Podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug and synthesis method
By introducing a bridge between disulfide bonds and lysine in podophyllum toxin and using polyethylene glycol coupling method, the podophyllum toxin-lysine-polyethylene glycol anti-tumor compound was constructed, and the ghost was solved The poor water solubility of calcified toxins and the problems of normal cytotoxicity have achieved effective targeting and improved proliferation activity of tumor cells.
Patent Information
- Application Number
- CN202210754816.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2022-06-15
- Filing Date
- 2022-06-30
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Podophyllotoxin has anti-tumor activity, but its water-solubleness is poor and has high cytotoxicity to normal cells, resulting in adverse reactions in tumor treatment, and there are fewer applications of lysine as "linker" in existing nanomedicine carriers.
By introducing a bridge between disulfide bonds and podophyllotoxin and using the coupling method of lysine and polyethylene glycol, podophyllotoxin-lysine-polyethylene glycol anti-tumor compound is formed, and lysine is used to form lysine The modification sites further introduce functional groups to build a dual-function or multifunctional delivery system.
The effective anti-tumor activity of podophyllotoxin is achieved and the toxicity to normal cells is reduced, its water solubility is improved, the targeted drug delivery ability to tumor cells is enhanced, and the tumors of MCF-7, K562 and HCT-116 are significantly improved. Proliferation activity of cells.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of organic synthesis and relates to a podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound and a synthesis method. Background Art
[0002] In recent years, with the development of many new anti-tumor drugs and preparations, traditional drug therapy has made great progress. A large number of emerging new anti-tumor drug molecules have shown significantly improved therapeutic effects and safety; the defects of some early dosage forms and molecules in in vivo metabolism, biocompatibility and pharmacological activity have been compensated by improved dosage forms and structural optimization. In this process, the important position of drug delivery system (DDS) in the design of anti-tumor drug preparations has gradually emerged. Most molecules with anti-tumor activity are not only difficult or unable to achieve the expected therapeutic effect without the assistance of drug delivery systems, but may even cause serious adverse reactions. With the rapid development of nanotechnology, the idea of nano drug carriers as a means of transporting natural products has gradually been included in the research scope of academia. In the field of anti-tumor research, nanoscale drug delivery systems have attracted much attention in the field of anti-tumor drug delivery due to their significantly enhanced cell permeability, extended in vivo circulation half-life due to the EPR effect, targeted tumor accumulation and even controlled release of drugs in tumor cells. Currently reported nano drug carriers include polymer micelles, liposomes and metal nanoparticles. Over the past few decades, nanocarrier-based anticancer drug delivery has evolved from blind systemic administration to targeted, high-precision treatment.
[0003] In the field of new drug design, there are precedents for amino acids and peptides as bridge structures that connect drugs to other molecules that can improve the drug's metabolic process and therapeutic effects in the body, but there are relatively few reports on lysine as a "linker".
[0004] Podophyllotoxin is a natural lignan extracted from the roots and rhizomes of plants of the genus Podophyllum in the family Botrytis cinerea. Its anti-tumor activity has been widely recognized. The anti-tumor effect of podophyllotoxin comes from its inhibition of tubulin polymerization, destruction of microtubule assembly, and induction of tumor mitotic arrest and apoptosis. However, due to its high cytotoxicity to normal cells (especially tissues with vigorous division), as well as its adverse effects such as causing severe gastrointestinal disorders, and its poor water solubility, it is difficult to be directly used in tumor treatment.
[0005] Among the many reports on the design of anti-tumor preparations, the drug delivery system based on polyethylene glycol (PEG) is considered to be an ideal choice for optimizing the release behavior of drug molecules. Since PEG contains both polar oxygen atoms and non-polar (CH2)2 units, PEG has good solubility in most polar and non-polar solvents, and is widely reported as an ideal means to improve the water solubility of hydrophobic drugs. Among PEG and its many derivatives, linear PEG long chains can be combined with other polymers to form amphiphilic polymers to construct new drug delivery systems (such as amphiphilic micelles, vesicles, nanoparticles, etc.), and can also improve the water solubility of drugs by directly conjugating drugs; the branched PEG structure, when used as the outer layer structure of the drug delivery system, has the function of protecting peptides or other biomacromolecules that are easily hydrolyzed by proteases, and reduces immunogenicity through the shielding effect of its "umbrella structure"; multi-arm PEG can be used as a polymer skeleton to construct a cross-linked network such as a hydrogel; and the comb-like PEG structure has been reported to be used to optimize gas separation performance.
