CN116410152B - Small molecular compound for inhibiting ASCT2, derivative thereof and application thereof - Google Patents

Abstract

A small molecule compound for inhibiting ASCT2, the molecular formula of the compound is C ₃₅ H ₃₉ N ₃ O ₃ The structural formula is as follows:compound C in the present invention ₃₅ H ₃₉ N ₃ O ₃ The derivative has an inhibiting effect on glutamine uptake, can obviously inhibit the glutamine uptake by cancer cells, can obviously inhibit the expression of ASCT2 protein, induces the oxidative stress and autophagy of breast cancer cells, has a good anti-tumor effect, and particularly has an excellent effect on inhibiting cell proliferation of the breast cancer cells.

Description

Small molecular compound for inhibiting ASCT2, derivative thereof and application thereof

Technical Field

The invention relates to the technical field of ASCT2 targeted inhibitors, in particular to a small molecular compound for inhibiting ASCT2, a derivative thereof and application thereof.

Background

Na ⁺ The dependent glutamine vector 2 (alanine-ser-ine-cysteine transporter, ASCT 2) is Na encoded by the SLC1A5 gene ⁺ The dependent transporter, which is a neutral amino acid transporter, glutamine is required to perform its biological function, first to enter the cell via ASCT 2. ASCT2 is mainly distributed on cell membranes, mediates the transportation of neutral amino acids such as glutamine, alanine, serine, cysteine and the like, and provides an important substrate for rapidly growing tumor cells. The increase in glutamine decomposition in cancer cells is related to the increase in expression of membrane transporter protein mediating glutamine cell uptake, and compared with normal tissue, ASCT2 is highly expressed in tumor tissue such as non-small cell lung cancer, breast cancer and liver cancer, and the demand of cancer cells for glutamine is greatly increased to support metabolic demand, and ATP and macromolecular substances such as synthetic proteins, nucleic acids and the like are produced. Inhibition of ASCT2 protein expression, prevention of intracellular glutamine uptake is a new tumor treatment modality and has been demonstrated to be effective on melanoma tumor cells, prostate cancer, breast cancer and acute myelogenous leukemia. At present, the research on ASCT2 functions is focused on the aspect of tumor proliferation, and the research discovers that inhibiting ASCT2 in prostate cancer, breast cancer cells and melanoma can reduce glutamine uptake, activate mTorrC 1 channels, inhibit cell growth and cell cycle processes, and therefore inhibit proliferation of tumor cells.

Because ASCT2 can be expressed abnormally or excessively in tumor tissues such as non-small cell lung cancer, breast cancer, liver cancer and the like. Meanwhile, the literature reports that ASCT2 is closely related to the survival time of clinical patients, so that the ASCT2 is considered to be a potential anti-tumor metabolic target. The first ASCT2 inhibitor developed was GPNA, a structural analogue of glutamine, which inhibits sodium-dependent amino acid transporters including ASCT 2. Because of the high levels of drug concentration required to inhibit ASCT2 transport function, they are currently only used as tool drugs in the field of tumor-based research. V-9302 is the first powerful small molecule inhibitor aiming at the glutamine transporter ASCT2, inhibits ASCT2 transport function mainly by combining with an ASCT2 allosteric region, finally generates good anti-tumor proliferation effect, and V-9302 can obviously inhibit proliferation of colon cancer cells, increase oxidative stress injury and promote death of tumor cells. This study opens the way to the development of inhibitors targeting ASCT2, targeted therapies for glutamine metabolism. However, it has been experimentally confirmed that V-9302 is not an ASCT2 specific inhibitor, and its target is not a single ASCT2 transporter, but acts on other transporters LAT1, etc.

Disclosure of Invention

The invention aims to provide a small molecular compound and a derivative thereof for targeted inhibition of ASCT2, which can inhibit the glutamine uptake capacity of ASCT2, can induce autophagy and oxidative stress of tumor cells after intracellular glutamine uptake is reduced, inhibit the growth of the tumor cells and finally obviously inhibit the proliferation of tumors.

Another object of the present invention is to provide the use of the small molecule compounds and derivatives thereof described above for targeted inhibition of ASCT 2.

The invention aims at realizing the following technical scheme:

a small molecule compound that inhibits ASCT2, characterized by: the molecular formula of the compound is C ₃₅ H ₃₉ N ₃ O ₃ The structural formula is as follows:

further, the small molecule compound for inhibiting ASCT2 has various derivatives with molecular formula of C ₃₄ H ₃₉ N ₄ O ₃ 、C ₃₃ H ₃₉ N ₅ O ₃ 、C ₃₂ H ₃₉ N ₆ O ₃ 。

Further, the derivative has a formula of C ₃₄ H ₃₉ N ₄ O ₃ When the structural formula comprises:

any one of them.

Further, the derivative has a formula of C ₃₃ H ₃₉ N ₅ O ₃ When the structural formula comprises:

any one of them.

Further, the derivative has a formula of C ₃₂ H ₃₉ N ₆ O ₃ When the structural formula comprises:

any one of them.

The compound and the derivative thereof have good glutamine uptake inhibition effect, particularly have tumor growth inhibition effect on breast cancer cells, and further research shows that the compound has effects of promoting oxidative stress level and inducing autophagy on the breast cancer cells under different concentrations, thereby achieving the effect of inhibiting proliferation of the cancer cells.

The application of the compound and the derivative thereof is characterized in that: the application in preparing antitumor drugs.

Preferably, the tumor is breast cancer.

The invention has the following technical effects:

compound C in the present invention ₃₅ H ₃₉ N ₃ O ₃ The derivative has an inhibiting effect on glutamine uptake, can obviously inhibit the glutamine uptake by cancer cells, can obviously inhibit the expression of ASCT2 protein, induces the oxidative stress and autophagy of breast cancer cells, has a good anti-tumor effect, and particularly has an excellent effect on inhibiting cell proliferation of the breast cancer cells.

Drawings

Fig. 1: nuclear magnetic hydrogen spectrogram of the compound ZR-001 in the invention.

Fig. 2: liquid chromatogram of compound ZR-001 in the invention.

Fig. 3: nuclear magnetic hydrogen spectrogram of the derivative Y1 in the invention.

Fig. 4: liquid chromatogram of derivative Y1 in the present invention.

Fig. 5: nuclear magnetic hydrogen spectrogram of the derivative Y4 in the invention.

Fig. 6: liquid chromatogram of derivative Y4 in the present invention.

Fig. 7: nuclear magnetic hydrogen spectrogram of the derivative Y6 in the invention.

Fig. 8: liquid chromatogram of derivative Y6 in the present invention.

Fig. 9: the compound ZR-001 and the derivatives thereof are combined with ASCT2 to inhibit ASCT2 transport function.

Fig. 10: the effect of the compound ZR-001 on ASCT2 genes and proteins in the invention.

Fig. 11: the compound ZR-001 can inhibit proliferation of breast cancer cells in vitro.

Fig. 12: the ZR-001 derivative can inhibit proliferation of breast cancer cells in vitro.

Fig. 13: the compound ZR-001 induces oxidative stress of breast cancer cells.

Fig. 14: the compound ZR-001 induces autophagy of breast cancer cells.

Fig. 15: the compound ZR-001 can inhibit proliferation of breast tumor in vivo.

Detailed Description

The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will be to those skilled in the art in light of the foregoing disclosure.

Example 1:

compound C ₃₅ H ₃₉ N ₃ O ₃ Is synthesized by the following steps:

to a solution of adamantane-1-carboxylic acid (300 mg,1.664 mmol) in DCM (3 mL) was added oxalyl chloride (0.211 mL,2.496 mmol) and N, N-dimethylformamide (0.013 mL,0.166 mmol), the resulting mixture was stirred at room temperature for 3h, the reaction mixture was concentrated under reduced pressure to give adamantane-1-carbonyl chloride (330 mg, yield 99.79%) as a yellow solid, 2- (4-aminophenyl) -1, 3-benzoxazol-5-amine (4 g,17.758 mmol) and triethylamine (7.405 mL,53.274 mmol) were added to THF (200 mL), the resulting solution was added to a solution of adamantane-1-carbonyl chloride (8.82 g, 44.3995 mmol) in dichloromethane (100 mL) at room temperature, the resulting mixture was stirred at room temperature under nitrogen protection for 12h, and the mixture was then filtered to give a yellow solid. For yellow solid (MeOH: H) ₂ O=1:1) was slurried 3 times, filtered, and the solid dried in vacuo to give N- {2- [4- (adamantan-1-amido) phenyl as a white solid]-1, 3-Benzoxazol-5-yl } adamantane-1-carboxamide (8.63 g,14.933mmol, yield 84.09%). HPLC purity: 95.1%. ESI-MS: m/z=550.1 [ m+1 ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d ₆ )δ9.45(s,1H),9.24(s,1H),8.12-8.10(m,3H),7.94-7.92(m,2H),7.66-7.60(m,2H),2.04-2.02(m,6H),1.97-1.93(m,12H),3.69-3.63(m,3H),1.73-1.71(m,12H)。

The chemical structure is as follows:

designated ZR-001. The hydrogen spectrogram and the liquid chromatogram are shown in fig. 1 and 2.

