Hsp90 and hdac dual-targeting inhibitors, and preparation method and application thereof

Abstract

The application discloses a compound which can be used for a novel HSP90 and HDAC double-target inhibitor, and a structural general formula is shown as formula I: wherein R1 is selected from an alkyl group or an aromatic group, R2 is selected from a C1-C3 alkyl group, and L is selected from a substituted phenyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a furanyl group, a thiophenyl group, a thiazolyl group and an isoxazolyl group. The application provides a novel compound which can be used for a novel HSP90 and HDAC double-target inhibitor, can simultaneously inhibit HSP90 and HDAC activities, can play a synergistic role in disease treatment, and has strong therapeutic activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antitumor drugs, in particular to HSP90 and HDAC dual-target inhibitors and synthesis thereof. BACKGROUND

[0002] HSP90, also known as cancer protein, was first discovered in mammals in 1986, and is one of the most widely tested targets for cancer treatment. HSP90 is a class of energy-dependent chaperone proteins that maintain cell differentiation, growth and survival by regulating the conformational stability, folding and function of its "client proteins" with the help of cooperative chaperones. HSP90 is equivalent to a "switch" in the human body, which is upstream of many cell signaling pathways. These client proteins are "sub-switches", and HSP90 can affect the survival of cancer cells by changing their conformation. Inhibiting the function of HSP90 can lead to instability of its client proteins and ultimately degradation through the proteasome pathway, and cut off the dependence of tumors on branch signals, thereby solving the drug resistance of HSP90 downstream targets.

[0003] HDAC (histone deacetylases), also known as histone deacetylase, is an important enzyme in the process of histone modification, which catalyzes and regulates histone deacetylation. Currently, 18 kinds of HDAC have been found in the human body, which belong to four categories (I, II, III and IV). Among them, 11 subtypes of I, II and IV are Zn2+-dependent proteins; 7 subtypes of Sir1-7 of class III are NAD+ catalytic active sites. HDAC inhibitors are potential proliferation inhibitors, which can induce apoptosis. Therefore, these drugs can be used as potential antitumor drugs.

[0004] HSP90 and HDAC are important targets for new drug research and development, and HSP90 is a substrate of HDAC6. The combination of inhibitors of the two targets shows strong synergistic effect. There are few literatures on HSP90 and HDAC dual-target inhibitors reported at present, most of which are single-target inhibitors, and only a small number of patents report dual-target inhibitors. For example, CN113121505A discloses a triazolone Hsp90 inhibitor and Hsp90 HDAC dual-target inhibitor, which can be used as an antifungal and antitumor drug. Since there are few types of inhibitors at present, it is possible that they will eventually fail to pass clinical trials, so it is of great significance to develop new HSP90 and HDAC dual-target inhibitors. SUMMARY

[0005] In order to solve the above technical problems, the first aspect of the present application provides a compound which can be used as a new HSP90 and HDAC dual-target inhibitor, the structure general formula is shown as formula I:

[0006]

[0007] wherein R1 is selected from alkyl or aryl, R2 is selected from C1-C3 alkyl, and L is selected from substituted phenyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, thienyl, thiazolyl, isoxazolyl.

[0008] Further, R1 is selected from linear alkyl, branched alkyl, substituted phenyl, furanyl, thienyl or pyridyl;

[0009] R2 is selected from methyl, ethyl or isopropyl;

[0010] L is selected from

[0011] Further, R3 is a single or multiple substituent on the phenyl ring, and R3 is selected from H atom, F atom, Cl atom, methyl or trifluoromethyl.

[0012] Further, R1 is isopropyl or cyclopropyl, and R2 is methyl, and the structure of the inhibitor is shown in Formula II or III:

[0013]

[0014]

[0015] Further, the atoms in the structure of the compound can be selected from all isotopes thereof, for example, each carbon atom is independently selected from 12 C, 13 C or 14 C; each hydrogen atom is independently selected from 1 H (protium), 2 H (deuterium) or 3 H (tritium); each nitrogen atom is independently selected from 14 N or 15 N; each oxygen atom is independently selected from 16 O, 17 O, 18 O; and each fluorine atom is independently selected from 18 F, 19 F.

[0016] The second aspect of the present application also provides a method for synthesizing the compound. According to different substituents, the synthesis method is as follows:

[0017] Synthesis Route I:

[0018]

[0019] Method and reagents: A: bromo isopropyl, anhydrous aluminum chloride, anhydrous dichloromethane, reflux, overnight; B: benzyl bromide, acetonitrile, reflux, overnight; C: lithium hydroxide, water, tetrahydrofuran, room temperature, overnight; D: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; E: HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; F: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; G: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0020] Synthesis route two:

[0021]

[0022] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0023] Synthesis route three:

[0024]

[0025] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0026] Synthesis route four:

[0027]

[0028] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0029] Synthesis route five:

[0030]

[0031] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0032] Synthesis Route Six:

[0033]

[0034] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0035] Synthesis Route Seven:

[0036]

[0037] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0038] Synthesis Route Eight:

[0039]

[0040] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0041] Synthesis Route Nine:

[0042]

[0043] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0044] Synthesis Route Ten:

[0045]

[0046] Method and reagent: A: bromocyclopropane, anhydrous aluminum chloride, anhydrous dichloromethane, reflux, overnight; B: benzyl bromide, acetonitrile, reflux, overnight; C: lithium hydroxide, water, tetrahydrofuran, room temperature, overnight; D: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; E: HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; F: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; G: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0047] The third aspect of the present application provides the use of the compound in the preparation of an HSP90 inhibitor, an HDAC inhibitor, or an HSP90 and HDAC dual-target inhibitor.

