TW201010988A - 5-(4-methanesulfonyl-phenyl)-thiazole derivatives for the treatment of acute and chronic inflammatory diseases - Google Patents

Abstract

The invention relates to the use of a compound of formula (I): or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, in the preparation of a medicament for the treatment of an acute or chronic inflammatory disease, by inhibiting the production of at least one pro-inflammatory cytokine selected from TNF-alpha and IFN-gamma, or by inmunomodulating the IL-8 chemokine and/or the IL-10 regulatory cytokine. The invention also relates to novel compounds of formula (I'): or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, as well as a pharmaceutical composition comprising said compound.

Description

201010988 Λ VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to compounds derived from 5-(4-methylsulfonyl-phenyl)-thiazole for use in the treatment of acute or chronic inflammatory diseases or conditions ( Use in pharmaceutical products such as rheumatoid arthritis. [Prior Art] 〇 Immunology is a scientific study that distinguishes between self and non-self. The disintegration of autologous tolerance is the origin of autoimmune diseases. In addition, other conditions such as transplantation, atherosclerosis, septic and non-septic acute or chronic inflammatory pathology and many other diseases that have not been considered autoimmune so far also exhibit pathological mechanisms mediated by immune cells. Most authors believe that the activation of the immune inflammatory effector response is based on two signals: - Signal 1 contains a receptor that stimulates the cell line antigen (TCR-CD3 T cell receptor complex) to identify the embedding in the primary A homologous antigen in a histocompatibility complex mites (MHC) molecule. In B cells, an extracellular soluble or membrane-bound antigen is cross-linked with the immunoglobulin CD79 (Ig/CD79) cell line receptor complex to deliver the signal 1 to the antibody secreting lymphocyte lineage - in addition to antigen delivery In addition to signal 1, signal 2 is required to avoid tolerance due to anergy or cell line deletion. Signal 2 is delivered by a costimulator, such as CD86 on the surface of specialized antigen-expressing cells (APCs), such as human monocytes and their descendants of the differentiation system. In the congenital immune inflammatory response, the risk signal is enhanced by the stimulating pattern of the microorganisms of the 201010988 recognizer (PRR) and autoregulatory antigens or mitogens. PRR signaling pathways such as TLR4 (the receptor for Gram-negative bacterial cell wall lipopolysaccharide or LPS) deliver high levels of costimulatory surface molecules (i.e., CD80 or CD86). Next, the co-stimulatory ligands CD80-CD86 and T help CD28 cross-linking on the cell surface. The combination of signal 1 (i.e., anti-CD3 antibody cross-linking) and signal 2 (i.e., CD28 is cross-linked by a particular antibody) experimentally mimics the natural conditions of an effective inflammatory effector immune response. It is noted that approved immunosuppressive drugs have several difficulties in inhibiting the activation of the proinflammatory cytokine cascade under conditions of high co-stimulation. The latter occurs in autoimmune diseases and many other serious acute or chronic inflammatory conditions. APC is not only the key to determining the type of immune response in the acquired immune system, because they profoundly affect the cell lineage that is the effector (also It is the T cell help (Th), cellular cytotoxic (Th), antibody production (B cell) response or tolerogenic response (low response or apoptosis). APC itself can also deliver an effector direct immune response. A well-documented example is that monocytes produce and release high amounts of tumor necrosis factor a (TNF-α) in cancer patients. In these patients, TNF-α promotes amplification and activation of vascular endothelial cells, accompanied by secretion of interleukin-8 (IL-8) and other proinflammatory cytokines, as well as procoagulant activity. At the same time, TNF-α promotes thrombosis and ischemic tumor necrosis, which results in the original definition of cachectin or TNF-α. However, TNF-α is a polytropic interleukin that is involved in many other disease states. Over the years, evidence shows that LPS can stimulate the secretion of large amounts of TNF-α by monocytes, which strongly leads to the occurrence of septic shock in 201010988. Recently, high levels of TNF-ct inappropriate secretion are considered to be treatments for autoimmune inflammatory diseases such as rheumatoid arthritis, Crohn's disease, uveitis, and psoriasis. Therefore, the latest strategy for the treatment of diseases using anti-TNF-α is now widely recognized, wherein reducing the amount of biologically active TNF-enable may lead to an improvement in the condition of the patient. In this regard, it may be possible to benefit from the rapid increase in the number of TNF-α antagonists, including atherosclerosis, metabolic group, encephalitis, viral hepatitis, glomerulonephritis, and tumors. Non-inflammatory reaction and septic shock. In addition to those produced by several somatic cells, TNF-α has a major source of production, monocytes and sputum lymphocytes. During the process of obtaining the response, APC is displayed under the guidance of pre-inflammatory to signal stimulation of IL-12 ρ70 (heterodimer of ρ35 and ρ40), and then the intercellular secretion characteristics of the activated Th cells. Polarization becomes a costimulatory molecule of the scorpion type. Thl cells exhibit a characteristic pathway of interleukin, in which the interferon r (IFN-r) secretion is a true footprint. These cells secrete high amounts of TNF-α. IFN-τ secreted by Th1 promotes many potentially important effects on inflammatory diseases. In one aspect, it further activates monocytes and gives the amount of TNF-[alpha] secretion caused by the stimulation. On the other hand, its TNF-α receptor is more strongly signaled downstream. In summary, IFN- has its own direct effects such as antiviral activity, but also the performance of MHC-II and by the force of D TNF-α still strong effect to make inflammation, target the symptoms of these alpha When two immune-impaired patients are Th-1, the increase in decomposition also causes the increase in r to cause inflammation and lesions in 201010988. The pre-inflammatory grade is initiated by IFN-r and TNF- 〇: production of TNF-[alpha] or Th1 cells of monocytes, which is expanded by other pro-inflammatory interleukin pathways, such as IL-8. IL-8 (regardless of its original nomenclature) is described as a chemokine produced by macrophages and other cell types, such as epithelial cells, which can also be synthesized by endothelial cells and is therefore also referred to as CXCL8. Although the neutrophil granulosa cell line is the primary target cell for IL-8, a wide variety of cells (endothelial cells, macrophages, hypertrophic cells, keratinocytes) also respond to this chemokine. The primary function of IL-8 is to induce chemotaxis of its target cells, such as neutrophil cells. In neutrophils, a series of cellular physiological responses required for migration and its major functional phagocytosis are also triggered, such as increased intracellular Ca2+, extracellular secretion (such as histamine release), and respiratory bursts. IL-8 can be secreted by any cell having a TLR associated with an innate immune response. The primary function of IL-8 is to recruit neutrophils to phagocytose the antigen that causes the antigenic pattern TLR. By delivering IL-8 target cells to the endothelium and other target tissues, in addition to its role in body defense, Q IL-8 is involved in the expansion and progression of many of the pathological effects of TNF-α. The movement and regression of white blood cells is regulated not only by chemotactic hormones and their receptors, but also by several adhesion molecules. Among these adhesion molecules, the selectin CD62L is considered to be a therapeutic target for preventing leukocytes from moving to the lymph nodes, and thus is considered as an evaluation parameter for the in vitro effect of arranging non-steroidal anti-inflammatory drugs (NSAIDs). Many immunosuppressive agents and anti-TNF-α molecules affect normal immune -8- 201010988 \ · defense mechanisms because they enhance cytotoxicity against immune cells or inhibit cell line expansion before successful effector immune response Proliferation mechanism. Given the importance of TNF-α secreted by extracellular space (whether soluble in the plasma membrane insertion or secretion), many studies have been designed to block extracellular TNF-α and TNF-receptors I and/or A therapeutic agent for the interaction of TNF-receptor II. The most suitable method is to use a soluble decoy φ TNF-receptor to capture TNF-α and prevent the interaction of the proinflammatory ligand with the cellular receptor due to the long time required for dissociation. The second strategy is to produce humanized anti-human TNF-α antibodies in a conventional manner or to produce bispecific single-chain molecules that are also labeled with other molecules associated with a given disease (eg, in rheumatoid arthritis). Anti-VEGF/anti-TNF-a). Although the above-mentioned molecules are TNF-α antagonists, they limit their mechanism of action to block extracellular secretion of TNF-α. A detailed but incomplete standard that can drive the production and secretion of TNF-α may be: (a) a molecule that drives transcription of TNF-a; (b) drives TNF-a-RNA transport from the nucleus to the cytoplasm and RNA. a molecule that cleaves; (c) a molecule that directs translation of TNF-α; (d) a molecule that regulates TNF-cz-mRNA stability; (e) a cell membrane that directs high-bass cells to the surface of the pre-TNF-α anchoring site Molecules; (f) molecules associated with secretion of TNF-α secreting molecules such as TNF-α converting enzyme (TACE) and (g) molecules that regulate the internalization of the surface form of pre-TNF-α and its signals. All of these are related to the cell labeling of TNF-α production and secretion. Although there are different ways to block the treatment of TNF-o: 201010988, if it can be found that selective blockade is not just TNF-α production, there are other key pro-inflammatory interleukins (such as IFN-r). The new drugs produced will be highly valued. SUMMARY OF THE INVENTION The authors of the present invention have surprisingly discovered that the compounds of formula (I) exhibit some highly interesting immunomodulatory effects which may be useful in controlling the pathological mechanisms of acute and chronic inflammatory diseases and therefore have potential clinical utility. Specifically, the compound used in the present invention can inhibit the production of TNF-α from peripheral blood mononuclear cells (PBMC) of patients with chronic inflammatory diseases such as rheumatoid arthritis, and also inhibits the passage of these cells. IFN-Τ secretion after T cell stimulation. Furthermore, the compounds of formula (I) have been able to modulate the bioreactive properties of secreted chemokines (i.e., IL-8) and regulatory interleukins (i.e., IL-10) and are regulated in different ways in health and disease states. The overall immunomodulatory effects of the compounds of formula (I) do not cause any toxic effects on monocytes from peripheral blood. In addition, activation and proliferative responses following stimulation of cell division are not regulated or even increased by these compounds. In this small molecule, it also has the inhibitory effect on several proinflammatory interleukins such as TNF-α and IFN-r and regulates IL-8 chemokines and/or IL-1 0 regulatory interleukins (all The potential of systemic and organ-specific autoimmune disorders, transplantation, acute and chronic inflammatory diseases, certain metabolic and degenerative diseases, and the pathophysiology of atherosclerosis, Compounds can be classified in the novel immunomodulatory 201010988 class of agents and are exemplified by a cascade of proinflammatory/regulatory interleukins and chemokines prior to various possible reprogramming levels of clinical and therapeutic importance. It is apparent that the immune cell line is sessile and enters the organ to patrol body tissue that is infiltrated in an inflammatory condition in which the immune cells accumulate in lesions distributed according to disease activity, target organ, and degree of damage. Since the compounds used in the present invention are expected to modulate several morphological forms of the pathophysiological process, development assays can be utilized to characterize the number of receptors, binding efficiency, receptor occupancy, and drug probe concentration. Since the white blood cells have a regression characteristic, detecting the therapeutic target can also obtain information about the location of the target cell and the lesion site. Thus, the compounds of formula (I) also exhibit a use as a biomarker for development in drug development, clinical trials and individualized medicine, which provides information not only on pharmacokinetics, distribution and administration, but also in preclinical and Important data on individualized response patterns in clinical trials. The latter may Φ result in a surrogate biomarker defining a validated, reliable, individualized clinical endpoint for administration to a patient in need of the prognostic and individualized assessment of an effective amount of a compound of formula (I) or a pharmaceutical composition thereof , to define the precise bio-developing technology known to those skilled in the art. According to a first aspect, the invention relates to a compound of formula (I): -11 - 201010988

Use of (D or a pharmaceutically acceptable salt, prodrug and/or solvate thereof for the manufacture of a medicament for the treatment of an acute or chronic inflammatory disease, wherein:

Ri is selected from hydrogen, substituted or unsubstituted CrCe alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic ; r2 is selected from substituted or unsubstituted C!-c6 alkyl, substituted or unsubstituted cycloalkyl or N(R'R"), wherein R' and R" are each hydrogen or A substituted or unsubstituted alkyl group; and R3 is an unsubstituted Ci-6 alkyl group. In a particular embodiment, the medicament for treating an acute or chronic inflammatory disease is effected by inhibiting the production of at least one inflammatory interleukin selected from TNF-[alpha] or IFN-r, or by immunomodulating IL -8 Chemokines and/or IL-10 regulatory interleukins. In another specific embodiment of the invention, the production of the inflammatory interleukin TNF-[alpha] prior to the inhibition is at least partially expressed in terms of the amount of TNF-a mRNA. In a particular aspect of the invention, the acute or chronic inflammatory disease is selected from the group consisting of acute and chronic seropositive or seronegative oligoarthritis and polyarthritis, spondyloarthropathy, glomerulonephritis, collagen disease, renal tubules Interstitial -12- 201010988 Nephritis, metabolic syndrome, atherosclerosis, osteoarthritis, asthma, chronic obstructive pulmonary disease, interstitial lung disease, multiple sclerosis, demyelinating disease, meningitis, encephalitis , meningoencephalitis, inflammatory radiculopathy and peripheral neuropathy, inflammatory bowel disease, cirrhosis, hepatitis, heart failure, ischemic disease, renal failure, inflammatory cystitis, benign prostatic hyperplasia, prostatitis, myocarditis, heart Encapsulation, uveitis, atopic dermatitis, eczema, measles, psoriasis, rose acne, allergic rhinitis, sepsis, septic shock, multiple organ failure, systemic autoimmune disease (such as systemic Lupus erythematosus), vasculitis, dermatomyositis, amyloidosis or sarcoma-like disease, organ-specific autoimmune diseases (such as myasthenia gravis), Goiter or insulitis, organ transplantation, infection and tumor-induced inflammation, TNF-α-dependent cell degeneration, necrosis, apoptosis, graft versus host disease, cachexia or autocrine and paracrine pathological cell growth. These therapeutic indications are at least abnormal immune response, immunoregulatory abnormalities, immune interference, immune pathogenesis, immunotherapy, immunosuppression or quarantine-free biological response. In another aspect, the invention relates to a compound of formula (Γ)

