Radioactive prodrug compound as well as preparation method and application thereof

Abstract

The invention discloses a radiation-activated prodrug as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The novel radiation-activated prodrugs (RAPs) are prepared by the optimization design of the structures of the sorafenib and the regorafenib (nitrogen-containing heterocyclic rings), so that the novel radiation-activated prodrugs (RAPs) can be used for preparing the novel radiation-activated prodrugs (RAPs) and the novel radiation-activated prodrugs (RAPs). External X-ray irradiation is utilized, the prodrug can be specifically activated in a tumor microenvironment, and a raw drug with strong anti-tumor activity is released, so that precise killing of tumors is realized. Meanwhile, the prodrug keeps relatively low systemic toxicity before being exposed to X-ray irradiation, so that complete compatibility with a radiotherapy scheme is ensured, and the potential of the prodrug serving as a tumor targeted therapy drug is shown. The radiation-activated prodrug has a structure as shown in a general formula I: # imgabs0 #, wherein I1: R = H; i2: R = F.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a radiation-activated prodrug compound and a preparation method and application thereof. Background Art

[0002] Cancer is one of the major global public health challenges, and its treatment strategies are constantly evolving. Tumor radiotherapy is a local treatment method that uses radiation to treat tumors. It has the advantages of being non-invasive, highly precise, and having a wide range of applications. Radiotherapy alone is limited by its total dose and radiotoxicity. Clinically, radiotherapy combined with chemotherapy is often used to treat cancer. Although combined therapy can improve patient prognosis, it still has limitations such as poor selectivity and large toxic side effects. Photosensitizers designed and developed using light as an external excitation condition produce high concentrations of reactive oxygen species under excitation at specific wavelengths, mediating tumor cell apoptosis or necrosis, which can effectively address the dose-limiting toxicity and drug resistance issues of traditional chemotherapy drugs. However, PDT relies on specific wavelengths (usually visible light or near-infrared light) for activation, and short-wavelength light has limited penetration depth in tissues, making it difficult to treat deep tumors or large lesions. Because radiation has precise spatiotemporal controllability and dose controllability, it is an ideal stimulation method for achieving controlled drug release in the treatment of deep tissue tumors. Using highly tissue-penetrating X-rays (or gamma rays) as an exogenous stimulus to control the precise release of drugs, the development of tumor-specific radiotherapy-activated prodrugs (RAPs) has important clinical significance for treating tumors while reducing the toxicity of combined therapies. The main principle of RAPs is to release functional molecules or therapeutic drug molecules through a covalent bond cleavage reaction mediated by active substances (including hydrated electrons, hydroxyl radicals, hydrogen radicals, etc.) produced by the radiolysis of water by ionizing radiation. Compared with traditional chemotherapy drugs, RAPs maintain low toxicity before exposure to the ionizing radiation irradiation area. When irradiated by ionizing radiation, chemical reactions occur, and the molecular structure is transformed into a highly active form, which then produces a therapeutic effect on the lesions in the irradiated area. This allows the release of drugs with a high degree of temporal and spatial precision, minimizes damage to normal tissues, and reduces the systemic toxicity of traditional chemotherapy drugs.

[0003] Studies have reported that the oxidized N-oxides of tertiary amines, as bioorthogonal reagents, exhibit excellent biocompatibility and stability in the bloodstream. Fluorescent probes and antitumor prodrugs designed based on N-oxides can be reduced to tertiary amines in response to exogenous stimuli in the hypoxic tumor microenvironment, exerting tumor-site-specific imaging and therapeutic effects. Sorafenib and regorafenib are multikinase inhibitors that simultaneously inhibit multiple kinases present within and on the cell surface, including RAF kinase, vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial growth factor receptor-3 (VEGFR-3), platelet-derived growth factor receptor-β (PDGFR-β), KIT, and FLT-3, exhibiting multiple anti-tumor proliferative effects. However, this broad range of kinase inhibitory activity leads to a high incidence of side effects, including skin reactions, digestive system reactions, and adverse cardiovascular events.

