Chromanol, quinone, or hydroquinone compounds for the treatment of sepsis

Abstract

The present invention relates to certain chromanol, quinone, or hydroquinone compounds and their derivatives for the treatment of sepsis and sepsis-induced organ dysfunction. Specifically, the present invention relates to chromanol compounds selected from S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone and S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone, and pharmaceutically acceptable salts thereof.

Description

[Technical Field]

[0001] I. The present invention relates to chromanol compounds and derivatives thereof for the treatment or prevention of sepsis. The present invention further relates to chromanol compounds and derivatives thereof for the treatment or prevention of organ dysfunction induced by sepsis. [Background technology]

[0002] II. Sepsis is a harmful systemic inflammatory response to infection. It is the leading cause of morbidity and mortality worldwide (Rudd et al., Lancet 2020;395:200-211). Sepsis is now defined as a life-threatening organ failure caused by dysregulation of the host's response to infection. The most severe forms of sepsis can lead to multi-organ failure and a serious disease state characterized by severe immune dysfunction and catabolism (Gotts & Matthey, BMJ 2016;353:il585).

[0003] Current treatments for sepsis, which focus on antibiotics, eradicating the source of infection, and supporting blood pressure, organ blood flow, and ventilation, have shown only limited effectiveness in reducing sepsis-related mortality. Despite efforts to improve current treatment strategies, the in-hospital mortality rate for sepsis in developed countries remains at approximately 20% (Seymour et al., N.Engl.J.Med.2017;376:2235-2244; Fleischmann-Struzek et al. Intensive Care Med.2018 https: / / doi.org / 10.1007 / s00134-018-5377-4).

[0004] Septic reactions typically begin with microbial infection. When microbial components such as lipopolysaccharides (LPS), peptidoglycans, lipoteichoic acid, and unmethylated CpG DNA are recognized by Toll-like receptors (TLRs), the innate immune response is rapidly activated, releasing various humoral mediators, including glucocorticoids, catecholamines, and proximal inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6. This pro-inflammatory state is defined as systemic inflammatory response syndrome (SIRS).

[0005] The excessive production of inflammatory cytokines and the induction of more distal mediators such as nitric oxide, platelet-activating factor, and prostaglandins are involved in endothelial changes and the induction of procoagulable states, leading to hypotension, insufficient organ perfusion, and necrotizing cell death associated with multiple organ failure syndrome (MODS).

[0006] Multiple organ failure syndrome (MODS) has been identified as one of the most fatal complications of sepsis. While many drugs targeting various stages of the systemic inflammatory response have been developed over the years, most have shown little to no efficacy in clinical trials.

[0007] WO2019 / 172766A1 describes the use of alkaline phosphatase for the prevention, treatment, cure, or improvement of symptoms of acute kidney injury caused by sepsis, for example.

[0008] WO2017 / 220810A1 describes the use of cilastatin in the treatment or prevention of sepsis in mammals, provided that cilastatin is administered in combination with another drug that is not a β-lactam antibiotic.

[0009] US6231894 describes the use of several different compounds for the treatment of disorders in which nitric oxide synthase contributes to the production of reactive oxygen species that cause tissue damage. Most of these compounds are arginine derivatives. One of the proposed compounds is BN80933, which is described as a nitric oxide synthase inhibitor that blocks the electron transfer reaction between nitric oxide synthase and NO-donating compounds. The structure of compound BN80933 is as follows: [ka]

[0010] Although sepsis is mentioned in US6231894, the compound appears to have little effect at the mitochondrial level, as shown in the experimental section below (Example 5).

[0011] New compounds are still needed for the treatment of sepsis.

[0012] The object of the present invention is to provide compounds for the treatment or prevention of sepsis, and in particular for the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection, such as renal dysfunction. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2019 / 172766 [Patent Document 2] International Publication No. 2017 / 220810 [Patent Document 3] U.S. Patent No. 6231894 [Non-patent literature]

[0014] [Non-Patent Document 1] Rudd et al.,Lancet 2020;395:200-211

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0015] III. The above object is satisfied by providing a specific chromanol, quinone or hydroquinone compound.

[0016] The above object is achieved by the present invention by providing a compound according to formula (I), (II), a hydroquinone analogue of formula (II), or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of sepsis.

Chemical Formula

[0017] In the present invention, the compound according to formula (II) includes a hydride quinone (i.e., hydroquinone) analog, but a quinone derivative is preferred from the viewpoint of stability.

[0018] In preferred embodiments, nitrogen may be an amine, quaternary amine, guanidine, or imine, and oxygen may be a hydroxyl, carbonyl, or carboxylic acid, and / or oxygen and nitrogen may together form an amide, urea, or carbamate group.

[0019] In a preferred embodiment, R1 in formula (I) is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen.

[0020] In preferred embodiments of either the compound according to formula (I) or formula (II), R2 and R3, together with the N atoms to which they are bonded, form a saturated ring that incorporates an additional N atom, which is either unsubstituted or substituted with an alcohol or an alkanol group having 1 to 4 carbon atoms, such as ethylol.

[0021] In another preferred embodiment, R2 is a hydrogen atom, and R3 comprises a saturated cyclic structure having 4 to 7 carbon atoms and 1 nitrogen atom, wherein the ring may be substituted with an alkyl group, an alcohol group, or a group having 1 to 4 carbon atoms, which may include an oxygen, carboxylic acid, or amine group.

[0022] In another preferred embodiment, the compound is a compound of formula (II), where R2 is a hydrogen atom and R3 comprises a cyclic structure having 4 to 6 carbon atoms and 1 nitrogen atom, the ring being unsubstituted or substituted with an alcohol or an alkanol group having 1 to 4 carbon atoms such as ethylol, preferably optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0023] In another preferred embodiment, the compound is a compound of formula (I), where R2 is a hydrogen atom and R3 comprises a saturated cyclic structure having 4 to 7 carbon atoms and 1 nitrogen atom, the ring being unsubstituted or substituted with an alcohol or an alkanol group having 1 to 4 carbon atoms such as ethylol, preferably optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0024] In another preferred embodiment, R3 is an aryl group or arylalkyl group optionally substituted with nitrogen or oxygen, and R3 comprises 6 to 10 carbon atoms, and R3 may contain one or more nitrogen atoms in the ring, and may contain a linear and / or branched aliphatic group optionally substituted with one or two nitrogen and / or oxygen atoms, comprising an aromatic ring structure.

[0025] More preferably, R2 and R3 do not contain aromatic rings.

[0026] In yet another preferred embodiment, the compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidine-3-yl)chroman-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-109), or a pharmaceutically acceptable salt thereof, either as a racemic mixture or as one of its enantiomers.

[0027] In the most preferred embodiment, the compound is the S-enantiomer of SUL-109, namely S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-138), or the S-enantiomer of SUL-121, namely S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-151), or a pharmaceutically acceptable salt thereof.

[0028] In preferred embodiments of the present invention, either compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da, more preferably less than 450 Da, and most preferably less than 400 Da (as a free base).

