A boron-containing compound and use thereof
By designing boron-containing compounds with specific structures and synthesizing them through amidation reactions, the problem of insufficient targeting of existing drugs has been solved, achieving highly efficient targeting and low toxicity of tumor cells, and improving the therapeutic effect of boron neutron capture therapy.
Patent Information
- Application Number
- CN202210923557.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-02
- Publication Date
- 2025-11-04
- Estimated Expiration
- 2042-08-02
AI Technical Summary
Existing boron-containing drugs lack sufficient targeting in boron neutron capture therapy, failing to effectively target tumor cells, resulting in poor treatment efficacy and limited indications.
A boron-containing compound has been developed that can bind to tumor cells with high affinity through a specific structural design, meeting the requirements of boron neutron capture therapy. This compound includes the definition of specific R1, R2, R3, R4, and R5 groups and is synthesized through an amidation reaction. The preparation method includes the use of reagents such as N,N,N',N'-tetramethylchloromethamphicanine hexafluorophosphate and 1-methylimidazole.
This compound exhibits low toxicity to tumor cells, effectively targets tumor cells, enhances the therapeutic effect of boron neutron capture therapy, meets the clinical requirement of being non-toxic to humans, achieves a concentration ratio of 4:1 to 3:1 between tumor and normal tissues, and maintains a boron drug concentration of 20 to 35 μg per gram of tumor tissue, ensuring a certain therapeutic concentration is maintained in the tumor tissue during treatment.
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Figure CN115353527B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a boron-containing compound and its use. BACKGROUND
[0002] Boron Neutron Capture Therapy (BNCT) is a new technique of binary radiotherapy targeting cancer treatment. The principle is that a boron-containing drug with specific affinity to tumor is injected into the patient's body, and then an ultrahot neutron beam is irradiated to the tumor site, so that the boron atoms in the tumor cells capture the thermal neutrons and form unstable compound nuclei. 10 B drug is injected into the patient's body, and the boron-containing drug is specifically concentrated in the tumor. Then, an ultrahot neutron beam is irradiated to the tumor site, 10 B atomic nucleus captures thermal neutrons to form an unstable compound nucleus 11 B, 11 B spontaneously splits into an α particle with a kinetic energy of 1.78 MeV and a 7 Li recoil nucleus (reaction cross section of 6.3%); or an α particle with a kinetic energy of 1.47 MeV and a 7 Li recoil nucleus and emits a photon with an energy of 0.48 MeV (reaction cross section of 93.7%). Due to the high energy of the released particles, it has the characteristics of high linear energy transfer (LET) and low oxygen enhancement ratio, so it can achieve the effect of high selectivity and high intensity killing tumor cells.
[0003] 7 The range of Li recoil nucleus and α particle in biological tissue is about 5 μm and 9 μm, which is smaller than the diameter of tumor cells 10 μm, so 7 The killing effect of Li and α particle is limited to the cells that have taken up 10 B and their immediately adjacent cells. On the other hand, 10 B (n, α) 7 Li reaction cross section reaches 3840 target barns, which is much larger than the reaction cross section of neutrons with normal tissue and blood composition nuclei. Therefore, the radiation damage effect of neutrons on normal tissues that have not taken up 10 B can be controlled at a safe dose level. Thus, this treatment is praised as cell-level radiotherapy, which combines the excellent characteristics of external radiotherapy and internal radiotherapy, and effectively avoids the side effects of conventional external radiotherapy and internal radiotherapy.
[0004] Compared with other tumor treatment technologies, BNCT has the following special advantages:
[0005] 1) Targeting of BNCT therapy
[0006] The boron-containing drug has targeting characteristics. The boron-containing drug has high affinity with tumor cells and is mainly absorbed by cancerous tissues, and the content of 10 B in tumor cells is greater than that in normal cells10 The content of B is thus much higher in tumor cells than in normal cells, and the therapeutic dose of tumor cells is much higher than that of normal cells.
[0007] 2) High linear energy transfer (LET) characteristics
[0008] Traditional radiotherapy x-rays, γ-rays, etc. are of low LET, i.e. the relative biological effectiveness (RBE) is low, and oxygen is needed to enhance the biological radiation effect. However, due to the rapid invasive growth of malignant tumors, the tumor tissue is often poorly supplied with blood, resulting in local hypoxia, and the treatment effect is relatively poor. α particles and 7 Li particles are high LET charged particles, and can kill tumor cells in both oxygen-rich and oxygen-poor conditions. Even for protons and carbon ions, the RBE of α particles generated by BNCT nuclear reactions is still the highest.
