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  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/0803—Compounds with Si-C or Si-Si linkages
  • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Coronary heart disease remains the leading cause of death in the industrialized countries. Despite recent declines in CHD mortality, CHD is still responsible for more than 500,000 deaths in the U.S. annually. It is estimated that CHD, directly and indirectly, costs the U.S. more than $100 billion a year.
• the primary cause of CHD is atherosclerosis, a disease characterized by the deposition of lipids in the arterial vessel wall, resulting in a narrowing of the vessel passages and ultimately hardening the vascular system.
• Serum lipoproteins are the carriers for lipids in the circulation. They are classified according to their density: chylomicrons, very low-density lipoproteins (VLDL) , low density lipoproteins (LDL) and high-density lipoproteins (HDL) . Chylomicrons mainly participate in transporting dietary triglycerides and cholesterol from the intestine to adipose tissue and liver.
• VLDL deliver endogenously synthesized triglycerides from liver to adipose and other tissues.
• LDL transports cholesterol to peripheral tissues and regulate endogenous cholesterol levels in those tissues .
• HDL transports cholesterol from peripheral tissues to the liver.
• Arterial wall cholesterol is derived almost exclusively from LDL. Brown and Goldstein, Ann. Rev. Biochem. 52, 223 (1983); Miller, Ann. Rev. Med. 31, 97 (1980)) .
• the development of atherosclerosis is rare. Accordingly, it is desirable to provide a method for reducing plasma cholesterol in patients with hypercholesterolemia or at risk of developing hypercholesterolemia.
• Elevated cholesterol levels are also associated with a number of disease states, including restenosis, angina, cerebral arteriosclerosis, and xanthoma. It is desirable to provide a method for reducing plasma cholesterol in patients with, or at risk of developing, restenosis, angina, cerebral arteriosclerosis, xanthoma, and other disease states associated with elevated cholesterol levels.
• Vascular cell adhesion molecule-1 VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules in the immunoglobulin superfamily that are upregulated in vascular endothelial and smooth muscle cells by cytokines, such as, for example, interleukin-1 (IL-1) , interleukin-4 (IL-4) and tumor necrosis factor- ⁇ (TNF- ⁇ ) .
• cytokines such as, for example, interleukin-1 (IL-1) , interleukin-4 (IL-4) and tumor necrosis factor- ⁇ (TNF- ⁇ ) .
• IL-1 interleukin-1
• IL-4 interleukin-4
• TNF- ⁇ tumor necrosis factor- ⁇
• Inhibitors of VCAM-1 and/or ICAM-1 have therapeutic applications for many types of chronic inflammatory disorders including atherosclerosis, asthma, rheumatoid arthritis, and autoimmune diabetes.
• atherosclerosis a chronic inflammatory disorder
• asthma a chronic inflammatory disorder
• rheumatoid arthritis a chronic inflammatory disorder
• autoimmune diabetes a chronic inflammatory disorder
• VCAM- 1 and ICAM-1 adhesion molecules
• VCAM-1 is also involved as a mediator in other chronic inflammatory disorders such as asthma, rheumatoid arthritis and autoimmune diabetes.
• a chronic inflammatory disorders such as asthma, rheumatoid arthritis and autoimmune diabetes.
• VCAM-1 and ICAM-1 are increased in asthmatics. Pilewski, J.M. et al. , Am. J. Respir. Cell Mol. Biol. 12, 1-3 (1995); Ohkawara, Y. et al., Am. J. Respir. Cell Mol. Biol. 12, 4-12 (1995) .
• VCAM-1 and ICAM-1 suppressed both early and late phase responses in an ovalbumin-sensitized rat model of allergic airway responses.
• Rabb H.A. et al. , Am. J. Respir. Care Med. 149, 1186-1191 (1994) .
• endothelial adhesion molecules including VCAM-1, in the microvasculature of rheumatoid synovium. Koch, A.E. et al, Lab. Invest. 64, 313-322 (1991); Morales-Ducret, J. et al. , Immunol. 149, 1421-1431 (1992) .
• VCAM-1 is expressed by cells both as a membrane bound form and as a soluble form.
• the soluble form of VCAM-1 has been shown to induce chemotaxis of vascular endothelial cells in vi tro and stimulate an angiogenic response in rat cornea. Koch, A.E. et al. , Nature 376, 517-519 (1995) .
• Inhibitors of the expression of soluble VCAM-1 have potential therapeutic value in treating diseases with a strong angiogenic component, including tumor growth and metastasis. Folkman, J. , and Shing, Y. , J. Biol. Chem. 10931-10934 (1992) .
• both promoters have been cloned and characterized.
• both promoters contain multiple DNA sequence elements which can bind the transcription factor, NF-kB.
• Iademarco M.F. et al . , J. Biol. Chem. 267, 16323-16329 (1992); Voraberger, G. et al . , J. Immunol. 147, 2777-2786 (1991).
• the NF-kB family of transcription factors is central in the regulation of several genes upregulated within sites of inflammation. The activation of NF-kB as a transcription factor involves dissociation from an inhibitory subunit, IkB, in the cytoplasm.
• NF-kB subunits translocate to the nucleus, bind to specific DNA sequence elements, and activate transcription of several genes, including VCAM-1 and ICAM- 1. Collins T. et al . , Lab. Invest. 68, 499-508 (1993) .
• VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors.
• redox reduction-oxidation
• the antioxidants pyrollidine dithiocarbamate and N- acetylcysteine inhibit cytokine-induced expression of VCAM- 1, but not ICAM-1 in vascular endothelial cells. Mauri, N. et al., J. Clin. Invest. 92, 1866-1874 (1993) . This would indicate that the inhibition of VCAM-1 expression by antioxidants involves some additional factors not involved in the regulation of ICAM-1 expression.
• 2, 6-Di-alkyl-4-silyl-phenols are disclosed as antiatherosclerotic agents by Parker et al . in U.S. Pat. No. 5,155,250, issued October 13, 1992. Furthermore, 2,6- Di-alkyl-4-silyl-phenols are disclosed as serum cholesterol lowering agents in PCT International Publ. No. WO 95/15760, published June 15, 1995.
• VCAM-1 and/or ICAM-1 It would be advantageous to control the release of VCAM-1 and/or ICAM-1, and to treat VCAM-1 and/or ICAM-1 mediated effects. It would also be advantageous to control or treat chronic inflammation, without production of concomitant side effects known to accompany the use of antiinflammatory steroids and non-steroidal antiinflammatory agents.
