CN114349739A

CN114349739A - Azo compound for degrading tubulin and synthesis method and application thereof

Info

Publication number: CN114349739A
Application number: CN202111528045.2A
Authority: CN
Inventors: 王尊元; 马臻; 黄文海; 沈正荣; 梁美好; 章迟啸; 曾申昕; 潘有禄
Original assignee: Hangzhou Medical College
Current assignee: Hangzhou Medical College
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-04-15

Abstract

The invention discloses an azo compound for degrading tubulin, a synthetic method and application thereof. The method takes azo micromolecules with tubulin inhibition as raw materials, and azo compounds for degrading the tubulin are obtained through further reaction. The preparation method of the azo compound for degrading the tubulin has the characteristics of low cost, simple method operation, mild conditions and the like. The azo compound for degrading the tubulin does not show pharmacological activity in dark environment, but shows affinity to the tubulin under the illumination of specific wavelength due to the specific photoisomerization performance, and further promotes the degradation of the tubulin, so that the azo compound can be applied to the preparation of medicaments for preventing or treating diseases caused by excessive increase of the tubulin.

Description

Azo compound for degrading tubulin and synthesis method and application thereof

Technical Field

The invention relates to an azo compound, in particular to an azo compound (based on a CRBN ligand) capable of degrading tubulin, a pharmaceutically usable salt thereof, a synthetic method thereof and application thereof in a medicament for selectively degrading the tubulin.

Background

Microtubules (microtubule) are dimers formed by two types of tubulin, alpha and beta, and are distributed in the form of networks or bundles in cells, and are involved in the formation of cytoskeleton, maintenance of cell morphology, cell contraction, transport of intracellular substances, cell division, and the like. Therefore, by inhibiting the polymerization of tubulin into microtubules in the cell division process or inhibiting the depolymerization of microtubules into tubulin, mitosis can not be performed or stopped, the interruption of the cell mitosis process can have greater influence on cells, so that the growth of the cells is inhibited, and the occurrence of apoptosis is finally induced, thereby achieving the purpose of inhibiting the proliferation and the growth of the cells. Since cancer cells differ most from normal cells in that their mitosis is frequent and uncontrolled, blocking tubulin synthesis is of particular importance in the treatment of malignant tumors.

Combretastatin (Combretastatin a4, CA4) was obtained from the south african shrub african windmill (combretam cafrum). The cis-stilbene structure of CA4 can target tubulin in body, inhibit its polymerization and further destroy tumor blood vessel, so that it has the function of resisting tumor. CA4 is effective in various tumor models and is a promising antitumor drug, however, CA4 has extremely poor water solubility, only 11.8 μ g/ml, so that the oral bioavailability is low; meanwhile, the clinical application value of the stilbene derivative is limited due to the characteristic that the stilbene derivative parent nucleus is easy to be destructed into trans form to cause activity disappearance. In order to overcome the defects of CA4, researchers have conducted extensive structure-activity relationship research on CA4 and developed a water-soluble phosphate prodrug CA4P, which is currently in phase III clinical trials.

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

In order to enhance the targeting property, the azobenzene optical switch is a good modification approach in view of the stilbene core structure of CA4 and the activity change of cis-trans isomers thereof. The azobenzene compound contains a special conjugated pi chemical structure, so that the azobenzene compound has photoisomerization performance, namely: the trans-isomer and the cis-isomer can be mutually converted after the light irradiation of a special wave band. In 2015, the international journal of apex "Cell" reports: borowjak et al synthesized several azotized derivatives of CA4, PSTs, using azobenzene in place of the stilbene fragment in CA4 (i.e., C-N-bond) (Borowjak et al 2015.Cell 162, 403-. The research finds that: because azo compounds have unique photoisomerization performance, PSTs exist in an inactive trans isomer (trans-) state in the dark or under normal daily irradiation; under the irradiation of light with certain wavelength, the inactive trans isomer can be quickly converted into cis isomer (cis-), which has biological activity, targets tubulin, inhibits the polymerization of the tubulin, and can realize targeted single cell death.

