A tyrosine skeleton-based hdac inhibitor, a synthesis method and application thereof
By designing HDAC inhibitors with a tyrosine backbone and optimizing their affinity for the HDAC active pocket, the problem of poor efficacy of existing HDAC inhibitors against solid tumors has been solved, achieving more efficient HDAC inhibition and anti-tumor effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE SECOND PEOPLES HOSPITAL OF SHANDONG PROVINCE (SHANDONG PROVINCIAL EAR NOSE & THROAT HOSPITAL SHANDONG PROVINCIAL INST OF EAR NOSE & THROAT)
- Filing Date
- 2026-04-23
- Publication Date
- 2026-07-03
AI Technical Summary
Existing HDAC inhibitors are not effective in treating solid tumors, with high IC50 values and a lack of subtype selectivity, resulting in insignificant efficacy.
We designed an HDAC inhibitor based on a tyrosine backbone by introducing substituents, such as halogens and methyl groups, onto the tyrosine benzene ring to optimize its affinity for the HDAC active pocket. We then connected a flexible alkyl chain and a terminal hydroxamic acid group via ether bonds to form stable chelation and hydrogen bonding.
The prepared compound b19 exhibited an HDAC inhibition IC50 of 26.8 nM, which was significantly superior to SAHA. In particular, its IC50 against solid tumors such as HeLa cells was 0.55 μM, enhancing its antitumor activity against solid tumors.
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Figure CN122079826B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug preparation technology, specifically relating to an HDAC inhibitor based on a tyrosine backbone, its synthesis method, and its application. Background Technology
[0002] Histone deacetylases (HDACs) are key enzymes in epigenetic regulation, and their overexpression is closely related to tumorigenesis and development. HDAC inhibitors increase histone acetylation levels by inhibiting HDAC activity, thereby activating tumor suppressor gene expression and inducing tumor cell apoptosis and cell cycle arrest.
[0003] Currently, HDAC inhibitors approved by the U.S. Food and Drug Administration (FDA), such as vorinostat and romidesin, are mainly used for hematologic malignancies, but their effects on solid tumors are limited, due to reasons including the complex microenvironment of solid tumors and poor selectivity for HDAC subtypes.
[0004] A typical HDAC inhibitor structure consists of three parts: a zinc ion-binding group (ZBG, such as hydroxamic acid), a linker, and a surface recognition region (Cap).
[0005] Vorinostat (SAHA), a marketed HDAC inhibitor, has a structure comprising an isohydroxamic acid group as ZBG, a flexible alkyl chain as a linker, and an aniline derivative as a cap. SAHA binds to the HDAC active site Zn via isohydroxamic acid. 2+ Chelation exerts inhibitory activity, but its role in the treatment of solid tumors is limited. 50 High concentrations (e.g., 1.94 μM in HeLa cells) and lack of subtype selectivity lead to poor treatment outcomes in solid tumors. Summary of the Invention
[0006] The purpose of this invention is to provide an HDAC inhibitor based on a tyrosine backbone, its synthesis method and application, in order to solve the above-mentioned technical problems.
[0007] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows:
[0008] A tyrosine-based HDAC inhibitor, the general structural formula of which is:
[0009] ;
[0010] Wherein, R1 is naphthyl, monosubstituted phenyl, or disubstituted phenyl;
[0011] The monosubstituted phenyl group is an ortho-substituted phenyl group, a meta-substituted phenyl group, or a para-substituted phenyl group;
[0012] In the ortho-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, or fluorine;
[0013] In the meta-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethyl;
[0014] In the para-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethoxy.
[0015] The disubstituted phenyl group is 3-bromo-2-chlorophenyl or 3-chloro-2-bromophenyl.
[0016] This invention also provides a method for preparing an HDAC inhibitor based on a tyrosine backbone, comprising the following steps:
[0017] S1. Using CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate as reactants, an affinity substitution reaction was carried out in the presence of anhydrous potassium carbonate to prepare (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate methyl ester.
