CN111116647B

CN111116647B - A kind of phosphoric acid compound with HDAC inhibitory activity and preparation method and use thereof

Info

Publication number: CN111116647B
Application number: CN201911213773.7A
Authority: CN
Inventors: 唐初
Original assignee: Xidian University
Current assignee: Xidian University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2021-08-24
Anticipated expiration: 2039-12-02
Also published as: CN111116647A

Abstract

一种具有HDAC抑制活性的磷酸类化合物及其制备方法与用途。本发明属于医药技术领域，本发明提供了一种式(I)所示的磷酸类化合物、或其构象异构、或其光学异构体。式I：

。通过试验证明了，本发明式(I)所示的磷酸类化合物能够有效的抑制组蛋白去乙酰化酶HDACs的活性，并且，本发明的化合物对肿瘤细胞的增殖活性也具有明显的抑制效果，此外，这些磷酸类化合物对正常乳腺上皮细胞MCF‑10A没有毒性。所以，本发明化合物在制备组蛋白去乙酰化酶抑制剂及应用中，包括抗肿瘤活性，具有非常大的潜力。本专利主要介绍这类化合物的结构、制备方法及在制备抗乳腺癌药物中的应用。A phosphate compound with HDAC inhibitory activity and its preparation method and use. The present invention belongs to the technical field of medicine, and provides a phosphate compound represented by formula (I), or its conformational isomer, or its optical isomer. Formula I:

. It has been proved by experiments that the phosphate compounds represented by the formula (I) of the present invention can effectively inhibit the activity of histone deacetylase HDACs, and the compounds of the present invention also have a significant inhibitory effect on the proliferation activity of tumor cells, Furthermore, these phosphonates were not toxic to normal mammary epithelial cells MCF‑10A. Therefore, the compounds of the present invention have great potential in the preparation of histone deacetylase inhibitors and their applications, including antitumor activity. This patent mainly introduces the structure, preparation method and application of such compounds in the preparation of anti-breast cancer drugs.

Description

Phosphoric acid compound with HDAC (Histone deacetylase) inhibitory activity as well as preparation method and application thereof

Technical Field

The invention relates to a phosphate compound with HDAC (histone deacetylase) inhibitory activity, and a preparation method and application thereof.

Background

Histone Deacetylase (HDAC) is a key enzyme for catalyzing the removal of acetyl group of lysine residue in histone and non-histone, and plays an important role in gene transcription, cell cycle progression, apoptosis, tumorigenesis, etc. Eyes of a userPreviously, HDACs were abnormally high expressed in a variety of diseases, such as tumors. Therefore, the HDAC targeted therapy is expected to become an effective therapeutic method, and the HDAC inhibitor is expected to be developed into a disease targeted therapeutic agent. Several hundred HDAC inhibitors (HDACi) have been designed and synthesized, and their structures are abundant, but HDACi is Zn²⁺Dependent form thereof with Zn²⁺The chelating types of (1) are mainly classified into four types: the first class, hydroxamic acids, represents the drug SAHA (approved for marketing in 2006); the second, cyclotetrapeptide, represents the drug Romidepsin (approved for marketing in 2009); the third class, the benzamides, represents the inhibitor Chidamide (marketed in 2014); the fourth class, fatty acids, represents inhibitors such as Valproic acid (phase III clinical trial) and the like.

Of these inhibitors, the HDACi activity of hydroxamic acid is generally greater than that of other types. However, the hydroxamic acids HDACi are unstable in vivo and are easily metabolized into toxic substances; furthermore, most HDACis are now a broad spectrum of HDACs, which are taken up and distributed widely among the various HDAC isoforms in the tissues, and this lack of selectivity often results in undesirable side effects such as fatigue, thrombocytopenia, nausea/vomiting and cardiotoxicity, due to the different isoforms' functions. Clinical data have shown that SAHA can cause neurotoxicity of thrombus formation, and FK228 has cardiotoxicity. These disadvantages severely limit the widespread clinical use of HDACis. Therefore, the development of new-structure, high-efficiency and low-toxicity HDACi is an urgent task.

Disclosure of Invention

In order to solve the above problems, the present invention provides a phosphate compound having HDAC inhibitory activity, and preparation thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a compound of formula I:

formula I

Wherein R is H, Me, OMe, F.

The invention also aims at the application of the compound or the isomer thereof in preparing HDAC inhibitor medicines.

The HDAC inhibitor medicine is used for treating diseases caused by abnormal activity of HDAC.

Further, the disease is any one or more of cell proliferation disease, autoimmune disease, inflammation, neurodegenerative disease, and viral disease.

Still further, the disease is cancer.

