CN118892486A - A small molecule compound that inhibits VEGF and TNF signals in vascular endothelial cells - Google Patents

Abstract

The invention provides a compound serving as a VEGF and TNF inhibitor and application thereof, and in particular discloses application of a compound shown in a formula (I) in preparing a medicament or a pharmaceutical composition, wherein the medicament or the pharmaceutical composition has excellent VEGF and TNF inhibition activity and can be used for treating or preventing VEGF and TNF regulated related diseases.

Description

A small molecule compound that inhibits VEGF and TNF signals in vascular endothelial cells

Technical Field

The present invention relates to the field of biomedicine, and in particular to a small molecule compound that inhibits vascular endothelial cell VEGF and TNF signals.

Background Art

Angiogenesis refers to the process of new capillaries growing from existing blood vessels. Angiogenesis can not only promote the body's growth, development, tissue and organ function, and wound repair, but also affect solid tumors, inflammation, diabetic retinopathy, as well as the course of many diseases such as myocardial infarction, heart failure, vascular atherosclerosis, rheumatoid arthritis, and skin diseases.

Endothelial cells in the body that have the potential to divide and are in a resting state are key cells throughout the entire process of angiogenesis. Under normal circumstances, there is a balance between factors that promote angiogenesis and those that inhibit angiogenesis, maintaining the normal growth of the vascular system. Once the balance is broken, angiogenesis can be enhanced or weakened. Endothelial cells can be stimulated by a series of factors to induce angiogenesis, among which vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. Developing tissue cells, tumor cells, cells in damaged tissues, or hypoxic cells all secrete VEGF. VEGF binds to receptors (VEGFR) located on the membrane of vascular endothelial cells, activating a series of signaling pathways, causing endothelial cells to sprout, proliferate, migrate, and form tubular structures, which then anastomose into new vascular networks. There are three known VEGF receptors: Flt-1 (VEGFR-1), KDR (VEGFR-2), and Flt-4 (VEGFR-3), all of which belong to receptor tyrosine protein kinases. It is currently believed that VEGFR2 is the main receptor type that controls angiogenesis and can promote the proliferation, survival, migration and differentiation of endothelial cells. VEGFR-1 lacks a transmembrane kinase domain and can act as a decoy receptor for VEGF, limiting the amount of free VEGF and preventing them from binding to VEGFR2, thereby inhibiting angiogenesis.

In addition, inflammation is also an important pathological factor that triggers angiogenesis. When the body encounters factors such as tumor occurrence, microbial infection, accumulation of body metabolites and harmful chemicals, abnormal immune response, and tissue cell death, an inflammatory response often occurs. The local microenvironment induces immune cells or surrounding tissue cells to secrete VEGF to promote angiogenesis, and also secretes tumor necrosis factor α (TNFα). TNFα is a type of cytokine with vasoactive activity, which not only causes inflammatory responses in endothelial cells, but also affects vascular function and the formation of new blood vessels. TNFα exerts its biological functions by binding to two receptors, TNFR1 and TNFR2. TNFR1 mediates classic proinflammatory responses, such as affecting the NF-κB pathway and causing the expression of various classic proinflammatory cytokines. In addition to participating in the activation of inflammatory signals, the activation of TNFR2 mainly promotes cell activation, migration, and proliferation. There is a dialogue relationship between the TNFα-TNFR and VEGF-VEGFR2 pathways, such as TNFα-TNFR can affect angiogenesis by inducing the expression of VEGF.

Angiogenesis and inflammation are complex regulated cellular events, and appropriate intervention may help prevent and treat many diseases. In the field of medicine, the discovery and development of highly effective angiogenesis inhibitors and TNF inhibitors has always been one of the hot topics in basic, clinical and translational medical research. The discovery of new and potent VEGF and TNFα signaling inhibitors may not only promote basic research in related fields, but also discover new first-line clinical drugs.

Summary of the invention

The purpose of the present invention is to provide a small molecule compound for inhibiting VEGF and TNF signals of vascular endothelial cells.