[0018] S2. Using methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate as a raw material, the tert-butyl group is removed under the action of trifluoroacetic acid to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid;
[0019] S3. Using (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid and aniline derivatives as raw materials, HATU as a condensing agent and triethylamine as an acid-binding agent, an amide condensation reaction was carried out to prepare an intermediate.
[0020] S4. Add the intermediate to potassium hydroxylamine solution and react at room temperature for 2-4 hours. After the reaction is complete, remove the organic solvent by rotary evaporation, then adjust the pH of the reaction system to 5-6, let it stand, filter to obtain the precipitate, and dry the precipitate under reduced pressure to obtain the HDAC inhibitor.
[0021] As a further improvement, step S1 is as follows: CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate are added to a dimethylformamide solution containing anhydrous potassium carbonate. Under stirring conditions, the reaction is carried out at 50°C for 8-10 hours. After the reaction is completed, the reaction is quenched with saturated sodium bicarbonate solution, and the mixture is extracted three times with ethyl acetate. The organic phases are combined, and the organic phases are purified by column chromatography and dried to obtain (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]methyl hexanoate.
[0022] As a further improvement, the molar ratio of anhydrous potassium carbonate, CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate is 2~4:1:1.
[0023] As a further improvement, step S2 is as follows: Methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate is added to a mixed solution of trifluoroacetic acid and dichloromethane, and reacted at 50°C for 8-10 h. After the reaction is completed, the reaction is quenched with saturated sodium bicarbonate solution, extracted three times with ethyl acetate, the organic phases are combined, separated by column chromatography and dried to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid.
[0024] As a further improvement, in the mixed solution, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1.5~2, and the molar ratio of (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate to trifluoroacetic acid is 1:5~10.
[0025] As a further improvement, step S3 is as follows: (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid is added to dichloromethane and stirred to dissolve. Then, triethylamine and HATU are added sequentially, and the mixture is stirred and activated at room temperature for 1-2 hours. Then, an aniline derivative is added and refluxed at 45°C for 8-10 hours. After the reaction is completed, the insoluble matter is removed by filtration, distilled water is added, and the mixture is extracted three times with ethyl acetate. The organic phases are combined, washed with saturated sodium chloride solution, dried, evaporated to dryness, separated and purified by column chromatography, and dried to obtain the intermediate.
[0026] As a further improvement, the molar ratio of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid, triethylamine, HATU, and aniline derivative is 1:1.2:1.2:1.
[0027] As a further improvement, in step S4, the molar ratio of potassium hydroxylamine to the intermediate in the potassium hydroxylamine solution is 5~10:1.
[0028] This invention also provides the application of a tyrosine-based HDAC inhibitor in the preparation of anti-solid tumor drugs.
[0029] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:
[0030] This invention provides an HDAC inhibitor based on a tyrosine backbone, its synthesis method, and its application. The prepared compound b19 exhibits HDAC inhibition IC50. 50The concentration reached 26.8 nM, exhibiting superior antitumor activity compared to SAHA, especially against solid tumors such as HeLa cells with IC50. 50 It is 0.55 μM.
[0031] This invention uses L-tyrosine as the backbone, with its aromatic ring serving as the Cap region, linked by a flexible alkyl chain via an ether bond, and terminated with a ZBG (hydroxamic acid) group. In the prepared compound, the tyrosine backbone provides a rigid structure, reducing entropy loss and enhancing hydrophobic interactions with the HDAC surface. Further optimization of the affinity for the HDAC active pocket is achieved by introducing substituents (such as halogens, methyl groups, etc.) onto the tyrosine benzene ring.
[0032] The compounds prepared in this invention form stable interactions with the active pockets of HDAC1 and HDAC2, and molecular docking shows interaction with Zn. 2+ It generates chelation, hydrogen bonding and hydrophobic interactions. Attached Figure Description
[0033] Figure 1 This describes the specific process for preparing the compounds of this invention;
[0034] Figure 2 This describes the specific preparation process of compounds b1~b19;
[0035] Figure 3 These are the molecular docking results of compound b19 with HDAC1 and HDAC2, where A and B are the molecular docking results of compound b19 with HDAC1, and C and D are the molecular docking results of compound b19 with HDAC2. Detailed Implementation
[0036] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0037] In this invention, nM is nmol / L and μM is μmol / L.
