Lipoxin compounds and their use in treating cell proliferative disorders

<u style="single">Background of the invention</u>Lipoxin is a group of biologically active mediators derived from arachidonic acid by the action of the lipoxygenase (LO) enzyme system (Serhan, CN and Samuelsson, B. (1984) Proc. Natl. Acad. Sci. USA). 81: 5335). Formation in the human cell type is initiated by 5-LO or 15-LO (Serhan, CN (1991) J. Bioenerg. Biomembr. 23: 105). The single cell type produces lipoxin at the nanogram level during cell-cell eicosanoid biosynthesis between human neutrophils-platelets and eosinophils (Serhan, CN and Sheppard, K.-A. (1990)). J.Clin.Invest.85: 772). LX is a conjugated tetraene-containing eicosanoid that regulates cellular events in various organ systems. Lipoxin A₄(LXA₄) And lipoxin B₄(LXB₄) Are the two major lipoxins. Each increases protein kinase C (PKC) activity in the nucleus of red leukemia cells at 10 nM (Beckman, BS et al. (1992) Proc. Natl. Acad. Sci. USA 201: 169). Each induces rapid vasodilation at the level of nM (Busija, DW et al. (1989) Am.J.Physiol.256: H468; Katoh, T. et al. (1992) Am.J.Physiol.263 ( Renal Fluid Electrolyte Physiol. 32): F436). The vasodilatory effect of lipoxin is well known in the literature. For example, a micromolar amount of LXA₄Administration by inhalation blocks bronchoconstriction in asthmatic patients (Chrisstie, PE et al. (1992) Am. Rev. Respir. Dis. 145: 1281). Ten^-10In the range of M, LXA₄Also stimulates cell proliferation and induces myeloid bone marrow colonization in combination with suboptimal concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF) (Stenke, L. et al. (1991) Biochem. .Biophys.Res.Commun.180: 255). LXA₄Also stimulates human mononuclear cell colonization (Popov, GK et al. (1989) Bull.Exp.Biol.Med.107:93). LXA₄Blocks the chemotaxis of polymorphonuclear leukocytes (Lee, TH et al. (1991) Biochem.Biophys.Res.Commun.180: 1416). A combination of equimolar amounts of lipoxin has been found to regulate polymorphonuclear neutrophil-mesangial cell interactions in glomerular inflammation (Brady, HR et al. (1990) Am.J. Physiol. 809). .. Activation of polymorphonuclear neutrophils (PMNs) involves the release of structurally and functionally abnormal mediators associated with the early stages of glomerular inflammation (Wilson, CB and Dixon, FJ (1986) (The Kidney,). BM Brenner and FC Rector, Pennsylvania, Philadelphia: Saunders, p.800-891)). Lipoxin acts as an antagonist to leukotrienes (LT) (these are mediators of inflammation). LXA₄LTC in asthma patients₄-Regulates induced airway obstruction (Christie, PE et al. (1992) Am.Rev.Respir.Dis.145:1281). LXA₄Is LTD₄-And LTB₄-In vivo inhibition of mediated inflammation in model animals (Badr, KF et al. (1989) Proc. Natl. Acad. Sci. 86: 3438; Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137: 571 ). LXA₄Previous exposure to (nM) is LTD₄Blocks vasoconstriction in the kidney (Katoh, T. et al. (1992) Am.J. Physiol.263 (Renal Fluid Electrolyte Physiol.32) F436)). Leukotriene-induced inflammation occurs in circulatory diseases, including, for example, arthritis, asthma, various types of shock, hypertension, kidney disease, allergic reactions, and myocardial infarction. Lipoxin is a potent small molecule that can be administered in vivo to treat many diseases, but these molecules are short-lived in vivo. A compound that has the same bioactivity as natural lipoxin but has a longer in vivo half-life would be a valuable drug.<u style="single">Abstract of the invention</u>The present invention is characterized by a substantially purified 15-epi-lipoxin compound. In one embodiment, the 15-epi-lipoxin compound is 15R-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid, in another embodiment. This acid has a 5S, 6R arrangement (15-Epi-LXA)₄). In another embodiment, the 15-epi-lipoxin compound is 15R-5,14,15-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid, which is , 5S, 14R arrangement (15-Epi-LXB₄). In yet another embodiment, the 15-epi-lipoxin compound is 15-hydroxyeicosatetraenoic acid (15-HETE), which has a 15R configuration. The invention also features lipoxin analogs that have active regions that are the same as or similar to natural lipoxins, but that have metabolic conversion regions that are more resistant to in vivo catabolism. Therefore, the lipoxin analogs disclosed in this application have the biological activity of natural lipoxins, but have a longer metabolic half-life. Some of the lipoxin analogs disclosed herein are further enhanced in vivo efficacy (higher binding affinity for lipoxin receptors or increased bioactivity compared to natural lipoxins). have. Like natural lipoxins, the small molecules disclosed in this application are highly potent and biocompatible (ie, non-toxic). However, unlike natural lipoxins, lipoxin analogs block, resist or slowly undergo metabolism and therefore have longer pharmacological activity. Moreover, the compounds disclosed herein are more lipophilic than natural lipoxins and are therefore more easily incorporated by biological membranes. Furthermore, the invention features a method of improving the unwanted proliferation of certain cells based on contacting them with an effective amount of a substantially purified 15-epi-lipoxin compound. In a preferred embodiment, these cells have cancer-like or neoplastic growth. In a more preferred embodiment, these cells are selected from the group consisting of epithelial cells, leukocytes, endothelial cells and / or fibroblasts. In one preferred embodiment of the invention, cells are contacted in vivo. In another embodiment, cells are contacted with exo-vivo. The invention also features a method of ameliorating cell proliferation disease in a patient by administering an effective amount of a substantially purified 15-epi-lipoxin compound. In other respects, the invention is a substantially purified 15-epi-lipoxy of the invention. It is characterized by a pharmaceutical composition having a compound and a pharmaceutically acceptable carrier. In a preferred embodiment, the 15-epi-lipoxin compound is an effective amount to prevent unwanted cell proliferation in a patient. In another embodiment, the pharmaceutical composition contains an effective amount of acetylsalicylic acid (ASA). The present invention is also related to diagnostic and research applications for these lipoxin compounds. Further features and advantages of the present invention will become apparent from the detailed description and claims below.<u style="single">[Simple explanation of drawings]</u>FIG. 1A is a graph showing prostaglandin endoperoxide synthase (PGHS) and lipoxygenase (LO) expression in human tumor cell line (A549 cells) alveolar type II epithelial cells. Cells, T-75 cm²In the flask, interleukin 1_β(IL-1_β) (1 ng / ml) was grown at 37 ° C for 24 hours in the presence or absence. Extracted total RNA (1 μg) is taken and is specific for reverse transcriptase (RT) and PGHS-1 and -2, 15-, 12- and 5-OL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was subjected to PCR using oligonucleotides. Radioactive bands were directly quantified by phosphor imager analysis, standardized to GAPDH expression, and IL-1._βExpressed as doubling of mRNA levels after exposure to. The inset in Figure 1A shows the expression of 15-LO mRNA in human lung tissue and peripheral blood monocytes (PBM), with ND meaning that 15-LO mRNA expression was not detected. Figure 1B shows [2] at 37 ° C for 20 minutes.³H] -Transparent IL-1 exposed to arachidonic acid (20 μM)_βTreated A549 cells (1.5 x 10)⁶Derived from cells / ml) [³It is a graph which shows the RP-HPLC profile of H] labeled monohydroxyeicosatetraenoic acid (HETE). Extract the product and methanol: H₂Chromatography was performed using a linear gradient of O: acetic acid (65: 35: 0.01; v / v / v) and methanol: acetic acid (99.9: 0.1, v / v) (flow rate of 1.0 ml / min). Arrows indicate standard simultaneous chromatography of synthesis. FIG. 2A is a graph showing the generation of 15-HETE. A549 cells (6 × 10)⁶Cells / flask) IL-1_βTreated with (1 ng / ml) for 24 hours, frozen and thawed (2 cycles), vehicle (0.1% v / v ethanol (EtOH)), acetylsalicylic acid (ASA), cytochrome P450 inhibitor (17-octadecinic acid (17-ODYA)) ), 5 μM) or 5-LO inhibitor (Rev-5901 isomer 5 μM) and incubated with arachidonic acid (20 μM) for 20 minutes at 37 ° C. In some experiments, cells were heat denatured prior to incubation (100 ° C, 60 minutes). Incubation was stopped by the addition of methanol (2 volumes) and the product was extracted for reverse phase (RP) -high performance liquid chromatography (HPLC). The data are mean ± SEM of 4-6 separate flasks.^*, P &lt;0.05 and^**, P &lt;0.01 (processed product vs. control). FIG. 2B is a graph showing the time course of 15-HETE formation from extrinsic origin. A549 cells (1.5 x 10)⁶Cells / ml), IL-1_βGrowing for 48 hours in the presence or absence of (1 ng / ml), and A₂₃₁₈₇Incubated in 4 ml HBSS with or without (5 μM) (37 ° C, 30 minutes). 15-HETE levels were measured by RIA. The results represent the mean ± SEM of three different experiments measured in duplicate.^*, P &lt;0.05 (processed product vs. control). Figure 3 is a graph showing the relative chirality of the 15-HETE triggered by the ASA. A549 cells (10⁷Cells / flask) IL-1_βExposure to (1 ng / ml) for 24 hours, treated with vehicle (0.1% v / v ethanol) () or ASA () for 20 minutes, then arachidonic acid (20 μM) and A₂₃₁₈₇Incubated in HBSS containing (5 μM) (37 ° C, 30 minutes). The product was chromatographed by RP-HPLC (as in Figure 1B), and the region containing 15-HETE was collected, extracted with chloroform and treated with diazomethane. Chiral analysis was performed using Bakerbond DNBPG (detailed in the method). The results represent two separate experiments showing similar results. The inset in Figure 3 shows the ratio of A549-derived 15R to 15S-HETE in the presence or absence of the ASA (filled bar). FIG. 4A is a graph showing the RP-HPLC chromatogram of the product from epithelial cell-polymorphonuclear neutrophil (PMN) co-stimulation. IL-1 dense A549 cells_βExposure to (1 ng / ml) for 24 hours, treatment with ASA (20 minutes) and arachidonic acid (20 μM, 60 seconds), and each incubated with freshly isolated PMN (1: 8 A549 cells: PMN cell ratio), then Ionophore A in 4 ml Hanks Balanced Salt Solution (HBSS)₂₃₁₈₇Stimulation at 37 ° C for 30 minutes at (5 μM). The product was extracted as described in the method section of Example 5 and run on RP-HPLC. The chromatogram was plotted at 300 nm, which shows an experiment with n = 6. FIG. 4B is a graph showing the online ultraviolet (UV) spectra of products from epithelial cell-PMN co-stimulation described in FIG. 4A. Substances eluted below peak B predominate in 15-epi-LXB₄Was identified as. FIG. 4C is a graph showing the online UV spectra of products from epithelial cell-PMN co-stimulation described in FIG. 4A. The material eluting below the illustrated peak predominates in 15-epi-LXA.₄Was identified as. FIG. 5A is a graph showing the ASA that regulates the formation of tetraene-containing lipoxin (lipoxin plus 15-epi-lipoxin) during epithelial cell-PMN co-stimulation. A549 cells were subjected to IL-1 prior to the addition of arachidonic acid (20 μM, 1 minute) and freshly isolated PMN (1: 5 A549: PMN cell ratio)._βExposed to (1 ng / ml, 24 hours) and treated with vehicle (0.1% v / v) or ASA. Simultaneous stimulation was performed as shown in FIG. 4A. The results represent mean ± SEM from 3-5 different donors. The inset in Figure 5A shows the effect of cell ratio on the production of tetraene-containing lipoxin (lipoxin plus 15-epi-lipoxin) in co-incubation of A549 cells with PMN in the absence () or presence () of ASA. Is shown. Figure 5B shows peptide leukotrienes (LTC) during epithelial cell-PMN co-stimulation according to the conditions outlined in Figure 5A.₄Plus LTD₄) Is a graph showing the ASA that regulates the formation. The inset in Figure 5B shows the peptide leukotriene (LTC) in a co-incubation with PMN of A549 cells in the absence () or presence () of the ASA.₄Plus LTD₄The effect of cell ratio on the production of) is shown. Figure 6A shows lipoxin A₄(LXA₄), Lipoxin B₄(LXB₄), Dexamethasone (DEX) and vehicle alone are graphs showing the effect of treatment over time on A549 cell count. A549 cells in a 96-well plate at vehicle (0.15% EtOH) or equimolar concentration (10)^-6M) LXA₄, LXB₄Alternatively, it was treated with DEX at 37 ° C for a maximum of 96 hours. At the indicated intervals, cells were harvested for the 3, (4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazolium bromide) MTT assay. The data are mean ± SEM of 3-7 experiments performed in quadruple. About compound vs. vehicle^*, P &lt;0.05 and^**, P &lt;0.005 is shown. Figure 6B shows LXA relative to the percentage inhibition of A549 cell proliferation at varying A549 cell concentrations.₄, LXB₄And it is a graph which shows the effect of DEX processing. A549 cells at the indicated concentrations, LXA₄, LXB₄Alternatively, it was exposed to DEX at 37 ° C for 72 hours. The result is the mean ± SEM of 5-8 experiments performed in quadruple. Results are expressed as a percentage of inhibition of growth against the vehicle. About compound vs. vehicle^*, P &lt;0.05^**, P &lt;0.025 and^***, P &lt;0.005 is shown. Figure 7A shows A549 cells LXA₄, LXB₄And LXA for DNA synthesis of the cells shown in the uptake of 3H-thymidine grown for 72 hours in the presence of DEX (varies in concentration in the range of 5 nM to 500 nM).₄, LXB₄And it is a graph which shows the effect of DEX. Twenty-four hours before the assay, methyl [³H] Thymidine (2 μCi / ml) was added to each well. Then, the cells, DPBS²⁺It was washed 4 times at (4 ° C), dissolved in 0.25N sodium hydroxide (NaOH), and the uptake of radioactivity was monitored. The values represent the mean ± SEM of three different experiments performed in quadruple. The result is for vehicle alone [³H] Expressed as a percentage of thymidine uptake. About compound vs. vehicle^*, P &lt;0.05 and^**, P &lt;0.005 is shown. Figure 7B shows A549 cells LXA₄, LXB₄LXA for inhibition of A549 cells when seeded in 12-well culture plates in the presence of DEX (1 μM) and cell counts were obtained at 72 hours by counting tripan-excluded cells.₄, LXB₄And it is a graph which shows the effect of DEX. The values represent the mean ± SEM of three different experiments. Results are expressed as a percentage of growth inhibition relative to buffer. About compound vs. vehicle^*, P &lt;0.05 is shown. FIG. 8 is a diagram showing the proposed biochemical pathway for 15-epi-lipoxin production. ASA-acetylated PGHS-2 and / or P450 activity contributes to 15R-HETE. The epithelial 15R-HETE undergoes intercellular conversion by leukocyte 5-LO to become a 15-epi-5 (6) -epoxytetraene intermediate, which is a 15-epi-LXA.₄And 15-Epi-LXB₄It is common to both.<u style="single">Detailed description of the invention</u>As used herein, the following terms are defined as: "lipoxin compound" is a natural lipoxin compound (lipoxin A).₄Or lipoxin B₄) And / or / lipoxin analog. The "lipoxin analog" means a compound having an "active region" that acts in the same manner as the active region of "natural lipoxin", but has a "metabolic conversion region" different from that of natural lipoxin. Lipoxin analogs include compounds that are structurally similar to natural lipoxin, compounds that share the same receptor recognition site, compounds that share the same or similar lipoxin metabolic conversion region as lipoxin, and are technically as analogs of lipoxin. Contains compounds found in. Lipoxin analogs include lipoxin analog metabolites. The compounds disclosed herein can contain at least one asymmetric center. At least one stereoisomer is possible in the presence of an asymmetric carbon atom, and all possible isomer types are intended to be included within the indicated structural representation. The optically active (R) and (S) isomers can be separated using conventional techniques known to those of skill in the art. The present invention is intended to include possible diastereoisomers as well as racemates and optically split isomers. Suitable lipoxin compounds for use in the subject invention are "15-epi-lipoxin compounds". As used herein, the "15-epi-lipoxin compound" is a lipoxin compound having an absolute configuration of R at 15 carbons. The term "15-epi-lipoxin compound" is intended to include precursors. The term "precursor" is intended to refer to a chemical intermediate that can be converted in vivo, exo vivo and / or in vitro to form the 15-epi-lipoxin compound of the invention. ing. The term "precursor" also contemplates a prodrug that is converted in vivo to the 15-epi-lipoxin compound of the invention (eg, RBSilverman, 1992, "The Organic Chemistry of Drug Design and Drug Action". , Academic Press, See Chapter 8). Examples of such prodrugs are in vivo, exo vivo and / or in vitro esters of hydroxides that can be hydrolyzed or converted to the 15-epi-lipoxin compounds of the invention and /. Alternatively, it includes, but is not limited to, a carboxyl group and / or a compound. The terms "corresponding lipoxin" and "natural lipoxin" refer to natural lipoxin or lipoxin metabolites. If the analog has activity on a lipoxin-specific receptor, the corresponding or native lipoxin is a normal ligand for that receptor. For example, analog LXA on differentiated HL-60 cells₄LXA with specific activity on specific receptors₄If analog, the corresponding lipoxin is LXA₄Is. If the analog has activity as an antagonist against other compounds (eg, leukotrienes), which is counteracted by the natural lipoxin, then the natural lipoxin is the corresponding lipoxin. The term "active region" means the region of the natural lipoxin or lipoxin analog involved in cell interactions in vivo. This active region can bind to the "recognition site" of the cellular lipoxin receptor, or of macromolecules or macromolecular complexes, including enzymes and their cofactors. Suitable lipoxin A₄Analog is natural lipoxin A₄C₅~ C₁₅Has an active region containing. Suitable lipoxin B₄Analog is natural lipoxin B₄C₅~ C₁₄Has an active region containing. The term "recognition site" or receptor is technically recognized and, in general, functional macromolecules (a group of cellular messengers such as hormones, leukotrienes and lipoxins initiate biochemical and physiological responses to these messengers. It is intended to refer to a complex of macromolecules) or a complex of macromolecules that must first interact with this macromolecule. As used in this application, the receptor can be isolated in intact or permeable cells or in tissues (including organs). Receptors can be from or in a living patient, or can be cloned. Receptors may be normally present or may be induced by disease status, injury or artificial means. The compounds of the present invention can bind reversibly, irreversibly, competitively, non-competitively, or non-competitively (with respect to the natural substrate of the recognition site). The term "metabolizing transformation region" is generally a portion of lipoxin, a metabolite of lipoxin or a lipoxin analog (including a metabolite of a lipoxin analog), where the enzyme or enzyme and its cofactors metabolize at least one. It is intended to refer to the part that attempts to convert (the enzyme or enzyme and cofactors are usually converted with lipoxins). This metabolic conversion region may or may not be susceptible to conversion. Non-limiting examples of the metabolic conversion region of lipoxin are C-13, 14 double bonds or C-15 hydroxyl groups, or LXA containing both.