• the present invention provides compounds of the formula
• R 1 and R 6 are each independently C j -C ⁇ alkyl;
• R 2 , R 3 and R 4 are each independently hydrogen or C,-C 6 alkyl;
• R is hydrogen or -C(0) - (CH 2 ) m -Q wherein Q is hydrogen or -COOH and m is an integer 1, 2, 3 or 4;
• Z is a thio, oxy or methylene group;
• A is a C 1 -C 1 alkylene group;
• R 5 and R 7 are each independently a C ⁇ C g alkyl or - (CH 2 ) n - (Ar) wherein n is an integer 0, 1, 2 or 3 ; and
• Ar is phenyl or naphthyl unsubstituted or substituted with one to three substituents selected from the group consisting of hydroxy, methoxy, ethoxy, halogen, trifluoromethyl, C j -C 6 alkyl, or -NR ⁇ R, where
• the present invention also provides a method of inhibiting the peroxidation of LDL lipid in a patient in need thereof comprising administering to said patient an effective antioxidant amount of a compound of formula (1)
• the present invention further provides a method for lowering plasma cholesterol level in a patient in need thereof by administration of a plasma cholesterol lowering amount of a compound of formula (1) .
• the present invention further provides a method for inhibiting the progression of atherosclerosis and/or a method for treating atherosclerosis in a patient in need thereof comprising administering to the patient an antiatherosclerotic amount of a compound of formula (1) .
• the present invention further provides a method of inhibiting cytokine-induced expression of vascular cell adhesion molecule-1 and/or intercellular adhesion molecule- 1 in a patient in need thereof comprising administering to the patient an effective vascular cell adhesion molecule-1 and/or intercellular adhesion molecule-1 inhibiting amount of a compound of formula (1) .
• the present invention further provides a method of treating a patient afflicted with a chronic inflammatory disease comprising administering to the patient a therapeutically effective amount of a compound of formula (1) .
• C ⁇ C j alkyl refers to a saturated hydrocarbyl radical of straight, branched or cyclic configuration made up of from one to six carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tertiarybutyl, n-pentyl, n-hexyl, cyclohexyl and the like.
• C j -C, alkylene refers to a saturated hydrocarbyldiyl radical of straight or branched configuration made up of from one to four carbon atoms.
• R 5 is a -(CH 2 )n-(Ar) radical
• the "-(CH 2 ) n -" moiety represents a saturated hydrocarbyldiyl radical of straight chain configuration.
• the term "n” is defined as an integer 0, 1, 2 or 3.
• the moiety "-(CH 2 ) n -" thus represents a bond, methylene, 1,2- ethanediyl or 1, 3-propanediyl.
• the "-(Ar)” moiety represents an aryl radical defined as a substituted or unsubstituted phenyl or napthyl group.
• the phenyl or napthyl can bear from 1 to 3 substituents in any position otherwise occupied by a hydrogen atom.
• substituents are selected from the group consisting of hydroxy, methoxy, ethoxy, chloro, fluoro and C 1 -C 6 alkyl group.
• - (CH 2 ) n - (Ar) are phenyl; napthyl; phenylmethyl; phenylethyl; 3, 4, 5-trihydroxyphenyl; 3,4,5- trimethoxyphenyl; 3 , 4, 5-triethoxyphenyl; 4-chlorophenyl; 4- methylphenyl; 3 , 5-di-tertiarybutyl-4-hydroxyphenyl; 4- fluorophenyl; 4-chloro-1-naphthyl; 2-methyl-1- naphthylmethyl; 2-naphthylmethyl; 4-chlorophenylmethyl; 4- tertiarybutylphenyl; 4-tertiarybutylphenylmethyl and the like.
• pharmaceutically acceptable acid addition salts is intended to apply to any non-toxic organic or inorganic acid addition salt of a suitable compound of formula (1), such as 2, 6-di-t-butyl-4 [ (4-N,N- dimethylaminophenyldimethylsilyl)methyloxy]phenol.
• suitable inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium mono- hydrogen orthophosphate and potassium hydrogen sulfate.
• organic acids which form suitable salts include the mono, di and tricarboxylic acids.
• Such acids are, for example, acetic, trifluoroacetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonic acid and
• 2-hydroxyethane sulfonic acid Such salts can exist in either the hydrated or substantially anhydrous form.
• the compounds of formula (1) can be prepared by utilizing procedures and techniques well known and appreciated by one of ordinary skill in the art.
• a general synthetic scheme for preparing compounds of formula (1) wherein Z is sulfur or oxygen is set forth in Scheme A, wherein all substituents, unless otherwise indicated, are previously defined.
• X chlorine, bromine, or iodine
• a phenol of structure la can be prepared by reacting the appropriate alkyl-4-mercaptophenol or alkylhydroquinone of structure 2 (or suitably protected derivatives) with a non-nucleophilic base, such as sodium hydride, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, and the like, and the appropriate haloalkylenesilane of structure 3, such as the appropriate chloroalkylenesilane, in a suitable aprotic solvent, such as acetonitrile, dimethylformamide or dimethylacetamide, or in an aqueous solvent, such as water/2-butanone.
• a non-nucleophilic base such as sodium hydride, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, and the like
• the appropriate haloalkylenesilane of structure 3 such as the appropriate chloroalkylenesilane
• a suitable aprotic solvent such as acetonitrile, dimethylformamide
• a phenol ester of structure lb can be prepared by acylating a phenol of structure la according to standard acylation techniques.
• a phenol of structure la is dissolved in a suitable aprotic solvent such as acetonitrile, dimethylformamide or dimethylacetamide, or an ethereal solvent such as diethyl ether or dioxane, and treated with a suitable base, such as triethylamine, N- methylmorpholine, sodium hydroxide or sodium hydride.
• a suitable aprotic solvent such as acetonitrile, dimethylformamide or dimethylacetamide, or an ethereal solvent such as diethyl ether or dioxane
• a suitable base such as triethylamine, N- methylmorpholine, sodium hydroxide or sodium hydride.
• An excess of O-acylating agent is then added at room temperature and the reaction is stirred at room temperature for 1 to 24 hours.
• O-acylating agents are acetyl chloride, propionyl chloride, monoethylsuccinyl- chloride, succinic anhydride, and the like.
• the product is then purified by techniques well known in the art, such as extractive methods and flash chromatography.
• additional treatment with a suitable base, such as sodium hydroxide with subsequent acidification with a suitable acid, such as hydrochloric acid, followed by extraction and flash chromatography may be performed to provide the phenol ester of structure lb.
• Silyl starting materials for various compounds of formula (1) such as (trimethylsilyl)methyl iodide, (trimethylsilyl)methyl bromide, (trimethylsilyl)methyl chloride, (1-chloropropyl) trimethylsilane, are described in Synthesis 4, 318-19 (1988) and J. Am. Chem. Soc. 105, 5665- 75 (1983) .
