CN111995629B

CN111995629B - Germacrene leaf derivative, pharmaceutical composition thereof and application thereof in medicine

Info

Publication number: CN111995629B
Application number: CN201910421119.9A
Authority: CN
Inventors: 王恒山; 朱彦葵; 王采奕; 梁东; 贾强
Original assignee: Seasons Biotechnology Taizhou Co ltd
Current assignee: Seasons Biotechnology Taizhou Co ltd
Priority date: 2019-05-11
Filing date: 2019-05-11
Publication date: 2021-06-01
Anticipated expiration: 2039-05-11
Also published as: CN111995629A

Abstract

The present invention relates to germacra leaf derivatives, pharmaceutical compositions thereof and their use in medicine. Specifically, the invention relates to germacra leaves compounds, a preparation method thereof and application of a pharmaceutical composition taking the compounds as active ingredients in preparation of VEGF (vascular endothelial growth factor) -induced human umbilical cord endocrine (HUVEC) proliferation selective inhibitor and low-toxicity drugs for resisting angiogenesis-related diseases such as retinopathy and the like.

Description

Germacrene leaf derivative, pharmaceutical composition thereof and application thereof in medicine

Technical Field

The invention belongs to the technical field of pharmaceutical compounds and medicines. In particular to germacra leaf compounds, a preparation method thereof and application of the compounds and pharmaceutical compositions thereof in preparing VEGF (vascular endothelial growth factor) -induced human umbilical endothelial cell secretion (HUVEC) proliferation selective inhibitors and low-toxicity drugs for resisting angiogenesis-related diseases such as retinopathy and the like.

Background

Retinopathy (retinopathy) is classified into many types, and is complicated, and the most common types include retinal detachment, maculopathy, ocular trauma, diabetic retinopathy, endophthalmitis, intrabulbar foreign bodies, congenital ocular diseases such as neonatal Retinopathy (ROP), and intraocular parasites, and the most common retinal detachment is taken as an example.

Diabetic retinopathy is one of the most common and serious microvascular complications of diabetes, and in the past, people have used general vasodilators, vitamins and other medicines for support therapy, but the effect is not exact. It is one of four major cases of blindness and the condition is further aggravated. Thus, the acute situation has conferred medical workers a worthless undertaking-finding new highly effective, low side-effect, low toxicity anti-angiogenic agents, particularly new agents capable of selectively treating obesity and diabetic retinopathy.

VEGF induces the secretion of endothelial cells to participate in the regulation of some physiological processes related to inflammation, is related to the occurrence and malignancy of various cancers, such as inflammatory reaction, tumor cell proliferation, infection, metastasis, new vessel proliferation and the like, and is an important target point for treating malignant tumors. There are 5 VEGF receptors currently found: VEGFR1(Flt1), VEGFR2(KDR/Klk1), VEGFR3(Flt4), NP1, and NP 2. Flt1, KDR, Flt4 are all receptor tyrosine protein kinases (PTKs). VEGFR2 is the most commonly expressed one

VEGF-mediated modulation of angiogenic pathways is considered an important goal in drug discovery programs for diseases associated with hyperactive angiogenesis. Several VEGF signaling pathway inhibitors are commercially available for the treatment of cancer or in clinical trials. In addition to their use as anti-cancer drugs, VEGF pathway inhibitors have been proposed as potential therapeutic agents for obesity and diabetic retinopathy.

Novel anti-angiogenic agents targeting the VEGF pathway, particularly for the treatment of diabetic retinopathy, require long-term administration. Therapeutic agents are required to be minimally cytotoxic or non-cytotoxic to avoid side effects on normal cells.

There are two types of major inhibitors acting on the VEGF-induced endothelial cell secretion machinery pathway: antibodies and small molecules.

Monoclonal antibodies: studies have shown that Bevacizumab (a monoclonal anti-VEGF-a antibody), an approved drug for the treatment of metastatic colon cancer patients, can reduce tumor size and can also increase prolactin levels. Is used for clinical tests of colorectal cancer, lung cancer, breast cancer and glioblastoma.

