Novel compositions and methods of treatment

Abstract

To provide a medical use of a compound for the treatment and / or management of disorders related to sepsis, septic shock, ocular inflammation, ocular neovascularization, etc.SOLUTION: Provided is a pharmaceutical composition containing the compound represented by formula (VI) or a pharmaceutically acceptable salt thereof. (n=0-1. R=benzyl, substituted benzyl. R1=COCH3, N-dimethyl maleimide. R2=cyclohexyl, p-nitrophenyl, piperidine nitroxy, piperidine-N-hydroxyl, p-methoxyphenyl.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 315,144, filed March 30, 2016, which is incorporated herein by reference.

[0002] The present invention relates to novel products, their variants, pharmaceutically acceptable salts and prodrugs, and methods for treating sepsis, septicemia, septic shock, ocular infections, ocular inflammation, ocular neovascularization, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel diseases (IBD), asthma, The medical use of such compounds for the treatment and / or management of chronic obstructive pulmonary disease, fever syndromes, cachexia, psoriasis, autoimmune diseases, heart disease, retinoblastoma, cancer, and / or any disorder associated with inflammation, immunomodulation, and microbial infection. [Background technology]

[0003] Sepsis has been identified as one of the five most expensive hospitalizations in the United States. Sepsis outcomes are particularly unfavorable in elderly, immunocompromised, and critically ill patients. In addition to its clinical challenges, treating sepsis imposes a significant economic burden on healthcare systems worldwide. More than 900,000 cases are estimated to occur annually in the United States alone, with total annual costs estimated at approximately $26 billion nationwide. Currently, sepsis is typically treated using one of three broad supportive (non-antibiotic) therapeutic approaches: (1) improved supportive care (i.e., oxygen therapy / ventilation strategies, fluid optimization / vasopressor use, early goal-directed therapy), (2) targeting bacterial virulence factors (i.e., antiendotoxin antibodies, endotoxin removal columns), or (3) targeting host response factors (i.e., corticosteroids, anticytokine drugs, anticoagulants). However, current therapies are not completely effective in patients with sepsis and septic shock. These treatments are even less effective in immunocompromised and elderly patients. Summary of the Invention [Means for solving the problem]

[0004] The present invention overcomes the shortcomings of the prior art by providing low molecular weight, water-soluble oligosaccharides (compounds 1-3) that exhibit antagonist activity against TLR4, useful for treating inflammatory conditions, such as the pathogenesis of age-related macular degeneration (AMD). Such inflammatory conditions include, but are not limited to, ocular inflammatory diseases and choroidal neovascularization. Specifically, the present invention relates to compounds (4, 15, 16, and 25) for the treatment of inflammation. In some embodiments, the inflammation may be caused by polymicrobial infection. In some embodiments, the compounds find use in the treatment of sepsis and severe sepsis, SIRS, and septic shock.

[0005] The compounds of the present invention have unexpectedly been found to have excellent anti-inflammatory activity in inhibiting inflammatory biomarkers such as TNF-α, IL-1β, and IL-6 in an LPS-induced human monocyte assay and in producing the anti-inflammatory cytokine IL-10 in monocytes. The compounds of the present invention protected mice from both lethal Gram-negative sepsis caused by E. coli and lethal polymicrobial sepsis in a cecal ligation and puncture model. Thus, the present disclosure provides a method for inhibiting inflammatory biomarkers, such as, but not limited to, TNF-α, IL-1β, and IL-6, in an LPS-induced human monocyte assay by administering the compounds disclosed herein to a patient in need thereof. Furthermore, the present disclosure provides a method for inhibiting inflammatory biomarkers, such as, but not limited to, TNF-α, IL-1β, and IL-6, in an LPS-induced human monocyte assay by administering the compounds disclosed herein to a patient in need thereof. The present invention provides a method for protecting mice from lethal Gram-negative sepsis caused by Escherichia coli by administering the compounds disclosed herein to a patient in need thereof. The compounds of the present invention have also been unexpectedly shown to possess broad-spectrum antibacterial activity against both Gram-positive bacteria (methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae), and a fungus (Candida albicans) primarily found in burns and septic wounds. Accordingly, the present disclosure provides methods for treating infections caused by both Gram-positive bacteria (methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae), and fungi (Candida albicans) by administering the compounds disclosed herein to a patient in need thereof. The compounds of the present invention have also unexpectedly been found to inhibit and eradicate biofilms formed by Staphylococcus aureus. Accordingly, the present disclosure provides methods for inhibiting or eradicating biofilms formed by microorganisms, such as, but not limited to, Staphylococcus aureus, by contacting a surface with a compound disclosed herein. The compounds of the present invention also protect mice against cecal ligation and puncture (CLP)-induced death and organ dysfunction. The compounds of the present invention were also unexpectedly found to have excellent activity against HMGB1-induced inflammation in mouse macrophages, reduced VEGF expression in retinal pigment epithelial cells (ARPE-19), and daily intraperitoneal injections of the compounds reduced the average size of CNV lesions compared to control mice treated with vehicle alone. The compounds disclosed herein were able to reduce the average size of CNV lesions in mice by about 60%. Thus, the present disclosure provides a method of protecting a subject in need of such treatment from an infection or an infection-related disorder by treating said subject with the compounds disclosed herein.

[0006] The foregoing brief summary broadly describes the features and technical advantages of certain embodiments of the present invention. Additional features and technical advantages are set forth in the following detailed description of the invention. The novel features believed to be characteristic of the present invention will be better understood from the detailed description of the invention when considered in conjunction with any accompanying drawings. However, the drawings provided herein are intended to aid in explaining the invention or to aid in developing an understanding of the invention, and are not intended as a definition of the scope of the invention. That is, the present invention relates to the following: 1. Formula (VI) TIFF0007774885000001.tif36162 (In the formula, n=0~1 R=benzyl, substituted benzyl, R1 = COCH3, N-dimethylmaleimide, R2 = cyclohexyl, p-nitrophenyl, piperidine nitroxy, piperidine-N-hydroxyl, p-methoxyphenyl) or a pharmaceutically acceptable salt thereof; 2. The composition according to claim 1, formulated into a sterile injectable aqueous or oily suspension, or a sterile topical gel, ointment or aqueous spray; 3. The composition according to claim 1, further comprising at least one of an anti-inflammatory agent or an antibacterial agent. 4. Use of the composition according to 1 above for treating sepsis, septic shock, toxemia, burns or wound infections, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (VI) or a pharmaceutically acceptable salt thereof, thereby treating said subject; 5. The use according to 4 above, wherein the administering comprises administering to a patient in need thereof a pharmaceutical composition comprising about 5.0 mg to about 100 mg of a compound of formula (VI) or a pharmaceutically acceptable salt thereof, thereby treating the patient. 6. The use according to 4 above, wherein the administering comprises administering to a patient in need thereof a pharmaceutical composition comprising about 10.0 mg to about 1000 mg of a compound of formula (VI) or a pharmaceutically acceptable salt thereof, thereby treating the patient. 7.Formula (V) TIFF0007774885000002.tif39160 (In the formula, n=0~5 R=H, C(O)CH3, C(O)-piperidine nitroxy, C(O)-piperidine N-hydroxyl R1 = alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, piperidine nitroxyl, piperidine N-hydroxyl or a pharmaceutically acceptable salt thereof; 8. The composition according to claim 7, which is formulated into a sterile injectable aqueous or oily suspension, or a sterile topical gel, ointment or aqueous spray; 9. The composition according to claim 7, further comprising at least one of an anti-inflammatory agent or an antibacterial agent. 10. Use of the composition according to 7 above for treating sepsis, septic shock, toxemia, burns or wound infections, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (V) or a pharmaceutically acceptable salt thereof; 11. The use according to the above item 10, wherein the pharmaceutical composition comprises about 5.0 mg to about 100 mg of the compound of formula (V) or a pharmaceutically acceptable salt thereof for a patient in need thereof, thereby treating the patient, or the pharmaceutical composition comprises about 10.0 mg to about 1000 mg of the compound of formula (V) or a pharmaceutically acceptable salt thereof for a patient in need thereof, thereby treating the patient; 12.Formula (II) TIFF0007774885000003.tif39151 (wherein n=5 to 7) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable vehicle containing PLA or PLGA microparticles. 13. The composition according to claim 12, which is formulated into a sterile injectable aqueous or oily suspension or as a sterile topical ophthalmic solution. 14. The composition according to claim 12, further comprising at least one of an anti-inflammatory agent or an antibacterial agent. 15. Use of a composition of formula (II) in a therapeutically effective amount of a pharmaceutical composition or a pharmaceutically acceptable salt thereof for treating ocular neovascularization, ocular inflammation; 16. The use according to 15 above, wherein the pharmaceutical composition comprises about 5.0 mg to about 100 mg of the compound of formula (II) or a pharmaceutically acceptable salt thereof for a patient in need thereof, thereby treating the patient, or the pharmaceutical composition comprises about 10.0 mg to about 1000 mg of the compound of formula (II) or a pharmaceutically acceptable salt thereof for a patient in need thereof, thereby treating the patient. [Brief explanation of the drawings]