Example 2

Preparation of derivative Y1:

step (1): 5-nitro-2-pyridinecarboxylic acid (3 g,17.8 mmol) was dissolved in thionyl chloride (25 mL) and DMF (0.028 mL, 0.356 mmol) was added dropwise with stirring. Heating to 80 ℃, refluxing and stirring for 8 hours. After completion, the mixture was concentrated in vacuo to give the yellow target compound 5-nitro-2-pyridinecarboxylic acid:

step (2): 2-amino-4-bromophenol (2 g,10.6 mmol) was dissolved by adding DMF (20 mL), TEA (7.39 mL,53.2 mmol) was added, 5-nitro-2-pyridinecarboxylic acid (2.18 g,11.7 mmol) was added in portions, and stirred at 20℃for 3h. The mixture was then poured into 200mL of water, acidified to ph=5 with HOAc, stirred and filtered. The filter cake was dried in vacuo to give a yellow powder N- (5-bromo-2-hydroxyphenyl) -5-nitropyridine-2-carboxamide:

(3) To N- (5-bromo-2-hydroxyphenyl) -5-nitropyridine-2-carboxamide (3.5 g,10.4 mmol) and B ₂ Pin ₂ To a solution of (9.20 g,36.2 mmol) in IPA (40 mL) was added tBuOK (2.67 g,23.8 mol), then the reaction was stirred at 90 ℃ for 5h, the mixture was acidified to ph=5.5 with HOAc, diluted with water (80 mL) and extracted with EA (2 x 100 mL). The organic layer was washed with brine (150 mL) and concentrated in vacuo. The concentrate was purified by reverse phase to give 5-amino-n- (5-bromo-2-hydroxyphenyl) pyridine-2-carboxamide as a white solid:

(4) To a solution of 5-amino-n- (5-bromo-2-hydroxyphenyl) pyridine-2-carboxamide (800 mg,2.60 mmol) in TsOH (1.12 g,6.49 mmol) in xylene (20 mL) was added a reaction solution of TsOH (1.12 g,6.49 mmol), the reaction was stirred at 140℃for 12h, concentrated in vacuo after the completion of the reaction, and purified by reverse phase to give 6- (5-bromo-1, 3-benzoxazol-2-yl) pyridin-3-amine as a white solid:

(5) To a solution of 6- (5-bromo-1, 3-benzoxazol-2-yl) pyridin-3-amine (250 mg,0.862 mmol) in Py (10 mL) was added adamantane-1-carbonyl chloride (514 mg,2.59 mmol). The reaction was stirred at 20deg.C for 12h, after which time the mixture was quenched with MeOH (0.5 mL), concentrated in vacuo, and purified by reverse phase to give N- [6- (5-bromo-1, 3-benzooxazol-2-yl) pyridin-3-yl ] adamantan-1-carboxamide as a white solid:

(6) N- [6- (5-bromo-1, 3-benzoxazol-2-yl) pyridin-3-yl ]Diamond-1-carboxamide (200 mg,0.442 mmol), diamond-1-carboxamide (1599 mg,0.884 mmol), cs2CO3 (433 mg,1.33 mmol), xantphos (51.2 mg,0.088 mmol) and Xantphos-pd-G ₂ (78.6 mg,0.088 mmol) and toluene (10 mL) were added and the mixture was taken up in N ₂ Deaeration is carried out for 3-4 minutes, and then the reaction is stirred at 100℃for 12h. After completion, the mixture was concentrated in vacuo, purified by silica gel chromatography (DCM/meoh=50/1), and the crude product was slurried with DCM/MeOH (1/1; 10 ml), stirred for 1h, filtered, and the filter cake dried in vacuo to give the derivative as a white solid:

MS(ESI)m/z＝551.5[M+1] ⁺ . The derivativeDenoted as Y1, its corresponding hydrogen and liquid chromatograms are shown in fig. 3 and 4, respectively.

Example 3:

preparation of derivative Y2:

(1) 6-chloropicolinic acid (1.8 g), 2-amino-4-nitrophenol (1.5 g) and 50mL of Dichloromethane (DCM) were mixed and stirred in an ice bath, then Dicyclohexylcarbodiimide (DCC) (3.3 g) and a solution of 4-Dimethylaminopyridine (DMAP) (0.62 g) in DCM were added dropwise and the reaction was continued at room temperature for 6h. After the reaction is completed, the reaction solution is filtered by suction, the filtrate is collected, dried by rotation, purified by a column by using petroleum ether and ethyl acetate as eluent, and dried to obtain 6-chloro-N- (2-hydroxy-5-nitrophenyl) pyridine carboxamide, wherein the reaction formula is as follows:

(2) 6-chloro-N- (2-hydroxy-5-nitrophenyl) pyridine carboxamide (7.2 g), diisopropyl azodicarboxylate (11.3 mL), triphenylphosphine (6.0 g) and THF (50 mL) were mixed and stirred at 60℃for 2h, the resulting reaction mixture was cooled to room temperature and concentrated under reduced pressure, ethanol was added and the resulting solid was collected by filtration as 2- (6-chloropyridin-2-yl) -5-nitrobenzo [ d ] oxazole, the reaction scheme was as follows:

(3) To a solution of 2- (6-chloropyridin-2-yl) -5-nitrobenzo [ d ] oxazole (2.1G) and (3 s,5 s) -adamantane-1-carboxamide (1.2G) in toluene (30 mL) was added Cs2CO3 (4.5G) and XantPhos Pd G2 (423 mg). After 3 minutes of purging with N2, the mixture was heated to 110 ℃ and stirred for 16h. The residue was purified by flash chromatography eluting with methanol in dichloromethane (1 r,3r,5 s) to give N- (5- (5-nitrobenzo [ d ] oxazol-2-yl) pyridin-2-yl) adamantane-1-carboxamide (350 mg) as a white solid, the reaction was as follows:

(4) A solution of 200mg (1 r,3R, 5S) -N- (6- (5-nitrobenzo [ d ] oxazol-2-yl) pyridin-3-yl) adamantane-1-carboxamide in 10mL MeOH/THF (1:1) containing 5% w/w Pd-C (10%) was hydrogenated (1 atm., balloon) overnight. The contents of the flask were passed through a short pad of celite and washed with EtOAc. The filtrate was evaporated under reduced pressure to give 130mg of crude (1 r,3R, 5S) -N- (5- (5-aminobenzo [ d ] oxazol-2-yl) pyridin-2-yl) adamantane-1-carboxamide as a solid, having the following formula:

(5) To a solution of (1 r,3r,5 s) -N- (6- (5-aminobenzo [ d ] oxazol-2-yl) pyridin-3-yl) adamantane-1-carboxamide (105 mg) in dichloromethane (10 mL), EDCl (89 mg), DMAP (25 mg) was added 1-AdaMantyl carboxylic acid (45 mg) at room temperature, followed by silica gel column chromatography to give (1 r,3r,5 s) -N- (5- (5- ((3 r,5r,7 r) -adamantane-1-carboxamide) benzo [ d ] oxazol-2-yl) pyridin-2-yl) adamantane-1-carboxamide (34 mg) as a yellow solid. MS (ESI) m/z= 551.2 (m+h+), having the structural formula:

designated Y2.

Example 4

Preparation of derivative Y3:

according to the same preparation route as Y2, 6-chloropicolinic acid was replaced with 5-chloropyrazine-2-carboxylic acid to finally prepare (1 r,3R, 5S) -N- (5- (5- ((3R, 5r,7 r) -adamantan-1-carboxamide) benzo [ d ] oxazol-2-yl) pyrazin-2-yl) adamantan-1-carboxamide (27 mg) as a yellow solid. MS (ESI) m/z= 552.2 (m+h+). The structural formula is as follows:

designated Y3.

Example 5

Preparation of derivative Y4:

step (1), 4-aminobenzoic acid (2 g, 14.284 mmol) in SOCl ₂ The solution in (20 mL) was stirred at 80℃for 12h. After completion, the mixture was concentrated in vacuo to give 4-aminobenzoyl chloride (2200 mg,14.141mmol, 96.96%) as a yellow solid 4-aminobenzoyl chloride of the formula:

Step (2) to a solution of 2, 5-dibromopyridin-3-amine (3 g,11.909 mmol) in pyridine (30 mL) was added 4-aminobenzoyl chloride (2.22 g, 14.2910 mmol). The mixture was then stirred at 20℃for 2h. The mixture was poured into water (300 mL), stirred and filtered. The filter cake was dried under vacuum to give 4-amino-N- (2, 5-dibromopyridin-3-yl) benzamide (3.2 g,8.625mmol, 72.42%) as a yellow solid.

Step (3), 4-amino-N- (2, 5-dibromopyridin-3-yl) benzamide (2 g,5.390 mmol) and Cs ₂ CO ₃ A suspension of (3.51 g,10.781 mmol) in DMSO (80 mL) was stirred at 130℃for 12h. After completion, the mixture was poured into water (500 mL) and extracted with EA (2X 200 mL). The organic layer was washed with brine (200 mL), and dried over Na ₂ SO ₄ Drying, filtering, and concentrating the filtrate in vacuum. The residue was triturated with MeOH (10 mL), sulphited and the filter cake dried in vacuo to give 4- { 6-bromo- [1,3 as a yellow solid]Oxazolo [5,4-b ]]Pyridin-2-yl } aniline (700 mg,2.413mmol, 44.76%) was reacted as follows:

step (4) pyridine (6 mL), adamantane-1-carbonyl chloride (308.17 mg,1.551 mmol) was added to a solution of 4- { 6-bromo- [1,3] oxazol [5,4-b ] pyridin-2-yl } aniline (300 mg,1.034 mmol), and the mixture was stirred at 20℃for 2h. The mixture was poured into water (60 mL), stirred and filtered; the filter cake was dried in vacuo to give N- (4- { 6-bromo- [1,3] oxazolo [5,4-b ] pyridin-2-yl } phenyl) adamantane-1-carboxamide (420 mg,0.743mmol, 71.83%) as a yellow solid.