A pharmaceutical composition for inhibiting histone deacetylase activity is a preparation prepared by taking the compound or crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

The present invention also provides a method for preparing a phosphate compound having HDAC inhibitory activity represented by the above structural formula I, comprising the steps of:

(1) dissolving aniline or its derivatives (p-toluidine, p-methoxyaniline, p-fluoroaniline) and 6-heptenoic acid in dichloromethane, and adding 2- (7-azobenzotriazol)N,N,N',N' -tetramethyluronium Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIEA), stirred at room temperature for 12 hours; after the reaction was complete, saturated NaHCO was added₃Solution, dichloromethane extraction, anhydrous Na₂SO₄Drying, evaporating to remove dichloromethane to obtain crude product, and purifying by column chromatography to obtain compound 3a (orN-phenyl-6-heptenylamine), 3b (NP-tolyl-6-heptenylamine), 3c (N-p-methoxyphenyl-6-heptenylamine), 3d (N-p-fluorophenyl-6-heptenylamine); the mass ratio of aniline or its derivative, 6-heptenoic acid, HATU and DIEA is 1: 1.2: 1.3: 3.

(2) Dissolving the compound 3a, 3b, 3c or 3d inN,N-Dimethylformamide (DMF), K is added₂CO₃And dibenzyl phosphite, heating to 50 ℃ and reacting for 2 hours; after the reaction was completed, 5% citric acid solution and ethyl acetate were addedExtracted with anhydrous Na₂SO₄Drying, evaporating to remove ethyl acetate to obtain crude product, and purifying by column chromatography to obtain (7-oxo-7-anilino) heptyl dibenzyl phosphate compounds 4a, 4b, 4c or 4 d; compounds 3a, 3b, 3c or 3d with K₂CO₃And the mass ratio of dibenzylphosphite is 1: 2: 1.6.

(3) Mixing (7-oxo-7-anilino) heptyl dibenzyl phosphate compounds 4a, 4b, 4c or 4d and 6M HCl, and heating to 50 ℃ for reaction for 12 hours; distilling under reduced pressure to remove newly generated methanol, adding 6M HCl, heating to 50 deg.C for reaction for 12 hr, distilling under reduced pressure to remove newly generated methanol, extracting with ethyl acetate, and extracting with anhydrous Na₂SO₄Drying, evaporating to remove ethyl acetate to obtain crude product, and purifying by column chromatography to obtain (7-oxo-7-anilino) heptyl phosphate compounds 5a, 5b, 5c or 5 d; the mass ratio of the (7-oxo-7-anilino) heptyl dibenzyl phosphate compound to the 6M hydrochloric acid was 1: 1.7.

The reaction flow is as follows:

the phosphate compound with HDAC inhibitory activity has stronger inhibitory activity on HDAC enzyme.

The obtained phosphoric acid compound with HDAC inhibitory activity has strong inhibitory activity on breast cancer MCF-7 and MDA-MB-231 cells, and has no toxicity on normal breast epithelial cells MCF-10A.

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

(1) the inhibitors of the invention bind zinc ions in the active pocket of HDAC enzymes using phosphate as a chelating group, unlike conventional hydroxamic acid, benzamide chelating groups. Thus, phosphate compounds are a novel class of HDAC inhibitors.

(2) The inhibitors of the invention are not toxic to normal mammary epithelial cells.

(3) The preparation method provided by the invention is simple, mild in condition and high in yield, provides a new drug research and development idea, improves the utilization efficiency and treatment effect of the drug for treating cancer, and has a remarkable effect on inhibiting solid tumors.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely a few embodiments of the invention and not all embodiments thereof. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making innovative efforts, fall within the scope of protection of the present invention.

Example 1: preparation of (7-oxo-7-anilino) heptyl phosphate (5a)

The chemical structure of (7-oxo-7-anilino) heptyl phosphate (5a) in this example is as follows:

the synthetic route of 5a in this example is as follows:

Nsynthesis of (3a) phenyl-6-heptenamine

Aniline (502.9 mg, 5.4 mmol) and 6-heptenoic acid (833.1 mg, 6.5 mmol) were dissolved in dichloromethane, followed by addition of 2- (7-azobenzotriazole) -N,N,N',N' -tetramethyluronium hexafluorophosphate (HATU, 2.662 g, 7.0 mmol) andN,Ndiisopropylethylamine (DIEA, 2.094 g, 16.2 mmol), stirred at room temperature for 12 h; after the reaction was complete, 25 mL of saturated NaHCO was added₃Solution, dichloromethane (3X 10 mL) extraction, anhydrous Na₂SO₄Drying and evaporation of dichloromethane gave the crude product which was purified by column chromatography to give 602.7 mg of compound 3a in 54.9% yield.