Or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, -13- 201010988 wherein: I is selected from the group consisting of hydrogen, substituted or unsubstituted Ci_c6 alkyl, substituted or unsubstituted naphthenic a substituted or unsubstituted aryl group or a substituted or unsubstituted heterocyclic group; and R3 is an unsubstituted Ci-Q alkyl group. In a third aspect, the object of the present invention is a pharmaceutical composition comprising a compound of the formula (I') as defined above, or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, And at least one pharmaceutically acceptable carrier, adjuvant and/or excipient. Another aspect of the invention pertains to a compound of formula (Γ) as defined above for use as a medicament. Finally, another aspect of the invention pertains to the use of a compound of formula (I) as defined above, particularly a compound of formula (U.S.), as a biomarker for development in development and pharmacological development techniques. These biomarkers can be used to identify immune lesions, target cells, and target molecules. DETAILED DESCRIPTION OF THE INVENTION In the context of the present invention, the following terms have the meanings as detailed below. The term "Mountain-6 alkyl" means a linear or branched hydrocarbon chain group consisting of carbon and hydrogen atoms. a saturated hydrocarbon having from one to six carbon atoms and linked to the remaining molecules by a single bond, such as methyl, ethyl, n-propyl, propyl, η-butyl, t-butyl, n-pentyl, etc. . The Cl-6 alkyl group may be optionally substituted by one or more substituents such as cycloalkyl, aryl, heterocyclyl • 14- 201010988 \ , halo, hydroxy, alkoxy, cyano, amine Base, nitro or alkylthio. The term "cycloalkyl" refers to a saturated or partially saturated diazet 3 to 8 membered cyclic group consisting solely of carbon and hydrogen atoms, such as cyclohexyl or cyclopentyl. Unless the specification specifically dictates otherwise, the term "cycloalkyl" is intended to include a cycloalkyl group which may be optionally substituted with at least one substituent selected independently from hydrogen, q-6 alkyl group, Halo, hydroxy, _N(R3)(R4), wherein ampules 3 and R4 are independently selected from the group consisting of hydrogen and linear or branched Cl.6 院 院. The term "aryl" refers to a 5 to 8 membered aromatic cyclic group of diazepam and is composed only of carbon and hydrogen atoms, such as phenyl or cyclooctatetraene. Unless the specification specifically dictates otherwise, the term "aryl" is intended to include an aryl group which is optionally substituted with at least one substituent selected independently from hydrogen, (: 丨-6 alkyl group, Halo, hydroxy, -N(R3)(R4), wherein R3 and R4 are independently selected from hydrogen and a linear or branched alkyl group. "Heterocyclyl" means a stable 3 to 8 membered cyclic group. It consists of a carbonium atom and one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For the purposes of the present invention, the heterocyclic ring may be partially or fully saturated or aromatic. Including, but not limited to, pyrrolidine, pyridine, thiophene, furan, etc. Unless otherwise specifically stated in the specification, the term "heterocyclyl" is intended to include a heterocyclyl group which is optionally substituted with at least one substituent, the substituent Is independently selected from the group consisting of hydrogen, Cu alkyl group, halogen group, hydroxyl group, _n(r3)(r4) ' wherein R3 and R4 are independently selected from hydrogen and linear or branched Cl.6 alkyl group. "Halo" means a desert, a chlorine, a confirmatory or a gas base. -15- 201010988 The term "acute and chronic seropositive or "Keep-negative oligoarthritis and polyarthritis" refers to synovitis diseases involving one or several joints with positive or negative rheumatoid factors, including rheumatoid arthritis and primary and secondary Glenn syndrome. The term "sponge joint disease" refers to an immunopathological HLA-B27-associated inflammatory disease involving the ankle joint and/or the spine and/or surrounding joints, including uveitis. "Acne nephritis" refers to an inflammatory lesion of the glomerulus. The term "tubular interstitial nephritis" refers to an inflammatory disease involving the renal tubules and the renal interstitium. The term "collagen disease" refers to a systemic inflammatory disease with pathological immune mechanisms. 'Including systemic lupus erythematosus (SLE), dermatomyositis, and scleroderma. "Inflammatory bowel disease" refers to gastrointestinal inflammatory disease with pathological immune mechanisms, with or without systemic features, including Crohn's disease. And ulcerative colitis. The term "obstructive pulmonary disease" refers to a bronchial disease that is reversible or irreversibly reduced in forced expiratory volume (FEV), including asthma and chronic obstructive pulmonary disease. "Pulmonary disease" refers to an inflammatory disease involving the interstitial lung. The term "demyelinating disease" refers to a pathogenic immune mechanism that causes myelinolysis to cause central nervous system inflammatory diseases, including multiple sclerosis and optic neuritis. "Inflammation, encephalitis, and meningoencephalitis" refers to inflammatory diseases of the meninges and/or other structures of the central nervous system. -16- 201010988 \ The term "inflammatory radiculopathy and peripheral neuropathy" refers to the inflammatory disease of the peripheral nervous system. The term "inflammatory inflammatory cystitis" refers to an inflammatory disease of the bladder. The term "benign prostatic hyperplasia" means non-malignant hypertrophy and/or hyperplasia of the prostate. The term "atopic dermatitis, eczema and urticaria" means With or without allergic skin disease associated with the immunopathological mechanism of IgE. Φ The term "psoriasis" refers to hyperkeratosis and erythematous skin reactions associated with the pathogenesis of the immune system. The term "rosal acne" refers to the common inflammatory condition of the skin characterized by erythema (redness and redness) in the center of the face. ), can also cross the cheeks, nose or forehead, less in the neck and chest. The term "allergic rhinitis" refers to intermittent (also known as seasonal) or persistent (also known as perennial) inflammatory nasal mucosal disease, which is associated with immunopathic pathogenesis. # The term "sepsis, septic shock and multiple organ failure" refers to a systemic inflammatory disease that is mediated by an abnormal immune response to a microbial agent or other virulence factor. The term "sarcoma-like and amyloidosis" refers to a spontaneous immune disease in which an organ and/or system is involved without a well-defined cause, in which an abnormal immune response is observed. The term "organ-specific autoimmune disease" refers to organ lesions of the immune system that are not defined by the causative agent, including myasthenia gravis, thyroiditis, pituitary gland, adrenalitis, and others. -17- 201010988 The term "organ transplantation" refers to the prevention and treatment of rejection of transplanted cells, tissues and organs. The term "inflammation caused by infection and tumor" refers to an abnormal immune response secondary to microbial agents or cancer stimulation. The term "TNF-α-dependent cell degeneration, apoptosis or necrosis" refers to tissue degradation or death caused by TNF-α. The term "graft versus host disease" refers to an inflammatory immune response caused by a transplanted cell. The term "cachexia" refers to a systemic anorexia or malnutrition and weight loss caused by an inflammatory or neoplastic disease. The term "atherosclerosis" refers to any arteriosclerosis caused by atheroma or by inflammatory cells (mostly macrophages) and accumulation of cell debris containing lipids on the arterial wall. The term "ischemic disease" refers to organ lesions, including cardiac and cerebral ischemia, secondary to tissue oxygenation and/or decreased blood flow. The term "autologous secretion and paracrine pathological cell growth" refers to a cell using TNF-a as a malignant or benign disease that regulates the cellular activation and proliferation of interleukins. Unless otherwise stated, the compounds used in the present invention are intended to include compounds having only one or more isotopically enriched atoms. For example, a compound having the present structure but substituting hydrogen with hydrazine or hydrazine, or substituting carbon with 13C- or 14C-enriched carbon or 15N-enriched nitrogen is within the scope of the present invention. The phrase "a pharmaceutically acceptable salt, solvate or prodrug thereof" relates to a salt or solvate which, when administered to a recipient, provides (directly or indirectly) a compound such as those described in this -18-201010988. Or a prodrug. However, it will be found that pharmaceutically unacceptable salts are also within the scope of the invention as they can be used in the preparation of pharmaceutically acceptable salts. Salts, prodrugs and derivatives can be prepared by the method known from the latest. "Pharmaceutically acceptable" is preferably a molecular entity which is physiologically tolerable when administered to a human or animal and which generally does not cause an allergic reaction or a similar untoward reaction such as gastric abnormalities, dizziness and the like. Composition. The term "pharmaceutically acceptable" means approved by the regulatory agency of the federal or state government or included in the United States Pharmacopoeia or other generally recognized pharmacopoeia for animals, especially humans. For example, a pharmaceutically acceptable salt of a compound previously described herein is synthesized from the previously described compound containing a basic or acidic unit using conventional chemical methods. The salt is usually prepared by, for example, reacting the free acidic or basic form of the compounds with a suitable base or acid in water or an organic solvent or a mixture of the two. Non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of the acid addition salt include, for example, mineral acid addition salts such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfates, nitrates, phosphates, and, for example, organic acid addition salts such as acetate, maleate, fumarate, Citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the base-added salt include, for example, inorganic salts such as sodium, potassium, calcium, ammonium, magnesium, aluminum, lithium, and, for example, organic base salts such as ethylenediamine, ethanolamine, N,N-dienylethanolamine, reduced glucosamine, and Basic amino acid salt. The term "prodrug" is defined herein as a chemical derivative that has undergone chemical derivation 19 · 201010988 to alter (for medical use) some of its physicochemical properties (such as solubility or bioavailability) by, for example, substituting or adding additional chemical groups. The chemical compound, for example an ester or ether derived from the active compound, produces the active compound of its own after it is administered to the individual. Examples of well-known methods for producing prodrugs from a given active compound are known to those skilled in the art and can be found, for example, in Krogsgaard-Larsen et al., Textbook of Drug Design and Discovery, Taylor & F r an ci s (Apri 1 2002). According to the invention, the term "solvate" is used to denote a compound of the invention in any form in which other molecules, most likely polar solvents, are bonded thereto via non-covalent bonds. Examples of the solvate include hydrates and alcoholates such as methanolate. It is particularly preferred that the prior drug system increases the bioavailability of the compound of the invention when administered to a patient (eg, allows the orally administered compound to be absorbed into the blood more quickly) or The original compound can increase the distribution of the original compound to the biological compartment (eg, the brain or lymphatic system). The compound used in the present invention may be in the form of a crystalline form of a free compound or a solvate (e.g., a hydrate), i.e., a polymorph, and it is to be understood that both forms are within the scope of the present invention. The method of solubilization is generally known in the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate. Salt 'solvates and prodrugs can be prepared by the latest known methods. It will be observed that pharmaceutically unacceptable salts, solvates or prodrugs are also included within the scope of the invention' because they can be used in the preparation of pharmaceutically acceptable salts, solvates or prodrugs. -20- 201010988 The compounds of formula (I) or their salts or solvates are preferably in pharmaceutically acceptable form or in substantially pure form. The pharmaceutically acceptable form is understood to be, in particular, a pharmaceutically acceptable purity excluding normal pharmaceutical additives such as diluents and excipients, and does not contain any substance which is considered to be toxic under normal dosage levels. The purity of the drug is preferably more than 50%, more preferably more than 70%, and still more preferably more than 90%. In a preferred embodiment, it is greater than 95% of the compound of formula (I) or a salt thereof, a lysate or a prodrug. The compound of the present invention shown by the above formula (I) may include an image isomer or an isomer (e.g., Z, E) depending on the presence of a chiral center. Various isomers, mirror image isomers, non-image isomers, and mixtures thereof are within the scope of the invention. In a particular embodiment, the compound of formula (I) may be fluorescent or luminescent for use in development and pharmacological development techniques, or as an alternative to biomarkers in drug development, clinical trials, and individualized medicine. Or Φ banner designation, which is imported using any method known to those skilled in the art. In a particular embodiment of the invention, the substituted, unsubstituted c,-c6 alkyl group. Preferably, Ri is a methyl group. In another specific embodiment, R2 is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl. Preferably, R2 is a substituted or unsubstituted cycloalkyl group. More preferably, the r2 is a cyclopentyl group. In another preferred embodiment, 'R2 is a substituted or unsubstituted CrQ alkyl group. Preferably, R2 is substituted by a heterocyclic group of Cl-C6 -21 - 201010988. More preferably, R2 is a CrQ alkyl group substituted with ethylene oxide. In another specific embodiment, r3 is methyl. In another more specific embodiment, the compound of formula (I) used in the present invention is selected from the following compounds, or a pharmaceutically acceptable salt, prodrug and/or solvate thereof.

◎ -22- 201010988

00 (9) a. ^Xk: H-Ij. Eight. (10) 5 Bu a\v 1 7—ch2-cn Ά, α0 △ (4) Cl N 八(15) α0 ^>-〇 . Eight. (10) O. 〇 C, \ ό (17) α0 cy^o (18) The compound of the formula (I) used in the present invention can be obtained by a synthetic method which can be obtained. For example, they can be prepared by the methods outlined in the following figures. -23- 201010988

This method first comprises an esterification reaction of the compound 4-R3-phenylthioacetic acid to give a methyl ester derivative. The reaction can be carried out using a methylating agent such as Mel in the presence of a salt such as NaHC03 or using MeOH as a methylating agent in the presence of an acidic medium. In the next step, the methyl ester derivative (optionally without any purification) is oxidized at the sulfur atom using an oxidizing agent (e.g., oxone), thereby obtaining the maple compound from the thioether group. Subsequent halogenation with a reagent such as NBS results in the formation of a halide at the alpha position of the ester group. The cyclization addition of this compound is carried out under heating with different thioguanamine to produce high-purity Ri-substituted 5-(4-R3-sulfonyl-phenyl)-4-hydroxythiazole. The thioguanamine used in this cyclization addition is obtained from the corresponding ones as described in /. MM. Chem (34) 2158-2165, 1991 and J. Org. Chem. (65), 13, 3P 73, The lanthanide of the Lawson reagent. The thioguanamine includes those wherein R! is selected from the group consisting of hydrogen, substituted or unsubstituted C^-Ce alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, as defined above Or a compound of a substituted or unsubstituted heterocyclic group. Introduction of the R2 group via a hydroxyl group can be carried out by any of the methods known to those skilled in the art from -24 to 201010988 to produce a compound of formula (I) for use in the present invention. In a particular embodiment, the hydroxyl group is reacted with a halide of the formula R2-Hal, preferably R2-Br, to provide a compound of formula (I). All of the reactants used in the reactions mentioned are commercially available. In another aspect, the invention provides a compound of formula (Γ)

cAo (Γ) or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, wherein:

Ri is selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic And

Rs is an unsubstituted (^-Ce alkyl group. In a specific embodiment, 1 is a substituted or unsubstituted Ci-C6 fluorenyl group. Preferably, 1 is a methyl group. In another embodiment, R3 is methyl. In another specific embodiment, the compound of the formula (Γ) of the present invention is selected from the following 'or a pharmaceutically acceptable salt, prodrug and/or solvate thereof物-25- 201010988

The present invention further provides a pharmaceutical composition for administration to a patient, the pharmaceutical composition comprising the novel compound of the formula (Γ) of the present invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and At least one pharmaceutically acceptable carrier, adjuvant and/or excipient. In a particular embodiment, the compound of formula (I), a salt, prodrug and/or solvate thereof, which is pharmaceutically acceptable from -26 to 201010988, will be in a therapeutically effective amount with one or more pharmaceutically acceptable The carrier, adjuvant and/or excipient are formulated together into a suitable pharmaceutical composition for administration in the prevention and/or treatment of an acute or chronic inflammatory condition. The phrase "carrier, adjuvant and/or excipient" is a molecular entity or substance that is administered with the active ingredient. The pharmaceutical carrier, adjuvant or excipient can be a sterile liquid such as water and oil (including those from petroleum or animal, Q plants or synthetic sources such as peanut oil, soybean oil, mineral oil, sesame oil and the like) ), excipients, disintegrants, wetting agents or diluents. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The pharmaceutical composition can be administered by any suitable method of administration, for example, orally or parenterally (e.g., subcutaneously, intraperitoneally, intravenously, Intramuscular, etc., rectal administration, etc., usually by oral administration because of the chronic characteristics of the disease being treated. Φ In certain embodiments, the pharmaceutical composition can be in the form of a medical form for oral administration, whether in solid or liquid form. Illustrative examples of oral administration of pharmaceutical forms include tablets, capsules, granules, solutions, suspensions and the like, and may contain conventional excipients such as binding agents, diluents, disintegrating agents, lubricants, wetting agents, and the like, And can be prepared by a conventional method. The pharmaceutical composition may also be in a suitable dosage form, such as a sterile lyophilized product, suspension or solution, suitable for parenteral administration; in this case, the pharmaceutical composition will include suitable excipients, such as Buffer, surfactant, etc. In any case, the excipient will be selected according to the selected form of administration. A review of the different forms of medicines and their preparations for the administration of the drug -27- 201010988 can be found in "Trtado de Farmacia Galinica'' by C. Fauli i Trillo, 1 0th Edition, 1993, Luzan 5, SA de Ediciones. For therapeutic use, the compound of formula (I) will preferably be in a pharmaceutically acceptable or substantially pure form, i.e., the compound of formula (I) has pharmaceutically acceptable excluding pharmaceutically acceptable excipients. Purity, and does not contain substances considered to be toxic at normal dosage levels. The purity of the compound of formula (I) is preferably above 50%, more preferably above 70%, more preferably above 90%. In a preferred embodiment, the purity is greater than 95%. The therapeutically effective amount of the compound of formula (I) to be administered will generally depend on the individual being treated, the severity of the disease the individual is suffering from. Other factors depending on the method of administration chosen, etc. Therefore, the dosages referred to in the present invention must be considered only as a reference principle for those skilled in the art, and the skilled person must adjust the dosage according to the aforementioned variation parameters. I) compounds can One or more times a day, for example 1, 2, 3 or 4 times a day, typically from 0.1 to 1000 mg/kg body weight per day, preferably 10 mg/kg body weight per day. Compounds of formula (I) The pharmaceutically acceptable salts, prodrugs and/or solvates thereof and pharmaceutical compositions comprising the same can be used in combination with other additional drugs for the treatment of acute and chronic inflammatory diseases. The additional drugs can form the same medicine. A portion of the composition, or in the form of a separate composition, is administered simultaneously or non-simultaneously with the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt, prodrug or solvate thereof. In the context of the present invention, the term "acute and chronic inflammatory diseases" -28-201010988 relates to any activation and pathology resulting from the pathogenesis of abnormal co-stimulatory lines involving any combination of inflammatory/immune cells and inflammatory interleukin cascades Disease, abnormality or condition. The main cell line involved are inflammatory/immune cells such as monocytes, macrophages, APCs, sputum cells, B cells, natural killer (NK) cells, plasma cells, granulosa cells, and mast cells, or are associated with the disease to be treated. A combination of the above subpopulations of cells. The term "interleukin" refers to the secretion of egg white that affects the function of other cells, particularly the interaction between cells that regulate the immune system or between cells involved in the inflammatory response. These interleukins are TNF-α (tumor necrosis factor-a), IFN-r (interferon-r), IL-8 (interleukin-8) chemokine and regulatory interleukin IL-10 ( Interleukin-10). The production of IL-10 is caused by high levels of TNF-[alpha], which promotes the formation of negative feedback to TNF-[alpha] production by blocking the transcription of proinflammatory interleukins. In a particular aspect of the invention, the acute or chronic inflammatory disease is selected from the group consisting of acute and chronic seropositive or seronegative oligoarthritis and multiple joint inflammation, spondyloarthropathy, glomerulonephritis, collagen disease, kidney Tubulointerstitial nephritis, metabolic syndrome, atherosclerosis, osteoarthritis, asthma, chronic obstructive pulmonary disease, interstitial lung disease, multiple sclerosis, demyelinating disease, meningitis, encephalitis, meningeal brain Inflammation, inflammatory radiculopathy and peripheral neuropathy, inflammatory bowel disease, cirrhosis, hepatitis, heart failure, ischemic disease, renal failure, inflammatory cystitis, benign prostatic hyperplasia, prostatitis, myocarditis, pericarditis , uveitis, atopic dermatitis, eczema 'urticaria, psoriasis, rose acne, allergic rhinitis, sepsis, septic shock, multiple organ failure, systemic autoimmune diseases (such as systemic -29- 201010988 Lupus erythematosus), vasculitis, dermatomyositis, amyloidosis or sarcoma-like disease, organ-specific autoimmune diseases (such as myasthenia gravis), Prostatitis or insulitis, inflammation caused by the organ transplantation, infections and tumors, TNF_ α-dependent cellular degeneration, necrosis, apoptosis, graft versus host disease, cachexia or autologous autocrine and paracrine pathological cell growth. In a preferred embodiment, the acute or chronic inflammatory disease is seropositive or seronegative chronic polyarthritis, more preferably rheumatoid arthritis. Another aspect of the invention is a method for treating an acute or chronic inflammatory disease, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutical composition thereof, as defined above, to a patient in need thereof. The term "treating" in the context of the present specification means administering a compound having the formula of the present invention to prevent, alleviate or eliminate the disease or one or more symptoms associated with the disease. "Treatment" also includes the prevention, alleviation or elimination of the physiological consequences of the disease. The term "alleviation" in the context of the present invention is considered to mean any improvement in the condition of the patient being treated, including subjectively (feeling or giving a feeling to the patient) and objectively (measuring parameters). Another aspect of the invention refers to the use of a compound of formula (I) as defined above as a developing biomarker in development and pharmacological development techniques; in particular, to identify immune foci, target cells and target molecules. In a particular embodiment, the invention refers to the use of a compound of formula (Γ) as defined above as a developing biomarker in development and pharmacological development techniques; preferably for identifying immune foci, target cells, and The target molecule. -30- 201010988 Pharmacological development technology will rapidly grow the appropriate preclinical (molecular, cell, organ and whole animal tracking and commemorative mechanism verification, efficacy test, etc.) and clinical (human medicine) in vivo development technology combination of biomarkers The scope extends to useful information generated by the compounds described herein and used as pharmaceuticals. The invention is further illustrated below using the embodiments. This explanation should not be construed as limiting the scope of the invention as it is defined in the scope of the claims. [Examples] Examples Synthesis Example 1. Synthesis of (4-methylphenylthio)-methyl acetate COOH NaHCO ;XT' 1.82 g (10 mmol) of 4-methylphenylthioacetic acid was dissolved in 30 ml of DMF, followed by 1.34 g (16 mmol) of NaHC03: the stirred mixture was stirred. 15 minutes. Next, 1.9 ml of ICH3 was added while stirring was continued for 24 hours at room temperature. After this time has elapsed, the mixture is poured onto water/ice. Since no sinking source occurred, 'addition of ether to extract. After the organic phase was washed with water and dried over anhydrous sodium sulfate, the organic phase was concentrated in a rotary evaporator to give I.92 g (9·7 mmoles of '201010988 97% yield) of colorless oil'. It has the structure of expectation. This product was used in the next step of the synthesis without purification because it gave a single spot on TLC (eluent dichloromethane/methanol 9/1). RMN ^(CDCh) : 7.2(s,4H) 3.7(s,3H) 3.5(s,2H) 2.4(s,3H CH3S) RMN 13C(CDC13) : 1 71.5 136.9 1 30.4 1 29.4 126.4 51.6 40.2 15.5

