TW581767B - Epoxyvibsanin B - Google Patents

Abstract

This invention relates to a new compound, epoxyvibsanin B, which can be used to treat an IL-12 overproduction-related disorder such as an inflammatory disease.

Description

581767 V. Description of the invention (l) Bright background Interleukin-1 2 (IL-1 2) is a key cytokine that causes the IL-1 2 receptor to produce a pro-inflammatory cytokine 'or promote specific lymphocyte response. One of its key functions is to promote the response of type 1 T helper cells (τ 匕 1) and thus achieve cellular immunity. Excessive production of IL-12 will cause excessive Thi reactions, which may cause inflammation, such as autoimmune diseases. Gately et al., 1 99 8 ′ Annual Review of Immunology 16 ′ 495. Therefore, it is an ideal target for drug intervention in the treatment of K syndrome caused by excessive proliferation of D cells. Trembleau seeks ‘1995’ Immunology Today a, 383 and

Adorini et al., 1 997, Chemical Immunology 68, 175. Some pharmacological methods, including the regulation of intracyclic cycloadenylic acid levels, suppression of pale western excitatory sounds, and nuclear factor kaba B, can inhibit the overproduction of IL-12 and its causes. Thl-type immune response. Hasko et al., 1 999, British Journal of Pharmacology 127, 1 295. It is necessary to discover a new compound for the treatment of IL-12-related overproduction. SUMMARY OF THE INVENTION The present invention is based on the discovery of a new compound from a class of plant extracts that have been screened to inhibit ILj production. One aspect of the present invention relates to a compound, Epoxyvibsanin B, whose structural formula is as follows ...

581767 V. Description of the invention (3) Detailed description of the invention According to the following steps, Capri f1 1 aceae will remove solid substances from Miami Capr i f0i aceae solvent, such as ethanol, filter flash evaporator to dry, and remove the agent, such as ethyl acetate. This resulted in another addition step after heavy extraction, the water and B were collected for each wash, washed once with reversed-phase chromatography, and then dehydrated and powdered. One step weighs a solid to the solid several times, and the dry solid extract is mixed to check the combined and subtractive extraction column. Water and the solid are collected. The compound of the present invention, epoxy veganin B, can Separated from vi brunum Awabuk i. (Florida, USA) Collected vibrunum Awabuki leaves are suspended in an organic 'and crushed with a blender. The resulting solution is filtered and dried at a temperature of less than 45 ° C under reduced pressure or used to obtain a solid extract. Another organic solvent was added to the solid extract to produce a liquid extraction plural-human. The liquid extracts were combined and the extracts were dried under reduced pressure. Re-extracting another organic solvent, such as methanol, produces another liquid extract. This combines all liquid extracts. This liquid was dried 'using a flash evaporator to produce a more concentrated solid extract. It was passed through a solid phase extraction column (SepPak Cl8). The eluent was used as the eluent, and the eluate was received in several portions. Inhibits the activity produced by I L 1 2. The active part was dried to obtain no m-body f. Then, use the eluent and follow the above steps to obtain more solid material. The mixture of acetic acid was used as a gas, and the soil pine was used as an eluent, and purified using a high-pressure liquid phase. The washing containing epoxy vesanin B was sub-dried under reduced pressure to obtain pure epoxy vesanin B. White epoxy vesanin B and a drug combination containing an effective amount of vesanin β

$ 6 pages \\ CHENLIN \ Lika \ FIS11186.ptd 581767 Description of the invention (4), which includes chemotherapeutics and effective surface treatment of human inflammatory arthritis. The pharmaceutical composition is used to treat IL- 12 Excessive production of related disorders, such as autoimmune diseases (autoimmune diseases such as rheumatoid psoriasis' type I diabetes and multiple sclerosis), are all in the hair circumference =. For administration to a patient in need of treatment for inflammation such as an autoimmune disease, an effective amount of epoxy veganin B is defined as the amount of the compound required to administer a therapeutic effect to a subject. Freire et al. Described the internal relationship (based on milligrams per square meter of body surface area) for animal doses in the Cancer Therapy Report (196, 50, 21). Volume can be approximately determined from the patient's height and weight. See, "Geigy Pharmaceuticals, Ardley, New York, 1970, 537." The effective amount of epoxy veganin b ranges from about 0.1 mg / kg to 50 mg / kg. Those skilled in the art believe that the effective dose will also vary depending on the route of administration, the use of excipients, and possibly other treatments such as the use of other anti-inflammatory agents. / By using two conventional methods, epoxy vesanin B can be formulated into a pharmaceutical form for other routes of administration. For example, it can be formulated as an oral capsule, gel pill or tablet. Capsules can contain any acceptable substance, such as gelatin or cellulose. A mixture of epoxy veganin B, a solid carrier and a lubricant can be compressed into tablets according to conventional procedures. Solid carriers include starch and sugar bentonite. Epoxyvasanin B can also be administered in the form of more shelled tablets or capsules. Hard shelled tablets or capsules contain binding agents such as lactose or mannitol, conventional fillers and tablets. Theta music composition can be administered parenterally, including oral topical, subcutaneous, intraperitoneal, intramuscular, and intravenous

