TWI875311B - Prodrug, nanoparticle comprising the same, and their uses thereof - Google Patents

Abstract

The present invention provides prodrug. The prodrug is represented by the formula (I). Furthermore, the present invention provides a nanoparticle, the nanoparticles comprising the prodrugs. The prodrug and the nanoparticle will be converted into a form with anti-tumor activity upon exposure to X-rays.

Description

Prodrug, nanoparticles containing the same and uses thereof

The present invention relates to the field of medicine, and in particular to a prodrug, nanoparticles containing the prodrug, and uses thereof.

According to the top ten causes of death in 2012 announced by the Ministry of Health and Welfare, "malignant tumors (cancer)" ranked first, which shows that cancer poses a huge threat to people's health. However, in traditional treatment methods, surgical resection is only applicable to the early stage of cancer. When the tumor spreads and invades other body tissues, drug treatment is needed to control the growth and spread of the tumor. However, the current drug treatment has limited drugs to choose from, and the side effects of the drugs also discourage many patients. Therefore, how to develop new anti-tumor drugs and reduce damage to other healthy cells or tissues while killing tumors is a problem that needs to be solved.

The purpose of the present invention is to solve the problems described in the prior art.

Based on the purpose of the present invention, the present invention provides a prodrug, the chemical structure of which is as follows: .

In one embodiment of the present invention, the use of the prodrug is for preparing an anti-tumor prodrug.

In one embodiment of the present invention, the use of the prodrug is to prepare a prodrug that inhibits the expression of BRD4 protein.

In one embodiment of the present invention, the use of the prodrug is used to prepare a drug for inhibiting c-Myc protein.

In addition, based on the purpose of the present invention, the present invention also provides a nanoparticle coated with the above-mentioned prodrug.

In one embodiment of the present invention, the average particle size of the nanoparticles is less than 80 nanometers.

In one embodiment of the present invention, the nanoparticles are liposomes.

In one embodiment of the present invention, the nanoparticles are used to prepare anti-tumor drugs.

In one embodiment of the present invention, the nanoparticles are used to prepare drugs for inhibiting the expression of BRD4 protein.

In one embodiment of the present invention, the nanoparticles are used to prepare drugs for inhibiting c-Myc protein.

In summary, the prodrug and nanoparticles provided by the present invention have anti-tumor effects.

《Preparation method of compound NPUST-3》:

Step (a): 5.3 g of compound A1 was placed in a round-bottom flask, and then 40 mL of anhydrous dichloromethane (DCM) was added. During the stirring process, 24 mL of 70% (v/v) trifluoroacetic acid (TFA) was added dropwise. The mixture was stirred at room temperature for 1 to 4 hours to allow tert-butoxycarbonyl (Boc) to evaporate under normal pressure to form a first reaction solution. The first reaction solution was transferred to a separatory funnel, and then extracted three times with deionized water. The organic phase was collected and dried with anhydrous sodium sulfate and filtered. Then, the mixture was subjected to silica gel column chromatography. The product was separated by HPLC and eluted with the mobile phase. The liquid obtained by elution with the mobile phase was used as the eluent. The eluent was then used to remove the solvent using a vacuum rotary evaporator to obtain 934.6 mg of a beige powdery product. The beige powdery product was compound A2. The IUPAC full name of compound A2 is (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid). The yield was 90%. Compound A1 was JQ1. The IUPAC full name of compound A1 was tert-butyl 2-[7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetate), JQ1 is a known BET bromodomain inhibitor, wherein the mobile phase used in the silica gel column chromatography is dichloromethane/methanol (CH ₂ Cl ₂ : MeOH), the volume ratio of dichloromethane to methanol in the mobile phase is 100 : 5, and the flow rate of the mobile phase is 5 mL/min; the nuclear magnetic resonance qualitative results of compound A2 are ¹ H NMR (CDCl ₃ , 300 MHz) δ: 12.51, 7.51, 7.75, 7.51, 7.75, 4.74, 2.35, 2.36, 2.14, 2.88. ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 136.0, 131.4, 155.6, 149.3, 120.3, 133.9, 136.6, 137.1, 128.9, 130.6, 128.9, 130.6, 168.6, 177.3, CH3: 10.4, 12.2, 10.5. The chemical reaction formula of step (a) is shown below: .