In the first aspect of the present invention, there is provided a use of a compound of formula (I), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof for preparing a medicament or a pharmaceutical composition, wherein the medicament or the pharmaceutical composition is selected from the group consisting of:

(i) treating or preventing diseases or conditions related to the regulation of VEGF and/or TNF,

(ii) VEGF signaling inhibitors, and/or

(iii) TNF signaling inhibitors;

in,

R ₁ is selected from the group consisting of hydrogen, deuterium, halogen, hydroxy, amino, cyano, nitro, C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkoxy, -C(O)NH ₂ , -C(O)OH, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted C3-8 oxacyclic, substituted or unsubstituted C5-7 cycloalkenyl, substituted or unsubstituted 4-8 membered heteroaryl, substituted or unsubstituted C8-14 heteroaromatic bicyclic or tricyclic ring system, substituted or unsubstituted C5-C7 saccharide;

The substitution refers to substitution by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxyl, cyano, nitro, azido, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, C _3-8 halocycloalkyl, phenyl, and naphthyl.

In another preferred embodiment, R ₁ is selected from the following group: hydrogen, deuterium, halogen, hydroxyl, amino, C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkoxy, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted C3-8 oxygen heterocycle, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 4-8 membered heteroaryl or substituted or unsubstituted C5-C7 saccharyl.

In another preferred embodiment, the substitution refers to substitution by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, and C _3-8 halocycloalkyl.

In another preferred embodiment, R ₁ is selected from the following group: hydrogen, halogen, hydroxyl, amino, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted C3-6 oxygen heterocycle, substituted or unsubstituted C3-6 cycloalkyl or substituted or unsubstituted 4-8 membered heteroaryl; the substitution refers to substitution by one or more substituents selected from the following group: halogen, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, C _3-8 halocycloalkyl.

In another preferred embodiment, R ₁ is selected from the following group: hydrogen, halogen, hydroxyl, amino, pyran, substituted or unsubstituted C3-6 oxygen heterocycle, substituted or unsubstituted C3-6 cycloalkyl or substituted or unsubstituted 4-6 membered heteroaryl; the substitution refers to substitution by one or more substituents selected from the following group: halogen, hydroxyl.

In another preferred embodiment, R ₁ is

In another preferred embodiment, the compound is

In another preferred embodiment, the VEGF and/or TNF-regulated related diseases are selected from the following groups: VEGF-induced endothelial cell angiogenesis, TNF-induced inflammatory response-related diseases or a combination thereof.

In another preferred embodiment, the VEGF and/or TNF-regulated related diseases are selected from the following groups: solid tumors, hemangiomas, inflammation, diabetic retinopathy, rheumatoid arthritis, autoimmune diseases or a combination thereof.

In another preferred embodiment, the compound or drug combination inhibits vascular endothelial cell angiogenesis induced by VEGF165 and its isoforms and inflammatory response induced by TNFα.

In another preferred embodiment, the VEGF-related disease is caused by signals such as FAK and AKT phosphorylation.

In another preferred embodiment, the drug or pharmaceutical composition is also used

(a1) Inhibit the function of vascular endothelial cells;

(a2) Inhibit VEGF downstream signals such as FAK and AKT phosphorylation;

(a3) inhibiting the expression of TNFR1/2 and/or VEGFR2 and/or increasing the expression of sVEGFR1;

(a4) inhibiting the expression of inflammatory molecules induced by TNF;

(a5) Inhibit TNF downstream signals such as IκBα and JNK.

In another preferred embodiment, the function of inhibiting endothelial cells is selected from the following group: inhibiting the number of sprouts of endothelial cells, inhibiting the length of sprouts of endothelial cells, inhibiting the migration of endothelial cells, inhibiting the sprouting of endothelial cells, or a combination thereof.

In another preferred embodiment, the drug or drug composition inhibits the function of vascular endothelial cells by activating Caspase-3 apoptosis signal.

In another preferred embodiment, the inflammatory molecule is selected from the following group: ICAM-1, VCAM-1, SELE, IL-6, or a combination thereof.

In a second aspect, the present invention provides a method for inhibiting the phosphorylation of FAK and AKT, comprising the steps of:

The compound of formula (I), its stereoisomer, tautomer or pharmaceutically acceptable salt thereof is contacted with vascular endothelial cells to inhibit VEGF-induced FAK and AKT phosphorylation

wherein _R1 is as described above.

