TW202402321A - A formyl peptide receptor 1 antagonist and uses thereof - Google Patents

Abstract

Disclosed herein is related to a novel formyl peptide receptor 1 (FPR1) antagonist having a chemical structure of formula (I), and its uses in manufacturing a medicament for the treatment and/or prophylaxis of diseases mediated by FPR1 (e.g., acute respiratory distress syndrome, ARDS).

Description

A type I methacrylate receptor antagonist and its use

This disclosure generally relates to the technical field of lung disease treatment, and specifically relates to a type I formyl peptide receptor (formyl peptide receptor 1, FPR1) antagonist. This disclosure also covers the following The antagonist is used for preparing a medicament that can treat and/or prevent diseases mediated by FPR1.

In recent years, acute respiratory distress syndrome (ARDS) has gradually received more attention from the medical community in the field of clinical critical care medicine. Although modern medical standards continue to improve, and medical equipment can provide patients with critical respiratory conditions with the most advanced Oxygen supply equipment, artificial respirators, and drugs are used for treatment. However, the mortality rate of ARDS patients is still high, which can be as high as about 40-50%. At the same time, patients are admitted to intensive care due to factors such as respiratory failure and multiple organ failure. In the ward, considerable medical manpower and medical resources are often consumed, so ARDS is a very difficult disease in the field of critical care medicine. At present, there is no effective treatment for ARDS. The current treatment is still mainly to provide supportive therapy, including the use of artificial respirators, hemodialysis to treat organ failure, or the administration of steroids, etc., the efficacy of which is very limited.

ARDS was first reported in 1967. Patients have some common clinical features, including: acute hypoxic respiratory failure. Chest X-ray examination will show severe infiltration of both lungs. Although the patient's hypoxic condition is not suitable for artificial respirators, The positive end-expiratory pressure (PEEP) treatment used will respond, but the mortality rate is very high. ARDS can have different causes, such as severe lung infection (pneumonia) or systemic infection (sepsis), trauma, multiple blood transfusions, severe burns, severe pancreatitis, following drowning or other inhalation events, drug reactions, or The large amounts of fluid used during resuscitation after pulmonary trauma may induce lung injury, leading to ARDS. In the microscopic pathological examination, in the early stage of ARDS, obvious macrophage and neutrophil infiltration can be seen in the lung tissue. In the middle and late stages of ARDS, some cells in the lung (such as type II alveolar cells, fibromuscular cells, etc.) cells, and immune cells, etc.), which will lead to fibrosis in the lungs and the loss of gas exchange function in the lungs, making the patient unable to leave the respirator. Generally speaking, ARDS often occurs within 72 hours after an acute lung injury event. At this time, although the direct damage to the lungs may have disappeared, activated white blood cells continue to destroy lung tissue.

On the surface of these activated white blood cells (including macrophages, neutrophils, eosinophils, and basophils, etc.), a group of receptors called formyl peptide receptors (FPR) can be found. ) molecules are overexpressed. The FPR can recognize N -formyl peptide, which is a G protein-coupled receptor (GPCR). Human FPR can be roughly divided into three types: type I FPR (FPR1), type II FPR (FPR2), and type III FPR (FPR3). Among them, FPR1 has received relatively much attention. It has been reported that FPR1 It is an important regulatory factor in the recruitment of neutrophils and the generation of pulmonary fibrosis. Therefore, a suitable FPR1 antagonist is considered to have the potential to be developed into a drug for the treatment of immune-related diseases (such as the above-mentioned ARDS), thereby improving the current treatment of ARDS.

In view of this, there is an urgent need in the relevant field to develop a novel drug that can act through FPR1, so as to develop a safe and effective treatment method for the treatment and/or prevention of immune-related diseases and/or disorders.

This summary is intended to provide a simplified summary of the disclosure to provide the reader with a basic understanding of the disclosure. This summary is not an extensive overview of the disclosure and it is not intended to identify key/critical elements of the embodiments of the invention or to delineate the scope of the invention.

The present disclosure is based, at least in part, on the unexpected discovery by the inventors that a compound (having the structural formula of formula (I)) isolated from a secondary metabolite of marine Bacillus sp. The body molecule FPR1 has a powerful regulatory effect. This compound can compete with an FPR1 ligand (also an FPR1 agonist) for binding to FPR1, thereby antagonizing the FPR1 agonist's message to FPR1. Transmitting signals can then be used to develop a drug that can treat diseases mediated by FPR1, and in particular, can be used to develop a drug that can treat ARDS related to FPR1 mediation.

Accordingly, one aspect of the present disclosure relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is administered in an effective amount for treatment and/or prevent acute respiratory distress syndrome (ARDS) in an individual in need of treatment: Among them, R ₁ is a C _6-12 linear or branched alkyl group.

According to embodiments of the disclosure, this is a C ₁₀ branched alkyl group. According to a specific operating embodiment of the present disclosure, the formula (I) has the chemical structural formula of formula (Ia): (Ia).

According to another specific operational embodiment of the present disclosure, the formula (I) has the chemical structural formula of formula (Ib): (Ib).

As mentioned above, the compound of formula (I) or a pharmaceutically acceptable salt thereof in the present disclosure is an FPR1 antagonist. According to an embodiment of the present disclosure, the compound of formula (I) of the present disclosure is a biologically active secondary metabolite extracted from a marine Bacillus species.

According to the preferred embodiment of the present disclosure, the effective amount of the compound of formula (I) or its pharmaceutically acceptable salts is about 0.01-100 mg/kg; more preferably, the compound of formula (I) or The effective amount of pharmaceutically acceptable salts is about 0.1-1 mg/kg.

According to a preferred embodiment disclosed in the present invention, the individual in need of treatment is a human being.

After referring to the following embodiments, those with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other objectives of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The embodiments cover features of multiple specific embodiments as well as method steps and their sequences for constructing and operating these specific embodiments. However, other specific embodiments may also be used to achieve the same or equivalent functions and step sequences.

I. definition

For convenience, specific terminology used in the specification, examples, and appended claims are concentrated here. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as commonly understood and customary by a person of ordinary skill in the art to which this invention belongs. Furthermore, unless there is any conflict with the context, the singular noun used in this specification shall include the plural form of the noun, and the plural noun used shall also include the singular form of the noun. Specifically, in this specification and claims, the singular form "a" (a, an, and the) includes plural references unless the context dictates otherwise. In addition, in this specification and the scope of the patent application, the expressions "at least one" and "one or more" have the same meaning. Both of them represent one, two, three or more. many. Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of molecular biology, synthetic chemistry, structural biology, and immunology, which are within the scope of the art. Such techniques have been described in detail in the open literature.

Notwithstanding that the numerical ranges and parameters defining the broader scope of the invention are approximations, the relevant numerical values in the specific embodiments are presented as precisely as possible. Any numerical value, however, inherently contains the standard deviation resulting from the individual testing methods used. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specified value or range. Alternatively, the word "about" means that the actual value falls within an acceptable standard error of the mean, as determined by a person of ordinary skill in the art to which this invention belongs. Except for experimental examples, or unless otherwise expressly stated, all ranges, quantities, numerical values and percentages used herein (such as to describe the amount of material, length of time, temperature, operating conditions, quantitative proportions and other similar ) are all modified by "approval". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent claims are approximate values and may be changed as required. At a minimum, these numerical parameters should be understood to mean the number of significant digits indicated and the value obtained by applying ordinary rounding. Herein, numerical ranges are expressed from one endpoint to the other point or between two endpoints; unless otherwise stated, numerical ranges stated herein include the endpoints.

The term "an effective amount" as used herein refers to an effective amount that can achieve the desired therapeutic results for cancer treatment within the necessary dose and time. For example, when treating cancer, the recombinant polypeptides, complexes, immune compositions, and methods of the present disclosure will be effective in preventing the spread and/or growth of cancer cells. An effective amount of an agent does not necessarily cure the disease or condition, but will provide treatment for the disease or condition, thereby delaying, hindering, or preventing the onset of the disease or condition, or alleviating the symptoms of the disease or condition. The specific effective or sufficient amount will vary depending on factors such as the specific condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or gender), the mammal or animal being treated type, duration of treatment, nature of concurrent therapies (if any), and specific formulations used. An effective amount may be expressed, for example, as the total mass of the active agent (e.g., in grams, milligrams, or micrograms), or as a ratio of the mass of the active agent to body weight (e.g., in milligrams per kilogram (mg/kg)). unit). An effective amount may be suitably divided into one, two or more doses for one, two or more administrations throughout a specified period. Preferably, an effective dose refers to a human equivalent dose (HED), which is the maximum safe dose for human individuals. The HED can be determined in accordance with the industry guidance "Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteers for Treatment in Initial Clinical Trials" issued by the U.S. Food and Drug Administration (FDA). Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the maximum safe dose for use in human subjects.