Synthesis of dibenzyl (4a) phosphate (7-oxo-7-anilino) heptyl

Will be provided withN-phenyl-6-heptenamine Compound 3a (406.6 mg, 2.0 mmol) was dissolved inN,N-dimethylformamide (DMF, 10 mL), K was added₂CO₃(552.8 mg, 4.0 mmol) and dibenzylphosphite (839.2 mg, 3.2 mmol) were heated to 50 ℃ for 2 hours; after the reaction was complete, 5% citric acid solution (15 mL) was added, and the mixture was extracted with ethyl acetate (3X 20 mL) and anhydrous Na₂SO₄Drying, evaporating to remove ethyl acetate to obtain crude product, and purifying by column chromatography to obtain 452.2 mg of compound 4a with yield of 48.6%.

Synthesis of (7-oxo-7-anilino) heptyl phosphate (5a)

(7-oxo-7-anilino) heptyl dibenzyl phosphate 4a (558.6 mg, 1.2 mmol) and 6M HCl (6 mL) were mixed and heated to 50 ℃ for 12 hours; distilling under reduced pressure to remove newly generated methanol, adding 6M HCl (6 mL), heating to 50 deg.C for reaction for 12 hr, distilling under reduced pressure to remove newly generated methanol, extracting with ethyl acetate, and extracting with anhydrous Na₂SO₄Drying, removing ethyl acetate by evaporation to obtain a crude product, and purifying by column chromatography to obtain 279 mg (7-oxo-7-anilino) heptyl phosphate 5a with a yield of 81.5%.¹HNMR (400 M, CDCl₃) δ7.55 (d, J = 7.6 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.11-7.18 (m, 1H), 2.40 (t, J = 7.2 Hz, 2H), 1.75-1.83 (m, 4H), 1.61-1.65 (m, 2H), 1.54 (d, J = 6.8 Hz, 4H). ¹³CNMR (100 M, CDCl₃) δ171.27, 137.94, 128.97, 124.26, 119.95, 58.65, 40.00, 37.46, 26.31, 25.40, 25.02.

Example 2: synthesis of [ 7-oxo-7- (4-methylanilino) ] heptyl phosphoric acid (5b)

The procedure is as in example 1, wherein the aniline is replaced by p-toluidine and the product is a white solid with a yield of 83.4%.¹HNMR (400 M, CDCl₃) δ7.41 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.86 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.72-1.78 (m, 2H), 1.52 (d, J = 6.8 Hz, 4H) 1.25-1.33 (m, 4H).

Example 3: synthesis of [ 7-oxo-7- (4-methoxyanilino) ] heptyl phosphoric acid (5c)

The procedure is as in example 1, wherein aniline is replaced by p-anisidine, and the product is a white solid with a yield of 86.2%.¹HNMR (400 M, CDCl₃) δ7.39 (d, J = 8.4 Hz, 2H), 7.24 (s, 1H), 7.12 (d, J= 8.0 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H), 2.30 (s, 3H), 1.71-1.76 (m, 4H), 1.51 (d, J = 6.4 Hz, 4H) 1.25-1.28 (m, 2H).

Example 4: synthesis of [ 7-oxo-7- (4-fluoroanilino) ] heptyl phosphoric acid (5d)

The procedure is as in example 1, the aniline being replaced by para-fluoroaniline, and the product being a white solid in 81.8% yield.¹HNMR (400 M, CDCl₃) δ7.63 (s, 1H), 7.44-7.44 (m, 2H), 7.00 (t, J = 8.8 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.6 Hz, 1H), 1.68-1.74 (m, 3H), 1.49 (d, J = 6.4 Hz, 3H), 1.24 (t, J = 6.4 Hz, 2H), 1.18 (d, J = 6.0 Hz, 1H).

Example 5: HDAC enzyme Activity test assay

The procedure was carried out using a commercially available HDAC enzyme assay kit (BPS Bioscience). The compound was first dissolved in 100% DMSO, then diluted with a buffer in a kit to a concentration of 25, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001. mu.M, and added to a 96-well plate, then 15. mu.L of Hela nuclear extract (15. mu.L of buffer was added to each well of the control group) was added to the reaction well plate, and after 15 min of reaction at room temperature, 10. mu.L of fluorogenic substrate solution was added to start the reaction, the final concentration of HDACs protein in the reaction was 6 nM, the final concentration of tryptin was 0.05. mu.M, and the final concentration of Ac-peptide was 8. mu.M, and after 1 hour of reaction at room temperature in the dark, the fluorescence intensity (emission wavelength 355 nM, absorption wavelength 460 nM) was measured with a microplate reader to calculate the inhibition ratio. Specific results are shown in table 1.

TABLE 1 HDACs inhibitory Activity results (IC) for representative Compounds of interest 5a-d synthesized according to the invention₅₀, nM)

Compounds	IC₅₀
		5a	169
5b	192
		5c	246
5d	295

The above experimental results show that: the synthesized compound shows a strong inhibition effect on the inhibition activity of HDACs enzyme.