[0038] Example 1: An HDAC inhibitor based on a tyrosine backbone, with the following general structural formula:
[0039] ;
[0040] Wherein, R1 is naphthyl, monosubstituted phenyl, or disubstituted phenyl;
[0041] The monosubstituted phenyl group is an ortho-substituted phenyl group, a meta-substituted phenyl group, or a para-substituted phenyl group;
[0042] In ortho-substituted phenyl groups, the substituents are methyl, bromine, chlorine, methoxy, or fluorine;
[0043] In meta-substituted phenyl groups, the substituents are methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethyl.
[0044] In para-substituted phenyl groups, the substituents are methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethoxy.
[0045] The disubstituted phenyl group is 3-bromo-2-chlorophenyl or 3-chloro-2-bromophenyl.
[0046] Specifically, based on the above general formula, the HDAC inhibitor with a tyrosine backbone is one of compounds b1 to b20, wherein, in compounds b1 to b19, the structural formula of R1 is:
[0047] ;
[0048] R2, R3, and R4 are shown in Table 1.
[0049] Table 1. R2, R3, and R4 groups in compounds b1-b19
[0050]
[0051] In compound b20, the structural formula of R1 is:
[0052] .
[0053] Example 2: A method for preparing an HDAC inhibitor based on a tyrosine backbone, specifically including the following steps:
[0054] S1. Using CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate as reactants, an affinity substitution reaction was carried out in the presence of anhydrous potassium carbonate to prepare (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate methyl ester.
[0055] 1 mol of CBZ-L-tyrosine tert-butyl ester and 1 mol of methyl 6-bromohexanoate were added to 6 mL of dimethylformamide solution containing 4 mol of anhydrous potassium carbonate. The mixture was stirred and reacted at 50 °C for 8 h. After the reaction was completed, stirring was stopped, and saturated sodium bicarbonate solution was added to quench the reaction. The mixture was then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product. The crude product was purified by column chromatography. The eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 5:1. After purification and drying, a pale yellow liquid (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]methyl hexanoate was obtained.
[0056] S2. Using methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate as a raw material, the tert-butyl group is removed under the action of trifluoroacetic acid to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid;
[0057] Take 10 mL of trifluoroacetic acid (0.13 mol) and 15 mL of dichloromethane to obtain a mixed solution;
[0058] 13 mmol of methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate was added to a mixed solution and reacted at 50 °C for 10 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution, and then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product, which was then purified by column chromatography with a 40:1 volume ratio of dichloromethane and methanol as the eluent. After purification and drying, (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was obtained.
[0059] S3. Using (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid and o-toluidine as raw materials, HATU as condensing agent and triethylamine as acid-binding agent, an amide condensation reaction was carried out to prepare the intermediate.
[0060] 1 mol of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was added to 15 mL of dichloromethane and stirred to dissolve. Then, 1.2 mol of triethylamine and 1.2 mol of ureonium salt condensing agent HATU were added sequentially, and the mixture was stirred and activated at room temperature for 1 h. Then, 1 mol of aniline derivative (o-toluidine) was added, and the mixture was refluxed at 45 °C for 8 h. After the reaction was completed, the insoluble matter in the reaction system was removed by filtration, 30 mL of distilled water was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and washed three times with saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate for 1 h, then evaporated to dryness, and purified by column chromatography with a dichloromethane and methanol mixture of 80:1 (v / v). After purification, the intermediate was dried.
[0061] The structural formula of the intermediate is:
[0062] ;
[0063] S4. Dissolve potassium hydroxide in anhydrous methanol to obtain solution A. Add hydroxylamine hydrochloride to anhydrous methanol to obtain solution B. Place solution B in an ice bath and slowly add solution A to it. Stir in the ice bath for 3 hours to obtain potassium hydroxylamine solution. The specific preparation method is the existing technology.
