Methods and Uses of Colchicine Derivatives

Abstract

The present invention provides a pharmaceutical composition containing a colchicine derivative for the treatment of inflammation. [Solution] In one embodiment, a pharmaceutical composition for the treatment of inflammation caused by neutrophils is provided, comprising a colchicine derivative represented by the following formula, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof. JPEG2026062757000069.jpg6062

Description

[Technical Field]

[0001] This application generally relates to colchicine derivatives, methods thereof, and uses thereof. [Background technology]

[0002] Inflammatory conditions affect millions of people worldwide, and targeted molecular medicine aims to develop safer and more effective drugs and therapies in this field. Colchicine is a widely used antimitotic agent in the treatment of inflammatory diseases because it can target molecular pathways involved in inflammation. For example, beneficial effects have been reported in the treatment of psoriatic arthritis (P. Seidemann, B. Fjellner, A. Johannesson, J. Rheumatol. 14 (1987) 777-779) and leukocytoclastic vasculitis (JP Callen, J. Am. Acad. Dermatol. 13 (1987) 193-200). Furthermore, recent studies have shown that colchicine inhibits leukocyte-endothelial cell adhesion (SJ Rosenman, AA Ganji, WM Gallatin, FASEBJ 5 (1991) 1603-1609) and T cell activation (YA Mekory, D. Baram, A. Goldberg, A. Klajman, Cell. Immunol. 120 (1989) 330-340) by binding to intracellular tubulin monomers, thereby preventing their polymerization (GO Borisy, EW Taylor, J. Cell. Biol. 34 (1967) 533-548). Common uses of colchicine include the treatment of gout and familial Mediterranean fever (FMF). In fact, patients with FMF typically receive lifelong colchicine therapy. However, the use of colchicine remains challenging due to its low therapeutic index between efficacy and side effects that limit treatment. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] Therefore, there is a need for the development of drugs, as well as for their use and / or methods of use that avoid or mitigate at least one of the aforementioned disadvantages, or provide a useful alternative. [Means for solving the problem]

[0004] In one embodiment, compounds of formula I, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, tautomers thereof, optical isomers thereof, or combinations thereof are provided: [ka] Here, Z is either O or S; X 1 R is selected from substituted or unsubstituted hydrocarbon groups, or substituted or unsubstituted heterogeneous groups. 2 and R 3 Each of these is independently selected from substituted or unsubstituted hydrocarbon groups, substituted or unsubstituted heterogeneous groups, substituted or unsubstituted carbocyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aromatic groups, or substituted or unsubstituted heteroaromatic groups.

[0005] In another embodiment, R 2 and R 3 Each of these is independently selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted alkenyl groups, substituted or unsubstituted alkynyl groups, substituted or unsubstituted aromatic groups, substituted or unsubstituted heteroaromatic groups, substituted or unsubstituted carbocyclic groups, or substituted or unsubstituted heterocyclic groups. In another embodiment, R 2 and R 3is independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted C1-C6 alkylcarbonyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl. In another aspect, R 2 and R 3 are each independently selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C1-C6 alkylcarbonyl, C1-C6 alkylene-O-alkyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl. In another aspect, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkylaryl. In another aspect, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl, or substituted or unsubstituted alkylaryl. In another aspect, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl. In another aspect, R 2 and R 3 0are each independently selected from substituted or unsubstituted C1-C6 alkyl. In another aspect, R 2 and R 3Each of these is independently selected from unsubstituted C1-C6 alkyl groups. In another embodiment, R 2 and R 3 Each is independently selected from methyl, ethyl, or propyl. In another embodiment, R 2 is methyl. In another embodiment, R 3 It is ethyl or propyl.

[0006] In another embodiment, X 1 X is a substituted or unsubstituted hydrocarbon group. In another embodiment, X 1 X is selected from substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, or substituted or unsubstituted alkynyls. In another embodiment, X 1 X is selected from substituted or unsubstituted C1-C6 alkyl groups. In another embodiment, X 1 X is selected from unsubstituted C1-C6 alkyl groups. In another embodiment, X 1 X is selected from methyl or ethyl. In another embodiment, X 1 is methyl. In another embodiment, X 1 is OR 10 And R 10 R is selected from substituted or unsubstituted hydrocarbon groups, or substituted or unsubstituted heterogeneous groups. In another embodiment, R 10 R is selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted alkenyl groups, substituted or unsubstituted alkynyl groups, substituted or unsubstituted aromatic groups, substituted or unsubstituted heteroaromatic groups, substituted or unsubstituted carbocyclic groups, or substituted or unsubstituted heterocyclic groups. In another embodiment, R 10is selected from substituted or unsubstituted alkyl, CH2OH, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl. In another embodiment, R 10 R is selected from substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, or substituted or unsubstituted alkynyls. In another embodiment, R 10 R is selected from substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C2-C6 alkenyl groups, or C2-C6 alkynyl groups. In another embodiment, R 10 R is selected from substituted or unsubstituted alkyl groups. In another embodiment, R 10 R is selected from substituted or unsubstituted C1-C6 alkyl groups. In another embodiment, R 10 R is selected from unsubstituted C1-C6 alkyl groups. In another embodiment, R 10 R is selected from methyl or ethyl. In another embodiment, R 10 It is methyl.

[0007] In another embodiment, X 1 These are heterogeneous groups, either substituted or unsubstituted. In another embodiment, X 1 -CR 4 R 5 R 6 Selected from, R 4 , R 5 , and R 6Each is independently selected from H, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterogeneous group. In another embodiment, R 4 , R 5 , and R 6 Each is independently selected from substituted or unsubstituted amide groups. In another embodiment, R 4 and R 5 Each is independently selected from H or substituted or unsubstituted alkyl, and R 6 NR(CO)CR 7 R 8 R 9 R is selected from H and substituted or unsubstituted alkyl groups, 7 , R 8 , and R 9 Each is selected from H, a halo group, and a substituted or unsubstituted alkyl group. In another embodiment, R 7 , R 8 , and R 9 X can be selected from fluoro groups. In another embodiment, X 1 The compound is -CH2NH(CO)CF3.

[0008] In another embodiment, Z is O. In another embodiment, Z is S. In another embodiment, the compound [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0009] In another embodiment, the compound [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0010] In another embodiment, the compound, [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0011] In another embodiment, the compound [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0012] In another embodiment, the compound [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0013] In another embodiment, the compound [ka] This includes its pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof.

[0014] In another embodiment, the compound is formula I and / or a pharmaceutically acceptable salt thereof. In another embodiment, the configuration at C7 is the S configuration. In another embodiment, the compound binds to β-tubulin at the colchicine binding site. In another embodiment, β-tubulin is β-VI, β-V, and / or β-I. In another embodiment, β-tubulin is β-VI. In another embodiment, the compound has a binding energy lower than that of colchicine. In another embodiment, the compound is less toxic than colchicine. In another embodiment, the compound targets neutrophils more specifically than colchicine. In another embodiment, the compound inhibits the increase in intracellular calcium concentration at lower doses than colchicine. In another embodiment, the compound inhibits the increase in intracellular calcium concentration at doses at least about 10 times lower (about one-tenth) than colchicine. In another embodiment, the compound inhibits the increase in intracellular calcium concentration at doses about 10 to about 100 times lower (about one-tenth to about one-hundredth) than colchicine. In another embodiment, the compound inhibits the increase in intracellular calcium concentration at a dose of approximately 0.1 μM. In another embodiment, the compound inhibits the production of inflammatory mediators at a lower dose than colchicine. In another embodiment, the compound inhibits the production of inflammatory mediators at a dose at least approximately 10 times lower (approximately one-tenth) than colchicine. In another embodiment, the compound inhibits the production of inflammatory mediators at a dose approximately 10 to 100 times lower (approximately one-tenth to approximately one-hundredth) than colchicine. In another embodiment, the compound inhibits the production of inflammatory mediators at a dose of approximately 0.1 μM. In another embodiment, the inflammatory mediator is selected from IL-8, IL-1, superoxide, or a combination thereof. In another embodiment, the compound exhibits a monotonic or non-monotonic dose-response with respect to the inhibition of at least one of intracellular calcium concentration and inflammatory mediator production. In another embodiment, the inflammatory mediator is selected from IL-8, IL-1, superoxide production, or a combination thereof. In another embodiment, the compound inhibits the recruitment of white blood cells.

[0015] In another embodiment, compounds described herein are provided for the treatment of inflammation. In another embodiment, inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or combinations thereof. In another embodiment, inflammation includes inflammation caused by neutrophils. In another embodiment, inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or combinations thereof. In another embodiment, inflammation caused by neutrophils is associated with cardiovascular disease. In another embodiment, cardiovascular disease is coronary atherosclerosis. In another embodiment, inflammation caused by neutrophils is associated with gout.

[0016] In another embodiment, the compounds described herein are provided for the treatment of gout. In another embodiment, the compounds have an inhibitory effect on immune function in response to inflammation induced by monosodium urate (MSU). In another embodiment, the inhibitory effect on immune function is brought about via mediators selected from intracellular calcium production, IL-1 production, IL-8 production, superoxide production, or a combination thereof. In another embodiment, the immune function relates to neutrophils. In another embodiment, the inhibitory effect is more potent than that of colchicine. In another embodiment, the inhibitory effect is at least about 10 times greater than that of colchicine. In another embodiment, the inhibitory effect occurs at a concentration of about 0.1 μM. In another embodiment, the compounds described herein are provided for the treatment of cardiovascular disease. In another embodiment, the cardiovascular disease is coronary atherosclerosis.

[0017] In another embodiment, a pharmaceutical composition comprising the compounds described herein is provided. In another embodiment, the composition further comprises an antigout agent. In another embodiment, the antigout agent is selected from nonsteroidal anti-inflammatory drugs (NSAIDS), intra-articular glucocorticoids, xanthine oxidase inhibitors, recombinant non-human uricase enzymes, uric acid excretion promoters, uric acid excretion agents, or a combination thereof. In another embodiment, the composition further comprises at least one pharmaceutically acceptable carrier and / or diluent. In another embodiment, the composition comprises two or more compounds described herein. In another embodiment, a pharmaceutical composition comprising the compounds described herein for the treatment of inflammation is provided. In another embodiment, inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or a combination thereof. In another embodiment, inflammation includes inflammation caused by neutrophils. In another embodiment, inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or a combination thereof. In another embodiment, inflammation caused by neutrophils is associated with cardiovascular disease. In another embodiment, the cardiovascular disease is coronary artery atherosclerosis. In another embodiment, the inflammation caused by neutrophils is associated with gout. In another embodiment, a pharmaceutical composition comprising the compounds described herein is provided for the treatment of gout.

[0018] In another embodiment, a method for treating inflammation in a mammal is provided, comprising administering a therapeutically effective amount of a compound described herein to a mammal. In another embodiment, two or more compounds described herein are present. In another embodiment, the compounds are administered orally and / or parenterally. In another embodiment, the compounds are administered intravenously and / or intraperitoneally. In another embodiment, a method for treating inflammation in a mammal is provided, comprising administering a therapeutically effective amount of a composition described herein to a mammal. In another embodiment, the composition is administered orally and / or parenterally. In another embodiment, the composition is administered intravenously and / or intraperitoneally. In another embodiment, inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or combinations thereof. In another embodiment, inflammation includes inflammation caused by neutrophils. In another embodiment, inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or combinations thereof. In another embodiment, inflammation caused by neutrophils is associated with cardiovascular disease. In another embodiment, cardiovascular disease is coronary atherosclerosis. In another embodiment, inflammation caused by neutrophils is associated with gout. In yet another embodiment, the mammal is a human.

[0019] In another embodiment, the use of a therapeutically effective amount of a compound described herein is provided for the treatment of inflammation in mammals. In another embodiment, there are two or more compounds described herein. In another embodiment, the compounds are administered orally and / or parenterally. In another embodiment, the compounds are administered intravenously and / or intraperitoneally. In another embodiment, the use of a therapeutically effective amount of a composition described herein is provided for the treatment of inflammation in mammals. In another embodiment, the composition is administered orally and / or parenterally. In another embodiment, the composition is administered intravenously and / or intraperitoneally. In another embodiment, inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or combinations thereof. In another embodiment, inflammation includes inflammation caused by neutrophils. In another embodiment, inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or combinations thereof. In another embodiment, inflammation caused by neutrophils is associated with cardiovascular disease. In another embodiment, cardiovascular disease is coronary atherosclerosis. In another embodiment, inflammation caused by neutrophils is associated with gout. In another context, mammals are humans.

[0020] In another embodiment, a method for treating gout in a mammal is provided, comprising administering a therapeutically effective amount of a compound described herein to the mammal. In another embodiment, there are two or more compounds described herein. In another embodiment, the compound is administered orally and / or parenterally. In another embodiment, the compound is administered intravenously and / or intraperitoneally. In another embodiment, a method for treating gout in a mammal is provided, comprising administering a therapeutically effective amount of a composition described herein to the mammal. In another embodiment, the composition is administered orally and / or parenterally. In another embodiment, the composition is administered intravenously and / or intraperitoneally. In another embodiment, the mammal is a human. In another embodiment, gout is selected from chronic gout and / or acute gout. In another embodiment, the treatment of gout comprises the treatment of at least one gouty symptom. In another embodiment, the at least one gouty symptom is selected from gouty attacks, gouty nodule formation, gouty arthritis, gout-associated inflammation, and / or gout-associated joint destruction. In another embodiment, the at least one gouty symptom is selected from gouty inflammation and / or pain associated with inflammation.

[0021] In another embodiment, the use of a therapeutically effective amount of a compound described herein is provided for the treatment of gout in a mammal. In another embodiment, there are two or more compounds described herein. In another embodiment, the compounds are administered orally and / or parenterally. In another embodiment, the compounds are administered intravenously and / or intraperitoneally. In another embodiment, the use of a therapeutically effective amount of a composition described herein is provided for the treatment of gout in a mammal. In another embodiment, the composition is administered orally and / or parenterally. In another embodiment, the composition is administered intravenously and / or intraperitoneally. In another embodiment, the mammal is a human. In another embodiment, gout is selected from chronic gout and / or acute gout. In another embodiment, the treatment of gout includes the treatment of at least one gouty symptom. In another embodiment, the at least one gouty symptom is selected from gouty attacks, gouty nodule formation, gouty arthritis, gout-associated inflammation, and / or gout-associated joint destruction. In another embodiment, the at least one gouty symptom is selected from gouty inflammation and / or inflammation-associated pain.

[0022] In another embodiment, a method for treating inflammation is provided, the method comprising administering a βVI-tubulin inhibitor. In another embodiment, the βVI-tubulin inhibitor is a compound or composition described herein. In another embodiment, the inflammation is associated with leukocyte infiltration. In another embodiment, the leukocyte infiltration includes neutrophil and / or monocyte infiltration. In another embodiment, the inflammation is associated with gout. In another embodiment, the inflammation is associated with atherosclerosis.

[0023] Other features and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description, it should be understood that the detailed description and specific examples between embodiments of the present invention are given for illustrative purposes only. [Brief explanation of the drawing]

[0024] Here, embodiments are described only by reference to the figures, as examples. [Figure 1] Figure 1 shows the synthesis scheme for preparing compounds (2) and (3). [Figure 2] Figure 2 shows the synthesis scheme for preparing compounds (4) and (5). [Figure 3] Figure 3 shows the synthetic scheme for preparing compound (6) to (38). Figure 3A shows the structure of colchicine with modifications to colchicine at the R position, from (50) to (54). [Figure 4] Figure 4 shows the structure of thiocolchicine with modifications to thiocolchicine at the R and R1 positions (39), (3a-c), (4a-c), and (5a-c). Figures 4A to 4D show examples of colchicine and thiocolchicine derivatives. [Figure 5]Figure 5 shows the differences in residues within the colchicine binding site. Figure 5A shows the residues contained in the binding surface of colchicine [pdb code 1SA0], shown as black letters in a standard β1-tubulin sequence. Differences between the three binding sites are shown in medium-gray letters, the remaining letters are gray, and dashed lines indicate identical positions between sequences. Figure 5B shows the solvent-accessible surface drawn on β-tubulin [pdb code 1SA0]. Residues constituting the colchicine binding surface are shown in black in the figure, residues showing differences between the three binding site models are shown as black bars, and colchicine is shown as a molecular structure, with the A ring and X and Y positions clearly visible.

[0025] [Figure 6] Figure 6 shows the calculated ΔG [kcalmol-1] for colchicine and its derivatives bound to type I (top), type II (middle), and type III (bottom) β-tubulin binding sites. The box plots for derivative ((3)-D-20) and colchicine (CH) were generated from energy assessments of 10 independent docking poses. Whiskers are shown with 5% and 95% confidence values. [Figure 7]Figure 7 shows the effects of colchicine and colchicine derivatives on calcium storage mobilization by human neutrophils stimulated with monosodium urate (MSU). Figures 7A and 7D show the effects of colchicine. Figure 7B shows the effects of colchicine derivative (91). Figure 7C shows the effects of colchicine derivative TPO. Figures 7E-J show the effects of colchicine derivatives 28a, 39, 47a, 89, 14, and 43 compared to colchicine (Figure 7D). Figures 7K-L show the effects of colchicine at various doses. Figures 7M-P show the effects of various doses of colchicine derivatives (43) (Figures 7M-N) and (47a) (Figures 7O-P) compared to various doses of colchicine (Figures 7K-L). Figures 7Q and 7R show the effects of colchicine derivatives (47a) and (43), respectively. Figure 7S shows a comparison of the inhibitory activity of the compounds tested in Figures 7A, 7B, 7Q, and 7R. Figures 7T and 7U show the effects of colchicine and its derivatives (91) and (43) on the fMLP-induced increase in cytoplasmic calcium concentration. Figures 7V and 7W show colchicine and its derivatives (91) and (43) relating to the increase in cytoplasmic calcium concentration induced by MSU.

[0026] [Figure 8]Figure 8 shows the effects of colchicine and colchicine derivatives on the release of IL-8 (Figures 8A to 8G and 8L) or IL-1 (Figures 8H to 8K) by human neutrophils stimulated with monosodium urate (MSU). Figure 8A shows the effect of colchicine derivative (43). Figure 8B shows the effect of colchicine derivative (47a). Figure 8C shows the effect of colchicine. Figure 8D shows colchicine derivative (91). Figure 8E shows colchicine derivative (47a). Figure 8F shows the effect of colchicine derivative (43). Figure 8G shows a comparison of the inhibitory activities of the compounds tested in Figures 8C to F. Figure 8H shows the effect of colchicine. Figure 8I shows colchicine derivative (91). Figure 8J shows the effect of colchicine derivative (43). Figure 8K shows a comparison of the inhibitory activities of the compounds tested in Figures 8H to J. Figure 8L shows the baseline levels of IL-8 production in human neutrophils incubated with colchicine derivatives (43) or (47a) in the absence of MSU.

[0027] [Figure 9] Figure 9 shows the effects of colchicine and colchicine derivatives on superoxide production by human neutrophils stimulated with monosodium urate (MSU). Figure 9A shows the effect of colchicine. Figure 9B shows the effect of colchicine derivative (91). Figure 9C shows the effect of colchicine. Figure 9D shows colchicine derivative (91). Figure 9E shows colchicine derivative (47a). Figure 9F shows the effect of colchicine derivative (43). Figure 9G shows a comparison of the inhibitory activity of the compounds from Figures 9C to F. Figure 9H shows the baseline level of superoxide production in human neutrophils stimulated with colchicine derivative (43) or (47a) in the absence of MSU. [Figure 10] Figure 10 shows the plasma concentrations of colchicine derivative (91) (Figure 10A) or colchicine derivative (43) (Figure 10B) over 2 hours in mice that were subcutaneously injected with either colchicine derivative (91) or colchicine derivative (43), respectively.

[0028] [Figure 11] Figure 11 shows the concentrations of colchicine derivative (91) (Figure 11A) or colchicine derivative (43) (Figure 11B) in circulating leukocytes of mice two hours after subcutaneous injection of either colchicine derivative (91) or colchicine derivative (43). [Figure 12] Figure 12 shows the effects of colchicine and colchicine derivatives (91) on leukocyte recruitment to the dorsal air pouch in mice injected with monosodium urate (MSU). [Figure 13] Figure 13 shows the therapeutic effects of colchicine and colchicine derivatives (91) on leukocyte recruitment to the dorsal air pouch in mice injected with monosodium urate (MSU). [Figure 14] Figure 14 shows the therapeutic effect of colchicine derivatives (43) on leukocyte recruitment to the dorsal air pouch in mice injected with monosodium urate (MSU).

[0029] [Figure 15] Figure 15 shows a Western blot of β-tubulin expression in human neutrophils. [Figure 16] Figure 16 shows how colchicine interacts with βIII tubulin. [Figure 17] Figure 17 shows how the colchicine derivative (91)(CCI) interacts with βIII tubulin. [Figure 18] Figure 18 shows how the colchicine derivative (89) interacts with βIII tubulin. [Figure 19] Figure 19 shows the effects of a high-fat diet and CCI on body weight in wild-type and LDLRKO mice. C57BL / 6 mice were fed either a control diet (CD) or a high-fat diet (HF) for 8 weeks and subcutaneously injected with 0.5 μmol / kg of CCI or vehicle (DMSO) three times a week. The mice's body weight was measured three times a week. The graph shows the body weight gain of each group of mice from week 2 to week 8.

[0030] [Figure 20] Figure 20 shows the effect of CCI on serum triglyceride levels in wild-type and LDLRKO mice fed a high-fat diet. C57BL / 6 mice were fed either a control diet (CD) or a high-fat diet (HF) for 8 weeks and received subcutaneous injections of 0.5 μmol / kg CCI or vehicle (DMSO) three times a week. Blood was collected after 8 weeks of diet, serum was prepared and frozen for analysis. [Figure 21] Figure 21 shows the effect of CCI on serum cholesterol levels in wild-type and LDLRKO mice fed a high-fat diet. C57BL / 6 mice were fed either a control diet (CD) or a high-fat diet (HF). They received subcutaneous injections of 0.5 μmol / kg CCI or vehicle (DMSO) three times a week for 8 weeks. Blood was collected after 8 weeks of dieting, serum was prepared, and frozen for analysis.

[0031] [Figure 22] Figure 22 shows the effect of CCI on the development of atherosclerosis in the aorta of LDLRKO mice fed a high-fat diet. Figure 22A: C57BL / 6 mice were fed either a control diet (CD) or a high-fat diet (HF) for 8 weeks and subcutaneously injected with 0.5 μmol / kg of CCI or vehicle (DMSO) three times a week. The aorta was harvested and dissected, and a frontal assay of aortic lesions was performed using Sudan IV staining. Sudan IV is a lipid-soluble dye that stains lipids, triglycerides, and lipoproteins. Figure 22B shows the percentage of the total area of ​​the aortic arch covered with plaque stained by the "frontal assay". Figure 22C shows the percentage of the total area of ​​the descending aorta covered with plaque stained by the "frontal assay". [Figure 23] Figure 23 shows the effect of CCI on cytokine serum levels in LDLRKO mice fed a high-fat diet: C57BL / 6 mice were fed either a control diet (CD) or a high-fat diet (HF) for 8 weeks and subcutaneously injected with 0.5 μmol / kg CCI or vehicle (DMSO) three times a week. Blood was collected after 8 weeks of diet, serum was prepared and frozen for analysis by Luminex assay. [Modes for carrying out the invention]

[0032] Detailed description of a specific embodiment References cited herein are incorporated by reference.

[0033] definition In describing the compounds, compositions, methods, and uses of the present invention, the following terms have the meanings set forth below unless otherwise specified. As used herein, the term “colchicine derivative” may include any of the derivatives described herein, and, for example, thiocolchicine derivatives, where appropriate. As used herein, the term “therapeutic dose” means the amount of an active compound or drug that elicits a biological or medical response in a tissue, system, or animal, such as a mammal (e.g., human), as determined by researchers, veterinarians, physicians, or other clinicians. Where given to treat a disorder, condition, and / or disease, it is the amount that, when administered to a subject, including mammals, can achieve the desired outcome, such as the treatment of symptoms.