Step (b): 534.7 mg of Compound A2, 346.7 mg of 2-[3-(2-aminoethoxy)propoxy]acetic acid tert-butyl ester (Amino-PEG3-CH2CO2-t-butyl ester) and 562.8 mg of HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) were placed in a round-bottom flask, and then 35 mL of anhydrous dichloromethane was added. 1.55 mL of N,N-diisopropylethylamine (N,N-Diisopropylethylamine, DIEA) to form a second reaction solution, and then the second reaction solution was placed at room temperature and stirred for 12 hours, and then the second reaction solution was transferred to a separatory funnel, and then extracted three times with deionized water and a saturated ammonium chloride aqueous solution, respectively. The organic phase was collected and dried and filtered with anhydrous sodium sulfate, and then separated by silica gel column chromatography to obtain a first crude product. The first crude product was washed with a saturated sodium carbonate aqueous solution and then concentrated by thin film distillation to obtain 573.7 mg of an oily liquid product. The oily liquid product is compound A3. The IUPAC full name of compound 3 is tert-butyl (S)-2-(3-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)propoxy)acetate, the yield is 80%, wherein the mobile phase used in the silica gel column chromatography is dichloromethane/methanol, the volume ratio of dichloromethane to methanol in the mobile phase is 100:3, and the flow rate of the mobile phase is 5 mL/min; the nuclear magnetic resonance qualitative results of compound A3 are ¹ H NMR (CDCl ₃ , 300 MHz) δ: 8.01(m, 3H), 7.51(m, 9H), 7.75(m, 2H), 7.51(m, 9H), 7.75(m, 2H), 4.72(d, J = 8.16, 1H), 4.33(d, J = 1.36, 2H), 3.35(m, 4H), 3.28, (m, 4H) 2.35, 2.36(m, 4H), 2.14(m, 1H), 2.83, 1.75(m, 1H), 1.42(d, J = 6.92, 3H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 136.0, 131.4, 155.6, 149.3, 120.3, 133.9, 136.6, 137.1, 168.6, 169.2, 173.3, 82.1, CH: 128.9, 130.6, 128.9, 130.6, 55.1, CH ₂ : 68.3, 70.2, 68.1, 68.4, 41.3, 42.1, 29.5, CH ₃ : 10.4, 12.2, 10.5, 28.7, 28.7. The chemical reaction formula of step (b) is shown below: .

Step (c): 735.6 mg of compound A3 was placed in a round-bottom flask, followed by 13 mL of anhydrous dichloromethane, and 14 mL trifluoroacetic acid, then placed at room temperature and stirred for 12 hours to evaporate tert-butyloxycarbonyl (Boc) at normal pressure to form a third reaction solution, then subjected to thin layer chromatography on the third reaction solution, and then removed the organic solvent in the third reaction solution by thin film distillation, and then added with anhydrous dichloromethane and transferred to a separatory funnel, then extracted with deionized water three times, and then the organic phase was collected and dried and filtered with anhydrous sodium sulfate, and then separated by silica gel column chromatography to obtain a second crude product, and the liquid washed with the mobile phase was used as the washing solution, and the washing solution was distilled by thin film distillation. After concentration, 542.6 mg of a light yellow powder product was obtained. The light yellow powder product was compound A4. The IUPAC full name of compound A4 is (S)-2-(3-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)propoxy)acetic acid, with a yield of 85%. The mobile phase used in the silica gel column chromatography was dichloromethane/methanol, the volume ratio of dichloromethane to methanol in the mobile phase was 100:5, and the flow rate of the mobile phase was 5 mL/min. The qualitative results of the nuclear magnetic resonance of compound A4 were ¹ H NMR (CDCl ₃ , 300 MHz) δ: 12.80(t, J = 5.40, 1H), 8.01(s, 1H), 7.51(d, J = 8.04, 4H), 7.75, 7.51(d, J = 8.16, 3H), 4.72(m, 1H), 4.31(s, 1H), 3.67(t, J = 5.84, 2H), 3.35(m, 4H), 3.28(m, 4H), 2.35(s, 3H), 2.36(t, J = 5.84, 2H), 2.14(m, 1H), 2.83, 1.75(m, 1H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 136.0, 131.4, 155.6, 149.3, 120.3, 133.9, 136.6, 137.1, CH: 128.9, 130.6, 128.9, 130.6, 55.1, CH ₂ : 67.4, 70.2, 68.1, 68.4, 41.3, 42.1, 29.5, CH ₃ :10.4, 12.2, 10.5. The chemical reaction formula of step (c) is shown below: .

