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WO1998016532A1 - Derives de triterpene a action immunosuppressive - Google Patents

Derives de triterpene a action immunosuppressive Download PDF

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Publication number
WO1998016532A1
WO1998016532A1 PCT/US1997/018651 US9718651W WO9816532A1 WO 1998016532 A1 WO1998016532 A1 WO 1998016532A1 US 9718651 W US9718651 W US 9718651W WO 9816532 A1 WO9816532 A1 WO 9816532A1
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WIPO (PCT)
Prior art keywords
alkyl
defined above
hydroxy
heteroaryl
aryl
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PCT/US1997/018651
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English (en)
Inventor
Robert K. Baker
Frank Kayser
Jianming Bao
Andrew Kotliar
William H. Parsons
Kathleen M. Rupprecht
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Merck & Co., Inc.
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Priority claimed from GBGB9626239.9A external-priority patent/GB9626239D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU48225/97A priority Critical patent/AU4822597A/en
Publication of WO1998016532A1 publication Critical patent/WO1998016532A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • 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
    • 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
    • A61K31/33—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
    • A61K31/70—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.
  • 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 hyperproliferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoi
  • a is: a single bond, or a double bond when R4 is absent;
  • b is: a single bond, or a double bond
  • R! and R ⁇ are independently: a) H, or b) (C ⁇ -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, CO2H, COCl -C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONR1R2, NR*R2, NR ⁇ COC ⁇ -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, COCi-C6
  • R ⁇ 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, (Cl -C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl -C6-alkyl, C02Cl-C6-alkyl, CONR ⁇ R 2 , NR ⁇ R 2 , NRlCOCl-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(Cl-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl
  • a is: a single bond
  • b is: a single bond
  • n 1 to 4.
  • R! 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, CO2H, COCl-C6-alkyl, C02Cl-C6-alkyl, CONRlR 2 , NRIR 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, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC] -
  • R3 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, (Cl -C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl -C6-alkyl, C02Cl-C6-alkyl, CONRLR 2 , NRIR 2 , NRlCOCl-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(Cl-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl -C6)-al
  • An embodiment of this embodiment are the compounds of structural Formula I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:
  • 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, (Cl -C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, C02Cl-C6-alkyl, CONRLR 2 , NR*R 2 , NR ⁇ COCl-C ⁇ -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.
  • 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 ;
  • R! 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, C02H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR 2 , NR IR 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, (Cl -C6)-alkoxy, phenyl, phenoxy, cyano, oxo, nitro, hydroxy, CHO, C
  • R 2 , NR ! COC 1 -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, heteroaryl, wherein 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, (Cl-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR 2 , NR1R 2 , NRiCOCl-C ⁇ -alkyl, any two adjacent substituents can be joined to form a 5-, 6- or 7-membered fused ring said ring
  • R 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, (Cl -C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C02Cl-C6-alkyl, CONRlR 2 , NRIR 2 , NRlCOCl-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(Cl -C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl -C6)-
  • An embodiment of the second embodiment are the compounds of structural Formula I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:
  • 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, (Cl -C6)-alkoxy, cyano, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C02Cl-C6-alkyl, CONRlR 2 NRIR 2 , NRlCOCl-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.
  • a third embodiment of this invention are the compounds of Formula I or pharmaceutically acceptable salts, crystal forms or hydrates such as
  • the compounds of the present invention have asymmetric centers and this invention includes all of the optical isomers and mixtures thereof.
  • compounds with carbon-carbon double bonds may occur in Z- and E- forms with all isomeric forms of the compounds being included in the present invention.
  • 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.
  • 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, nitro, hydroxy, CHO, C02H, COCl-C6-alkyl, C ⁇ 2Cl-C6-alkyl, CONRlR 2 , NRIR 2 , NRlCOCl-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. Also included within the scope of this invention are crystal forms, hydrates and solvates of the compounds of Formula I. As seen in Reaction Scheme A, compound la [(4, 6, 7, 15,16- pentakis(acetyloxy)-21 ,22-epoxy-1 -hydroxy-22-methyoxycarbonyl- [6oc,7 , 15 ⁇ , 16 ⁇ ,21 ⁇ ,22 ⁇ ]D:A-Freido-A-homo-27,30-dinor-24- oxaoleana-1 , 20(29)-dien-3-one] can be converted to a tetrahydrofuran (THF) analog in two steps.
  • THF tetrahydrofuran
  • the lactone is first ozonized under standard conditions.
  • the C20(29) olefin is also oxidized to the C20 ketone.
  • Reductive workup with an agent such as sodium borohydride gives the lactol-C20-hydroxy intermediate.
  • the purified lactol intermediate is then reacted with triethylsilane and a Lewis acid such as borontrifiuoride diethyl etherate to give the tetrahydrofuran (THF) analog.
  • THF tetrahydrofuran
  • the C20 hydroxy derivative can be converted back to the C20(29) olefin derivative by first oxidizing the hydroxy group to a C20 ketone.
  • a variety of reagents will achieve this including the Jones reagent (Cr ⁇ 3, H2SO4, H2 ⁇ ) in a solvent such as acetone.
