WO1998016531A1 - Derives de triterpene presentant une activite immunosuppressive - Google Patents

Derives de triterpene presentant une activite immunosuppressive Download PDF

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WO1998016531A1
WO1998016531A1 PCT/US1997/018334 US9718334W WO9816531A1 WO 1998016531 A1 WO1998016531 A1 WO 1998016531A1 US 9718334 W US9718334 W US 9718334W WO 9816531 A1 WO9816531 A1 WO 9816531A1
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alkyl
hydroxy
defined above
friedo
tetranor
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PCT/US1997/018334
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English (en)
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Robert K. Baker
Jianming Bao
Frank Kayser
Andrew Kotliar
William H. Parsons
Kathleen M. Rupprecht
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Merck & Co., Inc.
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Priority claimed from GBGB9625887.6A external-priority patent/GB9625887D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU49813/97A priority Critical patent/AU4981397A/en
Publication of WO1998016531A1 publication Critical patent/WO1998016531A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Graves ophthalmopathy and asthma.
  • the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.
  • autoimmune or a rejection process tissue destruction caused by inflammatory cells and the mediators they release.
  • Anti-inflammatory agents such as NSAID's act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease.
  • cytotoxic agents such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.
  • Cyclosporin A which was approved by the US FDA in 1983 is currently the leading drug used to prevent rejection of transplanted organs.
  • FK-506 Prograf
  • CsA and FK-506 act by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein.
  • CsA was approved by the US FDA for the treatment of severe psoriasis and has been approved by European regulatory agencies for the treatment of atopic dermatitis. Though they are effective in fighting transplant rejection, CsA and FK-506 are known to cause several undesirable side effects including nephrotoxicity, neurotoxicity, and gastrointestinal discomfort. Newer, safer drugs exhibiting less side effects are constantly being searched for in the field.
  • the present invention describes newly developed immunosuppressive compounds derived from the compounds described in Formulae 1 (a) through 1 (d) and which have the relative stereochemistry depicted above.
  • This invention relates to a class of triterpene derivatives of the general structural Formula I
  • the compounds of this invention are useful in the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.
  • pharmaceutical formulations comprising a compound of Formula I and a pharmaceutical carrier, as well as, pharmaceutical formulations comprising a compound of Formula I, a second immunosuppressive compound and a pharmaceutical carrier.
  • the present invention is related to compounds of formula I, including but not limited to those specified in the examples, which are useful in a mammalian subject for the treatment and prevention of the resistance by transplantation of organs or tissue, graft-versus-host diseases brought about by medulla ossium transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenile-onset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, infectious diseases caused by pathogenic microorganisms, inflammatory and hyperproHferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid
  • a is: a single bond, or a double bond when R4 is absent;
  • n 1 to 4.
  • X is: O or S
  • R1 and R 2 are independently: a) H, or b) (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, C ⁇ 2H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR 2 , NRlR2, NRlCOCl-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, C02H, COCl-C6
  • any two adjacent substituents can be joined to form a 5-, 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring;
  • R 3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; b) -(Cl -C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (Ci-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONR1R2 ?
  • n) -(Cl-C6)-alkyl, alkyl as defined above, o) -(C2-C6)-alkenyl, alkenyl as defined above, or p) CR1 R2, such that one and only one of R and R2 can be hydrogen.
  • a is: a single bond
  • b is: a single bond
  • n 1 to 4;
  • n 1 to 4.
  • r is: O or 1 ;
  • s is: O or 1;
  • X is: O or S
  • R1 and R2 are independently: a) H, or b) (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • (Cl-C6)-alkoxy vinyl, cyano, oxo, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR , NRlR 2 , NRlcOCl-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • n) -(Cl-C6)-alkyl, alkyl as defined above, o) -(C2-C6)-alkenyl, alkenyl as defined above, or p) CRl R , such that one and only one of Rl and R can be hydrogen.
  • An embodiment of this embodiment are the compounds of structural Formula I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:
  • C6-alkyl, CONR1R2, NRlR , NRlCOCl-C6-alkyl and any two of adjacent substituents can be joined to form a 5-, 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or
  • heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • a is: a single bond
  • b is: a double bond
  • n 1 to 4;
  • n 1 to 4.
  • r is: 0 or 1;
  • s is: 0 or 1;
  • X is: O or S
  • R! and R2 are independently: a) H, or b) (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • (Cl-C6)-alkoxy vinyl, cyano, oxo, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR 2 , N lR2, NRlCOCl-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • R 3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; b) -(Cl -C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I,
  • n) -(Cl-C6)-alkyl, alkyl as defined above, o) -(C2-C6)-alkenyl, alkenyl as defined above, or p) CRl R 2 , such that one and only one of R! and R 2 can be hydrogen.