Step (d): 673.3 mg of Compound A4, 574.3 mg of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride, and 745.5 mg of HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) were added to a round-bottom flask, and then 25 mL of anhydrous dichloromethane was added. 1.45% d-H-pyrrolidine was added dropwise while stirring. mLN,N-diisopropylethylamine (DIEA) was added to form a fourth reaction solution, and then the fourth reaction solution was placed at room temperature and stirred for 12 hours. The fourth reaction solution was transferred to a separatory funnel, and then extracted three times with deionized water and a saturated aqueous ammonium chloride solution, respectively. The organic phase was collected and dried with anhydrous sodium sulfate and filtered to obtain a third crude product. The third crude product was separated by silica gel column chromatography to obtain a third crude product. The third crude product was then subjected to rotary vacuum evaporation by a vacuum rotary concentrator to obtain 1102.3 mg of a white powder compound NPUST- 1. The IUPAC name of compound NPUST-1 is: (4R)-1-((S)-2-(tert-butyl)-15-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,14-dioxo-6,10-dioxa-3,13-diazapent adecanoyl)-4-hydroxy-N-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, with a yield of 75%. The compound NPUST-1 in the present invention is a drug with anti-tumor effect. However, if the compound NPUST-1 is directly administered, the compound NPUST-1 may damage other healthy cells or tissues during the transport process in the body. Therefore, the present invention further Steps (e), (f), (g) and (h) are used to convert the compound NPUST-1 into a prodrug. Since the prodrug does not have the ability to directly kill cells and tissues, compared with directly administering the compound NPUST-1, administering the prodrug is less harmful to healthy cells or tissues, thereby reducing the chance of affecting other healthy cells or tissues during transport in the body. The mobile phase used in the silica gel column chromatography method is dichloromethane/methanol, the volume ratio of dichloromethane to methanol in the mobile phase is 100:3, and the flow rate of the mobile phase is 5 mL/min; the nuclear magnetic resonance qualitative results of compound NPUST-1 are ¹ H NMR (CDCl ₃ , 300 MHz) δ: 8.27(t, J = 5.40, 1H), 8.32(d, J = 7.68, 1H), 8.01, 9.07(s, 1H), 7.47(d, J = 8.60, 2H), 7.51(d, J = 8.04, 4H), 7.80, 7.75, 7.47(d, J = 8.16, 3H), 5.37(d, J = 9.48, 1H), 4.72(m, 1H), 3.50 (m, 10H), 3.67(t, J = 5.84, 2H), 3.35(m, 4H), 4.94(m, 1H), 4.24(s, 1H), 3.28(m, 4H), 2.35(s, 3H), 2.36(s, 3H), 2.22, 2.14(m, 1H), 2.83, 1.75(m, 1H), 1.44(d, J = 6.92, 3H), 0.94(s, 9H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 136.0, 114, 131.4, 155.6, 149.3, 150.9, 120.3, 133.9, 136.6, 141.2, 137.1, 141.5, 168.6, 172.6, 162.2, 172.7, 173.3, 35.5, CH: 152.7, 68.2, 66.7, 128.9, 129.5, 130.6, 125.9, 128.9, 129.5, 130.6, 125.9, 55.1, 53.2, 57.9, CH ₂ : 57.0, 36.7, 71.3, 70.2, 67.7, 68.4, 41.3, 42.1, 29.1, CH ₃ : 10.4, 12.2, 12.2, 10.5, 21.5, 25.8, 25.8, 25.8. The chemical reaction formula of step (d) is shown below: .

Step (e): In a round-bottom flask, a mixed solution of acetone and water was added, wherein the volume ratio of acetone to water in the mixed solution of acetone and water was 2:1, and then 1.5 mg of 2,3,4,5,6-pentafluorobenzaldehyde and 0.532 g of sodium azide were added, and the mixture was refluxed at 80°C overnight to form a fifth reaction solution. After the fifth reaction solution was cooled to room temperature, the acetone was first removed by nitrogen blowing with a nitrogen blower, and then 30 mL of water was added, and then 50 The organic phase was extracted three times with 300 mL of ether. The organic phase was washed with a saturated sodium chloride aqueous solution, dried with anhydrous magnesium sulfate and filtered. The solvent was then removed by nitrogen blowing with a nitrogen blower. The product was separated by silica gel column chromatography to obtain 434.3 mg of a light yellow solid, which is compound B1. The IUPAC full name of compound B1 is 4-azido-2,3,5,6-tetrafluorobenzaldehyde. The yield was 25%. The mobile phase used in the silica gel column chromatography was petroleum ether/ethyl acetate (PE/AcOEt). The volume ratio of petroleum ether to ethyl acetate in the mobile phase was 40:1. The flow rate of the mobile phase was 5 mL/min. The qualitative results of the nuclear magnetic resonance of compound B1 were ¹ H NMR (CDCl ₃ , 300 MHz) δ: 10.36. ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 147.5, 144.9, 147.5, 144.9, 111.3, 113.0, 191.0. ¹⁹ F NMR (CDCl ₃ , 376 MHz) δ: -144.92, -150.97. The chemical reaction formula of step (e) is shown below: .

Step (f): 1 g of compound B1 was dissolved in 15 mL of acetic acid, and then 0.322 g of dimethylamine boron (Me ₂ NH-BH ₃ ) was added. The mixture was stirred at room temperature for 2 hours, 50 mL of water was added, and then three extractions were performed with 50 mL of ether. The organic phase was washed twice with 50 mL of 15% sodium carbonate aqueous solution and once with 50 mL of saturated sodium chloride aqueous solution, and then dried and filtered with anhydrous magnesium sulfate. The solvent was then removed by nitrogen blowing with a nitrogen blower, and then separated by silica gel column chromatography to obtain 856.4 mg of a light yellow solid, which was compound B2. The IUPAC full name of compound B2 is: (4-azido-2,3,5,6-tetrafluorophenyl) methanol, with a yield of 85%. The mobile phase used in the silica gel column chromatography was petroleum ether/ethyl acetate (PE/AcOEt), the volume ratio of petroleum ether to ethyl acetate in the mobile phase was 5:1, and the flow rate of the mobile phase was 5 mL/min. The nuclear magnetic resonance qualitative results of compound B2 were ¹ H NMR (DMSO-d6, 300 MHz) δ: 7.47, 4.61. ¹³ C NMR (DMSO-d6, 100 MHz) δ: 147.2, 134.8, 147.2, 134.8, 104.4, 117.9, 51.1. ¹⁹ F NMR (DMSO-d6, 470 MHz) δ: -145.39, -152.90. The chemical reaction formula of step (f) is shown below: .