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the method is in vitro.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a third aspect, the present invention provides a method for inhibiting angiogenesis of vascular endothelial cells, comprising the steps of:

The compound of formula (I), its stereoisomer, tautomer or pharmaceutically acceptable salt thereof is contacted with vascular endothelial cells to inhibit angiogenesis of vascular endothelial cells.

wherein _R1 is as described above.

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the method is in vitro.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In another preferred embodiment, the method is to inhibit endothelial cell angiogenesis by inhibiting the expression of VEGFR2 and/or TNFR1/2 and/or increasing the expression of sVEGFR1.

In another preferred embodiment, the method is to inhibit vascular endothelial cell angiogenesis by inhibiting VEGF downstream signals such as FAK and AKT phosphorylation.

In a fourth aspect, the present invention provides a method for inhibiting inflammatory response of vascular endothelial cells, comprising the steps of:

The compound of formula (I), its stereoisomer, tautomer or pharmaceutically acceptable salt thereof is contacted with vascular endothelial cells to inhibit TNF-induced inflammatory response.

wherein _R1 is as described above.

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the method is in vitro.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In another preferred embodiment, the method is to inhibit the inflammatory response of vascular endothelial cells by inhibiting the expression of TNFR1/2.

In another preferred embodiment, the method is to inhibit the inflammatory response of vascular endothelial cells by inhibiting IκBα and JNK signals downstream of TNFR.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the effect of eVTi on the angiogenesis ability of endothelial cells.

FIG2 shows the effect of eVTi on VEGF receptor expression.

FIG3 shows the effect of eVTi on the expression of inflammatory molecules induced by TNFα.

FIG4 shows the effect of eVTi on TNFα receptor expression.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventor unexpectedly discovered for the first time that eVTi can be used as a VEGF inhibitor and a TNF inhibitor in preventing and treating diseases or conditions related to VEGF and TNF regulation. Experiments have shown that the compounds of the present invention inhibit angiogenesis of vascular endothelial cells, inhibit phosphorylation of FAK and AKT, inhibit the expression of TNFR1/2 and/or VEGFR1/2, and/or inhibit the expression of inflammatory molecules. On this basis, the present invention was completed.

the term

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

As used herein, the term "isoform" refers to a form of a protein that has the same function but differs in amino acid sequence. Such differences may be due to alternative splicing of genes, post-translational modifications, or different gene expression patterns. Isoforms play an important role in biology, as they can affect protein function, stability, and intracellular localization.

eV

4-[3A,5A,10-TRIHYDROXY-9A-(HYDROXYMETHYL)-11A-METHYL-7-[(3,4,5-TRIHYDROXY-6-METHYLOXAN-2-YL)OXY]-HEXADECAHYDRO-1H-CYCLOPENTA[A]PHENANTHREN-1-YL]-2,5-DIHYDROFURAN-2-ONE, CAS No.: 2415529-52-9, its chemical structure is as follows:

The simplified molecular linear input specifications (Smiles) of compounds are as follows:

[H][C@@]12[C@@H](CC3(C)C(CCC3([C@]2([H])CCC2(CC(CCC12CO)OC1C(C(C(C(C) O1)O)O)O)O)O)C1COC(C＝1)＝O)O

Compound InChI Key:BUGNRCRUPAIYMD-WUNDEPHJSA-N

The basic properties of the compound are as follows:

Molecular weight: 568.66; logP: -0.8479; logD: -0.8479; logSw: -2.5965;

Number of hydrogen bond acceptors: 12; number of hydrogen bond donors: 7, polar surface area: 147.339.

Through screening, the applicant found that the compound can effectively inhibit VEGF165-induced angiogenesis in endothelial cells and TNFα-induced inflammatory response. It was further found that the compound can effectively inhibit the expression of VEGFR2, TNFR1 and TNFR2, and increase the expression of sVEGFR1, confirming that the compound is a dual-effect small molecule compound that can strongly inhibit VEGF and TNFα signals. Its targets are VEGF and TNFα membrane receptors. It is named dual-VEGF/TNFi. At the same time, the first letters of endothelial cell, VEGF, TNFα and inhibitor are selected to simplify its name to eVTi.