[0009] 3) Treatment effect is independent of the state of cancer cells
[0010] Chemotherapy, X-knife, γ-knife and ordinary radiotherapy generally have an effect on tumor cells in the proliferation phase (G1, S, G2 and M phase), but are not sensitive to tumor cells in the resting phase (G0 phase). G0 phase tumor cells can still grow, which is the root cause of tumor recurrence. BNCT based on high LET, α particles and 7 Li particles can kill tumor cells in the resting phase, and oxygen-poor tumor cells are more resistant to low LET γ-rays, electron beams and other conventional radiotherapy, but BNCT based treatment can kill oxygen-poor cancer cells as long as sufficient boron enters.
[0011] 4) Fine treatment scale
[0012] So far, in cancer therapy, the treatment effect range is fine to the micron level, and there is no second except for neutron capture therapy. The most precise minimally invasive surgery in surgical operation is limited to the millimeter level, and the Bragg peak focusing dose depth in heavy ion therapy is about 2.5-3.0 cm. The finer the treatment size, the smaller the side effects.
[0013] An ideal BNCT treatment boron-containing targeted drug should meet the following requirements:
[0014] No toxicity to the human body at a clinical dose;
[0015] 10 The B drug has a high affinity for tumor tissue, and the concentration ratio of tumor to normal tissue can reach 4: 1-3: 1;
[0016] The concentration of B drug in each gram of tumor tissue reaches 20-35 μg; 10
[0017] A certain therapeutic concentration can be maintained in tumor tissues during the treatment period.
[0018] Currently, the development of BNCT is relatively slow, which is closely related to the relatively slow development of boron-containing drugs. Up to now, only two boron-containing drugs, disodium mercaptododecaborate (BSH) and p-carboxyphenylalanine boronic acid (BPA), have been used in clinical practice. BPA was approved for marketing in Japan in 2020, becoming the first boron-containing drug approved for marketing in the world. As a key breakthrough in BNCT technology, BSH and BPA are far from the requirements in terms of effect, and there are problems such as insufficient tumor targeting specificity and insufficient affinity to tumor cells. Through in-depth research, it is found that the boron concentration ratio of tumor to normal tissue measured by BSH on human glioma samples is generally <1 (average 0.6), which is considered to be a non-specific boron carrier. The boron concentration ratio generated by BPA is generally not more than 2.4, it cannot penetrate into the internal components of tumor cells, and thus only transiently stays in the cells. In addition, there are groups of tumor cells that do not absorb BPA. BNCT is mainly used for the treatment of brain tumors, gliomas and melanomas in clinical practice, which is also mainly limited by the current clinical boron-containing drugs with few types and limited indications. Therefore, it is imperative to develop boron-targeted drugs with more indications, low toxicity and good targeting.
[0019] Fibroblast activation protein (FAP) is one of the specific markers of tumor-associated fibroblasts (TAF), has special biological characteristics, and is stably expressed in tumor stroma. The existing FAP inhibitors are mainly used for tumor treatment. However, there is no FAP-targeted drug that can be applied to boron neutron capture therapy. SUMMARY
[0020] The technical problem to be solved by the present application is that the existing compounds suitable for BNCT have a single structure, and therefore the present application provides a boron-containing compound and its application. The compound has low toxicity to tumor cells and can be used for targeted cancer treatment in boron neutron capture therapy.
