Novel immunodulating small molecules

Abstract

To provide compounds that can be used as adjuvants for prophylactic and therapeutic vaccines for infectious diseases and cancer, and provide therapeutic methods that use the compounds as immunotherapeutics for cancer, infectious diseases, allergy / asthma, lung injury, bronchopulmonary dysplasia either alone or in combination with existing therapies.SOLUTION: The present invention provides novel compositions and methods, comprising a compound of the formula I, wherein the amount of the compound is selected to either inhibit or activate the immune response.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 693,023, filed 2 July 2019, and all its contents are incorporated herein by reference.

[0002] Description of federally funded research This research was partially supported by NIH / NIAID Federal Grant 1 R43 AI129164-01 awarded to AyuVis Research.

[0003] The present invention generally relates to the field of novel immunomodulatory molecules, and more particularly to carbohydrate-derived Toll-like receptor modulators. In one embodiment, the present invention generally relates to compounds that can stimulate or modulate an immune response in a subject. More specifically, the present invention relates to novel combinations of antigens and small molecules used in vaccine therapy. In one embodiment, the compounds can be used as adjuvants for the prevention and treatment of vaccines for infectious diseases and cancer. In another embodiment, they can be used as immunotherapeutic agents, alone or in combination with existing therapies, for cancer, infectious diseases, allergies / asthma, lung injury, and bronchopulmonary dysplasia. In a further embodiment, the present invention generally relates to methods for identifying small molecule immunomodulators, and more specifically, to assays for the detection of immunostimulants and immunosuppressants by measuring immune markers such as cytokines, chemokines, and / or growth factors. [Background technology]

[0004] Without limiting the scope of this invention, the background art describes diseases related to inflammation and immunomodulation.

[0005] Immunity. When the immune system is sensitized to a foreign antigen, it responds by activating a protective response. This response is characterized by the coordinated interaction of both the innate and adaptive immune systems. These systems were once considered separate and independent, but it is now recognized that when combined, the two interdependent parts satisfy two mutually exclusive requirements: velocity (to which the innate system contributes) and specificity (to which the adaptive system contributes).

[0006] The innate immune system acts as the first line of defense against invading pathogens, blocking them until an adaptive response matures. It is triggered antigen-independently within minutes of infection in response to the broad conservation patterns of the pathogen (non-specific, but capable of distinguishing self from the pathogen). Crucially, it also generates an inflammatory and co-stimulatory environment (sometimes called a danger signal) that activates the adaptive immune system and directs (or polarizes) it to the most appropriate cellular or hormonal response to fight the infectious pathogen (discussed in more detail below).

[0007] The adaptive response is effective for days or weeks, but ultimately provides fine antigen specificity for the complete elimination of the pathogen and generates immunological memory. This is primarily mediated by T and B cells, which have undergone germline gene rearrangement and are characterized by sophisticated specificity and persistent memory. However, this also involves the recruitment of elements of the innate immune system, including specialized phagocytic cells (macrophages, neutrophils, etc.) and granulocytes (basophils, eosinophils, etc.) that engulf bacteria, viruses, and even relatively large protozoan parasites. Once the adaptive immune response matures, subsequent exposure to the pathogen results in rapid elimination (usually before symptoms of infection appear) because highly specific memory cells have developed and are rapidly activated by subsequent exposure to the same antigen.

[0008] Interaction of innate and adaptive responses. Here, the earliest events after pathogen invasion are thought to be influenced by the cellular components of the innate immune system. When commensal tissue macrophages and dendritic cells (DCs) encounter a pathogen, signals are generated by the interaction between pattern-recognition receptors (PRRs), which are shared by many microorganisms, and pathogen-associated molecular patterns (PAMPs). Therefore, upon activation, they initiate a response. Activated macrophages and DCs, in response to stimulation, release various cytokines (including cytokines and chemokines such as TNF-α, IL-1β, IL-6, IL-8, MIP-1α, and MIP-1β), which constitute a "danger signal" and release natural killer (NK) agents into the tissue. It induces the influx of killer cells, macrophages, and immature dendritic cells.

[0009] When an antigen is applied, activated dendritic cells (DCs) migrate to lymph nodes. Once there, they act as antigen-presenting cells (APCs), thus becoming part of the adaptive immune response. It activates (primarily naive B and T cells). Activated cells migrate to the site of infection (guided by "danger signals"), and upon arrival, further amplify the response by recruiting cells of the innate immune system (including neutrophils, basophils, monocytes, NK cells, and granulocytes). This cell transport is orchestrated by a large array of cytokines (particularly a subgroup of chemokines) and involves immune cells of many different types and tissue sources (Luster, A.). D., The role of chemokines in linking innate and adaptive immunity. Current Opinion in Immunology 2002, 14, 129-135).

[0010] Polarization of adaptive immune responses. Adaptive immune responses exert influence primarily through two independent mechanisms: cell-mediated (Type 1) immunity and antibody-mediated or hormone-mediated (Type 2) immunity.

[0011] Type 1 immunity involves the activation of T lymphocytes that act on infected cells carrying foreign antigens or stimulate other cells to act on infected cells. Therefore, this branch of the immune system effectively contains and kills cancerous or pathogen-infected cells (especially viruses). Type 2 immunity involves the production of antibodies against foreign antigens by B lymphocytes. This antibody-mediated branch of the immune system attacks and effectively neutralizes extracellular foreign antigens.

[0012] There is a growing recognition that both sides of the immune system are crucial in fighting disease, and that the type of immune response is just as important as its intensity or duration. Furthermore, since type 1 and type 2 responses are not necessarily mutually exclusive (often, an effective immune response requires both to occur in parallel), the balance between type 1 / type 2 responses (also known as the Th1:Th2 response ratio / balance, referring to the specific cytokines and effector cell subsets involved in regulating each response—see below) can also play a role in determining the effectiveness (and rebound) of immune defense.

[0013] In many cases, the immune response is strongly skewed towards a type 1 or type 2 response immediately after exposure to an antigen. The structure of this type 1 / type 2 skew or polarization is not yet fully understood, but it is known to involve a complex system of cell-mediated chemical messengers (cytokines, especially chemokines), and the type 1 / type 2 polarization (or balance) is at least partially determined by the nature of the initial PRR-PAMP interaction when innate immune system DCs and macrophages are first stimulated, followed by the cytokine environment in which the initial antigen stimulation of naive helper T cells occurs.

[0014] Two cytokines, in particular, appear to play an initial role in determining the pathways of the immune response. Interleukin-1 (IL-1) is secreted by macrophages and stimulates the differentiation of Th1 cells, which are helper cells that manage the type 1 response. Therefore, it drives a type 1 response. Another macrophage cytokine, interleukin-10 (IL-10), inhibits this response and instead drives a type 2 response.

[0015] Type 1 and Type 2 responses can be distinguished, in particular, based on certain phenotypic changes associated with the initial stimulation and subsequent polarization of naive helper T cells. These phenotypic changes are characterized, at least in part, by the nature of the cytokines secreted by polarized helper T cells.

[0016] Th1 cells produce so-called Th1 cytokines, which include one or more of TNF-α, IL-1, IL-2, IFN-gamma, IL-12, and / or IL-18. Th1 cytokines are involved in macrophage activation, and Th1 cells harmonize and organize the type 1 response. In contrast, Th2 cells produce so-called Th2 cytokines, which include one or more of IL-4, IL-5, IL-10, and IL-13. Th2 cytokines can promote the production of various antibodies and suppress the type 1 response.

[0017] The involvement of Th1 and Th2 cells, as well as cytokines, in the polarization of Type 1 and Type 2 immune responses has led to the use of the terms Th1 response and Th2 response to define Type 1 and Type 2 immune responses, respectively. Therefore, these terms are used interchangeably in this specification.

[0018] There is a growing recognition that the type of immune response is as important in therapy and prevention as its intensity or duration. For example, an excessive Th1 response can give rise to autoimmune diseases, inappropriate inflammatory responses, and transplant rejection. An excessive Th2 response can result in allergies and asthma. Furthermore, perturbation of the Th1:Th2 ratio is symptomatic of many immune diseases and disorders, and developing ways to alter the Th1:Th2 ratio is currently a priority.

[0019] Vaccine adjuvants. The immune response to certain antigens, which would otherwise be weakly antigenic, can be enhanced by the use of vaccine adjuvants. Such adjuvants activate the immune response to specific antigens and are thus a subject of significant interest in the medical community.

[0020] Research has enabled the development of vaccines with antigenic epitopes that were previously impossible to generate. For example, current available vaccine candidates include synthetic peptides that mimic streptococcal, gonococcal, and malaria antigens. These purified antigens are generally weak antigens and require adjuvants to induce protective immunity. However, conventional vaccine adjuvants have several drawbacks that limit their overall use and effectiveness. They are also not suitable for other uses despite being acceptable as vaccines.

[0021] Substances that stimulate immune cells in vitro also exhibit similar immune-stimulatory effects in vivo. These compounds, such as recombinant cytokines, pathogen products (e.g., toxins, lipids, proteins / peptides, carbohydrates, and nucleic acids), and other mammalian-derived immunostimulatory molecules (e.g., heat shock proteins, complement, immune complexes, and proteoglycans), all induce pro-inflammatory responses that can be measured both in vitro and in vivo.

[0022] Historically, classical adjuvants have been Freund's complete or incomplete (i.e., mycobacteria-free) adjuvants. Edmund Coley described the potential of Coley toxins in cancer immunotherapy. Other materials such as mineral oil and aluminum hydroxide can also be used as adjuvants, but these have always had disadvantages. For example, mineral oil produces tissue irritation and is known to be potentially carcinogenic. Alum is the only approved adjuvant in the United States, induces granulomas at the injection site, and does not effectively induce cell-mediated immunity. Furthermore, many of the currently available adjuvants have limited usefulness because they contain components that are not metabolizable by humans. In addition, most adjuvants may require time-consuming procedures and, in some cases, the use of sophisticated and expensive equipment to formulate the vaccine and adjuvant system, making them difficult to prepare. Immunological adjuvants are described in "Current Status of Immunological Adjuvants", Ann. Rev. Immunol., 1986, 4, pp. 369-388 and "Recent Advances in Vaccine Adjuvants and Delivery Systems" by Derek T O'Hagan and Nicholas M. Valiente. See also the disclosures of various vaccine adjuvants appearing in the patent literature, U.S. Patent Nos. 4,806,352, 5,026,543, and 5,026,546.

[0023]

[0024] TLR4 agonists as immunostimulants. The use of immunomodulatory strategies aimed at improving resistance to bacterial infections can be beneficial in various clinical scenarios in which the host is susceptible to infectious complications. These include patients with severe burns or major trauma, patients who have undergone major surgery, or patients who have received immunosuppressive therapy for cancer or organ transplantation. The appeal of interventions that can improve the host response to infection is further enhanced by the increasing incidence of antibiotic resistance among bacteria, particularly those that commonly cause hospital-acquired infections.

[0025] Immunocompromised patients are at higher risk of developing bacterial infections of the central nervous system (CNS) (Safdieh, JE; Mead, PA; Sepkowitz, KA; Kiehn, TE; Abrey, LE, Bacterial and fungal meningitis in patients with cancer. Neurology 2008, 70, 943-7). The list of pathogens indicates low pathogenicity in immunocompetent hosts. This includes many organisms. Furthermore, the distribution of pathogens also differs among immunocompetent hosts and is influenced by the nature of the immune deficiency. Patients with reduced B lymphocyte function or loss of splenic function have an increased risk of meningitis caused by encapsulating bacteria, while patients with impaired T lymphocyte-macrophage system are more susceptible to CNS infections caused by intracellular pathogens. One additional factor that increases susceptibility to this CNS infection is likely reduced local immune protection.

[0026] Bacterial lipopolysaccharide (LPS, endotoxin) is a component of the cell wall of Gram-negative bacteria and is known to have immunomodulatory properties (Broad, A.; Jones, DE; Kirby, JA, Toll-like receptor (TLR) response tolerance: a key physiological "damage limitation" effect and an important potential opportunity for therapy. Current medicinal chemistry 2006, 13, 2487-502). LPS is a component of Toll-like receptor 4. Recognized by TLR4 (Toll-Like receptor 4), it is expressed by various leukocytes and activates both TRIF and MyD88-dependent signaling pathways. TLR4 signaling activity induces the production of numerous pro-inflammatory mediators, including cytokines, chemokines, and nitric oxide, which contribute to key inflammatory features such as increased vascular permeability, edema formation, and leukocyte recruitment. Interestingly, prior exposure to LPS induces a state where post-sensitization with LPS or bacteria results in a significant decrease in pro-inflammatory mediator production (Cavaillon, JM; Adrie, C.; Fitting, C.; Adib-Conquy, M., Endotoxin tolerance: is there aclinical relevance?). Journal of endotoxin research 2003, 9, 101-7). Induced by initial stimulation with LPS. This altered immunophenotype has historically been called endotoxin resistance (Cross, AS, Endotoxintolerance - current concepts in historical perspective. Journal of endotoxin Research 2002, 8, 83-98). Induction of endotoxin resistance is usually achieved with lethal doses of LPS. Endotoxin pretreatment has been shown to be highly effective in reducing both post-night sensitization-related morbidity and mortality (Murphey, ED; Fang, G.; Sherwood, ER, Endotoxin pretreatment improves bacterial clearance and decreases mortality in mice challenged with Staphylococcusaureus. Shock (Augusta, Ga.) 2008, 29, 512-8). Because initial LPS stimulation attenuates the production of pro-inflammatory cytokines in response to LPS or bacterial sensitization, many researchers previously characterized LPS resistance as an immunosuppressive state. However, few studies have examined the effect of LPS treatment on the host response to live-cell infection. Studies have demonstrated that mice initially stimulated with LPS exhibit improved resistance to bacterial infections (Wy, CA; Goto, M.; Young, RI; Myers, TF; Muraskas, J., Prophylactic treatment of endotoxic shock with monophosphoryl lipid A innewborn rats. Biology of the neonate 2000, 77, 191-5; Wynn, JL; Scumpia, PO; Winfield, RD; Delano, MJ; Kelly-Scumpia, K.; Barker, T.; Ungaro, R.; Levy, O.; Moldawer, LL, Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists. Blood 2008, 112, 1750-8). However, the clinical applicability of LPS as a therapeutic or prophylactic agent is ruled out due to its toxicity and narrow therapeutic index in humans.

[0027] Monophosphoryl Lipid A (MPLA) is an endotoxin derivative used as a vaccine adjuvant in humans (Thoelen, S.; VanDamme, P.; Mathei, C.; Leroux-Roels, G.; Desombere, I.; Safary, A.; Vandepapeliere, P.; Slaoui, M.; Meheus, A., Safety and immunogenicity of ahepatitis B vaccine formulated with a novel adjuvant system. Vaccine 1998, 16, 708-14), and is TLR4 It is a gonist. Pre-treatment with MPLA prevents hypothermia, improves survival rates, and enhances bacterial clearance from systemically infected mice (Romero, CD; Varma, TK; Hobbs, JB; Reyes, A.; Driver, B.; Sherwood, ER, The Toll-Like Receptor 4 Agonist MonophosphorylLipid A Augments Innate Host Resistance to Systemic Bacterial Infection. Infection and Immunity 2011, 79, 3576-3587). The protective effect of MPLA is mediated by TL4. Post-infection treatment with MPLA enhances bacterial clearance but does not reduce the production of inflammatory cytokines or prevent hypothermia.