Step (5) of subjecting N- (4- { 6-bromo- [ 1.3)]Oxazoles [5,4-b ]]Pyridin-2-yl]Phenyl group]Phenyl) adamantane-1-carboxamide (300 mg,0.663 mmol), adamantane-1-carboxamide (237.77 mg,1.326 mmol), cs2CO3 (540.21 mg, 1.618 mmol) and RuPhos Pd G2 (10.30 mg,0.013 mmol) and dioxane (0.8 mL) were replaced 3-4 times with N2, and the mixture was stirred at 100℃for 2h. The mixture was concentrated in vacuo. The residue was diluted with water (50 mL) and extracted with DCM/MeOH (20/1; 3X50 mL). The organic layer was washed with brine (100 mL) and concentrated in vacuo. The residue was purified by reverse phase (CH ₃ CN/water (0.1% FA), to give N- {4- [6- (adamantane-1-amide) - [1,3 ] as a yellow solid]Oxazolo [5,4-b ]]Pyridin-2-yl]Phenyl } adamantane-1-carboxamide:

MS (ESI) m/z=551.1 (m+h+), derivativesDesignated Y4. The corresponding hydrogen and liquid chromatograms are shown in fig. 5 and 6.

Example 6

Preparation of derivative Y5:

step (1), according to the synthetic route of Y4, replacing 4-aminobenzoic acid with 4-bromobenzoic acid, and replacing 2, 5-dibromopyridin-3-amine with 2-chloro-5-iodopyridin-4-amine to prepare yellow solid 4-bromo-N- (2-chloro-5-iodopyridin-4-yl) benzamide:

step (2), 4-bromo-N- (2-chloro-5-iodopyridin-4-yl) benzamide (2.3 g) and Cs ₂ CO ₃ (3.8 g), 1, 10-phenanthroline (230 mg) and CuI (120 mg) form a suspension. The DMSO (80 mL) solution was stirred at 130deg.C for 12h, washed, concentrated in vacuo and dried to give 2- (4-bromophenyl) -6-chlorooxazolo [5,4-c ] as a yellow solid]Pyridine:

step (3) of reacting 2- (4-bromophenyl) -6-chlorooxazole [5,4-c ]]To a solution of pyridine (150 mg) and (3S, 5 s) -adamantane-1-carboxamide (120 mg) in toluene (5 mL) was added Cs ₂ CO ₃ (500 mg) and XantPhos Pd G2 (45 mg). By N ₂ After 3 minutes of purging, the mixture was heated to 110 ℃ and stirred for 12h. The residue was purified by preparative TLC eluting with methanol in dichloromethane 1:10 (v/v) to give (1 r,3R, 5S) -N- (4- (6- ((3R, 5r,7 r) -adamantane-1-carboxamide) oxazol [5,4-c ] as a white solid]Pyridin-2-yl) phenyl) adamantan-1-acetamide:

derivatives and their use as inhibitors of viral infectionDesignated Y5.

Example 7

Preparation of derivative Y6:

step (1): to a solution of 4-nitrobenzoic acid (0.8 g,4.787 mmol) in DCM (10 mL) was added DMF (0.037 mL,0.479 mmol) and oxalic acid dichloride (0.527 mL,6.223 mmol) at 0deg.C and the mixture was stirred at room temperature for 12h. The mixture was then concentrated under reduced pressure to give a residue. The residue was dissolved in DCM (10 mL). To a solution of 6-chloro-3-iodopyridin-2-amine (1.22 g,4.787 mmol) in pyridine (5 mL) was added the above solution at 0deg.C and the mixture was stirred at 25deg.C for 2h. The mixture was poured into water and extracted with dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with ethyl acetate in petroleum ether 0-20% (v/v). N- (6-chloro-3-iodopyridin-2-yl) -4-nitro-N- (4-nitrobenzoyl) benzamide (0.6 g,1.086mmol, 22.68%) was obtained as a yellow solid, the reaction formula was as follows:

The corresponding hydrogen profile of the reaction product is shown in FIG. 7.

Step (2): to a solution of N- (6-chloro-3-iodopyridin-2-yl) -4-nitro-N- (4-nitrobenzoyl) benzamide (600 mg,1086 mmol) in THF (6 mL) and MeOH (6 mL) was added 1M NaOH (6 mL) and the mixture stirred at 25℃for 1h. The mixture was poured into water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was used directly in the next step to give N- (6-chloro-3-iodopyridin-2-yl) -4-nitrobenzamide (400.00 mg,0.991mmol, 91.30%) as a yellow solid, the reaction scheme was as follows:

the hydrogen spectrum of the reaction product is shown in FIG. 7.

Step (3): to a solution of N- (6-chloro-3-iodopyridin-2-yl) -4-nitrobenzamide (400 mg,0.991 mmol) in a mixed solvent of THF (4 mL), meOH (4 mL) and H2O (1 mL), the mixture was heated to 80℃and stirred for 12H. The mixture was filtered through celite and washed with ethyl acetate. The solvent was removed under reduced pressure. Water and ethyl acetate were added to the resulting residue. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was used directly in the next step. 4-amino-N- (6-chloro-3-iodopyridin-2-yl) benzamide (210.00 mg,0.562mmol, 56.76%) was obtained as a white solid, according to the following reaction scheme:

Step (4): to a solution of 4-amino-N- (6-chloro-3-iodopyridin-2-yl) benzamide (210 mg,0.562 mmol) in toluene (1 mL) was added K2CO3 (233.08 mg,1.686 mmol), 1, 10-phenanthroline (121.56 mg,0.675 mmol) and CuI (117.76 mg, 0.6278 mmol). After 3 minutes of purging with N2, the mixture was heated to 110 ℃ and stirred for 12h. The reaction was quenched and the mixture was concentrated to give a residue, which was then added water and dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica eluting with methanol in dichloromethane 0-5% (v/v). 6- { 5-chloro- [1,3] oxazol [4,5-b ] pyridin-2-yl } pyridin-3-amine (60 mg,0.243mmol, 43.27%) was obtained as a yellow solid as follows:

the hydrogen spectrum of the reaction product is shown in FIG. 7.

Step (5) to a solution of (3 s,5 s) -adamantane-1-carboxylic acid (60 mg,0.333 mmol) in DCM (1 mL) was added DMF (0.003ml, 0.033 mmol) and oxalic acid dichloride (0.034 mL,0.399 mmol) at 0 ℃ and the mixture was stirred at room temperature for 12h. The mixture was then concentrated under reduced pressure to give a residue, which was dissolved in DCM (3 mL) to give a solution, which was added to a solution of 6- { 5-chloro- [1,3] oxazol [4,5-b ] pyridin-2-yl } pyridin-3-amine (82.11 mg,0.333 mmol) in pyridine (1 mL) at 0deg.C, and the mixture was stirred at 25deg.C for 2h. LCMS showed consumption of starting material and detection of the desired mass. The mixture was poured into water and a yellow solid precipitated. The mixture was filtered and the solid was used in the next step without purification. To give (3S, 5 s) -N- (4- { 5-chloro- [1,3] oxazol [4,5-b ] pyridin-2-yl } phenyl) adamantane-1-carboxamide as a yellow solid, the reaction scheme was as follows:

Step (6): to (3S, 5 s) -N- (4- { 5-chloro- [1, 3)]Oxazolo [4,5-b ]]To a solution of pyridin-2-yl } phenyl) adamantane-1-carboxamide (100 mg,0.245 mmol) and (3S, 5 s) -adamantane-1-carboxamide (52.74 mg, 0.254 mmol) in toluene (1 mL) was added Cs ₂ CO ₃ (239.63 mg, 0.730 mmol), xantPhos Pd G2 (21.77 mg,0.024 mmol), xantphosPd G2 (21.7 mg,0.023 mmol). After 3 minutes of purging with N2, the mixture was heated to 110 ℃ and stirred for 12h. The residue was purified by preparative TLC eluting with methanol in dichloromethane 1:10 (v/v). (3R, 5S) -N- (4- {5- [ (3R, 5S,7 s) -adamantan-1-amid-yl ] as a white solid]-[1,3]Oxazoles [4,5-b ]]Pyridin-2-yl } phenyl) adamantane-1-carboxamide (28.3 mg,0.051mmol, 20.96%) was reacted as follows:

the derivativeDesignated Y6.

The nuclear magnetic hydrogen spectrum of the derivative Y6 is shown in FIG. 7, and the liquid phase chromatogram is shown in FIG. 8.

Example 8

Preparation of derivatives Y7-Y15:

preparation of derivative Y7: the synthetic route of Y5 is adopted, 5-bromopyridine carboxylic acid is used for replacing 4-bromopyridine carboxylic acid, 2, 5-dibromopyridine-3-amine is used for replacing 2-chloro-5-iodopyridine-4-amine, and the rest steps are the same as the synthetic steps of Y5.

