Pyrrolidine amide derivatives, methods of making and using the same

Abstract

Provided are pyrrolidine amide derivatives, optical isomers thereof, and salts thereof, and methods of making and using the same, which are capable of preventing, ameliorating, and / or treating inflammatory conditions, including inflammatory bowel disease.

Description

[0001] This application is a divisional application of Chinese application filed on July 8, 2016, with application number 201680020220.8 and entitled "Pyrrolidineamide derivatives and methods for their preparation and use". Technical Field

[0002] This invention relates to pyrrolidine carboxamido derivatives, their optical isomers or pharmaceutically acceptable salts thereof, and methods for preparing and using them. Background Technology

[0003] Various compounds / compositions / methods, including but not limited to immunosuppressive drugs (e.g., infliximab), aminosalicylic acids (e.g., sulfasalazine), and steroids, have been proposed as methods to reduce cytokines and / or chemokines for the prevention and / or treatment of various diseases, including but not limited to inflammatory indications, cancer, and ophthalmic indications (Expert opinion on emerging drugs (2015) 20(3): 349-352; Cell. (2010) 19 March; 140(6): 883-899; Progress in Retinal and Eye Research 37(2013) 68e89, which are incorporated herein by reference). However, they are not satisfactory, at least because they are expensive and / or contain side effects and / or show low therapeutic efficacy (P&T 41 (2016), June 6; Gut 56 (2007): 725-732; World J Gastroenterol (2005); 11(16): 2462-2466, which are incorporated herein by reference). Therefore, new compounds, compositions and / or methods are still needed. Summary of the Invention

[0004] This invention is based on the discovery that certain pyrrolizamide derivatives can inhibit the expression and activity of inflammatory cytokines (e.g., IL-6) and / or chemokines, and can maintain sufficiently high concentrations in target tissues / cells while having minimal exposure to the blood. This invention is also based on the discovery that certain pyrrolizamide derivatives can inhibit NF-κB activity by stabilizing IκB. Furthermore, this invention is based on the discovery that certain pyrrolizamide derivatives can disrupt the formation of inflammatory signal transduction complexes mediated by myeloid differentiation primary response gene 88 (MyD88) and / or receptor interacting protein 1 (RIP1), which act downstream of signaling pathways including Toll-like receptor 2 / 4 and IL-1β.

[0005] In one aspect, the present invention provides compounds represented by the following Formula 1, their optical isomers, or pharmaceutically acceptable salts thereof.

[0006] [Formula 1]

[0007]

[0008] Where: n is 0, 1, or 2; A is -a 1 -, the -a 1 - An amino acid independently selected from the group consisting of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V), wherein the two ends of the amino acid are bonded to a carbonyl or amino group via an amide bond; R 1 For straight or branched C 1-36 Alkyl groups, including straight-chain or branched C-chains with at least one double bond. 2-36 Alkenyl group, or straight-chain or branched C group including at least one triple bond. 2-36 Alkyne group.

[0009] In another aspect, the present invention provides a method for preparing the compound, the optical isomer, and the salt.

[0010] In another aspect, the present invention provides compositions for the prevention, improvement, and treatment of various diseases (e.g., inflammatory indications, cancer, and ophthalmic indications). Each composition comprises at least one of the compounds, at least one of the optical isomers, or at least one of the salts as an active component.

[0011] In another aspect, the present invention provides methods for preventing, improving, or treating various diseases (e.g., inflammatory indications, cancer, and ophthalmic indications). All of these methods involve administering to a subject in need a composition comprising at least one of the compounds, at least one of the optical isomers, or at least one of the salts as the active group.

[0012] Compounds according to certain embodiments of the present invention can inhibit the degradation of IκB in the inflammatory signaling pathway mediated by MyD88 (myddosome complex) and / or RIP1, thereby preventing NF-κB from being transported into the cell nucleus, resulting in inhibition of the expression of cytokines and chemokines (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, and VCAM-1), and preventing inflammatory responses that would otherwise be caused by their expression.

[0013] Other aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the specific embodiments and the accompanying drawings. Attached Figure Description

[0014] Figure 1A The electrophoretic results show the inhibition of IL-6 expression by compounds according to embodiments of the present invention.

[0015] Figure 1B This is a diagram illustrating the inhibition of IL-6 expression by a compound according to an embodiment of the present invention.

[0016] Figure 2 The compound according to an embodiment of the invention is shown to inhibit the expression of cytokines and chemokines in the RAW264.7 cell line.

[0017] Figure 3 The compound according to an embodiment of the invention is shown to inhibit the expression of IL6 in the host cell line RAW264.7.

[0018] Figure 4 The compound according to an embodiment of the present invention is shown to inhibit the activity of NF-κB.

[0019] Figure 5 An embodiment of the compound according to the present invention is shown to inhibit NF-κB.

[0020] Figure 6 The compound shown in the embodiment of the invention inhibits the activity of NF-κB without affecting the signal transduction of TGF-β and BMP.

[0021] Figure 7 This illustrates the inhibition of the formation of protein complexes mediated by IRAK-1, MyD88, and / or RIP1 by compounds according to embodiments of the present invention, and illustrates the immunoprecipitation results of altering IκB concentrations by compounds according to embodiments of the present invention.

[0022] Figure 8This demonstrates that compounds according to embodiments of the invention can disrupt the formation of protein complexes mediated by IRAK-1, MyD88, and / or RIP1 in inflammatory signaling pathways.

[0023] Figure 9 The changes in the pretreatment concentration of the compounds according to embodiments of the present invention altered the concentration of IκB in RAW 264.7 macrophages and BMDM cells.

[0024] Figure 10 The graph shows the disease activity index score of the dosage of the compound according to the embodiment of the invention in an animal model with DSS-induced chronic colitis, in the case of oral administration of the compound according to the embodiment of the invention.

[0025] Figure 11A The disease activity index score is shown, representing the ability of compounds according to embodiments of the invention to inhibit acute colitis in an animal model with DSS-induced acute colitis.

[0026] Figure 11B The compound according to embodiments of the invention is shown to affect the expression levels of chemokines (CCL2, CCL20 and CXCL1) in an animal model of DSS-induced chronic colitis.

[0027] Figure 12-16 Images showing the shape of intestinal villi from the untreated group, the DDS-induced chronic colitis model group, and the group treated with compounds according to embodiments of the present invention.

[0028] Figure 17 This is a graph representing the recovery level of the colonic wall in the untreated group, the DDS-induced chronic colitis model group, the group treated with compounds according to embodiments of the present invention, and the group treated with sulfasalazine.

[0029] Figure 18 This is a graph showing the change in blood concentration over time of a compound according to an embodiment of the invention administered intravenously.

[0030] Figure 19 This is a graph showing the change in blood concentration over time of a compound according to an embodiment of the invention administered orally.

[0031] Figure 20A This is a Western blot image confirming whether the compounds according to embodiments of the present invention inhibit the MAPK / ERK signaling pathway.

[0032] Figure 20B This is a diagram depicting the signaling pathway of Toll-like receptors.

[0033] Figure 21This is a Western blot image confirming whether the compounds according to embodiments of the present invention inhibit the MAPK / ERK signaling pathway.

[0034] Figure 22A and Figure 22B The inhibitory levels of NF-κB activation by compounds and IRAK1 / 4 inhibitors according to embodiments of the present invention are shown respectively.

[0035] Figure 23A and Figure 23B These are immunoblot images confirming whether compounds and IRK1 / 4 inhibitors according to embodiments of the present invention alter IκB concentration.

[0036] Figure 24A and Figure 24B The images and figures are respectively images and graphs comparing the ability of compounds from embodiments of the present invention to inhibit the MAPK / ERK signaling pathway and the ability of IRAK1 / 4 inhibitors to inhibit the MAPK / ERK signaling pathway.

[0037] Figure 25A This is a Western blot confirming whether the compounds according to embodiments of the present invention inhibit the expression of Nox-4, VEGF, VEGFR1, VEGFR2, Ang-2, EPO and EPOR in ARPE-19 and can increase the expression of Ang-1 and Tie2.

[0038] Figure 25B This is a qRT-PCR image confirming whether the compound according to an embodiment of the present invention inhibits VEGF expression in HRMEC.

[0039] Figure 26 This image illustrates how a compound according to an embodiment of the present invention inhibits tube formation in HRMEC.

[0040] Figure 27A and Figure 27B This image illustrates how compounds according to embodiments of the present invention inhibit increased activated oxygen in a mouse model of STZ-induced type 1 diabetic retinopathy.

[0041] Figure 28A The figure illustrates the therapeutic effect of compounds according to embodiments of the present invention in MOG-induced EAE mice.

[0042] Figure 28B This is a graph showing how compounds according to embodiments of the present invention alter the weight of MOG-induced EAE mice.

[0043] Figure 29 This is a diagram illustrating the therapeutic effect of the compound according to an embodiment of the invention in a cecal ligation and puncture (CLP) model. Detailed Implementation

[0044] 1. Definition

[0045] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. General definitions of many terms used in this invention are provided to those skilled in the art by reference to the following references incorporated herein: Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms have the meanings assigned to them as follows.

[0046] Unless explicitly stated or obvious from the context, the term “or” as used herein is understood to mean “include”.

[0047] Unless explicitly stated or obvious from the context, the terms “a” and “described” are understood to be singular or plural as used herein. Thus, for example, reference to “compound” includes mixtures of such compounds; reference to “support” includes mixtures of two or more supports; and so on.

[0048] Unless explicitly stated or obvious from the context, as used herein, the term "approximately" is understood to mean within the range of normal tolerances in the art, for example, within 2 standard deviations of the mean. "Approximately" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "approximately".

[0049] The terms “active agent,” “drug,” and “drug formulation” are used interchangeably herein to refer to a chemical substance or compound that, when administered to a subject (e.g., any animal, including human or non-human animals) by any method and / or route, causes a desired pharmacological effect (e.g., a reduction in inflammation).

[0050] As used herein, the term "additive" can refer to any additional component that can be added to the compositions described herein. For example, if the additional component is pharmaceutically acceptable for the specific condition to be treated, the additive may include excipients (e.g., one or more excipients), antioxidants (e.g., one or more antioxidants), stabilizers (e.g., one or more stabilizers), preservatives (e.g., one or more preservatives), pH adjusters and / or buffers (e.g., one or more pH adjusters and / or buffers), osmotic pressure regulators (e.g., one or more osmotic pressure regulators), thickeners (e.g., one or more thickeners), suspending agents (e.g., one or more suspending agents), binders (e.g., one or more binders), thickeners (e.g., one or more thickeners), etc. For example, additives may also include processing aids, drug delivery modifiers, and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low-melting-point waxes, ion exchange resins, and any combination of two or more thereof. Other suitable pharmaceutically acceptable excipients are described by reference in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991) and “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), both incorporated herein by reference. The additives described herein may be used in any suitable amount.

[0051] As used herein, the term "administration" means oral administration, suppository administration, local contact administration, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, or subcutaneous administration, or administration of a sustained-release device, such as a small osmotic pump, to the subject. Administration is via any route, including parenteral and transmucosal routes (e.g., oral, nasal, pulmonary, rectal, oral cavity, vaginal, ocular, and transdermal routes).

[0052] The terms "derivative" and "analyte" are used interchangeably herein and refer to compounds and combinations thereof having the same core as the parent compound, but with a different bonding sequence, in the absence of or in the presence of one or more atoms and / or groups of atoms. For example, a derivative may differ from the parent compound in the presence of one or more substituents on the core, which may include one or more atoms, functional groups, or substructures. A derivative may also differ from the parent compound in the different bonding sequence between atoms within the core. Generally, derivatives can be conceived as being formed, at least theoretically, from a parent compound via chemical and / or physical processes.

[0053] As used herein, "antioxidant" can refer to any man-made or natural substance that can prevent or delay certain types of cell damage and / or oxidation. Antioxidants are found in many foods, including fruits and vegetables. Antioxidants can also be used as dietary supplements. Exemplary antioxidants may include: beta-carotene, lutein, lycopene, selenium, vitamin A, vitamin C, and vitamin E. Other antioxidants known to those skilled in the art may also be used. The antioxidants described herein may be used in any suitable amount.