[0028] TLR7 / 8 agonists as immunostimulants. Transmembrane Toll-like receptors (TLRs) have binding domains specific to different microbial and viral components (Takeda, Kaisho et al. 2003). TLR7 / 8 recognizes single-stranded viral RNs (Diebold, Kaisho et al.). (Heil, Hemmi et al. 2004). When receptor-ligand pairs ligate, intracellular signaling is initiated, and downstream transcription factors activate genes for pro-inflammatory cytokines (TNF, IL-1), interleukin type I (IFNα), and costimulatory molecules (CD80, CD86). (Lore, Betts et al. 2003, Russo, Cornella-Taracido et al. 2011, Desmet and Ishii 2012). Activation of these genes promotes dendritic cell (DC) maturation and facilitates antigen presentation and subsequent stimulation of the adaptive immune response. The precise immune response that occurs is determined by the appropriate stimulation and the resulting transcriptional and cellular changes. CD8 + Cell-mediated immunity carried out by T lymphocytes is possible via accessory Th1 cells and cytokines INFα, IL-2, and IL-12 (Pennock, White et al. 2013). However, excessive production of innate pro-inflammatory cytokines such as IL-1 and TNFα leads to both local and systemic inflammation. Therefore, the appropriate combination of these signals must be generated to create a safe adjuvant that enhances the immune response against a given antigen.

[0029] Several TLR7 / 8 agonists, including R848 and R837, are being investigated as potential vaccine adjuvants. While some of these agonists have found success as topical agents, when administered orally or intravenously, they do not reach effective concentrations. However, dose-limiting toxicity has been demonstrated. This profile renders current TLR7 / 8 agonists ineffective as systemic vaccine adjuvants (Pockros, Guyader et al. 2007). These drugs are more potent than CL075 (a TLR7 / 8 agonist) and are designed to induce lower inflammatory cytokine levels, thereby overcoming the problems of systemic adjuvants.

[0030] Acute Respiratory Distress Syndrome (ARDS) ARDS is a severe form of respiratory failure that develops in association with a variety of causes, including blood, systemic infection, inhalation of harmful drugs, bacterial infection, burns, and blast trauma, and accounts for 30-40% of all-cause mortality. In the United States, it is estimated that 200,000 individuals develop ARDS each year (Rubenfeld, GD; Caldwell, E.; Peabody, E.; Weaver, J.; Martin, DP; Neff, M.; Stern, EJ; Hudson, LD, Incidence and outcomes of acute lung injury. N Engl J Med 2005, 353, 1685-93; Villar, J.; Blanco, J.; Kacmarek, RM, Current incidence and outcome of the acute respiratory distress syndrome. Current opinion in critical care 2016, 22, 1-6). One of the main complications of trauma-based morbidity in combat is the development of ARDS, occurring in 8–82% of selected patient populations. These include patients with pulmonary contusions, severe trauma (trauma severity score > 25), head injuries, transfusion requirements of concern, and major orthopedic injuries such as long bone and pelvic fractures. The presence of ARDS is associated with a significant increase in morbidity, increased use of hospital resources, and up to a 4.3-fold increase in mortality (Salim, A.; Martin, M.; Constantinou, C.; Sangthong, B.; Brown, C.; Kasotakis, G.; Demetriades, D.; Belzberg, H., Acute respiratory distress syndrome). in the trauma intensive care unit: Morbid but not mortal. Arch Surg 2006, 141,655-8).

[0031] To date, there is no specific treatment for ARDS. Current therapies consist only of supportive care, using ventilators to support the lungs until full recovery is achieved. Therefore, new therapies for this condition are urgently needed.

[0032] Another such disease is bronchopulmonary dysplasia (BPD), the most common chronic respiratory disease in infants and young children, a devastating condition that disrupts the lung development program and often occurs as a consequence of premature birth. BPD occurs secondary to the interaction of genetic and environmental factors (hyperoxia, invasive mechanical ventilation, and sepsis) (Bhandari, A.; Bhandari, V., Pitfalls, problems, and progress inbronchopulmonary dysplasia. Pediatrics 2009, 123, 1562-73; Bhandari, A.; Bhandari, V., "New" BronchopulmonaryDysplasia: A clinical review. Clin Pulm Med 2011, 18, 137-143; Jensen, EA; Schmidt, B., Epidemiology of bronchopulmonary dysplasia. Birth defects research. Part A, Clinical and molecular teratology 2014, 100, 145-57).The definition of BPD has evolved over the past decade, but is currently defined as the "physiological" assessment of the need for oxygen (O2) supplementation over a 28-day period and the supplementation O2 requirement at 36 weeks postmenstrual (Bhandari, A.; Bhandari, V., "New" Bronchopulmonary Dysplasia: A clinical review. Clin Pulm Med 2011, 18,137-143; Trembath, A.; Laughon, MM, Predictors of bronchopulmonary dysplasia. Clin Perinatol 2012, 39,585-601; Bancalari, E.; Claure, N., Bronchopulmonary dysplasia: definitions and epidemiology. In Bronchopulmonary Dysplasia, Firsted.; Bhandari, V., Ed. Springer International Publishing: Switzerland, 2016; pp167-182). In the United States, 10,000 to 15,000 new cases of bipolar disorder (BPD) occur every year. Importantly, 97% of all BPD cases occur in infants with a birth weight of less than 1250 grams (Bhandari, A.; Bhandari, V., "New" Bronchopulmonary Dysplasia: A clinical review. Clin Pulm Med 2011, 18, 137-143). Despite many advances in neonatal ventilation techniques, the widespread use of surfactants and prenatal corticosteroids, and invasive fluid management, the incidence of BPD remains the same (Smith, VC; Zupancic, JA; McCormick, MC; Croen, LA; Greene, J.; Escobar, GJ; Richardson, DK, Trends in severe bronchopulmonary dysplasia rates between 1994 and 2002. J Pediatr 2005, 146, 469-73), and there has even been a slight increase (Bhandari, A.; Bhandari, V., "New" Bronchopulmonary Dysplasia: A clinical review. Clin Pulm Med 2011, 18, 137-143; Trembath, A.; Laughon, MM, Predictors of bronchopulmonary dysplasia. Clin Perinatol 2012, 39, 585-601). Managing BPD places a significant burden on healthcare services. In preterm infants, the single most expensive complication during infant hospitalization is bilateral pediatric disease (BPD), with an average cost of $116,000 per discharge (Russell, RB; Green, NS; Steiner, CA; Meikle, S.; Howse, JL; Poschman, K.; Dias, T.; Potetz, L.; Davidoff, MJ; Damus, K.; Petrini, JR, Cost of hospitalization for preterm and low birth weight infants in the United States. Pediatrics 2007, 120, e1-9). In addition, BPD is a serious complication that can lead to persistent health problems into adulthood. Related to pulmonary and neurodevelopmental sequelae (Bhandari, A.; Bhandari, V., "New" Bronchopulmonary Dysplasia: A clinical review. Clin Pulm Med 2011, 18, 137-143, Natarajan, G.; Pappas, A.; Shankaran, S.; Kendrick, DE; Das, A.; Higgins, RD; Laptook, AR; Bell, EF; Stoll,BJ; Newman, N.; Hale, EC; Bara, R.; Walsh, MC, Outcomes of extremely low birth weight infants with bronchopulmonary dysplasia: impact of the physiologic definition. Early Hum Dev 2012, 88, 509-15, Bhandari, A.; McGrath-Morrow, S., Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia. Semin Perinatol 2013, 37,132-7, Raju, TNK; Buist, AS; Blaisdell, CJ; Moxey-Mims, M.; Saigal, S., Adults born preterm: a review of general health and system-specific outcomes.Acta Paediatr 2017, 106, 1409-1437). Therefore, treatment and costs, as well as access to health services, in the life of those born prematurely. Understanding the long-term outcomes of BPD is important because it may have a significant impact.

[0033] Typical pathological features of BPD include hyperxia-induced pulmonary inflammation (Bhandari, V., Postnatal inflammation in the pathogenesis of bronchopulmonary dysplasia. Birth defects research. Part A, Clinical and molecular teratology 2014, 100, 189-201; Balany, J.; Bhandari, V., Understanding the Impact of Infection, Inflammation, and Their Persistence in the Pathogenesis of Bronchopulmonary Dysplasia. Front Med(Lausanne) 2015, 2, 90; Harijith, A.; Bhandari, V., Hyperoxia in the pathogenesis of bronchopulmonary dysplasia. In Bronchopulmonary Dysplasia, First ed.; Bhandari, V., Ed. Springer International Publishing: Switzerland, 2016; pp 3-26), increased cell death (Li, Z.; Choo-Wing, R.; Sun, H.; Sureshbabu, A.; Sakurai, R.; Rehan, V. K.; Bhandari, V., A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-beta1-mediated injury in the developing murine lung. BMC Cell Biol 2011, 12, 54, Choo-Wing R, S. M., Harijith A, Bowen B, Pryhuber G, Janer C, Andersson S, Homer RJ, Bhandari V, Hyperoxia and interferon-γ-induced injury in developing lungs occur via cyclooxygenase-2 and the 内质网应激依赖性途径《美国呼吸细胞与分子生物学杂志》2013年,第48卷,第749 - 757页、苏雷什巴布,A.;赛义德,M. A.;博杜帕利,C. S.;德霍达卡尔,M. V.;霍默,R. J.;米努,P.;班达里,V.,TGFβ1的条件性过表达通过TGFβR2促进发育中小鼠肺的炎症、凋亡和死亡。《呼吸研究》2015年,第16卷,第4期、苏雷什巴布,A.;赛义德,M.;达斯,P.;贾纳,C.;普赖胡伯,G.;拉赫曼,A.;安德森,S.;霍默,R. J.;班达里,V.,抑制RPTOR可通过增强自噬和减少新生小鼠的凋亡来预防高氧诱导的肺损伤。《美国呼吸细胞与分子生物学杂志》2016年)、血管新生因子的调节不全(班达里,V.;朱 - 温,R.;李,C. G.;优素福,K.;内德雷洛,J. H.;安巴拉瓦南,N.;马尔库斯,H.;霍默,R. J.;伊莱亚斯,J. A.,发育再 gulation of NO-mediated VEGF-induced effects in the lung. Am J Respir Cell Mol Biol 2008, 39, 420–30 Sun HC-WR, Sureshbabu A, Fan J, LengL, Yu S, Jiang D, Noble P, Homer RJ, Bucala R,Bhandari V murine lung. PLoS ONE 2013, 8. Sun H, C.-WR, Fan J, Leng L, Syed MA, Hare AA, Jorgensen WL, Bucala R, Bhandari V, Small molecularmodulation of macrophage migration inhibitory factor in the hyperoxia-inducedmouse model of bronchopulmonary dysplasia. Respir Res 2013, 14、Syed, MA; Choo-Wing , R. ; Homer , RJ ; Bhandari, V., Role ofNitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury inVascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs. PLoS One 2016, 11, e0147588). Neonatal dysregulation occurs (Balany, J.; Bhandari, V., Understanding the Impact of Infection, Inflammation, and Their Persistence in the Pathogenesis of Bronchopulmonary Dysplasia. Front Med (Lausanne) 2015, 2, 90). Respiratory distress syndrome (RDS) and hyperoxia are common prodromal symptoms of biparietal disease (BPD). Preterm birth The current standard-of-care treatment for RDS in neonates is exogenous surfactants and supplemental oxygen, but there is no specific effective method for the prevention or treatment of BPD (Bhandari, V., Drug therapy trials for the prevention of bronchopulmonary Dysplasia: Current and Future Targets. Front Pediatr 2014, 2, 76). According to the National Institute of Child Health and Human Development / National Heart, Lung and Blood Institute (NICHD / NHLBI), identifying potential drugs targeting BPD in premature infants is classified as a "research priority" (McEvoy, CT; Jain, L.; Schmidt, B.; Abman, S.; Bancalari, E.; Aschner, JL, Bronchopulmonary dysplasia: NHLBI Workshop on the Primary Prevention of Chronic Lung Diseases. Annals of the American Thoracic Society 2014, 11 Suppl3, S146-53). The National Institutes of Health (NIH) workshop focused on the primary prevention of chronic lung diseases with a focus on BPD. The workshop was organized by the NHLBI of the NIH (McEvoy, CT; Jain, L.; Schmidt, B.; Abman, S.; Bancalari, E.; Aschner, JL, Bronchopulmonary dysplasia: NHLBI Workshop on the Primary Prevention of Chronic Lung Diseases. Annals of the American Thoracic Society 2014, 11 Suppl 3, S146-53). It was noteworthy and disappointing that only two specific drugs—caffeine and inhaled nitric oxide (iNO)—were mentioned in terms of "promising near-future prospects for primary BPD prevention research," specifically "clinical research priorities and specific clinical trials for BPD prevention" (Bhandari, V., Drug therapy trials for the prevention of bronchopulmonary dysplasia: current and future targets. Front Pediatr 2014, 2, 76).

[0034] Reducing inflammation through the activation of the anti-inflammatory cytokine IL-10 is a therapeutic approach for acute lung injury (ALI), ARDS, and BPD, which are related to bacterial infections and other factors. It is characterized by the overproduction of pro-inflammatory cytokines TNF-α, IL-6, i-NOS, and ROS resulting from injury, excessive exposure to oxygen and harmful gases. These mediators induce pulmonary endothelial and epithelial damage, vascular leakage, edema, and vasodilation, subsequently leading to the development of ALI and ARDS (Densmore, JC; Signorino, PR; Ou, J.; Hatoum, OA; Rowe, JJ; Shi, Y.; Kaul, S.; Jones, DW; Sabina, RE; Pritchard, KA, Jr.; Guice, KS; Oldham, KT, Endothelium-derived microparticles induce endothelial dysfunction and acute lung injury. Shock 2006, 26, 464-71). IL-10, primarily produced by T helper 2 cells, B cells, monocytes, macrophages, and keratinocytes, is known to reduce the synthesis of pro-inflammatory cytokines and terminate inflammatory responses (Moore, KW; Rousset, F.; Banchereau, J., Evolving principles in immunopathology: interleukin 10 and its relationship to Epstein-Barr virus protein BCRF1. Springer seminars in immunopathology 1991, 13, 157-66). Low levels of IL-1 0 is found in patients with transfusion-related ALI (Kapur, R.; Kim, M.; Rebetz, J.; Rondina, MT; Porcelijn, L.; Semple, JW, Low levels of interleukin-10 in patients with transfusion-related acute lung injury. Annals of Translational Medicine 2017, 5, 339). Importantly, treatment with IL-10 is ischemia-reperfusion, lipopolysaccharide (LPS) (Bi, MH; Wang, BE; Zheng, XX; Li, M.; Mayer, K.; Zhang, SW, [The effect of recombinant interleukin-10 / Fc fusion protein on lipopolysaccharide-induced acute lung injury in mice]. (Zhongguowei zhong bing ji jiu yi xue = Chinese critical care medicine = Zhongguoweizhongbing jijiuyixue 2008, 20, 461-4), it mitigated bleomycin and ozone-induced lung injury, and the absence of endogenous IL-10 enhanced carrageenan-induced ALI. In addition, it has been reported that pre-incubation of cultured fetal rat type II alveolar cells with recombinant IL-10 before exposure to 65% hyperoxia reduces cell necrosis and increases cell proliferation (Lee, H.-S.; Kim, C.-K., Effect of recombinant IL-10 on cultured fetal rat alveolar type II cells exposed to 65%-hyperoxia. Respiratory Research 2011, 12, 68-68). As reported by Bhandari, V., Molecular mechanisms of hyperoxia-induced acute lung injury. Frontiers in bioscience: a journal and virtual library 2008, 13, 6653-61 and others (Li, HD; Zhang, QX; Mao, Z.; Xu, XJ; Li, NY; Zhang, H., Exogenous interleukin-10 attenuates hyperoxia-induced acute lung injury in mice. Experimental physiology 2015, 100, 331-40), exogenous IL-10 treatment is effective. This likely alleviated hyperxia-induced ALI in mice by regulating neutrophil recruitment, followed by the generation of cytokines, NO, and matrix metalloproteinases.

[0035] Acne vulgaris is a common disorder affecting 17 million people in the United States alone. While acne is rarely life-threatening, it can have a significant impact on the physical and mental state of patients. The pathogenesis of acne is multifactorial, involving hormonal, microbial, and immune mechanisms. One of the factors contributing to the pathogenesis of acne is Propionibacterium acnes, which can be significantly increased in the follicular sebaceous gland units of acne patients. It is part of Laura (Leyden, McGinley et al. 1975). P. acnes is a Gram-positive bacterium, but it is variable and weakly Gram-positive. It is rod-shaped and slightly curved, and is therefore described as diphtheriae-like or coryneform. Several distinctive features in the cell wall and outer envelope of P. acnes further distinguish it from other Gram-positive bacteria. P. acnes synthesizes phosphatidylinositol, which is impossible for almost all other bacteria but can be produced by virtually all eukaryotes. Unlike most Gram-positive bacteria, the peptidoglycan of P. acnes contains a cross-linking region between the peptide chain and L,L-diaminopimelic acid and D-alanine, where two glycine residues are combined with the amino and carboxyl groups of two L,L-diaminopimelic acid residues (Kamisango, Saiki et al. 1982).