  • An embodiment of the third embodiment are the compounds of structural Formula I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:
  • C6-alkyl, CONR ⁇ R 2 , NRlR 2 , NRlCOCl-C6-alkyl and any two of adjacent substituents can be joined to form a 5-, 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or
  • heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I,
  • Formula I or pharmaceutically acceptable salt, crystal form or hydrate such as:
  • alkyl includes those alkyl groups of a designated number of carbon atoms of either a straight, branched, or cyclic configuration.
  • alkyl include methyl, ethyl, propyl, isopropyl, butyl, sec-and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like.
  • Alkoxy represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge, such as methoxy, ethoxy, propoxy, butoxy and pentoxy.
  • alkanoyl is intended to include those alkylcarbonyl groups of specified number of carbon atoms, which are exemplified by formyl, acetyl, propanoyl and butyryl;
  • alkanoyloxy is intended to include those alkylcarbonyl groups of specified number of carbon atoms attached through an oxygen bridge, which are exemplified by formyloxy, acetoxy, propionoyloxy, and butyryloxy.
  • Alkenyl is intended to include hydrocarbon chains of a specified number of carbon atoms of either a straight- or branched- configuration and at least one unsaturation, which may occur at any point along the chain, such as ethenyl, propenyl, butenyl, pentenyl, dimethyl pentenyl, and the like, and includes E and Z forms, where applicable.
  • Hydrogen as used herein, means fluoro, chloro, bromo and iodo.
  • aryl is defined as a phenyl or naphthyl ring which is optionally substituted with the substituents listed above at any available carbon atoms.
  • the aryl may also be substituted with a fused 5-, 6-, or 7-membered ring containing one or two oxygens and the remaining ring atoms being carbon, the fused 5-, 6-, or 7-ring being selected from the group consisting of: dioxolanyl, dihydrofuranyl, dihydropyranyl, and dioxanyl.
  • heteroaryl as utilized herein is intended to include the following a 5 or 6-membered ring substituted with one or two heteroatoms selected from O, S, N, and is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl -C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONR R 2 , NRlR 2 , NR!COCl-C6-alkyl, any two adjacent substituents can be joined to form a 5-, 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring.
  • Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, and pyrrolyl which are substituted or unsubstituted as defined above.
  • the heteroaryl group may be optionally substituted with the substituents listed above at any available carbon atom or nitrogen atom (if present), but compounds bearing certain substitutents, directly substituted to a nitrogen may be relatively unstable and are not preferred.
  • the heteroaryl may also be fused to a second 5-, 6-, or 7-membered ring containing one or two oxygens selected from the remaining ring atoms being carbon, selected from the group consisting of: dioxolanyl, dihydrofuranyl, dihydropyranyl, and dioxanyl.
  • Pharmaceutically acceptable salts include both the metallic (inorganic) salts and organic salts; a list of which is given in Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985). It is well known to one skilled in the art that an appropriate salt form is chosen based on physical and chemical stability, flowability, hydro-scopicity and solubility.
  • pharmaceutically acceptable salts include, but are not limited to salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate or salts of an organic acid such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate or palmoate, salicylate and stearate.
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium (especially ammonium salts with secondary amines).
  • Preferred salts of this invention for the reasons cited above include potassium, sodium, calcium and ammonium salts.
  • crystal forms, hydrates and solvates of the compounds of Formula I As seen in Reaction Scheme A, compound la
  • the lactone group can be converted to a tetrahydrofuran (THF) analog in two steps.
  • the lactone is first ozonized under standard conditions. Reductive workup with an agent such as sodium borohydride gives the lactol intermediate.
  • the purified lactol intermediate is then reacted with triethylsilane and a Lewis acid such as borontrifluoride diethyl etherate to give the tetrahydrofuran (THF) analog.
  • the lactol hydroxy group can be replaced as demonstrated in Scheme C with a variety of substituents (reprsented as R ⁇ ).
  • DAST diethylaminosulfur trifluoride
  • a basic solvent such as collidine
  • the lactol hydroxy group is oxidized to its lactone derivative by use of a variety of reagents.
  • One that is particularly effective is pyridinium chlorochromate (PCC).
