An NQO1-activated prodrug, its preparation method and application

Abstract

This invention belongs to the field of nanoprodrug technology, specifically relating to an NQO1-activated prodrug, its preparation method, and its applications. The prodrug molecule of this invention can self-assemble into a stable nanodrug in an aqueous phase without the use of excipients. This nanodrug exhibits good structural stability, possesses both active and passive targeting capabilities, and achieves specific recognition of tumor cells, resulting in precise chemotherapy, improved treatment efficiency, and reduced toxic side effects on normal cells. Furthermore, the release of irinotecan can be monitored in real time using changes in its fluorescence. The preparation method is simple and holds promise for widespread application in the specific treatment of cancers with NQO1 overexpression.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanoprodrug. More particularly, it relates to a NQO1-activated prodrug and a preparation method and application thereof BACKGROUND

[0002] Cancer is one of the most common and most deadly diseases that threaten human life and health. Traditional methods for treating cancer include surgery, chemotherapy and radiotherapy. However, these treatments have strong side effects, are prone to drug resistance, lack of targeting to cancer cells, are prone to recurrence, and the treatment results are difficult to satisfy. Irinotecan is a broad-spectrum chemotherapy drug used to treat metastatic colon cancer, rectal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, etc. However, it has a short circulation time in the body, poor selectivity, and serious side effects such as delayed diarrhea, acute cholinergic syndrome (abdominal pain, sweating, tearing, drooling and pupil constriction), severe neutropenia, bone marrow suppression, intestinal obstruction, nausea, vomiting, hair loss, fatigue, etc.

[0003] In order to solve the above problems of irinotecan, the prior art often prepares irinotecan into nanoparticles, so that it has a more suitable half-life, prolongs the circulation time of the drug in the body and thus is not easily metabolized; passive targeting can also be achieved through enhanced permeability and retention effect (EPR), effectively increasing its selectivity to tumor cells and reducing its toxic side effects on normal cells. However, irinotecan has good hydrophilicity and cannot self-assemble into a nanodrug in an aqueous phase, and needs to be modified and combined with excipients to form a nanodrug, which results in a low drug loading rate (DL%) of the drug (<20%), so that the drug cannot reach an effective therapeutic concentration in tumor cells, and is prone to induce drug resistance in cancer cells. In order to reduce the use of excipients, Deyue Yan et al. coupled irinotecan with the anticancer drug chlorambucil to obtain a self-assembling nanodrug (Combination of Small Molecule Prodrug and Nanodrug Delivery: Amphiphilic Drug-Drug Conjugate for Cancer Therapy. Ping Huang, Dali Wang, Yue Su, Wei Huang, Yongfeng Zhou, Daxiang Cui, Xinyuan Zhu, Deyue Yan. J. Am. Chem. Soc. 2014, 136, 33, 11748-11756), but the nanodrug cannot achieve specific activation of the prodrug in tumor cells, making it difficult to achieve precise treatment. SUMMARY

[0004] The technical problem solved by the present application is to overcome the short in-vivo circulation time, poor selectivity, serious toxic side effects and reduced drug loading rate after using excipients of irinotecan, and to provide an NQO1 activated prodrug which can self-assemble into NQO1 activated nanomedicine with specific selectivity for tumor cells without adding excipients.

[0005] The present application aims to provide a preparation method of the NQO1 activated prodrug.

[0006] Another object of the present application is to provide an NQO1 activated nanomedicine.

[0007] Another object of the present application is to provide a preparation method of the NQO1 activated nanomedicine.

[0008] Another object of the present application is to provide an application of the NQO1 activated prodrug or the NQO1 activated nanomedicine in the preparation of an antitumor drug.

[0009] The above objects of the present application are achieved by the following technical solutions.

[0010] An NQO1 activated prodrug, the molecular formula of the NQO1 activated prodrug is C 46 H 52 O9N4, and the structural formula is shown in formula (I):

[0011]

[0012] NQO1 is a cytoplasmic two-electron oxidoreductase with highly specific catalytic properties, which can catalyze the reduction of various quinone compounds to the corresponding phenols. In the process of cancer treatment, it is found that quinone oxidoreductase 1 (NQO1) is overexpressed in various tumors, and is an ideal target for activating prodrugs.