[0036] P. acnes contributes to the inflammation of acne by inducing monocytes to secrete pro-inflammatory cytokines including TNF-α, IL-1β, and IL-8 (Vowels, Yang). (et al. 1995). In particular, IL-8 accompanied by other P. acnes-induced chemotactic factors can play an important role in attracting neutrophils to the follicular sebaceous gland unit. In addition, P. acnes releases lipases, proteases, and hyaluronidases that contribute to tissue damage (Hoeffler, Ko et al. 1976, Hoeffler 1977). For these reasons, P. acnes is inflammatory It is the primary target for acne treatment.

[0037] The mechanism by which P. acnes activates cytokine release in monocytes is unknown, but it is thought to be associated with pattern recognition receptor (PRR) 3 of the innate immune system. Recently identified Toll-like receptors (TLRs) are an example of PRRs. Toll receptors were first identified by Drosophila, and mammalian homologs have been found to mediate immune responses to microbial ligands (Me dzhitov, Preston-Hurlburt et al. 1997, Yang, Mark et al. 1998). TLR is grams While it has been suggested that Gram-positive and Gram-negative organisms can be distinguished (Underhill, Ozinsky et al. 1999), bacterial ligands of Gram-positive bacteria that can activate monocytes via TLR2 or TLR4 have been identified (Takeuchi, Hoshino et al. 1999).

[0038] Reported evidence suggests that P. acnes induces inflammatory cytokines in monocytes via a TLR2-dependent pathway. TLR2 expression in acne lesions indicates that TLR2 activation may contribute to inflammation at the site of disease activity. Therefore, there remains a need for improved compositions and methods to treat these conditions.

[0039] Psoriasis. Interleukin (IL)-10 is an important immunomodulatory cytokine. One of its main biological functions appears to be the limitation and termination of inflammatory responses. Surprisingly, relative deficiency of IL-10 expression has been found in psoriasis, a highly prevalent inflammatory skin disease characterized by a type 1 cytokine pattern. Induction of IL-10 expression has been found with conventional antipsoriasis therapies, suggesting that IL-10 may be a key cytokine in psoriasis and that the application of this cytokine may have therapeutic effects. In initial clinical trials of 3-7 weeks in patients with established psoriasis, IL-10 was well tolerated and clinically effective. In long-term trials in patients in remission, IL-10 therapy reduced the incidence of relapses and extended the asymptomatic period. Laboratory studies suggest that IL-10 exerts its antipsoriasis activity by affecting different cell populations, including antigen-presenting cells and T cells. IL-10 resulted in a persistent type 1 / type 2 cytokine balance shift. However, the direct effect of IL-10 on keratinocytes is unlikely to contribute to clinical response, as keratinocyte non-responsiveness to IL-10 was observed in vitro. IL-10 appears to have significant importance in psoriasis (Asadullah, Volk et al. 2002).

[0040] Until now, the cytokine IL-10 has been used therapeutically as a recombinant protein, i.e., as a large molecule. Therefore, it is quite expensive to produce, must be administered by injection, which is quite inconvenient for patients, and can induce neutralizing antibodies, which can limit its effectiveness and necessitate its exclusion from long-term use. Identifying molecules that mediate the effects of this cytokine, which are suitable for pharmacological intervention with small molecules, is therefore becoming increasingly interesting. Induction of IL-10 cytokine production by small molecular weight compounds represents a novel therapeutic approach.

[0041] Arthritis. Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation that often leads to joint destruction. It is now established that pro-inflammatory cytokines such as TNF-β, IL-1, GM-CSF, and IL-6 are all produced by the synovial membrane of RA patients and play an important role in the pathophysiology of this disease (Di Giovine, Nuki et al. 1988, Hirano, Matsuda et al. 1988, Miyasaka, Sato et al. 1988, Harris 1990).

[0042] Both IL-10 mRNA and protein are present in the synovial membrane of rheumatoid arthritis (RA) and osteoarthritis (OA). IL-10 plays a crucial role in the cytokine network of rheumatic synovial membrane, contributing immunomodulatory functions to inflammatory and, if possible, T-cell cytokine production. Conversely, IL-10 itself is regulated by IL-1 and TNF-α; therefore, IL-10 appears to be a key component of the complex cytokine network of rheumatic synovial membrane. Finally, we demonstrated that exogenous IL-10 inhibits both TNF-α and IL-1β production by RA synovial membrane cultures. These findings suggest the potential for novel therapeutic strategies involving IL-10 upregulators for the treatment of rheumatoid arthritis (Katsikis, Chu et al. 1994).

[0043] Lung cancer. Worldwide, lung cancer is the most common cause of cancer-related death in men and women. Lung cancer mortality rates have been rising in recent decades. Chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD) are risk factors for lung cancer. It has been confirmed that TLR4 is also actively involved in the immune response to cancer, so researchers hypothesized that TLR4 plays both a protective role in normal cells and a negative role in cancer cells (Starska, Forma et al. 2012). However, the available evidence is still not conclusive in linking TLRs to lung cancer. Studies of functional TLR-4 and mature TLR4 in mice have found that mice with the former had lower pulmonary capillary permeability, less weight loss, leukocyte inflammation, and primary tumorigenesis. Therefore, Bauer et al. The authors hypothesized that TLR4 inhibits lung carcinogenesis by inhibiting tumor progression (Bauer, Dixonet al. 2005). The researchers demonstrated that TLR4 activation can protect the lung from inflammation during any potential tumor formation (Bauer, Fostel et al.). (2009). In other areas, smokers have lower levels of T in the nasal mucosal epithelium compared to non-smokers. LR4 was observed and greatly reduced in patients with severe COPD (MacRedmond, Greene et al. 2007). This finding suggests a potential role of TLR4 in airway inflammation and lung cancer progression. In vitro studies have also found that TLR4 is constitutively expressed and upregulated in human lung cancer cells (Zhang, He et al. 2009). In one study, TL R4 levels were significantly associated with the production of immunosuppressive cytokines, the production of pro-angiogenic chemokines, and resistance to apoptosis by lung cancer cells (He, Liu et al. 2007). Despite the reported significance of TLR-9 in lung cancer progression (Droemann, Albrecht et al. 2005, Wang, Rayburn et al. 2006, Manegold, Gravenor et al. 2008, Ren, Wen et al. 2008), the correlation between TLR4 and tumor differentiation, rather than TLR-9, in lung cancer patients remains unresolved. A statistical correlation (P, 0.05) was reported (Zhang, He et al. 2009).

[0044] Neovascularization. Posterior segment neovascular ocular disease, exemplified by proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and retinopathy of prematurity (ROP), is a growing and significant health threat that requires new and effective therapies. Retinal neovascularization associated with PDR is a leading cause of blindness in working-age adults. Choroidal neovascularization (CNV) accounts for 200,000 new cases of exudative AMD annually in the United States, and this neovascular pathology is a leading cause of legal blindness in the non-Third World. The projected number of AMDs in 2020 was 196 million, and is expected to increase to 288 million by 2040 [Wong et al, The Lancet Global Health 2014, 2, e106-e116]. Pathological vascular involvement of ROP. Neonatal blindness is the leading cause of blindness in children under 7 years of age [Harrell et al, NeonatalNetwork 2007, 26, 371-378].

[0045] Toll-like receptor (TLR2 / 4) signaling may be associated with pathological changes in retinal diseases, including AMD eye, mediated by oxidized lipids, lipofuscin, and drusen components [Cho et al, Investigative Ophthalmology & Visual Science 2009, 50, 5614-5618]. Therefore, TLR4 may contribute to the pathogenesis of AMD through multiple mechanisms, including the release of TNF-α, interleukin 1-β, and other pro-inflammatory mediators. Activation of TLR4 suppresses Wnt signaling, leading to reduced expression and secretion of growth factors, and increased photoreceptor death in response to oxidative stress, and may also result in oxidative damage to the outer segments of photoreceptors. TLR4 has a direct effect on several inflammation-related signaling pathways, including MAPK, NFκ-B, and Jak1 / Stat1, and has been shown to mediate caspase-3, neuronal iNOS, and neurotoxicity via ERK1 / 2, JNK1 / 2, and p38. Interestingly, TLR4-mediated microglial activity by endogenous photoreceptor proteins in retinal inflammation may exacerbate retinal cell death. Finally, high-mobility groups in ischemic nerve tissue. Release of box-1 has been shown to initiate a TLR4-dependent response that contributes to retinal neovascularization [He et al, Arteriosclerosis, Thrombosis, and Vascular Biology. 2013; 33:330-338].

[0046] Therefore, there is a need for more effective treatments for inflammation, particularly for the pathogenesis of both dry and wet AMD. Accordingly, the compounds and methods described herein have demonstrated that inhibition of TLR2 / 4 activity has therapeutic value in ROP, DR, AMD, and other retinal diseases. The innovative compounds are small molecules that can synergistically inhibit angiogenesis and inflammation and accelerate phagocytosis, with therapeutic potential to treat these diseases. [Prior art documents] [Patent Documents]

[0047] [Patent Document 1] U.S. Patent No. 4,806,352 [Patent Document 2] U.S. Patent No. 5,026,543 [Patent Document 3] U.S. Patent No. 5,026,546 [Non-patent literature]

[0048] [Non-Patent Document 1] Luster, AD, The role of chemokines inlinking innate and adaptive immunity. Current Opinion in Immunology 2002, 14,129-135 [Non-Patent Document 2] "Current Status of Immunological Adjuvants", Ann. Rev. Immunol., 1986, 4, pp. 369-388 [Non-Patent Document 3] "Recent Advances in Vaccine Adjuvants and Delivery Systems" by Derek T O'Hagan and Nicholas M. Valiente [Non-Patent Document 4] Safdieh, JE; Mead, PA; Sepkowitz, KA; Kiehn, TE; Abrey, LE, Bacterial and fungal meningitis in patients with cancer. Neurology 2008, 70, 943-7 [Non-Patent Document 5] Broad, A.; Jones, DE; Kirby, JA,Toll-like receptor (TLR) response tolerance: a key physiological "damagelimitation" effect and an important potential opportunity for therapy.Current medicinal chemistry 2006, 13,2487-502 [Non-Patent Document 6] Cavaillon , JM ; Adrie,C.; Fitting, C.; Adib-Conquy, M., Endotoxin intolerance: is there a clinical relevance? Journal of Endotoxin Research2003, 9, 101-7

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[0049] The compounds of the present invention have a chemical structure obtained through an easily synthesized route compared to MPLA and other TLR4 agonists (Adanitsch, F.; Shi, J.; Shao, F.; Beyaert, R.; Heine, H.; Zamyatina, A., Synthetic glycan-based TLR4 agonists targeting caspase-4 / 11 for the development of adjuvants and immunotherapeutics. Chemical Science 2018, 9, 3957-3963). Unexpectedly, the compounds of the present invention target TLR4, TLR6, TLR7 and T These compounds are found to be LR8 agonists. They enhance bacterial and viral clearance in post-infection treatment, similarly reduce the production of inflammatory cytokines, and demonstrate a balance of Th1 and Th2 cytokine production by these compounds, which has not been shown in MPLA. Prior treatment with some of the compounds of the present invention stimulates human monocytes to become macrophages and increases intracellular phagocytosis of bacteria, thus possessing immunostimulatory activity. Subsequent treatment with some of the compounds of the present invention reduces inflammatory cytokines and prevents tissue damage in mouse models of BPD and ARDS, thus possessing anti-inflammatory activity. Unexpectedly, the compounds of the present invention are found to be TLR2, TLR4, TLR7, TLR8, and TLR9 antagonists. Treatment with some of these compounds of the present invention inhibits the proliferation of lung cancer cells and reduces neovascularization in epithelial cells, thus possessing anti-cancer and anti-angiogenic activity, respectively.

[0050] In one embodiment, the present invention relates to formula I:

[0051] [ka]

[0052] The compound comprises [wherein n=0 to 5, X=NH, O, S, CH2, Y=phenyl, or a phenyl group substituted with at least one methyl, a phenyl group substituted with at least one nitro, a phenyl group substituted with at least one nitrogen, a phenyl group substituted with at least one boron, or an aryl, substituted aryl, heteroaryl, 4-6 membered cycloalkyl, 4-6 membered heterocycloalkyl, R=H, C(O)R2, SO2R2, R1=H, C(O)R2, SO2R2, R2=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, NH2, NR3R4, R3, R4=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, 3-6 membered cycloalkyl, and Z=NH, O, S, CH2 or none]. In one embodiment, the amount of the compound is modified or selected to inhibit or activate the immune response. In one embodiment, the compound has the following formula.

[0053] [ka] TIFF2023109826000003.tif179169 TIFF2023109826000004.tif208170 TIFF2023109826000005.tif80128

[0054] In one embodiment, the compound inhibits TLR4, TLR2, or both TLR2 and TLR4 receptors and has the following formula and concentration.

[0055] [Table 1] TIFF2023109826000007.tif72163

[0056] In another embodiment, the compound inhibits an immune response at a first concentration and activates an immune response at a second concentration, and the compound is formulated into low or high-dose compositions comprising the following:

[0057] [Table 2]

[0058] In another embodiment, the compound has the following formula:

[0059] [ka]

[0060] To inhibit the immune response, the composition is formulated at low concentrations of 0.1 to 50 milligrams / kg.

[0061] In another embodiment, the compound has the following formula:

[0062] [ka]

[0063] To activate the immune response, the compound is formulated in compositions at high concentrations exceeding 50 milligrams / kg. In another embodiment, the compound is formulated in compositions to treat hyperinflammation selected from lung injury, lung cancer, irritable bowel disease, arthritis, psoriasis, acne, BPD, arthritis, necrotizing enterocolitis, or sepsis, and is formulated in low concentrations to inhibit the immune response. The following compounds are provided and selected.

[0064] [ka]

[0065] In another embodiment, the compound is a vaccine adjuvant, an antimicrobial agent. The compounds are formulated into compositions to activate the immune response as antibacterial, antiviral, or immunostimulators, and the compounds are selected from the following: It will be selected.

[0066] [ka]

[0067] In another embodiment, the compound is formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, or salts. In another embodiment, the compound is formulated into a pharmaceutical composition adapted for intrapulmonary, alveolar, rectal, parenteral, intravenous, topical, or oral administration. In another embodiment, the compound is formulated into an aerosol, nebulizer, or inhaler. In another embodiment, the compound is used in a composition further comprising one or more liposomes, polymers, surfactants, salts, or buffers. In another embodiment, the compound is used in a composition further comprising additional therapeutic agents selected from the group consisting of corticosteroids, bronchodilators, anticholinergics, vasodilators, diuretics, antihypertensives, acetazolamide, antibiotics, antivirals, immunosuppressants, and surfactants. In another embodiment, the compound is provided in an amount that competitively inhibits inflammation and activates macrophages to protect or limit lung tissue damage. In another embodiment, the compound is a TLR4 modulator and is provided in an amount that upregulates IL-10. In another embodiment, the compound is a TLR2, TLR4, TLR7, and TLR8 inhibitor and is provided in an amount that downregulates IL-1β. In yet another embodiment, the compound is a TLR9 inhibitor that downregulates IFN-α.