In the present invention, the terms "eVTi" and "compound of the present invention" are used interchangeably and refer to the compound of formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof;

in,

R ₁ is selected from the following group: hydrogen, deuterium, halogen, hydroxyl, amino, cyano, nitro, C1-6 alkyl, C3-8 cycloalkyl, C1-6 alkoxy, -C(O)NH2, -C(O)OH, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted C3-8 oxygen heterocycle, substituted or unsubstituted C5-7 cycloalkenyl, substituted or unsubstituted 4-8 membered heteroaryl, substituted or unsubstituted C8-14 heteroaromatic bicyclic or tricyclic ring system, substituted or unsubstituted C5-C7 saccharide;

The substitution refers to substitution by one or more substituents selected from the following group: deuterium, halogen, amino, hydroxyl, cyano, nitro, azido, C1-6 alkyl, C1-6 alkoxy, C3-8 cycloalkyl, C3-8 halocycloalkyl, phenyl, naphthyl.

In another preferred embodiment, R ₁ is selected from the following group: hydrogen, deuterium, halogen, hydroxyl, amino, C1-6 alkyl, C3-8 cycloalkyl, C1-6 alkoxy, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted C3-8 oxygen heterocycle, substituted or unsubstituted C3-6 cycloalkyl or substituted or unsubstituted 4-8 membered heteroaryl.

In another preferred embodiment, the substitution refers to substitution by one or more substituents selected from the following group: deuterium, halogen, amino, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C3-8 cycloalkyl, C3-8 halocycloalkyl.

In another preferred embodiment, the compound is

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds and a pharmaceutically acceptable carrier.

Since the compounds of the present invention have excellent VEGF and/or TNF inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases associated with VEGF and/or TNF inhibition.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

The main advantages of the present invention include:

(1) The compounds of the present invention effectively inhibit VEGF165-induced angiogenesis in vascular endothelial cells and TNFα-induced inflammatory response.

(2) The compounds of the present invention can effectively inhibit the expression of VEGFR2, TNFR1 and TNFR2, and increase the expression of sVEGFR1.

(3) The compounds of the present invention can inhibit the expression of inflammatory molecules such as ICAM-1, VCAM-1, SELE and IL-6 induced by TNFα.

(4) The compounds of the present invention target VEGFR and TNFR and are at the most upstream link of angiogenesis and inflammation. Therefore, they can act on a wide range of diseases related to VEGF and TNF signals and have a strong efficacy.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Example 1eVTi inhibits VEGF-induced angiogenesis in endothelial cells

1.1 Methods

(A) Effects of eVTi (0nM, 150nM, 300nM) on the ability of HUVECs to form tube-like structures;

(B) CCK-8 method was used to detect the effect of eVTi (0nM, 50nM, 100nM, 200nM, 300nM) on the proliferation ability of HUVEC;

(C-E) 3D microcarrier budding experiment. HUVEC infected with Ad-GFP were coated onto Cytodex 3 microcarrier beads (GE Healthcare) (approximately 400 cells per bead), and then the cell-containing microbeads were mixed with fibrinogen (2 mg/mL), and 1 U/mL thrombin and 150 μg/mL aprotinin were mixed in the fibrinogen. Then, different concentrations of eVTi were added to the upper culture medium and cultured for 3-4 days. Finally, the buds were observed under a fluorescence microscope, and the number of buds (D) and the length of buds (E) were quantified using Image J software after taking pictures.

(F) 3D cell spheroid budding experiment. A round-bottom 96-well plate was coated with 0.8% agarose, and 1500-2000 HUVEC infected with Ad-GFP and an equal amount of MGC803 gastric cancer cells not infected with adenovirus were seeded in the wells and cultured overnight to form cell spheres. The culture medium was discarded, and the cell spheres were resuspended with 0.5 ml of a fibrinogen (2 mg/mL) solution containing 1 U/mL thrombin and 150 μg/mL aprotinin, and placed in a 24-well plate. After solidification, a culture medium containing DMSO or 150 nM eVTi was added on top, and the culture was continued for 3-4 days, and finally photographed with a confocal microscope.

(G) HUVECs were treated with 150 nM eVTi for different time periods, proteins were extracted, and Western blotting was used to detect the activation of Caspase-3;

(H) HUVECs were treated with different concentrations of eVTi for 30 h, proteins were extracted, and Western blotting was used to detect the activation of Caspase-3.

All experiments were repeated three times with independent cell lines, **P<0.01 vs. DMSO.