[0029] In a preferred embodiment of the present invention, either compound according to formula (I) or formula (II) is for use in the treatment or prevention of sepsis in an organ system, the organ being the lungs, heart and blood vessels, liver, kidneys, brain, or intestines.

[0030] In a more preferred embodiment of the present invention, either compound according to formula (I) or formula (II) is intended for use in the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection, particularly renal organ dysfunction caused by sepsis.

[0031] The kidneys appear to be a critical organ in sepsis because acute kidney injury is the most frequently caused by sepsis. Similarly, the occurrence of acute kidney injury is strongly associated with other organ dysfunction and a three-fold higher in-hospital mortality rate. Furthermore, acute kidney injury increases the risk of developing chronic kidney disease after sepsis by nine times, associated with an increased risk of developing end-stage renal disease. Therefore, preventing or mitigating the severity of acute kidney injury is one of the main goals in the treatment of sepsis, but achieving this is shown to be extremely difficult.

[0032] The compounds for use according to the present invention in the prevention or treatment of sepsis are generally used as adjunctive therapy in addition to standard treatment of antibiotics and / or other care (see Gotts above).

[0033] Improved organ survival rates can prove highly beneficial not only for short-term survival in sepsis but also in the long term. The importance of long-term survival rates is increasing. While mortality rates have decreased over the past decade, the number of sepsis survivors at risk of increased long-term morbidity, particularly high risk of (fatal) cardiovascular events, has risen. As a result, the long-term survival rate after sepsis is less than 50% five years post-sepsis.

[0034] The treatments currently being invented are expected to significantly improve long-term survival rates.

[0035] IV. [Brief explanation of the drawing]

[0036] [Figure 1] This shows the xiphoid process temperature of mice after sepsis induction in a standard cecal ligation and puncture (CLP) model, with or without SUL-138. [Figure 2] This shows the plasma levels of cytokines (IL-6, TNFα, and IL-12) in mice treated with and not treated with SUL-138 after CLP induction. [Figure 3] By measuring NGAL and urea in plasma, which are biomarkers of renal function, we have demonstrated CLP-induced renal dysfunction or its prevention in mice in response to treatment with SUL-138. [Figure 4] Regardless of whether or not they were treated with SUL-138, CLP-induced renal inflammation in mice is indicated by the expression of RNA of numerous markers in the kidney. [Figure 5] This study demonstrates the effects of SUL-151 on survival rate and geotaxis after sepsis induction in Drosophila melanogaster. [Figure 6]This study demonstrates the efficacy of SUL-138 against LPS-induced mitochondrial dysfunction and cell death in vitro. [Figure 7A] We will compare NO production and superoxide production in cells and mitochondrial peroxides of BN-80933, SUL-138, or SUL-150. [Figure 7B] We will compare NO production and superoxide production in cells and mitochondrial peroxides of BN-80933, SUL-138, or SUL-150. [Figure 7C] We will compare NO production and superoxide production in cells and mitochondrial peroxides of BN-80933, SUL-138, or SUL-150. [Figure 7D] We will compare NO production and superoxide production in cells and mitochondrial peroxides of BN-80933, SUL-138, or SUL-150. [Figure 8A] This study compares the activation of endothelial inflammation by BN-80933, SUL-138, or SUL-150, and the survival rate of endothelial cells after hypothermic rewarming stress. [Figure 8B] This study compares the activation of endothelial inflammation by BN-80933, SUL-138, or SUL-150, and the survival rate of endothelial cells after hypothermic rewarming stress. [Figure 8C] This study compares the activation of endothelial inflammation by BN-80933, SUL-138, or SUL-150, and the survival rate of endothelial cells after hypothermic rewarming stress. [Figure 8D] This study compares the activation of endothelial inflammation by BN-80933, SUL-138, or SUL-150, and the survival rate of endothelial cells after hypothermic rewarming stress. [Modes for carrying out the invention]

[0037] V. The objective of the present invention, which is to provide a compound for the treatment or prevention of sepsis, and in particular a compound for the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection, such as renal dysfunction, is achieved by providing a compound according to the above formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of sepsis.

[0038] In a more preferred embodiment of the present invention, either compound according to formula (I) or formula (II) is intended for use in the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection.

[0039] Organs that are susceptible to damage and / or dysfunction may include the lungs, heart, and blood vessels, liver, kidneys, brain, or one or more of the intestines.

[0040] The compounds according to the present invention are particularly suitable for treating or preventing renal organ failure caused by sepsis.

[0041] Treatment or prevention with chromanol, quinone, or hydroquinone compounds according to the present invention is preferably part of a combination therapy with one or more other common means for treating sepsis.

[0042] R1 can be a substituent that is readily removed in the human body, and therefore the compound is a prodrug. R1 can be, for example, an amino acid derivative or an ester derivative, and generally has a molecular weight of less than 100 daltons.

[0043] In preferred embodiments, R1 in formula (I) is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen. The ester may contain one or more ether or alcohol groups. Suitable esters include acetate esters, butyrate esters, and 3-hydroxybutyrate esters.

[0044] In a preferred embodiment of option (i), either compound according to formula (I) or formula (II) is such that R2 and R3, together with the N atoms to which they are bonded, form a saturated ring having 3 to 6 carbon atoms and incorporating one additional N atom, which may be substituted with 1 to 4 carbon atoms that may contain oxygen, a carboxylic acid, or an amine group.

[0045] More preferably, R2 and R3, together with the N atom to which they are bonded, form a 5- to 7-membered ring containing one additional amine group, which may optionally be substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0046] In option (i) above, R2 and R3 together have 3 or more and 12 or fewer carbon atoms.

[0047] In option (ii) above, preferably, R3 has 3 to 12 carbon atoms.

[0048] In another preferred embodiment of option (ii), R2 is a hydrogen atom, and R3 comprises a cyclic structure having 3 to 6 carbon atoms and 1 nitrogen atom.

[0049] More preferably, in option (ii), R2 is a hydrogen atom, and R3 comprises a 5-7 membered ring containing one additional amine group, the ring being bonded to an amide nitrogen and optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0050] In either case, the ring (a cyclic structure formed by R2 and R3, or R3 alone) may be unsubstituted or substituted with an alkyl, alcohol, or alkanol group having 1 to 4 carbon atoms, such as ethylol.

[0051] In another preferred embodiment according to option (iii), R3 is an aryl group or arylalkyl group optionally substituted with nitrogen or oxygen, and R3 contains 10 or fewer carbon atoms. In a preferred embodiment, R3 may contain one or more nitrogen atoms in the ring and may contain one or two, preferably one, linear and / or branched aliphatic groups optionally substituted with carboxylic acid, ester, or amide groups.

[0052] In preferred embodiments of the present invention, either compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da, more preferably less than 450 Da, and most preferably less than 400 Da (as a free base).

[0053] In preferred embodiments, the compound for use according to the present invention is a chromanol compound according to formula I.

[0054] Certain chromanol compounds are described in WO2014 / 098586. Compounds described in detail have abbreviations that refer to SUL-XXX (where XXX is a two- or three-digit number). Many of these compounds are racemic mixtures, but some enantiomers have also been tested. Appropriate methods for preparing chromanol compounds according to the present invention are described in WO2014 / 098586 or WO2014 / 011047.