₄Is the part of. The term "detectable labeled molecule" includes fluorescent, phosphorescent and radioactively labeled molecules used to track or identify the compound or receptor recognition site to which the detectable labeled molecule binds. The labeled molecule can be detected by any of several methods known in the art. The term "labeled lipoxin analog" also refers to radioactive isotopes (eg, tritium (eg, tritium).³H), Juterium (H)²H), carbon (¹⁴It is understood to include C), compounds labeled with (but not limited to), or compounds labeled otherwise (eg, fluorescently). The compounds of the present invention are labeled or derivatized, for example, for kinetic binding experiments, for further elucidation of metabolic pathways and enzymatic mechanisms, or for characterization by methods known in the field of analytical chemistry. can do. The term "inhibits metabolism" means blocking or reducing the enzymatic activity that metabolizes natural lipoxin. This block or reduction is due to covalent binding, irreversible binding, reversible binding with the substantial effect of irreversible binding, or the enzyme in its usual manner for other lipoxin analogs (including lipoxin analog metabolites). , May be caused by any other means of blocking action on lipoxins or lipoxin metabolites. The term "resisting metabolism" is intended to include the inability to undergo at least one metabolically degradable conversion by at least one of the enzymes that metabolize lipoxin. LXA that resists metabolism₄Two non-limiting examples of analogs are 1) a structure that cannot be oxidized to the 15-oxo form, and 2) an enzymatic reduction to the 13,14-dihydro form that can be oxidized to the 15-oxo form. The structure is not easy to receive. The term "subject to slower metabolism" means having slower kinetics or taking more time to complete a series of metabolic transformations by at least one of the enzymes that metabolize lipoxin A. LXA undergoing metabolism more slowly₄An unrestricted example of analog is LXA for hydrogenation of C-15, as the analog is sterically impaired at C-16.₄It is a structure with even higher transition state energy. The term "tissue" refers to intact cells, blood, blood preparations such as plasma and serum, bones, joints, muscles, Intended to include smooth muscle and organs. The term "halogen" is meant to include fluorine, chlorine, bromine and iodine, or fluoro, chloro, bromo and iodo. The term "pharmaceutically acceptable salt" is intended to include technically recognized pharmaceutically acceptable salts. These non-toxic salts are usually hydrolyzed under physiological conditions and include organic and inorganic bases. Examples of salts include sodium, potassium, calcium, ammonium, copper and aluminum as well as primary, secondary and tertiary amines, basic ion exchange resins, purines, piperazines and the like. The term is intended to further include esters of lower hydrocarbon groups such as methyl, ethyl and propyl. The term "pharmaceutical composition" comprises at least one lipoxin analog or a pharmaceutically acceptable salt thereof as an active ingredient, and may also include a pharmaceutically acceptable carrier and optionally other ingredients. These compositions are suitable for oral administration, rectal administration, ocular administration, pulmonary administration, nasal administration, skin administration, topical administration, parenteral administration (subcutaneous administration, intramuscular administration and intravenous administration) or inhalation administration. Including. The most appropriate route in any particular case will depend on the nature and severity of the disease being treated and the nature of the active ingredient. These compositions can be provided in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. The medication regimen can be adjusted for the purpose of improving the therapeutic response. For example, several divided doses can be administered daily, or the dose can be reduced over time. One of ordinary skill in the art can usually determine the effective dosage and the appropriate regimen. Pharmaceutical compositions of lipoxin analogs can also refer to combinations comprising lipoxin, lipoxin analogs and / or lipoxin metabolites, including metabolites of lipoxin analogs. Non-limiting examples of combinations include lipoxin analog x, which inhibits one enzyme that metabolizes lipoxin and appropriately has specific activity with the lipoxin receptor recognition site, and lipoxin. It is a mixture containing a second lipoxin analog y that has specific activity with a receptor recognition site and appropriately inhibits or resists lipoxin metabolism. This combination inhibits one of the enzymes that metabolize x-garbage poxin, resulting in a longer tissue half-life, at least for y. Therefore, the lipoxin action mediated or counteracted by y is enhanced. The term "substantially pure or purified" lipoxin compound has less than about 20% (dry weight) of other biological macromolecules, preferably less than about 5% of other biological macromolecules. Defined to include natural or synthetic compounds having (although water, buffers and other small molecules, especially molecules with a molecular weight of less than 5000, may be present). The term "purified" as used herein is preferably in the range of at least 80% (dry weight), more preferably 95-99% by weight, most preferably at least 99. It means the presence of 8% by weight of the same type of biological macromolecule (although water, buffers and other small molecules, especially those with a molecular weight of less than 5000, may be present). The term "pure", as used herein, preferably has the same numerical limitations as "purified" immediately above. "Isolated" and "purified" do not include the natural material in its original state and are separated into components (eg, in an acrylamide gel) but are pure material or solution (eg, contaminating protein). Or it does not include natural substances not obtained as chromatographic reagents such as denaturants and polymers such as acrylamide or agarose). The term "patient" is intended to include living organisms susceptible to disease caused by inflammation, inflammatory response, vasoconstriction, myeloid suppression and / or unwanted cell proliferation. Examples of patients include humans, dogs, cats, cows, goats and mice. The term patient is also intended to include transgenic species. The term "cell proliferation disease" includes diseases involving unwanted proliferation of cells. Non-limiting examples of such diseases include tumors (eg, tumors of the brain, lungs (small and non-small cells), ovaries, prostate, mammary gland or large intestine) or other carcinomas or sarcomas (eg, leukemia, lymphoma). included. The term "improved" is intended to include the treatment, prevention, restriction and / or inhibition of unwanted cell proliferation and / or cell proliferation disorders. Lipoxin Compounds The present invention is based on the surprising finding that substantially pure 15-epi-lipoxin compounds improve unwanted cell proliferation in patients. The 15-epi-lipoxin compound of the present invention contains 15R-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid in a 5S, 6R configuration (15-epi-). LXA₄). The 15-epi-lipoxin compounds of the present invention also contain 15R-5,14,15-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid in a 5S, 14R configuration (15-epi). -LXB₄). The 15-epi-lipoxin of the present invention further comprises 15-hydroxyeicosatetraenoic acid (particularly in the 15R configuration). The present invention is based on the surprising finding that lipoxin is rapidly metabolized in a unique manner by cells that are in vivo. Other LO-induced products (eg, leukotrienes) are metabolized by ω oxidation followed by β-oxidation (Huwyler et al. (1992) Eur. J. Biochem. 206, 869-879). , Based on the unexpected finding that lipoxin is metabolized by a series of oxidation and reduction reactions that act on certain sites of the lipoxin molecule. For example, LXA₄Metabolism is caused, at least in part, by the oxidation of C-15 hydroxyl, 15-oxo-LXA.₄To produce a C-13,14 double bond reduction of 13,14-dihydro-15-oxo-LXA₄And further reduction of 13,14-dihydro-LXA₄Has been found to occur. LXB₄In and its natural isomers, similar oxidation occurs at the C-5 hydroxyl and reduction occurs at the C-6,7 double bond. Accordingly, the present invention features lipoxin analogs that have lipoxin activity but are chemically modified to prevent in vivo dehydrogenation and subsequent degradation. In these analogs, the C-1 to C-13 portions of natural lipoxin can or cannot be conserved. Variants of parts C-1 to C-13 contain different cis or transgeometry as well as substitutions. These disclosed compounds are represented below by the genus of structure, which is further subdivided into subgenus. The subgenus contained in each of the following two R groups is indicated by Roman numerals on the left side of the page. The lipoxins of the present application, which include an "active region" and a "metabolic conversion region" (both of these terms are defined herein), generally have the following structure:<img file="JP4060366B2_D0001.tif" />{In the formula, R₁Is<img file="JP4060366B2_D0002.tif" />May be, and R₂Is<img file="JP4060366B2_D0003.tif" />May be}. In one embodiment, the lipoxin analog of the present invention has the following structural formula I:<img file="JP4060366B2_D0004.tif" />{In the formula, X is R₁, OR₁Or SR₁Is; (here, R₁Is (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; (iii) cycloalkyl of 3 to 10 carbon atoms; (iv) aralkyl of 7 to 12 carbon atoms; ( v) Phenyl; (vi) Substituted phenyl<img file="JP4060366B2_D0005.tif" />(Here, Z_i, Z_iiiAnd Z_vAre each independently, -NO₂, -CN, -C (= O) -R₁, -SO₃Selected from the group consisting of H and hydrogen; here Z_iiAnd Z_ivAre independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls) (vii) detectable labeled molecules; or (viii) straight or branched chain alkenyl with 2-8 carbon atoms. ) In the formula, Q₁Is (C = O), SO₂Or (CN); in the formula, Q₃Is O, S or NH; in the formula, R₂And R₃One is hydrogen and the other is (a) H; (b) alkyl (straight or branched) with 1-8 carbon atoms; (c) cycloalkyl with 3-6 carbon atoms; (d) 2-8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms;₄Is (a) H; (b) alkyl (straight or branched chain) of 1 to 6 carbon atoms; in the formula, Y₁Or Y₂Is -OH, methyl or -SH, and in the equation, the other is (a) H (b) CH_aZ_{b b}(Here, a + b = 3, a = 0-3, b = 0-3; and Z is cyano, nitro or halogen); (c) Alkoxy (linear or fractional) of 2-4 carbon atoms Branched chain); or (d) alkoxy with 1 to 4 carbon atoms; or Y₁And Y₂Together (a) = N; or (b) = O; in the equation, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0006.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁, Methoxy and -SO₃Choose from a group of H; and here, Z_iias well as_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls)); (c) -R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; Here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms); (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0-3, b = 0 + 3, and any Z are independently selected from the group consisting of halogens)); (e) 1-8 carbon atoms and 1 ~ 6 Halogen atoms of haloalkyl (straight or branched); and in the formula, R₆Are (a) H; (b) alkyl (straight or branched chain) of 1 to 4 carbon atoms; (c) halogen; where C-1 amides, C-1 alkanoates, and pharmaceuticals Acceptable (5S, 6R, 15S) -Trihydroxy-7E, 9E, 11Z, 13E-Eicosatetraenoic acid (LXA)₄) Excluding C-1 salt; also LXA₄Excluding C-5, C-6 and C-15 alkanoates}. In one embodiment of the invention, the lipoxin analog has structure II:<img file="JP4060366B2_D0007.tif" />{In the formula, X is R₁, OR₁Or SR₁Is; (here, R₁Is (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; (iii) cycloalkyl of 3 to 10 carbon atoms; (iv) aralkyl of 7 to 12 carbon atoms; (v) ) Phenyl; (vi) substituted phenyl<img file="JP4060366B2_D0008.tif" />(Here, Z_i, Z_iiiAnd Z_vAre each independently, -NO₂, -CN, -C (= O) -R₁, Hydrogen and -SO₃Selected from the group consisting of H; here, Z_iiAnd Z_ivAre independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls) (vii) Detectable labeled molecules such as fluorescent labels without limitation; or (viii) linear chains of 2-8 carbon atoms. Or branched chain alkenyl) in the formula, Q₁Is (C = O), SO₂Or (C = N); in the formula, Q₃Is O, S or NH; in the formula, R₂And R₃One is hydrogen and the other is (a) H; (b) alkyl (straight or branched) with 1-8 carbon atoms; (c) cycloalkyl with 3-6 carbon atoms; (d) 2-8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms;₄Is (a) H; (b) alkyl (straight or branched chain) of 1 to 6 carbon atoms; in the formula, Y₁Or Y₂Is -OH, methyl, -H or -SH, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 to 3, b = 0 to 3Z are halogens containing cyano, nitro or F, Cl, Br, I); (c) 2 to 4 carbon atoms Alkoxy (straight or branched chain); (d) alkoxy with 1 to 4 carbon atoms; or Y₁And Y₂Together (a) = N; or (b) = O; in the equation, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0009.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁, Methoxy and -SO₃Choose from the group consisting of H; here, Z_iAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls)); (c) -R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; And here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms); (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; and (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 + 3, any Z is independently selected from the group consisting of halogens)); (e) 1 ~ 8 carbon atoms and 1 ~ 6 Halogen atom haloalkyl (straight or branched chain)}. In one embodiment of the invention, the lipoxin analog has structure III:<img file="JP4060366B2_D0010.tif" />{In the formula, X is R₁, OR₁Or SR₁Is; (here, R₁Is (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; (iii) cycloalkyl of 3 to 10 carbon atoms; (iv) aralkyl of 7 to 12 carbon atoms; (v) ) Phenyl; (vi) substituted phenyl<img file="JP4060366B2_D0011.tif" />(Here, Z_i, Z_iiiAnd Z_vAre each independently, -NO₂, -CN, -C (= O) -R₁, Hydrogen and -SO₃Selected from the group consisting of H; here, Z_iiAnd Z_ivAre independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls) (vii) detectable labeled molecules; or (viii) straight or branched chain alkenyl with 2-8 carbon atoms. ) In the formula, Q₁Is (C = O), SO₂Or (C = N); in the formula, Q₃Is O, S or NH; in the formula, R₂And R₃One is hydrogen and the other is (a) H; (b) alkyl (straight or branched) with 1-8 carbon atoms; (c) cycloalkyl with 3-6 carbon atoms; (d) 2-8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms;₄Is (a) H; or (b) alkyl (straight or branched chain) of 1 to 6 carbon atoms; in the formula, Y₁Or Y₂Is hydroxyl, methyl, hydrogen or thiol, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 to 3, b = 0 to 3Z are cyano, nitro or halogen [including F, Cl, Br, I]); (c) 2 to 4 carbons. Atomic alkyl (straight or branched chain); (d) alkoxy with 1-4 carbon atoms; or Y₁And Y₂Together (a) = N; or (b) = O; and in the equation, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; (iii) substituted phenyl.<img file="JP4060366B2_D0012.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁, Methoxy and -SO₃Choose from the group consisting of H; here, Z_iiAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls)); (c) -R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; Here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms); or (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 + 3, Z are independently selected from the group consisting of halogens)}. In another embodiment of the invention, the lipoxin analog has the following structural formula IV:<img file="JP4060366B2_D0013.tif" />{In the formula, X is R₁, OR₁Or SR₁Is; (here, R₁Is (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; (iii) cycloalkyl of 3 to 10 carbon atoms; (iv) aralkyl of 7 to 12 carbon atoms; (v) ) Phenyl; (vi) substituted phenyl<img file="JP4060366B2_D0014.tif" />(Here, Z_i, Z_iiiAnd Z_vAre each independently, -NO₂, -CN, -C (= O) -R₁, Methoxy, hydrogen and -SO₃Selected from the group consisting of H; here, Z_iiAnd Zi_ivAre independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls) (vii) detectable labeled molecules; or (viii) straight or branched chain alkenyl with 2-8 carbon atoms. ) In the formula, Q₁Is (C = O), SO₂Or (CN); in the formula, Q₃Is O, S or NH; in the formula, R₂And R₃One is hydrogen and the other is (a) H; (b) alkyl (straight or branched) with 1-8 carbon atoms; (c) cycloalkyl with 3-6 carbon atoms; (d) 2-8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms;₄Is (a) H; or (b) alkyl (straight or branched chain) of 1 to 6 carbon atoms; in the formula, Y₁Or Y₂Is -OH, methyl or -SH, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 to 3, b = 0 to 3Z are cyano, nitro or halogen); (c) Alkoxy (straight or branched chain) of 2 to 4 carbon atoms. (D) Alkoxy of 1 to 4 carbon atoms; or Y₁And Y₂Together (a) = N; or (b) = O; in the equation, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0015.