[0054] "Co-administration" means administering a compound or composition described herein simultaneously, either before or after the administration of an adjunct therapy, active agent, or additive described herein. The compounds or compositions disclosed herein may be administered alone or co-administered to a subject in need. Co-administration means including simultaneous or sequential administration of compounds, alone or in combination (more than one compound or agent). Formulations may also be combined with other active substances when needed.

[0055] In this disclosure, "comprising" and its variations, "containing" and "having" may have the meanings given to them by U.S. Patent Law and may mean "comprising" and its variations; "consisting substantially of" or "consisting substantially of" also have the meanings given to them by U.S. Patent Law, and the term is open-ended, allowing for the existence of additional basic or novel features as long as the listed basic or novel features are not altered by the presence of additional basic or novel features beyond the listed basic or novel features, but excluding prior art embodiments.

[0056] As used herein, “co-administration” includes at least partial overlap in duration. For example, when two agents (e.g., any biologically active agent or class of agents described herein) are administered in combination, their administration occurs within a specific desired timeframe. Administration of the agents can begin and end on the same day. Administration of one agent may also occur one day (or several days) prior to administration of the second agent, provided that both agents are taken at least once on the same day. Similarly, administration of one agent may precede administration of the second agent, provided that both agents are taken at least once on the same day. Active agents need not be taken simultaneously every day to constitute co-administration.

[0057] As used herein, an "effective amount" or "therapeutic effective amount" is an amount sufficient to influence the expected biological effects, including clinical outcomes, such as beneficial effects. Thus, an "effective amount" is dependent on the circumstances in which it is applied. Effective amounts can vary according to factors known in the art, such as the disease state, age, sex, and weight of the individual being treated. Several separate doses may be administered daily, or the dose may be proportionally reduced according to the urgency of the treatment situation. Furthermore, the compounds, compositions, or formulations of this disclosure may be administered frequently as needed to achieve therapeutic amounts.

[0058] As used herein, the term "gel" can refer to a liquid that does not flow easily and is not a solid (i.e., a semi-solid). Gels can be formed from naturally occurring or synthetic materials. Gels can exhibit birefringence and liquid crystal properties ranging from disordered to micro-ordered. Gels can be applied topically.

[0059] As used herein, the term "inflammatory bowel disease" has its usual medical meaning and refers to a group of inflammatory indications / diseases of the colon and small intestine. Exemplary inflammatory bowel diseases may include, but are not limited to, Crohn's disease, ulcerative colitis, Johns Hopkins disease, Bessett syndrome, collagenous colitis, metastatic colitis, indeterminate colitis, infectious colitis, ischemic colitis, lymphocytic colitis, and closely related conditions and gastrointestinal disorders.

[0060] As used herein, the term "inhibition" means to prevent, reduce, slow down, or stop. In one embodiment, the amount or rate of a process or reaction occurring in the presence of a compound, composition, or formulation is reduced by at least about 10% compared to the amount or rate in the absence of the compound, composition, or formulation, and the compound, composition, or formulation may be considered to inhibit the activity of at least one protein (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, VCAM-1, and NF-κB). In another embodiment, the amount or rate of a process or reaction occurring in the presence of a compound, composition, or formulation is reduced by at least about 20% compared to the amount or rate in the absence of the compound, composition, or formulation, and the compound, composition, or formulation may be considered to inhibit the process or reaction. In other embodiments, when compared to the amount or rate in the absence of the compound, composition, or formulation, the amount or rate of the process or reaction occurring in the presence of the compound, composition, or formulation is reduced by at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, or about 80%, and the compound, composition, or formulation is believed to inhibit the activity of one or more proteins (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, VCAM-1, and NF-κB). In yet another embodiment, the compound, composition, or formulation is believed to inhibit the activity of one or more proteins, i.e., prevent their development.

[0061] As used herein, “intermittent administration” includes a period of administration of the active agent (which may be considered the “first administration period”), followed by a period of no administration or administration at a lower maintenance dose (which may be considered the “interval period”), followed by a period of re-administration of the agent (which may be considered the “second administration period”). Typically, during the second phase of administration, the dose level of the agent will be matched to the dose level administered during the first administration period, but may be increased or decreased as medically necessary.

[0062] According to this disclosure, a “gel” is a type of gel that is a semi-solid system consisting of a suspension composed of small inorganic particles or large organic molecules interpenetrating through a liquid, wherein the structural adhesive matrix comprises a high portion of liquid, typically water.

[0063] As used herein, a “liquid” is a dosage form consisting of a composition in its liquid state. A liquid is pourable; it flows at room temperature and conforms to its container. Liquids exhibit Newtonian or pseudoplastic flow properties. In embodiments, a “semi-liquid” as used herein can have properties of both a liquid and another formulation (i.e., a suspension, emulsion, solution, cream, gel, and colloid, etc.).

[0064] "Myelodifferentiation primary response gene 88," or "MYD88," is a protein encoded by the MYD88 gene in humans. MyD88 plays a central role in both innate and adaptive immune responses. This protein serves as a fundamental signal transduction pathway in the interleukin-1 and Toll-like receptor signaling pathways. These pathways regulate the activation of numerous pro-inflammatory genes. The encoded protein consists of an N-terminal death domain and a C-terminal Toll-interleukin-1 receptor domain.

[0065] As used herein, the term "ointment" can refer to a highly viscous liquid or semi-liquid preparation that can be used for the therapeutic treatment of a condition, syndrome, or disease (e.g., inflammatory bowel disease).

[0066] As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. The type of carrier can be selected based on the intended route of administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile topical solutions or dispersions. Such media and agents are well known in the art for the use of pharmaceutically active substances. For the purposes of this disclosure, the use of any conventional media or agent in ophthalmic compositions is contemplated, except that it is incompatible with any conventional media or agent (e.g., of Formula I, its derivatives / analogs, or pharmaceutically acceptable salts thereof, solvents thereof, hydrates thereof, or polymorphs thereof).

[0067] As used herein, “pharmaceutical carrier” or “carrier” may also include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically aqueous pH buffer solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween. TM Polyethylene glycol (PEG) and Pluronics TM .

[0068] In addition, "pharmaceutically acceptable" means approved or permitted by a federal or state government regulatory agency or a corresponding agency in a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more specifically for use in humans.

[0069] As used herein, the terms "pH agent" or "buffer" can refer to compounds or buffers used as pH adjusters. These include, but are not limited to, glycerol buffers, citrate buffers, borate buffers, acetate buffers, gluconate buffers, phosphate buffers, or may also include citrate-phosphate buffers. pH agents or buffers can be used in any suitable amount.

[0070] As described herein, the term "preservative" can refer to a substance or chemical substance that prevents undesirable chemical changes to the compounds, compositions, or preparations described herein. Suitable preservatives may include, for example, benzalkonium chloride, thiomercuric acid, chlorobutanol, methylparaben, propylparaben, phenethyl alcohol, disodium edetate, sorbic acid, polyquaternium salts (Onamer MPolyquat), hexadecane, hexadecylpyridine chloride, benzyl bromide, EDTA, phenylmercuric nitrate, phenylmercuric acetate, thiomercuric acid, thimerosal, phenylmercuric acetate and borate, polymyxin B sulfate, methylparaben and propylparabens, quaternary ammonium chloride, sodium benzoate, sodium propionate and sodium perborate, and other reagents or combinations thereof known to those skilled in the art. Preservatives may be used in any suitable amount.

[0071] As used herein, the term "prevention" and its variations, as well as other grammatical equivalents, includes preventing the development, occurrence, inhibition, or avoidance of symptoms of a disease or condition, and reducing the occurrence of symptoms. Prevention can be complete (i.e., no detectable symptoms) or partial, resulting in fewer observed symptoms than would have been without treatment. The term also includes preventative benefits. The composition may be administered to patients at risk of having a particular disease or who report one or more physiological symptoms, even if a diagnosis of the disease has not yet been made.

[0072] The range provided here is understood as a shorthand for all values ​​within the range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange of values ​​that are groups of all intermediate decimal values ​​between the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as any group of intermediate decimal values ​​such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. For a subrange, specifically consider an "embedded subrange" extending from either end of the range. For example, an embedded subrange of the exemplary range 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction. A range may be expressed herein as from "about" one specific value and / or to "about" another specific value. When such a range is expressed, the other side includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation, it is understood that the specific value forms the other side by using the preceding "about". It is also understood that each endpoint of a range is meaningful with respect to the other endpoint and is independent of the other endpoint. It is also understood that there are multiple values ​​disclosed herein, and each value is also disclosed herein as an "about" specific value in addition to the value itself. It is also understood that throughout the application, data is provided in a variety of different formats, and this data represents a range of endpoints and starting points, as well as any combination of data points. For example, if specific data point "10" and specific data point "15" are disclosed, it is understood that values ​​greater than, greater than or equal to, less than, less than or equal to, equal to 10 and 15, and values ​​between 10 and 15 are considered disclosed. It is also understood that each unit between the two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0073] As used herein, “receptor-interacting protein” or “RIP1” describes a protein kinase that acts as a key regulator of cell survival and death. RIP1 and RIP2 also possess C-terminal domains belonging to the death domain superfamily, which allow recruitment of large protein complexes to initiate different signaling pathways.

[0074] As used herein, “salt,” “salt form,” or “pharmaceutically acceptable salt” can include base addition salts (formed from free carboxyl groups or other anionic groups) derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. These salts form acid addition salts with any free cation group and are typically formed with inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, or organic acids such as acetic acid, citric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts of this disclosure can include amine salts formed by protonation of an amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc. Salts of this disclosure also include amine salts formed by protonation of an amino group with a suitable organic acid such as p-toluenesulfonic acid, acetic acid, etc., for example. The excipients intended for use in the practice of this disclosure are excipients available to those skilled in the art, for example, excipients described herein by reference in United States Pharmacopeia Volume XXII and National Formulary Volume XVII, US Pharmacopoeia Convention, Inc., Rockville, Md. (1989).

[0075] According to this disclosure, a "semi-solid gel" is a semi-solid. The apparent viscosity of a semi-solid formulation can increase with increasing concentration.

[0076] As used herein, “sequential administration” includes the administration of two agents (e.g., the compounds or compositions described herein) occurring separately on the same day or not on the same day (e.g., over several consecutive days).

[0077] According to this disclosure, a "solution" can be a transparent, homogeneous liquid dosage form containing one or more chemical substances dissolved in a solvent or a mixture of miscible solvents. A solution is a liquid formulation containing one or more dissolved chemical substances dissolved in a suitable solvent or a mixture of miscible solvents. Because the molecules of the pharmaceutical substance are uniformly dispersed in a solution, using a solution as a dosage form generally provides assurance of uniform dosage at administration and good accuracy when the solution is diluted or further mixed.

[0078] As used herein, the term "solvent" refers to an aqueous or non-aqueous liquid solvent. The choice of solvent depends primarily on the composition's solubility in the solvent and the mode of application. Aqueous solvents may consist of water alone, or water plus one or more miscible solvents, and may contain dissolved solutes such as sugars, buffers, salts, or other excipients. More commonly used non-aqueous solvents are short-chain organic alcohols (such as methanol, ethanol, propanol), short-chain ketones (such as acetone), and polyols (such as glycerol). Solvents may be present in any suitable amount.

[0079] "Subject" or "patient" refers to a human or a non-human animal such as a mammal. "Subject" can include any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. Human subjects can be referred to as patients.

[0080] The term "suspension" as used here refers to a liquid dosage form containing solid particles dispersed in a liquid carrier.

[0081] As used herein, the term "syndrome" can refer to a group of symptoms that always occur together or a condition characterized by a group of related symptoms. A syndrome (e.g., inflammatory bowel syndrome) can be a group of medical signs and symptoms that are related to each other and are often associated with a specific disease. On the other hand, a disease can be a health condition with a clearly defined underlying cause. However, syndromes (derived from the Greek word "run together") produce many symptoms without a clear cause. They may suggest the possibility of an underlying disease, or even the possibility of a disease developing.