[0055] WO2017 / 060432A1 discloses amide derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinone-2-yl)-butanoic acid and methods for producing such compounds.

[0056] Hydrogenated quinone derivatives can be easily prepared by hydrogenating the quinone structure.

[0057] In yet another preferred embodiment, the compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidine-3-yl)chroman-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-109), or a pharmaceutically acceptable salt thereof, either as a racemic mixture or as one of its enantiomers.

[0058] In the most preferred embodiment, the compound is the S-enantiomer of SUL-109, namely S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-138), or the S-enantiomer of SUL-121, namely S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-151), or a pharmaceutically acceptable salt thereof.

[0059] The counterions in pharmaceutically acceptable salts may be counterions known in the art. Preferably, the compound has at least one basic nitrogen, an amine, that can be protonated. The counterions are preferably halogens such as chlorides, sulfates, citrates, and formates, most preferably chlorides.

[0060] These compounds are available as racemic mixtures or in substantially pure enantiomer forms. The compounds have one or more chiral centers, generally one or two.

[0061] Experiments have shown that enantiomer morphology is not a strong determinant of efficacy in sepsis. However, for general regulatory reasons, it is preferable that the compound be substantially enantiomerically pure. Substantially enantiomerically pure compounds have an enantiomer excess of about 95% or more, more preferably about 98%, and most preferably about 99% or more. These amounts also apply if the compound contains multiple chiral centers.

[0062] The compound is preferably used in an effective amount to achieve the treatment or prevention of sepsis.

[0063] The terms treatment or prevention include reducing the progression of sepsis, including improving the symptoms of sepsis and / or improving organ function.

[0064] Preferably, the compounds according to the present invention are for use in the treatment or prevention of sepsis in the organs of mammals, and the mammal is preferably a human.

[0065] In a more preferred embodiment of the present invention, either compound according to formula (I) or formula (II) is intended for use in the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection.

[0066] In its most preferred embodiment, the compound according to the present invention is intended for use in the treatment or prevention of renal impairment caused by infection.

[0067] Infections are considered to be caused by external factors and are contrasted with autoimmune diseases. Common causes of infections include the spread of bacteria, fungi, or viruses. Bacterial and fungal infections are the most common sources. A recent example of a virus causing dysregulation of the host's response to infection is COVID-19. In cases of hospitalization due to respiratory failure, the compounds according to the present invention can be administered prophylactically before sepsis develops.

[0068] The effect is generally observed at a concentration of approximately 1 μM in body fluids, but it is preferable to use a larger amount. A preferred amount is an in vivo or in vitro concentration of approximately 10 μM or more, more preferably approximately 20 μM or more. Generally, concentrations of approximately 200 μM or less are sufficient and safe in humans.

[0069] When used in humans, this means a dose of approximately 10 mg or more, assuming a distribution volume of 30 L, 100% efficacy, and a concentration of approximately 1 μM. A preferred amount yields a concentration of approximately 10 μM, for which a dose of approximately 100 mg or more would be appropriate. Therefore, a dosage form of approximately 20 mg or more, preferably 50 mg or more, and preferably 100 mg or more is suitable.

[0070] Generally, solid, oral dosage forms contain up to about 500 mg of the compound, preferably about 450 mg or less, to allow for the use of excipients.

[0071] For example, parenteral administration, such as intravenous administration, or other liquid administration methods, allow for the administration of larger quantities.

[0072] Examples of usable dosages are effective doses of 0.2 mg / kg or more of the compound of the present invention, preferably in the range of about 1 mg / kg to about 100 mg / kg, or in the range of about 2 mg / kg to about 40 mg / kg body weight, or in the range of about 3 mg / kg to about 30 mg / kg body weight, or in the range of about 4 mg / kg to about 15 mg / kg body weight. The compound of the present invention can be administered in a once-daily dose, or the total daily dose can be administered in two, three, or four divided doses per day.

[0073] The compounds described herein can be prepared as pharmaceutical compositions by incorporating pharmaceutically or physiologically acceptable excipient carriers and additives such as vehicles.

[0074] Suitable pharmaceutically or physiologically acceptable excipients, carriers, and vehicles include, for example, treatment agents, drug delivery modifiers, and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidone, low-melting-point waxes, and any combination of two or more thereof. Other suitable pharmaceutically acceptable excipients are described in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991).

[0075] The pharmaceutical composition preferably comprises a unit dose formulation, where the unit dose is a dose sufficient to produce a therapeutic effect. The unit dose may be a dose administered regularly in the course of treating or suppressing a disorder.

[0076] The compounds of the present invention may be administered rectally or topically by enteral, oral, parenteral, sublingual, or inhalation (e.g., as a mist or spray) in a dosage unit formulation comprising, if necessary, a conventional non-toxic, pharmaceutically or physiologically acceptable carrier, adjuvant, and vehicle. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intratarsal injection, or infusion methods. The compounds are mixed with a pharmaceutically acceptable carrier, adjuvant, and vehicle suitable for the desired route of administration.

[0077] Generally, oral administration is the preferred route of administration, and formulations suitable for oral administration are preferred formulations.

[0078] Since sepsis is often an acute illness, oral formulations are useful when a patient is at risk of developing sepsis, and such oral formulations are administered prophylactically to such patients.

[0079] However, especially in acute sepsis, patients are often too severe for oral administration such as tablets or pills, making intravenous injection and / or continuous intravenous infusion preferable. Furthermore, sepsis can substantially affect the oral availability of the given drug. Plasma level certainty can generally only be achieved by intravenous or other parenteral administration.

[0080] The compounds described herein for use may be administered in solid form, liquid form, aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injections, creams, solutions, suppositories, enemas, colon lavages, emulsions, dispersions, food premixes, and other appropriate forms. The compounds may also be administered in liposomal formulations.

[0081] Injectable formulations, such as sterile aqueous or oily suspensions for injection, can be prepared according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, for example, as a solution in propylene glycol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixatives have traditionally been used as solvents or suspension media. For this purpose, any mild fixative containing synthetic monoglycerides or diglycerides can be used. In addition, fatty acids such as oleic acid have been used in the preparation of injectable formulations.

[0082] Suppositories for rectal administration of drugs can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycol, which are solid at room temperature but liquid at rectal temperature and therefore melt in the rectum to release the drug.

[0083] Solid dosage forms for oral administration may include capsules, tablets, pills, granules, powders, and granules. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain additional substances other than the inert diluent, such as lubricants, such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. Tablets and pills may be further prepared with enteric coating.

[0084] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, liquids, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, cyclodextrins, sweeteners, flavorings, and fragrances.

[0085] The amount of active ingredient that can be combined with a carrier material to produce a single-dose formulation will vary depending on the host to which the active ingredient is administered and the specific mode of administration. The selected unit dose is typically manufactured and administered to provide a defined final concentration of the drug in the blood, tissue, organ, or other target area of ​​the body. The effective dose for a given situation can be readily determined by routine experimentation and is within the scope of the skill and judgment of a typical clinician or person skilled in the art.

