell recovery

[0082] Remove the cryovial of the cell line from liquid nitrogen and place it on a float in a 37°C water bath for rapid thawing. Transfer the thawed cell suspension into a pre-prepared 15mL centrifuge tube (containing 5mL of culture medium). Centrifuge at 1000rpm for 10 minutes. Discard the supernatant and resuspend the cells in 1mL of fresh culture medium. Transfer the cells to a culture flask and incubate in a 37°C, 5% CO2 incubator. After 24 hours, observe cell growth and replace the culture medium. Cells can be passaged when they reach 80% confluency.

[0083] (2) Cell passage

[0084] Remove the culture flask from the incubator, aspirate and discard the old culture medium, and rinse twice with 2 mL of PBS. Add 1 mL of trypsin solution, shake well, and incubate in a 37°C incubator for 1-3 minutes. Observe cell growth and terminate digestion by adding culture medium. Mix thoroughly by pipetting, then transfer to a suitable centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend in fresh culture medium, and transfer an appropriate amount of the suspension to a culture flask containing an appropriate amount of complete culture medium. Transfer to a constant temperature incubator and continue culturing.

[0085] (3) Cell cryopreservation

[0086] When the cell confluency reaches 80%, cryopreserve the cells. Aspirate the old culture medium, wash three times with PBS, and add 1 mL of trypsin solution for digestion. Once the cells have rounded, add fresh culture medium to terminate digestion. After mixing, transfer the suspension to a pre-prepared 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL of cell freezing solution, and resuspend by pipetting. Transfer the cell suspension to a pre-prepared cryovial that has been cooled to room temperature. Tighten the cryovial cap and place the cryovial in a -80°C freezer. After 24 hours, transfer the cells to a liquid nitrogen tank.

[0087] 3.3 Cell proliferation assay

[0088] The anti-proliferative activity of a series of conjugates was determined using the MTT assay. The mechanism of action is that exogenous MTT is reduced to water-insoluble blue-purple crystals that precipitate in living cells, whereas it does not precipitate in dead cells. Therefore, the amount of precipitated crystals can be used to assess cytotoxicity. Formazan in living cells is soluble in DMSO, and its absorbance is measured using a microplate reader to calculate the number of viable cells.

[0089] (1) Cell counting and plating

[0090] When the cell confluency reaches 80%, remove the culture flask from the incubator, discard the old culture medium, rinse twice with 2 mL of PBS, add 1 mL of trypsin solution, shake well, and incubate in a 37°C incubator for 1-3 minutes. After observing the cells become rounded under a microscope, add an appropriate amount of culture medium to terminate the digestion. After pipetting and mixing, transfer to a suitable centrifuge tube and centrifuge for 5 minutes (1000 rpm). Discard the supernatant and resuspend in fresh culture medium. Aspirate an appropriate amount of the cell suspension, transfer to a hemocytometer, and count.

[0091] Use a pipette to transfer an appropriate amount of equal cell suspension to a 96-well plate, add PBS to the outermost background plate of the well plate, and after plating, gently tap the side wall of the 96-well plate to evenly distribute the cells in the well plate, and observe the uniformity under a microscope, then place it in a 37°C constant temperature incubator for 24 hours.

[0092] (2) Dosing

[0093] For initial compound screening, dilute the sample with the appropriate culture medium based on the calculated sample results. Add 10 μL of the diluted sample to each well of a 96-well plate, achieving a final concentration of 10 μM. Each sample should be plated in triplicate. Add 10 μL of fresh culture medium to the blank control group. After sample addition, gently tap the 96-well plate and incubate at 37°C for 48 hours.

[0094] Determination of IC 50 Drug Addition: Dilute the sample using the calculated two-fold dilution method with the corresponding culture medium. Add 10 μL of the diluted sample to each well of the corresponding 96-well plate, so that the final concentration of the drug in each well is 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM. In triplicate wells, add 10 μL of fresh culture medium without drug to the control group. After sample addition, gently tap the 96-well plate and incubate at 37°C for 48 hours.