[0064] 1 mol of intermediate was added to a potassium hydroxylamine solution containing 5 mol of potassium hydroxylamine and reacted at room temperature for 4 h. After the reaction was completed, the organic solvent was removed by rotary evaporation. Then, the pH of the reaction system was adjusted to 5 with hydrochloric acid solution. After precipitation, the precipitate was allowed to stand, filtered, and dried under reduced pressure to obtain HDAC inhibitor b1.
[0065] In this embodiment, the preparation reaction route of b1 is as follows: Figure 1 As shown.
[0066] Example 3-21 Example 3-21 provides a method for preparing an HDAC inhibitor based on a tyrosine backbone. The specific steps are the same as in Example 1, except that the aniline derivative used in step S4 is different. The specific substances used are shown in Table 2.
[0067] Table 2. Aniline derivatives used in Examples 3-21
[0068]
[0069] like Figure 2 The figure shows the specific preparation process of compounds b1~b19 prepared in Examples 2-20.
[0070] Example 22 A method for preparing an HDAC inhibitor based on a tyrosine backbone, specifically including the following steps:
[0071] S1. Using CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate as reactants, an affinity substitution reaction was carried out in the presence of anhydrous potassium carbonate to prepare (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate methyl ester.
[0072] 1 mol of CBZ-L-tyrosine tert-butyl ester and 1 mol of methyl 6-bromohexanoate were added to a dimethylformamide solution containing 2 mol of anhydrous potassium carbonate. The mixture was stirred and reacted at 50 °C for 10 h. After the reaction was completed, stirring was stopped, and the reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product. The crude product was purified by column chromatography. The eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 5:1. After purification and drying, a pale yellow liquid (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]methyl hexanoate was obtained.
[0073] S2. Using methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate as a raw material, the tert-butyl group is removed under the action of trifluoroacetic acid to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid;
[0074] Take 10 mL of trifluoroacetic acid (0.13 mol) and 20 mL of dichloromethane to obtain a mixed solution;
[0075] 26 mmol of methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate was added to a mixed solution and reacted at 50 °C for 8 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution, and then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product, which was then purified by column chromatography with a 40:1 volume ratio of dichloromethane and methanol as the eluent. After purification and drying, (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was obtained.
[0076] S3. Using (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid and o-toluidine as raw materials, HATU as condensing agent and triethylamine as acid-binding agent, an amide condensation reaction was carried out to prepare the intermediate.
[0077] 1 mol of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was added to dichloromethane and stirred to dissolve. Then, 1.2 mol of triethylamine and 1.2 mol of ureonium salt condensing agent HATU were added sequentially, and the mixture was stirred and activated at room temperature for 2 h. Then, 1 mol of aniline derivative (o-toluidine) was added, and the mixture was refluxed at 45 °C for 10 h. After the reaction was completed, the insoluble matter in the reaction system was removed by filtration, distilled water was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and washed three times with saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate for 1 h, then evaporated to dryness, and purified by column chromatography with a dichloromethane and methanol mixture of 80:1 (v / v). After purification, the intermediate was dried.
[0078] S4. Add 1 mol of intermediate to a potassium hydroxylamine solution containing 10 mol of potassium hydroxylamine and react at room temperature for 2 h. After the reaction is complete, remove the organic solvent by rotary evaporation. Then adjust the pH of the reaction system to 6 with hydrochloric acid solution. After the precipitate is formed, let it stand, filter it, and dry the precipitate under reduced pressure to obtain the HDAC inhibitor.
[0079] Example 23 A method for preparing an HDAC inhibitor based on a tyrosine backbone, specifically including the following steps:
[0080] S1. Using CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate as reactants, an affinity substitution reaction was carried out in the presence of anhydrous potassium carbonate to prepare (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate methyl ester.
[0081] 1 mol of CBZ-L-tyrosine tert-butyl ester and 1 mol of methyl 6-bromohexanoate were added to a dimethylformamide solution containing 3 mol of anhydrous potassium carbonate. The mixture was stirred and reacted at 50 °C for 9 h. After the reaction was completed, stirring was stopped, and the reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product. The crude product was purified by column chromatography. The eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 5:1. After purification and drying, a pale yellow liquid (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]methyl hexanoate was obtained.