[0021] The present application provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof:
[0022]
[0023] wherein, R 1 is a single bond, C1-C 10 straight-chain alkylene, C2-C 10 branched alkylene, C5-C8 cycloalkylene, C6-C 10arylene-C1-C4alkylene, C1-C4alkylene-C6-C 10 arylene-C1-C4alkylene, or C6-C 10 arylene;
[0024] R 2 is hydroxy, C1-C5straight chain alkyl, C3-C6branched alkyl, C5-C8cycloalkyl, C6-C 10 aryl-C1-C4alkyl, C1-C4alkyl-C6-C 10 arylene-C1-C4alkylene, or C6-C 10 aryl;
[0025] R 3 is hydroxy, C1-C5straight chain alkyl, C3-C6branched alkyl, C5-C8cycloalkyl, C6-C 10 aryl-C1-C4alkyl, C1-C4alkyl-C6-C 10 arylene-C1-C4alkylene, or C6-C 10 aryl;
[0026] R 4 is H, F or Cl;
[0027] R 5 is H, F or Cl;
[0028] n1, n2 and n3 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0029] In one embodiment, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is described in which the definitions of the moieties are as described below, and the definitions of the remaining moieties are as described in any one embodiment (hereinafter referred to as "in one embodiment"): R 1 may be a single bond.
[0030] In one embodiment, R 2 may be hydroxy.
[0031] In one embodiment, R 3 may be hydroxy.
[0032] In one embodiment, R 4 may be F or Cl.
[0033] In one embodiment, R 4 may be F.
[0034] In one embodiment, R 5 may be F or Cl.
[0035] In an embodiment, R 5 may be F.
[0036] In an embodiment, the compound of formula I is compound I-1:
[0037]
[0038] The present application also provides a compound of formula 2, formula 3 or formula 4:
[0039]
[0040] wherein R 31 is C1-C4 alkyl;
[0041] In the compound of formula 3, R 32 is C1-C3 alkyl; R 33 is C1-C3 alkyl; R 34 is C1-C3 alkyl; R 35 is C1-C3 alkyl;
[0042] R1, R2 and R3 are as defined above.
[0043] In an embodiment, the compound of formula 2 is compound 2-1:
[0044]
[0045] In an embodiment, the compound of formula 3 is compound 3-1:
[0046]
[0047] In an embodiment, the compound of formula 4 is compound 4-1:
[0048]
[0049] The present application also provides a method for preparing the compound of formula I, which comprises the following steps: carrying out an amidation reaction of a compound of formula 4 with a compound of formula a in a solvent in the presence of an amidation reagent to obtain a compound of formula I, i.e.
[0050]
[0051] In an embodiment, the amidation reaction can be carried out under normal pressure.
[0052] In an embodiment, the amidation reaction can be carried out in the presence of oxygen.
[0053] In an embodiment, the amidation reagent can be a combination of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) and 1-methylimidazole.
[0054] wherein the solvent is a solvent commonly used in the art for such reactions.
[0055] In an embodiment, the solvent can be acetonitrile.
[0056] In an embodiment, the compound of Formula 4 can be compound 4-1 described above.
[0057] In an embodiment, the molar volume ratio of the compound of Formula 4 to the solvent can be 0.105 mol / L.
[0058] In an embodiment, the molar ratio of the compound of Formula 4 to N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) in the amidation reagent can be 0.666.
[0059] In an embodiment, the molar volume ratio of the compound of Formula 4 to 1-methylimidazole in the amidation reagent can be 1.575 mol / L.
[0060] In an embodiment, the reaction temperature can be 35°C.
[0061] In an embodiment, the reaction condition can be stirring for 2 hours.
[0062] In an embodiment, the preparation method can further comprise isolation of the reaction product, which can be in the order of acidification, extraction, washing, drying, column chromatography.
[0063] In an embodiment, the acidification can be acidification using trifluoroacetic acid. Preferably, 1% trifluoroacetic acid acidification.
[0064] In an embodiment, the extraction can be ethyl acetate extraction. Preferably, equal volume of ethyl acetate extraction.
[0065] In an embodiment, the washing can be sodium bicarbonate solution washing. Preferably, equal volume of sodium bicarbonate solution washing.
[0066] In an embodiment, the drying can be anhydrous sodium sulfate drying.
[0067] In an embodiment, the column chromatography uses a developing agent of petroleum ether, ethyl acetate.
[0068] In an embodiment, the acidification can be acidification with 1% trifluoroacetic acid; the extraction can be 3 times extraction with equal volume of ethyl acetate; the washing can be 2 times washing with equal volume of sodium bicarbonate solution; the drying can be drying with anhydrous sodium sulfate; and the column chromatography can use petroleum ether and ethyl acetate as the developing agent.