[0013] Irinotecan itself has good hydrophilicity, and the hydroxyl group in the lactone ring is connected to the carboxyl group of the NQO1 response group through an ester bond to obtain an amphiphilic prodrug molecule, so that it can self-assemble into stable nanomedicine in water without using excipients.

[0014] The 3-methyl-3-(2,4,5-trimethyl-3,6-dicyclohexanone-1,4-diene-1-yl) butyric acid (NQO1 response group) is used as the protecting group of the lactone ring hydroxyl of irinotecan, so that the NQO1 response release active irinotecan is achieved. On the one hand, the nano drug is specifically activated after entering the cancer cells with overexpression of NQO1, the quinone of the response group in the prodrug molecule is specifically reduced to phenol, the phenolic hydroxyl attacks the ester bond formed by the response group and irinotecan, so that the cyclization reaction occurs, and the active drug irinotecan is released, which can inhibit the activity of topoisomerase I for precise chemotherapy, thereby improving the treatment efficiency and reducing the toxic side effects; on the other hand, irinotecan itself has strong fluorescence, but after being modified by the response group to form a prodrug, the electron-deficient property of the quinone causes the fluorescence of irinotecan itself to be quenched, and when irinotecan is released, the fluorescence is restored, and the release process of irinotecan can be monitored in real time by using the change of the fluorescence of the NQO1 activated prodrug itself.

[0015] The application also protects a preparation method of the NQO1 activated prodrug, and the synthesis route is as follows:

[0016]

[0017] including the following steps:

[0018] Irinotecan, 3-methyl-3-(2,4,5-trimethyl-3,6-dicyclohexanone-1,4-diene-1-yl) butyric acid (NQO1 response group), a condensing agent and a catalyst are dissolved in an organic solvent to react completely, and then post-processing is performed, so that the product is obtained.

[0019] Preferably, the condensing agent is one of 1-(3-dimethylpropyl)-3-ethyl carbodiimide hydrochloride (EDC-HCl), dicyclohexyl carbodiimide (DCC), diethyl azodicarboxylate or carbonyl diimidazole.

[0020] More preferably, the condensing agent is 1-(3-dimethylpropyl)-3-ethyl carbodiimide hydrochloride (EDC-HCl).

[0021] Preferably, the catalyst is one of 4-dimethylamino pyridine (DMAP), pyridine, 4-pyrrolidinyl pyridine or triethylamine.

[0022] Preferably, the temperature of the reaction is 15-35℃.

[0023] Preferably, the time of the reaction is 20-30h.

[0024] More preferably, the time of the reaction is 24h.

[0025] Preferably, the method of post-treatment is column chromatography purification with eluent of dichloromethane:methanol = (10-30) :1 by volume ratio.

[0026] More preferably, the method of post-treatment is column chromatography purification with eluent of dichloromethane:methanol = 20:1 by volume ratio.

[0027] Preferably, the molar ratio of irinotecan to 3-methyl-3-(2,4,5-trimethyl-3,6-dicyclohexanone-1,4-diene-1-yl) butyric acid, condensing agent and catalyst is 1: (0.5-1) : (1.5-2.5) : (1.5-2.5).

[0028] More preferably, the molar ratio of irinotecan to 3-methyl-3-(2,4,5-trimethyl-3,6-dicyclohexanone-1,4-diene-1-yl) butyric acid, condensing agent and catalyst is 5:4:10:10

[0029] The present application also protects a NQO1-activated nano-drug obtained by self-assembly of molecules of NQO1-activated prodrugs.

[0030] Preferably, the NQO1-activated nano-drug is self-assembled from NQO1-activated prodrugs in an aqueous phase without using excipients.

[0031] The present application also protects a method for preparing a NQO1-activated nano-drug, comprising the following steps:

[0032] S1. Dissolve the NQO1-activated prodrug in dimethyl sulfoxide to obtain solution A;

[0033] S2. Drop the solution A obtained in step S1 into distilled water, stir, and separate to obtain.

[0034] Preferably, the volume ratio of dimethyl sulfoxide in step S1 to distilled water in step S2 is 1: (1-3).