  • Compound la can be reacted with ozone under conditions described in Reaction Scheme B. These conditions also cleave the C20 olefin to give the keto lactol intermediate. This intermediate can be worked up under conditions described in Reaction Schemes B or C to give C20 hydroxy THF analogs. The C20 hydroxy derivative can then be oxidized to the C20 ketone under a variety of conditions.
  • the Jones reagent chromic acid and sulfuric acid in H2 ⁇ ), pyridinium chlorochromate, or oxalyl chloride plus DMSO will achieve this conversion.
  • the lactone group can be rearranged to substituted THF ethers under acidic or basic conditions.
  • Stirring the lactone derivative in an alcoholic solvent such as methanol or ethanol with an acid such as H2SO4 or a base such as potassium carbonate (K2CO3) gives the rearranged THF ester with the ester group corresponding to the alcohol selected as solvent.
  • This reaction also causes the hydrolysis of the C4 acetate group which is reattached by stirring the hydroxy analog with acetic anhydride (Ac2 ⁇ ).
  • R5a_acetadehyde THF derivatives can be prepared by reacting the lactone starting material with reducing agents such as NaBH4 or LiEt3BH (Super hydride) in an alcoholic solvent.
  • reducing agents such as NaBH4 or LiEt3BH (Super hydride) in an alcoholic solvent.
  • R ⁇ a.ketone THF derivatives are prepared by reacting the lactone starting material with alkylmetal reagents such as methyl lithium in an aprotic solvent such as THF preferably at low temperatures such as -78°C.
  • R ⁇ ester, aldehyde and ketone groups of these THF analogs can be derivatized with a variety of procedures commonly known to practitioners.
  • the C20 olefin can be reduced to the methyl derivative by procedures described in Reaction Scheme A. It can also be oxidatively cleaved by reaction with osmium tetroxide (O3O4), preferably under catalytic conditions to give the diol. The diol is then cleaned with sodium meter periodate (NaI ⁇ 4) to give the ketone.
  • O3O4 osmium tetroxide
  • NaI ⁇ 4 sodium meter periodate
  • Tetrahydrofuran derivatives can be selectively de-acetylated at C4 to give the corresponding alcohol by reaction with an aqueous solution of HCl (preferably 2M to 3M concentration) in THF at 45°C.
  • De-acetylation can also be achieved by reacting with CH3(C1)A1[N(0CH3)CH3 (Weinreb reagent) in inert solvents such as THF, toluene or methylene chloride.
  • the C4 acetate can be selectively removed using HCl in THF.
  • the C4 hydroxy group can be oxidized to the corresponding ketone by a variety of oxidizing agents.
  • the Jones reagent chromic acid and sulfuric acid in H2O
  • pyridinium chlorochromate pyridinium chlorochromate
  • oxalyl chloride plus DMSO all will achieve this conversion.
  • the C4 hydroxy group can also be dehydrated to give the olefin. Reaction of the alcohol with tris- phenoxymethylphosphonium iodide in hexamethylphosphorous triamide (HMPT) at 75°C will achieve this conversion. REACTION SCHEME H
  • esters at C4 can be prepared by reaction of a preformed carboxylic acid chloride with the C4 alcohol derivative (Reaction Scheme F) in a basic solvent such as pyridine.
  • the acid chlorides when not purchased, are prepared by stirring the carboxylic acids in reagents such as oxalyl chloride or thionyl chloride.
  • Esters may also be prepared by reaction of the acid chloride and C4 alcohol with silver cyanide (AgCN) in an aprotic solvent such as HMPA.
  • AgCN silver cyanide
  • C4 sulfonate derivatives are prepared in a similar manner by reaction with sulfonyl chlorides.
  • C4 carbonate and carbamate derivatives are prepared by first reacting the C4 alcohol derivative with carbonyldiimidazole (CDI) to obtain the imidazolecarbonyl intermediate which is then reacted with an alcohol or amine (RlR ⁇ NH) to give the corresponding carbonate or carbamate derivatives.
  • CDI carbonyldiimidazole
  • RlR ⁇ NH an alcohol or amine
  • C4 ether derivatives can also be prepared.
  • the best procedure involves reacting an alcohol with trifluoromethanesulfonic anhydride (Tf2 ⁇ , triflic anhydride) in the presence of a hindered base such as 2,6-di-t-butyl pyridine to obtain the preformed triflate in dichloromethane at reduced temperature, preferably -78°C.
  • a hindered base such as 2,6-di-t-butyl pyridine
  • trite ⁇ ene alcohol the reaction mixture is warmed to room temperature and stirring is continued until reaction is complete.