[0082] As used herein, the term "treatment" or variations thereof and its grammatical equivalents include the relief, mitigation, improvement, or prevention of a disease, symptom (e.g., inflammatory bowel disease) or condition; prevention of complications; improvement or prevention of the underlying metabolic cause of a symptom; inhibition of a disease or symptom (e.g., preventing the development of a disease or condition, relieving a disease or symptom, causing the disease or symptom to subside, relieving symptoms caused by a disease or symptom, or stopping the symptoms of a disease or symptom), and is intended to include prevention. The term also includes obtaining a therapeutic benefit and / or a preventive benefit. A therapeutic benefit refers to the eradication or improvement of an underlying disease that is being treated. Additionally, a therapeutic benefit is achieved by eliminating or improving one or more physiological symptoms associated with an underlying disease, resulting in an observed improvement in the patient, although the patient may still have the underlying disease.

[0083] As used herein, “viscosity” refers to the resistance to the flow of a fluid. Viscosities may be used herein and include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, other reagents known to those skilled in the art, or combinations thereof.

[0084] The term "weight percentage" or "% (w / w)" refers to the percentage of a component in a solution based on the weight of the component and solvent. For example, a 1% (w / w) solution of a component will dissolve 1 g of the component in 100 g of solvent. The term "volume percentage" or "% (v / v)" refers to the percentage of a component in a solution based on the volume of the component and solvent. For example, a 1% (v / v) solution of a component will dissolve 1 ml of the component in 100 ml of solvent. The term "weight / volume percentage" or "% (w / v)" refers to the percentage of a component in a solution based on both the weight of the component and the volume of the solvent. For example, a 1.0% (w / v) solution of a component will dissolve 1 g of the component in 100 ml of solvent.

[0085] 2. Compounds

[0086] As discussed above, one aspect of the invention provides compounds represented by the following Formula 1, their optical isomers, or pharmaceutically acceptable salts thereof.

[0087] [Formula 1]

[0088]

[0089] Where: n is 0, 1, or 2; A is -a 1 -, the -a 1 - An amino acid independently selected from the group consisting of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V), wherein the two ends of the amino acid are bonded to a carbonyl or amino group via an amide bond; R 1 For straight or branched C 1-36 Alkyl groups, including straight-chain or branched C-chains with at least one double bond. 2-36 Alkenyl, or straight-chain or branched C including at least one triple bond 2-36Alkyne group.

[0090]

[0091] The term "compounds of the present invention" and equivalent expressions mean that compounds of the formula described above are included, including pharmaceutically acceptable salts and solvates, such as hydrates, solvates of pharmaceutically acceptable salts as permitted by the context.

[0092] According to some embodiments of the present invention, a 1 It can be R 1 It can be a straight chain or a branched chain C 1-36 alkyl.

[0093] Non-limiting examples of compounds include the following compounds:

[0094] [Formula A]

[0095]

[0096] [Formula B]

[0097]

[0098] [Formula C]

[0099]

[0100] [Form D]

[0101]

[0102] [Formula E]

[0103]

[0104] [Formula F]

[0105]

[0106] [Form G]

[0107]

[0108] [Formula H]

[0109]

[0110] [Formula I]

[0111]

[0112] [Form J]

[0113]

[0114] [Form K]

[0115]

[0116] [Form L]

[0117]

[0118] [Form M]

[0119]

[0120] [Formula N]

[0121]

[0122] [Form O]

[0123]

[0124] The compounds according to embodiments of the present invention are effective for the prevention or treatment of various diseases, including inflammatory indications, cancer, and ophthalmic indications. More specifically, the compounds are effective in inhibiting the expression of cytokines and / or chemokines (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, and VCAM-1). The compounds are also effective in inhibiting the degradation of IκB in the inflammatory signaling pathway mediated by MyD88 (myddosome complex) and / or RIP1, thereby preventing NF-κB from being transported into the cell nucleus. Furthermore, the effective concentration of the compounds in target cells / tissues is maintained for a sufficient period of time.

[0125] 3. Preparation method

[0126] Another aspect of the present invention provides a method for preparing a compound represented by Formula 1. As shown in Reaction Formula 1 below, the method comprises: reacting compound 2 with compound 3 to prepare compound 4 (step 1); hydrolyzing compound 4 in the presence of a base to prepare compound 5 (step 2); reacting compound 5 with compound 6 to prepare compound 7 (step 3); hydrolyzing compound 7 in the presence of a base to prepare compound 8 (step 4); reacting compound 8 with compound 9 to prepare compound 10 (step 5); and hydrolyzing compound 10 in the presence of a base to prepare the compound of Formula I (step 6).

[0127] [Reaction Formula 1]

[0128]

[0129] Among them, A and R 1 And n is the same as defined above, R 2 For straight or branched C 1-5 alkyl.

[0130] In some embodiments, in step 1, compound 2 may be combined with compound 3 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), hydroxybenzotriazole (HOBt), and a base. The base may be an organic or inorganic base. Non-limiting examples of organic bases include pyridine, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Non-limiting examples of inorganic bases include sodium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, and sodium hydride. These may be used in chemotactic doses or in excess, and may be used alone or in combination. Non-limiting examples of solvents that can be used to react compound 2 with compound 3 include ethers (e.g., tetrahydrofuran (THF), dioxane, diethyl ether, and 1,2-dimethoxyethane), alcohols (e.g., methanol, ethanol, propanol, and butanol), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetone, benzene sulfonates, toluene sulfonates, chlorobenzene sulfonates, xylene sulfonates, ethyl acetate, phenyl acetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, maleate, tartrate, methane sulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate. These solvents can be used alone or in combination.

[0131] The base in step 2 can be an organic or inorganic base. Similarly, non-limiting examples of organic bases that can be used in step 2 include pyridine, triethylamine, N,N-diisopropylethylamine (DIPEA), and 1,8-diazabicyclo[5.4.0]diene-7-ene (DBU). Non-limiting examples of inorganic bases include sodium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, and sodium hydride. These can be used stoichiometrically or in excess, alone or in combination. Non-limiting examples of solvents that can be used to react compound 4 with compound 5 include ethers (e.g., tetrahydrofuran (THF), dioxane, diethyl ether, and 1,2-dimethoxyethane), alcohols (e.g., methanol, ethanol, propanol, and butanol), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetone, benzene sulfonates, toluene sulfonates, chlorobenzene sulfonates, xylene sulfonates, ethyl acetate, phenyl acetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, maleate, tartrate, methane sulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate. These solvents can be used alone or in combination.

[0132] Steps 3 and 5 can be performed in the same or similar manner as step 1. Steps 4 and 6 can be performed in the same or similar manner as step 2.

[0133] Preparation of compound 2

[0134] An example of compound 2, represented by formula 2 below, which is the starting material for reaction 1, can be prepared by, for example, preparation method A described below.

[0135] [Equation 2]

[0136]

[0137] Where n is 0, 1, or 2; A is -a 1 -, the -a 1- An amino acid independently selected from the group consisting of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V), wherein the two ends of the amino acid are bonded to a carbonyl or amino group via an amide bond; R 1 For straight or branched C 1-36 Alkyl groups, including straight-chain or branched C-chains with at least one double bond. 2-36 Alkenyl group, or straight-chain or branched C group including at least one triple bond. 2-36 Alkyne group.

[0138] [Preparation Method A]

[0139] Compound 2 was prepared by combining the compound represented by formula a shown below with an amino acid selected from the group consisting of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V) to form an amide bond.

[0140] [Formula a]

[0141]

[0142] (R 1 (Same as defined in Equation 2)

[0143] 4. Composition / Formulation

[0144] Another aspect of the invention provides compositions for the prevention, improvement and treatment of various diseases (e.g., inflammatory indications, cancer and ophthalmic indications), said compositions comprising at least one compound, at least one optical isomer or at least one salt as an active component.

[0145] The compositions according to some embodiments can inhibit the expression of cytokines and / or chemokines including G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, and VCAM-1. The compositions according to other embodiments can inhibit NF-κB activity. The compositions according to other embodiments can inhibit the formation of MyD88-mediated inflammatory signal transduction complexes. The compositions according to other embodiments can inhibit the formation of RIP1-mediated inflammatory signal transduction complexes. The compositions according to other embodiments can inhibit the formation of Pellino-1-mediated inflammatory signal transduction complexes.

[0146] In some embodiments, the present invention provides compositions for the prevention, improvement, and treatment of inflammatory bowel disease (including closely related conditions), said compositions comprising at least one compound, at least one optical isomer, or at least one salt as an active ingredient. Inflammatory bowel disease may include, but is not limited to, ulcerative colitis, Behcet's disease, and Crohn's disease. The compositions may also include additives.

[0147] In some embodiments, the present invention provides a method for preventing, improving or treating multiple sclerosis, psoriasis, sepsis, geographic atrophy, wet age-related macular degeneration, dry age-related macular degeneration, diabetic retinopathy, infectious lung disease, bacterial pneumonia, viral pneumonia, diffuse large B-cell lymphoma, viral infection, autoimmune diseases, blood cancers including lymphoma, and tumors in internal organs, wherein the composition comprises at least one compound, at least one optical isomer, or at least one salt as an active component.

[0148] In some embodiments, the present invention provides a composition for preventing, improving or treating hair loss, the composition comprising at least one compound, at least one optical isomer or at least one salt as an active ingredient, wherein the active ingredient inhibits the expression of IL-6 in the scalp and hair follicles.

[0149] This invention includes formulations suitable for the administration of the compounds described herein. The compounds described herein may be incorporated in a formulation (including pharmaceutical compositions) with additives such as excipients (e.g., one or more excipients), antioxidants (e.g., one or more antioxidants), stabilizers (e.g., one or more stabilizers), preservatives (e.g., one or more preservatives), pH adjusters and / or buffers (e.g., one or more pH adjusters and / or buffers), osmolarity regulators (e.g., one or more osmolarity regulators), thickeners (e.g., one or more thickeners), suspending agents (e.g., one or more suspending agents), binders (e.g., one or more binders), viscous agents (e.g., one or more viscous agents), etc. In some embodiments, the formulation may include a combination of two or more additional components as described herein (e.g., 2, 3, 4, 5, 6, 7, 8 or more additional components). For example, in some embodiments, the additives include processing aids, drug delivery modifiers, and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low-melting-point waxes, ion exchange resins, and any combination of two or more thereof. Other suitable pharmaceutically acceptable excipients are described by reference in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991) and “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), which are incorporated herein by reference.

[0150] Formulations of pharmaceutical compositions suitable for administration by any medically acceptable method are included in this invention. Pharmaceutical formulations may include pharmaceutically acceptable carriers and pharmaceutically acceptable compounds (compositions) suitable for administration. For example, formulations of the compositions described herein may be suitable for oral administration. They may be formed in various forms including solutions, suspensions, semi-liquids, semi-solids, gels, emulsions, ointments, tablets, and creams. Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, silica gel, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically compatible carriers.

[0151] The compositions (formulations) can be administered via a variety of routes, including but not limited to oral, nasal, lung, rectal, oral cavity, vaginal, ocular, and transdermal routes. The mode of administration, frequency, and effective amount of the compositions (formulations) can be determined according to methods known in the art and / or those described herein (e.g., oral administration, 0.1-1,000 mg / day, once daily). For example, they can be administered alone or in combination. For example, they can be administered in combination, co-administered, and / or intermittently.

[0152] 5. Methods of using compounds, compositions, or formulations

[0153] Another aspect of the invention provides a method for preventing, improving, or treating various diseases (e.g., inflammatory indications, cancer, and ophthalmic indications), wherein the method administers a composition (or the compound or preparation described herein) to a subject in need.

[0154] In embodiments, the present invention provides a method for preventing, improving, or treating inflammatory bowel disease, the method comprising administering to a subject in need a composition (or a compound or preparation described herein) comprising at least one compound, at least one optical isomer, or at least one salt as an active ingredient.

[0155] In another embodiment, the present invention provides a method for preventing, improving, or treating a disease or syndrome, the method comprising administering to a subject in need a composition comprising at least one compound, at least one optical isomer, or at least one salt as an active ingredient. The disease or syndrome may involve the formation of a Pellino-1-induced inflammatory signal transduction complex comprising MyD88, RIP1, or both. The disease or syndrome may include, but is not limited to, multiple sclerosis, psoriasis, sepsis, geographic atrophy, wet age-related macular degeneration, dry age-related macular degeneration, diabetic retinopathy, infectious lung diseases, bacterial pneumonia, viral pneumonia, diffuse large B-cell lymphoma, viral infections, autoimmune diseases, blood cancers including lymphoma, and tumors in internal organs (e.g., liver, lung, intestine, prostate, pancreas, etc.).