581767 V. Description of Invention (5) Shooting. 5% glucose solution in parenteral preparation, or its preparation. Cosolvents, such as cyclodextrin, can be used for delivery of therapies. The following special cases are only for the remainder of the disclosure. Further consideration may be required, based on this. Therefore, the research papers cited here. Aqueous solutions, isotonic saline solutions or active agents containing the well-known pharmaceutically acceptable chelate ^ or other solubilizing / excipient pharmaceutical excipients well known in the art. =, In any way, it is not limited = believe that experienced technicians in the field, Wu Chen's narrative can be used to the fullest extent. All publications are specifically embodied in the whole. For example, > Hengp suspended 2 kg of fresh CaprifoHaceae vibrUnum Awabuki leaves collected from Miami, Florida in 3 liters of ethanol, and crushed with a blender and soaked at room temperature for 2-3 hours. The resulting solution was filtered to remove solids, and the filtrate was dried with a flash evaporator at a temperature below 45 ° C to produce approximately 100 g of a solid extract. This solid extract was then extracted three times with 0.5 liters of ethyl acetate. The obtained liquid extracts were combined and concentrated 'under reduced pressure to obtain an enriched solid extract. 0.3 L of methanol was added to the solid extract to produce a liquid extract. This step was repeated three times. The liquid extracts were combined and dried with a flash evaporator to obtain another solid extract (20 g). Subsequently, 0.2 g of the solid extract was passed through a solid-phase extraction column (36 to 8 L (: 18, 2 cm \ 4 cm). Rinse with 100 ml of a 50% aqueous methanol solution, and then use 100 ml After washing with 50% acetonitrile in water, the extraction column was eluted with 80% acetonitrile in water. Fractions containing epoxy veganin B were collected and

\\ CHENLIN \ Lika \ FIS11186.ptd Page 8 581767 V. Description of the invention (6) Drying under reduced pressure gives a solid. Extraction peptone was then eluted again with an eluent to obtain additional epoxy vesanin B. 10-50% acetonitrile-water as eluent 'was used to further purify the solid (100 mg) using a high-pressure liquid chromatography column (Waterser inner diameter 50 X 300 mm). The components containing epoxy veganin were collected and concentrated under reduced pressure to give 5.2 mg of a white powder. (, Melting point: 100-102 ° C); lmax221nin (acetonitrile); high resolution electron bombardment mass spectrum m / z 41 7. 2626 (C25H3705) ΜΗ NMR, see Table 1. , Said 〇

All determination of epoxy vesanin B (30 0MHz 1 Chemical shift Proton number Coupling mode Coupling constant Marked position (δ ppm) (Hz)

1. 05 3H S

\\ CHENLIN \ Lika \ FIS11186.ptd Page 9 581767 V. Description of the invention (7) 1. 39 1H ddd 4.6, 11.7 ^ 15.1 11a 1 · 26 1H m 12b 1. 57 3H S 17 1. 66 3H S 16 1 . 80 2H m 13 1. 45 3H s 25 1. 97 1H dd 11 and 7 1 β 2 · 62 1H dd 11 and 7 1 a 2. 14 3H d 1. 2 24 1. 89 3H s 19 4. 17 1H d 14 18a 4. 48 1H dd 1 4 and 1 · 2 18b 3. 19 1H dd 4 · 7 and 1 8 5 a 3. 39 1H d 4. 7 and 1 3 5 β 2. 74 1H dd 1 8 and 1 3 6 5. 75 1H dd 11 · 7 and 7 2 5 · 05 1H t 7 14 5. 07 1H d 13. 5 8 5. 32 1H dd 16 · 4 and 13. 5 9 5 · 69 1H d 16. 4 10 5 · 70 1H br s 22 IL-1 2 Standard program for suppressing monocytes in human peripheral blood (PBMC) by speech test