[0034] The compounds of the present invention may have an asymmetric center, a chiral axis, and a chiral plane (as described, for example, in E.L. Eliel and SH. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and may occur as racemates, racemic mixtures, and individual diastereomers. All possible isomers, including optical isomers, and mixtures thereof are included in the present invention. Furthermore, the compounds disclosed herein may exist as tautomers, and both tautomer forms are intended to be included in the scope of the present invention, even if only one tautomer structure may be shown. Generally, references to specific elements such as hydrogen (H) imply, where appropriate, that all isotopes of that element are included. When the term “alkyl group” is used alone or within other terms such as “haloalkyl group” and “alkylamino group,” it encompasses, for example, linear or branched carbon radicals having 1 to about 20 carbon atoms, or in specific embodiments, 1 to about 12 carbon atoms. In other embodiments, the alkyl group is a “lower alkyl” group having 1 to about 6 carbon atoms. Examples of such groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, and hexyl. In more specific embodiments, the lower alkyl group has 1 to 4 carbon atoms.

[0035] The term “alkenyl group” encompasses linear or branched carbon radicals having at least one carbon-carbon double bond. The term “alkenyl group” can also encompass conjugated and unconjugated carbon-carbon double bonds or combinations thereof. An alkenyl group can, for example, have 2 to about 20 carbon atoms, or in certain embodiments, 2 to about 12 carbon atoms. In embodiments, an alkenyl group is a “lower alkenyl” group having 2 to about 4 carbon atoms. Examples of alkenyl groups, but not limited to these, include ethenyl, propenyl, allyl, propenyl, butenyl, and 4-methylbutenyl. The terms “alkenyl group” and “lower alkenyl group” encompass groups having “cis” and “trans” orientations, or “E” and “Z” orientations. The term "alkynyl group" refers to a linear or branched carbon radical having at least one carbon-carbon triple bond. The term "alkynyl group" can encompass conjugated and unconjugated carbon-carbon triple bonds or combinations thereof. An alkynyl group can, for example, have 2 to about 20 carbon atoms, or in certain embodiments, 2 to about 12 carbon atoms. In embodiments, an alkynyl group is a "lower alkynyl" group having 2 to about 10 carbon atoms. Some examples are lower alkynyl groups having 2 to about 4 carbon atoms. Examples of such groups include propargyl and butynyl.

[0036] The term "halo" refers to halogens such as fluorine, chlorine, bromine, or iodine atoms. The term "haloalkyl group" encompasses groups in which one or more alkyl carbon atoms are substituted with halos as defined above. Specifically, this includes polyhaloalkyl groups, including monohaloalkyl groups, dihaloalkyl groups, and perhaloalkyl groups. As an example, a monohaloalkyl group may have one of the following atoms in its group: iodine, bromo, chloro, or fluoro. Dihalo and polyhaloalkyl groups may have two or more identical halo atoms or combinations of different halo groups. "Lower haloalkyl groups" encompass groups having 1 to 6 carbon atoms. In some embodiments, lower haloalkyl groups have 1 to 3 carbon atoms. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.

[0037] The term “hydroxyalkyl group” encompasses, for example, linear or branched alkyl groups having 1 to about 10 carbon atoms, any one of which may be substituted with one or more hydroxyl groups. In embodiments, a hydroxyalkyl group is a “lower hydroxyalkyl” group having 1 to 6 carbon atoms and one or more hydroxyl groups. Examples of such groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxyhexyl. The term “alkoxy group” encompasses linear or branched oxy-containing groups, each having an alkyl moiety of 1 to about 10 carbon atoms, for example, but not limited to these. In embodiments, the alkoxy group is a “lower alkoxy” group having 1 to 6 carbon atoms. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, and tert-butoxy. In certain embodiments, the lower alkoxy group has 1 to 3 carbon atoms. The “alkoxy” group may be further substituted with one or more halo atoms, such as fluoro, chloro, or bromo, to provide a “haloalkoxy” group. In other embodiments, the lower haloalkoxy group has 1 to 3 carbon atoms. Examples of such groups include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy, and fluoropropoxy.

[0038] The terms “aromatic group” or “aryl group” refer to an aromatic group having one or more rings, such rings may be joined together in a pendant-like manner or may be fused. In certain embodiments, an aromatic group has one, two, or three rings. Monocyclic aromatic groups may contain 4 to 10 carbon atoms in the ring, typically 4 to 7 carbon atoms, more typically 4 to 6 carbon atoms. Typical polycyclic aromatic groups have two or three rings. Polycyclic aromatic groups with two rings typically have 8 to 12 carbon atoms in the ring, preferably 8 to 10 carbon atoms. Examples of aromatic groups, but not limited to these, include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl, or acenaphthyl. The term "heteroatom" refers to an atom other than carbon. Typically, heteroatoms are selected from the group consisting of sulfur, phosphorus, nitrogen, and oxygen atoms. Groups containing multiple heteroatoms may include different heteroatoms.

[0039] The terms “heteroaromatic group” or “heteroaryl group” refer to aromatic groups having one or more rings, such rings may be joined together in a pendant-like manner or fused, and the aromatic group has at least one heteroatom. Monocyclic heteroaromatic groups may contain 4 to 10 member atoms in the ring, typically 4 to 7 member atoms, and more typically 4 to 6 member atoms. Typical polycyclic heteroaromatic groups have two or three rings. Polycyclic aromatic groups with two rings typically have 8 to 12 member atoms, and more typically 8 to 10 member atoms in the ring. Examples of heteroaromatic groups include, but are not limited to, pyrrole, imidazole, thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline, quinazoline, and quinoxaline. The term "carbocyclic group" refers to a saturated or unsaturated carbocyclic hydrocarbon ring. Carbocyclic groups are not aromatic. Carbocyclic groups can be monocyclic or polycyclic. Polycyclic carbocyclic groups can be condensed, spiro, or bridging ring systems. Monocyclic carbocyclic groups can contain 4 to 10 carbon atoms, typically 4 to 7 carbon atoms, more typically 5 to 6 carbon atoms within the ring. Bicyclic carbocyclic groups can contain 8 to 12 carbon atoms, usually 9 to 10 carbon atoms, within the ring.

[0040] The term "heterocyclic group" refers to a saturated or unsaturated cyclic structure containing a carbon atom and one or more heteroatoms within the ring. Heterocyclic groups are not aromatic. Heterocyclic groups can be monocyclic or polycyclic. Polycyclic heterocyclic groups can be condensed, spiro, or bridging cyclic systems. Monocyclic heterocyclic groups may contain 4 to 10 member atoms within the ring (i.e., both carbon atoms and at least one heteroatom), typically 4 to 7, more typically 5 to 6. Bicyclic heterocyclic groups may contain 8 to 18 member atoms within the ring, typically 9 or 10. Representative heterocyclic groups include, by example, pyrrolidine, imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine, thiomorpholine, piperazine, and 3-pyrroline. The term "heterogeneous group" refers to a saturated or unsaturated chain of non-hydrogen member atoms, including a carbon atom and at least one heteroatom. Heterogeneous groups typically have 1 to 25 member atoms. More typically, the chain contains 1 to 12 member atoms, 1 to 10, and most typically 1 to 6. The chain can be linear or branched. A typical branched heterogeneous group has one or two branches, more commonly one branch. Heterogeneous groups are typically saturated. Unsaturated heterogeneous groups may have one or more double bonds, one or more triple bonds, or both. A typical unsaturated heterogeneous group has one or two double bonds or one triple bond. More typically, an unsaturated heterogeneous group has one double bond.

[0041] The term "hydrocarbon group" or "hydrocarbyl group" refers to a chain of 1 to 25 carbon atoms, typically 1 to 12 carbon atoms, more typically 1 to 10 carbon atoms, and most typically 1 to 8 carbon atoms. Hydrocarbon groups can have a linear or branched chain structure. Typical hydrocarbon groups have one or two branches, typically one branch. Typically, hydrocarbon groups are saturated. Unsaturated hydrocarbon groups may have one or more double bonds, one or more triple bonds, or a combination thereof. Typical unsaturated hydrocarbon groups have one or two double bonds or one triple bond. More typically, unsaturated hydrocarbon groups have one double bond. When the term "unsaturated" is used in combination with any group, that group may be fully unsaturated or partially unsaturated. However, when the term "unsaturated" is used in combination with a specific group as defined herein, the term maintains the limitations of that specific group. For example, an unsaturated "carbocyclic group" based on the limitations of "carbocyclic groups" as defined herein does not include aromatic groups.

[0042] The term "carboxyl group" or "carboxyl group," whether used alone or in conjunction with other terms such as "carboxyalkyl group," refers to -(C=O)-O-. The term "carbonyl group," whether used alone or in conjunction with other terms such as "aminocarbonyl group," refers to -(C=O)-. The term "alkylcarbonyl group" refers to a carbonyl group substituted with an alkyl group. In certain embodiments, a "lower alkylcarbonyl group" has a lower alkyl group as described above bonded to a carbonyl group. The term “aminoalkyl group” encompasses linear or branched alkyl groups having one to about ten carbon atoms, any one of which may be substituted with one or more amino groups. In some embodiments, the aminoalkyl group is a “lower aminoalkyl” group having one to six carbon atoms and one or more amino groups. Examples of such groups include aminomethyl, aminoethyl, aminopropyl, aminobutyl, and aminohexyl.

[0043] The term "alkylaminoalkyl group" encompasses aminoalkyl groups having an alkyl group independently substituted with a nitrogen atom. In certain embodiments, the alkylaminoalkyl group is a "lower alkylaminoalkyl" group having an alkyl group with 1 to 6 carbon atoms. In other embodiments, the lower alkylaminoalkyl group has an alkyl group with 1 to 3 carbon atoms. Suitable alkylaminoalkyl groups can be mono- or dialkyl-substituted, such as N-methylaminomethyl, N,N-dimethylaminoethyl, and N,N-diethylaminomethyl. The term “aralkyl group” encompasses aryl-substituted alkyl groups. In embodiments, the aralkyl group is a “lower aralkyl” group having an aryl group bonded to an alkyl group having 1 to 6 carbon atoms. In other embodiments, the lower aralkyl group phenyl is bonded to an alkyl moiety having 1 to 3 carbon atoms. Examples of such groups include benzyl, diphenylmethyl, and phenylethyl. The aryl in the aralkyl group may be further substituted with halo, alkyl, alkoxy, haloalkyl, and haloalkoxy groups.

[0044] The term "arylalkenyl group" encompasses aryl-substituted alkenyl groups. In embodiments, an arylalkenyl group is a "lower arylalkenyl" group having an aryl group bonded to an alkenyl group having 2 to 6 carbon atoms. Examples of such groups include phenylethenyl. The aryl in the arylalkenyl may be further substituted with halo, alkyl, alkoxy, haloalkyl, and haloalkoxy groups. The term "arylalkynyl group" encompasses aryl-substituted alkynyl groups. In embodiments, an arylalkynyl group is a "lower arylalkynyl" group having an aryl group bonded to an alkynyl group having 2 to 6 carbon atoms. Examples of such groups include phenylethynyl. The aryl in the aralkyl group may be further substituted with halo, alkyl, alkoxy, haloalkyl, and haloalkoxy. The terms benzyl and phenylmethyl are interchangeable.

[0045] The term "alkylthio group" encompasses groups containing a linear or branched alkyl group of 1 to 10 carbon atoms bonded to a divalent sulfur atom. In certain embodiments, lower alkylthio groups have 1 to 3 carbon atoms. An example of an "alkylthio" is methylthio(CH3S-). The term “alkylamino group” refers to amino groups substituted with one alkyl group and two alkyl groups, including the terms “N-alkylamino” and “N,N-dialkylamino.” In embodiments, the alkylamino group is a “lower alkylamino” group having one or two alkyl groups of 1 to 6 carbon atoms bonded to a nitrogen atom. In other embodiments, the lower alkylamino group has 1 to 3 carbon atoms. Suitable “alkylamino” groups may be mono- or dialkylaminos such as N-methylamino, N-ethylamino, N,N-dimethylamino, and N,N-diethylamino. The term "arylamino group" refers to an amino group substituted with one or two aryl groups, such as N-phenylamino. The "arylamino" group may be further substituted at the aryl ring portion of the group. The term "heteroarylamino" refers to an amino group substituted with one or two heteroaryl groups, such as N-thienylamino. A "heteroarylamino" group may be further substituted at the heteroaryl ring portion of the group.

[0046] The term "aralkylamino group" refers to an amino group substituted with one or two aralkyl groups. In other embodiments, phenyl-C1-C3-alkylamino groups such as N-benzylamino may be present. The "aralkylamino" group may be further substituted with the aryl ring portion of the group. The term "alkylaminoalkylamino group" refers to an alkylamino group substituted with one or two alkylamino groups. In embodiments, a C1-C3-alkylamino-C1-C3-alkylamino group is present.

[0047] The term "arylthio group" encompasses aryl groups of 6 to 10 carbon atoms bonded to a divalent sulfur atom. An example of "arylthio" is phenylthio. The term "aralkylthio group" encompasses the above aralkyl groups bonded to a divalent sulfur atom. In certain embodiments, phenyl-C1-C3-alkylthio groups exist. An example of "aralkylthio" is benzylthio. The term "aryloxy group" encompasses aryl groups that are optionally substituted with an oxygen atom, as defined above. An example of such a group is phenoxy. The term "aralkoxy group" encompasses oxy-containing aralkyl groups bonded to another group via an oxygen atom. In certain embodiments, the aralkoxy group is a "lower aralkoxy" group having an optionally substituted phenyl group bonded to a lower alkoxy group, as described above. The term "cycloalkyl group" includes saturated carbocyclic groups. In certain embodiments, cycloalkyl groups include C3-C6 rings. In embodiments, compounds containing cyclopentyl, cyclopropyl, and cyclohexyl exist.

[0048] The term "cycloalkenyl group" includes carbocyclic groups having one or more carbon-carbon double bonds: conjugated or unconjugated, or a combination thereof. "Cycloalkenyl" and "cycloalkyldienyl" compounds are included in the term "cycloalkenyl." In certain embodiments, the cycloalkenyl group includes a C3-C6 ring. Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cycloheptadienyl. The "cycloalkenyl" group may have one to three substituents, such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, and lower alkylamino. The terms “suitable substituent,” “substituent,” or “substituted” as used in conjunction with the groups described herein refer to chemically and pharmaceutically acceptable groups, i.e., parts that do not negate the therapeutic activity of the compounds of the present invention. It is understood that the substituents and substitution patterns of the compounds of the present invention can be selected by those skilled in the art to provide compounds that are chemically stable and readily synthesizable by the art and the methods described below. If the substituent itself is substituted with multiple groups, it is understood that these multiple groups may be on the same carbon / member atom or on different carbon / member atoms, as long as a stable structure is obtained. Some exemplary examples of suitable substituents include cycloalkyl, heterocyclyl, hydroxyalkyl, benzyl, carbonyl, halo, haloalkyl, perfluoroalkyl, perfluoroalkoxy, alkyl, alkenyl, alkynyl, hydroxy, oxo, mercapto, alkylthio, alkoxy, aryl or heteroaryl, aryloxy or heteroaryloxy, aralkyl or heteroaralkyl, aralkoxy or heteroaralkoxy, HO-(C=O)-, amide, amino, alkyl- and dialkylamino, cyano, nitro, carbamoyl, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylcarbonyl, aryloxycarbonyl, alkylsulfonyl, and arylsulfonyl. Typical substituents include aromatic groups, substituted aromatic groups, hydrocarbon groups containing alkyl groups such as methyl groups, substituted hydrocarbon groups such as benzyl, and heterogeneous groups containing alkoxy groups such as methoxy groups.

[0049] The term "condensation" means that two or more carbon / member atoms are common to two adjacent rings, for example, that the rings are "condensed rings". pharmaceutically acceptable salts of the compounds of the present invention include, for example, conventional non-toxic salts of the compounds of the present invention formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and trifluoroacetic acid. pharmaceutically acceptable salts of the compounds of the present invention can be synthesized from the compounds of the present invention, including basic or acidic moieties, by conventional chemical methods. Generally, salts of basic compounds are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric amount or excess of the desired salt-forming inorganic or organic acid in a suitable solvent or a variety of solvent combinations. Similarly, salts of acidic compounds are formed by reaction with a suitable inorganic or organic base.

[0050] The present invention comprises pharmaceutically acceptable salts, solvates, and prodrugs of the compounds of the present invention and mixtures thereof. The term “condition” refers to a physical condition in a mammal (as a whole or as one or more of its parts) that does not conform to a standard physical condition relating to the health status of a mammal. Conditions described herein include, but are not limited to, disorders and diseases, wherein the term “disorder” refers to a mammalian condition relating to a functional abnormality of the mammal or any part thereof, and the term “disease” refers to a mammalian condition that impairs the normal function of the mammal’s body or any part thereof, usually manifesting by distinguishing between signs and symptoms. Typically, the compounds and compositions described herein are useful for treating inflammatory conditions, which typically have inflammatory components caused by neutrophils.

[0051] The term "neutrophil-induced" inflammation means that the inflammation is related to neutrophils. Given the multifactorial nature of inflammation, using this term implies that neutrophils are drivers or mediators of inflammation, not necessarily the sole drivers or mediators, but contribute to at least part of the pathogenesis of inflammation. For example, other immune cells such as monocytes and / or macrophages can often also be drivers of inflammation. Neutrophil-induced inflammation can be selected from neutrophil-induced inflammatory diseases, neutrophil-induced inflammatory disorders, neutrophil-induced inflammatory conditions, or a combination thereof. For example, neutrophil-induced inflammation may refer to a condition in which cytokines secreted by neutrophils have pathological effects, such as IL-1 and / or IL-8.

[0052] Examples of such conditions include psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult dyspnea syndrome, thrombosis, glomerulonephritis, rheumatoid arthritis, osteoarthritis, meningitis, stroke including ischemic and hemorrhagic stroke, neurotraumatic / occlusive head injury, endotoxemia and / or toxic shock syndrome, inflammatory responses induced by endotoxins or inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, multiple sclerosis, cachexia, bone resorption, psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis, rubella arthritis, acute synovitis, diabetes mellitus, pancreatic β-cell disease, Alzheimer's disease, pseudogout, cardiovascular disease, and other acute or chronic inflammatory disease conditions such as vasculitis. Typically, the condition is gout, pseudogout, cardiovascular disease, vasculitis, or atherosclerosis. As used herein, “treatment,” “to treat,” or “therapy” refers to an approach to obtain a beneficial or desired clinical outcome. For the purposes described herein, beneficial or desired clinical outcomes may include, but are not limited to, relief of symptoms, reduction of disease severity, stable (i.e., non-exacerbating) state of disease, delay or slowing of disease progression, and / or improvement or mitigation of the condition, whether detectable or undetectable. Thus, “treatment” or “therapy” can be considered an intervention undertaken with the intention of altering the condition of a disorder. Specifically, treatment or therapy may directly prevent, slow, or otherwise reduce the condition of a disease or disorder, or may make the subject more sensitive to treatment or therapy with other therapeutic agents.

[0053] The term “chronic gout” includes gout present in individuals with recurrent or prolonged gout attacks (also known as “gout flares”), gouty nodule formation, chronic inflammatory arthritis, and / or gout-related joint destruction. The term "acute gout" includes gout present in a person who has or is suffering from at least one gouty symptom, such as a gout attack. The terms "gout-associated inflammation" or "gouty arthritis" refer to local or systemic inflammation (which may be asymptomatic) resulting from an immune response to uric acid crystals. The term “administer” (e.g., “administer” the compound) in relation to the compounds of the present invention means introducing the compound or a prodrug of the compound into a system of an animal in need of treatment. When the compound or its prodrug of the present invention is provided in combination with one or more other active agents (e.g., an antigout agent), “administer” and its variants are understood to include the simultaneous and sequential introduction of the compound or its prodrug and the other agents, respectively.

[0054] The terms "treating gout" or "treatment of gout" refer to the administration of medication to mammals suffering from a gouty condition, and refer to the effect of reducing the gouty arthritis condition by limiting inflammation and / or reducing inflammation-related pain. The term "pseudogout," also known as calcium pyrophosphate crystal deposition (CPPD) disease, refers to a type of arthritis that causes spontaneous, painful swelling in the joints. It occurs when CPP crystals form in the synovial fluid, causing inflammation and pain. The terms "comprising," "having," and "including," and their various inflectional endings, all mean something that includes the constituent elements shown but excludes other elements; they are unrestricted (open-ended).

[0055] The term "consists of" has a closed-end or restrictive meaning, and "essentially consists of" means that it contains the specified components, excluding materials present as impurities, unavoidable materials present as a result of the processes used to provide the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "essentially consists of" includes any known pharmaceutically acceptable additives, excipients, diluents, carriers, etc. Typically, a composition consisting essentially of a set of components will contain less than 5% by weight, typically less than 3% by weight, and more typically less than 1% by weight of an unspecified component. When introducing elements disclosed herein, the articles “a” (one), “an” (one), “the” (the), and “said” (the foregoing) are intended to mean that one or more elements exist. Where used herein, the term “and / or” includes any and all combinations of one or more of the related listed items.

[0056] Any component defined herein as being included may be expressly excluded from the claimed invention by means of a proviso or negative limitation. For example, in certain embodiments, certain functional groups may be expressly excluded from the compounds described herein. Finally, as used herein, terms of degree such as “substantially,” “about,” and “almost” mean a reasonable amount of deviation from the modified term so as not to significantly alter the final result. These terms of degree should be interpreted as including a deviation of at least ±5% of the modified term, provided that this deviation does not negate the meaning of the word being changed. Furthermore, all ranges described herein include any point at the end and middle of the range, whether explicitly stated or not. Thus, as used herein, phrases such as "between X and Y" and "about between X and Y" should be interpreted as including X and Y. As used herein, phrases such as "about between X and Y" mean "about between X and about Y". As used herein, phrases such as "about X to Y" mean "about X to about Y".

[0057] Colchicine derivatives Colchicine derivatives, compositions containing the derivatives, methods of administering the same, and their use are provided for the treatment of inflammation.

[0058] Colchicine derivatives are compounds of formula I: [ka] Here, Z is either O or S; X 1R is selected from substituted or unsubstituted hydrocarbon groups, or substituted or unsubstituted heterogeneous groups; 2 and R 3 Each is independently selected from substituted or unsubstituted hydrocarbon groups, substituted or unsubstituted heterogeneous groups, substituted or unsubstituted carbocyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aromatic groups, or substituted or unsubstituted heteroaromatic groups; they are represented by their pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof. In other embodiments, R 2 and R 3 If both are methyl, then X 1 It is not methyl.

[0059] In a particular embodiment of formula I, R 2 and R 3 Each of these is independently selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted alkenyl groups, substituted or unsubstituted alkynyl groups, substituted or unsubstituted aromatic groups, substituted or unsubstituted heteroaromatic groups, substituted or unsubstituted carbocyclic groups, or substituted or unsubstituted heterocyclic groups. In more specific embodiments, R 2 and R 3 Each is independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted C1-C6 alkylcarbonyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl. In other embodiments, R2 and R 3 are each independently selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C1-C6 alkylcarbonyl, C1-C6 alkylene-O-alkyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl. In more specific embodiments, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkylaryl. In further embodiments, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl, or substituted or unsubstituted alkylaryl. In additional embodiments, R 2 and R 3 are each independently selected from substituted or unsubstituted alkyl. In further embodiments, R 2 and R 3 are each independently selected from substituted or unsubstituted C1-C6 alkyl.

[0060] In one embodiment, X 1 is a substituted or unsubstituted hydrocarbon group. In further embodiments, X 1 is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl. In certain embodiments, X 1 is selected from substituted or unsubstituted C1-C6 alkyl, such as substituted or unsubstituted alkyl especially unsubstituted C1-C6 alkyl. In more specific embodiments, X 1 is selected from methyl or ethyl.

[0061] In another embodiment, X 1 is a substituted or unsubstituted heterogeneous group. In further embodiments, X 1 is selected from -CR 4 R 5 R 6 where R 4 R 5 R6 is independently selected from H, a substituted or unsubstituted hydrocarbon group, and a substituted or unsubstituted heterogeneous group. In particular, R 4 R 5 R 6 can each be independently selected from substituted or unsubstituted amide groups. In certain embodiments, R 4 and R 5 are each independently selected from H and substituted or unsubstituted alkyl, and R 6 is -NR(CO)CR 7 R 8 R 9 where R is selected from H and substituted or unsubstituted alkyl, and R 7 R 8 and R 9 are each selected from H, a halo group, and substituted or unsubstituted alkyl. R 7 R 8 and R 9 can be selected from halo. More specifically, R 7 R 8 and R 9 can be selected from fluoro groups.

