CN114349657B

CN114349657B - Azo microtubulin inhibitor and preparation method and application thereof

Info

Publication number: CN114349657B
Application number: CN202111529543.9A
Authority: CN
Inventors: 王尊元; 马臻; 黄文海; 沈正荣; 曾申昕; 梁美好; 章迟啸; 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: 2023-12-22
Anticipated expiration: 2041-12-14
Also published as: CN114349657A

Abstract

The invention discloses an azo compound for inhibiting tubulin, a preparation method and application thereof. The invention takes substituted aniline as raw material, and after diazotization reaction of sodium nitrite, the azo micromolecule is obtained by coupling reaction of the substituted aniline and substituted phenol in an organic solvent. The azo compound for inhibiting 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 special photoisomerization property, and further damages the balance of the tubulin, which can be applied to the preparation of medicines for preventing or treating diseases caused by excessive growth of the tubulin.

Description

Azo microtubulin inhibitor and preparation method and application thereof

Technical Field

The invention relates to an azo compound, in particular to an azo compound capable of inhibiting tubulin and pharmaceutically usable salt thereof, a preparation method thereof and application thereof in medicaments for inhibiting tubulin.

Background

Microtubules (microtubules) are dimers of two types of alpha and beta tubulin, are distributed in a network or bundle shape in cells, and participate in the formation of cytoskeleton, maintenance of cell morphology, cell contraction, intracellular material transport, 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, the mitosis can not be carried out or stagnated, and the interruption of the cell mitosis process can have larger influence on cells, so that the growth of the cells is inhibited, and finally the generation of apoptosis is induced, thereby achieving the purpose of inhibiting the proliferation and the growth of the cells. Given that cancer cells differ most from normal cells in that mitosis of cancer cells is abnormally frequent and uncontrolled, blocking tubulin synthesis is particularly important for the treatment of malignant tumors.

It has now been found that a large number of natural, synthetic and semisynthetic compounds have the effect of affecting the microtubule system, interfering with its normal function, and are collectively known as tubulin inhibitors because most of them bind to tubulin. These tubulin inhibitors have found wide application in medicine, such as anti-tumor, antifungal, insect repellent, etc. Tubulin has become an important target for research and development of novel antitumor drugs, and tubulin inhibitors have also become important antitumor drugs that are effective in clinical use. The action mechanism of the inhibitor is that in tumor cells which are rapidly divided, the inhibition of tubulin polymerization leads the spindle body not to be formed or promotes the tubulin polymerization, leads the spindle body not to be restored to a microtubule network again so as to interfere the normal mitosis process of the cells, leads the mitosis process of the cells to be interrupted and to be stopped in a period, thereby leading the tumor cells to be apoptotic and playing an anti-tumor role. The microtubule plays a very key role in the growth and development of tumor cells, and particularly, the effectiveness of the targeted drugs of taxol, colchicine, vinblastine and the like in clinical treatment of tumors makes the microtubule an ideal target for anti-tumor drug research. Table 1 lists some of the marketed tubulin inhibitors, and many of them are currently under development at various stages.

TABLE 1 microtubulin inhibitors which are partly marketed

Combretastatin A4, CA4 was obtained from african pinus (Combretum caffrum) in south africa. CA4 has cis-stilbene structure which can simulate Colchicine, thus it can target microtubulin in vivo, inhibit its polymerization, and further destroy tumor blood vessel, thereby playing an anti-tumor role. CA4 is effective on various tumor models, and is a very promising antitumor drug, however, the water solubility of CA4 is very poor, and only 11.8 mug/ml, so that the oral bioavailability is lower; meanwhile, the characteristics that the stilbene parent nucleus is easy to become trans, so that the activity disappears limit the clinical application value. In order to overcome the defect of CA4, scientific researchers have conducted extensive structure-activity relationship research on CA4, and developed a water-soluble phosphate prodrug CA4P of CA4, which is currently in clinical trials in stage III.

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

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides an azo compound with a tubulin inhibiting effect, pharmaceutically usable salts thereof, a synthesis method thereof and application thereof in medicines for degrading tubulin.