Example 2. Synthesis of (4-methylsulfonyl-phenyl)-acetic acid

30_7 g (50 mmol) of an oxygenated solution (in 80 ml of water) was added dropwise to a compound containing 3.4 g (17.3 mmol) of the compound obtained in Example 1. The solution was maintained in a water/ice bath in milliliters of methanol. After the addition was completed, stirring was continued for 5 hours to allow the temperature to rise to room temperature. Next, a part of the solvent was concentrated under reduced pressure, and the precipitated solid was washed repeatedly with water. After drying, the solid weighed 3 grams. The filtered water was extracted with methylene chloride. The organic phase was washed with water and dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to give a yield of more than 4 g (17 m. ) the product. This solid was melt-decomposed at 57 ° C and its purity was determined by HPLC to be 99%. Spectroscopic data confirmed the expected structure: RMN ^(CDCls) : 7.9(d,2H) 7.5(d,2H) 3.7(s,s 3 + 2H) -32- 201010988 3 .1 (s,3H) RMN 13C( CDC13) : 1 70.80 1 40.1 3 1 39 1 30.3 3 1 27.63 52.3 1 44.49 40.82 Example 3. Synthesis of bromo-(4.methylsulfonyl-phenyl)-methyl acetate

Br

❹ 20 Dissolve 20 g (87.62 mmol) of the ester in 300 ml of carbon tetrachloride. Add 19 g (1 〇 5 mmol) of bromide amber imine, 2 g (12.18) Monomolar azobisisobutyronitrile and 0.1 ml of bromine. The reaction mixture was heated at 80 ° C for 3 hours, then cooled, filtered and washed with dichloromethane. The filtrate was washed with water and then with brine. The solution was dried over anhydrous sodium sulfate and concentrated in vacuo to give <RTI ID=0.0>>> Concentration of the purer fraction yielded 19.63 g (72.94% yield) of the brominated derivative compound.

Melting point: 8 1.8 - 8 3 . 3 °C NMR ^(CDCh) : 7.9 (d, 2H) 7.75 (d, 2H) 5.4 (s, lH) 3.8 (s, 3H) 3.1 (s, 3H) NMR 13C ( CDC13): 168.0(C) 141.6(C) 141.1(C) 129.8(CH) 127.8(CH) 53.7(CH) 44.7(CH3) and 44.2(CH3) ppm -33- 201010988 Example 4. Synthesis of 2-methyl -5-(4-methylsulfonyl-phenyl)_4_yl-thiazole

16.7 g (54.3 mmol) of the vinegar obtained in Example 3 was dissolved in 400 ml of toluene, followed by the addition of 19 ml of susceptine and 4. 〇 8 g (5 4.34 mmol) of thio Guanamine. The reaction mixture was stirred at 8 (rc (water bath temperature) while stirring for 2 hours. Then, the mixture was allowed to stand for cooling, and the precipitated solid was filtered and washed with water (2×50 ml) and then ether (2×30 ml). Drying in vacuo to give 7 g (2 6 mmol, 47.86% yield) of a cream solid, m.p. 2 166-226 ° C. Spectroscopic data confirm the structure of the desired product. RMN ^(d^MSO) <5 : 1 1 · 8 (s, 1 Η) 7.8 (m, 4H) 3 · 1 (s, 3 Η) 2.6 (s, 3H) ppm RMN 13C (d6 DMSO): 162.9 (C) 159.4 (C) 146.2(C) 143.0(C) 128_0(CH) 126.1 (CH) 104.4(C) 44_2(CH3) 1 9.9(CH3) ppm Example 5. Synthesis of 4-cyclopentyloxy-5-(4-methylsulfonate) Acid-phenyl)_2 methyl-thiazole

201010988 The 7. gram (26 millimolar) hydroxythiazole obtained in Example 4 and 8.8 grams (63.67 millimoles) of potassium carbonate were dissolved in 2 mM of dmF. 14 ml (130 mmol) of bromocyclopentane was added dropwise. The reaction mixture was heated at 80 ° C for 3 hours' followed by cooling, the mixture was poured onto ice / water and extracted with ethyl acetate. The organic phase was washed with water (3 χ 1 mL) and dried over anhydrous sodium sulfate and concentrated by heptane/ether (1/1) to yield 12 g of crude material. The product was purified by flash chromatography using heptane / ethyl acetate (2/1) as eluent to afford 6.7 g (19.85 m.

Melting point: 124.6-125.2 °C NMR 1H (CDC13): 7.9 (dd, 4H) 5.4 (m, lH) 3.0 (s, 3H) 2.6 (s, 3H) 1 . 7 - 2.0 (m, 8 H) ppm NMR 13C(CDC13) : 162.6(C) 159.5(C) 138.3(C) 137.1(C) 128.1(CH) 126.8(CH) 109.1(C) 83.7(CH) 45.0(CH3) 33.5(CH2) 24.1(CH2) 20.4 (CH3) ppm Bioassay Materials and Methods 1. Individuals The test was performed by venipuncture to collect heparinized peripheral blood from healthy volunteers and patients with rheumatoid arthritis. Healthy volunteers are blood donors under routine control within the hospital business. Patients with rheumatoid arthritis included in this trial meet the diagnostic criteria of the American College of Rheumatology and present clinically active disease at the time of enrollment, and at least 6 patients treated with oral methotrexate at -35-201010988 20 mg/week month. The extent of disease activity is defined as follows: a) DAS2823.2 and/or b) 6 or more swollen joints and 6 or more painful joints. Exclusion criteria were as follows: a) active infectious disease at the time of inclusion; b) tumor disease prior to enrollment; c) other systemic or non-complete remission at least one year prior to inclusion in the visit Organ-specific autoimmune disease; <0 suffering from severe kidney, heart or liver conditions, regardless of the degree of change caused by rheumatoid arthritis such as major diseases; e) because it is not associated with major diseases The condition causes a serious deterioration of the overall condition; f) has received treatment for corticosteroids, immunosuppressive agents, cytostatic agents or any other drug with known activity in the immune system within one year prior to the inclusion of the visit, but the above doses Except for methotrexate; g) pregnancy at the time of the visit or in the postpartum period. The trial was approved by the Commission of Inquiry of the University of Alcala, Madrid, Spain. 2. Material - MultiGuard Barrier Pipette Tip (Sorenson, Salt Lake City, Utah, USA) - MicroAmp® PCR 0.2 ml reaction tube (Applied Biosystems, Inc., Catalog No. N8010540, USA) - Fast Optical 96-well Barcode Reaction Plate (Applied Biosystems) - Discarded Cell Scrape (Costar, Inc., 3010, USA) 201010988 - Tissue Culture Plate, 6-well Flat-bottomed Low Evaporation Cap (New Jersey, USA) Becton Dickinson Labware 353046 Falcon) - Tissue culture plate, 96-well flat bottom low evaporation cap (Berton Dickens, Franklin Lake, New Jersey, USA 074 1 7- 1 886) Becton Dickinson Labware 3 53 073 Falcon) - Non-sterilized plate, 96-well V-plate (Soria Greiner Grena, Madrid, Spain) - Discarded Pasteur pipette (German trademark) - Volume adjustable micropipettes 20, 200, 1000 and 5000 microliters (Gilson, France) - Eppendor f) Multichannel pipette 4780 (Hamburg, Germany) - Ai Bend sterile straw tip (Hamburg, Germany) - Virgin propylene sterile pipette tip (Daslab, Madrid, Spain) ❹ - Aseptic plastic test tubes 5 and 10 ml ( Madrid Das Labs, Spain - Aseptic centrifuge tubes 15 and 50 ml (Franklin Lake BD Falcon, USA) - Cell count polystyrene tube 5 ml (B Frank Falcon, USA) - Heparin blood collection tube 10 ml (Belgium, Belgium) Company (Terumo Europe) Venojet) • Blood Prize Separation Tube 1 〇 ml (British Plymouth BDVacutainer) -37- 201010988 -Pipetboy plus Pipette Aspirator (Flow Lab.) -Millex -GS 22 micron sterile filter (Millipore, France) - Transparent coverslip (Hirschman, Germany) - Neubauer calculation disk (Saaringia, Germany) -Herasafe HS12 vertical laminar flow venting cabinet (Heraeus, Germany) ▲ -Heracell 150 carbon dioxide cell incubator (Herly, Germany) -Beckman refrigerated centrifuge - Multifuge 3 SR Centrifuge (Herly, Germany) - 7 (TC Freezer (S electa), Trassa, Spain - Olympus CHS-2 microscope (Olympus, Tokyo, Japan) Company) - Flow Cytometry FACScalibur (Bakerton Dickens, Inc., Mountain View, USA), Analysis Software BD Cell quest Pro v. Version 5.5.1 - BD FACS Array Bioanalyzer - Enzyme Immunoassay Reader (Fro Laboratory Titertek Multiscan Plus) - Beckman radioactive beta counter - AutoMACS sorter (Myltenyi Biotec, Bergisch Gladbach, Germany) - Nanodrop spectrophotometer (Wars, MA, USA) Thermo Scientific -38- 201010988 Thermo Scientific) -GeneAmp 9700 PCR System (Applied Biosystems) -7900 HT Fast Instant Q-PCR System (Applied Biosystems) 3. For separation and identification Reagents for Cell Subsets - Simple Chloride Brine (SSF) Apiroserum (Ibys, Madrid, USA) 〇- Lymphoprep Lymphocyte Separation Solution (Nyegaard, Norway) coll-Hypaque Gradient Solution) _Trypan Blue (Flucke, Germany) - PBS FACS FLOW Solution (Beckton Dickens) - HEPES Buffer (Sigma and Panreac) Company's reagent) -Methyl-3H thymidine (3[H]-T), specific activity 60 rit / millimole (ITISA American Radiochemical Company, Madrid, Spain) ^ - CliniMACS CD 14 Reagent (Mitsubishi Bio Technology company) - Monoclonal antibodies for immunofluorescence and flow cytometry: Table 1 Table 1 · Individual antibodies for this test CD* Ac Mo Cell subgroup subtypes Fluorescent pigment 62L SK11 White blood cells L- Selectin IgG2a phycoerythrin (PE) 3 SK7 T lymphocyte TCR/CD3 antigen receptor IgGl FITC 14 M5E2 LBP-LPS monocyte TLR4 co-receptor IgG2a phycoerythrin (PE) * in "The 7th human leukocyte Definition of Differentiation Antigens Conference-39-201010988 4. Reagents for cell culture - 1% added by RPMI-1640 (Cambrex BioSciences B4800, Belgium) L-glutamic acid 200 mM and 25 mM Hepes (US plus Complete medium of Irvine Froe Laboratories - Fetal Bovine Serum (FCS) (Gibco, Grand Island, NY) - Medium: heated to 57 °C using complete medium supplemented with 10% FCS 45 minutes to remove all of the serum complement-phytohemagglutinin M (PHA) (Spain Madrid Sigma product lot number 115K4132 number L-8902) -LPS (California 92921 San Diego, United Kingdom Invigo Gene (InvivoGene) large intestine Bacillus 0111: strain B4) - antibiotic mixture. Ampicillin sodium 10 mg/ml (Beitham A., trade name Britapen, Spain), Jiantianmycin sulfate 1.6 mg/ml (Dr. Esteve SA, Madrid, Spain) A mixture of Tamadit Laboratories and amphotericin B 0.5 μg/ml (Esplugues, Squibb, Fungizona, Spain) was added to the medium. - Anti-CD3 monoclonal antibody (Orthopharmaceutical Corporation, Orthoclone OKT3, USA) - Anti-CD28 monoclonal antibody (Minini, Spain 201010988 (Menarini), trade name Cion 15E8) 5. Reagents for mRNA isolation, reverse transcription, and real-time quantitative PCR (Q-PCR) - Illustra RNAspin mRNA Purification Kit (GE Health Care, UK, Buckinghamshire, Cat. No. 25-0 500-72) - High Yield cDNA Inverse Transcription kit (Applied Biosystems Catalog number ❹ 4368814) -TaqMan® Gene Expression Universal Mixture (Applied Biosystems Cat. No. 4369016) - TaqMan® Gene Performance Analysis for Human TNF-α ID No. HS001 74128_ml [FAM] Biosystems catalog number 4331182) - Human ACTB (cold-actin) endogenous control [VIC] (Applied Biosystems Catalog No. 43263 1 5E) 〇 6. Obtained biological samples a) Venous blood: Monocyte lineage from the elbow The venous blood collected by venipuncture is obtained. 50 ml of blood was collected and stored in a lithium heparin tube (Venojet), followed by dilution with saline 1/1 (vol/vol), all under sterile conditions. b) Collect human cell subtypes. To isolate the PBMC, the remaining blood components were separated by a density gradient on Ficoll. This method is based on differences in the density of blood cells. In a 50 ml centrifuge tube containing diluted and heparinized blood, carefully add 15 ml of Ficoll- -41 - 201010988