Step (g): 200 mg of compound NPUST-1, 201 mg of 4-nitrophenyl carbonate and 24 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 8.0 mL of dichloromethane and stirred at room temperature for 12 hours. The solvent was removed by nitrogen blowing using a nitrogen blower to obtain a fourth crude product, which was then separated by silica gel column chromatography to obtain 116.0 mg white solid. The white solid is compound NPUST-2. The IUPAC full name of compound NPUST-2 is: (3R,5S)-1-((R)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15diazaheptadecanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl) ethyl)carbamoyl) pyrrolidin-3-yl (4- nitrophenyl) carbonate with a yield of 49%. The mobile phase used in the silica gel column chromatography was dichloromethane/methanol, the volume ratio of dichloromethane to methanol in the mobile phase was 100:3, and the flow rate of the mobile phase was 5 mL/min. The qualitative results of the nuclear magnetic resonance of compound NPUST-2 were ¹ H NMR (DMSO-d6, 300 MHz) δ: 9.07(s, 1H), 8.27(m, 3H), 8.32(m, 3H), 8.01, 7.51(m, 2H), 7.52(m, 2H), 8.30 (m, 3H), 7.80(m, 2H), 7.75(m, 2H), 7.47(m, 9H), 7.51(m, 9H), 7.52(m, 9H), 8.30(m, 3H), 7.80(m, 2H), 7.75(m, 2H), 7.47(m, 9H), 5.14 (m, 1H), 4.72(d, J = 8.16, 1H), 4.40(d, J = 1.36, 2H), 3.70(dd, J = 12.08, 3.12, 1H), 3.50(t, J = 5.96, 2H), 3.67(m, 8H), 3.35(m, 4H), 4.94, 3.28(m, 4H), 2.45(s, 3H), 2.35(m, 4H), 2.36(m, 4H), 2.22(m, 1H), 2.14(m, 1H), 2.83, 1.75 (s, 3H), 1.44 (d, J = 6.96, 3H), 0.94 (s, 9H). ¹³ C NMR (DMSO-d ⁶ , 100 MHz) δ: 136.0, 114, 131.4, 155.6, 149.3, 150.9, 120.3, 133.9, 136.6, 157.4, 144.7, 141.2, 137.1, 141.5, 168.6, 153.0, 172.6, 162.2, 172.7, 173.3, 35.5, CH: 128.9, 122.5, 125.3, 129.5, 130.6, 125.9, 128.9, 129.5, 130.6, 125.9 , 55.1, 152.7, 75.4 , 66.9, 53.2 , 57.9, CH2: 71.3, 70.2, 67.7, 68.4, 41.3, 10.4, 12.2, 10.5, 42.1, 29.1, 53.4, 34.3, CH3: 21.5, 25.8. The chemical reaction formula of step (g) is shown below: .

Step (h): 100 mg of compound NPUST-2, 66 mg of compound B2 and 37 mg of 4-dimethylaminopyridine were dissolved in 5 mL of dimethylformamide (DMF), and then stirred at 50°C for 12 hours. The mixture was then evaporated by rotary vacuum evaporation using a rotary concentrator to remove the solvent to obtain a fifth crude product. The fifth crude product was then separated by silica gel column chromatography to obtain 56.0 mg of a white solid, which is compound NPUST-3. The IUPAC full name of compound NPUST-3 is: 4-azido-2,3,5,6-tetrafluorobenzyl ((3R)-1-((S)-2-(tert-butyl)-15-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,14-dioxo-6,10-dioxa-3,13-diazapentadecanoyl)-5-((1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl) carbonate with a yield of 78%. The compound NPUST-3 is the precursor drug of the present invention. The mobile phase used in the silica gel column chromatography is methanol, the volume ratio of dichloromethane to methanol in the mobile phase is 100:5, and the flow rate of the mobile phase is 5 mL/min; the qualitative results of the nuclear magnetic resonance of compound NPUST-3 are ¹ H NMR (DMSO-d6, 300 MHz) δ: 9.07 (s, 1H), 8.27(t, J = 4.84, 1H), 8.32(d, J = 7.44, 1H), 8.01(t, J = 4.84, 1H), 7.51(m, 7H), 7.75(m, 7H), 7.47(m, 7H), 5.14(m, 1H), 4.40 (m, 3H), 3.70(m, 10H), 2.45(s, 3H), 4.72(m, 3H), 5.13(m, 1H), 3.50(m, 10H), 3.67(m, 10H), 3.35(m, 6H), 4.94 (m, 3H), 4.24(m, 16H), 3.28(m, 6H), 2.35(s, 3H), 2.36(s, 3H), 2.22(m, 1H), 2.14(m, 1H), 2.83(s, 3H), 1.75(s, 3H), 1.44(d, J = 6.80, 3H, 0.94(s, 9H). ¹³ C NMR (DMSO-d6, 100 MHz) δ: 147.2, 134.8, 147.2, 134.8, 104.4, 117.9, 51.1. ¹⁹ F NMR (DMSO-d6, 470 MHz) delta: -143.03, -152.23. Chemical formula: C ₅₇ H ₆₁ ClF ₄ N ₁₂ O ₉ S ₂ , Molecular weight: 1233.75, m/z: 1232.38 (100.0%), 1233.38 (62.7%), 1234.37 (41.1%), 1234.38 (24.9%), 1235.38 (22.6%), 1236.38 (9.0%), 1235.37 (8.0%), 1233.37 (6.0%), 1236.37 (4.6%), 1235.39 (3.9%), 1237.38 (2.4%), 1237.37 (2.1%), Elemental Analysis: C, 55.49; H, 4.98; Cl, 2.87; F, 6.16; N, 13.62; O, 11.67; S, 5.20. The chemical reaction formula of step (h) is shown below: .