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁, Methoxy and -SO₃Choose from the group consisting of H; here, Z_iiAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls)); (c) R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; Here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms); (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; or (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 + 3, and Z are independently selected from the group consisting of halogens)); or (e) 1 ~ 8 carbon atoms and 1 ~ It is a haloalkyl (straight or branched chain) of 6 halogen atoms; and R₆Is (a) H; (b) alkyl (straight or branched chain) of 1 to 4 carbon atoms; or (c) halogen}. In another embodiment of the invention, the lipoxin analog has the following structural formula V:<img file="JP4060366B2_D0016.tif" />{In the formula, R₁Is (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; (iii) cycloalkyl of 3 to 10 carbon atoms; (iv) aralkyl of 7 to 12 carbon atoms; (v) ) Phenyl; (vi) substituted phenyl<img file="JP4060366B2_D0017.tif" />(Here, Z_i, Z_iiiAnd Z_vAre each independently, -NO₂, -CN, -C (= O) -R₁, Hydrogen and -SO₃Selected from the group consisting of H; here, Z_iiAnd Z_ivAre independently selected from the group consisting of halogens, methyls, hydrogens and hydroxyls) (vii) detectable labeled molecules; or (viii) straight or branched chain alkenyl with 2-8 carbon atoms. ) In the formula, n = 1 ~ 10; in the formula, R₂, R_3aAnd R_3bAre independently selected from the following; (a) H; (b) alkyl of 1 to 8 carbon atoms (straight or branched chain); (c) cycloalkyl of 3 to 6 carbon atoms; (d) 2 ~ 8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; and R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms; in the equation, Y₁Or Y₂Is -OH, methyl, hydrogen or -SH, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 to 3, b = 0 to 3Z are cyano, nitro or halogen); (c) Alkoxy (straight or branched chain) of 2 to 4 carbon atoms. (D) Alkoxy of 1 to 4 carbon atoms; or Y₁And Y₂Together (a) = N; or (b) = O; in the equation, Y₃Or Y₄Is -OH, methyl, hydrogen or -SH, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 ~ 3 any Z is cyano, nitro or halogen); (c) Alkoxy (straight or minute) of 2-4 carbon atoms Branched chain); (d) Alkoxy of 1 to 4 carbon atoms; or Y₃And Y₄Together (a) = N; or (b) = O; in the equation, Y₅Or Y₆Is -OH, methyl, hydrogen or -SH, and in the equation, the other is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 ~ 3 any Z is cyano, nitro or halogen); (c) Alkoxy (straight or minute) of 2-4 carbon atoms Branched chain); (d) Alkoxy of 1 to 4 carbon atoms; or Y₅And Y₆Together (a) = N; or (b) = O; in the equation, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0018.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁And -SO₃Choose from the group consisting of H; here, Z_iiAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens, methoxys and hydroxyls)); (c) R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; and here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) or substituted phenyl with 0-8 carbon atoms); (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; or (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 + 3, and Z are independently selected from the group consisting of halogens)); or (e) 1 ~ 8 carbon atoms and 1 ~ It is a haloalkyl (straight or branched chain) of 6 halogen atoms; however, C-1 amide, C-1 alkanoate, and pharmaceutically acceptable (5S, 14R, 15S) -trihydroxy-6E, 8Z, 10E, 12E-Eicosatetraenoic acid (LXB)₄) Excluding C-1 salt; also LXB₄Excluding C-5, C-6 and C-5 alkanoates}. In another embodiment of the invention, the lipoxin analog has the following structural formula VI:<img file="JP4060366B2_D0019.tif" />In the formula, R_aIs selected from the following groups; (a) H; or (b) alkyl of 1 to 8 carbon atoms;_{b b}Choose from the following group:<img file="JP4060366B2_D0020.tif" />In another preferred embodiment of the invention, the lipoxin analog has the following structural formula VII:<img file="JP4060366B2_D0021.tif" />In the formula, R_aIs selected from the following groups; (a) H; or (b) alkyl of 1 to 8 carbon atoms;_{b b}And R_cAre independently selected from the following groups: (a) H; (b) hydroxyl, or thiol; (c) methyl or halomethyl (-CF)₃And -CH₂(Including F); (d) Halogen; (e) Alkoxy of 1 to 3 carbon atoms (including methoxy);_dAnd R_eAre independently selected from the following groups; (a) H; (b) hydroxyl, or thiol; (c) methyl or halomethyl (-CF)₃And -CH₂(Including F); (d) Halogen; (e) Alkoxy of 1-3 carbon atoms (including methoxy); or (f) Alkoxy or haloalkyl (straight or branched chain) of 2-4 carbon atoms; C-1 amide, C-1 alkanoate, and pharmaceutically acceptable (5S, 6R, 15S) -trihydroxy-7E, 9E, 11Z, 13E-eicosatetraenoic acid (LXA)₄) C-1 salt; LXA₄Excludes C-5, C-6 and C-15 alkanoates. In another preferred embodiment of the invention, the lipoxin analog has the following structural formula VIII:<img file="JP4060366B2_D0022.tif" />In the formula, R_aIs selected from the following groups; (a) H; or (b) alkyl of 1 to 8 carbon atoms;_{b b}And R_cAre independently selected from the following groups: (a) H; (b) hydroxyl, or thiol; (c) halomethyl (CF)₃Including); (d) Halogen; (e) Alkoxy of 1-3 carbon atoms (straight or branched chain); or (f) Alkoxy of 1-3 carbon atoms;_dAnd R_eIndependently select from the following groups (a) H; (b) hydroxyl, or thiol; (c) methyl or halomethyl (-CF)₃And -CH₂(Including F); (d) Halogen; (e) Alkoxy of 1-3 carbon atoms (including methoxy); or (f) Alkoxy or haloalkyl (straight or branched chain) of 2-4 carbon atoms. In another preferred embodiment of the invention, the lipoxin analog has the following structural formula IX:<img file="JP4060366B2_D0023.tif" />In the formula, R_aIs selected from the following groups; (a) H; or (b) alkyl of 1 to 8 carbon atoms;_{b b}And R_cAre independently selected from the following groups: (a) H; (b) hydroxyl, or thiol; (c) halomethyl (CF)₃And CH₂Including F); (d) Halogen; (e) Alkoxy of 1-3 carbon atoms (straight or branched chain); (f) Alkoxy of 1-3 carbon atoms;₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 4 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0024.tif" />(Here, Z_i, Z_iiiAnd Z_v, Independently, hydrogen, -NO₂, -CN, -C (= O) -R₁And -SO₃Choose from the group consisting of H; here, Z_iiAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens, methoxys and hydroxyls)); (c) R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is an alkyl (straight or branched chain) or substituted phenyl with 0-8 carbon atoms); (d) -C (R)_iii) (R_iv)-R_i(Here, R_iiiAnd R_ivIndependently select from the group consisting of (i) H; or (ii) CH_aZ_{b b}(Here, a + b = 3, a = 0 ~ 3, b = 0 + 3, and Z are independently selected from the group consisting of halogens)); or (e) 1 ~ 8 carbon atoms and 1 ~ It is a haloalkyl (straight or branched chain) of 6 halogen atoms. In another preferred embodiment of the invention, these compounds have the following structural formula X:<img file="JP4060366B2_D0025.tif" />In the formula, R_aIs selected from the following groups; (a) H; or (b) alkyl (straight or branched chain) of 1 to 8 carbon atoms;_{b b}And R_cAre independently selected from the following groups: (a) H; (b) hydroxyl, or thiol; (c) halomethyl (eg, CF)₃(Including); (d) Halogen; (e) Alkoxy of 1-3 carbon atoms (straight or branched chain); (f) Alkoxy of 1-3 carbon atoms (including methoxy); Other suitable of the present invention In a specific example, these compounds have the following structural formula XI:<img file="JP4060366B2_D0026.tif" />In the formula, R_aIs (i) hydrogen; (ii) alkyl (straight or branched chain) of 1 to 8 carbon atoms; or (iii) detectable labeled molecule; in the formula, n = 1 to 10; in the formula, Y₂, R_3aAnd R_3bAre independently selected from the following; (a) H; (b) alkyl of 1 to 8 carbon atoms (straight or branched chain); (c) cycloalkyl of 3 to 6 carbon atoms; (d) 2 ~ 8 carbon atom alkenyl (straight or branched chain); or (e) R_aQ₂R_{b b}(Here, Q₂Is -O- or -S-; R_aIs an alkylene (straight or branched chain) with 0-6 carbon atoms; and R_{b b}Is an alkyl (straight or branched chain) of 0-8 carbon atoms; in the equation, Y₁Is -OH, methyl or -SH, in the formula, Y₂Is (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0 to 3, b = 0 to 3Z are halogens); or (c) alkyl (straight or branched) of 2 to 4 carbon atoms; During the ceremony, Y₃And Y₅Independently select from the group consisting of the following; (a) H; (b) CH_aZ_{b b}(Here, a + b = 3, a = 0-3, b = 0-3 any Z is cyano, nitro or halogen); or (c) alkyl (linear or) 2-4 carbon atoms. Branched chain); in the formula, Y₄And Y₆Are independently selected from the group consisting of the following; (a) H; (b) alkyl (straight or branched chain) of 2 to 4 carbon atoms; (c) alkoxy (linear or branched chain) of 1 to 4 carbon atoms. Or branched chain); or (d) hydroxyl or thiol; and in the formula, R₅Is (a) alkyl (straight or branched chain) of 1-9 carbon atoms; (b)-(CH)₂)_n-R_i(Here, n = 0 ~ 3 and R_iIs (i) cycloalkyl of 3 to 10 carbon atoms; (ii) phenyl; or (iii) substituted phenyl.<img file="JP4060366B2_D0027.tif" />(Here, Z_iiAnd Z_ivAre each independently selected from the group consisting of halogens, methyls, hydrogens, methoxys and hydroxyls)); (c) R_aQ_aR_{b b}(Here, Q_a= -O- or -S-; here, R_aIs an alkylene (straight or branched chain) of 0-6 carbon atoms; here, R_{b b}Is<img file="JP4060366B2_D0028.tif" />(d) Haloalkyl (straight or branched) of 1-8 carbon atoms and 1-6 halogen atoms; however, C-1 amides, C-1 alkanoates, and pharmaceutically acceptable (5S) , 14R, 15S) -Trihydroxy-6E, 8Z, 10E, 12E-Eicosatetraenoic acid (LXB)₄) Excluding C-1 salt; also LXB₄C-5, C-6 and C-5 position alkanoate (acetate) excluded}. In the most preferred embodiment of the invention, the compounds of the invention have the following structural formulas:<img file="JP4060366B2_D0029.tif" /><img file="JP4060366B2_D0030.tif" />In the formula, R'is H or CH₃And in the formula, C^*The substituent in is the R configuration. In another preferred embodiment of the invention, the compound of the invention has the following structural formula:<img file="JP4060366B2_D0031.tif" /><img file="JP4060366B2_D0032.tif" />Method for Producing Lipoxin Compound A suitable compound can be produced particularly as described in Example 1 below. After standard chemical methods such as selective hydrogenation, Pd (0) -Cu (I) coupling, Witchhi-type coupling, Sharpless epoxidation and coupling of major intermediates, the other compounds of the invention Can be produced by a combined strategy for lipoxin (LX) and prostaglandin analog synthesis to produce stable LX analogs of the invention using asymmetric reduction of (Webber, SE). Etc. (1988) Adv.Exp.Med.Biol.229:61; Raduchel, B. and Vorbruggen, H. (1985) Adv.Prostaglandin Thromboxane Leukotriene Res.14:263; and Nicolaou, KC et al. (1991) Angelw Chem.Int.Ed.Engl.30: 1100). Geometric variants can be achieved, for example, as described in US Pat. No. 4,576,758 and Nicolaou, KC (1989) J. Org. Chem. 54: 5527. Subgenus in Diagram I, as shown below<u style="single">1</u>LX analog compounds containing, can be prepared as three major fragments (A, B and C), which can then be combined to form the entire molecule.<img file="JP4060366B2_D0033.tif" />Precursor fragment<u style="single">2</u>Substituent R to select from hydrogen, phenyl, halogen or methyl for the synthesis of epoxy alcohols for₂, R₃And R₄Can occur using. Each of these epoxy alcohols can be converted to a phenylurethane derivative such as 3.<img file="JP4060366B2_D0034.tif" />PhNCO, pyrimidines and CH₂Cl₂And then SN²Using a Lewis acid catalyst from the opening, the vicinal diol was placed at C-6 in the (R) configuration required for binding, and also in the (S) configuration established for bioactivity and binding at the recognition site. Give 1,2-cyclic carbonate contained in C-5. These alcohols, then<u style="single">4</u>Precursor like<u style="single">A</u>Protect to generate fragments.<img file="JP4060366B2_D0035.tif" />these<u style="single">A</u>Fragments are now fragmented, as in Webber, SE et al. (1988) Adv.Exp.Med.Biol.229:61.<u style="single">B</u>Intermediate, phosphonium bromide<u style="single">5</u>Combined with, in grams, combined<u style="single">A</u>+<u style="single">B</u>Fragment product<u style="single">6</u>Can be generated.<img file="JP4060366B2_D0036.tif" />Fragment of Diagram I<u style="single">C</u>Intermediates are produced in parallel with the preparation of AB conjugate. these<u style="single">C</u>In the fragment, Y₁And / or Y₂Substituents of are methyl, methoxy, hydrogen, cyano, nitro or halogen (see Specific Example 3). Thus, having 15-methyl and / or, for example, 16-methyl or 16-phenoxy derivatives, these substituted LXA.₄Makes analogs less susceptible to dehydrogenation. Therefore, it has suitable resistance to enzymatic oxidation and / or dehydrogenation.<u style="single">C</u>Fragments are transformed by key site protection and subsequent bromination, fragments<u style="single">C</u>Vinyl bromide products, for example<u style="single">6b</u>Can be given, which is the catalytic amount of P (Ph)₃)₄And with CuI<u style="single">6</u>LXA of genus I combined with₄Produces a complete analog backbone structure. This scheme will be further described by the following examples.<img file="JP4060366B2_D0037.tif" />The compounds of the present invention within the subgenus formulas II and III can be synthesized in a similar manner. The compounds of the genera II and III are first fragments.<u style="single">A</u>Substituted compounds of were individually prepared and each individually prepared fragment as shown in Diagram I.<u style="single">B</u>Combined with<u style="single">7</u>That is,<u style="single">A</u>₁+<u style="single">B</u>₁Fragment (X, Q as shown₁, R₂, R₃And R₄Is generated by generating).<img file="JP4060366B2_D0038.tif" />C with acetylene groups C-14,15 and ω-C-20 terminal substitutions₁Fragment<u style="single">8</u>Are produced as described above for the prostaglandin analogs of structure 6, respectively, and converted to their corresponding vinyl bromide products (KCN JAC 1985, Webber) with individual substituted fragments C.₁Or<u style="single">8</u>Species are produced, these are catalytic amounts of P (Ph)₃)₄And with CuI<u style="single">20</u>Coupling to acetylene LXA₄Suitable for producing analog class combined products. Each final product is then subjected to gradient RP-HPLC with rapid diode array detection for purification (as in Serhan, CN, Methods in Enzymology). LXA₄The presence of modifications in C-15 to C-20 can alter metabolism by dehydrogenase and oxidase by steric hindrance, producing stable prostaglandin analogs with C-15 to ω terminal substitutions. (Raduchel, B. and Vorbruggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14: 263 and Vorbruggcn, H. et al. (Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids (Roberts, SM, SM, Scheinmann, F. ed.) Oxford: Pergamon Press)). Cyclo LXA of the present invention within the range of genus IV₄The compound can be prepared by the following method.<img file="JP4060366B2_D0039.tif" />This class of parent compounds is also subjected to a similar total synthesis strategy and structure.<u style="single">30</u>Three major fragments inside<u style="single">A</u>、<u style="single">B</u>as well as<u style="single">C</u>To assign. Precursors of Fragment A were prepared by the pathway used in Nicolaou, KC (1989) J. Org. Chem. 54: 5527, 7-cis, 11-trans-LXA.₄In the synthesis of methyl esters<u style="single">10</u>Can be manufactured.<img file="JP4060366B2_D0040.tif" />Fragment B in 30 is a precursor<u style="single">11</u>Alternatively, it is obtained as produced by (Vorbruggen et al., P.353) via saligenin-([O-hydroxybenzyl alcohol]). This benzyl alcohol<u style="single">11</u>In the presence of NaH in DMF<u style="single">10</u>And react (1: 1)<u style="single">12</u>give. This key intermediate is silylated in BSTFA and then coupled with the individual fragments designed for the C precursor.<img file="JP4060366B2_D0041.tif" />Then<u style="single">13</u>Add 4.0 equivalents of bromite precursor to a given individual design of vinyl brominated fragment C, AgNO₃Processed with, then 7.0 equivalents of KCN, EtOH / THF / H₂It can be combined by processing with O (1: 1: 1) (025 ° C, 2-4 hours). The individual products are then CHed for 2-3 hours.₂Cl₂The individual compounds belonging to Genus IV are catalyzed in Lindlar for mild hydrogenation by selective catalyst in. Each can be saponified in LiOH / THF to give the corresponding free acid after separation by RP-HPLC.<img file="JP4060366B2_D0042.tif" />The inventions of these genus IV compounds are described below: 15 (±) methyl-cyclo-LXA₄Further described in the synthesis of methyl esters and corresponding free acids. The compounds of the present invention within the range of genus V can be made by the following methods. LXB₄Some systems studied with are showing that some sites in natural compounds are required for biological activity (Serhan, CN (1991) J. Bioenerg. And Biomembr. 23:105. ). These sites contain the C-14 alcohol in the (R) configuration and the tetraene double bond at positions C-8, 9 in the cis configuration. In addition, based on the metabolic studies that gave rise to the present invention, some key additional sites are LXB.₄It has been identified as necessary to protect the biological activity of. These protect the C-15 alcohol from dehydrogenase activity (ie, 5-oxo-LXB).₄Blocking the formation); maintaining both the Δ8 bond and the 14 (R) alcohol; and including the reduction of the Δ6-7 double bond and the inhibition of β / ω oxidation of the resulting compound.<img file="JP4060366B2_D0043.tif" />Thus, the genus V (<u style="single">14</u>) Is the LXB required for biological activity₄The region is retained, but the region that can be used for metabolic degradation is modified. Again, retrosynthetic analysis<u style="single">14</u>Gives priority to the three key fragments A, B and C designed in. Key intermediate LXB₄The coupling that produces the members of the analog class is LXA₄And its analogs outlined standard techniques ie selective hydrogenation (producing 8-cis geometry); with unique substitutions<u style="single">A</u>as well as<u style="single">B</u>Pd (0) -Cn (I) couplings for binding fragments; Witchhi-type couplings for binding C fragments with the required substitutions and Sharpless epoxides for producing 14 (R) vicinal alcohols. (References Nicolaou, KC et al. (1991) Angew.Chem.Int.Ed.Engl.30: 1100), References Weber, SE et al. (1988) Adv.Exp.Med.Biol.229:61 Ed. See Wong, PK and Serhan, CN and those cited therein). Thus, LXA₄Analog and natural LXB₄A particular analog can be obtained by using a similar strategy for the structure of.<img file="JP4060366B2_D0044.tif" />H, CH₃, OCH₃, Phenyl, halo-substituted phenyl R₂, R₆, R₇Have a substitution in<u style="single">15</u>of<u style="single">A</u>Fragments by standard methods, for example<u style="single">16</u>(In the equation, the increasing chain length = CH₂).<img file="JP4060366B2_D0045.tif" /><img file="JP4060366B2_D0046.tif" />Compound<u style="single">16</u>To the trimethylsilylacetylene intermediate as in (Nicolaou, KC et al. (1991) Angew.Chem.Int.Ed.Engl.30: 1100-16).<u style="single">17</u>Via vinyl bromide<u style="single">15</u>Convert to (<u style="single">17</u>By Pinanil-9BBN, then n-BuN₄Reduced in THF by NF<u style="single">18</u>This is subjected to bromination after protecting essential parts such as alcohol.<u style="single">15</u>(Give Fragment A).<u style="single">14</u>Fragment inside<u style="single">C</u>Is R as shown₅Compounds with substitutions<u style="single">19</u>Generate from.<img file="JP4060366B2_D0047.tif" />Acetylene alcohol<u style="single">19</u>Is then reduced in LAH and then subjected to Sharpless asymmetric epoxidation.<u style="single">20</u>Is produced and isolated by RP-HPLC to give the (+) isomer, which is used to obtain the required LXB.₄Produces analog C-14 alcohol in (R) configuration, compound<u style="single">20</u>, R₄And R₅The corresponding aldehyde after protection with PCC in the substituents of the alcohol as well as in methylene chloride.