[0004] Therefore, it is necessary to develop a prodrug of sorafenib and regorafenib to reduce the in vivo toxicity of sorafenib and regorafenib, and to achieve targeted radioreduction release of the original drug at the tumor site, providing patients with a more efficient and safer treatment plan. Summary of the Invention

[0005] The purpose of the present invention is to solve the defects in the prior art and provide a novel radiation-activated prodrug compound and its preparation method and application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect of the present invention, a radiation-activated prodrug compound is provided, wherein the radiation-activated prodrug compound has the following structure:

[0008]

[0009] Wherein, R=H or F.

[0010] Specifically, I1:R=H; I2:R=F.

[0011] The structural formula of I1(R=H) is:

[0012]

[0013] The structural formula of I2(R=F) is:

[0014]

[0015] In a second aspect of the present invention, a method for preparing the above-mentioned radiation-activated prodrug compound is provided, wherein the original drug and 3-chloroperbenzoic acid are dissolved in dichloromethane, reacted at room temperature until the reaction is complete, and the solvent is removed by rotary evaporation. The resulting product is purified by column chromatography to obtain a white solid, which is the radiation-activated prodrug compound; wherein the original drug is sorafenib or regorafenib.

[0016] The radioactive prodrug compound I1 (R=H) was prepared by dissolving the known compound sorafenib and 3-chloroperbenzoic acid in dichloromethane and reacting them at room temperature for 72 hours. After completion of the reaction, the solvent was removed by rotary evaporation. The resulting product was purified by column chromatography to yield I1 as a white solid.

[0017] The radioactive prodrug compound I2 (R=F) was prepared by dissolving the known compound regorafenib and 3-chloroperbenzoic acid in dichloromethane and reacting them at room temperature for 72 hours. After completion of the reaction, the solvent was removed by rotary evaporation. The resulting product was purified by column chromatography to yield I2 as a white solid.

[0018] The third aspect of the present invention provides a use of the above-mentioned radiation-activated prodrug compound in the preparation of a radiation-activated prodrug, wherein the radiation-activated prodrug is used to specifically release the original drug under radiation conditions.

[0019] The fourth aspect of the present invention provides the use of the above-mentioned radiation-activated prodrug compound in the preparation of a drug for treating tumors.

[0020] In some embodiments of the present invention, the tumor is one of liver cancer, colon cancer and lung cancer.

[0021] This invention optimizes the pyridine ring (nitrogen-containing heterocycle) of sorafenib and regorafenib to create an N-oxide prodrug. Under external X-ray irradiation, the prodrug is specifically activated in the tumor microenvironment, releasing the original drug with potent anti-tumor activity, thereby achieving precise tumor destruction. Furthermore, the prodrug maintains low systemic toxicity before exposure to X-rays, ensuring full compatibility with radiotherapy regimens and demonstrating its potential as a targeted tumor therapy.

[0022] The radiation-activated prodrug provided by the present invention has the following advantages over the prior art:

[0023] The compounds of the present invention can be used to generate water and electrons (e - aq ) is specifically reduced to the original drug with potent anti-tumor activity.

[0024] The compounds of the present invention are activated by X-ray irradiation. Cytotoxicity tests on multiple tumor cells have shown that the compounds of the present invention can significantly inhibit tumor cell proliferation under hypoxic conditions and exhibit significant cytotoxicity. Furthermore, the prodrug remains in a low-activity state before exposure to X-ray irradiation.

[0025] The compounds of the present invention were injected into tumor-bearing mice via the tail vein and then irradiated with X-rays at the tumor site. Compared with the PBS group, the compounds of the present invention significantly inhibited tumor growth. The anti-tumor therapeutic effect was fully verified.