  • Ethers may also be prepared by heating a mixture of trite ⁇ ene C4 alcohol, the appropriate alkylhalide and an excess of silver oxide (Ag2 ⁇ ) in an aprotic inert solvent such as DMF.
  • Amines at C4 can be prepared from the C4 ketone described in Reaction Scheme G by reaction with an amine NHRlR 2 in a variety of solvents with a reducing agent such as sodium cyanoborohydride.
  • THF derivatives containing a C20(29) olefin can be selectively hydrogenated across this double bond to give the methyl analog by procedures described in Reaction Scheme A.
  • THF analogs When THF analogs contain a C20 olefin, it can be selectively converted to the corresponding ketone by sequential reaction with osmium tetroxide or ruthenium tetroxide and sodium periodate.
  • the C20 ketone can be conveniently reduced to its corresponding alcohol by using tetramethylammonium triacetoxyborohydride in a solvent such as THF at room temperature.
  • a solvent such as THF at room temperature.
  • the C20 keto-THF and C20-hydroxy-THF derivatives can also be prepared by methods described in Reaction Scheme D.
  • the C20-29 olefin can be converted to the C20 hydroxymethyl derivative.
  • One procedure involves reaction with diborane in THF followed by oxidative workup with tri-N- methylamine-N-oxide (Me3NO).
  • the C20,C29 bis-alcohol can also be prepared by use of catalytic osmium tetroxide followed by oxidative workup with a reagent such as Me3NO.
  • the C20 ketone derivative can be reacted to produce olefins with a variety of well known olefination agents.
  • a particularly useful reagent is the Homer-Emmons-type of olefination reagent [(RO)2P(0)CR'R"]. This produces a mixture of geometric isomers.
  • the C20 ketone can be reacted with nucleophiles (R29a'-M+) to give C20 substituted hydroxy derivatives.
  • nucleophiles R29a'-M+
  • Grignard reagents R2 a']yigBr
  • alkyllithium reagents R ⁇ 9a j
  • aprotic solvents such as diethyl ether or THF.
  • Amines at C20 can be prepared from the C20 ketone by reaction with an amine R 1 R ⁇ NH in a variety of solvents with a reducing agent such as sodium cyanoborohydride.
  • the C20 hydroxy derivative (Reaction Scheme I) or the C20 hydroxymethyl derivative (Reaction Scheme J) can be converted to ether, ester, carbonate, carbamate, sulfonate and other related derivatives by procedures commonly practiced in the art and as described in Reaction Scheme I.
  • the C20 hydroxymethyl derivative may also be derivatized as a methanesulfonate ester or triflate ester by standard procedures.
  • the methansulfinate or triflate can then be reacted with an amine NR1R 2 H to give amine derivatives.
  • the present invention is related to compounds of formula I, including but not limited to those specified in the examples, which are useful in a mammalian subject for the treatment and prevention of immunemediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum ***, limb, muscle, nervus, duodenum, small-bowel, pancreatic-islet-cell, including xeno transplants, etc.; graft-versus-host diseases brought about by medulla ossium transplantation; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenile- onset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, and the like;
  • Further uses may include the treatment and prophylaxis of inflammatory and hyperproHferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases (autoimmune and otherwise) such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis
  • renal diseases such as interstitial nephritis, Good- pasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy
  • nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy
  • endocrine diseases such as hyperthyroidism and Basedow's disease
  • hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia
  • bone diseases such as osteoporosis
  • respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia
  • skin disease such as dermatomyositis, leukoderma vulgaris, ichthy
  • the compounds of the invention are useful for the treatment and prevention of hepatic disease such as immunogenic diseases (for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g., chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g.
  • immunogenic diseases for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis
  • partial liver resection e.g.
  • necrosis caused by toxin, viral hepatitis, shock, or anoxia B-virus hepatitis, non-A/non-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and "acute-on- chronic" liver failure (acute liver failure on chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, particularly HCMV infection, and antiinf ammatory activity; and
  • the compounds of the present invention may also be used in the treatment of immunodepression or a disorder involving immunodepression, such as AIDS, cancer, senile dementia, trauma (including wound healing, surgery and shock) chronic bacterial infection, and certain central nervous system disorders.