Unless otherwise indicated, a "therapeutically effective amount" of a compound means an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease, or to delay or minimize one or more associated symptoms of the disease dosage. A therapeutically effective amount of a compound is an amount of a therapeutic agent that provides a therapeutic benefit when used alone or in combination with other agents that may be used to treat or manage a disease. The term "a therapeutically effective amount" may also refer to an amount sufficient to increase the overall therapeutic effect or reduce or prevent symptoms, or to increase the efficacy of other therapeutic agents.

Unless otherwise indicated, "a prophylactically effective amount" of a compound means an amount sufficient to prevent the occurrence or recurrence of a disease or condition, or one or more symptoms associated with the disease or condition. "A prophylactically effective amount" of a compound refers to the amount of a therapeutic agent used alone or in combination with other drugs that can provide preventive benefits by preventing the occurrence of a disease. The term "preventatively effective dose" can also refer to an amount sufficient to increase the overall preventive effect or improve the efficacy of other preventive agents.

The term "treat" (treat, treatment or treating) as used herein may refer to a curative or palliative measure. Specifically, the word "treatment" used herein refers to the compound of formula (I) of the present disclosure or its pharmaceutically acceptable salts, or a pharmaceutical composition or method containing the compound of formula (I). Administered to a subject suffering from ARDS, having symptoms associated with ARDS, or having a disease or condition secondary to ARDS, in order to achieve partial or complete alleviation of one or more symptoms or symptoms of ARDS, The purpose of improving, alleviating, delaying onset, inhibiting disease progression, reducing severity, and/or reducing incidence.

The term "subject" or "patient" refers to a compound of formula (I) or a pharmaceutically acceptable salt thereof in this disclosure, or a drug containing the compound of formula (I). Compositions or methods for treating animals, including human species. The term "individual" or "patient" refers to both males and females unless a gender is specifically indicated. Accordingly, the term "individual" or "patient" includes any mammal that may benefit from treatment for ARDS. Illustrative "individuals" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. In an exemplary embodiment, the individual is a mouse. In another exemplary embodiment, the individual is a human.

The term "administered" (administered, administratoring or administration) is used interchangeably in this disclosure to refer to the administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound containing the formula (I). I) A pharmaceutical composition or method of administering a compound to an individual in need thereof.

II. Detailed description of the invention

Accordingly, the present disclosure relates to a compound extracted from a secondary metabolite of a marine Bacillus species, with a chemical structural formula of formula (I). The compound has a powerful regulatory effect on FPR1, and it promotes the regulation of FPR1 by interacting with FPR1. The agonist competes for binding with FPR1, thereby blocking the activation of FPR1 by the FPR1 agonist, thereby achieving the purpose of inhibiting FPR1 activation, and can then be used to treat diseases related to FPR1 activation, especially ARDS related to FPR1 activation. Therefore, the compounds of the present disclosure are potential FPR1 antagonists and can be used to develop lead compounds that can treat or prevent diseases and/or disorders (eg, ARDS) mediated via FPR1.

1. Treatment

Accordingly, one aspect of the present disclosure relates to a method for treating and/or preventing an individual suffering from, or suspected of suffering from, a disease and/or disorder mediated via FPR1. The method of the present disclosure includes administering a therapeutically effective amount or a prophylactically effective amount of a compound of formula (I) to the above-mentioned individual, thereby reducing, slowing, and/or preventing symptoms of the disease and/or disorder mediated via FPR1: .

The present disclosure also encompasses a method for treating or preventing diseases and/or disorders mediated via FPR1. The method includes administering to a subject a therapeutically effective amount or a prophylactically effective amount of a compound of formula (I) above, so as to slow, alleviate, and/or prevent the condition of a disease and/or disorder mediated via FPR1.

Specifically, in the above-mentioned compound of formula (I), R ₁ is a straight-chain or branched saturated hydrocarbon group (alkyl) having 6 to 12 carbon atoms ("C _6-12 alkyl" ( C _6-12 alkyl)). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 6 carbon atoms (“C ₆ alkyl”). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 7 carbon atoms (“C ₇ alkyl”). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 8 carbon atoms (“C ₈ alkyl”). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 9 carbon atoms ("C ₉ alkyl"). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 10 carbon atoms (“C ₁₀ alkyl”). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 11 carbon atoms (“C ₁₁ alkyl”). In certain embodiments, _{R 1} is a straight or branched chain alkyl group having 12 carbon atoms (“C ₁₂ alkyl”).

Exemplary C _6-12 alkyl groups include, but are not limited to, hexane (C ₆ , hexane) (for example, n-hexane (n-hexane), 2-methylpentane (2-methylpentane), 3-methyl Pentane (3-methylpentane), 2,3-dimethylbutane (2,3-dimethylbutane), 2,2-dimethylbutane (2,2-dimethylbutane)); heptane (C ₇ , heptane ) (for example, n-heptane (n-heptane), 2-methylhexane (2-methylhexane), 3-methylhexane (3-methylhexane), 2,2-dimethylpentane (2,2 -dimethylpentane), 2,3-dimethylpentane (2,3-dimethylpentane), 2,4-dimethylpentane (2,4-dimethylpentane), 3,3-dimethylpentane (3, 3-dimethylpentane), 3-ethylpentane (3-ethylpentane), 2,2,3-trimethylbutane (2,2,3-trimethylbutane)); octane (C ₈ , octane) (for example, 2-methylheptane (2-methylheptane), 3-methylheptane (3-methylheptane), 4-methylheptane (4-methylheptane), 2,2-dimethylheptane (2,2- dimethylhexane; neo-octane), 3,3-dimethylhexane (3,3-dimethylhexane), 3-ethylhexane (3-ethylhexane), 2,3-dimethylhexane (2,3-dimethylhexane), 2,4-dimethylhexane (2,4-dimethylhexane), 2,5-dimethylhexane (2,5-dimethylhexane), 3,4-dimethylhexane Alkane (3,4-dimethylhexane), 2,3,4-trimethylpentane (2,3,4-trimethylpentane), 2,2,3-trimethylpentane (2,2,3-trimethylpentane) , 2,2,4-trimethylpentane (isooctane), 2,3,3-trimethylpentane (2,3,3-trimethylpentane), 2-methyl-3-ethylpentane (2-methyl-3-ethylpentane), 3-methyl-3-ethylpentane (3-methyl-3-ethylpentane), 2,2,3,3- Tetramethylbutane (2,2,3,3-tetramethylbutane)); nonane (C ₉ , nonane) (for example, n-nonane (n-nonane)); decane (C ₁₀ , decane) (for example, n-decane (n-decane), 2-methylnonane (2-methylnonane), 3-methylnonane (3-methylnonane)); undecane (C ₁₁ , hendecane) (for example, n-undecane (n-hendecane)); or dodecane (C ₁₂ , dodecane) (for example, n-dodecane (n-dodecane)).

According to a specific embodiment of the present disclosure, R ₁ is 3-methylnonane (IA-1), and the compound of formula (I) of the present disclosure has the chemical structural formula of the following formula (Ia): (Ia).

According to another specific embodiment of the present disclosure, R ₁ is 2-methylnonane (IA-2), and the compound of formula (I) of the present disclosure has the chemical structural formula of the following formula (Ib): (Ib).

Unless otherwise indicated, the alkyl groups in each embodiment may be unsubstituted, that is, "unsubstituted alkyl" (unsubstituted alkyl), or substituted with one or more substituents (for example, fluorine, etc. Halogen or -OH) is substituted, that is, "substituted alkyl" (substituted alkyl). In certain embodiments, R ₁ is unsubstituted C _6-12 alkyl (eg, unsubstituted C ₁₀ alkyl). In certain embodiments, R ₁ is substituted C _6-12 alkyl (eg, C ₁₀ alkyl substituted with, for example, -CF ₃ or -CH ₂ OH).

