A microtubule protein degradation agent based on VHL ligand and its application

Abstract

The present invention discloses a tubulin degrading agent based on VHL ligand and its application. The present invention uses azo small molecules with tubulin inhibitory effect as raw materials, connects with VHL ligand, and obtains a tubulin degrading agent. The preparation method of the present invention has the characteristics of low cost, simple method operation, mild conditions, etc. The tubulin degrading agent of the present invention does not show pharmacological activity in a dark environment, but when a specific wavelength of light is applied to the tumor site, it will promote tubulin degradation, thereby overcoming the systemic distribution defect of small molecule tubulin inhibitors.

Description

Technical Field

[0001] The present invention relates to a microtubule protein degradation agent, in particular to a microtubule protein degradation agent based on the photoisomerization property of azo compounds, and a pharmaceutically usable salt thereof, a synthesis method thereof and an application of the agent in drugs for selectively degrading microtubule protein. Background Art

[0002] Microtubules are dimers formed by two types of tubulin, α and β, and are distributed in a network or bundle in the cell. They participate in the formation of the cytoskeleton, the maintenance of cell morphology, cell contraction, intracellular material transport, and cell division. Therefore, by inhibiting the polymerization of tubulin into microtubules during cell division, or inhibiting the depolymerization of microtubules into tubulin, mitosis will inevitably be unable to proceed or stagnate. The interruption of the cell mitosis process will inevitably have a greater impact on the cell, inhibiting its growth, and ultimately inducing the occurrence of cell apoptosis, thereby achieving the purpose of inhibiting cell proliferation and growth. Given that the biggest difference between cancer cells and normal cells is that the mitosis of cancer cells is abnormally frequent and uncontrolled, blocking the synthesis of tubulin is particularly important for the treatment of malignant tumors, and is also widely used in antifungal and anthelmintic treatments.

[0003] Combretastatin A4 (CA4) is extracted from the South African shrub Combretum caffrum. The cis-stilbene structure of CA4 can target tubulin in vivo, inhibit its polymerization, and further destroy tumor blood vessels, thereby playing an anti-tumor role. CA4 is effective in a variety of tumor models and is a very promising anti-tumor drug. However, CA4 has extremely poor water solubility, only 11.8μg / ml, which makes its oral bioavailability low; at the same time, its stilbene parent nucleus is easily converted to trans, resulting in the loss of activity, which also limits its clinical application value. In order to overcome the defects of CA4, researchers have conducted extensive structure-activity relationship studies on it and developed its water-soluble phosphate prodrug CA4P, which is currently in Phase III clinical trials.

[0004] However, currently, tubulin inhibitors including CA4 are non-specific, their biological activity cannot be limited to specific tumor sites, and they are also toxic to normal cells and healthy cells. This non-specificity can cause serious systemic side effects in cancer treatment, such as cardiotoxicity and neurotoxicity, which greatly limits the dosage and route of administration of such inhibitors; at the same time, the generation of acquired drug resistance also greatly weakens their clinical therapeutic value.

[0005] In order to enhance its targeting, azobenzene photoswitch is a good modification approach in view of the diphenylethylene core structure of CA4 and the activity changes of its cis-trans isomers. Azobenzene compounds contain a special conjugated π chemical structure, so they have photoisomerization properties, that is, after irradiation with light in a special band, the trans isomer and the cis isomer can be mutually converted. In 2015, the international top journal "Cell" reported that Borowjak et al. used azobenzene to replace the diphenylethylene fragment in CA4 (i.e., replacing C=C with N=N bond) and synthesized several azo derivatives PSTs of CA4 (Borowjak et al 2015.Cell 162,403-411). The study found that due to the unique photoisomerization properties of azo compounds, PSTs exist in the inactive trans-isomer state in the dark or under normal daily illumination; under irradiation with light of a certain wavelength, the inactive trans-isomer can be quickly converted into the biologically active cis-isomer (cis-), targeting microtubules, inhibiting their polymerization, and achieving targeted single-cell death.

[0006] This azo derivative of CA4 can be transported in vivo without toxicity and activated at the target site, thus solving the targeting problem of tubulin inhibitors to a certain extent. However, since it still exerts its pharmacological effect by inhibiting tubulin, it needs a certain dose to bind and occupy the active site of tubulin for a long time to inhibit its activity, so it still cannot solve the inherent acquired drug resistance defect of tubulin inhibitors. Summary of the invention

[0007] The purpose of the present invention is to provide a tubulin degrading agent based on VHL ligand, and a pharmaceutically acceptable salt thereof and the use thereof in drugs for selectively degrading tubulin in view of the deficiencies of the prior art.