[0048] The fourth aspect of the present application provides the use of the compound in the preparation of a drug for treating a tumor, an organ fibrosis, or an inflammatory bowel disease.

[0049] Further, the tumor is selected from lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, multiple myeloma, B-cell lymphoma, or leukemia, etc.

[0050] The organ fibrosis is selected from lung fibrosis, liver fibrosis, kidney fibrosis, or cystic fibrosis, etc.

[0051] The inflammatory bowel disease is selected from ulcerative colitis, Crohn's disease.

[0052] The fifth aspect of the present application provides a drug for treating a tumor, an organ fibrosis, or an inflammatory bowel disease, comprising the compound of the present application and a pharmaceutical excipient.

[0053] Compared with the prior art, the present application has at least the following beneficial effects:

[0054] The present application designs a new HSP90 and HDAC dual-target inhibitor, which is a brand new compound, can inhibit the activities of HSP90 and HDAC, and has a synergistic effect in disease treatment, and thus has strong therapeutic activity. DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below with specific embodiments. It should be understood that the specific examples described in the following embodiments of the present application are only exemplary descriptions of the specific embodiments of the present application, and are intended to explain the present application, but do not constitute a limitation on the present application.

[0056] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. These ranges and values should be construed as approximately.

[0057] Example 1

[0058] The structure of the representative compounds of the HSP90 and HDAC dual-target inhibitor of the present application is shown in the following table:

[0059]

[0060]

[0061]

[0062] Example 2

[0063] The synthesis method of the 30 representative compounds in Example 1 is as follows:

[0064] Among them, the compounds HH-1, HH-2, HH-3, HH-4, HH-5, HH-6 adopt synthesis route one:

[0065]

[0066] Methods and reagents: A: bromo isopropyl, anhydrous aluminum chloride, anhydrous dichloromethane, reflux, overnight; B: benzyl bromide, acetonitrile, reflux, overnight; C: lithium hydroxide, water, tetrahydrofuran, room temperature, overnight; D: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; E: HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; F: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; G: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0067] The compound HH-7 adopts synthesis route two:

[0068] Synthesis route two:

[0069]

[0070] Methods and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0071] The compound HH-8 adopts synthesis route three:

[0072] Synthesis route three:

[0073]

[0074] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0075] Compound HH-9, using synthetic route four:

[0076] Synthetic route four:

[0077]

[0078] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0079] Compound HH-10, using synthetic route five:

[0080] Synthetic route five:

[0081]

[0082] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0083] Compound HH-11, using synthetic route six:

[0084] Synthetic route six:

[0085]

[0086] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1 M aqueous hydrochloric acid, room temperature.

[0087] Compound HH-12, using synthetic route seven:

[0088] Synthetic route seven:

[0089]

[0090] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0091] Compound HH-13, using synthetic route eight:

[0092] Synthetic route eight:

[0093]

[0094] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0095] Compound HH-14, HH-15, using synthetic route nine:

[0096] Synthetic route nine:

[0097]

[0098] Method and reagents: A: methylamine hydrochloride, triethylamine, anhydrous methanol, sodium borohydride, ice bath then room temperature; B: compound 3, HOBt, EDCI, anhydrous dichloromethane, room temperature, overnight; C: boron trichloride, anhydrous dichloromethane, ice bath then room temperature, 3 hours; D: potassium hydroxylamine, anhydrous methanol, 1M aqueous hydrochloric acid, room temperature.

[0099] HH-16 to HH-30, the only difference from HH-1 to HH-15 is whether the substituent of resorcinol is isopropyl or cyclopropyl, only need to replace bromo isopropyl with bromo cyclopropyl, the other synthetic routes are exactly the same, thus can use synthetic routes one to nine respectively.