The compound of formula (I) of the present disclosure can be extracted from the secondary metabolite (culture fluid) of a marine Bacillus species by the method disclosed in the operational examples of the present disclosure. In addition, the compound of formula (I) of the present disclosure contains one or more stereocenters, so the compound of formula (I) of the present disclosure may exist in the form of a racemic mixture of mirror image compounds or a mixture of non-mirror image compounds. Accordingly, this disclosure also encompasses such compounds in their pure stereochemical form, as well as mixtures thereof. Stereoisomers can be synthesized asymmetrically or purified by separation from each other using crystallization, chromatography, or resolving agents. A preferred method for separating mirror image compounds is preparative HPLC. Alternatively, the mirror image compounds can be separated from each other by reacting the racemic mixture with an optically active separating agent in the presence of an appropriate solvent. Depending on the optical activity of the separating agent, one of the two mirror image compounds can be separated into a highly optically active insoluble salt, while the other mirror image compound remains in solution.

Accordingly, the present disclosure further encompasses stereo compound mixtures of the compounds of Formula (I); also encompasses configurational isomers (i.e., cis or trans isomers) of the compounds of Formula (I) of the present disclosure, and regardless of which Whether the compound exists in the form of a pure compound or a mixture of stereoisomers is within the scope of this disclosure.

According to an embodiment of the present disclosure, the compound of formula (I) of the present disclosure (including compounds of formula (I-a) or (I-b)) is an FPR1 antagonist, and the disease and/or disorder mediated via FPR1 can be ARDS.

The methods of the present disclosure are also contemplated for use with other known drugs or therapies for the treatment of diseases and/or disorders mediated by FPR1 (e.g., ARDS), that is, by administering the methods of the disclosure to an individual in need of treatment. (I) The compound is administered before, simultaneously with, or after another known agent or therapy useful in the treatment of ARDS. The conventional agent may be a known FPR1 antagonist (for example, cyclosporine A, cyclosporine H, N-(N-aralkyl-L-tryptamine acyl) )-D-phenylalanine (N-(N-aroyl-L-tryptophanyl)-D-phenylalanine), 3,4-dihydroisoquinolin-2(1H)-yl-3-phenylurea (3,4 -dihydroisoquinolin-2(1H)-yl-3-phenylurea), etc.); known ARDS therapeutic drugs (e.g., corticosteroids (e.g., hydrocortisone), triamcinolone acetonide, Dexamethasone , halometasone, etc.), surfactant, N - acetylcysteine, statins (for example, fluvastatin) fluvastatin), atorvastatin (atorvastatin), cerivastatin (cerivastatin), pitavastatin (pitavastatin), simvastatin (simvastatin), etc.), β-agonists (β-agonists) (for example, Doba dobutamine, clenbuterol, salbutamol, methoxyphenamine, etc.); known anti-inflammatory drugs (e.g., alcofenac, aceclofen) Aceclofenac, sulindac, tolmetin, etodolac, fenopren, thiaprofenic acid, meclofenamic acid), meloxicam, tenoxicam, lornoxicam, nabumetone, acetaminophen, phenacetin , ethenzamide, sulpyrine, mefanamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, etc.); the conventional therapy is a respiratory treatment, for example, using a respirator, high frequency Ventilation (high frequency ventilation), extracorporeal membrane oxygenation (extra-corporeal membrane oxygenation), liquid or partial liquid ventilation (liquid or partial liquid ventilation), nitric oxide (nitric oxide) therapy, etc. For example, in the process of treating ARDS, in addition to administering the compound of formula (I) of the disclosure to the individual in need of treatment, the method of the disclosure further includes administering a respiratory therapy to the individual, and/or other Drugs known to treat ARDS.

According to the preferred embodiment disclosed by the present invention, the compound of formula (I) is administered to the individual in need of treatment at a dosage of about 0.01-100 mg/kg, for example, at a dosage of about 0.01, 0.05, 0.1, 0.2, 0.3 ,0.4,0.5,0.6,0.7,0.8,0.9,1.0,2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10,11,12,13,14,15,16,17,18,19 ,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 ,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69 ,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94 , 95, 96, 97, 98, 99, or 100 mg/kg; preferably, it is administered to the individual in need of treatment at a dosage of about 0.1-50 mg/kg, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 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 mg/kg; more preferably, about 0.1-20 mg/kg is administered to the individual in need of treatment, for example, about 0.1, 0.2 , 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 ,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18 , 18.5, 19, 19.5, or 20 mg/kg; more preferably, it is administered to the individual in need of treatment at a dosage of about 0.1-10 mg/kg, such as about 0.1, 0.2, 0.3, 0.4 ,0.5,0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5 , 7, 7.5, 8, 8.5, 9, 9.5, 10, or 10 mg/kg; more preferably, it is administered to the individual in need of treatment at a dosage of about 0.1-1 mg/kg, such as Approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/kg is administered. According to a specific embodiment of the present disclosure, the compound of formula (I) is administered to the individual in need of treatment at a dosage of approximately 0.5 mg/kg. According to another embodiment of the present disclosure, the compound of formula (I) is administered to the individual in need of treatment at a dosage of approximately 1 mg/kg.

Compounds of Formula (I) of the present disclosure may be administered via appropriate routes known to those skilled in the art, such as orally, transmucosally (eg, nasal, sublingual, intrabuccal, intravaginal, intrarectally) or transdermally, or Administration is by injection (e.g., intradermal, subcutaneous, intravenous, intramuscular, intraarterial). In general, the most appropriate route of administration will vary depending on a variety of factors, such as the nature of the drug (e.g., its stability in the circulation), and/or the individual's physiological conditions (e.g., Whether the individual is tolerant to intravenous administration) and other factors.

It is understandable that medical staff can determine the exact dosage of the compound of formula (I) of the present disclosure within the scope of reasonable medical judgment to achieve a certain medical effect. The effective amount administered to a particular individual will depend on various factors, including the disease and severity of the disease being treated; the activity of the particular active ingredient employed; the composition employed; and the species, age, weight, and general health of the individual. , gender and dietary habits, the severity of side effects or drug abnormalities; the time, route of administration and rate of excretion of the active ingredient; the duration of treatment; and other factors commonly known in the medical field. Also, an effective amount may be encompassed in a single dose (eg, a single intravenous injection), or in multiple doses (eg, multiple intravenous injections). When multiple doses are administered to an individual, the frequency with which the multiple doses are administered to the individual may also be adjusted depending on the actual situation. The above factors and adjustment methods are all well known and commonly used by those with ordinary knowledge in this field.

An individual suitable for treatment by the methods of the present disclosure is a mammal, preferably the individual is a human.

2. pharmaceutical composition

The present disclosure also contemplates providing a pharmaceutical composition for treating diseases and/or disorders mediated via FPR1. The pharmaceutical composition contains an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier.

Generally speaking, if the total weight of the pharmaceutical composition is taken as 100%, the compound of formula (I) accounts for approximately 0.01-99% (weight%) of the total weight of the pharmaceutical composition; in certain embodiments, the compound of formula (I) The compound I) accounts for about at least 0.1% (weight%) of the total weight of the pharmaceutical composition. In specific embodiments, the compound of formula (I) accounts for at least 5% (weight%) of the total weight of the pharmaceutical composition; in other In embodiments, the compound of formula (I) accounts for at least 10% (wt%) of the total weight of the pharmaceutical composition; in other embodiments, the compound of formula (I) accounts for at least 25% of the total weight of the pharmaceutical composition. (weight%).

In certain embodiments, the pharmaceutical composition of the present disclosure further includes a known antagonist of FPR1. According to another embodiment of the present invention, the pharmaceutical composition further includes a known ARDS therapeutic drug. According to yet another embodiment of the present invention, the pharmaceutical composition further includes an anti-inflammatory drug.