Hipaque (density 1.077 g/ml). After 45 minutes of centrifugation (400 x g), three layers (red blood cells, Ficoll and plasma) separated by two interfaces were obtained: The PBMC system contained an interface layer between the diluted plasma and Ficoll. It can be collected by suction using a Pasteur pipette. The cells collected in this manner were resuspended in SSF and centrifuged (300 x g for 10 minutes); the supernatant was discarded (washing step), and the cell pellet was resuspended in RPMI 164 培养基 medium containing 10% PBS. c) Purification of PBMC monocytes: Freshly circulating mononuclear cell lines were isolated from PBMC using paramagnetic microspheres with CD14-specific antibodies using an automated magnetic cell sorter. The subunit of the CD14-based LPS receptor complex, which is located on the surface of conventional circulating human monocytes, whose performance is developmentally restricted to monocytes rather than other PBMC subpopulations, allowing for A fast and efficient single-step positive purification of human monocytes using a commercially available AutoMACS sorter (D-5 1429, Bergisch Gladbach Biotech, Germany) to remove them from the remaining PBMC subtypes Sorted out. This procedure is carried out in accordance with the manufacturer's detailed instructions. Briefly, 1〇8 PBMC was resuspended in complete medium in 5 ml sterile plastic tubes and incubated with magnetic microspheres with CD14 antibody (CliniMACS CD14 reagent, Meitian Biotech Co., Ltd.) for 20 minutes at 4 °C. After that, it was added to the Aut〇MACSTM Cell Sorter (Mitsubishi Biotech) and used a double positive selection sorting program. Monocytes are first placed in the two columns of AutoMACS, and other cell populations are eluted and separated from mononuclear cells. The monocytes were then eluted and released after release of the paramagnetic field applied to the column and collected in 5 ml sterile plastic tubes. -42- 201010988 7. Counting and viability tests The cell concentration and viability of all cell suspensions were determined using a 0.1% diluted trypan blue and Neubauer disk microscopy. The percentage of viable cells is based on the ability of the dye to be discharged. The trial was continued only when the cell viability was above 95%. φ 8. Serum Interferon ELISA Quantification In order to quantify the serum concentration of interleukin, the collected blood was stored in a test tube containing anticoagulant and no anticoagulant (using anterior venous puncture and arterial catheter to collect blood, respectively) . The blood was allowed to aggregate in the laboratory at room temperature, followed by centrifugation to separate the serum (60 o xg for 20 minutes). The supernatant was collected, filtered, and aliquoted and stored at -7 0 °C. The concentration of different interleukins was determined using the commercial kits described in Table 2. Table 2. F g industry E LI S A kit for this test. Interleukin quantitative supernatant (pick/ml) PM (kiloton) Company TNF-a 3.83 pg/ml 17 Bender

The supernatant was thawed back to room temperature and reacted with individual anti-interleukin antibodies that were fixed at the bottom of the plate and cultured at different times. Next, the cleaning is carried out to the substances recommended by the relevant company to allow the colorimetric reaction to occur, which is proportional to the amount of interleukin present in the supernatant. The results were determined by the enzyme immunoassay reader Multiscan plus (Fuertek Multiscan Plus). These results are compared with the standard curve ratio -43-201010988, which is obtained from the analysis of the known concentration of the interleukin 0 standard curve provided by the company and the subsequent interpolation method by the software Delta Soft II version 4.1 ( Biometallics (Inc.) is carried out on an Apple Macintosh computer. The interleukin concentration was converted to a molar number to calculate the molecular ratio and the number of soluble molecules in each case. 9. Immunophenotypic assay Direct immunofluorescence technique using monoclonal antibodies of two colors of FITC or phycoerythrin (PE). The FITC system is excited by 488 nm wavelength laser light to emit 525 nm wavelength phosphors, which can be detected by commercial flow cytometry and easily distinguished from laser light and PE emission wavelengths. The PE system is excited by a 488 nm laser light and emits a phosphor of 570 nm, which can be detected by a flow cytometer and easily distinguished. Direct labeling using the antibodies described in Table 1: PBMC labeling: This method was labeled using a 96-well V-plate. 5x1 04 cells were added to each well, followed by centrifugation at 400 xg for 5 minutes. After washing, the cells were resuspended and 5 μl of the corresponding monoclonal antibody was added. The cells were resuspended again and the plates were incubated for 20 minutes in the dark at 4 °C. Then 150 μl of PBS was added and washed twice. Finally, the cells were resuspended in 150 μl of PBS and collected in a tube of a cytometer, and PBS was added to make a final volume of 0.3 ml. For calculations, a FACSCalibur flow cytometer equipped with an argon laser (adjusted to a wavelength of 488 nm) was used to induce FITC and phycoerythrin fluorescence. • 44- 201010988 This instrument provides information on size (FSC) and cell complexity (SSC) in addition to the fluorescent channel. Test cells unlabeled and other unlabeled monoclonal antibodies of the same type as the monoclonal antibodies (18〇1_ FITC, PE, TC, IgG2a FITC, PE, TC) used in the test were used as negative controls. group. Analysis of flow cytometry data using Flow Jo software V8.4 (Tree Star, Inc., Ashland, OH) to evaluate peripheral blood mononuclear cells isolated by AutoMACS purity. 10. Cell culture and functional assays General culture conditions: All cell cultures are run under sterile conditions in a vertical laminar exhaust cabinet, using sterile disposable materials or sterilized with ethylene oxide or a sterilization device. The culture was stored in a 3 7 ° C incubator with 5% carbon dioxide and 95% relative humidity. 11. 3[H]-thymidine proliferation assay When stimulated by mitogens, lymphocytes undergo blast-like transformation and cell differentiation. The method for quantifying cell proliferation is a measurement of 3[H]-thymidine nucleoside cleavage into DNA resynthesis, and detection of the dried extract from the cell culture (the sulfhydryl group is added) is detected before the end and collection thereof. Beta radiation. In the different experiments in which the proliferation was carried out, the purified cell preparation was cultured in a 96-well flat bottom plate at a concentration of 5×10 4 cells/well (200 μl final volume) in the presence of various concentrations of various mitogens. 4 days - 45 - 201010988 and repeated three times. The response to a particular stimulus depends on the density and the type of cell being studied, as well as the culture time and concentration of the mitogen. One [H]-thymidine was added to each well 20 to 24 hours before the completion of the cell culture; the culture was collected by a glass filter using a specific culture collector. DNA synthesis is expressed in counts per minute (cpm). Each test was repeated three times, and the data with an average 値 variability of more than 10% of the three replicates was rejected because it may indicate technical error or culture contamination. The culture was performed with a fixed amount of cells per well and a fixed volume of 200 microliters. All mitogens and interleukins were first tested in a dose response curve and time response. 1 Tables 3 and 4 show the catalog number and lot number of each reagent used in the development of this experiment. Samples were obtained from a BD FACS Array Bioanalyzer (FACSArray, Bakerton Dickens, San Jose, Calif.) and the results were analyzed on a PC PC with a software FCAP Array (Becktondi Rehabilitation). Briefly, the experimental procedure involves pre-cleaning the plate with a wash buffer. After pouring, the mixture of captured microspheres was resuspended by shaking. Standards and samples are added to the appropriate 201010988 dilution. After incubation for 1 hour with permanent magnet stirring, PE detection reagent was added and cultured again at room temperature for 2 hours. After washing, sampling was performed on a flow cytometer. Table 3. Kit CB A human soluble protein Multiplex Flex Set system reagent catalog number batch number universal buffer kit 500 determination 58265 95098 assay diluent 51-90003992 86192 capture microsphere dilution 51-90003804 90262 detection reagent dilution 51- 90003993 95811 Washing buffer 51-90003798 85964 Instrument setting microsphere A1 51-90003855 87987 Instrument setting microsphere A9 51-90003858 95933 Instrument setting microsphere Π 51-90003851 89231 Instrument setting microsphere F9 51-90003854 94229 PE instrument setting microsphere F1 51-9005038 94498 ΓΕ positive control detection agent 51-9005065 92154 CBA Flex Set kit sensitivity IL-8 (A9) 558277 85109 1.2 picogram / ml IL-10 (B7) 558274 97196 0.13 picogram soar TNF- a(D9) 558273 85106 0.7Pick/3⁄4L IFN-r (E7) 558269 81562 1.8Pick/3⁄4L Table 4. Kits included in the design of Kit CBA Flex Set Reagents Standards Reagents Capture Microsphere Fluorescent Reagents (ΡΕ) IL-8(A9) 84854 87670 77948 IL-10(B7) 70333 96280 95934 TNF-a(D9) 65908 87667 77294 IFN- γ (E7) 80390 87664 77136

13. Purification of mRNA -47- 201010988

The mRNA isolation was performed using the Illustrat RNAspin purification kit according to the manufacturer's detailed instructions (catalog number 24-50-72). Briefly, 5x106 cells were collected by centrifugation, buffered with RA1 and vigorously shaken to dissolve the cells. A volume of 70% ethanol was added to the lysate, and the mixture was vigorously mixed and loaded onto an RNA-binding column. The column was centrifuged at 8000 xg for 30 seconds to allow mRN A to adhere to the column matrix. The salt in the column was removed by adding membrane desalting buffer (MDB) and centrifugation at 1 000 G for 1 minute. The removal of contaminating DNA was carried out by adding 95 μl of the DNase reaction mixture to the substrate and incubating for 15 minutes at room temperature. The column matrix was washed once with buffer RA2 and twice with buffer RA3. Finally, the purified RN A was eluted in 100 μl of H20 and quantified using a Nano drop spectrophotometer using Nanodrop 1.2 software and immediately stored at -80 °C.

14. Reverse transcription of mRNA into cDNA mRNA is reverse transcribed into a cDNA line using a high yield cDNA reverse transcription kit (Applied Biosystems Catalog No. 43 688 1 4) according to the manufacturer's detailed instructions. Briefly, mix 0.5 μg of total mRNA with 2 μl of 10x RT buffer, 0.8 μl of 25 fold dNTP mix (100 millimolar), 2 microliters of 10 times RT in a 0.2 ml PCR tube. A random primer, 1 μl of MultiScribe reverse transcriptase, 4.2 μl of nuclease-free water, and a final volume of 20 μl. The reverse transcription reaction was performed in a GenAmp 97〇0 Thermal Cycler using the following program: 251 10 minute step, 37 ° C 120 minute step and finally -48- 201010988 8 5 t: 5 seconds step. 15. Real-time Q-PCR analysis of human TNF-α mRNA expression-specific quantitative PCR system using TNF TaqMan® gene expression analysis (Applied Biosystem ID No. 118 00 1 74 1 28_1111) and Ding & 91^11© gene expression universal mixture The liquid (Applied Biosystems Catalog No. 4369016) was carried out according to the manufacturer's instructions. The human cold-φ actin endogenous control (catalog number 4326536E) was used to quantify the TNF-[alpha] fold regulation of transcriptional activation of the TNF-[alpha] gene. Briefly, the reaction was performed on a Fast Optical 96-well barcode reaction plate (Applied Biosystems Catalog No. 43469 06) with a final volume of 20 μl/well. The reaction mixture included in each well is 5 μl of cDNA, 10 μl of 2-fold Taqman® Gene Universal Mix, 2 μl of 20-fold TNF-α (and/or stone-actin) TaqMan® Gene performance analysis with 3 μl of nuclease-free water. The optical plate was reacted in a 7900 HT Rapid Instant Q-PCR System (Applied Biosystems Catalog No. 435 1 4〇5) using the following thermal cycling conditions: Step 1: Maintain a temperature of 50 °C for 2 minutes; 2 steps: maintain a temperature of 95 ° C for 10 minutes; 35 cycles: temperature 95 ° C 1 〇 seconds, temperature 60 ° C 1 minute. All samples were repeated as technical controls to avoid operational errors. The relative quantification of the expanded response was performed using RQ Manager 1.2 software analysis included in the Q-PCR instrument (Applied Biosystems). Statistical Analysis The results of the experimental tests are expressed as the difference between the average 値 and the mean 与 and the estimated error -49- 201010988 (according to the type of distribution). For statistical analysis, the characteristics of the distribution are first analyzed using the normal distribution of shaPiro_ Whilk. Mann-Whitney's nonparametric U test was used to establish comparisons. In all cases, a significance of less than 5% is significant (Ρ <〇·〇5). Statistical analysis was performed using software SPSS 1 1 ·0 (SPSS Corporate Headquarters, 11th Floor, 233, Southwark Avenue, Chicago, Illinois 60606). Results of immunomodulatory effects of compounds 4, 5, 6, 9, 10, 12, 15, 16 and 17 Production of TNF-α in peripheral blood mononuclear cells (PBMC) in healthy volunteers and patients with rheumatoid arthritis , immunomodulatory effects of IFN-r and other interleukins. Example 6. Effect of Compounds 5 and 12 on TNF-α Production First, the effects of Compounds 5 and 12 on the production of TNF-α by PBMCs in healthy volunteers in the presence or absence of LPS were investigated. PBMCs from healthy volunteers (5xl04 cells/well, 200 μl) were plated in 96-well flat-bottomed low-evaporation cap tissue culture plates (Berton Dickens Laboratory Supplies, Franklin Lake, New Jersey, USA 074 1 7- 1 886) 353072 Falcon) Parallel repeated secondary culture, using added fetal bovine serum (formerly US, Ji Boco No. 261 40-079) and a single solvent of 1 CT6 molar or adding 10_6 mol, 1〇_7 mol or 1 (Complete medium for the compound shown in Γ8 Moer (RPΜΙ-1640 containing glutamic acid, Belgian Β4800 Vervier Can Blakesson-50-201010988 Material Technology Company No. BE-12-70F), at LPS (10 μg/ml; cultured in E. coli LPS 01ιι: Β4 strain of Invitrogen, 92021, San Diego, CA, USA), cultured for 24 hours in the presence and absence of culture. The culture supernatant was frozen at ·20° (:, Species-specific ELISA for quantification of TNF-α concentration (enzyme-linked immunosorbent assay for quantitative detection of tumor necrosis factor-α: A TNF- a ELISA BM S223/4TEUCE, Likouzhou 94010 Berlin Gemband Medical Systems Table 5. Health in the presence of Compound 5 The results of PBMC production of TNF-α. The results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results are shown in bold, and their corresponding ρ 値 is included in _ brackets (each experiment Conditions compared to the carrier data. TNF-α production of healthy volunteers PBMC (Pick/3⁄4 liters) n-D 纲10·6 莫ΙΟ·7莫耳ΙΟ"8-stage medium 141.4±277.2 847.15 ±206.7 (0.893) 263.21140.72 (0.225) 736.2±265 (0.893) LPS (10 mg/3⁄4 liter) 500.3±226.9 7421228.88 (0.893) 256.91146.49 (0.043) 679.11279.1 (0.500) as shown in Table 5 When Compound 5 was present in culture at a concentration of 10.7 moles, TNF-α production (p < 0.05) of PBMCs stimulated by LPS was significantly reduced. Figure 2 shows Compound 12 at 10.6. The concentration of TNF-α in the supernatant of PBMC cultures stimulated by LPS is reduced in a statistically significant manner when molar or 1 〇 7 molar concentrations are present in the culture (ρ<〇.〇) 5) In addition, 1〇_6 mol of compound 12 also reduces the spontaneous production of TNF-α in PBMC. -51 - 201010988 Table 6. Compounds In the presence of substance 12, PBMC of healthy volunteers produced TNF-α:. Results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results are shown in bold, and their corresponding P値 is included in parentheses (the experimental strips are more complex than the carrier). n=10 TNF-α production of healthy volunteers PBMC [pick/ml) Carrier ΚΓ6 mol 1〇-7 molar medium 1118.00±800.00 551.60±324.17 (0.009) 1121.00±100.48 (0.114) LPS (10 μg /ml) 3084.631450.00 1064.91+52.82 (0.005) 1938.00+487.74 (0.028) The immunomodulatory effects of Compound 12 are further characterized by the cellular production of PBMC, which is stimulated by LPS or The strain antibody anti-CD3 binds to anti-CD2 8 to activate lymphocytes. This test was performed in PBMC cultures from healthy volunteers and patients with rheumatoid arthritis in the presence or absence of a titration dose of Compound 12. PBMC (5xl04 cells/well) from 13 healthy volunteers were added to the highest solvent concentration (1 (Τ6 mol), or 10.6 mol and 1 (Γ7 mol of compound 12 of 200 μl of complete medium) In the presence or absence of LPS (10 μg/ml liter), culture (repeated twice) for 24 hours. The culture supernatant was frozen at -20 °C, and the specified interleukin concentration was simultaneously measured. Specific reagents (CBA Flex Multiplex Set, BDTM Flow Cellular Microspheres, CBA: IL-8, IL-10, TNF-α, and IFN-γ, California 9 2 1 2 1 San Diego Beckton Dickens Biotech Pharmaceuticals Inc. was quantified using a BDFACS Array Bioanalyzer (California 9 2 1 2 San Diego BD Biotech Company Catalog No. 340128). The concentration of Mox and 10.7 Mo in the medium was combined -52 - 201010988 Event 12 reduced the TNF-α production of PBMC in healthy volunteers stimulated by LPS in a statistically significant manner (p<〇.〇5). Conversely, whether it was monoclonal antibody anti-CD3 and anti-CD28 Stimulation or TNF-α secretion of PBMC spontaneously (without exogenous stimuli) Subject to modulation by Compound 12. Table 7. Production of TNF- in healthy volunteers in the presence of Compound 12. (Results are expressed as mean 値± standard error of the secreted interleukin. Statistically significant results are in bold, Their corresponding 卩値 are included in parentheses ( _ each experimental condition compared to the carrier data). _ η = 13 TNF-α production of healthy volunteer PBMC (Pick / 3⁄4 liter) Carrier HT6 Mo 1〇·7 Mo Er 1〇·8 Mourn Medium 163.84130.00 148.31150.00 (0.422) 197.81±40.00 (0.249) 168.49±16.00 (0.65) LPS (10 μg/3⁄4 liter) 923.93+160.00 602.12±100.00 (0.002 746.841125.00 (0.046) 738.81±100.00 (0.055) Anti-CD3 (12.5 ng liters) + anti-CD28 (l/3xl05) 435.81±60.00 334.21163.00 (0.116) 404.11 soil 97.00 (0.753) 355.62±65.00 (0.422 In another experiment, Φ PBMC (5χ104 cells per well) from 7 patients with rheumatoid arthritis were added at the highest solvent concentration (1〇·6 mol) or 10_6 mol, 1 (Γ7 mol) And 1 (Γ8 mol of compound 12 in 200 μl of complete medium, in LPS (10 μg/ml) or anti-CD3 (12.5 Ng/升) (Orthoclone OKT3, Riden Otto Pharmaceuticals, New Jersey, USA) + Anti-CD28 (l/3xl05) (Cion 15E8, Menarini, Spain) culture in the presence or absence of (repeated twice) 24 hour. The culture supernatant was frozen at -20 °C, and the concentration of TNF-α was quantified by a BD FACS array bioanalyzer according to the manufacturer's instructions using a CBA Flex Set specifically measuring the concentration of the interleukin. -53- 201010988 In PBMC of patients with rheumatoid arthritis stimulated by LPS, the production of TNF-α was significantly inhibited by concentration 1 (Γ6 mol of compound 12 (ρ < 0.05) (Fig. 4). Compound 12 contained in the compound does not inhibit the spontaneous TNF-α production of PBMC in rheumatoid arthritis patients, nor does it inhibit the production of TNF-α after stimulation with monoclonal antibodies against CD3 and anti-CD28 in brackets. (The experimental conditions are compared with the carrier data.) n=7 TNF-α production of PBMC in patients with rheumatoid arthritis (pick/ml) Carrier 10-6 mol 1〇-7 mol 10 -8 molar medium 100.64140.00 177.32±95.00 (0.735) 191.33180.00 (0.735) 131.95±85.00 (0.735) LPS (10 micrograms soar) 700.39±180.00 383.75±75.00 (0.05) 626.571150.00 (0.237) 683.401120. 00 (0.499) Anti-CD3 (12_5 Nike/3⁄4 L) + Anti-CD28 (l/3xl05) 648.72±90.00 532.07±80.00 (0.31) 568.47±97.00 (0.612) 695.81179.00 (0.398) Table 8. In the presence of compound 12 TNF-α was produced in patients with rheumatoid arthritis. The results were expressed as the mean 値± standard error of the secreted interleukin. Statistically significant results were obtained. Bold, they correspond to the Ρ Zhi include ©