[0062] In a further embodiment, X 1 is OR 10 where R 10 is selected from a substituted or unsubstituted hydrocarbon group or a substituted or unsubstituted heterogeneous group. In a further embodiment, R 10 is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group. In particular, R 10The following are selected from substituted or unsubstituted alkyl, CH2OH, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl.

[0063] In other embodiments, R 10 R is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl. In other embodiments, R 10 This is selected from substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C2-C6 alkenyl groups, or C2-C6 alkynyl groups. In a further embodiment, R 10 R is selected from substituted or unsubstituted C1-C6 alkyl groups, particularly substituted or unsubstituted alkyl groups such as unsubstituted C1-C6 alkyl groups. In a more specific embodiment, R 10 It is selected from methyl or ethyl.

[0064] In certain embodiments, the colchicine derivative includes the compound of formula IA: [ka]

[0065] In the case of formula IA, R 2 , R 3 and X 1 The above may apply to equation I. In certain embodiments, the colchicine derivative includes the compound of formula IB: [ka]

[0066] In the case of formula IB, R 2 , R 3 and X 1 The above may apply to equation I. In other embodiments, the colchicine derivative includes compounds of formula IC: [ka]

[0067] In the case of formula IC, R 3 and X 1 The above may apply to equation I. In other embodiments, the colchicine derivative includes the compound of formula ID: [ka]

[0068] In the case of expression ID, R 3 and X 1 The above may apply to equation I. In certain embodiments of the formulas I and IA to ID, X 1 is methyl or methoxy. In another embodiment, R 3 R is selected from substituted or unsubstituted alkyl groups. In further embodiments, R 3 R is selected from substituted or unsubstituted C1-C6 alkyl groups, more specifically, 3 It is ethyl.

[0069] Colchicine derivatives described herein may be in the form of their pharmaceutically acceptable salts, hydrates, solvates, tautomers, optical isomers, or combinations thereof. In more specific embodiments, compounds of formulas I and IA to ID have an S configuration at C7. Specific examples of compounds of formulas I and IA to ID are shown in Figures 1 to 4 and 4A to 4D.

[0070] The specific compounds described herein can be prepared, for example, as follows: a) Compounds of formula IV [ka] Reacting with ROCl [ka] This forms a substituted or unsubstituted alkyl group, where R can be selected from substituted or unsubstituted alkyl groups, and X 1 This may be as defined above.

[0071] The specific compounds described herein can also be prepared as follows: a) Compounds of formula IV [ka] to R 2 React with Br [ka] It forms X. 1 and R 2 This may be as defined above.

[0072] The specific compounds described herein can also be prepared as follows: a) Compounds of formula VII [ka] to R 2 React with Br [ka] It forms X. 1 and R 3 This may be as defined above.

[0073] More specific X 1 The base is, for example, formula VI or VIII HO(CO)CR 4 R 5 R 6 It can be added by reacting with -(CO)X 1 is -(CO)OR, and R 4 R 5 R 6 Each of these is independently selected from H, a substituted or unsubstituted hydrocarbon group, and a substituted or unsubstituted heterogeneous group. In particular, R 4 R 5 R 6 Each of these can be independently selected from substituted or unsubstituted amide groups. In certain embodiments, R 4 and R 5 Each of these is independently selected from H, a substituted or unsubstituted alkyl group, and R 6 is -NR(CO)CR 7 R 8 R 9 And here, R 7 , R 8 , and R 9 Each of these is selected from H, a halo group, and a substituted or unsubstituted alkyl group. 7 , R 8 , and R 9 This can be selected from halo groups. More specifically, R 7 , R 8 , and R 9 The group can be selected from fluorogroups.

[0074] Certain compounds described herein can also be prepared, for example, as follows: a) Compounds of formula VIA [ka] This was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), hydroxybenzotriazole (HOBt), and CF3NHCH2COOH (F3CglyOH). [ka] It forms R 2 This can be as defined above.

[0075] The specific compounds described herein can also be prepared as follows: a) Compounds of formula VIIA [ka] Protect the hydroxyl group [ka] Forms a protective group (PG): b) The compound of formula VIIB is reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), hydroxybenzotriazole (HOBt), and CF3NHCH2COOH (F3CglyOH), followed by deprotection: [ka] It forms.

[0076] The specific compounds described herein can be prepared, for example, as follows: a) React the compound of formula XX with RO(C=O)Cl: [ka] [ka] It forms R 2 , R 3 and R 10 This can be as defined above.

[0077] The specific compounds described herein can also be prepared as follows: a) Protect the hydroxyl group of the compound of formula XXII [ka] [ka] Forms a protective group (PG): b) Compound XIIB is R 10 React with O(C=O)Cl, then deprotect: [ka] It forms.

[0078] Generally, the compounds described herein can be prepared by using reactions and standard procedures known in the literature or illustrated herein. The compounds described herein are useful in treating inflammatory conditions, diseases, and / or disorders, such as gout. The gout being treated may be, for example, chronic gout and / or acute gout. In particular, the compounds described herein can treat at least one gout symptom, such as a gout attack or gout-related joint destruction. For example, the compounds described herein can limit gouty inflammation and / or reduce inflammation-related pain. Gout is known as one of the most painful and common forms of inflammatory arthritis. Its prevalence (3–6% in Western countries) is increasing worldwide due to rising conditions that promote major risk factors for hyperuricemia, such as gout, obesity, and renal failure. Monosodium urate (MSU) crystals are the etiology of gouty arthritis, forming in the joints and soft tissues when the concentration of uric acid in circulation exceeds its solubility (>6 mg / mL), triggering a powerful innate immune response that causes unbearable pain.

[0079] The initial events of a gout attack include MSU-induced activation of macrophages in the tissue and cell death within the joint. These cellular events lead to the release of pro-inflammatory cytokines that promote and amplify the inflammatory response. IL-1 and IL-8 have been identified as cytokines in the pathogenesis of gout. IL-1 modulates the initial molecular events of a gout attack, including increased expression of adhesion molecules (such as E-selectin) on the surface of endothelial cells and the release of chemokines. IL-8 is one of the most potent chemotactic substances for neutrophils, promoting a massive influx of neutrophils that triggers the typical symptoms of gout, including severe pain and swelling in the affected joint. Recruited neutrophils are activated by MSU and release inflammatory cytokines, reactive oxygen species (ROS), proteases, and neutrophil extracellular traps (NETs) that amplify the inflammatory response. At high concentrations of neutrophils, NETs interact with MSU to form complexes called aggregated NETs, ​​which contribute to the resolution of the gout attack.

[0080] Treatment for gout targets two key aspects of its pathogenesis: the development of MSU crystals and inflammation. Drugs used for the former include xanthine oxidase inhibitors (XOIs), while those for the latter include colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), or corticosteroids. Colchicine is also used for the prevention of gout attacks upon initiation of uric acid-lowering therapy, for some patients intolerant to XOIs, and for prolonged prevention. While the frequency and occurrence of gout attacks are unpredictable, it is not uncommon for patients to suffer from gout attacks for approximately four years after initiating uric acid-lowering therapy. On average, 60% of patients experience a gout attack within one to two years of their first attack.

[0081] Treating gout is challenging because many patients suffer from multiple comorbidities associated with relative contraindications to available anti-inflammatory drugs. Furthermore, most anti-inflammatory drugs lack specificity to the molecular mechanisms underlying MSU-induced inflammation. In contrast, colchicine exhibits some specificity to the molecular pathways involved in gout, as it inhibits MSU-induced neutrophil activation but does not inhibit specific neutrophil responses induced by bacterial peptide fMLP. Nevertheless, the therapeutically safe dose range of colchicine is very narrow, and it exhibits significant gastrointestinal toxicity, which is a major reason for poor compliance with this drug. The colchicine derivatives described herein are less toxic and more specific to hematopoietic cells such as neutrophils; and to inflammatory cells abundant in gout attacks. PCT Publication No. WO2011022805 (Tuszynski et al.) demonstrated that colchicine, an analogue of an antimitotic agent, is less toxic for the treatment of cancer.

[0082] Advantageously, the anti-inflammatory activity of the colchicine derivatives described herein is not only comparable to that of colchicine, but can also be preserved at doses where colchicine lacks anti-inflammatory properties (for example, the colchicine derivatives described herein may provide anti-inflammatory effects at lower doses than colchicine (e.g., at least about 10 times lower), and are therefore, in some respects, more potent than colchicine). As shown in the following examples, compounds (91), 43, and 47a can suppress the increase in intracellular calcium concentration at a low dose of 0.1 μM. In contrast, colchicine induced similar inhibition of calcium recruitment at a concentration of 10 μM (Figures 7A, E, K, and L). Therefore, unexpectedly, colchicine derivatives (91), (47a), and (43) can significantly reduce the mobilization of intracellular calcium stores at concentrations approximately 10 to 100 times lower (approximately 1 / 10 to 1 / 100) than colchicine; at doses in which colchicine has been shown to be ineffective (Figure 7).

[0083] The efficacy of colchicine for other inflammatory diseases has been demonstrated in the treatment of relapsing pericarditis and familial Mediterranean fever (FMF) (Slobodnock et al. The American Journal of Medicine (2015)). Patients with FMF, a genetic disorder primarily affecting one in 200 to 1,000 people in a population originating in the Mediterranean region, benefit from lifelong treatment with colchicine. Colchicine is the gold standard treatment for FMF (https: / / www.fmffoundation.org / fmf). For pericarditis, current European guidelines recommend administering 2 mg of colchicine daily for 1 to 2 days, followed by a maintenance dose (Slobodnock et al. The American Journal of Medicine (2015)). Other secondary markets for colchicine derivatives include, but are not limited to, diseases involving neutrophil-mediated inflammation such as pseudogout, coronary atherosclerosis, vasculitis, or combinations thereof. In particular, with regard to pseudogout, colchicine has been suggested for the prevention and acute treatment of CPP arthritis (Slobodnock et al. The American Journal of Medicine (2015)). Furthermore, in coronary artery atherosclerosis, in which a complex immune-inflammatory pathway involving neutrophils is involved in the development, growth, and instability of atherosclerotic plaques, colchicine has been reported to suppress the blood levels of inflammatory mediators and prevent cholesterol crystal-induced neutrophil-mediated inflammation, which is involved in the progression and instability of atherosclerosis (Nidorf et al. 2014).With respect to the results presented herein, CCI and other colchicine derivatives are expected to be used in the treatment of gout and other inflammatory diseases, conditions, and / or disorders, such as recurrent pericarditis, FMF, psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult dyspnea syndrome, thrombosis, glomerulonephritis, rheumatoid arthritis, osteoarthritis, meningitis, stroke including ischemic and hemorrhagic stroke, neurotraumatic / occlusive head injury, endotoxemia and / or toxic shock syndrome, inflammatory responses induced by endotoxins or inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, multiple sclerosis, cachexia, bone resorption, psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis, rubella arthritis, acute synovitis, diabetes mellitus, pancreatic β-cell disease, Alzheimer's disease, pseudogout, cardiovascular disease, and other acute or chronic inflammatory disease conditions such as vasculitis. Typically, the condition is gout, pseudogout, cardiovascular disease, vasculitis, or atherosclerosis.

[0084] Regarding the treatment of gout, colchicine is most specific to the pathogenesis of this inflammatory disease because it weakens most of the inflammatory activity of neutrophils, the major white blood cells involved in gout attacks. Nevertheless, the administration of this alkaloid remains challenging due to the low therapeutic index between efficacy and treatment-limiting side effects. In fact, and unexpectedly, the colchicine derivatives described herein can offer increased specificity to neutrophils compared to colchicine and also possess similar anti-inflammatory activity at low doses. In other words, the colchicine derivatives described herein were found to produce the same anti-inflammatory effect as colchicine, but surprisingly, they were significantly more potent. These unexpected results offer the advantage that their increased specificity reduces the potential for toxicity and undesirable secondary effects, while allowing the colchicine derivatives described herein to be administered at lower doses. This is particularly important for gout patients suffering from chronic kidney disease, as most anti-inflammatory drugs used to treat gout attacks, including colchicine, are contraindicated in these patients. The same applies to patients with advanced liver dysfunction. The colchicine derivatives described herein can be administered at lower doses, have higher specificity, and thereby reduce the potential for toxicity; therefore, they can be associated with fewer troublesome side effects caused by colchicine administration, such as gastrointestinal complications.

[0085] The compounds of the present invention can be administered to mammals, typically animals such as humans, alone or in combination with pharmaceutically acceptable carriers or diluents in pharmaceutical compositions, and optionally in combination with known adjuvants such as alum, according to standard pharmaceutical practices. The compounds can be administered orally or parenterally, including via intravenous, intramuscular, intraperitoneal, and subcutaneous routes of administration. As described above, the compounds of the present invention can be administered orally. For oral use of the compounds or compositions according to the present invention, the selected compounds may be administered, for example, in the form of tablets or capsules, or as aqueous solutions or suspensions. In the case of oral tablets, commonly used carriers include lactose and corn starch, and lubricants such as magnesium stearate are commonly added. In the case of oral administration in capsule form, useful diluents include lactose and dried corn starch. If an aqueous suspension is required for oral use, the active ingredient is combined with emulsifiers and suspending agents. Specific sweeteners and / or flavorings may be added as needed. For intramuscular, intraperitoneal, subcutaneous, and intravenous use, it is usually necessary to prepare a sterile solution of the active ingredient and buffer it by appropriately adjusting the pH of the solution. For intravenous use, it is necessary to control the total concentration of the solute to make the formulation isotonic.

[0086] The compounds of the present invention can also be administered in combination with and / or concurrently with other therapeutic agents selected for their particular usefulness for treating gout. For example, the compounds of the present invention can be administered simultaneously or consecutively in combination with and / or concurrently with antigout agents. Examples of antigout agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular glucocorticoids, xanthine oxidase inhibitors, recombinant non-human uricase enzymes, uric acid excretion promoters, uric acid excretion agents, and combinations thereof. This compound may also be useful in conjunction with other therapies, such as when administered concurrently with components for treating other relevant indications. Xanthine oxidase inhibitors include compounds that lower serum uric acid levels by inhibiting the enzyme xanthine oxidase. Examples of xanthine oxidase inhibitors include, but are not limited to, febuxostat, propolis, oxypurinol, thisopurine, or inositol and allopurinol.

[0087] Recombinant non-human uricase enzymes include rasburicase or pegroticase. Uric acid excretion promoters or uric acid excretors refer to compounds that accelerate the rapid excretion of uric acid accumulated in the body by preventing the reabsorption of uric acid into the renal bloodstream, resulting in a net increase in excretion. Examples of such uric acid excretion promoters or uric acid excretors include probenecid, benzbromarone, sulfinpyrazone, guaifenesin, losartan, atorvastatin, amlodipine, adrenocorticotropic hormone, or fenofibrate. NSAIDs include, but are not limited to, diclofenac, indomethacin, naproxen, sulindac, lumiracoxib, or Cox-2 selective inhibitors. Cox-2 selective inhibitors include, but are not limited to, etricoxib, celecoxib (SC-58635), 5-bromo-2-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-thiophene (DUP)-697), floxlid (CGP-28238), meloxicam, 6-methoxy-2-naphthylacetate (6-MNA), MK-966 (Vioxx), nabumetone (6-MNA prodrug), nimeslide, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS-398), SC-5766, SC-58215, or 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-1-one (T-614).

[0088] Anti-inflammatory agents may include corticosteroids. Corticosteroids include prednisone, methylprednisolone, prednisolone, dexamethasone, fluticasone propionate, 6α,9α-difluoro-17-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioate S-fluoromethyl ester, and 6α,9α-difluoro-11β-hydroxy-16α-methyl Ru-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioate S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone ester, 17-propionate ester or 17,21-dipropionate ester, budesonide, flunisolide, mometasone ester, furoate ester, triamcinolone acetonide, lorofreponide, ciclesonide, propionyl Butyxocoate, RPR-106541, ST-126, fluticasone propionate, 6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(-4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioate S-fluoromethyl ester and 6α,9α-difluoro-17α-[(2-furanylcarbonyl) This includes, but is not limited to, oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioate S-fluoromethyl ester, or 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioate S-fluoromethyl ester.

[0089] When prescribed as a fixed dose, such combination products shall use the compounds of the present invention within the dose ranges described below and other pharmaceutically active agents within their approved dose ranges. Alternatively, the compounds described herein may be used sequentially with known pharmaceutically acceptable agents if a combination formulation is unsuitable. When the compounds according to the present invention are administered to human subjects, the daily dose is usually determined by the prescribing physician, and the dose generally varies depending on the individual patient's age, weight, and response, as well as the severity of the patient's symptoms. In one exemplary use, an appropriate amount of the compound is administered to a mammal being treated for gout. Doses may range from approximately 0.001 mg / kg body weight to over 100 mg / kg body weight per day; from approximately 0.001 mg / kg body weight to approximately 500 mg / kg body weight per day; from approximately 0.001 mg / kg body weight to approximately 250 mg / kg body weight per day; or from 0.001 mg / kg body weight to approximately 100 mg / kg body weight per day. These dosages can be administered more specifically orally. Any combination of dosages can be used. These combinations can be used sequentially or simultaneously.

[0090] β-tubulincolchicine binding site Models for the five most common human β-tubulin isotypes have been determined, and the colchicine binding site has been identified herein as the most promising for isotype-specific drug design. Using this binding site as a template, the colchicine derivatives described in PCT Publication No. WO2011022805 were designed to preferentially bind to the β-tubulin isotype of interest, based on the fact that there is inherent variability between isotypes and that the colchicine binding site on each β-tubulin isotype differs in both geometric and biochemical properties. As described therein, colchicine analogs were generated that preferentially bind to β-III tubulin, an isotype of tubulin overexpressed in cancer cells, and these derivatives were found to be more potent than Taxol in inhibiting tumor growth at low doses. Using the approach described above, we modified the structure of colchicine to enhance its ability to bind to the β-VI tubulin isotype. β-VI tubulin is important as a target because it is one of the major β-tubulin isotypes expressed in immune cells such as neutrophils (see Figure 15). Furthermore, binding to β-VI tubulin minimizes off-target effects from non-hematopoietic cells because this isotype is specific to hematopoietic cells. The β-VI tubulin isotype is highly distinct from others, particularly in its colchicine binding domain, has a narrow distribution in the human body, and offers a high level of specificity and selectivity for the drug it binds to. The colchicine derivatives described herein preferentially bind to βVI and βV and have low affinity for βI tubulin. The calculation method described herein provides colchicine derivatives that may be unexpectedly active at much lower concentrations than colchicine, exhibiting increased specificity for cells involved in inflammatory conditions such as gout inflammation, and avoiding the undesirable side effects of colchicine.

[0091] Colchicine binding was investigated. The sequence of residues constituting the colchicine binding site showed the greatest variation (77.8% identity) among all human tubulin isotypes (Huzil JT et al., Nanotechnology. 2006:17:S90-S100). This binding site has been previously shown to interact with several natural compounds, including colchicinoids, benzimidazole (Laclette JP et al., Biochem Biophys Res Commun. 1980; 92:417-23; Tahir SK, Biotechniques. 2000; 29:156-60; Russell GJ et al., Biochem. Mol. Biol. Int. 1995; 35:1153-9; and Hoebeke J. et al., Biochem Biophys. Res. Commun. 1976; 69:319-24) and podophyllotoxin (Ravelli RB et al., Nature. 2004; 428:198-202), and is designed to accommodate several binding conformations (Garland DL, Biochemistry. 1978; 17:4266-72; Sackett DL et al. (al., Biochemistry, 1993; 32:13560-5; Andreu JM et al., Biochemistry. 1982; 21:6465-76; Chaudhuri AR et al., J. Mol. Biol., 2000; 303:679-92). Colchicine possesses very potent anti-mitotic activity, which is observed only at toxic or near-toxic levels, limiting its use as a treatment for gout. It is used herein as a standard for comparison with similar compounds that exhibit high selectivity for tubulin isotypes expressed in hematopoietic stem cells. Using computational screening, colchicine derivatives that were likely to have superior anti-inflammatory properties were identified based on β-tubulin isotype affinity (particularly affinity for β-VI). The anti-inflammatory properties of these derivatives (e.g., derivatives 91, 47a, and 43) were then validated using in vitro and in vivo tests described herein. For example, colchicine derivatives with higher affinity for certain β-tubulin isotypes, such as compound 91 described herein (see, e.g., Figure 7), were found to be superior to colchicine in their effects on inflammatory cells (e.g., inhibition of calcium mobilization at concentrations approximately 10 to 100 times lower (approximately 1 / 10 to 1 / 100) without the disadvantages of colchicine toxicity). The anti-inflammatory properties of the derivatives described herein are outlined in the following examples.

[0092] While there is a wealth of structural information regarding the interaction between tubulin and several ligands, the conformation of tubulin decays over time, and drug binding itself can induce significant conformational changes within the protein (Luduena RF et al., Biochem. 1995; 34:15751-9; Chaudhuri AR et al., J. Mol. Biol., 2000; 303:679-92; and Schwarz PM et al., Biochem. 1998; 37:4687-92). Therefore, modeling predictions using specific, fixed conformations of the binding site may be unreliable. This is particularly true for colchicine binding, where unbound β-tubulin shows a complete lack of the colchicine binding cavity (Nogales E. et al., Nature. 1995; 375:424-7). To overcome this limitation, first, three representative models of colchicine binding sites found across all human β-tubulin isotypes were created. Second, a systematic docking procedure was performed, which attempts to sample the conformational space of colchicine binding sites using a simulated annealing method. To design a model system that can enhance the specificity of β-tubulin isotypes expressed in hematopoietic cells using computational modeling techniques, several modifications were made to colchicine. To investigate the differences between isotypes, the cavities beneath the bound colchicine in the crystal structure were examined. In particular, several C3-demethylthiocolchicine derivatives and C1-demethylcolchicine derivatives were synthesized.

[0093] Ultimately, tubulin isotype-specific drugs should have fewer side effects than their currently prescribed counterparts. This is because they bind to and destroy microtubules only in cells expressing specific β-tubulin isotypes associated with inflammation. These results also suggest that modeling is likely to produce better drugs, and that rational drug design is possible using tubulin. The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the present invention. Modifications of form and substitution of equivalents are considered as they may suggest or provide a favorable situation. Certain terms are used herein, but such terms are intended in a descriptive sense and are not intended to limit. [Examples]

[0094] Example 1 - Synthesis and Analysis of Colchicine Derivatives Materials and methods All compounds used in the study, including colchicine and N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (1), were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).

[0095] Synthesis of colchicine compounds For the synthesis scheme, please refer to Figures 1-3. N-[(7S)-2,3,10-trimethoxy-1-((methyl)carbonyloxy)-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (2) and N-[(7S)-1-hydroxy-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (3). The synthesis of (2) and (3) was adopted from Blade-Font (A. Blade-Font, Afinidad, 36 (1979) 329-331) and is shown in Figure 1. N-[(7S)-1-((ethyl)carbonyloxy)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (4) and N-[(7S)-1-(((methyl)ethyl)carbonyloxy)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (5). One millimolar of (2) was dissolved in 2.5 mL of sodium hydroxide solution. The solution was cooled to 0°C. One millimolar of CH3CH2COCl or (CH3)CH(CH3)COCl was dissolved in 3.5 mL of acetone and added to compound (4) or (5). After the solution was allowed to stand for 15 hours, 25 mL of alkaline water was added. The resulting product was extracted using chloroform and dried over magnesium sulfate. The synthesis of (4) and (5) is shown in Figure 2.

[0096] N-[(7S)-1-(ethoxy)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(6); N-[(7S)-1-(ethoxy-1-methyl)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(7); N-[(7S)-2,3,10-trimethoxy-1-(2-methylpropoxy)-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(7a); N-[(7S)-1-(butoxy)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(7b); N-[(7S)-1-((buta(3-ene)oxy)-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(7c); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-(propanoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(8); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((propa(2-ene)oxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(9); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((phenyl)methoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(10); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-(((3-methoxy)propane)oxy)(3-methoxy))-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(11); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((phenyl(3-chloro))methoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(12); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((pyridine(3))yl)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(13); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((phenyl(2-chloro))methoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(14); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-(((phenyl(4-chloro))methoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(15); N-[(7S)-2,3,10-trimethoxy-1-((methyl)cyclohexane)-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide(16).