The stationary phase used in the "silica gel column chromatography" in the above steps (a) to (h) is silica gel (product name: Silica gel pore size 60 A, 40-63 um, technical (grade), manufacturer: Sigma, item number: 717185).

The "NMR characterization" in the aforementioned steps (a) to (h) is to perform qualitative analysis on the products obtained in each step using a Varian 500 M NMR spectrometer.

《X-ray treatment of prodrugs》

Step (i): Compound NPUST-3 is dissolved in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution containing 10 mM compound NPUST-3, and then the 10 mM dimethyl sulfoxide solution of compound NPUST-3 is diluted with phosphate buffered saline (PBS) containing 0.1% polysorbate 80 (0.1% Tween-80) to obtain a mixed solution containing 50 μM compound NPUST-3, and then the mixed solution containing 50 μM compound NPUST-3 is degassed with argon, and then the mixed solution containing 50 μM compound NPUST-3 is irradiated with X-ray using an X-ray biological irradiator (company name: Rad Source; model: RS2000 Pro), wherein X The parameters of the X-ray bioirradiator were set to 225 kV, 17.7 mA, and 112 Gy/min. The compound NPUST-3 after X-ray irradiation was called compound X-NPUST-3. Compound X-NPUST-3 was then analyzed by ultra-high performance liquid chromatography (UPLC). In addition, the aforementioned compounds NPUST-1 and NPUST-3 were also analyzed by ultra-high performance liquid chromatography. Specifically, the analysis was performed on an ACQUITY UPLC H-Class PLUS instrument equipped with a Waters PDA detector and a Waters Acquity QDA mass spectrometer. The chromatographic column was ACQUITY UPLC BEH C18 (2.1 × 50 mm), the flow rate was 0.3 mL/min, the detection wavelength was 250 nm, and the mobile phase was 5% (v/v) The methanol aqueous solution was eluted with a 95% (v/v) methanol aqueous solution for 8 minutes, and eluted with a 95% (v/v) methanol aqueous solution for 2 minutes. In addition, the aforementioned methanol aqueous solution contained 0.1% (v/v) formic acid. In the analysis results of the ultra-high performance liquid chromatography method, the retention time of compound NPUST-3 was 16.587 minutes, while the retention time of compound NPUST-1 and compound X-NPUST-3 was 15.828 minutes. Therefore, it can be known that the present invention can cleave compound NPUST-3 into compound NPUST-1 (i.e., X-NPUST-3) by irradiating compound NPUST-3 with X-rays to cause compound NPUST-3 to undergo a cleavage reaction. The chemical reaction formula of step (i) is shown below: .

The present invention uses X-rays to cleave compound NPUST-3 into compound NPUST-1, which not only allows compound NPUST-3, which has low toxicity to healthy cells, to reach the tumor tissue first through intracellular circulation or direct injection into the tumor tissue, but then uses X-rays to irradiate the tumor site to cleave compound NPUST-3 into compound NPUST-1, allowing compound NPUST-1 to directly kill the tumor tissue, thereby inhibiting the growth of the tumor and achieving an anti-tumor effect, while at the same time reducing damage to other healthy tissues of the body and reducing the occurrence of systemic toxicity.