[0035] Preferably, in step S2, the stirring is magnetic stirring; the stirring speed is 50-2000 rpm; the stirring temperature is 20℃-80℃; and the stirring time is 1-60 min.

[0036] More preferably, in step S2, the stirring time is 10 min.

[0037] Preferably, in step S2, the separation method is dialysis.

[0038] Preferably, the molecular weight cut-off of the dialysis bag used in the dialysis process is <1000.

[0039] Preferably, the dialysis time is 12 to 36 hours.

[0040] More preferably, the dialysis time is 24 hours.

[0041] Preferably, the water change frequency for dialysis is once every 1 to 5 hours.

[0042] More preferably, the water change frequency for the dialysis is once every 3 hours.

[0043] This invention also protects the use of an NQO1-activated prodrug and an activated nanodrug in the preparation of antitumor drugs.

[0044] The present invention has the following beneficial effects:

[0045] The prodrug molecule of this invention can self-assemble into a stable nanodrug in an aqueous phase without the use of excipients. This nanodrug has good structural stability, combines active and passive targeting functions, and achieves specific recognition of tumor cells, thus achieving precise chemotherapy, improving treatment efficiency, and reducing toxic side effects on normal cells. At the same time, the release of irinotecan can be monitored in real time by utilizing the fluorescence change of irinotecan. Moreover, the preparation method is simple and it is expected to be widely used for the specific treatment of cancers with NQO1 overexpression. Attached Figure Description

[0046] Figure 1 NQO1-activated prodrug molecule 1 HNMR spectrum.

[0047] Figure 2 NQO1-activated prodrug molecule 13 C10 NMR spectrum.

[0048] Figure 3 This is the mass spectrum of an NQO1-activated prodrug molecule.

[0049] Figure 4 This is a dynamic laser scattering pattern of NQO1-activated nanomedicine.

[0050] Figure 5 This is a critical aggregation concentration curve for NQO1-activated nanomedicines.

[0051] Figure 6 This is a transmission electron microscope image of the NQO1-activated nanomedicine.

[0052] Figure 7 The graph shows the changes in particle size and polydispersity index of NQO1 activated nanoparticles over time.

[0053] Figure 8The fluorescence spectrum of the NQOl-activated nano-drug at different times under the action of NQOl.

[0054] Figure 9 The fluorescence intensity change curve of the NQOl-activated nano-drug at 436 nm at different times under the action of NQOl.

[0055] Figure 10 The drug release curve of the NQOl-activated prodrug under the action of NQOl and without the action of NQOl.

[0056] Figure 11 The cytotoxicity curve of the NQOl-activated prodrug on different cell lines. DETAILED DESCRIPTION

[0057] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0058] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0059] Example 1: Synthesis of NQOl-activated prodrug

[0060] In a round-bottom flask, irinotecan (300 mg, 0.511 mmol), 3-methyl-3-(2,4,5-trimethyl-3,6-dihydroxycyclohexa-1,4-dien-1-yl)butanoic acid (102.2 mg, 0.408 mmol), 1-(3-dimethylpropyl)-3-ethyl carbodiimide hydrochloride (EDC-HCl, 195.92 mg, 1.022 mmol) and 4-dimethylaminopyridine (DMAP, 124.89 mg, 1.022 mmol) were added, and 16 mL of dichloromethane was added, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, silica gel powder was added and dried, and silica gel column chromatography (dichloromethane:methanol = 20:1) was carried out to obtain NQOl-activated prodrug in the form of light yellow solid, with a yield of 67.7%.

[0061] 1 The H NMR spectrum results are shown in Figure 1

[0062] 1 ​H NMR (400 MHz, DMSO-d6, ppm) δ 8.19 (s, 1H), 7.99 (s, 1H), 7.68 (s, 1H), 6.88 (s, 1H), 5.45 (s, 2H), 5.32 (s, 2H), 4.21 (d, J = 7.0 Hz, 2H), 3.18 (s, 4H), 3.12-2.86 (m, 3H), 2.56 (s, 3H), 2.05 (s, 5H), 1.84 13 (s, 3H), 1.74 (m, 5H), 1.54 (m, 6H), 1.41 (s, 6H), 1.29 (t, J = 7.6 Hz, 5H), 0.85 (t, J = 7.4 Hz, 3H).