[0008] The object of the present invention is achieved through the following technical solution: an azo compound for degrading tubulin has the following structure:

[0009] LigandA-Linker-LigandB

[0010] Wherein: Ligand A is an azobenzene small molecule having the following structure:

[0011]

[0012] Wherein, R1 and R2 are independently selected from hydrogen, C1-C12 alkyl, C1-C12 carbonyl, C1-C12 alkenyl, C1-C12 haloalkyl, C1-C12 oxaalkyl, C1-C12 azaalkyl, aryl, substituted aryl;

[0013] R3, R4, R5, R6, R7, R8, R9, R 10 Independently selected from hydrogen, hydroxyl, amine, halogen, C1-C12 alkenyl, C1-C12 alkyl, C1-C12 haloalkyl, C1-C12 oxaalkyl, C1-C12 azaalkyl, keto(acyl), aryl, substituted aryl;

[0014] X and Y are independently hydrogen, oxygen, sulfur or nitrogen;

[0015] The R1 or R2 position can be connected to the Linker independently.

[0016] Linker is a connecting chain, including nonlinear chain, aliphatic chain, aromatic chain, heteroaromatic ring structure chain, connected to LigandA and LigandB through covalent bonds. The structure of some connecting chains is shown in the following general formula:

[0017]

[0018] in:

[0019] n is selected from an integer between 0-30.

[0020] LigandB is a ligand that can recruit ubiquitin E3 ligases, including but not limited to ligands of Von Hippel-Lindau (VHL), Cereblon (CRBN), Murine Double Mimute 2 (MDM2) and Cellular inhibitors of apoptosis proteins 1 (cIAP1). The corresponding ligand structures are exemplified as follows:

[0021]

[0022] Wherein X is CH2 or C=O, Y is independently oxygen or nitrogen, and R is the linker position.

[0023] Further, the LigandA compound is selected from the following structures:

[0024] R1 and R2 are independently selected from hydrogen, carbonyl, acetyl, propionyl, butyryl, benzoyl, substituted benzoyl, benzyl, substituted benzyl, phenyl, substituted phenyl, methyl, ethyl, propyl, butyl, trifluoromethyl;

[0025] R3, R4, R5, R6, R7, R8, R9, R10 are independently selected from hydrogen, hydroxyl, amine, halogen, methyl, ethyl, propyl, butyl, trifluoromethyl, methoxy, ethoxy, propoxy, aryl, substituted aryl;

[0026] X and Y are independently oxygen, sulfur or nitrogen;

[0027] R1 or R2 is connected to Linker independently;

[0028] The LigandB is selected from the VHL ligand:

[0029]

[0030] n is selected from an integer between 1-15.

[0031] Furthermore, the VHL ligand-based tubulin degrading agent of the present invention is selected from the following structures:

[0032]

[0033] wherein R1 is selected from hydrogen, acetyl, propionyl, butyryl, benzoyl, substituted benzoyl, benzyl, substituted benzyl, phenyl, substituted phenyl, methyl, ethyl, propyl, butyl, trifluoromethyl;

[0034] X and Y are independently oxygen, sulfur or nitrogen;

[0035] n is selected from an integer between 0-15.

[0036] Furthermore, the VHL ligand-based tubulin degrading agent of the present invention is selected from the following structures:

[0037] R1 is selected from hydrogen, methyl, ethyl, propyl, butyl, trifluoromethyl;

[0038] X and Y are independently oxygen or nitrogen;

[0039] n is selected from an integer between 1-15.

[0040] Furthermore, the VHL ligand-based tubulin degrading agent of the present invention is a compound with the following structure:

[0041]

[0042] n is selected from an integer between 1-6.

[0043] The present invention also provides a method for preparing a microtubule protein degrader based on a VHL ligand, specifically: firstly prepare or purchase a LigandA compound and a LigandB compound according to a literature method, and connect them with Lingker using different chemical reactions according to the different substituents of R1 and R2. Examples are as follows:

[0044]

[0045] The VHL ligand-based microtubule protein degradation agent described in the present invention can be used alone or prepared into a pharmaceutically acceptable salt by conventional methods. The pharmaceutically acceptable salt is hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurine, citrate, succinate, or a mixed salt thereof.