[0156] In another embodiment, the present invention provides a method for preventing, improving, or treating geographic atrophy, wet age-related macular degeneration, dry age-related macular degeneration, or diabetic retinopathy, the method comprising administering to a subject in need a composition comprising at least one compound, at least one optical isomer, or at least one salt as an active ingredient. The compound, optical isomer, and salt can have pharmacological effects on retinal pigment epithelial cells. In retinal pigment epithelial cells, they can inhibit the expression of at least one protein selected from the group consisting of Nox-4, VEGF, VEGFR1, VEGFR2, Ang2, EPO, and EPOR. In retinal pigment epithelial cells, they can increase the expression of Ang1, Tie2, or both.

[0157] Example

[0158] The invention will be illustrated in more detail by way of the following examples. These examples are presented for illustrative purposes only, and the invention is not limited to these examples.

[0159] Example 1: Preparation of compounds

[0160] Example 1.1: (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetamido)propionic acid (Pal-PPGY-OH)

[0161]

[0162] Step 1: Preparation of (S)-methyl 1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid ester

[0163] A mixed solution was prepared by mixing (S)-1-palmitoylpyrrolidine-2-carbonyl acid (10.0 g, 28.3 mmol), EDCI (5.96 g, 31.1 mmol), HOBt (4.20 g, 31.1 mmol), and triethylamine (11.8 mL, 84.9 mmol) in dichloromethane. Proline methyl ester hydrochloride (5.15 g, 31.1 mmol) was added to the mixed solution. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium bicarbonate solution, and extracted three times with ethyl acetate. All organic layers were washed with salt solution and three times with 1N HCl. The product was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give (S)-methyl 1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid ester (11.4 g, 87% yield).

[0164] 1H-NMR (300MHz, CDCl3) δ4.69-4.65 (m, 1H), 4.54-4.58 (m, 1H), 3.83-3.93 (m, 1H), 3.58-3.72 (m, 5H), 3.45-3.53 (m, 1H), 1.89-2.31 (m, 10H), 1.60-1.64(m, 2H), 1.25(m, 24H), 0.88(t, J=6.87Hz, 3H).

[0165] MS(ESI), calculated value C 27 H 48 N₂O₄ 464.4, found value m / z 465.2 (M+H) + ).

[0166] Step 2: Preparation of (S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid

[0167] The (S)-methyl 1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid ester (15.0 g, 32.3 mmol) prepared in step 1 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (2.58 g, 64.6 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. 1 N HCl was added to adjust the pH to 1.0. The aqueous layer was extracted three times with ethyl acetate. All organic layers were dried over anhydrous magnesium sulfate and concentrated to give (S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (13.2 g, 91% yield) as a white solid.

[0168] MS(ESI), calculated value C 26 H 46 N₂O₄ 450.3, found value m / z 451.1 (M+H) + ).

[0169] Step 3: Ethyl 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetic acid Ester (ethyl 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2- Preparation of carboxamido (acetate)

[0170] The (S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (12 g, 26.6 mmol) prepared in step 2 was mixed with dichloromethane. Glycine ethyl ester hydrochloride (4.09 g, 29.3 mmol), EDCI (5.62 g, 29.3 mmol), HOBt (3.96 g, 29.3 mmol), and triethylamine (11.1 mL, 79.8 mmol) were added to the mixture. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated to give ethyl 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-amide)acetate (11.1 g, 78% yield).

[0171] MS(ESI), calculated value C 30 H 53 N3O5 535.4, found value m / z 536.5 (M+H) + ).

[0172] Step 4: Preparation of 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetic acid Preparation

[0173] The ethyl 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetate (12 g, 22.4 mmol) prepared in step 3 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (1.79 g, 44.8 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. 1 N HCl was added to adjust the pH to 1.0. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetic acid (9.5 g, 84% yield).

[0174] MS(ESI), calculated value C 28 H 49 N3O5 507.4, found value m / z 508.2 (M+H) + ).

[0175] Step 5: (S)-Methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl) Preparation of pyrrolidine-2-acylaminoacetamidopropionate

[0176] The 2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetic acid (10 g, 19.7 mmol) prepared in step 4 was mixed with tetrahydrofuran. Tyrosine methyl ester (4.23 g, 21.7 mmol), EDCI (4.16 g, 21.7 mmol), HOBt (2.93 g, 21.7 mmol), and triethylamine (8.19 mL, 59.1 mmol) were added to the mixture. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium bicarbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried with anhydrous magnesium sulfate, concentrated, and purified by MPLC to give (S)-methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetamido)propionate (8.4 g, yield 62%).

[0177] MS(ESI), calculated value C 37 H 60 N4O7684.4, found value m / z 685.2 (M+H) + ).

[0178] Step 6: (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyridine Preparation of pyrrolidine-2-acylamino)acetamido)propionic acid

[0179] The (S)-methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetamido)propionate (4.9 g, 7.16 mmol) prepared in step 5 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (0.86 g, 21.5 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. 1 N HCl was added to adjust the pH to 1.0. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-acylamino)acetamido)propionate (4.5 g, 93% yield).

[0180] 1H-NMR (300MHz, MeOD) δ7.04 (d, J=8.31Hz, 2H) 6.70 (d, J=8.37Hz, 2H), 4.38-4.68 (m, 3H), 3.44-4.04 (m, 6H), 2.91-3.13 (m, 2H), 1.81-2.38 (m, 10H), 1.54-1.60 (m, 2H), 1.30 (m, 24H), 0.88 (t, J=6.63Hz, 3H).

[0181] MS(ESI), calculated value C 37 H 58 N4O7670.4, found value m / z 671.3 (M+H) + ).

[0182] Example 1.2: (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-acylamino)acetamido)propionic acid (Pal-PGY-OH)

[0183]

[0184] Step 1: Preparation of (S)-methyl-1-palmitoylpyrrolidine-2-carboxylic acid ester

[0185] Palmitic acid (7 g, 27.3 mmol), EDCI (5.78 g, 30.0 mmol), HOBt (4.05 g, 30.0 mmol), and triethylamine (11.4 mL, 81.9 mmol) were mixed with dichloromethane. Proline methyl ester hydrochloride (4.97 g, 30.0 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1 N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give (S)-methyl-1-palmitoylpyrrolidine-2- Carboxyl esters (9.6g, yield 96%).

[0186] 1 H-NMR (300MHz, CDCl3) δ4.46-4.50 (m, 1H), 3.47-3.75 (m, 5H), 1.90-2.36 (m, 6H), 1.59-1.69 (m, 2H), 1.25 (m, 24H), 0.88 (t, J=6.84Hz, 3H)

[0187] MS(ESI), calculated value C 22 H 41 NO3367.3, found value m / z368(M+H)+ ).

[0188] Step 2: Preparation of (S)-1-palmitoylpyrrolidine-2-carboxylic acid

[0189] The (S)-1-palmitoylpyrrolidine-2-carboxylic acid ester (10.0 g, 27.2 mmol) prepared in step 1 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (3.26 g, 81.6 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. The pH was adjusted to 1.0 with 1 N HCl. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give (S)-1-palmitoylpyrrolidine-2-carboxylic acid (8.6 g, 89% yield) as a white solid.

[0190] 1 H-NMR (300MHz, CDCl3) δ4.59-62(m, 1H), 3.42-3.59(m, 2H), 2.46-2.53(m, 1H), 2.33-2.38(m, 2H), 1.93-2.01(m, 3H), 1.62-1.69(m, 2H), 1.25(m, 24H), 0.88(t, J=6.90Hz, 3H)

[0191] MS(ESI), calculated value C 21 H 39 NO3353.3, found value m / z354.2(M+H) + ).

[0192] Step 3: Preparation of (S)-ethyl 2-(1-palmitoylpyrrolidine-2-acylamino)acetate

[0193] The (S)-1-palmitoylpyrrolidine-2-carboxylic acid (10 g, 28.3 mmol), glycine ethyl ester hydrochloride (4.34 g, 31.1 mmol), EDCI (5.76 g, 31.1 mmol), HOBt (4.20 g, 31.1 mmol), and triethylamine (15.7 mL, 113 mmol) prepared in step 2 were mixed with dichloromethane. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated to give (S)-ethyl 2-(1-palmitoylpyrrolidine-2-acylamino)acetate (10.7 g, 86% yield).

[0194] MS(ESI), calculated value C 25 H 46 N₂O₄ 438.3, found value m / z 439.1 (M+H) +).

[0195] Step 4: Preparation of (S)-2-(1-palmitoylpyrrolidine-2-acylamino)acetic acid

[0196] The (S)-ethyl 2-(1-palmitoylpyrrolidine-2-acylamino)acetic acid ester (12 g, 27.4 mmol) prepared in step 3 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (2.20 g, 54.7 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. The pH was adjusted to 1.0 with 1 N HCl. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give (S)-2-(1-palmitoylpyrrolidine-2-acylamino)acetic acid (10.2 g, 91% yield) as a white solid.

[0197] MS(ESI), calculated value C 23 H 42 N₂O₄ 410.3, found value m / z 411.3 (M+H) + ).

[0198] Step 5: (S)-Methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-amido)acetyl Preparation of aminopropionate

[0199] The (S)-2-(1-palmitoylpyrrolidine-2-amide)acetic acid (7 g, 27.3 mmol) prepared in step 4 was mixed with dichloromethane. Tyrosine methyl ester (5.86 g, 30.0 mmol), EDCI (5.78 g, 30.0 mmol), HOBt (4.05 g, 30.0 mmol), and triethylamine (11.4 mL, 81.9 mmol) were added to the mixture. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium bicarbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, concentrated, and purified by MPLC to give (S)-methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-amido)acetamido)propionate (9.8 g, 61% yield).

[0200] 1H-NMR (300MHz, CDCl3) δ7.25-7.50 (m, 3H), 6.93 (d, J=8.34Hz, 2H) 6.70 (d, J=8.34Hz, 2H), 4.70-4.77 (m, 1H), 4.39-4.43 (m, 1H), 3.95-4.21 (m, 1H), 3.41-3.72 (m, 5H), 2.92-3.12 (m, 2H), 1.91-2.35 (m, 7H), 1.57-1.61 (m, 2H), 1.25 (m, 24H), 0.88 (t, J=6.87Hz, 3H).

[0201] MS(ESI), calculated value C 33 H 53 N3O6587.4, found value m / z588.1 (M+H) + ).

[0202] Step 6: (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-amido)acetamide Preparation of hydroxypropionic acid

[0203] The (S)-methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-amido)acetamido)propionate (2.85 g, 4.85 mmol) prepared in step 5 was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (0.58 g, 14.6 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. The pH was adjusted to 1.0 with 1 N HCl. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give (S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-amido)acetamido)propionate (2.2 g, 79% yield) as a white solid.

[0204] 1 H-NMR (300MHz, MeOD) δ7.03 (d, J=8.40Hz, 2H), 6.70 (d, J=8.40Hz, 2H), 4.58-4.61 (m, 1H), 4.33-4.56 (m, 1H), 3.58-4.37 (m, 4H), 2.96-3.15 (m, 2H), 1.92-2.39 (m, 6H), 1.55-1.62 (m, 2H), 1.29 (m, 24H), 0.91 (t, J=6.87Hz, 3H).

[0205] MS(ESI), calculated value C 32 H 51 N3O6 573.4, found value m / z 574.2 (M+H) + ).

[0206] Example 1.3: Palmitoyl-L-alanyl-L-prolyl-glycyl-L-tyrosine (Pal-APGY-OH)

[0207] The compound is prepared according to reaction formula 2 below.

[0208] [Reaction 2]

[0209]

[0210] Compound (1) (10 g, 46.5 mmol), compound (2) (7.15 g, 51.2 mmol), EDCI·HCl (9.82 g, 51.2 mmol), HOBt (6.92 g, 51.2 mmol), and triethylamine (19.4 mL, 140 mmol) were mixed with dichloromethane. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give compound (3) as a viscous liquid (yield 91%).

[0211] LC-MS (ESI): Calculated value C 14 H 24 N₂O₅ 300.2, found value m / z 301.2 (M+H) + ).