《Preparation method of nanoparticles》

10 mg of compound NPUST-3, 10.0 mg of phosphatidyl choline (PC), and 10.0 mg of phosphatidyl ethanolamine (PE) were placed in a centrifuge tube, and then 1,000 μL of dimethyl sulfoxide (DMSO) was added to dissolve the phosphatidyl choline and phosphatidyl ethanolamine in the centrifuge tube to form a nanoparticle material solution. Then, the nanoparticle material solution was dropped into 900 μL of ultrapure water under the action of 90 MHz and 1,100 W ultrasound in an ice bath environment. Then, the nanoparticle material solution was added dropwise using a 3500 molecular weight cut-off (MWCO) HPLC. The Da dialysis bag is dialyzed three times with deionized water, and then the solution containing the nanoparticles coated with the compound NPUST-3 in the dialysis bag is concentrated by centrifugation using an ultrafiltration centrifuge tube to obtain the nanoparticles coated with the compound NPUST-3. Hereinafter, the "nanoparticles coated with the compound NPUST-3" will be referred to as "NNPUST-3".

《Particle size distribution analysis of prodrugs》

In the "Particle Size Distribution Analysis of Prodrugs", NNPUST-3 was prepared in pure water at a concentration of 1 mg/mL for two batches, hereinafter referred to as the first batch and the second batch. The particle size distribution and polymer dispersity index (PDI) of the nanoparticles in each batch were then measured using a particle size analyzer and a Zeta potential analyzer. The experimental results of the particle size distribution and the polymer dispersity index are expressed as "mean ± standard deviation". The unit of the particle size distribution of the first batch is "nanometer (nm)". The particle size distribution measured in the analysis was 69.2±3.4 nm, and the PDI was 0.15±0.04, while the particle size distribution of the second batch was 73.0±0.5 nm, and the PDI was 0.16±0.02, indicating that the average particle size of NNPUST-3 prepared from the two batches was less than 80 nm, and the particle size distribution was concentrated.

《Serum stability and surface charge testing of prodrugs》

In the "Serum Stability and Surface Charge Test of Prodrug", in order to confirm the stability of the prepared NNPUST-3, NNPUST-3 was prepared with 10% (v/v) fetal bovine serum (FBS) to a concentration of 1.0 mg/mL, and then placed at room temperature. The particle size distribution and polymer dispersibility index at each time point were measured by particle size and Zedar potential analyzers at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, respectively. The experimental results of the particle size distribution and polymer dispersibility index are expressed as "mean ± standard deviation". The unit of the particle size distribution of the first batch is "nanometer (nm)". The experimental results are shown in Table 1. The experimental results show that in the case of 10% (v/v) fetal bovine serum simulating the human blood environment, the particle size distribution of NNPUST-3 does not change significantly, and the particle size distribution is also quite concentrated, showing good stability. In addition, the zeta potential was also measured in this experiment. NNPUST-3 and compound NPUST-3 were prepared at 0.1 mg/mL using HEPES buffer solution, and they were called NNPUST-3 group and NPUST-3 group respectively. Then, the zeta potential was measured using a particle size and zeta potential analyzer. The experimental results of zeta potential are expressed as "mean ± standard deviation". The unit of zeta potential is "millivolt (millivolt, mV), of which the zeta potential of the NPUST-3 group was -1.3±0.08 mV, while that of the NNPUST-3 group was -3.8±0.47 mV. The experimental results showed that the absolute value of the average value of the zeta potential of the NNPUST-3 group was greater than the absolute value of the average value of the zeta potential of the NPUST-3 group, indicating that NNPUST-3 has better dispersibility than compound NPUST-3, and because NNPUST-3 is in the form of nanoparticles, it is easier to reach the tumor site through the systemic circulation.

Table 1: Particle size distribution and polymer dispersibility index analysis results of NPUST-3 and NNPUST-3 groups time NPUST-3 Group NNPUST-3 Group Particle size distribution (nm) Polymer Dispersibility Index Particle size distribution (nm) Polymer Dispersibility Index Hour 0 72.86 ±1.01 0.13 ±0.03 94.36 ± 1.05 0.14 ±0.01 Hour 2 67.14 ±1.45 0.15 ±0.02 84.78 ± 1.05 0.14 ±0.04 Hour 4 72.24 ±1.03 0.13±0.02 81.95 ± 1.19 0.14 ±0.02 8th hour 70.38 ±1.15 0.14 ±0.05 76.23 ± 1.42 0.14 ±0.03 12th hour 70.74 ± 1.04 0.14 ±0.03 70.11 ± 2.03 0.15 ±0.01 Hour 24 73.00 ±1.02 0.15±0.04 69.2 ± 3.4 0.16 ± 0.02