The final preparation yielded (1 r,3R, 5S) -N- (6- (6- ((3R, 5r,7 r) -adamantane-1-carboxamide) oxazol [5,4-b ] pyridin-2-yl) pyridin-3-yl) adamantane-1-carboxamide (29 mg) as a white solid. MS (ESI) m/z= 552.3 (m+h+). The structural formula of the derivative is as follows:

Designated Y7.

Preparation of derivative Y8:

according to the preparation route of Y7, 2-chloro-5-iodopyridin-4-amine was used in place of 2, 5-dibromopyridin-3-amine, and the remaining steps were the same, to finally prepare 2- (5-bromopyridin-2-yl) -5-chlorooxazolo [4,5-b ] pyridine as a yellow solid. The structural formula of the derivative is as follows:

designated Y8.

Preparation of derivative Y9:

according to the preparation route of Y7, 6-chloro-3-iodopyridin-2-amine was used in place of 2, 5-dibromopyridin-3-amine, and the remaining steps were the same, to finally prepare 2- (5-bromopyridin-2-yl) -5-chlorooxazolo [4,5-b ] pyridine as a yellow solid. The structural formula of the derivative is as follows:

designated Y9.

Preparation of Y10:

according to the Y7 preparation route, 6-chloronicotinic acid replaces 5-bromopicolinic acid, and the rest steps are the same, and (1 r,3R, 5S) -N- (5- (6- ((3R, 5r,7 r) -adamantane-1-carboxamide) oxazol [5,4-b ] pyridin-2-yl) pyridin-2-yl ] adamantane-1-carboxamide of a white solid is prepared, MS (ESI) m/z= 552.2 (m+H+), the structural formula of the derivative is as follows:

designated Y10.

Preparation of Y11:

according to the Y7 synthetic route, 6-chloronicotinic acid was substituted for 5-bromopicolinic acid, 2-chloro-5-iodopyridin-4-amine was substituted for 2, 5-dibromopyridin-3-amine, and the remaining steps were identical, to prepare (1 r,3r,5 s) -N- (5- (6- ((3 r,5r,7 r) -adamantan-1-carboxamide) oxazol [5,4-c ] pyridin-2-yl) pyridin-2-yl ] adamantan-1-carboxamide (22 mg) MS (ESI) m/z=552.4 (m+h+), the derivative had the following structural formula:

Y11。

Preparation of Y12:

according to the Y7 synthetic route, 6-chloronicotinic acid was substituted for 5-bromopyridine carboxylic acid, 6-chloro-3-iodopyridin-2-amine was substituted for 2, 5-dibromopyridin-3-amine, and the remaining steps were identical, giving (1 r,3r,5 s) -N- (5- (5- ((3 r,5r,7 r) -adamantan-1-carboxamide) oxazol [4,5-b ] pyridin-2-yl-adamantan-2-carboxamide (22 mg). MS (ESI) m/z= 552.3 (m+h+). This derivative had the following structural formula:

designated Y12.

Preparation of Y13:

according to the Y7 synthetic route, 5-chloropyrazine-2-carboxylic acid was substituted for 5-bromopyridine carboxylic acid, the remaining steps were identical, affording (1 r,3R, 5S) -N- (5- (6- ((3R, 5r,7 r) -adamantane-1-carboxamide) oxazolo [5,4-b ] pyridin-2-yl) pyrazin-2-yl) adamantane-1-carboxamide (21 mg) as a white solid. MS (ESI) m/z= 553.3 (m+h+). The structural formula of the derivative is as follows:

designated Y13.

Preparation of Y14:

according to the Y7 synthetic route, 5-chloropyrazine-2-carboxylic acid was substituted for 5-bromopyridine carboxylic acid, 2-chloro-5-iodopyridin-4-amine was substituted for 2, 5-dibromopyridin-3-amine, and the remaining steps were identical, giving (1 r,3R, 5S) -N- (5- (6- ((3R, 5r,7 r) -adamantan-1-carboxamide) oxazol [5,4-c ] pyridin-2-yl) pyrazin-2-yl) adamantan-1-carboxamide (21 mg) as a white solid. MS (ESI) m/z= 553.2 (m+h+). The structural formula of the derivative is as follows:

Designated Y14.

Preparation of Y15:

according to the Y7 synthetic route, 5-chloropyrazine-2-carboxylic acid instead of 5-bromopyridine carboxylic acid, 6-chloro-3-iodopyridin-2-amine instead of 2, 5-dibromopyridin-3-amine, and the remaining steps were identical, giving (1 r,3R, 5S) -N- (5- (5- ((3R, 5r,7 r) -adamantan-1-carboxamide) oxazol [4,5-b ] pyridin-2-yl) pyrazin-2-yl) adamantan-1-carboxamide (23 mg) as a white solid. MS (ESI) m/z=553.4 (m+h+). The structural formula of the derivative is as follows:

designated Y15.

Effect verification of the above compounds and their derivatives, cell lines for testing:

SKBR3 cells: human breast cancer cell lines, growing on the wall, are purchased from Shanghai cell banks of the national academy of sciences.

MDA-MB-468 cells: human breast cancer cell lines, growing on the wall, are purchased from Shanghai cell banks of the national academy of sciences.

MDA-MB-231 cells: human breast cancer cell lines, growing on the wall, are purchased from Shanghai cell banks of the national academy of sciences.

BT-549 cells: human breast cancer cell lines, growing on the wall, are purchased from Shanghai cell banks of the national academy of sciences.

HCC-1806 cells: human breast cancer cell lines, grown adherent, were purchased from Shanghai Saibuten Biotechnology Co.

HCC-1937 cells; human breast cancer cell lines, growing on the wall, are purchased from Shanghai cell banks of the national academy of sciences.

Example 9: binding of ZR-001 and its derivatives to ASCT2 inhibits ASCT2 transport function

The experimental steps are as follows:

computer virtual screening

First the PDB (http:// www.rcsb.org /) downloads the ASCT2 protein (PDB: 5 llm) PDB file. All hetero atoms are removed for subsequent molecular docking. ASCT2 proteinThe docking grid has been maximized for ZR-001 docking. Prior to virtual screening, PDB files (5 llm) are converted into macromolecules in PDBQT format. The location of the grid (ligand docking search space) is as described above. Autodock vina1.1.2 was then used for subsequent molecular docking. Interaction of the protein with the ligand can be observed using the 1.7.4.5 version of Pymol. Spike protein proximity hit ligandIs highlighted as a potential interaction residue involved in protein-ligand interactions.

FIG. 9-A shows that ZR-001 interacts with GLY220, CYS175, VAL218, LEU179, SER197, MET221, ASN222, ILE223, ILE183, PHE176, LEU224, VAL429 amino acid positions in ASCT2 protein.

Surface plasmon resonance (surface Plasmon resonance, SPR)

Principle of: biacore is a novel bioanalytical sensing technology developed based on the principle of surface plasmon resonance (surface Plasmon resonance, SPR). The 3 core parts of this technology are the sensor chip, the SPR optical detection system and the microfluidic cartridge. In experiments, a biomolecule was immobilized on the dextran surface of the sensor and the molecule that interacted with it was dissolved on the chip surface through which the solution flowed. SPR detector can record a sensor graph according to the change of the whole process of combining and dissociating molecules in the tracking solution and molecules on the surface of the chip, and provide dynamics and affinity data. The Biacore technology has the advantages of no need of labeling, high sensitivity, rapid detection, real-time quantitative test and the like, and has been widely used for researching interactions of biomolecules such as proteins, nucleic acids, polypeptides, small molecular compounds and the like. We used Biacore T200, which is currently authoritative in the field of molecular interactions, to conduct affinity detection on ZR-001 and ASCT2 proteins.

ASCT2 protein and compound ZR-001 surface plasmon interaction assay was performed using Biacore T200.

1) The ASCT2 ubiquitination labeled protein is coupled by using an SA chip, the ASCT2 label protein is coupled by using a CM5 chip, a 1 channel is used as a control channel, and a No. 2 channel is coupled with the target protein;

2) Selecting a multi-cycle kinetic detection method, using PBS-P containing 5% DMSO as a Running Buffer, setting the combination and dissociation time to 90s, and flushing residual small molecular compounds in a channel by 50% DMSO;

3) The concentration of each small molecule ranged from 0.15625. Mu.M to 10. Mu.M (2-fold dilution starting from 10. Mu.M, 6 concentration gradients were set) and 3 additional zero concentrations were added to verify reproducibility. The assay for each compound was repeated three times.

FIG. 9-B shows that the binding KD of ASCT2 protein to ZR-001 is 9.93 μm, which shows that ZR-001 can stably bind to ASCT2 protein.

Measurement of glutamine uptake

(1) Uptake of isotopes

Breast cancer cells were cultured and 1X 10 cells were grown when the number of cells was sufficient ⁵ cell/well inoculated in 24-well plate, and adhered overnight; the MEM medium was prepared with isotope solution 5. Mu. Ci/mL ³ H]L-glutamine (Perkin Elmer), adding the prepared isotope solution into a 24-well plate, incubating for 15min at 37 ℃, and removing the isotope solution after the incubation, wherein the adding and removing time of the isotope solution needs to be strictly controlled in the process, so that the incubation time of the cells in each well is precisely 15min. Pre-treating the medicine pretreatment group, and then incubating the medicine pretreatment group by spot plates; then the 24-well plate is washed three times by using 1 XPBS solution, the time for adding and removing the 1 XPBS solution is strictly controlled in the first time, and the time is not controlled in the second time and the third time; if the next experimental run cannot be performed immediately, the 24-well plate should be stored at-20 ℃.