The azo derivative of CA4 can be transported in vivo without toxicity and activated at a target site, thereby solving the targeting problem of the tubulin inhibitor to a certain extent. However, the drawback of acquired resistance inherent in tubulin inhibitors has not been addressed because they still inhibit tubulin to exert a pharmacological effect, require a certain dose to bind and occupy the active site of tubulin for a long period of time to inhibit its activity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an azo compound capable of degrading tubulin, a pharmaceutically usable salt thereof, a synthesis method thereof and application thereof in a medicament for selectively degrading tubulin.

The purpose of the invention is realized by the following technical scheme: an azo compound for degrading tubulin, which has the following structure:

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

x, Y, W is independently oxygen, or nitrogen;

n is selected from an integer between 0 and 16.

More specifically, the azo compound is selected from the following structures:

r1 is selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, trifluoromethyl;

x, Y, W is independently oxygen, sulfur or nitrogen;

n is selected from an integer between 1 and 12.

More specifically, the azo compound is selected from the following structures:

r1 is selected from hydrogen, methyl, butyl, pentyl, hexyl;

x, Y, W is independently oxygen, or nitrogen;

n is selected from an integer between 1 and 12.

The azo compound for degrading tubulin can be used alone or prepared into pharmaceutically acceptable salts by a conventional method, wherein the pharmaceutically acceptable salts are hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurate, citrate, succinate or mixed salts thereof.

The synthesis method of the azo compound is characterized by comprising the following steps:

1) reacting bromo acid with lenalidomide in a solvent in the presence of anhydrous potassium carbonate to obtain a white solid bromide;

2) reacting white solid bromide with sodium azide in a solvent to obtain azide, carrying out Click reaction on the azide and an azo compound in the presence of catalytic amount of Cu salt, and separating to obtain the target product with the structure shown in the formula I.

The invention also provides a preparation method of the azo compound for degrading the tubulin, which comprises the following steps: the LigandA a and LigandB compounds were first prepared or purchased according to literature methods and attached to linker using different chemistries depending on the substituents R1, R2. Examples are as follows:

the reaction steps are as follows:

the commercial bromoacid reacts with the commercial lenalidomide in a solvent (DMF) in the presence of anhydrous potassium carbonate to obtain white solid bromide. The bromide reacts with sodium azide in a solvent (DMF) to obtain the azide. Carrying out Click reaction on the azide and a micromolecular azo compound in the presence of catalytic amount of Cu salt, and separating to obtain a target product.

The invention also provides an application of the azo compound and the pharmaceutically acceptable salt thereof in pharmacy, which comprises the following specific steps: the pharmaceutical preparation is used for preparing a pharmaceutical preparation for preventing or treating diseases caused by over-expression of tubulin, wherein the diseases comprise breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma and the like, and also comprise acute gout, arthralgia, familial mediterranean fever and liver cirrhosis.

Compared with the prior art, the invention has the following beneficial effects:

based on the rapid development of ProTAC (Proteolysis-targeting chimaaras, a proteolytic targeting chimera), the invention solves the problem of acquired drug resistance inherent in small molecule inhibitors in a protein degradation mode: the azo compound and the pharmaceutically available salt thereof provided by the invention do not need to occupy active sites for a long time like an inhibitor, but only need to be combined with tubulin for a short time to form a ternary complex to induce the protein degradation effect, and the function can be restored only by re-synthesis after the protein is degraded, so that the generation of drug resistance is greatly delayed; it is also effective for those proteins that have developed resistance to drugs. Therefore, the method can be applied to the preparation of medicaments for preventing or treating diseases caused by over-expression of tubulin.

Description of the drawings:

FIG. 1 is a HRMS diagram of the target product HAzo-C11-DA synthesized in example 5;

FIG. 2 is a graph showing the degradation effect of the target product HAzo-C11-DA protein.

Detailed Description

The structure, preparation method and application in preparing pharmaceutical preparations for preventing or treating diseases caused by over-expression of tubulin will be further described in the following without limiting the invention.