[0086] The present invention will be further described using the following embodiments. The embodiments are shown with reference to the figures.

[0087] VI. [Examples]

[0088] The efficacy of the compounds according to the present invention for the treatment or prevention of sepsis was tested in vivo in mice and Drosophila, and in vitro in HUVA, HUVEC, or NRK cells. (Examples 1-4)

[0089] Example 1; Mouse experiment Animal testing was approved by the Institutional Animal Care and Use Committee of the University Medical Center Groningen (IvD nr.16593).

[0090] Male C57 / BL6J mice were housed at room temperature in a 12-hour-12-hour light-dark cycle. The animals were free-feeding with standard animal laboratory feed and had free access to drinking water at all times.

[0091] A standard cecal ligation and puncture (CLP) model was used to induce sepsis. Animals were anesthetized by subcutaneous injection of xylazine / ketamine (100 / 10 mg / kg), followed by administration of buprenorphine (0.1 mg / kg) as an analgesic. After anesthesia was confirmed by lack of response to paw pinching and eye reflexes, the abdomen was shaved, washed, and disinfected with povidone-iodine solution before a 1 cm midline incision was made. The cecum was ligated with 6-0 sutures at half the distance between the distal pole and the base of the cecum and punctured once with a 21 gauge needle ("penetrating" from the mesentery to the anti-mesentery direction), which is recognized as a model of "moderate" sepsis. A small amount of stool (2-3 mm) was then pushed out to ensure wound patency. After repositioning the cecum to prevent fecal matter from spilling onto the edges of the wound, the abdominal muscle tissue was sutured, and the abdomen was closed with short nodular sutures to the skin. Next, 1 ml of saline solution (warmed, 0.9% NaCl subcutaneous injection) was administered to compensate for the relative volume loss expected due to the development of sepsis.

[0092] The mice recovered at 26-28°C. A broad-spectrum antibiotic (imipenem / cilastatin, 100 mg / kg subcutaneous injection) was administered along with an analgesic (buprenorphine, 0.1 mg / kg body weight, subcutaneous injection) 2 and 10 hours after surgery.

[0093] A group of manipulated animals with a cecum located but not punctured served as a decoy.

[0094] Furthermore, the study included a group of animals that received time-synchronized anesthesia but did not undergo surgery, serving as a control group.

[0095] Mice in the SUL-138 treatment group were injected with SUL-138 (dissolved in physiological saline, 5 mg / kg, subcutaneous injection) 2 hours before and 8 hours after surgery, while mice in the other groups were injected with the same amount of physiological saline at these times.

[0096] The xiphoid process temperature of mice was measured 8 and 24 hours after the procedure. The results are shown in Figure 1 and explained below.

[0097] The mice were euthanized 24 hours after the procedure. At the time of euthanasia, the EDTA-anticoagulated blood was separated into plasma by centrifugation at 10 mm with 1,600 g of blood, allowed to coagulate for 30 minutes, and then separated into serum by centrifugation at 3,000 g for 10 minutes.

[0098] For further analysis, plasma, serum, and organs were flash-frozen in liquid nitrogen.

[0099] To quantify the severity of sepsis relative to systemic inflammation and the efficacy of SUL-138 treatment, plasma levels of TNFα, IL-6, and IL-12 were measured using Mouse DuoSet ELISA (DY410, DY406, and D419, RnD-Systems, respectively) according to the manufacturer's instructions. Briefly, an ELISA plate (DY990, RnD Systems) was coated overnight with capture antibody diluted in 100 μL PBS. The plate was washed three times with washing buffer (0.05% Tween20 in PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4), followed by blocking in 300 μL of reagent diluent (1% probumin w / v in PBS) for 1 hour. Washing was repeated, and samples were added to the wells. Plasma samples were diluted 10-fold for TNFα and IL-12, and 100-fold for IL-6 with reagent diluents. After incubation at room temperature for 2 hours, the plates were washed, and then 100 μL of detection antibody diluted with reagent diluents was added to each well. The plates were incubated again at room temperature for 2 hours and then washed. Finally, 100 μL of substrate solution (DY999, RnD Systems) was added, incubated in the dark for 20 minutes, and then 50 μL of stop solution (2 M H2SO4) was added. Optical density (OD) was measured using a microplate reader set to 450 nm, and the reading at 540 nm was subtracted as a correction to improve accuracy. The results are shown in Figure 2 and are explained below.

[0100] Furthermore, serum levels of NGAL and urea were measured. NGAL and urea are common biomarkers for kidney damage in mice. The results are shown in Figure 3 and explained below.

[0101] To evaluate the expression of pro-inflammatory cytokines and adhesion molecules in the kidney, such as IL-6, TNF-α, IL-1β, and ICAM, RNA was isolated from approximately 30 mg of kidney tissue using Nucleospin RNA (Machery-Nagel, Duren, Germany), quantified using a nanodrop spectrophotometer ND-1000, and converted to copy DNA (cDNA) using 0.5 μg of RNA from each sample. For RNA isolation from cells, the same kit was used, but with a slight modification: TRIzol and chloroform were used as the lysis buffer instead of the kit's lysis buffer. Oligonucleotide primers were designed using NCBI Primer Blast and Clone Manager (see Appendix) and validated by evaluating efficiency, melting temperature and curves using qRT-PCR, as well as the size of naive and enzymatic digests on gel electrophoresis. qRT-PCR amplification was performed using the following thermal profiles. Specifically, the reaction consisted of 40 cycles: 2 mm at 95°C, followed by 15 seconds at 95°C, 30 seconds at 58°C, and 30 seconds at 72°C. All reactions were carried out in triplicate, using the standard curve for each primer. The results are shown in Figure 4 and explained below.

[0102] result Figure 1 shows the xiphoid process temperature of mice after CLP induction with and without SUL-138 compared to an unchallenged control. Figure 1 shows that xiphoid process temperature recovers after 24 hours with SUL-138 treatment, while the CLP procedure results in a decrease in xiphoid process temperature at 8 and 24 hours. Statistical significance is calculated at 24 hours between the unchallenged control to CLP / saline and the CLP / SUL-138 group to CLP / saline, using an unpaired one-sided Student's t-test. An asterisk (*) in the figure indicates a statistically significant difference.

[0103] Figure 2 shows that plasma levels of inflammatory cytokines (IL-6, TNF-α, and IL-12) decreased to levels close to those of the untreated control after treatment with SUL-138. Statistical significance was calculated using an unpaired, one-sided Student's t-test between the control (CLP / saline), the false (CLP / saline), and the CLP / Sul-138 group. TNF-α levels were significantly lower, but the difference was not statistically significant by the t-test.

[0104] Figure 3 shows that CLP-induced renal dysfunction is eliminated by treatment with SUL-138. Serum urea (A) and NGAL (B) levels are significantly increased in sepsis, but this is eliminated by treatment with SUL-138. Serum urea and NGAL levels in animals treated with SUL-138 before CLP were no different from those in sham surgery animals. Significant differences are calculated using an unpaired one-sided Student's t-test between control to CLP / saline, sham to CLP / saline, and CLP / SUL-138 to CLP / saline.