(2) Detection of radioactivity

Respectively adding 220 mu L of NaOH solution with the concentration of 1N into each hole of a 24-hole plate to lyse the cells for 30min, and taking 20 mu L of the cells from each hole for BCA quantification after the completion of the lysis; after 20. Mu.L was removed, 200. Mu.L of 1N HCl solution was added to each well for neutralization; after neutralization, 2mL of scintillation liquid is added into each well, and the mixture is transferred to a 96-well plate after being fully and uniformly mixed, and then radioactivity can be detected. The absolute value of glutamine uptake is obtained.

(3) BCA protein quantification

Firstly, measuring the absorbance of each hole of a blank 96-hole ELISA plate under the 595nm condition by using an ELISA reader; preparing 8 concentrations of protein standard substances, namely 0, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL; respectively adding the protein solution and the protein standard substance which are taken out from each hole after the completion of the cleavage in the radioactivity detection into a 96-hole ELISA plate; brandford was added to each plate and incubated for 30min, and absorbance was measured at 595nm using a microplate reader. And obtaining standard yeast according to the protein standard substance, and calculating the protein concentration of each sample according to the standard yeast. The final absolute radioactivity/protein concentration gives the relative glutamine uptake values for each sample.

FIGS. 9C-D show that the inhibition rates of the breast cancer SKBR3 cell lines were 5% and 34% at ZR-001 at 1. Mu.M and 10. Mu.M, respectively, and 36% and 46% at V9302 at 1. Mu.M and 10. Mu.M, respectively, compared to the control group without the administration; compared to the control group, the inhibition rates of breast cancer MDA-MB-468 cells were 28% and 38% at ZR-001 administration and 1. Mu.M and 10. Mu.M, respectively, while the inhibition rates of V-9302 administration were 21% and 44% at 1. Mu.M and 10. Mu.M, respectively. The results indicate that ZR-001 has a concentration-dependent inhibition of glutamine uptake by breast cancer cells.

FIG. 9E-F shows that derivatives Y1-Y15 of 10. Mu.M ZR-001 have an inhibitory effect on glutamine uptake in breast cancer cell lines SKBR3 and MDA-MB-468, as compared to the control.

Example 10: experiment of influence of ZR-001 on ASCT2 Gene and protein expression

The experimental steps are as follows:

real-time quantitative PCR

(1) Total RNA extraction

Discarding the six-well plate culture medium, slowly adding pre-cooled 1 XPBS along the side wall of the six-well plate for washing twice, discarding the residual PBS, adding 1 mL/well of Trizol lysate, ice-bathing for 10min, carefully blowing down the lysed cell fragments by a pipette, sucking the lysate, adding the lysate into an EP tube which is prepared in advance and has no enzyme of 1.5mL, and preserving at-80 ℃. Taking out the lysate from-80 ℃, adding 200 mu L of chloroform into each tube, shaking for 30s by vortex, so that the mixed solution becomes emulsion, and standing on ice for 5min; centrifuge 12000g/min at preset temperature of 4deg.C for 15min, gently take out the EP tube, and the solution in the tube is obviously layered: the upper layer is colorless RNA extract, the middle layer is white DNA precipitation, the lower layer is pink protein extract, carefully absorbing 500 mu L of supernatant, adding equal volume of isopropanol, mixing for 10 times upside down, and standing for 10min at room temperature; centrifuging at 12000g/min at 4deg.C for 10min, wherein white precipitate can be seen at the bottom of EP tube, and discarding supernatant; 1mL of DEPC water is added to prepare 75% ethanol, the mixture is gently inverted, the precipitate is washed to float but not to be dispersed, then 75% ethanol is carefully sucked away, the EP tube is dried at room temperature for 20min to the bottom to precipitate to transparent color, then 20 mu L of DEPC treatment water is added, the metal bath at 50 ℃ is dissolved for 10min, and the concentration of RNA is detected after complete mixing.

(2) RNA reverse transcription

After total RNA was dissolved uniformly, RNA concentration was determined using NanoDrop 2000 and diluted to 0.5. Mu.g/. Mu.L with DEPC treated water. The PCR octal was added to a system of 2. Mu.L of DEPC-treated water 10. Mu. L, RNA dilution and 4 XgDNA 4. Mu.L, sealed, and subjected to instantaneous centrifugation, and then the mixture was subjected to reverse transcription in a Bio-Rad My Cycler PCR apparatus according to the procedure shown in Table 1 to obtain cDNA.

Table 1: reverse transcription reaction procedure

Temperature (temperature)	Time
		25℃	10min
42℃	30min
		85℃	5min
4℃	∞

(3) Real-time fluorescent quantitative PCR

mu.L of cDNA obtained by reverse transcription was diluted to 250. Mu.L with DEPC water to prepare cDNA dilution, which was mixed well for use. Taking an enzyme-free PCR plate, adding 11.4 mu L of primer (with the concentration of 5 mu M) and 8.6 mu L of cNDA diluent into each well SYBR Green Master Mix (premixed ROX Reference Dye 1) to prepare a polymerase chain reaction system with the volume of 20 mu L of each well, and adding each sample into each well in a three-fold way; and sealing the compound hole by using a fluorescent quantitative PCR optical sealing plate film, and instantaneously centrifuging the mixed system. The PCR procedure was set up according to Table 2 and samples were run for detection.

TABLE 2

FIGS. 10A-B show that ZR-001 has no effect on the expression level of mRNA for SLC1A5, indicating that ZR-001 does not affect transcription of the SLC1A5 gene.

Western Blot (immunoblot assay):

(1) Protein sample preparation

Firstly, uniformly spotting cells in a 6-hole plate, and adding ZR-001 with different concentrations for 48 hours after the cells are attached; cell samples were taken out of the carbon dioxide incubator, observed for cell density under a microscope, and washed twice with 1 XPBS buffer pre-chilled at 4℃after media was aspirated off. The suspension cells were centrifuged and washed twice with pre-chilled 1 XPBS buffer at 4 ℃. Add 100. Mu.L of the cell lysate of premixed phosphatase inhibitor and protease inhibitor and lyse smoothly on ice for 20min. After scraping the cells with a clean spatula, the collected 1.5mL centrifuge tube was centrifuged in a high-speed refrigerated centrifuge at 12000rpm for 15min at 4 ℃, and then the cell lysate was transferred to the 1.5mL centrifuge tube with a gun. The whole operation was performed as much as possible on ice and the volume of the lysate was quantified with a pipette. Suspension was then directly lysed in a 1.5mL centrifuge tube. And (5) placing the centrifuged supernatant into a centrifuge tube for preservation at the temperature of-20 ℃.

(2) Protein quantification (BCA method protein quantification)

A gradient diluted Bovine Serum Albumin (BSA) standard was prepared, prepared in a clean 1.5mL centrifuge tube, numbered. mu.L of the protein sample and the standard were precisely aspirated and diluted 10-fold to 70. Mu.L with ultrapure water. BCA working fluid (working fluid a: working fluid b=50:1 (v/v)) was prepared as needed, and mixed uniformly for use. The diluted protein sample and the standard substance diluent (20. Mu.L) are added into a 96-well plate (3 multiple wells of each sample), and 200. Mu.L of the mixed BCA working solution is added, and the mixture is gently shaken and homogenized. The 96-well plate was placed in an oven at 37℃for 15min. OD values were measured at a full wavelength microplate reader at excitation wavelength of 562 nm. And (3) establishing a standard curve, calculating the protein concentration, and adjusting the concentration of other samples (diluted by using cell lysate) according to the minimum concentration in the sample to be detected, so as to ensure that the loading amounts of all the samples are the same. Respectively adding 5×loading buffer (1/4 of the protein sample volume) into the sample, vortex mixing, heating in a metal bath at 100deg.C for 10min, and preserving at-20deg.C or-80deg.C.

(3) SDS-polyacrylamide gel electrophoresis

Firstly, preparing the gel, and determining the concentration of the separation gel according to the molecular weight of the target protein (shown in table 3); cleaning the glass plate for glue preparation, drying in an oven at 80 ℃, fixing the glass plate on a glue preparation frame, adding three distilled water for leak detection, and taking down and fixing again if liquid leakage occurs; discarding the triple distilled water in the glass plate, then mixing the mixed solution of the separating glue according to proportion, shaking uniformly (as shown in table 4), pouring the mixed solution into the glass plate until the upper edge of the glue mixing clamp and the position between the plate openings are immediately sealed by the triple distilled water, standing at room temperature until the mixed solution is solidified, wherein the time is about 30 minutes; removing three distilled water used for liquid sealing after the separation gel is solidified, and sucking residual water by using filter paper; then the prepared concentrated glue is gently vibrated and evenly mixed, poured above the separating glue, quickly inserted into comb teeth, kept stand at room temperature until the concentrated glue is solidified for about 15 minutes, soaked in triple distilled water after solidification and stored at 4 ℃ for standby; taking out the glue which is prepared in advance during electrophoresis, fixing the glue on an electrophoresis clamp, and adding a newly prepared 1 multiplied by electrophoresis buffer solution; taking out the protein sample, thawing at room temperature, and fully vortex and centrifuging; pulling out comb teeth for loading, adding recovered 1 Xelectrophoresis buffer solution into an outer tank after loading, starting electrophoresis, wherein the electrophoresis is divided into two stages, namely a first stage of 80V constant-pressure electrophoresis for 40min; the second stage 120V constant voltage electrophoresis for 1h; the 1 Xwet stock at-25℃was removed and thawed at room temperature.