Pharmaceutically acceptable carriers are those ingredients that are compatible with other ingredients in the pharmaceutical composition formulation and are biologically acceptable. The pharmaceutical composition containing the compound of formula (I) of the present disclosure can be made into a dosage form suitable for oral, transmucosal or transdermal, or injection use. Examples of dosage forms include, but are not limited to, tablets (chewable), lozenge halves, capsules (eg, soft gelatin capsules), cachets, lozenges, suspensions, suppositories, ointments, Ointments, pastes, powders, dressings, plasters, solutions, patches, sprays (e.g., nasal sprays or inhalants), gels; liquid dosage forms suitable for oral or transmucosal use (e.g., suspensions (aqueous) or oily suspension), emulsifier (oil-in-water or water-in-oil emulsifier), solution or elixir); a liquid dosage form suitable for injection; or a sterile solid that can be reconstituted into a liquid dosage form suitable for injection (e.g., crystalline or amorphous solids).

The formulations of the pharmaceutical compositions of the present disclosure are suitable for administration by a variety of different routes. For example, known techniques can be used to coat tablets with a coating (or enteric coating) that can delay disintegration and absorption in the gastrointestinal tract to achieve sustained release over a longer period of time (i.e., long-acting dosage form) . For example, substances that can delay release such as glyceryl monostearate or glyceryl distearate can be added. Or coating the tablet with an appropriate coating to form a penetrating therapeutic agent. Similarly, the compounds of the present invention can also be made into liposomes to prevent them from being destroyed by degrading enzymes, help them to be transported in the circulation system, and to effectively transport drugs across cell membranes and into cells.

Dissolving agents, emulsifiers, surfactants (for example, cyclodextrin (α-cyclodextrin or β-cyclodextrin)), non-aqueous solvents (including, but not limited to, ethanol, isopropanol, carbonic acid, etc.) may also be used. Ethyl ester, ethyl acetate, benzyl alcohol, benzyl benzoate, ethylene glycol, 1,3-butyldiol, dimethylformamide, dimethyl sulfoxide (DMSO), biodegradable Tolerant oils (e.g., cottonseed oil, nut oil, corn oil, olive oil, rapeseed oil, sesame oil), glycerin, tetrahydrofuranol, polyethylene glycol, fatty acid esters of sorbitol, and mixtures thereof (e.g., A mixture of DMSO and sesame oil)) is used to encapsulate poorly water-soluble drugs into liquid dosage forms.

The composition, shape and form of dosage forms vary depending on their route of administration. For example, a dosage form used to treat an acute condition may contain a higher amount of the active agent than a dosage form used to treat a chronic condition; similarly, an injectable dosage form may contain a higher amount of the active agent than an oral dosage form. Smaller amounts of active agent. Such adjustments of drug dosage or carrier in response to different dosage forms are knowledge familiar to those with ordinary knowledge in the art.

2.1 Oral dosage form

Oral dosage forms of pharmaceutical compositions of the present disclosure may be tablets, lozenges, capsules, or liquid (eg, syrup) dosage forms. Such dosage forms contain a predetermined amount of the active ingredient and may be manufactured by methods well known in the art and well documented in the existing published literature. Typical oral dosage forms are manufactured according to traditional pharmaceutical methods by mixing the active ingredient with at least one excipient, where the selection of excipients can be determined according to the preparation form. Since oral administration is the easiest way to take it, tablets and capsules are the most common forms. If necessary, it can be coated with an appropriate coating using standard solution or non-solution coating methods. Generally speaking, it can be manufactured by uniformly mixing the active ingredient with a liquid carrier, a solid carrier, or both, and then shaping them into an appropriate shape. A disintegrating agent can also be added to help it dissolve quickly. Or add lubricant to aid its manufacturing.

2.2 Transmucosal or transdermal dosage forms

Transmucosal or transdermal dosage forms include, but are not limited to, ophthalmic solutions, sprays, creams, lotions, ointments, gels, solutions, emulsions, and other known dosage forms. Excipients useful in transmucosal or transdermal dosage forms are well known in the art. Additionally, other transdermal agents (i.e., agents used to assist in the delivery of active agents across the skin or mucous membranes) may be used concurrently or sequentially, depending on the specific tissue being treated. In addition, the pH value of the pharmaceutical composition, the polarity, ionic strength or tension of the solvent carrier can also be adjusted depending on the tissue to be applied to improve its delivery effect. For example, stearate can be added to change the lipophilicity or lipophilicity of the active ingredient. Hydrophobic to aid drug delivery.

2.3 Injectable dosage form

Injectable dosage forms are generally sterile and include, but are not limited to, ready-to-use injectable solutions, solids or powders that can be readily dissolved in a pharmaceutically acceptable injectable carrier (eg, physiological saline), or emulsions. Pharmaceutically acceptable carriers for injection are well known in the art, including water, isotonic sodium chloride solution, physiological saline, Ringer's solution, and glucose solution; water-miscible carriers, such as ethanol, polyethylene glycol, Alcohol, propylene glycol; non-aqueous solvents, such as corn oil, cotton wool, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, benzoyl benzoate.

3. set

The present disclosure also encompasses a manufacture or kit comprising a compound of Formula (I) of the present disclosure for the treatment or prevention of diseases and/or disorders mediated via FPR1 (eg, ARDS). According to one embodiment, the kit includes a container for loading the compound of formula (I) of the present disclosure. Suitable containers can be bottles, vials, syringes, blister packs, etc., and the container material can be glass or plastic. Alternatively, the set may further include a second container to hold a pharmaceutically acceptable buffer (eg, physiological saline, Ringer's solution, or glucose solution). Other materials necessary from a commercial or usage perspective may also be included, such as other buffers, diluents, fillers, needles or syringes, etc. The kit may also include a label affixed to the container or instructions embedded inside the container.

The kit may also include instructions and instructions to guide users on how to use the compound of formula (I) of the present disclosure, or, if necessary, include instructions for a second method of treating or preventing diseases mediated through FPR1 and/or or unbalanced pharmaceutical formulations. Specifically, if the kit includes a first formula (which includes the compound of formula (I) of the present disclosure) and a second formula, it may further include instructions on how to use the first and third formulas simultaneously, sequentially or separately. Two recipes. According to one embodiment, the kit at least includes: (1) a first container containing a compound of formula (I) of the present disclosure; and optionally (2) a second container containing a second therapeutic agent, It can be a known FPR1 antagonist, ARDS treatment drug, or anti-inflammatory drug; and (3) an instruction manual to guide the user on how to use the kit. The instructions can be in the form of a foldout, video tape, CD, VCD, or DVD.

Multiple experimental examples are provided below to illustrate certain aspects of the present invention to facilitate those with ordinary knowledge in the technical field to which the present invention belongs to implement the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that one skilled in the art, after reading the description set forth herein, can fully utilize and practice the present invention without undue interpretation. The entire texts of all published documents cited here are deemed to be part of this specification.

Example

Materials and methods

1. cell culture

Human peripheral blood samples are separated from human neutrophils through dextran precipitation, Ficoll Paque Plus density gradient centrifugation and red blood cell hypotonic lysis, and then the isolated Human neutrophils were resuspended in Hank's balanced salt solution (HBSS) containing 2% human serum albumin (HSA) and 1 mM calcium, and the cells were counted. And adjust to the required concentration for subsequent experiments.

2. FPR1 combination test

The cell membrane of human neutrophils contains the receptor for fMLF, FPR1, which binds to FPR1 and is fluorescently labeled fNLFNYK to interact with the test substance (IALB-E, IA-1 or IA-2). The competitive combination of entities. Therefore, receptor binding analysis is used to further clarify whether the test substance interacts with the receptor. Human neutrophils (2×10 ⁶ cells/ml) are reacted with a specific concentration of test substance (0-5 micromolar concentration (μM)) or fMLF (10 μM) at 4°C. Approximately 5 minutes, followed by calibration with or without the FPR1-specific ligand fNLFNYK (2 nanomolar (nM)) for 30 minutes, and binding was immediately measured with a FACScan flow cytometer.

3. animal testing

An animal model of acute respiratory distress syndrome (ARDS) induced by LPS in mice. BALB/c male mice about 7-8 weeks old were housed in an animal room with a 12-hour light-dark cycle and had free access to standard feed and drinking water. All animal experiments were conducted in compliance with the guidelines issued by the Animal Care and Use Committee of Chang Gung Memorial University.