Nitrogen heterocyclic small molecules: n- (2-chloro-5-methoxyphenyl) -6-methoxy-7- [ (1-methyl-4-piperidinyl) methoxy ] - (M475271) is a novel anilinoquinazoline derivative which is shown to inhibit tumor growth in vivo. Although M475271 is a molecule found to have a cell migration-targeting effect in clinical antitumor experimental studies, it was developed as a tyrosine kinase inhibitor. The non-specific or non-selective inhibitor affects multiple immune targets, has high toxicity and has extremely limited clinical application value. Therefore, the research and development of a new generation of inhibitors with high specificity of VEGF-induced endothelial cell secretion pathway has important practical significance.

Germacranolide is mainly present in [10+5] ring-type sesquiterpenes of the genus Artemisia of the family Compositae, and is mainly in the cardiovascular and oncological fields in terms of activity. At present, no report exists on the application of the compound in the medicines for treating diabetes.

We have prepared novel germacranolide derivatives with novel anti-angiogenic properties without significant cytotoxicity. Are a novel class of compounds with enhanced anti-angiogenic activity and minimal cytotoxicity. This structure effectively inhibits the formation of clear blood vessels in diabetic retinopathy.

Research findings indicate the potential of this compound as a lead structure for the development of anti-angiogenesis, as a drug with novel functions and as a probe for elucidating novel biological mechanisms associated with angiogenesis.

Disclosure of Invention

According to one aspect of the present application, there is provided a compound having formula (I) or a pharmaceutically acceptable salt thereof:

wherein

R₁Independently selected from hydrogen, phenyl, optionally substituted phenyl derivatives, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 acyl, optionally substitutedC1-C6 alkenyl, optionally substituted C1-C6 alkynyl;

R₂independently selected from hydrogen, phenyl, optionally substituted phenyl derivatives, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 acyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C6 alkynyl.

In one embodiment of the present application, wherein said R is₁Is hydrogen or C1-C6 acyl.

In one embodiment of the present application, wherein said R is₂Hydrogen, C1-C6 alkyl.

According to another aspect of the present application, there is provided a compound having formula (II) or a pharmaceutically acceptable salt thereof:

in one embodiment of the present application, wherein said R is₃Is hydrogen or C1-C6 acyl.

In one embodiment of the present application, wherein said R is₄Hydrogen and hydroxyl.

According to another aspect of the present application, there is provided a pharmaceutical composition comprising a compound having activity herein or a pharmaceutically acceptable salt thereof and combinations thereof and one or more pharmaceutically acceptable carriers, diluents, excipients or combinations thereof.

The pharmaceutical composition can be prepared into certain dosage forms, and the administration route is preferably oral administration, parenteral administration (including subcutaneous, intramuscular and intravenous), rectal administration and the like. For example, dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, powders, lozenges, syrups or suspensions; formulations suitable for parenteral administration include aqueous or non-aqueous solutions or emulsions for injection; dosage forms suitable for rectal administration include suppositories with hydrophilic or hydrophobic carriers. The dosage forms may also be formulated as desired to provide rapid, delayed or modified release of the active ingredient.

The pharmaceutical composition comprises a pharmaceutically acceptable carrier, such as a solid carrier including, but not limited to: diluents such as starch, modified starch, lactose, powdered cellulose, microcrystalline cellulose, anhydrous calcium hydrogen phosphate, tricalcium phosphate, mannitol, sorbitol, sugar, and the like; binders such as acacia, guar gum, gelatin, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyethylene glycol, copovidone, and the like; disintegrants such as starch, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, crospovidone, croscarmellose sodium, colloidal silicon dioxide, and the like; lubricants, such as stearic acid, magnesium stearate, zinc stearate, sodium benzoate, sodium acetate, and the like; glidants such as colloidal silicon dioxide and the like; complex-forming agents, such as various grades of cyclodextrins and resins; release rate controlling agents such as hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, methyl methacrylate, waxes, and the like. The pharmaceutically acceptable carrier also includes liquid carriers, specifically including but not limited to: aqueous solvent, alcoholic solvent or oil such as sterile water, physiological saline solution, glucose solution, mannitol solution, vegetable oil, cod liver oil, ethanol, propanol, glycerol, etc., and carrier such as polyethylene glycol, polypropylene glycol, etc. can also be used. Alternative other pharmaceutically acceptable carriers include, but are not limited to, film forming agents, plasticizers, colorants, flavoring agents, viscosity modifying agents, preservatives, antioxidants, osmotic agents, buffering agents, and the like. Each carrier must be acceptable, compatible with the other ingredients of the formulation and not injurious to the patient.