1.2 Results

The applicant observed the effect of eVTi on the function of vascular endothelial cells and found that 150nM and 300nM eVTi can significantly inhibit the ability of vascular endothelial cells to form tube-like structures (A in Figure 1), as well as the proliferation of vascular endothelial cells (Figure 1B). Using the 3-D cell spheroid sprouting experiment, it was found that 150nM and 300nM eVTi can significantly inhibit the number and length of sprouts of vascular endothelial cells (C-E in Figure 1). The applicant then mixed the gastric cancer cell line MGC803 and endothelial cells to form mixed cell spheres in agarose-coated culture wells, and found that 150nM eVTi can significantly inhibit the migration and sprouting of endothelial cells in the mixed cell spheres (F in Figure 1). Moreover, with the extension of the 150nM eVTi treatment time (G in Figure 1), or with the increase of the eVTi dose (H in Figure 1), the cultured vascular endothelial cells showed activation of Caspase-3 apoptosis signals. These research data show that eVTi exhibits significant anti-angiogenic effects at a concentration of 100nM and is a potent inhibitor of vascular endothelial cell function.

Example 2

2.1 Methods

The applicant then analyzed the effect of eVTi on VEGFR expression in HUVECs, treated endothelial cells with 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 2 μM, 5 μM, and 10 μM, and detected using qPCR technology.

(A-B) HUVECs were treated with different concentrations (0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM) of eVTi for 16 h, and the mRNA expression levels of VEGFR2 (A) and sVEGFR1 (B) were analyzed by RT-qPCR;

(C) HUVECs were treated with different concentrations (0nM, 50nM, 100nM, 200nM, 300nM, 400nM) of eVTi for 16 hours, and the mRNA expression of VEGFR2 was analyzed by RT-qPCR; and HUVECs were treated with different concentrations (0nM, 10nM, 20nM, 30nM, 40nM, 50nM) of eVTi for 16 hours, and the mRNA expression of sVEGFR1 was analyzed by RT-qPCR;

(D) After adding 150 nM eVTi to HUVECs for 16 h, fresh medium was replaced twice to remove eVTi, and then the mRNA expression of VEGFR2 and sVEGFR1 was analyzed at 8 and 24 h;

(E) HUVECs were treated with different concentrations (0 nM, 10 nM, 150 nM, 300 nM, 500 nM, 1 μM) of eVTi for 16 h, and the expression of VEGFR2 protein was analyzed by western blotting;

(F) HUVECs were treated with different concentrations (0 nM, 50 nM, 100 nM, and 250 nM) of eVTi for 16 h, and the expression of sVEGFR1 protein was analyzed by western blotting;

(G) After HUVECs were treated with 150 nM eVTi for 16 h, the cells were stimulated with 25 ng/ml VEGF for 15 min, and the phosphorylation of FAK and AKT was analyzed by western blotting.

All experiments were repeated three times with independent cell lines, *P<0.05 vs. DMSO, **P<0.01 vs. DMSO.

2.2 Results

eVTi can inhibit the mRNA expression of VEGFR2 in a dose-dependent manner (A in Figure 2), but promote the mRNA expression of sVEGFR1 (B in Figure 2); further reducing the dose of eVTi, analysis found that as low as 100nM eVTi can inhibit VEGFR2 expression, and as low as 50nM eVTi can promote sVEGFR1 expression (C in Figure 2). In order to observe whether the regulatory effect of eVTi on sVEGFR1 and VEGFR2 is reversible, after adding 150nM eVTi for 16 hours, fresh culture medium was replaced twice to remove the added eVTi, and total RNA was extracted at 8 hours and 24 hours, respectively, to analyze the expression of VEGFR. We found that the regulation of eVTi on sVEGFR1 and VEGFR2 is reversible (D in Figure 2). Subsequently, protein immunoblotting was used to confirm that eVTi has a very significant regulatory effect on the expression of VEGFR2 (E in Figure 2) and sVEGFR1 (F in Figure 2) proteins. Correspondingly, eVTi can significantly inhibit the phosphorylation of FAK and AKT stimulated by VEGF165 (Figure 2G). It can be seen that eVTi inhibits VEGF signaling and VEGF-induced endothelial cell angiogenesis by inhibiting VEGFR2 and increasing sVEGFR1.

Example 3

3.1 Methods

Applicants analyzed the effect of eVTi on the expression of inflammatory molecules induced by TNFα.