[0006] In the field of medicinal chemistry, disulfide bonds have received extensive attention due to their unique chemical properties. By introducing disulfide bonds in drug molecule design, the parent structure can be functionalized or structurally improved from multiple perspectives according to the different design purposes. For example, disulfide bond pairing is an effective means to stabilize the conformation of polypeptides. In the design of anti-tumor drug molecules and formulations, disulfide bonds are more used as a way to give molecules redox responsiveness and enhance the ability to target tumor cells.
[0007] Regarding the selection of "linker", lysine is selected in the present invention as the central "linker" structure to support and connect other functional structures. Its main characteristics are simple structure, rich modification sites, stable chemical properties, low price and non-toxicity. Summary of the invention
[0008] The present invention provides a podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound and a synthesis method. The present invention creates a bridging method between a disulfide bond and podophyllotoxin, and expands the coupling method of polyethylene glycol to a certain extent. Finally, the distal amino group is selectively exposed on lysine as a potential modification site, and other functional groups can be further introduced to form a bifunctional or multifunctional delivery system. The compound can be used for the treatment of tumors and can be applied to the preparation of anti-tumor drugs.
[0009] The purpose of the present invention is achieved through the following technical solutions:
[0010] The structure of a podophyllotoxin-lysine-polyethylene glycol antitumor compound is as follows:
[0011]
[0012] Furthermore, the anti-tumor activity of the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug was evaluated at the cell level, and the results showed that the compound had anti-tumor activity at the in vitro level.
[0013] The above-mentioned podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound is obtained by the following synthetic route:
[0014]
[0015] The modification steps of lysine intermediates are as follows:
[0016]
[0017] The steps of modifying the intermediate of anti-tumor drug (podophyllotoxin) are as follows:
[0018]
[0019] The steps for modifying the polyethylene glycol intermediate are as follows:
[0020]
[0021] The synthetic steps for connecting each fragment are as follows:
[0022]
[0023] The advantages and positive effects of the present invention are as follows:
[0024] 1. The present invention synthesizes for the first time a prodrug molecule with podophyllotoxin as the anti-tumor active part, uses lysine as the delivery system, and the distal amino group can be used as a potential modification site. Other functional groups can be further introduced to form a bifunctional or multifunctional delivery system, creating a bridging method for disulfide bonds and podophyllotoxin, and expanding the coupling method of polyethylene glycol.
[0025] 2. The podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug described in the present invention has anti-tumor properties, can be used for the treatment of non-alcoholic fatty hepatitis, and can also be used in the preparation of anti-non-alcoholic fatty hepatitis drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 is the 1H-NMR spectrum of compound 1.
[0027] Figure 2 1H-NMR spectrum of compound 2.
[0028] Figure 3 1H-NMR spectrum of compound 3.
[0029] Figure 41H-NMR spectrum of compound 4.
[0030] Figure 5 This is the 1H-NMR spectrum of compound 5.
[0031] Figure 6 This is the 1H-NMR spectrum of compound 6.
[0032] Figure 7 This is the 1H-NMR spectrum of compound 9.
[0033] Figure 8 is the 1H-NMR spectrum of compound 10.
[0034] Fig. 9 is the 1H-NMR spectrum of compound 11. Specific implementation methods
[0035] Unless otherwise specified, the raw materials used in the present invention are conventional commercial products; the methods used in the present invention are conventional methods in the art unless otherwise specified.
[0036] The structural formula of the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound of the present invention is as follows
[0037] The podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug was evaluated for its anti-tumor activity at the cell level, and the results showed that the compound had anti-tumor activity at the in vitro level.