• Additional methods for preparing suitable silanes include a Grignard reaction.
• R 7 is a phenyl moiety containing a methoxy substituent
• 4- bromoanisole is reacted with magnesium metal to form the Grignard reagent and the reagent is reacted with chlorodimethyl chloromethyl silane to give chloromethyldimethyl-4-methoxy phenyl silane.
• anisole may be lithiated by reaction with n- butylithium and the lithio compound formed is reacted with chlorodimethyl chloromethyl silane to give chloromethyl dimethyl-2-methoxyphenyl silane.
• R 7 is a phenyl moiety containing a -NR ⁇ R g substituent, such as dimethylamine
• 4-bromo-N,N- dimethylaniline is reacted with magnesium metal to form the Grignard reagent and the reagent is reacted with chlorodimethyl chloromethyl silane to give 4-N,N- dimethylaminophenyl (dimethyl) chloromethylsilane.
• R 7 is a phenyl moiety containing a trifluoromethyl substituent
• bromo benzotrifluoride is reacted with magnesium metal to form the Grignard reagent and the reagent is reacted with chlorodimethyl chloromethyl silane to give dimethyl (chloromethyl) trifluoromethylphenylsilane.
• R 5 and R 7 are both phenyl
• about two molar equivalents of phenyl magnesium bromide is reacted with about one molar equivalent of dichloromethyl chloromethyl silane to give methyl diphenylchloromethyl silane.
• the 1-phenol functionality of a compound of structure 2 may react with the compounds of structure 3 under the conditions of the reaction
• the 1- phenol functionality of compound of structure 2 may be blocked with standard phenol blocking agents which are well known and appreciated in the art.
• standard phenol blocking agents which are well known and appreciated in the art.
• blocking groups are well known to one of ordinary skill in the art. In general, blocking groups should be selected which adequately protect the phenol in question during subsequent synthetic steps and which are readily removable under conditions which will not cause degradation of the desired product.
• Suitable phenol protecting groups are ethers, such as methoxymethyl, 2-methoxyethoxymethyl, tetrahydro-pyranyl, t-butyl and benzyl; silyl ethers, such as trimethylsilyl and t-butyldimethylsilyl; esters, such as acetate and benzoate; carbonates, such as methylcarbonate and benzylcarbonate; as well as sulfonates, such as methanesulfonate and toluenesulfonate.
• Step a Preparation of chloro (diphenyl)methylsilane: A solution of bromobenzene (31.5mL, 0.3 mol) was cooled in dry THF (450mL) to -60°C. To this solution was added 2.5M n-butyllithium (120mL, 0.3 mol) dropwise while keeping the reaction temperature below -55°C. Once the addition was complete, chloromethyl (dichloro)methylsilane (18.9mL, 0.15 mol) was added at such a rate as to keep the reaction temperature below -55°C. The mixture was then warmed to room temperature and ethyl acetate (5mL) was added to quench any unreacted n-butyllithium.
• Step c Preparation of 2, 6-Di-t-butyl-4- ⁇ (diphenyl- methylsilyDmethyloxylPhenol (MDL 104.599) : A solution of diphenyl (iodomethy1)methylsilane (9.17g, 27.1 mmol) and 2,6- di-t-butylbenzhydroquinone (6.0g, 27 mmol) in dry acetonitrile (250mL) was thoroughly degassed with nitrogen. To this solution was added potassium carbonate (4.5g, 32.6 mmol) and the mixture refluxed under nitrogen for 3 days. The reaction mixture was cooled, filtered and evaporated.
• diphenyl (iodomethy1)methylsilane 9.17g, 27.1 mmol
• 2,6- di-t-butylbenzhydroquinone 6.0g, 27 mmol
• potassium carbonate 4.5g, 32.6 mmol
• Step a Preparation of chloromethyl (dimethyl) -4-N.N- dimethvlaminophenvlsilane: Magnesium turnings (9.7g, 0.4g atom) were stirred with a Teflon paddle overnight under nitrogen. This "activated" magnesium was suspended in dry THF (lOOmL) and a crystal of iodine was added. To this suspension was added a solution of 4-bromo-N,N- dimethylaniline (80. Og, 0.4 mol) in THF (400mL) at such arate as to maintain a gentle reflux. Once the addition was complete, stirring was continued ( ⁇ 2hrs.) until nearly all of the magnesium was consumed.
• Step b Preparation of 4-N,N-dimethylaminophenyl- (dimethyl) iodomethylsilane: A solution of chloromethyl-
• Step c Preparation of 2, 6-Di-t-butyl-4- T (4-N.N- dimeth ⁇ laminophenyldimethylsilyl)methyloxy1phenol (MDL 104, 556) : A solution of 4-N,N-dimethylaminophenyl- (dimethyl) iodomethylsilane (444g, 1.39 mol) and 2,6-di-t- butyl-benzhydroquinone (309g, 1.39 mol) in dry acetonitrile (1.5L) was thoroughly degassed with nitrogen. To this solution was added cesium carbonate (435g, 1.39 mol) and the mixture refluxed under nitrogen for 24 hours.
• Step a Preparation of dimethvl-4-trifluoromethyl- phenylchloromethylsilane: Magnesium metal shavings (9.7g, 0.4 mol) were placed in a 3-neck flask and stirred overnight under a nitrogen atmosphere with an overhead stirrer to activate the metal. THF (lOOmL) and a crystal of iodine were added and a solution of 4-bromobenzotrifluoride (90g, 0.4 mol) in THF (500mL) was added at a rate which maintained reflux.
• Step b Preparation of 2 , 6-Di-t-butyl-4- r (dimethyl-4- trifluoromethylphenylsilyl) methyloxylPhenol (MDL 105.975) : A solution of dimethyl (iodomethyl) -4-trifluoromethylphenyl- silane (12.0g, 35 mmol) and 2, 6-di-t-butylbenzhydroquinone (6.5g, 29.2 mmol) in dry acetonitrile (200mL) was thoroughly degassed with nitrogen. To this solution was added potassium carbonate (4.8g, 35 mmol) and the mixture refluxed under nitrogen for 36 hours. The reaction mixture was cooled, filtered and evaporated.