[0007] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] 1 is a graph showing the results of evaluation of Compound 1, a TLR4 antagonist of the present invention, in a mouse endotoxemia and sepsis model, demonstrating that Compound 1 protects mice from lethal Gram-negative sepsis caused by Escherichia coli. [Figure 2] 1 is a graph showing the results of evaluation of Compound 4, a TLR4 antagonist of the present invention, in a cecal ligation and puncture (CLP) model, demonstrating that Compound 4 protects mice from CLP-induced polymicrobial sepsis and death. [Figure 3] Upon treatment with Compound 4, histopathology of major organs demonstrates that the compound of the present invention reverses the major pathological changes, with tissues resembling those in the sham group. [Figure 4] It was demonstrated that compound 4 of the present invention effectively down-regulates inflammatory cytokines in vivo in CLP mice. [Figure 5] 1 is a graph showing the results of evaluation of compounds 15 and 25 of the present invention in a cecal ligation and puncture (CLP) model, demonstrating that both compounds protect mice from CLP-induced polymicrobial sepsis and death. [Figure 6]1 shows the evaluation of the TLR4 antagonist Compound 2 of the present invention in a laser-induced CNV mouse model of wet AMD. Compound 2 reduced choroidal neovascularization by approximately 60% compared to the positive control. [Figure 7A] ~ [Figure 7B] A series of compounds of the present invention are shown to effectively bind to the TLR4 receptor and not to the TLR2 receptor. [Figure 8A] ~ [Figure 8B] We demonstrate that compounds of the invention inhibited LPS-induced production of inflammatory mediators in human monocytes. [Figure 9] We demonstrate that compounds of the present invention inhibit HMGB1-induced production of an inflammatory mediator (TNF-α) in mouse bone marrow-derived macrophages. [Figure 10] We demonstrate that compounds of the present invention inhibit HMGB1-induced production of inflammatory mediators (TNF-α, i-NOS) and upregulate the M2 biomarker CXCR4 in mouse macrophages. [Figure 11] The compounds of the present invention are shown to produce the anti-inflammatory cytokine IL-10. [Figure 12] 1 shows that compounds of the present invention (1 and 2) reduced HMGB1-induced production of VEGF in ARPE-19 cells. [Figure 13] It is shown that the compounds of the present invention have broad spectrum antibacterial activity. [Figure 14] It is demonstrated that compounds of the present invention inhibited biofilm formation. [Figure 15] The broad spectrum antibacterial activity of the compounds of the present invention is shown to be due to disruption of cell membranes. [Figure 16] It is demonstrated that the compounds of the present invention do not bind to plasma proteins, serum proteins. [Figure 17] It is shown that the compounds of the present invention are not toxic to fibroblasts. [Figure 18] 1 shows a synthetic scheme for preparing compound 11. [Figure 19] A synthetic scheme for preparing compounds 22-23 is provided. [Figure 20] A synthetic scheme for preparing compounds 24-25 is provided. DETAILED DESCRIPTION OF THE INVENTION

[0008] In sepsis caused by Gram-negative bacteria, lipopolysaccharide (LPS) activates the immune system through the signaling receptor Toll-like receptor 4 (TLR4), initiating the production of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and reactive oxygen species (ROS)) that contribute to excessive inflammation. Therefore, some researchers have attempted to develop antagonists that block either TLR activation or downstream signaling pathways to inhibit the storm of inflammatory molecules. However, with the exception of the LPS analog eritoran, which failed in clinical trials likely due to poor clinical trial design, no targeted molecules have progressed beyond experimental and preclinical studies (Opal, S.M.; Laterre, P.; Francois, B.; et al., Effect of eritoran, an antagonist of md2-TLR4, on mortality in patients with severe sepsis: The access randomized trial. JAMA 2013, 309, 1154-1162). The underlying mechanisms of action of most other classes of compounds are not fully understood (Leon, Carlos G.; Tory, Rita; Jia, Jessica; Sivak, Olena; Wasan, Kishor M. a. Pharmaceutical Research (2008), 25(8), 1751-1761; Savva Athina; Roger Thierry From Frontiers in Immunology (2013),4387, Language: English, Database: MEDLINE).

[0009] The compounds of the present invention synergistically inhibit inflammatory microbial infections and have therapeutic potential for treating sepsis, septicemia, and septic shock. It is a small molecule that can upregulate M2 biomarkers such as

[0010] Posterior ocular neovascular diseases, exemplified by proliferative diabetic retinopathy (PDR), exudative age-related macular degeneration (AMD), and retinopathy of prematurity (ROP), pose an immense and growing health threat and require effective new therapies. Retinal neovascularization associated with PDR is the leading cause of blindness in working-age adults. Choroidal neovascularization (CNV) accounts for 200,000 new cases of exudative AMD each year in the United States, and this neovascular condition is the leading cause of legal blindness in non-Third World countries. The estimated number of AMD patients in 2020 was 196 million, and is expected to increase to 288 million by 2040 (Wong, W.L.; Su, X.; Li, X.; Cheung, C.M.G.; Klein, R.; Cheng, C.-Y.; Wong, T.Y., Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. The Lancet Global Health 2014, 2, e106-e116). Pathological angiogenesis associated with retinopathy of prematurity (RPP) is the leading cause of blindness in children under the age of 7 (Harrell, S.N.; Brandon, D.H., Retinopathy of Prematurity: The Disease Process, Classifications, Screening, Treatment, and Outcomes. Neonatal Network 2007, 26, 371-378).

[0011] Multiple lines of evidence suggest that Toll-like receptor 4 (TLR4) signaling may be associated with pathological changes in retinal diseases, including AMD eyes due to oxidized lipids, lipofuscin, and drusen components (Cho, Y.; Wang, J.J.; Chew, E.Y.; Ferris, F.L.; Mitchell, P.; Chan, C.-C.; Tuo, J., Toll-like Receptor Polymorphisms and Age-Related Macular Degeneration: Replication in Three Case-Control Samples. Investigative Ophthalmology & Visual Science 2009, 50, 5614-5618). Once activated, TLR4 mediates the production of TNF-α, interleukin-1β, and other pro-inflammatory cytokines. TLR4 may contribute to the pathogenesis of AMD through multiple mechanisms, including the release of mediators. TLR4 activation suppresses Wnt signaling, reduces growth factor expression and secretion, and increases photoreceptor death in response to oxidative stress, potentially causing oxidative damage to the photoreceptor outer segments. TLR4 has been shown to directly affect several inflammation-related signaling pathways, including MAPK, NFκ-β, and Jak1 / Stat1, and to mediate neurotoxicity via caspase-3, neuronal iNOS, ERK1 / 2, JNK1 / 2, and p38. Interestingly, TLR4-mediated microglial activation by endogenous photoreceptor proteins during retinal inflammation can exacerbate retinal cell death. Finally, release of high-mobility group box 1 in ischemic neural tissue has been shown to initiate TLR4-dependent responses that contribute to retinal neovascularization (He, C.; Sun, Y.; Ren, X.; Lin, Q.; Hu, X.; Huang, X.; Su, S.-B.; Liu, Y.; Liu, X., Angiogenesis mediated by toll-like receptor 4 in ischemic neural tissue. Arterioscler Thromb Vasc Biol. 2013, 33(2):330-8).

[0012] Therefore, there is a need for more effective treatments for inflammation, especially for both dry and wet AMD pathogenesis.Therefore, the compounds and methods described herein demonstrate that inhibiting TLR4 activity has therapeutic value in AMD and other retinal diseases.The compounds of the present invention are small molecules that can synergistically inhibit angiogenesis, inflammation, and promote phagocytosis, and have therapeutic potential for treating AMD.

[0013] However, prior to the present disclosure, there appear to be no published reports of the use of chitohexaose (compound 1), chitohepatose (compound 2) and chitooctaose (compound 3) and their derivatives as TLR4 antagonists to inhibit inflammation and angiogenesis in ocular indications such as dry / wet AMD, diabetic retinopathy, or any chronic ocular inflammation.

[0014] In one embodiment, the principles of the present disclosure provide a compound of formula (I):

[0015] [ka] (In the formula, R=H, C(O)R1, alkyl, benzyl, substituted benzyl, R1 = CH3, alkyl, piperidine nitroxyl R2 = H, C(O)R1, or An acyloxyalkyl carbamate of the formula: R2 = C(O)OCHR3OC(O)OR4, piperidine nitroxyl R3=H, CH3, C2H5, isopropyl R4 = optionally substituted alkyl group X=O, NH, S R5 = alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, heteroalkyl, heterocycloalkyl, piperidine nitroxyl, piperidine N-hydroxylamine (n=0 to 7) The present invention provides a compound of the formula:

[0016] In another embodiment, the present disclosure provides a compound of formula (II)

[0017] [ka] (wherein n=2 to 7) The present invention provides a compound of the formula:

[0018] Additional compounds of the present disclosure include the following structures shown below:

[0019] [ka]

[0020] Compounds (2, 3) of the present disclosure showed unexpectedly potent activity in inhibiting LPS- and HMGB1-induced inflammatory biomarkers (TNF-α, IL-1β, and IL-6) in mouse bone marrow-derived macrophages and human monocytes. Compounds 1 and 2 reduced VEGF production in ARPE-19 cells. Compound 2 showed a significant reduction in CNV size in a laser-induced mouse model of wet AMD. Compounds (1-3) of the present invention can be synthesized using the synthetic scheme shown in Figure 19 in conjunction with knowledge available in the prior art and can be modified as needed.

[0021] In another embodiment, the present invention provides a compound of formula (III)

[0022] [ka] (In the formula, n=0~5 R=COR1 R1=CH3, alkyl) The present invention provides a compound of the formula:

[0023] Additional compounds of the present invention include the following structures shown below:

[0024] [ka]

[0025] Compound (4) of the present invention showed unexpectedly excellent activity in inhibiting LPS-induced inflammatory biomarkers (TNF-α, IL-1β, and IL-6) in human monocytes. As shown in Figures 2-4, compound 4, administered intravenously (IV) at 10 mg / kg, demonstrated excellent efficacy in protecting mice in a cecal ligation and puncture (CLP) sepsis model against organ dysfunction and death, and statistically significantly downregulated inflammatory cytokines such as TNF-α, IL-1β, and IL-6.

[0026] The compounds of the present invention (4-6) can be synthesized using the reported procedures described in Mohamed R. E et al., Carbohydrate research, 2001, 331, 129-142.

[0027] In another embodiment, the present invention provides a compound of formula (IV)

[0028] [ka] 1. (In the formula, R=H and C(O)R1 R1 = piperidine nitroxyl or piperidine N-hydroxylamine (n=0 to 7) The present invention provides a compound of the formula:

[0029] Additional compounds of the present invention include the following structures shown below:

[0030] [ka]

[0031] [ka]

[0032] Compound (11) of the present invention showed unexpectedly excellent activity in inhibiting LPS-induced inflammatory biomarkers (TNF-α, IL-1β, and IL-6) in human monocytes. Thus, the compound finds use as an anti-inflammatory compound. Compounds (7-11) of the present invention can be synthesized using the synthetic scheme shown in Figure 18, in combination with knowledge available in the prior art.