[0069] In an embodiment, the compound of formula I can be a compound of formula I-2:
[0070] The present application also provides a preparation method of the compound of formula I-2, which comprises the following steps: (1) reacting a compound of formula 1 with a compound of formula D in a solvent to obtain a compound of formula 2;
[0071] (2) reacting the compound of formula 2 with a compound of formula C in a solvent to obtain a compound of formula 3;
[0072] (3) reacting the compound of formula 3 in a solvent to obtain a compound of formula 4-1;
[0073] (4) amidating the compound of formula 4-1 with a compound of formula A in the presence of an amidation reagent in a solvent to obtain the compound of formula I-2, and
[0074]
[0075] The solvent is a solvent commonly used in the art for such reactions.
[0076] In an embodiment, in the step (1), the reaction can be carried out at normal pressure; the reaction can be carried out in the presence of oxygen; the solvent can be methanol; the compound of formula D can be methanol; the reaction can be carried out in the presence of dichlorosulfoxide; the reaction temperature can be 80°C; the reaction time can be 4 hours; the reaction can be quenched by adding water; and the reaction can further comprise separation of the reaction product, which can be carried out by sequentially adjusting the PH of the reaction solution and filtering.
[0077] In an embodiment, in the step (1), the molar volume ratio of the compound of formula 1 to the solvent can be 0.8 mol / L; the molar volume ratio of the compound of formula 1 to the compound of formula D can be 0.8 mol / L; the molar volume ratio of the compound of formula 1 to dichlorosulfoxide can be 6.6 mol / L; and the PH of the reaction solution can be adjusted by sodium bicarbonate.
[0078] In a certain embodiment, in step (2), the reaction can be carried out under normal pressure; the reaction can be carried out under inert atmosphere; the solvent can be 1,4-dioxane; the compound of formula 2 is compound 2-1 described above; the compound of formula C can be pinacol borane; the reaction can be carried out in the presence of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium and potassium acetate; the reaction temperature can be 80℃; the reaction time can be 12 hours; the reaction can further comprise isolation and purification of the reaction product, which can be filtration of the reaction solution, dilution, extraction, washing, drying, concentration and purification in sequence.
[0079] In a certain embodiment, in step (2), the molar volume ratio of the compound of formula 2 to the solvent can be 0.35 mol / L; the molar ratio of the compound of formula 2 to the compound of formula C can be 0.67; the molar ratio of the compound of formula 2 to [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium can be 20; the molar ratio of the compound of formula 2 to potassium acetate can be 0.5; the extraction in the isolation and purification is ethyl acetate extraction; the purification in the isolation and purification is column chromatography purification.
[0080] In a certain embodiment, in step (2), in the column chromatography purification in the isolation and purification, the developing agent is petroleum ether and ethyl acetate.
[0081] In a certain embodiment, in step (3), the reaction can be carried out under normal pressure; the reaction can be carried out in the presence of oxygen; the solvent can be 1,4-dioxane; the compound of formula 3 is compound 3-1 described above; the reaction can be carried out in the presence of hydrochloric acid; the reaction temperature can be 85℃; the reaction time can be 12 hours; the reaction can further comprise isolation and purification of the reaction product, which can be concentration of the reaction solution, beating and filtration in sequence.
[0082] In a certain embodiment, in step (3), the molar volume ratio of the compound of formula 3 to the solvent can be 0.42 mol / L; the concentration of hydrochloric acid is 6 mol / L; the molar volume ratio of the compound of formula 3 to hydrochloric acid can be 0.3 mol / L; the beating in the isolation and purification is beating with petroleum ether and ethyl acetate.
[0083] In a certain embodiment, the reaction conditions in step (4) can refer to the reaction conditions in the preparation method of the compound of formula I described above.
[0084] The application also provides a pharmaceutical composition comprising substance X and a pharmaceutically acceptable excipient; the substance X is the compound of formula I described above or a pharmaceutically acceptable salt thereof.
[0085] The application also provides a use of a substance X in the preparation of a medicament for treating tumors; the substance X is the compound as shown in the formula I or a pharmaceutically acceptable salt thereof; and the medicament is a medicament for treating tumors.
[0086] In a certain embodiment, the medicament for treating tumors is a radiotherapy targeting medicament.
[0087] In a certain embodiment, the radiotherapy targeting medicament is a boron neutron capture therapy medicament.