  • the lactol hydroxy group from the lactol-C20-hydroxy derivative of Reaction Scheme A can be replaced with a variety of substituents (R ⁇ a and Rb).
  • DAST diethylaminosulfur trifluoride
  • a basic solvent such as collidine
  • F fluoro analog
  • trisubstituted aluminum reagents such as triethyl aluminum
  • the lactol-C20-hydroxy derivative can be oxidized to its ketolactone derivative by use of a variety of reagents.
  • One that is particularly effective is pyridinium chlorochromate (PCC) or the Jones reagent.
  • PCC pyridinium chlorochromate
  • the C20 keto group can then be converted to the C20(29) olefin by procedures described in Reaction Scheme B.
  • 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 ⁇ ).
  • the lactone can also be reacted with dilute acid such as 2N HCl in THF to give the carboxylic acid derivative which can be derivatized to give amides, esters and other analogs.
  • R ⁇ a -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.
  • alkylmetal reagents such as methyl lithium
  • THF aprotic solvent
  • the R ⁇ a carboxylic acid, ester, aldehyde and ketone groups of these THF analogs can be derivatized with a variety of procedures commonly known to practitioners.
  • 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 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 H2 ⁇
  • 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.
  • HMPT hexamethylphosphorous triamide
  • esters at C4 can be prepared by reaction of a preformed carboxylic acid chloride with the C4 alcohol derivative (Reaction Scheme E) 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 2 NH) to give the corresponding carbonate or carbamate derivatives.
  • CDI carbonyldiimidazole
  • RlR 2 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 2,6-di-t- butylpyridine to obtain the preformed triflate in dichloromethane at reduced temperature, preferably -78°C.
  • Tf2 ⁇ trifluoromethanesulfonic anhydride
  • 2,6-di-t- butylpyridine 2,6-di-t- butylpyridine
  • 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 NHR 1 R 2 in a variety of solvents with a reducing agent such as sodium cyanoborohydride.
  • 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 hype ⁇ roliferative 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, he ⁇ etic keratitis, conical cornea, dystrophia epitheli
  • 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 pu ⁇ ura, 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 antiinflammatory 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.
  • 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 hype ⁇ roliferative 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 epitheliali
  • 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 pu ⁇ ura, 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 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 T- lymphocytes.
  • 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.
  • K + channels 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 K v 1.3 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 1.3 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 sco ⁇ ion venom, no specific inhibitor of the Kv l .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, 11, 637, 1993). Since the compounds of the embodiments of this invention produce blockade of K v 1.3, they will also inhibit T-cell activation.
  • 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 K v 1.3.
  • 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 a 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,
  • 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 resetted T cells were then lysed with ammonium chloride lysing buffer (GIBCO, 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 inco ⁇ oration. 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 Kyi.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 (glucose), 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: CAPSULES
  • 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, 1 1 milligrams of starch and 98.8 milligrams of lactose.
  • Appropriate coatings may be applied to increase palatability or delay abso ⁇ tion.
  • 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.
  • TLC systems such as E. Merck silica gel 60F254, methylene chloride- ethyl acetate 1 :1 , Rf 1(a) 0.4, Rf 1 (b) 0.3; Whatman KCl 8, methanol- water 9: 1 , Rf 1(a) 0.65, Rf 1(b) 0.75 and by HPLC using a Zorbax RxC8 column, acetonitrile-water 3:2, k' 1 (a) 4.15, k' 1(b) 3.30; and by NMR.
  • Mass spectra were recorded on JEOL SX-102A (electron impact, EI,903V) and JEOL HX1 10 (Fast Atom Bombardment, FAB) mass spectrometers. Exact mass measurements were performed at high resolution (HR-EI) using perfluorokerosene (PFK) as the internal standard. Trimethyisilyl derivatives were prepared with a 1 : 1 mixture of BSTFA-pyridine at room temperature The FAB spectrum was run in a matrix of dithiothreitol (20/80).
  • the compound of Formula 1(a) runs underivatized by El.
  • the molecular ion is observed a m/z 788 and three successive loses of acetic acid are observed.
  • the base peak is observed a m/z 334.
  • the compound does not silylate.
  • Scanning HR-EI indicated a molecular formula of C40H52O16. A table of the critical HR-EI data is given below.
  • 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).
  • Component 1(d) had a retention time of 10.5 min. and a molecular weight of 744 which is observed a m/z: 745 (M+H), 762 (M+NH3), 786 (M + H + MeCN).
  • Component 1(c) has a retention time of 11.8 and a molecular weight of 746 which is observed at m/z: 747 (M+H), 764 (M+NH3) and 788 (M + H + MeCN).
  • 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.
  • a simplified purification process allows for rapid fraction- ation 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.
  • Tributyl phosphine (0.07 ml, 0.25 mmole) was added dropwise to give a deep red solution. After the disappearance of the starting alcohol (TLC-control, ⁇ 2.5h), 0.5 ml of a 30% solution of hydrogen peroxide in water were added. The mixture was stirred for 14h at room temperature. The solution was diluted with 20ml dichloromethane, 10ml of 10% hydrochloric acid was added and the layers were separated. The organic layer was washed with saturated aqueous sodium chloride then was dried over MgS04 and concentrated.