[0095] (3) OD value detection

[0096] After 48 hours of treatment, add 20 μL of MTT solution (5 mg / mL) to each well and continue incubation at 37°C for another 3 hours. Terminate the culture, gently remove the culture medium with a pipette, add 100 μL of DMSO to each well, gently shake on a shaker to completely dissolve the crystals, and measure the absorbance at 490 nm using a microplate reader. Calculate the relative cell viability using the formula: Relative viability (%) = OD 加药组平均值 / OD 对照组平均值 ×100%

[0097] 4. Results and Discussion

[0098] Activity detection results of the coupled compound NO-SS-AurB

[0099] As shown in Figures 1-3, cell viability was measured 48 hours after administration of the conjugates NO-SS-AurB (6a-8g) at a 10 μM concentration. The conjugates exhibited significant inhibitory activity against MDA-MB-231 cells, significantly more potent than the positive-acting drug paclitaxel and the parent drug AurB. They also exhibited significant inhibitory activity against 4T1 cells, but showed no significant activity against lung cancer A549 cells. These results demonstrate that the conjugates exhibited selectivity against triple-negative breast cancer cells, selectively inhibiting their viability while lacking significant inhibitory activity against lung cancer cells. In contrast, the NO donor exhibited strong inhibitory activity against all three cell lines, demonstrating a lack of selectivity. In summary, the activity of the synthesized compounds is consistent with the original design, enhancing the anti-TNBC cell activity and selectivity of the parent drug AurB and significantly increasing the selectivity of the NO donor for TNBC cells. Compounds 6a-8g were observed for their inhibitory activity against triple-negative breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells.

[0100] IC50 determination results of the coupled compound NO-SS-AurB

[0101] Compounds 6b, 6g, 7b, 7d, 7f, 7g, 8b, 8d, 8f, 8g and 2 (NO donor) were selected to determine IC50 values ​​against MDA-MB-231 and 4T1 cells. The experimental results are shown in Table 6 and Figure 4.

[0102] Table 6 Inhibitory effect of each coupling compound on MDA-MB-231 and 4T1 cells

[0103] Note: ND=Not Determined; NA=Not available; 2*=control group (NO donor)

[0104] Among all compounds, 6g, 7g, 8g, and 2 demonstrated superior activity and stability, with IC50 values ​​of 4.32μM, 1.41μM, 2.42μM, and 1.32μM, respectively, against MDA-MB-231. However, their inhibitory activity against 4T1 was less pronounced than that of compound 7g. Compound 7g exhibited an IC50 value similar to that of control 2 against MDA-MB-231 cells, but exhibited significantly improved selectivity compared to control 2. These results demonstrate that these compounds exhibit selective inhibitory effects against TNBC cells, while control 2 exhibited strong inhibitory activity against both cell lines, indicating that the conjugate significantly enhanced the selectivity of control 2.

[0105] Example 3 selected three tumor cell lines, MDA-MB-231 cells, 4T1 cells and A549 cells, and performed preliminary activity screening tests on the 20 synthesized NO-SS-AurB conjugates. Preliminary experimental results showed that the synthesized conjugates could significantly inhibit the proliferation of MDA-MB-231 cells at the same concentration, and the inhibitory effect was significantly stronger than that of the positive drug paclitaxel and the mother drug AurB. It also showed a good inhibitory effect on 4T1 cells, but had no obvious cell activity on lung cancer A549 cells. Further IC50 results showed that among all compounds, 6g, 7g, 8g and NO donor had better activity and stability, with IC50 of 4.32μM, 1.41μM, 2.42μM and 1.32μM, respectively. Among them, compound 7g showed a strong inhibitory effect and selectivity on MDA-MB-231 cells, IC 50 =1.41M, which improved the activity and selectivity of the parent drug AurB against TNBC cells and significantly increased the selectivity of the NO donor for TNBC cells. In summary, the experimental results show that this series of conjugates based on self-elimination synthesis significantly improved the activity and selectivity of AurB against triple-negative breast cancer cells.