[0026] Compared with the original drug, the compound of the present invention has significantly reduced blood toxicity and liver toxicity, showing good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Graph showing the radiation dose-dependent response test results of the radiation-activated prodrug of the present invention;

[0028] Figure 2 Graph showing the results of a radiation-specific activation release test of the radiation-activated prodrug of the present invention;

[0029] Figure 3 This is an in vivo anti-tumor activity test of the radiation-activated prodrug obtained in Example 1 of the present invention;

[0030] Figure 4 This is a biosafety test of the radiation-activated prodrug obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings so that those skilled in the art can better understand the advantages and features of the present invention and thus more clearly define the scope of protection of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0032] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0034] The preparation method of the radioactive prodrugs of the present invention comprises dissolving the known compound sorafenib and 3-chloroperbenzoic acid in dichloromethane, reacting the mixture at room temperature for 72 hours, and then oxidizing the mixture to obtain the novel radioactive prodrugs I1 and I2. The synthesis route is shown below.

[0035]

[0036] Example 1: Preparation of 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]formyl]amino]phenoxy]-N-methylpyridine-2-carboxamide 1-oxide (I1)

[0037] 4-[4-[[[[4-Chloro-3-(trifluoromethyl)phenyl]amino]formyl]amino]phenoxy]-N-methylpyridine-2-carboxamide (464 mg, 1 mmol) and 3-chloroperoxybenzoic acid (5 mmol) were dissolved in 10 ml of dichloromethane. The reaction was allowed to proceed at room temperature for 72 hours. After completion of the reaction, the solvent was removed by rotary evaporation. The resulting product was purified by column chromatography to afford I1 as a white solid in 80% yield.

[0038] Spectral data of I1: 1 HNMR (400MHz, DMSO-d6) δ11.41(d,J=4.9Hz,1H,NH),9.22(s,1H,NH),9.01(s,1H,NH),8.37(d,J=7.2Hz,1H,ArH),8.11(d,J =2.5Hz,1H,ArH),7.67–7.54(m,5H,5ArH),7.29–7.23(m,1H,ArH),7.18(d,J=8.6Hz,2H,2ArH),2.85(d,J=4.8Hz,3H,CH3). 13 C NMR(101MHz,DMSO-d6)δ159.81,157.09,152.91,148.67,142.87,140.81,139.77,137.55, 132.44,127.32,127.01,124.63,123.55,122.81,121.55,120.96,117.24,114.12,26.30.

[0039] Example 2: Preparation of 4-[4-[[[4-chloro-3-(trifluoromethyl)phenyl]amino]formyl]-3-fluorophenoxy]-N-methylpyridine-2-carboxamide 1-oxide (I2)

[0040] [4-[[[[4-Chloro-3-(trifluoromethyl)phenyl]amino]formyl]amino]phenoxy]-N-methylpyridine-2-carboxamide (482 mg, 1 mmol) and 3-chloroperoxybenzoic acid (5 mmol) were dissolved in 10 ml of dichloromethane. The reaction was allowed to proceed at room temperature for 72 hours. After completion of the reaction, the solvent was removed by rotary evaporation. The resulting product was purified by column chromatography to afford I2 as a white solid in 83% yield.

[0041] Spectral data of I2: 1 H NMR (400MHz, DMSO-d6) δ11.38(q,J=4.8Hz,1H,NH),9.54(s,1H,NH),8.76(s,1H,NH),8.42(d,J=7.2Hz,1H,ArH),8.22– 8.10(m,2H,2ArH),7.71(s,3H,3ArH),7.63–7.51(m,4H,4ArH),7.10(d,J=8.9Hz,1H,ArH),2.88(d,J=4.9Hz,3H,CH3). 13 CNMR(101MHz,DMSO-d6)δ166.57,159.78,156.40,152.63,143.00,140.92,139.47,133 .81,133.36,132.59,131.14,129.30,128.40,123.45,117.65,117.09,114.74,26.35.

[0042] Example 3: Radioactive Properties of Compounds of the Invention

[0043] The responsiveness of the compounds of the present invention to different radiation doses was detected by high performance liquid chromatography.