[0088] Unless otherwise specified, the terms used in the present application have the following meanings:
[0089] The term "alkyl" refers to a straight chain or branched alkyl group having a specified number of carbon atoms (e.g., C1-C4). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, s-butyl, and the like.
[0090] The term "alkylene" refers to a straight chain or branched divalent alkyl group having a specified number of carbon atoms (e.g., C1-C4) that connects to other groups through two sites. Alkylene groups include, but are not limited to, and the like.
[0091] The term "cycloalkyl" refers to a saturated monocyclic ring group consisting of only carbon atoms having a specified number of carbon atoms (e.g., C5-C8). Cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
[0092] The term "aryl" refers to a cyclic group consisting of only carbon atoms having a specified number of carbon atoms (e.g., C6-C 10 ), which is monocyclic or polycyclic, and each ring has aromaticity (complying with Huckel's rule). Aryl groups include, but are not limited to, phenyl, naphthyl, and the like.
[0093] The term "arylene" refers to a divalent cyclic group consisting of only carbon atoms having a specified number of carbon atoms (e.g., C6-C 10 ), which is monocyclic or polycyclic, and each ring has aromaticity (complying with Huckel's rule), which connects to other groups through two sites. Arylene groups include, but are not limited to, and the like.
[0094] The term "pharmaceutically acceptable salt" refers to a salt of a compound that is produced by reaction of the compound with a pharmaceutically acceptable (relatively non-toxic, safe, suitable for patient use) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of the pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of the pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, methanesulfonate salts, and the like. See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, 2002) for further details.
[0095] The above-mentioned preferred conditions can be combined in any manner without departing from the common general knowledge of the skilled person, thereby obtaining preferred embodiments of the present application.
[0096] The reagents and raw materials used in the present application are commercially available.
[0097] The positive progress effect of the present application is that the compound has low toxicity to tumor cells and can target cancer treatment by boron neutron capture therapy. DETAILED DESCRIPTION
[0098] The present application will be further described by way of examples, but the present application is not limited to the examples. The experimental methods in the following examples, for which no specific conditions are indicated, are carried out according to conventional methods and conditions, or according to the instructions of the commercial products.
[0099] The A549 cells, HepG2 cells, U87MG cells, SCC9 cells, and A375 cells in the following examples are from the Shanghai Cell Bank of the Chinese Academy of Sciences;
[0100] In the following examples
[0101] The CCK-8 cell viability detection kit is produced by Shanghai Donguan Biological Technology Co., Ltd.
[0102] The PBS is produced by Shanghai Shuangmo Biological Technology Co., Ltd.
[0103] The DMEM high-sugar culture solution is produced by Shanghai Donguan Biological Technology Co., Ltd.
[0104] Example 1 Preparation of the compound I-1 of the present application
[0105]
[0106] 1. Preparation of compound I-1
[0107] The preparation route of I-1 is shown as follows:
[0108]
[0109] First step: Compound 1 (50 g, 198 mmol) was dissolved in methanol (250 ml), and dichlorosulfoxide (30 ml) was slowly added at room temperature. The reaction solution was reacted at 80 °C for 4 hours. The reaction solution was quenched with water (50 ml), and the pH was adjusted to 7 with sodium bicarbonate solution. Filtration was performed, and the solid was collected. White compound 2-1 was obtained with a yield of 93%.
[0110] The identification data of compound 2-1 are as follows: LC-MS: M (C 11 H8BrNO2) = 266.09 (m / z), [M+H] + 267.2
[0111] Second step: Compound 2-1 (12.0 g, 45.1 mmol), pinacol diboronic acid (BDP) (17.2 g, 67.6 mmol) were dissolved in 1,4-dioxane (130 ml), and then [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (Pd(dppf)Cl2) (1.65 g, 2.25 mmol), potassium acetate (8.85 g, 90.2 mmol) were added at room temperature under nitrogen protection. The reaction solution was reacted at 80 °C for 12 hours under nitrogen protection. After filtration, the reaction solution was collected, diluted with water (50 ml), and extracted twice with ethyl acetate (100 ml / time). The organic phase was combined, washed twice with saturated brine (30 ml / time), and dried with anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography purification was performed (developing agent: petroleum ether, ethyl acetate). The product 3-1 was obtained as a yellow solid with a yield of 92%.