Dissolve the test substance (IALB-E or IA-1) in 50 μl of physiological saline containing 10% DMSO and 10% Tween-20, and adjust the final concentration of the test substance to 5 mg/kg or 10 mg/kg, injected intravenously into mice. After 1 hour, the mice were anesthetized with Zoletil ^® 50 (containing zolazepam and tiletamine; 30 mg/kg) and xylazine (5 mg/kg). , and 2 mg/kg of LPS (E. coli O111:B4; Sigma-Aldrich) was administered intratracheally by spray; the mice in the control group were administered physiological saline. Regarding the post-treatment after administration of the test substance IALB-E, 5 mg/kg or 10 mg/kg IALB-E was injected into the mice via intravenous injection 1 hour after LPS was administered to the mice. Then, 6 hours after LPS was administered to the mice, the mice were sacrificed by cervical dislocation and their lung tissues were removed. The lung tissues were fixed with 10% formalin and embedded in paraffin blocks for subsequent analysis. Observation of tissue sections.

4. Histopathological and immunohistochemical analysis

Lung tissue was fixed with 10% formalin and embedded in paraffin blocks. Tissue sections with a thickness of approximately 5 μm were cut out and then stained with hematoxylin-eosin (H&E). In terms of immunohistological staining, primary antibodies against Ly6g or anti-elastase are first used for identification, and then secondary antibodies that can recognize the primary antibodies are used for identification. Finally, 3,3’-diaminobenzidine (DAB) color matrix was used for staining, and then hematoxylin was used for contrast staining. In the statistical analysis section, when analyzing the H&E staining area, the ratio of the nuclear staining area to the total staining area is calculated, and when analyzing the Ly6g or elastase staining area, the immunohistochemistry positive staining area is calculated relative to the total staining area. Ratio of stained area.

5. Measuring elastase release

Take methoxysuccinyl-alanine-alanine-proline-valine-p-nitroanilide ( N -methoxysuccinyl-Ala-Ala-Pro-Val- p- nitroanilide, MeOSuc-AAPV-PNA) as matrix to measure elastase release. Briefly, MeOSuc-AAPV-PNA (100 μM) was first added to the culture medium, and human neutrophils (6 × 10 ⁵ cells/ml) were allowed to equilibrate therein for about 5 minutes, and then mixed with the specific concentration to be tested. The substance (IALB-A, IALB-B, IALB-C, IALB-D, IALB-E, IA-1 or IA-2) acts for about 2 minutes. Next, cells were treated with cytochalasin B (CB; 0.5 μg/ml) for about 3 minutes, and then with specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), MMK1 (0.3 μM), IL-8 (100 nanograms/ml), LTB ₄ (0.1 μM), or NaF (20 mM), etc., activate cells for about 10 minutes or 30 minutes, and measure the change in absorption value at 405 nanometer wavelength; IALB-F group The experimental conditions were the same, but no agonist was added. In another set of experiments, cells were treated with the test substance (IA-1 or IA-2) at a specific concentration (1 or 2.5 μM), and then activated with fMLF at a specific concentration (10 ⁰ -10 ⁴ nM). The steps are the same as above. The experimental results are expressed as the percentage of elastase release.

6. Measuring superoxide anion (O ₂ ^- ) Generation

The O ₂ ^- content is determined by the amount of ferricytochrome c reduction that can be inhibited by superoxide dismutase ( SOD). Briefly, after adding 0.6 mg/ml ferrocytochrome c to the cell culture medium, the test substance (IALB-A, IALB-B, IALB-C, IALB-D, IALB-E, IA-1 or IA-2) treats human neutrophils (6× ¹⁰ cells/ml or 3× ¹⁰ cells/ml) for approximately 2 minutes. Next, the neutrophils were treated with cytochalasin B (1 μg/ml) for about 3 minutes, followed by specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), and MMK1 (0.3 μM). , PMA (0.01 μM), NaF (20 mM), etc., activate the cells for about 10-30 minutes; the experimental conditions of the IALB-F group were the same, but no agonist was added. Changes in absorbance at a wavelength of 550 nm due to reduction of ferrocytochrome c were continuously monitored with constant stirring in a dual-beam, 6-slot positioning spectrometer (Hitachi U-3010), measured as an extinction coefficient (ε = 21.1 mM ^-1 /10 mm) to calculate the amount of O ₂ ^-generated . In another set of experiments, cells were treated with the test substance (IA-1 or IA-2) at a specific concentration (1 or 2.5 μM), and then activated with fMLF at a specific concentration (10 ⁰ -10 ⁴ nM). The steps are the same as above.

7. Measure CD11b Performance

To measure the expression of CD11b on the cell surface, mix human neutrophils in the suspension at 37°C, preheat for 5 minutes and then add a specific concentration (5 μM) of the test substance (IA-1 or IA-2). ) to treat cells, then add cytochalasin B (1 μg/ml) and react for 3 minutes, and then react with cells with specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), MMK1 (0.3 μM), etc. 5 After minutes, place on ice to stop the reaction. After centrifugation, the supernatant was removed, and the cells were resuspended in a balanced salt solution containing 5% bovine serum albumin (BSA), and fluorescein isothiocyanate (FITC)-labeled antibody was added. -CD11b antibody was reacted on ice in the dark, and finally balanced salt solution was added to terminate the reaction. Flow cytometry was used to detect the changes in fluorescence expression to further understand whether the test substance would affect the expression of CD11b on the cell membrane of human neutrophils after stimulation.

8. Measure intracellular calcium ions (Ca ²⁺ ) Release amount

Human neutrophils stained with fluo-3/AM (2 µM) were added to the test compound and reacted for 5 minutes, and then fMMYALF (0.1 µM) was used to stimulate the reaction. Finally, Triton X-100 was added to obtain the maximum fluorescence value (F _max ), and ethylene glycol tetraacetic acid was added to obtain the minimum fluorescence value (F _min ). The changes in fluorescence intensity were recorded with a fluorescence spectrophotometer, and the excitation wavelength and divergence wavelength were set to 488 nm and 520 nm respectively. The change in intracellular Ca ²⁺ concentration is calculated by the following equation: [Ca ²⁺ ] _i (nM) = 400 × (F - F _min ) / (F _max - F), where 400 is the calcium ion of fluo-3 Dissociation constant, F is the fluorescence value that stimulates intracellular calcium ions.

9. Statistical analysis

All experiments were performed in at least three replicates (except for the animal experiment in Figures 2A-2D in which IALB-E was used to treat LPS-induced ARDS in mice with two replicates), and the experimental results are expressed as mean ± SEM. Statistical analysis was performed using Student's t test, and P < 0.05 was considered statistically significant. All statistical analyzes were performed using Sigma Plot software.

Example 1 Extracts from marine Bacillus culture fluid have anti-inflammatory effects

1.1 Preparation of extracts from marine Bacillus culture broth

This embodiment aims to study the therapeutic effect of the extract of the marine Bacillus culture fluid. For this purpose, the extract of the marine Bacillus culture fluid is prepared. After centrifuging a culture medium of marine Bacillus to remove bacteria, the supernatant was extracted with ethyl acetate (EA) at room temperature to obtain a crude extract IALB (1.322 g). Next, IALB was filtered through a filter membrane with a pore size of 0.45 μm, and then preparative HPLC purification was performed. The filtrate was loaded into a silica gel column (Sunniest C18), and eluted with methanol/0.1% formic acid (FA) at a flow rate of 20 ml/min into 6 parts, each of which was the elution fraction of part 1. (labeled as IALB-A; 28.7 mg), fraction 2 of the eluate (labeled as IALB-B; 80.1 mg), fraction 3 of the fraction (labeled as IALB-C; 46.1 mg), part 3 The eluate of fraction 4 (labeled IALB-D; 25.1 mg), the eluate of fraction 5 (labeled IALB-E; 43.3 mg), and the eluate of fraction 6 (labeled IALB -F; 359.9 mg). After obtaining the elution fractions of the extracts, the biological activities of the extract fractions are analyzed.

1.2 Characteristic analysis of liquid separation of extracts from marine Bacillus culture fluid

First, we explored whether the extracted extracts had anti-inflammatory effects. Specifically, we explored the ability of these extracted extracts to inhibit the production of superoxide anions and the release of elastase by human neutrophils. Among them, IALB- The cells in the A-IALB-E group were treated with IALB-A, IALB-B, IALB-C, IALB-D or IALB-E, and then activated by CB sensitization (primed) and fMLF stimulation; and the IALB-F group After treatment with IALB-F, the cells were only sensitized by CB and no longer activated by fMLF stimulation; the relevant experimental results are shown in Table 1.