Further, the effect of Compound 12 on the production of TNF-α in the presence or absence of LPS in the presence of mononuclear cells purified from PBMC from 9 healthy volunteers was examined. PBMCs from various healthy volunteers were isolated and their mononuclear cells were purified by automated magnetic activated cell sorting (MACS). Resuspend 1〇8 PBMC in complete medium in 5 ml sterile plastic test tubes and magnetic microspheres with CD 14 antibody at 4 °C (CliniMACS CD14 reagent, Bios technology of Bergisch-Gradbach, Germany) Company D-5 1 429)) - Cultured, added to Aut〇MACSTW -54- 201010988 Cell Sorter (Mitsubishi Biotech) to purify their CD 1 4 + PBMC subpopulations (known monocytes) ). The isolated monocytes were stained with FITC-labeled CD3 and PE-labeled CD14-specific antibodies and assayed in 5 ml cell count polystyrene tubes using direct immunofluorescence and multicolor flow cytometry. Purity. Quantification of the surface distribution of the designated T cells (CD3) and monocyte (CD14) surface receptors was performed in a FACScalibur analyzer (Beckondi Rex). Analysis of the results © using the Flow Jo flow cytometer software (Treestar Inc., Oregon, USA). The purity of the isolated PBMC CD3-CD14+ monocytes is routinely > 98% of viable cells, monocytes according to their particular forward scattered light (FSC) and side scatter light (SCC) The characteristics were circled to allow for the definition of distinct monocyte clusters and the remaining lymphocytes (Figure 5). Mononuclear cells (1·25χ105 cells/well, 5 ml) were cultured in a 6-well flat-bottomed low-evaporation cover tissue culture plate (353046 Falcon, Becktondi Rex), supplemented with fetal bovine serum and concentration 1 (Γ6 mol The solvent alone or the complete medium of 1 〇 6 mol of the chelate 12 was added and cultured in the presence or absence of 10 g/ml of LPS for the period of time indicated by the kinetic experiments. The supernatant and cells were simultaneously stored in the kinetic experiments as shown in Figures 6 and 7 to determine the expression of the TNF-α gene product using the amount of mRNA in the cells and the amount of protein in the supernatant. The supernatant is stored frozen at -20 ° C, and the concentration of the secreted TNF-α protein is BD FACS array bioanalyzer using BD TNF-α CBA reagent (Becktondi Rehabilitation Bioscience Pharmaceutical Co., Ltd.) (Beckton Dickens) was measured according to the manufacturer's instructions. The adherent mononuclear cell line was collected from the culture plates using a disposable cell-55-201010988 spatula (Costa Corporation, Massachusetts, 021 39, USA). Cellular mRNA immediately Isolation, mRNA quantification and reverse transcription reactions and quantitative instant polymerase chain reaction (Q-PCR) assays for the determination of TNF-α genes in the 7900 HT Rapid Instant Q-PCR System (Applied Biosystems, Foster City, CA) mRNA expression. Figure 5 shows that highly enriched monocytes (98.3 0 ± 0.17%) were used in this 9 healthy volunteers to analyze mRNA and protein expression of TNF-α in this PBMC subpopulation. Kinetics of secretion. As expected, Figure 6 shows that mRNA expression of TNF-α is strongly induced in human mononuclear cells stimulated by LPS (4 8.35 + 17.46 times, ρ < 0 00 1), in the baseline period In the in vitro condition (〇 hours), the kinetics of the very early activation gene showed a peak at 2.5 hours, while the transient transcription produced a rapid decrease, so the mRNA of TNF-α decreased to near the baseline period at 24 hours. Level (Figure 6 square). In the absence of LPS stimulation, TNF-a gene expression was not activated, and TNF-α specific mRNA maintained its in vitro level in this in vitro system (Figure 6 diamond). I have to pay attention to Compound 12 significantly reduced the peak induction of LPS-stimulated TNF-a mRNA from healthy volunteers and the TaqMan® gene specific for human TNF-cr in the 7900 HT rapid real-time Q-PCR system. Performance analysis measurements (33.72 ± 15.88%, ρ < 〇 · 〇 1) (Figure 6 triangle). In addition, the amount of mRNA expressed by the TNF-α gene was also significantly reduced after 5 and 8 hours of LPS stimulation of the purified monocytes. (27.14±9·07%, p<0.01; and 36.45±11.58%, respectively, ρ<〇.〇1). We conclude that Compound 12 significantly reduced LPS-stimulated TNF- ct mRNA induction in PBMC pure 201010988 monocytes from healthy volunteers, and induced changes during all of the trials without affecting TNF - The peak kinetics of up- and down-regulation of alpha gene products. The very low TNF-α gene expression in human monocytes that have not been stimulated with LPS is consistent with the rapid kinetics of mRNA induction. The secretion of TNF-α protein is initiated rapidly, but is accompanied by translation from mRNA to protein. The @delay time required for the complete biological process of handling, secretion and release into the medium, such that the expected peak yield occurs 8 hours after PBMC purified monocytes in the control medium lacking Compound 12 by LPS (Figure 7 square). Mononuclear cell culture containing only a solvent carrier but no LPS and Compound 12 did not induce secretion of TNF-[alpha] (Fig. 7 diamond). It is noted that Compound 12 significantly reduced peak production of the LPS-stimulated TNF-α secreted protein (8 hours), with purified monocyte lines from healthy volunteers and in a FACS array bio-biological analyzer. The CBA reagent kit for human TNF-α was measured (31.52±14.38%, ^ρ<〇·〇1) (Fig. 7 triangle). As shown above in the kinetics of TNF-a mRNA-induced kinetics, the amount of TNF-a secreted protein was lower from the time point of 0 hour stimulation of the production kinetics monitoring after the culture. In fact, statistical analysis showed that Compound 1 2 also significantly inhibited TNF-α secreted protein from purified PBMC monocytes from healthy volunteers stimulated by LP S at 2.5 hours, 5 hours, and 24 hours (45.56 soil 16.25%, respectively) , ρ <0.01; 41.33 ± 13.37 〇 / 〇, ρ <0.01; and 51.17 ± 15.62%). As the accompanying data showed that Compound 12 significantly inhibited the amount of mRNA and protein expressed by TNF-α-based-57-201010988 in PBMC-purified monocytes from nine healthy donors, we will continue to study Yes, whether there is a statistically significant correlation between the amount of inhibition of TNF-a mRNA induction by Compound 12 and the amount of TNF-α secreted protein, and regression analysis was performed at their individual peaks. It is noted that Compound 12 was found to have a significant correlation with the immunomodulatory effects between the mRNA expressed by the TNF-cr gene product of the purified monocytes and the secreted protein (r2 = 0.7952). Taken together, the above results show that the inhibition of TNF-a mRNA induction by LPS-stimulated purified monocytes by Compound 12 is a mechanism which can be used to explain the substantial part of the immunomodulatory effect of Compound 12 on the inhibition of TNF-α secretion. Example 7. Effect of Compounds 4, 5, 6, 9, 10, 12, 15, 16 and 17 on IFN-R Production PBMCs from healthy volunteers (5X104 cells per well) were spiked with only the highest solvent concentration ( 1〇_δ莫耳), or add 10·6 moles, 10_7 moles or 1 (Γ8 moles of test compound in 200 μl of complete medium, in LPS (10 μg/ml) or anti-CD3 (12.5 Nai)克/ml) + anti-CD28 (l/3 xlO5) in the presence and absence of culture (repeated twice) for 24 hours. The culture supernatant was frozen at -20 ° C, specifically for the determination of IFN-r Concentration of CBA Flex Set quantifies the concentration of rFN_7 using a Bd FACS array bioanalyzer according to the manufacturer's instructions. 201010988 Table 9. PBMC production of IFN- in healthy volunteers in the presence of compound 16. Results The median ± standard error of the secreted interleukin is expressed. The statistically significant results are shown in bold, and their corresponding p値 is included in parentheses (each experimental condition is compared with the carrier) 〇n=5 Healthy volunteer PBMC IFN- «Tau production system (Pick / ml) Carrier ίο·6 Mo Er 1〇-7 Mo Er 1〇·8 Mo Medium 94.5124.5 45.46±6.89 (0.043) 50.215.2 (0.043) 44.3±3.56 (0.043) LPS (10 μg/«L) 115+25.1 68.38±24.83 (0.138) 62.84±4 (0.043) 69.77110.34 (0.043 Anti-CD3 (12.5 Ng/3⁄4 liter) + Anti-CD28 (l/3xl05) 256611819.3 1075±1239.04 (0.043) 169111921.1 (0.138) 1097±1665.03 (0.080) Concentration 1〇_7mol or 1(Γ8莫耳Compound 16 significantly inhibited the IFN-R production of PBMC stimulated by LPS (ρ < 0.05). In addition, 1 (Τ6 molar concentration inhibition induced by monoclonal antibody anti-CD3 and anti-CD2 8 IFN- 7 production (ρ < 0.05). 10_6 Mo, 10.7 Mo and 10.8 More Compound 16 significantly inhibited the spontaneous secretion of IFN-r from healthy B BMC in healthy volunteers (Ρ<〇·〇5 10 Table 10. Production of IFN-r by PBMCs from healthy volunteers in the presence of Compound 4. Results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results are shown in bold, they are Corresponding P値 includes _ in parentheses (data for each experimental condition compared to the carrier). __ n=5 Health Physician PBMC IFN- r production (Pick / 3⁄4 liter) Carrier ΗΓ 6 Mo 1 -7 Mo 10 8 8 Mo medium 94.5 ± 24.5 56.38 ± 9.98 (0.080) 74.51 ±10.75 (0.225) 57.54±2.59 (0.080) LPS (10 μg/ml) 115±25.1 67.82+4.62 (0.043) 45.2411.29 (0.043) 46.25+3.45 (0.043) Anti-CD3 (12.5 Ng/ml) + resistant CD28(l/3xl05) 2566±1819.3 1084 soil 672.2 (0.043) 2622 soil 1283.57 (0.686) 3797 soil 775.88 (0.500) -59- 201010988 Concentration 1 (Γό莫耳, 1 (Γ7莫耳 and 1 (Γ8莫耳之Compound 4 significantly inhibited the IFN- r production of PBMC stimulated by LPS (p<0.05). In addition, Compound 1 at a concentration of 1CT6 Moline also significantly inhibited IFN induced by monoclonal antibody against CD3 and anti-CD28. - r production (Ρ <〇·〇5). Compound 4 did not significantly regulate the spontaneous secretion of IFN-^ from healthy volunteers PBMC. Table 11. PBMC of healthy volunteers in the presence of Compound 5 produced IFN-r The results were obtained by the secretory cell @η=5 healthy volunteer PBMC IFN-r (pick/ml) carrier nr6 mol HT7 mol 10_8 molar medium 94.5±24.5 34.8±2.9 (0.043) 30.21±5.64 (0.043) 49.7±60 (0.500) LPS (10 μg/ml) 115±25.1 32.56±2.42 (0.043) 35.33±12.15 (0.043) 81.4137.9 (0.500) Anti-CD3 (12.5 NEK/ 3⁄4 liters) + anti-CD28 (l/3xl05) 2566±1819_3 193411002.79 (0.043) 13201803.97 (0.080) 152811271.3 (0.500) median soil standard error expression. Statistically significant results are shown in bold, they correspond to P値 is included in parentheses (each experimental condition is compared to the carrier). _ _ concentration 1 (Γ6 mol or 1CT7 mol compound 5 significantly inhibits IFN-r production by PBMC stimulated by LPS in healthy volunteers (ρ < 0.05) In addition, 1 (Γ6 molar concentration of Compound 5 also significantly inhibited the production of IFN-r induced by monoclonal antibodies against CD3 and anti-CD28 (ρ < 0.05). The concentration of 10_6 Mo and 1 (Γ7 molar compound 16 also inhibited the spontaneous secretion of IFN-r from healthy volunteers PBMC (Ρ<〇·〇5). -60- 201010988 Table 12. Healthy volunteers in the presence of Compound 6 PBMC produced IFN-r. The results were expressed as the median ± standard error of the secreted interleukin. Statistically significant results were shown in bold, and their corresponding p値 was included in parentheses (compared to each experimental condition) Information on the carrier) n=5 IFN- r production of PBMC in healthy volunteers (Pick / 3⁄4 liter) Carrier HT6 Mo 1 -7 Mole ι 〇 8 molar medium 94.5 ± 24.5 70.9816. 37 (0.080) 55.7±18.4 (0.043) 77.44±15.5 (0.043) LPS (10 μg/« liter) 115±25.1 80.6 Soil 6.89 (0.043) 104.2120.1 (0.080) 82.8±16.2 (0.080) Anti-CD3 (12.5克/3⁄4 liters) + anti-CD28 (l/3xl05) 256611819.3 850±2396.16 (0.500) 147112031.5 (0.893) 328212584.7 (0.225) Concentration 1 (Γ6 mol of compound 6 significantly inhibited IFN of PBMC in healthy volunteers stimulated by LPS -r production system (p < 0.05). It significantly reduced the spontaneous secretion of IFN·7 in healthy volunteers PBMC at 1〇7 mol and 1 (Γ8 mol) (Ρ&lt 〇·〇5). Table 13. IFN-r production by PBMCs from healthy volunteers in the presence of Compound 9. Results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results are in bold It is indicated that their corresponding p値 is included in parentheses (data of each experimental condition compared to the carrier). n=5 IFN- r production of PBMC in healthy volunteers (Pick/3⁄4 liter) Carrier ίο·6 Moer 1〇·7 Molar 10-8 More Medium 94.5±24.5 75.80112.86 (0.080) 66.20±9.80 (0.043) 45.50±9.20 (0.043) LPS (10 μg/ml) 115125.1 62.60±24.88 (0.138) 46.80115 .10 (0.080) 61.00±12.10 (0.080) Anti-CD3 (12.5 NEK/3⁄4 liter) + Anti-CD28 (l/3xl05) 2566± 1819.3 701.00 Soil 1875.04 (0.138) 1176.00 Soil 1954.90 (0.500) 2764.00 Soil 1281.70 (0.686) Concentration 1 (Γ7 mol and 1 (Γ8 mol of compound 9 significantly inhibited -61 - 201010988 spontaneous secretion of IFN-r from PBMC of 5 healthy volunteers (p<0.05) Table 14. In the presence of compound 10 PBMC from healthy volunteers produced IFN-[τ. The results are expressed as the median soil standard error of the secretory interleukin. Statistically significant results are shown in bold and their corresponding p値 is included in parentheses (data for each experimental condition compared to vehicle). n=5 IFN-γ production of PBMC in healthy volunteers (Pick/3⁄4 liter) Carrier 10_6 Mo 1〇-7 Mo 1〇-8 Moule medium 94.5±24.5 84.15129.43 (0.225) 113.32+21.96 (0.500) 126.10133.60 (0.686) LPS (10 μg/ml) 115125.1 66.20115.91 (0.080) 124.88120.47 (0.080) 114.60±27.40 (0.043) Anti-CD3 (12.5 Ng/3⁄4 liter) + Anti-CD28 (l /3xl05) 256611819.3 1345±1379.13 (0.043) 1841±2135.94 (0.893) 214911224.50 (0.225) Concentration 1 〇 _8 mol of compound 1 〇 significantly inhibited IFN-r production of PBMC stimulated by LPS (P<;〇.〇5). In addition, at a concentration of 1 (Γ6 mol, it also significantly inhibited the production of PBMC IFN-r stimulated by monoclonal antibodies against CD3 and anti-CD28 (Ρ <〇.〇5). Compound 10 does not regulate health volunteers. Spontaneous secretion of IFN-r from PBMC. -62- 201010988 Table 15. Production of IFN-7 by PBMCs from healthy volunteers in the presence of compound 17. Results are expressed as the median ± standard error of the secreted interleukin Statistically significant results are shown in bold, and their corresponding p値 is included in parentheses (data for each experimental condition compared to vehicle). η=5 IFN- r production of healthy volunteer PBMC (Pick/毫升) Carrier ΗΓ6 Mo ΙΟ·7 Mo ΗΓ 8 Moule Medium 94.5±24.5 20.60122.80 (0.080) 75.15115.44 (0.043) 67.40113.80 (0.138) LPS (10 μg/ml) 115±25.1 33.10±22.20 (0.080) 84.60±15.29 (0.043) 90.00±43.90 (0.500) Anti-CD3 (12.5 Nike )) + Anti-CD28 (l/3xl05) 2566±1819.3 2389±2480.70 (0.893) 272111241.75 (0.500) 123811237.60 (0.345) Concentration 1〇_7 mol of compound 17 significantly inhibited IFN-r of LPS-stimulated PBMC from 5 healthy volunteers ρ(ρ<0·05). It significantly inhibited the spontaneous secretion of IFN-r from volunteer PBMC at 10-7 mol. Table 16. The production of IFN-γ by PBMC in healthy volunteers in the presence of compound 15. The result was secretion. Interleukin quantification error is indicated. Statistically significant results are shown in bold, and their corresponding Ρ値 includes ® _ in brackets (each experimental condition is compared to the carrier). _ n=5 IFN-γ production of healthy volunteer PBMC (Pick/3⁄4 liter) Carrier 10·6 Molar 7 Moer 10-8 Mo culture medium 94.5124.5 78.60±1.60 (0.225) 119.52±5.88 (0.893) 89.40± 18.80 (0.893) LPS (10 μg / 3⁄4 liter) 115125.1 73.30 ± 7.80 (0.043) 90.49124.35 (0.225) 41.70114.00 (0.043) Anti-CD3 (12.5 NEK / 3⁄4 liter) + Anti-CD28 (l/3xl05) 2566 ±1819.3 249011654.30 (0.345) 3831±1547.58 (0.686) 316511514.10 (0.686) Concentration 1 (Γ8 mol of compound 15 significantly inhibited IFN- r production from LPS-stimulated PBMC from 5 healthy-63-201010988 volunteers (P<〇.〇5). It did not significantly regulate the spontaneous or IFN-r stimulation of PBMC in healthy volunteers. Table Π. In the presence of Compound 12, PBMC of healthy volunteers produced IFN-γ. The results are expressed as the mean standard error of the secretory interleukin. The statistically significant results are shown in bold, and their corresponding P値 includes __ in parentheses (data for each experimental condition compared to the carrier). _ n=13 IFN- 7 production of healthy volunteer PBMC (Pick / 3⁄4 liter) Carrier ΙΟ "6 Mo Er 1〇-7 Molar 10_8 Mo culture medium 283.78±90.00 189.72140.00 (0.279) 296.28190.00 (0.807) 279.16±60.00 (0.807) LPS (10 micro Wei liters) 860.42±105.00 649.61±120.00 (0.011) 919.53 soil 130.00 (0.552) 1049.85i230.00 (0.382) Anti-CD3 (12.5 NEK/3⁄4 liter) + anti-CD CD28(l/3 xlO5) 11802.22 soil 1300.00 7927.17± 1003.00 (0.019) 11552.08士1600.00 (0.917) 10254.66 soil 1500.00 (0.196) Concentration 1 〇_6 mol of compound 1 2 significantly inhibited by LP S stimulation or by single plant Anti-CD3 and anti-CD28-stimulated healthy volunteers PBMC _ 〇 IFN- τ production system (p < 〇. 〇 5) (Figure 8). Compound 12 did not modulate the spontaneous secretion of IFN-r from healthy volunteer PBMC. PBMC from 8 rheumatoid arthritis patients (5x1 04 cells per well) were added at the highest solvent concentration (10_6 mol), and 1 〇·6 mol, 1 〇·7 mol and 1〇_8 Moultaneous compound 12 in 200 μl of complete medium, cultured in the presence and absence of LPS (10 μg/ml) or anti-CD3 (12.5 Ng/ml) + anti-CD28 (l/3xl05) (repeated Second) 24 hours. The culture supernatant was frozen at -200 ° C, and the concentration of IFN-r was measured by a BD FACS array bioanalyzer using a CBA Flex Set specifically for measuring the concentration of -64-201010988 IFN-7 according to the manufacturer's instructions. Compound 12, which is present at a concentration of 1 (Γ6 mol, 1 (Γ7 mol and 1 〇·8 mol), significantly reduced PBMC from rheumatoid arthritis patients when stimulated with monoclonal antibodies against CD3 and anti-CD28 The induced 1?]^-7 production (ρ < 0.05) (Fig. 9). Compound 12 did not regulate spontaneous IFN-r secretion or LPS-induced secretion of PBMC from rheumatoid arthritis patients. In the presence of Compound 12, PBMC of rheumatoid arthritis patients produced IFN-7" The results are expressed as the mean 値± standard error of the secreted interleukin. Statistically significant results are shown in bold, and they correspond. _P値 is included in parentheses (data for each experimental condition compared to vehicle). n=8 rheumatoid 1 IFN- τ production of PBMC in patients with gangfire (Pick/ml) Carrier HT6 Mo Ear HT7 Moule 8 More Medium 145.49i28.00 193.68145.00 (0.779) 203.72150.00 (0.208) 235.10160.00 (0.674) LPS (10 μg / 3⁄4 liter) 461.14185.00 205.26 ± 46.00 (0.484) 146.50133.00 (0.208) 248.73±35.00 (0.889) Anti-CD3 (12.5 Ng/ml) + Anti-D28 (1/3x105) 8406.11⁄2 1000.00 5115.27 Soil 600.00 (0.05 6411.7士700.00 (0.05) 7122.07土950 (0.017) Example 8. Effect of compounds 5, 6, 12 and 16 on the production of interleukin 8 (1 L-8) Further study of compounds 5, 6, 12 and 16 The effect of IL-8 production by PBMC from healthy volunteers was evaluated at the same time as LPS stimulation and the presence and absence of monoclonal antibody anti-CD3 and anti-CD28. PBMC from healthy volunteers (5xl04 cells per well) Add only the highest solvent concentration (1〇_ 6m), or add 1〇_6mol, 1〇_7mol or 1〇_8mol test-65- 201010988 Compound in 200 μl of complete medium , cultured (repeated twice) in the presence and absence of LPS (10 μg / ml) or anti-CD3 (12.5 Ng / ml) + anti-CD28 (l / 3xl05) for 24 hours. The culture supernatant was frozen The concentration of IL-8 was quantified by a BD FACS array bioanalyzer at -20 ° C using a CBA Flex Set dedicated to determining IL-8 concentration according to the manufacturer's instructions. Table 19. PBMC of healthy volunteers in the presence of Compound 16 Production of IL-8. The results are expressed as the median ± standard error of the secretory cell. Statistically significant results are shown in bold and their corresponding P値 is included in parentheses (data for each experimental condition compared to vehicle). η=5 IL-8 production of PBMC in healthy volunteers (pick/ml) Carrier HT6 Molex 10.7 Molar 10-8 More medium 80961±2255 65232129577 (0.138) 53645130467 (0.043) 55043129843 (0.080) LPS( 10 μg / 3⁄4 liter) 119800 soil 23353 66988 ± 29703 (0.138) 65232 ± 32229 (0.138) 60278 ± 33874 (0.225) Anti-CD3 (12.5 Ng / ml) + anti-CD28 (1/3 χ 105) 90653 ± 16263 59496126929 (0.345 56850128160 (0.686) 59496126425 (0.345) Compound 16 significantly reduced IL-8 production from 5 donor PBMCs under all stimulation conditions and test concentrations of the compounds. In addition, at 1 (Γ7 molar concentration, it significantly inhibited the spontaneous secretion of IL-8 from healthy volunteers PBMC (Ρ<〇.〇5). 201010988 Table 20. Production of PBMC in healthy volunteers in the presence of Compound 5 The case of IL-8. The results are expressed as the median ± standard error of the secreted interleukin. The statistically significant results are shown in bold, and their corresponding p値 is included in the brackets (the experimental conditions are compared with the Information of the agent) n=5 IL-8 production of PBMC in healthy volunteers (Pick / 1 ! liter) Carrier 10·6 Mo Er 1〇·7 Mo Er 10·8 Mo Er Medium 8096112255 48453±25626 ( 0.043) 62698±28354 (0.138) 58725128788 (0.138) LPS (10 μg/ml) 119800±23353 51626133558.86 (0.043) 59886+33157 (0.138) 58725132519 (0.138) Anti-CD3 (12.5 Ng/ml) + anti-CD28 (1 /3χ105) 90653+16263 70202±27558 (0.500) 62698127532 (0.686) 61072127502 (0.686) Compound 16 significantly reduced IL-8 production from 5 donor PBMCs under all stimulation conditions and test concentrations of the compound. At 1 (Γ7 molar concentration, it significantly inhibited IL-8 spontaneously in healthy volunteer PBMC Secretion (Ρ < 〇. 〇5). Table 21. Production of IL-8 by PBMCs in healthy volunteers in the presence of Compound 6. Results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results They are indicated in bold and their corresponding p値 is included in Φ _ brackets (data for each experimental condition compared to vehicle). n=5 IL-8 production of healthy volunteer PBMC (Pick/3⁄4 liter) Carrier 10-6 Moer·7 Mo Er 1〇·8 Moule Medium 80961±2255 56528 Soil 20562.02 (0.043) 680623121088 (0.225) 115156±17433 (0.500) LPS (10 μg/ml) 119800123353 120889±18073 (0.345) 120889119096 (0.345) 120889±19725 (0.225) Anti-CD3 (12.5 NEK / «L) + anti-CD28 (l/3xl〇5) 90653116263 67547116971 (0.893) 73902±16943 (0.500) 65317+19671 (0.500) -67- 201010988 Compound 6 significantly reduced IL-8 production by 5 donors of PBMC under conditions of CD3 + CD28 antibody stimulation and spontaneous release at the test concentrations of all compounds. In addition, under the latter conditions, it significantly inhibited the spontaneous secretion of IL-8 in healthy volunteers by a concentration of 1 (Γό莫耳(ρ<0·05) ° Table 22. Production of PBMC in healthy volunteers in the presence of Compound 12 The results of IL-8 were obtained. The results were expressed as the mean 値± standard error of the secreted interleukins. Statistically significant results are shown in bold, and their corresponding P値 is included in parentheses (each experimental condition is compared to the carrier) Information)) η=13 IL-8 production of PBMC in healthy volunteers (Pick/3⁄4 liter) Carrier ίο·6 Mo ΗΓ7 Mo Er 1〇-8 Mo Er Medium 72814.17 Soil 10647.16 45634.31 土6311.13 (0.016) 53732.66±7 952.75 (0.006) 57773.60 soil 6667.57 (0.087) LPS (10 μg / 3⁄4 liter) 173877.36 soil 8744.49 168467.15 soil 9596.96 (0.807) 162075.59 soil 10258.41 (0.249) 169609.11 soil 9060.90 (0.861) anti-CD3 (12.5 Ng / ml ) + anti-CD28 (1/3x105) 107008.56 soil 8351.21 99779.72 soil 20000 (0.311) 97968.61 士10220.21 (0.055) 92989.29 土8463.47 (0.028) at 1 (Γ6莫耳, 1 (Γ7莫耳和1(Γ8莫耳的浓度 concentration) Compound 12 significantly inhibits the spontaneous production of IL-8 (ρ≪ 0.05) (Fig. 10). 1 (Γ7 Mo and 1 (Γ8 molar concentration of compound 12 significantly inhibited IL-8 production induced by monoclonal antibodies against CD3 and anti-CD28. However, no observation was observed. LPS stimulates the effects of IL-8 production of PBMC. Also investigates the effects of PBMC on the production of IL-8 in patients with rheumatoid arthritis. PBMC from 8 patients with rheumatoid arthritis (5 per well) X104 cells) were added at the highest solvent concentration (1 (Γ6 mol), and ΐ(Γ6莫尔-68-201010988, 10_7 mol and 1 (Γ8 mol of compound 12 in 200 μl of complete medium in LPS ( 10 μg/ml) or anti-CD3 (12_5 Ng/ml) + anti-CD28 (1/3 xlO5) in the presence and absence of culture (repeated twice) for 24 hours. The culture supernatant was frozen at -20 °C, using a CBA Flex Set specifically for the determination of IL-8 concentration, the concentration of IL-8 was determined by a BD FACS array bioanalyzer according to the manufacturer's instructions. A concentration of 1 (Γ6 mol and 1〇_8 mol) Compound 12 present in culture significantly regulates LPS-induced IL-8 production in PBMC from rheumatoid arthritis patients, but does not regulate spontaneous and monogenic resistance The combination of anti-CD3 induced CD28 and IL-8 production of manufactured (FIG. 11). All results show that modulation of IL-8 by Compound 12 may show a clear regulatory pattern under significant health and defined disease conditions. Table 23. IL-8 production in patients with rheumatoid arthritis. The results are expressed as the mean 値± standard error of the secreted interleukin. Statistically significant results are shown in bold, and their corresponding ρ値 is included in parentheses (data for each experimental condition _ collision carrier). n=8 IL-8 production of PBMC in patients with rheumatoid arthritis (pick / 3⁄4 liter) Carrier KT6 Mo 1 -7 Mo 1 -8 Mole medium 57877.64 soil 11143.52 98012.08 soil 8395.97 (0.208 96498.50土10784.62 (0.161) 93977.67 soil 9243.38 (0.123) LPS (10 μg / 3⁄4 liter) 150759.65 soil 22966.06 185536.78 soil 22000.00 (0.012) 174156.23 soil 13385.66 (0.208) 184S41J7 soil 21500.00 (0.012) anti-CD3 (12.5 Ng / ml ) + anti-CD28 (1/3χ105) 118017.97 soil 21116.00 124576.09 soil 14605.02 (0.263) 109607.00 soil 18310.15 (0.889) 121587.88 soil 16117.25 (0.575) Example 9. Effects of Compounds 5, 12 and 16 on the Production of Interleukin 10 (IL-10) -69-201010988 Studies Compounds 5, 12 and 16 for healthy volunteers and patients with rheumatoid arthritis (Compound 12) PBMC, the effect on IL-10 production in the presence and absence of LPS stimulation and the combination of monoclonal antibody anti-CD3 and anti-CD28. PBMCs from healthy volunteers (5xl04 cells per well) were spiked with only the highest solvent concentration (10_6 mol), and 1 mol, 1 〇-7 mol or 1 (Γ8 mol of test compound 200 μl) Culture (repeated twice) in the presence or absence of LPS (10 μg/ml) or anti-CD3 (12.5 Ng/ml) + anti-CD28 (l/3xl〇5) in complete medium for 24 hours. The supernatant was frozen and the concentration of human IL-10 was determined using a CBA Flex Set according to the manufacturer's instructions. Table 24. The production of IL-10 by PBMCs of healthy volunteers in the presence of Compound 16. The results are expressed as the median ± standard error of the secreted interleukin. Statistically significant results are shown in bold, and their corresponding p値 includes _ in parentheses (data for each experimental condition compared to the carrier). n=5 IL-10 production of PBMC of Health Zhiyuan (Pick/«L) Carrier I.·6 Mo Er 1〇-7 MoΗΓ8 Mo Er Medium 367±188 328±266 (0.225) 4181238 (0.138) 307±271 (0.686) LPS (10 μg/ml) 425±314 494±337 (0.686) 4861351 (0.225) 4411376 (0.138) Anti-CD3 (12.5 g/ml) + anti-CD28 (l/3xl05) 366±217 3171297 (0.138) 432±255 (0.043) 379±237 (0.043) Addition of compound 16 to culture does not regulate spontaneous and LPS induction from healthy donor PBMC According to this method, at 1 (Γ7 mol and 1 (Γ8 mol concentration, it significantly up-regulates or down-regulates the IL-in the healthy volunteer PBMC after stimulation with CD3 + CD28) 10 points 201010988 secretion (p < 0.05), which contrasted with the observation that ΠΓ 6 mole significantly inhibited IFN- r production in the multiplex analysis under the same conditions (p < 0.05). Table 25. Health in the presence of compound 5 The PBMC of volunteers produced IL-10. The results were expressed as the median ± standard error of the secreted interleukin. The statistically significant results were shown in bold, and their corresponding p値 was included in the _I tongue arc. (The experimental conditions are compared with the carrier data.) n=5 IL-10 production of PBMC in healthy volunteers (Pick/3⁄4 liter) Carrier ΗΓ6 Mo Er KT7 Mo Er 1〇-8 Mo Er Medium 367.4± 187.5 299.60 soil 240.8 (0.893) 333.40 soil 211.71 (0.500) 339.10 soil 420.30 (0.893) LPS (10 μg / ml) 424.9 ± 31 3.9 427.20 soil 310.49 (0.500) 402.35 soil 370.67 (0.686) 441.30 505.40 (0.686) anti-CD3 (12.5 ng / ml) + anti-CD28 (l / 3xl05) 365.50 ± 217 418.00 soil 196.46 (0.893) 391.45 soil 241.22 (0.043 ) 394.30 soil 390.40 (0.345)