《Anti-tumor testing in vivo》

In the in vivo anti-tumor test, 15 female BALB/c nude mice were selected. The age of these 15 female BALB/c nude mice was 4-5 weeks and the weight was 18-20 g. In this experiment, the mammary fat pads of these 15 female BALB/c nude mice were subcutaneously injected with human cancer cell suspension, and the same position was also injected with a mixture of DMEM high glucose medium and matrix gel, in which the volume ratio of DMEM high glucose medium and matrix gel was 1:1, and the human cancer cell suspension was 100 μL containing 10 ⁷ MDA-MB-231 cells (ATCC HTB-26) cell suspension was used to form human breast cancer transplant tumors in 15 female BALB/c nude mice to obtain 15 female BALB/c nude mice with human breast cancer transplant tumors. The female BALB/c nude mice with human breast cancer transplant tumors are referred to as "experimental mice" below. When the volume of the human breast cancer transplant tumors in the experimental mice reached 100 ^mm3 , the 18 experimental mice were randomly divided into 3 groups, each with 5 experimental mice. Each experimental mouse in each group was injected once through the tail vein every two days. Each group was injected 5 times through the tail vein. The solutions injected during the tail vein injection treatment in three groups were phosphate buffer solution, 10 mg/kg Compound NPUST-1 and 10 mg/kg NNPUST-3. In the "In vivo Antitumor Test", the group injected with phosphate buffer solution was named "@a", the group injected with compound NPUST-1 was named "@b", and the group injected with NNPUST-3 was named "@c". Before the tail vein injection, the compounds NPUST-1 and NNPUST-3 in group @b and group @c were weighed and mixed with physiological saline before being injected into the tail vein of the experimental mice. After each tail vein injection, group @c waited for 30 minutes to allow NNPUST-3 to reach the tumor site through the circulation of the experimental mice, and then used an X-ray biological irradiator (company name: Rad Source; model: RS2000 Pro) The experimental mice in group @c were subjected to three consecutive X-ray irradiations, with an interval of 1 minute between irradiations. The parameters of the X-ray biological irradiator were set at 230 kV, 17.7 mA, and 11.2 Gy/min. In the "In vivo anti-tumor test", the first tail intravenous injection treatment is regarded as the first day, so the completion of the fifth tail intravenous injection is the ninth day, and the tumor volume of each group of experimental mice is continuously monitored and recorded until the 21st day after the five tail intravenous injections. The experimental results are shown in Table 2. The experimental results in Table 2 show the tumor volume of group @a, group @b and group @c. The tumor volume is presented in the form of "mean ± standard deviation". The experimental results in Table 2 show the average of group @b and group @c from the 11th day. The tumor volumes were all smaller than the average tumor volume of group @a, and the average tumor volume of group @c was smaller than the average tumor volume of group @b, indicating that the compound NPUST-1 of the present invention can indeed inhibit tumor volume, and NNPUST-3 can also inhibit tumor growth after X-ray irradiation, that is, it has anti-tumor technical efficacy, and the injection of NNPUST-3 in the form of nanoparticles combined with X-ray irradiation has a more obvious effect of inhibiting tumor volume than direct injection of compound NPUST-1.

Table 2: Experimental results of in vivo anti-tumor tests Group time @a (mm ³ ) @b (mm ³ ) @c (mm ³ ) Day 1 45 ±16 35 ±11 23 ±12 Day 3 52 ±28 86 ±29 61 ±22 Day 5 66 ±32 124 ±33 87 ±28 Day 7 98 ±39 236 ±37 126 ±22 Day 9 205 ±52 297 ±42 208 ±28 Day 11 468 ±85 442 ±47 199 ±32 Day 13 599 ±212 539 ±55 226±36 Day 15 673 ±221 564 ±67 277 ±40 Day 17 825 ±236 609 ±89 392 ±45 Day 19 1190 ±268 694 ±100 434 ±52 Day 21 1400 ±340 870 ±120 521 ±62