[0082] S2. Using methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate as a raw material, the tert-butyl group is removed under the action of trifluoroacetic acid to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid;
[0083] Take 10 mL of trifluoroacetic acid (0.13 mol) and 18 mL of dichloromethane to obtain a mixed solution;
[0084] 20 mmol of methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate was added to a mixed solution and reacted at 50 °C for 9 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution, and then extracted three times with ethyl acetate. The organic phases were combined to obtain the crude product, which was then purified by column chromatography with a 40:1 volume ratio of dichloromethane and methanol as the eluent. After purification and drying, (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was obtained.
[0085] S3. Using (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid and o-toluidine as raw materials, HATU as condensing agent and triethylamine as acid-binding agent, an amide condensation reaction was carried out to prepare the intermediate.
[0086] 1 mol of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid was added to dichloromethane and stirred to dissolve. Then, 1.2 mol of triethylamine and 1.2 mol of ureonium salt condensing agent HATU were added sequentially, and the mixture was stirred and activated at room temperature for 1.5 h. Then, 1 mol of aniline derivative (o-toluidine) was added, and the mixture was refluxed at 45 °C for 10 h. After the reaction was completed, the insoluble matter in the reaction system was removed by filtration, distilled water was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and washed three times with saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate for 1 h, then evaporated to dryness, and purified by column chromatography with a dichloromethane and methanol mixture of 80:1 (v / v). After purification, the intermediate was dried.
[0087] S4. Add 1 mol of intermediate to a potassium hydroxylamine solution containing 8 mol of potassium hydroxylamine, and react at room temperature for 3 h. After the reaction is complete, remove the organic solvent by rotary evaporation. Then adjust the pH of the reaction system to 5.5 with hydrochloric acid solution. After the precipitate is formed, let it stand, filter, and dry the precipitate under reduced pressure to obtain the HDAC inhibitor.
[0088] The HDAC inhibitory activity of b1-b20 and SAHA was determined by fluorescence substrate assay, and the IC50 values of each compound were obtained. 50 Meanwhile, the topological polar surface area (tPSA) and lipid-water partition coefficient (cLogP) were obtained through chemical structure calculations, and the results are shown in Table 3.
[0089] Table 3. HDAC enzyme inhibitory activity of the compounds
[0090]
[0091] As can be seen from Table 3, all target products have inhibitory activity at the nM (nmol / L) level.
[0092] Monosubstituted aniline compounds, as aniline derivatives, exhibit better inhibitory activity when substituted at the ortho position. The ortho substituent has a certain spatial volume, which can form stable van der Waals interactions and hydrophobic stacking with hydrophobic amino acid residues (such as Phe583 and Phe643) in the active pocket of HDAC enzymes. This steric hindrance effect helps to stabilize the binding conformation of the inhibitor and the enzyme, prolong the residence time, and thus improve the inhibitory efficiency.
[0093] In contrast, while para-substitution can also produce hydrophobic effects, its steric hindrance effect is weaker and it cannot provide the same level of conformational stability.
[0094] Furthermore, ortho-substitution, being closer to the amide group, can more effectively regulate the electron density of the amide nitrogen through its electron-donating effect, enhancing the ability to form hydrogen bonds with key residues (such as Tyr308 and Tyr745) at the HDAC active site. This redistribution of electron cloud density enhances the electrostatic interaction between the inhibitor and the enzyme catalytic center, thereby increasing the binding affinity.
[0095] The introduction of halogen atoms can significantly increase the lipid-water partition coefficient (logP) of the compound, making the molecule more lipid-soluble. This may be the reason why ortho-substitution has better HDAC inhibition activity.
[0096] However, compounds with fluorine atoms as substituents do not exhibit this pattern. This may be because the fluorine atom is too small to provide sufficient steric hindrance to stabilize the binding conformation of the inhibitor with the HDAC enzyme, nor can it form effective van der Waals interactions with the hydrophobic residues in the enzyme's active pocket.