• 6-Di-t-butyl-4- r (dimethyl-3-trif luorome thylphenylsilyl ) methyloxyl phenol (MDL 105 . 726 )
• Step a Preparation of chloromethyl (dimethyl) -3- trifluoromethylphenvlsilane: Magnesium turnings (9.7g, 0.4g atom) were stirred with a Teflon paddle overnight under nitrogen. This "activated" magnesium was suspended in dry THF (lOOmL) and a crystal of iodine was added. To this suspension was added a solution of 3-bromo-benzotrifluoride (56mL, 0.4 mol) in THF (400mL) at such arate as to maintain a gentle reflux. Once the addition was complete, stirring was continued ( ⁇ 2hrs.) until nearly all of the magnesium was consumed.
• Step b Preparation of dimethyl (iodomethyl) -3- trifluoromethvlphenvlsilane: A solution of chloromethyl (dimethyl) -3-trifluoromethyl-phenylsilane (25.3g, 0.1 mol) and sodium iodide (15.3g, 0.102 mol) in 2- butanone (400mL) was refluxed overnight. The solution was then filtered and evaporated. The resulting liquid was then redissolved in ethyl acetate (500mL) , washed with water (3x250mL) , saturated aqueous sodium chloride (3x250mL) , dried with anhydrous magnesium sulfate, filtered and evaporated. The resulting title compound as a pale orange liquid (33.2g, 97% yield) was sufficiently pure (-96%) to use as is.
• Step c Preparation of 2 , 6-Di-t-butyl-4- T (dimethyl-3- trifluorometh ⁇ lphen ⁇ lsilyl)methyloxylphenol (MDL 105.726) :
• a solution of dimethyl (iodomethyl) -3-trifluoro methylphenylsilane (15.5g, 45 mmoDand 2, 6-di-t- butylbenzhydroquinone (lOg, 45 mmol) in dry acetonitrile (250mL) was thoroughly degassed with nitrogen.
• potassium carbonate 6.2g, 45 mmol
• the ether layer was washed with water and evaporated to dryness to give 12.3g of a waxy solid.
• the solid was combined with 200mL methanol and heated to reflux.
• Sodium hydroxide (5.0g in 20mL water) was added and the reaction refluxed for 30 minutes then diluted with water and allowed to cool.
• the aqueous suspension was acidified with cone, hydrochloric acid and 10.6g of a solid was collected which was recrystallized from hexan to give 9.3g of a white crystalline powder, mp 146-147°C, 2, 6-Di-t-Butyl-4- [ (Dimethyl-phenylsilyl)methylthio]phenol Succinic Acid Ester.
• the oil was chromatographed on silica gel (chloroform) to give 3.4g of an oil.
• the oil was combined with 50mL methanol and heated to reflux.
• Sodium hydroxide (0.6g in lOmL water) was added and the reaction refluxed for 30 minutes then diluted with water and allowed to cool .
• the aqueous suspension was acidified with cone, hydrochloric acid and extracted with ether.
• the ether layer was separated and evaporated to dryness to give 3. Og of a white solid foam.
• Step b Preparation of 2.3.6-Trimethvl-4- r (dimethylphen ⁇ lsilvl)methvloxvlphenol acetic acid ester (MDL 103.157) : 4-Acetoxy-2, 3 , 5-trimethylphenol (8.1g, 41.7 mmol), chloromethyldimethylphenylsilane (7.7g, 41.7 mmol), lithium bromide (3.6g, 41.7 mmol), potassium carbonate
• acetonitrile 250mL was added iodomethyldimethyl (4- methoxy)phenyl silane (31.26g, 102 mmol), cesium carbonate (36.6g, 112.2 mmol) , and t-butylhydroquinone (18.6g, 112.2 mmol, Aldrich Chemical Co., Milwaukee, WI 53233) .
• the mixture was heated to 90°C, and stirred for 42 hours.
• the mixture was cooled to room temperature, and filtered.
• the acetonitrile was removed in vacuo, and the residue taken up in ethyl acetate (500mL) .
• Step a Preparation of chloromethylbis (4-methoxy- phenyl)methylsilane: A solution of 4-bromoanisole (50mL, 0.4 mol) in THF (500mL) was added to a suspension of "activated" magnesium (9.7g, 0.4 g atom) in dry THF (lOOmL) containing a crystal of iodine. Then a solution of chloromethyl (dichloro)methylsilane (25.5mL, 0.2 mol) in dry THF (lOOmL) was subsequently added, all in a manner according to example 2, step a, to provide a pale yellow oil. The pale yellow oil was distilled at 200°C at 5mm Hg to remove lower boiling impurities. GC/MS confirmed structure and purity (-87%) of the title compound (51.9g, 85% yield) .
• Step b Preparation of iodomethylbis (4-methoxyphenyl) - methylsilane: A solution of chloromethylbis (4- methoxyphenyl)methylsilane (51.9g, 0.169 mol) and sodium iodide (25.5g, 0.17 mol) in 2-butanone (400mL) was refluxed overnight. The solution was then filtered and evaporated. The resulting liquid was redissolved in ethyl acetate (500mL), washed with water (3x250mL) , saturated aqueous sodium chloride (3x250mL) , dried with anhydrous magnesium sulfate, filtered and evaporated.
• ethyl acetate 500mL
• Step c Preparation of 2 , 6-Di-t-butyl-4- f (methyl-di-p- methoxyphenvl-silvl)methyloxvlphenol : A solution of iodomethylbis (4-methoxyphenyl)methylsilane (53.7g, 0.135 mol) and 2 , 6-di-t-butylbenzhydroquinone (30. Og, 0.135 mol) in dry acetonitrile (500mL) was thoroughly degassed with nitrogen. To this solution was added potassium carbonate (20.
• Step a Preparation of dimethylbenzylchloromethyl- silane: A solution of benzylbromide (68.4g, 0.4 mol) in THF (400mL) was added to a suspension of "activated" magnesium (9.7g, 0.4 mol) in dry THF (100ml) containing a crystal of iodine. Then a solution of dimethylchloro- methylsilane (52.7ml, 0.4 mol) in dry THF (200ml) was subsequently added, all in a manner according to example 2, step a to provide the title compound. Yield 67%, bp 60- 80°C at 5mm Hg.
• Step b Preparation of 2-t-but ⁇ l-4- r (dimethylbenzylsilyl)methyloxy]phenol (MDL 108.804) : A mixture of dimethylbenzylchloromethylsilane (55.2g, 0.3 mol) and sodium iodide (52g, 0.35 mol) in acetonitrile (600mL) was heated at reflux for 24 h, solvent (20mL) was distilled off to remove any water, and the mixture was cooled to ambient temperature. To the cooled mixture was added 2-t-butylbenzhydroquinone (49.8g, 0.3 mol) and cesium carbonate (50g, 0.15 mol) .