The aim of the invention is realized by the following technical scheme: an azo compound with tubulin inhibiting effect, having the following structure:

wherein 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, alkenyl, allyl, alkynyl, propargyl, and propargyl;

r3, R4, R5, R6, R7, R8, R9, R10 are independently selected from hydrogen, hydroxy, amino, halogen, methyl, ethyl, propyl, butyl, trifluoromethyl, methoxy, ethoxy, propoxy, aryl, substituted aryl;

x, Y is independently hydrogen, oxygen, sulfur or nitrogen.

Further, the azo compound with tubulin inhibition effect is selected from the following structures:

r1 and R2 are independently selected from hydrogen, carbonyl, acetyl, methyl, ethyl, propyl, butyl, trifluoromethyl, alkenyl, allyl, alkynyl, propargyl and propargyl;

r3, R4, R5, R6, R7, R8, R9, R10 are independently selected from hydrogen, hydroxy, amino, halogen, methoxy, ethoxy, propoxy;

x, Y is independently hydrogen, oxygen or nitrogen.

More particularly, the azo compound with tubulin inhibition effect is selected from the following structures:

the azo compound for degrading the tubulin can be used singly or can be 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, taurine, citrate, succinate or mixed salts thereof.

The preparation method of the azo microtubulin inhibitor comprises the following steps:

diazotizing substituted aniline of formula 1, and coupling with substituted benzene compound containing active XH group of formula 2 to generate azo compound of formula 3; then with Z-R ₁ Performing condensation reaction to obtain a target compound of the formula I;

wherein R is ₁ -R ₁₀ X, Y are the same as in formula I; z is halogen atom Br or I.

The invention relates to a preparation method of azo compounds with tubulin inhibition effect, which specifically comprises the following steps:

diazotizing substituted aniline, and coupling with substituted benzene compound containing active XH group to generate azo compound; depending on the structural requirements, further condensation reactions may be required to obtain the target compounds. Wherein R1 to R10, X, Y are the same as before; z is halogen atom Br or I.

The invention also provides application of the azo compound and pharmaceutically acceptable salt thereof in pharmacy, which comprises the following specific steps: for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by overexpression of tubulin. The diseases are breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma and the like, and also comprise neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, cataract, parkinson's disease, creutzfeldt-Jakob disease and Huntington's disease, and further comprise other diseases such as acute gout, arthralgia, familial mediterranean fever, liver cirrhosis and the like.

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

the azo compound for inhibiting the tubulin exists in a trans isomer with high stability in a dark environment, and does not show pharmacological activity; but, due to its characteristic photoisomerization properties, it turns into cis-isomer under light of a specific wavelength, shows affinity to tubulin, destroys the balance of tubulin, inhibits its polymerization, and further destroys the growth of cells. Therefore, the azo compound for inhibiting the microtubulin can be applied to the preparation of medicaments for preventing or treating diseases caused by excessive growth of the microtubulin, including tumors such as breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma and the like, neurodegenerative diseases such as Alzheimer disease, amyotrophic lateral sclerosis, cataract, parkinson's disease, creutzfeldt-Jakob disease and Huntington's disease, and other diseases such as acute gout, arthralgia, familial mediterranean fever, liver cirrhosis and the like.

Description of the drawings:

FIG. 1. UV-visible absorption spectrum of Azo-F under 368nm (blue light) irradiation;

FIG. 2. UV-visible absorption spectrum of Azo-F at 625nm (red light);

FIG. 3 HPLC spectra of Azo-F compounds without irradiation;

FIG. 4. Spectra of Azo-F compounds after 20min exposure to blue light;

FIG. 5. Spectra of Azo-F compounds after 100min exposure to blue light;

FIG. 6 shows a molecular docking diagram (panel a) and a partial enlargement (panel b) of a compound and a protein.

Detailed Description

The structure, the preparation method and the use of the pharmaceutical preparation for preventing or treating diseases caused by the overexpression of tubulin according to the present invention are further described below with reference to examples, but the present invention is not limited thereto.

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

the thermometer is uncorrected; bruker DRX400 nuclear magnetic resonance apparatus; agilent 5975 mass spectrometer; bruker Vector 22 IR spectrometer.