[0068] In another embodiment, one or more compounds can each directly bind to the active site of TLR4. In another embodiment, the compound binds to TLR2, TLR6, TLR7, and TLR8, but not to TLR1 and TLR5. In another embodiment, the compound is formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, or salts. In another embodiment, the compound is formulated into a pharmaceutical composition suitable for intrapulmonary, intraalveolar, intrarectal, parenteral, intravenous, topical, or oral administration. In another embodiment, the compound is formulated in an aerosolized form. In another embodiment, the compound is suitable for use in a nebulizer or inhaler. In another embodiment, the composition further comprises one or more liposomes, polymers, surfactants, salts, or buffers. In another embodiment, the composition further comprises additional therapeutic agents selected from the group consisting of corticosteroids, bronchodilators, anticholinergics, vasodilators, diuretics, antihypertensives, acetazolamide, antibiotics, antivirals, anticancer drugs, immunosuppressants, and surfactants. In another embodiment, the compound is suitable for lung tissue injury. To protect or limit lung tissue damage, the compound competitively inhibits inflammation and activates alternative pathways of macrophages. In another embodiment, the compound selectively kills lung cancer cells but does not kill normal lung epithelial cells. In another embodiment, the compound targets peripheral blood monocytes and human umbilical vein endothelial cells (HUVECs). It is not cytotoxic. In another embodiment, the compound reduces VEGF-induced angiogenesis in HUVEC.

[0069] A method for treating lung injury, which involves identifying the subject who requires treatment for lung injury, and applying formula (Formula I):

[0070] [ka]

[0071] A method comprising providing an effective amount of a compound having [wherein n=0 to 5, X=NH, O, S, CH2, Y=phenyl, or a phenyl group substituted with at least one methyl, a phenyl group substituted with at least one nitro, a phenyl group substituted with at least one nitrogen, a phenyl group substituted with at least one boron, an aryl, a substituted aryl, a heteroaryl, a 4-6 membered cycloalkyl, a 4-6 membered heterocycloalkyl, R=H, C(O)R2, SO2R2, R1=H, C(O)R2, SO2R2, R2=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, NH2, NR3R4, R3, R4=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, a 3-6 membered cycloalkyl, and Z=NH, O, S, CH2 or none]. In one embodiment, the amount of the compound in the formulation or composition is modified or selected to inhibit or activate an immune response.

[0072] In one embodiment, the compound is 1, 2, 3, or 13. In another embodiment, the subject is human. In another embodiment, the lung injury is acute respiratory distress syndrome (ARDS), adult respiratory distress syndrome (adult RDS), hyperoxygen-induced lung injury, or This is bronchopulmonary dysplasia (BPD). In another embodiment, lung disorders include chronic obstructive pulmonary disease (COPD), exacerbated COPD, cystic fibrosis, asthma, severe asthma, and exacerbated asthma. The conditions are: breath, allergic asthma, acute lung injury, idiopathic pulmonary fibrosis, airway remodeling, obstructive bronchiolitis syndrome, or lung cancer. In another embodiment, the compound is formulated into a pharmaceutical composition suitable for intrapulmonary, alveolar, rectal, parenteral, intravenous, topical, or oral administration. In another embodiment, the compound is administered by a nebulizer or inhaler. In another embodiment, the compound is inhaled in an aerosolized form. In another embodiment, the compound is inhaled in an aerosolized form containing droplets less than 10 micrometers in diameter, the droplets containing the compound in a suitable pharmacokinetically acceptable liquid carrier. In another embodiment, the compound is inhaled in a powder form containing particles less than 10 micrometers in diameter. In another embodiment, the subjects are premature infants born during a gestation period of approximately 24 to approximately 32 weeks. In another embodiment, the subjects are infants whose birth weight is approximately 1500 grams or less. In another embodiment, the subjects are infants In another embodiment, the birth weight of infants is approximately 1000 grams or less. In another embodiment, the subject is an infant, and at least one additional agent or therapy is selected from the group consisting of oxygen therapy, mechanical ventilation, and bronchodilators. In another embodiment, the method further comprises administering an additional therapeutic agent selected from the group consisting of corticosteroids, bronchodilators, anticholinergics, vasodilators, diuretics, antihypertensives, acetazolamide, antibiotics, immunosuppressants, surfactants, and supplemental oxygen. In another embodiment, the compound is provided in an amount sufficient to protect or limit lung tissue damage, competitively inhibit inflammation, and activate an alternative pathway for macrophage activation. In another embodiment, the compound is a TLR4 modulator that upregulates IL-10. In another embodiment, the compound is a TLR2, TLR4, TLR7, and TLR8 inhibitor that downregulates IL-1β. In another embodiment, the compound is a TLR9 inhibitor that downregulates IFN-α. In another embodiment, the compound inhibits cancer cell proliferation and induces cancer cell death. In another embodiment, the compound inhibits angiogenesis. In yet another embodiment, the compound is provided in an amount sufficient to competitively inhibit inflammation and angiogenesis and protect against angiogenesis-related injury of the eye.

[0073] A method for suppressing inflammation and upregulating the anti-inflammatory cytokine IL-10 by inhibiting the TLR2, TLR4, TLR7, TLR8, and TLR9 pathways, wherein the method involves identifying subjects requiring treatment for lung injury, and applying formula I to the subjects:

[0074] [ka]

[0075] [wherein n=0~5, X=NH, O, S, CH2, Y=phenyl, a phenyl group substituted with at least one methyl, a phenyl group substituted with at least one nitro, a phenyl group substituted with at least one nitrogen, a phenyl group substituted with at least one boron, aryl, substituted aryl, heteroaryl, 4-6 membered cycloalkyl, 4-6 membered heterocycloalkyl, R=H, C(O)R2, SO2R2, R1=H, C(O)R2, SO2R A method comprising providing an effective amount of a compound having [R2 = ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, NH2, NR3R4 = ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, 3-6 membered cycloalkyl, and Z = NH, O, S, CH2 or none], wherein the compound suppresses inflammation in vivo by inhibiting the TLR4 / TLR2 pathway and upregulating the anti-inflammatory cytokine IL-10. In a particular embodiment, the amount of the compound is formulated in an amount selected to inhibit or activate the immune response described in the table above. In one embodiment, the composition improves the lung barrier function in vivo. In another embodiment, the subjects are premature infants born during a gestation period of about 24 to about 32 weeks. In another embodiment, the subjects are infants whose birth weight is about 1500 grams or less. In another embodiment, the subjects are infants whose birth weight is about 1000 grams or less. In another embodiment, the subject is an infant, and at least one additional drug or therapy is selected from the group consisting of oxygen therapy, mechanical ventilation, and bronchodilators. In another embodiment, the compound is a nebulizer or inhaler. In another embodiment, the compound is inhaled in an aerosolized form. In another embodiment, the compound is inhaled in an aerosolized form containing droplets less than 10 micrometers in diameter, the droplets containing the compound in a suitable pharmacoposityally acceptable liquid carrier. In another embodiment, the compound is inhaled in a powder form containing particles less than 10 micrometers in diameter. In another embodiment, the subject has a lung disorder selected from chronic obstructive pulmonary disease (COPD), exacerbating COPD, cystic fibrosis, asthma, severe asthma, exacerbating asthma, allergic asthma, acute lung injury, idiopathic pulmonary fibrosis, airway remodeling, or bronchiolitis obliterans syndrome. In another embodiment, the subject has sepsis or acute lung injury (ALI). In another embodiment, the compound is formulated into pharmaceutical compositions suitable for intrapulmonary, intraalveolar, intrarectal, parenteral, intravenous, topical, or oral administration.

[0076] Formula I:

[0077] [ka]

[0078] Compounds of the following type: [wherein n=0 to 5, X=NH, O, S, CH2, Y=phenyl, or a phenyl group substituted with at least one methyl group, a phenyl group substituted with at least one nitro group, a phenyl group substituted with at least one nitrogen group, a phenyl group substituted with at least one boron group, aryl, substituted aryl, heteroaryl, 4-6 membered cycloalkyl, 4-6 membered heterocycloalkyl, R=H, C(O)R2, SO2R2, R1=H, C(O)R2, SO2R2, R2=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, NH2, NR3R4, R3, R4=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, 3-6 membered cycloalkyl, and Z=none].

[0079] In one embodiment, the compound is selected from the following:

[0080] [ka]

[0081] In another embodiment, the present invention provides a method for increasing bacterial phagocytosis or bacterial clearance by stimulating the Th1 immune response by agonizing TLR4, TLR6, TLR7, and TLR8, and for identifying targets that require stimulation of the Th1 immune response. To increase bacterial phagocytosis or bacterial clearance, and to target formula I:

[0082] [ka]

[0083] [In the formula, n = 0 to 5, X = NH, O, S, CH2, Y = phenyl, at least The present invention provides an effective amount of a compound having [a phenyl group substituted with one methyl group, a phenyl group substituted with at least one nitro group, a phenyl group substituted with at least one nitrogen group, a phenyl group substituted with at least one boron group, an aryl group, a substituted aryl group, a heteroaryl group, a 4-6 membered cycloalkyl group, a 4-6 membered heterocycloalkyl group, where R=H,C(O)R2,SO2R2, R1=H,C(O)R2,SO2R2, R2=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, NH2,NR3R4, R3,R4=ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, 3-6 membered cycloalkyl group, and Z=none], wherein the amount is sufficient to stimulate a Th1 immune response, increase bacterial or viral phagocytosis, or increase cell or viral clearance. In one embodiment, bacterial clearance is the treatment of at least one of systemic bacterial infection, sepsis, pulmonary infection, atopic dermatitis, or skin wound infection. In one embodiment, viral clearance is to treat at least one of systemic viral infection, sepsis, pulmonary viral infection, atopic dermatitis, or skin wound infection. In a particular embodiment, the amount of the compound is formulated in an amount selected to inhibit or activate the immune response listed in the table above. In another embodiment, the compound is a TLR2, TLR4, TLR7, and TLR8 inhibitor that downregulates IL-1β. In another embodiment, the compound is a TLR9 inhibitor that downregulates IFN-α. In another embodiment, the compound inhibits cancer cell proliferation and induces cancer cell death. In another embodiment, the compound inhibits angiogenesis. In another embodiment, the compound is provided in an amount sufficient to competitively inhibit inflammation and angiogenesis and protect against angiogenesis-related injury of the eye. [Brief explanation of the drawing]

[0084] To better understand the features and advantages of the present invention, embodiments for carrying out the invention are referred to herein with reference to the accompanying drawings. [Figure 1]This table shows compounds that exhibit TLR2 and TLR4 inhibitory and activating activity in THP-1 cells at low and high concentrations. [Figure 2] Compounds possessing TLR4 agonist activity have been shown to upregulate the inflammatory cytokine IL-1β, indicating stimulation of innate immunity. [Figure 3] This exhibits compounds with TNF-α upregulatory activity. [Figure 4] This shows the regulation of IL-10 production in monocytes. [Figure 5] This paper presents compound 8, which, in response to the TLR4 ligand LPS, regulates IL-1β production in a concentration-dependent manner in monocytes. [Figure 6] Compound 8 is shown to regulate IL-1β production in monocytes in a concentration-dependent manner in response to the TLR7 / 8 ligand CL075. [Figure 7] Compounds 8 and 32 that regulate IL-10 production in monocytes in a concentration-dependent manner are shown. [Figure 8] Compound 8 is shown to regulate IL-10 production in monocytes in a concentration-dependent manner in response to the TLR4 ligand LPS. [Figure 9] Compound 8, which upregulates IL-6 production in monocytes in response to the TLR6 ligand Pam2CSK4, is presented. [Figure 10] This paper presents compound 8, which regulates IL-6 production in monocytes in a concentration-dependent manner in response to the TLR1 / 2 ligand Pam3CSK4. [Figure 11] This paper presents compound 8, which downregulates INF-α production in monocytes in response to the TLR9 ligand ODN2216. [Figure 12] This demonstrates that AVR compounds do not have cytotoxicity to human peripheral blood monocytes (hPBMCs). [Figure 13] This demonstrates that AVR compounds do not have cytotoxicity against primary human type I alveolar (AT-1, Alveolar Type-I) lung epithelial cells. [Figure 14] The study demonstrates the cytotoxic activity of AVR compounds against human lung adenocarcinoma A549 cells. [Figure 15] This demonstrates that AVR compounds do not have cytotoxicity to human umbilical vein endothelial cells (HUVECs). [Figure 16] This study demonstrates that AVR compounds reduce the increase in the number of VEGF-induced closed-tube structures in HUVEC. [Figure 17] This study demonstrates that AVR compounds reduce the increase in the number of VEGF-induced branched tube structures in HUVEC. [Figure 18] This study demonstrates that treatment with AVR compounds increased the phagocytic activity of Pseudomonas aeruginosa in THP-1 cells. [Figure 19] This study demonstrates that treatment with compound 8 increased intracellular death of Pseudomonas aeruginosa in THP-1 cells. [Figure 20] This table shows the minimum and partial inhibitory concentrations of the compound against both Gram-positive and Gram-negative bacteria, as well as its synergistic activity with colistin. [Figure 21] This study demonstrates that compounds possessing TLR4 agonist activity reduce the CFU count of Pseudomonas aeruginosa in a mouse skin wound model. [Figure 22] Figures 22A, 22B, 22F, and 22G show total cell and neutrophil cell counts in LPS-treated bronchoalveolar lavage (BAL) fluid and hyperxia-induced lung injury in mice treated with or not treated with compounds 1 and 8. Figures 22C-E and 22H-J show ELISA assays for IL-6, IL-1β, and IL-10 in LPS-treated BAL fluid and hyperxia-induced lung injury in mice treated with or not treated with compounds 1 and 8. [Figure 23]Figures 23A, 23B, 23D, and 23E show pulmonary edema in the lungs (measured by total protein and Evans blue dye concentrations in BAL fluid). Figures 23C and 23F show lung injury scores in mice treated with compounds 1 and 8 or the control. Data from both Figures 22A-J and 23A-F are expressed as mean ± SE (n=6-8, *p=≥0.05, **p=≥0.01, and ***p=≥0.001). Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey analysis. [Figure 24] This study demonstrates that compounds 1 and 8, when formulated as saline solutions, prevent hyperoxia-induced bipolar disorder (BPD) in offspring mice through both intraperitoneal (IP) and intravenous (IV) administration. [Figure 25] Figures 25A-25C show improvements in chord length, septal thickness, and radial alveolar count in mouse lungs in groups treated with room air (RA), bipolar disorder (BPD), and either compound 1 or compound 8 via IP injection. n=4-8, ***p=≥0.001, ANOVA. [Figure 26] Figures 26A-G show, by ELISA, that treatment with compound 8 at 10 mg / kg reduces inflammatory cytokines and chemokines (MIP-2, MCP-1, IL-17, INF-Υ, TNF-α, IL-1β, and IL-6). Figure 26H shows, by ELISA, that treatment with compound 8 at 10 mg / kg increases the anti-inflammatory cytokine IL-10. n=4-8, ***p=>0.001, ANOVA. [Figure 27] The slow drug release profiles of PLGA-encapsulated nanosuspends (3NP, 8NP, and 32NP) in saline solution are shown. [Figure 28] This study demonstrates that the nano-suspending formulations 3NP, 8NP, and 32NP prevent hyperoxygen-induced bipolar disorder (BPD) in offspring mice via intranasal inhalation. [Figure 29] The synthesis scheme for preparing compounds 32 and 35 is shown. [Figure 30] The synthesis scheme for preparing compound 17 is shown. [Figure 31] The synthesis scheme for preparing compound 8 is shown. [Figure 32] The synthesis scheme for preparing compound 31 is shown. [Figure 33] The synthesis scheme for preparing compound 17 is shown. [Figure 34] The synthesis scheme for preparing compounds 2 and 3 is shown. [Figure 35] The synthesis scheme for preparing compounds 6 and 7 is shown. [Modes for carrying out the invention]

[0085] While various embodiments of the present invention are described in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely examples of specific methods of creating and using the invention and do not define the scope of the present invention.

[0086] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings that are generally understood by those skilled in the art relating to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but are particularly This includes general classifications for which specific examples may be used. The terminology used herein is used to describe specific embodiments of the invention, but its use is not limited to the invention except as outlined in the claims.