[0097] 1 mmol of compound (2) was dissolved in 2.5 mL of sodium hydroxide solution, and the solution was cooled to 0°C. 1 mmol of bromide derivative (e.g., 1-bromoethane in case (6), 2-bromopropane in case (7), 1-bromo-2-methylpropane in case (7a), 1-bromo-butane in case (7b), 4-bromobuta-1-ene in case (7c), 1-bromopropane in case (8), 3-bromopropa-1-ene in case (9), (bromomethyl)benzene in case (10), 1-methoxy-2-bromoethane in case (11), 1-bromomethyl 3-(bromomethyl)-3-chlorobenzene, 3-(bromomethyl)pyridine in the case of (13), 1-bromomethyl-2-chlorobenzene in the case of (14), 1-bromomethyl-4-chlorobenzene in the case of (15), and (bromomethyl)cyclohexane in the case of (16) were dissolved in 3.5 mL of acetone. Each solution was left to stand for 15 hours. Next, 25 mL of alkaline water was added. The compounds were extracted using chloroform and dried over magnesium sulfate. The synthesis of (6-16) is shown in Figure 3.

[0098] General preparation procedure for N-deacetyl-N-(N-trifluoroacetylaminoacyl)colchicine: 3 mmol of the derivatives (6-16) in methanol (50 mL) and 2N HCl (25 mL) were heated at 90°C with stirring for 1 day. The reaction mixture was cooled and neutralized with NaHCO3. The product was extracted with methylene chloride and washed with brine. The extract was dried over Na2SO4 and concentrated. The deacetylated compounds (17-27) were crystallized from CH2Cl2. One millimolar of the deacetylated compound (17-27) and one millimolar of [(trifluoroacetyl)amino]acetic acid were dissolved in six mL of dichloromethane at room temperature. One millimolar of dicyclohexylcarbodiimide was added. After two hours, the suspension was cooled to 0°C and filtered. Products (28-38) were chromatographed using a silica gel column eluted with dichloromethane / methanol (1:0 to 0:1). Crystallization of (28-38) was carried out using dichloromethane:ethyl ether (1:1).

[0099] analytical analysis (2)C(23)H(25)O(7)N(1); Calculated value M, 427, Measured value EIMS m / e 427.1 (M + ); (3) C(21)H(23)O(6)N(1); Calculated value M, 385, Measured value EIMS m / e 385.1 (M + ); (4) C(24)H(27)O(7)N(1); Calculated value M, 441, Measured value EIMS m / e 441.1 (M + ); (5) C(25)H(29)O(7)N(1); Calculated value M, 455 Measured value EIMS m / e 455.0 (M + ); (6) C(23)H(27)O(6)N(1); Calculated value M, 413, Measured value EIMS m / e 413.1 (M + ); Elemental analysis calculated values: C% 66.83, H% 6.55, N% 23.22. Measured values: C% 66.82, H% 6.54, N% 23.22; (7) C(24)H(29)O(6)N(1); Calculated value M, 427, Measured value EIMS m / e 427.1 (M + ); Elemental analysis calculated values: C% 67.44, H% 6.77, N% 3.22, Measured values: C% 67.41, H% 6.73, N% 3.21; (8) C(24)H(29)O(6)N(1); Calculated value M, 427, Measured value EIMS m / e 427.1 (M +); Elemental analysis calculated values: C% 67.44, H% 6.79, N% 32.78, Measured values: C% 67.44, H% 6.80, N% 32.77; (9) C(24)H(27)O(6)N(1); Calculated value M, 425, Measured value EIMS m / e 425.1 (M + ); Elemental analysis calculated values: C% 67.76, H% 6.35, N% 3.29 Measured values: C% 67.77, H% 6.33, N% 3.28; (10) C(28)H(28)O(6)N(1); Calculated value M, 475, Measured value EIMS m / e 475.2 (M + ); Elemental analysis calculated values: C% 70.88, H% 5.91, N% 2.95. Measured values: C% 70.87, H% 5.92, N% 2.93; (11) C(24)H(29)O(7)N(1); Calculated value M, 443, Measured value EIMS m / e 443.1 (M + ); Elemental analysis calculated values: C% 65.01, H% 6.54, N% 3.16. Measured values: C% 65.02, H% 6.53, N% 3.11; (12) C(28)H(27)O(6)N(1)Cl(1); Calculated value M, 509, Measured value EIMS m / e 509.1 (M + ); Elemental analysis calculated values: C% 71.04, H% 6.13, N% 2.93. Measured values: C% 71.05, H% 6.12, N% 2.95; (13) C(27)H(28)O(6)N(2); Calculated value M, 476, Measured value EIMS m / e 476.1 (M + ); Elemental analysis calculated values: C% 68.06, H% 5.88, N% 5.88, Measured values: C% 68.09, H% 5.86, N 5.89%; (14) C(28)H(28)O(6)N(1)Cl(1); Calculated value M, 509, Measured value EIMS m / e 509.1 (M +); Elemental analysis calculated values: C% 66.01, H% 5.50, N% 2.94, Cl% 6.87 Measured values: C% 66.03, H% 5.51, N% 2.95, Cl% 6.88; (15) C(24)H(29)O(7)N(1); Calculated value M, 509, Measured value EIMS m / e 509.1 (M + ); Elemental analysis calculated values: C% 65.01, H% 6.09, N% 3.16, Cl% 7.90, Measured values: C% 65.02, H% 6.07, N% 3.10, Cl% 7.92; (16) C(28)H(34)O(6)N(1); Calculated value M, 495, Measured value EIMS m / e 495.2 (M +); Elemental analysis calculated values: C% 70.02, H% 7.09, N% 2.91. Measured values: C% 70.04, H% 7.08, N% 2.93; (17) C(21)H(25)O(5)N(1); Elemental analysis calculated values: C% 67.92, H% 7.27, N% 3.77. Measured values: C% 67.93, H% 7.28, N% 3.78; (18) C(22)H(27)O(5)N(1) Elemental analysis calculated values: C% 68.57, H% 7.01, N% 3.77. Measured values: C% 68.59, H% 7.03, N% 3.79; (19) C(22)H(27)O(5)N(1); Elemental analysis calculated values: C% 68.63, H% 7.04, N% 3.78. Measured values: C% 68.62, H% 7.05, N% 3.79; (20) C(22)H(25)O(5)N(1); Elemental analysis calculated values: C% 68.92, H% 6.52, N% 3.65. Measured values: C% 68.94, H% 6.53, N% 3.67; (21) C(26)H(26)O(5)N(1); Elemental analysis calculated values: C% 72.22, H% 6.01, N% 3.24. Measured values: C% 72.21, H% 6.04, N% 3.23; (22)C(22)H(27)O(6)N(1); Elemental analysis calculated values: C% 65.83, H% 6.73, N% 3.49. Measured values: C% 65.82, H% 6.73, N% 3.48; (23) C(26)H(25)O(5)N(1)Cl(1); Elemental analysis calculated values: C% 66.95, H% 5.36, N% 3.02, Cl 7.51. Measured values: C% 66.93, H% 5.34, N% 3.01, Cl 7.53; (24) C(22)H(26)O(5)N(1); Elemental analysis calculated values: C% 81.25, H% 6.77, N% 3.64. Measured values: C% 81.26, H% 6.78, N% 3.66; (25)C(26)H(26)O(5)N(1)Cl(1); Elemental analysis calculated values: C% 66.80, H% 5.56, N% 2.99, Cl% 7.49; Measured values: C% 66.81, H% 5.55, N% 2.98, Cl% 7.48; (26)C(22)H(27)O(5)N(1); Elemental analysis calculated values: C% 77.92, H% 7.01, N% 3.63, Measured value: C% 77.93, H% 7.03, N% 3.65; (27)C(26)H(32)O(5)N(1); Elemental analysis calculated value C% 71.23, H% 7.30, N% 3.19 Measured value: C% 71.22, H% 7.32, N% 3.20; (28)C(25)H(27)O(7)N(2)F(3); Elemental analysis calculated value C% 57.25, H% 5.15, N% 5.18, F% 10.85, Measured value: C% 57.25, H% 4.99, N% 5.34, F% 10.86; (29) C(26)H(29)O(7)N(2)F(3); Elemental analysis calculated values: C% 57.99, H% 5.39, N% 5.20, F% 10.59. Measured values: C% 56.38, H% 5.3, N% 5.3, F% 10.87; (30) C(26)H(29)O(7)N(2)F(3); Elemental analysis calculated values: C% 57.99, H% 5.39, N% 5.20, F% 10.59, Measured values: C% 57.58, H% 5.32, N% 5.28, F% 10.59; (31) C(26)H(27)O(7)N(2)F(3); Elemental analysis calculated values: C% 57.99, H% 5.39, N% 5.20, F% 10.56, Measured values: C% 57.99, H% 5.88, N% 5.28, F% 10.55; (32)C(30)H(28)O(7)N(2)F(3); Elemental analysis calculated values ​​C% 59.92, H% 4.66, N% 4.65, F% 9.46, Measured values: C% 59.71, H% 4.65, N% 4.37, F% 9.49; (33) C(26)H(29)O(7)N(2)F(3); Elemental analysis calculated values ​​C% 57.99, H% 5.39, N% 5.20, F% 10.59 Measured values: C% 56.38, H% 5.21, N% 4.68, F% 9.55; (34)C(30)H(27)O(7)N(2)Cl(1)F(3); Elemental analysis calculated values: C% 56.77, H% 4.28, N% 4.13, F% 8.41; Measured values: C% 56.74, H% 4.29, N% 4.12, F% 8.43; (35) C(26)H(27)O(7)N(2)F(3); Elemental analysis calculated value: C% 58.20, H% 4.86, N% 4.69, F% 9.56, Measured value: C% 58.12, H% 4.87, N% 4.69, F% 9.57; (36)C(30)H(28)O(7)N(2)Cl(1)F(3); Elemental analysis calculated value C% 58.06, H% 4.15, N% 4.12, F% 8.41 Measured value: C% 58.06, H% 4.14, N% 4.13, F% 8.40; (37)C(26)H(28)O(7)N(2)Cl(1)F(3); Elemental analysis calculated value C% 54.54, H% 4.87, N% 4.73, F% 9.25, Measured value: C% 54.53, H% 4.88, N% 4.72, F% 9.26; (38) C(30)H(34)O(7)N(2)F(3); Elemental analysis calculated values: C% 60.91, H% 5.75, N% 4.73, F% 9.64; Measured values: C% 60.79, H% 5.67, N% 4.63, F% 9.67.

[0100] Synthesis of thiocolchicine compounds (Figure 4) Thiocorchicine, N-[(7S)-1,2,3-trimethoxy-10-methylsulfanil-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (39): Colchicine (1) (1 mmol) was dissolved in 10 mL of methanol / dimethylformamide (1:1) at 70-80°C. The solution was cooled to room temperature and sodium methanethiolate (2 mmol) was added. The mixed solution was stirred overnight. Water (20 mL) was added, and the reaction mixture was extracted with CH2Cl2 (10 mL), dried over Na2SO4, and concentrated. Product (39) was obtained in 71% yield by crystallization of the residue from ethyl ether / acetone (1:1).

[0101] N-[(7S)-3-hydroxy-1,2-dimethoxy-3-hydroxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (40): 1 mmol of thiocolchicine (39) was dissolved in 10 mL of methanol, and 30 mL of 0.2 N hydrochloric acid was added. The methanol was evaporated, the mixture was cooled, and sodium hydroxide solution was added until the pH reached 11. The resulting alkaline solution was extracted with chloroform to remove non-phenolic substances. The sodium hydroxide solution (red) was acidified with hydrochloric acid and extracted with chloroform. After drying and concentration, the yield of (40) was 58%. N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-(propa(2-ene)oxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (41), N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (42), and N-[(7S)-3-propoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]acetamide (43): 1 mmol of compound (40) was dissolved in 2.5 mL of 1N sodium hydroxide solution. The resulting solution was cooled to 0°C, and compound (41) was obtained with 3-bromopropa-1-ene (1 mmol), compound (42) with 1-bromoethane (1 mmol), or compound (43) with 1-bromopropane (1 mmol). These compounds were dissolved in 3.5 mL of acetone and added to the cooled solution. After allowing the solution to stand for 15 hours, 25 mL of alkaline water was added. The resulting product was extracted using chloroform and dried over magnesium sulfate. The yield of (41) was 68%, and the yield of (42) was 71%.

[0102] Preparation of N-deacetyl-N-(N-trifluoroacetylaminoacyl)thiocolchicine : N-[(7S)-3-hydroxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]amine(44); N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-(propa(2-ene)oxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]amine(45); N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]amine(46); N-[(7S)-3-hydroxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]-N-[(trifluoroacetyl)glycyl]acetamide(47); N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-(propa-2-enoxy)-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]-N-[(trifluoroacetyl)glycyl]acetamide(48); N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl]-N-[(trifluoroacetyl)glycyl]acetamide (49).

[0103] Each derivative (44-46) and (47-49) was prepared by the same method. One millimolar of a suitable derivative (40), (41), or (42) was dissolved in methanol (20 mL) containing 2N HCl (10 mL) and heated at 90°C with stirring for 24 hours. The reaction mixture was cooled, neutralized with NaHCO3, and extracted with CH2Cl2. The extract was dried over Na2SO4 and concentrated. Crystallization occurred from (1:1)CH2Cl2 / CH3OH. The yields of the deacetylated compounds (44), (45), and (46) were 58%, 63%, and 71%, respectively. One millimolar of the deacetylated compound (44), (45), or (46) and one millimolar of N-trifluoroacetyloamino acid were dissolved at room temperature, and six mL of dichloromethane was added with stirring. One millimolar of dicyclohexylcarbodiimide was added to the suspension, and after two hours, the mixture was cooled to 0°C and filtered. Each compound (47), (48), or (49) was crystallized from a dichloromethane:ethyl ether (1:1) solution. The yields of (47), (48), and (49) were 64%, 67%, and 75%, respectively.

[0104] Analysis of compounds (39), (40-42), (44-46), and (47-49) Colchicine (1): Mp 275 °C; (39): Mp 250°C -252°C; Elemental analysis calculated values ​​C(22)H(25)N(1)O(5)S(1): C% 63.60, H% 6.06, N% 3.37, S% 7.72; Measured values: C% 63.71, H% 6.15, N% 3.42, S% 7.79; (40): Mp 306°C; Elemental analysis calculated values ​​C(21)H(23)O(5)N(1)S(1): C% 62.8, H% 5.8, N% 3.5, S% 8.0, Measured values: C% 62.9, H% 5.8, N% 3.3, S% 7.5; Calculated value M, 401.1, Measured value EIMS m / e 401.1 (M + ); (41): Mp 306°C; Elemental analysis calculated values ​​C(24)H(27)O(5)N(1)S(1), C% 65.3, H% 6.12, N% 3.17, S% 7.24, Measured values: C% 65.07, H% 6.59, N% 3.21, S% 7.28; Calculated value M, 454.5, Measured value EIMS 454.5 (M + Na + ); 442.5; (42): Mp 273°C; Elemental analysis calculated values: C(23)H(27)O(5)N(1)S(1), C% 64.33, H% 18.64, N% 3.26, S% 7.45, Measured values: C% 64.4, H% 18.9, N% 3.27, S% 7.61; Calculated value M, 452.6, Measured value EIMS 452.6 (M +Na + ); (44): Mp 281°C; Elemental analysis calculated value C(19)H(21)O(4)N(1)S(1), C% 63.51, H% 5.91, N% 3.88, S% 8.92, Measured value: C% 63.55, H% 5.83, N% 3.75, S% 8.93; (45): Mp 254°C; Elemental analysis calculated value C(22)H(25)O(4)N(1)S(1), C% 65.8, H% 6.77, N% 3.52, S% 7.99, Measured value: C% 65.83, H% 6.49, N% 3.63, S% 8.31; (46): Mp 276°C; Elemental analysis calculated value C(21)H(25)O(4)N(1)S(1), C% 65.81, H% 6.50, N% 3.6, S% 8.24, Measured values: C% 65.12, H% 6.54, N% 3.57, S% 8.27; (47): Mp 284°C; Elemental analysis calculated values: C(23)H(23)O(6)N(2)S(1)F(3), C% 55.42, H% 4.61, N% 2.92, S% 6.42, F% 11.44 Measured values: C% 55.43, H% 4.62, N% 2.91, S% 6.42, F% 11.44; (48): Mp 324°C; Elemental analysis calculated values: C(26)H(27)O(6)N(2)S(1)F(3), C% 56.52, H% 4.89, N% 5.07, S% 5.79, F% 10.32. Measured values: C% 56.52, H% 4.87, N% 7.01, S% 5.79, F% 10.32; (49): Mp 256°C; Elemental analysis calculated values: C(25)H(27)O(6)N(2)S(1)F(3), C% 57.03, H% 5.13, N% 5.32, S% 6.08, F% 10.87. Measured values: C% 53.67, H% 4.5, N% 5.32, S% 6.05; F% 10.85.

[0105] Specific synthesis of colchicine derivatives Compound (2) [ka] A solution of 1 (30.0 g) and sodium thiomethoxide (30.0 mL) in water (2000 mL) was stirred overnight at room temperature. The reaction mixture was extracted with dichloromethane, and the organic layer was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (20.0 g, 65%).

[0106] Compounds (6), (17), and (28) [ka] To a solution of 1 (1.0 g, 2.51 mmol) and acetyl chloride (3 mL), tetrachloride (1 mL) was added, and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of methanol / water containing 2 (crude) and lithium hydroxide (4 equivalents) was stirred at room temperature for 1 hour. The aqueous phase was extracted and concentrated to obtain the crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps). A mixture of 3 (800 mg, 2.01 mmol), bromoethane (450 mg, 4.16 mmol), and potassium carbonate (1.2 g, 8.31 mmol) in DMF (20 mL) was stirred at 90°C for 2 hours. The reaction mixture was poured into water, extracted with ethyl acetate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.5 g, 60%).

[0107] A mixture of 4 (700 mg, 1.69 mmol), (Boc)2O (3.7 g, 16.95 mol), and DMAP (83 mg, 0.68 mmol) in THF (15 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and sodium methoxide (365.0 mg, 6.76 mmol) in methanol (15 mL) was stirred at room temperature for 2 hours. Water was then added, and the mixture was extracted with dichloromethane. The extract was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.6 g). A solution of 6 (600 mg, 1.27 mmol) and trifluoroacetic acid (5 mL) in dichloromethane (5 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated to give the product (0.45 g, 96%). A solution of 7 (50 mg, 0.13 mmol), EDCI (39 mg, 0.20 mmol), HOBT (27 mg, 0.20 mmol), F3CGlyOH (28 mg, 0.16 mmol) and triethylamine (54 mg, 0.54 mmol) in dichloromethane (3 mL) was stirred at room temperature overnight. The reaction mixture was washed with water, dried and concentrated to give a crude product. The crude product was purified by chromatography to give the desired product (22 mg, 31%).

[0108] Compounds (11), (22), and (33) [Chemical formula] Acetyl chloride (3 mL) was added to a solution of 1 (1.0 g, 2.51 mmol) in tetrachloride (1 mL), and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of 2 (crude) and lithium hydroxide (4 equivalents) in methanol / water was stirred at room temperature for 1 hour. The aqueous phase was extracted and concentrated to give a crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps).

[0109] A mixture of 3 (800 mg, 2.01 mmol), 1-bromo-2-methoxyethane (580 mg, 4.16 mmol) and potassium carbonate (1.15 g, 8.31 mmol) in DMF (20 mL) was stirred at 75 °C for 3 hours. The reaction mixture was poured into water, extracted with ethyl acetate and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give the desired product (0.5 g, 54%). A mixture of 4 (500 mg, 1.13 mmol), (Boc)2O (2.5 g, 11.29 mmol), and DMAP (55 mg, 0.45 mmol) in THF (10 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and sodium methoxide (244.0 mg, 4.52 mmol) in methanol (15 mL) was stirred at room temperature for 2 hours. Water was then added, and the mixture was extracted with dichloromethane. The extract was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.45 g). A solution of 6 (0.6 g, 1.20 mmol) and trifluoroacetic acid (5 mL) in dichloromethane (5 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated to obtain the product (0.45 g, 94%). Solutions of 7 (65 mg, 0.16 mmol), EDCI (46 mg, 0.24 mmol), HOBT (32 mg, 0.24 mmol), F3CGlyOH (42 mg, 0.24 mmol), and triethylamine (65 mg, 0.65 mmol) in dichloromethane (3 mL) were stirred overnight at room temperature. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (25 mg, 28%).

[0110] Compounds (13), (24), and (35) [ka] To a solution of 1 (1.0 g, 2.51 mmol), acetyl chloride (3 mL) and tetrachloride (1 mL) were added, and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of 2 (crude) in methanol / water and lithium hydroxide (4 equivalents) was stirred at room temperature for 1 hour. The aqueous phase was extracted and concentrated to obtain the crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps).

[0111] A mixture of 3 (1.0 g, 2.6 mmol), 3-(chloromethyl)pyridine (0.64 g, 3.9 mmol), and potassium carbonate (1.08 g, 7.8 mmol) in DMF (20 mL) was stirred at 90°C for 8 hours. The reaction mixture was poured into water, extracted with ethyl acetate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.7 g, 58%). A mixture of 4 (700 mg, 1.47 mmol), (Boc)2O (3.2 g, 14.71 mol) and DMAP (72 mg, 0.59 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was purified by silica gel column chromatography to obtain the product (0.7 g, 87%). A solution of 5 (0.7 g, 1.22 mmol) and sodium methoxide (131.0 mg, 2.43 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 6 (crude) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.3 g). Solutions of 7 (50 mg, 0.13 mmol), EDCI (44 mg, 0.23 mmol), HOBT (31 mg, 0.23 mmol), F3CGlyOH (39 mg, 0.23 mmol), and triethylamine (47 mg, 0.46 mmol) in dichloromethane (3 mL) were stirred overnight at room temperature. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (22 mg, 32%).

[0112] Compounds (40), (44), and (47) [ka]

[0113] A mixture of 1 (4.0 g) in 120 mL of phosphoric acid was stirred overnight at room temperature. The mixture was poured onto ice, and the pH was adjusted to 5 by adding a 15% sodium hydroxide aqueous solution, followed by several extractions with dichloromethane. The combined organic layer was concentrated to obtain the crude product. The crude product was purified by crystallization with acetone to obtain the title compound (1.8 g, 67%). A mixture of 2 (600 mg, 1.50 mmol), (Boc)2O (3.3 g, 14.96 mmol), and DMAP (73 mg, 0.60 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 3 (crude) and sodium methoxide (120.0 mg, 2.3 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 4 (crude) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g).

[0114] To a solution of 5 (50 mg, 0.14 mmol) and imidazole (9 mg, 0.14 mmol) in dichloromethane (3 mL) cooled to 0°C, tert-butyldimethylsilyl chloride (21 mg, 0.14 mmol) was added. The resulting mixture was stirred at room temperature for 10 minutes. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (30 mg, 45%). Solutions of 6 (30 mg, 0.06 mmol), EDCI (24 mg, 0.13 mmol), HOBT (17 mg, 0.13 mmol), F3CGlyOH (22 mg, 0.13 mmol), and triethylamine (26 mg, 0.26 mmol) in dichloromethane (3 mL) were stirred overnight at room temperature. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step without further purification. A solution of 7 (crude) in THF (3 mL) was treated with TBAF (28 mg, 0.11 mmol). The resulting mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated and purified by chromatography to afford the desired product (20 mg).

[0115] Compound 47a

Chemical formula

[0116] A mixture of 2 (15.0 g, 36.0 mmol), (Boc)2O (79.0 g, 361.0 mmol) and DMAP (1.8 g, 14.0 mmol) in THF (220 mL) was refluxed overnight. The reaction mixture was washed with water, dried and concentrated to give the crude product, which was used directly in the next step. A solution of 3 (crude) and sodium methoxide (4.0 g, 74.0 mmol) in methanol (400 mL) was stirred at room temperature for 2 hours. Then water was added and the mixture was extracted with dichloromethane. The extract was concentrated to give the crude product. The crude product was purified by silica gel column chromatography to afford the desired product (15.0 g). A solution of 4 (15.0 g, 31.8 mmol) and trifluoroacetic acid (20 mL) in dichloromethane (20 mL) was stirred at room temperature for 1 hour. The reaction solution was concentrated to give the product (11.0 g, 85%). A solution of 5 (11.0 g, 29.0 mmol), EDCI (11.3 g, 59.0 mmol), HOBT (2.0 g, 59.0 mmol), F3COGlyOH (7.6 g, 44.0 mmol) and triethylamine (11.9 g, 118.0 mmol) in dichloromethane (200 mL) was stirred at room temperature overnight. The reaction mixture was washed with water to give the crude product. The crude product was purified by silica gel column chromatography to afford the desired product (12.0 g, 77%).