[0063] 13 The C NMR spectrum results are shown as follows: Figure 2

[0064] 13 C NMR (101 MHz, DMSO-d6, ppm) δ 191.77, 190.46, 187.09, 177.76, 171.40, 167.44, 156.95, 153.07, 151.78, 150.54, 149.23, 146.94, 146.74, 145.73, 145.49, 142.89, 139.07, 138.40, 131.92, 128.83, 127.59, 126.59, 119.30, 115.44, 94.87, 76.32, 66.74, 61.86, 61.83, 55.38, 50.11, 46.94, 38.37, 30.60, 28.82, 26.28, 22.69, 12.21, 7.97.

[0065] The ESI-MS spectrum results are shown as follows: Figure 3

[0066] MS (ESI): m / z [H+M] 819.45, the molecular weight of the prodrug is 818.39, and the test result is the same as the theoretical value.

[0067] In summary, the structure of the prodrug is correct through nuclear magnetic resonance hydrogen spectrum, carbon spectrum characterization, structure analysis and mass spectrometry analysis.

[0068] Example 2: Synthesis of NQO1-activated nanodrug

[0069] 5 mg of NQO1-activated prodrug was dissolved in 2 mL of dimethyl sulfoxide, slowly dropped into 4 mL of distilled water under stirring condition, stirred for 15 minutes, dialyzed with dialysis bag (molecular weight <1000) for 24 hours, and the water was changed every 3 hours to obtain the nanodrug, which was stored in the refrigerator.​​

[0070] Example 3: Synthesis of NQOl-activated nanodrug

[0071] 5 mg of NQOl-activated prodrug was dissolved in 2 mL of dimethyl sulfoxide, slowly added dropwise into 6 mL of distilled water under stirring, stirred for 20 minutes, dialyzed in a dialysis bag (molecular weight cut-off < 1000) for 24 hours, and the water was changed every 5 hours to obtain the nanodrug, which was stored in a refrigerator.

[0072] Experimental Example 1: Particle size test of NQOl-activated nanodrug

[0073] The nanodrug obtained in Example 2 was subjected to dynamic laser scattering analysis by using a nanoparticle size and Zeta potential analyzer to test the particle size and polydispersity index (PDI) value of the nanodrug in an aqueous solution system, and the test results are shown in Figure 4 The average particle size was 99.15 nm and the average PDI was 0.125, indicating that the NQOl-activated nanodrug had good particle size distribution and excellent dispersibility.

[0074] Experimental Example 2: Determination of critical aggregation concentration (CAC) of NQOl-activated nanodrug

[0075] Pyrene was used as a fluorescent probe, and 3 ul (6 x 10 -4 mol / L) of pyrene acetone solution was added to 3 mL of NQOl-activated prodrug aqueous solution with different concentrations (10 -4 -2 mg / mL). The concentration of pyrene acetone solution in each sample was kept at (6 x 10 - 7 The fluorescence emission spectrum data was recorded, the emission wavelength was 335 nm, and the logarithmic value of the concentration of NQOl-activated prodrug was taken as the abscissa, and the fluorescence intensity of pyrene at 384 nm and 373 nm was taken as the ordinate to plot a graph, and the critical aggregation concentration was obtained by the turning point of the curve: according to Figure 5 the two-line equation in the above formula, the X value at the intersection was -2.8972, i.e. the logarithmic value of the concentration was -2.8972, and the critical aggregation concentration of the nanodrug was 1.267 ug / mL.

[0076] Experimental Example 3: Morphology characterization of NQOl-activated nanodrug

[0077] The nanodrug obtained in Example 2 was subjected to transmission electron microscopy test, and the results are shown in Figure 6 The particle morphology of the nanodrug was circular with a diameter of about 95 nm, which was consistent with the results obtained by dynamic laser scattering in Experimental Example 1.