[0033] In another embodiment, the present invention provides a compound of formula (V)

[0034] [ka] (In the formula, n=0~5 R=H, C(O)CH3, C(O)-piperidine nitroxy, C(O)-piperidine N-hydroxyl R1 = alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, piperidine nitroxyl, piperidine N-hydroxyl The present invention provides a compound of the formula:

[0035] Additional compounds of the present invention include the following structures shown below:

[0036] [ka]

[0037] Compounds (12-25) of the present invention demonstrated inhibition of LPS-induced inflammatory biomarkers (TNF-α, IL-1β, and IL-6) in human monocytes and upregulated the M2 biomarker IL-10, an anti-inflammatory cytokine. Compound 12 also demonstrated broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria and fungi. Compound 12 unexpectedly inhibited biofilm formation by MSSA and MRSA. Compound 25, when administered intravenously (10 mg / kg dose), demonstrated high survival and organ protection in a CLP mouse model of sepsis. Thus, the compounds described herein, in some embodiments, find use as anti-inflammatory molecules. In some embodiments, the compounds are anti-infective or antibacterial.

[0038] [ka] During the ceremony, n=0~1 R=benzyl, substituted benzyl, R1 = COCH3, N-dimethylmaleimide, R3 = cyclohexyl, p-nitrophenyl, piperidine nitroxy, piperidine-N-hydroxyl, p-methoxyphenyl.

[0039] Additional compounds of the present invention include the following structures shown below:

[0040] [ka]

[0041] The compounds of the present invention showed inhibition of LPS-induced inflammatory biomarkers (TNF-α, IL-1β, and IL-6) in human monocytes and upregulated IL-10. Compound 15 also exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria and fungi. Compound 15 unexpectedly inhibited biofilm formation by MSSA and MRSA. Compound 15 demonstrated high survival and organ protection in a CLP mouse model of sepsis when administered intravenously (5.0 mg / kg dose). The compounds of the present invention can be synthesized using the synthetic scheme shown in Figure 19, which was developed by the present inventors in conjunction with knowledge available in the prior art.

[0042] Furthermore, certain embodiments include pharmaceutically acceptable salts of the compounds according to the present invention. Pharmaceutically acceptable salts include, but are not limited to, soluble or dispersible forms of the compounds according to the present invention that are suitable for treating diseases without undue undesired effects, such as allergic reactions or toxicity. Representative pharmaceutically acceptable salts include, but are not limited to, acid addition salts such as acetate, citrate, benzoate, lactate, or phosphate, and base addition salts such as lithium, sodium, potassium, or aluminum.

[0043] formulation In some embodiments, the compounds of the present disclosure are incorporated into parenteral formulations. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, and intraarterial injection using various infusion techniques. As used herein, intraarterial and intravenous injection include administration via a catheter. Preferred for certain indications are administration methods that allow rapid access to the tissue or organ to be treated, such as intravenous injection for the treatment of endotoxemia or sepsis.

[0044] The compounds of the present disclosure are administered at a dose that adequately inhibits LPS activation of target cells, and generally, these doses are preferably 50-3000 mg / patient, or 100-2500 mg / patient, or 200-2000 mg / patient, or 500-1000 mg / patient, or 750-1000 mg / patient, more preferably 500-750 mg / patient, and most preferably 250-500 mg / patient. The dose is preferably administered once daily for 28 days, more preferably once daily for 28 days. Preferably, twice daily for 14 days, or most preferably, three times daily for 7 days.

[0045] Pharmaceutical compositions containing the active ingredient may be in any form suitable for the intended method of administration.

[0046] The aqueous suspensions of the present invention contain the active substance in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), and condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate.

[0047] The pharmaceutical compositions of the present invention are preferably in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to well-known techniques using the above-mentioned suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solution, or prepared as lyophilized powders. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any mild, fixed oil, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids, such as oleic acid, can also be used in the preparation of injectables.

[0048] In some embodiments, the formulation comprises PLA or PLGA microparticles, which may be further mixed with Na2HPO4, hydroxypropyl methylcellulose, polysorbate 80, sodium chloride, and / or edetate disodium.

[0049] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders of the kind described above.

[0050] However, it will be understood that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the particular compound used, the age, weight, general health, and sex of the individual being treated, the time and route of administration, excretion rate, other drugs previously administered, and the severity of the particular disease being treated.

[0051] In some embodiments, the compositions of the present disclosure also contain about 80% to about 99.5%, preferably about 90 or 95% to about 98.5%, of a compatible non-aqueous, pharmaceutically acceptable topical vehicle. Some vehicles are described in U.S. Pat. No. 4,621,075, which is incorporated herein for this disclosure. Preferably, these vehicles are water-free, although the compositions of the present invention may contain up to about 5% water without significantly adversely affecting the formation of the desired gel. These non-aqueous vehicle components are also well known in the pharmaceutical industry. These non-aqueous vehicle components include (but are not limited to) hydrocarbon oils and waxes, lanolin and lanolin derivatives, silicone oils, monoglycerides, diglycerides, and triglyceride esters, fatty alcohols, fatty acid alkyl and alkenyl esters, dicarboxylic acid alkyl and alkenyl diesters, polyhydric alcohols and their ether and ester derivatives, short-chain alcohols and ketones, such as wax esters and beeswax derivatives, and emollients. Preferred vehicles incorporate methanol, ethanol, n-propanol, isopropanol, butanol, polypropylene glycol, polyethylene glycol, and mixtures of these components. Particularly preferred vehicles include ethanol, n-propanol, and butanol, especially ethanol. These preferred solvents can also be combined with other components, such as diisopropyl sebacate, isopropyl myristate, methyl laurate, silicones, glycerin, and mixtures of these components, to provide non-aqueous vehicles useful in the present invention. Of these additional components, diisopropyl sebacate is particularly useful. Indeed, a preferred medium includes a mixture of ethanol and diisopropyl sebacate in a weight ratio of about 4:1 to about 1:4. A preferred medium contains about 15% to about 35% diisopropyl sebacate and about 65% to about 85% ethanol.

[0052] The compositions of the present invention may further contain compatible auxiliary ingredients conventionally used in the formulation of topical pharmaceutical compositions at use levels established in the art. These auxiliary ingredients may include, but are not limited to, pharmaceutically active ingredients (auxiliary antibacterial or anti-inflammatory ingredients, such as steroids), or ingredients used to enhance the formulation itself (excipients, dyes, fragrances, skin penetration aids, stabilizers, preservatives, antioxidants, etc.). Because the compositions of the present invention are capable of gel formation without the need for conventional gelling agents, such agents are preferably not included. Examples of such agents include pharmaceutically acceptable acidic gelling agents, such as Carbopol compounds commercially available from BFGoodrich Chemicals, Cleveland, Ohio. Carboxy polymers are included.

[0053] The gel compositions of the present invention can be formulated by conventional mixing of the above ingredients, with gel formation occurring within about 2 minutes to about 16 hours after mixing, depending on the ingredients used.

[0054] In one embodiment, a cream, lotion, or gel packaged in a standard trigger spray container adheres firmly to the target area after a regular dose of cream is sprayed from the container. This is described in WO 98 / 51273, incorporated herein by reference. Accordingly, in one aspect, the present disclosure provides a pharmaceutical non-aerosol spray composition for topical application, comprising a compound described herein, alone or in combination. The compound is present in the cream, lotion, or gel in an amount ranging from 0.1% to 20% by weight, or in some embodiments, from 1% to 15% by weight, or in some embodiments, from 2% to 10% by weight. The compounds used in the present invention can be incorporated into a neutral hydrophilic matrix cream, lotion, or gel. In a first preferred embodiment, the cream or lotion matrix for topical application is characterized by a polyoxyethylene alkyl ether. In a second preferred embodiment, the gel is characterized by a high molecular weight polymer of cross-linked acrylic acid. Polyoxyethylene alkyl ethers are nonionic surfactants widely used as emulsifiers for water-in-oil and oil-in-water emulsions, primarily in topical pharmaceutical formulations and cosmetics. This is characterized in the present invention as the base for a trigger-sprayable non-aerosol cream or lotion.The cross-linked acrylic acid polymer (carbomer) used to form the gel is another object of the present invention.

[0055] Thus, a particularly suitable base for a non-aerosol spray is a cream or lotion containing 1-25% polyoxyethylene alkyl ether, 3-40% moisturizer, and 0.1-1% preservative(s), the remainder to reach 100% being purified water. Suitably, the polyoxyethylene alkyl ether is polyoxyl 20 cetostearyl ether (Atlas G-3713), polyoxyl 2 cetyl ether (ceteth-2) , Polyoxyl 10 cetyl ether (ceteth-10), Polyoxyl 20 cetyl ether (ceteth-20), Polyoxyl 4 lauryl cetyl ether (laureth-4), Polyoxyl 23 lauryl cetyl ether (laureth-23), Polyoxyl 2 oleyl ether (oleth-2), Polyoxyl 10 oleyl ether (oleth-10), Polyoxyl 20 oleyl ether (oleth-20), Polyoxyl 2 stearyl ether (steareth-2), Polyoxyl 10 stearyl ether (steareth-10), Polyoxyl 20 stearyl ether (steareth-20), and Polyoxyl 100 stearyl ether (steareth-100). Suitable humectants may be one or any combination selected from the group consisting of propylene glycol, polyethylene glycol, sorbitol, or glycerin. Suitable preservatives are one or any combination selected from the group consisting of methylparaben, propylparaben, benzyl alcohol, benzoic acid, sodium benzoate, sorbic acid and its salts or phenylethyl alcohol.