Analytical data for the samples were determined by the following instruments:

the thermometer is not corrected; bruker DRX400 nmr; agilent model 5975 mass spectrometer; bruker Vector22 infrared spectrometer.

EXAMPLE 1 Synthesis of Azo-H

A100 ml three-necked flask is selected, a magnetic rotor is added, a thermometer and a constant pressure dropping funnel are arranged on the magnetic stirrer. Weighing 1.83g (10mmol) of 3,4, 5-trimethoxyaniline, adding 5ml of 95% ethanol solution, cooling to-10 ℃ with glycerol at-20 ℃, opening a magnetic stirrer, and stirring for dissolving, wherein the reaction solution is bright yellow.

Measuring 2ml of 37% concentrated hydrochloric acid, dropwise adding into the reaction solution, and periodically replacing a cooling liquid during the dropwise adding period to ensure that the temperature of the reaction solution is lower than 0 ℃ and the reaction solution is changed into beige turbid liquid.

Sodium nitrite 1.39g (20mmol) was weighed out to prepare a 5ml aqueous solution, which was similarly cooled in a cooling liquid. After the hydrochloric acid is dropwise added, dropwise adding a sodium nitrite solution, and paying attention to the temperature during the dropwise adding process to ensure that the reaction temperature is always lower than 0 ℃. After the addition, the solution appeared orange-yellow. The reaction was stirred for 1h while maintaining the temperature below 0 ℃.

8g of sodium hydroxide is weighed and dissolved in 50ml of water to prepare a sodium hydroxide solution.

O-propargyloxyphenol (9mmol) was weighed, 5ml of the previously prepared sodium hydroxide solution was added, and after dissolution, the solution was cooled to-10 ℃.

Dropwise adding the solution into the diazotization reaction solution, and adjusting the pH value to 7 after the dropwise adding is finished. The cooling liquid was removed and the reaction was stirred at room temperature for 2 h. Separating, extracting with ethyl acetate, and concentrating to obtain a product as a brown yellow solid.¹H-NMR(CDCl₃,ppm):7.20(s,2H,Ph),6.55(d,2H,Ph),3.95(s,6H,OCH3),3.94(s,3H,OCH3).¹³C-NMR(CDCl₃,ppm):158.7,158.6,156.1,153.4,149.2,140.4,100.5,61.1,56.2.

EXAMPLE 2 Synthesis of Azo-Me

0.55g of Azo-H prepared in example 1, 0.3g of potassium carbonate and excess methyl iodide were added to 2mL of DMF, and the reaction was stirred at room temperature of 25 ℃ overnight. After the reaction, water was added and extracted with ethyl acetate to obtain a tan solid.¹H-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).

Example 3 Synthesis of Azo-Pr

Azo-Pr was obtained as described in example 2, substituting methyl iodide with propyl bromide. MS (ESI) M/z [ M + H ]]⁺＝384.

Example 4 Synthesis of Azo-Bu

The procedure as described in example 2 was followed, substituting methyl iodide with butyl bromide to give a tan solid.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.

Example 5 Synthesis of HAzo-C11-DA

Commercially available bromoundecanoic acid (1.3 g) was dissolved in DMF, and thionyl chloride (2 mL) was added thereto, followed by reaction at 90 ℃ for 2 hours. After removing excess SOCl2 under reduced pressure, 1g of commercially available lenalidomide and 2g of anhydrous potassium carbonate were added at room temperature, and the mixture was reacted at 60 ℃ overnight. After the reaction is finished, white solid bromide is obtained after extraction and concentration and is directly used for the next reaction.

105mg of bromide was weighed out and dissolved in 2mL of DMF, and 40mg of sodium azide was added thereto to react at 60 ℃ overnight. After the reaction, a large amount of water is added, and white solid azide is obtained after centrifugation.¹H NMR(400MHz，CDCl ₃)δ(ppm)8.59(1H,s,NH),7.88(1H,s,NH),7.69(2H,t,Ph),7.46(1H,t,Ph),5.10(1H,d,CH),4.36(2H,s,CH2),3.26(2H,t,CH2),2.78(2H,m,CH2),2.41(2H,t,CH2),2.20(2H,dd,CH2),1.71(2H,t,CH2),1.59(2H,t,CH2),1.28-1.35(12H,m,CH2).