<u style="single">21</u>Convert to. (Nicolaou, KC, etc. (1991) Angew.Chem.Int.Ed.Engl.30: 1100)<img file="JP4060366B2_D0048.tif" />Phosphonium salt<u style="single">22</u>Can be prepared as in the references (Weber, SE et al. (1988) Adv.Exp.Med.Biol.229: 61, Ed.Wong, PK and Serhan, CN), and used herein, R.₄And R₅Designed permutations that have permutations<u style="single">23</u>Have on the basis of<u style="single">23</u>By witch-type coupling that gives<u style="single">BC</u>Fragment coupling can occur. this<u style="single">23</u>The double bond of the cis is I₂To<u style="single">14</u>Is isomerized using as a catalyst to give the parental precursor form of<u style="single">24</u>A trans isomer such as can be given.<img file="JP4060366B2_D0049.tif" />Then<u style="single">24</u>of<u style="single">15</u>Coupling to is done by Pd (0) -Cu (I) coupling,<u style="single">14</u>Acetylene precursor (<u style="single">25</u>(Indicated by) is given. Individual LXB after selective Lindlar-catalyzed hydrogenation₄Analogs can be further purified using rapid diol array detection by RP-HPLC using the tetraene backbone as a convenient method of isolating individual products (Serhan, CN (1990) Meth. Enzymol. 187: 167). Usefulness The compounds of the present invention have the biological activity of natural LX, but are more resistant to degradation or prevent degradation of natural LX. Therefore, thisThe disclosed compounds have medicinal utility for treating or preventing many diseases associated with inadequate or inappropriate LX-mediated cellular responses in patients. Based on the antiproliferative effects of the disclosed 15-epi-lipoxin compounds, the present invention comprises a pharmaceutical composition comprising an effective amount of a substantially purified 15-epi-LX compound in cells and a pharmaceutically acceptable carrier. Provides a method of improving unwanted cell proliferation by contact with. The cells can be contacted in vivo and / or in vitro. Alternatively, cells can be removed from the patient; contacted with the substantially purified 15-epi-lipoxin compound of the invention with Exo-Vivo and transplanted into the patient. The invention also provides a method of ameliorating cell proliferation disease in a patient, comprising administering an effective amount of a substantially purified 15-epi-LX compound. The effective amount is usually the amount required to ensure adequate exposure to the target cell population. Such amounts usually depend on the nature of the 15-epi-LX compound, the mode of administration, the severity of unwanted cell proliferation or cell proliferation disease and other factors considered by those skilled in the art in determining medication regimens. To do. The target cells to be contacted may be those with cancer-like and / or neoplastic growth. Alternatively, the target cells may be those that proliferate abnormal cells in response to stimuli such as restenosis; And / or the target cell may consist of transformed cells having genetic properties altered from the genetic properties of the original cell. Suitable target cells include epithelial cells, leukocytes, endothelial cells and / or fibroblasts. Based on the stimulatory effect of LX on selected cells, the invention also provides a method of treating a patient with myelosuppressive disease by administering to the patient a pharmaceutical composition comprising an effective amount of the LX analog. .. This effective amount is usually the amount required to ensure adequate exposure to the target cell population. Such amounts usually depend on the nature of the analog, the mode of administration, the severity of myelosuppression and other factors considered by those skilled in the art in determining the medication regimen. Therapeutic applications for cell-proliferating LX analogs also include removing cells from the patient, stimulating cell growth in vitro, and returning the promoted cell specimen to the patient in whole or in part. Additional therapeutic agents (eg, cytokines such as GM-CSF) can optionally be used with LX during stimulation or with the introduction of cell preparations. In other embodiments, the compounds of the invention are used to treat or prevent an inflammatory or inflammatory response. LXA₄Blocks the activation of leukocytes, the mediator of inflammation. LXA₄Induced effects include inhibition of leukocyte migration, production of reactive oxygen species and formation of pro-inflammatory mediators involved in tissue swelling. (Raud, J. et al. (1991) Adv.Exp.Med.Biol.314: 185.Cell-Cell Interactions in the Release of Inframmation Mediators vol.314) LXB₄Shows radioprotective effects such as prevention of diarrhea and ataxia in an in-vivo assay using mouse hematopoietic stem cells (Walken, TLJr., (1988) J. Radiat. Res. 29: 255). Leukocyte-mediated inflammatory or inflammatory responses cause or contribute to a variety of illnesses, including various forms of asthma and arthritis. Inflammatory responses to physical trauma, radiation exposure and other physical wounds are included within the invention. In another embodiment, the compounds of the invention are used to treat or prevent inflammation by counteracting the effects of leukotrienes. LXA₄Is LTB₄It blocks induced inflammation and blocks both plasma leakage and leukocyte migration in an in-vivo assay in the hamster's cheek pouch. (Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137: 571) Plasma leakage and leukocyte migration are key events in both wound healing and inflammation. LXA₄Also LTD₄LTD on mesangial cells, which negates the induced renal hemodynamic effects and is partly responsible for the regulation of renal hemodynamics in the kidney₄Block the combination of. (Badr.KF et al. (1989) Proc.Natl.Acad.Sci.USA 86: 438) Administering the compounds of this invention, sulfide peptide leukotrienes such as LTD₄, LTC₄And LTB₄Can counteract the effects of. Leukotriene-mediated vasoconstrictor responses include asthma, anaphylactic reaction, allergic reaction, shock, inflammation, rheumatoid arthritis, gout, psoriasis, allergic rhinitis, adult respiratory disorder syndrome, Crohn's disease, endotoxin shock, traumatic shock, hemorrhagic shock Diseases such as intestinal ischemic shock, renal glomerular disease, benign prostatic hypertrophy, inflammatory bowel disease, myocardial ischemia, myocardial infarction, circulatory shock, brain injury, systemic erythematosus, chronic kidney disease, cardiovascular disease and hypertension Is related to. In other embodiments, the compounds of the invention are used to treat or prevent a vasoconstrictor response or condition. LX is endothelium-dependent vasodilation (LXA)₄) (Lefer, AM et al. (1988) Proc. Natl. Acad. Sci. USA 85: 8340) and induce cerebral arteriole dilatation in vivo in newborn pigs (LXA₄And LXB₄) (Busija, DW et al. (1989) Am.J.Physiol.256: 468). In addition, LXA₄Induced rapid arteriole dilation in vivo in hamster cheeks (Dahlen, S.-E. et al. (1987) Acta Physiol.Scand.130: 643), induced in rat renal hemodynamics (Badr, KF et al. (1987) Biochem.Biophys.Res.Commun.145: 408). Vascular contractile responses and symptoms include renal blood flow dynamics (including glomerular disease), cardiovascular disease (including hypertension, myocardial infarction, myocardial ischemia and vascular disease), and gastrointestinal disease. Causes and contributes to or is associated with illnesses and symptoms. Methods of screening LX analogs or other compounds to identify those with a tissue half-life longer than the corresponding native LX are also included in the invention. Using this method, it is possible to determine whether a compound blocks metabolism compared to natural LX and resists it or receives it more slowly. This method prepares at least one enzyme that metabolizes LX and contacts the compound with its enzyme preparation to see if the compound blocks the enzyme's metabolism and resists it or receives it more slowly. It is done by measuring. Cells with LX recognition sites such as polymorphonuclear neutrophils, peripheral blood monocytes, and differentiated HL-60 cells are in suitable sources of enzyme preparations. The LX recognition site can be naturally present or can be artificially induced (due to medical condition or injury). Artificially induced LXA₄A non-limiting example of a recognition site is the induction of such site in differentiated HL-60 cells. In one embodiment, the preparation of the enzyme involved harvesting cells and repeating lysis by freeze-thaw three times and then ultracentrifuging to produce 100,000 g of supernatant. Cell-free 100,000 g pellets can also be used. In addition, the enzyme preparation may include any enzyme that is not involved in the metabolism of natural LX, but that performs conversions to LX similar to or equal to those performed by enzymes that naturally metabolize LX. Non-limiting examples of suitable enzymes are 15-hydroxyprostaglandin dehydrogenase, cytochrome P-450 monogenase (derived from human leukocytes and rat and human liver microsomes). Typing of LX metabolites included standard techniques such as extraction, chromatography and quantitative HPLC followed by trimethylsilyl derivatization, O-methoxym derivatization and gas chromatography / mass spectrum analysis. Details of the experiment of this specific example are described in Example 1 below. The LX analog can also be screened for binding activity to the LX receptor recognition site, for example by contacting the compound with the receptor recognition site and measuring whether and to what extent the compound binds. Examples of kinetic binding assays include homologous substitution, competitive binding, isotherm and equilibrium binding assays. The receptor recognition site may be present in the normal state or it may be induced by medical condition, injury or artificial means. For example, retinoic acid, PMA or DMSO can be used to induce differentiation into HL-60 cells. Differentiated HL-60 cells are LXA₄Expresses a specific receptor recognition site. Examples of other cells that can be screened for LX specificity include PMNs, epithelial cells and peripheral blood monocytes. The choice of competitive ligand depends on the nature of the recognition site, the structure of the natural substrate, the structural or functional analogs of the natural substrate known in the art, and other factors considered by those skilled in the art in making such decisions. Dependent. Such ligands also include antagonists of known receptors. The compounds of the present invention are prepared by standard techniques known in the field of radiochemistry or synthetic chemistry.²H,³H,¹³C and¹⁴It can be radioactively labeled with an isotope containing C. In a specific example of this method, the induced LXA₄The structural specificity of the recognition site, LXB₄, LTC₄, LTB₄And trihydroxyheptanic acid methyl ester were used for evaluation. Details of the experiment of this specific example are described in Example 2 below. Furthermore, the compounds of the present invention can be used to exert a certain effect on a specific cell type as a model for the development of inflammation or injury. For example, LXA₄Is intracellular Ca²⁺Stimulates chemotaxis without fluidization, lipid reconstruction and aggregation in human PMN (Palmblad, J. et al. Biochem. Biophys. Res. Communi. (1987) 145: 168; Lee, TH et al. Clin. Sci (1989) 77: 195; Nigam, S. et al. J. Cell. Physiol. (L990) 143: 512; Luscinskas, FW et al. (1990) Biochem. Pharmacol. 39: 355). LXA₄Also, LTB₄And FMLP-induced response eg IP₃It also blocks both generations. LXB₄Also stimulates lipid reconstruction. LXA₄Activates isolated PKCs and is specific for the γ-subspecies of PKCs found in the cerebrospinal cord. (Hansson, A. et al. Biochem. Biophys. Res. Communi. (1986) 134: 1215; Shearman, MS et al. FEBS Lett. (1989) 245: 167); Publications Nicolaou, KC et al. Angew. Chem. Int. Ed. Engl. (1991) 30: 1100 and the references cited therein are incorporated herein by reference. The present invention will be further described by the following examples, which should not be construed as further limiting. All references cited in all parts of this application, including background, and the content of the issued patent are incorporated herein by reference.<u style="single">Example</u>Example 1: Synthesis of lipoxin analog compounds<img file="JP4060366B2_D0050.tif" /><u style="single">Preparation of methyl ester precursor of compound 1:</u>N-Propylamine (0.05 ml, 0.61 mmol) and Pd (PPh3) 4 in a solution of 3-methyl-3-trimethylsiloxy-1-bromo-1-octene (130 mg, 0.44 mmol) to benzene (1.5 ml) (20 mg, 0.02 mmol) was added to protect the solution from light. Then, it was degassed by the freeze-thaw method and stirred at room temperature for 45 minutes. (7E, 9E, 5S, 6R) Methyl 5,6-di (tert-butyldimethylsiloxy) Dodeca-7,9-diene-11-inoate (183 mg, 0.44 mmol) (Compound 12) and copper iodide (14 mg, 0.07 mmol) was added and the solution was degassed again by the freeze-thaw method. The mixture was stirred at room temperature for 3 hours, inactivated with saturated aqueous NH4Cl and extracted with ether. It was then washed with saline and dried over DDL4 to evaporate the solvent. Flash column chromatography (silica, 3% ether hexane) gave the pure compound as a colorless liquid (171 mg, 57% yield). To a solution of this compound (171 mg, 0.25 mmol) in THF (0.5 ml) was added n-BuN4F (0.9 ml, 0.90 mmol) and the mixture was stirred at room temperature. The reaction was completed in 2 hours, at which point it was poured into water and extracted with ether. The ether extract was washed with brine and dried over Na2SO4 to evaporate the solvent. Flash column chromatography (silica, 4% MeOH / CH2Cl2) gave methyl ester (24 mg) with some of the corresponding lactones. HPLC retention time: 9.39 minutes (microsolve reverse phase, 4.6 mmx25 cm, C-18 column, MeOH / H2O = 70: 30, flow rate = 1 ml / min, UV detector = 300 nm). UV in MeOH: λmax 283, 294, 311 nm. 1H-NMR (500MHz, CDCl3): δ6.53 (dd.15.2,10.9Hz, 1H), 6.32 (dd, J = 15.1,11.OHz, 1H), 6.17 (d, J = 15.9Hz,<u style="single">Preparation of methyl ester precursor of compound 2:</u>A solution of the methyl ester precursor of Compound 1 (3 mg in CH2Cl2 (1 ml)) was mixed with Lindlar catalyst (1 mg) and placed in a hydrogen atmosphere. The mixture was stirred in the dark at room temperature and then subjected to HPLC until about 80% conversion (1 hour). Pure methyl ester was obtained by filtration through the sea light, evaporation of the solvent, and separation by HPLC. HPLC retention time: 10.02 min (microsolve reverse phase, 10 mmx25 cm, C-18 column, MeOH / H2O = 70: 30, flow rate = 4 ml / min, UV detector = 300 nm). UV in MeOH: ηmax 287, 301, 315nm.<u style="single">Preparation of methyl ester precursor of compound 3:</u>This compound was made in the same manner as the preparation of the methyl ester precursor of compound 1 (from 3-cyclohexyl-3-trimethylsiloxy-1-bromo-1-octene). Desilylation of this compound was also carried out in the same manner to obtain a methyl ester. HPLC retention time: 8.02 min (microsolve reverse phase, 4.6 mmx25 cm, C-18 column, MeOH / H2O = 70: 30, flow rate = 1 ml / min, UV detector = 300 nm). UV in MeOH: λmax 282, 293, 311 nm. 1H-NMR (360MHz, CDCl3): δ6.56 (dd.15.4,10.9Hz, 1H), 6.33 (dd, J = 15.2,10.9Hz, 1H), 6.13 (dd, J = 15.8,6.5Hz, 1H) , 5.81 (dd, J = 15.2,6.4Hz, 1H), 5.80 (d, J = 15.6Hz, 1H), 5.73 (dd, J = 15.4, 2.1Hz, 1H), 4.15 (br, 1H), 3.93- 3.90 (m, 1H), 3.67 (br, 1H) 3.65 (s, 3H), 2.34 (t, 2H), 1.82-1.65 (m, 10H), 1.46-1.38 (m, 3H), 1.26-1.01 (m) , 5H).<u style="single">Preparation of methyl ester precursor of compound 4:</u>Selective hydrogenation of the methyl ester precursor of compound 3 followed by HPLC purification gave the methyl ester precursor of compound 4. HPLC retention time; 9.72 min (microsolve reverse phase, 10 mmx25 cm, C-18 column, MeOH / H2O = 70: 30, flow rate = 4 ml / min, UV detector = 300 nm). UV in MeOH: λmax 288, 301, 315nm. 1H-NMR (250MHz, C6D6): δ6.66-6.89 (m, 2H), 5.95-6.24 (m, 4H), 5.55-5.66 (m, 2H), 3.82 (m, 1H), 3.73 (m, 1H) ), 3.41 (m, 1H) 3.31 (s, 3H, OCH3), 2.08 (t, 2H, CH2COO), 1.00-1.81 (m, 18H). This methyl ester can be converted to the corresponding alcohol using standard techniques.<u style="single">Synthesis of 15 (R) -15-methyl-LXA4 and 15 (±) methyl-LXA4</u>Webber, SE et al. (1988) Adv.Exp.Med.Biol.229: 61; Nicolaou, KCet al. (1991) Angew.Chem Int.Ed.Engl.30: 1100; and Vorbruggen, H.et al .: "Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids" (Roberts, SM, Schein mann, F.eds.) As Oxford, Pergamon Press, using Friedel-crafts acylation of bis (trimethylsilyl) acetylene with hexanoyl chloride. , Approximately 1 g of acetylene ketone a is produced and reduced with (-)-pinile-9-BBN to give (S) alcohol in CH3N2 to produce methyl at C-15.<img file="JP4060366B2_D0051.tif" />Instead, treat keto groups with CH3MgBr, as in Vorbruggen, H. et al .: "Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids" (Roberts, SM, Scheinmann, F.eds.) Oxford, Pergamon Press. Then (6070 ° C), add 2,6-lutidine (5.2 ml) and tert-butyldimethylsilyl trifurate (6.9 ml) in sequence, and add 15 of b in dry CH2Cl2 (~ 20 ml) at 0 ° C. (±) Methyl (2-5 g) can be obtained. The reaction is mixed for 1 hour, then diluted with 100 ml of ether for aqueous extraction and dried over DDL4.<img file="JP4060366B2_D0052.tif" />The product c is then combined with d,<img file="JP4060366B2_D0053.tif" />This is Nicolaou, KCet al. (1991) Angew.Chem.Int.Ed.Engl.30: 1100; Nicolaou, KCet al. (1989) J.Org.Chem.54:5527 and Webber, SBet al. (1988) ) Generate as Adv.Exp.Med.Biol.229:61. Structure d from Fragment A of Scheme I was suspended in 4.0 eq AgNO3 containing EtOH: THF: H2O (1: 1: 1) followed by 7.0 eq KCN at 0-25 ° C for 2 hours. A C-methyl ester protected 15-methyl-LXA4 analog was produced, concentrated and saponified in THF at 4 ° C with LiOH (2 drops, 0.1 M) for 12-24 hours to accommodate. Obtain free acid.<u style="single">Synthesis of 16-dimethyl-LXA4</u><img file="JP4060366B2_D0054.tif" />This compound uses a similar strategy to bind d above to e with reference to above, to bind f to form a 15-phenyl-LXA4 analog, or to bind g to 17-. It is produced by producing an m-chlorophenoxy-LXA4 analog.<img file="JP4060366B2_D0055.tif" />Appropriate C fragments of Diagram I (ie, e, f, g, h) are Raduchel, B and Vorbruggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14: for corresponding known prostaglandin analogs, respectively. Manufacture as reviewed in 263. At h, R = H; Cl, methoxy or halogen.<u style="single">13,14-Synthesis of acetylene LXA4 and halogen-containing analogs</u><img file="JP4060366B2_D0056.tif" />A2B2 generated from the fragment of Diagram II is used to produce the corresponding C2 fragment for binding. Structures j and k were generated as Nicolaou, K Cet al. (1989) J. Org. Chem. 54: 5527, Raduchel, B and Vorbruggen, H. (1985) Adv. Prostaglandin Thrombox ane Leukotriene Res. 14: It is methylated as in 263 and combined with 7 to give these LX analogs. These materials may be subjected to RP-HPLC and purified with reference to the above.