Example 1 Synthesis of Azo-H

A100 ml three-necked flask was selected, added with a magnetic rotor, fitted with a thermometer and a constant pressure dropping funnel, and placed on a magnetic stirrer. 1.83g (10 mmol) of 3,4, 5-trimethoxyaniline was weighed, 5ml of 95% ethanol solution was added, the mixture was cooled to-10℃with glycerol at-20℃and the magnetic stirrer was turned on to stir and dissolve the mixture, and the reaction solution was bright yellow.

2ml of 37% concentrated hydrochloric acid is measured and added into the reaction liquid dropwise, and the cooling liquid is replaced periodically during the dripping period, so that the temperature of the reaction liquid is ensured to be lower than 0 ℃, and the reaction liquid is converted into beige turbid liquid.

1.39g (20 mmol) of sodium nitrite was weighed out and prepared as a 5ml aqueous solution, which was likewise cooled in a cooling liquid. After the hydrochloric acid is added dropwise, the sodium nitrite solution is added dropwise, and the temperature is also noted during the dropwise, so that the reaction temperature is always lower than 0 ℃. After the addition, the solution appeared orange-yellow. The reaction was continued with stirring for 1h while maintaining the reaction solution at a temperature below 0 ℃.

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

0.85g (9 mmol) of phenol was weighed, 5ml of sodium hydroxide solution prepared before addition was added, and after dissolution, it was cooled to-10℃with a cooling liquid.

And (3) dropwise adding the solution into diazotization reaction liquid, and regulating the pH value to be 7 finally after the dripping is finished. The cooling liquid was removed, and the reaction was stirred at room temperature for 2h. The mixture was separated, extracted with ethyl acetate and concentrated, and the product was an orange flaky solid. ¹ H-NMR(CDCl ₃ ,ppm):7.85(d,2H,Ph),7.22(s,2H,Ph),6.95(d,2H,Ph),3.95(s,6H,OCH3),3.93(s,3H,OCH3). ¹³ C-NMR(CDCl ₃ ,ppm):158.6,153.4,148.7,146.8,140,124.9,115.9,100.1,61.1,56.2.MS(ESI)m/z:[M+H] ⁺ ＝288.

Example 2 Synthesis of Azo-F

The same procedure as in example 1 was followed using difluorophenol as starting material and the product was a tan solid. ¹ H-NMR(CDCl ₃ ,ppm):7.20(s,2H,Ph),6.55(d,2H,Ph),3.95(s,6H,OCH ₃ ),3.94(s,3H,OCH ₃ ). ¹³ C-NMR(CDCl ₃ ,ppm):158.7,158.6,156.1,153.4,148.7,146.8,140,124.9,115.9,100.1,61.1,56.2.MS(ESI)m/z:[M+H] ⁺ ＝324.

EXAMPLE 3 Synthesis of Azo-Cl

The same procedure as in example 1 was followed using dichlorophenol as a starting material and the product was a black solid. ¹ H-NMR(CDCl ₃ ,ppm):7.12(2H,s,Ph),6.85(2H,s,Ph),3.88(9H,s,OCH ₃ ). ¹³ C-NMR(CDCl ₃ ,ppm):155.8,153.5,148.3,141.2,141.1,128.4,116.6,100.6,61.2,56.3.MS(ESI)m/z:[M+H] ⁺ ＝356.

EXAMPLE 4 Synthesis of Azo-H

The same procedure as in example 1 was followed using o-propargyloxyphenol as starting material, the product being a tan solid. ¹ H-NMR(CDCl ₃ ,ppm):7.20(s,2H,Ph),6.55(d,2H,Ph),3.95(s,6H,OCH3),3.94(s,3H,OCH3). ¹³ C-NMR(CDCl ₃ ,ppm):158.7,158.6,156.1,153.4,149.2,140.4,100.5,61.1,56.2.