• 2-t-butylbenzhydroquinone 49.8g, 0.3 mol
• cesium carbonate 50g, 0.15 mol
• the mixture was heated at 85- 90°C under an inert atmosphere for 3 days, cooled and poured into a mixture of water/ethyl acetate (IL each) .
• the organic layer was isolated, dried and evaporated.
• the residue was heated on a Kugelrohr apparatus at 90°C for 3 h.
• the residue was chromatographed (twice with 9/1 hexane/ethyl acetate, then once 1/1) .
• the residue was a liquid (10.5g, 10%) .
• a phenol of structure lc can be prepared according to Scheme B in a two-step process.
• step a the appropriate haloalkylenesilane of structure 3 is reacted with magnesium metal in a suitable aprotic solvent, such as ethyl ether, in order to form the magnesium halide salt.
• the magnesium halide salt (Grignard reagent) is then reacted with the appropriate alkyl-4-hydroxy-benzaldehyde of structure 4 (or a suitably protected derivative) to give the alcohol of structure 5.
• step b the alcohol of structure 5 can be reduced to the desired phenol of structure lb by a variety of reduction techniques and procedures as are well known and appreciated in the art.
• the alcohol of structure 5 can be reduced by means of a Birch reduction by reacting it with sodium in liquid ammonia.
• a phenol ester of structure Id can be prepared by acylating a phenol of structure lc according to standard acylation techniques as described previously in Scheme A.
• Step a Mix magnesium turnings (240mg, lOmmol) and anhydrous ethyl ether under an inert atmosphere. Add a solution of chloromethyltrimethylsilane (1.9g, lOmmol) in anhydrous ethyl ether. Stir until the magnesium metal dissolves. Add a solution of 2, 3, 5-trimethyl-4- hydroxybenzaldehyde (1.7g, lOmmol) in anhydrous ethyl ether. Stir until reaction is complete. Cool the reaction mixture to 0°C and add saturated ammonium chloride solution. Separate the ether layer, wash with water and dry (MgSOJ . Evaporate to give 4-hydroxy-2, 3 , 5-trimethyl- ⁇ - [ (trimethylsilyl)methyl]benzenemethanol and purify by silica gel chromatrography.
• Step b Mix sodium metal (520mg, 22.6mmol) and liquid ammonia (13mL) . To this solution add, by dropwise addition, a solution of 4-hydroxy-2, 3 , 5-trimethyl- ⁇ - [ (trimethylsilyl)methyl]benzenemethanol (2.37g, lOmmol) in ethyl alcohol (0.5g) and ethyl ether (5ml) . After the blue color disappears, cautiously add water (13mL) , extract with ethyl ether, dry (MgSOJ , and evaporate the solvent. Purify the residue by silica gel chromatography to yield the title compound.
• compounds of formula (1) wherein Z is methylene can be prepared according to the procedure set forth in Scheme C, wherein all substituents, unless otherwise indicated, are previously described.
• a phenol of structure lb can be prepared by first reacting the appropriate haloalkylenesilane of structure 3 with magnesium metal in an suitable aprotic solvent, such as ethyl ether, in order to form the magnesium halide salt.
• the magnesium halide salt (Grignard Reagent) is then reacted with the appropriate alkyl-4- hydroxy-benzylhalide of structure 6 (or a suitably protected derivative) to give the desired phenol of structure lc.
• a phenol ester of structure Id can be prepared by acylating a phenol of structure lc according to standard acylation techniques as described previously in Scheme A.
• the 1-phenol functionality of the alkyl-4-hydroxy- benzylhalide of structure 6 in Scheme C may be blocked prior to the Grignard reaction with a standard phenol blocking agent as described previously in Scheme A.
• a solution of diphenyl (methyl) chloromethylsilane (1.85g, lOmmol, Example 1) in anhydrous ethyl ether is reacted with a mixture of magnesium turnings (240mg, lOmmol) and anhydrous ethyl ether and subsequently reacted with a solution of 4-bromomethyl-2, 6-di-t-butylphenol (2.9g, lOmmol, Maybridge # MB 00185) in anhydrous ethyl ether in a manner analogous to the procedure described in Example 26 to provide the title compound.
• Preferred compounds of formula (1) are those in which R is hydrogen, acetyl or succinyl; R ⁇ is methyl or tertiarybutyl; R 2 and R 3 are each independently hydrogen, methyl or tertiarybutyl; R ⁇ is hydrogen or methyl; R 6 is methyl; A is methylene; and R 5 and R 7 are each independently methyl or -(CH 2 ) n -(Ar) where n is 0 or 1 and Ar is phenyl unsubstituted or substituted with one to three substituents selected from the group consisting of hydroxy, methoxy, ethoxy, halogen, trifluoromethyl, C j -C 6 alkyl, or -NR 8 R 9 , wherein R ⁇ and R 9 are each independently hydrogen or methyl . More preferred are the compounds: 2, 6-Di-t-butyl-4- [ (diphenylmethylsilyl)methyloxy]phenol
• the term "patient” refers to a warm- blooded animal or mammal which is in need of treatment for a chronic inflammatory disease, atherosclerosis, hypercholesterolemia or which is in need of inhibiting cytokine-induced expression of vascular cell adhesion molecule-1 and/or intercellular adhesion molecule-1. It is understood that guinea pigs, dogs, cats, rats, mice, hamsters, rabbits and primates, including humans, are examples of patients within the scope of the meaning of the term.
• Atherosclerosis is a disease state characterized by the development and growth of atherosclerotic lesions or plaque.
• An effective antiatherosclerotic amount of a compound of formula (1) is an amount which is effective in inhibiting the development or growth of atherosclerosis in a patient in need thereof.
• successful treatment of a patient for atherosclerosis is understood to include effectively slowing, interrupting, arresting, or stopping atherosclerotic lesion or plaque development or growth and does not necessarily indicate a total elimination of atherosclerosis. It is further understood and appreciated by those of ordinary skill in the art that successful treatment for atherosclerosis can include prophylaxis in preventing atherosclerotic lesion or plaque formation.
• Peroxidation of LDL lipid such as the unsaturated fatty acid portions of LDL cholesteryl esters and phospholipids, is known to facilitate the deposition of cholesterol in macrophages which subsequently are deposited in the vessel wall and are transformed into foam cells.
• the identification of those patients who are in need of inhibition of peroxidation of LDL lipid is well within the ability and knowledge of one of ordinary skill in the art. For example, those individuals who are in need of treatment for atherosclerosis as defined hereinabove, are also patients who are in need of inhibition of peroxidation of LDL lipid.
• An effective antioxidant amount of a compound of formula (1) is an amount which is effective in inhibiting the peroxidation of LDL lipid in a patient's blood.