[0100] Taking HH-16 as an example, the synthetic route ten is as follows:

[0101] Synthetic route ten:

[0102]

[0103] Example 3

[0104] The compounds synthesized by the synthetic method of Example 2 were subjected to high resolution mass spectrometry structure analysis, and it was confirmed that the compounds in Example 1 were synthesized, and the high resolution mass spectrometry results are shown in the following table:

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Example 4

[0111] The 30 compounds of Example 1 were tested for inhibitory activity against HSP90 and HDAC6 using the following assay methods:

[0112] For HDAC6: All enzymatic reactions were carried out for 30 minutes at 37°C. The 50ul reaction mixture contained 25mM Tris, pH 8.0, 1 mM MgCl2, 0.1 mg / ml BSA, 37 mM NaCl, 2.7 mM KCl, HDAC6 and enzyme substrate. Compounds were diluted in 10% DMSO and 5ul of the dilution was added to 50ul of the reaction such that the final concentration of DMSO was 1% in all reactions. The assay was performed by quantifying the amount of fluorescent product in solution after the enzyme reaction. Fluorescence was then analyzed on a SpectraMax M5 microtiter plate reader with excitation at 350-360 nm and emission at 450-460 nm. IC 50 values were calculated using Prism GraphPad software using nonlinear regression fitting of the standard dose-response.

[0113] For HSP90: A dilution series of test compounds was prepared in 10% DMSO in assay buffer and 10ul of the dilution was added to 100ul of the reaction such that the final concentration of DMSO was 1% in all reactions. The reaction was carried out at room temperature for 3 hours in 100ul of a mixture containing assay buffer, 5nM FITC-labeled geldanamycin and the test compound. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader with excitation at 485 nm and emission at 530 nm. Activity determinations were carried out in duplicate at each concentration. Fluorescence intensity was converted to fluorescence polarization using Tecan Magellan 6 software. The fluorescence polarization in the absence of compound in each data set was defined as 100% activity. The fluorescence polarization (FPb) value in each data set in the absence of protein and compound was defined as 0% activity. From this the percent activity in the presence of compound was calculated and IC 50 values were calculated using Graphpad Prism.

[0114] The detection results are shown in the following table, and it can be seen that 30 compounds can simultaneously produce good inhibition effect on HSP90 and HDAC6.

[0115]

[0116] Example 5

[0117] The proliferation inhibition activity of 30 compounds of Example 1 on A549 lung cancer cells and HFL-1 lung fibroblasts was detected, and commercial products SAHA (purchased from Tao Technology https: / / www.tsbiochem.com) and pirfenidone (purchased from Tao Technology https: / / www.tsbiochem.com) were used as control groups. The detection method used MTT method, and the specific operation steps were as follows: trypsin digestion of logarithmic phase cells, centrifugal collection after termination, preparation of cell suspension, and adjustment of cell count to 5-10×10 4 / milliliter. Then the cell suspension was prepared, mixed gently, 100 microliters were added to each well, and the density of the test cells was 5000-10000 / well. The inoculated cell culture plate was placed in a culture incubator for culture until the cell monolayer was fully covered on the bottom of the well (96-well flat-bottom plate), and then compounds with concentration gradient (gradient was 0.01, 0.1, 1, 10, 100 micromolar) were added. 5% CO2, 37℃ incubation for 72 hours, and the effect of the drug was observed under an inverted microscope. 10 microliters of MTT solution (5mg / ml, i.e. 0.5% MTT) was added to each well, and the culture was continued for 4 hours. The culture was terminated, and the crystal was prepared for dissolution. 150 microliters of dimethyl sulfoxide was added to each well, and the crystal was fully dissolved by shaking on a shaker for 10 minutes. The absorbance value of each well was measured at OD490nm in an enzyme-linked immunoassay instrument, and the IC 50 value was calculated according to the absorbance value.

[0118] The detection results are shown in the following table, and it can be seen that 30 compounds can simultaneously A549 lung cancer cells and HFL-1 lung fibroblasts have good inhibition effect on proliferation, and have great potential as anticancer drugs and organ fibrosis drugs.

[0119]

[0120]

[0121] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A compound, characterized in that, Its general structural formula is shown in Formula I: （I）， Wherein, R1 is isopropyl or cyclopropyl; R2 is methyl; L is selected from substituted phenyl, pyridinyl, pyrazinyl, pyrimidinyl, furanyl, thiophenyl, thiazolyl, and isoxazolyl; The substituents of the substituted phenyl group are selected from F atoms, Cl atoms, methyl groups, or trifluoromethyl groups.

2. Use of the compound of claim 1 in the preparation of HSP90 inhibitors, HDAC inhibitors, or dual-target inhibitors of HSP90 and HDAC.

3. Use of the compound of claim 1 in the preparation of a medicament for treating tumors, organ fibrosis, or inflammatory bowel disease.

4. The use as described in claim 3, characterized in that, The tumor is selected from lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, multiple myeloma, B-cell lymphoma, or leukemia.

5. The use as described in claim 3, characterized in that, The organ fibrosis is selected from pulmonary fibrosis, liver fibrosis, kidney fibrosis, or cystic fibrosis.

6. The use as described in claim 3, characterized in that, The inflammatory bowel disease mentioned is selected from ulcerative colitis or Crohn's disease.

7. A medicament for treating tumors, organ fibrosis, or inflammatory bowel disease, comprising the compound of claim 1 and medical excipients.

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