[0044] The specific method is to prepare a 10 mM stock solution of the compound of the present invention in DMSO and dilute it with oxygen-free water to a final concentration of 200 μM. These solutions are irradiated with different doses of X-rays. The reaction mixture is analyzed by high performance liquid chromatography. The results are as follows: Figure 1 shown.

[0045] in, Figure 1 A is a radiation dose-dependent response diagram of the invention compound I1. Radiation reduction of the invention compound I1 occurs immediately at 10 Gy, and the production of sorafenib is detected by HPLC. When the radiation dose reaches 40 Gy, I1 is almost completely reduced to sorafenib. Figure 1 B is the radiation dose-dependent response fitting curve of the invention compound I1. Figure 1C is the HPLC result of the radiation dose-dependent response of the inventive compound I2. When the radiation dose reaches 40 Gy, I2 is almost completely reduced to regorafenib. Figure 1 D is the radiation dose-dependent response fitting curve of the inventive compound I2.

[0046] Example 4: Specific responsiveness of the compounds of the present invention to radiation

[0047] The specific response of the compound of the present invention to X-ray was detected by high performance liquid chromatography. Due to the presence of various factors affecting the tumor microenvironment, it is necessary to exclude other physiological substances such as various metal ions (Na + , K + 、Cu 2+ 、Fe 2+ 、Zn 2+ ) and enzymes (NQO1, AzoR, LAP) on radioreduction. Compound I1 of the present invention was added to the corresponding biological analytes for incubation at room temperature, followed by HPLC determination.

[0048] like Figure 2 As shown in Figure A, after different interfering ions were added to the solution of the compound I1 of the present invention, there was no obvious change in the generation of sorafenib; however, after X-ray irradiation, the generation of sorafenib increased significantly, proving that the compound I1 of the present invention has good response specificity to X-rays.

[0049] Figure 2 B is a solution of the compound I1 of the present invention, in which different bioreductants are added to simulate the redox environment in the biological environment, and the e generated by radiation - aq Compared with the strong reducing effect of these biological reducing agents, the reduction of compound I1 of the present invention by these biological reducing agents is negligible. This indicates that compound I1 of the present invention has good response specificity to X-rays.

[0050] Example 5: Activity test of the compounds of the present invention in different tumor cells.

[0051] The toxicity of compounds I1 and I2 of the present invention to tumor cells (human lung cancer cell line A549, human colon cancer cell line HT29, and human liver cancer cell line HepG2) was evaluated in vitro using a methylthiazolium tetrazolium (MTT) colorimetric assay. HepG2, A549, and HT29 cells in the logarithmic growth phase were trypsinized and resuspended in a medium containing 10% fetal bovine serum. The cells were plated at 1×10 4 / well were seeded on a 96-well plate, 100 μL of culture medium was added to each well, and the incubation continued for 24 hours. The old culture medium was then replaced with fresh culture medium containing different concentrations of drugs, and 100 μL was added to each well. The I1+X-ray group and the I2+X-ray group were placed in a transparent box and incubated for 2 hours using an AnaeroPack (D-07) hypoxia pack to simulate the tumor hypoxia environment. They were then irradiated with 20 Gy of X-rays and continued to incubate in normoxia for 72 hours. Finally, MTT (5 mg / ml) was added to the 96-well plate and reacted in an incubator for 4 hours. The absorbance of each well was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay, and the cell proliferation inhibition rate was calculated.

[0052] After a series of tumor cell anti-proliferation activity tests, it was found that compounds I1 and I2 of the present invention had certain inhibitory activity on the proliferation of HepG2, A549 and HT29 tumor cells under hypoxic conditions, and the inhibitory activity on tumor cells was significantly enhanced after X-ray irradiation.

[0053] Table 1 In vitro cytotoxicity of the compounds of the present invention (IC 50 ,μM)

[0054]

[0055] Example 6: Tumor inhibition test of the compounds of the present invention in tumor-bearing mice.