[0117] Compounds (40), (41), (45), and (48)

Chemical formula

[0118] A solution of 4 (crude) and sodium methoxide (120.0 mg, 2.21 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was then used directly. A solution of 5 (crude) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g). Solutions of 6 (50 mg, 0.13 mmol), EDCI (48 mg, 0.25 mmol), HOBT (34 mg, 0.25 mmol), F3CGlyOH (43 mg, 0.25 mmol), and triethylamine (63 mg, 0.63 mmol) in dichloromethane (3 mL) were stirred overnight at room temperature. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (25 mg, 36%).

[0119] Compounds (40), (42), (46), and (49) [ka]

[0120] A mixture of 1 (4.0 g) in 120 mL of phosphoric acid was stirred overnight at room temperature. The mixture was poured onto ice, and the pH was adjusted to 5 by adding a 15% sodium hydroxide aqueous solution, followed by several extractions with dichloromethane. The combined organic layer was concentrated to obtain the crude product. The crude product was purified by crystallization with acetone to obtain the title compound (1.8 g, 67%). A mixture of 2 (50 mg, 0.12 mmol), bromoethane (21 mg, 0.19 mmol), and potassium carbonate (52 mg, 0.37 mmol) in acetone (3 mL) was refluxed for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (35 mg, 65%). A mixture of 3 (500 mg, 1.16 mmol), (Boc)2O (2.5 g, 11.63 mol), and DMAP (57 mg, 0.47 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 4 (crude) and sodium methoxide (122.0 mg, 2.26 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and trifluoroacetic acid (10 ml) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g). Solutions of 6 (50 mg, 0.13 mmol), EDCI (49 mg, 0.26 mmol), HOBT (35 mg, 0.26 mmol), F3CGlyOH (44 mg, 0.26 mmol), and triethylamine (65 mg, 0.65 mmol) in dichloromethane (3 mL) were stirred overnight at room temperature. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (25 mg, 36%).

[0121] Compounds (6a), (17a), and (28a) [ka]

[0122] A mixture of 1 (20.0 g, 0.05 mmol), (Boc)2O (109.3 g, 0.50 mol), and DMAP (2.4 g, 0.02 mol) in THF (300 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of compound 2 (crude) and sodium methoxide (5.4 g, 0.1 mol) in methanol (400 mL) was stirred at room temperature for 2 hours. Water was then added, and the mixture was extracted with dichloromethane. The extract was concentrated to obtain the crude product, which was purified by silica gel column chromatography (20.0 g, 87%). A solution of 3 (2.95 g, 6.46 mmol) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated to obtain the product (2.1 g, 91%). A solution of 4 (200 mg, 0.56 mmol), DCC (138 mg, 0.67 mmol), DMAP (82 mg, 0.67 mmol), and triethylamine (115 mg, 1.12 mmol) in dichloromethane (5 mL) was stirred overnight at room temperature. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (110 mg, 39%).

[0123] Compound (83) [ka] To a solution of 1 (1.0 g, 2.51 mmol), acetyl chloride (3 mL) and tetrachloride (1 mL) were added, and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of 2 (crude) in methanol / water and lithium hydroxide (4 equivalents) was stirred at room temperature for 1 hour. The aqueous phase was extracted and concentrated to obtain the crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps).

[0124] A mixture of 3 (800 mg, 2.01 mmol), bromoethane (450 mg, 4.16 mmol), and potassium carbonate (1150 mg, 8.31 mmol) in DMF (20 mL) was stirred at 90°C for 2 hours. The reaction mixture was poured into water, extracted with ethyl acetate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.5 g, 60%). A mixture of 4 (700 mg, 1.69 mmol), (Boc)2O (3.7 g, 16.95 mol), and DMAP (83 mg, 0.68 mmol) in THF (15 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and sodium methoxide (365.0 mg, 6.76 mmol) in methanol (15 mL) was stirred at room temperature for 2 hours. Water was then added, and the mixture was extracted with dichloromethane. The extract was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.6 g). A solution of 6 (600 mg, 1.27 mmol) and trifluoroacetic acid (5 mL) in dichloromethane (5 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated and the product was divided into nine (0.45 g, 96%). To a solution of 7 (50 mg, 0.13 mmol) and triethylamine (27 mg, 0.27 mmol) in dichloromethane (3 mL), methyl carbonochloridate (19 mg, 0.20 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (15 mg, 26%).

[0125] Compound (84) [ka] To a solution of 1 (1.0 g, 2.51 mmol), acetyl chloride (3 mL) and tetrachloride (1 mL) were added, and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of 2 (crude) in methanol / water and lithium hydroxide (4 equivalents) was stirred at room temperature for 1 hour. The aqueous phase was extracted and concentrated to obtain the crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps).

[0126] A mixture of 3 (800 mg, 2.01 mmol), 1-bromo-2-methoxyethane (580 mg, 4.16 mmol), and potassium carbonate (1.15 g, 8.31 mmol) in DMF (20 mL) was stirred at 75°C for 3 hours. The reaction mixture was poured into water, extracted with ethyl acetate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.5 g, 54%). A mixture of 4 (500 mg, 1.13 mmol), (Boc)2O (2.5 g, 11.29 mmol), and DMAP (55 mg, 0.45 mmol) in THF (10 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and sodium methoxide (244.0 mg, 4.52 mmol) in methanol (15 mL) was stirred at room temperature for 2 hours. Water was then added, and the mixture was extracted with dichloromethane. The extract was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.4 g). A solution of 6 (0.6 g, 1.20 mmol) and trifluoroacetic acid (5 ml) in dichloromethane (5 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated to obtain the product (0.45 g, 94%). To a solution of 7 (50 mg, 0.12 mmol) and triethylamine (25 mg, 0.25 mmol) in dichloromethane (3 mL), methyl carbonochloridate (18 mg, 0.19 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (16 mg, 28%).

[0127] Compound (85) [ka] To a solution of 1 (1.0 g, 2.51 mmol), acetyl chloride (3 mL) and tetrachloride (1 mL) were added, and the mixture was stirred at room temperature for 40 hours. The crude product was used directly in the next step. A solution of 2 (crude) and lithium hydroxide (4 equivalents) in methanol / water was stored at room temperature for 1 hour. The aqueous phase was extracted and concentrated to obtain the crude product. The product was obtained by recrystallization (0.2 g, 21%, 2 steps).

[0128] A mixture of 3 (1.0 g, 2.6 mmol), 3-(chloromethyl)pyridine (0.64 g, 3.9 mmol), and potassium carbonate (1.08 g, 7.8 mmol) in DMF (20 mL) was stirred at 90°C for 8 hours. The reaction mixture was poured into water, extracted with ethyl acetate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the desired product (0.7 g, 58%). A mixture of 4 (700 mg, 1.47 mmol), (Boc)2O (3.2 g, 14.71 mol) and DNP (72 neg, 0.59 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was purified by silica gel column chromatography to obtain the product (0.7 g, 87%). A solution of 5 (0.7 g, 1.22 mmol) and sodium methoxide (131.0 mg, 2.43 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 6 (crude) and trifluoroacetic acid (10 ml) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.3 g). To a solution of 7 (50 mg, 0.12 mmol) and triethylamine (35 mg, 0.35 mmol) in dichloromethane (3 mL), methyl carbonochloridate (16 mg, 0.17 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (12 mg, 21%).

[0129] Compound (89) [ka] A mixture of 1 (4.0 g) in 120 mL of phosphoric acid was stirred overnight at room temperature. The mixture was poured onto ice, and the pH was adjusted to 5 by adding a 15% sodium hydroxide aqueous solution, followed by several extractions with dichloromethane. The combined organic layer was concentrated to obtain the crude product. The crude product was purified by crystallization with acetone to obtain the title compound (1.8 g, 67%). A mixture of 2 (600 mg, 1.50 mmol), (Boc)2O (3.3 g, 14.96 mmol), and DMAP (73 mg, 0.60 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was then used directly.

[0130] A solution of 3 (crude) and sodium methoxide (120.0 mg, 2.3 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was then used directly. A solution of 4 (crude) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g). To a solution of 5 (50 mg, 0.14 mmol) and lm (9 mg, 0.14 mmol) in dichloromethane (3 mL) cooled to 0°C, tert-butyldimethylchlorosilane (21 mg, 0.14 mmol) was added. The resulting mixture was stirred at room temperature for 10 minutes. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (30 mg, 45%). To a solution of 6 (100 mg, 0.13 mmol) and triethylamine (64 mg, 0.64 mmol) in dichloromethane (3 mL), methyl carbonochloridate (40 mg, 0.42 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (50 mg, 45%). To a solution of 7 (50 mg, 0.09 mmol) in tetrahydrofuran (3 mL), TBAF (29 mg, 0.11 mmol) was added. The resulting mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated and purified by chromatography to obtain the desired product (20 mg, 51%).

[0131] Compound(90) [ka] A mixture of 1 (4.0 g) in 120 mL of phosphoric acid was stirred overnight at room temperature. The mixture was poured onto ice, and the pH was adjusted to 5 by adding a 15% sodium hydroxide aqueous solution, followed by several extractions with dichloromethane. The combined organic layer was concentrated to obtain the crude product. The crude product was purified by crystallization with acetone to obtain the title compound (1.8 g, 67%).

[0132] A mixture of 2 (50 mg, 0.12 mmol), 3-bromopropa-1-ene (23 mg, 0.19 mmol), and potassium carbonate (52 mg, 0.37 mmol) in acetone (3 mL) was refluxed for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (30 mg, 55%). A mixture of 3 (500 mg, 1.13 mmol), (Boc)2O (2.5 g, 11.31 mol) and DMAP (55 mg, 0.45 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 4 (crude) and sodium methoxide (120.0 mg, 2.21 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and trifluoroacetic acid (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g). To a solution of 6 (50 mg, 0.13 mmol) and triethylamine (25 mg, 0.25 mmol) in dichloromethane (3 mL), methyl carbonochloridate (24 mg, 0.25 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (20 mg, 35%).

[0133] Compound(91) Synthesis Route A [ka] A mixture of 1 (4.0 g) in 120 mL of phosphoric acid was stirred overnight at room temperature. The mixture was poured onto ice, and the pH was adjusted to 5 by adding a 15% sodium hydroxide aqueous solution, followed by several extractions with dichloromethane. The combined organic layer was concentrated to obtain the crude product. The crude product was purified by crystallization with acetone to obtain the title compound (1.8 g, 67%). A mixture of 2 (50 mg, 0.12 mmol), bromoethane (21 mg, 0.19 mmol), and potassium carbonate (52 mg, 0.37 mmol) in acetone (3 mL) was refluxed for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (35 mg, 65%).

[0134] A mixture of 3 (500 mg, 1.16 mmol), (Boc)2O (2.5 g, 11.63 mol), and DMAP (57 mg, 0.47 mmol) in THF (20 mL) was refluxed overnight. The reaction mixture was washed with water, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 4 (crude) and sodium methoxide (122.0 mg, 2.26 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water, extracted with dichloromethane, dried, and concentrated to obtain the crude product, which was used directly in the next step. A solution of 5 (crude) and triethylamine (10 mL) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain the product (0.4 g). To a solution of 6 (50 mg, 0.13 mmol) and triethylamine (25 mg, 0.25 mmol) in dichloromethane (3 mL), methyl carbonochloridate (24 mg, 0.25 mmol) was added at 0°C. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was washed with water and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain the desired product (20 mg, 35%).

[0135] Synthetic Route B [ka] Step 1 - Acid hydrolysis [ka]

[0136] THC (1 g) was dissolved in warm concentrated 85% phosphorous acid (40 ml) and stirred for approximately 12 hours. The solution was extracted with chloroform approximately 5 times. The resulting chloroform fraction was washed with water and then concentrated using a rotary evaporator to obtain the product from hydrolysis. The hydrolysis product was dissolved in acetone, and an aqueous solution of K2CO3 was added. The resulting solution was concentrated, then acetone was added again, and the solution was subsequently concentrated to obtain the potassium salt (3-demethylthiocolchicine).

[0137] Step 2 - Synthesis of ether (3-ethoxythiocolchicine) [ka] The potassium salt (3-demethylthiocolchicine) from Step 1 was dissolved in acetone (75 ml), and ethyl iodide in a 2-molar excess was added. The resulting solution was stirred and refluxed (boiled) for about 5 hours. The solution was concentrated, acetonitrile was added, and the solution was concentrated again to remove the remaining ethyl iodide to obtain precipitated 3-ethoxythiocolchicine.

[0138] Step 3 - Hydrolysis of amide bonds [ka] 3-Ethoxythiocolchicine was dissolved in 5M HCl (100 ml), refluxed (boiled), stirred while boiling, and monitored by TLC. After about 5 hours, the reaction mixture was extracted seven times with chloroform. The resulting chloroform fraction was washed with water and evaporated to dryness. The product was dissolved in methanol and concentrated three times to remove trace amounts of water. The product was dissolved in acetonitrile and evaporated to dryness to obtain the amine (3-ethoxydeacetylthiocolchicine).

[0139] Step 4 [ka] Dried amine (3-ethoxydeacetylthiocolchicine) was dissolved in THF (50 ml), and TEA (triethylamine) (1.5 ml) was added. Methyl chloroformate (0.5 g) was added to this solution, and the mixture was stirred at RT (room temperature) for approximately 5 hours and monitored by TLC. The resulting solution was evaporated to dryness, dissolved in chloroform, and extracted twice with 0.2 M HCl, then once with water. The resulting chloroform fraction was evaporated to dryness, dissolved in acetonitrile, and evaporated again to dryness. The resulting product (CR-42-024=(91)) was purified by silica gel flash chromatography. The eluent was a hexane / ethyl acetate mixture containing 0-5% hexane.

[0140] T In several experiments described below, colchicine derivatives TPO (Kerekes P, Sharma PN, Brossi A, Chignell CF, Quinn FR (1985) Synthesis and biological effects of novel thiocolchicines. 3. Evaluation of N-acyldeacetylthiocolchicines, N-(alkoxycarbonyl)deacetylthiocolchicines, and O-ethyldemethylthiocolchicines. New synthesis of thiodemecolcine and antileukemic effects of 2-demethyl- and 3-demethylthiocolchicine.) J Med Chem The efficacy of (91) against inflammation associated with gout was determined using (28:1204-1208, which is incorporated herein by reference in its entirety). [ka] T

[0141] Example 2 - In vitro study of gout inflammation A series of in vitro experiments were conducted using the colchicine derivative (91) (referred to as "CCI" in some figures) in MSU-induced neutrophil activation. Experiments were also performed to demonstrate the effects of colchicine derivatives with structures similar to (91), such as CH-35(43) and CR42-003(47a). The structure of colchicine, the scaffold used to generate the compounds, was modified at the positions shown in Table 1 below to generate CCI(91), CH-35(43), and CR-42-003(47a). In summary, as will be described in more detail below, human neutrophils were isolated from healthy donors and stimulated with MSU, the causative agent of gout, in the presence or absence of (91), (43), and (47a). The major neutrophil responses evaluated were cytoplasmic calcium levels (Figure 7), production of pro-inflammatory cytokines (such as IL-8 or IL-1) (Figure 8), and superoxide production (Figure 9). [ka]

[0142] [Table 1]

[0143] The compounds were selected using an algorithmic approach to balance the need for high-affinity interactions of the compounds with the target isoform (in this case, βVI) and the lowest possible affinity for off-target tubulin isoforms in sensitive organs or tissues. Therefore, compounds with improved specificity / selectivity for the desired target were selected. Key parameters used in this approach to identify chemical modifications likely to enhance colchicine's affinity for the β-tubulin isotype within the target cell include: (i) different affinities of colchicine to β-tubulin isotypes, and (ii) quantitative and qualitative differences in the expression of these β-tubulin isotypes between different cell types. Detailed knowledge of drug docking and the modes of interaction between ligands and individual residues of tubulin has made it possible to determine modifications to improve the specificity and selectivity of selected tubulin isotypes through expression in target cell types. It should be noted that the computational work is based on homology modeling of human tubulin isotypes, using bovine tubulin as the scaffold from which equilibrated human structures are generated. Due to the very high sequence similarity between human and bovine tubulin, the reliability of the results obtained is very high. The structures and predicted binding energies of the three compounds shown in Table 1, as well as the details of the chemical synthesis of the three compounds studied herein, and their closely structurally related colchicine derivatives are described herein and can also be found in U.S. Patent No. 9,458,101, the contents of which are incorporated by reference.

[0144] Materials and methods material Antibodies against human β-tubulin isotypes β-I (MAB8527) and β-III (MAB1195) were purchased from R&D System, β-II (ab155311) and β-V (ab82366) were obtained from Abcam, and β-IVb (WH0010383M2) and anti-PI3 kinase p85 (ABS1856) antibodies were purchased from Sigma-Aldrich. β-VI (LS-C338196) antibody was obtained from LS Bio, and horseradish peroxidase-labeled donkey anti-mouse immunoglobulin (IgG) (715-035-150) and horseradish peroxidase-labeled donkey anti-rabbit immunoglobulin (IgG) (711-035-152) were obtained from JacksonImmunoresearch. Rat anti-mouse CD45Fitc (11-0451-82) and Fura-2-acetoxymethyl ester (Fura-2AM) were purchased from Invitrogen. Colchicine, dextran T500, aprotinin, leupeptin, and cytochrome C were obtained from Sigma-Aldrich. The Western Lightning Chemiluminescence Plus ECL kit was obtained from PerkinElmer, and Ficoll-Paque was obtained from Wisent Bioproducts. Triclinic MSU crystals were synthesized in-house. Endotoxin contamination was ruled out by Limulus amoeba cell lysate assay.

[0145] Isolation of human neutrophils Neutrophils were isolated from the venous blood of healthy adult volunteers. Briefly, venous blood was collected in a tube containing isocitrate, red blood cells were precipitated in 2% dextran, and neutrophils were aseptically purified by centrifugation in a Ficoll-Paque cushion. Contaminated red blood cells were removed by hypotonic lysis, and neutrophils were purified with Mg containing 1.6 mM CaCl2. 2+ It was resuspended in HBSS that did not contain [the substance]. Stimulation of human neutrophils with MSU and colchicine or colchicine derivatives to determine intracellular calcium mobilization. Human neutrophils (1 x 10) 7Cells (1 / ml) were pre-incubated with 1 μM Fura-2AM and colchicine at the indicated concentrations (Figure 7A), (91)(CCI)(Figure 7B), TPO (Figure 7C), or diluent (DMSO) before the addition of 1 mg / ml MSU. Similar experiments were further conducted with further colchicine derivatives. In particular, human neutrophils were stimulated with 10 μM colchicine (Figure 7D), D1=(28a)(Figures 7E and 7J), D2=(39)(Figure 7F), D3=(47a)(Figures 7G and 7J), and D4=(89)(Figure 7H) before the addition of MSU (1 mg / ml). In further experiments, human neutrophils were stimulated with 10 μM colchicine, 1 μM CH-22=(14), 3 μM CH-35=(43), or diluent DMSO (Figures 7I and 7J). In further experiments, human neutrophils were stimulated with 0.1 μM or 10 μM colchicine (Figures 7K and 7L), 0.01 μM, 0.1 μM, 1 μM, or 10 μM derivatives (43) (Figures 7M and 7N), or 0.01 μM, 0.1 μM, 1 μM, or 10 μM derivatives (47a) (Figures 70 and 7P). In further experiments, human neutrophils were stimulated with 0.1 μM, 1 μM, or 10 μM derivatives (43) (Figure 7Q), or 0.1 μM, 1 μM, or 10 μM derivatives (47a) (Figure 7R), followed by the addition of 1 mg / ml MSU. Figure 7S shows plots of the potency of the compounds at the concentrations indicated for the compounds tested in Figures 7A, 7B, 7Q, and 7R. Neutrophils (1×10 7 Incubate cells ( / ml) with 1 μF Mura-2 AM and specified concentrations of (91)(CCI), TPO, 28a, 39, 47a, 89, 14, 43, or colchicine at 37°C for 30 minutes, wash once with HBSS, and then drain 5 × 10⁻⁶ cells. 6 The cells were resuspended at a concentration of cells / ml and transferred to a temperature-controlled (37°C) cuvette compartment of a spectrofluorometer (Jobin Yvon's Fluorolog-SPEX).

[0146] Measurement of calcium levels in human neutrophils Intracellular calcium concentration is measured using a spectrofluorometer and expressed as the area under the curve (up to 100 seconds after MSU injection). Next, changes in cytoplasmic calcium were measured using two excitation wavelengths, 340 and 380 nm, and an emission wavelength of 510 nm, after the addition of MSU or HBSS (negative control). Free intracellular calcium concentration was estimated from the ratio of fluorescence values ​​obtained at 340 nm and 380 nm. The results are calculated as the area under the curve of intracellular calcium concentration as a function of time (0–100 seconds after addition of stimulation). Determination of the specificity of the response to MSU: fMLP-induced increase in cytoplasmic calcium in human neutrophils. Human neutrophils (1 x 10) 7 Cells ( / ml) were isolated as described above, pre-incubated with 1 μM Fura-2AM and the indicated concentrations of colchicine, CCI(91), or CH-35(43), and then 10- 7 M fMLP (Figures 7T and 7U) or 1 mg / ml MSU (Figures 7V and 7W) was added. Intracellular calcium concentration was measured using a spectrofluorometer and expressed as the area under the curve.

[0147] Stimulation of human neutrophils with MSU and colchicine or colchicine derivatives to determine CXCL8 / IL-8 or IL-1 release. Human neutrophils were isolated as described above and incubated with the indicated concentrations of colchicine (43) (Figure 8A), (47a) (Figure 8B), or DMSO at 37°C for 30 minutes. Next, MSU (1 mg / ml) or buffer (RPMI) was added to the cells and incubated for a further 3 hours at 37°C. The cells were centrifuged (400xg for 2 minutes), the supernatant was collected, and centrifuged again at 16,000xg for 5 minutes. In further experiments, human neutrophils were incubated with the indicated concentrations of colchicine (Figure 8C), CCI (91) (Figure 8D), CR42-003 (47a) (Figure 8E), or (43) (Figure 8F), followed by incubation with 1 mg / ml MSU for 3 hours. Figure 8G shows plots of the potency of the compounds at the indicated concentrations tested in Figures 8C-F. The baseline levels of IL-8 production in the presence of CR42-003 (47a) and CH-35 (43) were also determined before MSU stimulation (Figure 8L). In the case of IL-1β, human neutrophils in white RPMI (2x10) 7Cells ( / ml) were primed with 250 U / ml TNFα and incubated with the indicated concentrations of colchicine (Figure 8H), CCI (91) (Figure 8I), and CH-35 (43) (Figure 8J). Then, 1 mg / ml MSU was added, and the mixture was incubated at 37°C for 3 hours. Figure 8K shows a comparison of the potency of the compounds tested in Figures 8H-J at the indicated concentrations. Next, the cells were centrifuged, and the supernatant was collected as described above. Extracellular CXCL8 / IL-8 or IL-1β was quantified by ELISA (Invitrogen). All samples were measured twice.

[0148] Measurement of CXCL8 / IL-8 or IL-1 levels in human neutrophils The amount of CXCL8 / IL-8 or IL-1 released by stimulated neutrophils was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits from Invitrogen (human IL-8 site set, no. CHC1303 and IL-1β (no. 88-7261-88)). Stimulation of human neutrophils with MSU and colchicine or colchicine derivatives to determine superoxide production. Human neutrophils were isolated as described above and incubated with the indicated concentrations of colchicine (Figure 9A), (91)(CCI)(Figure 9B), or diluent (DMSO) at 37°C for 30 minutes, followed by stimulation with 1 mg / ml MSU for 10 minutes at 37°C in the presence of 125 μM cytochrome C or buffer (HBSS). In further experiments, human neutrophils were incubated with the indicated concentrations of colchicine (Figure 9C), CCI (91)(Figure 9D), (47a)(Figure 9E), or (43)(Figure 9F) at 37°C for 30 minutes, followed by stimulation with 1 mg / ml MSU. Figure 9G shows plots of the potency of the compounds at the indicated concentrations for the compounds tested in Figures 9C-9F. The baseline levels of ROS production in the presence of (47a) and (43) were also determined before MSU stimulation (Figure 9H). The results are expressed as the ratio of superoxide produced by the MSU control.