[0038] The synthesis route of the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound of the present invention is shown in the following formula:
[0039]
[0040] The modification steps of the lysine intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug of the present invention are shown in the following formula:
[0041]
[0042] The modification steps of the anti-tumor drug (podophyllotoxin) intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug of the present invention are shown in the following formula:
[0043]
[0044] The modification steps of the polyethylene glycol intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug of the present invention are shown in the following formula:
[0045]
[0046] The synthetic steps for connecting each fragment are as follows:
[0047]
[0048] The present invention provides a podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound, the synthesis method of which is as follows:
[0049] The present invention first uses lysine with two amine groups selectively protected and carboxyl groups exposed as the starting material, and constructs a lysine intermediate 2 with propargyl alcohol through an esterification reaction, wherein the alkynyl group is used to connect the PEG module. Secondly, an activated ester end group and a disulfide bond structure are introduced at position 4 of podophyllotoxin to obtain a podophyllotoxin intermediate 6, wherein the activated ester structure is used to connect the lysine module. Afterwards, an azide group is introduced into the long chain of polyethylene glycol monomethyl ether 5000 to obtain a polyethylene glycol intermediate 8 with an azide group at one end, wherein the azide group is used to connect the lysine module. Finally, the above modules are connected in sequence to obtain a prodrug molecule 11, as follows:
[0050] Synthesis of intermediate 1:
[0051] A (500 mg, 1.07 mmol, 1.0 eq.) and propargyl alcohol ethoxylate (104.12 μL, 106.84 mg, 1.07 mmol, 1.0 eq.), 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PYBOP) (555.33 mg, 1.07 mmol, 1.0 eq.) were dissolved in 5 mL of dichloromethane, and N,N-diisopropylethylamine (DIEA) (352.74 μL, 275.84 mg, 2.14 mmol, 2.0 eq.) was added dropwise, and the mixture was reacted at room temperature for 3 h. After the reaction was completed by TLC, the mixture was extracted with 30 mL × 3 dichloromethane, the organic phase was washed with saturated NaCl aqueous solution, and dried over anhydrous Na2SO4 for 30 min. The intermediate 1 was purified by 300-400 mesh silica gel column chromatography (eluent system petroleum ether: ethyl acetate = 1:1) to obtain 386 mg of intermediate 1 with a yield of 65.7%.
[0052] Synthesis of intermediate 2:
[0053] Intermediate 1 (5.17 g, 9.63 mmol) was dissolved in trifluoroacetic acid (TFA) dichloromethane solution (40 mL TFA + 40 mL CH2Cl2) and reacted at room temperature for 1.5-2 h. After the reaction was completed by TLC detection, the system was concentrated by vacuum distillation and purified by 300-400 mesh silica gel column chromatography (gradient elution, eluent system petroleum ether: ethyl acetate = 5: 1-1: 10) to obtain 3.62 g of intermediate 2 with a yield of 86.0%.
[0054] Synthesis of intermediate 3:
[0055] A 30 mL acetonitrile solution of 3-mercaptopropionic acid (409.8 μL, 500 mg, 4.71 mmol, 1.0 eq.) was slowly added dropwise to a 10 mL acetonitrile solution of D (1.56 g, 7.07 mmol, 1.5 eq.) over 30 min, and the mixture was reacted at room temperature for 3 h. After the reaction was completed by TLC, the system was concentrated by vacuum distillation. Purification by 300-400 mesh silica gel column chromatography (eluent system petroleum ether: ethyl acetate = 5:1) gave 489 mg of intermediate 3, with a yield of 48.4%.
[0056] Synthesis of intermediate 4:
[0057] Podophyllotoxin (B) (2.85 g, 6.88 mmol, 1.0 eq.), intermediate 3 (1.78 g, 8.25 mmol, 1.2 eq.), dicyclohexylcarbodiimide (DCC) (1.70 g, 8.25 mmol, 1.2 eq.), 4-dimethylaminopyridine (DMAP) (168.04 mg, 1.38 mmol, 0.2 eq.) were dissolved in 540 mL of dichloromethane and reacted at room temperature for 3 h. A white precipitate gradually precipitated during the reaction. After TLC detection, the reaction was filtered after completion. The filtrate was concentrated by vacuum distillation and washed with 0.1 M dilute hydrochloric acid, saturated NaHCO3 aqueous solution, and saturated NaCl aqueous solution in turn, and dried over anhydrous Na2SO4 for 30 min. Purification by 300-400 mesh silica gel column chromatography (eluent system petroleum ether: ethyl acetate = 5:1) gave 3.94 g of intermediate 4 with a yield of 91.6%.