《Protein degradation ability test in organisms》

In the "Protein degradation ability test in vivo", 15 female BALB/c nude mice were selected. The age of these 15 female BALB/c nude mice was 4 to 5 weeks and the weight was 18 to 20 g. In this experiment, the mammary fat pads of these 15 female BALB/c nude mice were subcutaneously injected with human cancer cell suspension, and the mixture of DMEM high glucose medium and matrix gel was also injected at the same location. The volume ratio of DMEM high glucose medium and matrix gel was 1:1, and the human cancer cell suspension was 100 μL containing 10 ⁷ MDA-MB-231 cells (ATCC HTB-26) cell suspension was used to form human breast cancer transplant tumors in 15 female BALB/c nude mice to obtain 15 female BALB/c nude mice with human breast cancer transplant tumors. The female BALB/c nude mice with human breast cancer transplant tumors are referred to as "experimental mice" below. When the volume of the human breast cancer transplant tumors in the experimental mice reached 100 ^mm3 , the 18 experimental mice were randomly divided into 3 groups, each with 5 experimental mice. Each experimental mouse in each group was injected once through the tail vein every two days. Each group was injected 5 times through the tail vein. The solutions injected during the tail vein injection treatment in three groups were phosphate buffer solution, 10 mg/kg Compound NPUST-1 and 10 mg/kg NNPUST-3. In the "Protein Degradation Ability Test in the Organism", the group injected with phosphate buffer solution was named "@d", the group injected with compound NPUST-1 was named "@e", and the group injected with NNPUST-3 was named "@f". Before the tail vein injection, the compounds NPUST-1 and NNPUST-3 in group @e and group @f were weighed and mixed with physiological saline before being injected into the tail vein of the experimental mice. Group @f waited 30 minutes after each tail vein injection, and then used an X-ray biological irradiator (company name: Rad Source; model: RS2000 Pro) irradiated the experimental mice of group @f three times in a row with an interval of 1 minute. The parameters of the X-ray biological irradiator were set to 230 kV, 17.7 mA and 11.2 Gy/min. Finally, when each group completed five tail intravenous injections and group @f also completed three consecutive X-ray irradiations, the experimental mice of each group were dissected and the human breast cancer transplanted tumors on the experimental mice were collected. Then, the human breast cancer transplanted tumors were immersed in tissue fixative and then protein extraction reagent (product name: Optiblot SDS-PAGE Sample Preparation Kit, brand: Abcam, model number: ab133414) was used to extract the protein in the transplanted tumor. The protein extracted from the transplanted tumor was then placed in a centrifuge at 11,000 at 4°C. The supernatant was taken out after centrifugation at 400 rpm for 15 minutes, and the protein in the supernatant was quantified using BCA protein quantification reagent. The same amount of supernatant was mixed with protein sample loading buffer for gel electrophoresis, followed by Western blotting. The gel used in the Western blotting was Bis-Tris SurePAGE gel purchased from Genscript. The gel was then transfected to a 0.45 μm poly(vinylidene fluoride) (PVDF) membrane purchased from Merck Millipore. The transfected PVDF membrane was then blocked with a blocking agent, which was QuickBlock Western blocking agent purchased from Beyotime. buffe, and then the blocked polyvinylidene difluoride membrane was reacted with primary antibodies, including wasabi peroxidase-labeled anti-BRD4 protein antibody (product name: HRP Anti-BRD4 antibody, brand: Abcam, model: ab197609), wasabi peroxidase-labeled anti-c-Myc protein antibody (product name: HRP Anti-Myc tag antibody, brand: Abcam, model: ab62928), and wasabi peroxidase-labeled anti-GAPDH protein antibody (product name: HRP Anti-GAPHD antibody, brand: Abcam, model: ab9484). The dilution of each antibody was carried out according to the corresponding product instructions, and then the developer (product name: ECL Western Blotting Substrate Kit , brand: Abcam, model: ab65623) was used for development, and the image quantification of the development results was performed using ImageJ software. The results are shown in Table 3. Table 3 shows the image quantification results, wherein the results in Table 3 are based on the average value of the image quantification results of c-Myc protein, BRD4 protein and GAPDH protein of group @d. The experimental results of Western blot method show GAPDH protein, BRD4 protein and c-Myc protein of groups @d, @e and @f. The experimental results of Western blot method show that the expression levels of BRD4 protein and c-Myc protein in group @f are lower than those of group @d and group @e, indicating that NNPUST-3 of the present invention is indeed formed after being irradiated with X-ray to cleave the compound NPUST-3 coated by NNPUST-3 into compound NPUST-1. The expression of BRD4 protein and c-Myc protein was inhibited. Compared with group @d as the control group, the expression of BRD4 protein in group @e was only 67% of that in group @d, and the expression of c-Myc protein in group @e was only 72% of that in group @a. In addition, the expression of BRD4 protein in group @f was only 24% of that in group @d, and the expression of c-Myc protein in group @f was only 24% of that in group @d. The above results show that the compound NPUST-1 in the present invention can significantly inhibit the expression of BRD4 protein and c-Myc protein, and the group of NNPUST-3 in the present invention combined with X-ray irradiation has a better effect in inhibiting the expression of BRD4 protein and c-Myc protein than the group of direct injection of compound NPUST-1, which once again verifies that the present invention has anti-tumor efficacy.

Table 3: Experimental results of in vivo protein degradation test Group protein @d @e @f BRD4 1±0.04 0.67±0.09 0.24±0.11 c-Myc 1±0.03 0.72±0.09 0.42±0.14 GAPDH 1±0.02 1.12±0.18 0.9±0.24

In summary, the present invention proves that the prodrug and nanoparticles synthesized by the present invention can indeed be applied in anti-tumor applications, and the anti-tumor effect of the prodrug coated with nanoparticles is better.

without.

without

Claims (10)

A prodrug having the following chemical structure: . A use of the prodrug as described in claim 1, which is used to prepare an anti-tumor prodrug. A use of the prodrug as claimed in claim 1, for preparing a prodrug for inhibiting the expression of BRD4 protein. A use of the prodrug as claimed in claim 1, for preparing a drug for inhibiting c-Myc protein. A nanoparticle coated with the prodrug as described in claim 1. The nanoparticles as described in claim 5, wherein the average particle size of the nanoparticles is less than 80 nanometers. The nanoparticle as described in claim 5, wherein the nanoparticle is a liposome. A use of the nanoparticles as described in claim 5, which is used to prepare anti-tumor drugs. A use of the nanoparticles as described in claim 5, which is used to prepare a drug for inhibiting the expression of BRD4 protein. A use of the nanoparticles as described in claim 5, which is used to prepare a drug for inhibiting c-Myc protein.