EXAMPLE 5 Synthesis of Azo-Me

The Azo-H prepared in example 4 was 0.55g, potassium carbonate 0.3g and an excess of methyl iodide were added to 2mL of DMF and the reaction stirred at room temperature overnight. After the reaction, water was added and extracted with ethyl acetate to give a brown yellow solid. ¹ H-NMR(CDCl ₃ ,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 6 Synthesis of Azo-Pr

Azo-Pr was obtained by substituting bromopropane for methyl iodide as described in example 5. MS (ESI) M/z: [ M+H ]] ⁺ ＝384.

EXAMPLE 7 Synthesis of Azo-Bu

The procedure as described in example 5 was followed using bromobutane instead of iodomethane to give a tan solid. MS (ESI) m/z:[M+H] ⁺ ＝398.

Example 8 preparation of Azo-Bu hydrochloride

46mg of Azo-Bu synthesized in example 7 was dissolved in acetone, and dry HCl gas was slowly introduced to supersaturation with stirring. White crystals are precipitated in an ice-water bath to obtain hydrochloride with a yield of 80 percent.

EXAMPLE 9 photoisomerization property study (taking the product of example 2 as an example)

1) Ultraviolet-visible absorption spectrum

Precisely weighing the product (Azo-F) of the example 2, adding the product into a 100mL volumetric flask, and adding ethanol to fix the volume; absorbing 2mL into another volumetric flask to prepare 0.002mol/L solution, testing under U3010 UV-Vis spectrophotometer (Hitachi, cell optical path is lcm, japan), and measuring ultraviolet-visible absorption spectrum at room temperature under 200-700nm with solvent as reference. Maximum absorption was found at 368 nm. The cuvette was placed under 368nm illumination and scanned every 15min to see its spectral appearance under illumination.

The results are shown in FIG. 1. It can be seen that the absorption intensity gradually becomes weaker with the increase of the irradiation time, whereas the case of the sub-absorption peak around 260nm is contrary, and the absorption intensity gradually becomes stronger with the increase of the irradiation time.

In the same way, azo-F was scanned once every 15min under the irradiation of 625nm (red light).

The results are shown in FIG. 2. It can be seen that as the irradiation time is prolonged, the absorption intensity thereof at 368nm becomes gradually strong, and the sub-absorption peak around 260nm becomes gradually weak.

It can be seen that the effect of light of different wavelengths on such azo compounds is different, which also illustrates to some extent that such azo compounds counteract photoisomerization.

2) Research on photoisomerization phenomenon before and after blue light irradiation

After the prepared Azo-F compound was irradiated with blue light (wavelength range 450 to 470 nm) for a predetermined period of time, changes in the peak pattern and the content before and after irradiation with light were measured by HPLC (Shimadzu A20 type).

HPLC spectra of Azo-F compounds without irradiation are shown in FIG. 3. The compound is mainly shown as two peaks in the solution, the peaks are 3.894min and 11.159min respectively, and the content is 41.48% and 50.86% respectively, which indicates that the cis-trans conformations exist in the solution and a certain equilibrium state is achieved.

The spectrum of the Azo-F compound after exposure to blue light for 20min is shown in FIG. 4. It can be seen that the peak at 3.816min was significantly reduced, the content was reduced to 15.75%; the other main peak around 11min has a content greatly increased to 78.62%. It is shown that after 20min of irradiation, the cis-trans conformations of the azobenzene compound are mutually converted, wherein the trans-conformations are more dominant.

The spectrum after continuing to irradiate for 100min is shown in fig. 5. It can be seen that the peak at 3.816min is significantly reduced and substantially vanished by 100min blue light irradiation; the other major peak around 11min had a significant increase in content, reaching 93.9%, indicating substantially complete conformation conversion of azobenzene.

EXAMPLE 10 photoisomerization performance study (taking the product of example 7 as an example)

7.96mg of the product of example 7 (Azo-Bu) was placed in a 10mL volumetric flask A, and the chromatographic methanol was dissolved and the volume was fixed. Another 100. Mu.L of the solution in flask A was placed in flask B of 10mL and the chromatographic methanol was used to determine the volume. After the preparation, 1mL of the solution in volumetric flask B was placed in 6 different EP tubes, respectively, and numbered. The prepared EP tube was placed in a thin layer chromatography scanner and irradiated with red light (620-630 nm) and one of the tubes was taken out at 15min,30min,1h,2h,4h,8h and placed in LC-MS to see the ratio change of cis-to trans-structure (cis-polar is large, early peak).