[0087] This invention includes novel compounds and / or quantities thereof whose immunostimulatory or inhibitory activity has been evaluated. Furthermore, immunostimulatory or inhibitory activity was also evaluated for structurally specific TLR7 / 8 agonist / antagonist compounds. The compounds of this innovation are small molecules that can modulate the TLR pathway, upregulate IL-10, and downregulate inflammatory cytokines, with therapeutic potential to treat BPD, ARDS, ILD, and COPD. The compounds of this innovation are small molecules that can inhibit the TLR2 pathway, with therapeutic potential to treat psoriasis, acne, and other inflammatory diseases. The compounds of this innovation are small molecules that upregulate IL-10, with therapeutic potential to treat rheumatoid arthritis (RA) and osteoarthritis (OA). The compounds of this innovation are small molecules that can inhibit the TLR4 pathway, with therapeutic potential to treat lung cancer.

[0088] The present invention includes compositions and methods using carbohydrate-derived Toll-like receptor antagonists to simultaneously suppress pneumonia while improving pulmonary endothelial barrier function useful for BPD, ARDS, COPD, cystic fibrosis, and pneumonia. One such compound is of the following formula (Formula I):

[0089] [ka]

[0090] [In the formula, n = 0 to 5, X = NH, O, S, CH2. Y = 4-6 member cycloalkyl, phenyl group substituted with at least one methyl group, phenyl group substituted with at least one nitro group, phenyl group substituted with at least one nitrogen group, phenyl group substituted with at least one boron group, aryl, substituted aryl, heteroaryl, cycloalkyl, R=H, C(O)R2, SO2R2. R1 = H, C(O)R2, SO2R2. R2 = alkyl, substituted alkyl, aryl, substituted aryl, NHR3. R3 = H, ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, Z = NH, O, S, CH2, or none. It holds.

[0091] The inventors have produced the following representative compounds.

[0092] [ka]

[0093] The compounds of the present invention have found specific uses in the delivery of low-density particles to the pulmonary system and in the delivery of large-sized drugs. Biodegradable particles have been developed for the controlled release and delivery of compounds as disclosed herein. Langer, R., Science, 249: 1527-1533 (1990).

[0094] The airway consists of the upper airway, which includes the oropharynx and larynx, followed by the trachea, and then the lower airway, which includes the bronchi and bronchioles from its branching points. The upper and lower airways are called the guided airways. Next are the terminal bronchioles. The bronchi divide into respiratory bronchioles, which then lead to the alveoli or deep lungs, which are the final respiratory regions. The present invention can be formulated to deliver to any part of the airway, for example, Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract," in Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313, 1990, the relevant portion of which is incorporated herein by reference. In one non-limiting example, the deep lungs or alveoli are the primary target of the inhaled therapeutic aerosol for systemic drug delivery of the present invention.

[0095] Inhaled aerosols are used to treat localized lung disorders, including asthma and cystic fibrosis, and have potential for systemic delivery of the compounds of the present invention. Pulmonary drug delivery strategies present many challenges in delivering macromolecules, including excessive loss of inhaled drugs in the oropharyngeal cavity (often more than 80%), inadequate control of deposition sites, non-reproducibility of therapeutic outcomes due to variations in respiratory patterns, often too rapid drug absorption with the potential to produce local toxic effects, and phagocytosis by lung macrophages.

[0096] Considerable attention has been paid to the design of therapeutic aerosol inhalers and the design of dry powder aerosol surface textures to improve the efficiency of inhalation therapy. The inventors recognize the need to avoid particle aggregation, that is, the phenomenon of particle aggregation significantly reducing the efficiency of inhalation therapy, which is required for efficient and consistent deep lung delivery.

[0097] In one example of a pulmonary delivery formulation, particles containing the active compound of the present invention can be used by topical and systemic inhalation therapy to provide controlled release of the therapeutic agent. The particles containing the active compound enable the slow release of the therapeutic aerosol, prolonging the retention of the administered drug in the airways and acini, and reducing the rate of drug release into the bloodstream. Reduced use and increased dose consistency lead to increased patient adherence to medication.

[0098] The human lungs can remove or rapidly decompose deposited aerosols that can be hydrolytically cleaved within a range of minutes to hours. In the upper respiratory tract, ciliated epithelium contributes to a “mucociliary escalator,” thereby sweeping particles from the airways into the oral cavity. In the deeper parts of the lung, alveolar macrophages are well known to be able to phagocytose particles immediately after deposition. The particles containing the active compound provided herein enable dry powder inhalation therapy that is effective for both short-term and long-term release of therapeutic agents, either locally or systemically, with minimal aggregation. The increased consistency of the particle size is expected to reduce particle clearance by the lung's natural mechanisms until the drug is effectively delivered.

[0099] PLGA-encapsulated nanosuspends with an extended drug release profile. Nanoparticle formulation. Nanoparticle formulation can be carried out via single or double emulsion technology. For example, in single emulsion technology, 10 mg of the compound was dissolved in 3 ml of chloroform containing 100 mg of PLGA to form an oil phase. This solution was then added dropwise to 20 ml of 5% PVA solution (aqueous phase) and emulsified at 50 W for 5 minutes to form compound-supported nanoparticles. The final emulsion was stirred overnight to evaporate the solvent. The nanoparticles were washed, collected by ultracentrifugation, and lyophilized before use.

[0100] In an example of double emulsion technology, 30 mg of poly(D,L-lactide-co-glycolide) (PLGA) was dissolved in 1 mL of chloroform at 4°C. Simultaneously, 2 mL of 2% w / v poly(vinyl alcohol) (PVA, poly(vinyl alcohol)) / distilled deionized water was dissolved. A solution was formed. PVA was solubilized in water and added to the PVA solution with 1 mL of ethanol or methanol as a non-solvent. The active compound was then added to the PVA / ethanol solution at a concentration of 1 mM and stirred. For example, a storage solution of the activator, e.g., 10 mg / mL, was formed by dissolving curcumin in water under alkaline conditions using 0.5 M NaOH. The activator was then added at concentrations of 0.5, 1.0, and 2.0 mg / mL per 150 microliters of aqueous solution. Add to PLGA / chloroform solution at the specified concentration. Primary emulsion formation is performed by vortexing the activator-PLGA / chloroform solution for 20 seconds, followed by tip sonication at 55W for 1 minute using a Branson Sonifier Model W-350 (Branson, Danbury, CN). Then, the primary emulsion is mixed with BS3 / PVA / E The tanol solution is added to initiate the formation of a secondary emulsion. The secondary emulsion is completed by vortexing for 20 seconds and tip sonication at 55W for 2 minutes. The stabilized and activated nanoparticles are then transferred to a 1.5 mL Eppendorf tube. Centrifuge at 18,000 g for 5 minutes. Remove chloroform and residual PVA supernatant by aspirating, and remove the particles in, for example, 1 mL of phosphate-buffered saline (PBS). The nanoparticles were resuspended in pH 7.2 by tip sonication. After resuspending, the nanoparticles were allowed to stand at -80°C for 1 hour and then freeze-dried overnight. Freeze-drying can be performed under a vacuum of 250 μT using the ATR FD 3.0 system (ATR, St. Louis, MO). After freeze-drying, the nanoparticles were stored at 4°C. When using, weigh the nanoparticles and store them in an Eppendorf container. The sample was placed in a tube and resuspended in 1 mL of PBS, pH 7.4.

[0101] The present invention comprises compositions and methods using carbohydrate-derived Toll-like receptor 2 and 4 (TLR2 / 4) antagonists that suppress skin inflammation and are useful for acne and related bacterial infections. Examples of inflammatory conditions that can be treated by the present invention include sepsis, ankylosing spondylitis, psoriasis, psoriatic arthritis, Behçet's disease, arthritis, inflammatory bowel disease (IBD), and inflammatory bowel disease. This includes disease) and / or allergies. Some of these diseases are related to a "cytokine storm" and may be treated with compositions provided herein in the amounts taught herein. Such compounds are of the following formula (Formula I):

[0102] [ka]

[0103] [In the formula, n = 0 to 5, X = NH, O, S. Y = a phenyl group substituted with at least one methyl group, a phenyl group substituted with at least one nitro group, a phenyl group substituted with at least one nitrogen group, a phenyl group substituted with at least one boron group, phenyl, aryl, substituted aryl, heteroaryl, cycloalkyl, R=H, C(O)R2, SO2R2. R1 = H, C(O)R2, SO2R2. R2 = alkyl, substituted alkyl, aryl, substituted aryl, NHR3. R3 = H, ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, Z = NH, O, S, CH2, or none. It holds.

[0104] The inventors have produced the following representative compounds.

[0105] [ka]

[0106] The present invention comprises compositions and methods using carbohydrate-derived Toll-like receptor agonists that stimulate the Th1 immune response, increase bacterial phagocytosis, and enhance bacterial clearance, which are useful for systemic bacterial infections, sepsis, pulmonary infections, atopic dermatitis, and skin wound infections. One such compound is of the following formula I:

[0107] [ka]

[0108] [In the formula, n = 0 to 5, X = NH, O, S. Y = a phenyl group substituted with at least one methyl group, a phenyl group substituted with at least one nitro group, a phenyl group substituted with at least one nitrogen group, a phenyl group substituted with at least one boron group, phenyl, aryl, substituted aryl, heteroaryl, cycloalkyl, R=H, C(O)R2, SO2R2. R1 = H, C(O)R2, SO2R2. R2 = alkyl, substituted alkyl, aryl, substituted aryl, NHR3. R3 = H, ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, Z = none] It holds.

[0109] The inventors have produced the following representative compounds.

[0110] [ka]

[0111] In some embodiments, the compounds of this disclosure are incorporated into parenteral formulations. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, and intra-arterial injections by various infusion techniques. Intra-arterial and intravenous injections, as used herein, include administration via a catheter. Preferred for certain indications are administration methods that allow for rapid delivery to the tissue or organ being treated, such as intravenous injection for the treatment of endotoxemia or sepsis.

[0112] The compounds of this disclosure are administered in doses that provide appropriate inhibition or activation of TLRs in target cells, and these doses are generally preferably 0.25 to 50 mg per patient, or per patient. 1.0-100 mg per person, or 5.0-200 mg per patient, or per patient 100-500 mg per dose, more preferably 0.25-50 mg per patient, most preferably The appropriate dose is 1.0 to 100 mg per patient. The dosage is preferably once a day. The treatment period is 28 days, more preferably 14 days with two doses per day, and most preferably 7 days with three doses per day.

[0113] The pharmaceutical composition containing the active ingredient may be in any form suitable for the intended method of administration. Techniques and compositions for producing dosage forms useful for use in the present invention are referenced in the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed. This information is found in one or more of the following: Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, 2000, and updates thereto; and Martindale, The ExtraPharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999), all of which are incorporated by reference, and relevant portions are incorporated herein by reference.

[0114] The present invention includes compositions and methods for producing and generating aerosols for the delivery of activators described herein in specific doses. In one embodiment, the compound is formulated to be aerosolized by an aerosol generating device. Typical embodiments of the present invention include liquid compositions having predetermined physical and chemical properties that facilitate the formation of aerosol formulations. Such formulations typically include three or four basic parameters, for example, (i) an active ingredient, (ii) a liquid carrier for the active ingredient, and (iii) aerosol properties. A solid substance and optionally (iv) at least one excipient. These components are combined This provides a therapeutic composition having enhanced properties for delivery to the user by generating an inhalable aerosol for delivery to the lungs.

[0115] The aqueous suspension of the compound of the present invention contains the active substance mixed with an excipient suitable for the production of the aqueous suspension. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, as well as dispersants or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensates of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensates of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), and condensates of ethylene oxide and partial esters derived from fatty acids and hexitol anhydride (e.g., polyoxyphenylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl n-propyl p-hydroxybenzoate.

[0116] The pharmaceutical compositions of the present invention may be in the form of sterile injectable preparations, such as sterile aqueous or oily suspensions for injection. These suspensions can be formulated according to known techniques using the appropriate dispersants or wetting agents and suspending agents described above. The sterile injectable preparations may also be prepared as sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol, or as lyophilized powders. Acceptable media and solvents that may be used include water, Ringer's solution, and sodium chloride isotonic solutions. In addition, sterile fixatives can conventionally be used as solvents or suspension media. For this purpose, any non-irritating fixatives containing synthetic mono or diglycerides can be used. In addition, fatty acids such as oleic acid can similarly be used in the preparation of injectable preparations.

[0117] In some embodiments, the formulation comprises PLA or PLGA microparticles and may be further mixed with Na2HPO4, hydroxypropyl methylcellulose, polysorbate 80, sodium chloride and / or disodium edetate.

[0118] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formed solution isotonic with the intended recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and require only the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. It can be stored in a lye (freeze-dried) state. Immediate injection solutions and suspensions can be prepared from the types of sterile powders previously described.

[0119] However, it is understood that the specific dose level for any particular patient depends 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, the elimination rate, other medications previously administered, and the severity of the particular disease being treated.

[0120] In some embodiments, the compositions of this disclosure also contain about 80% to about 99.5%, preferably about 90 or 95% to about 98.5%, of suitable non-aqueous, pharmaceutically acceptable topical media. Some of these media are described in U.S. Patent No. 4,621,075, which is incorporated herein by reference. While these media are preferably water-free, the compositions of the present invention may contain up to about 5% water without having a significant adverse effect on the formation of the desired gel. These non-aqueous media components are also well known in the pharmaceutical art and include, but are not limited to, hydrocarbon oils and waxes, short-chain alcohols and ketones and emollients, lanolin and lanolin derivatives, silicone oils, monoglycerides, diglycerides and triglyceride esters, fatty alcohols, alkyl and alkenyl esters of fatty acids, alkyl and alkenyl diesters of dicarboxylic acids, polyhydric alcohols and their ethers and ester derivatives, wax esters and beeswax derivatives. Preferred media incorporate methanol, ethanol, n-propanol, isopropanol, butanol, polypropylene glycol, polyethylene glycol, and mixtures thereof. Particularly preferred media include ethanol, n-propanol, and butanol, and especially ethanol. These preferred solvents can also be combined with other components such as diisopropyl sebacate, isopropyl myristate, methyl laurate, silicone, glycerin, and mixtures thereof to provide non-aqueous media, which are also useful in the present invention. Of these additional components, diisopropyl sebacate is particularly useful. In fact, preferred media include mixtures of ethanol and diisopropyl sebacate in a weight ratio of about 4:1 to about 1:4. Preferred media contain about 15% to about 35% diisopropyl sebacate and about 65% to about 85% ethanol.

[0121] The compositions of the present invention may additionally contain suitable auxiliary components conventionally used in the formulation of topical pharmaceutical compositions at usage levels established in the art. These auxiliary components may include, but are not limited to, pharmaceutically active substances (e.g., supplemental antimicrobial or anti-inflammatory components, e.g., steroids) or components used to enhance the formulation itself (e.g., excipients, dyes, fragrances, skin penetration enhancers, stabilizers, preservatives, and antioxidants). Examples of such agents include pharmaceutically acceptable acidic carboxypol compounds such as Carbopol compounds, which are commercially available from BF Goodrich Chemicals, Cleveland, Ohio. It contains rimer.

[0122] In one embodiment, the compound of the present invention is formulated into a cream, lotion, or gel packaged in a general trigger spray container. It may also adhere firmly to the desired area like a normal cream after being sprayed from the container. This is described in International Publication No. 98 / 51273, which is incorporated herein by reference. Accordingly, in one embodiment, the present disclosure provides a pharmaceutical product that can be incorporated into a topical non-aerosol spray composition comprising the compounds described herein alone or in combination. The compounds are present in an amount ranging from 0.1% to 20% by weight, 1 to 15% by weight in some embodiments, and 2 to 10% by weight in some embodiments, of the cream, lotion, or gel. The compounds of the present invention can be incorporated into a neutral hydrophilic cream, lotion, or gel matrix. In a preferred embodiment, the topical cream or lotion matrix is ​​characterized by a polyoxyethylene alkyl ether. In a second preferred embodiment, the gel is characterized by a high molecular weight polymer of crosslinked acrylic acid. Polyoxyethylene alkyl ether is a nonionic surfactant widely used in pharmaceutical topical formulations and cosmetics, mainly as an emulsifier for water-in-oil and oil-in-water emulsions. It is characterized in the present invention as a base for a non-aerosol trigger-sprayable cream or lotion. A crosslinked acrylic acid polymer (carbomer) used to form the gel is another object of the present invention.