[0149] Measurement of superoxide levels in human neutrophils Superoxide generation was measured using a cytochrome C assay reduction. The difference between the optical density readings at 550 nm and 540 nm, read within the first 10 minutes, was multiplied by the extinction coefficient of cytochrome C to obtain 1 x 10⁻⁶. 6 The number of nmolO2 produced by each cell was determined. The result was nmolO2 / 1x10 6 It is expressed as cells / ml. Western blot analysis Neutrophil suspension (2 x 10 7 Cells ( / ml) were directly transferred to an equal volume of 2× Boiling Modified Laemmli Sample Buffer (1× Buffer: 62.5 mM Tris·HCl (pH 6.8), 4% (wt / vol) sodium dodecyl sulfate) (SDS), 5% (vol / vol) β-mercaptoethanol, 8.5% (vol / vol), glycerol, 2.5 mM orthovanadate, 10 μg / ml leupeptin, 10 μg / ml aprotinin, and 0.025% bromophenol blue) and boiled for 7 minutes. Proteins were separated by SDS-PAGE on a 10% acrylamide gel under reducing conditions and transferred to a polyvinylidene fluoride (PVDF) membrane. Blocking agents and antibodies were diluted in Tris-buffered saline Tween 20 (TBST) solution (25 mM Tris·HCl, pH 7.8, 190 mM NaCl, 0.15% vol vol Tween-20). Primary and secondary antibodies were used at the manufacturer's recommended concentrations. PVDF membranes were incubated in blocking solution (5% wt / vol powdered milk in TBST) before immunoblotting with anti-β-tubulin isotype-specific antibodies. Horseradish peroxidase-labeled donkey anti-mouse IgG and donkey anti-rabbit IgG were diluted in TBST solution. Antibodies were detected using chemiluminescent reagents with a maximum exposure time of 5 minutes. All presented immunoblots were controlled to have equal protein loading using anti-PI3 kinase p85 antibody.

[0150] result (91)(CCI) inhibits MSU-induced calcium mobilization at lower concentrations than colchicine. Since one of the earliest molecular events in neutrophils initiated by MSU is the mobilization of intracellular calcium stores, the effects of (91)(CCI), (43), and (47a) were evaluated in this early signaling event. Briefly, as described in more detail above, human neutrophils were incubated with the fluorescent calcium indicator Fura-2, then incubated in the concentration range of (91)(CCI), (43), or (47a) and stimulated with MSU. Fura-2 monitors the increase in cytoplasmic calcium caused by release from intracellular stores. The concentrations of (91)(CCI), (43), or (47a) tested ranged from 0.1 to 10 μM. For comparison, the same experiment was performed using the same concentrations of colchicine. As shown in Figures 7A–C, (91)(CCI) significantly inhibits the increase in intracellular calcium concentration at a low dose of 0.1 μM, while TPO shows inhibition at higher concentrations. In contrast, colchicine can induce significant inhibition of calcium mobilization only at a concentration of 10 μM. Thus, (91) can significantly reduce the mobilization of intracellular calcium stores at a concentration about 1 / 100th that of colchicine, and is therefore about 100 times more potent than colchicine in inhibiting calcium mobilization. Other colchicine derivatives 28a, 39, 47a, 14, and 43 showed neutrophil-inhibitory effects with respect to the decrease in human neutrophil calcium levels compared to colchicine (Figure 7E), with the exception of (89)(Figure 7H) (Figures 7E–J). This is indicated by the increasing slope of the graphed line (after the initial spike) and indicates an increase in the concentration of free calcium in the cytoplasm (Figures 7D–I).

[0151] Notably, when compared to colchicine (Figures 7K and 7L), similar findings to those observed with colchicine derivatives (91) were seen with colchicine derivatives (43) (Figures 7M and 7N) and (47a) (Figures 7O and 7P) at a low concentration of 0.1 μM. Therefore, colchicine derivatives such as 91, 47a, and 43 demonstrate the ability to inhibit the increase in calcium mobilization of human neutrophils induced by MSU at concentrations far lower than those required for colchicine (e.g., about 1 / 100th) (Figure 7). In further experiments, the derivatives were tested in the concentration range of 0.01–10 μM, as shown in Figures 7B and 7Q-S. For comparison, the same experiments were performed using the same concentrations of colchicine. CCI(91) (Figure 7B) and CH-35(43) (Figure 7R) were found to significantly reduce the increase in cytoplasmic calcium at a concentration of 0.1 μM. As shown in Figure 7R, CH-35(43) was able to significantly suppress the increase in intracellular calcium concentration at 0.1 μM, 1 μM, and 10 μM. CH-35(43) showed a classical monotonic dose-response, and its effect was concentration-dependent. In contrast, CCI(91) showed a non-monotonic dose-response and did not significantly affect the MSU-induced increase in cytoplasmic calcium at a concentration of 10 μM. As shown in Figure 7S, a comparison of the inhibitory activity of the derivatives and colchicine at 0.1 μM confirmed that CCI(91) and CH-35(43) are more potent than colchicine in significantly reducing the increase in cytoplasmic calcium induced by MSU. Therefore, CCI(91) and CH-35(43) maintain inhibitory activity at lower concentrations compared to colchicine.

[0152] To evaluate the specificity of CCI(91) for MSU-induced neutrophil activation, we determined whether CCI(91) could inhibit the neutrophil response to an unrelated stimulus. Bacterial stimulation was chosen because neutrophil activation in response to this stimulus affects host survival. Briefly, human neutrophils were incubated with 10 μM CCI(91) and then activated with the bacterial peptide fMLF. As shown in Figures 7T and 7U, neutrophil activation by fMLF induced a significant increase in cytoplasmic calcium concentration. However, in the presence of CCI(91), the cytoplasmic calcium response was unaffected. These results demonstrate that the inhibition of neutrophil activation by CCI(91) exhibits some degree of selectivity compared to MSU.

[0153] (91)(CCI) inhibits MSU-induced CXCL8 / IL-8 or IL-1 release at lower concentrations than colchicine. The ability of (91)(CCI), (43), or (47a) to weaken calcium mobilization in response to MSU facilitated the determination of their impact on downstream neutrophil responses to this signaling event. Since early signaling events such as calcium mobilization depend on MSU-induced activation of Src kinase, it was also determined whether Src-dependent production of CXCL8 / IL-8 induced by MSU is inhibited by (91)(CCI). Furthermore, since IL-1 induces the expression of adhesion molecules and the synthesis of pro-inflammatory cytokines on endothelial cells, which together promote massive neutrophil mobilization, the ability of CCI(91) and CH-35(43) to suppress IL-1 production was also evaluated. Briefly, as described in more detail above, neutrophils were incubated with specified concentrations of (91)(CCI), (43), (47a), and colchicine or DMSO before stimulation with MSU or before incubation for 3 hours in phenol-free RPMI (negative control). Next, the cell-free supernatant was collected, and the amount of CXCL8 / IL-8 or IL-1 released by activated neutrophils was determined by ELISA.

[0154] As shown in Figures 8A-B, a significant decrease in IL-8 release by neutrophils pre-incubated with colchicine derivative (43) (Figure 8A) or colchicine derivative (47a) (Figure 8B) was observed even at a low concentration of 0.1 μM. Further experiments, as shown in Figures 8D–F, revealed a significant reduction in IL-8 release by neutrophils pre-incubated with CCI(91), CR42–003(47a), and CH–35(43) from low concentrations of 0.1 μM to 10 μM (Figures 8D–F). In contrast, colchicine only significantly inhibited IL-8 release at 1 and 10 μM (Figure 8C). A comparison of the inhibitory activity of the derivatives at 0.1 μM (Figure 8G) revealed that CCI(91) was more potent than CH–35(43) and CR42–003(47a) in reducing the MSU-induced increase in IL-8 production at concentrations where colchicine could no longer weaken this neutrophil effector function.

[0155] In vitro assays have shown that CCI(91) and CH-35(43) attenuate the increase in cytoplasmic calcium and the production of ROS (see below) in response to MSU; therefore, this assay was performed using CCI(91) and CH-35(43) for IL-1 production. The effects of CCI(91) and CH-35(43) on MSU-induced synthesis of IL-1 were determined using the same experimental approach described for IL-8 with slight modifications. Two stimuli are required for the production and secretion of mature IL-1. Therefore, human neutrophils were primed with TNF-α before stimulation with MSU. A decrease in IL-1 production was observed in cells treated with 10 μM CCI(91), CH-35(43), or colchicine (Figure 8H-J). Notably, CCI(91) was the most potent compound at a threshold concentration of 0.1 μM, while CH-35(43) was effective at 1 μM (Figures 8I and 8J). Compared to colchicine, both derivatives were more potent (Figure 8K). Together, the compounds exhibit high efficacy in inhibiting MSU-induced IL-1 production in neutrophils. Furthermore, as shown in Figure 8L, the amount of IL-8 measured in the supernatant of neutrophils incubated with CR42-003(47a) or CH-35(43) alone was similar to the amount in the negative control (neutrophils contained in HBSS). This suggests that neither CR42-003(47a) nor CH-35(43) exhibits a nonspecific effect on neutrophils determined by IL-8 release.

[0156] Superoxide levels of human neutrophils stimulated with colchicine or colchicine derivatives An additional neutrophil effector function known to be calcium-dependent and activated by MSU, which may damage inflamed joints, is the production of ROS. Since (91)(CCI), (43), and (47a) inhibit calcium mobilization, (91)(CCI), (43), and (47a) may also modulate MSU-induced ROS production in neutrophils. Briefly, as detailed above, human neutrophils were incubated with (91)(CCI), (43), (47a), or colchicine for comparison purposes before stimulation with MSU. As shown in Figures 9A-B, (91)(CCI) caused a significant reduction in MSU-induced superoxide production. (91)(CCI) inhibits superoxide production at a concentration of 0.1 μM, while the minimum dose at which colchicine can inhibit MSU-induced superoxide production is 1 μM. (91)(CCI) therefore inhibits MSU-induced superoxide production in human neutrophils at a concentration approximately 10 times lower (about one-tenth) than colchicine, and is therefore approximately 10 times more potent than colchicine in inhibiting MSU-induced superoxide production in human neutrophils.

[0157] Further experiments, as shown in Figures 9D–9F, demonstrated that CCI(91), CR42–003(47a), and CH–35(43) significantly reduced MSU-induced superoxide production at a concentration of 0.1 μM. In contrast, colchicine inhibitory ability has a high threshold of 1 μM (Figure 9C). A comparison of the inhibitory activity of the derivatives and colchicine at 0.1 μM (Figure 9G) revealed that CCI(91) and CH–35(43) were significantly more potent than colchicine and CR42–003(47a) in reducing the increase in MSU-induced ROS production. Furthermore, as shown in Figure 9H, the amount of superoxide measured when neutrophils were incubated with CR42–003(47a) or CH–35(43) alone was similar to the amount in the negative control (neutrophils contained in HBSS). This suggests that neither CR42-003(47a) nor CH-35(43) exhibits nonspecific effects on neutrophils, as determined by superoxide generation.

[0158] Example 3 - ADMET Prediction Based on the in vitro findings described above, and based on the divergent effect on the inflammatory response of human neutrophils observed between derivatives (91) and (89), ADMET predictions were determined among these derivatives to assess the use of (91) as a potential drug for the treatment of gout before evaluating the function of derivative (91) in vivo. ADMET Predictor 7.2 (Simulations Plus, CA) is industry-standard pharmacokinetic prediction software. It was run on a composite structure to predict ADMET properties. Table 2 below shows the ADMET risk indicators. Each risk indicator is a score indicating that a higher value indicates a higher risk of the compound failing as a drug due to pharmacokinetic or toxicity problems. CYP_Risk is a collection of metabolic responsibility models. TOX_MUT_Risk is a collection of mutagenicity models for S. typhimurium. TOX_Risk is a collection of toxicity responsibility models. ADMET_Risk is an overall risk score combining multiple factors. Specific factors contributing to the score are noted. This analysis is a reliable in-silico proxy of in vivo operation. For each risk indicator, the risk score for (89) was found to be higher than the score for (91). Therefore, (89) is predicted to be more likely to fail as a drug due to ADMET factors.

[0159] [Table 2] Therefore, the ADMET prediction indicates that (89) has a significantly higher toxicity risk compared to (91), making it a candidate that is more likely to fail in animal studies.

[0160] Example 4 - In vivo research Having demonstrated the in vitro effects of colchicine derivatives (see above), a series of experiments were conducted using CCI (compound 91) in MSU-induced in vivo inflammation. In summary, mice were injected with MSU to induce inflammation similar to gout, as will be described in more detail below. The mice were further injected with CCI (compound 91) or CH-35 (compound 43) alone or before or after MSU injection to determine the plasma half-life of CCI (compound 91; Figure 10A), the plasma half-life of CH-35 (compound 43; Figure 10B), whether CCI (compound 91) or CH-35 (compound 43) was taken up by circulating leukocytes (Figures 11A and B, respectively), and the effects of CCI (compound 91) or CH-35 (compound 43) versus colchicine (Figures 12, 13, and 14) on the MSU-induced inflammatory response were compared.

[0161] A. Determination of the plasma half-life of colchicine derivatives in mice Measurement of the experimental model used and the concentration of CCI or CH-35 in plasma. Mice were subcutaneously injected with 5 μmol / kg of (91)(CCI) or CH-35(43) and sacrificed at 15, 30, 45, 60, or 120 minutes post-injection. Blood was collected by cardiac puncture using 35% Tyrode's Buffer pH 6.5 and 20% citrate-dextrose solution (ACD), and plasma was obtained by centrifugation at 2500 × g for 15 minutes at RT. The concentrations of the compounds in the plasma (Figures 10A and B) were determined by mass spectrometry. The concentration of (91)(CCI) or CH-35(43) is expressed as the average amount of (91)(CCI) or CH-35(43) measured in the plasma (ng / ml) of five mice harvested at the same time point.

[0162] B. Determination of the concentration of colchicine derivatives in circulating leukocytes Measurement of CCI or CH-35 concentrations in circulating leukocytes and the experimental model used. Mice were subcutaneously injected with 5 μmol / kg of (91)(CCI) or CH-35(43) and sacrificed at 15, 30, 45, 60, or 120 minutes post-injection. Blood was collected by cardiac puncture and centrifuged to obtain circulating leukocytes. The concentration of the compound in the leukocytes (Figures 11A and B) was determined by mass spectrometry. The concentration of (91)(CCI) or CH-35(43) is expressed as the average amount (ng / ml) of (91)(CCI) or CH-35(43) measured in circulating leukocytes harvested at the same time point from five mice.

[0163] C.(91)(CCI), when administered 5 minutes before MSU injection, inhibits MSU-induced leukocyte mobilization in an air pouch model. Experimental model used and measurement of white blood cells in the air pouch The anti-inflammatory activity of colchicine and colchicine derivatives was evaluated in vivo in wild-type mice (CD-1 mice). Air pouches were created on the dorsal side of mice over 7 days by subcutaneously injecting air. Seven days after the initial air injection, 10 μl / g of HBSS containing the indicated amount of (91)(CCI), colchicine, or DMSO (vehicle) was subcutaneously injected 5 minutes before injecting MSU (1.5 mg / ml) or diluent (PBS) into the air pouch. Seven hours after MSU administration, the exudate from the air pouch was collected by flushing twice with 2 ml of PBS + 0.5 M EDTA and once with 1 ml of PBS + 0.5 M EDTA, and the number of recruited leukocytes was measured by flow cytometry. Leukocytes were stained with anti-CD45 and anti-Ly6G antibodies.

[0164] D.(91) or (43), when administered 1.5 hours after MSU injection, inhibits MSU-induced leukocyte mobilization in an air pouch model. Experimental model used and measurement of white blood cells in the air pouch The anti-inflammatory activity of colchicine and colchicine derivatives was evaluated in vivo in wild-type mice. Air pouches were created on the dorsal side of mice over 7 days by subcutaneously injecting air. Seven days after the initial air injection, 1.5 hours after injecting MSU (1.5 mg / ml) or diluent (PBS) into the air pouch, 10 μl / g of HBSS containing the indicated amounts of (91), (43), colchicine, or DMSO (vehicle) was subcutaneously injected. Seven hours after MSU administration, the exudate from the air pouch was collected by flushing twice with 2 ml of PBS + 0.5 M EDTA and once with 1 ml of PBS + 0.5 M EDTA, and the number of recruited leukocytes was measured by flow cytometry. Leukocytes were stained with anti-CD45 and anti-Ly6G antibodies.

[0165] result Half-life of (91)(CCI) or (43)(CH-35) in circulation and its concentration in leukocytes Before testing the anti-inflammatory activity of CCI or CH-35(43) in vivo, their half-lives in mouse plasma and their concentrations in circulating leukocytes over 2 hours were determined. As shown in Figure 10A, mass spectrometry of the plasma sample revealed that the concentration of (91)(CCI) peaked at 15 minutes (80 ng / ml) and then decreased to less than 1 / 4 of this concentration by 60 minutes (t 1 / 2 (13.3 mins). A decrease in the concentration of (91)(CCI) was observed, but it persisted in the plasma for at least 2 hours. Similarly, the peak concentration of circulating CH-35(43) reached 15 minutes after infusion (Figure 10B). However, in contrast to CCI(91), the amount of CH-35(43) in the plasma decreased to undetectable levels approximately 50 minutes after drug injection. Thus, the concentration of CH-35(43) decreased significantly in the plasma within 1 hour after administration. Taken together, these results indicate that CCI(91) and CH-35(43) have very short half lives, suggesting rapid uptake into different compartments. As shown in Figure 11A, in circulating leukocytes, the concentration of (91)(CCI) peaked at 15 minutes and persisted in these cells until the last time point analyzed (2 hours). Similarly, as shown in Figure 11B, in circulating leukocytes, the concentration of CH-35(43) peaked at 15 minutes and persisted in these cells until the last time point analyzed (2 hours). Therefore, CH-35(43) may persist in circulating leukocytes for at least 2 hours after subcutaneous injection.

[0166] (91)(CCI) or (43)CH-35 reduces MSU-induced inflammation in vivo. The in vivo anti-inflammatory activity of (91)(CCI) or CH-35(43) was evaluated in an air porch model of MSU-induced inflammation. This model was chosen because the dorsal air porch shares the same profile and essential cellular characteristics as the inner layer of the synovial membrane of the joint (e.g., fibroblasts and macrophages) and leukocyte recruitment. As shown in Figure 12, MSU induced leukocyte recruitment to the air porch within 7 hours, which was inhibited by colchicine at a concentration of 5 μmol / kg. Even at approximately one-tenth the concentration, (91)(CCI) was able to inhibit leukocyte recruitment, even when injected before MSU administration. As shown, the ability of CCI(91) to inhibit leukocyte recruitment at a significantly lower dose of 0.5 μmol / kg compared to 5 μmol / kg of colchicine was demonstrated, although this dose of colchicine was not significant in this series. At a dose of 0.5 μmol / kg, colchicine lost its ability to significantly inhibit MSU-induced leukocyte recruitment in vivo. Conversely, CCI(91) may not be more effective than colchicine at a concentration of 5 μmol / kg due to its non-monotonic dose-response curve.

[0167] To determine the therapeutic potential of (91)(CCI), the air pouch experiment was modified, and 0.5 μmol / kg (91)(CCI) was injected after adding MSU to the air pouch. As shown in Figure 13, a significant decrease in leukocyte recruitment was observed under these experimental conditions as well, indicating that MSU-induced inflammation can be suppressed even when (91)(CCI) is administered after MSU has already triggered the inflammatory process. In contrast, colchicine at a concentration of 0.5 μmol / kg failed to significantly inhibit MSU-induced leukocyte recruitment in vivo (Figure 13). Rather, surprisingly, (91)(CCI) inhibits MSU-induced leukocyte recruitment at a concentration approximately one-tenth that of colchicine. In further experiments, CH-35(43) was similarly tested, as shown in Figure 14, and compared to the results obtained with CCI(91) shown in Figure 13. The results in Figure 14 show that CH-35(43) is weaker than CCI(91) in inhibiting leukocyte influx into the air pouch. The lowest concentration of CH-35(43) that significantly suppressed leukocyte recruitment was 2.5 μmol / kg. Therefore, CH-35(43) may inhibit leukocyte recruitment during MSU-induced inflammation. In summary, these results indicate that both CH-35(43) and CCI(91) can inhibit leukocyte recruitment in vivo at lower concentrations than colchicine, while CCI(91) is the most potent compound for inhibiting MSU-induced leukocyte recruitment in vivo.

[0168] Example 5 - Expression profiles of beta-tubulin isotypes in neutrophils method Neutrophil suspension (2 x 10 7Cells / ml were directly transferred to an equal volume of 2× Boiling Modified Laemmli Sample Buffer (1× Buffer: 62.5 mM Tris·HCl (pH 6.8), 4% (wt / vol) sodium dodecyl sulfate (SDS), 5% (vol / vol) β-mercaptoethanol, 8.5% (vol / vol), glycerol, 2.5 mM orthovanadic acid, 10 μg / ml leupeptin, 10 μg / ml aprotinin, and 0.025% bromophenol blue), and boiled for 7 minutes. Proteins were separated by SDS-PAGE on a 10% acrylamide gel under reducing conditions and transferred to a polyvinylidene fluoride (PVDF) membrane. Blocking agents and antibodies were diluted in Tris-buffered saline Tween 20 (TBST) solution (25 mM Tris·HCl, pH 7.8, 190 mM NaCl, 0.15% vol vol Tween-20). Primary and secondary antibodies were used at the manufacturer's recommended concentrations. PVDF membranes were incubated in blocking solution (5% wt / vol powdered milk in TBST) before immunoblotting with anti-β-tubulin isotype-specific antibodies. Horseradish peroxidase-labeled donkey anti-mouse IgG and donkey anti-rabbit IgG were diluted in TBST solution. Antibodies were detected using chemiluminescent reagents with a maximum exposure time of 5 minutes. All presented immunoblots were controlled to have equal protein loading using anti-PI3 kinase p85 antibody.

[0169] result The expression profiles of β-tubulin isotypes in leukocytes have been investigated at the mRNA level. While βVI mRNA expression is primarily limited to hematopoietic cells, β-tubulin I, IV, and V mRNAs are ubiquitous, and β-II and β-III mRNAs are limited to the brain. Using a rational design approach, colchicine derivatives were developed based on different binding affinities of colchicine to various β-tubulin isotypes to determine whether the mRNA expression profiles of these isotypes in neutrophils correlate with protein-level expression. In short, newly isolated human neutrophils were lysed and analyzed by Western blotting using commercially available antibodies against β-I, α / β-II, β-III, β-IV, β-V, and β-VI tubulins. Note that βII and βIV each have two variants, called βIIa, βIIb and βIVa, βIVb, respectively, but their sequences and structures are very similar. As shown in Figure 15, human neutrophils express beta I, alpha / beta II, beta IV, and beta VI, but not beta III and beta V. The band observed for βV is nonspecific (confirmed with positive control (data not shown)). Since β-VI expression is mainly limited to hematopoietic cells, a colchicine derivative with higher affinity for this β-tubulin isotype was selected for the analysis described herein. To identify the β-tubulin isotype most likely to confer the ability of CH-35(43) to inhibit the MSU-induced response at low concentrations, the binding free energies (kcal / mol) of colchicine, CCI(91), CR42-003(47a), and CH-35(43) were compared in Table 3 below.

[0170] [Table 3]

[0171] As shown in Table 3 above, CH-35(43) has significantly lower binding free energies for βIVb and βVI compared to colchicine and CR42-003(47a). These data suggest that the high affinity of CH-35(43) for βIVb and βVI makes it highly likely that CH-35(43) will be activated at lower concentrations compared to colchicine and CR42-003(47a) in the tested assays. Furthermore, CCI(91) also shows high affinity for βIVb and βVI compared to colchicine and CR42-003(47a). The difference between CCI(91) and CH-35(43) is that CH-35(43) exhibits a monotonic dose relationship, which does not apply to CCI(91). The binding free energy data suggest that this may be due to the higher affinity of CH-35(43) for βIVb or βVI, or both, as it is the only two β-tubulin isotypes expressed in neutrophils with significantly higher binding free energy for CH-35(43) compared to CCI(91).