  • Further uses may include the treatment and prophylaxis of inflammatory and hyperproHferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases
  • autoimmune and otherwise such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.; pollen allergies, reversible obstructive airway disease, which includes condition such as asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyper-responsiveness), bronchitis and the like; inflammation of mucous and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory
  • renal diseases such as interstitial nephritis, Good- pasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy
  • nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy
  • endocrine diseases such as hyperthyroidism and Basedow's disease
  • hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia
  • bone diseases such as osteoporosis
  • respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia
  • skin disease such as dermatomyositis, leukoderma vulgaris, ichthy
  • organ injury such as ischemia- reperfusion injury of organs (such as heart, liver, kidney and digestive tract) which occurs upon preservation, transplantation or ischemic disease (for example, thrombosis and cardiac infraction): intestinal diseases such as endotoxin-shock, pseudomembranous colitis and colitis caused by drug or radiation; renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency; pulmonary diseases such as toxinosis caused by lung-oxygen or drug (for example, paracort and bleomycins), lung cancer and pulmonary emphysema; ocular diseases such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring and corneal alkali bum; dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement
  • the compounds of the invention are useful for the treatment and prevention of hepatic disease such as immunogenic diseases (for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g., chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g.
  • immunogenic diseases for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis
  • partial liver resection e.g.
  • necrosis caused by toxin, viral hepatitis, shock, or anoxia B-virus hepatitis, non-A/non-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and "acute-on- chronic" liver failure (acute liver failure on chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, particularly HCMV infection, and antiinflammatory activity; and immunodepression or a disorder involving immunodepression, such as AIDS, cancer, senile dementia, trauma (including wound healing, surgery and shock), chronic bacterial infection, and certain central nervous system disorders.
  • An embodiment of the invention is a method for the treatment of autoimmune diseases.
  • Another embodiment of the invention is a method for the prevention of rejection of foreign organ transplants comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula I.
  • autoimmune or a rejection process tissue destruction caused by inflammatory cells and the mediators they release.
  • Anti-inflammatory agents such as NSAID's and corticosteroids act principally by blocking the effect or secretion of these mediators, but do nothing to modify the immunologic basis of the disease.
  • cytotoxic agents such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.
  • Cyclosporin A which was approved by the US FDA in 1983, is currently the leading drug used to prevent rejection of transplanted organs. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. Though cyclosporin A is effective in fighting transplant rejection, it is nephrotoxic and is known to cause several undesirable side effects including kidney failure, abnormal liver function and gastrointestinal discomfort.
  • the present invention provides for immunosuppressant agents which are inhibitors of a voltage dependent potassium channel, K ⁇ l .3, that is found on human
  • Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K + channels, in their role as modulators of the plasma membrane potential in human T -lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In regulating membrane potential, K+ channels play a role in the regulation of intracellular Ca ++ homeostasis, which has been found to be important in T-cell activation. The biochemical characterization of K+ channels is underdeveloped, due to the paucity of selective high affinity probes.
  • Functional voltage-gated K + channels can exist as multimeric structures formed by the association of either identical or dissimilar subunits. This phenomena is thought to account for the wide diversity of K+ channels. However, subunit compositions of native K+ channels and the physiologic role that particular channels play are, in most cases, still unclear.
  • the Kvl .3 channel is a voltage-gated potassium channel that is found in neurons, blood cells, osteoclasts and T-lymphocytes.
  • the Chandy and Cahalan laboratories proposed a hypothesis that blocking the K ⁇ l .3 channel would elicit an immunosuppressant response. (Chandy et al., J. Exp. Med. 160, 369, 1984; Decoursey et al., Nature, 307, 465, 1984).
  • the K+ channel blockers employed in their studies were non-selective. Until research with the peptide margatoxin, a peptide found in scorpion venom, no specific inhibitor of the Kvl.3 channel existed to test this hypothesis.
  • Margatoxin blocks only K v 1.3 in T-cells, and has immunosuppressant activity in both in vitro and in vivo models. (Lin et al., J. Exp. Med, 111, 637, 1993). Since the compounds of the embodiments of this invention produce blockade of
  • Also within the scope of this invention is a method of treating a condition in a mammal, the treatment of which is effected or facilitated by K v 1.3 inhibition, comprising the administration of a pharmaceutical composition comprising a suitable pharmaceutical carrier and a compound of Formula (I), in an amount that is effective at inhibiting Kvl.3.
  • a combination therapy comprising a compound of formula I and one or more immunosuppressant agents.