The pharmaceutical compositions may be prepared using methods well known to those skilled in the art. In preparing the pharmaceutical composition, the germacra lactone and/or lactam derivative solvate or crystal thereof or crystal form thereof of the present invention is used as a pharmaceutically active ingredient to be mixed with one or more pharmaceutically acceptable carriers, and optionally one or more other active ingredients. For example, solid formulations may be prepared by direct mixing, granulating and the like, and liquid formulations may be prepared by mixing, dissolving and the like.

Of particular mention are tablets or capsules which may be prepared by mixing the pharmaceutically active ingredient with one or more of the aforementioned pharmaceutically acceptable carriers and subsequently compressing the resulting mixture in a conventional tableting machine or into capsule shells to make capsules. Examples of the carrier include: anhydrous dibasic calcium phosphate, PVP-VA copolymer, microcrystalline cellulose, sodium starch glycolate, corn starch, mannitol, potato starch, talc, magnesium stearate, gelatin, lactose, gums and the like, and carriers which are usually used for the above purpose, such as coloring agents, flavoring agents, preservatives and the like, may also be used.

Particular mention is made of the wet granulation process of solid formulations, exemplified by the wet granulation of tablets, which is: blending the dry solids (pharmaceutical active ingredient, filler, binder, etc.), wetting with a wetting agent such as water or alcohol (e.g., ethanol), and forming the wetted solids into agglomerates or granules, continuing wet granulation until the desired uniform particle size is obtained, and subsequently drying the granulated product. Typically, vortioxetine salt of the present invention or its crystals or crystalline forms, lactose, corn starch and copovidone are mixed with water in a high shear machine, after the formation of granules, these granules are screened with a suitable mesh number of screens and dried, and the resulting dried granules are then mixed with microcrystalline cellulose, croscarmellose sodium and magnesium stearate and then tableted. Alternatively, wet granulation of the compounds of the present invention can be achieved using mannitol, corn starch and copovidone, which is mixed with microcrystalline cellulose, sodium starch glycolate and magnesium stearate and then tableted. Alternatively, wet granulation of the compounds of the present invention can be achieved using anhydrous dibasic calcium phosphate, corn starch, and copovidone, which is mixed with microcrystalline cellulose, sodium starch glycolate, and magnesium stearate, and then tableted. The copovidone is PVP-VA copolymer. Alternatively, the active ingredient, mannitol and microcrystalline cellulose are mixed in a fluidized bed granulation dryer, an aqueous solution of hydroxypropyl cellulose is sprayed to the mixture to obtain a granulated fine powder, and the obtained granules are mixed with microcrystalline cellulose, sodium starch glycolate and magnesium stearate and then tableted. Alternatively, the tablets are coated with a suitable coating material, for example by spraying a solution containing the coating material onto the tablets.

Of particular mention are oral suspensions, an advantage of such administration forms is that patients may not have to swallow solid forms, particularly elderly, children or patients with oral, throat impairment who may have difficulty swallowing solid forms. Suspensions are two-phase systems formed by dispersing solid particles of a pharmaceutically active ingredient in a liquid, which remains in its original solid form in an aqueous or alcoholic carrier. Other components of oral suspensions may include buffers, surfactants, viscosity modifiers, preservatives, antioxidants, coloring agents, flavoring agents, taste-masking agents, and the like, as is well known to those skilled in the art.

According to another aspect of the application, the application provides an active compound or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition of the application in preparing a VEGF-induced human umbilical endothelial cell secretion (HUVEC) proliferation selective inhibitor and a low-toxicity medicament for resisting angiogenesis-related diseases such as retinopathy.

Drawings

Figure 1 is a micrograph of matrigel tubule formation experiment of

compounds

1, 2, 3, 4a, 4b, 5, 6.

