WO1994018225A1 - 15-oxasterols utilises en tant qu'agent traitant l'hypocholesterolemie - Google Patents

15-oxasterols utilises en tant qu'agent traitant l'hypocholesterolemie Download PDF

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Publication number
WO1994018225A1
WO1994018225A1 PCT/US1994/001191 US9401191W WO9418225A1 WO 1994018225 A1 WO1994018225 A1 WO 1994018225A1 US 9401191 W US9401191 W US 9401191W WO 9418225 A1 WO9418225 A1 WO 9418225A1
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WIPO (PCT)
Prior art keywords
compound
compounds
mixture
added
solution
Prior art date
Application number
PCT/US1994/001191
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English (en)
Inventor
Soo Sung Ko
Original Assignee
The Du Pont Merck Pharmaceutical Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Du Pont Merck Pharmaceutical Company filed Critical The Du Pont Merck Pharmaceutical Company
Priority to AU61692/94A priority Critical patent/AU6169294A/en
Publication of WO1994018225A1 publication Critical patent/WO1994018225A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom

Definitions

  • This invention relates to novel 15-oxasterols, pharmaceutical compositions thereof, and methods of using said compositions in mammals to inhibit the biosynthesis of cholesterol.
  • Ko and Trzaskos International Patent Application (PCT) WO 91/13903, published on September 19, 1991, disclose a class of 15-oxasterol cholesterol biosynthesis inhibitors useful as cholesterol lowering agents.
  • the application of Ko and Trzaskos discloses 15-oxa, thia and azasterols substituted at the 14- position of the sterol nucleus with alkyl, alkenyl or alkynyl, which can themselves be substituted with oxygen, sulfur, nitrogen and other functional groups.
  • WO 91/13903 encompasses the compounds of this invention, however, the compounds of this invention are neither exemplified nor specifically claimed in WO 91/13903. Applicants have now discovered that the compounds of the present invention exhibit remarkable and unexpected potency as cholesterol biosynthesis inhibitors, and are unexpectedly superior to compounds disclosed in WO 91/13903. ⁇ F.T ⁇ TTF. ⁇ DESCRIPTION OF THF INVENTION
  • This invention provides 15-oxasterol compounds which exhibit remarkable and unexpected potency as cholesterol biosynthesis inhibitors.
  • the compounds of this «invention are 15-oxasterols of formula 1:
  • This invention also provides pharmaceutical compositions comprising compounds of formula 1.
  • this invention provides a method of treating hypercholesterolemia by orally administering a compound of formula 1.
  • terapéuticaally effective amount means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human subject that is being sought by a clinician or researcher.
  • the compounds of formula 1 are prepared using methods disclosed in Schemes 1 and 2. As shown in Scheme 1, the starting material, 4, 4-dimethyl-5a- cholesta-8, 14-dien-3b-ol (I) [Bloch and Gautschi, J.
  • the aldehyde- carrying chain was shortened by converting the aldehyde to an enamine (IV) and reacting the enamine with ozone followed by dimethylsulfide in dichloromethane at -78 °C to give a new enone-aldehyde (V) .
  • the aldehyde was treated with sodium borohydride to provide the corresponding enone- alcohol (VI) , which serves as the key intermediate for the 15-oxasterols of this invention.
  • compounds of formula 1 are then prepared by nucleophillic addition to the enone- alcohol (VI) using either a Grignard reagent or an alkyl lithium reagent in an inert solvent such as diethyl ether, tetrahydrofuran or in a mixture of inert solvents; and treating the product with an acid in an alcoholic solvent such as methanol or ethanol.
  • the primary hydroxyl group of the enone-alcohol (VI) can be masked with a suitable protecting group such as a silyl ether prior to the nucleophillic addition when necessary.
  • the compounds of Examples 2 and 3 are prepared by this method.
  • the vinyl group of (VII) can be hydrogenated to afford the corresponding ethyl compound which is the compound of the formula 1 wherein R is ethyl (Example 2), or it can be treated with ozone to yield the corresponding aldehyde (VIII) .
  • the aldehyde can then be reacted with either ethyl magnesium bromide or ethyllithium to afford the compound of formula 1 wherein R is CHOHCH2CH3 (Example 1) .
  • the compound of Example 1 compound can be prepared by reacting the aldehyde (VIII) with vinyl magnesium bromide or vinyllithium to provide Compounds IXa and IXb, and then reducing the vinyl group by catalytic hydrotjenation.
  • Step 1 Preparation of 4, 4-Dimethyl-5 ⁇ -cholest-8-ene- 3 ⁇ ,14 ⁇ ,15 ⁇ -triol (Compound II) .