After adding 150nM eVTi to HUVEC for 16 hours, the cells were stimulated with 2ng/ml TNFα for 4 hours, and the expression of ICAM-1 (A), VCAM-1 (B), SELE (C) and IL-6 (D) was analyzed by qPCR. All experiments were repeated three times on independent cells, *P<0.05 vs. DMSO; **P<0.01 vs. DMSO.

3.2 Results

Pretreatment of HUVEC with 100 nM eVTi can significantly inhibit the expression of inflammatory molecules such as ICAM-1 (A in Figure 3), VCAM-1 (B in Figure 3), SELE (C in Figure 3) and IL-6 (D in Figure 3) induced by TNFα. These data suggest that eVTi is a highly effective inhibitor of TNFα in HUVEC.

Example 4

4.1 Methods

(A-B) HUVECs were treated with different concentrations (0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM) of eVTi for 16 h, and the mRNA expression levels of TNFR1 (A) and TNFR2 (B) were analyzed by RT-qPCR;

(C) After 150 nM eVTi was added to HUVECs for 16 h, fresh medium was replaced twice to remove eVTi, and then the mRNA expression of TNFR1 and TNFR2 was analyzed at 8 and 24 h;

(D) HUVECs were treated with different concentrations (0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM) of eVTi for 16 h, and the expression of TNFR1 and TNFR2 proteins was analyzed by western blotting;

(E) After 16 hours of treatment with 150 nM eVTi, endothelial cells were stimulated with 2 ng/ml TNFα for 10 minutes, and the expression of IκBα and pJNK was analyzed by western blotting.

All experiments were repeated three times in independent cell lines. *P<0.05 vs. DMSO; **P<0.01 vs. DMSO

4.2 Results

eVTi can significantly inhibit the expression of TNFR1 (A in Figure 4) and TNFR2 (B in Figure 4) in HUVEC at a concentration of 50-100 nM. Moreover, 16 hours after the addition of eVTi, the added eVTi was removed by replacing the culture medium twice, and the expression of TNFR1/2 was analyzed. We found that the regulation of TNFR1 and TNFR2 by eVTi was reversible (C in Figure 4). By protein immunoblotting, we confirmed that 50-100 nM eVTi can significantly inhibit the expression of TNFR1 and TNFR2 proteins in HUVEC (D in Figure 4). Correspondingly, eVTi can significantly inhibit the degradation of IκBα induced by TNFα, as well as the phosphorylation of JNK (E in Figure 4). It can be seen that eVTi inhibits the TNFα signal and the regulatory effect of TNF on inflammation by inhibiting the expression of TNFR1 and TNFR2 in vascular endothelial cells.

The applicant also detected the effect of eVTi on the expression of VEGFR and TNFR in brain microvascular endothelial cells of bEnd.3 mice. qPCR experiments confirmed that eVTi did not significantly affect their expression, suggesting that the regulatory effect of eVTi on VEGFR and TNFR may be affected by species.

It can be seen that eVTi at a concentration level of 50 to 150 nM significantly inhibits the expression of VEGFR2, TNFR1 and TNFR2 in human vascular endothelial cells, significantly increases the expression of sVEGFR1, and thus significantly inhibits VEGF signaling and TNFα signaling. eVTi is a dual-effect small molecule compound that potently inhibits VEGF and TNFα signals in vascular endothelial cells, and may have good application prospects in the prevention and treatment of clinical diseases related to angiogenesis and vascular inflammation, such as hemangiomas, various solid tumors that depend on angiogenesis, rheumatoid arthritis, diabetic retinopathy and other diseases.

discuss

This application verifies that the compounds of the present invention can simultaneously affect the expression of VEGFR and TNFR receptors and have a dual inhibitory effect. They can not only be used in the treatment of clinical diseases, but can also be used as VEGFR and TNFR signal inhibitors in scientific research.

The compounds of the present invention can act on angiogenesis. Relevant studies have shown that one new vascular endothelial cell may be required for every 10-100 tumor cells, so inhibiting the growth of vascular endothelial cells may amplify the inhibitory effect on tumor cells; since the mutation rate of vascular endothelial cells is low and it is not easy to develop drug resistance, in theory, the compounds applied for in this project can more widely treat all solid tumors that rely on angiogenesis. It is particularly important that hemangioma, a common clinical disease, is a type of tumor lesion caused by abnormal proliferation of vascular endothelial cells, which is mainly related to VEGF-induced angiogenesis and abnormal vascular proliferation. The compounds discovered in this project have strong anti-angiogenesis ability, especially in terms of VEGF signaling, and have a strong inhibitory effect, which may be an effective drug for this tumor.