Table 1 Effect of extract separation on superoxide anion generation and elastase release Sample (5 μg/ml) Superoxide anion generation Elastase release ^IC _50a inhibition% Increase% Salience ^IC _50a inhibition% Increase% Salience IALB-A ＞10 7.82 ± 2.39 N/A ^b * ＞10 1.44 ± 1.57 N/A ^b IALB-B ＞10 7.36 ± 1.45 N/A ^b ** ＞10 2.03 ± 1.08 N/A ^b IALB-C ＞10 13.81 ± 2.52 N/A ^b ** ＞10 1.55 ± 2.82 N/A ^b IALB-D ＞10 31.81 ± 6.06 N/A ^b * ＞10 5.53 ± 4.59 N/A ^b IALB-E 1.60±0.49 97.83 ± 0.82 N/A ^b *** 2.29 ± 0.85 106.58 ± 5.66 N/A ^b *** IALB-F N/A ^b N/A ^b 106.85 ± ^7.04c *** N/A ^b N/A ^b 63.25 ± ^5.34c *** Note 1: * P ＜ 0.05; ** P ＜ 0.01; *** P ＜ 0.001, compared with DMSO control group. Note 2: ^aThe required concentration (μg/ml) to achieve half inhibitory concentration (IC ₅₀ ). Note 3: ^b N/A: means not applicable here; ^c value means promotion: means it will not inhibit, but will increase the generation of superoxide anions and the release of elastase under the action of CB; the value is based on fMLF/ CB functions as a 100% conversion.

As can be seen from Table 1, IALB-E has the ability to strongly inhibit the production of superoxide anions and the release of elastase by human neutrophils. Therefore, IALB-E will be further explored to confirm whether the mechanism of the above ability is related to that of FPR1. Information transmission related. The experimental results are shown in Figure 1. IALB-E (0-5 μg/ml) will significantly inhibit the binding of another FPR1-specific ligand (fNLFNYK) to FPR1 in a dose-dependent manner, representing IALB-E There is an active ingredient in it, and this active ingredient is likely to block the activation of human neutrophils through FPR1.

1.3 IALB-E for treatment ARDS Efficacy

Based on the above results, IALB-E was further explored and a living animal model was used to confirm whether IALB-E has therapeutic effect on ARDS. The relevant experimental results are shown in Figures 2A-2D. It can be seen from the immunohistochemical analysis results in Figure 2A that whether IALB-E is administered in advance (pretreatment) before LPS stimulation is administered to mice, or IALB-E is administered after LPS stimulation is administered to mice. E (post-processing), both conditions can significantly reduce the number of human neutrophils infiltrating lung tissue (either the number of nuclear-stained cells in H&E staining (Figure 2B)) or anti-Ly6g responders (Picture 2C)), and because the number of human neutrophils is reduced, the release of elastase can be greatly reduced (Picture 2D), thus protecting lung tissue from damage. The corresponding statistical analysis results of staining area As shown in Figures 2B-2D. The above experimental results clearly confirm that IALB-E has the ability to effectively prevent and treat ARDS in mice.

Example 2 IA-1 and IA-2 Has anti-inflammatory effects

2.1 separate and identification IA-1 and IA-2

It can be seen from Example 1 that the active ingredient in IALB-E has the ability to strongly inhibit the production of superoxide anions and the release of elastase by human neutrophils. In order to clarify the active ingredient, ultra-high performance liquid chromatography- Mass spectrometry (ultra-high performance liquid chromatography-mass spectrometry, UPLC-MS) analyzed IALB-E, thereby obtaining that the active ingredient is a compound of the following formula (Ia) (i.e. IA-1) and a compound of formula (Ib) (i.e. IA -2), and its structure was confirmed by total ion chromatogram (TIC) (data not shown) and mass spectrum (data not shown). (Ia) (Ib)

2.2 IA-1 and IA-2 Effect on elastase release

The purpose of this test is to confirm the ability of IA-1 and IA-2 to inhibit the release of elastase from human neutrophils. The relevant experimental results are shown in Figures 3A-3F (for IA-1) and Figures 4A-4F ( For IA-2).

As shown in Figures 3A-3B, IA-1 can inhibit the release of elastase from human neutrophils activated by the exogenous FPR1 agonist fMLF or the endogenous FPR1 agonist fMMYALF, and its IC ₅₀ is respectively 0.72 ± 0.11 μM and 0.84 ± 0.30 μM. However, IA-1 does not have the ability to inhibit the release of elastase from human neutrophils activated by the FPR2 agonist MMK1 (Figure 3C). It is clear that IA-1 is not able to inhibit the release of elastase from human neutrophils. Elastase is selective and can specifically inhibit the release of elastase caused by activation of cells by the FPR1 (but not FPR2) signaling pathway. In addition, this experiment also explored three other factors that can activate human neutrophils, including IL-8, LTB ₄ , NaF, etc. These factors will also promote the release of elastase after activating human neutrophils. The experimental results are shown in Figures 3D-3F. IA-1 also has varying degrees of ability to inhibit the release of elastase, with the IC ₅₀ of IL-8 being 3.36 ± 0.46 μM. In summary, IA-1 does have the effect of inhibiting the release of elastase from activated human neutrophils, and therefore has anti-inflammatory effects.

Similar experimental results can also be seen in the experimental results related to IA-2 inhibiting the release of elastase from activated human neutrophils. As shown in Figures 4A-4B, IA-2 can inhibit elastase release from human neutrophils activated by fMLF or fMMYALF, with _IC50 of 0.85 ± 0.28 μM and 0.94 ± 0.19 μM respectively. Similarly, IA-2 does not have the ability to inhibit the release of elastase from human neutrophils activated by MMK1 (Figure 4C). It can be seen that IA-2 is effective in inhibiting the release of elastase from human neutrophils. It is selective and can specifically inhibit the release of elastase caused by activation of cells by the FPR1 (but not FPR2) signaling pathway. In addition, for three other factors that can activate human neutrophils, including IL-8, LTB4, NaF, etc., IA-2 also has varying degrees of ability to inhibit the release of elastase caused by activation of these factors. (Figures 4D-4F). In summary, IA-2 does have the effect of inhibiting the release of elastase from activated human neutrophils, and therefore has anti-inflammatory effects.

2.3 IA-1 and IA-2 Effect on superoxide anion generation

This experiment explores the ability of IA-1 and IA-2 to inhibit the production of superoxide anions by human neutrophils. The relevant experimental results are shown in Figures 5A-5E (for IA-1) and Figures 6A-6E (for IA-1). IA-2).

As shown in Figures 5A-5B, IA-1 can inhibit the production of superoxide anion by human neutrophils activated by fMLF or fMMYALF, with _IC50 of 0.81 ± 0.14 μM and 0.52 ± 0.05 μM, respectively. However, IA-1 does not have the ability to inhibit the production of superoxide anions by human neutrophils activated by MMK1 (Figure 5C), indicating that IA-1 inhibits the production of superoxide anions by human neutrophils. It is selective and can specifically inhibit the generation of superoxide anion caused by activation of cells by the FPR1 (but not FPR2) signaling pathway. In addition, this experiment also tested two other factors that can induce the release of superoxide anions from human neutrophils, including PMA and NaF. The experimental results are shown in Figures 5D-5E. IA-1 also has varying degrees of ability to inhibit the generation of superoxide anions. In summary, IA-1 does have the effect of inhibiting the production of superoxide anions by activated human neutrophils, and therefore has anti-inflammatory effects.

Similar experimental results can also be seen in the experimental results related to IA-2 inhibiting the production of superoxide anion by activated human neutrophils. As shown in Figures 6A-6B, IA-2 can inhibit the production of superoxide anion by human neutrophils activated by fMLF or fMMYALF, with _IC50 of 0.72 ± 0.07 μM and 0.52 ± 0.07 μM, respectively. Similarly, IA-2 does not have the ability to inhibit the production of superoxide anion by human neutrophils activated by MMK1 (Figure 6C), which means that IA-2 has no ability to inhibit the production of superoxide anion by human neutrophils. The anion is selective and can specifically inhibit the production of superoxide anion caused by activation of the FPR1 (but not FPR2) signaling pathway in cells. In addition, for PMA and NaF, IA-2 also has varying degrees of ability to inhibit the generation of superoxide anions induced by these factors (Figure 4D-4E). In summary, IA-2 does have the effect of inhibiting the production of superoxide anions by activated human neutrophils, and therefore has anti-inflammatory effects.