Claims

1. A releasable NO-aurovertin B conjugate having the following general structural formula:

2. The coupling object according to claim 1, wherein: The structural formula is:

3. The coupling object according to claim 2, wherein: The structural formula is:

4. A method for preparing the NO-aurovertin B conjugate according to claim 1, characterized in that: The method comprises the following steps: Synthesis of nitric oxide donors and their derivatives: Steps (i) and (ii): Phenylthioacetic acid was added to a round-bottom flask, glacial acetic acid was added, and 30% hydrogen peroxide was slowly added dropwise. After completion of the dropwise addition, the reaction was allowed to react at room temperature and monitored by TLC. After completion of the reaction, the reaction was allowed to cool to room temperature and then slowly added dropwise with fuming nitric acid. The reaction solution was then refluxed in an 80°C water bath. After completion of the reaction, the reaction solution was cooled to room temperature and placed in an explosion-proof refrigerator overnight. After a large amount of white needle-like crystals precipitated, the solution was filtered and washed with a small amount of water. The solid was collected, dried, and weighed to obtain Product 2. Step (iii): Product 2 was placed in a round-bottom flask and dissolved in tetrahydrofuran. The mixture was reacted in an ice bath. NaOH solution was added and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC. Upon completion of the reaction, the mixture was extracted with water and ethyl acetate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:acetone, and concentrated under reduced pressure to obtain compounds 4a-4e. Step (iv): Product 2 was placed in a round-bottom flask, dichloromethane was added, and the reaction was allowed to proceed in an ice bath. DBU was added and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC. Upon completion of the reaction, the product was extracted with water and ethyl acetate in small amounts several times, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography, eluted with dichloromethane:methanol, and concentrated under reduced pressure to obtain compound 4f-4g. Synthesis of NO-SS-AurB conjugates: Step (a): Dissolve 2,2-dithiodiacetic acid in dichloromethane, add DMAP and DCC, react at room temperature, add AurB, and react at room temperature. Monitor the reaction by TLC, wait for the reaction to terminate, hang to dryness, add ether for filtration, take the filtrate and concentrate under reduced pressure to obtain a crude product, the crude product is chromatographed on a crude silica gel column, eluted, and concentrated under reduced pressure to obtain a yellow oil. Take 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid) and DMAP and dissolve them in dichloromethane, react at room temperature, add AurB and EDCI, and react at room temperature. After the reaction is terminated, extract with water and dichloromethane, and concentrate under reduced pressure to obtain a crude product. The crude product is chromatographed on a crude silica gel column, eluted, and concentrated under reduced pressure to obtain a yellow oil, 5X1, 5X2, 5X3; Step (b): Dissolve 5X1, 5X2, 5X3, and DMAP in an appropriate amount of dichloromethane. After reaction at room temperature, add 4a-4e and EDCI. The reaction was continued at room temperature and monitored by TLC. After termination, the crude product was extracted and concentrated under reduced pressure to obtain the crude product. The crude product was chromatographed on a crude silica gel column and eluted. The product was concentrated under reduced pressure to obtain a yellow oil, 6a-6e, 7a-7e, and 8a-8e. Step (c): Dissolve 5X1, 5X2, 5X3, and HOBT in an appropriate amount of dichloromethane. After reaction at room temperature, add 4f-4g and EDCI. The reaction was continued at room temperature and monitored by TLC. Upon completion, the crude product was extracted and concentrated under reduced pressure to obtain the crude product. The crude product was analyzed by chromatography using a gradient elution method and concentrated under reduced pressure to obtain white solids (6f-6g, 7f-7g, and 8f-8g).

5. The method for preparing the NO-aurovertin B conjugate according to claim 4, wherein: In the step (iii), the concentration of the NaOH solution is 25%, the amount used is 2.5 times that of the product 2, and the reaction time at room temperature is 2.5-3.5 hours.

6. The method for preparing the NO-aurovertin B conjugate according to claim 4, wherein: In the step (a), the ratio of the amounts of 2,2-dithiodiacetic acid, DMAP, and DCC catalyst is 1.1:0.5:

2.

7. The method for preparing the NO-aurovertin B conjugate according to claim 4, wherein: In the step (a), the ratio of the amounts of 3,3-dithiodipropionic acid (4,4-dithiodibutyric acid), DMAP, AurB, and EDCI is 1.1:0.5:1:

1.

8. The method for preparing the NO-aurovertin B conjugate according to claim 4, wherein: In the step (b), during the separation and purification process, the Rf value is reduced to below 0.

1.

9. The method for preparing the NO-aurovertin B conjugate according to claim 4, wherein: In the step (c), the crude product is chromatographed on an RP-18 reverse phase column using a gradient elution of methanol: water = 40:60→100:

0.

10. Use of the NO-aurovertin B conjugate according to claim 1 in the preparation of active and selective drugs against TNBC.

Images