[0097] Of all the compounds, compound b19 exhibited the best HDAC inhibition activity, with an IC50 value of [missing value]. 50 The value was 26.8 nmol / L, which was better than the HDAC inhibitory activity of the drug control SAHA (47.1 nmol / L). This may be due to the synergistic steric hindrance effect of the ortho and meta halogen atoms, which made the binding conformation of the inhibitor and the enzyme more stable and prolonged the residence time.
[0098] Furthermore, the synergistic electronic effect of ortho- and meta-substitution can more effectively regulate the electron density of amide nitrogen and enhance the ability to form hydrogen bonds with key residues (such as Tyr308 and Tyr745) at the HDAC active site.
[0099] The antitumor activities of compounds b1-b20 and SAHA against cervical cancer cells (HeLa), breast cancer cells (MDA-MB-231), pharyngeal squamous cell carcinoma cells (FaDu), and non-small cell lung cancer cells (A549) were determined using the CCK-8 assay. The specific methods are as follows:
[0100] (1) Cell seeding: Dilute the cell suspension at 5×10 3 The cells were seeded in 96-well plates using the middle 10 columns × 6 rows. Nine columns were seeded with cells, and one column was used as a blank well with only culture medium added. The edge wells were filled with 1×PBS. All solutions were 100 μL / well. It is important to ensure that no air bubbles are generated during the seeding process and that the cell suspension is evenly distributed at the bottom of the well. After adding the culture medium, the wells were incubated in a 5% CO2, 37°C incubator for 24 hours until the cells adhered completely.
[0101] (2) Drug administration: Different drugs were prepared into eight concentrations of 0.7813 μmol / L, 1.5625 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L by serial dilution. The 96-well plate was taken out and the supernatant was discarded (PBS wells were left untouched). The blank wells and well 0 were given DMEM / MEM without serum. The drug administration was 100 μL per well. After the drug was added, the plate was incubated in a 5% CO2, 37℃ incubator for 48 h.
[0102] (3) Add detection reagent: After the incubation is over, dilute the CCK-8 reagent with serum-free DMEM / MEM to prepare a concentration of 10% by volume. Take out the 96-well plate, discard the supernatant (do not leave PBS), and add the prepared CCK-8 solution, 100 μL per well.
[0103] (4) Absorbance detection: Place the 96-well plate into the reading chamber of the microplate reader in the specified orientation. Set the detection wavelength of the microplate reader to 450 nm and perform three independent replicate experiments. Calculate the IC50 using GraphPad Prism 9.5 based on the ratio of absorbance of the cells treated with the test compound to that of the blank control group. 50 The results are shown in Table 4.
[0104] Table 4. Results of antitumor activity of b1-b20 and SAHA
[0105]
[0106] As shown in Table 4, all target compounds exhibited micromolar-level inhibitory activity in the aforementioned solid tumor cell lines, demonstrating broad-spectrum antitumor potential. Among the compounds, compound b19 showed the best antiproliferative activity, with inhibitory effects in both HeLa and FaDu cells superior to the positive control drug SAHA. Particularly in HeLa cells, compound b19 exhibited a significantly higher IC50 value. 50 The value reached 0.55 μmol / L, significantly lower than the IC50 of SAHA. 50 The value (1.94 μmol / L) showed a stronger inhibitory effect.
[0107] like Figure 3 As shown, the molecular docking results of compound b19(S)-6-(4-(3-((3-bromo-2-chlorophenyl)amino)-2-(3-hydroxyurea)-3-oxopropyl)phenoxy)-N-hydroxyhexanamide with HDAC1 and HDAC2 are shown. Among them, A and B are the molecular docking results of compound b19 with HDAC1, and C and D are the molecular docking results of compound b19 with HDAC2. The results show that compound (S)-6-(4-(3-((3-bromo-2-chlorophenyl)amino)-2-(3-hydroxyurea)-3-oxopropyl)phenoxy)-N-hydroxyhexanamide has a high affinity for HDAC1 and HDAC2.
[0108] The molecular docking software used in this invention is Autodock and Pymol.