<img file="JP4060366B2_D0057.tif" /><u style="single">14,15-Synthesis of acetylene LXA4</u><img file="JP4060366B2_D0058.tif" />The designed bound A2B2 fragment can be made from the binding of fragments A1 and B1 shown in Route II and the structure of 7 or 4 can be carried out as described above to bind to fragment C2. The l precursor of the C2 fragment can be prepared as Raduchel, B and Vorbruggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14: 263 for prostaglandin analogs.<img file="JP4060366B2_D0059.tif" />Precursor m prepared in the above [Nicolaou, K Cet al (1989) J. Org. Chem. 54: 5527] was added to benzene with l having Me2Al in 1.2 to 0.05 equivalents of Pd (PPh3) 4, 0.16 equivalents. Add to CuI, n-PrNH2 at room temperature for 2-3 hours to give n.<img file="JP4060366B2_D0060.tif" />The alcohol protecting group TBDMS = R was removed with 10 equivalents of HF-pyr, THF, 0-25 ° C (4 hours) and then contacted with 3.0 equivalents of Et3N, MeOH, 25 ° C for 15 minutes to LXs. [Serhan, CN (1990) Meth. Enzymol. 187: 167 and Nicolaou, K Cet al . (1989) J.Org.Chem.54:5527]. After mild treatment with 5% by weight Lindlar catalyst, the extracted material may be saponified with LiOH in THF to produce the free acid of the target molecule, which Serhan, CN (1990) ) Meth. Enzymol. 187: 167 can be further purified by the RP-HPLC gradient mobile phase.<u style="single">Synthesis of 15 (±) methyl-cyclo-LXA4</u><img file="JP4060366B2_D0061.tif" />Compound o as a SiMe3 derivative can be dissolved in degassed benzene (20 ml) and placed in a 100 ml round bottom flask in an atmosphere rich in argon (~ 1 ml). To this is added 3.0 equivalents of vinyl bromide fragment with reference to: This coupling reaction can be followed by injecting an aliquot of this suspension into RP-HPLC, which is carried out in catalytic amounts of Pd (PPh3) 4 and CuI and followed by UV abundance with a high performance scanning diode. .. The traveling line travels at 23 ° C for 1-3 hours, after which the material is extracted at ethyl acetate: H2O = 4: 1v / v and concentrated by evaporation on a rotary evaporator. Methyl esters can be saponified in LiOH / THF to give a quantitative yield of free carboxylic acid. Other derivatives can be prepared using Fragment A with different portions of Fragment B, as described above, which have been substituted to give, for example, dimethyl or other derivatives. This can be obtained by using readily available ketone p and treating with CH3MgBr to produce q, which also uses conventional techniques such as Pd (O) -CuI coupling, as described above. Can be used to bind fragment A. An increase in chain length from C-15 is also obtained.<img file="JP4060366B2_D0062.tif" /><u style="single">Synthesis of 5-methyl-LXB4 and 4,4-dimethyl-LXB4</u>5-Methyl-LXB4 inhibits or prolongs the formation of 5-oxo-LXB4. Using the general scheme outlined above, the A fragment can be constructed to carry 5-methyl in the r precursor of vinyl bromide, which can be carried by Pd (O) -CuI coupling. , Bind to the bound B + C fragment.<img file="JP4060366B2_D0063.tif" />Vinyl bromide r can be obtained from s having either a dimethyl or hydrogen substituent at its C-4 position. Protected precursor t containing fragment B + C is available in reference [Nicolaou, KCet. Generate as reported in al. (1991) Angew.Chem.Int.Ed.Engl.30: 1100-16]. Compound t is converted to s or 28 by binding to the illustrated vinyl bromide. In this way, the target molecule can be produced by adding 1.0 equivalent (1 g) of r together with t injected into Et2NH at 1.2 equivalent to a degassed round bottom flask containing Et2NH as a solvent. Pd (Ph3P) 4 is added in 0.02 equivalents to give the 8 (9) -containing acetylene precursor methyl ester of s. The material was extracted, suspended in quinoline (0.5 eq) in CH2Cl2, subjected to evaporation on a rotary evaporator, and hydrogenated with Lindlar catalyst (10%; 25 ° C) and H2 stream. It selectively reduces the acetylene double bond at position 8. The formation of the tetraene component of the methyl ester of 5-methyl-LXB4 or 4-dimethyl-LXB4 methyl ester can be followed by RP-HPLC to assess the completion of reduction (ie 1-3 hours). The product is then treated with LiOH in 25 μl of THF and the methyl # ester is saponified to the corresponding free acid by adding H2O at 024 ° C for 8-24 hours. Example 2: Metabolism of lipoxin A4 by human promyelocytic leukemia cells and monocytes: Half-life test HL-60 cells from the US Standards Collection (Rockville, MD), other cell culture reagents from GIBCO (Grand) I bought it from Island, NY). Belsen (EDTA) was purchased from Whittaker Bioproducts (Walkersville, MD). Synthetic 11,12-acetylene LXA4 methyl esters and LXs from Cascade Biochemical (Reading, UK), 15 (S) -15-m-PGE1, PGE1 and 5-HETE from Cayman Chemical Co. (Ann Arbor,) It was from MI). [11, 12-3H] LXA4 was manufactured from 11,12-acetylene LXA4 using Lindlar catalyst as custom tritiumization (NET-259, lot number 0 2793-275, New England Nuclear, Boston, MA). .. The tritized product was isolated using RP-HPLC [Fiore et al. (1992) J. Biol. Chem. 267: 16168; Serhan, CN (1990) Meth. Enzymol. 187: 167]. Methoxyamine and NAD were obtained from Sigma Chemical Company (St. Louis, MO). Manganese dioxide and Adams reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI). Human PMNs were obtained from healthy volunteers by gradient centrifugation of fresh heparinized venous blood [Boyum, A. (1986) Scand.J. Clin.Lab.Invest. 21:77]. HL-60 cells were seeded in RPMI fortified with penicillin (100 U / ml), streptomycin (100 μ / ml), fetal bovine serum (10%) (Hyclone, Logan, UT) and warmed in a 250 ml plastic flask. Placed (37 ° C in 5% CO2 atmosphere). Individual flasks containing 5x10-7 HL-60 cells were heated in the presence or absence of phorbol 12-millistate 13-acetate (PMA) (10 or 16 nM, 24-27 hours) and Collins. Adhesion was followed for induction of macrophage-like phenotype, as per SJ (1978) Blood 70: 1233. Peripheral blood monocytes were obtained after plate culture of fresh mononuclear cells at 37 ° C for 1 hour in a plastic Petri dish containing glucose (1 mg / ml) and PBS [Goldyne, ME et al. (1984). J.Biol.Chem.259: 8815]. The non-attached cells were removed and the attached mononuclear cells were gently resuspended using Versen (7 ml / plate) and washed with PBS. PMN (&gt; 98%), adherent monocytes (&gt; 95%) and HL-60 cells were counted by light microscopy while suspended in PBS for warming, and &lt;2-3% in each case was trypan. It was transparent to blue. For some experiments, cell-free supernatants were prepared from HL-60 cells treated with PMA (16nM) for 24-72 hours. After collection, the differentiated cells were washed, then lysed by freeze-thaw and centrifuged (100,000 g, 1 hour). Incubation with eicosanoids was stopped with cold methanol containing either PGB2 or 5-HETE as an internal standard (5-HETE was used when quantifying 15-oxo-ETE). The product was extracted using Sep-PakC18 and routinely chromatographed as per Serhan, CN (1990) Meth. Enzymol. 187: 167. The RP-HPLC system consists of an LKB gradient dual pump equipped with an Altex Ultrasphere-ODS column (4.6 mmx25 cm), a flow rate of 1 ml / min, methanol / H2O / acetic acid (65: 35: 0.01), and ω metabolites of LTB4 (65: 35: 0.01). That is, it consisted of elution (0-20 minutes) with methanol / acetic acid (99.99 / 0.1) on the linear gradient (20-45 minutes) used to quantify 20-COOH and 20-OH-LTB4) and LXA4. .. The average restoration of the internal standard was 82.2 and SD was 7.9 (n = 13). Compounds I-IV were separated on 100,000 g using an Altex Ultrasphere-ODS column (10 mmx25 cm) eluted with methanol / H2O / acetic acid (60: 40: 0.01, v / v / v) at a flow rate of 3.0 ml / min. Formation of 15-oxo-ETE by Qing [Agins, A Pet al. (1987) Agents Actions 21: 397; Xun, See CQ. et al. (1991) Biochem. J. 279: 553; and Sok, DE. et al. (1988) Biochem. Biophys. Res. Communi. 156: 524] after RP-HPLC. Elution with H2O / acetic acid (70: 30: 0.01, v / v / v) was quantified using an ODS column (4.6 mmx25 cm) traced at 280 nm at a flow rate of 1 ml / min. Monocyte-derived products are also chromatographed by chromatography using a Hypersil column (5IL, 4mmx300mm), eluting with methanol / H2O / acetic acid (60: 40: 0.01, v / v / v) and a flow rate of 1 ml / min. Attached to. The online spectrum was recorded using a diode array detector (Hewlett-Packard 1040M series type II) equipped with software (DOS series) called HPLC3D ChemStation. Spectrum was obtained using a 4 nm step, Bw = 10 nm, range = 235-360 nm, with a sampling interval of 1.28 seconds. GC / MS was performed on a Hewlett-Packard 5971A mass-selective detector quadrupole equipped with a workstation called HPG1030A and GC5890. The column was HP Ultra2 (crosslinked 5% phenylmethyl silicone gum phase; 25mx0.2mmx0.33μm) and injection was performed in splitless mode in bis (TMS) trifluoroacetamide (BSTFA). The temperature program started at 150 ° C and reached 250 ° C in 10 minutes and 325 ° C in 20 minutes. Standard saturated fatty acid methyl esters (C16-C26) gave the following retention times (minutes: seconds, average of n = 6): C16, 8.03; C18, 9.77; C20, 12.22; C22, 16.11; C24, 20.72; C26, 23.62. Using this, Serhan, CN (1990) Meth.Enzymol.187: As shown in 167, the C value of each LX-derived metabolite was calculated. Diazomethane was produced and the methyl ester product was treated with BSTFA (Pierce Chemical Co., Rockford, IL) to obtain a Me3Si derivative. O-methoxym derivatives of methyl esters were prepared as per Kelly, RW and Abel, MH (1983) Biomed. Mass Spectrom. 10: 276. Contact hydrogenation was performed with Adams' reagent (Aldrich, Milwaukee, WI) in methanol (1 ml) by saturating Platinum Oxide IV (1-2 mg) with a stream of hydrogen foam (20 minutes, room temperature). After extraction, the material was treated with diazomethane and then BSTFA (overnight; room temperature). Results Metabolism of LXA4: Intact neutrophils from the peripheral blood of healthy donors did not significantly metabolize foreign LXA4, but cells from the same donor rapidly converted LXA4 by ω oxidation. In contrast, PMA-treated HL-60 cells with monocyte / macrophage-like characteristics rapidly converted LXA4. Within the first 60 seconds of contact,&gt; 70% of LXA4 was metabolized. In the absence of PMA treatment, neither intact HL-60 cells (undifferentiated) nor their cell-free supernatant (100,000 g) converted LXA4 (n = 3). Differentiated HL-60 cells warmed with LXA4 converted this eicosanoid to several products. Labeled LXA4 was converted to four main products with tritium (referred to as compounds I-IV), which were collected and further analyzed. Manufactured as per 276. Contact hydrogenation was performed with Adams' reagent (Aldrich, Milwaukee, WI) in methanol (1 ml) by saturating Platinum Oxide IV (1-2 mg) with a stream of hydrogen foam (20 minutes, room temperature). After extraction, the material was treated with diazomethane and then BSTFA (overnight; room temperature). Results Metabolism of LXA4: Intact neutrophils from the peripheral blood of healthy donors did not significantly metabolize foreign LXA4, but cells from the same donor rapidly converted LXA4 by ω oxidation. In contrast, PMA-treated HL-60 cells with monocyte / macrophage-like characteristics rapidly converted LXA4. Within the first 60 seconds of contact,&gt; 70% of LXA4 was metabolized. In the absence of PMA treatment, neither intact HL-60 cells (undifferentiated) nor their cell-free supernatant (100,000 g) converted LXA4 (n = 3). Differentiated HL-60 cells warmed with LXA4 converted this eicosanoid to several products. Labeled LXA4 was converted to four main products with tritium (referred to as compounds I-IV), which were collected and further analyzed. Manufactured as per 276. Contact hydrogenation was performed with Adams' reagent (Aldrich, Milwaukee, WI) in methanol (1 ml) by saturating Platinum Oxide IV (1-2 mg) with a stream of hydrogen foam (20 minutes, room temperature). After extraction, the material was treated with diazomethane and then BSTFA (overnight; room temperature). Results Metabolism of LXA4: Intact neutrophils from the peripheral blood of healthy donors did not significantly metabolize foreign LXA4, but cells from the same donor rapidly converted LXA4 by ω oxidation. In contrast, PMA-treated HL-60 cells with monocyte / macrophage-like characteristics rapidly converted LXA4. Within the first 60 seconds of contact,&gt; 70% of LXA4 was metabolized. In the absence of PMA treatment, neither intact HL-60 cells (undifferentiated) nor their cell-free supernatant (100,000 g) converted LXA4 (n = 3). Differentiated HL-60 cells warmed with LXA4 converted this eicosanoid to several products. Labeled LXA4 was converted to four main products with tritium (referred to as compounds I-IV), which were collected and further analyzed. 000g) also did not convert LXA4 (n = 3). Differentiated HL-60 cells warmed with LXA4 converted this eicosanoid to several products. Labeled LXA4 was converted to four major products with tritium (referred to as Compounds I-IV), which were collected and further analyzed. 000g) also did not convert LXA4 (n = 3). Differentiated HL-60 cells warmed with LXA4 converted this eicosanoid to several products. Labeled LXA4 was converted to four major products with tritium (referred to as Compounds I-IV), which were collected and further analyzed.<u style="single">Structure of compounds I ~ IV</u>: Retention time on RP-HPLC was determined using demarcation 3H labeled elution images and pooled from several incubators to obtain quantities that would allow structural studies of these compounds. Labeled samples were chromatographed, individually collected from within these regions and subjected to GC / MS analysis. Selected ion tracking of the products obtained after treatment with diazomethane and BSTFA showed that compounds I-IV each gave a different retention time than LXA4, but m / z 203 [-CH (OSiMe3), respectively. )-(CH2) 3-COOCH3] showed outstanding ions, revealing that carbons 1-5 of LXA4 (carbon of carboxyl is number 1) were not modified. The methyl ester, a trimethylsilyl derivative of LXA4, showed predominant ions at m / z 203 (basic peak) and 173 along with its molecular ion at 582 in its electron impact spectrum (M + 4). Other ions for the diagnostic value of this derivative of LXA4 are m / z 171 (203-32), 409 (M-173), 379 (M-203), 482 (M-100) and 492 (M-90). Observed [Serhan, CNet al. (1984) Proc. Natl. Acad. Sci. USA 81: 5335; and Serhan, CN (1990) Meth. Enzymol. 187: 167]. It is noteworthy that LXs are generally known to give extremely weak molecular ion peaks [Serhan, CNet al. (1984) Proc. Natl. Acad. Sci. USA 81: 5335]. Nevertheless, the compound labeled I &amp; II also has m / z 173 (Me3SiO).₊= CH- (CH2) 3-CH3) has predominant ions, indicating that the carbon 15-20 fragments of these LXA4 inducible products are intact, but in compounds III and IV, m / z 173 is apparent. It wasn't. Therefore, the conclusion that compounds I-IV are metabolites of LXA4 is their physical properties (HPLC and GC / MS), the finding that they have tritium labeling, and HL-60 cells not treated with PMA. Based on the absence of these products in the warming with. Next, we focused on compounds III and IV. This is because it seemed to represent a structural modification of LXA4 with carbon 15-20 fragments. Since ω oxidation (hydroxylation at carbon 20) is one possibility, the ions that can be attributed to the respective 20-OH and 20-COOH of LXA4 after derivative formation, namely m / z 261 and 217 (m / z 261 and 217 (). Me3SiO₊= CH- (CH2) 4-CH2OSiMe3 and Me3SiO₊= CH- (CH2) 4-CO2Me) was scanned with the obtained GC-MS data image. Neither III nor IV showed predominant ions at either m / z 261 or 217, and these products did not appear to be the result of ω oxidation. The mass spectrum (C value: 24.3) of the Me3Si derivative, that is, the methyl ester of compound III was obtained. The predominant ions in that spectrum are m / z 203 (basic peak, CH (OSiMe3)-(CH2) 3-COOHCH3), 171 (203-32; elimination of CH3OH), 215 [(M-203) -90, trimethyl. Exclusion of silanol (Me3SiOH)] and 99 (O = C- (CH2) 4-CH3) were observed. Lower intensity ions are m / z 508 (M)₊) And 418 (M-90; lack of Me3SiOH). The presence of these ions suggested that the material co-eluted with 3H-labeled Compound III was a 15-oxo derivative of LXA4. This is evidence of some strains, namely m / z 173 (Me3SiO).₊Substantial lack of predominant ions at = CH- (CH2) 4-CH3), presence of m / z99 (O = C- (CH2) 4-CH3), absence of tetraene chromophore, and UVλmax at 335-340 nm This is supported by the emergence of a new chromophore. The tetraenone chromophore was produced by treating LXA4 with MnO2 in chloroform, as used in the prostaglandin conversion [Anggard, E. and Samuelsson, B. (1964) J. Biol. Chem. 239: 4097]. confirmed. The mass spectra of catalytic hydrogenated products are m / z 203 (basic peak), m / z 99 (66%), m / z 313 (M-203 or M-CH (OSiMe3)-(CH2) 3-COOHCH3; 35. A C value of 25.1 was given with the dominant ions at%) and m / z 171 (36%) and the absence of dominant ions at m / z 173. The weaker ion is m / z 516 (M₊) And m / z 426 (M-90). Therefore, the upward shift of 8amu, and the fragmentation of this saturated derivative, is consistent with the production of the corresponding 15-oxo derivative. To further verify this LXA4 inducible product, an aliquot of the material eluting under the peak labeled III was treated with diazomethane and then methoxymed [Bergholte, JM et al. (1987) Arch.Biochem.Biophys. .257: 444], processed by BSTFA. Its spectrum (C value 25.4) is at m / z 203 (basic peak), 171 (203-32; lack of CH3OH) and 229 [M-128 or CH3O-N = C- (CH2) 4CH3- (2x90)]. Showed the predominant ion of. Lower intensity ions are m / z 537 (M)₊), 466 (M-71, αt cleavage ion M-CH2 (CH2) 3CH3), 481 (M-56 or M-CH2 = CH-CH2-CH3, McClaferty rearranged ion), 431 [M-106 ( Probably C7H5N₊) Lack], 401 [M-136 (elimination of Me3SiOH + CH3 + · OCH3)] and 460 (elimination of M-77, NOCH3 + MeOH). Again, the alcohol-containing C-15 fragment (Me3SiO)₊The ion at m / z 173, which appears to be of -CH- (CH2) 4-CH3) origin, was virtually absent in its spectrum. Thus, the ions present correspond to the methyl ester, an O-methoxym derivative produced from a 15-oxo-containing derivative of LXA4. Taken together, the predominant ions observed in these different derivatives suggest that the material eluting under the peak labeled III was the 15-oxo product of LXA4 (ie, 15-oxoLXA4). .. The mass spectra of the Me3Si derivative (C value 26.0), which is the methyl ester of compound IV, are m / z 203 (basic peak, CH (OSiMe3)-(CH2) 3-COOHCH3), 171 (203-32; lack of CH3OH), The predominant ions at 99 (O = C- (CH2) 4-CH3) and 307 (M-203 or M-CH (OSiMe3)-(CH2) 3-COOHCH3) were shown. Lower intensity ions are m / z 510 (M)₊), 420 (M-90, lack of trimethylsilanol) and 208 (M- (99 + 203)). Its UV spectrum shows triple-line absorption with maximum values at 259, 269 and 280 nm, consistent with conjugated triene chromophores. The presence of these ions, and the UV spectrum, suggest that IV was a dihydro-15-oxo metabolite of LXA4. This basic structure shows the presence of ions at m / z 99, which coincides with the keto group at carbon 15 and m / z 203 as the basic peak indicating that the alcohol groups at carbon 5 and 6 remain intact. Supported by the existence of. In addition, the absence of the trienone chromophore (λcal = 310 nm) indicates that the lack of double bonds was present at positions Δ13-14, giving the observed triene chromophore. Taken together, these results indicate that Compound IV was 13,14-dihydro-15-oxo-LXA4. Methyl ester, a Me3SiO derivative of compound II (C value-25.4), is m / z 203 (basic peak; CH (OSiMe3)-(CH2) 3-COOHCH3), 173 (Me3SiO).