[0046] The present invention also provides a VHL ligand-based tubulin degradation agent and its pharmaceutically acceptable salt for pharmaceutical use, the specific application being: for preparing a pharmaceutical preparation for preventing or treating diseases caused by excessive tubulin expression, wherein the diseases caused by excessive tubulin expression are tumors such as breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, etc., and also include neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, cataracts, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, and other diseases such as acute gout, joint pain, familial Mediterranean fever, cirrhosis, etc. Preferably, the diseases are breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, etc., and also include acute gout, joint pain, familial Mediterranean fever, cirrhosis, etc.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] Based on the rapid development of PROTAC ( Pro Teolysis- ta rgeting c himaeras, protein cleavage targeted chimera) technology, the present invention solves the problem of acquired drug resistance in the form of protein degradation: the microtubule protein degrader based on VHL ligand and its pharmaceutically acceptable salt provided by the present invention do not need to occupy the active site for a long time like microtubule protein inhibitors, but only need to bind to microtubule protein for a short time to induce protein degradation effect. Since the protein needs to be resynthesized to restore its function after being degraded, this greatly delays the generation of drug resistance; it is still effective for those proteins that have already developed drug resistance. Therefore, the microtubule protein degrader based on VHL ligand and its pharmaceutically acceptable salt provided by the present invention may be used in the preparation of drugs for preventing or treating diseases caused by excessive expression of microtubule protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 .Mass spectrum of BuAzo-PEG1-VHL;

[0050] Figure 2 .Mass spectrum of BuAzo-PEG2-VHL;

[0051] Figure 3 .Mass spectrum of BuAzo-PEG4-VHL;

[0052] Figure 4 .A diagram showing the effect of BuAzo-PEG1-VHL compound on microtubule protein degradation. DETAILED DESCRIPTION

[0053] The structure, preparation method and application of the present invention in preparing a pharmaceutical preparation for preventing or treating diseases caused by excessive tubulin expression are further described below in conjunction with the examples, but the present invention is not limited thereto.

[0054] In the following examples, unless otherwise specified, all percentages are by mass.

[0055] The analytical data of the samples were measured by the following instruments:

[0056] Thermometer uncalibrated; Bruker DRX400 NMR; Agilent 5975 mass spectrometer; Bruker Vector 22 infrared spectrometer.

[0057] Example 1 Synthesis of Azo-H

[0058]

[0059] Select a 100ml three-necked flask, add a magnetic rotor, install a thermometer and a constant pressure dropping funnel, and place it on a magnetic stirrer. Weigh 1.83g (10mmol) of 3,4,5-trimethoxyaniline, add 5ml of 95% ethanol solution, cool to -10℃ with -20℃ glycerol, turn on the magnetic stirrer, stir and dissolve, and the reaction solution is bright yellow.

[0060] Measure 2 ml of 37% concentrated hydrochloric acid and add it dropwise to the above reaction solution. During the addition, the coolant is replaced regularly to ensure that the temperature of the reaction solution is below 0°C. The reaction solution turns into a beige turbid liquid.

[0061] Weigh 1.39g (20mmol) of sodium nitrite and prepare 5ml of aqueous solution, which is also placed in the coolant for cooling. After the hydrochloric acid is added dropwise, add the sodium nitrite solution dropwise. During the addition, pay attention to the temperature to ensure that the reaction temperature is always below 0°C. After the addition is completed, the solution is orange-yellow. Continue to stir the reaction for 1h, while maintaining the temperature of the reaction solution below 0°C.

[0062] Weigh 8 g of sodium hydroxide and dissolve it in 50 ml of water to prepare a sodium hydroxide solution.

[0063] Weigh o-propargyloxyphenol (9 mmol), add 5 ml of the previously prepared sodium hydroxide solution, dissolve it, and cool it to -10°C with a coolant.

[0064] The above solution was added dropwise to the diazotization reaction solution. After the addition was completed, the pH value was adjusted to 7. The cooling liquid was removed and the reaction was stirred at room temperature for 2 hours. The liquid was separated, extracted with ethyl acetate and concentrated to obtain a brown-yellow solid. 1 H-NMR(CDCl3,ppm):7.20(s,2H,Ph),6.55(d,2H,Ph),3.95(s,6H,OCH3),3.94(s,3H,OCH3). 13 C-NMR (CDCl3, ppm): 158.7, 158.6, 156.1, 153.4, 149.2, 140.4, 100.5, 61.1, 56.2.

[0065] Example 2 Synthesis of Azo-Me

[0066]

[0067] 0.55 g of Azo-H prepared in Example 1, 0.3 g of potassium carbonate and excess methyl iodide were added to 2 mL of DMF and stirred at room temperature for overnight reaction. After the reaction, water was added and the mixture was extracted with ethyl acetate to obtain a brown-yellow solid. 1 H-NMR(CDCl3,ppm):7.66(1H,dd,Ph),7.63(1H,d,Ph),7.23(2H,s,Ph),7.02(1H,d,CH),4.87(2H,f,CH2),3.97(9H,s,3×OCH3),3.93(3H,s,OCH3).