50mg of an azide and 50mg of an Azo-H compound obtained in example 1 and a catalytic amount of CuBr (about 2mg) were weighed out and placed in a reaction flask, and after 2mL of DMSO was added and dissolved, a small amount of water was added and the reaction was carried out at room temperature and 25 ℃ for 24 hours with stirring. After the reaction is finished, water is added, EA is used for extraction, column chromatography separation is carried out, and white solid HAzo-C11-DA is obtained after concentration. HRMS: found [ M + H]⁺811.3749, Calc C42H51N8O9 811.3779. The HRMS spectrum of the product is shown in figure 1.

Example 6 Synthesis of HAzo-C6-DA

By the same procedure as in example 5, commercially available bromohexanoic acid was used in place of bromoundecanoic acid, and separation was carried out by column chromatography to obtain HAzo-C6-DA as a white solid. MS: [ M + H ]]⁺＝740。

Example 7 Synthesis of HAzo-C8-DA

In the same manner as in example 5, commercially availableAnd (3) replacing bromoundecanoic acid with bromooctanoic acid, and performing column separation to obtain white solid HAzo-C8-DA. MS: [ M + H ]]⁺＝768。

Example 8 preparation of HAzo-C11-DA hydrochloride

46mg of HAzo-C11-DA synthesized according to claim 5 was dissolved in acetone, and dried HCl gas was slowly introduced into the mixture while stirring until supersaturated. White crystals were precipitated in an ice-water bath as the hydrochloride salt in a yield of 80%.

EXAMPLE 9 study of the Photoisomerizing Properties of Small molecule Compounds (example 4)

7.96mg of the target product is placed in a 10mL volumetric flask A, and the chromatographic methanol is dissolved to fix the volume. Another 100. mu.L of the solution in the volumetric flask A is placed in a 10mL volumetric flask B, and the volume is determined by the chromatographic methanol. After the preparation, 1mL of the solution in the volumetric flask B is taken and placed in 6 different EP tubes respectively, and the solutions are numbered. The prepared EP tube is placed in a thin layer chromatography scanner and irradiated by red light (620-630nm), one of the tubes is taken out at 15min, 30min, 1h, 2h, 4h and 8h and is placed in an LC-MS to check the change of the ratio of cis-structure to trans-structure (the cis-polarity is large, the peak emergence is early).

The operation is the same as above, the wavelength is changed, yellow light (580-. The results are shown in Table 1.

TABLE 1 cis-trans isomer content (%, area normalization) under different wavelength illumination

As can be seen, under the irradiation of four wavelengths of red light (620-630nm), yellow light (580-585nm), green light (520-530nm) and blue light (450-470nm), the interconversion between the cis-structure and the trans-structure of the two azobenzene compounds can not be obviously caused. While the purple light of 395nm and 405nm can obviously cause the structural interconversion of the two azobenzene compounds.

Example 10 antiproliferative activity of small molecule compounds:

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 for four days in triplicate with a series of individual compound dilutions. During four days of compound treatment, one group of cells was grown in the dark, while the other group was grown under pulsed UV light (390-400nm, 10 seconds per 0.5 h). At the end of compound treatment, 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) reagent was added to each well and incubated with cells for 1 hour at 37 ℃. The crystals formed in each 96 well were then dissolved using DMSO. Optical density values were measured at wavelengths of 570nm and 630nm using a BioTek Synergy H4(BioTek Instruments, Winooski, VT) and the difference between the two optical density values was used to analyze relative cell viability in each 96 well. IC (integrated circuit)₅₀Values were calculated by Graphpad Prism software using a sigmoidal dose response plot.

The results are shown in table 2 below:

TABLE 2 IC in darkness/UV (395nm)₅₀Value (MTT method determination)

^aNot detected within the experimental test concentration

As can be seen, the azo derivative of CA4 shows weaker cell proliferation inhibition effect on Hela \ MCF-7 cells under dark conditions, but shows stronger cell proliferation inhibition effect under the illumination of specific wavelength, and the inhibition activity of the compound 8 with the strongest activity on Hela cells is 7.5 times stronger than that of the positive control CA 4.