  • Step 2 Preparation of 4, 4-Dimethyl-3 ⁇ -hydroxy-15-oxo- 5 ⁇ -secocholest-8-en-14-one (Compound III) .
  • Step 3 Preparation of 4, 4-Dimethyl-3 ⁇ , 16-dihydroxy- 14, l6-seco-D-nor-cholest-8-en-14-one (Compound VI) .
  • the enamine was dissolved in dichloromethane (1750 ml) and the solution was cooled to -78 °C . Ozone was 0 bubbled through the solution and reaction was followed by thin layer chromatography (TLC) using silica gel plate and a 7:93 mixture of acetone and toluene. Near the end point of the reaction was added few drops of Sudan Red 7B in dichloromethane and ozone was continued 5 to bubble through until the color become light pink.
  • TLC thin layer chromatography
  • Step 1 Preparation of 4, -Dimethyl-15-oxa-14-vinyl-5 ⁇ - cholest-8-en-3 ⁇ -ol (Compound VII) .
  • Step 2 Preparation of 3 ⁇ -Hydroxy-15-oxa-lanost-8-en-32- al (Compound VIII) .
  • Step 3 Preparation of 15-Oxa-32-vinyl-lanost-8-ene- 3 ⁇ ,32-diol (Compounds IXa and IXb) .
  • Step 4 Preparation of 32-Ethyl-15-oxa-lanost-8-ene- 3 ⁇ ,32-diol (Example 1 ) .
  • HMGR HMGR Suppression Assay
  • HepG2 cells (a human hepatoma cell line) used in the assay were obtained from the American Type
  • the cells were harvested by washing cultures with 10 ml Hank's Balanced Salt Solution (2x) and incubating with 0.125% Trypsin in Versene (0.02% EDTA) for approximately one minute. After the cells are visibly rounded and loosened from the flask, 9 ml of the above medium is added. The cells are transferred to a 100 mM culture dish and syringed up and down through a 20 gauge needle to break up aggregated clumps of cells. Cell cultures are plated at 0.1 x 10 6 /ml and 0.075 x 10 6 /ml in two 24 well plates, respectively, aliquoting 1 ml cells/well.
  • the cells are allowed to attach for 24 hours (48 hours for the lower density plate) before washing 2x with HBSS and refed with 1 ml of the above medium with 1% Cabosil delipidated serum instead of Fetal Bovine Serum.
  • the cells are treated with the test compounds after 24 hours of exposure to the delipidated serum media.
  • Chemicals are routinely prepared as alO mM solution in 100% ethanol or dimethyl sulfoxide (DMSO) . All test compounds are added at 50 mM, 25 mM, 10 mM, 1 mM at a final concentration of 0.45% solvent, 0.25% Bovine Serum Albumin (BSA) suspension per well.
  • the solvent BSA is sonicated for 10 seconds to ensure maximum solubilization before addition to the cells.
  • Control wells receive solvent/BSA at the same concentrations as the drug treated cultures. After incubation for one hour at 37 °C, 5% CO2, 20 mCi/ml 3 H-MVAL is added per well in ethanol/medium so the final concentration of solvent is 1.2%. After 22 hours of incubation with the test compounds 2.5 mCi of 14 C-acetate is added per well for an additional 2 hours so that the final concentration of ethanol is 1.6%. Two known cholesterol biosynthesis inhibitors are incubated with each assay, namely 25-hydroxycholesterol and Lovastatin, to determine the reliability and validity of each assay.
  • the cultures are harvested by aspirating the media and washing twice with ice cold 0.5 M Tris, 0.15 M NaCl, PH 7.4 to remove excess radiolabel not incorporated into the cells.
  • Stop Reagent (1 ml of 15% potassium hydroxide, 85% methanol, 100 mg/ml butylated hydroxytoluene (BHT) is added to each well and the plate is sonicated in a mild water sonicating bath to release the cells from the bottom of the well.
  • BHT butylated hydroxytoluene
  • the digested cell extracts are transferred to 15 ml extraction tubes. Each well is rinsed with an additional 1 ml of the Tris/NaCl buffer which is added to the appropriate extraction tube. An aliquot (100 ml) is removed for protein determination at this point if desired.
  • the cell extracts are saponified at 80 °C for 30 minutes. After cooling 8 ml petroleum ether is added and the tubes are twirled on a rotary extractor for 5-10 minute's to extract the sterols into the organic solvent phase. The top organic phase is removed and passed through a Silica Seppak (Waters) which binds all sterols and free fatty acids. Sterols are eluted with a 5 ml diethyl ether:hexane (1:1) rinse. This sterols extraction is automated using Millilab (Waters) to ensure reproducibility and accuracy from sample to sample.
  • Millilab Waters
  • the eluted sterols are dried under nitrogen gas and resuspended in 150 ml ethanol. A 15 ml aliquot is removed from each sample and added to a scintillation vial filled with formula 989 (NEN) . The samples are counted on the dual label 3 H: 14 C program on the Beckman scintillation counter Model LS 7800. The incorporation of both radiolabeled precursors into sterols is compared between the treated and non- treated (control) cultures and expressed as "% control" for each precursor. Test compounds are classified as being “active” or “inactive” from these results and IC 50 values were determined for the active compounds.