[0123] Therefore, a base particularly suitable for non-aerosol sprays is a cream or lotion containing 1-25% polyoxyethylene alkyl ether, 3-40% humectant, and 0.1-1% one or more preservatives, with the remainder being purified water to make 100%. Appropriately, the polyoxyethylene alkyl ether may be one or any combination selected from the group consisting of polyoxyl 20 cetostearyl ether (Atlas G-3713), poloxyl 2 cetyl ether (ceteth-2), poloxyl 10 cetyl ether (ceteth-10), poloxyl 20 cetyl ether (ceteth-20), poloxyl 4 lauryl cetyl ether (laureth-4), poloxyl 23 lauryl cetyl ether (laureth-23), poloxyl 2 oleyl ether (oleth-2), poloxyl 10 oleyl ether (oleth-10), poloxyl 20 oleyl ether (oleth-20), poloxyl 2 stearyl ether (steareth-2), poloxyl 10 stearyl ether (steareth-10), poloxyl 20 stearyl ether (steareth-20), and poloxyl 100 stearyl ether (steareth-100). A suitable humectant may be one or any combination selected from the group consisting of propylene glycol, polyethylene glycol, sorbitol, and glycerin. A suitable preservative may be one or any combination selected from the group consisting of methylparaben, propylparaben, benzyl alcohol, benzoic acid, sodium benzoate, sorbic acid, and their salts and phenylethyl alcohol.

[0124] Another base suitable 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% one or more preservatives, with the remainder being purified water to make 100%. Preferably, the carbomer may be one or any combination selected from the group consisting of carbomer 934, carbomer 940, and carbomer 941. Suitable humectants, preservatives, and purified water for the gel are the same as those for the cream or lotion. Other sprayable formulations are described in U.S. Pre-Grant Patent Publication No. 2005 / 00255048, which is expressly incorporated herein by reference.

[0125] Ophthalmic preparations (topical and intravitreal administration): The compounds of the present invention typically constitute a small percentage of the overall ophthalmic formulation. 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. Furthermore, the compounds of the present invention are also typically 5.0 w / v% or less, more typically 3.0 w / v% or less, and even more typically 1.5 w / v% or less of the ophthalmic composition.

[0126] Ophthalmic compositions typically include an ophthalmic medium suitable for delivering the compound to the eye. It is intended that ophthalmic compositions may be configured for topical or intravitreal application to the eye, and that the ophthalmic medium may vary depending on the method of application. Generally, for topical or intravitreal application, it is preferable that the ophthalmic composition is aqueous and 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% of water (e.g., purified water).

[0127] In intravitreous application, particularly when the ophthalmic composition is applied to the eye by syringe, the ophthalmic composition may consist solely of, for example, water and the compound of the present invention, or essentially consist of water and the compound of the present invention. For sustained drug release, PLGA and PLA macroparticle formulations of the compound of the present invention are used as described in Shelke et al [DrugDeliv Transl Res. 2011, (1): 76-90]. Naturally, the ophthalmic composition may also contain other components, such as Na2HPO4, hydroxypropyl methylcellulose, polysorbate 80, sodium chloride, and disodium edetate.

[0128] If the medium is water alone or essentially water for topical application, this may be especially true if the topical application is performed immediately after the water is combined with the test compound, or if the composition is packaged to prevent contamination. However, if the ophthalmic composition is applied as a large dose over a long period of time (e.g., as droplets from an eye dropper once, twice, three or four or more times a day over several days), the ophthalmic composition may contain additional components, such as antimicrobial or preservative agents or systems, surfactants, buffers, isotonic agents, antioxidants, viscosity modifiers, or any combination thereof.

[0129] For topical application, the compositions of the present invention typically contain an antimicrobial agent. Potential antimicrobial agents include, but are not limited to, hydrogen peroxide, chlorine-containing preservatives, such as benzalkonium chloride. However, according to a preferred embodiment, the compositions of the present invention contain any nonpolymeric quaternary antimicrobial agent, such as benzalkonium chloride (BAK). It contains none or substantially none. The most preferred antimicrobial agents in pharmaceutical compositions are polymeric quaternary ammonium compounds.

[0130] As used herein, the phrase “substantially absent” means, when referring to a component of an ophthalmic composition, that the ophthalmic composition may be entirely absent from that particular component or is intended to contain only a very small amount of that particular component.

[0131] The polymeric quaternary ammonium compounds useful in the compositions of the present invention are those that have antimicrobial effects and are acceptable for ophthalmic use. Preferred compounds of this type are described in U.S. Patents Nos. 3,931,319, 4,027,020, 4,407,791, 4,525,346, 4,836,986, 5,037,647 and 5,300,287, and in PCT International Publication No. 91 / 09523 (Dziabo et al.), which are referenced below. This is expressly incorporated herein. The most preferred polymeric ammonium compound is polyquaternium-1, also known as POLYQUAD® or ONAMERM®. It has a number-average molecular weight of 2,000 to 30,000. Preferably, the number-average molecular weight is 3,000 to 14,000.

[0132] Polymeric quaternary ammonium compounds are commonly used in the suspensions of the present invention in amounts exceeding approximately 0.00001 w / v%, more typically exceeding approximately 0.0003 w / v%, and even more typically exceeding approximately 0.0007 w / v% of the suspension. Furthermore, polymeric quaternary ammonium compounds are commonly used in the compositions of the present invention in amounts less than approximately 3 w / v%, more typically less than approximately 0.003 w / v%, and even more typically less than approximately 0.0015 w / v% of the composition.

[0133] The antimicrobial agents of the compositions of the present invention additionally or alternatively include antimicrobial systems, such as borate / polyol complex systems. As used herein, the term “borate” refers to boric acid, salts of boric acid, borate derivatives and other pharmaceutically acceptable borates or combinations thereof. Most suitable are boric acid, sodium borate, potassium borate, calcium borate. These include um, magnesium borate, manganese borate, and other such borates. Borates interact with polyols such as glycerol, propylene glycol, sorbitol, and mannitol to form borate-polyol complexes. The type and ratio of such complexes are determined by the number of OH groups of the polyol relative to adjacent carbon atoms that are not in trans configuration with each other. The weight / volume percentages of the polyol and borate components are understood to include these amounts, whether they are part of the complex or not.

[0134] As used herein, the term “polyol” includes any compound having at least one hydroxyl group for each of two adjacent carbon atoms that are not in trans configuration with each other. Polyols can be linear or cyclic, substituted or unsubstituted, or mixtures thereof, as 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, and include, but are not limited to, mannitol, glycerin, xylitol, sorbitol, and propylene glycol.

[0135] When used, the borate / polyol complex antimicrobial system (i.e., borate and polyol together) typically contains at least 0.05 w / v%, more typically at least 0.5 w / v%, more preferably at least 1, and even preferably at least 1.2 w / v%, and also typically less than 5 w / v%, more typically less than 2.2 w / v%, and more preferably less than 1.6 w / v%. The ratio (weight to weight ratio) of borate to polyol in the composition is typically 1:1 to 1:10, more typically 1:2 to 1:4 (e.g., about 1:3).

[0136] Tyroxapol, polysorbate 80, and polyoxyl hydrogenated castor oil are preferred surfactants. Tyroxapol is a very preferred surfactant. When used, the surfactant is present in the composition at a concentration of at least 0.01 w / v%, more typically at least 0.025 w / v%, and more preferably at least 0.1 w / v%, and typically less than 5 w / v%, more typically less than 2.0 w / v%, and more preferably less than 1.0 w / v%.

[0137] The compositions of the present invention used for topical application are typically formulated for topical use to be suitable for the eye. Ophthalmic compositions intended for direct application to the eye are formulated to have a pH and isotonicity suitable for the eye. 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. Particularly desirable pH ranges are 6.0 to 7.8, and more specifically 6.4 to 7.6. The compositions contain 200 to 400 or 450 milliosmoles per kilogram (mOsm / kg). ), more preferably having an osmolality of 240 to 360 mOsm / kg.

[0138] A preferred composition of the present invention is a multi-dose ophthalmic composition. For example, the composition is in an eye dropper and can be topically applied to the eye in one or more drops, such as once, twice, three times or more than four times a day. In this case, the composition preferably has antimicrobial activity sufficient for the composition to meet the USP preservative effectiveness requirements of aqueous pharmaceutical compositions and other preservative effectiveness criteria. There are two preservative effectiveness criteria in the European Pharmacopoeia, "A" and "B".

[0139] The criteria confirmed above for USP27 are substantially the same as the requirements described in previous editions of USP, particularly USP24, USP25 and USP26, which are hereby incorporated by reference. As an additional advantage, these ophthalmic compositions containing the TLR4 antagonist compounds of the present invention are suitable for topical application to the eye. The formulations described herein are the antimicrobial agents, pain relievers, etc. described above, but may also contain additional active ingredients, including but not limited to those described above. be contained.

[0140] Hyperoxia-induced inflammation is a cornerstone of BPD pathogenesis (Bhandari, V., Drug therapy trials for the prevention of bronchopulmonary dysplasia: current and future targets. Front Pediatr 2014, 2, 76) (i.e., secondary to the production of reactive oxygen species or ROS) (Harijith, A.; Bhandari, V., Hyperoxia in the pathogenesis of bronchopulmonary dysplasia. In Bronchopulmonary Dysplasia, First ed.; Bhandari, V., Ed. Springer International Publishing: Switzerland, 2016; pp 3-26), Thus, various inflammatory molecules such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and inducible nitric oxide synthase (iNOS) are involved in and / or associated with the development of BPD (Bhandari, A.; Bhandari, V., Biomarkers in bronchopulmonary dysplasia. Paediatr Respir Rev 2013, 14, 173-9). Toll-like receptor 4 (TLR-4) , involved in this method (Menden, HL; Xia, S.; Mabry, SM; Navarro, A.; Nyp, M.) F.; Sampath, V., Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Regulates LPS-Induced Inflammation and Alveolar Remodeling in the Developing Lung. Am JRespir Cell Mol Biol 2016, 55, 767-778; Yao, L.; Shi, Y.; Zhao, X.; Hou, A.; Xing, Y.; Fu, J.; Xue, X., Vitamin D attenuates hyperoxia-induced lung injury through downregulation of Toll-like receptor 4. Int J Mol Med 2017, 39, 1403-1408), and in particular, is involved in prenatal sepsis / inflammation leading to BPD (Glaser, K.; Speer, CP, Pre and Postnatal inflammation in the pathogenesis of bronchopulmonary dysplasia. In Bronchopulmonary Dysplasia, First ed.; Bhandari, V., Ed. Springer International Publishing: Switzerland, 2016; pp 55-77), is one such signaling pathway.

[0141] Chitin and chitosan are high molecular weight oligosaccharides with diverse biological activities, including hypocholesterolemic, antimicrobial, immunostimulatory, tumorigenesis-preventing, calcium and iron absorption-accelerating, anti-inflammatory, and antioxidant properties (Xia, W.; Liu, P.; Zhang, J.; Chen, J., Biological activities ofchitosan and chitooligosaccharides. FoodHydrocolloids 2011, 25, 170-179). These polysaccharides are known to activate alternative pathways and inhibit sepsis caused by Gram-negative bacteria (Okawa, Y.; Kobayashi M Fau - Suzuki, S.; Suzuki S Fau - Suzuki, M.; Suzuki, M., Comparativestudy of protective effects of chitin, chitosan, and N-acetyl chitohexaose against Pseudomonas aeruginosa and Listeriamonocytogenes infections in mice、Qiao, Y.; Bai, X.-F.; Du, Y.-G., Chitosan oligosaccharides protect micefrom LPS challenge by attenuation of inflammation and oxidative stress.International Immunopharmacology 2011, 11, 121-127 Solov; #039; eva, T.; Davydova, V.; Krasikova, I.; Yermak, I., MarineCompounds with Therapeutic Potential in Gram-Negative Sepsis. Marine Drugs2013, 11, 2216-2229). However, the manipulation of the chemical core structures of these high molecular weight polysaccharides to achieve optimal drug-like properties has not been studied by any group.

[0142] This invention provides for the first time a dual-acting small molecule capable of producing alternatively activated macrophages and competitively inhibiting LPS-induced inflammation, thereby resulting in organ protection and limiting tissue damage. One such compound is Compound 1, which was designed and confirmed to bind differently to its target. Instead of binding to the TLR4-MD2 complex, it directly binds to the active site of TLR4, and therefore inhibits downstream components. In addition, a novel series of compounds have been designed and validated through SAR studies, for example, in an in vivo model system using neonatal cells with BPD, TLR4 also... The inhibitory compounds are 1, 3, 8, and 32.

[0143] Chitin and chitosan possess excellent properties for ideal drug delivery (Janes, KA; Fresneau, MP; Marazuela, A.; Fabra, A.; Alonso, M. a. J., Chitosan nanoparticles delivery systems for doxorubicin. Journal of Controlled Release 2001, 73,255-267; Williams, J.; Lansdown, R.; Sweitzer, R.; Romanowski, M.; LaBell, R.; Ramaswami, R.; Unger, E., Nanoparticle drug delivery system for intravenous delivery). of topoisomerase inhibitors. Journal of Controlled Release 2003, 91, 167-172, Li, N.; Zhuang, C.; Wang, M.; Sun, X.; Nie,S.; Pan, W., Liposome coated with low Molecular weight chitosan and its potential use in ocular drug delivery. (International Journal of Pharmaceutics 2009, 379, 131-138). LMW chitosan is systemically toxic. These are natural molecules that do not possess any properties. They have the ability to be delivered as polymer nanoparticles and are excellent candidates for drug-like targets. A computer model of the binding of N-hexaacetylchitohexaose to the TLR4 active site was presented in our previous publication (Panda, SK; Kumar, S.; Tupperwar, NC; Vaidya, T.; George, A.; Rath, S.; Bal, V.; Ravindran, B., ChitohexaoseActivates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia. PLoS Pathog 2012, 8, e1002717). Preliminary results. Based on molecular docking, the inventors designed and synthesized several compounds as described above and screened them by in vitro assays. Based on optimal physicochemical properties, the inventors selected compounds 1, 3, 8, and 32 for study in a developmentally appropriate hyper-oxygen-exposed BPD mouse model.

[0144] The novel compound is innovative in both concept and methodology. Since the multifactorial pathology of ARDS cannot be completely countered by monotherapy, we have developed a bifunctional molecule that upregulates the compensatory anti-inflammatory cytokine IL-10 via binding to TLR4, improving lung barrier function, and suppresses inflammation caused by inflammatory cytokines (TNF-α, IL-1β, IL-6). Furthermore, as a result of the synthesis of various compounds based on the structure-activity relationship (SAR) method and formula I used herein, Those skilled in the art can systematically modify the structures of compounds in this amino sugar series to produce additional effective analogs. Therefore, the present invention includes SAR compositions and methods for producing derivatives of 1-O-substituted amino sugar analogs that are active against the excessive inflammation caused in sepsis, ARDS, and ALI. Figure 1 is a table showing compounds that exhibit TLR2 and TLR4 inhibitory and activating activity in THP-1 cells at low and high concentrations.

[0145] As shown in Table 1, compounds 1, 2, 3, 6, 7, 8, 14, 32, and 35 are potent TLR2 / 4 antagonists, while compounds 17, 23, 28, 29, 30, 38, 39, and 40 are TLR4 agonists. Compounds 8, 32, and 35 exhibit concentration-dependent TLR4 regulatory activity. Compounds with TLR4 agonist activity show upregulation of the inflammatory cytokines IL-1β (Figure 2) and TNF-α (Figure 3), indicating stimulation of innate immunity and a shift in the Th1:Th2 ratio towards Th1. This class of TLR4 agonists may be useful as vaccine adjuvants and in retract sepsis, where the patient's immune system is suppressed and they are susceptible to secondary infections. Boosting the immune system is useful not only in cancer but also in HIV patients with impaired innate immunity.