[0068] Example 3 Synthesis of Azo-Pr

[0069]

[0070] According to the method described in Example 2, methyl iodide was replaced by propyl bromide to obtain Azo-Pr. MS (ESI) m / z: [M+H] + =384.

[0071] Example 4 Synthesis of Azo-Bu

[0072]

[0073] As described in Example 2, using butyl bromide instead of methyl iodide, a brown solid was obtained. 1HNMR (400MHz, CDCl3) δ7.56 (td, J=4.6, 2.2Hz, 1H), 7.15 (s, 1H), 6.94 (d, J=9.2Hz, 1H), 4.78 (d, J=2.4Hz, 1H), 4.05 (t, J=6.6Hz, 1H), 3.90 (s, 3H), 3.86 (s, 1H), 3.50 (t, J=6.7Hz, 1H), 2.47 (t, J=2.4Hz, 1H), 1.89-1.73 (m, 2H), 1.50 (s, 2H).MS (ESI) m / z: [M+H] + =398.

[0074] Example 5 Synthesis of BuAzo-PEG1-VHL

[0075]

[0076] Add 119.4 mg (0.3 mmol) of the Azo-Bu compound obtained in Example 4 to a three-necked flask, then add 167.1 mg (0.3 mmol) of the commercially available N3-PEG1-VHL compound, add 15 mL of tetrahydrofuran to dissolve, add 4.8 mg of copper sulfate and 16 mg of sodium ascorbate, and stir the reaction at room temperature under a nitrogen atmosphere for 16 h. Monitor the entire process with TLC (EA: EtOH = 50: 3) until the reaction is completed. The reaction solution was extracted with EA (2×25 mL), the organic phase was washed with saturated brine, the liquids were separated, the ethyl acetate layers were collected and combined, and dried over anhydrous sodium sulfate overnight. Filter and dry under reduced pressure to obtain the crude product. Separate by column chromatography (EA: EtOH = 50: 3), and the solvent was concentrated to dryness under reduced pressure to obtain the product BuAzo-PEG1-VHL. MS: Found: [M+H] + =956.4,[M+Na] + =979.4, Cal: C48H61N9O10S = 956.117. See the attached mass spectrum Figure 1 .

[0077] Example 6 Synthesis of BuAzo-PEG2-VHL

[0078]

[0079] According to the same method as in Example 5, the commercially available N3-PEG2-VHL compound was used as the raw material to perform a click chemistry reaction to obtain the product BuAzo-PEG2-VHL. MS: Found: [M+H] + =1000.4,[M+Na] + =1023.4,Cal:

[0080] C50H65N9O11S=1000.17. Mass spectrum is attached Figure 2 .

[0081] Example 7 Synthesis of BuAzo-PEG4-VHL

[0082]

[0083] According to the same method as in Example 5, the commercially available N3-PEG4-VHL compound was used as the raw material to perform a click chemistry reaction to obtain the product BuAzo-PEG4-VHL. MS: Found: [M+Na] + =1110.4, Cal: C54H73N9O13S = 1087.5. See the attached mass spectrum Figure 3 .

[0084] Example 8 Preparation of BuAzo-PEG2-VHL Hydrochloride

[0085] 46 mg of the synthesized BuAzo-PEG2-VHL was added to acetone and dissolved, and dry HCl gas was slowly introduced into the mixture under stirring until it was supersaturated. The mixture was placed in an ice-water bath to precipitate white crystals, which were its hydrochloride salt, with a yield of 70%.

[0086] Example 9 Study on the Photoisomerization Performance of Small Molecule Compounds (Taking the Product of Example 4 as an Example)

[0087] Take 7.96 mg of the target product and place it in a 10 mL volumetric flask A, dissolve it in chromatographic methanol, and make up to volume. Take another 100 μL of the solution in volumetric flask A and place it in a 10 mL volumetric flask B, and make up to volume with chromatographic methanol. After the preparation is completed, take 1 mL of the solution in volumetric flask B and place it in 6 different EP tubes and number them. Place the prepared EP tube in a thin layer chromatography scanner and irradiate it with red light (620-630 nm), and take out one of the tubes at 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h and place it in LC-MS to check the changes in the ratio of cis and trans structures (cis has high polarity and early peak).

[0088] The operation was the same as above, but the wavelength was changed, and the samples were illuminated for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h with yellow light (580-585 nm), green light (520-530 nm), blue light (450-470 nm), violet light (405 nm), and violet light 395 nm, respectively, and the data were recorded. The results are shown in Table 1.