[0105] Figure 4 shows that CLP-induced kidney inflammation is significantly reduced by treatment with SUL-138. CLP-induced sepsis upregulated the expression of pro-inflammatory cytokines and adhesion molecules in the kidney, including IL-6, TNF-α, IL-1β, and ICAM. Treatment of animals with SUL-138 before induction of CLP-induced sepsis completely prevented the increase in IL-6 expression (see Figure 4A). TNF-α, IL-1β, or ICAM (Figure 4B-D) were lower than in untreated CLP animals, but the differences were not statistically significant when using unpaired one-sided Student's t-tests.

[0106] Consideration The decrease in body temperature in mice after sepsis induction indicates a loss of metabolic homeostasis, while the recovery of mice treated with SUL-138 24 hours later suggests the restoration of metabolic homeostasis despite sepsis.

[0107] Elevated plasma levels of IL-6 and IL-12 indicate systemic inflammation induced by CLP, but in animals treated with SUL, both cytokines were significantly reduced after CLP, indicating a decrease in inflammation levels.

[0108] Elevated levels of NGAL and urea following CLP induction indicate acute renal dysfunction. NGAL and urea are biomarkers of renal function in mice. Figure 3 shows that treatment with SUL-138 eliminates or at least significantly reduces CLP-induced renal dysfunction in mice.

[0109] RNA expression of IL-6, TNF-α, IL-1β, and ICAM in the kidney increased after CLP induction, indicating a localized inflammatory response in the kidney. Treatment with SUL-138 significantly reduced IL-6 expression, while other markers were reduced by SUL-138 treatment.

[0110] Example 2; Drosophila melanogaster experiment W1118 flies were reared at 25°C in a 12-hour-12-hour light / dark cycle. The flies were stored in vials containing approximately 5 mL of standard yeast cornmeal medium. The flies kept as stock were inverted into new vials weekly. The yeast cornmeal medium used was prepared according to the instructions in Stocker and Gallant's "Drosophila Methods and Protocols," consisting of 100 grams of yeast, 75 grams of glucose, 8 grams of agar, 55 grams of cornmeal, and 10 grams of wheat flour per liter of water. After boiling, phosphoric acid and propionic acid were added to the mixture to prevent bacterial growth.

[0111] Staphylococcus aureus (S. aureus) was cultured overnight in 2.5% trypsin soy broth (TSB) under aerobic conditions, using a Staphylococcus aureus glycerol stock maintained at -80°C, with continuous rotation (200 rounds per minute) at 37°C. For each experiment, a fresh culture was prepared one day prior to the experiment. For bacterial infections, the optical density (OD) was measured at 600 nm the following day. Clean TSB was used as OD=0.00, and the bacterial culture was diluted to OD=2.20 with PBS. Next, 1.0 mL of the bacterial culture (OD=2.20) was centrifuged at 14,000 g for 1 minute, the supernatant was discarded, and the bacterial pellet was resuspended in 1.0 mL of PBS. For appropriate experiments, serial dilutions (5-fold and 25-fold dilutions with optical densities of approximately 1.05 and 0.25, respectively, in PBS) were used.

[0112] Male flies (3-5 days old) were anesthetized on a CO2 pad immediately before injection. A tungsten needle (0.25 mm diameter, Fine Science Tools, 10130-10) was dipped in a bacterial suspension, and the flies were pinned to the side of their thorax (the scutellum before thoracic suture). Dummy flies were administered only PBS, while control flies were left unhandled and only anesthetized on a CO2 pad. Dummy flies were injected first to ensure hemolymphatic coating of the needle and to prevent accidental bacterial injection into the dummy flies. A subgroup of flies was treated with antibiotics. Linezolid was administered orally, dissolved at a concentration of 500 μg per 1 mL of fly medium. After pinning the intervention group with various concentrations of Staphylococcus aureus, the flies were placed in vials containing linezolid medium.

[0113] After 48 hours, flies were introduced into non-antibiotic vials. To verify the efficiency of inducing bacterial infection, 10 flies were homogenized in 250 μL PBS to determine the bacterial load. After short-term centrifugation, the supernatant was plated on Luna-Bertam (LB) agar plates at 1 / 10, 1 / 100, and 1 / 1000 dilutions and checked after 24 hours.

[0114] Linezolid was administered orally by dissolving 500 μg of the substance per 1 mL of fly culture medium. After pinning, control flies, sham-operated flies, and infection intervention groups containing various concentrations of Staphylococcus aureus were placed in vials containing linezolid medium. After 48 hours, the flies were transferred to non-antibiotic vials.

[0115] Compound SUL-151 was administered by intrapleural injection simultaneously with bacterial injection. The intervention group received 3 mM SUL-151, while flies infected with the vehicle received 1% DMSO, dissolved in the bacterial solution immediately before bacterial injection.

[0116] Negative geotaxis was studied as a marker of fly health. Therefore, a group of 15 flies were transferred to empty styrene vials (9.5 cm tall). Up to nine vials were then placed in a 3D-printed vial holder designed for negative geotaxis. The flies were allowed to acclimate for 5 minutes. Next, the flies were gently tapped towards the bottom of the vials by lightly tapping the vial holder three times against a worktop. A digital camera was used to take a photograph 5 seconds after the last tap. This experiment was repeated 5 times with a 1-minute rest between trials. ImageJ was used to determine the distance each fly traveled within 5 seconds, and the 5 trials were averaged for each group.

[0117] Furthermore, the survival of the flies was checked 24 hours after infection by visual inspection, counting the number of dead flies in the vial.

[0118] result Figure 5 shows the effect of SUL-151 on survival rate (A) and geotaxis (B) after sepsis induction in Drosophila melanogaster. Survival rate at 24 hours was 80% in flies treated with SUL-151, compared to only 42% in sepsis flies without SUL treatment (A). Geotaxis improved at both 24 and 48 hours after sepsis induction in flies pre-treated with SUL (B). Significant differences are calculated using an unpaired one-sided Student's t-test between sepsis / DMSO versus control and sepsis / DMSO versus sepsis / SUL-151.

[0119] Consideration Injection of 3 mM SUL-151 at the time of sepsis induction resulted in improved trajectory at 24 and 48 hours post-sepsis induction, and mortality was significantly reduced at 24 hours. Both measurements demonstrate the efficacy of SUL151 against infection-induced inflammation.

[0120] Example 3; Endothelial cells experiment Human umbilical vein endothelial cells (HUVECs) were obtained from the RuG / UMCG Endothelial Cell Facility. Primary isolates from the umbilical cord were mixed and then cultured in HUVEC culture medium consisting of RPMI 1640 (Lonza, art.nr.BE12-115F) supplemented with 20% heat-inactivated fetal bovine serum (ThermoFisher Scientific, art.nr.10082147), 2 mM 1-glutamine (Life Technologies, art.nr.25030), 5 U / ml heparin (Leo Pharmaceutical Products), 1% penicillin / streptomycin (Sigma-Aldrich, art.nr.P4333), and 50 μg / ml EC growth factor supplementation with (Sigma-Aldrich, art.nr.E2759).