[0146] When THP-1 (human monocyte) cells are treated with 100-200 μM of compounds 8, 14, 17, 23, 29, and 32, they produce increased IL-1β and TNF-α responses and decreased anti-inflammatory cytokine IL-10 (Figure 4).

[0147] Surprisingly, two of these compounds, 8 and 32, were found in human peripheral blood after 48 hours. In monocytes, IL-10 production is dose-dependently increased (stimulating Th2 macrophages at concentrations of 0.1–1.0 μM) or decreased (at concentrations of 10–100 μM) (Figures 7 and 8).

[0148] Two of these compounds, 8 and 32, stimulated monocyte differentiation into macrophages, increased phagocytosis of Pseudomonas aeruginosa (Figure 18), and increased intracellular bacterial death (Figure 19). These compounds have potential for use as vaccine adjuvants and in combination with existing antibiotics for prophylactic treatment in patients with conditions such as cystic fibrosis.

[0149] Compound 8 modulates TLR4 activity in a concentration-dependent manner. At low concentrations (0.1 μM to less than 1 μM), ELISA detection revealed that compound 8, alone or in combination with the TLR4 ligand LPS, downregulated IL-1β protein levels in hPBMCs after 24 hours, demonstrating TLR4 antagonist activity. However, at a concentration of 1.0 μM, compound 8, alone or in combination with LPS, significantly stimulated IL-1β production, exhibiting TLR4 agonist activity (Figure 5).

[0150] Compound 8 modulates TLR7 / 8 activity in a concentration-dependent manner. At low concentrations (0.1 μM to less than 1 μM), ELISA detection revealed that compound 8, alone or in combination with the TLR7 / 8 ligand CL075, downregulated IL-1β protein levels in hPBMCs after 24 hours, demonstrating TLR7 / 8 antagonist activity. However, at a concentration of 1.0 μM, compound 8, alone or in combination with CL075, significantly stimulated IL-1β production, exhibiting TLR7 / 8 agonist activity (Figure 6).

[0151] Compound 8 modulates TLR1 / 2 activity in a concentration-dependent manner. At low concentrations (0.1 μM to less than 1 μM), ELISA detection revealed that compound 8, in combination with the TLR1 / 2 ligand Pam3CSK4, downregulated IL-6 protein levels in hPBMCs after 24 hours, demonstrating TLR1 / 2 antagonist activity. However, at concentrations of 1.0, 10, and 100 μM, compound 8, in combination with CL075, significantly stimulated IL-6 production, exhibiting TLR1 / 2 agonist activity (Figure 10).

[0152] When compound 8 is detected by ELISA, in combination with the TLR6 ligand Pam2CSK4, it stimulates TLR6 activity in hPBMCs 24 hours after treatment, thereby upregulating the production of IL-6 cytokines (Figure 9).

[0153] When compound 8 is detected by ELISA, in combination with the TLR9 ligand ODN2216, it antagonizes TLR9 activity in hPBMCs 48 hours after treatment, thereby downregulating IFN-α production (Figure 11).

[0154] Compounds 1, 2, 7, 8, 14, 17, and 32 were tested for cytotoxicity in hPBMCs at concentrations of 0.1, 1.0, 10, 100, and 1000 μM. Cells were treated with the compounds and incubated for 24 hours. Cell viability was measured using the MTT cell breeding kit (Promega). The compounds were evaluated using the samples. All compounds were non-toxic to hPBMC (Figure 12).

[0155] Compounds 8, 17, 29, and 35 were tested for cytotoxicity in normal lung epithelial cells (AT-1) at concentrations of 1 mM and 2 mM. Cells were treated with the compounds and incubated for 48 hours. Cell viability was assessed using a triptan blue live / death assay. All compounds were non-toxic to AT1 cells, except for the positive control, Taxol, which was toxic (Figure 13).

[0156] Compounds 8, 17, 29, and 35 were tested for cytotoxic activity in A549 lung adenocarcinoma cells at concentrations of 1 mM and 2 mM. The cells were treated with the compounds and incubated for 48 hours . Cell viability was evaluated using the trypan blue live / dead assay. Compounds 8, 17, and 35 were toxic to A549 cells and were excellent in killing cancer cells, while the standard anticancer drug Taxol was not (Figure 14).

[0157] Selected TLR2 / 4 antagonist compounds 2, 6, 7, 8, 14, 17, and 32 were evaluated for cytotoxicity in HUVEC at a concentration of 100 μM. The cells were treated with the compounds and incubated for 24 hours. Cell viability was evaluated using the MTT cell proliferation kit (Promega ). All compounds were not toxic to HUVEC (Figure 15).

[0158] Selected TLR2 / 4 antagonist compounds 2, 6, 7, 8, 14, 17, and 32 were evaluated for anti-angiogenic activity using the Tube Formation kit (Cat#3470-096-K). VEGF (100 ng / mL) was used as a positive control. All compounds significantly decreased VEGF-induced tube formation, as shown in Figures 16 and 17. These compounds have potential applications in diseases related to pathological angiogenesis such as solid tumors, retinopathy of prematurity, diabetic retinopathy, and AMD.

[0159] Screening against selected Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii (A. baumanni)) and Gram-positive bacteria (MRSA) (using the liquid dilution assay), compounds 1 and 8 showed bactericidal activity at MICs of 50 - 200 mg / L . Unexpectedly, when combined with the standard antibiotic colistin, the MICs of the compounds decreased to 3.1 - 6.25 μg / mL (Figure 20), demonstrating synergistic and additive activities. 90 ​

[0160] Based on their anti-inflammatory and antimicrobial activity, compounds 1 and 8 were evaluated in a mouse study of skin wound infections. Both compounds 1 and 8, when administered topically at a dose of 100 μM, effectively closed Pseudomonas aeruginosa (ATCC-10145GFP)-infected skin wounds in C57BL6 mice (n=4) and reduced tissue CFU (Figure 18).

[0161] The dorsal surfaces of 5 C57BL / 6 mice (8-10 weeks old, n=4) were injured. 10 6 25 μl of a PBS suspension containing Pseudomonas aeruginosa at a CFU / mL concentration was pipetteed and placed on the wound, allowing absorption over at least 3 minutes. Mice were then treated with either 25 μl of colistin (4 μg / ml) or compound 1 or 8 (100 μg / ml). Subsequently, infected wounds were treated with colistin or the test compound on alternate days for the next 13 days. On day 13, the Pseudomonas aeruginosa + colistin and Pseudomonas aeruginosa + compound 1 or 8 wounds reached complete closure, while the crust of the Pseudomonas aeruginosa + medium wounds did not close.

[0162] Following infection with Pseudomonas aeruginosa (13 days post-infection), bacterial counts in untreated control animals were significantly different from those obtained from animals treated with Pseudomonas aeruginosa + colistin and Pseudomonas aeruginosa + compound 1. Compound 8 also showed a statistically significant reduction in bacterial counts compared to controls (Figure 21).

[0163] Histological examination of H&E-stained wounds isolated 13 days post-injury revealed Pseudomonas aeruginosa + medium injection wounds exhibiting a delayed healing phenotype with incomplete re-epithelialization and focal inflammation of the wound bed. The results showed that re-epithelialization and granulation tissue were superior to those of Pseudomonas aeruginosa (PA,P. Aeruginosa) compared to the control. ) + colistin and PA + compound 1 group showed significant enhancement (p=0.050). The same trend was observed in compound 8 groups, but the difference was statistically insignificant on the borderline (p=0.073). Consequently, less focal inflammation was observed in the compound treatment groups compared to the PA group.

[0164] Two TLR4 modulators, compounds 1 and 8, were delivered by intraperitoneal (IP) injection to improve LPS and hyperxia-induced pneumonia in mice.

[0165] We used male C57BL / 6 mice (Jackson Laboratories) weighing 25-28 grams at 12-15 weeks of age, with up to 5 mice per cage.

[0166] The hyperoxygen-induced ALI model (ARDS model) consisted of exposing adult mice to 100% oxygen for 48 hours, followed by injection of the test compounds. Adult mice (N=5) were housed in a plexi chamber with a constant and continuous supply of 100% oxygen for 48 hours. After 4 hours of oxygen exposure, compounds 1 and 8 were injected as IP (10 mg / kg), the mice were returned to their cages, and removed again 12 hours later for a second dose of repeated injection. The mice were euthanized after 48 hours, lung tissue was collected, and (BAL) fluid was collected.

[0167] LPS-induced ALI model: Adult mice (N=5) were injected with a single dose of LPS (100 μg per mouse in 100 μl volume, intratracheally), followed by IP injection of compound 1 or 8 (10 mg per kg of body weight) 4 and 12 hours after LPS injection. Mice were then euthanized 24 hours later, lung tissue was collected, and BAL fluid was gathered. The lungs were sectioned after H / E staining for histopathological evaluation, and total cell counts (neutrophils and macrophages) were detected using BAL fluid, and proteins (exuded due to leakage) were measured. Pulmonary lysates were prepared, and specific markers were quantified by Western blotting after treatment with compound 1 or 8 following hyperoxygen exposure. Both compounds demonstrated significant protection against both hyperoxygen-induced and LPS-induced lung injury in mouse models mimicking ARDS and bacterial pneumonia, respectively.

[0168] Figures 22A, 22B, 22F, and 22G show total cell and neutrophil counts in LPS bronchoalveolar lavage (BAL) fluid and hyperxia-induced lung injury in mice treated with or not treated with compounds 1 and 8. Figures 22C, 22D, 22E, 22H, 22I, and 22J show ELISA assays for IL-6, IL-1β, and IL-10 in LPS BAL fluid and hyperxia-induced lung injury in mice treated with or not treated with compounds 1 and 8.

[0169] Figures 23A, 23B, 23D, and 23E show pulmonary edema in the lungs (measured by total protein and Evans blue dye concentrations in BAL fluid). Figures 23C and 23F show lung injury scores in mice treated with compounds 1 and 8, or the control. Data from both Figures 22 and 23 are expressed as mean ± SE (n=6-8). * p=0.05 or more, ** p = greater than 0.01 and *** (p = greater than 0.001). Statistical significance was assessed using one-way ANOVA, followed by post-hoc Tukey analysis.

[0170] Western blotting results showed junctional adhesion proteins VE-cadherin, β-catenin, and Src in the lungs of mice treated with compound 1 or 8 or the control. .

[0171] Next, the inventors demonstrated the protective effects of compounds 1, 3, 8, and 32 in a BPD lung injury model. To prove this concept and demonstrate its feasibility, the inventors tested compounds 1, 3, 8, and 32 in a mouse model of BPD, and all compounds prevented lung injury in neonates with BPD. Mouse and human neonates go through similar stages of lung development, but they differ in the duration of each stage and its temporal relationship to the gestation period. The lungs of mice born at term are in the terminal saccular stage and have sufficient surfactant, which can be considered somewhat similar to human preterm neonates at the same stage of lung development who have been exposed to full prenatal steroid replacement (which is known to enhance surfactant production). Regarding the imitation of the human condition, the most successful hyperoxygen models involve hyperoxygenation during the terminal saccular stage of lung development. This model is limited to exposure to oxygen. The mouse BPD model involves exposing neonatal mice (postnatal day 1 or PN1) to 100% oxygen until PN4, followed by a further 10 days of recovery in room air (RA). This model replicates the human model in a clinical scenario.

[0172] Compound 1 or 8 was injected intraperitoneally (IP) or intravenously (IV) into neonates of PN2 and PN4 at doses of 5 mg / kg or 10 mg / kg, respectively (Figure 24). After the recovery period, these lungs were collected and processed for quantitative PCR, Western blotting, histopathology, and ELISA.

[0173] Inflammatory cytokines decreased in lung tissue and serum after injection of compounds 1 and 8. Since inflammation is a characteristic of BPD, the inventors then determined whether the compounds could suppress inflammation. ELISA confirmed that after treatment with compound 8, there was a decrease in the expression of several pro-inflammatory cytokines, accompanied by a simultaneous increase in the anti-inflammatory marker IL-10 (Figure 26A-H).

[0174] To test the feasibility of intranasal delivery of compounds with a sustained drug-release profile and to avoid systemic exposure, compounds 3, 8, or 32 were encapsulated in PLGA nanoparticles. The nanoparticle size was 350–400 nm. Drug loading and drug-release profiles were determined using our previously published method (Le et al, Nat Sci, Rep, 2017). The nano-suspends of the present invention exhibited a slow drug-release profile (Figures 27A–C).

[0175] Compounds 3, 8, or 32, encapsulated in PLGA nanoparticles and referred to as 3NP, 8NP, and 32NP, were administered intranasally (IN) as nanosuspends to neonatal PN2 and PN4 cells, respectively (Figure 28). After a recovery period, these lungs were collected and processed for quantitative PCR, Western blotting, histopathology, and ELISA.

[0176] Lung morphology is improved in neonates with BPD after treatment with the test compound. BPD is characterized by simplified, enlarged alveoli, inflammation, increased cell death, decreased cell proliferation, atypical capillary arrangement and variable interstitial cellularity, and / or linear changes. Characterized by fibrosis. Lung structure is restored after treatment with compounds 3, 8, and 32 in both saline and nanosuspend formulations via the IV and IN pathways, respectively. This is determined by measuring the inter-alveolar distance, septal wall thickness, and radial alveolar count (RAC). In BPD, alveolar area increases (alveolar Characterized by an increase in the diameter of the sac, a thickening of the septum (hence hindering gas exchange), and a decrease in RAC, a parameter used by pulmonologists to assess the complexity of terminal respiratory units, as a result of inhibited lung development. All of these changes return to normal healthy lung parameters after treatment with the test compound (Figure 25A-C).

[0177] Activation of Toll-like receptor 2 in acne induces an inflammatory cytokine response (J Immunol. 2002August 1; 169(3): 1535-1541), and therefore, blocking TLR2 activity is beneficial in treating skin conditions caused by Gram-positive bacteria, including Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes.

[0178] THP-1 monocyte cells (1 × 10⁻⁶) 5 The cells were stimulated with 200 nM PMA for 48 hours and treated with different concentrations of the test compound. TLR2 activity was measured in the cell lysates after 24 hours using ELISA.

[0179] Synthesis of compound 32 (Figure 29). 2-Acetamide-3,4,6-tri-O-acetyl- 2-Deoxy-α-D-Glucopyranosilloride 41: To a stirred solution of commercially available 2-acetamido-2-deoxy-β-D-glucose (25 g, 0.113 mmol), acetyl chloride (50 mL) was added, and the reaction mixture was stirred overnight. The reaction mixture was further diluted with CH2Cl2 (100 mL) and poured into an ice water mixture. The organic phase was extracted with ice-cold saturated aqueous solution of NaHCO3 (2 × 150 mL) and ice-cold water (150 mL). The organic phase was dried, concentrated, and subjected to short column elution using siRNA in hexane (15-50%) as an eluent to obtain pure product 41 as a solid product in 68% yield. The NMR spectra were consistent with those reported in the literature (Sauerzapfe, B.; Namdjou, DJ; Schumacher, T.; Linden, N.; Krenek, K.; Kren, V.; Elling, L., Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus. Journal of Molecular Catalysis B: Enzymatic 2008, 50, 128-140).

[0180] p-Pinacolomboronatephenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside 42:CH2Cl2 (100 mL) and 1N A solution of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosylloride 2 (5.0 g, 13.67 mmol), tetrabutylammonium bisulfate (4.65 g, 13.67 mmol), and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (3.15 g, 1.05 equivalents) in a mixture of NaOH (50 mL) was vigorously stirred for 1 hour. The mixture was extracted with CH2Cl2 (2 × 125 mL) and washed with water and brine. The combined organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column with toluene (15-50%) in hexane as an eluent to obtain pure product 42 as a solid product in 48% yield.

