Substituted phenoxyacetohydrazide compounds, methods of making and use as anti-inflammatory inhibitors of stat3

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a substituted phenoxy acetic hydrazine compound, a preparation method thereof and application of the compound as an anti-inflammatory inhibitor of STAT3. The compound has a structure as shown in formula I. It is verified through experiments that the compound is an allosteric small-molecule compound combined with a STAT3 CCD structure domain, has high affinity, high membrane permeability and high selectivity. In a cell and a mouse acute lung injury model, the inhibitor is used to inhibit an inflammatory response of acute lung injury, so as to achieve the effect of relieving acute lung injury. The application provides a theoretical basis for drug treatment of development of a new STAT3 allosteric inhibitor for relieving an inflammatory response of acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular, the present application relates to a substituted phenoxy acetic hydrazide compound, a preparation method thereof and an application thereof as an anti-inflammatory inhibitor of STAT3. BACKGROUND

[0002] Acute lung injury (ALI) usually leads to acute respiratory distress syndrome, which is a severe respiratory disease caused by diffuse alveolar damage, leading to hypoxia respiratory failure, and can develop into multiple organ failure, with high morbidity and mortality in critical patient groups, seriously threatening human health and having few effective treatment methods, so it is becoming increasingly important to take effective protective measures for ALI. One of the main pathological features of ALI is acute inflammatory response, in which lung macrophages are polarized into M1 type and secrete IL-1β, IL-6, TNF-α and other pro-inflammatory cytokines, leading to aggravated lung injury. During the acute inflammatory period of ALI, inflammatory cytokines produced by lung macrophages form a complex signal network that regulates each stage of the inflammatory response of ALI. Therefore, inhibiting the inflammatory response of acute lung injury is of great significance for the treatment of ALI.

[0003] Signal transduction and transcription factor (STAT3) belongs to the STATs family, which is activated by the binding of extracellular cytokines to the corresponding receptors on the cell surface to initiate the intracellular JAK-STAT3 signaling pathway, and is the mediator of many inflammatory responses, playing an important role in macrophage immune response. In the early stage of acute lung injury, STAT3 in macrophages is excessively activated, and the secretion of IL-1β, IL-6, TNF-α and other inflammatory factors reaches a peak at 8 hours after injury, suggesting that STAT3 plays a crucial role in the initial stage of ALI inflammation. Studies have shown that the activation of STAT3 in ALI animal models exacerbates the inflammatory response and the degree of lung injury. Therefore, inhibiting STAT3 activity can effectively reduce or improve the inflammatory response in the early stage of acute lung injury, so studying new inhibitors targeting STAT3 can serve as a basis for clinical drugs to alleviate the inflammatory response of acute lung injury.

[0004] The coiled-coil (CCD) domain of STAT3 protein plays an important role in regulating the early activation and function of STAT3. The CCD domain is involved in the regulation of STAT3 activation by allosteric regulation, including receptor recognition binding, transfer to the nucleus, and regulating the function of STAT3 in a SH2 domain-independent manner. Allosteric regulation refers to the binding of a drug molecule to the allosteric site of a biological macromolecule, which together with the endogenous molecule bound to the active site, coordinates the function of the biological macromolecule. Compared with drugs acting on the active site, allosteric site drugs are a new drug action mode, which has the advantages of strong target selectivity and low drug side effects.

[0005] STAT3 protein has six subdomains: N-terminal domain, coiled-coil domain, DNA binding domain, linker domain, SH2 domain, and C-terminal transcriptional activation domain. A large number of literatures have reported inhibitors acting on the SH2 domain of STAT3, such as polypeptides, polypeptide analogs, and small molecule compounds, but these inhibitors have poor membrane permeability, low bioavailability, and other shortcomings, and have not been applied to clinical application. In view of the problems of STAT3 inhibitors, it is urgent to develop a class of small molecules that bind to other domains outside the SH2 domain of STAT3.

[0006] The CCD domain of STAT3 protein plays an important role in the early activation and function of STAT3. The CCD domain regulates STAT3 and receptor recognition binding in a SH2 domain-independent manner, affects the transfer of phosphorylated STAT3 to the nucleus, and participates in the transcriptional activity of the C-terminal of STAT3. Therefore, the STAT3 CCD domain can be used as a new domain for drug design and small molecule compound screening of STAT3. In acute lung injury inflammatory response, the STAT3 signaling pathway plays an important function, but there is no clinical drug for STAT3 inhibitors. The CCD domain of STAT3 regulates the activation and transcriptional activity of STAT3 by allosteric regulation. Therefore, a class of allosteric inhibitors targeting STAT3 CCD can be developed to reduce the occurrence and development of acute lung injury inflammation by inhibiting the activity of STAT3. Allosteric inhibitors have the characteristics of "high selectivity and low toxicity". Therefore, we aim to develop a class of STAT3 inhibitors that can alleviate acute lung injury and have "high selectivity and low toxicity". SUMMARY

[0007] One of the purposes of the present application is to provide a substituted phenoxyacetyl hydrazine compound.