The relevant IC ₅₀ experimental results regarding the effects of IA-1 and IA-2 on inhibiting the production of superoxide anions and the release of elastase from human neutrophils are summarized in Tables 2 and 3.

Table 2 IC ₅₀ of IA-1 for superoxide anion generation and elastase release agonist Superoxide anion generation Elastase release IC ₅₀ (μM) ^a IC ₅₀ (μM) ^a fMLF 0.81 ± 0.14 0.72 ± 0.11 fMMYALF 0.52 ± 0.05 0.84 ± 0.30 MMK1 ＞10 ＞10 NaF ＞10 ＞10 PMA ＞10 ND IL-8 ND 3.36 ± 0.46 LTB ₄ ND ＞10 Note 1: ^aThe required concentration to reach half inhibitory concentration (IC ₅₀ ). Note 2: ND: does not reach detectable concentration.

Table 3 IC ₅₀ of IA-2 for superoxide anion generation and elastase release agonist Superoxide anion generation Elastase release IC ₅₀ (μM) ^a IC ₅₀ (μM) ^a fMLF 0.72 ± 0.07 0.85 ± 0.28 fMMYALF 0.52 ± 0.07 0.94 ± 0.19 MMK1 ＞10 ＞10 NaF ＞10 ＞10 PMA ＞10 ND IL-8 ND ＞10 LTB ₄ ND ＞10 Note 1: ^aThe required concentration to reach half inhibitory concentration (IC ₅₀ ). Note 2: ND: does not reach detectable concentration.

2.4 IA-1 and IA-2 for FPR1 The ability to combine

Because IA-1 and IA-2 can inhibit human neutrophil activation induced by the exogenous FPR1 agonist fMLF or the endogenous FPR1 agonist fMMYALF, it is speculated that IA-1 and IA-2 inhibit human neutrophil activation. The activation of neutrophils is through the message transmission pathway of FPR1. In order to truly understand the mechanism of action of IA-1 and IA-2, we tested whether IA-1 and IA-2 would bind to FPR1. The relevant experimental results are shown in Figures 7A-7B, showing the binding abilities of IA-1 and IA-2 to FPR1 respectively. From the results in Figure 7A-7B, it is found that IA-1 and IA-2 (0-5 μM) can significantly inhibit the binding of another FPR1-specific ligand (fNLFNYK) to FPR1 in a dose-dependent manner, which is It means that IA-1 and IA-2 block a series of message transmission pathways related to FPR1 by binding to FPR1, thereby achieving their effect of inhibiting the activation of human neutrophils.

2.5 IA-1 and IA-2 for FPR1 Effects on human neutrophil activation

In order to further verify that IA-1 and IA-2 indeed inhibit the activation of human neutrophils through the FPR1 signaling pathway, the following experiments were conducted to prove this. The relevant experimental results are shown in Figures 8A-8B (for IA-1 ) and Figures 9A-9B (for IA-2). It is found from the results in Figures 8A-8B that as the concentration of fMLF increases, the ability of human neutrophils activated by fMLF to release elastase (Figure 8A) or generate superoxide anions (Figure 8B) increases, that is, A fixed concentration of IA-1 (1 or 2.5 μM) showed a decreasing inhibitory effect on the activation of fMLF. Similarly, from the results in Figures 9A-9B, it can be seen that the release of elastase (Figure 9A) or the generation of superoxide anions (Figure 9B) from human neutrophils activated by fMLF will also increase as the concentration of fMLF increases. increased, and a fixed concentration of IA-2 (1 or 2.5 μM) also showed a decreasing inhibitory effect on the activation of fMLF. The above results confirm that IA-1 and IA-2 can indeed inhibit the activation of human neutrophils induced by fMLF (via the FPR1 signaling pathway).

2.6 IA-1 and IA-2 for CD11b performance impact

Since activated human neutrophils express a surface adhesion factor CD11b, which participates in the inflammatory response by regulating the adhesion and migration of the cells themselves, the purpose of this experiment is to confirm the role of IA-1 and IA-2 in inhibiting The ability of human neutrophils to express CD11b is shown in Figures 10A-10C (for IA-1) and Figures 11A-11C (for IA-2).

As shown in Figures 10A-10C, IA-1 can inhibit expression of CD11b in human neutrophils activated by fMLF or fMMYALF (Figures 10A-10B), but it cannot inhibit human neutrophils activated by MMK1. Expression of CD11b (Figure 10C), it is clear that IA-1 specifically inhibits the expression of CD11b caused by activation of cells by the FPR1 (but not FPR2) signaling pathway. Similarly, as shown in Figures 11A-11C, IA-2 can inhibit human neutrophil expression CD11b (Figures 11A-11B) activated by fMLF or fMMYALF, but not human neutrophils activated by MMK1. Neutrophils express CD11b (Figure 11C). From this, it can be seen that IA-2 also specifically inhibits the expression of CD11b caused by activation of cells by the FPR1 (but not FPR2) signaling pathway. In summary, IA-1 and IA-2 do have the effect of inhibiting the expression of CD11b by activated human neutrophils, thereby inhibiting the activation of human neutrophils from participating in the inflammatory response, and therefore have anti-inflammatory effects.

2.7 IA-1 and IA-2 Effect on the release of intracellular calcium ions

Since activated human neutrophils will increase the intracellular calcium ion concentration to promote the activation of more human neutrophils and participate in the inflammatory response, this experiment aimed to confirm the role of IA-1 and IA-2 in inhibiting human neutrophils. The ability of neutrophils to release calcium ions within cells, the relevant experimental results are shown in Figure 12 (for IA-1) and Figure 13 (for IA-2).

As shown in Figures 12-13, IA-1 and IA-2 (5 μM) can inhibit the effects of fMLF (0.1 μM; Figure 12-13 left column) or fMMYALF (0.1 μM; Figure 12-13 right column), respectively. ) activates human neutrophils to release calcium ions. Specific experimental data are provided in Tables 4 and 5 respectively. Based on the above results, it has been clearly confirmed that IA-1 and IA-2 do have the effect of inhibiting the release of calcium ions in activated human neutrophil cells, thereby inhibiting the activation of human neutrophils from participating in the inflammatory response. Therefore, they have Anti-inflammatory effect.

Table 4 Experimental data of IA-1 for measuring intracellular calcium ion release medicine Peak [Ca ²⁺ ] _i (nM) Salience t _1/2 (second) Salience 0.1 μM fMLF 303.00 ± 11.61 25.14 ± 3.56 5 μM IA-1 + 0.1 μM fMLF 149.31 ± 16.48 *** 4.52 ± 1.28 *** 0.1 μM fMMYALF 290.71 ± 10.72 21.06 ± 3.00 5 μM IA-1 + 0.1 μM fMMYALF 73.03 ± 8.03 *** 6.00 ± 0.73 ** Note: ** P ＜ 0.01; *** P ＜ 0.001, compared with the control group.

Table 5 Experimental data of IA-2 for measuring intracellular calcium ion release medicine Peak [Ca ²⁺ ] _i (nM) Salience t _1/2 (second) Salience 0.1 μM fMLF 304.37 ± 10.58 24.42 ± 7.05 5 μM IA-2 + 0.1 μM fMLF 187.67 ± 12.66 *** 4.86 ± 0.56 *** 0.1 μM fMMYALF 293.14 ± 5.39 25.14 ± 2.25 5 μM IA-2 + 0.1 μM fMMYALF 97.18 ± 10.05 *** 5.04 ± 0.41 *** Note: *** P < 0.001, compared with the control group.

2.8 IA-1 for treatment ARDS Efficacy

From the above experimental results, it can be seen that IA-1 and IA-2 have significant inhibitory effects on human neutrophil activation. Therefore, IA-1 was used as an example for further discussion, and it was confirmed that in living animal models, IA-1 It does have therapeutic efficacy for ARDS, and the relevant experimental results are shown in Figures 14A-14D. It can be seen from the immunohistochemistry analysis results in Figure 14A that LPS stimulation alone causes human neutrophils to accumulate in the lung tissue in large numbers (regardless of the number of nuclear stained cells in H&E staining (Figure 14B), Or anti-Ly6g responders (Fig. 14C)). However, preadministration of IA-1 before LPS stimulation in mice can avoid massive infiltration of human neutrophils into lung tissue, and because humans The number of neutrophils in the lung tissue is reduced, which can significantly reduce the release of elastase (Figure 14D), thus protecting the lung tissue from damage. The corresponding statistical analysis results of staining area are as shown in Figure 14B-14D As shown in the figure. The above experimental results clearly confirm that IA-1 has the ability to effectively prevent and treat ARDS in mice.