  • Chromatography is performed at a flow rate of 1.5 ml per minute at 45 °C.
  • the compounds of this invention have greater potency in cholesterol biosynthesis inhibition than any of the compounds specifically disclosed and tested in WO 91/13903.
  • the compounds of this invention exhibited cholesterol biosynthesis inhibition activity approximately 2 to 80 fold higher than the most active compounds specifically disclosed in WO 91/13903 which have been tested.
  • Applicant has shown the compounds of the present invention to be unexpectedly superior to those disclosed and tested in PCT application WO 91/13903 in an identicle assay.
  • Compounds of this invention can be administered to treat said deficiencies by means that produces contact of the active agent with the agent's site of action in the body of a mammal.
  • the compounds can be administered by any conventional means available for use in conjunction with pharmaceuticals either as individual therapeutic agent or in 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.
  • the dosage administered will vary depending on the use and known factors such as pharmacodynamic character of the particular agent, and its mode and route of administration; the recipient's age, weight, and health; nature and extent of symptoms; kind of concurrent treatment; frequency of treatment; and desired effect.
  • the compounds of this invention can be orally administered daily at a dosage of the active ingredient of 0.002 to 200 mg/kg of body weight. Ordinarily, a dose of 0.01 to 10 mg/kg in divided doses one to four times a day, or in sustained release formulation was effective in obtaining the desired pharmacological effect.
  • Dosage forms (compositions) suitable for administration contain from about .1 mg to about 1000 mg of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5 to 95% by weight based on the total weight of the composition.
  • the active ingredient can be administered orally is solid dosage forms, such as capsules, tablets and powders; or in liquid forms such as elixirs, syrups, and/or suspensions.
  • the compounds of this invention can also be administered parenterally in sterile liquid dose formulations.
  • Gelatin capsules can be used to contain the active ingredient and a suitable carrier such as but not limited to lactose, starch, magnesium stearate, steric acid, or cellulose derivatives. 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 time.
  • a suitable carrier such as but not limited to lactose, starch, magnesium stearate, steric acid, or cellulose derivatives. 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 time.
  • Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste, or used to protect the active ingredients from the atmosphere, or to allow selective disintegration of the tablet in the gastrointestinal tract.
  • Liquid dose forms for oral administration can contain coloring of flavoring agents to increase patient acceptance..
  • water, pharmaceutically acceptable oils, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols, such as propylene glycol or polyethylene glycol 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, butter substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or in combination, are suitable stabilizing agents.
  • citric acid and its salts, and EDTA are also used.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", A. Osol, a standard reference in the field.
  • a large number of units capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 mg of powdered active ingredient, 150 mg lactose, 50 mg cellulose, and 6 mg magnesium stearate .
  • a mixture of active ingredient in a digestible oil such as soybean, cottonseed oil, or olive oil is prepared and injected by means of a positive displacement was pumped into gelatin to form soft gelatin capsules containing 100 mg of the active ingredient.
  • the capsules were washed and dried.
  • Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit was 100 mg active ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch, and 98.8 mg lactose.
  • Appropriate coatings may be applied to increase palatability or delayed adsorption.
  • the compounds of this invention may also be used as reagents or standards in the biochemical study of neurological function, dysfunction, and disease.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Steroid Compounds (AREA)