[0008] Another purpose of the present application is to provide a preparation method of the above-mentioned substituted phenoxyacetyl hydrazine compound.

[0009] It is another object of the present application to provide a composition comprising the substituted phenoxyacetohydrazide compound described above.

[0010] It is another object of the present application to provide the use of the substituted phenoxyacetohydrazide compound described above as a STAT3 inhibitor.

[0011] It is another object of the present application to provide the use of the substituted phenoxyacetohydrazide compound described above in the manufacture of a medicament for treating inflammation of acute lung injury.

[0012] Definitions of Terms

[0013] The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs unless specifically defined otherwise.

[0014] As used herein, the term "treatment" is intended to refer to procedures for obtaining a desired pharmacologic and / or physiologic effect. A subject is successfully "treated" if, as a result of receiving a therapeutic amount of a compound or a pharmaceutically acceptable salt, isomer, or pharmaceutical composition thereof according to the methods described herein, the subject exhibits an observable and / or detectable reduction in or improvement in one or more signs and symptoms. It will also be appreciated that the treatment of a disease state or condition as described herein includes not only treatment of the disease state or condition but also prophylaxis of the disease state or condition.

[0015] Technical Subject One

[0016] The present application provides a substituted phenoxyacetohydrazide compound having the structure of Formula I:

[0017]

[0018] Technical Subject Two

[0019] The present application also provides a method for synthesizing the compound of Formula I, comprising the following steps:

[0020]

[0021] Reaction conditions: (a) hydrazine hydrate 85%, ethanol, 80 °C (b) pyridine, ethanol, 80 °C

[0022] Step (a): Compound 1 is dissolved in anhydrous ethanol, hydrazine hydrate is added, heated to 75-85°C, after the reaction is completed, cooled to room temperature, poured into ice water, and white solid compound 2 is precipitated;

[0023] Step (b): Compound 2 and compound 3 are dissolved in anhydrous ethanol, pyridine is added, heated to 75-85°C, after the reaction is stopped, cooled to room temperature, and white solid is precipitated, filtered to obtain.

[0024] As a further improvement of the present application, the hydrazine hydrate in step (a) is 85% hydrazine hydrate.

[0025] Technical Subject Three

[0026] The present application provides a composition comprising a substituted phenoxyacetic hydrazide compound of Formula I.

[0027] Further, the "pharmaceutical composition" can further comprise one or more pharmaceutically acceptable carriers or excipients, prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wine, decoction, lozenges, mixtures, suppositories, injections, inhalants or sprays, etc.

[0028] As used herein, "pharmaceutically acceptable carriers or excipients" include diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifying agents, sweeteners, flavoring agents, taste-masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, viscosity-increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers, and one skilled in the art will understand that certain pharmaceutically acceptable excipients can be used in more than one function and in alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

[0029] For example, when used orally, it can be prepared into oral preparations such as tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, oral disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, and pills, etc. The tablets can contain fillers (such as sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (such as gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (such as cellulose derivatives such as sodium carboxymethyl cellulose, polyvinylpyrrolidone), lubricants (such as talc, calcium stearate, magnesium stearate, sperm oil, boric acid, sodium benzoate, leucine), stabilizers (methyl paraben, propyl paraben, etc.), flavoring agents (such as commonly used sweeteners, souring agents, and spices, etc.).

[0030] When used parenterally, it can be prepared into injections, including sterile powders for injection and injection solvents. The carriers or excipients used include sterile water, Ringer's solution, and isotonic sodium chloride solution, and appropriate additives such as antioxidants, buffers, bacteriostatic agents, solubilizers, co-solvents, pH adjustors, osmotic pressure adjustors can also be added according to the nature of the drug. The solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjustors can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjustors can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing lyophilized powder injections, mannitol, glucose, etc. can also be added as supporting agents. When used for rectal administration, the drug can be prepared into suppositories, etc.

[0031] When used for pulmonary administration, the drug can be prepared into inhalants or sprays, etc. There are many resources available to those skilled in the art, which describe pharmaceutically acceptable excipients and which can be used to select appropriate pharmaceutically acceptable excipients, such as the books "Remington's Pharmaceutical Sciences", "Chinese Pharmaceutical Yearbook", "Pharmaceutics", etc.