Fig. 2 is a graph of the results obtained from matrigel tubule formation experiments quantifying tubule length for

compounds

1, 2, 3, 4a, 4b, 5, 6.

FIG. 3 shows the results of microscope photographs of matrigel tubule formation experiments of Compound 2 at concentrations of 20, 40, and 80uM, respectively.

FIG. 4 is a graph of the results obtained from matrigel tubule formation experiments quantifying tubule length for Compound 2 at concentrations of 20, 40, and 80uM, respectively.

Figure 5 is a graph of the effect of compound 2 on the VEGFR2 signaling pathway.

Detailed Description

In order to better understand the essence of the present invention, the following test examples, examples and formulation examples of the present invention will be used to illustrate the preparation method and pharmacological effects of germacranolide and lactam derivatives represented by the general formula (I) and the general formula (II) of the present invention, but the technical scheme of the present invention is not limited thereto, and any scheme that can be made without creative efforts of those skilled in the art using the scheme similar to the technical scheme of the present invention is considered to be within the technical scheme of the present invention.

The instrument and method for data acquisition:

1D and 2D NMR were measured on a Bruker AM-400 NMR spectrometer using the deuterated reagent manufactured by Sigma Aldrich, TMS as internal standard, delta units in ppm and J units in Hz.

HRESI-MS was measured on a Thermo-Fisher Scientific active mass spectrometer.

Reagents used for the experiment:

silica gel for sample mixing is 100-200 meshes, silica gel for chromatography is 300-400 meshes, and thin layer plates (with thickness of 0.4-0.5mm) are produced in Qingdao ocean chemical factories;

the analytically pure solvents are all products of Shantou Wen Longgong chemical Co., Ltd; the chromatographically pure solvent is manufactured by Fisher corporation, USA.

Preparation example: preparation of germacranolide 4 and germacranolide 5

The sage extract (the extraction method is shown in the pharmacopoeia 2010 edition of the people's republic of China) is dissolved by acetone and ethanol in sequence, insoluble substances are filtered, the filtrate is subjected to silica gel sample mixing column chromatography, petroleum ether/acetone gradient elution, and then the mixture is purified by methods such as normal phase column chromatography, reverse phase column chromatography, petroleum ether/acetone recrystallization and the like repeatedly to obtain 4 (petroleum ether: ethyl acetate is 10: 1, Rf is 0.4) monomers and germacranolide 5 (petroleum ether: ethyl acetate is 5: 1, Rf is 0.3) monomers.

Example 1: preparation of Compound 4a/4b

To compound 4(100mg) was added acetic anhydride-pyridine (1: 1, 2.0mL) and the reaction was refluxed with acetylation at room temperature for 24 hours, and as a result of the reaction completion by TLC detection, H2O (2mL) was added to the reaction solution to quench the reaction, followed by extraction with EtOAc three times, and the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo and purified by semi-preparative reverse phase HPLC (MeOH-H2O, 70: 30) to give the acetylated product 4a1(45mg) and 4b1(20 mg). LiOH (2.2eq.), THF/H2O (3/1) were then added separately and deacetylated at room temperature under reflux for 4 hours, and the reaction mixture was concentrated in vacuo and purified by semi-preparative reverse phase HPLC (CH3CN-H2O, 55: 45) to give the deacetylated products 4a (13.6mg) and 4b (11.6mg), respectively.

Compound 4 a: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 377.1594[ M-H [ ]]^-(calcd for C₂₀H₂₅O₇377.1600), the molecular formula of the compound is presumed to be C₂₀H₂₆O₇。¹H-NMR(400MHz，Acetone)δ6.92(s，1H)，6.86(m，1H)，6.41(dd，J＝1.4，6.0Hz，1H)，3.58(d，J＝6.0Hz，1H)，2.96(s，1H)，2.95(m，1H)，2.18(m，1H)，2.08(m，1H)，2.04(d，J＝1.5Hz，3H)，1.91(m，1H)，1.88(m，3H)，1.84(dd，J＝1.0，7.1Hz，3H)，1.48(m，1H)，1.41(s，3H)，1.30(m，1H)，1.10(s，3H)。