Addition of Compound 5 to the culture produced minimal changes in IL-10 production from 5 donor PBMCs, regardless of the amount of interleukin secretion induced by LPS stimulation or CD3 + CD28 antibody in all tested concentrations of the compound. Very slight increases are occasionally reduced. According to this method, IL-10 secreted by PBMC stimulated by CD3+CD28 was increased (p<0.05) at a concentration of 1 (Γ7 mol). -71 - 201010988 Table 26. Health in the presence of Compound 12 The PBMC of volunteers produced IL-10. The results were expressed as the mean 値± standard error of the secreted interleukins. The statistically significant results were shown in bold, and their corresponding p値 included _ in brackets (each experiment) Conditions compared to the carrier data) 〇n=13 IL-10 production of PBMC in healthy volunteers (pick/ml) Carrier 1〇_6 Molar 10-7 Molar 10_8 Mo culture medium 490.46 Soil 87.16 559.06 Soil 112.20 (0.753) 609.10± 108.74 (0.701) 578.66 soil 106.20 (0.861) LPS (10 μg/3⁄4 liter) 2342.20 soil 382.02 2420.68 soil 455.50 (0.507) 2392.99 soil 427.86 (0.507) 23 89.63 soil 366.01 (0.701) anti-CD3 ( 12.5 Nike / 3⁄4 liter) + anti-CD28 (l/3xl05) 1042.64 soil 162.36 1239.47 soil 251.24 (0.507) 1132.69 ± 213.32 (0.382) 1122.51 soil 211.03 (0.507) On the other hand 'make 8 rheumatoid arthritis diseases PBMC (5xl04 cells per well) with the highest solvent concentration (10·6 mol) And 1 (Γ6 mol, 10_7 mol and 1 〇·8 mol of compound 12 in 200 μl of complete medium, in LPS (10 μg/ml) or anti-CD3 (12.5 Ng/ml) + anti-CD2 8 (1 / 3 X1 05) cultured (repeated twice) in the presence and absence of 24 hours. The culture supernatant was frozen at -20 Torr, using a CB A Flex Set dedicated to the determination of IL-10 concentration. The concentration of il-10 was measured by a BDF AC S array bioanalyzer according to the manufacturer's instructions. -72- 201010988 Table 27. Production of IL-10 by PBMC in rheumatoid arthritis patients in the presence of Compound 12. Results The average 値 値 误差 误差 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Arthritis patients with PBMC IL-10 production (pick / 3⁄4 liters) n = 8 carriers 10 · 6 moles 10 · 7 moles HT8 molar medium 452.69 ± 107.47 682.50 soil 113.65 (0.674) 798.92 soil 144.46 (0.401) 468.14± 65.80 (0.263) LPS (10 μg/3⁄4 liter) 2388.64 soil 300.00 2787.12 soil 416.91 (0.069) 2597.85 soil 513.19 (0.327 3000.00 soil 400.00 (0.025) anti-CD3 (12.5 Ng/ml) + anti-CD28 (l/3xl05) 836.39 soil 189.09 976.96 soil 187.99 (0.484) 805.41 soil 141.23 (1.000) 872.06 soil 150.87 (0.674) Compound 12 for health Volunteer p BMC had no effect on IL-10 production in the presence and absence of both test stimuli (Figure 12). However, Compound 12 was present in seven cultures at a concentration of 1 〇·8 mol, and significantly increased the IL-10 production of PBMC in patients with rheumatoid arthritis after LPS puncturing (p<0.05). Compound 12 had no significant effect on the spontaneous or monoclonal antibody anti-CD3 and anti-CD28 stimulation of IL-10 production in patients with rheumatoid arthritis (Fig. 13). Other Effects on Cells of the Immune System Example 10. Effect of Compounds 4, 5, 6, 9, 12, and 16 on PBMC Proliferative Response Studies Compounds 4, 5, 6, 9, 12, and 16 in Plant Hemagglutinin (PHA) 1 μg/ml; PHA 2 μg/ml plus exogenous IL-2 additive (25 IU/ml), and monoclonal antibody anti-CD-3 (12.5 Ng/-73-201010988 ml) and anti-CD The effect of a combination of -28 (1:320.00 0) on the proliferative response of PBMC from healthy volunteers in the presence or absence of stimulation. PBMC (50,000 cells per well) were cultured in complete medium for 4 days in a 37t and 5% carbon dioxide atmosphere (cell incubator HERA Cell 150, Deutsche Sölberg 63 5 05 Thermo Fisher Scientific). The culture conditions were stimulated with or without phytohemagglutinin (PHA, Madrid Sigma number L-8902) 10 μg/ml; PHA 2 μg/ml plus exogenous IL-2 additive (25 IU/mL, human recombination) IL-2 Macrolin, Chiron Iberica, Madrid, Spain, batch number SA753228/4), and monoclonal antibodies against CD-3 (12.5 ng/ml) and anti-CD-28 (1:320.000). Methyl-3H thymidine was added during the last 8 hours of culture (ITISA American Radiochemical Company, Madrid, Spain), methyl 3 thymidine nucleoside 60 mus / millimolar. The cells were collected on a glass fiber filter and embedded in scintillation medium (1 450-441 Meltilex A, de Wallac Oy, Ref #1450-441). DNA intrusion radioactivity was quantified using a beta particle counter (Wallac Oy). As a control group for cell viability, these were observed under microscope (Zeiss Axiovert 40CFL, Göttingen, Germany) based on the density and morphology of cell cultures containing medium but no other signals. The effect of the study molecule on the possible mitogenic division of PBMC was measured and quantified, and the cell viability after culture was evaluated. -74- 201010988 Table 28. Proliferative response of the PBMCs from 6 healthy volunteers to the indicated stimuli in the presence or absence of titrated compound 16 (referred to herein as a drug). Results are expressed as median ± standard error of methyl-3H thymidine uptake (cpm: counts per minute). Statistically significant results are shown in bold (none in this table) and their corresponding P 値 are included in parentheses (data for each experimental condition compared to vehicle). _ F-30125-RR 10_6 Mo 1 (Τ 7 Mo 1 〇 8 Mo 10_9 Mo 1 (Τ 1 () Mo medium carrier 4173 ± 750 31341731 10751559 786 ± 781 42111395 Drug 2355 + 668 (0.249) 2101 ±1006 (0.917) 1932±421 (0.463) 20401287 (0.345) 2456±212 (0.345) PHA-P (10 μg/3⁄4 liter) Carrier 132842 Soil 12078 133400 Soil 16114 149886 Soil 21502 151690 Soil 17207 125060 Soil 15270 Drug 101935 Soil 16298 (0.753) 150870 soil 19554 (0.463) 148142 soil 19742 (0.917) 142231 soil 12403 (0.600) 151052 soil 16849 (0.075) PHA-P (2ug / ml) + IL2 (25 units / 3⁄4 liters) carrier 75594 soil 7429 92185± 11446 85481 soil 9600 87486 soil 9689 72575 soil 7773 drug 71695± 8080 (0.463) 95472士8034 (0.463) 85565 soil 9936 (0.753) 92553 soil 8892 (0.753) 96128 soil 9283 (0.075) TCE (micro-cell ratio 2:1) Carrier 107000 soil 13172 177461 soil 25149 174961 ± 24024 178210 soil 23219 165498 soil 25428 drug 100537 soil 17868 (0.917) 1815001 28947 (0.116) 189561 soil 27195 (0.463) 170159 soil 19945 (0.917) 183824 ± 23708 (0.173 )