The wavelength was changed as above, and the sample was irradiated with yellow light (580-585 nm), green light (520-530 nm), blue light (450-470 nm), violet light (405 nm), and violet light 395nm for 15min,30min,1h,2h,4h,8h, respectively, and data were recorded.

Table 2: content of cis-trans isomer under different wavelength illumination (%), area normalization method

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

EXAMPLE 11 molecular docking

Molecular docking is a common and effective method for current computer-aided drug design, which guides us to screen compounds with good adaptability on the active site of proteins and reveals ideal binding patterns of small molecules. The CDOCKER program provides a refined docking approach for a multitude of ligands with a single target protein, and this grid-based docking approach utilizes the CHARMM force field, where the protein remains immobilized while allowing ligand to change conformation, with results obtained in terms of-CDOCKER energy and-CDOCKER interaction, with higher values for greater favorable binding between the protein and ligand. Before pharmacological activity detection, the synthesized small molecular compound is subjected to molecular docking with tubulin by using computer-aided drug design software Discovery studio 4.0 of Chuangteng company.

The docking results are shown in fig. 6. It can be seen that, in order of-CDocker_interaction_energy, azo-Bu and Azo-Pr each have a plurality of conformations scored higher than that of the small molecule CA-4 of the original protein (the name 5LYJ of its binding protein is shown), and the top-ranked conformations are consistent with the conformations of the small molecules in the original protein, and are cis structures, indicating that the designed compounds may have similar or stronger tubulin inhibition as CA-4.

Example 12 antiproliferative activity of small molecule compounds:

HeLa cells (as a model of human cervical adenocarcinoma) and H157 cells (a model of lung 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 three times with a series of dilutions of the individual compounds for four days. During four days of compound treatment, one group of cells grew in the dark and the other group of cells grew under pulsed ultraviolet light (390-400 nm, 10 seconds every 0.5 h). At the end of compound treatment, 3- (4, 5-dimethyltetrazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) reagent was added to each well and incubated with cells for 1 hour at 37 ℃. Crystals formed in each 96 well were then dissolved using DMSO. Optical density values were measured at wavelengths 570nm and 630nm using 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. IC50 values were calculated by Graphpad Prism software using a sigmoidal dose response plot.

The results are shown in Table 3 below:

IC under dark/ultraviolet (395 nm) ₅₀ Value (MTT method measurement)

^a No detection was observed within the experimental test concentrations

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

In conclusion, the azo derivative of CA4 provided by the invention has unique photoisomerization property, and can exist in an inactive or less active trans-isomer (trans-) state in dark; under the irradiation of light with a certain wavelength, the inactive trans isomer can be quickly converted into the cis isomer (cis-) with biological activity, the polymerization of the targeted tubulin is inhibited, and the targeted single cell death can be realized. The azo derivative of CA4 can be transported in vivo in a nontoxic manner and activated at a target site, so that the targeting problem of a tubulin inhibitor is solved to a certain extent.

Claims

1. An azo small molecular compound is characterized by having the following structure:

。

2. the pharmaceutically acceptable salt of the azo small molecule compound of claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride, a hydrobromide, or a mixed salt thereof.

3. The use of an azo small molecule compound according to claim 1 for the preparation of a pharmaceutical formulation for the prevention or treatment of diseases caused by tubulin overexpression.

4. The use of an azo small molecule compound according to claim 2 in the preparation of a pharmaceutical formulation for the prevention or treatment of diseases caused by tubulin overexpression.

5. The use according to claim 3 or 4, wherein the condition caused by tubulin overexpression is breast cancer, leukemia, skin cancer, cervical cancer, esophageal cancer, lung cancer, glioma, alzheimer's disease, amyotrophic lateral sclerosis, cataract, parkinson's disease, creutzfeldt-jacob disease, huntington's disease, acute gout, joint pain, familial mediterranean fever or cirrhosis.