\\ CHENLIN \ Lika \ FIS11186.ptd Page 10 581767 V. Description of the invention (8) Obtained by leukopak. These cells were diluted to three million per milliliter and stored in fetal bovine serum (10%), cyanogenin (100 units / ml), streptomycin (100 μg / ml) and L-glutamic acid Salt (2 litres) in rpmi medium. Human r interferon (IFN-r, Boehringer Mannheim, Cat. No. 104 0 5 9 6) was diluted with supplemented medium containing cells to 60 units / mL. Take 100 μl of the obtained cell culture, add it to each well of a 96-well, U-shaped bottom microtiter plate, and incubate overnight in a humidified, 37 ° C, 7% carbon dioxide incubator. Epoxyvasanin B in 4X RPMI culture medium storage solution was added to each well to a final concentration of 1 μg / ml, and then lipopolysaccharide (lps, saare) in 4X RPMI culture medium storage solution was added. Serratia arscencens Sigma; Cat. No. L-4766) was inserted into it with a final concentration of 1 μg / ml. Gently rotate the titration plate and continue incubating for 16 hours. I F N-r and L PS stimulate P B MC to produce IL-1 2. Supernatants of cell cultures in each well were collected and quantified using an interlayer EL I SA using an anti-human IL-12 antibody (R & D system; catalog number mAb 6 11 and catalog number BAF 2 1 9). IL -1 2 p 7 0 secreted in the supernatant. A control experiment was also performed without epoxy visartan B. Compared with the control experiments, the results showed that epoxy veganin B inhibited 90% of IL-12 production. Two groups of cells, PBMC and human pre-monocytic leukemia monocytes (THP-1 cells) were used to further examine the inhibitory effect on IL-12 production in parallel experiments. As described above, 50,000 PBMC cells were placed in each well of a 96-well titer plate, and PBMCs were stimulated to produce IL with IFN-r (200 units / ml) and LPS (1 μg / ml). -12. In parallel, add 800,000 Ding liver-1 cells to each well of a 96-well titer plate, and use 1? ^!-7

\\ CHENLIN \ Lika \ FIS11186.ptd Page 11 581767 V. Description of the invention (9) (200 00 units / ml) and Staphylococcus aureus cowan j (〇. 5%, SAC or Pansorbin, available from Calbiochem (B15921)) stimulates THP-1 to produce IL-12. The supernatant of the cell culture in each well was collected and analyzed for IL-12 p7 using a sandwich ELISA using the antibodies described above. The results show that cyclooxysanin B inhibits IL-12 production in PBMC and THP-1 cells. IC5G is about lnM for PBMC and about 2OnM for TP-1 cells. Epoxyvasanin B is 10 times more active in inhibiting IL-12 production than the known anti-inflammatory compound dexamethasone. Cytotoxicity experiments Cytotoxicity tests were performed on epoxy veganin b using Cell Titer 96 aqueous non-radioactive kit (promega, serial number G542 1). Phenazine methyl sulfate (Sigma; catalog number p 5812) was added as an electron donor to the cells containing PBMC (50, 000 cells per well) and epoxy veganin b (final concentration 1 μg / ml) 3-(4,5-Difluorenylthiazolyl-2yl) -5- (fluorenylphenyl) -2- (4-sulfophenyl) -2H-tetrazole (MTS). The cells were cultured in a humidified, 37 ° C, 7% carbon dioxide incubator for 2 hours. The supernatant of the cell culture was collected and its absorbance at 490 nm was recorded. Cell viability was determined based on the level of μτS (which reacts with mitochondrial dehydrogenase in living cells) and compared with data obtained from a control experiment without epoxy vesanin B. The cytotoxicity test of epoxy visartan B on THP-1 cells was further performed. The results showed that epoxy veganin B had similar cytotoxicity to PBMC and TIP-1 cells. It has 5 CC50 for PBMC and 10 CC50 for THP-1 cells. —— "IISI! 1! IIIII Ϊ IIII! I! II i 1 Discussion — \\ CHENLIN \ Lika \ FIS11186.ptd Page 12 V. Description of the invention (10) Fine primal motherhood of oxyvasanin B Lower than the anti-inflammatory compound dexamethasone. Examples Each feature of the M μ in this description; all features can be arbitrarily combined. Superseded by the public character in this note. ^ Alternative features that are used for the same, equivalent, or similar purpose are only one type of phase & f 'unless otherwise specifically indicated', each of the disclosures is: so; an example of a phase. Xi Ningshi Shuangnn Stone Technical personnel experienced in this field can easily _ the basic characteristics of X, without departing from the idea and scope of the present invention 2 = 2, various changes and improvements can be made to the present invention, Adapt it to the uses and conditions of work 1. For example, compounds similar in structure to epoxy veganin β can be manufactured and screened (as in the example above) and used in the practice of the present invention. Therefore, other embodiments are also within the scope of patent application.

581767 Schematic description \\ CKENLIN \ Lika \ FIS11186.ptd page 14

Claims (1)

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