  • immunosuppressant agents within the scope of this invention include, but are not limited to, IMUREK® azathioprine sodium, brequinar sodium, SPANIDIN® gusperimus trihydrochloride (also known as deoxyspergualin), mizoribine (also known as bredinin), CELLCEPT® mycophenolate mofetil, NEORAL® Cyclosporin A (also marketed as different formulation of Cyclosporin A under the trademark SANDIMMUNE®), PROGRAF® tacrolimus (also known as FK-506) and RAPIMMUNE® sirolimus (also known as rapamycin), leflunomide (also known as HWA-486), glucocortcoids, such as prednisolone and its derivatives, antibody therapies such as orthoclone (OKT3) and Zenapax and antithymyocyte globulins, such as
  • MNC Peripheral blood mononuclear
  • LSM ficoll-hypaque
  • SRBC neuraminidase treated sheep red blood cells
  • the cell suspension was immediately distributed into 96 well round-bottom microculture plates (Costar) at 200 ⁇ l/well. The various dilutions of test compound were then added in triplicate wells at 25 ⁇ l/well, incubated for 30 min at 37°C. lonomycin (125 ng/ml), and PMA (1 or 5 ng/ml), were added to the appropriate wells. The culture plates were then incubated at 37°C in a humidified atmosphere of 5% C02 - 95% air for 18-24 hours.
  • the supernatants were removed, and assayed for IL-2 with an IL-2 capture ELISA, using monoclonal anti-IL-2, and biotinylated goat anti-IL-2 antibodies (unconjugated antibodies purchased from R&D System, Minneapolis, MN).
  • the ELISA was developed with streptavidin conjugated peroxidase (Zymed, San Francisco, CA) and substrate for peroxidase (Sigma). Mean OD and units of IL-2 of the replicate wells were calculated from standard curve, created with recombinant IL-2 (Collaborative Biomedical Products, Bedford, MA) and the results were expressed as concentration of compound required to inhibit IL-2 production of T cells by 50%.
  • MNC Peripheral blood mononuclear cells
  • LSM Organon Teknika, Durham, NC
  • complete media RPMI 1640 medium with 5% fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, and 1 % penn-strep, obtained from GIBCO, Grand Island, NY
  • the sheep red blood cells (SRBC) of these rosetted T cells were then lysed with ammonium chloride lysing buffer (GEBCO, Grand Island, NY). After washing 2X with complete media, these purified T cells were also resuspended at 2-2.5 x lO ⁇ cells/ml in complete media. The various dilutions of the compound were added in triplicates at 50 ul/well of a 96 well flat-bottom microculture plate (Costar, Cambridge, MA). T cell suspension was then immediately distributed into the wells at 100 ul/well. After incubating the cells with compound for 30 min.
  • T lymphocytes were assessed by measurement of tritiated thymidine incorporation. During the last 18- 24 hrs. of culturing, the cells were pulse-labeled with 2 ⁇ Ci/well of tritiated thymidine (NEN, Cambridge, MA).
  • the cultures were harvested on glass fiber filters using a multiple sample harvester (MACH-II, Wallac,Gaithersburg, MD). Radioactivity of filter discs corresponding to individual wells was measured by standard liquid scintillation counting methods (Betaplate Scint Counter, Wallac). Mean counts per minute of replicate wells were calculated and the results were expressed as concentration of compound required to inhibit tritiated thymidine uptake of T cells by 50%.
  • CHO cells transfected with K v 1.3 channels at site densities of approximately 40,000 sites/cell are plated into 96 well culture plates and maintained in Iscove's Modified Dulbecco's Medium (IMDM, with L-Glutamine and HEPES, JRH Biosciences). Cells are incubated overnight with 86 Rb + (3 ⁇ Ci/ml, Dupont-NEN) in the glutamine supplemented IMDM.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the K ⁇ l.3 channels are opened by depolarization of the cells with High K Buffer (final concentrations, in mM, 63.25 KC1, 68.25 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES, pH adjusted to 7.2 with NaOH) also containing test compounds.
  • High K Buffer final concentrations, in mM, 63.25 KC1, 68.25 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES, pH adjusted to 7.2 with NaOH
  • To measure 86 Rb+ efflux through the channels aliquots of 100 ⁇ l are taken from each well after a given time and added to plates containing 100 ⁇ l MicroScint-40 (Packard) for counting by liquid scintillation techniques. MicroScint-40 (100 ⁇ l) is then added to each well of the cell plate to determine the remaining 86 Rb + activity.
  • IC50 39 amino acid peptide that is a potent blocker of K 1.3 channels
  • these compounds are useful in the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.
  • the compounds of this invention can be administered for the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses according to the invention by any means that effects contact of the active ingredient compound with the site of action in the body of a warm- blooded animal.
