Cross-Reference to Related Applications

This application is a continuation-in-part (CIP) application of United States patent application Serial No. 07/959,898, filed October 13, 1992, which is a continuation-in-part application of United States patent application Serial No. 07/667,686, filed March 8, 1991 , now abandoned, and a continution-in-part (CIP) application of PCT Application Serial No. PCT/US92/01773, filed March 3, 1992.

Field of the Invention

The present invention relates to a class of compounds useful in the treatment of diseases associated with undesirable cholesterol levels in the body, and particularly diseases of the cardiovascular system, such as atherosclerosis. Compounds of the present invention may also be useful in treating fungal infections.

Only about 7% of the total body cholesterol circulates in the plasma, where it has been linked to atherosclerosis. The remaining 93% is located in cells, where it performs vital structural and metabolic functions. Excluding the diet, which accounts for approximately one-third of the total body cholesterol, the cells obtain the necessary cholesterol by endogenous biosynthesis (Figure 1) or by removing low density lipoprotein (LDL) from the bloodstream. Approaches to the control of plasma cholesterol levels have been varied, however it has been shown that inhibiting endogenous cholesterol biosynthesis forces the cells to rely more on LDL uptake to satisfy their cholesterol requirements. Increased LDL uptake by cells, especially liver cells, has been shown to lower plasma cholesterol levels.

Squalene synthase is a microsomal enzyme that catalyzes the reductive dimerization of two molecules of famesyl diphosphate to form squalene. While farnesyl diphosphate serves as the precursor to several other biologically important compounds, squalene is utilized only for cholesterol biosynthesis. Consequently, this is the first totally committed step in the biosynthesis of cholesterol (see Figure 1). Inhibition at this step would stop only de novo cholesterol synthesis while allowing other essential pathways to isopentenyl tRNA, the prenylated proteins, ubiquinone, and dolichol to proceed unimpeded.

Inhibition of HMG-CoA reductase, an enzyme positioned early in the cholesterol biosynthetic pathway, results in a decrease of de novo cholesterol biosynthesis and an accompanying up-regulation of LDL receptors. However due to a large induction in the amount of the HMG-CoA reductase enzyme, the effect of this inhibition is blunted somewhat and the maximum LDL cholesterol reductions attainable are limited. Since inhibition of squalene synthase does not cause the same amount of enzyme induction (HMG-CoA reductase or squalene synthase), its inhibition results in a greater reduction of de novo cholesterol biosynthesis. This translates into more up-regulation of LDL receptors than is seen with an HMG-CoA reductase inhibitor and greater efficacy for lowering circulating LDL levels.

Reported Developments

The literature describes the cholesterol biosynthetic pathway and possible means for the inhibition of squalene synthase. In a series of papers including J. Am. Chem. Soc, 1982, 104, 7376-7378 and J. Am. Chem. Soc, 1989, 111, 3734-3739, C. Dale Poulter, gj a! disclose that ammonium substituted cyclopropyl polyene compounds mimic the topological and electrostatic properties of the primary cation and tertiary cation of presqualene diphosphate. and in the presence of phosphate buffer, inhibit squalene synthase. Scott A. Biller eisi in J. Med. Chem., 1988, 31, 1869-1871 disclose that a series of stable, non-ionizable analogues of farnesyl diphosphate, comprising phosphomethylene phosphate polyene compounds, inhibit squalene synthase.

Paul E. Schurr and Charles E. Day in in Lipids, Vol. 12, No. 1,22-28 describe a compound known as U-41 ,792, 1-[p-(1-adamantyloxy)phenyl]- piperidine, which is stated to cause a reduction in lower density lipoproteins, and is designated by the authors as having hypobetalipoproteinemia activity

Patent Cooperation Treaty Publication WO 92/15579 is directed to multicyclic tertiary amine polyaromatic squalene synthase inhibitors containing a multiazacyclic ring. United States Serial No. 07/997,818, filed December 29, 1992, is directed to cycloalkyi amine bis-aryl squalene synthase inhibitors. United States Serial No. 08/65,966 is directed to aliphatic amino bis-aryl squalene synthase inhibitors. International Patent Application Number PCT/US93/12638, filed December 29, 1993, is directed to cycloalkyi amine bis-aryl squalene synthase inhibitors. United States Serial No. 08/083,117, filed June 25, 1993, is directed to amino bi- and tri-carboxylic alkane bis-aryl squalene synthase inhibitors. Each of these applications is assigned to the same assignee as the present application.

United States Patent 5,135,935 assigned to Merck and Co., is directed to squalene synthase inhibitors which are aryl-oxadiazole-quinuclidines. Patent Cooperation Treaty Publication Numbers WO 92/12159, 92/12158, 92/12157, 92/12156, 92/12160 and 92/15579' all of which are assigned to Glaxo Group Ltd., are directed to bridged cyclic ketal derivatives for lowering the level of blood plasma cholesterol.

Patent Cooperation Treaty Publication Numbers: WO 93/09115 and WO

93/13096, both of which are assigned to Imperial Chemical Industries PLC, and WO 93/21184, WO 93/21183, WO 93/24486 and WO 94/03451, assigned to Zeneca Limited, are all directed to quinuclidinyl-containing squalene synthase inhibitors. All have publication dates on or after May 13, 1993 and none of these publications disclose quinolinyl phenyl 3-hydroxyquinuclidines.