Table 3: optimal separation range of separation gels of different concentrations

SDS-PAGE separation gel concentration	Optimal separation range
		6% glue	50-150kD
8% glue	30-90kD
		10% glue	20-80kD
12% glue	12-60kD
		15% glue	10-40kD

Table 4: separating gel/concentrating gel formula

After the addition of the above liquid, 100. Mu.L of 10% APs, 10. Mu.L of TEMED, 30. Mu.L of 10% APs, 5. Mu.L of TEMED were added to the separating gel; and (5) immediately mixing and pouring glue.

(4) Wet transfer

After electrophoresis, carefully prying the glass plate, taking out gel after electrophoresis separation, cutting off the gel where the required target protein is located according to a pre-dyeing Marker position, clamping a wet transfer clamp according to the sequence of positive electrode (white) -sponge-filter paper-PVDF membrane-gel-filter paper-sponge-negative electrode (black), placing the PVDF membrane in methanol for activation for 30s before use, taking care to empty bubbles between the gel and the PVDF membrane in the wet transfer clamp process, then placing the gel into a wet transfer tank according to the direction corresponding to the positive electrode and the negative electrode, adding 1X wet transfer liquid and an ice box pre-cooled at 4 ℃ in advance, carrying out ice bath in the wet transfer tank to maintain a low temperature state, and transferring the membrane under the condition of 0.32A constant current. The wet-transfer time was determined according to the protein molecular weight at 1 min/kDa.

(5) Blocking and incubation of antibodies

After the wet transfer is finished, carefully taking out the PVDF membrane protein strips, placing the PVDF membrane protein strips into a protein incubation box, adding the protein strips into a blocking solution (5% BSA), and placing the protein strips in a shaking table for blocking for 1h under the condition of room temperature; after the sealing is finished, recovering sealing liquid, adding primary anti-dilution liquid, and placing the mixture in a shaking table for incubation at 4 ℃ for 12 hours; after the primary antibody incubation is finished, the primary antibody diluent is recovered, 1 XTBST is added, and the primary antibody is placed on a shaking table for washing for 5min each time for 3 times; discarding 1 XTBE, adding secondary antibody diluent, placing in a shaking table to incubate for 1h at 4 ℃, recovering secondary antibody diluent after the secondary antibody incubation is finished, adding 1 XTBE, placing in a shaking table to remove unbound secondary antibody diluent for 5min each time, and 4 times; after the second antibody is washed, exposure is carried out.

(6) Chemiluminescent imaging

ECL luminous liquid (A liquid: B liquid=1:1 (v/v)) is prepared for exposure, PVDF membrane protein strips are carefully taken out and placed on an imaging plate in a Bio-Rad full-automatic gel imager, a program is opened, parameters such as focal length brightness and the like are adjusted, a template is saved, luminous liquid prepared in advance is dripped, and the data is saved by clicking exposure.

FIGS. 10C-D show that the protein levels of ASCT2 were gradually decreased in breast cancer MDA-MB-231 cell lines when ZR-001 was administered at 1. Mu.M, 3. Mu.M, 10. Mu.M, and 30. Mu.M, respectively, as compared to the control group; compared with the control group, the protein level of ASCT2 was gradually reduced when ZR-001 was administered at 0.3. Mu.M, 1. Mu.M, 3. Mu.M, and 10. Mu.M. The results indicate that ZR-001 down-regulates ASCT2 protein levels in energy dose-dependent induced breast cancer cell lines.

Example 11: effect of ZR-001 and its derivatives on proliferation of breast cancer cells

The experimental steps are as follows:

MTT experiment:

washing TNBC cell line in exponential growth phase with sterile 1×PBS once, adding 0.25% pancreatin (containing EDTA) for digestion, blowing off cells, transferring cell suspension into clean sterilized centrifuge tube, l000 rpm for 5min, discarding supernatant, adding small amount of complete culture medium for blowing off cell precipitate, counting and diluting cells to (2-4) ×10 ⁴ individual/mL; sucking the cell diluted suspension with a row gun, inoculating on a 96-well plate at 180 μL/well, and standing at 37deg.C with 5% CO ₂ Is cultured in an incubator; after 24h of cell inoculation, adding a tested drug according to 20 mu L/hole, setting 3 compound holes for each concentration, and continuously culturing for 96h; adding 20 mu LMTT solution into each hole, and incubating for 4 hours in an incubator; carefully pipette the supernatant with a 1mL syringe, add 150 μldmso to each well, shake the 96-well plate to dissolve the crystals well; measuring OD value at 570nm wavelength in enzyme labeling instrument, calculating cell inhibition rate of each drug concentration with solvent hole as reference, drawing inhibition rate curve, and calculating IC ₅₀ Values. The cell inhibition rate was calculated as follows:

FIG. 11A shows that, in the breast cancer cell lines MDA-MB-231 cells, BT-549 cells, HCC-1806, HCC-1937, MDA-MB-436, HCC-38, when ZR-001 was administered at 0.1. Mu.M, 0.3. Mu.M, 1. Mu.M, 3. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M, ZR-001 was able to dose-dependently inhibit survival of breast cancer cells, and IC ₅₀ 3.421. Mu.M, 1.214. Mu.M, 2.897. Mu.M, 4.475. Mu.M, 11.98. Mu.M, 4.967. Mu.M, respectively.

Plate clone formation experiments:

(1) Taking cells with good growth state and in logarithmic phase, digesting and centrifuging, counting after cell resuspension, taking cell suspension with corresponding volume in a complete culture medium, blowing and mixing uniformly, and adding the cell suspension into a 6-hole plate. 2mL of cell suspension per well contained 1000 cells. Shaking six pore plates up and down, left and right, placing at 37deg.C constant temperature, 5% CO ₂ Is cultured in an incubator;

(2) After 24h of incubation, fresh complete medium was changed and randomly divided into 5 groups: a negative control group, a dosing group (low, medium, high dose), a positive control group; continuing to culture in the dressing box, changing the culture medium every 3-4 days, and stopping culturing until the single cell mass contains at least 50 cells;

(3) Taking out the six-hole plate, discarding the supernatant, washing once with 1 XPBS, adding 4% paraformaldehyde into each hole, fixing at room temperature for 30min, discarding the fixing solution, adding 1mL of 0.2% crystal violet dye solution into each hole, and dyeing at room temperature for 30min in a dark place; and finally, washing the bottom of the six-hole plate by using tap water with small flow, washing crystal violet dye liquid, and taking a picture after air drying.

FIGS. 11B-C show that MDA-MB-231 cells have growth inhibition rates of 11.5%, 60.5%, 100% and 100% at ZR-001, 1. Mu.M, 3. Mu.M, 10. Mu.M, and 0.1. Mu.M, respectively; the growth inhibition rates of BT-549 cells at ZR-001 at 1. Mu.M, 3. Mu.M, 10. Mu.M, and doxorubicin at 0.1. Mu.M were 78.7%, 99%, 100%, and 100%, respectively; HCC-1806 cells showed growth inhibition rates of 65.2%, 99%, 100% and 100% at ZR-001 at 1. Mu.M, 3. Mu.M, 10. Mu.M and doxorubicin at 0.1. Mu.M, respectively; the growth inhibition rates of HCC-1937 cells at ZR-001 at 1. Mu.M, 3. Mu.M, 10. Mu.M, and doxorubicin at 0.1. Mu.M were 54.2%, 84.9%, 100%, and 100%, respectively. The results show that ZR-001 is able to significantly inhibit the clonogenic capacity of breast cancer cells. Growth curve experiment:

(1) Taking cells with good growth state and in logarithmic phase, digesting and centrifuging,cells were counted after resuspension and diluted to 6X 10 ³ Taking cell suspension with required volume per mL, re-suspending in complete culture medium, blowing uniformly, inoculating 1000 cells per well into 96-well plate at 180 μL/well, standing at 37deg.C under 5% CO ₂ Is cultured in an incubator;

(2) The randomization was divided into 5 groups: the negative control group, the administration group (low, medium and high dose) and the positive control group are continuously cultured in the dressing case;

(3) OD values were measured using CCK-8 kit, once every 24h for 6 consecutive days.

FIG. 11D shows that MDA-MB-231 cells showed growth inhibition of 16%, 46%, 54%, 66% at ZR-001, 3. Mu.M, 10. Mu.M, and 30. Mu.M, respectively, and that doxorubicin, a positive drug, showed a growth inhibition of 74% at 1. Mu.M, respectively, compared to the control group; the growth inhibition rates of BT-549 cells at 1. Mu.M, 3. Mu.M, and 10. Mu.M were 11%, 44%, 73%, and 91% for doxorubicin at 1. Mu.M, respectively, compared to the control group. The results indicate that ZR-001 can significantly inhibit the growth of breast cancer cells.

The results in FIGS. 12A-B show that the ZR-001 derivatives Y1-Y15 significantly inhibited the growth of breast cancer cells MDA-MB-231 and BT-549 after 5 days of action compared to the control.

EdU cell proliferation assay:

firstly, taking cells with good growth state in the log phase, performing experiments, digesting the cells with 0.25% pancreatin, re-suspending the cells with complete culture medium, uniformly spotting the cells into 96-well plates with the density of 2 multiplied by 10 ³ cells/mL, volume 90. Mu.L/well, and then drug treatment was performed after incubation in a carbon dioxide incubator for 24 h. After 72h of treatment, edU staining was performed using EdU labeling.