• Hypercholesterolemia is a disease state characterized by levels of serum cholesterol or of LDL cholesterol which are elevated by a clinically significant amount over that considered normal by those of ordinary skill in the art.
• the identification of those patients who are in need of treatment for hypercholesterolemia is well within the ability and knowledge of one skilled in the art.
• individuals who have serum cholesterol levels or LDL cholesterol levels, as determined by clinical laboratory tests, which are substantially and chronically elevated over that considered normal by those of ordinary skill in the art are patients in need of treatment for hypercholesterolemia.
• individuals who are at risk of developing hypercholesterolemia can also be patients in need of treatment for hypercholesterolemia.
• a clinician skilled in the art can readily identify, by use of clinical tests, physical examination and medical/family history, those patients who are suffering from hypercholesterolemia and those who are at risk of developing hypercholesterolemia and thus readily determine if an individual is a patient in need of hypercholesterolemia.
• chronic inflammatory disease refers to diseases or conditions characterized by persistent inflammation in the absence of an identifiable irritant or microbial pathogen.
• Inflammatory diseases for which treatment with a compound of formula (1) will be particularly useful include: asthma, chronic inflammation, rheumatoid arthritis, autoimmune diabetes, transplant rejection and tumor angiogenesis.
• a "therapeutically effective amount" of a compound of formula (1) is an amount which is effective, upon single or multiple dose administration to the patient, in providing relief of symptoms associated with chronic inflammatory diseases .
• an "effective vascular cell adhesion molecule-1 and/or intercellular cell adhesion molecule-1 inhibiting amount" of a compound of formula (1) is an amount which is effective, upon single or multiple dose administration to the patient, in providing relief of symptoms associated with vascular cell adhesion molecule-1 and/or intercellular adhesion molecule-1 mediated conditions.
• relief of symptoms of a chronic inflammatory disease or vascular cell adhesion molecule-1 mediated conditions refers to decrease in severity over that expected in the absence of treatment and does not necessarily indicate a total elimination or cure of the disease. Relief of symptoms is also intended to include prophylaxis.
• the therapeutically effective amount or dose a number of factors are considered by the attending diagnostician, including, but not limited to: the species of the mammal; its size, age, and general health; the specific disease involved; the degree of or involvment or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant cirmumstances.
• a therapeutically effective amount, an effective antioxidant amount, a plasma cholesterol lowering amount, an effective antiatherosclerotic amount or an effective VCAM-1 and/or ICAM-1 inhibiting amount of a compound of formula (1) will generally vary from about 1 milligram per kilogram of body weight per day (mg/kg/day) to about 5 grams per kilogram of body weight per day (gm/kg/day) .
• a daily dose of from about 1 mg/kg to about 500 mg/kg is preferred.
• the compounds of this invention are inhibitors of VCAM-1 and/or ICAM-1 expression. It is believed that the compounds of this invention exert their inhibitory effect through inhibition of VCAM-1 and/or ICAM-1 upregulation by cytokines and thereby prevent or provide relief of symptoms for chronic inflammatory diseases including asthma, chronic inflammation, rheumatoid arthritis, autoimmune diabetes, and the like; atherosclerosis and hypercholesterolemia.
• chronic inflammatory diseases including asthma, chronic inflammation, rheumatoid arthritis, autoimmune diabetes, and the like; atherosclerosis and hypercholesterolemia.
• the present invention is not limited by any particular theory or proposed mechanism to explain its effectiveness in an end-use application.
• a compound of formula (1) can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and parenteral routes.
• the compound can be administered orally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, and the like.
• Oral administration is generally preferred.
• One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the disease state to be treated, the stage of the disease, and other relevant circumstances. Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990) .
• a compound of formula (1) can be administered in the form of pharmaceutical compositions or medicaments which are made by combining a compound of formula (1) with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the chosen route of administration, and standard pharmaceutical practice.
• the pharmaceutical compositions or medicaments are prepared in a manner well known in the pharmaceutical art.
• the carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art.
• the pharmaceutical composition may be adapted for oral or parenteral use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like.
• compositions may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets .
• a compound of formula (1) may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of a compound of formula (1) , the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit.
• the amount of the active ingredient present in compositions is such that a unit dosage form suitable for administration will be obtained.
• the tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders, such as macrocrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; and sweetening agents, such as sucrose or saccharin may be added or flavoring agents, such as peppermint, methyl salicylate or orange flavoring.
• a liquid carrier such as polyethylene glycol or a fatty oil.
• dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
• tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
• a syrup may contain, in addition to the active ingredient, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
• a compound of formula (1) may be incorporated into a solution or suspension.
• These preparations should contain at least 0.1% of a compound of the invention, but may be varied to be between 0.1 and about 50% of the weight thereof.
• the amount of the active ingredient present in such compositions is such that a suitable dosage will be obtained.
• the solutions or suspensions may also include one or more of the following adjuvants depending on the solubility and other properties of a compound of formula (1) : sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose.
• the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic .
• HAVEC human umbilical vein endothelial cells
• HASMC human aortic smooth muscle cells
• the cultures were maintained in growth medium (EGM or SMGM2 , Clonetics, San Diego, CA) for two days prior to addition of cytokines or drugs .
• Cytokines plus or minus compounds were added for 20 to 24 hours prior to analysis for adhesion molecule levels.
• Tumor necrosis factor (Genzyme,
• HBSS Hanks buffered saline solution
• the primary antibody (anti-human VCAM-1 from Upstate Biotechnology, Inc., Lake Placid, NY or anti-human ICAM-1 from Immunotech, Inc., Westbrook, ME) was added to each well (1 ⁇ g/mL in HBSS plus 5% newborn calf serum, GIBCO-BRL, Gaithersburg, MD) and incubated at 37°C for 1 hr.
• the wells were washed twice with HBSS, then 100 ⁇ L of a 1/1000 dilution of goat anti-mouse IgG conjugated to horse radish peroxidase (BioRad, Hercules, CA) in HBSS plus 5% newborn calf serum was added to each well and incubated for 1 hr at 37°C.
• TMB substrate BioRad, Hercules, CA
• IC 50 values were determined from curves of absorbance values obtained from serial dilutions of compounds (dissolved in dimethyl sulfoxide) .
• the IC 50 value is defined as the drug concentration that inhibits the cytokine-induced adhesion molecule expression by 50%. Maximal values for adhesion molecule expression in cytokine-induced cultures was subtracted from the basal level of adhesion molecule expression (minus cytokines) in the cultures to determine the level of induction. VCAM-1 was typically induced about 5-7 fold. ICAM-1 was typically induced 5-10 fold. Each drug concentration was tested in quadruplicate wells. Single point tests of compounds at 50 ⁇ M were assayed as described for IC 50 determinations, except that the data represent the level of inhibition without correction for basal expression. (Basal adhesion molecule expression was 10-20% of the total induced expression) .