[0212] Compound (3) (12 g, 40 mmol) was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (6.40 g, 160 mmol) was added to the mixture. The resulting mixture was stirred overnight at room temperature and concentrated. The pH was adjusted to 1.0 with 1 N HCl. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give compound (4) as a white solid (96% yield).

[0213] LC-MS (ESI): Calculated value C 12 H 20 N₂O₅ 272.1, found value m / z 273.1 (M+H) + ).

[0214] Compound (4) (6.25 g, 23 mmol), compound (5) (4.85 g, 25.3 mmol), EDCI·HCl (4.85 g, 25.3 mmol), HOBt (43.42 g, 25.3 mmol), and triethylamine (TEA, 12.8 mL, 96 mmol) were mixed with dichloromethane. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed twice with aqueous sodium bicarbonate solution, washed three times with salt solution, and washed three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, and concentrated to give compound (6) as a white solid (yield 87%).

[0215] LC-MS (ESI): Calculated value C 22 H 31 N3O7449.2, found value m / z 450.2 (M+H) + ).

[0216] Compound (6) (8 g, 17.8 mmol) was dissolved in ethyl acetate. An excess of dioxane in 4N HCl was added at room temperature. The resulting mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure to give compound (7) as a white solid.

[0217] LC-MS (ESI): Calculated value C 17 H 23 N3O5 349.2, found value m / z 350.2 (M+H) + ).

[0218] Compound (7) (0.25 g, 0.65 mmol), compound (8) (Boc-alanine, 0.12 g, 0.65 mmol), EDCI·HCl (0.25 g, 1.30 mmol), HOBt (0.18 g, 1.30 mmol), and triethylamine (0.36 mL, 2.60 mmol) were mixed with dichloromethane. The resulting mixture was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed twice with aqueous sodium bicarbonate solution, washed three times with salt solution, and washed three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, concentrated, and purified by MPLC (dichloromethane / 2-propanol) to give compound (9) (yield 3%).

[0219] LC-MS (ESI): Calculated value C 25 H 36 N4O8 520.3, found value m / z 520.7 (M+H) + ).

[0220] Compound (9) (0.11 g, 0.21 mmol) was dissolved in ethyl acetate. An excess solution of dioxane in 4N HCl was added at room temperature, and the mixture was stirred for 4 hours at room temperature. The resulting mixture was concentrated under reduced pressure to give compound (10) as a white solid.

[0221] LC-MS (ESI): Calculated value C 20 H 28 N4O6 420.2, found value m / z 420.6 (M+H) + ).

[0222] Compound (10) (0.04 g, 0.09 mmol), EDCI·HCl (0.03 g, 0.16 mmol), HOBt (0.02 g, 0.16 mmol), and triethylamine (0.04 mL, 0.32 mmol) were added to a solution of palmitic acid (0.02 g, 0.08 mmol) in dichloromethane. The resulting product was stirred overnight at room temperature, concentrated under reduced pressure, diluted with aqueous sodium carbonate solution, and extracted three times with ethyl acetate. The entire organic layer was washed with salt solution and three times with 1N HCl. The organic layer was washed with salt solution, dried over anhydrous magnesium sulfate, concentrated, and purified by MPLC (dichloromethane / 2-propanol) to give compound (11) (58% yield).

[0223] LC-MS (ESI): Calculated value C 36 H 58 N4O7 658.4, found value m / z 659.1 (M+H) + ).

[0224] Compound (11) (0.03 g, 0.05 mmol) was mixed with tetrahydrofuran. An aqueous solution of sodium hydroxide (0.008 g, 0.20 mmol) was added. The resulting mixture was stirred overnight at room temperature and concentrated. The pH was adjusted to 1.0 with 1 N HCl. The aqueous layer was extracted three times with ethyl acetate. The entire organic layer was dried over anhydrous magnesium sulfate and concentrated to give compound (12) as a white solid (93% yield).

[0225] 1H NMR (500MHz, CD3OD) δ7.05 (d, J=8.50Hz, 2H), 6.70 (d, J=8.50Hz, 2H), 4.59-4.65 (m, 2H), 4.39-4.41 (m, 1H), 3.96-3.99 (m, 1H), 3.84-3.89 (m, 1H), 3.65-3.76 (m, 2H), 3.10-3.14 (m, 1H), 2.97-3.01 (m, 1H), 2.20-2.24 (m, 3H), 2.09-2.14 (m, 1H), 1.96-2.03 (m, 2H), 1.58-1.60 (m, 3H), 1.31-1.36 (m, 30H), 0.92 (t, J = 7.15 Hz, 3H). LC-MS (ESI): Calculated C 35 H 56 N4O7 644.4, found value m / z 644.6 (M+H) + ).

[0226] Compounds of Examples 1.4 to 1.15 were prepared using compound (7) as the starting material in the same manner as described in Example 1.3. The NMR data of these compounds are shown below.

[0227]

[0228] Example 1.4: Palmitoylglycyl-L-prolycylglycyl-L-tyrosine (pal-GPGY-OH) 1 ¹H NMR (500MHz, CD₃OD) δ 7.03 (d, J = 8.50Hz, 2H), 6.70 (d, J = 8.50Hz, 2H), 4.58–4.63 (m, 1H), 4.39–4.42 (m, 1H), 3.90–4.08 (m, 4H), 3.59–3.74 (m, 3H), 3.09–3.12 (m, 1H), 2.96–3.00 (m, 1H), 1.99–2.31 (m, 7H), 1.56–1.66 (m, 3H), 1.24–1.35 (m, 26H), 0.92 (t, J = 7.05Hz, 3H). LC-MS (ESI): Calculated C 34 H 54 N4O7 630.4, found value m / z 630.8 (M+H) + ).

[0229]

[0230] Example 1.5: ((9Z,12Z)-octadecane-9,12-dienoyl)glycyl-L-prolylglycyl-L-tyrosine (linoleyl-GPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.03 (d, J=8.50Hz, 2H), 6.71 (d, J=8.50Hz, 2H), 5.31-5.41 (m, 4H), 4.58-4.62 (m, 1H), 4.39-4.42(m, 1H), 3.99-4.08(m, 2H), 3.70-3.73(m, 1H), 3.59-3.67(m, 1H), 3.09-3.12(m, 1H), 2.96-3.00(m, 1H), 2.78-2.79(m, 3H), 2.25-2.31 (m, 1H), 2.18–2.22 (m, 2H), 1.99–2.13 (m, 7H), 1.56–1.64 (m, 3H), 1.31–1.42 (m, 18H), 0.93 (t, J = 7.10 Hz, 3H). LC-MS (ESI): Calculated C 36 H 54 N4O7654.4, found value m / z655(M+H) + ).

[0231]

[0232] Example 1.6: Palmitoyl-L-phenylalanyl-L-prolyl-glycyl-L-tyrosine (pal-FPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.27-7.28 (m, 5H), 7.07 (d, J=8.45Hz, 2H), 6.71 (d, J=8.30Hz, 2H), 4.59-4.63 (m, 1H), 4.40-4.42 (m, 1H), 3.99-4.02 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.78 (m, 1H), 3.52-3.55 (m, 1H), 3.09–3.16 (m, 2H), 2.86–3.02 (m, 3H), 1.95–2.22 (m, 8H), 1.44–1.62 (m, 4H), 1.31 (m, 25H), 0.92 (t, J = 7.10 Hz, 3H). LC-MS (ESI): Calculated value C 41 H 60 N4O7720.4, found value m / z 721.1 (M+H) + ).

[0233]

[0234] Example 1.7: Hexanoyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (hexanoyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.05 (d, J=8.50Hz, 2H), 6.70 (d, J=8.50Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.44 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.76 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.31-2.41 (m, 2H), 2.20-2.29 (m, 2H), 1.92-2.12 (m, 5H), (m, 8H), 1.57-1.66 (m, 3H), 1.31-1.38 (m, 5H), 0.93 (t, J = 7.00 Hz, 3H). LC-MS (ESI): Calculated C 27 H 38 N4O7 530.3, found value m / z 530.7 (M+H) + ).

[0235]

[0236] Example 1.8: Octanoyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (octanoyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.05 (d, J=8.50Hz, 2H), 6.71 (d, J=8.50Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.42 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.18-2.34 (m, 4H), 1.92-2.12 (m, 5H), 1.57-1.61 (m, 2H), 1.32-1.35 (m, 10H), 0.92 (t, J = 7.00 Hz, 3H). LC-MS (ESI): Calculated value C 29 H 42 N4O7 558.3, found value m / z 558.5 (M+H) + ).

[0237]

[0238] Example 1.9: Decanoyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (decanoyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.03 (d, J=8.55Hz, 2H), 6.71 (d, J=8.40Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.42 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.18-2.39 (m, 5H), 1.92-2.14 (m, 6H), 1.57-1.61 (m, 2H), 1.32-1.36 (m, 14H), 0.92 (t, J = 7.15 Hz, 3H). LC-MS (ESI): Calculated value C 31 H 46 N4O7 586.3, found value m / z 586.8 (M+H) + ).

[0239]

[0240] Example 1.10: Stearoyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (stearoyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.03 (d, J=8.55Hz, 2H), 6.71 (d, J=8.25Hz, 2H), 4.66-4.68 (m, 1H), 4.55-4.57 (m, 1H), 4.40-4.44(m, 1H), 3.96-4.02(m, 1H), 3.84-3.89(m, 1H), 3.71-3.76(m, 1H), 3.48-3.68(m, 4H), 3.08-3.12(m, 1H), 2.97-3.02(m, 1H), 2.14-2.42 (m, 5H), 1.90–2.14 (m, 6H), 1.57–1.61 (m, 2H), 1.32–1.35 (m, 30H), 0.92 (t, J = 7.15 Hz, 3H). LC-MS (ESI): Calculated value C 39 H 62 N4O7 698.5, found value m / z 698.5 (M+H) + ).

[0241]

[0242] Example 1.11: Hex-5-enoyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (5-hexenoyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.05 (d, J=8.55Hz, 2H), 6.70 (d, J=8.25Hz, 2H), 5.78-5.89 (m, 1H), 4.98-5.08 (m, 3H), 4.66-4.69(m, 1H), 4.54-4.57(m, 1H), 4.40-4.44(m, 1H), 3.97-4.01(m, 1H), 3.84-3.90 (m, 1H), 3.73-3.76 (m, 1H), 3.47-3.68 (m, 4H), 3.08-3.12 (m, 1H), 2.97-3.01 (m, 1H), 2.33-2.42 (m, 2H), 2.20-2.30 (m, 2H), 2.06-2.15 (m, 4H), 1.92-2.04 (m, 4H), 1.67-1.76 (m, 3H). LC-MS (ESI): Calculated C 27 H 36 N4O7528.3, found value m / z529(M+H) + ).

[0243]

[0244] Example 1.12: Oleyl-L-prolyl-L-prolyl-glycyl-L-tyrosine (oleyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.03 (d, J=8.50Hz, 2H), 6.71 (d, J=8.50Hz, 2H), 5.34-5.38 (m, 2H), 4.66-4.68 (m, 1H), 4.55-4.57 (m, 1H), 4.40-4.42 (m, 1H), 3.96-4.01 (m, 1H), 3.84-3.90 (m, 1H), 3.73-3.77 (m, 1H), 3.53-3.69 (m, 4H), 3.08-3.12 (m, 1H), 2.97-3.01 (m, 1H), 2.18-2.39 (m, 5H), 2.06-2.12 (m, 2H), 1.92-2.05 (m, 3H), 1.58-1.61 (m, 3H), 1.31-1.35 (m, 25H), 0.92 (t, J = 7.0 Hz, 3H). LC-MS (ESI): Calculated value C 39 H60 N4O7 696.4, found value m / z 697.3 (M+H) + ).

[0245]

[0246] Example 1.13: ((9Z,12Z)-octadecane-9,12-dienoyl)-L-prolyl-L-prolyl-glycyl-L-tyrosine (linoleyl-PPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.05 (d, J=8.45Hz, 2H), 6.71 (d, J=8.45Hz, 2H), 5.32-5.41 (m, 4H), 4.66-4.68 (m, 1H), 4.55-4.57(m, 1H), 4.40-4.43(m, 1H), 3.96-4.01(m, 1H), 3.84-3.89(m, 1H), 3.73-3.77(m, 1H), 3.53-3.68(m, 4H), 3.08-3.12(m, 1H), 2.97-3.01 (m, 1H), 2.80 (t, J = 6.40 Hz, 2H), 2.19–2.39 (m, 5H), 1.93–2.13 (m, 10H), 1.59–1.61 (m, 3H), 1.31–1.40 (m, 15H), 0.92 (t, J = 6.59 Hz, 3H). LC-MS (ESI): Calculated value C 39 H 58 N4O7 694.4, found value m / z 695.2 (M+H) + ).