[0112] The identification data of compound 3-1 are as follows: LC-MS: M (C 17 H 20 BNO4) = 313.16 (m / z), [M+H] + 314.1
[0113] Third step: Compound 3-1 (13.0 g, 41.5 mmol) was dissolved in 1,4-dioxane (100 ml), and hydrochloric acid (6 mol / L, 138 ml) was slowly added. The reaction solution was stirred at 85 °C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The crude product was slurried with ethyl acetate / petroleum ether (1:5, 50 ml) at room temperature for 30 minutes, and filtration was performed to obtain yellow compound 4 with a yield of 95%.
[0114] The identification data of compound 4-1 are as follows: LC-MS: M (C 10 H8BNO4) = 216.99 (m / z), [M+H] + 218
[0115] Fourth step: compound 4-1 (8 g, 31.5 mmol), compound a (20 g) was dissolved in acetonitrile (300 ml), slowly added N, N, N', N'-tetramethylchloroformamidum hexafluorophosphate (TCFH) (13.3 g, 47.3 mmol), 1-methylimidazole (20 ml). The reaction solution was stirred at 35°C for 2 hours. The pH of the reaction solution was adjusted to 5 with 1% trifluoroacetic acid, and the reaction solution was extracted with ethyl acetate three times (500 ml / time). The organic phase was washed with sodium bicarbonate solution twice (200 ml / time), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and then purified by column chromatography (developing agent: petroleum ether, ethyl acetate). The product I-1 was obtained as a white solid with a yield of 48%.
[0116] 2, structure identification
[0117] The LC-MS, NMR identification data of compound I-1 are as follows:
[0118] LC-MS: M (C 17 H 15 BF2N4O4) = 388.14 (m / z), [M+H] + 389.2.
[0119] 1 H NMR: (400 MHz, DMSO-d6): δ 9.09-9.06 (m, 1H), 9.01 (s, 1H), 8.64 (s, 1H), 8.28-8.27 (m, 2H), 8.04 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.57 (s, 1H), 5.18 (d, J = 9.6 Hz, 1H), 4.36-4.16 (m, 4H), 2.94-2.82 (m, 2H).
[0120] F NMR: (400 MHz, DMSO-d6): δ -95.153, -103.648.
[0121] Example 2: Cytotoxicity of compound I-1 on A549 cells
[0122] A549 cells in the logarithmic growth phase were inoculated on a 96-well plate at 1*10 4HepG2 cells in logarithmic growth phase were inoculated on 96-well plates at 1*10 4 cells per well, and cultured overnight in DMEM high-sugar culture medium. The medium was aspirated, and the cells were washed twice with PBS (0.01 mol / L). Compound I-1 was added to fresh culture medium to prepare a series of concentration gradient solutions containing compound I-1, and the concentration of compound I-1 was 0, 100, 250, 500, and 750 μg / ml, respectively. 100 μl was added to the cell well plate to be tested, and after the cells were treated with drugs for 48 hours, the cell culture medium was aspirated with a pipette, the cells were washed twice with PBS (0.01 mol / L), 100 uL of CCK8 working solution was added, and the culture plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was measured by an enzyme marker, and the effect of the drug on cell viability was calculated.
[0123] The test results are shown in Table 1. The low cytotoxicity of compound I-1 is an important indicator of BNCT, which can maximize the boron concentration in tumor cells. The compound I-1 described in the present application exhibits low toxicity to tumor cells, and the IC 50 50 of 320.2 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for boron neutron capture therapy.
[0124] Table 1 Inhibition rate of different concentrations of compound on A549 cells
[0125] Compound I-1 concentration (μg / ml) Inhibition rate against A549 cells 0 0.00％ 100 30.78％ 250 45.19％ 500 70.73％ 750 77.29％
[0126] Example 3: Cytotoxicity of compound I-1 on HepG2 cells
[0127] HepG2 cells in logarithmic growth phase were inoculated on 96-well plates at 1*10 4 cells per well, and cultured overnight in DMEM high-sugar culture medium. The medium was aspirated, and the cells were washed twice with PBS (0.01 mol / L). Compound I-1 was added to fresh culture medium to prepare a series of concentration gradient solutions containing compound I-1, and the concentration of compound I-1 was 0, 100, 250, 500, and 750 μg / ml, respectively. 100 μl was added to the cell well plate to be tested, and after the cells were treated with drugs for 48 hours, the cell culture medium was aspirated with a pipette, the cells were washed twice with PBS (0.01 mol / L), 100 uL of CCK8 working solution was added, and the culture plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was measured by an enzyme marker, and the effect of the drug on cell viability was calculated.