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Abstract

Les composés de la formule (I) se révèlent efficaces comme agents immunosuppresseurs.
PCT/US1997/018651 1996-10-16 1997-10-10 Derives de triterpene a action immunosuppressive WO1998016532A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020267A1 (fr) * 1997-10-17 1999-04-29 Merck & Co., Inc. Furanyle, derives de triterpene tetracyclique ayant une activite immunosuppressive
US6022890A (en) * 1997-11-14 2000-02-08 Merck & Co., Inc. Immunosuppressant tetracyclic triterpenes
US6051590A (en) * 1999-05-13 2000-04-18 Merck & Co., Inc. Immunosuppressant tricyclic compounds
US6083980A (en) * 1997-10-17 2000-07-04 Merck & Co., Inc. Furanyl, tetracyclic triterpene derivatives with immunosuppressant activity
US6100293A (en) * 1997-10-17 2000-08-08 Merck & Co., Inc. Tetracyclic triterpene derivatives with immunosuppressant activity
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection

Citations (1)

* 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

Patent Citations (1)

* 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 (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020267A1 (fr) * 1997-10-17 1999-04-29 Merck & Co., Inc. Furanyle, derives de triterpene tetracyclique ayant une activite immunosuppressive
US6083980A (en) * 1997-10-17 2000-07-04 Merck & Co., Inc. Furanyl, tetracyclic triterpene derivatives with immunosuppressant activity
US6100293A (en) * 1997-10-17 2000-08-08 Merck & Co., Inc. Tetracyclic triterpene derivatives with immunosuppressant activity
US6022890A (en) * 1997-11-14 2000-02-08 Merck & Co., Inc. Immunosuppressant tetracyclic triterpenes
US6051590A (en) * 1999-05-13 2000-04-18 Merck & Co., Inc. Immunosuppressant tricyclic compounds
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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