[0078] Experimental Example 4: Stability test of NQOl-activated nanodrug​

[0079] The nanodrugs obtained in Example 2 were subjected to dynamic laser scattering analysis every three days until the 30th day. The results, as shown in Table 1, indicate that the particle size and polydispersity index of the nanodrugs changed little. This indicates that the nanodrugs have good stability and can be stored for a long time. Figure 7

[0080] Experimental Example 5: Fluorescence responsive analysis of NQOl-activated nanodrugs on NQOl

[0081] The responsive group in the NQOl-activated prodrug molecule can quench the fluorescence of irinotecan through a photoinduced electron transfer mechanism. Under the action of NQOl, the responsive group is removed, and the fluorescence of irinotecan is restored, so the release process of irinotecan itself can be monitored by the increase in fluorescence of irinotecan.

[0082] The specific experimental method is as follows: 0.01 mmol / L, pH = 7.4 PBS buffer solution was used to prepare a mixture of the nanodrugs obtained in Example 2 and reduced coenzyme I (NADH), with their concentrations being 5 uM and 100 uM, respectively. NQOl enzyme was added to make its concentration 60 ug / mL, and the mixture was incubated at 37°C. The fluorescence spectrum of the reaction system was tested at different time periods using 365 nm excitation light. The test results, as shown in Table 2, indicate that the fluorescence intensity of the solution gradually increased with time, the fluorescence emission wavelength was strongest at 436 nm, and the fluorescence intensity reached a maximum value at about 20 minutes. It can be seen that the nanodrugs have good responsiveness to NQOl. Figure 8 9

[0083] Experimental Example 6: Drug release test of NQOl-activated nanodrugs

[0084] The PBS solution of the nanodrugs obtained in Example 2 was placed in a dialysis bag, and NQOl was added. At different time points (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 28, and 40 minutes), 10 uL of the solution outside the dialysis bag was taken out for high performance liquid chromatography test. The release amount was calculated according to the standard curve of irinotecan. The results, as shown in Table 3, indicate that in the absence of NQOl, the nanodrugs do not release active drugs irinotecan, and in the presence of NQOl, the nanodrugs can quickly release irinotecan. This result is in good agreement with the results in Experimental Example 5. Figure 10

[0085] Experimental Example 7: Toxicity analysis of NQOl-activated nanodrugs on different cell lines

[0086] ​​​​The toxicity of the nano-drug obtained in Example 2 to different cell lines was studied by MTT method. The cells used were human lung cancer cell line (A549, high expression of NQOl), human prostate cancer cell (LNCaP, moderate expression of NQOl), human pancreatic cancer cell (SW1990, moderate expression of NQOl), and normal cell (L929, low expression of NQOl). The implementation was as follows: under the condition of 37°C and 5% carbon dioxide, each cell was cultured in a 96-well plate at a density of about 5000 cells per well, after 24 hours of culture, different concentrations of nano-drug (0-10 ug / mL) were added, and pure culture medium was used as negative control, and then the culture was continued under the condition of 37°C and 5% carbon dioxide for 48 hours; 0.5 mg / mL of tetrazolium salt (MTT) solution was added to each well, and the culture was continued under the above condition for 4 hours; then 150 μL of dimethyl sulfoxide was added to each well, and oscillated for 5 minutes, and finally the 96-well plate was placed in an enzyme marker for detection, wherein the detection wavelength was 570 nm. The results are shown in Table 1. Figure 11 As shown in Table 1, the survival rate of normal cells all reached more than 90%, while the survival rate of the remaining cancer cells decreased significantly with the increase of concentration. It is shown that the nano-drug prepared by the present application has excellent specific selectivity and basically no toxic side effects.

[0087] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. An NQOl-activated nanodrug, characterized in that, The NQO1-activated nano-drug is obtained by self-assembly of molecules of NQO1-activated prodrug; the molecular formula of the NQO1-activated prodrug is C 46 H 52 O9N4, as shown in formula (I): （Ⅰ）。 2. The preparation method of the NQO1 activated nanomedicine according to claim 1, characterized in that, Comprising the following steps: S1. Dissolve the NQO1-activated prodrug in dimethyl sulfoxide to obtain solution A; S2. Drop solution A obtained in step S1 into distilled water, stir, and separate to obtain.

3. The preparation method according to claim 2, characterized in that, In step S2, the separation method is dialysis.

4. Use of the NQO1-activated nanoparticle drug of claim 1 in the preparation of an antitumor drug.

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