# US 9,000,168 B2 · Methods for obtaining cyclopamine > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,000,168 B2 - Document ID: US9000168B2 - Status: Granted - Kind: B2 - Application number: 12/737,297 - Filed: 2009-07-03 - Published: 2011-06-30 - Granted: 2015-04-07 - Earliest claimed: 2008-07-04 - Assignees: Radient Technologies Inc. - Applicants: Steven Splinter; Satyasagar Kadali - Inventors: Steven Splinter; Satyasagar Kadali - Web page: https://patentgrep.com/patent/US9000168B2 ## CPC - C07J 21/00: Normal steroids containing carbon, hydrogen, halogen or oxygen having an oxygen-containing hetero ring spiro-condensed with the cyclopenta(a)hydrophenanthrene skeleton - C07D 307/94: spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins - C07D 311/96: spiro-condensed with carbocyclic rings or ring systems - C07J 43/00: Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton - C07J 69/00: Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by contraction of only one ring by one atom and expansion of only one ring by one atom - C07J: STEROIDS (seco-steroids C07C) - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) ## Abstract The present invention relates to a process for extracting, purifying and isolating cyclopamine from cyclopamine-containing biomass comprising the steps of contacting cyclopamine-containing organic matter with an extractant (e.g., aqueous extractant) capable of extracting cyclopamine, separating the extracted organic matter from the cyclopamine/extract solution, concentrating the cyclopamine/extract solution leaving a concentrated aqueous solution, contacting the concentrated aqueous solution with an organic extractant capable of selectively extracting cyclopamine from the aqueous phase, concentrating the cyclopamine-containing organic phase, providing an enriched extract containing cyclopamine, separating cyclopamine from the enriched extract using column chromatography, providing at least one fraction containing cyclopamine, and crystallizing select chromatography fractions to provide purified cyclopamine. ## Claims ### Claim 1 A process for extracting, purifying and isolating cyclopamine from cyclopamine-containing biomass comprising the steps of: Mixing the cyclopamine-containing organic matter with an aqueous solvent, which solvent is transparent to microwaves; extracting cyclopamine using microwave-assisted technology to obtain a cyclopamine/extract solution separating the extracted organic matter from the cyclopamine/extract solution; concentrating the cyclopamine/extract solution leaving a concentrated aqueous solution; contacting the concentrated aqueous solution with an organic extractant capable of selectively extracting cyclopamine from the aqueous phase; concentrating the cyclopamine-containing organic phase, providing an enriched extract containing cyclopamine; separating cyclopamine from the enriched extract using column chromatography, providing at least one fraction containing cyclopamine; and crystallizing select chromatography fractions to provide purified cyclopamine. ### Claim 2 A process for obtaining cyclopamine from cyclopamine-containing biomass, the improvement comprising the preparation of a second residue following the initial extraction of cyclopamine-containing organic matter which is more highly concentrated in cyclopamine, said improvement comprising the steps of: extracting the cyclopamine-containing organic matter into an aqueous solvent capable of extracting cyclopamine using microwave-assisted technology to obtain a cyclopamine/extract solution; separating the extracted organic matter from the cyclopamine/extract solution; concentrating the cyclopamine/extract solution to form a first residue; partitioning the residue between an aqueous phase and an organic solvent or solvent mixture capable of dissolving the cyclopamine to produce an aqueous phase and an organic phase; separating the organic phase from the aqueous phase and recovering the organic phase substantially free of the aqueous phase; forming a second residue from the organic phase enriched in cyclopamine. ### Claim 3 (depends on 2) The process according to claim 2, wherein the second residue is optionally subjected to a finishing treatment to further purify or isolate cyclopamine. ### Claim 4 (depends on 3) The process according to claim 3, wherein the finishing treatment comprises at least one of liquid-liquid extraction, precipitation, crystallization, or chromatography treatment. ### Claim 5 (depends on 4) The process according to claim 4, wherein the finishing treatment comprises dissolving the second residue in an organic solvent or solvent mixture, precipitating impurities from the solvent or solvent mixture, separating the precipitated impurities from the remaining cyclopamine-containing organic solution, and concentrating the cyclopamine-containing organic solution to provide a more concentrated residue. ### Claim 6 (depends on 5) The process according to claim 5, wherein the organic solvent is methanol, acetonitrile, or a mixture of ethanol and acetonitrile. ### Claim 7 (depends on 2) The process according to claim 2, wherein the aqueous phase from the separation step is recovered substantially free of the organic phase and subjected to a further finishing treatment to recover steroidal alkaloids. ### Claim 8 (depends on 7) The process according to claim 7, wherein the steroidal alkaloids recovered are jervine, veratramine and cyclopasine. ### Claim 9 (depends on 1) The process according to claim 1, wherein the biomass contains Veratrum Californicum biomass. ### Claim 10 (depends on 1) The process according to claim 1, wherein the biomass is subjected to an initial drying step. ### Claim 11 (depends on 10) The process according to claim 10, wherein the biomass is dried to a residual moisture content of less than 20% by weight. ### Claim 12 (depends on 1) The process according to claim 1, wherein the extractant is an ethanol-water mixture. ### Claim 13 (depends on 12) The process according to claim 12 wherein the extractant is an ethanol-water mixture composed of between 50 and 95 percent v/v ethanol. ### Claim 14 (depends on 12) The process according to claim 12 wherein the extractant is and ethanol-water mixture composed of between 60 and 70 percent v/v ethanol. ### Claim 15 (depends on 1) The process according to claim 1, wherein the cyclopamine-containing organic matter is mixed with a suitable base before contacting with an extractant capable of extracting cyclopamine. ### Claim 16 (depends on 15) The process according to claim 15, wherein the base is sodium carbonate. ### Claim 17 (depends on 15) The process according to claim 15, wherein cyclopamine-containing organic matter is mixed with between the 0.05 and 0.20 kilograms of sodium carbonate before contacting with an extractant capable of extracting cyclopamine. ### Claim 18 (depends on 2) The process according to claim 2, wherein the organic solvent is ethyl acetate. ### Claim 19 (depends on 2) The process according to claim 2, wherein the organic solvent mixture is a mixture of hexane and ethyl acetate. ### Claim 20 (depends on 2) The process according to claim 2, wherein the organic solvent is a mixture comprised of approximately 70% hexane/30% ethyl acetate by volume.