[0121] Primary HUVEC cells were cultured in a 75 cm² tissue culture flask (Corning art.nr.430720U) at 37°C under 5% CO2 / 95% air conditions. HUVEC cells were used for experiments up to passage 8. Experiments were performed in 6-well (Corning art.nr.3506) or 96-well culture plates (Corning art.nr.3596) at 80% confluence. Cells were stimulated with LPS E. coli 0111:B4 (Sigma-Aldrich art.nr.L2630) at different concentrations. Cells were detached with trypsin (Sigma-Aldrich art.nr.25300054). All compounds were dissolved in Hanks equilibrium salt solution (Lonza art.nr.10-527F).

[0122] HUVECs were pre-incubated with 10 micrograms / ml of SUL-138 added one hour prior to LPS stimulation. Such pre-incubation is standard practice for in vitro models.

[0123] Membrane potential was measured using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide). Mitochondrial membrane potential was measured after 30 minutes.

[0124] Cells and mitochondria were incubated and measured at 548 / 574 nm excitation / luminescence rates using a Synergy H4 microplate reader (Bio-Tek) according to the manufacturer's protocol.

[0125] result Figure 6 shows that SUL-138 protects against LPS-induced mitochondrial dysfunction and cell death in vitro.

[0126] Figure 6A shows the respiration of HUVECs, demonstrating a reduction in decoupled state (CCCP) with 10 ug / ml LPS compared to controls. LPS-challenged HUVECs treated with SUL-138 nearly recover uncoupled respiration.

[0127] Figure 6B shows the LPS-induced increase in mitochondrial oxidative stress as measured by MitoSOX. Since MitoSOX measures ROS production in mitochondria, SUL-138 reduces LPS-induced mitochondrial oxidative stress.

[0128] Figure 6C shows that cell viability decreased after 48 hours with LPS in HUVEC, while it recovered after 48 hours with SUL-138. Cell death was measured by cyquant.

[0129] Significant differences are calculated using an unpaired one-sided Student's t-test between LPS and the control and LPS / SUL-138.

[0130] Consideration Experiments conducted on LPS-treated endothelial cells showed that sepsis at the cellular level involved mitochondrial dysfunction, which was restored by the compounds according to the present invention, while overall cell viability was significantly increased by the use of SUL-138.

[0131] While not bound by theory, the inventors believe that the compounds according to the present invention can be used to treat or prevent sepsis because they protect cells from LPS-induced mitochondrial dysfunction and cell death.

[0132] Example 4 (In vitro test using several SUL compounds) The following compounds were tested. [Table 1-1] [Table 1-2]

[0133] Experimental Design HUVEC cell culture Human umbilical cord endothelial cells (HUVEC, Lonza CC-2519) were maintained in RPMI1640 medium coated with 1% gelatin, containing 20% ​​fetal bovine serum, 2 mM glutamine (Sigma-Aldrich), 1% penicillin-streptomycin solution (Sigma-Aldrich), and 50 μg / ml bovine pituitary extract (Invitrogen). When the culture reached a confluence of 70%, the HUVECs were subculturized by trypsin treatment. In all experiments, HUVECs were 0.6-10 5 cells / cm 2 The seeds were sown and left to adhere for 24 hours.

[0134] NRK52E cell culture Rat kidney epithelial cells (ATCC #CRL-1571) were maintained in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Sigma-Aldrich). NRK52E cells were passaged by trypsin treatment when the culture reached a 70% confluence. In all experiments, NRK52E cells were passed 0.6-10 times. 5 cells / cm 2 The seeds were sown and left to adhere for 24 hours.

[0135] Inflammation activation after endotoxin exposure HUVEC and NRK52E cells were pre-incubated with SUL compound (10 μM) for 30 minutes, followed by exposure to LPS for 24 hours under standard culture conditions. Cells were lysed with triZOL reagent (Invitrogen), and total RNA was isolated according to the manufacturer's instructions. 1 μg of total RNA per sample was reverse transcribed using the FirstAid Reverse Transcription Kit (ThermoFisher). 5 ng of copy DNA equivalent to total RNA was amplified using a ViiA7 real-time PCR system (ThermoFisher, Waltham, MA) with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) and tumor necrosis factor alpha-specific primers, interleukin-1 beta, interleukin-6, and beta-actin as loading controls. Amplification was performed for 40 cycles of 30 seconds at 95°C followed by 1 minute at 60°C. Relative mRNA expression was...

number

[0136] Statistical evaluation All experiments were conducted in at least three sets for each condition. Data obtained from individual experiments were used for evaluation using GraphPad Prism 9.0 (GraphPad Software Inc, Ca). Statistical significance was calculated using ANOVA and subsequent Bonferroni post-hoc analysis. A probability value (p) less than 0.05 was considered significant.

[0137] result Inflammatory activation of the endothelium and epithelium.

[0138] Endothelial inflammation activation plays a crucial role in regulating the inflammatory process during sepsis. Endothelial cells, triggered by endotoxins or other pro-inflammatory molecules, initiate the production of inflammatory cytokines, which then recruit and replenish inflammatory cells, triggering an inflammatory response.

[0139] Endothelial cells and renal epithelial cells exhibit very low baseline gene expression of inflammatory cytokines, which increases significantly upon exposure to endotoxins such as LPS. Pretreatment of endothelial cells or renal epithelial cells with SUL compounds (10 μM in all 30 mm of cells) inhibits the induction of mRNA expression of the pro-inflammatory cytokine TNFα and the interleukins IL-1β and IL-6, with varying degrees of efficacy, prior to LPS challenge, as shown in the following table. [Table 3]

[0140] This table clearly shows that compounds not according to the present invention exhibit an inhibition rate of approximately 35% or less, while compounds according to the present invention exhibit an inhibition rate of approximately 40% or more.

[0141] Furthermore, a comparison between SUL-150 and SUL-151 shows that the effect is independent of the enantiomer configuration. A comparison between SUL-138 and SUL-138M2 shows that the compound according to formula 2 is not very desirable, but it does exhibit activity.

[0142] Example 5 and Comparative Experiment A In this study, the mechanisms of action of SUL compounds SUL-138 and SUL-150 (as described herein) were compared with those of BN-80933 (Comparative A, see the introduction above for structure) within the scope of in vitro experiments focusing on NO release, inhibition of cytoplasmic and mitochondrial oxidative stress, inflammatory signaling, and cell survival after hypothermic rewarming stress. These in vitro assays are considered appropriate indicators of their mechanisms of action because they were used before the efficacy of the specified compounds was established.

[0143] Experimental Design HUVEC cell culture Human umbilical cord endothelial cells (HUVEC, Lonza CC-2519) were maintained in RPMI1640 medium coated with 1% gelatin, containing 20% ​​fetal bovine serum, 2 mM glutamine (Sigma-Aldrich), 1% penicillin-streptomycin solution (Sigma-Aldrich), and 50 μg / ml bovine pituitary extract (Invitrogen). When the culture reached a concentration density (confluence) of 70%, the HUVECs were subculturized by trypsin treatment. In all experiments, HUVECs were subculturized to 0.6-10 5 cells / cm 2 The seeds were sown and left to adhere for 24 hours.