[0089] Table 1. Content of cis-trans isomers under different wavelengths of light (%)

[0090]

[0091] It can be seen that under the irradiation of red light (620-630nm), yellow light (580-585nm), green light (520-530nm), and blue light (450-470nm), the cis-trans structure of the two azobenzene compounds cannot be obviously converted. However, 395nm and 405nm violet light can obviously cause the structural conversion of the two azobenzene compounds.

[0092] Example 10 Antiproliferative activity of small molecule compounds:

[0093] HeLa cells (as a model of human cervical adenocarcinoma) and MCF-7 cells (a model of breast cancer) were maintained in DMEM / F12 medium supplemented with 10% FBS under standard cell culture conditions. 3,000 cells per well were seeded into two sets of 96-well plates, and the cells were incubated with a series of dilutions of individual compounds in triplicate for four days. During the four-day compound treatment, one set of cells was grown in the dark, while the other set of cells was grown under pulsed UV light (390-400 nm, irradiated for 10 seconds every 0.5 h). At the end of the compound treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to each well and incubated with the cells for 1 hour at 37°C. DMSO was then used to dissolve the crystals formed in each 96 well. Optical density values ​​were measured at wavelengths of 570 nm and 630 nm using a BioTek Synergy H4 (BioTek Instruments, Winooski, VT), and the difference between these two optical density values ​​was used to analyze the relative cell viability in each 96-well. 50 Values ​​were calculated by Graphpad Prism software using sigmoidal dose-response plots.

[0094] The results are shown in Table 2:

[0095] Table 2. IC in dark / UV (395nm) 50 Value (determined by MTT method)

[0096]

[0097]

[0098] a Not detected in experimentally tested concentrations

[0099] It can be seen that this type of CA4 azo derivatives showed a weak cell proliferation inhibitory effect on Hela\MCF-7 cells under dark conditions, but showed a strong cell proliferation inhibitory effect under light of a specific wavelength. The most active compound 8 had an inhibitory activity on Hela cells that was 7.5 times stronger than the positive control CA4.

[0100] Example 11 Degradation of tubulin by the target compound (taking the product of Example 5 as an example):

[0101] The MCF-7 cell line was given different concentrations of BuAzo-PEG1-VHL compound and placed under light with a wavelength of 390-400 nm, irradiated for 10 seconds every 0.5 h for a total of 4 h. The cells were collected and lysed with lysis buffer (50 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM sodium orthovanadate, 50 μg / ml aprotinin, 1 mM phenylmethysulfonylfluoride, and 10 mM Tris-HCl; pH 7.4) for 30 min on ice. The cells were centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was used for protein quantification and SDS-PAGE was performed. After electrophoresis, the samples were transferred to nitrocellulose membranes and then subjected to immunoreaction, which was followed by blocking at room temperature for 1 hour, incubation with primary EGFR antibody at 4°C overnight, incubation with secondary antibody at room temperature for 2 hours, washing with TBST for 2 hours, and adding ECL solution for accurate incubation for 1 minute. The membranes were drained and exposed to light for several minutes in a dark box, developed, and fixed. The X-ray films were scanned with a scanner and the grayscale values ​​of the bands were read using MetaMorph software. Statistical analysis was performed using GAPDH as an internal reference.

[0102] See attached for the results Figure 4 .

[0103] It can be seen that the product BuAzo-PEG1-VHL of Example 5 of the present invention can show degradation activity on tubulin at different concentrations and shows good dose dependence. Given that this type of compound is designed based on the PROTAC principle, other compounds with different chain lengths can also show degradation activity on tubulin.

[0104] The VHL ligand-based microtubule protein degradation agent provided by the present invention does not show pharmacological activity in a dark environment, but due to its unique photoisomerization property, it shows affinity for microtubule protein under light of a specific wavelength and further promotes the degradation of microtubule protein. This may be used in the preparation of drugs for preventing or treating diseases caused by excessive growth of microtubule protein, including tumors such as breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, etc., as well as neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, cataracts, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, and other diseases such as acute gout, joint pain, familial Mediterranean fever, cirrhosis, etc.

Claims

1. A microtubule protein degradation agent based on VHL ligand, characterized in that: A compound of the following structure: 。 2. The pharmaceutically acceptable salt of the VHL ligand-based tubulin degrading agent according to claim 1, characterized in that: The pharmaceutically acceptable salt is hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurate, citrate, succinate, or a mixed salt thereof.

3. Use of the VHL ligand-based tubulin degrader according to claim 1 in the preparation of a pharmaceutical preparation for preventing or treating breast cancer.

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