[0109] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. An HDAC inhibitor based on a tyrosine backbone, characterized in that, The general structural formula of the HDAC inhibitor is: ； Wherein, R1 is naphthyl, monosubstituted phenyl, or disubstituted phenyl; The monosubstituted phenyl group is an ortho-substituted phenyl group, a meta-substituted phenyl group, or a para-substituted phenyl group; In the ortho-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, or fluorine; In the meta-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethyl; In the para-substituted phenyl group, the substituent is methyl, bromine, chlorine, methoxy, fluorine, or trifluoromethoxy. The disubstituted phenyl group is 3-bromo-2-chlorophenyl or 3-chloro-2-bromophenyl.
2. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 1, characterized in that, Includes the following steps: S1. Using CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate as reactants, an affinity substitution reaction was carried out in the presence of anhydrous potassium carbonate to prepare (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate methyl ester. S2. Using methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate as a raw material, the tert-butyl group is removed under the action of trifluoroacetic acid to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid; S3. Using (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid and aniline derivatives as raw materials, HATU as a condensing agent and triethylamine as an acid-binding agent, an amide condensation reaction was carried out to prepare an intermediate. S4. Add the intermediate to potassium hydroxylamine solution and react at room temperature for 2-4 hours. After the reaction is complete, remove the organic solvent by rotary evaporation, then adjust the pH of the reaction system to 5-6, let it stand, filter to obtain the precipitate, and dry the precipitate under reduced pressure to obtain the HDAC inhibitor.
3. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 2, characterized in that, Step S1 is as follows: CBZ-L-tyrosine tert-butyl ester and methyl 6-bromohexanoate are added to a dimethylformamide solution containing anhydrous potassium carbonate. Under stirring conditions, the reaction is carried out at 50°C for 8-10 h. After the reaction is completed, the reaction is quenched with saturated sodium bicarbonate solution, and the mixture is extracted three times with ethyl acetate. The organic phases are combined, and the organic phases are separated and purified by column chromatography and dried to obtain (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]methyl hexanoate.
4. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 3, characterized in that, The molar ratio of anhydrous potassium carbonate, CBZ-L-tyrosine tert-butyl ester, and methyl 6-bromohexanoate is 2~4:1:
1.
5. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 2, characterized in that, Step S2 is as follows: Methyl (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate is added to a mixed solution of trifluoroacetic acid and dichloromethane, and reacted at 50°C for 8-10 h. After the reaction is completed, the reaction is quenched with saturated sodium bicarbonate solution, extracted three times with ethyl acetate, the organic phases are combined, separated by column chromatography and dried to obtain (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid.
6. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 5, characterized in that, The molar ratio of (S)-6-[4-(2-((benzyloxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)phenoxy]hexanoate to trifluoroacetic acid is 1:5~10, and the volume ratio of trifluoroacetic acid to dichloromethane in the mixed solution is 1:1.5~2.
7. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 2, characterized in that, Step S3 is as follows: (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid is added to dichloromethane and stirred to dissolve. Then, triethylamine and HATU are added sequentially, and the mixture is stirred and activated at room temperature for 1-2 hours. Then, aniline derivatives are added and refluxed at 45°C for 8-10 hours. After the reaction is completed, the insoluble matter is removed by filtration, distilled water is added, and the mixture is extracted three times with ethyl acetate. The organic phases are combined, washed with saturated sodium chloride solution, dried, evaporated to dryness, separated and purified by column chromatography, and dried to obtain the intermediate.
8. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 7, characterized in that, The molar ratio of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((6-methoxy-6-oxohexyl)oxy)phenyl)propionic acid, triethylamine, HATU, and aniline derivatives is 1:1.2:1.2:
1.
9. The method for preparing the HDAC inhibitor based on the tyrosine backbone according to claim 2, characterized in that, In step S4, the molar ratio of potassium hydroxylamine to the intermediate in the potassium hydroxylamine solution is 5~10:
1.
10. The use of the tyrosine-based HDAC inhibitor of claim 1 in the preparation of anti-solid tumor drugs.