[0144] RAW264.7 cell culture Mouse macrophage RAW264.7 cells (ATCC#TIB-71) were maintained in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Sigma-Aldrich). When the culture reached a concentration density (confluence) of 70%, the RAW264.7 cells were subculturized by trypsin treatment. In all experiments, RAW264.7 cells were subculturized to 0.5-10 5 cells / cm 2 The seeds were sown and left to adhere for 24 hours.

[0145] Measurement of NO gas Cellular NO production was estimated by measuring nitrate concentrations in the culture medium using the Measure-IT® High-Sensitivity Nitrite Assay Kit (#M36051, ThermoFisher) according to the manufacturer's instructions. The produced NO is unstable, with a half-life of 2–30 seconds, and rapidly reacts with oxygen molecules to form nitrite. Nitrite is further oxidized to nitrate in the cell culture medium. Therefore, the nitrate concentration in the conditioned medium is suitable for evaluating cellular NO production. 50 μl of conditioned medium from a single 96-well plate was used for each assay and normalized to protein concentration. All experiments were performed in 4-cycle configurations.

[0146] Measurement of oxidative stress in LPS-induced cells and mitochondria.

[0147] The generation of cellular and mitochondrial oxidative stress was measured using fluorescent probes for cellular and mitochondrial superoxide, i.e., dihydroethidium (DHE) and MitoSox (ThermoFisher), respectively. HUVECs were incubated at 30 mm with BN-80933, SUL-138, or SUL-150 (all 10 μM). Next, HUVECs were exposed to LPS (100 ng / ml) for a further 24 hours. At the end of incubation, 2.5 μM DHE or MitoSox was applied to each sample. HUVECs were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. Nuclear staining was performed with 5 μM 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), and the fluorescence intensity of the samples was recorded using a CLARIOStar Plus plate reader (BMG Labtech) with an appropriate filter set. Fluorescence recordings for DHE and MitoSox were corrected for DAPI fluorescence. All experiments were performed using a quad-sequence setup.

[0148] Inflammation activation after endotoxin exposure HUVEC cells were lysed with triZOL reagent (Invitrogen), and total RNA was isolated according to the manufacturer's instructions. 1 μg of total RNA per sample was reverse transcribed using the FirstAid Reverse Transcription Kit (ThermoFisher). The data was then processed using a ViiA7 real-time PCR system (ThermoFisher, Waltham, MA) with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) and tumor necrosis factor alpha-specific primers (TNFα; sense 5'-CAGCCTCTTCTCCTTCCTGAT-3', antisense 5'-GCCAGAGGGCTGATTAGAGA-3'), interleukin-1 beta (IL-1β; sense 5'-AAGCTGGAATTTGAGTCTGC-3', antisense 5'-ACACAAATTGCATGGTGAAG-3'), interleukin-6 (IL-6; sense 5'-AGCTCAATAAGAAGGGGCCTA-3', antisense 5'-TGAGAAACCCTGGCTTAAGTAGA-3'), and beta-actin (ACTB; sense 5'-CCAACCGCGAGAAGATGA-3', antisense 5'-CCAGAGGCGTACAGGGATAG-3') were used as loading controls to amplify copy DNA equivalent to 5 ng of total RNA. Amplification was performed for 40 cycles of 30 seconds at 95°C followed by 1 minute at 60°C. Relative mRNA expression was calculated according to the δδCt method.

[0149] Quantification of cell viability after hypothermic reheating stress.

[0150] HUVECs were pretreated with BN-80933, SUL-138 or SUL-150 (all at 10 μM) for 1 hour in a humidified incubator at 37 °C. Following pretreatment, HUVECs were placed in the refrigerator (2 - 8 °C) for 24 hours and then returned to 37 °C for 3 hours of rewarming. After 1 hour of rewarming, the cell culture medium was replaced with preheated and filtered 0.4 mg / mL neutral red (#N4638, Sigma - Aldrich) in the culture medium. After the remaining 2 - hour rewarming period, HUVECs were washed with PBS and NRU was solubilized in 100 μL of absorbance solution (50% ethanol, 1% acetic acid in dH2O). Absorbance was measured at a wavelength of 550 nm using a BioTek ELx808 plate reader. The recorded OD 550nm values were normalized against control samples that were maintained at 37 °C at all times and were assumed to be 100% viable.

[0151] Statistical evaluation All experiments were performed in at least triplicate for each condition. Data obtained from individual experiments were used for evaluation with GraphPad Prism 9.0 (GraphPad Software Inc, Ca). All datasets were normalized against vehicle controls. ANOVA followed by Bonferroni post - analysis was used to calculate statistical significance. Probability values (p) less than 0.05 were considered significant.

[0152] Results< 1. Endothelial NO production after endotoxin (LPS) challenge Basal endothelial NO production, measured by nitrate levels in the conditioned medium of HUVECs, was on average 239 pmol / mg protein (Figure 7A). BN - 80933 suppressed endothelial NO production by approximately 38% (p < 0.001), while the SUL compounds did not alter basal NO production (Figure 7A).

[0153] Endotoxins suppress endothelial NO production by limiting eNOS activity. HUVEC cells were pre-treated with a vehicle (DMSO), BN-80933, SUL-138, or SUL-150 (all 10 μM), and then exposed to endotoxin (LPS 100 ng / ml) for 24 hours. Nitrate levels in conditioned medium from LPS-exposed endothelial cells were an average of 160 pmol / mg protein (approximately 33% decrease compared to non-LPS-exposed HUVEC cells, p=0.002), which was lower than the baseline control level. BN-80933 further suppressed nitrate levels in conditioned medium to an average of 90 pmol / mg protein (p=0.008) (Figure 7A). In contrast, SUL-138 (p=0.008) and SUL-150 (p=0.004) mitigated the LPS-induced decrease in nitrate levels, and their levels did not change from basal nitrate levels.

[0154] 2. Macrophage NO production after endotoxin (LPS) challenge Under physiological conditions, inflammatory cells produce very limited amounts of NO, but under immunological stress such as endotoxin challenge, NO production by inducible nitric oxide synthase (iNOS) is promoted. Basal NO production, as measured by nitrate levels in the conditioned medium of RAW264.7 macrophages, was minimal, averaging 5 pmol / mg protein (Figure 7B). None of BN-80933, SUL-138, or SUL-150 affected basal extracellular nitrate production.

[0155] LPS induces macrophage NO production by activating iNOS. RAW264.7 macrophages were pre-treated with a vehicle (DMSO), BN-80933, SUL-138, or SUL-150 (all 10 μM), and then exposed to endotoxin (LPS 100 ng / ml) for 24 hours. Nitrate levels in conditioned medium from LPS-exposed RAW264.7 macrophages were an average of 83 pmol / mg protein (approximately a 16-fold increase compared to unexposed RAW264.7 cells, p=0.001). BN-80933, consistent with its function as an NOS inhibitor, slowed the LPS-induced increase in extracellular nitrate levels (p<0.001, Figure 7B). Neither SUL-138 nor SUL-150 strongly suppressed the LPS-induced increase in nitrate production by RAW264.7 macrophages (Figure 7B).