[0058] Synthesis of intermediate 5:
[0059] A solution of 3-mercapto-1-propanol (94.13 μL, 100.44 mg, 1.09 mmol, 1.0 eq.) in 24 mL of acetonitrile was slowly added dropwise to an 8 mL solution of intermediate 4 (1.0 g, 1.63 mmol, 1.5 eq.) in acetonitrile over 30 min, and the reaction was allowed to proceed at room temperature for 3 h. After the reaction was completed by TLC, the system was concentrated by vacuum distillation. Purification by 300-400 mesh silica gel column chromatography (eluent system: dichloromethane: methanol = 250:1) gave 357 mg of intermediate 5, with a yield of 55.3%.
[0060] Synthesis of intermediate 6:
[0061] The intermediate 5 (350 mg, 590.54 μmol, 1.0 eq.) was dissolved in 25 mL of anhydrous dichloromethane, and triethylamine (Et3N) (91.94 μL, 66.93 mg, 661.41 μmol, 1.12 eq.) was added dropwise, and a solution of p-nitrophenyl chloroformate (166.64 mg, 826.76 μmol, 1.4 eq.) in 25 mL of anhydrous dichloromethane was added dropwise, and the reaction was allowed to proceed overnight at room temperature under argon protection. After the reaction was completed by TLC detection, the system was concentrated by vacuum distillation. Purification was performed by 300-400 mesh silica gel column chromatography (eluent system petroleum ether: ethyl acetate = 10:1 to separate excess p-nitrophenyl chloroformate, and eluent system dichloromethane: methanol = 250:1 to purify the product) to obtain 365 mg of the intermediate 6, with a yield of 81.6%.
[0062] Synthesis of intermediate 7:
[0063] The raw material C (5g, 1mmol, 1.0eq.) was dissolved in 50mL of anhydrous dichloromethane, triethylamine (694.99μL, 505.95mg, 5mmol, 5.0eq.) was added dropwise under an ice-water bath, and methylsulfonyl chloride (MsCl) (386.96 μL, 572.70mg, 5.00mmol, 5.0eq.) was added dropwise, and the mixture was reacted at room temperature for 2-3h under argon protection. After the reaction was completed by TLC detection, it was washed twice with a saturated NaHCO3 aqueous solution, dried over anhydrous Na2SO4 for 30min, recrystallized twice with cold ether and dichloromethane, and the solvent was removed by distillation under reduced pressure to obtain 4.92g of intermediate 7 with a yield of 91.2%.
[0064] Synthesis of intermediate 8:
[0065] Intermediate 7 (5.2 g, 1.04 mmol, 1.0 eq.) and sodium azide (NaN3) (135.22 mg, 2.08 mmol, 2.0 eq.) were dissolved in 20 mL of anhydrous N,N-dimethylformamide (DMF) and reacted at 50°C for 48 h under argon protection. After the reaction was completed by TLC, the mixture was recrystallized twice from cold ether and dichloromethane, and the solvent was removed by distillation under reduced pressure to obtain 3.24 g of intermediate 8 with a yield of 62.8%.
[0066] Synthesis of intermediate 9:
[0067] Dissolve intermediate 2 (120 mg, 274.91 μmol, 1.0 eq.) and intermediate 6 (624.96 mg, 824.73 μmol, 3.0 eq.) in 36 mL of anhydrous dichloromethane, add 240 μL of triethylamine, and react overnight at room temperature under argon protection. The reaction system changes from a colorless or light yellow transparent solution to a yellow transparent solution. After the reaction is completed by TLC detection, the system is concentrated by vacuum distillation and purified by 300-400 mesh silica gel column chromatography (eluent system petroleum ether: ethyl acetate = 1:1) to obtain 104.5 mg of intermediate 9, with a yield of 36.0%.
[0068] Synthesis of intermediate 10:
[0069] Dissolve 0.5 mL of morpholine in 0.5 mL of dichloromethane, add intermediate 9 (40 mg, 37.91 μmol), and react at room temperature for 2 h. After the reaction is completed by TLC, wash with 0.1 M dilute hydrochloric acid, saturated NaHCO3 aqueous solution, and saturated NaCl aqueous solution in sequence, and dry over anhydrous Na2SO4 for 30 min. Purify by 300-400 mesh silica gel column chromatography (gradient elution, eluent system dichloromethane: methanol = 250: 1-50: 1) to obtain 27.0 mg of intermediate 10, with a yield of 85.5%.