₊= CH- (CH2) 4-CH3), 171 (203-32) and 508 (M)₊) Gives a peak. Its molecular ions were two mass units higher than the LXA4 derivative. These ions, and absorption: the 259 nm, 269 and 282 nm triplet bands of λmax MeOH suggest that Compound II was a dihydro derivative of LXA4. The methyl ester from HL-60 cells, the Me3SiO derivative of compound I, gave two products in GC. The major one (C value = 25.0) gave its mass spectrum an ion similar to LXA4, but its molecular ions were at m / z 586, m / z 555 (M-31) and 496 (M-). Ions were also present in 90), suggesting that two of the four double bonds were reduced (not shown). However, the same product was not observed in peripheral blood monocytes (see above), so the material derived from HL-60 cells from peak I was not further characterized in this experiment. The structures I-IV also show that LXA4 is not metabolized by ω oxidation by healthy leukocytes, both are dehydrogenated with carbon-15 alcohols, and conjugated tetraene is converted to a triene structure. Taken together, these findings indicate that LXA4 is known to react similarly to NAD-dependent 15-prostaglandin dehydrogenase (5-PGDH), a prostanoid as a substrate [Anggard]. , E. and Samuelsson, B. (1964) J. Biol. Chem. 239: 4097, and Hansen, HS (1976) Prostaglandins 12: 647. The activity of 15-PDGH has recently been shown to be induced in HL-60 cells [Xun, CQ.et al. (1991) Biochem.J. 279: 553], apparently using 15-HETE as a substrate. Use with 92% efficiency compared to PGE2 [Agins, AP. et al, (1987) Agents Actions 21: 397]. In fact, 100,000 g supernatant prepared from PMA-treated HL-60 cells converted 15-HETE to 15-oxo-ETE, indicating the presence of post-differentiation dehydrogenase activity. LXA4 competed for catalysis of 15-HETE and showed Ki = 8.2 ± 2.6 μM (SEM, n = 6) calculated from the Lineweaver-Burk plot. At equimolar concentrations of LXA4 and 15-HETE, LXA4 inhibited 15-oxo-ETE formation by almost 50%. Relative conversion to LX with 100,000 g supernatant showed that LXA4, 11-trans-LXA4 and LXB4 were converted, but 15-methyl-PGE1 was not. Taken together, these results suggest that LXA4&gt; 11-trans-LXA4&gt; LXB4 is the substrate for 15-PGDH, or equivalent enzyme systems. PMA induces the differentiation of HL-60 cells into monocyte-macrophage-like cell lines [Collins, SJ (1987) Blood 70: 1233], thus preserving peripheral blood monocytes to metabolize LX. I decided. LX has a strong effect on monocytes [Stenke, L. et al. (1991b) Biochem.Biophys.Res.Commun 180: 255], and these cells do not ω-oxidize eicosanoids [Goldyne, ME et al. . (1984) J.Biol.Chem 259: 8815]. LLXA4 has 15-oxo-LXA4, conjugated triene when contacted with both suspensions of intact monocytes (n = 5) and permeable cells (freeze-thaw or saponinized, n = 5). It was converted to the products 13,14-dihydro-LXA4 and 13,14-dihydro-15-oxo-LXA4. Similar to differentiated HL-60, monocytes rapidly converted LXA4 within 30 minutes (&gt; 60%). The temporal relationship for the formation of these metabolites in both intact and permeable monocytes is similar, suggesting that the metabolite 15-oxo-LXA4 is a transient intermediate. In the monocyte suspension warmed with 3H-LXA4 (d = 33), 13,14-dihydro-15-oxo-LXA4 and 13,14-dihydro-LXA4 are the main products with radiolabeling, respectively. Met. A product eluted before 13,14-dihydro-LXA4 was observed at 15.5-17 minutes, which also showed triene chromophores, resulting from the cis-trans isomerization that occurred during the treatment 13,14. It is noteworthy that it appeared to be the 11-trans isomer of -dihydro-LXA4. The 11-cis binding of undenatured LXA4 is unstable and easily isomerizes to total trans during extraction and isolation [Romano, M. and Serhan, CN (1992) Biochemistry 31: 8269]. Example 3: Binding affinity for lipoxin receptor analogs Human promyelocytic leukemia cells (HL-60) were purchased from the American Reference Strain Collection (Rockville, MD). RPMI and cell culture reagents were obtained from GIBCO (Grand Island, NY). Synthetic LXA4, trihydroxyheptanoic acid (methyl ester), LXB4, LTD4, LTC4 and LTB4 were obtained from Biomol (Plymouth Meeting, PA) and SKF104353 was obtained from Smith Kline and Frech Laboratories. ONO4057 was obtained from ONO Pharmaceutical Co., Ltd (Osaka, Japan). [14,15-3H] LTB4 (32.8mCi / mmol), [1-14C] arachidonic acid (50.2mCi / mmol), 32PγATP (3,000mCi / mmol), [9,10-3H (N)] palmitic acid ( 30.0 mCi / mmol) and [9,10-3H (N)] myristic acid (30. 7mCi / mmol) was purchased from New England Nuclear (DuPont Co., Boston, MA). 11,12-Acetylene LXA4 was obtained from Cascade Biochemical (Reading, UK). The microcentrifuge tube filter (0.45 μm cellulose acetate) is from PGC Scientific (Gaithersburg, MD), and the silicone oil (d = 1.05 and d = 0.963) is Harwick Chemical Corp. (Akron), respectively. , OH) and Thomas Scientific (Swedesboro, NJ). Nitroblue tetrazolium, PMA, DMSO, protease, retinoic acid and actinomycin D were purchased from Sigma (St. Louis, MO). The island activating protein (IAP) was obtained from LIST Biological Lab., Inc. (Campbell, CA). Plastic instruments, Whatman LK6D TLC plates and solvents (HPLC grade) were obtained from Fisher (Springfield, NJ). Purchased from MO). The island activating protein (IAP) was obtained from LIST Biological Lab., Inc. (Campbell, CA). Plastic instruments, Whatman LK6D TLC plates and solvents (HPLC grade) were obtained from Fisher (Springfield, NJ). Purchased from MO). The island activating protein (IAP) was obtained from LIST Biological Lab., Inc. (Campbell, CA). Plastic instruments, Whatman LK6D TLC plates and solvents (HPLC grade) were obtained from Fisher (Springfield, NJ).<u style="single">[11,12-3H] Manufacture of LXA4</u>Titration of: 11,12-acetylene LXA4 methyl ester was performed under custom titration service (NET-259: 92-2326) by New England Nuclear (Boston, MA). Briefly, 11,12-acetylene methyl ester is characterized by UV absorption and reverse phase HPLC as in [Nicolaou, KC et al. (1985) J. Am. Chem. Soc. 107: 7517] and chloride. The tritium atmosphere was contacted in methylene at room temperature. This warming was stirred in the presence of Lindlar catalyst (1.0 mg, from Fluka Chemicals) for ~ 1 hour. The resulting mixture was stored in methanol and isolated using RP-HPLC. The tritized product was chromatographed as a methyl ester using a gradient HPLC system equipped with a photodiode array high performance spectrum detector [Serhan, CN, Methods in Enzymoloty: Arachidonate Related Lipid Mediators: Murphy, RC, Fitzpatrick, F. (eds.), Vol 187. Orlando, FL, Academic, (1990) p.167]. This mixture contains both [11,12-3H] LXA4 and [11,12-3H] -11-trans-LXA4 methyl ester (~ 1: 3 ratio) as determined by co-eluting with synthetic standards. ) Contained. After RP-HPLC, fractions containing [11,12-3H] LXA4 were collected and extracted into ethyl acetate. The free acid was produced by LiOH saponification [Fiore, S. et al. (1992) J. Biol. Chem. 267: 16168]. Materials from these fractions showed over 90% radioactivity when injected into UV electrochemical detection HPLC with a product with tetraene co-eluted with synthetic LXA4. Materials eluted with a retention time of reference LXA4 in two HPLC systems were sampled for binding experiments. The specific activity calculated for [11,12-3H] LXA4 was 40.5 Ci / mmol.<u style="single">Cell culture and differentiation</u>: HL-60 cells are seeded in RPMI fortified with 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum (Hyclone, Logan, UT) and placed in a 250 ml flask with a 5% CO2 atmosphere. It was warmed at 37 ° C. Then, in individual flasks containing ~ 50x106 cells / ml, dimethyl sulfoxide (DMSO) (1.12% by volume, 120 hours), retinoic acid (RA) (1 μM, 120 hours) or holbol millistate acetate (PMA). ) (20 nM, 48 hours) was given. Ca2 before performing the binding test₊And Mg2₊Cells were washed twice in phosphate buffered saline (PBS) free of water, counted, and suspended in Tris buffer (10 mM) at pH 7.4 as 20x106 cells / ml [Fiore, S. et al. et al. (1992) J. Biol. Chem. 267: 16168]. As shown in [Imaizumi, M. and Breitman, TR (1986) Blood 67: 1273], reduction with nitroblue tetrazolium was performed to follow the induction of the polymorphonuclear phenotype, and the induction of the macrophage-like phenotype was performed. Cell attachment was determined [Collins, SJ (1978) Blood 70: 1233]. Endothelial cells (HUVEC) of human umbilical veins maintained in the third passage of culture were obtained from Dr. M. Gimbrone (Birmingham and Women's Hospital Department of Biology).<u style="single">Isolation of PMN, platelets and RBC from peripheral blood</u>: Human PMN was obtained by modified Boyum method [Boyum, A (1986) Scand.J.Clin.Lab.Invest.21: 77] from fresh heparinized blood after venipuncture of healthy normal volunteers. .. Suspensions in PBS were followed for cell number and viability by their ability to eliminate trypan blue, both above 98%. Red blood cells were obtained from 10 ml of heparinized blood after 3 centrifuges in PBS (2,500 rpm, 21 ° C for 10 minutes). As previously described [Serhan, CNand Sheppard, K.-A. (1990) J.Clin.Invest.85: 772], using blood aspirated in acid citrate dextrose (9: 1v / v). , Platelets were isolated.<u style="single">Ligand binding</u>The binding of: 3H-LXA4 and 3H-LXB4 was basically carried out as in Fiore, S. et al. (1992) J. Biol. Chem. 267: 16168. Cell cells in 10 mM Tris buffer (pH 7.4, Ca2)₊2.5mM, Mg2₊After suspension in 1.2 mM), aliquots (0.5 ml) were warmed with 3H-ligand alone (0.3 nM) or in the presence of increasing concentrations of homoligand and other compounds (3-300 nM) (4). 20 minutes at ° C). The incubator was rapidly centrifuged on silicone oil (d = 1.028) (60 seconds, 12,000 g) and the radioactivity associated with the cells was determined by liquid scintillation counting (Wallac 1409, Pharmacia, Piscataway, NJ). Binding experiments with HUVEC cells were performed on a 12-well plate with 3.5xl05 cells / well. After 10 minutes, the wells were washed twice with PBS and cell-associated labels were collected by adding glacial acetic acid (0.5 ml). The results obtained from these tests were submitted for subsequent analysis using a program called Ligand (Elsevier-Biosoft, Cambridge, UK).<u style="single">PLD activity</u>: Human PMN and HL-60 cells (50x106 cells / ml) prepared as described above in PBS with 3H-myristic acid or 3H-palmitic acid (8 μCi / 50x106 cells) for 40-60 minutes at 37 ° C. It was kept warm. Cellular uptake spanned 60-80% of the added labels to the total phospholipid group of PMN and HL-60 cells, 7.1 ± 4.2% (n = 10; mean ± SD) and 32.6 ± 10.3% (n, respectively). = 6; mean ± SD) was captured. Incubation was performed at 37 ° C (10x106 cells / PBS 1 ml). As Billah, MM et al. (1989) J. Biol. Chem. 264: 17069, an agent for the formation of phosphatidylethanol (PEt) in 50 μl PBS or EtOH at 1:10 (v / v). : Added as PBS. Stop warming at the time indicated by adding 3.5 ml of ice-cold CHCl3 / MeOH (2/5, v / v) containing 1-14C-arachidonic acid (5,000 ppm), which was used here as the internal standard. Then, the restoration of the extract was quantified. [Serhan, CNand Samples were extracted using modified Bligh and Dyer extractions as in Sheppard, K.-A. (1990) J. Clin. Invest. 85: 772]. The organic phase concentrated in 50 μl of CHCl3 / MeOH (8/2, v / v) was combined with the organic phase of ethyl acetate / isooctane / acetic acid / water (110/50/20/100, v / v / v / v). Spotted on TLC plate of linear K6D unfolding for 50 minutes [Billah, MMet al. (1989) J.Biol.Chem.264: 17069]. In this system, phosphatidylic acid (PA) gives Rf = 0.1 ± 0.04, PET gives Rf = 0.1 ± 0.04; n: 38 ± SD, and other phospholipids (remaining at the origin) or triglycerides (Rf =). It was clearly separated from 0.75-0.90). Lipids were identified by co-elution with reference standards, visualized with iodine vapor, also spotted on each TLC plate and chromatographed. Regions corresponding to PA, PET and internal standards were scraped and quantified by liquid scintillation counting. PA and PA and cells labeled with [1-14C] arachidonic acid (0.25 μCi / 30x106 PMN) and 32PγATP (20 μCi / 50x106 PMN) in addition to 3H-myristic acid or 3H-palmitic acid labeling The formation stimulated by LXA4 for PEt production was followed. In these experiments, PA was degraded using ethyl acetate / isooctane / acetic acid (45/15/10, v / v / v) as the solvent system [Bocckino, SBet]. al. (1989) Anal.Biochem.180: 24], Rf = 0.46 ± 0.03 (n = 15) was given. All values reported in Tables 4 and 5 for PEt formation were calculated by subtracting that measured in the presence of the agonist and 0.5% EtOH from the dpm obtained in the presence of the agonist alone.<u style="single">Impact of IAP and staurosporine on LXA4-induced PLD activity</u>: Cells were contacted with either IAP or staurosporine prior to the PLD activity assay performed as described (see above). IAP treatment of PMN was performed as previously described [Nigam, S. et al. (1990) J. Cell Physiol. 143: 512] and HL-60 cells were 37 ° C. in the presence or absence of IAP. After warming for 2 hours in [Kanaho, Y.et al. (1992) J. Biol. Chem. 267: 23554]. Aliquots (107 cells / 0.5 ml cells) were added to 0.4 ml buffer. The warmed cells were then contacted with either 100 μl of excipient (PBS, EtOH, 0.04%) or LXA4 (10-7M and 10-9M) in the presence or absence of 0.5% EtOH. .. Staurosporine (100 nM) was added to the cell suspension at 37 ° C for 5 minutes prior to the addition of LXA4. Results Synthesis After producing [11,12-3H] LXA4, its specific binding to promyelocytic cells (HL-60) was characterized and described as [14,15-3H] LTB4 specific binding. A direct comparison was carried out. When the typical phenotypic markers were followed, untreated HL-60 cells showed low levels of specific binding to both 3H-LXA4 and 3H-LXB4 ligands. Differentiation induced by 5-day contact with DMSO (1.12%) or retinoic acid (1 μM) was accompanied by a 3- to 5-fold increase in specific binding to both radioligands. PMA-treated cells exhibiting macrophage-like phenotypes, ie, NBT-negative cells attached to plastic [Imaizumi, M. and Breitman, TR (1986) Blood 67: 1273; Collins, SJ (1978) Blood 70: See also 1233] also led to the appearance of specific binding to both 3H-LXA4 and 3H-LXB4 ligands. Equilibrium bonding with 3H-LXA4 at 4 ° C was reached in 10 minutes and remained virtually unchanged for the next 20 minutes. Actinomycin D (2 μg / ml) was added with PMA warming to assess whether induction of specific binding to both 3H-ligands required de novo synthesis. Actinomycin D inhibits the PMA-induced increase in specific binding to both labeled eicosanoids, suggesting inhibition of de novo protein synthesis as well as the also inhibited appearance of the specific binding site. The impact of protease and glycosidase treatment was assessed in two differentiated HL-60 cells and human PMN for specific binding of 3H-LXA4. Protease treatment reduced specific binding and provided additional evidence to support the protein component of the specific binding site of LXA4. Results from isothermal binding tests on differentiated HL-60 cells and [11,12-3H] LXA4 (0.1-30 nM) show that the specific binding site for [11,12-3H] LXA4 is KD = 0.6 ± It was shown to give 0.3 nM. Non-linear parts of the Scatchard plot were observed for LXA4-specific binding by human PMN [Fiore, S. et al. (1992) J. Biol. Chem. 267: 16168]. The results obtained here for LTB4-specific binding by HL-60 cells, namely KD = 0.12nM, were recently reported by Harada [Xie, M. et al. (1991) J. Clin. Invest. 88:45]. It basically matches the value obtained, that is, KD = 0.23nM. To further characterize the interaction of 3H-LXA4 with its specific binding site, differentiated HL-60 cells, LXA4, LXB4, LTB4, LTC4, and leukotriene receptor antagonist SKF 104353 [LTD4 antagonist; Harada , Y. (1990) Hiroshima J. Med-Sci.39; In 89], a competitive binding experiment was conducted, and ONO-4057 [LTB4 antagonist; Gleason, JGet al. (1987) J. Med. Chem. 30: 959] was assessed as a potential competitive ligand. .. Neither LXB4, LTB4, or trihydroxyheptanoic acid (methyl ester) (300nM) could substitute for the specific binding of 3H-LXA4 to differentiated HL-60 cells, whereas LTC4 was logarithmic 3 When added in excess by molar number, a ~ 30% reduction in specific binding occurred. The finding that LXA4 (300nM) was unable to compete for 3H-LTB4 (0.3nM) binding to differentiated HL-60 cells indicates that LXA4 and LTB4 interact with specific taxa-specific binding sites. Suggest. The leukotriene receptor antagonists SKF 104353 and ONO-4057 did not replace 3H-LXA4 by differentiated HL-60 cells, whereas SKF l04353 and LTD4 compete for specific 3H-LXA4 binding by HUVEC. It was effective. HUVEC showed a KD of 11,0 ± 2.6 nM and a Bmax of 2.5x10-10M for 3H-LXA4, and substantially the same values were calculated for LTD4 competition. In the case of 3H-LTB4, HUVEC did not specifically bind to LTB4, but non-specific cell association with this 3H-ligand was evident (n = 3; Not shown). Specific association of 3H-LXA4 was unclear in some other cell types examined. Here, the cultured cell lines of washed platelets, RBCs, β cells (Raji), or T cells (Jurkat) did not show specific binding to 3H-LXA4. Taken together, these results indicate that LXA4 interacts with a unique binding site in differentiated HL-60 cells that is not sensitive to any leukotriene receptor antagonist (SKF 104353 or ONO-4057). Shown. In HUVEC, the specific binding of 3H-LXA4 is sensitive to both LTD4 and SKF 104353, but not in ONO-4057, and the specific binding of 3H-LXA4 in this cell type is presumed. It suggests that it may reflect its interaction with the LTD4 receptor. LXA4 rapidly stimulates phosphatidic acid formation in human neutrophils [Nigan, S. et al. (1990) J. Cell Physiol. 