[0172] discussion Among the drugs used to treat gout, colchicine is the most specific to the pathogenesis of this inflammatory disease, as it weakens most of the inflammatory action of neutrophils, the major white blood cells involved in gout attacks. Nevertheless, its administration remains challenging due to a low therapeutic index between efficacy and treatment-limiting side effects. Using a rational drug design approach, colchicine analogs (91) (CCI) were developed that inhibit MSU-induced inflammation in vivo and MSU-induced neutrophil responses in vitro at concentrations approximately 1 / 10 to 1 / 100 of colchicine. Notably, other colchicine derivatives 43 and 47a were similarly effective in reducing neutrophil responses at the same doses as 91, as described herein. CCI(91) and (43) retained anti-inflammatory activity in vitro and in vivo at doses approximately 1 / 10 to 1 / 100 of that of colchicine. Furthermore, these compounds exhibited anti-inflammatory properties when administered after the onset of an MSU-induced inflammatory response, suggesting their therapeutic potential.

[0173] The ability of (91)(CCI) to attenuate MSU-induced neutrophil activation in vitro and in vivo at lower concentrations than colchicine can be partially explained by its tubulin isotype specificity. In silico analysis of (91)(CCI) revealed that it preferentially binds to β-VI tubulin, one of the β-tubulin isotypes expressed in neutrophils. Furthermore, the β-VI tubulin isotype is highly distinct from others, particularly in its colchicine binding domain, providing a high level of specificity and selectivity for the drug it binds to. In contrast to (91)(CCI), colchicine exhibits the highest affinity for β-IV tubulin, a ubiquitously expressed β-tubulin isotype. Colchicine can inhibit MSU-induced activation and leukocyte recruitment, but is associated with undesirable side effects, most likely due to its ability to bind to β-tubulin isotypes expressed by many different cell types. Therefore, the specific binding of (91)(CCI) to β-VI tubulin is likely to minimize off-target effects in non-hematopoietic cells and maximize activity in neutrophils, since this isotype is not expressed in these cells.

[0174] In vitro analysis revealed that (91)(CCI) inhibits the increase in cytoplasmic calcium, one of the most upstream signaling events activated by MSU. The ability of (91)(CCI) to inhibit this molecular event at concentrations where colchicine is no longer effective (approximately 1 / 100th of the original dose) strongly suggests that the β-tubulin isotype preferentially bound by (91)(CCI) most likely plays a more significant role in MSU-induced intracellular calcium storage mobilization. (91)(CCI) preferentially binds to βVI and βV, and has a lower affinity for βI-tubulin, as predicted by in silico analysis. Similar reasoning can be extended to downstream effector functions of intracellular calcium mobilization, such as the production of superoxide induced by MSU. Direct evidence is provided herein that (91)(CCI) inhibits superoxide production by human neutrophils in response to MSU at doses approximately 10 times lower (about one-tenth) than colchicine. Similarly, MSU-induced IL-8 release is also suppressed by (91)(CCI). (91)(CCI) is particularly effective in inhibiting IL-8 release at concentrations up to approximately 10 times lower (about one-tenth) than colchicine. (91)(CCI) can be administered at low doses to treat MSU-induced inflammation, thereby reducing the risk of drug-related side effects. Inhibition of IL-8 production by (91)(CCI) at low concentrations is highly relevant to gout, as IL-8 is one of the most potent chemotactic substances for neutrophils. Similarly, suppression of superoxide production is also relevant to the pathogenesis of gout, as superoxide causes adjunctive damage to joints.

[0175] In vitro observations revealed that (91)(CCI) inhibits the MSU-induced neutrophil response, and subsequently, the ability of CCI to attenuate MSU-induced inflammation in vivo was determined. What is shown herein is that (91)(CCI) inhibits MSU-induced leukocyte mobilization when administered before or after MSU. The latter indicates that (91)(CCI) is therapeutically usable because its anti-inflammatory activity was maintained when injected approximately 1 hour and 30 minutes after MSU administration in in vitro experiments. In the experiments described herein, the minimum dose at which (91)(CCI) maintained its anti-inflammatory activity in vivo was 0.5 μmol / kg. To obtain an estimate of the human equivalent dose of a drug used in mice, in some embodiments, the concentration used in mice can be divided by approximately 12.3 (Nair, AB and Jacob, S. 2016. A simple practice guide for dose conversion between animals and human, J Basic Clin Pharma: 7: 27-31). The dose of colchicine that effectively attenuates MSU-induced leukocyte recruitment in mice is 5 μmol / kg (Chia, EW, Grainger, R. and Harper, JL 2008. British Journal of Pharmacology: 153: 1288-95). This dose is 10 times higher than the human dose given to gout patients and is therefore considered the human equivalent dose. By extrapolation and based on the experiments described herein, it is expected that (91)(CCI) can attenuate human MSU-induced inflammation at doses approximately 10 times lower (about one-tenth) than the doses of colchicine currently used to treat gout attacks.

[0176] Human neutrophils were targeted due to the massive influx of these cells into the joints during gout attacks, and the expression of beta-tubulin isotypes in these leukocytes was determined. Neutrophils express β-I, β-II, β-IV, and β-VI tubulins, but not β-III and β-V tubulins. This protein expression pattern correlates well with β-tubulin isotype mRNA expression profiles reported by others. β-VI tubulin mRNA is limited to hematopoietic cells and organs containing numerous leukocytes, such as bone marrow, thymus, and fetal liver. However, β-III and β-V tubulin mRNA is undetectable in leukocytes. The levels of β-I, β-II, and β-IV tubulin isotype mRNA are significantly lower than those of β-VI in leukocytes. The functional importance of the different β-tubulin isotypes in both neutrophils and most other cell types remains unclear, although there is evidence for both functional redundancy and specificity. Regarding the former, the ability of most β-tubulin isotypes to polymerize into heterogeneous microtubules supports functional redundancy. Regarding the latter, the different phenotypes of the group of diseases known as tubulin disorders suggest that tubulin isotypes play different functional roles. Tubulin disorders can be caused by mutations in various β-tubulin isotypes. Furthermore, tissue-specific and developmental stage-specific expression of β-tubulin isotypes also suggests non-redundant and functional roles of these proteins.

[0177] Although the function of β-VI tubulin in leukocytes remains unclear, this isotype was targeted because its leukocyte expression profile is thought to reflect some degree of functional specificity to leukocytes, and β-VI tubulin-specific agents may exhibit fewer off-target effects on fewer non-hematopoietic cells. The potency of three derivatives predicted to have significantly higher relative affinity for β-VI tubulin than colchicine was tested to inhibit MSU-induced human neutrophil activation in vitro and in vivo. The effector functions investigated in vitro play a role in the pathogenesis of gout, namely the release of IL-1 and IL-8 and the production of superoxide. Regarding IL-8, a highly effective neutrophil chemotactic, neutralizing antibodies against this cytokine significantly reduced MSU-induced neutrophil influx in a rabbit model. On the other hand, IL-1 plays a role in gout by inducing the expression of adhesion molecules and cytokines by endothelial cells and other cell types in the joints (such as synovial cells). Regarding superoxide, this reactive oxygen species is associated with collateral tissue damage in joints. The data provided herein suggest that CCI(91) and CH-35(43) at significantly lower concentrations than colchicine, approximately 1 / 10 to 1 / 100 in vitro, at which point MSU-induced ROS production and IL-8 release are reduced. The ability of these derivatives to reduce these neutrophil responses suggests they may target key molecular events that trigger gout attacks.

[0178] In vitro observations facilitated the determination of whether CCI(91) and CH-35(43) can attenuate MSU-induced inflammation in vivo with significantly higher potency than colchicine. Using an air pouch model of inflammation, the data provided herein suggest that the inhibitory activity of CCI(91) and CH-35(43) in vitro reflects their ability to attenuate MSU-induced inflammation in vivo. With respect to CCI(91), when infused before MSU administration, it inhibits leukocyte recruitment into the air pouch to which MSU is infused at a threshold dose approximately 10 times lower (about one-tenth) than colchicine. However, it should be noted that at high concentrations, CCI(91) has a less significant effect in inhibiting leukocyte influx. Similar non-monotonous trends were observed in in vitro assays of calcium and ROS. This non-monotonic dose-response most likely reflects the binding of CCI(91) to ligands other than β-tubulin isotypes, as has been reported with other drugs that exhibit non-monotonic dose-responses due to off-target effects such as endocrine disruptors.

[0179] We evaluated the therapeutic potential of CCI (91) because it demonstrated to be more potent than colchicine in significantly reducing leukocyte recruitment in response to MSU. Subcutaneous administration of CCI (91) 1.5 hours after MSU injection significantly reduced leukocyte recruitment to the air pouch. Therefore, CCI (91) can also suppress inflammation induced by MSU after initiation. Surprisingly, colchicine shared this property with CCI (91), but lost its anti-inflammatory activity at 0.5 μmol / kg, the concentration at which CCI (91) maintains its activity. Similar observations were made for CH-35 (43), despite its higher concentration of 2.5 μmol / kg compared to CCI (91). In contrast to CCI (91), CH-35 (43) showed a monotonic dose-response both in vitro and in vivo. In summary, these observations indicate that CCI(91) is more potent than CH-35(43) in suppressing MSU-induced inflammation in vivo. Although CCI(91) and CH-35(43) have short half lives, both derivatives can attenuate MSU-induced leukocyte recruitment when administered 7 hours before the peak of leukocyte recruitment. This can be partially explained by the uptake and retention of these compounds by leukocytes, particularly neutrophils. Colchicine preferentially accumulates in neutrophils compared to mononuclear cells. Retention of CCI(91) was observed in circulating leukocytes up to 2 hours post-injection (preliminary data). A complete characterization of the pharmacokinetics of CCI(91) and CH-35(43) may provide further insight into the mechanism of action of these compounds.

[0180] Because neutrophils play a crucial role in protecting the host from infection, targeting them to suppress inflammation has been challenging. CCI(91) was found to selectively inhibit MSU-induced inflammation while suppressing specific neutrophil antimicrobial responses, such as increased cytoplasmic calcium and reactive oxygen species generation (data not shown). Using the bacterial peptide fMLF, evidence was provided of neutrophils' ability to respond to this bacterial peptide in the presence of CCI(91). This observation suggests that CCI(91) should not increase the risk of bacterial infection while attenuating neutrophil stimulation to MSU, and that innate immunity mediated by the formyl peptide receptor is not entirely dependent on βVI tubulin. Either βVI tubulin plays a redundant role in bacterial defenses shared with other beta-tubulin isotypes, or βVI tubulin is not required at all for these defenses. It should be noted that CCI(91) and CH-35(43) are also presumed to have increased affinity for other beta-tubulin isotypes.

[0181] In summary, a new, less toxic anti-inflammatory drug has been developed that may suppress MSU-induced inflammation and alleviate symptoms in gout patients during a gout attack. The development of such drugs addresses a major unmet clinical need in gout patients. Colchicine has a very narrow therapeutic index and is associated with adverse side effects due to drug interactions. Furthermore, gout occurs more frequently in patients with comorbidities (e.g., chronic kidney disease (CKD)). Approximately 54% of gout patients suffer from CKD. This makes the use of colchicine difficult, as patients with CKD require careful administration of colchicine because, due to renal impairment, colchicine accumulates more readily to toxic levels in these patients. Comorbidities also significantly limit the selection of non-colchicine medications that can be used to alleviate inflammation and pain associated with acute gout attacks. Patients who cannot take current treatments due to intolerance and comorbidities are a driving force for newer, less toxic treatment approaches. While there are alternative anti-inflammatory drugs such as NSAIDs and corticosteroids, these compounds themselves are also associated with well-established side effects. Therefore, the findings presented here indicate that lower effective doses of CCI broaden its therapeutic range and reduce the adverse side effects associated with colchicine and other anti-inflammatory drugs. An estimated 12% of gout patients are resistant to all available treatments (unpublished observations). (91)(CCI) is active at very low doses, so it is unlikely that (91)(CCI) will cause toxicity through interactions with other drugs. This hypothesis is supported by acute inflammatory toxicity studies in rats that showed (91)(CCI) is less toxic than colchicine and other colchicine derivatives such as (89) mentioned above. The maximum tolerated dose of (91)(CCI) is three times that of colchicine. Such a drug is of particular interest to gout patients, especially those with CKD who cannot eliminate drugs due to impaired renal function. (91)(CCI) is a safer anti-inflammatory drug than colchicine and a safer alternative for gout patients.

[0182] Example 6 - Tubulin binding study Materials and methods Preparation of the tubulin model The consensus sequence for the human β-tubulin isotype has been previously described (Huzil JT et al., Nanotechnology. 2006:17:S90-S100). The residues constituting the colchicine binding site were determined by examining the B chain within the 1SA0 pdb coordinate system (Ravelli RB et al., Nature. 2004; 428:198-202). Using PyMol v1.0 (Delano WL. The PyMOL Molecular Graphics System. 2002), residues with atoms within 6 Å of colchicine were selected. From this subset of residues, the minimum set of contact residues found within the colchicine binding site was defined (Figures 5A and 5B). Examination of the primary sequences of βI, βIIa, βIIb, βIII, βIVa, βIVb, and βV based on this reduced contact set placed the tubulin isotypes on one of three colchicine binding sites: type I (βI and βIV), type II (βII), and type III (βIII and βV) (Figure 5A). Next, using a template β-tubulin structure obtained from 1SA0 B-chain coordinates (Ravelli et al., 2004, Nature, 428, 198-202), we constructed a model by replacing appropriate residues from a standard conformational isomer library using the mutation function found in PyMol v1.0 (Delano WL. The PyMOL Molecular Graphics System. 2002). Minimization of each binding site model was performed using the CHARMm (Chemistry at HARvard Molecular Mechanics) molecular force field ((Brooks BR, Brooks CLr, Mackerell AD.J. et al., CHARMM: The biomolecular simulation program.) J. Comput. Chem. 2009) with the GROMACS molecular dynamics (MD) package (version 3.2.1) (Lindahl F. et al., GROMACS 3.0: A package for molecular simulation and trajectory analysis.) J Mol. Mod. 2001; 7:306-17). The convergence criterion for the steepest descent and conjugate gradient minimization was set at a gradient of 0.05 kcalmol-1 Å-1. Following minimization, a short simulated annealing run (100 ps) was performed in a well-solvated periodic box (100 x 100 x 100 Å). Unconstrained charges were balanced with sodium ions, and long-range electrostatics were calculated using the particle mesh Ewald (PME).

[0183] Colchicine derivatives The structure of colchicine bound to tubulin was extracted from pdb structure file 1SA0 (Ravelli RB et al., Nature. 2004; 428:198-202) and imported into MarvinSketch (ChemAxon, Hungary). Derivatization of the C1 and C3 methoxy groups (Figures 2-4) was achieved by constructing the modifications using a 3D drawing tool. Each derivative was then exported as an MDL Molfile (Symyx Technologies, USA) in 3D coordinates. Parameterization and minimization of colchicine Colchicine and its derivative structures were prepared and parameterized using the CHARM force field (Brooks BR, Brooks CLr, Mackerell AD.J. et al., CHARMM: The biomolecular simulation program. J. Comput. Chem. 2009) implemented in Discovery Studio v2.1 (Accelrys, Inc., USA). A vacuum minimization step was performed before reintroducing each derivative into type I, II, and III binding site models. Since the initial colchicine coordinates were obtained from the crystal structure, harmonic constraints (10 kcalmol) were applied to the carbon atoms in each of the three rings. -1 The following was set: The mean squared deviation (RMS) gradient is 0.05 kcalmol. -1 Å -1 Hydrogen was added until the bond order was fixed and atomic positions were optimized using the CHARMm force field and the adopted basis set Newton-Raphson (ABNR) protocol. Specific colchicine derivatives were prepared slightly differently. Individual systems were placed in a TIP3 waterbox using GROMACS and minimized. After a short equilibration, system energies for three separate conditions were obtained. The energy of the solvated tubulin-colchicine complex E(P+L) was subtracted from the energy obtained from the tubulin-colchicine system in which colchicine was not bound to the colchicine binding site E(PL). In the case of E(PL), a large waterbox was used to avoid introducing non-binding interactions between colchicine and tubulin.

[0184] Computational Colchicine Screening The docking of 20 colchicine derivatives to type I, II, and III binding sites was performed using CDOCKER (Wu G. et al., J. Comput Chem. 2003; 24:1549-62), as implemented in Discovery Studio v2.1 (Accelrys, Inc., USA). Briefly, a simulated annealing MD approach using the CHARM force field was used to perform conformational searches of the derivatives (Brooks BR, Brooks CLr, Mackerell AD.J. et al., CHARMM: The biomolecular simulation program. J. Comput. Chem. 2009). The selection of input site spheres was defined across the entire colchicine binding site. Each derivative was then heated to a temperature of T=700K and annealed to T=300K. Ten such cycles were performed for each of the 20 colchicine derivatives, generating 600 pauses. Each conformation was then subjected to local energy minimization using the ABNR method described above. Bond energy evaluation Using MM-GBSA (Molecular Mechanics-Generalized Born Surface Area), the binding energy of each system was evaluated using vacuum electrostatics, and solvation was estimated using the Generalized Born model. Binding energy was calculated by obtaining the total potential energy of the system and subtracting the energies of the derivative and empty dimers.

number

number

[0185] Drug binding to purified tubulin isotypes Tubulin was purified from bulk microtubule protein by phosphocellulose chromatography (Fellous A., et al., Eur. J. Biochem. 1977; 78:167-74). Subsequently, αβII and αβIII tubulin dimers were purified by immunoaffinity chromatography using monoclonal antibodies as previously described (Banerjee A. et al., J. Biol. Chem. 1992; 267:13335-9; and Baneljee A. et al., J. Biol. Chem. 1988; 263:3029-34). For dynamic fluorescence measurements, 500 μL aliquots of tubulin (0.1 mg / ml) were incubated at 37°C in a quartz fluorescence cuvette (path length 0.5 cm) in the presence of a range of drug concentrations. Kinetics were performed under pseudo-primary conditions using a large excess of the drug compared to tubulin. The excitation and emission wavelengths used were 380 nm and 437 nm, respectively. The corrected fluorescence values ​​are plotted as a function of time (t) on the curve:

number

[0186] result Isotype sequencing analysis The tertiary structure of tubulin can be divided into three distinct domains: Domain I (residues 1-198), Domain II (residues 199-373), and Domain III (residues 374-428) (Nogales E. et al., Nature. 1995; 375:424-7). The βI, βIIa, βIIb, βIII, βIVa, βIVb, and βV isotypes share 87.4%, 88.1%, and 96.3% identity within these domains, respectively. For the residues involved in paclitaxel binding (Nogales E. et al., Nature. 1995; 375:424-7), the sequence identity was 91.7%, which was higher than expected compared to the overall identity among β-tubulin isotypes. This trend of higher-than-average values ​​continues for the Vinca binding site (Gigant B. et al., Nature. 2005:435:519-22) (92.3% identity) and the GDP binding site (Nogales E. et al., Nature. 1995; 375:424-7) (100% identity). The colchicine-binding site (Ravelli RB et al., Nature. 2004; 428:198-202) was found to consist of 18 residues: V236, C239, L246, A248, K252, L253, N256, M257, T312, V313, A314, A315, V316, N348, K350, T351, A352, and I368 (Figure 5A). In contrast to the paclitaxel and Vinca-binding sites, only 77.9% identity was shared among the seven β-tubulin isotypes examined.

[0187] Generally, the binding site is primarily nonpolar, with a slight positive charge introduced to the outer lip of the surface by residues K252 and K350. Specific substitutions within the colchicine binding surface were found to be C236S (βIII and βV), A315T (βIII and βV), V316I (βII), and T351V (βIII and βV) (Figure 5A). Based on the isotype distribution of substitutions within this site, β-tubulin isotypes were classified into three classes. Type I binding sites are characterized by standard βI sequences and, in most cases, contain βII and βIV isotypes. Type II binding sites are identical to type I sites, except for the V316I substitution found only within the βII isotype. Type III binding sites exhibit the greatest variation (C236S, A315T, and T351V) and contain βIII and βV isotypes. When substitutions found within the Type II and Type III binding sites were mapped to the βI-tubulin structure (Lowe J. et al., J. Mol. Biol. 2001; 313:1045-57), it was observed that all were located within the region surrounding the colchicine A ring (Figure 5B). None of these substitutions altered the surface charge, however C239S and A315T altered the surface polarity interacting with the A ring, particularly the three nonpolar phenolmethoxy groups.

[0188] Colchicine derivatives As outlined in Figures 2-4, several modifications were made to the basic colchicine and thiocolchicine scaffolds. These modifications consisted of alkane / alkene, ester / ether, aromatic modifications to C1-demethylcolchicine and C3-demethylcolchicine (Figures 3 and 3A), or alkane / alkene modifications to C3-demethylthiocolchicine (Figure 4). Specific modifications were selected to investigate the spatial and chemical differences between classes of isotype binding sites. Modifications made at C1 were designed to probe the differences observed between residues 315, 316, and 351, while modifications made at C3 were primarily designed to investigate nonpolar cavities beneath colchicine, which are observed in cocrystals (Ravelli RB et al., Nature. 2004; 428:198-202).

[0189] docking of colchicine derivatives The basic strategy employed to computationally investigate colchicine derivatives involved generating several ligand orientations, followed by MD-based simulated annealing, and a final purification step incorporating steepest descent and conjugation gradient minimization. Using CDOCKER (Accelrys, Inc., USA), a total of 10 replicas were generated for each colchicine derivative and randomly distributed around the center of the binding site model. Following the initial orientation of the derivatives, each was subjected to final purification by MD-based simulated annealing and minimization, yielding 10 docking poses for each derivative and colchicine in each of the three binding site models. The final step of the docking procedure was to score the refined docking poses using the Discovery Studio Score Ligand Poses protocol. Note that the average energy value of the 10 poses from each experiment was used to create the binding energy score. This procedure yielded 630 ligand conformational isomers, whose energy evaluations were performed.

[0190] Determination of bond energy The binding energy was determined by calculating the total potential energy of each complete system determined in the docking step, and then subtracting the energies of the binding ligand and apodimer (Tables 4 to 6). When the average binding energy of each colchicine derivative was plotted, the trend was consistent across all models, with no significant differences between type I, type II, or type III binding sites (Figure 6; CH represents colchicine). However, in all models, ester / ether and aromatic derivatives at position C1 showed higher binding energies compared to colchicine, while alkane / alkene and thiocolchicine derivatives at positions C1 and C3 showed superior binding affinity (Tables 4 and Figure 6). These plots also show the range of binding energies for each derivative, suggesting the overall appropriateness of the docking fit (Figure 6). Specifically, derivatives showing higher binding energies than colchicine tended to have a wider distribution of binding energies, while derivatives with lower overall binding energies had a narrower distribution. This trend appeared to correlate with the polarity of each functional group at the C1 position. To investigate the role these modifications played in vitro, all colchicine derivatives were synthesized and tested using a tubulin-binding assay.

[0191] These calculations clearly show that modification of the colchicine amide group increases the binding with tubulin (Tables 5 and 6). These results also suggest that, on average, the modifications made to certain derivatives ((40), (42), (43)) had the lowest energy. [Table 4]

[0192] [Table 5]

[0193] [Table 6]

[0194] Example 7 - Binding kinetics To determine which aspect of (91)(CCI) may contribute to the above unexpected results, a docking experiment (previously described in PCT publication number WO2011022805 and incorporated herein by reference) was performed to determine that the binding energy between the colchicine derivative and tubulin isotype β-III is X 1 Regarding positional modifications, we investigated whether they might contribute to the effects observed in vitro and in vivo. The 3D structures of colchicine derivatives shown in Table 7 below were docked to the colchicine binding site of βIII tubulin (structurally identical to the beta-VI colchicine binding site) using the Autodock4 program under conditions of flexible ligands and rigid receptors. AutoDock4 is designed to predict how drug candidates will bind to receptors of known 3D structures and consists of two main programs: autodock performs the docking of ligands to a set of grids describing the target protein, and autogrid pre-calculates these grids. The initial ligand structures were first minimized using the Amber12:EHT force field (MOE2013.0802) and then fully optimized based on the theory of RHF / cc-pVDZ levels from GAMESS-US version 2010-10-01. The βIII tubulin sequence data (TBB3_HUMAN) and homology model obtained from UniProt ID (Q13509) were constructed for βIII tubulin based on the tubulin structure in the RCSB protein databank (1SA0.pdb) using MOE2013.0802.