[0070] Synthesis of prodrug molecule 11:
[0071] Intermediate 10 (20 mg, 24.01 μmol, 1.0 eq.) and intermediate 8 (120.05 mg, 24.01 μmol, 1.0 eq.) were dissolved in 10 mL of ultra-dry tetrahydrofuran (THF), and cuprous bromide (CuBr) (3.44 mg, 24.01 μmol, 1.0 eq.) was added, and pentamethyldiethylenetriamine (PMDETA) (5 μL, 4.16 mg, 24.01 μmol, 1.0 eq.) was added dropwise. The mixture was reacted at 50°C for 30 to 45 min under argon protection, and the reaction system gradually changed from a light yellow solution to a brown solution. After the reaction was completed by TLC detection, the reaction solution was filtered, concentrated by vacuum distillation, diluted with dichloromethane, and purified by neutral alumina column chromatography to remove Cu salts in the reaction system. The obtained organic phase was concentrated by vacuum distillation and extracted with dichloromethane 20 mL×3 and water, and the organic phase was washed with a saturated NaCl aqueous solution and dried over anhydrous Na2SO4 for 30 min. After distillation under reduced pressure, the product was recrystallized with dichloromethane and cold ether, and the solvent was removed by distillation under reduced pressure to obtain the prodrug molecule 11.
[0072] The relevant tests of the podophyllotoxin-lysine-polyethylene glycol prodrug of the present invention are as follows:
[0073] The present invention synthesizes podophyllotoxin-lysine-polyethylene glycol prodrug for the first time, and uses the MTT method to evaluate the anti-tumor activity of the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug at the cell level for the first time.
[0074] Take cells in the logarithmic growth phase and adjust the cell density to 5×10 4 cells / mL were inoculated on a 96-well plate, 100 μL per well, blank wells and control wells were set up at the same time, and 3 parallel wells were set up for each compound concentration. Cultured in a 37°C, 5% CO2 incubator (suspension cells were cultured for 2 hours, adherent cells were cultured for 24 hours). Compounds with a final concentration of 1 μM and positive control CPT were added, 0.5 μL per well. Blank wells are pure culture medium wells without cells, DMSO and compounds, and control wells are cells with the same concentration of DMSO. After treatment according to the above scheme, the well plate was placed in a 37°C, 5% CO2 constant temperature incubator for 48 hours. Thereafter, 20 μL of 5 mg / mL MTT solution (prepared with PBS, sterilized by 0.22 μm filter membrane) was added to each well, placed in a 37°C, 5% CO2 constant temperature incubator for another 4 hours, and the culture was terminated. For suspension cells, add 100 μL of isopropanol hydrochloride directly to each well. For adherent cells, carefully remove the culture supernatant in the wells and add 100 μL of DMSO to each well. Place in an incubator at 37°C for 10 minutes to allow the purple crystals to fully dissolve. Use an ELISA reader (select 578 and 630 nm for suspension cells and 490 and 630 nm for adherent cells) to measure the absorbance (OD) value of each well. Calculate the cell survival rate according to the measured OD value using the following formula.
[0075] Cell survival rate (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of empty group) × 100%.
[0076] The activity test results are shown in Table 1. The results show that at 1 μM, the prodrug of podophyllotoxin-lysine-polyethylene glycol significantly improves the proliferation activity of MCF-7, K562 and HCT-116 tumor cells compared with podophyllotoxin.
[0077] Table 1 Antitumor activity test results of podophyllotoxin and podophyllotoxin-lysine-polyethylene glycol prodrug
[0078]
[0079] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.
Claims
1. A podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound, characterized in that: The structure is as follows: 。 2. The method for synthesizing the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound according to claim 1, characterized in that: Here are the steps: 。 3. The method for synthesizing the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound according to claim 2, characterized in that: The steps of modifying the lysine intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug are as follows: .
4. The method for synthesizing the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound according to claim 2, characterized in that: The steps of modifying the anti-tumor drug podophyllotoxin intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug are as follows: .
5. The method for synthesizing the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug compound according to claim 2, characterized in that: The steps of modifying the polyethylene glycol intermediate in the podophyllotoxin-lysine-polyethylene glycol anti-tumor prodrug are as follows: .