To summarize the above, the relevant experimental data of the present disclosure clearly prove that the biologically active extract extracted from the marine Bacillus culture fluid and the active ingredients IA-1 and IA-2 isolated therefrom can indeed pass through the barrier. Interrupting the FPR1 message transmission pathway achieves a significant effect in inhibiting the activation of human neutrophils, and also shows significant therapeutic efficacy in treating ARDS in mice. Based on the above results, the present disclosure provides a novel drug that can act through FPR1 for safe and effective treatment and/or prevention of ARDS.

Although the above embodiments disclose specific examples of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs can, without departing from the principles and spirit of the present invention, Various changes and modifications can be made to it, so the protection scope of the present invention shall be defined by the appended patent application scope.

without

In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described as follows:

Figure 1 illustrates the results obtained according to an embodiment of the present disclosure, illustrating an extract of a marine Bacillus culture solution obtained after purification by preparative high performance liquid chromatography (PHPLC). The elution fraction of part 5 (labeled IALB-E) is useful for blocking N -formyl-norleucine-leucine-phenylalanine-norleucine-tyrosine-lysine ( N -formyl-Nle-Leu-Phe-Nle-Tyr-Lys, fNLFNYK; FPR1 ligand) has the ability to bind to its receptor FPR1, where N -formyl-L-methionyl-L-leucine- L-phenylalanine ( N -formyl-methionyl-leucyl-phenylalanine, fMLF; exogenous FPR1 agonist, an analog of fNLFNYK) is the positive control group; microvolume molar concentration (μM); nanovolume Molar concentration (nM);

Figures 2A-2D are mouse experimental results obtained according to an embodiment of the present disclosure, illustrating the effect of IALB-E on inhibiting the release of elastase induced by stimulating the lungs of mice with lipopolysaccharide (LPS). Effect, where Figure 2A is the immunohistochemistry analysis result; Figures 2B-2D are the statistical analysis results of Figure 2A respectively, of which Figure 2B is the statistical analysis result of hematoxylin-eosin (hematoxylin-eosin, H&E) staining area , Figure 2C shows the statistical analysis results of staining area for Ly6g (lymphocyte antigen 6 complex locus G6D (lymphocyte antigen 6 complex locus G6D), a human neutrophil marker), and Figure 2D shows the results of elastase Statistical analysis results of staining area; pre-treatment (pre-treatment): administer IALB-E before administration of LPS; post-treatment (post-treatment): administer IALB-E after administration of LPS;

Figures 3A-3F are results obtained according to one embodiment of the present disclosure, illustrating the effect of the compound anteiso-C13-surfactin (labeled IA-1) on stimulating human neutrophils with specific agonists. Effects of leukocyte-induced elastase release with specific agonists: fMLF (exogenous FPR1 agonist; Figure 3A); formyl-methionine-methionine-tyramine Acid-alanine-leucine-phenylalanine (formyl-Met-Met-Tyr-Ala-Leu-Phe, fMMYALF; endogenous FPR1 agonist; Figure 3B); MMK1 (FPR2 agonist; Figure 3C Figure); IL-8 (CXCR1/2 agonist; Figure 3D); LTB ₄ (TLR agonist; Figure 3E); sodium fluoride (NaF, G protein agonist; Figure 3F ); millimolar concentration (mM);

Figures 4A-4F are results obtained according to one embodiment of the present disclosure, illustrating the effect of the compound iso-C13-surfactin (labeled IA-2) on stimulating human neutrophils with a specific agonist. Effect of induced elastase release, the specific agonists are: fMLF (Figure 4A); fMMYALF (Figure 4B); MMK1 (Figure 4C); IL-8 (Figure 4D); LTB ₄ (Figure 4E); NaF (Figure 4F);

Figures 5A-5E illustrate the results obtained according to one embodiment of the present disclosure, illustrating the effect of compound IA-1 on the generation of superoxide anion induced by stimulating human neutrophils with a specific agonist, wherein The specific agonists are: fMLF (Figure 5A); fMMYALF (Figure 5B); MMK1 (Figure 5C); phorbol myristate acetate (PMA); protein kinase C (protein kinase C, PKC) activator (Figure 5D); NaF (Figure 5E);

Figures 6A-6E are results obtained according to one embodiment of the present disclosure, illustrating the effect of compound IA-2 on the generation of superoxide anions induced by stimulating human neutrophils with a specific agonist, wherein the specific agonist They are: fMLF (Figure 6A); fMMYALF (Figure 6B); MMK1 (Figure 6C); PMA (Figure 6D); NaF (Figure 6E);

Figures 7A-7B are results obtained according to one embodiment of the present disclosure, illustrating the ability of IA-1 (Figure 7A) or IA-2 (Figure 7B) to block the binding of fNLFNYK to its receptor FPR1, wherein fMLF is the positive control group;

Figures 8A-8B are results obtained according to one embodiment of the present disclosure, illustrating the effect of Compound IA-1 on inhibiting elastase release (Figure 8A) or superoxidation induced by stimulating human neutrophils with a specific concentration of fMLF. The influence of anion generation (Figure 8B);

Figures 9A-9B are results obtained according to one embodiment of the present disclosure, illustrating the effect of Compound IA-2 on inhibiting elastase release (Figure 9A) or superoxidation induced by stimulating human neutrophils with a specific concentration of fMLF. The influence of anion generation (Figure 9B);

Figures 10A-10C illustrate the results obtained according to one embodiment of the present disclosure, illustrating the effect of compound IA-1 on inhibiting the expression of the surface adhesion factor CD11b induced by stimulating human neutrophils with a specific agonist, wherein the specific agonist The agents are: fMLF (Figure 10A); fMMYALF (Figure 10B); MMK1 (Figure 10C);

Figures 11A-11C are results obtained according to one embodiment of the present disclosure, illustrating the effect of compound IA-2 on inhibiting the expression of CD11b induced by stimulating human neutrophils with specific agonists, wherein the specific agonists are respectively : fMLF (Figure 11A); fMMYALF (Figure 11B); MMK1 (Figure 11C);

Figure 12 is a result obtained according to one embodiment of the present disclosure, illustrating the effect of Compound IA-1 on inhibiting the increase in intracellular calcium ion (Ca ²⁺ ) concentration induced by stimulating human neutrophils with a specific agonist. The specific agonists are: fMLF (left column); fMMYALF (right column);

Figure 13 is a result obtained according to one embodiment of the present disclosure, illustrating the effect of compound IA-2 on inhibiting the increase in intracellular calcium ion concentration induced by stimulating human neutrophils with a specific agonist, wherein the specific agonist They are: fMLF (left column); fMMYALF (right column); and

Figures 14A-14D are mouse experimental results obtained according to an embodiment of the present disclosure, illustrating the effect of IA-1 on inhibiting the release of elastase induced by stimulating the lungs of mice with lipopolysaccharide, wherein Figure 14A is an immune Histochemical analysis results; Figures 14B-14D are the statistical analysis results of Figure 14A respectively, Figure 14B is the statistical analysis results of H&E staining area, Figure 14C is the statistical analysis results of Ly6g staining area, and Figure 14D is the elasticity Statistical analysis results of enzyme staining area.

Claims (7)

The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is administered in an effective amount to treat and/or prevent acute respiratory distress syndrome in an individual , ARDS): Among them, R ₁ is a C _6-12 linear or branched alkyl group. The use as described in claim 2, wherein the formula (I) is (Ia). The use as described in claim 2, wherein the formula (I) is (Ib). The use as described in claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is a type I formyl peptide receptor (formyl peptide receptor 1, FPR1) antagonist. The use as claimed in claim 1, wherein the compound of formula (I) is extracted from a biologically active secondary metabolite of marine Bacillus sp. The use as described in claim 1, wherein the effective amount is 0.1-10 mg/kg. The use as described in claim 1, wherein the individual is a human being.

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