Example 6: antitumor Activity test of phosphoric acid Compounds

The breast cancer MCF-7, MDA-MB-231 and the normal epithelial cells MCF-10A of the breast are cultured in a DMEM liquid medium containing 10 percent fetal calf serum and phenol red. When the cell density reaches 80-90%, digesting the cells, and suspending the cells in a phenol red-free DMEM medium containing 10% fetal calf serumSpread to 96 well cell culture plates. After the cells are completely attached to the wall, the original culture solution is discarded, and 100 mul of fresh compound solution prepared by DMEM medium containing 10% fetal calf serum is added into each hole, wherein the compound concentration gradient is as follows: 1X 10^-8M, 1×10^-7M, 5×10^-7M, 1×10^-6 M, 1×10^-5 M, 5×10^-5M, 1×10^-4And M. After 3 days of drug treatment, the plates were removed and 20. mu.L of 5 mg/mL MTT medium was added to each well and placed at 37^oC、5% CO₂Incubate in the incubator for 4 hours. After that, the liquid was aspirated from each well, and then 100. mu.L of Dimethylsulfoxide (DMSO) was added to each well, and the mixture was shaken on a micro-stirrer for 15 minutes to dissolve the crystals sufficiently. And (3) reading a plate on a microplate reader to test the absorbance value (OD) of each hole, and selecting the wavelength at 490 nm as the dominant wavelength and the wavelength at 630 nm as the reference wavelength. The growth inhibition of tumor cells by different drug concentrations was calculated as follows:

performing linear regression analysis on the concentration of the drug by using the inhibition rate, and calculating IC by using a linear equation₅₀The specific results are shown in Table 2.

TABLE 2 results of MCF-7, MDA-MB-231 and MCF-10A cytostatic Activity (IC) of representative Compounds of interest 5a-d synthesized according to the invention₅₀, μM)

Compounds	MCF-7	MDA-MB-231	MCF-10A
				5a	6.9	5.5	> 100
5b	7.5	6.2	> 100
				5c	10.8	9.3	> 100
5d	14.3	7.6	> 100

The above experimental results show that: the synthesized compound shows good anti-breast cancer activity not only for hormone-dependent (MCF-7) breast cancer, but also for non-hormone-dependent (MDA-MB-231) breast cancer. Compound 5a (IC)₅₀= 5.5 μ M) exhibited the strongest inhibitory activity against non-hormone dependent (MDA-MB-231) breast cancer.

Claims

1. a preparation method of a phosphoric acid compound with HDAC inhibitory activity, is characterized in that, comprises the following steps:

(1) Dissolve aniline, p-toluidine, p-methoxyaniline or p-fluoroaniline and 6-heptenoic acid in dichloromethane, then add 2-(7-azobenzotriazole) -N , N , N ', N' -tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were stirred at room temperature for 12 hours; after the reaction was completed, saturated NaHCO ₃ solution was added, extracted with dichloromethane, no Dry with water over Na ₂ SO ₄ , evaporate dichloromethane to obtain the crude product, and obtain compound 3a, 3b, 3c or 3d after purification by column chromatography; aniline, p-toluidine, p-methoxyaniline or p-fluoroaniline and 6-heptane Substances of alkenoic acid, 2-(7-azobenzotriazole) -N , N , N ', N' -tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine The ratio is 1: 1.2: 1.3: 3; wherein the structural formula of compound 3a, 3b, 3c or 3d is as follows:

;

(2) Dissolve compound 3a, 3b, 3c or 3d in N , N -dimethylformamide, add K ₂ CO ₃ and dibenzyl phosphite, heat to 50°C and react for 2 hours; after the reaction is over, add 5% citric acid solution, extracted with ethyl acetate, dried over anhydrous Na ₂ SO ₄ , evaporated to remove ethyl acetate to obtain crude product, purified by column chromatography to obtain compound 4a, 4b, 4c or 4d; compound 3a, 3b, 3c Or the material ratio of 3d to K ₂ CO ₃ and dibenzyl phosphite is 1: 2: 1.6; wherein the structural formula of compound 4a, 4b, 4c or 4d is as follows:

;

(3) Compound 4a, 4b, 4c or 4d was mixed with 6 M HCl, heated to 50 °C and reacted for 12 hours; the newly formed benzyl alcohol was distilled off under reduced pressure, extracted with ethyl acetate, and anhydrous Na ₂ SO ₄ After drying, ethyl acetate was evaporated to obtain crude product, which was purified by column chromatography to obtain phosphoric acid compounds 5a, 5b, 5c or 5d with HDAC inhibitory activity; the substance ratio of compounds 4a, 4b, 4c or 4d and 6 M hydrochloric acid is 1:1.7; the structural formula of compound 5a, 5b, 5c or 5d is as follows:

.