In addition, retinal neovascularization is caused by a variety of causes. The choroidal neovascularization sprouts cross Bruch's membrane and proliferate under and/or on the retinal pigment epithelium to form fibrovascular tissue, which is a variety of fundus diseases. VEGF is closely related to the growth of retinal neovascularization and is a drug target. Anti-VEGF and other therapies have been applied to the treatment of retinal and choroidal neovascular diseases. The compounds discovered in this project have strong anti-angiogenic ability, especially in terms of VEGF signals, and have a strong inhibitory effect. This compound may play a good role in the treatment of retinal and choroidal neovascular diseases in the link of angiogenesis.

Moreover, TNF-α inhibitors are the first choice for treating autoimmune diseases, including rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA). The compounds discovered in this project have a strong ability to inhibit TNF signals. The compounds may target the downstream signaling of TNF and exert their effects on treating TNF-related diseases such as rheumatoid arthritis.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

1. Use of a compound of formula (I), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, for the preparation of a medicament or a pharmaceutical composition for use in a pharmaceutical composition selected from the group consisting of:

(i) Treating or preventing diseases or disorders associated with VEGF and/or TNF modulation,

(Ii) VEGF signaling inhibitors, and/or

(Iii) TNF signaling inhibitors;

wherein,

R ₁ is selected from the group consisting of: hydrogen, deuterium, halogen, hydroxy, amino, cyano, nitro, C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkoxy, -C (O) NH ₂, -C (O) OH, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted naphthalenyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted C3-8 oxa, substituted or unsubstituted C5-7 cycloalkenyl, substituted or unsubstituted 4-8 membered heteroaryl, substituted or unsubstituted C8-14 heterobicyclic or tricyclic ring system, substituted or unsubstituted C5-C7 glycosyl;

The substitution refers to substitution with one or more substituents selected from the group consisting of: deuterium, halogen, amino, hydroxy, cyano, nitro, azido, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, C _3-8 halocycloalkyl, phenyl, naphthyl.

2. The use according to claim 1, wherein R ₁ is selected from the group consisting of: hydrogen, deuterium, halogen, hydroxy, amino, C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkoxy, pyran, substituted or unsubstituted phenyl, substituted or unsubstituted C3-8 oxa, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 4-8 membered heteroaryl or substituted or unsubstituted C5-C7 glycosyl.

3. The use according to claim 1, wherein the compound is

4. Use according to claim 1, wherein the VEGF and/or TNF mediated related disease is selected from the group consisting of: VEGF-induced vascular endothelial cell angiogenesis, TNF-induced inflammatory response, or a combination thereof.

5. Use according to claim 1, wherein the VEGF and/or TNF mediated related disease is selected from the group consisting of: solid tumors, hemangiomas, inflammation, diabetic retinopathy, rheumatoid arthritis, autoimmune diseases or combinations thereof.

6. The use of claim 1, wherein the compound or pharmaceutical combination inhibits VEGF165 and isoforms thereof-induced vascular endothelial cell angiogenesis and tnfα -induced inflammatory responses.

7. The use according to claim 1, wherein the VEGF-related disease is caused by signals such as FAK and AKT phosphorylation.

8. A method of inhibiting FAK and AKT phosphorylation, comprising the steps of:

contacting a compound of formula (I), a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, with vascular endothelial cells, thereby inhibiting VEGF-induced FAK and AKT phosphorylation

Wherein R ₁ is as previously described.

9. A method of inhibiting vascular endothelial cell angiogenesis comprising the steps of:

contacting a compound of formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, with vascular endothelial cells, thereby inhibiting vascular endothelial cell angiogenesis

Wherein R ₁ is as previously described.

10. A method of inhibiting vascular endothelial inflammatory response comprising the steps of:

Contacting a compound of formula (I), a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, with vascular endothelial cells, thereby inhibiting a TNF-induced inflammatory response

Wherein R ₁ is as previously described.