[0128] The test results are shown in Table 2. The low cytotoxicity of compound I-1 is an important indicator of BNCT, which can maximize the boron concentration in tumor cells. The compound I-1 described in the present application exhibits low toxicity to tumor cells, and the IC 50421.4 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for boron neutron capture therapy.
[0129] Table 2 Inhibition rate of different concentrations of the compound on HepG2 cells
[0130] Compound I-1 concentration (μg / ml) Inhibition rate against HepG2 cells 0 0.00％ 100 15.50％ 250 22.14％ 500 56.64％ 750 71.86％
[0131] Example 4: Cytotoxicity of compound I-1 on U87MG cells
[0132] U87MG cells in the logarithmic growth phase were inoculated on a 96-well plate at 1*10 4 cells per well, and were cultured in DMEM high-glucose medium overnight. The medium was aspirated, and the cells were washed twice with PBS (0.01 mol / L). Compound I-1 was added to fresh medium to prepare a series of concentration gradients of the compound I-1 solution, and the concentration of the compound I-1 was 0, 100, 250, 500, and 750 μg / ml, respectively. 100 μl was added to the cell well plate to be tested, and after the cells were treated with the drug for 48 hours, the cell culture medium was aspirated, the cells were washed twice with PBS (0.01 mol / L), 100 uL of CCK8 working solution was added, and the culture plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was determined by an enzyme marker, and the effect of the drug on cell viability was calculated.
[0133] The test results are shown in Table 3. The low cytotoxicity of the compound I-1 is an important indicator of BNCT, which can ensure the maximum concentration of boron in tumor cells. The compound I-1 described in the present application exhibits low toxicity to tumor cells, and the IC 50 273.9 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for boron neutron capture therapy.
[0134] Table 3 Inhibition rate of different concentrations of the compound on U87MG cells
[0135] Compound I-1 concentration (μg / ml) Inhibition rate against U87MG cells 0 0.00％ 100 20.53％ 250 43.50％ 500 62.43％ 750 71.63％
[0136] Example 5: Cytotoxicity of compound I-1 on SCC9 cells
[0137] SCC9 cells in the logarithmic growth phase were inoculated on a 96-well plate at 1*10 4A375 cells in logarithmic growth phase were inoculated on 96-well plates at 1*10 4 The cells were washed twice with PBS (0.01 mol / L) and then incubated with a fresh culture medium containing compound I-1. A series of solutions containing compound I-1 were prepared at different concentrations, i.e., 0, 100, 250, 500, and 750 μg / ml. Then, 100 μl of each solution was added to the cell culture plate. After 48 hours of drug treatment, the cell culture medium was removed, the cells were washed twice with PBS (0.01 mol / L), and 100 uL of CCK8 working solution was added. The plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was measured by a microplate reader, and the effect of the drug on the cell viability was calculated.
[0138] The test results are shown in Table 4. The low cytotoxicity of compound I-1 is an important indicator of BNCT, which can maximize the boron concentration in tumor cells. The IC 50 The IC50 of compound I-1 for SCC9 cells was 187.8 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for BNCT.
[0139] Table 4 Inhibition rate of different concentrations of compound on SCC9 cells
[0140]
[0141]
[0142] Example 6: Cytotoxicity of compound I-1 on A375 cells
[0143] A375 cells in logarithmic growth phase were inoculated on 96-well plates at 1*10 4 The cells were washed twice with PBS (0.01 mol / L) and then incubated with a fresh culture medium containing compound I-1. A series of solutions containing compound I-1 were prepared at different concentrations, i.e., 0, 100, 250, 500, and 750 μg / ml. Then, 100 μl of each solution was added to the cell culture plate. After 48 hours of drug treatment, the cell culture medium was removed, the cells were washed twice with PBS (0.01 mol / L), and 100 uL of CCK8 working solution was added. The plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was measured by a microplate reader, and the effect of the drug on the cell viability was calculated.