[0056] Another suitable base for non-aerosol sprays is a gel containing 0.1-2.0% carbomer, 0.1-1% alkaline solution, 3-40% humectant, and 0.1-1% preservative or preservatives, with the remainder comprising purified water to reach 100%. Suitably, the carbomer may be one or any combination selected from the group consisting of Carbomer 934, Carbomer 940, or Carbomer 941. Suitable humectants, preservatives, and purified water for gels are the same as for creams or lotions. Other sprayable formulations are described in U.S. Patent Application Publication No. 2005 / 00255048, which is expressly incorporated herein by reference.

[0057] Ophthalmic preparations (topical and intravitreal): The compounds of the present invention typically comprise a small percentage of the total ophthalmic composition. The compounds of the present invention are typically at least 0.01 w / v%, more typically at least 0.1 w / v%, and even more typically at least 0.5 w / v% of the ophthalmic composition. The compounds of the present invention are also typically at most 5.0 w / v%, even more typically at most 3.0 w / v%, and even more typically at most 1.5 w / v% of the ophthalmic composition.

[0058] Ophthalmic compositions also typically contain a suitable ophthalmic vehicle for delivering the compound to the eye. Ophthalmic compositions can be configured for topical or intravitreal application to the eye, and it is believed that the ophthalmic vehicle may vary depending on the application method. Generally, for either topical or intravitreal application, the ophthalmic composition is aqueous and preferably contains a substantial amount of water. Typically, the composition contains at least 30 w / v%, more typically at least 80 w / v%, and even more typically at least 90 w / v% water (e.g., purified water).

[0059] For intravitreal application, especially when the ophthalmic composition is applied to the eye with a syringe, the ophthalmic composition can comprise or consist essentially of water and the compound of the present invention. For sustained drug release, PLGA or PLA microparticle formulations of the compound of the present invention are used as described by Shelke et al. [Drug Deliv Transl Res. 2011, (1): 76-90]. Of course, the ophthalmic composition can also contain other ingredients, such as NaHPO, hydroxypropylmethylcellulose, polysorbate 80, sodium chloride, and edetate disodium.

[0060] The vehicle may be water alone for topical application, particularly if the topical application is carried out immediately after the water is combined with the test compound or if the composition is packaged in a manner that prevents contamination. However, when the ophthalmic composition is applied as an ophthalmic composition that is administered multiple times over an extended period of time (e.g., as drops from an eye dropper, once, twice, three or more times daily over multiple days), the ophthalmic composition will likely contain additional ingredients such as antimicrobial agents or preservatives or systems, surfactants, buffers, isotonicity agents, antioxidants, viscosity modifiers, combinations thereof, and the like.

[0061] For topical application, the compositions of the present invention typically contain an antibacterial agent. Possible antibacterial agents include, but are not limited to, hydrogen peroxide, chlorine-containing antiseptics such as benzalkonium chloride, or others. However, according to a preferred embodiment, the compositions of the present invention contain a non-polymeric quaternary antibacterial agent such as benzalkonium chloride (BAK). The most preferred antimicrobial agents in the pharmaceutical composition include polymeric quaternary ammonium compounds.

[0062] As used herein, the phrase "substantially free" when referring to a component of an ophthalmic composition means that the ophthalmic composition may be completely devoid of that particular component or may contain only trace amounts of that particular component.

[0063] The polymeric quaternary ammonium compounds useful in the compositions of the present invention are those that have antibacterial effects and are ophthalmically acceptable. Preferred compounds of this type are described in U.S. Patent Nos. 3,931,319; 4,027,020; 4,407,791; 4,525,346; 4,836,986; 5,037,647; and 5,300,287; and PCT Publication WO 91 / 09523 (Dziabo et al.), which are expressly incorporated herein by reference. The most preferred polymeric ammonium compounds are polyquaternium 1, also known as POLYQUAD™ or ONAMERM™, having a number-average molecular weight of 2,000 to 30,000. Preferably, the number-average molecular weight is 2,000 to 30,000. The molecular weight is 3,000 to 14,000.

[0064] Polymeric quaternary ammonium compounds are generally used in suspensions of the invention in amounts greater than about 0.00001% w / v, more typically greater than about 0.0003% w / v, and even more typically greater than about 0.0007% w / v of the suspension. Furthermore, polymeric quaternary ammonium compounds are generally used in compositions of the invention in amounts less than about 3% w / v, more typically less than about 0.003% w / v, and even more typically less than about 0.0015% w / v of the composition.

[0065] The antimicrobial agent of the present compositions can additionally or alternatively include an antimicrobial system such as a borate / polyol complex system. As used herein, the term "borate" refers to boric acid, borate salts, boric acid derivatives, and other pharmaceutically acceptable borates, or combinations thereof. Most suitable are boric acid, sodium borate, potassium borate, calcium borate, magnesium borate, manganese borate, and other borate salts. Borates interact with polyols, such as glycerol, propylene glycol, sorbitol, and mannitol, to form borate-polyol complexes. The type and ratio of such complexes depends on the number of OH groups of the polyol on adjacent carbon atoms that are not in the trans configuration relative to each other. It should be understood that the weight / volume percentages of polyol and borate components include their amounts, whether or not they are part of a complex.

[0066] As used herein, the term "polyol" includes any compound having at least one hydroxyl group on each of two adjacent carbon atoms that are not in a trans configuration relative to one another. Polyols can be linear or cyclic, substituted or unsubstituted, or mixtures thereof, so long as the resulting complex is water-soluble and pharmaceutically acceptable. Examples of such compounds include sugars, sugar alcohols, sugar acids, and uronic acids. Preferred polyols are Sugars, sugar alcohols and sugar acids include, but are not limited to, mannitol, glycerin, xylitol, sorbitol and propylene glycol.

[0067] When used, the borate / polyol complex antimicrobial system (i.e., the borate and polyol together) typically constitutes at least 0.05 w / v%, more typically at least 0.5 w / v%, and sometimes at least 1 or at least 1.2 w / v% of the composition, and typically constitutes less than 5 w / v%, more typically less than 2.2 w / v%, and sometimes less than 1.6 w / v% of the composition. The ratio of borate to polyol (weight to weight) in the composition is typically 1:1 to 1:10, more typically 1:2 to 1:4 (e.g., about 1:3).

[0068] Tyloxapol, polysorbate-80, and polyoxyl hydrogenated castor oil are preferred surfactants. Tyloxapol is a highly preferred surfactant. When used, the surfactant is typically present at a concentration of at least 0.01 w / v%, more typically at least 0.025 w / v%, and sometimes at least 0.1 w / v% of the composition, and typically at a concentration of less than 5 w / v%, more typically less than 2.0 w / v%, and sometimes less than 1.0 w / v% of the composition.

[0069] Compositions of the present invention for topical application are typically formulated to be compatible with the eye. Ophthalmic compositions intended for direct application to the eye are formulated to have an ophthalmic compatible pH and isotonicity. The compositions typically have a pH in the range of 4 to 9, preferably 5.5 to 8.5, and most preferably 5.5 to 8.0. A particularly desirable pH range is 6.0 to 7.8, and more specifically 6.4 to 7.6. The compositions have an osmolality of 200 to 400 or 450 milliosmoles per kilogram (mOsm / kg), more preferably 240 to 360 mOsm / kg.

[0070] Preferred compositions of the present invention are multi-dose ophthalmic compositions, e.g., the compositions are in eye drops and can be administered topically to the eye as one or more drops once, twice, three or more times daily, where the compositions preferably have sufficient antimicrobial activity to meet USP (United States Pharmacopeia) preservative effectiveness requirements as well as other preservative effectiveness standards for aqueous pharmaceutical compositions.

[0071] Preservative potency standards for multi-dose ophthalmic solutions in the United States and other countries / regions are listed in the table below:

[0072] [Table 1]

[0073] The European Pharmacopoeia has two preservative effectiveness standards, "A" and "B".

[0074] The criteria described above for USP 27 are substantially identical to the requirements described in previous editions of the USP, particularly USP 24, USP 25, and USP 26.

[0075] As an added advantage, these ophthalmic compositions containing the TLR4 antagonist compounds of the present invention are suitable for topical application to the eye.

[0076] The formulations described herein may also contain additional active ingredients such as, but not limited to, antibacterial agents, analgesics, etc., as described above.

[0077] As such, the compounds and formulations described herein find use in treating a variety of ocular inflammatory diseases, including, but not limited to, AMD, sepsis and severe sepsis, SIRS, and septic shock. The method involves administering an effective amount of the antimicrobial and anti-inflammatory compositions described herein to a patient in need thereof to treat the disease or disorder. Medical uses of such compounds include sepsis, neonatal sepsis, toxemia, septic shock, burns and wounds, infective endocarditis, biofilm inhibition, ocular infections, ocular inflammation, and ocular neovascularization. The present invention is directed to the treatment and / or management of: diabetes, diabetic retinopathy, retinopathy of prematurity, uveitis, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel disease (IBD), asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, fever syndromes, cachexia, psoriasis, autoimmune diseases, heart disease, retinoblastoma, cancer, and / or any disorder associated with inflammation, immunomodulation, and microbial infection.