[0056] A HepG2 tumor-bearing mouse model was established to evaluate the tumor therapeutic potential of the compound I1 of the present invention. The mice were randomly divided into a PBS group, a sorafenib group, a compound I1 of the present invention group, and a compound I1+X-ray group. Sorafenib and the compound I1 of the present invention were both administered by intravenous injection. Two hours after administration, mice in the compound I1+X-ray group received local tumor irradiation (4Gy). Tumor volume and body weight were monitored every 3 days for 21 consecutive days. At the end of the experiment, the tumor inhibition treatment effect of each group was evaluated, and the results are shown as follows: Figure 3 shown.

[0057] in Figure 3 A is the experimental scheme for the tumor inhibition of compound I1 of the present invention. Figure 3 B is the average tumor volume of mice. Figure 3 C is a tumor image collected from a tumor-bearing mouse. Figure 3 D is the average tumor weight of mice. Figure 3 E is the change in mouse body weight during the experiment.

[0058] The results showed that the compound I1 of the present invention had a certain inhibitory effect on tumor growth in the case of single treatment (inhibition rate was 21%). After receiving radiation, the inhibition rate of tumor growth reached 82% ( Figure 4B, 4C, 4D). This indicates that the compound I1 of the present invention has a strong tumor-suppressing effect after systemic administration and local activation by X-ray irradiation. Figure 3 E shows that during the entire treatment period, the body weight of the mice in the compound I1 group of the present invention did not change significantly, while the mice in the sorafenib group showed a significant weight loss, indicating that the in vivo toxicity of the compound I1 of the present invention is lower than that of sorafenib.

[0059] Example 7: Safety evaluation of the compounds of the present invention in vivo.

[0060] To evaluate the safety of compound I1 of the present invention in vivo, blood samples were collected from mice after treatment and a complete blood cell count analysis and biochemical index test were performed. The results are as follows: Figure 4 shown. Figure 4 A is the white blood cell count (WBC); Figure 4 B is the absolute neutrophil count (ANC); Figure 4 C is the red blood cell count (RBC); Figure 4 D is the hemoglobin level (HGB); Figure 4 E is the hematocrit (HCT) Figure 4 F is platelet count (PLT); Figure 4 G is the aspartate aminotransferase level (AST); Figure 4 H is the alanine aminotransferase level (ALT); Figure 4 I is the alkaline phosphatase level (ALP).

[0061] The results showed that compared with the PBS group, the complete blood cell count of the sorafenib group was significantly reduced, and the liver function index was significantly increased, showing obvious blood toxicity and liver toxicity ( Figure 4 A-4I). However, no obvious abnormalities were observed in the complete blood cell count and liver function indexes of the compound I1 group of the present invention, indicating that the compound I1 of the present invention has good safety in vivo.

[0062] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A radiation-activated prodrug compound, characterized in that The radiation-activated prodrug compound has the structure shown in Formula I: Wherein, R is H or F.

2. A method for preparing the radiation-activated prodrug compound according to claim 1, characterized in that: The preparation method comprises the following steps: The original drug and 3-chloroperbenzoic acid were dissolved in dichloromethane, reacted at room temperature until the reaction was complete, and the solvent was removed by rotary evaporation. The resulting product was purified by column chromatography to obtain a white solid, which was the radioactive prodrug compound; Wherein, the original drug is sorafenib or regorafenib.

3. Use of the radiation-activated prodrug compound according to claim 1 in the preparation of a radiation-activated prodrug, wherein the radiation-activated prodrug is used to specifically release the prodrug under radiation conditions.

4. Use of the radiation-activated prodrug compound according to claim 1 in the preparation of a drug for treating tumors.

5. The use according to claim 4, characterized in that The drug is a drug that specifically releases the original drug during radiotherapy to produce a synergistic tumor treatment effect.

6. The use according to claim 4 or 5, characterized in that The tumor is one of liver cancer, colon cancer and lung cancer.

Images