[0247]

[0248] Example 1.14: Palmitoyl-L-valine-L-prolyl-L-prolyl-glycyl-L-tyrosine (Pal-VPPGY-OH) 1H NMR (500MHz, CD3OD) δ7.04 (d, J=8.35Hz, 2H), 6.71 (d, J=8.35Hz, 2H), 4.67-4.70 (m, 1H), 4.52-4.58 (m, 1H), 4.39-4.43 (m, 1H), 3.77-4.05 (m, 4H), 3.64-3.73 (m, 2H), 3.49-3.60 (m, 1H), 3.01-3.11 (m, 2H), 1.87-2.31 (m, 15H), 1.61-1.63 (m, 3H), 1.31 (m, 24H), 1.01 (d, J = 6.58 Hz, 3H), 0.98 (d, J = 6.58 Hz, 3H), 0.92 (t, J = 6.96 Hz, 3H). LC-MS (ESI), calculated value C 42 H 67 N5O8769.5, found value m / z 770.7 (M+H) + ).

[0249]

[0250] Example 1.15: Decanoyl-L-valine-L-prolyl-L-prolyl-glycyl-L-tyrosine (decanoyl-VPPGY-OH) 1 H NMR (500MHz, CD3OD) δ7.05 (d, J=8.45Hz, 2H), 6.71 (d, J=8.45Hz, 2H), 4.67-4.70 (m, 1H), 4.50-4.57 (m, 1H), 4.39-4.43(m, 1H), 3.95-4.05(m, 2H), 3.80-3.91(m, 1H), 3.65-3.70(m, 2H), 3.64-3.73 (m, 2H), 3.53-3.57 (m, 1H), 3.01-3.10 (m, 2H), 1.89-2.31 (m, 12H), 1.61-1.63 (m, 3H), 1.31 (m, 24H), 1.01 (d, J = 6.68 Hz, 3H), 0.97 (d, J = 6.55 Hz, 3H), 0.92 (t, J = 7.05 Hz, 3H). LC-MS (ESI), calculated value C 36 H 55 N5O8685.4, found value m / z 686.6 (M+H) + ).

[0251] Table 1 describes the compounds according to Examples 1 to 1.15.

[0252] Table 1: Compounds according to Examples 1 to 1.15

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] Example 2: Experiment

[0260] Example 2.1: Inhibiting IL-6 expression

[0261] To evaluate the inhibitory effect of the compounds of the present invention on IL-6 expression, the following experiments were conducted.

[0262] First, RAW264.7 macrophages were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured at 37°C under a 5% CO2 atmosphere using 10% fetal bovine serum (FBS) and Dalberg modified Eagle medium (DMEM) containing 1% penicillin / streptomycin.

[0263] Cultured RAW264.7 macrophages were transferred into 6-well cell culture dishes. After 24 hours, the cells were pretreated for 30 minutes with 100 nM compounds 1.1, 1.2, and smaducin-6, respectively, and then further treated with lipopolysaccharide (LPS) for 2 hours. Cells were collected and RNA was extracted. cDNA (complementary deoxyribonucleic acid) was synthesized from 2 μg of extracted RNA, and reverse transcription polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (RT-PCR) were performed. Samples from RT-PCR were confirmed by electrophoresis on agarose gels and quantified using a densitometer. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control for the interleukin-6 gene.

[0264] Figure 1A Images showing the inhibition of interleukin-6 expression by compounds 1.1 and 1.2 are presented. Figure 1B This is a graph showing the quantitative inhibition of interleukin-6 expression by compounds 1.1 and 1.2. Compared to untreated samples, treatment of cells with the compounds according to the invention suppressed the expression of interleukin-6 induced by LPS treatment.

[0265] In addition, RAW264.7 macrophages cultured as described above were also divided into 6-well plates. After 24 hours, they were pretreated with 100 nM compound 1.1 for 30 minutes and then further treated with LPS for 2 hours. The cell cultures were collected using a cytokine array (mouse cytokine array C3, RayBiotech), and the changes in the amounts of cytokines and chemokines were quantified by densitometer. The cytokines and chemokines detected by the compounds of the present invention include G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40 / 70, MIG, TNF-α, and VCAM-1. Compared with untreated samples, when cells were treated with the compounds of the present invention, the expression of the above cytokines and chemokines induced by LPS treatment was suppressed to a statistically significant level. Figure 2 ).

[0266] RAW264.7 macrophages were treated with different concentrations of compound 1.1 from 50 pM to 500 nM for 30 min, followed by treatment with 100 ng / mL LPS for 2 h. The induction of interleukin-6 expression was quantified as described above, and the results were as follows: Figure 3 As shown in the figure. In RAW264.7 macrophages, when interleukin-6 expression was suppressed to 50% or less (i.e., IC50), 50 In the case of ), the concentration of compound 1.1 is approximately 1.6 nM.

[0267] Example 2.2: Inhibition of NF-κB activity

[0268] To evaluate whether the compounds of the present invention specifically inhibit LPS-induced NF-κB signaling, the following experiments were conducted.

[0269] Specifically, 5x NF-κB-Luc reporter plasmid was transfected into RAW264.7 cells using Effectene (Qiagen, USA). The transfected cells were pretreated with 100 nM compound 1.1 for 30 min and then further treated with LPS (100 ng / ml) for 2 h. Luciferase activity in the cells was measured. Figure 4 A graph depicting the relative inhibitory activities of compounds 1.1 and 1.2 on NF-κB activation is shown. When cells were treated with compounds 1.1 and 1.2 according to the invention, LPS-induced NF-κB activation was inhibited. Figure 4 ).

[0270] In addition, the 5xNF-κB-Luc reporter plasmid was transfected into RAW264.7 macrophages and treated with the compounds of the present invention as described above. Twenty-four hours after transfection, cells were pretreated for 30 minutes with different concentrations of compound 1.1 (100 pM, 1 nM, and 100 nM), reference compound 1 (a compound from which palmitic acid has been removed), and smaducin-6, followed by treatment with 100 ng / mL LPS for 2 hours. Luciferase activity in the cells was measured, and the results are shown in Figure 5.

[0271] like Figure 5 As shown, when cells were pretreated with compound 1.1 (similar to cells pretreated with smaducin-6), the inhibition of LPS-induced NF-κB activity increased with increasing dose of compound 1.1. In contrast, when cells were pretreated with DMSO or reference compound 1, NF-κB activity was not inhibited in a dose-dependent manner.

[0272] Example 2.3: Signal Path Selectivity

[0273] To evaluate the signaling pathway selectivity of compound 1.1 according to the present invention, the following experiments were performed. The experiments were conducted to test whether compound 1.1 specifically inhibits signaling pathways induced by individual inducers.

[0274] Specifically, 5x NF-κB-Luc reporter plasmid, SBE-Luc reporter plasmid, and BRE-Luc reporter plasmid were transfected into Raw264.7 macrophages. After 24 hours, cells transfected with NF-κB-Luc reporter plasmid were treated with 100 ng / mL LPS, cells transfected with SBE-Luc reporter plasmid were treated with 5 ng / mL TFG-β1, and cells transfected with BRE-Luc reporter plasmid were treated with 100 ng / mL BMP6 for 2 hours.

[0275] Figure 6 This is a graph showing the relative inhibitory activities of compound 1.1 on different signaling pathways. (See figure.) Figure 6 As shown, when cells are treated with compound 1.1 according to the invention, LPS treatment-induced NF-κB activation is inhibited. However, BMP6 treatment-induced BRE activation or TFG-β1 treatment-induced SBE activation is not inhibited by compound 1.1. Therefore, compound 1.1 selectively inhibits the activation of the NF-κB signaling pathway.

[0276] Specifically, recent studies have reported that TFG-β and BMP signaling pathways are specifically associated with mucosal wound healing and other aspects in the treatment of inflammatory bowel disease (Nature 449(2007), 361-365, Am J Path, 162(2), (2003), Nature Immunol. 6, (2005), 507-514, J Cell Physiol. 196(2): (2003); 258-64) and Nature Protocols, 8(3), (2013) 627-637, which are incorporated herein by reference), and that TFG-β may be a very important factor in controlling the inflammatory state in the intestine (dendritic cell regulation) (J. Clin. Invest., 111(2003), 1297-1308, Immunity, 10(1999). References include 39-49, Eur. J. Immunol., 36 (2006), 864-874, Immunity, 25 (2006), 319-329, Cell 118 (2004), 229-241, and J. Immunol. 179 (2007), 2690-2694, which are incorporated herein by reference. Therefore, the compounds of the present invention do not inhibit the activation of the TFG-β and BMP signaling pathways, indicating that these compounds have a significant therapeutic effect on inflammatory bowel disease.

[0277] Example 2.4: Disruption of signaling complex formation and degradation of IκB inhibitor (IκB)

[0278] To test whether the compounds of the present invention can disrupt the formation of protein complexes in inflammatory signaling pathways mediated by MyD88 and / or RIP1 (e.g., Toll-like receptor (TLR) signaling pathway protein complexes), an immunoprecipitation assay was performed. Simultaneously, the following experiment was performed to measure the degradation of IκB due to changes in the concentration of measured IκB.

[0279] Specifically, the disruption of the formation of inflammatory signaling pathway protein complexes was confirmed by immunoprecipitation using antibodies corresponding to proteins involved in the formation of Toll-like receptor (TLR) signaling pathway protein complexes (e.g., IRAK1, TRAF6, MyD88, RIP1, and Pellino-1). As a control, the relative expression levels of β-actin from total cell lysates were compared and presented using Western blotting.

[0280] RAW264.7 macrophages were retreated with compounds 1.1, 1.2, and smaducin-6, respectively, and further treated with LPS. These RAW264.7 macrophages were collected and lysed in lysis buffer (PBS containing 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 2 mM MTT, 1 mM Na3O4V, 2 mM EGTA, and 1 mM protease inhibitor (PMSF)) and centrifuged at 13000 rpm for 10 minutes. For immunoprecipitation assays, the supernatant was incubated at 4°C for 12 hours with protein A agarose beads and the antibody corresponding to the protein, followed by washing the beads three times with lysis buffer. The immunoprecipitated material was dissociated from the beads by adding 2x sample buffer and boiling. The prepared sample was loaded onto an SDS-polyacrylamide gel (SDS-polyacrylamide gel). Figure 7 ).

[0281] To measure changes in IκB concentration, RAW264.7 macrophages were pretreated with compounds 1.1, 1.2, and smaducin-6 at 100 nM, followed by LPS treatment. Extracted cell lysates were used for Western blotting with an IκBα antibody, using β-actin as a reference. Figure 7 ).

[0282] Figure 7 Immunoprecipitation images showing the disruption of the MyD88 or RIP1-mediated inflammatory signaling pathway protein complex by compounds 1.1 and 1.2 are presented, further demonstrating the changes in IκB concentration induced by these compounds. When cells were treated with the compounds according to the invention, the disruption of the formation of the signaling pathway protein complex (mediated by MyD88 or RIP1) was verified compared to a control of total cell lysate (Figure 7). Cells treated with the compounds of the invention were stabilized by dephosphorylation of IκB compared to a β-actin reference expression level.

[0283] When cells were treated with the compounds of the present invention, the formation and activity of the MyD88 protein complex and the RIP1 protein complex were substantially disrupted and inhibited compared with the control of total cell lysate. Figure 7These results indicate that the compounds of the present invention can be used to treat diseases related to Pellino-1. In addition to the aforementioned inflammatory bowel disease, the compounds of the present invention can effectively prevent or treat geographic atrophy, wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD), diabetic retinopathy, multiple sclerosis (MS), lung inflammation, bacterial pneumonia, viral pneumonia, diffuse large B-cell lymphoma (DLBCL, GCB type or ABC type), and hair loss (Journal of Clinical Investigation, 124(11)(2014), 4976-4988, J Virology, 86(12), (2012), 6595-6604, Nature Medicine, 19(5), (2013), 595-602, J. Immunol., 187(2011)1-14, J. Ind. Derm., 132(2012), 43-49, Med. Intlamm., (2010), Article ID 928030, Hair The Transplant, 4(2014), 4:1, Exp. Derm., 17(2007), 12-19 and DDT Dis. Mech. 5(2009), e163-171, which are incorporated herein by reference.