[0156] 3. Superoxide production in cells and mitochondria HUVEC is cytoplasm (i.e., DHE) and mitochondrial (i.e., MitoSox) O2 - It generates a baseline level of fluorescence, which is significantly enhanced by LPS exposure (Figures 7C, 7D). Pretreatment with BN-80933, SUL-138, and SUL-150 (all at 10 μM for 30 minutes) mitigates the induction of cytoplasmic oxidative stress with comparable effect (Figure 7C). SUL-138 and SUL-150 also eliminated the LPS-induced increase in mitochondrial oxidative stress, which was unaffected by pretreatment with BN-80933 (Figure 7D) (Figure 7C).

[0157] These data show that BN-80933, SUL-138, and SUL-150 all possess specific antioxidant capabilities, but these capabilities vary depending on the cellular compartment, etc.

[0158] 4 Endothelial inflammation activation Endothelial cells exhibit low baseline gene expression of inflammatory cytokines, which dramatically increase in various sizes upon exposure to endotoxins (Figures 8A-C). Pretreatment with BN-80933, SUL-138, and SUL-150 (all at 10 μM for 30 minutes) similarly eliminates the induction of mRNA expression of the pro-inflammatory cytokine TNFα (Figure 8A). In particular, pretreatment of HUVEC with BN-80933 does not eliminate LPS-induced mRNA expression of interleukins IL-1β and IL-6 (Figures 8B, 8C), but SUL-138 and SUL-150 also eliminate the LPS-induced increase of IL-1β and IL-6 (Figures 8A-8C).

[0159] 5. Endothelial cell survival rate after hypothermic rewarming stress To investigate different mechanisms of action, endothelial cells were exposed to hypothermia as a model of mitochondrial damage. Endothelial cells subjected to a 24-hour cooling-reheating cycle at 4°C followed by a 3-hour cooling-reheating cycle at 37°C showed significantly reduced cell viability (approximately 61%) compared to normal-temperature control cells (Figure 8D). Pretreatment with BN-80933 did not affect the hypothermia-related decrease in cell viability (approximately a 55% decrease), but both SUL-138 and SUL-150 maintained cell viability at a level just below that of normal-temperature control cells (approximately a 15% decrease). This indicates that the mechanisms of action of SUL-138 and SUL-150 are different from those of BN-80933.

[0160] Consideration Investigating the mechanisms of action of BN-80933, SUL-138, and SUL-150 in relation to endotoxin-induced cell damage and hypothermic rewarming stress leads to the following conclusions. 1. Unlike SUL-138 or SUL-150, BN-80933 is a common inhibitor of nitric oxide synthase (NOS) enzyme in endothelial cells and macrophages. 2. BN-80933, SUL-138, and SUL-150 have cytoplasmic antioxidant properties. 3. SUL-138 and SUL-150 have mitochondrial antioxidant properties, but BN-80933 does not. 4. SUL-138 and SUL-150 inhibit endotoxin-induced endothelial cell activation for all TNFα, IL-1β, and IL-6. BN-80933 inhibited only TNFα. 5. SUL-138 and SUL-150 eliminate reheating damage to endothelial cells due to hypothermia, but BN-80933 does not.

[0161] In summary, these data indicate that BN-80933 and SUL compounds have very different mechanisms of action and are active in different cellular compartments. BN80933, in contrast to SUL compounds, is inactive in mitochondria.

[0162] conclusion Examples demonstrate that SUL-138 restores xiphoid process temperature 24 hours after signs of CLP in mice. Treatment with SUL-138 reduces plasma cytokine levels after sepsis induction. In particular, CLP-induced renal dysfunction is prevented or significantly reduced by treatment with SUL-138 in mice, as demonstrated by the restoration of NGAL and urea function.

[0163] The examples also show that SUL-151 reduces mortality in septic Drosophila melanogaster and improves geotaxis at 24 and 48 hours after sepsis induction.

[0164] In endothelial cells of sepsis patients, SUL-138 restores mitochondrial dysfunction.

[0165] BN-80933 and SUL compounds have significantly different mechanisms of action and are active in different cellular compartments. In contrast to SUL compounds, BN80933 is inactive in mitochondria.

[0166] Further in vitro experiments have shown that many SUL compounds are active in in vitro models of sepsis, indicating that the current findings are applicable to the group of compounds.

[0167] The combined in vivo and in vitro evidence indicates that the compounds defined by this invention are effective in treating sepsis and organ dysfunction caused by infection-induced inflammation.

Claims

1. A pharmaceutical composition for use in the treatment or prevention of sepsis, comprising a compound according to formula (I) or (II), a hydroquinone analog according to formula (II), or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 【Chemistry 2】 -In the formula, R1 represents hydrogen or a prodrug portion that can be removed in biological tissue, -i. R2 and R3, together with the N atom to which they are bonded, form a 5- to 8-membered saturated ring that incorporates an additional N atom, and this ring is either unsubstituted or substituted with an alcohol or an alkanol group having 1 to 4 carbon atoms. ii. Alternatively, a pharmaceutical composition in which R2 is a hydrogen atom, and R3 comprises a saturated cyclic structure having 4 to 7 carbon atoms and 1 nitrogen atom, and this ring can be substituted with an alkyl group, an alcohol group, or a group having 1 to 4 carbon atoms that may include an oxygen, carboxylic acid, or amine group.

2. The pharmaceutical composition for use according to claim 1, wherein R1 is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen.

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the compound is a compound according to formula I.

4. The pharmaceutical composition for use according to claim 3, wherein R2 and R3, together with the N atom to which they are bonded, form a 5- to 7-membered ring comprising one additional amine group, and this ring is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

5. The pharmaceutical composition for use according to claim 1, wherein the compound is a compound according to formula II, in which R2 is a hydrogen atom, and R3 comprises a cyclic structure having 4 to 6 carbon atoms and 1 nitrogen atom, and this ring is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

6. The pharmaceutical composition for use according to claim 1, wherein the compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidine-3-yl)chroman-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-109), or a pharmaceutically acceptable salt thereof, as a racemic mixture or as one of its enantiomers.

7. The pharmaceutical composition for use according to claim 6, wherein the compound is the S-enantiomer of SUL-109: S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazine-1-yl)methanone (SUL-138), or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition for use according to claim 6, wherein the compound is the S-enantiomer of SUL-121: S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazine-1-yl)methanone (SUL-151), or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition for use according to any one of claims 1 to 5, wherein the compound according to formula (I) or formula (II), as defined in options (i) and (ii), has a molecular weight of less than 500 Da, preferably less than 450 Da, and most preferably less than 400 Da.

10. A pharmaceutical composition for use according to any one of claims 1 to 9, wherein the use is for the treatment or prevention of organ dysfunction caused by dysregulation of the host's response to infection.

11. The pharmaceutical composition for use according to claim 10, wherein the organ is one or more of the lungs, heart and blood vessels, liver, kidney, brain, or intestines, preferably the kidney.

12. A pharmaceutical composition for use according to any one of claims 1 to 11, wherein the treatment or prevention is carried out in combination with one or more common means for treating sepsis.

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