[0144] The test results are shown in Table 5. The low cytotoxicity of compound I-1 is an important indicator of BNCT, which can ensure the maximum concentration of boron in tumor cells. The compound I-1 described in the present application exhibits lower toxicity to tumor cells, and the IC50 of compound I-1 to A375 cells is 688.5 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for boron neutron capture therapy. 50 The low cytotoxicity of compound I-1 is an important indicator of BNCT, which can ensure the maximum concentration of boron in tumor cells. The compound I-1 described in the present application exhibits lower toxicity to tumor cells, and the IC50 of compound I-1 to A375 cells is 688.5 μg / ml, which meets the requirement of low toxicity of boron-containing drugs for boron neutron capture therapy.
[0145] Table 5 Inhibition rate of different concentrations of compounds to A375 cells
[0146] Compound I-1 concentration (μg / ml) Inhibition rate against A375 cells 0 0 100 23.49％ 250 34.18％ 500 41.81％ 750 56.76％
Claims
1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, characterized in that: ； wherein R 1 is a single bond; R 2 is hydroxyl; R 3 is hydroxyl; R 4 is F or CI; R 5 is F or CI; The compound of formula I is not .
2. A preparation method of the compound as shown in formula I according to claim 1, comprising the following steps: carrying out amidation reaction of a compound as shown in formula 4 with a compound as shown in formula a in the presence of an amidation reagent in a solvent to prepare a compound as shown in formula I, and ； wherein R 1 is a single bond; R 2 is hydroxyl; R 3 is hydroxyl; R 4 is F or CI; R 5 is F or CI.
3. The process for the preparation of a compound of formula I according to claim 2, characterized in that, The preparation method of the compound as shown in formula I meets one or more of the following conditions: a) the amidation reaction is carried out at normal pressure; b) the amidation reaction is carried out in the presence of oxygen; c) the amidation reagent is a combination of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) and 1-methylimidazole; d) the solvent is acetonitrile; e) the compound of Formula 4 is ; f) the molar volume ratio of the compound as shown in formula 4 to the solvent is 0.105 mol / L; g) the reaction temperature is 35°C; h) the reaction condition is stirring for 2 hours; i) the preparation method further comprises separation of the reaction product, and the separation comprises acidification, extraction, washing, drying and column chromatography in sequence.
4. The process according to claim 3 for the preparation of a compound of formula I, wherein The preparation method of the compound as shown in formula I meets one or more of the following conditions: a) the molar ratio of the compound as shown in formula 4 to N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) in the amidation reagent is 0.666; b) the molar volume ratio of the compound as shown in formula 4 to 1-methylimidazole in the amidation reagent is 1.575 mol / L; c) the acidification is acidification with trifluoroacetic acid; d) the extraction is ethyl acetate extraction; e) the washing is sodium bicarbonate solution washing; f) the drying is anhydrous sodium sulfate drying; g) the column chromatography uses petroleum ether and ethyl acetate as developing agents.
5. The process for the preparation of a compound of formula I according to claim 4, characterized in that, The preparation method of the compound as shown in formula I meets one or more of the following conditions: a) the acidification is acidification with 1% trifluoroacetic acid; b) the extraction is equal volume ethyl acetate extraction; c) the washing is equal volume sodium bicarbonate solution washing.
6. A pharmaceutical composition comprising substance X and a pharmaceutical adjuvant, characterized in that, The substance X is a compound as shown in formula I or a pharmaceutically acceptable salt thereof; ； wherein R 1 is a single bond; R 2 is hydroxyl; R 3 is hydroxyl; R 4 is F or CI; R 5 is F or CI.
7. The pharmaceutical composition of claim 6, wherein The compound as shown in formula I is compound I-1: 。 8. Use of a substance X for the manufacture of a medicament, characterized in that The substance X is a compound as shown in formula I or a pharmaceutically acceptable salt thereof; and the drug is a drug for treating tumors. ； wherein R 1 is a single bond; R 2 is hydroxyl; R 3 is hydroxyl; R 4 is F or CI; R 5 is F or CI; The drug for treating tumors is a radiotherapy targeting drug.
9. Use according to claim 8, wherein the compound is ###0002### The compound as shown in formula I is compound I-1: 。 10. Use of a substance X according to claim 8 for the preparation of a medicament, characterized in that, The radiotherapy targeting drug is a boron neutron capture therapy drug.