Compound 4 b: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 377.1588[ M-H [ ]]^-(calcd for C₂₀H₂₅O₇377.1600), the molecular formula of the compound is presumed to be C₂₀H₂₆O₇。¹H-NMR(400MHz，Acetone)δ6.90(m，1H)，6.66(s，1H)，6.57(s，1H)，3.13(s，1H)，2.99(m，1H)，2.91(s，1H)，2.12(m，1H)，2.07(m，1H)，1.94(s，3H)，1.82(m，3H)，1.79(dd，J＝1.0，7.1Hz，3H)，1.66(s，3H)，1.63(m，1H)，1.60(m，1H)，1.58(s，3H)，1.54(m，1H)。

Example 2: preparation of Compound 5a/5b

Oriented foodAcetic anhydride-pyridine (1: 1, 2.0mL) was added to Compound 5(40mg) and the mixture was refluxed with acetylation at room temperature for 24 hours, and after completion of the reaction as detected by TLC, H was added to the reaction mixture₂The reaction was quenched with O (2ml) and extracted three times with EtOAc, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the reaction mixture was concentrated in vacuo and purified by semi-preparative reverse phase HPLC (MeOH-H2O, 70: 30) to give the acetylated products 5a1(18mg) and 5b1(16 mg). LiOH (2.2eq.), THF/H2O (3ml/1ml) were then added separately to the reaction mixture to deacetylate at room temperature under reflux for 4 hours. The reaction mixture was concentrated in vacuo and purified by semi-preparative reverse phase HPLC (CH3CN-H2O, 55: 45), respectively, to give the deacetylated products 5a (10.6mg) and 5b (8.6 mg).

Compound 5 a: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 379.1747[ M-H [ ]]^-(calcd for C₂₀H₂₇O₇379.1757), the molecular formula of the compound is presumed to be C₂₀H₂₈O₇。¹H-NMR(400MHz，Acetone)δ7.01(s，1H)，6.29(m，1H)，3.54(d，J＝6.0Hz，1H)，2.96(s，1H)，2.93(m，1H)，2.51(dq，J＝7.0，13.9Hz，1H)，2.17(m，1H)，2.08(m，1H)，2.06(d，J＝1.6Hz，3H)，1.89(m，1H)，1.75(m，1H)，1.52(m，1H)，1.45(m，1H)，1.45(m，1H)，1.39(s，3H)，1.16(d，J＝7.0Hz 3H)，1.08(s，3H)，0.96(t，J＝7.5Hz，3H)。

Compound 5 b: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 379.1747[ M-H [ ]]^-(calcd for C₂₀H₂₇O₇379.1757), the molecular formula of the compound is presumed to be C₂₀H₂₈O₇。¹H-NMR(400MHz，Acetone)δ6.82(s，1H)，6.28(s，1H)，3.10(s，1H)，2.92(m，1H)，2.90(m，1H)，2.41(dq，J＝7.0，13.9Hz，1H)，2.13(m，1H)，2.07(m，1H)，1.94(s，3H)，1.70(m，1H)，1.65(s，3H)，1.64(s，3H)，1.59(m，1H)，1.56(m，1H)，1.42(m，1H)，1.34(m，1H)，1.11(d，J＝7.0Hz，3H)，0.87(t，J＝7.5Hz，3H)。

Example 3: preparation of Compound 1

Step 1: in N₂Compound 5b (20mg) prepared in example 2 and NaN were added under an atmosphere₃(1eq.) of THF/H₂Pd (PPh) was added to O (2ml/0.2ml)₃)₄(5mg), followed by heating to 45 ℃ for 1 hour, TLC detection after completion of the reaction, cooling to room temperature, diluting with water, extracting with EtOAc three times, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, concentrating, and column chromatography (petroleum ether: ethyl acetate: 8: 1) to give the azide compound (18 mg).

Step 2: the azide compound (18mg) obtained in step 1 was weighed out and dissolved in THF/H₂O (2ml/0.2ml), then PPh was added thereto₃(2eq.) after heating to 45 ℃ for 14 h, reaction was complete by TLC and 28% NH was added₄After further reaction for 4 hours with OH (2ml), the reaction mixture was cooled to room temperature, diluted with water, extracted three times with EtOAc, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate 4: 1) to obtain a reduced compound (14 mg).