[0284] Myeloid-derived macrophages (BMDM) cells were pretreated with various concentrations of compound 1.1, reference compound 1, and smaducin-6 at 100 pM, 1 nM, and 100 nM, respectively, followed by further treatment with 100 ng / mL LPS. The extracted cell lysates were used for Western blotting as described above, and the results are shown in Figure 8.

[0285] Compared with cells pretreated with smaducin-6, in cells pretreated with compound 1.1, the formation of the MyD88 and RIP1-mediated inflammatory signaling pathway complex (e.g., Toll-like receptor (TLR)) was also disrupted in a dose-dependent manner with increased concentrations of compound 1.1. Figure 8 Simultaneously, cells pretreated with DMSO or reference compound 1 formed an inflammatory signaling pathway complex mediated by MyD88 and RIP1 in a dose-dependent manner.

[0286] Similarly, compared to the control of total cell lysates, the formation of the MyD88 and RIP1-mediated inflammatory signaling pathway complex was substantially disrupted when cells were pretreated with compound 1.1 of the present invention. Experiments using BMDM cells with the compounds of the present invention demonstrated that the compounds of the present invention can effectively prevent and treat various diseases associated with MyD88, prevent and treat diseases related to Pellino-1 expression, such as viral infections (respiratory viral infections, viral pneumonia), bacterial pneumonia, autoimmune diseases, blood cancers including lymphoma, tumors in various internal organs (e.g., liver, lung, intestine, prostate, pancreas, etc.), and prevent and treat multiple sclerosis (MS).

[0287] like Figure 9 As shown, RAW 264.7 macrophages (top) and BMDM cells (bottom) were pretreated with various concentrations of compound 1.1 (100 pM, 1 nM, and 100 nM), reference compound 1, and smaducine-6, respectively, followed by further treatment with 100 ng / ml LPS. The results were compared with reference β-actin expression. With increasing doses of pretreatment compound 1.1 and smaducin-6, IκB expression in cells increased. Simultaneously, because IκB is shown to be phosphorylated, it was degraded in cells pretreated with DMSO or reference compound 1. Therefore, compound 1.1 of the present invention inhibits IκB degradation.

[0288] Example 2.5: Evaluating the correlation between dose and disease activity index

[0289] To evaluate the disease activity index of the compounds of the present invention in an animal model of chronic colitis induced by sodium dextran sulfate (DSS), the following experiments were conducted.

[0290] Mice (7-8 weeks old, female, C57BL / 6) were fed 2% DDS polymer (approximately 50,000 Da MW) in drinking water for 5 to 7 days, inducing colitis every 2 to 15 days. Then, starting from day 3 after DDS administration, compound 1.1 was orally administered to mice already suffering from DDS-induced colitis at doses of 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg daily for 11 days. Mouse weight, diarrhea, and fecal hematochezia were examined daily, and the disease activity index was measured. Figure 10 ).

[0291] Figure 10 This is a graph showing the disease activity index scores in an animal model of DSS-induced chronic colitis based on orally administered doses of compounds 1.1 and 1.2. Figure 10As shown, when compound 1.1 of the present invention is administered at different doses ranging from 50 mg / kg to 400 mg / kg, the disease activity index of compound 1.1 of the present invention decreases with increasing dose, and the decrease in the disease activity index saturates at a dose of 200 mg / kg. Therefore, the compounds of the present invention increase their activity proportionally in a dose-dependent manner.

[0292] Example 2.6: Inhibition of enteropathy activity

[0293] To evaluate the inhibitory activity of the compounds of the present invention in an animal model of inducible acute colitis (induced by DSS), the following experiments were conducted.

[0294] Mice (7-8 weeks old, female, C57BL / 6) were fed 2% DSS polymer (approximately 50,000 Da MW) in drinking water for 7-8 days to induce colitis. Then, sulfasalazine was administered daily at 500 mg / kg and compound 1.1 at 100 mg / kg for 14 days. Mouse weight, diarrhea, and bloody stools were examined daily, and the Disease Activity Index (DAI) was measured. Figure 11A ).

[0295] DAI measurements are as follows:

[0296] 1) Weight loss (0 points: no weight loss; 1 point: 1-5% weight loss; 2 points: 6-10% weight loss; 3 points: 11-20% weight loss; 4 points: more than 20% weight loss);

[0297] 2) Diarrhea (0 points: normal stool; 2 points: loose stool; 4 points: diarrhea); and

[0298] 3) Blood in stool (0 points: normal stool; 2 points: mild blood in stool; 4 points: severe blood in stool).

[0299] Figure 11A This is a graph representing the disease activity index score of the compounds administered in an animal model of DSS-induced acute colitis. Compared to the anti-inflammatory sulfasalazine, the compounds of the present invention exhibit sufficient disease activity index scores at reduced doses, and therefore are more effective in treating colitis. Figure 11A ).

[0300] At the same time, such as Figure 11BAs shown, mice with DSS-induced chronic colitis were orally administered sulfasalazine at a dose of 500 mg / kg and compound 1.1 at a dose of 100 mg / kg for 10 days. After 10 days of administration, colonic tissue was obtained from the mice, and the expression of chemokines (CCL20, CCL2, and CX3CL1) in the tissue was measured using real-time polymerase chain reaction (real-time PCR) as described above. Compound 1.1 is a potent chemokine blocker that inhibits the chemotaxis of pathogenic immune cells towards inflamed tissue.

[0301] Example 2.7: Histological analysis of colonic villi

[0302] To confirm the therapeutic effect of compound 1.1 of the present invention on DDS-induced chronic colitis, images were taken of the colic villi from the untreated group, the DDS-induced chronic colitis model group, and the group treated with compound 1.1. Figures 12 to 16 ).

[0303] Figures 12 to 14 Images are shown of colic villi from the untreated group, the DDS-induced chronic colitis model group, and the group treated with compound 1.1. Figure 12 to Figure 14 As shown, the villi of the group treated with compound 1.1 were similar to those of the untreated group compared to the villi of the DSS-induced chronic colitis model group.

[0304] Figure 15 Images of colon tissue obtained from the untreated group, the DDS-induced chronic colitis model group, the group treated with compound 1.1 (100 mpk), and the group treated with sulfasalazine (500 mpk), an anti-inflammatory drug used to treat colitis, are shown. Figure 16 Images of the colonic mucosa morphology obtained from the untreated group, the DDS-induced chronic colitis model group, the group treated with compound 1.1 (100 mpk), and the group treated with sulfasalazine (500 mpk), an anti-inflammatory drug used for the treatment of colitis, are shown, and the mucosa was stained with Alcian blue. Figures 15 to 16 As shown, treatment of a DSS-induced chronic colitis model with compound 1.1 reduced the histological damage that could be found in inflamed tissue. Furthermore, compared to the untreated group and the group treated with sulfasalazine, the group treated with compound 1.1 exhibited greater mucosal recovery by blocking the entry of inflamed cells into the tissue.

[0305] Figure 17This is a graph showing the recovery level of the colonic wall in the untreated group, the DDS-induced chronic colitis model group, the treatment group using compound 1.1 (100 mpk), and the treatment group using sulfasalazine (500 mpk).

[0306] Specifically, as described in Nature Protocols, 8(3), (2013)627-637, FITC-glucan was orally administered at a dose of 44 mg per 100 g body weight on day 8 of treatment in a DDS-induced chronic colitis model. Four hours after administration, blood was collected from the heart of the mice in a volume of 300-400 μL. The amount of FITC-glucan released in the blood was measured using a spectrophotometer.

[0307] Figure 17 Quantitative analysis of FITC-glucan released from the blood revealed that the restoration of tight junctions or intestinal wall function caused by embolism of the colonic mucosa was comparable to that achieved with current sulfasalazine treatment. Therefore, the compounds of this invention have a significant effect on the restoration of intestinal epithelial barrier and tight junction function in chronic colitis tissues.

[0308] Example 2.8: Blood drug concentration

[0309] To evaluate the changes in blood concentration of compound 1.1 of the present invention after intravenous and oral administration, the following experiment was conducted.

[0310] Compound 1.1 was administered intravenously (5 mg / kg) orally (50 mg / kg) to mice. Plasma concentrations of compound 1.1 were measured 24 hours after administration. Figure 18 and Figure 19 ).

[0311] The concentration changes of compound 1.1 at different time intervals during intravenous administration were measured. Figure 18 The concentration decreased to 1 / 100 of its original concentration within 1-2 hours after administration. Very low concentrations of compound 1.1 were detected in the blood.

[0312] The changes in plasma concentration of compound 1.1 at different time points during oral administration were measured. Figure 19 Compound 1.1 was not detected in the blood 30 minutes after oral administration.

[0313] Table 2 shows the pharmacokinetic parameters of compound 1.1.

[0314] Table 2: Pharmacokinetic parameters of compound 1.1

[0315]

[0316] Example 2.9: Organizational Distribution

[0317] To evaluate the tissue distribution of compound 1.1 according to the present invention, the following experiments were performed.

[0318] Compound 1.1 was orally administered to rats at a dose of 10 mg / kg. The concentrations of compound 1.1 were determined in small intestinal tissue, large intestinal tissue, appendix tissue, retrimentum in the small intestine, and retrimentum in the large intestine 2 and 8 hours later. The results are shown in Table 3 below.

[0319] Table 3: Concentration of compound 1.1 after oral administration

[0320]

[0321]

[0322] ND: Not detected

[0323] Quantitative range: Intestinal 8-2000 ng / kg, Retrimentum 30-1000 ng / mL

[0324] As shown in Table 3, compound 1.1 was distributed in quantitative ranges in intestinal tissues such as the small intestine, large intestine and appendix at 2 hours and 8 hours after application, and compound 1.1 was also distributed in internal tissues at 8 hours after application.

[0325] Therefore, when administered orally, the effective concentration of the compound of the present invention is maintained continuously in the intestinal tissue even after 8 hours. Because the compound of the present invention is a small molecule drug, it can be readily absorbed into the intestine, thereby easily achieving its effective concentration. Therefore, by oral administration, the compound described herein can be used to effectively treat inflammatory bowel disease.

[0326] Example 2.10: Inhibition of MAPK signaling pathway activation

[0327] To test whether compounds 1.1 and 1.2 of the present invention inhibit the MAPK / ERK signaling pathway, Western blot analysis was performed (Figure 20).

[0328] RAW246.7 macrophages were pretreated with compounds 1.1, 1.2, and reference compound 1 at a concentration of 100 nM for 30 min, followed by further treatment with LPS for 0 h, 0.5 h, 1 h, and 2 h. Phosphorylation of MAPK signaling pathway proteins (ERK1 / 2, JNK, p38) from cells treated with compounds 1.1 and 1.2 was activated at 0.5 h in the same pattern as with reference compound 1, and gradually decreased at 1 h or 2 h in the same pattern as with reference compound 1. β-actin was used as a control. Changes in phosphorylation concentration of each protein were measured by immunoblotting using an antiphosphorylation antibody. Reference compounds 1, 1.1, and 1.2 did not inhibit phosphorylation of MAPK signaling pathway proteins.

[0329] Figure 21 Images showing the inhibition of phosphorylation of MAPK signaling pathway proteins (ERK1 / 2, JNK, p38) are presented. BMDM cells were pretreated with different concentrations of DMSO, reference compound 1, compound 1.1, and smaducin-6 at 100 pM, 1 nM, and 100 nM, followed by further treatment with LPS for 2 hours. Western blot analysis was then performed. Compound 1.1 according to the invention is similar to DMSO and reference compound 1; despite increased doses, compound 1.1 was not associated with inhibition of phosphorylation of MAPK / ERK signaling pathway proteins.