143: 512]. PET and PA were followed with both PMN and HL-60 to determine if 3H-LXA4 binding provides PLD activation. The results showed that in these cells LXA4 stimulates PLD activity with a similar temporal response. PMN contacted with LXA4 (10-10M) rapidly produced PET, an ethanol capture product, within 60 seconds, but by 5 minutes it had decayed to baseline levels. In the absence of added EtOH, PET was not formed at statistically significant levels. For PEt, biphasic concentration dependence was obtained in both PMN and differentiated HL-60 cells. Apparent maximal response was observed in the concentration range of ~ 10-9 ~ 10-10M, with a second peak of activity observed at 10-7M LXA4. Below 10-8M, both the chemotactic peptides FMLP and LXA4 showed similar strengths, but PMNs from several donors appear to be slightly stronger. Met. To assess the potential contribution of other biosynthetic pathways, LXA4-induced PA formation was also examined with PMN labeled with both 32PγATP and [1-14C] arachidonic acid. 32P-labeled PA was only apparent at statistically significant levels 30 minutes after contact with LXA4 (10-7M). Similar results were obtained with the formation of 14C-labeled PAs derived from 14C arachidonic acid-labeled precursors. These findings indicate that LXA4 can also stimulate other pathways of PA formation in PMN, but only 30 minutes after contact. Only differentiated HL-60 cells preserved with LXA4 (expressing a specific binding site for 3H-LXA4) rapidly produced PET, which was apparent within 30 seconds. Undifferentiated HL-60 cells (10-9M) warmed with LXA4 did not rapidly produce PET. Concentration dependence by these cells also showed a biphasic response by LXA4, giving an apparent maximum at 10-9M. Next, we investigated signaling events that may be involved in LXA4-mediated PLD activation. Therefore, PMN and HL-60 cells were contacted with either IAP or staurosporine. The results show that LXA4-mediated PLD activity induced within the lower concentration range (10-9 to 10-10M) is sensitive to IAP treatment in both cell types, as well as higher LXA4 concentrations (10). PLD activity at -7M) indicates that it was inhibited by staurosporine. Thus, in both cell lines with concentrations below 10-8M, LXA4 interacts rapidly with the specific binding site that induces PLD activity, thus providing a functional response, but in the concentration range of LXA4 below μM. Within, it may also stimulate additional processes that can lead to activation of PLDs. Example 4: Testing of Lipoxin Bioactivity Some of the suitable LX analogs (structurally shown above as Compounds 1-8) were produced by total synthesis as described in Example 1. After the production and isolation of these compounds via HPLC, the neutrophil adhesion assay and epithelial cell translocation assay were first performed to determine the presence or absence of biological activity retention [Nash, S. et al. ( 1987) J.Clin.Invest.80: 1104-1113; Nash, S. et al. (1991) J.Clin.Invest.87: 1474-1477; Parkos, CA et al. (1991) J.Clin.Invest. 88: 1605-1612; Parkos, CA et al. (1992) J.Cell. Biol. 117: 757-764; Madara, J Let al. (1992) J. Tiss. Cult. Meth. 14: As described in 205-216]. Compounds 1-8 (10-7-10-10M) were found to inhibit the adhesion of neutrophils to endothelial cells and their transfer in epithelial cells. Acetylene precursors (Compounds 1, 3, 5 and 7) have been found to be more physically stable than their tetraene counterparts. Compound 7 showed no biological activity in the assay, without the alcohol group at position C15, or any other modification in this series. Therefore, the substituent at position C15 of LX appears to be required for at least the biological activity of the LXA4 analog. 15-Methyl-LXA4 (Compound 2) was also found to inhibit leukotriene B4-inspired polymorphonuclear (PMN) adhesion to human endothelial cells with an IC50 of ~ 1M. LX analogs 1-8 were found to inhibit migration with greater or equal efficacy than synthetic LXA4. Compound 7 was found to be essentially inactive within the concentration range of LXA4 or other analog-induced inhibition. The results of these neutrophil-containing bioassays show that LXA4 analogs with modifications at positions C15-C20 can retain their biological effects and inhibit PMN translocation and adhesion events. The "biological half-life" of compounds 1-8 was assessed using phorbol ester-treated human promyelocytic leukemia (HL-60) as described in Example 2. These cells converted more than 95% of LXA4 within 5 minutes of addition to cell warming. In this system, LXA4 was rapidly converted to 15-oxo-LXA4. However, in the same assay, 15-methyl-LXA4 (Compound 2) and cyclohexyl-LXA4 (Compound 4) were quantitatively recovered in a warming medium for up to 2 hours. These results indicate that modification at carbon 20 to carbon 15 interferes with further metabolism of LXA4 by leukocytes. In addition, the stability of the acetylene methyl ester LXA4 (Compound 1) was assessed after extraction and reverse phase HPLC for 60 minutes of in vitro whole blood warming (37 ° C). Later, it was recovered basically intact. Taken together, these results indicate that LX analogs retain their biological activity in vitro and are resistant to further metabolism. Example 5: Effect of 15-epilipoxins on cell proliferation<u style="single">Materials and methods</u>Synthesis (5S, 6R, 15R), -Trihydroxy-7,9,13-trans-11-cis-Eicosatetraenoate: Carboxymethyl ester (15-Epi-LXA4 methyl ester) is produced by total organic synthesis. It was manufactured and donated by Professor NA Petasis (Department of Chemistry, University of Southern California). The free acid of 15-epi-LXA4 was obtained by saponification of 15-epi-LXA4-methyl ester with LiOH (0.1M) in tetrahydrofuran at 4 ° C for 24 hours. A reference sample called synthetic eicosanoids was obtained from Cascade Biochemical Limited (Reading, Berkshire, UK). Inhibitors of 5-LO (isomer of Rev 5901) and cytochrome P450 (17-octadecanoic acid, 17-ODYA) activity were obtained from Biomol (Plymouth Meeting, PA). Radiolabeled ([32P]) dCTP and ([3H]) arachidonic acid, as well as methylthymidine, are DuPont. Obtained from NEN (Boston, MA). Ionophore (A23187), ASA, 3, (4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyltetrazolium = bromide) (MTT) and guanidinium isothiocyanate are Sigma Chemical Co. (St. Louis). Purchased from Louis, MO). Recombinant human interleukin 1β (IL-1β) was obtained from R &amp; D Systems (Mineapolis, MN). Dulbeccoline Phosphate Buffered Saline (pH 7.4) (DPBS2) containing both CaCl2 (0.6 mM) and MgCl2 (1.0 mM)₊), Fetal bovine serum (FBS), penicillin and streptomycin were obtained from Bio Whittaker (Walkersville, MD). Hank's Equilibrium Salt Solution (HBSS) and F-12K nutrient mixture were obtained from Gibco Laboratories (Grand Island, NY). High Performance Liquid Chromatography (HPLC) Grade Solvents, and Cesium Chloride, Purchased from JT Baker (Phillipsburg, NJ), Methyl Formate from Eastman Kodak Co (Rochester, NY), Sep-Pak C18 Cartridges, Waters Obtained from Associates (Milford, MA). Diazomethane is Aldrich Chemical Manufactured from N-methyl-N'-nitro-N-nitroguanidine purchased from Company (Milwaukee, WI). N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) was obtained from Pierce (Rockford, IL). First-strand cDNA synthesis kits and other molecular biology reagents were obtained from Promega (Madison, WI). Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA). Cell Isolation and Culture Human type II epithelial A549 cells from human lung cancer and human skin fibroblasts (chest) were obtained from the US Reference Strain Collection (Rockville, MD). The A549 cell line was a useful cell line because it originated from human alveolar cell carcinoma and could be easily assessed and maintained in culture without contamination with tissue macrophages [Lieber, M. et al. (1976) "A continuous tumer-cell line from a human lung with the characteristics of type II alveolar epithelial cells. carcinoma with proper ties of type II alveolar epithelial cells) ", Int.J. Cancer]. Fibroblasts were used within their limited viability framework and their passage number was recorded (results reported for cells from the 3rd to 5th generation). Epithelial A549 cells were seeded in a T-75 cm2 tissue culture flask and maintained in F-12K medium fortified with 10% heat-inactivated FBS, penicillin (50 U / ml) and streptomycin (50 g / ml). Human PMNs from healthy donors who did not receive ASA or other medication for at least 2 weeks were obtained by Ficoll-Hypaque gradient centrifugation and dextran sedimentation [Boyum, A. (1986), Mononuclear cells from human blood. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1g) , Scand.J.Clin.Lab.Invest.21 (Suppl.97): 77-89], DPBS at pH 7.4₊Suspended in. Survival rates for A549 cells and PMNs were determined by their ability to eliminate trypan blue and were 95 ± 2 and 97 ± 1%, respectively. These values did not change significantly during the reported warming.<u style="single">Preservation conditions</u>: IL-1β-treated (1 ng / ml, 24 hours) A549 cells (1.5x106 cells / ml) were used as carriers (1.5x106 cells / ml) for warming associated with permeable A549 cells (prepared by fast freeze-thaw cycle) 0.1% EtOH), ASA (continued to be used at 500M), 5M 17-ODYA, an inhibitor of cytochrome P450 [Muerhoff, ASet al. (1989) "Prostaglandin in rabbit lung, and fatty acid ω and ( ω-1) Oxidation: Prostaglandin and fatty acid ω and (ω-1)-oxidation in rabbit lung: acetylenic fatty acid mechanism-based inactivators as specific inhibitors) , J.Biol.Chem.244: 749-756], or 5 mM Rev Pretreated with one of the 5901 isomers, 5-LO inhibitors, and subjected to two cycles of fast freezing in a dry ice-acetone bath and thawing at room temperature (total cycle &lt;20 minutes), DPBS2₊It was warmed with arachidonic acid (20M) in 4 ml at 37 ° C for 20 minutes. In experiments related to radiolabeled arachidonic acid, the addition of [3H] -arachidonic acid (0.25 Ci / ml) + unlabeled arachidonic acid (20M) initiated 20 minutes of warming at 37 ° C. For time course experiments related to the production of 15-HETE from endogenous sources (see Figure 2B), intact A549 cells were contacted with IL-1β (1 ng / ml) for up to 48 hours, followed by a carrier. After treatment with (containing 0.1% EtOH) or ASA for 20 minutes, ionophore A23187 (5 μM) in 4 ml of HBSS was added at 37 ° C for 30 minutes. In co-incubation experiments, dense A549 cells were contacted with IL-1β (1 ng / ml, 24 hours), washed in HBSS, at 37 ° C, carrier alone or ASA for 20 minutes, and arachidonic acid. Treated with acid (20M) for 60 seconds. Simultaneous incubation was performed by adding PMN to a monolayer of A549 cells and then co-stimulating with ionophore A23187 (5 μM) in 4 ml of HBSS at 37 ° C for 30 minutes.<u style="single">Analysis of eicosanoids</u>: Incubation was stopped with 2 volumes of cold MeOH containing prostaglandin B2 (200 ng) and the product was extracted using a Sep-Pak C18 cartridge. The material eluted during the methyl formate fraction is concentrated under N2 flow and scanned (in methanol) on the UV-visible material with a model 8452 spectrophotometer (Hewlett. Packard Co., Palo Alto, CA). , Reversed phase (RP) -injected into an HPLC system. This system includes dual pump gradients (LKB, Bromma, Sweden), diode array detectors (Hewlett-Packard 1040M Series II) and HPLC 3D. It consisted of software called ChemStation. The collected UV data were reproduced at 300 nm and the conjugated tetraene was tracked for triene at 270 nm and for mono HETE at 234 nm. All UV spectra were obtained with a sampling interval of 0.96 seconds using step = 4 nm, Bw = 10 nm, and range = 220-360 nm. Monohydroxyeicosanoids from A549 cells (ie, 5-, 12- and 15-HETE) at 1.0 ml / min using Ultrasphere-ODS columns (5 μm, 4.6 mmx25 cm) (Beckman Instruments, Fullerton, CA). Then, MeOH / H2O / acetic acid (65: 35: 0.01; v / v / v) is used as the first phase (t0 to 20 minutes), and MeOH / acetic acid (99.9 minutes) is used as the second phase (20 to 45 minutes). It was eluted with: 0.1; v / v) and analyzed. The R- and S-enantiomers of 15-HETE were reported by Hawkins et al. (1988) [Hawkins et. al. (1988) "Resolution of enantiomers of hydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquid chromatography", Anal.Biochem.173: It was partitioned and identified using a chiral HPLC system similar to 456-462]. Briefly, the RP-HPLC material that elutes under the peak of 15-HETE is extracted with chloroform, converted to methyl ester by etheric diazomethane treatment, and then Bakerbond DNBPG (shared valence) chiral column (5 μm, 4.6 mm x 25 cm). ) (JT Baker, Phillipsburg, NJ) was used to elute with n-hexane / 2-propanol (100: 0.4; v / v) at a flow rate of 0.8 ml / min. When indicated, the production of 15-HETE from endogenous sources was followed by radioimmunoassay (RIA). Antibodies were induced against 15S-HETE (Per Septive Diagnostics, Cambridge, MA), which had a cross-reactivity of 0.1% at 50% B / B0 for 5-HETE. For analysis of LXs (including LXs and 15-epi-LXs) from A549 cells-PMN, MeOH / H2O / acetic acid (60: 40: 0.01; v / v / v) at a flow rate of 0.6 ml / min ) Waters Bondapak C18 (3.9x300nm) column eluting with isocratic mobile phase, or Altex Ultrasphere eluting with MeOH / H2O / acetic acid (65: 35: 0.01; v / v / v) at a flow rate of 3 ml / min. Simultaneous warming was performed using one of the ODS columns (5 μm, 10 mm x 25 cm). Peptide leukotrienes (LTC4 and LTD4) eluted in the MeOH fraction from the Sep-Pak cartridge extract at 1 ml / min with MeOH / H2O / acetic acid (65: 35: 0.01; v / v / v), pH 5. It was divided using a Beckman Ultrasphere-ODS column eluting at 7. Incubation of PMN with 15R-HETE was stopped with MeOH and the methyl formate fraction of the Sep-Pak C18 extract product was fractionated with MeOH / H2O / acetic acid (65: 35: 0.01; v /) at a flow rate of 3 ml / min. It was injected into an Altex Ultrasphere ODS column (5 μm, 10 mm x 25 cm) eluted with v / v). Materials under peaks that absorb at 300 nm were collected individually after RP-HPLC, and Claria, J. and Serhan, CN (1995) "Aspirin has previously been described by human epithelial cell-leukocyte interactions. Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interaction) , Proc.Natl.Acad.Sci.USA 92: 9475-9479 (expressly incorporated herein by reference), 5971A mass-selective quadrupole detector It was analyzed by gas chromatography-mass spectrometry (GC-MS) using a Hewlett-Packard 5890 GC series type II equipped with.<u style="single">Reverse Transcription (RT) and PCR:</u>Total RNA was obtained from A549 cells by the guanidinium isothiocyanate-cesium chloride method and cDNA was generated by RT. PGHS-1 and PGHS-2 [(5'-TGC CCA GCT CCT GGC CCG CCG CTT-3'(sense), 5'-GTG CAT CAA CAC AGG CGC CTC TTC-3' (antisense)) and (5' '-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3' (sense) and (5'-AGA TCA TCT CTG CCT GAG TAT CTT-3'(antisense))], 15-LO [5'-ATG GGT CTC TAC CGC ATC CGC GTG TCC ACT-3'(sense) and 5'-CAC CCA GCG GTA ACA AGG GAA CCT GAC CTC-3'(antisense)], 12-LO [Funk, CD and FitzGerald, GA (1991) " Eicosanoid forming enzyme mRNA in human tissues) , J.Biol.Chem.266:12508-12513], [5'-AGT TCC TCA ATG GTG CCA AC-3'(sense) and 5'-ACA GTG TTG GGG TTG GAG AG-3'(anti) Sense)], and 5-LO [5'-GAA GAC CTG ATG TTT GGC TACC-3'(Sense) and 5'-AGG GTT CTC ATC TCC Oligonucleotide primers were constructed from the published sequence of CGG-3'(antisense)]. Samples of PGHS-1, PGHS-2 and GAPDH were amplified during 25 cycles of denaturation at 94 ° C for 1 minute, annealing at 58 ° C for 2 minutes, and extension at 72 ° C for 3 minutes. 5-, 12- and 15-LO were amplified over 35 cycles at 94 ° C (1 min), 55 ° C (2 min) and 72 ° C (2.5 min). PCR products were analyzed by electrophoresis on a 2% agarose gel and their identity was followed by restriction enzyme analysis. To detect specific PCR-amplified targets, 0.5 Ci [32P] dCTP (3,000 Ci / mmol) was added to the PCR mixture and the product was programmed with Image-Quant (Molecular Dynamics, San Lorenzo, CA). ) Was used and quantified by a phosphorimager.<u style="single">Cell proliferation:</u>Microculture 3, (4,5-dimethylthiazyl-2-yl) 2,5 (diphenyl-tetrazolium = bromide) (MTT) assay [Marshan, NJ, et al. (1995) "Cell proliferation and function A critical assessment of the use of microculture tetrazolium assay to measure cell growth and function, Growth Regul. 5: 69-84] was used to investigate the effects of LX and other eicosanoids on cell proliferation. A549 and fibroblasts from exponential phase maintenance cultures were counted in 100 μl volumes of culture and dispensed into 96-well culture plates for replication (~ 2,000 cells / well). After 24 hours at 37 ° C, the culture was removed and fresh culture (100 μl) containing either compound (5 to 1,000 nM) or carrier (culture + 0.15% ethanol) was examined under each condition. In addition to 4 replicas per, culture plates were then warmed to 96 at 37 ° C. in a 5% CO2 atmosphere. At the end of this period, 25 μl (5 mg / ml) of MTT prepared just before in HBSS was added to the wells and the plate was warmed at 37 ° C for 4 hours. The dye solution is aspirated, the wells are washed once with HBSS, and the dye ingested by the cells is extracted to 100 μl of isopropyl alcohol: 1N HCl (96: 4, v / v) and microplate reader (Molecular Devices,). Menlo Quantified at 570 nm using Park, CA). In some experiments, cells were grown in 12-well culture plates, treated as described above, and counted using a Neubawell hemocytometer. Survival was routinely assessed using the trypan blue exclusion test. For A549 cells and fibroblasts, a linear relationship was established with respect to MTT value and cell number within the range of the experiments shown (r = 0.995, P &lt;0.005). A549 cells grown for 72 hours in the presence of compound (5 to 1,000 nM) or carrier (0.1% EtOH) were lysed with 0.25N NaOH and bio-Rad (Richmond, CA) using bovine serum albumin as a standard. Cellular protein content was determined by applying the microassay method. The average cellular protein content in resting A549 cells was 46.6 ± 1.5 pg / cell.<u style="single">Thymidine uptake and DNA synthesis:</u>A549 cells (~ 2x104 cells / ml) were seeded on 96-well plates, colonized for 24 hours and grown for an additional 72 hours in the presence of compound (5 ~ 1,000 nM) or carrier (culture + 0.1% ethanol). Twenty-four hours prior to the assay, 2 Ci / ml methyl- [3H] thymidine (specific activity 6.7 Ci / mmol) was added to each well [Cybulsky et al. (1992) "Eicosanoids are epithelial in glomerular epithelial cells. See Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells, Am.J, Physiol.262 (Renal Fluid Electrolyte Physiol. 