• Table 1 summarizes the ability of various compounds of this invention to inhibit VCAM-1 using human aortic smooth muscle cells (HASMC) .
• HASMC human aortic smooth muscle cells
• Table 2 summarizes the ability of various compounds of this invention to selectively inhibit VCAM-1 or to inhibit both VCAM-1 and ICAM-1 using proliferating human umbilical vein endothelial cells (HUVEC) .
• the cells were coincubated. with tumor necrosis factor-alpha along with the indicated compounds about 20 to 24 hr before assaying cell surface adhesion molecule expression.
• VCAM-1 and/or ICAM-1 Inhibition of VCAM-1 and/or ICAM-1 in Human Umbilical Vein Endothelial Cells (HUVEC )
• In vivo activity of these compounds can also be assessed in other models of inf lammation predicted to involve elevated VCAM-1 levels .
• One such model for respiratory diseases such as asthma
• This model of pulmonary inflammation is IgE mediated and involves eos inophillia ( as does the asthmatic human) .
• the bronchial alveolar lavage (BAL) fluid obtained from experimental animals can be assessed for a number o_f parameters, including soluble adhesion molecule expression and leukocyte accumulation.
• Adhesion molecule expresssion can be assessed by immunohistochemistry within the tissues, especially the lung, of experimental animals.
• the effect of the claimed compounds, such as MDL 29,353 should be to suppress the upregulation of VCAM-1 expressionand inhibit eosinophil accumulation in the BAL fluid.
• the inhibitors could be tested in a rat model of adjuvant arthritis, which has been previously shown to respond to anti-ICAM-1 monoclonal antibodies. Iigo, Y. et al. , J. Immunol.147. 4167-4171 (1991) . In this model, adhesion molecule expression would be assessed in the limbs (joints) of experimental animals.
• autoimmune diabetes For autoimmune diabetes, one could test the compounds for their ability to delay the onset or prevent adoptive transfer of disease in the NOD mouse model. Heinke, E.W. et al., Diabetes 42, 1721-1730 (1993); Baron, J.L. et al. , J. Clin. Invest. 93, 1700-1708 (1994) . Furthermore, one can monitor the level of VCAM-1 expression in the tissues (e.g. pancreas) as well as monitor the development of diabetes in the experimental animal . Therapeutic potential for transplant rejection can be assessed by monitoring cardiac allograft survival (Balb/c hearts transplanted into C3H/He recipients. Isobe, M. et al. , J. Immunol.
• Activity of the compounds can be assessed by their effect on the number of lung metastases which develop, as well as their effect on VCAM-1 expression in the lung as described above for the mouse respiratory model.
• a model for evaluating anti- angiogenic compounds which can be used to test the compounds involves monitoring the vascular response to a mixture of angiogenic factors mixed with basement membrane proteins injected subcutaneously in mice. Passaniti, A. et al., Lab. Invest. 67, 519-528 (1992) .
• Angiogenesis is scored by the number of vessels recruited into the matrigel and by the hemoglobin content of the gels.
• Adhesion molecule expression and accumulation of leukocyte can be determined by immunohistochemical methods as in all of the above examples .
• Control chow was sprayed with ethanol. Rabbits were fed 100 grams food per day for 7 days (0.6% MDL 103,491 were fed for 14 days) ; water was available ad libitum. On day 7, rabbits (fasted overnight) were bled ( ⁇ 2 mL) from a marginal ear vein; 0.6% MDL 103,491 treated rabbits were tested on day 14. They were euthanized by carbon dioxide overdose. The total body and liver weights were recorded in grams. Food consumption was recorded as grams • day "1 • rabbit "1 . Aliquots of fresh serum were used for clinical chemistries, lipoprotein cholesterol determination, thiobarbituric acid reactive substances (TBARS) and compound and metabolite concentrations in serum. Livers ( ⁇ 5 gram aliquots) were frozen at -20°C for compound and metabolite concentration determination at a later time.
• TBARS thiobarbituric acid reactive substances
• TBARS are a qualitative indication of the oxidation of lipids in a sample.
• the oxidation of serum lipids is initiated with CuS0 4 , resulting in the formation of aldehydes, such as malondialdehyde (MDA) .
• MDA malondialdehyde
• the absorbance of the aldehydes can be detected at 530-540 nm.
• TBARS values which are lower than control serum values indicate the relative ability of a compound to inhibit the oxidation.
• TBARS were measured as follows: 50 ⁇ L of serum were mixed with 50 ⁇ L of 0.9% saline and 400 ⁇ L of a 5 mM CuSO, solution and incubated at 37°C for 5 hr. The reactions were stopped by addition of 1.0 mL of 20% trichloroacetic acid. Then 1.0 mL of 0.67% thiobarbituric acid in 0.05 N sodium hydroxide was added, mixed, and the samples incubated for 30 min at 90°C. Samples were centrifuged briefly to pellet undissolved material, and the supernatants were transferred to a 96-well microtiter plate. Absorbances were measured at 540 nm using a Biotek model EL311 microplate reader.
• nmoles of MDA produced were calculated from a standard curve of 0 to 10 nmoles of MDA prepared from malonaldehyde bis(dimethylacetal) . Serum samples from treated rabbits were compared to serum samples from control rabbits that received no MDL compound.
• Serum and liver concentrations of parent compounds and the metabolites, bisphenol and diphenoquinone, were determined by reverse phase HPLC using a Waters 990 Powerline system. Livers (1 gram) were homogenized with 5.0 mL PBS, pH 7.4, using a Polytron tissue homogenizer at setting 5 for 20-30 seconds. Serum or liver homogenates were extracted as follows: 100 ⁇ L of either serum or homogenate were added to 2.0 mL diethyl ether : ethanol (3:1) while vortexing the tube. The sample tubes were capped and centrifuged for 10 min at 5°C at 3500 rpm in a Beckman GPKR centrifuge with a GH 3.7 rotor.
• Lipoprotein fractions were separated on a Sepharose 6HR column (1 x 30 cm, Pharmacia) attached to a Waters Powerline HPLC system. Fifty ⁇ L of serum were injected onto the column and eluted with phosphate buffered saline, pH 7.4, at a flow rate of 0.5 mL/min.