[0032] The compounds of the present application and their pharmaceutically acceptable salts can be prepared into ordinary preparations, but also into sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle drug delivery systems.

[0033] Technical Subject Four

[0034] The present application also provides the use of a substituted phenoxyacetyl hydrazide compound represented by the structure of formula I as a STAT3 inhibitor.

[0035] Further, the application is to act on the STAT3 CCD domain.

[0036] Technical Subject Five

[0037] The application also provides the application of the substituted phenoxy acetyl hydrazine compound shown in formula I in the preparation of a drug for treating acute lung injury inflammation.

[0038] The application has the following beneficial effects:

[0039] The application develops a substituted phenoxy acetyl hydrazine compound, which is verified to act on the STAT3 CCD domain, and determines the effect of reducing the inflammatory response of acute lung injury. At present, the STAT3 inhibitor is to act on the SH2 and BDB domains, and has weak selectivity. The CCD domain is a sequence at the N terminal of STAT3 protein, which can play a role in regulating the function of the SH2 domain through the mutual conformational regulation effect between the various domains of STAT3, so the CCD domain can be used as a new STAT3 inhibitor binding site to develop STAT3 inhibitors.

[0040] The compound provided by the application is an allosteric small molecule compound combined with the STAT3 CCD domain, which has high affinity, high membrane permeability and high selectivity. In the cell and mouse acute lung injury model, it is confirmed that the inhibitor can inhibit the inflammatory response of acute lung injury, so as to relieve acute lung injury. This provides a theoretical basis for drug treatment for developing new STAT3 allosteric inhibitors to relieve the inflammatory response of acute lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The hydrogen spectrum of the compound K134 is shown in the figure;

[0042] Figure 2 The carbon spectrum of the compound K134 is shown in the figure;

[0043] Figure 3 The mass spectrum of the compound K134 is shown in the figure;

[0044] Figure 4 The affinity result graph of the compound K134 and STAT3 is shown in the figure, wherein: A) the IC50 of K134 and STAT3 is detected by fluorescence polarization experiment, B) the affinity Kd of K134 and STAT3 is detected by SPR;

[0045] Figure 5 The morphological characteristics of the lung tissue of the acute lung injury model mouse are shown in the figure;

[0046] Figure 6 The morphological characteristics of the lung tissue of the acute lung injury model mouse are shown in the figure;

[0047] Figure 7 To reduce the total cell number of the compound K134 in the alveolar lavage fluid of the acute lung injury model mice;

[0048] Figure 8 To reduce the protein levels of immune factors IL-1β, TNF-α, and IL-6 in the alveolar lavage fluid of the compound K134 in the acute lung injury model mice;

[0049] Figure 9 To inhibit the activation of STAT3 in the lung tissue of the compound K134 in the acute lung injury model mice;

[0050] Figure 10 To determine the effect of the compound K134 on the proliferation of AC16 cells. DETAILED DESCRIPTION

[0051] The present application will be described in detail below in conjunction with specific examples, which are not intended to limit the scope of the present application, but to provide guidance for those skilled in the art to prepare and use the compounds and compositions of the present application.

[0052] Example 1 Preparation

[0053] The synthetic route is as follows:

[0054]

[0055] Step (a): Compound 1 was dissolved in anhydrous ethanol, 2 equivalents of hydrazine hydrate (85%) were added, and the reaction was heated to 80°C. The reaction was monitored by TLC, and after the reaction was completed, it was cooled to room temperature. The reaction solution was poured into ice water, and white solids were precipitated. The solids were filtered, washed with anhydrous ethanol and water, and dried to obtain compound 2.

[0056] Step (b): Compound 2 and compound 3 (1.1 eq) were dissolved in anhydrous ethanol, and super-dry pyridine (0.1 eq) was added. The reaction was heated to 80°C overnight, and the reaction solution was cooled to room temperature. White solids were precipitated, filtered, and the filter cake was washed with anhydrous ethanol and dried to obtain compound K134 (compound of formula I), the structure of which is shown in Figures 1-3 .

[0057] Example 2 Determination of binding affinity to STAT3

[0058] The affinity of the small molecule K134 prepared in Example 1 to STAT3 and STAT3 truncated CCD was detected by fluorescence polarization experiment and SPR method, and the whole kinetic process of molecular interaction was observed.