And step 3: in N₂To the reduced compound from step 2 (14mg), DMF (4ml), solid NaHCO₃(8eq.) DPPA (4eq.) was added, and after 24 hours at 0 deg.C, TLC checked for completion, cooled to room temperature, diluted with water, extracted three times with EtOAc, the organic phases combined, washed with saturated brine, dried over anhydrous sodium sulfate, the reaction mixture concentrated in vacuo and purified by semi-preparative reverse phase HPLC ((MeOH-H2O, 47: 53)) to give compound 1(8.6 mg).

Compound 1: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 378.1912[ M-H [ ]]^-(calcd for C₂₀H₂₈O₆N, 378.1917), the compound is presumed to be of formula C₂₀H₂₉O₆N。¹H-NMR(400MHz，Acetone)δ7.91(s，1H)，6.32(dd，J＝1.3，6.0Hz，1H)，5.24(s，1H)，3.45(d，J＝6.0Hz，1H)，2.93(d，J＝10.9Hz，1H)，2.84(s，1H)，2.48(dd，J＝6.9，13.7Hz，1H)，2.15(m，1H)，1.98(d，J＝1.3Hz，3H)，1.93(d，J＝14.7Hz，1H)，1.88(d，J＝13.7Hz，1H)，1.74(m，1H)，1.54(m，1H)，1.45(t，J＝3.1Hz，1H)，1.37(s，3H)，1.30(m，1H)，1.15(d，J＝7.0Hz，3H)，1.10(s，3H)，0.96(t，J＝7.4Hz，3H)。

Example 4: preparation of Compound 2

Step 1: in N₂Compound 4b (20mg) prepared in example 1 and NaN were added under an atmosphere₃(1eq.) of THF/H₂Pd (PPh) was added to O (2ml/0.2ml)₃)₄(5mg), followed by heating to 45 ℃ for 1 hour, TLC detection after completion of the reaction, cooling to room temperature, diluting with water, extracting with EtOAc three times, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, concentrating, and column chromatography (petroleum ether: ethyl acetate: 8: 1) to give the azide compound (16 mg).

Step 2: the azide compound (16mg) obtained in step 1 was weighed out and dissolved in THF/H₂O (2ml/0.2ml), then PPh was added thereto₃(2eq.) after heating to 45 ℃ for 14 h, reaction was complete by TLC and 28% NH was added₄After further reaction for 4 hours with OH (2ml), the reaction mixture was cooled to room temperature, diluted with water, extracted three times with EtOAc, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate 4: 1) to obtain a reduced compound (12 mg).

And step 3: in N₂To the reduced compound from step 2 (12mg), DMF (4ml), solid NaHCO₃(8eq.) DPPA (4eq.) is added, after 24 hours at 0 ℃ the reaction is complete by TLC, cooled to room temperature, diluted with water, extracted three times with EtOAc, the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, the reaction mixture is concentrated in vacuo and passed through semi-preparative reverse phase HPLC (MeOH-H)₂O, 47: 53) to give Compound 2(7 mg).

Compound 2:a white powder; HRESI-MS spectrum shows excimer ion peak M/z 400.1714[ M + Na ]]⁺(calcd for C₂₀H₂₇O₆NNa, 400.1736), presumably of the formula C₂₀H₂₇O₆N。¹H-NMR(400MHz，Acetone)δ7.92(s，1H)，6.89(m，1H)，6.34(dd，J＝1.4，6.0Hz，1H)，5.17(s，1H)，3.49(d，J＝6.0Hz，1H)，2.94(d，J＝10.2Hz，1H)，2.83(s，1H)，2.15(m，1H)，1.96(d，J＝1.4Hz，3H)，1.94(s，1H)，1.90(d，J＝3.8Hz，1H)，1.87(s，3H)，1.83(dd，J＝1.0，7.1Hz，3H)，1.47(m，1H)，1.39(s，3H)，1.32(m，1H)，1.10(s，3H)。

Example 5: preparation of Compound 3

Compound 2(20mg), Ag, prepared in example 4 was added under an atmosphere of N2₂O (185.4mg, 0.8mmol) and MgSO₄(100mg) dissolved in CH₃CN (5mL) and the solution was cooled to 0-4 ℃. CH3I (0.1mL) was added dropwise to the solution and the reaction mixture was stirred at room temperature. The solution was filtered and the solvent removed in vacuo to give the crude product, which was purified by semi-preparative reverse phase HPLC (MeOH-H)₂O, 45: 55) to give Compound 3(12 mg).