• Cholesterol reagent (Roche Diagnostics, kit # 44334, diluted with 20 mL water and then with 20 mL of 0.9% saline) was added at 0.2 mL/min to the post column eluant and incubated in a knitted PFTE Kratos reaction coil (Applied Biosystems) at 37°C for 5 min. Absorbance was measured at 500 nm. The lipoprotein subfractions were quantitated as follows:
• Rabbits were fed x 7 days (except MDL 103,491 at 0.6% was for 14 days) .
• Diet % (weight MDL compound/weight food) x (100)
• LW/BW (liver weight / body weight in grams)
• CHOL total cholesterol mg/dL
• LDL Low Density lipoprotein cholesterol mg/dL
• HDL High Density lipoprotein cholesterol mg/dL
• TRIG triglycerides, mg/dL
• TBARS thiobarbituric acid reactive substances, expressed as nmole MDA TABLE 4
• Rabbits were fed x 7 days (except MDL 103,491 at 0.6% was for 14 days) .
• Diet % (weight MDL compound/weight food) x (100)
• parent parent compound concentration as ⁇ g/g liver
• Liver Bis bisphenol concentration as ⁇ g/g liver
• Liver Quin diphenoquinone concentration as ⁇ g/g liver
• Male Sprague-Dawley rats, 50-100 g, (Harlan Laboratories, Indianapolis, IN) were housed in groups of 5, fed ad libitum water and Purina Rodent chow (#5002) with or without MDL compound as a dietary admixture for 4 days. Dietary admixtures (0.3%) were made by mixing 1.2 grams of an MDL compound with 400 grams of Purina rodent chow (#5002) .
• the MDL compound was mixed with approximately 50 grams of food using a mortar and pestle. This was added to the remainder of the food and mixed for 3 hours on a rotary mixer.
• non-fasted rats were anesthetized with carbon dioxide, and blood was collected by cardiac puncture. Rats were sacrificed by cervical dislocation. Body weights and liver weights were recorded in grams. Food consumption was recorded as grams • day "1 • rat "1 . Deaths were recorded as mortality.
• Aliquots of fresh serum were used for clinical chemistries, thiobarbituric acid reactive substances (TBARS) and conjugated diene measurements. Aliquots of serum ( ⁇ 0.5mL) and whole livers were frozen at -20°C for compound and metabolite concentration determination at a later time.
• TBARS thiobarbituric acid reactive substances
• Serum and liver concentrations of parent compound and the metabolites, bisphenol and diphenoquinone, were determined by reverse phase HPLC using a Waters 990 Powerline system. Livers (1 gram samples) were homogenized with 5. OmL PBS, pH 7.4, using a Polytron tissue homogenizer at setting 5 for 20-30 seconds. Serum or liver homogenates were extracted as follows: 100 ⁇ L of either serum or homogenate were added to 2. OmL diethyl ether : ethanol (3:1) while vortexing the tube. The sample tubes were capped and centrifuged for 10 min at 5°C at 3500 rpm in a Beckman GPKR centrifuge with a GH 3.7 rotor. The supernatants were transferred to clean tubes and dried under N 2 .
• TBARS values which are lower than control serum values indicate the relative ability of a test compound to inhibit the oxidation of lipids in a sample.
• TBARS were measured as follows: 100 ⁇ L of serum were mixed with 400 ⁇ L of a 5mmol CuS0 4 solution and incubated at 37°C for 3 hr. The reactions were stopped by addition of 1.
• Conjugated diene lag phase is another indicator of the oxidation of lipids. Lipids exposed to Cu " form conjugated dienes that absorb ultraviolet light in the range of 230 to 235 nm. The lag phase of diene formation gives an indication of the amount of oxidation of the lipids. A lag phase longer than control samples indicate inhibition of the oxidation. Conjugated diene lay phase was dtermined using a Varian DMS200 spectrophotometer (fitted with a constant temperature, 5 cuvette sample changer) at 30°C. Twenty (20) ⁇ L of pooled serum were added to cuvettes containing 3. OmL phosphate buffered saline, pH 7.5, and mixed.
• Tables 5, 6 and 7 below present summary data from the individual experiments of this testing procedure.
• Table 5 presents measurements of the serum chemistries in the male Sprague-Dawley rats
• Table 6 presents the animal parameters
• Table 7 provides the drug or metabolite concentrations in both the serum and the liver.
• ALT alanine aminotransferase
• CHOL total cholesterol, mg/dL
• GLU glucose, mg/dL
• TBARS thiobarbituric acid reactive substances, expressed as nmoles MDA
• the data in Table 6 have been normalized according to the formula presented in Table 5.
• LW/BW (liver weight/body weight in grams)
• Rabbits (Hazelton, - 2.0-2.3 kg) 1% cholesterol enriched rabbit chow (Purina # 5322) with or without 0.4% of an MDL compound. Solubilize the MDL compound in 100% ethanol, spray on the chow, and dry overnight in a chemical fume hood. Alternatively, the MDL compounds can be incorporated into the rabbit food by Purina. Control chow is sprayed with ethanol. Feed rabbits 100 grams food per day for 70 days and allow water to be made availabe ad libitum.
• Rabbits (fasted overnight) are bled (- 2mL) from a marginal ear vein periodically to monitor serum cholesterol levels. Euthanize rabbits on day 70 by carbon dioxide overdose. Record total body and liver weights in grams. Record food consumption as grams • day "1 . Use aliquots of fresh serum for clinical chemistries, lipoprotein cholesterol determination, thiobarbituric acid reactive substances (TBARS) and compound and metabolite concentrations is serum. Freeze livers ( ⁇ 5 gram aliquots) at -20°C for compound and metabolite concentration determination at a later time.
• TBARS thiobarbituric acid reactive substances
• aldehydes such as malondialdehyde (MDA) .
• MDA malondialdehyde
• thiobarbituric acid detect the absorbance of the aldehydes at 530-540 nm.
• Measure TBARS as follows: mix 50 ⁇ L of serum with 50 ⁇ L of 0.9% saline and 400 ⁇ L of a 5mmol CuS0 4 solution and incubate at 37°C for 5 hr. Stop the reactions by addition of 1. OmL of 20% trichloroacetic acid.
• total serum cholesterol x (% area under the curve for each subfraction) .
• the compounds of formula (1) can be used as chemical antioxidant additives in organic materials normally subject to oxidative deterioration, such as, for example, rubber, plastics, fats, petroleum products and the like.
• a preservative amount of a compound of formula (1) which is sufficient in concentration to inhibit oxidative deterioration of the material to be protected, is admixed with the material subject to oxidation.
• the preservative amount of a compound of formula (1) will generally vary from about 0.01% to about 1.0% by weight.

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