Compound 16 present in the culture did not significantly regulate the proliferative response of PBMC from 6 healthy volunteers in the presence or absence of various schizophrenic stimuli (Table 28). High doses of IL-10 are known to inhibit CD3 + CD28 antibody-induced proliferative responses, suggesting that regulation of IL-1 levels is not the primary mechanism by which the experimental conditions of the assay are based. Unlike many general immunosuppressive agents, immunosuppressive compounds that negatively affect T cell proliferation. -75- 201010988 Table 29. Proliferative response of the PBMC from an anonymous health volunteer to the indicated stimulus in the presence or absence of a titrated amount of Compound 4 (herein referred to as a drug). Results are expressed as median ± standard error of methyl-3Η thymidine uptake (cpm: counts per minute). Statistically significant results are shown in bold (none in this table) and their corresponding p ί are included in parentheses (data for each experimental condition _ suspected carrier). F-30217-RS 10·6 Mo Er HT7 Mo Er 1〇-8 Mo Er 1〇_9 Mo Er 10_10 Mo Er Medium Carrier 4173±750 3134±731 1075±559 786±781 421±1395 Drug 3184 Soil 2296 (0.345) 2652士1544 (0.753) 2577土1162 (0.116) 2830 soil 1015 (0.116) 2528±417 (0.345) ΡΗΑ-Ρ (10 μg / 3⁄4 liter) Carrier 132842 soil 12078 133400 soil 16114 149886 soil 21502 151 690 17207 125060 soil 15270 drug 98922 soil 11888 (0.463) 112615 soil 13048 (0.173) 112835 ± 12645 (0.173) 1225041 13667 (0.173) 1189791 9448 (0.917) PHA-P (2ug / ml) + IL2 (25 units / 3⁄4 liters) Carrier 75594 soil 7429 92185 soil 11446 85481 soil 9600 87486 ± 9689 72575 soil 7773 drug 70821 soil 4932 (0.463) 76154 soil 6307 (0.173) 72169 ± 6899 (0.345) 86532 ± 6706 (0.917) 77816 soil 5686 (0.463) TCE ( Micro-cell ratio 2:1) Carrier 107000 soil 13172 177461 soil 25149 174961 soil 24024 178210 soil 23219 1654兕± 25428 Drug 113360 soil 18427 (0.753) 183187 soil 22197 (0.600) 173897 soil 20637 (0.753) 159689 soil 22900 (0.463 ) 182127 soil 19440 (0.600)

Compound 4 present in the culture did not significantly regulate the proliferative response of PBMC from 6 healthy volunteers in the presence or absence of various schizophrenic stimuli (Table 29). This means that this compound does not resemble many conventional immunosuppressive agents as immunosuppressive compounds that negatively affect T cell proliferation. -76-201010988 Table 30. Proliferative response of the PBMCs from 6 healthy volunteers to the indicated stimuli in the presence or absence of titrated Compound 5 (herein referred to as the drug). The results are expressed as the median ± standard error of methyl _3H thymidine uptake (cpm: counts per minute). Statistically significant results are shown in bold 'their corresponding P値 are included in parentheses (each experimental condition is compared to the carrier).__ F-30218-RS 1〇-6莫耳1〇·7mol 10_8 Mol 10.9 Mo Er 1 (Τ1() More Medium Carrier 4173±750 31341731 10751559 7861781 421±1395 Drug 28281391 (0.116) 2474+581 (0.753) 28961615 (0.075) 23281962 (0.249) 2680±731 (0.345) ΡΗΑ-Ρ (10 μg/ml) Carrier 132842 Soil 12078 133400 Soil 16114 149886 Soil 21502 151690 Soil 17207 125060 Soil 15270 Drug 78622 Soil 12398 (0.116) 131549 Soil 11837 (0.753) 151166士15659 (0.753) 147416士11912 (0.345 ) 147352土15286 (0.075) PHA-P(2ug/ml) +IL2 (25 units/3⁄4 liters) Carrier 75594 soil 7429 92185 soil 11446 85481 soil 9600 87486 soil 9689 72575 soil 7773 drug 45222 soil 6660 (0.075) 78290 soil 6748 (0.173) 79679 soil 5527 (0.345) 83402 soil 4557 (0.345) 83135 ± 4607 (0.173) TCE (micro-powder ratio 2:1) Carrier 107000 soil 13172 177461 soil 25149 174961士24024 178210 soil 23219 165498 soil 25428 drug 57761 soil 15198 (0.028) 157875± 19319 (0.600) 184083 soil 21986 (0.753) 183725 soil 21655 (0.249) 171383 soil 19208 (0.917) Only the compound 5 in culture with a maximum test dose of 1 〇 6 moles significantly inhibited PBMC from 6 healthy volunteers. The proliferative response of CD3 + CD28 antibody when stimulated (Ρ <〇·〇5, Table 30). This feature distinguishes this compound from other structurally related compounds, which have the same structural and functional properties. -77- 201010988 Table 31 PBMC from 6 healthy volunteers responded to the proliferative response of the indicated stimulus in the presence or absence of titrated Compound 6 (herein referred to as the drug). Results are expressed as median ± standard error of methyl-3H thymidine uptake (cpm: counts per minute). Statistically significant results are shown in bold (none in this table) and their corresponding p _ 値 are included in parentheses (data for each experimental condition compared to vehicle). F-30230-RS HT6 Molar 10·7 Molar 10_8 MoΙΟ-9 MoerΙΟ-10 Moter Medium Carrier 4173±750 3134+731 1075+559 786±781 421±1395 Drug 2432±411 (0.173 2388±388 (0.463) 2252+217 (0.173) 2144±164 (0.345) 24781223 (0.345) PHA-P (10 μg/3⁄4 liter) Carrier 132842 Soil 12078 133400 Soil 16114 149886 Soil 21502 1516901 17207 125060 Soil 15270 Drug 111292土13373 (0.753) 123381⁄2 10812 (0.753) 123872 soil 12805 (0.345) 123708 soil 12430 (0.345) 124177 soil 13284 (0.753) PHA- P (2ug/ml) + IL2 (25 units / ml) carrier 75594 soil 7429 92185士11446 85481土9600 87486土9689 72575土7773 drug 75230 earth 4053 (0.753) 84630 soil 7105 (0.345) 83203 soil 7576 (0.753) 74878 soil 9183 (0.463) 72810 soil 10656 (0.249) TCE (micro-face ratio 2 :1) Carrier 107000 soil 13172 177461 ± 25149 174961 soil 24024 1782101 23219 165498 soil 25428 drug 102354 soil 16275 (0.600) 164461 ± 17856 (0.917) 177780 soil 20099 (0.345) 1787 (B ± 19511 (0.345) 166529 ± 18415 ( 0.463)