Abstract

L'invention concerne des composés, des compositions de ces derniers et des méthodes d'utilisation desdits composés et compositions dans le traitement de hypercholestérolémie chez un mammifère. Les composés selon l'invention inhibent la biosynthèse du cholestérol et sont capables de réduire la formation de cholestérol chez les mammifères. Les composés de l'invention ont la formule (I) dans laquelle R représente CH2CH3, CHOHCH2CH3 ou CH2CH=CH2.
PCT/US1994/001191 1993-02-11 1994-02-08 15-oxasterols utilises en tant qu'agent traitant l'hypocholesterolemie WO1994018225A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61692/94A AU6169294A (en) 1993-02-11 1994-02-08 15-oxasterols as hypocholesterolemics

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1655493A 1993-02-11 1993-02-11
US08/016,554 1993-02-11

Publications (1)

Publication Number Publication Date
WO1994018225A1 true WO1994018225A1 (fr) 1994-08-18

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AU (1) AU6169294A (fr)
WO (1) WO1994018225A1 (fr)
ZA (1) ZA94961B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058549A1 (fr) * 1998-05-13 1999-11-18 Novo Nordisk A/S Composes regulateurs de la meiose
US6407086B2 (en) 1998-05-13 2002-06-18 Novo Nordisk A/S Meiosis regulating compounds
US6794375B2 (en) 2000-01-28 2004-09-21 The Procter & Gamble Co. Palatable arginine compounds and uses thereof for cardiovascular health

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013093A1 (fr) * 1990-03-02 1991-09-05 Bio-Technology General Corp. CLONAGE ET PRODUCTION DE POLYPEPTIDES HUMAINS DE DOMAINE DE LIAISON DE GPIb DE FACTEUR DE VON WILLEBRAND ET LEURS PROCEDES D'UTILISATION

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013093A1 (fr) * 1990-03-02 1991-09-05 Bio-Technology General Corp. CLONAGE ET PRODUCTION DE POLYPEPTIDES HUMAINS DE DOMAINE DE LIAISON DE GPIb DE FACTEUR DE VON WILLEBRAND ET LEURS PROCEDES D'UTILISATION

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058549A1 (fr) * 1998-05-13 1999-11-18 Novo Nordisk A/S Composes regulateurs de la meiose
US6407086B2 (en) 1998-05-13 2002-06-18 Novo Nordisk A/S Meiosis regulating compounds
US6884796B2 (en) 1998-05-13 2005-04-26 Novonordisk A/S Meiosis regulating compounds
USRE39678E1 (en) * 1998-05-13 2007-06-05 Novo Nordisk A/S Meiosis regulating compounds
US6794375B2 (en) 2000-01-28 2004-09-21 The Procter & Gamble Co. Palatable arginine compounds and uses thereof for cardiovascular health

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Publication number Publication date
ZA94961B (en) 1995-08-11
AU6169294A (en) 1994-08-29

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