(1) EdU markers

The volume ratio of the cell culture medium is 1000:1 (reagent a) and preparing a proper amount of 30 mu M of EdU culture medium; the medium in the 96-well plate was replaced and cultured in the incubator for 90min. The cells were then washed with PBS 1-2 times for 5min each.

(2) Cell immobilization

Adding 50 mu L of cell fixative (namely PBS containing 4% paraformaldehyde) into each hole, incubating for 30min at room temperature, and discarding the fixative; then adding 50 mu L of 2mg/mL glycine into each hole, and after a decoloration shaking table is incubated for 5min, discarding glycine solution; then adding 100 mu LPBS into each hole, washing for 5min by a decolorizing shaker, and discarding PBS; finally, adding 100 mu L of penetrating agent (PBS of 0.5% Triton X-100) into each well, and performing decolorization and shaking table incubation for 10min; PBS was washed 1 time for 5min.

(3) Apollo staining

First, 100. Mu.L of 1 XApollo staining solution (Table 5) was added to each well, and after incubation for 30min with a shaking table at room temperature under dark conditions, the staining solution was discarded. Then adding 100 μl of penetrant (PBS of 0.5% TritonX 100), decolorizing and shaking table for 2-3 times, 10min each time, and discarding penetrant; immediately after each well, 100 mu L of methanol is added for cleaning 1-2 times for 5min each time; PBS was washed 1 time, 5min each.

Table 5: apollo dyeing reaction liquid

Order of preparation	Apollo dyeing reaction liquid	500μL
			1	Deionized water	469μL
2	Apollo reaction buffer (reagent B)	25μL
			3	Apollo catalyst solution (reagent C)	5μL
4	Apollo fluorescent dye solution (reagent D)	1.5μL
			5	Apollo buffer additive (reagent E)	5mg

(4) DNA staining

Deionized water is used for preparing the water-based paint according to the volume ratio of 100:1, preparing a proper amount of 1 Xhoechst 33342 reaction solution, preserving in a dark place, adding 100 mu L of 1 Xhoechst 33342 reaction solution into each hole, incubating for 30min at a dark place at room temperature in a decoloration shaker, and discarding the staining reaction solution; then each well was washed 1-3 times with 100. Mu.L PBS.

(5) Photographing: photographs were taken using an inverted fluorescence microscope.

FIG. 11E-H shows that MDA-MB-231 cells showed growth inhibition of 22%, 10%, 42.4%, 62.6% to 100% at ZR-001, 1. Mu.M, 3. Mu.M, 10. Mu.M, 30. Mu.M, and 1. Mu.M doxorubicin, respectively; the growth inhibition rates of BT-549 cells at ZR-001 at 1. Mu.M, 3. Mu.M, 10. Mu.M, and doxorubicin at 0.5. Mu.M were 14.6%, 22%, 37%, and 100%, respectively. The results show that ZR-001 can remarkably inhibit DNA replication activity of breast cancer cells, and meanwhile, the administration test is carried out on MCF10A cells, so that the inhibition effect on normal cells is very small under the treatment drug concentration.

Cell cycle arrest assay:

(1) Taking cells in log phase, performing digestion, centrifugal resuspension, performing cell count, and adjusting cell density to 5×10 ⁴ Inoculating the cells/mL into six-hole plates, culturing 2mL of complete culture medium in each hole, culturing in a carbon dioxide incubator, and performing drug treatment after 24 hours;

(2) After 72h of drug treatment, collecting cell supernatant in a 1.5mL centrifuge tube, digesting cells with 0.25% pancreatin, and collecting the digested cells in the same centrifuge tube for centrifugation at 800rpm for 5min;

(3) Adding 1mL of pre-cooled 1 XPBS to resuspend cells, collecting the cells in 1.5mL of EP tube, centrifuging at 800rpm for 5min, discarding the supernatant, adding 300 mu L of pre-cooled 1 XPBS into each tube, adding 700 mu L of pre-cooled absolute ethyl alcohol to make the final content of the ethyl alcohol be 70%, gently blowing off the cells, and fixing the cells in a refrigerator at 4 ℃ for 12h;

(4) Centrifuging the cell sample fixed for 12 hours at 1000rpm for 5min, gently sucking and removing the supernatant, adding 1mL of precooled 1 XPBS for washing twice, and gently blowing off the cells by using a pipette; each sample was stained with 500 μl/RNaseA staining working solution (PI: rnasea=9:1) at room temperature for 30min in the dark; and (5) detecting by a flow cytometer after the dyeing is finished and the mixing is uniform.

FIG. 11I-J shows that MDA-MB-231 cells had a 10.8% increase in the G0/G1 phase cell fraction when ZR-001 was administered at 10. Mu.M, as compared to the placebo group. Whereas the G2/M phase cell fraction was reduced by 12.51%. The results indicate that ZR-001 can induce breast cancer cells to generate G1 phase retardation, thereby inhibiting proliferation of the breast cancer cells.

Example 12: ZR-001 induces oxidative stress in breast cancer cells

The experimental steps are as follows:

reactive oxygen species detection

(1) Cell preparation

The cells were spotted on 12 well plates, after complete adherence, the density reached between 40-50%, medium was discarded, serum-free medium was exchanged, and after 72h drug treatment, probes were loaded.

(2) In situ loading probe

DCFH-DA was diluted 1:1000 in serum-free medium to a final concentration of 10. Mu. Mmol/L. The cell culture broth was removed and a suitable volume of diluted DCFH-DA was added. The volume of addition is preferably sufficient to cover the cells, usually at least 500. Mu.L of diluted DCFH-DA is added to one well of a 12-well plate and incubated in a cell incubator at 37℃for 30min. Cells were washed three times with 1×pbs.

(3) Flow detection

Cells were digested with pancreatin from 12 plates in 1.5mLEP tubes, centrifuged at 800rpm for 5min, the supernatant was discarded, washed twice with 1mL of 1 XPBS, the supernatant was discarded after centrifugation, and 1mL of 1 XPBS was added to EP tubes, after blow-down was completed, the cell suspension was aspirated into flow tubes and checked on-line.

FIGS. 13A-B show that MDA-MB-231 cells increased ROS by 47%, 65%, 74% and 100% respectively after ZR-001 treatment with 3. Mu.M, 10. Mu.M, 30. Mu.M, and positive control, rosup, respectively; BT-549 cells increased ROS by 2%, 24%, 62%, 75% and 79% after ZR-001 dosing at 0.3 μm, 1 μm, 3 μm, 10 μm, and positive control, rosp treatment, respectively. The result shows that ZR-001 can obviously promote the oxidative stress level of the breast cancer cells, and meanwhile, the promotion effect of the ZR-001 on the oxidative stress level of normal cells is weaker.

Example 13: ZR-001 induces autophagy of breast cancer cells

The experimental steps are as follows:

transmission electron microscope scanning

(1) Taking human breast cancer cells in logarithmic phase, digesting, centrifuging, inoculating into 12-hole plate containing 12-hole climbing plates at proper density, standing at 37deg.C with 5% CO ₂ Is cultured in an incubator; after 24h, the medium was discarded after changing fresh complete medium and adding different concentrations of test drug for 48h, and washed twice with 1 XPBS. Then the cells were digested with pancreatin and placed in a 1.5mL EP tube, centrifuged at 800rpm at 4 ℃ for 5min, washed once with 1 x PBS and the supernatant discarded;

(2) Adding glutaraldehyde solution, and fixing at 4deg.C;

(3) Washing with phosphate buffer solution for 4 times, each time for one hour;

(4) Fixing with 1% osmium acid at 4deg.C for 2h;

(5)ddH ₂ o is washed for 3 times, each time for 10min;

(6) Dyeing with 2% uranium acetate aqueous solution for 2h;

(7) Preparing gradient ethanol solution, sequentially dehydrating, adding 50%, 70%, 90%, 100% ethanol every 15min, and dehydrating at 4deg.C for 15min;

(8) Soaking the sample in 100% acetone for dehydration for 2 times, each for 20min;

(9) 100% acetone: treating the embedding medium (1:1) for 2 hours at room temperature;

(10) After preparing a label and an embedding mould, placing a sample immersed in the complete embedding liquid for 12 hours in a 37 ℃ incubator, placing an embedding plate, placing a 45 ℃ oven for 12 hours, and keeping the 60 ℃ oven for 48 hours for embedding;

(11) Ultrathin sections were performed with the sections Leica EM UC 7;

(12) After washing 3 times with lead nitrate for 5min, the sample cells are observed by an electron microscope, and the ultrastructure of the sample cells is observed by a FEI Tencai G2 transmission electron microscope.

The results in FIG. 14A show that, compared with the blank, the increase of autophagosomes was confirmed by performing transmission electron microscopy after MDA-MB-231 cells were treated with 30. Mu.M ZR-001, and the increase of the bilayer membrane structure was found to be remarkable and the cytoplasmic structure was contained in the bilayer membrane. The results indicate that ZR-001 can induce autophagosome formation.