[0078] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the invention. [Example]

[0079] A series of compounds of the present invention effectively bind to the TLR4 receptor and do not bind to the TLR2 receptor: ELISA plates were coated with human monocyte lysate (isolated from commercially available LeukoPak blood samples) followed by a series of compounds (10 μM) as shown in Figure 6. These were then incubated with human monocyte lysate and then probed with anti-TLR4 and anti-TLR2 antibodies. The plate was developed using an anti-human IgG-HRP positive control. Compounds 1–3 effectively bind to the TLR4 receptor but not to the TLR2 receptor. Larger chain length analogs exhibited stronger antagonistic activity (Figure 7A). Similarly, other analogs were evaluated in the TLR4 antagonist assay, as shown in Figure 7B. Briefly, 0.5 million cells were grown in RPMI containing 10% FBSO / N in two 24-well plates. The next day, the medium was removed without disturbing the bottom layer, and different concentrations of compounds were added to a total volume of 0.5 ml of RPMI and incubated for 48 hours. Cells were harvested, and the harvested medium was analyzed using a human TLR4 ELISA kit according to the manufacturer's instructions (Raybiotech). did. [Example]

[0080] Compounds were tested to inhibit the production of inflammatory mediators in human monocytes: To understand the structural requirements and constraints for probing the TLR4 binding pocket for optimal potency and efficacy, we tested the ability of chitooligomers to inhibit LPS-induced inflammation in human monocytes (Figure 8A-B). Compounds 1-4 statistically significantly inhibited the LPS-induced cytokines TNF-α, IL-1β, and IL-6 at 10 μM concentrations. Compounds 2 and 4 (10 μM) were found to be more potent than chitohexaose (compound 1) (10 μM) in terms of inhibition of LPS-mediated induction of inflammatory cytokines (p<0.001 for LPS vs. Chtx, while p<0.0001 for LPS vs. compounds 2 and 4). The protocol was performed as described (Panda, S.K.; Kumar, S.; Tupperwar, N.C.; Vaidya, T.; George, A.; Rath, S.; Bal, V.; Ravindran, B., Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia. PLoS Pathog 2012, 8, e1002717). Human monocytes were treated with a series of compounds (10 μM). The cells were stimulated with LPS for 48 hours. TNF-α, IL-1β, and IL-6 in the culture supernatant were quantified according to the manufacturer's instructions. Similarly, other analogs were evaluated for their ability to inhibit LPS-induced TNF-α production in human monocytes. Compounds 12, 15, 16, 39, 40, 41, 42, and 25 were found to be potent inhibitors of TNF-α at 100 μM concentrations (Figure 8B). [Example]

[0081] Compounds 1, 2, and 26 inhibit LPS-induced production of inflammatory mediators (TNF-α) in mouse bone marrow-derived macrophages: Bone marrow-derived mouse macrophages were treated with 100 μM of the test compounds for 8 hours. Proinflammatory cytokine protein levels, such as TNF-α, were measured by real-time RT-PCR. LPS treatment (10 ng / ml) was used as a positive control (Figure 9). [Example]

[0082] Compound 26 inhibits HMGB1-induced production of inflammatory mediators (TNF-α, i-NOS) and upregulates the M2 biomarker CXCR4 in mouse macrophages: The expression of both TNF-α and iNOS was inhibited by compound 26 in macrophages. Interestingly, the M2 macrophage marker CXCR4 was upregulated by compound 26, suggesting a potential effect on macrophage polarization and suggesting immunomodulatory activity. Mouse bone marrow-derived macrophages were treated with HMGB1 for 8 hours with or without 100 μM compound 26. The mRNA levels of TNF-α, iNOS, and CXCR4 were measured by real-time RT-PCR and normalized to the expression levels of control cells (Figure 10). [Example]

[0083] The compounds of the present invention produce the anti-inflammatory cytokine IL-10: Interleukin-10 (IL-10) is a cytokine with potent anti-inflammatory properties that limits the host immune response to pathogens, thereby preventing host damage and playing a central role in maintaining normal tissue homeostasis. Dysregulation of IL-10 is associated with enhanced immunopathology in response to infection and an increased risk of developing many autoimmune diseases. Here, we evaluated compounds of the present invention for upregulating IL-10 levels in PBMCs using an ELISA assay (Figure 11). Briefly, 500,000 cells were grown in RPMI containing 10% FBS O / N in two 24-well plates. The next day, the medium was removed without disturbing the bottom layer, and different concentrations of compounds (0, 1, 10, 100 μM) were added to a total volume of 0.5 ml in RPMI. This was then incubated for 48 hours. Cells were harvested, and the harvested culture medium was analyzed using an ELISA kit according to the manufacturer's instructions (Raybiotech). [Example]

[0084] Chitohexaose (compound 1) protected mice from lethal Gram-negative sepsis caused by Escherichia coli: In an in vivo mouse model, chitohexaose (compound 1) protected mice from lethal Gram-negative sepsis caused by Escherichia coli. A previously reported bacterial sepsis model [Roger et al., Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2348-52] was employed to test the efficacy of compound 1 (Figure 1). 2 x 10 CFU of Escherichia coli (ATCC-25922) were inoculated into the mice. was injected intraperitoneally into BALB / c mice with or without Compound 1 (250 μg / animal). E. coli-mediated sepsis induced death, but mice co-treated with Compound 1 were protected from sepsis-induced death (40%). The above results demonstrated that bacteria-induced sepsis and death were inhibited and delayed for a certain period, which may represent a therapeutic window. [Example]

[0085] Compounds 4, 15, and 25 protected mice from CLP-induced polymicrobial infection and sepsis. To prove the concept and demonstrate feasibility, we tested compound 4 in a mouse CLP model. The CLP model consists of cecal perforation, which releases fecal material into the peritoneal cavity and induces a polymicrobial infection, leading to an exacerbated immune response. This model mimics the clinically relevant human condition. A previously reported CLP sepsis protocol (Toscano et al., Journal of Visualized Experiments: 2011, (51), 2860) was employed to test the efficacy of compound 4. C57BL / 6 mice (Jackson Laboratories, 10–12 weeks old, N = 15) in the CLP group and the CLP plus saline (0.5%) and antibiotic (primaxin, 5 mg / kg) group were intravenously injected with compound 4 (10 mg / kg) 16 and 40 hours after surgery. As a result, CLP-mediated sepsis leads to organ dysfunction. The present inventors also investigated the efficacy of the standard point-of-care antibiotic primaxin (500 mg / kg bw) in treating mice with sepsis-induced thrombocytopenia (500 mg / kg bw) and thrombocytopenia (500 mg / kg bw). demonstrated that compound 4, in combination with 100 mg / kg of sucralose, protected mice (approximately 93%) from sepsis-induced death (14 / 15) while delaying clinical symptoms such as hypothermia, shivering, huddling, anorexia, decreased movement, and increased heart and respiratory rates.

[0086] Compound 25 (10 mg / kg) and compound 15 (5.0 mg / kg) also protected mice (approximately 60%) from sepsis-induced death, as shown in Figure 5. We also demonstrated that in combination with the standard point-of-care antibiotic primaxin (5 mg / kg), both compounds protected mice (approximately 60%) from sepsis-induced death while simultaneously delaying clinical symptoms such as hypothermia, shivering, huddling behavior, anorexia, decreased movement, and increased heart and respiratory rates. [Example]

[0087] Histopathology of organ tissues in mice after CLP Hematoxylin and eosin (H / E) staining of various organs from sham-, CLP-, and compound 4-treated mice was performed. Briefly, after tissue collection, they were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, and sectioned at 4 μm for routine hematoxylin and eosin staining. CLP mice exhibited microthrombi and congestion in the heart, lungs, liver, kidneys, and brain, increased germinal center size in the spleen, villous necrosis in the intestine, and loss of testicular epithelium. Treatment with compound 4 reversed all of these changes to a significant extent, and the tissues resembled those in the sham group (Figure 3). [Example]

[0088] Post-CLP serum biomarker testing: Plasma collected from the tail vein 48 hours after surgery was stored at -30°C and analyzed for TNF-α, IL-6, and IL-β levels in the sham, CLP, CLP + Compound 4, CLP + Primaxin, CLP + Primaxin + Compound 4, and control (saline-injected) groups. Compound 4, alone or in combination with the antibiotic Primaxin, statistically significantly reduced TNF-α, IL-1β, and IL-6 levels compared with the CLP mice (n = 4, p < 0.0005) (Figure 4). Briefly, the above cytokines were estimated by sandwich ELISA using commercially available ELISA kits. ELISA plates were coated with capture antibodies and subsequently incubated with test samples and appropriate standards. They were then probed with biotin-labeled secondary antibodies and avidin-peroxidase. Color was developed using TMB, and the optical density (OD) was recorded. [Example]

[0089] The compounds of the present invention have broad spectrum antibacterial activity: The compounds were screened against selected Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), Gram-positive bacteria (MRSA), and a fungus (Candida albicans) primarily found in burn and septic wounds. Most of them exhibited antibacterial activity with MICs ranging from 50 to 200 mg / L (Figure 13). All prepared compounds met Clinical Laboratory Standards Institute (CLSI) guidelines for broth microdilution susceptibility testing. According to the guidelines, the minimum inhibitory concentration (MIC) assay was performed using both the test compound and the control substance. Compounds were dissolved in DMSO, diluted to the appropriate concentration, and added to a 96-well microdilution tray. Brain Heart Infusion Broth (BHI) was used for testing with bacterial strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Candida albicans. Compounds were diluted from 200 μg / ml to 0 μg / ml. Serial dilutions down to 0.0625 μg / ml were plated onto 96-well plates inoculated with approximately 1 x 105 CFU of each organism. The MIC endpoint was determined by the growth of the organism in microdilutions. The lowest concentration of test compound or control compound that completely inhibited the activity was determined for each compound after 24 hours. [Example]

[0090] Compounds of the invention inhibited biofilm formation: Based on the in vitro antibacterial MIC data, we selected three compounds, 1, 12, and 15, for further testing against MRSA. All three compounds showed better activity against MRSA and MSSA strains compared to colistin (a standard-of-care antibiotic), which was used as a positive control (Figure 14). Furthermore, as previously described (Ceri, H.; Olson, ME; Stremick, C.; Read, RR; Morck, D.; Buret, A., The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms. Journal of Clinical Microbiology 1999, 37, 1771-1776), the biofilms were significantly more effective than the control. The biofilm inhibition and eradication activities of compound 15 against MRSA were tested. The minimum biofilm eradication concentrations (MBIC) of compound 15 against MRSA were superior to those of colistin. Thus, these compounds with activity against S. aureus biofilms have a significant impact on the control of difficult-to-eradicate biofilm-mediated intravascular infections [Li et al., J InfectDis. 2016 Nov 1;214(9):1421-1429; Archer, NK; Mazaitis, MJ; Costerton, JW; Leid, JG; Powers, ME; Shirtliff, ME, Staphylococcus aureus biofilms: Properties, regulation, and roles in human disease. Virulence 2011, 2, 445-459]. Briefly, the Calgary Biofilm Device (CBD) technology was used for biofilm susceptibility to compounds. The CBD generates 96 equivalents of biofilm for antibiotic susceptibility assays using standard 96-well technology. Susceptibility to a standard panel of compounds and antibiotics was determined by the National Committee for Clinical Laboratory Standards (NCCLS) as previously described. [Example]