Compound 3: a light yellow powder; HRESI-MS spectrum shows excimer ion peak M/z 414.1880[ M + Na ]]⁺(calcd for C₂₁H₂₉O₆NNa, 414.1893), presumably of the formula C₂₀H₂₉O₆N。¹H-NMR(400MHz，Acetone)δ6.93(m，1H)，6.14(dd，J＝1.2，6.0Hz，1H)，3.49(d，J＝4.0Hz，1H)，2.99(s，3H)，2.96(d，J＝10.8Hz，1H)，2.81(d，J＝14.8Hz，1H)，2.16(m，1H)，2.0(s，3H)，1.96(d，J＝7.2Hz，1H)，1.93(s，1H)，1.88(s，3H)，1.84(d，J＝7.2Hz，3H)，1.49(d，J＝3.2，10.8Hz，1H)，1.42(s，3H)，1.32(m，1H)，1.26(s，1H)，1.11(s，3H)。

Example 6: preparation of Compound 2a

In N₂Compound 2 prepared in example 4 (20mg) was weighed out and dissolved in THF/MeOH/H under an atmosphere₂O (2ml/1ml/0.5ml), AcONa (3eq.) is added thereto, after 48 hours of reflux reaction at room temperature, the reaction is detected by TLC, after dilution with water, extraction is carried out three times with EtOAc, the organic phases are combined, washing is carried out with saturated brine, drying is carried out with anhydrous sodium sulfate, the reaction mixture is concentrated in vacuo and subjected to semi-preparative reverse phase HPLC (MeOH-H)₂O, 45: 55) to give Compound 2a (12 mg).

Compound 2 a: a white powder;¹H-NMR(400MHz，Acetone)δ5.34(d，J＝5.6Hz，1H)，5.30(m，1H)，5.00(s，1H)，2.81(s，3H)，2.80(m，1H)，2.79(m，1H)，2.07(m，1H)，1.99(m，1H)，1.80(d，J＝1.3Hz，3H)，1.60(s，3H)，1.54(s，3H)，1.50(m，1H)，1.21(m，1H)，1.19(d，J＝12.2Hz，1H)。

example 7: preparation of Compound 6

Step 1: in N₂Compound 4a (20mg) prepared in example 1 was weighed out and dissolved in THF/H under an atmosphere₂O (2.4ml/0.1ml), to which NaBH is then added₄(5eq.) after 2 h at 0 ℃ following completion of the TLC check, cooling to room temperature, diluting with water, extracting three times with EtOAc, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, concentrating the reaction mixture in vacuo and purifying by silica gel column chromatography to give the reduced compound (18 mg).

Step 2: in N₂The reduced compound of step 1(18mg) was weighed out and dissolved in THF/MeOH/H under an atmosphere₂O (2ml/1ml/0.5ml), AcONa (3eq.) is added to the mixture, after the mixture is refluxed for 48 hours at room temperature, the reaction is detected by TLC to be finished, and after the mixture is diluted by water, the mixture is extracted by EtOAc three timesThe organic phases are combined, washed with brine, dried over anhydrous sodium sulfate, the reaction mixture is concentrated in vacuo and purified by semi-preparative reverse phase HPLC (MeOH-H)₂O, 45: 55) to give Compound 6(12 mg).