31): F639-F646] .. After instant labeling, cold DPBS2 in each well₊The cells were washed 4 times with NaOH, the cells were lysed with NaOH (0.25 standard), and the radioactivity was measured. Student's t-test was used for statistical analysis, and the difference was considered significant at the P value (0.05). Results Eicosanoids are formed by initial oxygenation of arachidonic acid by the PGHS or LO enzymatic pathway [Samuelsson, B. et al. (1987) "Leukotrienes and Lipoxins: Structural, Biosynthetic and Biological Effects (Leukotrienes and) Lipoxins: structures, biosynthesis, and biological effects) ", Science 237: 1171-1176]. PGHS-1 and -2 from A549 cells grown in the presence or absence of IL-1β to assess which eicosanoid-producing enzymes are present and / or regulated by cytokines in A459 cells, Also, 5-, 12- and 15-LO mRNA levels were followed by RT-PCR and then analysis on a phosphorescence device. As shown in Figure 1A, mRNA levels for PGHS-2 increased significantly (~ 2-fold) after stimulation of A549 cells with IL-1β. In contrast, mRNA levels for PGHS-1 and 5-LO did not change significantly after cell contact with cytokines (Fig. 1A). A549 cells could not show either 15- or 12-LO expression before and after cytokine induction (Fig. 1A). Performing RT-PCR in parallel with RNA in human lung tissue and peripheral blood monocytes, where the absence of 15-LO mRNA in A549 cells is known to be a positive and negative source of 15-LO mRNA, respectively. Further confirmed by [Funk, CDand FitzGerald, GA (1991) "Eicosanoid forming enzyme mRNA in human tissues) , J. Biol. Chem. 266: 12508-12513] (see illustration in Figure 1A). To characterize the aspects of the monohydroxy product produced by airway epithelial cells, IL-1β-stimulated A549 cells (1.5x106 cells / ml) were permeable and warmed with arachidonic acid to form. The product was extracted and analyzed by RP-HPLC. Chromatographic images revealed the presence of a major product with strong UV absorption at 234 nm co-eluted with synthetic 15-HETE. In addition, when [3H] -arachidonic acid was added to IL-1β-treated A549 cells, the radiolabeled material was recovered under a peak that co-eluted with 15-HETE (Fig. 1B). In these experiments, neither 5- or 12-HETE formation was consistently observed. ASA treatment of A549 cells led to a marked increase in the formation of 15-HETE, but 17-ODYA, a reported inhibitor of eicosanoid metabolism by P450 [Muerhoff, ASet]. al. (1989) "Prostaglandins in rabbit lungs, and ω and (ω-1) oxidation of fatty acids: Prostaglandins and fatty acids based on the mechanism of acetylene fatty acids as specific inhibitors. ω and (ω-1)-oxidation in rabbit lung: acetylenic fatty acid mechanism-based inactivators as specific Inhibition of A549 cells permeable to inhibitors) , J. Biol. Chem. 244: 749-756] resulted in a ~ 50% reduction in 15-HETE (Fig. 2A). 17-ODYA is a potent inhibitor of eicosanoid metabolism by P450s and does not selectively inhibit the activity of either cyclooxygenase or LO [see Muerhoff et al. And supplier support]. The 5-LO inhibitor (Rev-5901 isomer) did not alter the amount of 15-HETE produced by A549 cells in a statistically significant manner. Heat-denatured A549 cells reduced the amount of 15-HETE by ~ 90%, suggesting an enzymatic component in its formation. Production of 15-HETE from the endogenous source of arachidonic acid was also obtained from A549 cells treated with IL-1β (1 ng / ml) for 24 hours (25.0 ± 10.0 ng / 107 cells). Taken together, these results indicate that 15-HETE is the major monohydroxy product produced by A549 cells, with acetylated PGHS-2 and cytochrome P450 contributing to this biosynthesis, respectively. Suggest to do. To investigate the temporal course of 15-HETE production from the endogenous source of arachidonic acid, intact A549 cells are contacted with IL-1β (1 ng / ml) for up to 48 hours and are present in the cell supernatant. The amount of immunoreactive 15-HETE was followed using specific RIA. In the resting state, A549 cells produced significant levels of immunoreactive 15-HETE (19.1 ± 10. 5ng / 107 pieces, n = 3, d = 2). These values did not change with the addition of ionophore A23187 (5M) in the absence of IL-1β (Fig. 2B). Also, in the absence of ionophore stimulation, addition of IL-1β to A549 cells for up to 48 hours did not result in increased 15-HETE production (Fig. 2B). In stark contrast, the addition of IL-1β + stimulation of A549 cells with A23187 led to a marked increase in 15-HETE production (Fig. 2B). Highest levels were found 24 hours of contact with cytokines, after which levels of 15-HETE declined. Due to interest in the stereochemistry of alcohol in 15-HETE produced by A549 cells, the individual relative amounts of R and S enantiomers of 15-HETE produced by IL-1β-treated A549 cells were chiral. It was investigated using phase HPLC analysis [see method section]. PGHS-2, along with cytochrome P450, is an enzyme other than 15-LO that can convert arachidonic acid to 15-HETE, respectively. 15-HETE derived from PGHS-2 acetylated with ASA has its carbon (C) -15 alcohol group mainly in the R configuration [Holtzman, M Jet al. (1992) "In cultured epithelial cells." Identification of a pharmacologically distinct prostagalndin H synthase in cultured epithelial cells, J. Biol. Chem. 267: 21438-21445]. Here, 15-HETE produced by IL-1β-activated A549 cells was converted to its methyl ester and subjected to SP-HPLC chiral analysis. 15-HETE from activated A549 cells had 65% R and 35% S configurations (Fig. 3). Pretreatment of A549 cells with ASA (20 minutes, 37 ° C) resulted in a 3-fold increase in the amount of 15R-HETE, whereas the formation of 15S-HETE remained unchanged (Figure). 3). In the presence of ASA, 15R-HETE accounted for 85% of the total 15-HETE product produced by A549 cells. These results indicate that in the presence of ASA, the majority of 15-HETE produced by IL-1β-activated A549 cells was in the R configuration. Transcellular eicosanoid biosynthesis is an important tool for the generation of new mediators as well as the amplification of lipid mediators [Marcus, AJ (1995) "Aspirin as prophylaxis against colorectal cancer", New Engl.J.Med.333: 656-658]. Simultaneous stimulation of human endothelial cells and PMN after ASA treatment leads to the formation of a new taxon of bioactive eicosanoids [Claria, J. and Sehan, CN (1995) "Aspirin is due to human epithelial cell-leukocyte interaction" Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interaction , Proc.Natl.Acad.Sci.USA 92: 9475-9479]. These novel eicosanoids have been identified as 15-epi-LX, and their biosynthesis involves leukocyte conversion of ASA-induced endothelium-derived 15R-HETE. From these results, the formation of novel eicosanoids by transcell biosynthesis may also occur during epithelial cell-PMN interactions. To test this hypothesis, dense A549 cells were contacted with IL-1β (1 ng / ml, 24 hours), treated with ASA and co-stimulated with PMN. FIG. 4A shows a representative HPLC image of the material obtained from stimulated cells in contact with the ASA, revealing the presence of four major products with strong UV absorbance when plotted at 300 nm. did. Online spectral analysis of these products showed the triplet absorption bands characteristic of conjugated tetraene-containing chromophores, each showing the basic structure of LX (maximum at 301 nm, and 288). And shoulders at 316 ± 2 nm) (Figs. 4B and 4C). Chromatographic images of LXA4 and 15-epi-LXA4 were identified based on co-elution with synthetic standards and the presence of characteristic chromophores. In these simultaneous warmings, 15-epi-LXA4 accounted for ~ 88% of the total amount of LXA4 detected, which was produced by IL-1β-activated A549 cells in contact with the ASA15. -Most of the HETE is consistent with the finding that it was primarily an R configuration (see Figure 3 and 4A, 4B and 4C). In this RP-HPLC system, 15-epi-11-trans-LXA4 and LXB4 co-eluted like 11-trans-LXA4 and 15-epi-LXB4 (not shown). These LX isomers are not further partitioned by HPLC and are shown in the image below the peaks labeled as peaks A and B, respectively (Fig. 4A). The compounds below peaks A and B were indeed split as OTMS, a methyl ester derivative in GC-MS (shown below). The product is ~ 8: It was predominant in those 15R epimers in a ratio of 2 (n = 3). Compound C (Fig. 4A) was not co-eluted with any of the previously identified LXs and was also present in EP-HPLC images from activated PMNs warmed with 15R-HETE (data not shown, n = 5). ). The material eluted under compound C matched the physical properties of compound III recently isolated from endothelial cell-PMN interactions [Claria, J. and Serhan, CN (1995) "Aspirin is a human epithelial cell. -Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interaction, Proc. Natl. Acad. Sci. USA 92: 9475-9479]. The complete stereochemistry of compound C is awaiting determination, but UV spectral data, and chromatographic mobility, indicate that it can be in the 15-epimer form of 7-cis-11-trans-LXA4. Suggests [Nicolaou, K Cet al. (1989) "Identification of novel 7-cis-11-trans lipoxin A4 produced by human neutrophils: total synthesis, spasm-inducing activity, and other geometries of LXA4 and B4. Identification of a novel 7-cis-11-translipoxin A4, generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of LXs A4 and B4 , Biochim.Biophys.Acta 1003: 44-63]. Therefore, the LXs produced during epithelial (A549 cell) -PMN co-stimulation after ASA treatment were predominantly 15-epi-LX (Fig. 4A). In addition to being able to produce LX, co-preservation of activated PMN with A549 cells also produces significant amounts (~ 8 times more than LX with tetraene) of peptide leukotrienes (pLT; LTC4 and LTD4) in the absence of ASA. (Figures 5A and 5B). The amounts of both LX and pLT produced during these simultaneous warmings depend on the proportion of individual cells (illustrations in Figures 5A and 5B). Contact of airway epithelial A549 to ASA prior to PMN addition (20 minutes) leads to increased LX formation and decreased pLT (FIGS. 5A and 5B). None of the separately warmed PMNs or A549 cells produce detectable levels of LX or pLT in the presence or absence of the ASA (Figures 5A and 5B, and data not shown). Taken together, these results indicate that during the A549 cell-PMN interaction, both LX and pLT originate in the transcellular pathway. LX is a vasodilator, a potent regulator that inhibits leukocyte responses such as chemotaxis, adhesion to endothelial cells, and transepithelial translocation [Serhan, CN (1994) "Lipoxin Biosynthesis". , And its impact in inflammatory and vascular events, Biochim.Biophys.Acta 1212: 1-25]. In contrast, pLT not only acts both as a vasoconstrictor and pro-inflammatory, but also stimulates the proliferation of several cell types, including fibroblasts, smooth muscle and glomerular epithelial cells [Baud]. , L. et al. (1985) "Leukotrien C4 binds to human glomerular epithelial cells and promotes their proliferation in vitro", J. Clin. Invest .76: 374-377]. LX reverses the vasoconstrictor action of LTD4 on rat renal hemodynamics and inhibits LTC4-stimulated hematopoiesis [Serhan, CN (1994) "Lipoxin biosynthesis and its effects on inflammatory and vascular events (" Lipoxin biosynthesis and its impact in inflammatory and vascular events) ", Biochim.Biophys.Acta 1212: 1-25]. Because ASA promotes 15-epi-LX formation and inhibits pLT biosynthesis (Figures 5A and 5B), these eicosanoids play a reverse regulatory effect on cell proliferation in human cancers and are protective agents of ASA. May contribute to the introduction. Therefore, the effects of these LO products on epidermal growth (Figs. 6A and 6B) were tested and their effects were tested by the soluble microculture tetrazolium (MTT) assay [Alley, MC, et al. (1988) "Microculture". Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay, Cancer Res. 48: 589-601] was used to compare with that of the well-established inhibitor dexamethasone. These experiments were performed with synthetic LXA4 and LXB4 available in sufficient quantities for bioassay, rather than the major LX produced by these cells, 15-epi-LX. As shown in Figures 6A and 6B, LXA4 and LXB4 inhibited the proliferation of A549 cells in a time (A) and dose (B) -dependent manner. LXA4 and LXB4, like dexamethasone [1 μM], inhibited A549 cell proliferation 72-96 hours after treatment (Fig. 6A). After 72 hours, LXA4 shared the antiproliferative properties observed for dexamethasone in these cells (Fig. 6A) [Croxtall, JD and Flower, RJ (1992) "Lipocorten I is a dexamethasone from the A549 adenocarcinoma cell line." (See Lipocortin I mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line), Proc. Natl. Acad. Sci. USA 89: 3571-3575]. The semi-maximum inhibition value (IC50) for LXA4 was ~ 80 nM compared to that of dexamethasone ~ 7 nM. 0. LXB4 at concentrations of 5 and 1 μM was three times more active than either LXA4 or dexamethasone (Fig. 6B). Interestingly, both LXA4 and LXB4 showed essentially equal efficacy in inhibiting the growth of A549 cells when each was added to cells repeatedly (ie, at 24-hour intervals) for 3 consecutive days (data shown). However, it is suggested that n = 3, d = 4), LX can be inactivated by these epithelial cells. Results from additional experiments using direct cell counting (Figure 7B) and measurements of total cellular protein content (data not shown, n = 3, d = 4) are parallel to those obtained by the MTT assay. Therefore, the antiproliferative effect of LX on A549 cells was confirmed. Moreover, inhibition of DNA synthesis as determined by 3H-thymidine uptake occurred when A549 cells were contacted with 50 nM or higher concentrations of LXB4 or dexamethasone for 72 hours (Fig. 7A). After warming A549 cells with the compounds, cell viability was found to be ~ 98% as determined by the trypan blue exclusion assay, and the concentrations of these compounds used in these experiments. It was shown that it is not harmful to cells within the range. The 15-hydroxyepimeric forms of LXA4 and LXB4 (15-epi-LXA4 and 15-epi-LXB4, respectively), which were the dominant forms of LX isolated from these cells, are shown in Table 1 below. As you can see, it also turned out to be a potent inhibitor of epidermal cell proliferation.<img file="JP4060366B2_D0064.tif" />Cells (2,000 A549 cells / well) are grown in 96-well plates and carrier (0.15% by volume in EtOH / F-12K culture solution) or equimolar (10-7M) LXA4, 15-epi- Contact with LXA4, LXB4, 15-epi-LXB4 or dexamethasone at 37 ° C for 72 hours. Values represent ± SEM from 3-7 experiments performed quadruple and are expressed as% of inhibition of cell proliferation.^*The P value means a statistical difference when compared with cells alone. &amp; 15-P &lt;0.001 for Epi-LXB4. At equimolar levels (100 nM), 15-epi-LXA4 inhibited A549 cell proliferation to the same extent as LXA4. On the other hand, 15-epi-LXB4 isolated from conversion of 15R-HETE by activated PMN and added back to A549 cells showed stronger antiproliferative activity than LXB4 (for ~ 34% growth inhibition). ~ 80%, P &lt;0.001; Table 1). The compound was characterized by UV, HPLC and GC-MS (C value: 23.3). The characteristic ions for the OTMS methyl ester were relatively predominant ions at m / z 173 (basic peak), 203, 289, 379, and 482 (M).₊-100) [Molecular ions could not be obtained due to insufficient amount]. Therefore, the dominant material below the peak labeled B in Figure 4B shows a shorter C value consistent with that of 15-epi-LXB4, from LXB4 to OTMS, the methyl ester in GC-MS analysis. Was isolated as. The mass spectra of 15-epi-LXB4 and LXB4 were essentially identical (not shown), but their C values were distinct. Materials eluted below the peak shown as C (isolated from activated PMN warmed with 15R-HETE) also showed a mild inhibitory effect on epithelial cell proliferation (18.2 ± 2% growth inhibition). , N = 3, d = 4). In stark contrast, 15-epi-trans-LXA4, 11-trans-LXB4, 8,9-acetylene LXB4, peptide leukotrienes (LTC4 and LTD4), and LX precursors tested at 10-6 to 10-9M, respectively. (15S- and 15R-HETE) were unable to significantly inhibit the proliferation of A549 cells (data not shown, n = 3-5, d = 4). These results indicate that LX and 15-epi-LX exert stereoselective effects on inhibiting cell proliferation in A549 cells. LXA4 and LXB4 were tested on human skin fibroblasts to determine if they were antiproliferative for this cell type. At 100 nM, both LXA4 and LXB4 inhibited fibroblast proliferation. LXB4 showed 38.0 (7.5% inhibition) and LXA4 showed 10.7 (1.8%), contrasted with dexamethasone (29.7 (0.4%)) as a positive control (n = 3). Presence of an active cytochrome P450 enzyme system in A549 cells of the human respiratory tract [Vogel, et al. (1994) "Transforming growth factor-β1 in hibits TCDD-induced cytochrome P450IA1 expression in human lung cancer A549" cells) , Arch.Toxicol.68: 303-307], combined with the result that inhibition of P450 inhibits 15-HETE production in these cells, as well as heat denaturation (Fig. 2A). It is suggested that this enzymatic system in epithelial cells also contributes to the biosynthesis of 15-HETE and the production of 15-epi-lipoxins by the transcellular pathway (Fig. 8). Taken together, these findings (Figures 1-4) can initiate the formation of 15-epi-lipoxin during airway epithelial cell-PMN interactions (Figure 8) two distinct enzymatic pathways (ie, ie). Confirm the presence of ASA-acetylated PGHS-2 and cytochrome P450). Also, because ASA can induce the P450 enzyme [Pankow, D. et al. (1994) "Acetylsalicylic acid-inducer of cytochrome P-450 2E1?", Arch.Toxicol.68: 261-265], it should be noted that these two independent pathways may work together to produce 15-epi-lipoxin. 303-307], coupled with the result that inhibition of P450 inhibits 15-HETE production in these cells as well as heat denaturation (Fig. 2A), this enzymatic system in epithelial cells also 15 It is suggested that it contributes to the biosynthesis of -HETE and the production of 15-epi-lipoxin by the transcellular pathway (Fig. 8). Taken together, these findings (Figures 1-4) can initiate the formation of 15-epi-lipoxin during airway epithelial cell-PMN interactions (Figure 8) two distinct enzymatic pathways (ie, ie). Confirm the presence of ASA-acetylated PGHS-2 and cytochrome P450). Also, because ASA can induce the P450 enzyme [Pankow, D. et al. (1994) "Acetylsalicylic acid-inducer of cytochrome P-450 2E1?", Arch.Toxicol.68: 261-265], it should be noted that these two independent pathways may work together to produce 15-epi-lipoxin. 303-307], in combination with the result that inhibition of P450 inhibits 15-HETE production in these cells as well as heat denaturation (Fig. 2A), this enzymatic system in epithelial cells also 15 It is suggested that it contributes to the biosynthesis of -HETE and the production of 15-epi-lipoxin by the transcellular pathway (Fig. 8). Taken together, these findings (Figures 1-4) can initiate the formation of 15-epi-lipoxin during airway epithelial cell-PMN interactions (Figure 8) two distinct enzymatic pathways (ie, ie). Confirm the presence of ASA-acetylated PGHS-2 and cytochrome P450). Also, because ASA can induce the P450 enzyme [Pankow, D. et al. (1994) "Acetylsalicylic acid-inducer of cytochrome P-450 2E1?", Arch.Toxicol.68: 261-265], it should be noted that these two independent pathways may work together to produce 15-epi-lipoxin.<u style="single">Equivalent</u>One of ordinary skill in the art will recognize or be able to recognize or confirm a myriad of equivalents to the particular procedure described herein, without the use of more than routine experiments. Such equivalents are considered to be within the scope of the invention and are covered by the following claims.<img file="JP4060366B2_D0065.tif" /><img file="JP4060366B2_D0066.tif" /><img file="JP4060366B2_D0067.tif" /><img file="JP4060366B2_D0068.tif" /><img file="JP4060366B2_D0069.tif" />