[0195] [Table 7]

[0196] result The results of the docking experiment described above are shown in Table 8 below. [Table 8]

[0197] As shown in Table 8 above, a comparison of colchicine and (91)(CCI) with (89) showed that the OH group increased the binding energy to tubulin from -5.97 and -6.2 to -5.42 kcal / mol. Therefore, the presence of the OH group may cause an increase in binding energy and a decrease in affinity to tubulin. Consequently, (91)(CCI) lacking the OH group decreased the binding energy and increased affinity to tubulin, and thus increased the functional response in the in vitro and in vivo results presented herein.

[0198] Example 8 - Mouse model of atherosclerosis Introduction Atherosclerosis is a chronic inflammatory disease in which arterial stenosis occurs due to neointima-induced lesions (1). Immune cells such as neutrophils play an important role in the onset, progression, and instability of atherosclerotic lesions (2). When these lesions rupture, they cause multiple cardiovascular complications such as myocardial infarction and stroke. Because inflammation plays a crucial role in atherosclerosis, suppressing inflammation is a treatment approach that has attracted much attention in this field. For example, drugs that inhibit IL-1β improve the outcome of atherosclerosis. This study concluded that atherosclerosis could be treated using colchicine derivatives tested in a gout model, because neutrophil-mediated inflammation is involved in the pathogenesis of both diseases. The advantage of using these derivatives to treat atherosclerosis is that they are active at significantly lower doses than colchicine, and consequently, are likely to be less toxic. Considering this data, the colchicine derivatives described herein are expected to play a role in other inflammation-mediated diseases, such as those involving neutrophil-mediated inflammation.

[0199] method Animals and food LDLR knockout mice were randomly divided into four groups and given either a control (CD) diet (groups 1 and 2) or a high-fat diet (groups 3 and 4). The high-fat (HF) diet contained 0.2% total cholesterol, 21% total fat (42% kcal from fat), over 60% total fatty acids, and was high in sucrose (34% by weight). The animals and their feed were weighed weekly. Treatment by CCI Mice in groups 2 and 4 were administered 0.5 μmol / kg of CCI subcutaneously (sc) three times a week for 8 weeks. This dose of CCI was selected because it is the lowest dose that significantly reduces leukocyte recruitment in the gout air porch model. Plasma lipid measurement After feeding mice a special diet for eight weeks, blood was collected. Serum was prepared from the collected blood and frozen for analysis. Serum triglycerides and cholesterol were measured in the mouse serum by our Multidisciplinary and Microbiology Laboratory Service. Quantification of macroscopic lesion area Frontal assays and Sudan IV staining of aortic lesions were performed on aortas collected from all mice. The proportion of lipid-stained area in aortic branches and descending aorta was compared between mice using the ImageJ "target area" tool.

[0200] Cytokine measurement After feeding mice a special diet for eight weeks, blood was collected. Serum was prepared from the collected blood and frozen for analysis. Cytokine levels in the serum were measured by the Luminex assay. The cytokines assayed were eotaxin (CCL11), CCL21, G-CSF (CSF-3), RANKL, VEGF-A, IL-1 beta, IL-6, and MCP-1 (CCL2). These cytokines were selected based on cytokine signatures reported to reflect the development of atherosclerosis in ApoEKO mice. The LDLR KO model was used because the inflammatory aspect of atherosclerosis is better represented in this model.

[0201] result Figure 19 shows that mice treated with CCI and fed a CD or HF diet gained the same amount of body weight over 8 weeks as mice that were not treated with the drug and were fed the same diet. This indicates that the mice tolerated the drug for 8 weeks using a dose of CCI that attenuates MSU-induced leukocyte recruitment in vivo. The mice behaved normally and showed no signs of distress. Compared to mice fed a CD diet, LDLR KO mice fed an HF diet for 8 weeks showed a significant increase in serum triglyceride levels (Figure 20). Triglyceride levels were lower in CCI-treated mice fed an HF diet for 8 weeks than in mice fed an HF diet alone. Compared to mice fed a CD diet, LDLR KO mice fed an HF diet for 8 weeks showed a significant increase in serum cholesterol levels (Figure 21). Cholesterol levels were lower in CCI-treated mice fed an HF diet than in mice fed an HF diet alone. Figure 22A shows that mice fed the HF diet for 8 weeks developed atherosclerotic lesions (stained in red). The percentage of stained lesion area in the aortic arch or descending aorta of CCI-treated mice fed the HF diet was lower than that of mice fed the HF diet alone. The percentage of the total area of ​​the aortic arch covered with stained plaque was determined for each mouse using the "frontal assay" (Figure 22B). Lesions covered a smaller percentage of the total area of ​​the aortic arch in LDLR KO mice fed a high-fat diet and our compound (HF+CCI) than in mice fed the HF diet (HF). The percentage of the total area of ​​the descending aorta covered with stained plaque was determined for each mouse using the "frontal assay" (Figure 22C). Lesions covered a smaller percentage of the total area of ​​the descending aorta in LDLR KO mice fed a high-fat diet and our compound (HF+CCI) than in mice fed the HF diet (HF). The majority of mice in the HF+CCI group had lesions covering less than 1% of the total area of ​​the descending aorta. In contrast, in the HF diet group, lesions covered 1.2–8% of the area. Except for RANKL, elevated levels of all cytokines were observed in the serum of mice fed the HF diet for 8 weeks compared to mice given CD (Figure 23). RANKL levels decreased after 8 weeks on the HF diet. CCI did not significantly affect the levels of these cytokines in the serum of LDLR KO mice fed the HF diet for 8 weeks.

[0202] discussion Atherosclerosis is a chronic inflammatory disease of the arterial walls (1). Several inflammatory cells and mediators contribute to the onset and progression of this disease (2-4). Therefore, the inflammatory components of this disease are potential therapeutic targets. We tested the ability of colchicine derivative CCI to delay the progression and / or onset of atherosclerosis in an LDLR KO mouse model. In summary, our findings indicate that CCI may influence the onset and / or progression of atherosclerosis by reducing several variables associated with this chronic inflammatory disease, including serum levels of cholesterol and triglycerides, and the percentage of aortic area stained for atherosclerotic plaques. Furthermore, this study provides further evidence that CCI is well tolerated by mice for at least two months. Due to its lower toxicity compared to colchicine and its tendency to reduce several reliable indicators of atherosclerosis, CCI is a promising alternative treatment for this disease in humans.

[0203] References 1. Shapiro, MD and Fazio, S. From Lipids to Inflammation. 2016. Circulation Research 118:732-749. 2. Hartwig, H; Silvestre R; Daemen M; Lutgens, E and Soehnlein, O. Neutrophils in atherosclerosis. Hamostaseologie 2015; 35: 121-127. 3. Stefan Mark Nidorf and Peter Lindsay Thompson. Why Colchicine Should Be Considered for Secondary Prevention of Atherosclerosis: An Overview. 2019. Clinical Therapeutics 41: 41-48. 4. Lin B, Pillinger M, Shah B, et al. Use of colchicine in atherosclerotic heart disease. 2018. Curr Res Integr Med 3(S1):2-4.

Claims

1. Compounds of formula I for the treatment of inflammation: 【Chemistry 1】 Here: Z is either O or S; X １ This is selected from substituted or unsubstituted hydrocarbon groups, or substituted or unsubstituted heterogeneous groups; R ２ and R ３ Each of these is independently selected from substituted or unsubstituted hydrocarbon groups, substituted or unsubstituted heterogeneous groups, substituted or unsubstituted carbocyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aromatic groups, or substituted or unsubstituted heteroaromatic groups; The pharmaceutically acceptable salt thereof, its hydrate, its solvate, its tautomer, its optical isomer, or combination thereof.

2. R ２ and R ３ The compound according to claim 1, wherein each of the following is independently selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group.

3. R ２ and R ３ each independently is selected from substituted or unsubstituted alkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted C １ -C ６ alkylcarbonyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl, the compound according to claim 1.

4. The aforementioned R ２ and R ３ However, each is either a substituted or non-substituted C. １ -C ６ Alkyl, substituted, or unsubstituted C ２ -C ６ Alkenyl, substituted or unsubstituted C １ -C ６ Alkylcarbonyl, C １ -C ６ The compound according to claim 1, independently selected from alkylene-O-alkyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl.

5. R ２ and R ３ The compound according to claim 1, wherein each of the elements is independently selected from a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted alkylaryl.

6. R ２ and R ３ The compound according to claim 1, wherein each of the elements is independently selected from substituted or unsubstituted alkyls, or substituted or unsubstituted alkylaryls.

7. R ２ and R ３ The compound according to claim 1, wherein each of the elements is independently selected from substituted or unsubstituted alkyl groups.

8. The aforementioned R ２ and R ３ However, each is either a substituted or non-substituted C. １ -C ６ A compound according to claim 1, independently selected from alkyl groups.

9. R ２ and R ３ Each of these is an unsubstituted C １ -C ６ A compound according to claim 1, independently selected from alkyl groups.

10. R ２ and R ３ The compound according to claim 1, wherein each of the elements is independently selected from methyl, ethyl, or propyl.

11. R ２ The compound according to any one of claims 1 to 10, wherein is methyl.

12. R ３ The compound according to any one of claims 1 to 11, wherein is ethyl or propyl.

13. X １ The compound according to any one of claims 1 to 12, wherein is a substituted or unsubstituted hydrocarbon group.

14. X １ The compound according to claim 13, wherein the compound is selected from a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or a substituted or unsubstituted alkynyl.

15. X １ However, C is either substituted or non-substituted. １ -C ６ A compound according to claim 14, selected from alkyl groups.

16. X １ is non-substituted C １ -C ６ A compound according to claim 14, selected from alkyl groups.

17. X １ The compound according to claim 16, wherein is selected from methyl or ethyl.

18. X １ The compound according to claim 17, wherein is methyl.

19. X １ OR １０ And R １０ The compound according to any one of claims 1 to 12, wherein is selected from substituted or unsubstituted hydrocarbon groups, or substituted or unsubstituted heterogeneous groups.

20. R １０ The compound according to claim 19, wherein the group is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group.

21. R １０ However, substituted or unsubstituted alkyl, CH ２ The compound according to claim 19, selected from OH, substituted or unsubstituted haloalkyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted cyanoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted alkylcycloalkyl, substituted or unsubstituted alkylcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkylheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted alkylheterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, alkylene-O-alkyl, alkylene-O-cycloalkyl, alkylene-O-heterocycloalkyl, alkylene-O-alkylene-cycloalkyl, or alkylene-O-alkylene-heterocycloalkyl.

22. R １０ The compound according to claim 19, wherein the compound is selected from a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or a substituted or unsubstituted alkynyl.

23. R １０ However, C is either substituted or non-substituted. １ -C ６ Alkyl, or substituted or unsubstituted C ２ -C ６ Alkenyl, or C ２ -C ６ A compound according to claim 19, selected from alkynyl.

24. R １０ The compound according to claim 19, wherein the alkyl group is selected from substituted or unsubstituted alkyl groups.

25. R １０ However, C is either substituted or non-substituted. １ -C ６ A compound according to claim 19, selected from alkyl groups.

26. R １０ is non-substituted C １ -C ６ A compound according to claim 19, selected from alkyl groups.

27. R １０ The compound according to claim 19, wherein is selected from methyl or ethyl.

28. R １０ The compound according to claim 27, wherein is methyl.

29. X １ The compound according to any one of claims 1 to 12, wherein is a substituted or unsubstituted heterogeneous group.

30. X １ ga-CR ４ R ５ R ６ Selected from, R ４ , R ５ , and R ６ The compound according to claim 29, wherein each is independently selected from H, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterogeneous group.

31. R ４ , R ５ , and R ６ The compound according to claim 30, wherein each of the groups is independently selected from substituted or unsubstituted amide groups.

32. R ４ and R ５ are each independently selected from H or substituted or unsubstituted alkyl, R ６ is -NR(CO)CR ７ R ８ R ９ where R is selected from H and substituted or unsubstituted alkyl, R ７ , R ８ , and R ９ are each independently selected from H, a halo group, and substituted or unsubstituted alkyl, the compound according to claim 30.

33. R ７ , R ８ , and R ９ The compound according to claim 32, wherein is selected from fluoro groups.

34. X １ ga-CH ２ NH(CO)CF ３ The compound according to claim 33.

35. A compound according to any one of claims 1 to 34, wherein Z is O.

36. A compound according to any one of claims 1 to 34, wherein Z is S.

37. The compound 【Chemistry 2】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

38. The compound 【Transformation 3】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

39. The compound 【Chemistry 4】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

40. The compound 【Transformation 5】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

41. The compound 【Transformation 6】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

42. The compound 【Transformation 7】 The compound according to claim 1, which is a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a tautomer thereof, an optical isomer thereof, or a combination thereof.

43. The compound according to any one of claims 1 to 42, wherein the compound is formula I and / or a pharmaceutically acceptable salt thereof.

44. The compound according to any one of claims 1 to 43, wherein the configuration at C7 is an S configuration.

45. The compound according to any one of claims 1 to 44, wherein the compound binds to β-tubulin at a colchicine binding site.

46. The compound according to claim 45, wherein β-tubulin is β-VI, β-V, and / or β-I.

47. The compound according to claim 46, wherein β-tubulin is β-VI.

48. The compound according to any one of claims 45 to 47, wherein the compound has a binding energy lower than that of colchicine.

49. A compound according to any one of claims 1 to 48, wherein the compound is less toxic than colchicine.

50. The compound according to any one of claims 1 to 49, wherein the compound targets neutrophils more specifically than colchicine.

51. The compound according to any one of claims 1 to 50, wherein the compound inhibits the increase in intracellular calcium concentration at a lower dose than colchicine.

52. The compound according to claim 51, wherein the compound inhibits the increase in intracellular calcium concentration at a dose at least about one-tenth that of colchicine.

53. The compound according to claim 52, wherein the compound inhibits the increase in intracellular calcium concentration at a dose of about one-tenth to about one-hundredth of that of colchicine.

54. The compound according to any one of claims 1 to 53, wherein the compound inhibits the increase in intracellular calcium concentration at a dose of about 0.1 μM.

55. A compound according to any one of claims 1 to 54, wherein the compound inhibits the production of inflammatory mediators at a lower dose than colchicine.

56. The compound according to claim 55, wherein the compound inhibits the production of inflammatory mediators at a dose at least about one-tenth that of colchicine.

57. The compound according to claim 55, wherein the compound inhibits the production of inflammatory mediators at a dose of about one-tenth to about one-hundredth of that of colchicine. 。

58. A compound according to any one of claims 1 to 57, wherein the compound inhibits the production of inflammatory mediators at a dose of about 0.1 μM.

59. The compound according to any one of claims 55 to 58, wherein the inflammatory mediator is selected from IL-8, IL-1, superoxide, or a combination thereof.

60. The compound according to any one of claims 1 to 50, wherein the compound exhibits a monotonic or non-monotonic dose-response with respect to inhibition of at least one of intracellular calcium concentration and inflammatory mediator production.

61. The compound according to claim 60, wherein the inflammatory mediator is selected from IL-8, IL-1, superoxide production, or a combination thereof.

62. A compound according to any one of claims 1 to 61, wherein the compound inhibits the recruitment of leukocytes.

63. The compound according to any one of claims 1 to 62, wherein the inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or a combination thereof.

64. The compound according to claim 62 or 63, wherein the inflammation includes inflammation caused by neutrophils.

65. The compound according to claim 64, wherein the inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or a combination thereof.

66. The compound according to claim 64, wherein the inflammation caused by neutrophils is associated with cardiovascular disease.

67. The compound according to claim 66, wherein the cardiovascular disease is coronary artery atherosclerosis.

68. The compound according to claim 64, wherein inflammation caused by neutrophils is associated with gout.

69. A compound according to any one of claims 1 to 62 for the treatment of gout.

70. The compound according to any one of claims 1 to 62, wherein the compound has an inhibitory effect on immune function in response to monosodium urate (MSU)-induced inflammation.

71. The compound according to claim 70, wherein the inhibitory effect on immune function is mediated by a mediator selected from intracellular calcium production, IL-1 production, IL-8 production, superoxide production, or a combination thereof.

72. The compound according to claim 71, wherein the immune function relates to neutrophils.

73. The compound according to any one of claims 70 to 72, wherein the inhibitory effect is stronger than that of colchicine.

74. The compound according to claim 73, wherein the inhibitory effect is at least about 10 times greater than that of colchicine.

75. The compound according to any one of claims 70 to 74, wherein the inhibitory effect is produced at a concentration of approximately 0.1 μM.

76. A compound according to any one of claims 1 to 62 for the treatment of cardiovascular disease.

77. The compound according to claim 76, wherein the cardiovascular disease is coronary artery atherosclerosis.

78. A pharmaceutical composition comprising the compound described in any one of claims 1 to 62.

79. The composition according to claim 78, further comprising an antigout agent.

80. The composition according to claim 79, wherein the antigout agent is selected from nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular glucocorticoids, xanthine oxidase inhibitors, recombinant non-human uricase enzymes, uric acid excretion promoters, uric acid excretion agents, or a combination thereof.

81. The composition according to any one of claims 78 to 80, further comprising at least one pharmaceutically acceptable carrier and / or diluent.

82. The composition according to any one of claims 78 to 81, wherein the composition comprises two or more compounds according to any one of claims 1 to 62.

83. A composition according to any one of claims 78 to 82 for the treatment of inflammation.

84. The composition according to claim 83, wherein inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or a combination thereof.

85. The composition according to claim 83 or 84, wherein the inflammation includes inflammation caused by neutrophils.

86. The composition according to claim 85, wherein the inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or a combination thereof. 。

87. The composition according to claim 85, wherein the inflammation caused by neutrophils is associated with cardiovascular disease.

88. The composition according to claim 87, wherein the cardiovascular disease is coronary artery atherosclerosis.

89. The composition according to claim 85, wherein inflammation caused by neutrophils is related to gout.

90. Composition according to any one of claims 78 to 81 for the treatment of gout

91. A method for treating inflammation in a mammal, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 62 to the mammal.

92. The method according to claim 91, wherein two or more compounds described in any one of claims 1 to 62 are present.

93. The method according to claim 91 or 92, wherein the compound is administered orally and / or parenterally.

94. The method according to claim 91 or 92, wherein the compound is administered intravenously and / or intraperitoneally.

95. A method for treating inflammation in a mammal, comprising administering to a mammal a therapeutically effective amount of the composition according to any one of claims 78 to 82.

96. The method according to claim 95, wherein the composition is administered orally and / or parenterally.

97. The method according to claim 95, wherein the composition is administered intravenously and / or intraperitoneally.

98. The method according to any one of claims 91 to 97, wherein the inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or combinations thereof.

99. The method according to any one of claims 91 to 98, wherein the inflammation includes inflammation caused by neutrophils.

100. The method according to claim 99, wherein the inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or a combination thereof.

101. The method according to claim 99, wherein inflammation caused by neutrophils is associated with cardiovascular disease.

102. The method according to claim 101, wherein the cardiovascular disease is coronary artery atherosclerosis.

103. The method according to claim 99, wherein inflammation caused by neutrophils is associated with gout.

104. The method according to any one of claims 91 to 103, wherein the mammal is a human.

105. Use of a therapeutically effective amount of the compound according to any one of claims 1 to 62 for the treatment of inflammation in mammals.

106. The use according to claim 105, wherein two or more compounds described in any one of claims 1 to 62 are present.

107. The use according to claim 105 or 106, wherein the compound is administered orally and / or parenterally.

108. The use according to claim 105 or 106, wherein the compound can be administered intravenously and / or intraperitoneally.

109. Use of a therapeutically effective amount of the composition according to any one of claims 78 to 82 for the treatment of inflammation in mammals.

110. The use according to claim 109, wherein the composition is administered orally and / or parenterally.

111. The use according to claim 109, wherein the composition can be administered intravenously and / or intraperitoneally.

112. The use according to any one of claims 105 to 111, wherein inflammation is selected from inflammatory diseases, inflammatory conditions, inflammatory disorders, or combinations thereof.

113. The use according to any one of claims 105 to 112, wherein the inflammation is neutrophil-induced inflammation.

114. The use according to claim 113, wherein the inflammation caused by neutrophils is inflammation associated with pseudogout, gout, cardiovascular disease, vasculitis, or a combination thereof.

115. The use according to claim 113, wherein the inflammation caused by neutrophils is associated with cardiovascular disease.

116. The use according to claim 115, wherein the cardiovascular disease is coronary artery atherosclerosis.

117. The use according to claim 113, wherein inflammation caused by neutrophils is associated with gout.

118. The use according to any one of claims 105 to 117, wherein the mammal is a human.

119. A method for treating gout in a mammal, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 62 to a mammal.

120. The method according to claim 119, wherein two or more compounds described in any one of claims 1 to 62 are present.

121. The method according to claim 119 or 120, wherein the compound is administered orally and / or parenterally.

122. The method according to claim 119 or 120, wherein the compound is administered intravenously and / or intraperitoneally.

123. A method for treating gout in a mammal, comprising administering to a mammal a therapeutically effective amount of the composition according to any one of claims 78 to 82.

124. The method according to claim 123, wherein the composition is administered orally and / or parenterally.

125. The method according to claim 123, wherein the composition is administered intravenously and / or intraperitoneally.

126. The method according to any one of claims 119 to 125, wherein the mammal is a human.

127. The method according to any one of claims 119 to 126, wherein the gout is selected from chronic gout and / or acute gout.

128. The method according to any one of claims 119 to 127, wherein the treatment of gout includes the treatment of at least one gout symptom.

129. The method according to claim 128, wherein the at least one gout symptom is selected from gout attack, gouty nodule formation, gouty arthritis, gout-related inflammation, and / or gout-related joint destruction.

130. The method according to claim 128, wherein the at least one gout symptom is selected from gouty inflammation and / or inflammation-related pain.

131. Use of a therapeutically effective amount of the compound according to any one of claims 1 to 62 for the treatment of gout in mammals.

132. The use according to claim 131, wherein two or more compounds according to any one of claims 1 to 62 are present.

133. The use according to claim 131 or 132, wherein the compound is administered orally and / or parenterally.

134. The use according to claim 131 or 132, wherein the compound can be administered intravenously and / or intraperitoneally.

135. Use of a therapeutically effective amount of the composition according to any one of claims 78 to 82 for the treatment of gout in mammals.

136. The use according to claim 135, wherein the composition is administered orally and / or parenterally.

137. The use according to claim 135, wherein the composition can be administered intravenously and / or intraperitoneally.

138. The use according to any one of claims 131 to 137, wherein the mammal is a human.

139. The use according to any one of claims 131 to 138, wherein the gout is selected from chronic gout and / or acute gout.

140. The use according to any one of claims 131 to 139, wherein the treatment of gout includes the treatment of at least one gout symptom.

141. The use according to claim 140, wherein the at least one gout symptom is selected from gout attack, gouty nodule formation, gouty arthritis, gout-related inflammation, and / or gout-related joint destruction.

142. The use according to claim 140, wherein the at least one gout symptom is selected from gouty inflammation and / or pain associated with inflammation.

143. A method for reducing the motility and / or recruitment of hematopoietic cells, comprising administering a βVI-tubulin inhibitor.

144. The method according to claim 143, wherein the βVI-tubulin inhibitor is a compound according to any one of claims 1 to 62.

145. The method according to claim 143 or 144, wherein the hematopoietic cells are leukocytes such as neutrophils and / or monocytes.

146. A method for treating inflammation, including the administration of a βVI-tubulin inhibitor.

147. The method according to claim 146, wherein the βVI-tubulin inhibitor is a compound according to any one of claims 1 to 62.

148. The method according to claim 146 or 147, wherein inflammation is associated with leukocyte infiltration.

149. The method according to claim 148, wherein the leukocyte infiltration includes neutrophil and / or monocyte infiltration. The method.

150. The method according to any one of claims 146 to 149, wherein the inflammation is related to gout.

151. The method according to any one of claims 146 to 149, wherein the inflammation is related to atherosclerosis.

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