[0091] The broad spectrum antibacterial activity of the compounds of the present invention is due to the disruption of cell membranes: The effects of AVR compounds on exponentially growing MRSA and membrane integrity were evaluated. MRSA resuspended in phosphate-buffered saline were exposed to antibiotic MICs and AVR compounds 1, 12, and 15 in brain heart infusion broth for 30 minutes and 1 hour, and the absorbance of leaked cellular material in the culture filtrate was measured by detecting the optical density at 260 nm (Figure 15). [Example]

[0092] The compounds of the present invention do not bind to plasma or serum proteins. One of the major difficulties in designing drug candidates for systemic, oral, or topical use is their poor plasma / tissue bioavailability due to drug binding to plasma and serum proteins. Therefore, we formulated the 10% ... The effect of serum on compound activity was tested by microdilution MICs in 100% swine flu vaccines (Hurdle, JG; Lee, RB; Budha, NR; Carson, EI; Qi, J.; Scherman, MS; Cho, SH; McNeil, MR; Lenaerts, AJ; Franzblau, SG; Meibohm, B.; Lee, RE, A microbiological assessment of novel nitrofuranylamides as anti-tuberculosis agents. Journal of Antimicrobial Chemotherapy 2008, 62, 1037-1045). Results showed that all compounds were active against MRSA and did not bind to serum proteins, suggesting that they had good bioavailability in target tissues (Figure 16). [Example]

[0093] The compounds of the present invention are not toxic to fibroblasts: Cytotoxicity and therapeutic index were also assessed by incubating the MEF mouse fibroblast cell line (ATCC, Manassas, VA) with antibiotics. MEF mice were exposed to colistin and AVR compounds for 24 hours and subsequently assessed by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, ThermoFischer, MA] assay as previously described. The cytotoxicity of all AVR compounds in fibroblast cell lines is shown in Figure 17, demonstrating that AVR compounds selectively inhibit MRSA growth without impairing fibroblast proliferation, which is critical in the wound healing process. [Example]

[0094] Compounds 1 and 2 reduced VEGF production in ARPE-19 cells: The RPE, central to photoreceptor survival and function, is a major source of the angiogenic factor VEGF and therefore plays a central role in the regulation and development of choroidal neovascularization leading to AMD (Spilsbury, K.; Garrett, KL; Shen, W.-Y.; Constable, IJ; Rakoczy, PE, Overexpression of Vascular Endothelial Growth Factor (VEGF) in the Retinal Pigment Epithelium Leads to the Development of Choroidal Neovascularization. The American Journal of Pathology 2000, 157, 135-144; Betts, BS; Parvathaneni, K.; Yendluri, BB; Grigsby, J.; Tsin, ATC, Ginsenoside-Rb1 Induces ARPE-19 Proliferation and Reduces VEGFRase. ISRN Ophthalmology 2011, 2011, 184295). We investigated whether compounds 1 and 2 can inhibit HMGB1 (the endogenous ligand for TLR4). As part of our preliminary results to evaluate the efficacy of compounds 1 and 2, we induced VEGF production in ARPE-19 cells. Figure 12 shows that compounds 1 and 2 (50 μg / mL) effectively reduced HMGB1-induced VEGF production in ARPE-19 cells with statistical significance (p<0.01). 2×10 ARPE-19 cells were seeded in complete medium containing 10% serum in a 24-well plate for 24 hours and then maintained in serum-free medium for an additional 24 hours. Cells were treated with 0 μg / mL (medium) or 100 ng / mL HMGB1 for 24 hours, with or without 50 μg / mL of test compound. Supernatants were collected and assayed using a human VEGF ELISA kit from Peprotech according to the manufacturer's instructions. RPE cells are located adjacent to the choriocapillaris and other major vasculature of the eye. Therefore, these findings suggest that compounds 1 and 2 may have significant effects on inhibiting choroidal and retinal capillary neovascularization (through inhibition of TLR4 and reduction of VEGF), which contributes to the development of AMD and retinopathy. [Example]

[0095] Compound 2 reduced choroidal neovascularization by approximately 60% compared to positive controls in a laser-induced CNV mouse model of wet AMD: To demonstrate the in vivo antiangiogenic effects of AVR compounds, we tested the compounds in a laser-induced CNV mouse model (Figure 5). Stem-connected Iridex Oculight GL 532 nm diode laser (Mountain View, CA) Laser CNV was induced in C57BL / 6 mice (10-12 weeks old) using a laser. The parameters used to reproducibly obtain a successful laser spot (confirmed by the formation of a gas bubble, indicating rupture of Bruch's membrane) were 350 mW, 75 msec, and a 50 μm spot size. Four laser spots were applied, 2-3 papillary diameters from the optic nerve. On days 2, 4, and 6 after laser irradiation, mice were treated with either PBS (negative control), compounds 1, 2, and 26, or a positive control (anti-VEGF antibody) (n = 4-6 mice per group). Compounds 1, 2, and 26 and BSS (vehicle) were administered intraperitoneally once daily, starting 1 day before laser irradiation and continuing for 10 days after laser irradiation. By the end of the experiment, mouse eyes were examined for fluorescein fundus angiography and / or optical coherence tomography (OCT). CNV lesions were visualized by MRI (microscopy and coherence tomography). The animals were then sacrificed, and RPE / choroid / sclera flat mounts were prepared and stained with both FITC-conjugated isolectin B4 and anti-ICAM-2 antibodies to quantitatively measure the size of CNV. Two experiments were performed to test the effects of 1, 2, and 26 on laser-induced CNV. As shown in Figure 5, daily intraperitoneal injection of 200 μg of compound 2 reduced the average size of CNV lesions to approximately 60% of that of control mice treated with vehicle alone (balanced salt buffer), comparable to the positive control. [Example]

[0096] Synthesis of compound 11: To chitotriose (10 mg, 0.015 mmol) in 5 mL of methanol was added a methanol solution of 4-carboxy-TEMPO (4.5 mg, 0.026 mmol). The mixture was stirred at room temperature for 48 hours and cooled at 2°C for 12 hours. Ether was added to precipitate a white solid, which was filtered and dried to give 4.0 mg of a white powder. LC / MS = 684 (M+1), 1H NMR (DMSO-D6, 500 MHz): δ 1.15 (s, 12H). , 1.35~1.55(m,4H), 1.97~2.05(bs,10H), 2.43(m,1H), 2.80~3.23(m,4H), 3.25~3.66(m, 11H), 4.07(s,2H), 4.41(s,2H), 4.65(s,2H), 5.18(d,1H), 5.52(bs,2H), 8.15(d,1H,NH).

[0097] Synthesis of compound 30: To a stirred suspension of D-glucosamine 29 (100 g, 0.55 mol) in EtOH (500 mL) was added NaOEt (30 g, 0.55 mol). After 10 min, the mixture was treated with dimethylmaleic anhydride (0.5 equiv.) and stirred for 20 min. Triethylamine (65.2 mL, 0.465 mol) was added, and the reaction mixture was treated again with the remaining dimethylmaleic anhydride (0.5 equiv.). The reaction mixture was warmed to 60 °C with stirring for 2 h, and the EtOH was evaporated and dried. The residue was treated with pyridine and acetic anhydride and stirred at room temperature for 20 h. The reaction was monitored by TLC, the solvent was evaporated, and the residue was poured onto ice, extracted with chloroform (3 × 1 L), washed with 1 L of aqueous hydrochloric acid (3%), saturated sodium bicarbonate solution (1 L), and distilled water (1 L), and dried over anhydrous sodium sulfate. The residue was purified by silica gel chromatography using 20-30% EtOAc in petroleum ether as the eluent to give compound 30 (80 g, ca. 37%). H NMR (400 MHz, CD Cl3):δ 1.91(3H,s), 1.94(6H,s), 2.01(s,3H), 2.03(s,3H), 2.09(s,3H), 3.92~3.96(m,1H), 4.0~4.12(dd,1H), 4.18~4.23(dd,1H), 4.30~ 4.33(dd,1H,J=4.4Hz), 5.14(t,1H,J=9.2Hz), 5.69(t,1H,J=9.2Hz), 6.34(d,1H,J=8.8Hz).

[0098] Synthesis of compound 31: To a solution of 30 (100 g, 0.219 mol) in DMF was slowly added hydrazine hydrate (12 ml, 0.219 mol) at 23 °C, and the mixture was stirred at the same temperature for 5-6 h and monitored by TLC. The reaction mixture was diluted with ethyl acetate (2 L), washed with water (3 × 1 L), brine (1 L), dried over NaSO, and evaporated to give compound 31 (65 g, ca. 71%). H NMR (400 MHz, CDCl): δ 1.85 (s, 3H), 1.9 0(s,6H), 1.98(s,3H), 2.05(s,3H), 3.79~3.84(m,1H), 3.95~4.06(dd,1H), 4.10~4.13(dd,1 H), 4.20~4.24(dd,1H), 5.06(t,1H,J=9.6Hz), 5.42(d,1H,J=8.4Hz), 5.60(t,1H,J=9.6Hz).