MDC staining

(1) Cell preparation

Taking human breast cancer cells in logarithmic phase, digesting, centrifuging, inoculating into 12-hole plate containing 12-hole climbing plates at proper density, standing at 37deg.C with 5% CO ₂ Is cultured in an incubator; after 24h, the fresh complete medium was changed and the test drugs at different concentrations were added for 12h, 24h, 48h respectively, the medium was discarded and 1 XPBS was added for two washes.

(2) Nuclear dyeing

After fixing with 4% paraformaldehyde for 15min, 200. Mu.L of Hoechest33342 dye solution was added to each well and after staining for 30min, the wells were washed three times with 1 XPBS.

(3) Autophagosomes

At 1 XWash Buffer: MDC staining fluid volume ratio = 9:1, diluting the dyeing liquid to the working concentration, calculating the volume of the dyeing liquid required by one experiment, preparing MDC dyeing working liquid, and lightly mixing; 200 mu LMDC staining working solution is added to each hole, after being stained for 30min at room temperature in a dark place, the staining solution is discarded, and the solution is washed three times with 1 XPBS.

(4) Sealing sheet

Slide glass was then dropped with 2.5. Mu.L of fluorescence quencher, and the slide glass was inverted and stored at 4 ℃.

(5) Photographing

As shown in fig. 14B-C, MDA-MB-231 cells showed a significant increase in perinuclear green-like fluorescent particles in MDA-MB-231 cells after 12h and 24h of administration of 30 μm ZR-001 and a significant increase in perinuclear green-like fluorescent particles after 24h of treatment of cells with 20 μm CQ as a positive drug; compared to the blank control, HCC-1937 cells showed a significant increase in perinuclear green-colored fluorescent particles in MDA-MB-231 cells after 12h and 24h of administration of 30. Mu.M ZR-001, and in perinuclear green-colored fluorescent particles after 24h of treatment of cells with the positive drug 20. Mu.M CQ. The results indicate that ZR-001 can induce autophagosome increase in breast cancer cells.

Western blots (immunoblotting) results are shown in FIGS. 14D-E, and compared with the blank, the protein levels of LC-3 BII were up-regulated in MDA-MB-231 cells after 6h, 12h, 24h, and 48h of administration of 30. Mu.M ZR-001, and in LC-3 BII after 24h of treatment of MDA-MB-231 cells with 20. Mu.M CQ as a positive drug; compared to the placebo group, the protein level of LC-3 BII was up-regulated in HCC-1937 cells in a time-dependent manner after 12h, 24h, 48h of administration of 30. Mu.M ZR-001, and in a 24h of treatment of HCC-1937 cells with the positive drug 20. Mu.M CQ. The results indicate that ZR-001 can induce the autophagy of breast cancer cells to be enhanced.

Immunofluorescence

(1) Cell treatment

Taking human breast cancer cells in logarithmic phase, digesting, centrifuging, inoculating into 12-hole plate containing 12-hole climbing plates at proper density, standing at 37deg.C with 5% CO ₂ Is cultured in an incubator; after 24h, the fresh complete medium was changed and the test drugs at different concentrations were added for 12h, 24h, 48h respectively, the medium was discarded and 1 XPBS was added for two washes. (2) Fixing

After fixing the slide with pre-chilled paraformaldehyde (4%) at 700. Mu.L per well for 30min at room temperature, the slide was rinsed 3 times with 1 XPBS for 3min each.

(3) Penetrating through

The slide was rinsed 3 times with 0.5% Triton X-100 permeabilization for 15min, 3min each time, with 1 XPBS.

(4) Closure

Blocking with 5% BSA for 1h.

(5) Incubation of primary antibody

After primary antibody was diluted with 5% blocking solution in a ratio of 1:200 (please determine mass ratio or volume ratio), 20 μl was dropped onto the membrane, the slide was back-buckled at 4 ℃ above overnight, and then placed back into 12-well plate with front side facing up, and the slide was rinsed 3 times with 1 x PBS.

(6) Incubation of secondary antibody

The secondary antibody was diluted with 1 XPBS in a ratio of 1:400 (please determine mass or volume ratio) while Hochest was 1:1000 (please confirm the mass ratio or volume ratio) to the secondary antibody diluent, drip 20 muL to the membrane, back-off the climbing film on the membrane and incubate for 1h at normal temperature, put back to 12-orifice plate, right side up, 1 XPBS dip slide 3 times.

(7) Anti-fluorescence quenching agent sealing piece

Slide glass was then dropped with 2.5. Mu.L of fluorescence quencher, and the slide glass was inverted and stored at 4 ℃.

(8) Photographing

As a result of photographing by a confocal laser microscope, as shown in FIG. 14F-G, the MDA-MB-231 cells showed an increase in the time-dependence of the green-like fluorescent spots in the MDA-MB-231 cells after 12 hours, 24 hours, and 48 hours of administration of 30. Mu.M ZR-001, and an increase in the green-like fluorescent spots in the cells after 24 hours of treatment of the MDA-MB-231 cells with 20. Mu.M CQ as a positive drug, compared with the blank group; compared to the blank, the green spot in HCC-1937 cells increased in time-dependent manner after 12h, 24h, 48h of administration of 30. Mu.M ZR-001, and in cells after 24h of treatment of HCC-1937 cells with 20. Mu.M CQ as a positive drug. The results indicate that ZR-001 can induce the autophagy of breast cancer cells to be enhanced.

Western blots results are shown in FIGS. 14H-I, and CQ can further induce protein expression of LC3 BII compared to the ZR-001-alone acting group after MDA-MB-231 cells were treated with 30. Mu.MZR-001, 20. Mu.M autophagy lysosomal inhibitor CQ for 48H, and 30. Mu.M ZR-001 and 20. Mu.M autophagy lysosomal inhibitor CQ simultaneously for 48H, respectively; after 24h treatment of HCC-1937 cells with 30. Mu.M ZR-001, 20. Mu.M ZQ, and 24h simultaneous treatment with 30. Mu.M ZR-001 and 20. Mu.M CQ, respectively, CQ can further induce protein expression of LC3 BII compared to the ZR-001-alone-acting group, which indicates that ZR-001 increases autophagy in breast cancer cells.

Example 14: ZR-001 inhibits proliferation of breast tumors in vivo

The experimental steps are as follows:

construction of nude mice xenograft tumor model

(1) Method for molding and administration

Inoculating MDA-MB-231 cells in vigorous growth period, and 2.0X10 of each mouse ⁶ 100. Mu.L of the cell suspension was inoculated subcutaneously in the right armpit of nude mice under aseptic conditions. The diameter of the transplanted tumor is measured by a vernier caliper for the xenograft tumor of the nude mice, and the tumor grows to 100mm ³ Animals were then randomly grouped. The effect of ZR-001 after administration was dynamically observed using a method of measuring tumor volume. Wherein ZR-001 and V9302 are administered intraperitoneally, paclitaxel is administered by tail vein injection, ZR-001 is administered once a day, V9302 is administered once a week, and paclitaxel is administered twice a week. Tumor volumes were measured 3 times per week, with simultaneous weighing of mice for each measurement, and mice were sacrificed on day 21 of dosing. V9302 was prepared with 2% DMSO physiological saline, and ZR-001 was prepared with corn oil containing 10% DMSO.

(2) Detection index and calculation method

a) Tumor Volume (TV), the calculation formula is: tv=1/2×a×b ² Wherein a and b respectively represent length and width;

b) Relative tumor volume (relative tumor volume, RTV), the calculation formula is: rtv=t _V1 /T _V0 . Wherein T is _V0 For administration in separate cages, i.e. (d) ₀ ) Tumor volume, T _Vt Tumor volume at each measurement;

c) The calculation formula of the relative tumor proliferation rate T/C (%) is as follows: T/C (%) =t _RTV /C _RTV ×100％

T _RTV : treatment group RTV; c (C) _RTV : negative control RTV. The test results were expressed as relative tumor proliferation rate T/C (%) as an anti-tumor agentAn evaluation index of tumor activity;

the results in FIGS. 15A-F show that the tumor volume and tumor weight of the 25mg/kg ZR-001, 50mg/kg ZR-001 and 75mg/kg V-9302 and 10mg/kg paclitaxel administration groups were significantly reduced compared to the control group, and that the inhibition rates of the 25mg/kg ZR-001, 50mg/kg ZR-001, 75mg/kg V-9302 and 10mg/kg paclitaxel administration groups on tumor growth were 36%, 48%, 44% and 87%, respectively, compared to the control group.

Claims (7)

1. A derivative of a small molecule compound that inhibits ASCT2, characterized by: the molecular formula of the derivative is C ₃₄ H ₃₉ N ₄ O ₃ The structural formula is selected from:

any one of them.

2. A derivative of a small molecular compound for inhibiting ASCT2, which is characterized in that the molecular formula of the derivative is C ₃₄ H ₃₉ N ₄ O ₃ The structural formula is selected from:

any one of them.

3. A derivative of a small molecular compound for inhibiting ASCT2, which is characterized in that the molecular formula of the derivative is C ₃₄ H ₃₉ N ₄ O ₃ The structural formula is selected from:

any one of them.

4. Use of a derivative according to any one of claims 1-3 for the preparation of an antitumor drug.

5. Application of small molecular compound for inhibiting ASCT2 in preparation of antitumor drugs, wherein molecular formula of compound is C ₃₅ H ₃₉ N ₃ O ₃ The structural formula is as follows:

。

6. the use according to claim 4, wherein: the tumor is breast cancer tumor.

7. The use according to claim 5, wherein: the tumor is breast cancer tumor.