Compound 6 present in the culture did not significantly regulate the proliferative response of PBMC from 6 healthy volunteers to any of the indicated stimuli (p&lt;〇.〇5; Table 31). This means that this compound does not resemble many general immunosuppressive agents as immunosuppressive compounds that negatively affect T cell proliferation. -78-201010988 * Table 32. Proliferative response of PBMCs from healthy volunteers to the indicated stimuli in the presence or absence of titrated Compound 9 (herein referred to as the drug). The results are expressed as the median soil standard error of methyl-3H thymidine uptake (cpm: counts per minute). Statistically significant results are shown in bold (none in this table). Their corresponding P値 is included in parentheses (data for each experimental condition compared to the carrier). __ F-40221-RS 1〇-6莫耳1(Τ7莫耳10_8莫耳1〇_9molecule medium carrier 4173±750 31341731 1075±559 786±781 421±1395 drug 3902±412 (0.846 21451287 (0.249) 24321231 (0.249) 27851248 (0.345) 24771229 (0.345) PHA-P (10 μg / 3⁄4 liter) Carrier 132842 Soil 12078 133400 ± 16114 149886 Soil 21502 1516901 17207 125060 ± 15270 Drug 91169 ± 14336 (0.345) 122456士24310 (0.173) 143737土10540 (0.345) 146143 soil 7862 (0.600) 143807 soil 10375 (0.345) PHA- P (2ug/ml) + IL2 (25 units / 3⁄4 liters) carrier 75594 soil 7429 92185 soil 11446 85481士9600 87486土9689 72575土7773 Drug 69428士9105 (0.917) 75861土16776 (0.173) 83875土8722 (0.917) 88996 soil 5048 (0.917) 93063 soil 7327 (0.116) TCE (microsphere / cell ratio 2:1) Carrier 107000 soil 13172 177461 soil 25149 174961 soil 24024, 178210 ± 23219 165498 soil 25428 drug 100953⁄4 19317 (0.917) 140678 soil 29168 (0.345) 162960 soil 20292 (0.753) 164158 soil 16818 (0.345) 1665291 21592 (0.600) exists in culture Compound 9 does not significantly inhibit health The proliferative response of PBMC to any of the indicated stimuli (p&lt;0.05; Table 32) indicates that this compound is not an immunosuppressive compound that negatively affects T cell proliferation like many common immunosuppressive agents. Compound 12 did not significantly regulate the proliferative response of PBMCs in healthy volunteers in the presence or absence of various schizophrenic stimuli (Figures 14a and 14b). -79- 201010988 Example 1 1. Compound 12 releases CD62L to PBMC The effect of the study compound 12 on the release of CD62L by lymphocytes and monocytes from PBMCs from healthy volunteers was affected. When Compound 12 was present at a concentration of 1 〇·6 mol, the release of CD62L (CD62L-phycoerythrin (PE), cion BD SK11 catalog number 341012, BD Biotech) was significantly increased (p&lt;〇.〇5) . It is noted that the decrease in geometric mean fluorescence intensity (MFI) of the CD62L-PE marker detected after CD62L release occurs selectively in lymphocytes, but no significant difference is observed in monocytes ( Figures 15a and 15b). Table 33. Average fluorescence intensity of CD62L expression on lymphocyte surface. P値 corresponds to the data of each experimental condition compared to the carrier. η=10 lymphocytes (mean 标准+standard error)/statistically significant carrier ΗΓ6 mol 1〇·7 mol MFI (P) 112.68113.57 90.01±8.93 (0.47) 116.90+11.02 (0.333) Table 34. The average fluorescence intensity of CD62L on the surface of nuclear cells. P値 corresponds to the data of each experimental condition compared to the carrier. _ n=10 monocytes (mean 标准 + standard error) / statistically significant carrier HT6 mol 1〇·7 mol MFI (P) 65.34±8.06 63.90±8.52 (0.721) 72.30110.21 (0.878) Example 12. Intravenous toxemia · Compound 12 for oral activity of TNF-α serum -80- 201010988 Concentration CD1 male mice were used with a body weight of 30 to 35 g and a specific pathogen-free environment. These animals were fasted (14 to 16 hours; 12 in each group; 6 in each cage) and 6 nutrient solutions of recommended sugar (8 %) and NaCl (0.2%). The oral test treatment was administered at a volume ratio of 10 ml per kg (carrier Tween 80 0. 1% Ν &amp;&lt;: 1 〇 · 9%). After 1 hour, by intraperitoneal injection of lipopolysaccharide from Escherichia coli serotype 055: Β5 (Sigma L-2880) (dissolved in sterile saline at a dose of 1 mg/kg and 1 〇ml/kg) to induce Rat toxemia. After 90 minutes of LPS injection (ip), the mice were anesthetized with isoflurane and 0.5 to 0.8 ml of blood was collected by cardiac puncture; the blood sample was centrifuged (6000 rpm; 10 minutes; 4 °C), taking two Serum samples were frozen at -70 °C until serum TNF-α concentrations were measured (EIA: mouse TNF/TNFSF1 A Quantikine from the R&amp;D system). The results obtained are summarized in Tables 3 and 16 . ❹ Table 35. TNF-α (Pick/3⁄4 liter) product carrier rolipram (rolipram) 12 doses of sputum) 10 ml / kg (oral) 5 mg / kg (oral) 100 mg / Kg (orally) 200 mg/kg (oral) η 10 9 8 7 Average 値3211.85 696.83 1386.51 956.42 Standard error 517.53 65.70 233.06 109.06 Inhibition (%) --- 78.30 56.83 70.35 Ρ値 (one-way anova) — P&lt; 0.001 P &lt; 0.01 P &lt; 0.01 -81 - 201010988 Compound 12 orally administered at a dose of 100 and 200 mg / kg / 10 ml showed activity in this experimental model of Escherichia coli LPS (ip) induced endotoxemia; It reduced serum TNF-[alpha] levels in a statistically significant manner compared to the vehicle group. Example 13. Anti-arthritic activity of Compound 12 in a mouse collagen-induced arthritis model 8 week old DBA/U male rats were housed in an appropriate environment. On the third day, the animals received a subcutaneous injection of 100 micrograms of bovine collagen type II emulsified in complete Freund's adjuvant (1/1) at the base of the tail. On the 16th day after the initial immunization, the mice were reinforced by the same procedure as above, but this time the collagen (1/1) was emulsified with incomplete Freund's adjuvant. On days 19 and 29, the animals received 40 micrograms of LPS (E. coli 0111: B4) intraperitoneally. Pharmacological treatment began on the 20th day and lasted for nearly a month. The arthritis index was assessed three times a week using a dual measurement system: all foot thickness (digital vernier calipers) and clinical scores for each limb (visual assessment to level 4). At the end of the experiment, serum samples were taken to determine IL-6 concentration (EIA of BD Biotech). The results obtained are summarized in Table 36 and Figure 17. -82- 201010988 Table 36 Treatment blank control dexamethasone compound 12 dose (mg/kg) 2(ip) lOO(ip) 300(po) η 4 12 12 12 12 average thickness (mm) 2.10 2.59 1.94** 2.30* 2.35* % recovery 132.65 51.02 48.98 Clinical score 0 0.3854 0++ 0.2188+ 0.2404 % recovery 100 43.23 37.62 IL-6 (pick / 3⁄4 liters) 9.37 49.13 1.86** 48.01 26.13* *ρ&lt;0·05 ' **ρ&lt ; 〇·〇1 compared with the control group, one-way ANOVA +p&lt;0.05,++ρ&lt;〇.〇1 compared with the control group, the Claska Valis test was administered intraperitoneally at 100 mg/kg and 300 mg/kg. Oral Compound 12 showed a significant anti-arthritic effect in a mouse collagen-induced arthritis model. [Simplified illustration] Figure 1: Selected microspheres for flow cytometry microarray φ (CBA) development of different fluorescence characteristics. Figure 2: Effect of Compound 12 on the production of TNF-α by PBMCs in healthy volunteers (n=10) stimulated by LPS. The rectangular column represents the median and standard error of the secondary culture of each sample under different experimental conditions. The asterisk indicates that the corresponding data for each experimental condition is statistically significantly different from the excipient (Ρ&lt;〇.〇5). Figure 3: Effect of Compound 12 on the production of TNF-α by PBMCs in healthy volunteers (n=13) stimulated by LPS or TCR/CD3 + CD28. The rectangular column represents the average 値 -83- 201010988 and standard error of the sample obtained by secondary culture under different experimental conditions. The asterisk indicates that the corresponding data for each of the different experimental conditions is statistically significantly different from the vehicle (P&lt;0. 〇5) ° Figure 4: Compound 1 2 for patients with rheumatoid arthritis (n = 7) The effect of PBMC on the production of TNF-〇: The rectangular column represents the average enthalpy and standard error obtained by secondary culture of each sample under different experimental conditions. The asterisk indicates that the corresponding data for each of the different experimental conditions is statistically significantly different from the excipient (P&lt;〇.〇5). Figure 5: Quantification of AutoMACS sorting mononuclear cells isolated from healthy volunteer PBMCs using a FACSCalibur analyzer. The left panel shows a scatter plot of the FSC (cell size) xSSC (granularity) distribution showing the percentage of total viable cells (98.5%) included in the circle of selected monocyte populations. The right panel shows the pattern of T lymphocyte and monocyte marker expression, showing that 99.6% of the cells in the monocyte gate defined in the left panel have CDH + CD3 · typical phenotypes of normal human circulating monocytes. The baseline background (quadrant) was set using a homozygous specific control group; even if not all of the cells (99.8%) were in the upper left "monocyte quadrant". The data shown is the median results of purified PBMC purified monocytes from 9 independent volunteers. Figure 6: Effect of Compound 12 on the kinetics of TNF-a mRNA expression in LPS-stimulated monocytes purified from healthy volunteer PBMC. The curve indicates TNF-a mRNA at the indicated time points after incubation with solvent vehicle (♦ rhombohedron) or LPS stimulated culture in the presence of (〇 triangle) or in the absence (square) of 1 〇 6 mol. Kinetics of changes in the amount of expression compared to the internal/9-actin mRNA housekeeping gene control 201010988 'A gene product was calculated at 79 〇〇ΗΤ fast real-time Q-PCR system at each time point. The time points of the kinetics (0 hours, 25 hours, 5 hours, 8 hours, and 24 hours) are shown on the horizontal axis. The tnF-α/call-actin mRNA expression is shown on the vertical axis compared to the induced fold. The data shown are from the median results of the TNF-a mRNA performance kinetics test for 9 independent volunteers. Figure 7: In LPS-stimulated PbmC purified ❹ mononuclear cells from healthy volunteers' Effect of Compound 12 on kinetics of TNF-α protein secretion. The curve indicates that the secretion is expressed at the indicated time points after incubation with solvent excipients (♦ diamonds) or LPS stimulation in the presence of (▲ triangles) or in the absence of (squares) of 1 〇 6 moles of compound 12 Kinetics of changes in TNF-α protein. The time points of the kinetics (〇 hours, 2 · 5 hours, 5 hours, 8 hours, and 24 hours) are shown on the horizontal axis. The amount of TNF-α protein measured by interleukin CBA quantification in the bD FACSarray Bioanalyzer was calculated from each time point to be displayed on the vertical axis. The displayed Q material was based on the median results of the TNF-α protein kinetics test for 9 independent volunteers. Figure 8: Effect of Compound 12 on the production of IFN-r by PBMC from healthy volunteers (η = ι3). Rectangular bars represent the average enthalpy and standard error of each sample subjected to secondary culture under different experimental conditions. The asterisk indicates that the corresponding data for the experimental conditions shown are statistically significantly different (p&lt;0.05) compared to the vehicle. Figure 9: Effect of Compound 12 on IFN-r production by PBMC from rheumatoid arthritis patients (n = 8). The rectangular column represents the average enthalpy and standard error of each sample under different experimental conditions of -85-201010988. The asterisks represent statistically significant differences in the corresponding data for each of the different experimental conditions compared to the vehicle (p&lt;0.05). Figure 10: Effect of Compound 12 on IL-8 production by PBMC from healthy volunteers (n = 13). The rectangular column represents the average enthalpy and standard error obtained by secondary culture of each sample under different experimental conditions. The asterisk represents the statistically significant difference in the corresponding data for each experimental condition compared to the vehicle (p&lt;0.05). Figure 11: Effect of Compound 12 on the production of IL-8 by PBMC from patients with rheumatoid arthritis (n = 8). Rectangular bars represent the average enthalpy and standard error of each sample subjected to secondary culture under different experimental conditions. Asterisks represent statistically significant differences in the corresponding data for each experimental condition compared to vehicle (p&lt;0.05). Figure 12: Effect of Compound 12 on the production of IL-10 by PBMC from healthy volunteers (n=13). The rectangular column represents the average enthalpy and standard error obtained by secondary culture of each sample under different experimental conditions. No statistical differences were found. Figure 13: Effect of Compound 12 on IL-10 production by PBMC from patients with rheumatoid arthritis (n=8). Rectangular bars represent the average enthalpy and standard error of each sample subjected to secondary culture under different experimental conditions. The asterisk indicates that the corresponding data is statistically significantly different from the vehicle (P<0.05). Figure 14: PHA, PHA plus IL-2 or monoclonal antibody anti-CD-3 (T3) and anti-CD-28 The effect of Compound 12(a) 201010988 and excipient (b) on the proliferative response of PBMC from healthy volunteers (n = 8) in the presence or absence of combined stimulation. Figure 15: Effect of Compound 12 on lymphocyte U) and monocyte (b) release of CD62L from PBMC from healthy volunteers. Figure 16: Effect of Compound 12 on the amount of serum TNF-[alpha]. Figure 17: Anti-arthritic activity of Compound 12 was evaluated by mean paw thickness (a) and clinical score (b) in a mouse collagen-induced arthritis model.

-87-

Claims (1)

201010988 VII. Patent application scope: 1. A compound of formula (I) Or a pharmaceutically acceptable salt, prodrug and/or solvate thereof for use in the manufacture of a medicament for the treatment of an acute or chronic inflammatory disease, wherein: @ Ri is selected from substituted or unsubstituted CrCe alkyl , substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic; R 2 is selected from substituted or unsubstituted alkyl, substituted or Unsubstituted cycloalkyl or N(R'R"), wherein R' and R" are each hydrogen or substituted or unsubstituted C^-Ce alkyl; and R3 is unsubstituted Ci- 6 alkyl. 2. The use of the first aspect of the patent application, wherein the medicament for treating acute or chronic inflammatory diseases is caused by inhibiting at least one inflammatory interleukin selected from the group consisting of TNF-〇: or IFN-r Producing, or by immunomodulating, IL-8 chemokines and/or IL-10 regulatory interleukins. 3. The use of the second aspect of the patent application, wherein the production of the inflammatory interleukin TNF-α is achieved at least in part in the expression amount of TNF-a mRNA. 4. The use of any one of claims 1 to 3, wherein the acute or chronic inflammatory disease is selected from the group consisting of acute and chronic seropositive or serum-88-201010988 negative oligoarthritis and polyarthritis, spine Arthropathy, glomerulonephritis, collagen disease, tubulointerstitial nephritis, metabolic syndrome, atherosclerosis, osteoarthritis, asthma, chronic obstructive pulmonary disease, interstitial lung disease, multiple sclerosis, Demyelinating disease, meningitis, encephalitis, meningoencephalitis, inflammatory radiculopathy and peripheral neuropathy, inflammatory bowel disease, cirrhosis, hepatitis, heart failure, ischemic disease, renal failure, inflammatory cystitis, Benign prostatic hyperplasia, prostatitis, myocarditis, pericarditis, uveitis@, atopic dermatitis, eczema, urticaria, psoriasis, rose acne, allergic rhinitis, sepsis, septic shock, multiple organ failure Systemic autoimmune disease (such as systemic lupus erythematosus), vasculitis, dermatomyositis, amyloidosis or sarcoma-like disease, organ-specific autoimmune disease (such as myasthenia gravis), thyroiditis or insulitis, organ transplantation, infection and tumor-induced inflammation, TNF-α-dependent cell degeneration, necrosis, apoptosis, graft-versus-host disease, cachexia or autocrine and adjacent Secretion of pathological cell growth. 〇 5. The use of the fourth aspect of the patent application, wherein the chronic inflammatory disease is seropositive or seronegative chronic polyarthritis. 6. The use of the fifth aspect of the patent application, wherein the chronic polyarthritis is rheumatoid arthritis. 7. The use of any one of claims 1 to 3 wherein Ri is substituted or unsubstituted alkyl. 8. For the purposes of application No. 7 of the patent scope, 1 is a methyl group. 9. The use of any one of claims 1 to 3 wherein R2 is a substituted or unsubstituted cycloalkyl group. -89- 201010988 10. The use of the ninth aspect of the patent application, wherein R2 is a cyclopentyl hydrazone, wherein the R3 is a methyl group, as claimed in any one of claims 1 to 3. 12. The use of any one of claims 1 to 3, wherein the compound of formula (I) is selected from the group consisting of: -90- 201010988 201010988 Or a pharmaceutically acceptable salt, prodrug and/or solvate thereof. 13. A pharmaceutical composition for the treatment of an acute or chronic inflammatory disease, the pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as defined in claim 1 of the scope of the patent application. 14. A compound of the formula (Γ) Or a pharmaceutically acceptable salt, prodrug and/or solvate thereof, wherein: R! is selected from substituted or unsubstituted CKC6 alkyl, substituted or unsubstituted cycloalkyl, substituted or An unsubstituted aryl group or a substituted or unsubstituted heterocyclic group; and R3 is an unsubstituted Ci-CU alkyl group. 1 5 · A compound of claim 14 wherein R! is a substituted or unsubstituted c, -C 6 alkyl group of -92 - 201010988. 16. A compound according to claim 15 wherein Ri is a methyl group. 1 7 · If the compound of claim 14 is applied, the compound is selected from the following: Or a pharmaceutically acceptable salt, prodrug and/or solvate thereof. A pharmaceutical composition comprising a compound of the formula (I) as defined in any one of claims 14 to 17 or a pharmaceutically acceptable salt, prodrug thereof and/or A solvate, and at least one carrier, adjuvant, and/or excipient that is acceptable on the pharmaceutical-93-201010988. 19. The compound of the formula (1,) according to any one of claims 14 to 17, which is used as a medicament. 20. Use of a compound of formula (I) as defined in claim 1 of the patent application as a developing biomarker in development and pharmacological development techniques for the identification of immune foci, target cells and target molecules. 21. Use of a compound of formula (I,) according to any one of claims 14 to 17 as a developing biomarker in development and pharmacological development techniques. -94-