Compound 6: a white powder; HRESI-MS spectrum shows excimer ion peak M/z 279.1227[ M-H [ ]]^-(calcd for C₁₅H₁₉O₅279.1212), the molecular formula of the compound is presumed to be C₁₅H₂₀O₅。¹H-NMR(400MHz，Acetone)δ5.44(d，J＝5.6Hz，1H)，5.31(m，1H)，5.02(s，1H)，2.87(s，3H)，2.82(m，1H)，2.79(m，1H)，2.08(m，1H)，2.00(m，1H)，1.83(d，J＝1.3Hz，3H)，1.63(s，3H)，1.59(s，3H)，1.53(m，1H)，1.26(m，1H)，1.20(d，J＝12.2Hz，1H)。

Example 8: VEGF pathway Activity assay

Experiment for inhibition of matrigel tubule formation in diabetic retinopathy: the day before the experiment 96-well plates were placed in a 4 ℃ refrigerator and Matrigel was placed in the 4 ℃ refrigerator from-20 ℃ overnight. Sucking 60ul of the matrigel liquid by a gun head, flatly paving the liquid at the bottom of a 96-well plate, and then putting the plate in an environment at 37 ℃ for 30min to fully form the matrigel. HUVECs were digested, centrifuged, resuspended in low serum (0.5%) EBM medium, and the cell concentration was adjusted to 2X 10⁴And/ml. The amount of cell suspension per well was 200ul in a 96 well plate plated with gel, VEGF and equal amounts of blank medium were added to the control group, and compounds 1, 2, 3, 4A, 4B, 5, 6(80um) and VEGF were added to the experimental group. The 96-well plate was placed in a cell incubator, and after 4 hours, the plate was taken out and photographed under a microscope for observation and recording, and the results are shown in FIG. 1.

The results of culturing the single-cell suspension of HUVECs in a 96-well plate coated with Matrigel in advance by using VEGF as a tube formation promoting factor show that obvious tube formation can be observed after the HUVECs are cultured on the surface of the Matrigel for 4 hours in the presence of exogenous VEGF, and the tube formation capability of the HUVECs is obviously inhibited after the HUVECs are added with the

compounds

2, 4b and 6. Inhibition of tubule formation in HUVECs by

compounds

2, 4b, 6 after comparison of the length of tubules formed with the length obtained for the VEGF-treated control group. The results are shown in FIG. 2.

The results in experiments on inhibition of matrigel tubule formation in diabetic retinopathy show that significant tubule formation is observed after HUVECs are cultured on the surface of matrigel for 4h in the presence of exogenous VEGF, and the tubular forming capability of HUVECs is relatively most strongly inhibited after compound 2 is added. The experiment was then repeated with different concentrations of Compound 2 to obtain IC₅₀Control groups were supplemented with VEGF (VEGF) and an equal amount of blank medium (VEGF-), respectively, at 40.4 μ M. The results obtained by photographing under microscope observation are shown in FIG. 3, and the results obtained by quantifying the length of the tubules are shown in FIG. 4.

Example 9: study of VEGF pathway

The results of western blot detection are consistent with the results of tubule formation, and the compound 2 inhibits the expression of VEGFR2(p-VEGFR2) activated by VEGF, the expression level of VEGFR2 is down-regulated, and the phosphorylation of VEGFR2 is also inhibited. Cells were treated with different concentrations of compound 2 for 30min and then VEGF (50ng/mL) for 20 min. Beta-actin was used as a loading control and the results are shown in FIG. 5.

It will be appreciated by those skilled in the art that modifications or variations may be made to the present invention in light of the above teachings. Such modifications and variations are intended to be included herein within the scope of this disclosure and the appended claims.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein

R₁Independently selected from hydrogen, C1-C6 alkyl, C1-C6 acyl, C1-C6 alkenyl, C1-C6 alkynyl;

R₂independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl.

2. The compound of claim 1, wherein R is₁Is hydrogen or C1-C6 acyl.

3. The compound of claim 1, wherein R is₂Hydrogen, C1-C6 alkyl.

4. A pharmaceutical composition comprising a compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, and combinations thereof, and one or more pharmaceutically acceptable carriers, excipients, or combinations thereof.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is an oral formulation or an injectable formulation.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is selected from solid oral formulations of tablets, capsules, granules, pills or powders.

7. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 4 to 6, in the manufacture of a medicament for the selective inhibition of VEGF-induced proliferation of human umbilical cord endothelial cell secretion (HUVEC) and retinopathy.