[0099] Synthesis of compound 32: To a stirred DCM solution of compound 31 (35 g, 0.084 mol) and imidazole (14.4 g, 0.211 mol) at 23 °C, TBDMSCl (15.2 g, 0.101 mol) was added portionwise, stirred at the same temperature for 16 h, monitored by TLC, diluted with DCM (1 L), washed with water (2 × 1 L), brine (500 mL), dried over NaSO, and concentrated to give the crude product. The residue was purified by silica gel chromatography using 15–20% EtOAc in petroleum ether as the eluent to give compound 32 (27 g, ca. 61%). 1HNMR(400MHz,CDCl3):δ 0.01(s,3H), 0.05(s,3H), 0.76(s,9H), 1.91(s,3H), 1.93(s,6H), 2.01(s,3H), 2.07(s,3H), 3.80~3.83(m,1H), 3.98~4. 03(dd,1H), 4.10~4.14(dd,1H), 4.20~4.24(dd,1H), 5.05(t,1H,J=9.6Hz), 5.36(d,1H,J=8.4Hz), 5.66(t,1H,J=9.6Hz).

[0100] Synthesis of compound 33: To a stirred solution of compound 32 (27 g, 0.051 mol) in MeOH (100 mL) was added NaOMe (2.76 g, 0.052 mol) portionwise at 23 °C and stirred at the same temperature for 3–4 h. The reaction was monitored by TLC, and the MeOH was concentrated under reduced pressure, diluted with 50 mL of water, and the pH was adjusted to 6.5–7.0. The solid was filtered and dried to give pure compound 33 (16 g, ca. 77%). 1HNMR(400MHz,CDCl3):δ 0.03(s,3H), 0.05(s,3H), 0.76(s,9H), 1.94(s,6H), 3.45~3.47(m,1H), 3.57~ 3.61(m,1H), 3.77~3.90(m,3H), 4.21(t,1H,J=8.0Hz), 5.23(d,1H,J=8.0Hz).

[0101] Synthesis of compound 34: A suspension of compound 33 (30 g, 0.074 mol) and dibutyltin oxide (37.25 g, 0.149 mol) in toluene (400 mL) was heated under reflux for 12 h. Tetrabutylammonium iodide (55.2 g, 0.149 mol) and benzyl bromide (25.5 g, 0.149 mol) were added, and the mixture was gently refluxed for 3 h. The reaction mixture was cooled and concentrated to give the crude product. The residue was purified by silica gel chromatography using 15-20% EtOAc in petroleum ether to give compound 34 (25 g, ca. 60%). 1HNMR(400MHz,CDCl3):δ 0.06(s,3H), 0.02(s,3H), 0.73(s,9H), 1.83~1.93(bs,6H), 3.56~3.61(m,1H), 3.72~3.80(m,2H), 3.86~3.89(dd,1H), 4.09~4.14( dd,1H), 4.53(d,1H,J=12Hz), 4.56~4.63(dd,2H), 4.69~4.76(dd,2H), 5.16(t,1H,J=8.0Hz), 7.15~7.24(m,5H), 7.33~7.37(m,5H).

[0102] Synthesis of compound 35: To a mixture of compound 31 (5 g, 0.012 mol) and CClCN (2.06 g, 0.014 mol) in dry CHCl was added DBU (0.37 g, 0.002 mol) and stirred at room temperature for 15-16 h. The reaction mixture was concentrated to give the crude product. This crude product was purified by silica gel chromatography using 25-35% EtOAc in petroleum ether to give compound 35 (3.8 g, 55%). H NMR (400 MHz) ,CDCl3):δ 1.91(s,3H), 1.93(s,6H), 2.01(s,3 H), 2.07(s,3H), 3.93~4.01(m,1H), 3.98~4.03(dd,1H), 4.33~4.40(m,2H), 5.20(t,1H,J=9.2Hz), 5.73(t,1H,J=9.2Hz), 6.45(d,1H,J=9.2Hz), 8.67(s,1H).

[0103] Synthesis of compound 36: A mixture of compound 35 (4 g, 0.007 mol) and compound 34 (3.3 g, 0.0057 mol) was placed in an oven-dried round-bottom flask containing activated molecular sieve powder (4 A0). The RB was then filled with argon twice, and dry DCM (10 mL) was added and stirred at 23 °C for 2 h. The resulting reaction mixture was then cooled to -10 °C, and a 0.1 M solution of TfOH in DCM was added and stirred for an additional 16 h. The reaction mixture was concentrated under reduced pressure to give a crude mixture, which was purified by silica gel chromatography using EtOAc (25-35%) in petroleum ether to give compound 36 (2 g, 30%). H NMR (400 M) indicated a chromatographically resolved ... Hz, CDCl3):δ -0.005(s,3H), -0.11(s,3H), 0.7 1(s,9H), 1.76(bs,6H), 1.90(s,3H), 1.95(bs,6H), 1.98(s,3H), 3.36~3.44(m,3H), 3.4 8~3.55(m,3H), 3.80~3.83(m,2H), 3.90~3.93(dd,1H), 4.04~4.12(m,3H), 4.14~4.20(dd ,1H), 4.42(d,1H,J=12.4Hz), 4.57~4.64(dd,2H), 4.81(d,1H,J=12.4Hz), 5.03~5.08(m, 2H), 5.36(d,1H,J=8.4Hz), 5.61(t,1H,J=9.2Hz), 7.13~7.19(m,5H), 7.33~7.39(m,5H).

[0104] Synthesis of compound 37: To a solution of compound 36 (5.5 g, 0.0056 mol) in dry THF (50 mL) was added AcOH (0.36 mL, 0.0063 mol) and cooled to −5° C. Then, a THF solution of 1 M T BAF (6.3 mL, 0.0063 mol) at −5° C. was added, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution, extracted with DCM, and concentrated under reduced pressure to give crude compound 37 (3 g, crude), which was used in further reactions without purification. 1H NMR (400 MHz, CDCl3): δ 1.76 (s, 6H), 1.89 (s, 3H), 1.95 (bs, 6H), 1.98 (s, 3H), 3.38-3.55 (m, 3H), 3.62-3.66 (m, 1H), 3.76-3.80 (m, 1H), 3.90-3.94 (m, 1H), 4.06-4.22 (m, 6H), 4.14-4.20 (dd, 1H), 4.4 0(d,1H,J=12.8Hz), 4.57~4.64(dd,2H), 4.85(d,1H,J=12.8Hz), 5.01~5.07(m,2H), 5. 33(d,1H,J=8.4Hz), 5.55~5.63(t,1H,J=9.2Hz), 7.11~7.20(m,5H), 7.29~7.42(m,5H).

[0105] Synthesis of compound 38: To a mixture of compound 37 (4 g, 0.0045 mol) and CClCN (0.54 mL, 0.0054 mol) in dry CHCl was added DBU (0.13 mL, 0.0009 mol) and stirred at room temperature for 15–16 h. The reaction mixture was concentrated to give the crude product. The crude product was purified by silica gel chromatography using 25–35% EtOAc in petroleum ether to give compound 38 (1 g, 20%).

[0106] Synthesis of compounds 39-42: A mixture of compound 38 (1 g, 0.89 mmol) and R-OH (0.7 equiv.) was placed in an oven-dried round-bottom flask containing activated molecular sieve powder (4 A0). The RB was then filled with argon twice, and dry DCM (20 mL) was added and stirred at room temperature for 2 h. The resulting reaction mixture was then cooled to -10 °C and 0.1 MtfOH (1.3 mL, 0.13 mmHg) was added. A solution of 39-42 (approximately 40-45%) was added to the reaction mixture in DCM and stirred for another 16 hours. The reaction mixture was concentrated under reduced pressure to give a crude mixture. The crude product was purified by silica gel chromatography using 25-35% EtOAc in petroleum ether to give compounds 39-42 (approximately 40-45%). Got it.

[0107] Compound 39:1H NMR (400MHz, CDCl3):δ 1.76(s,6H), 1.89(s,3H), 1.95(bs,6H), 1.98(s,3H), 3.49~3.51(m,2H), 3.64~3.71 (m,2H), 3.94~3.96(m,1H), 4.15~4.19(m,6H), 4.40(m,1H), 4.56~4.65(m,2H), 4.86 ~4.91(m,1H), 5.06~5.09(m,1H), 5.33(d,1H,J=8.4Hz), 5.55~5.63(t,1H,J=9.2Hz) , 6.91(d,2H,J=8.4Hz), 7.12~7.21(m,5H), 7.29~7.42(m,5H), 8.06(d,2H,J=8.4Hz). LC / MS:M+=982.

[0108] Compound 40:1H NMR(400MHz,CDCl3):δ 1.21~1.48(m,10H), 1.76(bs,6H), 1.89(s,3H), 1.95~1.96(s,9H), 1.98(s,3H), 3.36~3.38(m ,1H), 3.43~3.46(m,3H), 3.59(d,1H,J=8.4Hz), 3.89~3.92(m,2H), 4.04~4.08(m,4H), 4.41(d, 1H,J=10.0Hz), 4.60(s,2H), 4.84(d,1H,J=10.0Hz), 4.93(d,1H,J=6.4Hz), 5.04(t,1H,J=7.2 Hz), 5.35(d,1H,J=6.4Hz), 5.57~5.61(t,1H,J=7.2Hz), 7.13~7.19(m,5H), 7.28~7.40(m,5H). LC / MS:M+-2=943.

[0109] Compound 41: ¹H NMR (400 MHz, CDCl₃): δ 1.21–1.35 (m, 8H), 1.45–1.62 (bs, 8H), 1.76 (bs, 6H), 1.90 (s, 3H), 1.95–2.22 (m, 12H), 3.43–3.46 (m, 4H), 3.60–3.71 (m, 1H), 3.94 (d, 1H), 4.04–4.12 (m, 4H), 4.20 (m, 1H), 4.55 ~4.59(m,1H), 4.62~4.70(m,2H), 4.84(d,1H,J=10.0Hz), 4.93(d,1H,J=6.4Hz), 5.30(t, 1H,J=7.2Hz), 5.35(m,1H), 5.61(t,1H,J=7.2Hz), 7.13~7.19(m,5H), 7.28~7.40(m,5H). LC / MS:M+-2=1015.