[0330] Example 2.11: Comparison with IRAK1 / 4 inhibitors

[0331] As described in Example 2.3, 5x NF-κB-Luc reporter plasmid was transfected into RAW246.7 macrophages. After 24 hours, the cells transfected with the NF-κB-Luc reporter plasmid were pretreated for 30 minutes with compound 1.1 (100 nM), an interleukin-1 receptor-associated kinase-1 / 4 (IRAK1 / 4) inhibitor (25 μM, CAS 509093-47-4), and smaducin-6 (100 nM), followed by a further 2 hours of treatment with LPS (100 ng / ml). Subsequently, luciferase activity in the cells was measured.

[0332] Figure 22A Compound 1.1 and the IRAK1 / 4 inhibitor are shown. Figure 22B Inhibition of NF-κB activation. At high concentrations of IRAK1 / 4 inhibitor (e.g., 25 μM), NF-κB activation was inhibited, similar to that of compound 1.1.

[0333] The changes in IκB concentration in the cell lysate of RAW246.7 macrophages were measured (Figure 23). RAW246.7 macrophages were pretreated with compound 1.1 (100 nM), an IRAK1 / 4 inhibitor (25 μM), and smaducin-6 (100 nM) and then further treated with LPS (100 ng / ml). Similar to compound 1.1, the IRAK1 / 4 inhibitor also inhibited IκB degradation (Figure 23).

[0334] Figure 24 shows a graph and image comparing the inhibition of the MAPK / ERK signaling pathway by compound 1.1 and the IRAK1 / 4 inhibitor. Specifically, RAW246.7 macrophages were pretreated for 30 minutes with DMSO, compound 1.1 (100 nM), the IRAK1 / 4 inhibitor (25 μM), and smaducin-6 (100 nM), followed by further treatment with LPS (100 ng / ml) for two hours. Figure 24A Specifically, immunoblotting results are shown to evaluate whether phosphorylation of MAPK signaling pathway proteins (such as ERK, JNK, and p38) was inhibited by the above treatment. β-actin was used as a reference. Compounds 1.1 and smaducin-6 did not inhibit either the MAPK signaling pathway or protein phosphorylation. In contrast, IRAK1 / 4 inhibitors inhibited at least one or more MAPK signaling pathways.

[0335] In addition, the following experiments were performed to examine for undesirable side effects (e.g., the fact that the IRAK1 / 4 inhibitor inhibits AP-1 transcriptional signaling in the LPS-induced MAPK pathway, which is the opposite of compound 1.1).

[0336] As described in Example 2.3, RAW246.7 macrophages were transfected with the AP-1-Luc reporter plasmid. Twenty-four hours later, the transfected cells with the AP-1-Luc reporter plasmid were pretreated for 30 minutes with compound 1.1 (100 nM), an IRAK1 / 4 inhibitor (25 μM), and smaducin-6 (100 nM), followed by a further treatment with LPS (100 ng / ml) for 2 hours. Luciferase activity in the cells was measured, and the results are shown in Figure 24B. Compound 1.1 of the present invention selectively inhibits the activation of the NF-κB signaling pathway but does not inhibit the MAPK signaling pathway, which is important for cellular biological activity. However, the IRAK1 / 4 inhibitor inhibits the MAPK signaling pathway, which leads to the inhibition of the AP-1 transcription factor, resulting in undesirable side effects when applied to biological subjects.

[0337] The results in Examples 2.1 to 2.11 above indicate that the compounds of the present invention can (1) inhibit LPS-induced expression of interleukin-6 and NF-κB activity; (2) disrupt the inflammatory signaling pathway mediated by MyD88 and RIP1; and (3) provide a similar disease activity index at a lower dose than that of sulfasalazine, a current anti-inflammatory drug for colitis. Furthermore, when the compounds of the present invention are administered orally, blood concentrations are low, while effective concentrations are maintained in cells and / or tissues. Specifically, in intestinal tissues, effective concentrations are maintained even 8 hours after administration. Therefore, the compounds of the present invention are intended for the treatment of inflammatory diseases in intestinal tissues, and specifically, are effective for the prevention, relief, and treatment of inflammatory bowel diseases such as ulcerative colitis, Behcet's disease, and Crohn's disease.

[0338] Example 2.12: Effects on retinal pigment epithelial cells

[0339] Compound 1.1 affects angiogenesis-related factors or inhibitory factors.

[0340] ARPE-19 cells were treated with 5.5 mM glucose as a control. For the experimental group, ARPE-19 cells were treated with 30 mM glucose for 48 hours to induce a hyperglycemic state, and simultaneously treated with DMSO, compound 1.1 (10 nM), and compound 1.1 (50 nM). Changes in the expression of Nox-4, VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR proteins were measured by Western blot analysis.

[0341] Ang1 and Tie-2, which control bleeding by enhancing blood vessels, increase with increasing concentration of compound 1.1. Figure 25A However, treatment with compound 1.1 reduced the expression of Nox4, a factor that produces ROS (reactive oxygen species), VEGF, VEGFR1, VEGFR2, Ang2, an Ang1 antagonist, and EPO and EPOR, factors that contribute to diabetic retinopathy.

[0342] In addition, HRMEC cells were treated with 5.5 mM glucose as a control. For the experimental group, HRMEC cells were treated with 30 mM glucose for 48 hours to induce a hyperglycemic state, and simultaneously treated with DMSO, compound 1.1 (10 nM), and compound 1.1 (50 nM). The expression changes of Nox-4, VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR proteins were quantitatively determined by qRT-PCR (quantitative RT-PCR). Treatment with compound 1.1 was used to reduce the expression of VEGF precursors, which are stimulators of angiogenesis. Figure 25B ).

[0343] To evaluate the effect of compound 1.1 on tube formation during angiogenesis, HRMEC cells (8 × 10³) were cultured on matrix gel-coated microslides and treated with 20 ng / ml VEGF for 4 h to induce tube formation. Simultaneously, the control group was treated with DMSO, and the experimental group was treated with 50 nM compound 1.1 and 1 μM calcein AM. Cells were observed using a fluorescence microscope.

[0344] Figure 26 Images of tube formation in cells observed using a fluorescence microscope are shown, demonstrating that compound 1.1 inhibits tube formation. Indeed, in ARPE-19 cells of human retinal pigment epithelium, compound 1.1 was observed to inhibit the expression of Nox-4, VEGF, VEGFR1, VEGFR2, Ang2, EPO, and EPOR proteins according to its concentration gradient, and the expression of Ang1 and Tie-2 was increased. Thus, compound 1.1 is effective for the prevention, relief, or treatment of ophthalmic indications such as diabetic retinopathy. Furthermore, by disrupting the formation of the Myd88 signaling pathway complex, compound 1.1 is effective for the prevention, relief, or treatment of geographic atrophy, wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD), etc. (Cell. 149(4), (2012); 847-859).

[0345] Example 2.13: Effects on diabetic retinopathy

[0346] Mice were administered streptozotocin (STZ) daily at a dose of 50 mg / kg for 5 days to obtain a mouse model of induced diabetic retinopathy. From day 20 to day 24 of administration, the experimental group of mice was injected into one eye three times at two-day intervals with a dose of 0.2 μg. After 50 days of administration, DR samples untreated with compound 1.1 and DR samples treated with compound 1.1 were collected.

[0347] Retinal tissue collected from each mouse group was stained with 5 μM ethyl dithioester as described above, and the reactive oxygen species rate was measured for each group. Figure 27B Compared to untreated DR samples, DR samples injected with compound 1.1 showed a reduced reactive oxygen species rate. Therefore, compound 1.1 of the present invention can be effectively used for the prevention, relief, or treatment of ocular indications such as diabetic retinopathy from biological experiments using mice.

[0348] Example 2.14: Effects on multiple sclerosis

[0349] To confirm the effect of compound 1.1 on multiple sclerosis, mice (10 weeks old, female) were sensitized with MOG35-55 / CFU and PTX to obtain an experimental autoimmune encephalomyelitis mouse model. The experimental methods are described by reference in Oncotarget, Vol. 7 (2016) No. 13, 15382-15393, which is incorporated herein by reference.

[0350] like Figure 28A As shown, compound 1.1 was administered subcutaneously at doses of 1 mg / kg and 40 mg / kg for 25 days, every other day. As a control group, 10,000 units of interferon-β were administered subcutaneously for 25 days, every other day. Clinical scores were measured and presented. Therefore, compound 1.1 showed efficacy in treating multiple sclerosis at a minimum dose of 1 mg / kg compared to interferon-β, a conventional drug currently used for the treatment of multiple sclerosis.

[0351] Furthermore, when weight change was measured in each experimental group, the group treated with compound 1.1 did not experience weight loss compared to the EAE disease model group. Figure 28B In other words, compound 1.1 exhibits the same or higher therapeutic efficacy and safety compared to the main drugs currently used to treat multiple sclerosis (e.g., interferon-β), and is therefore effective for the prevention, mitigation or treatment of multiple sclerosis in experimental autoimmune encephalomyelitis models.

[0352] Example 2.15: Effects on sepsis

[0353] To confirm the effect of compound 1.1 on sepsis, 10 mice in each group (7 weeks old, male) were anesthetized and the appendix was exposed via abdominal incision. The lower part of the ileocecal valve of the exposed appendix was tied together and a single hole was made using a 22-gauge syringe needle. The treated appendix was reinserted into the abdominal cavity and sutured to obtain a mouse model of sepsis-induced sepsis (cecal ligation and puncture model, CLP model). The experimental methods are described by reference in EMBO Mol Med. (2015) Mar 12; 7(5): 577-92, incorporated herein by reference.

[0354] Two hours after the CLP step, treated mice were subcutaneously injected with compound 1.1 at a dose of 1 mg / kg, three times at 12-hour intervals. As a control, smaducin-6 was subcutaneously injected at a dose of 12 mg / kg using the same method as compound 1.1. Survival rates were measured in each group. Figure 29 Following abdominal incision and anastomosis, the negative control group received no treatment. After preparation of the cecal ligation and puncture model, the CLP+PBS control group was treated with phosphate-buffered saline (PBS) instead of the drug compound, receiving alternative drug therapy. Therefore, compound 1.1 (1 mg / kg) was effective in treating sepsis with a 60% survival rate, compared to high doses of smadudin-6 (e.g., 12 mg / kg) in patients who had not previously received low doses of conventional drugs for sepsis. In other words, compound 1.1 was effective in the cecal ligation and puncture (CLP) model, indicating that compound 1.1 is effective for the prevention, relief, or treatment of sepsis.

[0355] <Formulation 1> Granules

[0356] 2g of compound of formula 1

[0357] 1g lactose

[0358] Particles are prepared according to methods known in the art.

[0359] <Formulation 2> Tablets

[0360]

[0361] Tablets are prepared according to methods known in the art.

[0362] <Formulation 3> Capsules

[0363]

[0364]

[0365] Capsules are prepared according to methods known in the art.

[0366] <Formulation 4> Injection

[0367]

[0368] The injection is prepared according to methods known in the art.

[0369] References

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[0410] All publications cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference, as if each individual publication were specifically and independently identified by reference, as fully elucidated.

Claims

1. A compound represented by Formula 1 below, its optical isomer, or a pharmaceutically acceptable salt thereof, [Formula 1] 。 2. A method for preparing the compound of claim 1 represented by formula 1, said method comprising the steps represented by the reaction formula 1 below: Compound 2 is reacted with compound 3 to prepare compound 4; Compound 4 was hydrolyzed in the presence of a base to prepare compound 5; Compound 5 is reacted with compound 6 to prepare compound 7; Compound 7 was hydrolyzed in the presence of a base to prepare compound 8; Compound 8 is reacted with compound 9 to prepare compound 10; as well as Compound 10 was hydrolyzed in the presence of a base to prepare the compound according to claim 1; [Reaction Formula 1] ； in, n is 1; A is -a 1 -;as well as a 1 yes The a 1 The two ends are bonded to the carbonyl or amino group of the compound via amide bonds; and R 1 It is a straight chain C 15 Alkyl, R 2 For straight or branched C 1-5 alkyl.

3. A composition for the prevention, improvement or treatment of chronic colitis or acute colitis, said composition comprising, as an active ingredient, the compound, optical isomer or salt of claim 1.

4. A composition for preventing, improving, or treating diabetic retinopathy, wherein, The composition comprises, as an active component, the compound, optical isomer, or salt according to claim 1.

5. A composition for the prevention, improvement, or treatment of sepsis or multiple sclerosis, wherein, The composition comprises, as an active component, the compound, optical isomer, or salt according to claim 1.

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