Example 11 degradation of tubulin (exemplified by the product of example 5):

MCF-7 cell lines were given different concentrations of HAzo-C11-DA and exposed to light at a wavelength of 390-400nm for 10 seconds every 0.5h for a total of 4 h. The cells were collected, lysed on ice for 30min after adding a lysate (50mM NaCl,5mM EDTA, 0.5% SDS, 0.1mM sodium orthovanadate, 50. mu.g/ml aprotinin, 1mM phenylthiononyl fluoride, and 10mM Tris-HCl; pH 7.4), centrifuged at 12000rpm for 15 minutes at 4 ℃, and the supernatant protein was quantitated and subjected to SDS-PAGE; after electrophoresis, transferring the sample to a nitrocellulose membrane, then carrying out immunoreaction, sequentially carrying out room temperature sealing for 1 hour, incubating overnight at 4 ℃ primary antibody, incubating for 2 hours at room temperature secondary antibody, washing the membrane for 2 hours by TBST, adding ECL solution, and carrying out accurate warm bath for 1 minute; draining the film, exposing in a dark box for several minutes, developing and fixing; the X-ray film was scanned by a scanner and then the grayscale values of the bands were read by MetaMorph software, and statistical analysis was performed with GAPDH as an internal reference.

The results are shown in FIG. 2.

It can be seen that the product HAzo-C11-DA of example 5 of the present invention showed a slight degradation effect on tubulin at various concentrations, and the lack of activity may be related to the failure to obtain a suitable cleavage site and also to the length of the linker chain. Since the compounds are designed based on the ProTAC principle, other compounds with different chain lengths can also show the degradation activity on tubulin.

The azo-based compound degrading tubulin according to the present invention shows no pharmacological activity in dark environment, but shows affinity to tubulin under light of a specific wavelength due to its characteristic photoisomerization property, and further promotes tubulin degradation, which may be applied in the preparation of a drug for preventing or treating diseases caused by excessive increase of tubulin, including tumors such as breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, etc., and also including neurodegenerative diseases such as alzheimer's disease, amyotrophic lateral sclerosis, cataract, parkinson's disease, creutzfeldt-jakob disease, huntington's disease, and also including other diseases such as acute gout, joint pain, familial mediterranean fever, liver cirrhosis, etc.

Claims

1. An azo compound for degrading tubulin, which is characterized by having a structure shown in formula I:

wherein R is₁Selected from the group consisting of hydrogen, acetyl, propionyl, butyryl, benzoyl, substituted benzoyl, benzyl, substituted benzyl, phenyl, substituted phenyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, trifluoromethyl;

x, Y, W is independently oxygen, sulfur or nitrogen;

n is selected from an integer between 0 and 16.

2. The azo-based compound of claim 1, wherein R is₁Selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, trifluoromethyl;

x, Y, W is independently oxygen, sulfur or nitrogen;

n is selected from an integer between 1 and 12.

3. The azo-based compound of claim 1, wherein R is₁Selected from hydrogen, methyl, butyl, pentyl, hexyl;

x, Y, W is independently oxygen, or nitrogen;

n is selected from an integer between 1 and 12.

4. The pharmaceutically acceptable salt of the azo-based compound of any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurate, citrate, succinate, or a mixed salt thereof.

5. A method for synthesizing an azo compound according to any one of claims 1 to 3, comprising the steps of:

6. Use of an azo compound according to any one of claims 1 to 3 for the preparation of a pharmaceutical preparation for the prevention or treatment of a disease caused by overexpression of tubulin.

7. Use of the azo compound of claim 4 in the preparation of a pharmaceutical formulation for the prevention or treatment of a disease caused by overexpression of tubulin.

8. The use according to claim 6 or 7, wherein the disease caused by overexpression of tubulin is breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, cataract, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, acute gout, joint pain, familial Mediterranean fever or liver cirrhosis.