  • administration can be oral, topical, including transdermal, ocular, buccal, intranasal, inhalation, intravaginal, rectal, intracisternal and parenteral.
  • parenteral refers to modes of administration which include subcutaneous, intravenous, intramuscular, intraarticular injection or infusion, intrasternal and intraperitoneal.
  • the compounds can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism and includes mammals and birds.
  • the dosage administered will be dependent on the age, health and weight of the recipient, the extent of disease, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • a daily dosage of active ingredient compound will be from about 1 -500 milligrams per day. Ordinarily, from 10 to 100 milligrams per day in one or more applications is effective to obtain desired results.
  • These dosages are the effective amounts for the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, troches, dragees, granules and powders, or in liquid dosage forms, such as elixirs, syrups, emulsions, dispersions, and suspensions.
  • the active ingredient can also be administered parenterally, in sterile liquid dosage forms, such as dispersions, suspensions or solutions.
  • dosages forms that can also be used to administer the active ingredient as an ointment, cream, drops, transdermal patch or powder for topical administration, as an ophthalmic solution or suspension formation, i.e., eye drops, for ocular administration, as an aerosol spray or powder composition for inhalation or intranasal administration, or as a cream, ointment, spray or suppository for rectal or vaginal administration.
  • Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water a suitable oil, saline, aqueous dextrose
  • sugar and related sugar solutions and glycols such as propylene glycol or polyethylene gycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are also used.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propylparaben, and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • the compounds of the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers.
  • the compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device.
  • the preferred delivery system for inhalation is a metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons.
  • MDI metered dose inhalation
  • an ophthalmic preparation may be formulated with an appropriate weight percent solution or suspension of the compounds of Formula I in an appropriate ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye.
  • Useful pharmaceutical dosage-forms for administration of the compounds of this invention can be illustrated as follows:
  • a large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.
  • a mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient.
  • the capsules are washed and dried.
  • a large number of tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose.
  • Appropriate coatings may be applied to increase palatability or delay absorption.
  • a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol. The solution is made to volume with water for injection and sterilized.
  • An aqueous suspension is prepared for oral administration so that each 5 milliliters contain 100 milligrams of finely divided active ingredient, 100 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.
  • the same dosage forms can generally be used when the compounds of this invention are administered stepwise or in conjunction with another therapeutic agent.
  • the dosage form and administration route should be selected depending on the compatibility of the combined drugs.
  • coadministration is understood to include the administration of the two agents concomitantly or sequentially, or alternatively as a fixed dose combination of the two active components.
  • the bioactive methylene chloride extract was dried down to give 12 mg of residue. This was first fractionated by preparative thin layer chromatography (TLC) on a 20 cm by 20 cm E. Merck silica gel 60F254 plate of 1mm thickness using methylene chloride-ethyl acetate 1 : 1 (v/v) as solvent, then by high performance liquid chromatography (HPLC) using a Zorbax RxC8 4.6 mm x 25 cm column, operated at 50°C and eluted with a 50 minute gradient of acetonitrile: water (1 :1 , v/v) to 100% acetonitrile, delivered at 1 ml/min, to afford 4 mg of compound 1(a) and 1 mg of 1(b).
  • TLC preparative thin layer chromatography
  • HPLC high performance liquid chromatography
  • the IH NMR spectra of compound of Formula(a) is provided as Figure 1.
  • the spectra was recorded at 400 MHz in CD2CI2 on a Varian Unity 400 NMR spectrometer at 25°C. Chemical shifts are in ppm relative to TMS at zero ppm using the solvent peak at 55.32 as the internal standard.
  • the carbon count of 40 is in agreement with the molecular formula C40H50OI6 derived by scanning HR EI-MS.
  • Analogs of the compounds of Formula 1 (a) and 1 (b) could be detected in the crude extract and fractions thereof when the process of Example 1 was carried out on a larger scale.
  • 50 g of ethanol extract were partitioned as described in Example 1 using 900 ml of each solvent at each step.
  • Partial purification of the methylene chloride extract was achieved by column chromatography on E. Merck silica gel 60 (120 ml), eluting with a step gradient of ethyl acetate in methylene chloride.
  • the step gradient was designed so that the column was washed first with 100% methylene chloride and then with methylene chloride- ethyl acetate mixtures of 9:1 , 8:2, 3:2, 2:1 , 1 :1 , 1 :2, 2:8 and 1 :9.
  • the column was washed with 100% ethyl acetate.
  • Fractions eluted with methylene chloride-ethyl acetate 3:2 were enriched in compound of Formula 1(a) and 1 (b).