[0181] p-Pinacolomboronatephenyl 2-acetamido-2-deoxy-β-D-glucopyranoside 32: The above p-pinacolomboronatephenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside 42 (2.45 g, 4.45 mmol) was suspended in anhydrous MeOH (70 mL), and a methanol solution of NaOMe (1 M solution, 4.25 equivalents) was added. The mixture was stirred at room temperature for 15 minutes until dissolution was complete. Dowex 50WX2-200 (5 g, pre-washed with methanol) was added, and after 15 minutes, the mixture was filtered. Further removal was performed. The solution was evaporated to dryness under vacuum. The white solid was dissolved in CH2Cl2, and MTBE was added to precipitate a white solid. The solid was filtered and dried under high vacuum to obtain compound 32 in 89% yield. LC / MS = 423.9(M+1); 11H NMR (CD3OD, 500 MHz): δ 1.41 (s, 12H), 2.01 (s, 3H), 3.41 - 3.45 (m, 1H), 3.58 - 3.64 (m, 2H), 3.68 - 3.80 (m, 1H), 3.90 - 4.15 (m, 2H), 5.19 (d, 1H), 7.10 (d, 2H), 7.65 (d, 2H).

[0182] p - Boronic acid phenyl 2 - acetamido - 2 - deoxy - β - D - glucopyranoside 35: The above - mentioned compound p - pinacolyl boronate phenyl 2 - acetamido - 3,4,6 - tri - O - acetyl - 2 - deoxy - β - D - glucopyranoside 42 (1.5 g, 2.73 mmol) was treated with NaIO4 (2.5 equivalents) and NH4OAc (1.5 equivalents), and stirred at room temperature for 24 hours. The pH of the reaction mixture was adjusted to 3 by the addition of 1N HCl, and further stirred for 30 minutes. The reaction mixture was extracted with DCM, washed with brine, dried over anhydrous Na2SO4, and filtered. The combined filtrates were concentrated to obtain crude boronic acid, which was purified by silica gel column using EtOAc in hexane (0 - 50%) as the eluent to obtain the product 43 as a solid product in a yield of 75%. Then, this product was treated with NaOMe according to the procedure for 32 above to obtain compound 35 as a white solid in a yield of 91%. The NMR and MS spectra confirmed the structure of the above - mentioned product. Structure was confirmed. 1 1H NMR (D2O + DMSO - d6, 500 MHz): δ 1.21 (s, 3H), 2.6 - 2.8 (m, 4H), 3.15 (m, 2H), 3.68 - 3.80 (m, 1H), 4.35 (m, 1H), 6.25 (d, 2H), 7.01 (d, 2H).

[0183] Synthesis of compound 17 (Figure 30). (2R,3S,4S,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-(benzo[c][1,2,5]oxadiazole-5-yloxy)tetrahydro-2H-pyran-3,4-diyldiacetate 44: Following the procedure for compound 32 above, compound 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosilloride 41 (1.0 g, 2.73 mmol) was reacted with commercially available benzo[c][1,2,5]oxadiazole-5-ol (1.05 equivalents) to obtain intermediate 44 as a white solid in 65% yield. The NMR spectrum confirmed the structure of the above product.

[0184] N-((2S,3R,4S,5S,6R)-2-(benzo[c][1,2,5]oxadiazole-5-yloxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide 17: Intermediate 44 (0.25 g, 0.53 mmol) was converted to compound 17 as a white solid in 92% yield. NMR and MS spectra confirmed the structure of the product. LC / MS = 339.9 (M+1); 1 H NMR (CD3OD, 500MHz): δ2.01(s,3H), 3.41~3.45(m,1H), 3.58~3.64(m,2H), 3.68~3. 80(m,1H), 3.90~4.15(m,2H), 5.25(d,1H), 7.21(d,1H), 7.32(s,1H), 7.83(s,1H).

[0185] Synthesis of compound 8 (Figure 31). p-nitrophenyl 2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside intermediate 45: Following the procedure for compound 42, compound 2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosilloride 41 (1.0 g, 2.73 mmol) was coupled with commercially available p-nitrophenol (1.05 equivalents) to obtain intermediate 45 as a white solid in 89% yield. 1 1H NMR

[0186] Following the same procedure used for the synthesis of p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside 8:32, the above intermediate 45 (0.25 g, 0.53 mmol) was converted to compound 8 as a pure white solid in 92% yield. LC / MS = 343.1 (M+1); 1 H NMR(D2O)+CD3OD,500MHz): δ2.01(s,3H), 3.46(t,1H), 3.60~3.65(m,2H), 3. 68~3.81(dd,1H), 3.90~4.15(m,2H), 5.24(d,2H), 7.20(d,2H), 8.23(d,2H).

[0187] Synthesis of compound 14 (Figure 33). p-cyanophenyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside intermediate 48: Following the procedure for compound 42, compound 2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosilloride 41 (1.0 g, 2.73 mmol) was coupled with commercially available p-cyanophenol (1.05 equivalents) to obtain compound 48 as a white solid in 65% yield. 1 H NMR(CD3OD,500MHz):δ1.65(s,3H), 2.18(s,12H), 3.88(m,1H), 4.15~4.22(m,2H) ), 4.25(m,1H), 5.19(m,1H), 5.45(m,2H), 5.80(m,1H), 7.15(d,2H), 7.65(d,2H).

[0188] p-Cyanophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside 14 Following the procedure for compound 32, the above intermediate 48 (0.25 g, 0.53 mmol) was converted to compound 14 as a pure white solid in 91% yield. LC / MS = 323.1(M+1); 1H NMR (D2O+CD3OD,600MHz): δ2.01(s,3H), 3.52(t,1H), 3.60~3.68(m,2H), 3. 75~3.81(dd,1H), 3.90~4.15(m,2H), 5.24(d,2H), 7.20(d,2H), 7.75(d,2H).

[0189] Synthesis of compound 31 (Figure 32). 4-ethylcarboxylatephenyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside intermediate 46: Following the procedure for compound 42, compound 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosilloride 41 (1.0 g, 2.73 mmol) was coupled with commercially available 4-hydroxyethyl benzoate (1.05 equivalents) to obtain intermediate 46 as an oil in 89% yield.

[0190] Following the procedure for 4-carboxylate phenyl-2-acetamido-2-deoxy-β-D-glucopyranoside compound 31:compound 32, the above intermediate 46 (0.25 g, 0.53 mmol) was converted to compound 31 as a pure white solid in 90% yield. 1 H NMR (D2O, 500MHz): δ1.85(s,3H), 3.41~3.45(m,1H), 3.58~3.64(m,3H), 3.80~3.95(m,2H), 5.19(d,2H), 6.95(m.2H), 7.85(m,2H).

[0191] 2-Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosideazide intermediate 49 (Figure 34): 2-Acetamide-3,4,6-tri-O-A Cetyl-2-deoxy-β-D-glucopyranosylloride 41 was converted to sugar azide 49 according to a literature protocol, which was then converted to amine 50 as a white solid in 89% yield using TPP and H2O.

[0192] Disaccharide compound 2: Compound 31 (0.197 g, 0.72 mmol), EDCI (0.179 g, 1.44 mmol), HOBt (0.089 g, 0.866 mmol), and DIPEA (0.195 g, 1.8 mmol) were added to a stirred solution of amine 50 (0.25, 0.72 mmol) in anhydrous DMF, and the reaction mixture was stirred overnight. The reaction mixture was quenched by adding water, and all solvent was removed under high vacuum. The crude solid mass was purified by silica gel column chromatography using MeOH in siRNA (0-20%) as the eluent to obtain pure product 2 as a solid in 89% yield. 1 H NMR (CD3OD+D2O, 500MHz): δ1.90(d,3H), 2.0~2.11(m,12H), 3.45~3.58(m,2H), 3.60~3.68(m,1H), 3.68~3.80(m,1H), 3.82~4.05(m ,3H), 4.110~4.21(m,1H), 4.35~4.45(m,1H), 5.08~5.10(m,2H), 5.20(m,1H), 5.42(m,1H), 5.45(m,1H), 7.12(d,2H), 7.80(d,2H).

[0193] Compound 2 (0.21 g, 0.313 mmol) was converted to compound 3 as a pure white solid in 95% yield, following the procedure for disaccharide analog compound 3:32. LC / MS = 544.2 (M + 1); 1H NMR (CD3OD + D2O, 600 MHz): δ 1.80 (d, 6H), 3.30~3.45 (m, 3H), 3.45~3.60 (m, 4H), 3.60~3.68 (m, 2H), 3.71~3.90 (m, 4H), 5.10 (m, 2H), 6.90 (d, 2H), 7.55 (d, 2H).

[0194] Compound 23 was synthesized following the same procedure as described for the synthesis of compound 17. 1 H NMR (CD3OD, 500MHz): δ1.01(s,12H), 1.65(s,3H), 3.20(m,1H), 3.31(m,1H), 3.40~3.51(m, 2H), 3.60(m,1H), 3.75(m,1H), 5.20(d,1H), 6.8(d,1H), 7.05(t,1H), 7.21(d,1H), 7.8(bs,1H).

[0195] Disaccharide analog compound 6 (Figure 35). -NO2 is reduced to an amine using Fe and NH4Cl. Using the well-known literature procedure described above, compound 45 was converted to amine 51 as a semi-solid product in 91% yield, and this was directly coupled with compound 31 using the same procedure as described above for compound 2 to obtain disaccharide amide compound 6 in 89% yield. 1 H NMR (CD3OD+D2O, 500MHz): δ1.80~2.2(m,15H), 3.40~3.65(m,4H), 3.75~3.85(m,1H), 3.85~4.25(m,4H), 5.08~5.10(m,2H), 5.20(m,1H), 5.42(m,1H), 5.45(m,1H), 7.0~7.20(dd,4H), 7.60(d,2H), 7.90(d,2H).

[0196] Compound 6 was converted to compound 7 as a pure white solid in 92% yield, following the same procedure as described for 32. 1 H NMR (CD3OD+D2O, 500MHz): δ1.80~2.2(2s,6H), 3.30~3.45(m,7H), 3.55~3. 85(m,5H), 5.08~5.10(m,2H), 7.0~7.20(dd,4H), 7.60(d,2H), 7.90(d,2H).

[0197] Any embodiment discussed herein is intended to be carried out with respect to any method, kit, reagent, or composition of the present invention, and vice versa. Furthermore, the methods of the present invention can be achieved using the compositions of the present invention.

[0198] The specific embodiments described herein are for illustrative purposes only and should not be understood as limiting the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art can recognize or confirm numerous equivalents to the specific procedures described herein using experiments that do not exceed the routine procedures. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0199] All publications and patent applications described herein represent the skill level of a person skilled in the art to which the present invention relates. All publications and patent applications are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually incorporated by reference.

[0200] The use of the words “a” or “an,” when used with the term “comprising” in the claims and / or specification, may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or,” unless it is explicitly indicated that it refers only to substitutes or that the substitutes are mutually exclusive; however, this disclosure supports the definitions of substitutes only, as well as “and / or.” Throughout this application, the term “about” is used to indicate that a value includes inherent variation errors in the variability present between the device, the method used to determine the value, or the subject of study.

[0201] When used herein and in the claims, the terms “comprising” (and any form of comprising, e.g., “comprise” and “comprises”) and “having” ( And any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") is inclusive or open and excludes any additional unquoted elements or method steps. Not to remove. In any embodiment of the compositions and methods provided herein, “comprising” means “consisting essentially of” or “from.” It may be replaced with "consisting of". When used in this specification, the phrase " "Consisting essentially of" refers to a specific integer or step, and a sequence of numbers. It is required that the features or functions of the claimed invention are not substantially affected. When used herein, the term "consisting" is used to indicate that only the integers (e.g., features, elements, characteristics, properties, methods / method steps or limitations) or groups of integers (e.g., groups of features, elements, characteristics, properties, methods / method steps or limitations) are present.

[0202] The term “or any combination thereof,” as used herein, refers to all permutations and combinations of the items listed prior to the term. For example, “A, B, C or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where the order is important in a particular context, at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, what is clearly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will typically understand that there is no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0203] When used herein, approximate terms, such as “about,” “substantial,” or “substantially,” are understood to mean a state that, when modified, does not necessarily need to be absolute or complete, and is considered by those skilled in the art to be close enough to justify specifying an existing state. The extent to which these specifications may be modified depends on how large the possible changes are and whether those skilled in the art still recognize that the modified feature still possesses the required characteristics and capabilities of the unmodified feature. Generally, numerical values ​​in these specifications that are the subject of the foregoing considerations but modified by approximate terms such as “about” may vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated value.

[0204] All compositions and / or methods disclosed and claimed herein can be prepared and carried out without excessive experimentation based on this disclosure. Although the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the compositions and / or methods, as well as the steps or sets of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. All such similar substitutions and modifications will be apparent to those skilled in the art and will be recognized as being within the spirit, scope and concept of the invention as defined in the appended claims.

[0205] To assist the Japan Patent Office and any reader of any patent issued pursuant to this application in interpreting the claims attached herein, the applicants have decided not to use the words “method (means for)” or “ste.” Unless the term "step for" is explicitly used in a particular claim, it should be noted that none of the attached claims are intended to invoke Sections 112-6 to 35, 112(f), or equivalent of the Patent Act, as they exist as of the filing date.

[0206] In each claim, each dependent claim may depend on the independent claim, as well as on any prior dependent claim of any claim, insofar as the prior claim provides a basis for appropriate prior art of the terms and elements of the claim.

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Claims

1. A composition for regulating the immune response of immune cells, comprising at least one compound selected from compounds 8, 17, 28, 29, 30 or 35, formulated to activate the TLR4 receptor, the TLR2 receptor, or both the TLR2 receptor and the TLR4 receptor, respectively, and having the following formula and concentration: Table 1

2. A compound having the formula: 【Chemistry 1】 and formulated into a composition at a concentration ranging from 0.1 to 50 mg / kg to activate an immune response.

3. 1. The compound of formula: 【Chemistry 2】 and is formulated into a composition at a concentration greater than 50 milligrams / kg to activate an immune response.

4. A pharmaceutical composition comprising an amount of a compound that inhibits the immune response of immune cells as a vaccine adjuvant, antimicrobial, antibacterial, antiviral or immunostimulant, wherein the compound is selected from the following: 【Chemistry 4】 inhibits TLR4 in vitro at concentrations of 75-100 μM; 【Transformation 6】 inhibits TLR2 in vitro at concentrations of 1-10 μM and 75-100 μM; 【Transformation 7】 inhibits TLR4 in vitro at a concentration of 1-10 μM; or 【Chemistry 9】 It inhibits TLR4 in vitro at concentrations of 1-10 μM.

5. A composition for modulating the immune response of immune cells, comprising at least one compound selected from compounds 8, 17, 28, 30 or 35, formulated to inhibit the TLR4 receptor, the TLR2 receptor, or both the TLR2 receptor and the TLR4 receptor, respectively, and having the following formula and concentration: Table 2

6. 6. The composition of claim 5, formulated into a pharmaceutical composition for treating hyperinflammation selected from lung injury, lung cancer, irritable bowel disease, arthritis, psoriasis, acne, BPD, arthritis, necrotizing enterocolitis, or sepsis, and provided at a concentration to inhibit an immune response, wherein the compound is selected from: 【Chemistry 1】

7. 6. The composition of claim 5, formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, or salts.

8. 6. The composition of claim 5, formulated into a pharmaceutical composition adapted for pulmonary, intraalveolar, rectal, parenteral, intravenous, topical, or oral administration.

9. 6. The composition of claim 5, formulated into an aerosol, nebulizer, or inhaler.

10. The composition of claim 5 , further comprising one or more liposomes, polymers, surfactants, salts, or buffers.

11. 6. The composition of claim 5, further comprising an additional therapeutic agent selected from the group consisting of corticosteroids, bronchodilators, anticholinergics, vasodilators, diuretics, antihypertensives, acetazolamide, antibiotics, antivirals, immunosuppressants, and surfactants.

12. 6. The composition of claim 5, wherein the compound is provided in an amount that competitively inhibits inflammation and activates macrophages to protect against or limit lung tissue injury.

13. 6. The composition of claim 5, wherein the compound is a TLR4 modulator and is provided in an amount that upregulates IL-10.

14. 6. The composition of claim 5, wherein the compound is a TLR2, TLR4, TLR7 and TLR8 inhibitor and is provided in an amount that downregulates IL-1β.

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