[0110] Compound 42: ¹H NMR (400 MHz, CDCl₃): δ 1.76 (s, 6H), 1.90 (s, 3H), 1.95 (s, 3H), 1.98 (s, 6H), 2.01 (s, 3H), 3.43–3.49 (m, 3H), 3.62–3.64 (m, 1H), 3.72 (s, 3H), 3.91–3.94 (m, 1H), 4.07–4.21 (m, 5H), 4.42 (d, 1H, J = 12.4 Hz), 4.5 9(s,2H), 4.85(d,1H,J=12.4Hz), 5.05(t,1H,J=10.8Hz), 5.34~5.37(m,2H), 5.60(t,1H,J=1 0.8Hz), 6.69(d,2H,J=9.2Hz), 6.77(d,2H,J=9.2Hz), 7.11~7.21(m,5H), 7.29~7.42(m,5H). LC / MS:M+=986.

[0111] Synthesis of compound 15: Compound 15 was synthesized from compound 39 (0.5 g) by removing the NDMM group using hydrazine hydrate in HCl, followed by the introduction of an NHAc group by treatment with AcO. Finally, the -OAc group was removed with NaOCH / MeOH at room temperature, as previously described [Mohamed RE et al., Carbohydrate research, 2001, 331, 129-142], to give compound 15 (22 mg of a white solid). H NMR (400 MHz, CDCl): δ 1.98 (s, 6H), 3.43–3.49 (m, 3H), 3.62–3.64 (m, 1H), 3.72 (s, 3H), 3.91–3.94 (m, 1H), 4.23–4.42 (m, 3H). H), 4.62(m,4H), 5.90(d,1H,J=10.8Hz), 6.42(d,1H,J=10.8Hz), 6.91(d,2 H,J=8.4Hz), 7.12~7.21(m,5H), 7.29~7.42(m,5H), 8.06(d,2H,J=8.4Hz). LC / MS:M+=710.

[0112] Synthesis of Compound-43: To a stirred suspension of D-glucosamine 29 (200 g, 0.93 mol) in pyridine was added acetic anhydride (720 mL, 7.4 mol). The reaction mixture was stirred for 12 h (monitored by TLC) and warmed to 60 °C. The reaction mixture was concentrated under reduced pressure. The residue was poured onto ice, extracted with chloroform (3 × 1 L), washed with 1 L of aqueous hydrochloric acid (3%), saturated sodium bicarbonate solution (1 L), distilled water (1 L), and dried over anhydrous sodium sulfate. The crude mixture 43 was carried on to the next step without further purification.

[0113] Synthesis of compound 44: To a solution of crude mixture 43 (30 g, 77.09 mmol) in THF, methylamine in MeOH (2 M, 77 mL, 154.1 mmol) was slowly added at room temperature and stirred for 12 hours at the same temperature while monitoring by TLC. The reaction mixture was diluted with ethyl acetate (2 L), washed with water (3 × 1 L), brine (1 L), dried over NaSO, and evaporated to give compound 44 (20 g).

[0114] Synthesis of Compound-45: A mixture of compound 44 (20 g, 0.057 mol), CClCN (11 mL, 0.114 mol), and DBU (2.5 mL, 0.0014 mol) in dry CHCl was stirred at room temperature for 15-16 h. The reaction mixture was concentrated to give the crude product. This crude product was purified by silica gel chromatography using 25-35% EtOAc in petroleum ether to give compound 45 (5 g). H NMR (400 MHz, CDCl) :δ 1.91(s,3H), 1.93(s,3H), 2.01(s,3H), 2.07 (s,3H), 3.93~4.01(m,1H), 3.98~4.03(dd,1H), 4.33~4.40(m,2H), 5.20(t ,1H,J=9.2Hz), 5.73(t,1H,J=9.2Hz), 6.45(d,1H,J=9.2Hz), 8.67(s,1H).

[0115] Synthesis of Compound-24: To a stirred suspension of compound 45 (2 g, 5.14 mmol) and 4-OH-TEMPOL (0.7 g, 4.11 mmol) in DCM, molecular sieve powder was added and stirred at room temperature for 2 h. Triflic acid (0.11 g, 0.77 mmol) was added and stirred at room temperature for 4 h. The RM (reaction mixture) was distilled and purified by column chromatography eluting with 5% MeOH in DCM to give compound 24 (300 mg). H NMR (400 MHz, CD Cl3):δ 1.14(s,12H), 1.41~1.66(m,4H), 1.91( s,3H), 1.93(s,3H), 2.01(s,3H), 2.07(s,3H), 4.15~4.36(m,4H), 5.30(d,1H,J=9.2Hz), 5.73(t,1H,J=9.2Hz), 6.45(d,1H,J=9.2Hz). TLC-based :10% MeOH in DCM, Rf :0.3.

[0116] Synthesis of compound 25: To a stirred solution of compound 24 (0.3 g, 0.79 mmol) in MeOH at room temperature, NaOMe (0.04 g, 0.63 mmol) was added and stirred at the same temperature for 3-4 hours. The reaction mixture was monitored by TLC, and the MeOH was concentrated and neutralized with 1M HCl in dioxane. The RM was concentrated and purified by column chromatography using 7% MeOH in DCM as the eluent to give compound 25 (50 mg). H NMR (400 MHz, CDCl ) :δ 1.14(s,12H), 1.41~1.66(m,4H), 1.93(s,3H ), 4.15~4.36(m,4H), 5.30(d,1H,J=9.2Hz), 5.73(t,1H,J=9.2Hz), 6.45(d,1H,J=9.2Hz). LC / MS:M+=374. TLC system: 10% MeOH in DCM, Rf: 0.2. [Example]

[0117] Topical / intravitreal preparations The following table presents exemplary ranges for topical or intravitreal ophthalmic compositions according to the present invention:

[0118] [Table 2] [Example]

[0119] Injection The following table represents an exemplary range of intravenous (IV) compositions according to the invention: A compound of the invention is dissolved in a majority of water (35°C-40°C) and the pH adjusted to 4.0-7.0 with hydrochloric acid or sodium hydroxide as needed. The batch is then made up to volume with water and filtered through a sterile micropore filter into sterile 10 mL amber glass vials (Type 1) and sealed with sterile closures and seals.

[0120] [Table 3] [Example]

[0121] The following table presents exemplary ranges of topical gel, lotion and spray compositions according to the present invention:

[0122] Gel formulation: The following table represents an exemplary range of gel compositions according to the present invention: Carbomer 934 is uniformly dispersed in approximately 40% of the total amount of water. Ammonia solution is gradually added to the dispersion with stirring to form a transparent gel. In a separate container, methylparaben is dissolved in propylene glycol, and then 10.0 g of Compound 15 is dispersed in this solution to form a homogeneous suspension. The suspension is gradually added to the gel with stirring to obtain a homogeneous, white, opaque gel.

[0123] [Table 4]

[0124] Cream or lotion formulations: The following table shows exemplary ranges for cream or lotion compositions according to the present invention: Methylparaben is dissolved in approximately 80% of the total amount of propylene glycol. Poloxyl 2 cetyl ether is added to this solution with stirring. In a separate processing vessel, 20% of propylene glycol, a portion of purified water, and 10.0 g of Compound 15 are mixed to form a uniform suspension. The suspension is gradually added to the first processing vessel with moderate stirring until a homogeneous, soft, white cream is obtained. The cream is passed through a colloid mill to bring the batch to the target mass.

[0125] [Table 5]

[0126] Non-aerosol spray formulations: The following table shows an exemplary range of non-aerosol spray compositions according to the present invention: 5.00 g of polyoxyl 10 oleyl ether was dissolved in 188.75 g of castor oil with gentle stirring. 25.0 g of Compound 15 and 25.0 g of zinc oxide were suspended in the slurry with moderate stirring, followed by 1.25 g of fumed silica gel. The slurry was mixed under high shear until uniform and smooth, and packaged in a spray bottle.

[0127] The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to the particular embodiments of the process, manufacture, composition of matter, compound, means, methods, and / or steps described herein. Various modifications, substitutions, and variations can be made to the disclosed materials without departing from the spirit and / or essential characteristics of the invention. Accordingly, those skilled in the art will readily appreciate from the disclosure that subsequent modifications, substitutions, and / or variations that perform substantially the same function or achieve substantially the same result as the embodiments described herein may be utilized in accordance with such related embodiments of the present invention. Accordingly, it is intended that the following claims include within their scope such modifications, substitutions, and variations of the processes, manufacture, compositions of matter, compounds, means, methods, and / or steps disclosed herein.

[0128] [Table 6]

[0129] To the extent that the above description and accompanying drawings disclose any additional subject matter, the invention is not dedicated to the public and no one or more of the following claims any such additional inventions. reserves the right to file more than one application. References

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Claims

【Request Item 1】 【Chemistry 1】 wherein R=C(O)alkyl, C(O)-piperidinenitroxy, alkyl, benzyl, substituted benzyl. or 【Chemistry 2】 10. A pharmaceutical composition for treating sepsis, septic shock, toxemia, burns or wound infections, comprising a compound selected from the group consisting of:

2. 10. The pharmaceutical composition of claim 1 formulated as a sterile injectable aqueous or oleaginous suspension.

3. 10. The pharmaceutical composition of claim 1, formulated as a sterile topical gel, ointment, or aqueous spray.

4. 10. The pharmaceutical composition of claim 1, further comprising an anti-inflammatory or antibacterial agent. 【Request Item 5】 【Chemistry 3】 where R = H, C(O)alkyl, C(O)-piperidinenitroxy, alkyl, benzyl, substituted benzyl. or 【Chemistry 4】 or a pharmaceutically acceptable salt thereof, for treating sepsis, septic shock, toxemia, burns or wound infections, thereby treating a subject.

6. 6. The medicament of claim 5, wherein administering comprises administering to a patient in need thereof a medicament comprising about 5.0 mg to about 100 mg of the compound or a pharmaceutically acceptable salt thereof, thereby treating the patient.

7. 6. The medicament of claim 5, wherein administering comprises administering to a patient in need thereof a medicament comprising about 10.0 mg to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof, thereby treating the patient.

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