  • the 13c NMR spectra obtained for the compound of Formula 1 (c) using the conditions previously described is as follows: 15.1 (2x), 16.9, 19.8, 20.8, 20.91, 20.94, 21.9, 22.3, 35.6, 40.6, 42.2, 43.9, 45.0, 47.7, 50.8, 53.5, 55.6, 61.8, 63.5, 66.0, 67.6 (2x), 69.8, 70.0, 73.9, 75.0, 75.6, 119.3, 123.7, 139.0, 144.4, 167.8, 169.2, 169.5, 170.1 , 170.4, 171.4 ppm.
  • the carbon count of 38 is in agreement with the molecular formula C38H50O16 derived by scanning HR EI-MS.
  • a simplified purification process allows for rapid fractionation of even larger amounts of crude extract and the preparation of gram amounts of the compounds of Formula 1 (a) and 1(b).
  • the ethanol extract is first dissolved at 20 grams per
  • Volume of elution for the compound of Formula 1(a) ranges from about 2 to about 3.5 column volumes of solvent; that for the compound of Formula 1(b) is about 3 to about 4.5 column volumes.
  • the reaction vessel was then pressurized with H2 to 15 psi (latm) and shaken for 65h at 25 °C. After that time the solvent was removed under reduced pressure. The residue was dissolved in a small amount of ethyl acetate/hexanes (2:1) (ca. 1 mL) and filtered through 30g of silica gel eluting with 500 ml of ethyl acetate / hexanes (2: 1). The first fractions, containing the Wilkinson-catalyst (approx. 50 mL) were discarded. The fractions containing the product were combined.
  • Step A 4,6,7, 15, 16-pentakis(acetyloxy)-21 ,22-epoxy- 1 ,18- dihydroxy-22-methoxycarbonyl[6 ,7 , 15 ⁇ , 16 ⁇ ,21 ⁇ ,22 ⁇ ] ⁇
  • Step B 4,6,7, 15, 16-pentakis(acetyloxy)-21 ,22-epoxy- 18-hydroxy- 22-methoxycarbonyl[6 ,7oc, 15 ⁇ , 16 ⁇ ,21 ⁇ ,22 ⁇ ]D: A-Friedo- 2.3.27.30-tetranor-24-oxaoleanane
  • Step A 4,6,7, 15,16-pentakis(acetyloxy)-21 ,22-epoxy-l ,18,20-tris- hydroxy-22-methoxycarbonyl[6 ,7oc,15 ⁇ ,16 ⁇ ,21 ⁇ ,22 ⁇ ]-
  • D A-Friedo-2,3,27,30-tetranor-24-oxaoleanane
  • Step B 4,6,7, 15, 16-pentakis(acetyloxy)-21 ,22-epoxy- 18,20- dmydroxy-22-methoxycarbonyl[6 ⁇ ,7 ⁇ ,15 ⁇ ,16 ⁇ ,21 ⁇ ,22 ⁇ ]- D:A-Friedo-2.3.27.30-tetranor-24-oxaoleanane
  • Step C 4,6,7, 15, 16-pentakis(acetyloxy)-21 ,22-epoxy- 18-hydroxy- 20-oxo-22-methoxycarbonyl[6 ⁇ ,7 ⁇ ,15 ⁇ ,16 ⁇ ,21 ⁇ ,22 ⁇ ]D:A- Friedo-2.3.27.30-tetranor-24-oxaoleanane
  • reaction mixture was stirred for 3 hr whereupon it was quenched with 10 ml of sat. sodium bicarbonate solution.
  • the mixture was extracted with dichloromethane and the combined organic fractions were washed with brine, dried over sodium sulfate, filtered through silica gel and evaporated in vacuo.
  • the product was purified by HPLC (Waters RCM, Prep Nova-Pak HR Silica, 2 25X100 mm cartridges) to give 48.5 mg (94%) of the title compound.

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Abstract

L'invention a pour objet des composés représentés par la formule (I), qui sont utilisés comme agents immunosuppresseurs.
PCT/US1997/018334 1996-10-16 1997-10-10 Derives de triterpene presentant une activite immunosuppressive WO1998016531A1 (fr)

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EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection

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Publication number Priority date Publication date Assignee Title
US5599950A (en) * 1994-04-22 1997-02-04 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S) Preparation process of ginkgolide B from ginkgolide C

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599950A (en) * 1994-04-22 1997-02-04 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S) Preparation process of ginkgolide B from ginkgolide C

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection

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