[0059] The results show that the IC 50, the addition of K134 at different concentrations will significantly change the polarization value of STAT3 binding with FITC-pYLPQTV-NH2, as shown in Figure 4 Figure 1A, the K134 concentration-dependent inhibition of the binding of the phosphorylated fluorescent peptide segment to STAT3, IC 50 is 15.35 μM. The affinity of STAT3 CCD to K134 was determined by SPR method, K d is 4.68 μM, as shown in Figure 4 Figure 1B.

[0060] Example 3 verifies that the inhibitor reduces the inflammatory response of acute lung injury in mice

[0061] 1. Constructing a LPS-induced acute lung injury mouse model

[0062] BALB / c, male, 6-8 weeks, 18-20 g, 40 were randomly divided into Sham group, LPS group, solvent control group, inhibitor group, after conventional anesthesia of mice, the LPS group, solvent control group and inhibitor group were instilled with 3 mg / kg LPS (dissolved in 50 μl of normal saline) in the airway, the Sham group of mice were instilled with the same volume of normal saline in the trachea. After the drug entered the airway, immediately through the upright rotation and other means, so that the drug was evenly distributed in the lung tissue.

[0063] Each group was given different treatment 2 hours and 12 hours after operation:

[0064] sham group and LPS group: no treatment.

[0065] Solvent control group: each mouse was given 100 μL of normal saline, DMSO volume 1%, castor oil volume 5%.

[0066] Inhibitor group: each was given 30 mg / kg K134, solvent was 100 ul of normal saline, DMSO volume 1%, castor oil volume 5%.

[0067] After the operation was completed, the wound was sutured, and the alveolar lavage fluid and lung tissue were taken after 24 hours. In the acute lung injury model, after LPS induction, the lung tissue of mice had characteristics such as the appearance of transparent membrane in the layer of neutrophils, lymphocytes, macrophages, vascular endothelial cells, atelectasis and the like, see Figure 5 .

[0068] 2. Improvement of the inflammatory response of the inhibitor to acute lung injury in lung tissue

[0069] The tissue was taken 24 hours after the wound was sutured after the operation, embedded in paraffin, and fixed on a microtome. The embedded wax block was cut into thin slices, generally 5-8 microns thick, and the slices were stained with hematoxylin and eosin. The severity of lung injury was evaluated by measuring the accumulation of neutrophils in the alveoli or interstitial space, the formation of hyaline membranes, the presence of protein fragments in the alveolar interstitial space, and the thickening of the alveoli. From Figure 6 It was observed from HE staining that the morphology of the lung tissue of the LPS-stimulated acute lung injury mice was significantly destroyed, and the K134 group showed that the alveolar tissue morphology was restored and the inflammatory cell infiltration was reduced, indicating that K134 could alleviate the inflammatory response caused by acute lung injury and improve the morphology of the lung tissue.

[0070] 3. Inhibition of inflammatory factors in acute lung injury lung by the inhibitor

[0071] The protein levels of IL-1β, IL-6, and TNF-α in the bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice were detected by ELISA. The results are shown in Figure 7 and Figure 8 It was found that the number of inflammatory cells in the LPS group increased significantly, and the K134 group could significantly reduce the number of cells, indicating that the inhibitor could alleviate inflammation. K134 could significantly reduce the amount of IL-1β, IL-6, and TNF-α, indicating that K134 could inhibit the inflammatory response of the acute lung injury model.

[0072] 4. Inhibitor improves the inflammatory response of acute lung injury by reducing STAT3 activity

[0073] The inhibitory effect of the inhibitor on the phosphorylation of STAT3 in the lung tissue sections of acute lung injury was detected by immunohistochemistry. The inhibitor reduced the phosphorylation levels of STAT3 Tyr-705 and Ser-727 in the lung tissue. The immunohistochemistry results are shown in Figure 9 The phosphorylation levels of STAT3 in the LPS and NC groups were high, and the phosphorylation of STAT3 Ser-727 and Tyr-705 in the K134 treatment group was reduced, indicating that K134 improved acute lung injury by reducing STAT3 phosphorylation.

[0074] Example 3 Safety Evaluation

[0075] AC16 is a normal cardiac muscle cell. We used the CCK8 method to detect the growth inhibition of K134 on AC16 cells, and from Figure 10 It can be seen that K134 has little effect on the growth of AC16 cells, indicating that K134, as a type of STAT3 CCD-targeted inhibitor, has the advantage of low cell-level toxicity and side effects.

Claims

1. The application of a substituted phenoxyacetylhydrazine compound, as shown in Formula I, acting on the CCD domain of STAT3, in the preparation of a medicament for treating acute lung injury and inflammation: 。

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