The present invention is directed to a class of novel quinolinyl phenyl 3- hydroxyquinuclidines which exhibit squalene synthase inhibition properties.

Summary of the Invention

The compounds of this invention comprise 3-hydroxyquinuclidines bonded directly or through a chain to a phenylene group bonded directly or through a chain to a quinolinyl group. The compounds of this invention possess properties which reduce levels of serum cholesterol in the body without significantly reducing mevalonic metabolite synthesis and thus provide a therapeutic agent having fewer side effects than agents which act by inhibiting the HMG-CoA reductase enzyme.

This invention relates also to pharmacological compositions and method of treatment for lowering serum cholesterol levels using the compounds of this invention.

The compounds of the present invention are more specifically described by Formula I:

Formula I

B and D are independently CR'R', O, S, NR', SO, S0 ₂ , C=0, R'C-=CR' _t C≡C or a bond;

R\ R-i, R2. R3, R4. Rδ, Rδ. 7 and Re are independently hydrogen or alkyl,

R is hydrogen, alkyl, hydroxy, keto, alkoxy, acyloxy, halo, haloalkyl, amino, mono- and di-alkylamino or acylamino; and a, b, d and e are 0-4;

or a pharmaceutically acceptable salt thereof.

Description of the Drawing

Figure 1 is a schematic diagram of the biosynthetic pathway of cholesterol.

Detailed Description and Preferred Embodiments

As employed above and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Alkyl", either alone or with various substituents defined herein, means a saturated aliphatic hydrocarbon, either branched- or straight-chained. Preferred alkyl

is "loweralkyi" having about 1 to about 6 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, butyl, sec-butyl, t-butyl, amyl and hexyl.

"Alkoxy" refers to an alkyl-O-group.

The preferred acyloxy group is acetoxy.

"Halo" means a halogen. Preferred halogens include chloride, bromide and fluoride. The preferred haloalkyl group is trifluoromethyl.

The preferred acylamino group is acetylamino.

The preferred compounds of this invention are described according to Formula I wherein:

B and D are independently CR'R', O, R'C=CR', NR' or a bond;

R\ Rι, R2. R3. R4. R5. Re. R7 and Re are independently hydrogen or alkyl,

R is hydrogen, alkyl, hydroxy, alkoxy, halo or haloalkyl; and

a, b, d and e are 0-2.

Those compounds which are still more preferred are described by formula II

Formula II where:

R is hydrogen, alkyl, hydroxy, alkoxy, halo or haloalkyl;

while the most preferred compounds are described by Formulae III and IV:

Formula III

Formula IV

The compounds of this invention may be prepared by employing procedures known in the literature starting from known compounds or readily preparable intermediates.

Since the compounds of this invention have certain substituents which are necessarily present, the introduction of each substituent is, of course, dependent on the specific substituents involved and the chemistry necessary for their formation. Thus, consideration of how one substituent would be affected by a chemical reaction when forming a second substituent would involve techniques familiar to the skilled artisan. This would further be dependent on which ring was involved.

It is convenient to synthesize these molecules by employing condensation reactions at the above-described reactive B and D sites of the molecule. Exemplary general procedures are shown below.

Thus, in order to prepare those compounds where B or D is O, S or NR' the following reactions or combination of reactions are employed:

where L is a leaving group, preferably halo, tosylate or mesyJate.

The 3-hydroxyquinuclidine is protected with the usual protecting groups such as hydroborane complex on the amine or a protecting group if needed on the amine and hydroxy groups hydroxy which are removed at the appropriate time by known methods.

Where B and D are O or S, any base normally employed to deprotonate an alcohol or thiol may be used, such as sodiun hydride, sodium hydroxide, triethyl amine, sodium bicarbonate or diisopropylethylamine.

Reaction temperatures are in the range of -78°C to reflux depending on the reactants involved. (Preferably 0°C to room temperature). Reaction times vary from about 2 to about 96 hours. The reaction is usually carried out in a solvent that will dissolve both reactants and is inert to both as well. Solvents include, but are not limited to, diethyl ether, tetrahydrofuran, N,N-dimethyl formamide, dimethyl sulfoxide, dioxane and the like.

In the case for the most preferred compounds the following reactions may be carried out.

Reaction of 6-trimethylstannylquinoline with dihalobenzene in the presence of catalyst (Pd) forms the 6-(4-halophenyl)quinoline which in turn when converted to the corresponding 6-(4-lithiophenyl)quinoline and reacted with 3-quinuclidinone results in the desired 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]quinuclidine.

The reaction to form 6-(4-halophenyl)quinoline is preferably carried out in solvents such as dimethylformamide, tetrahydrofuran or dioxane in the presence of a catalyst such as Pd(OAc)2 or Pd(PPh3)4 and at a raised temperature to insure condensation. The final product is then prepared by converting the 6-(4- halophenyl)quinoline to the corresponding lithium compound by reaction with t- butyllithium at decreased temperatutes (preferably -100°C) followed by reaction with 3-quinuclidinone.

Following the same procedures the 3-hydroxy-3-[4-(quinolin-3-yl)phenyl]- quinuclidines may be prepared.

A further reaction sequence may be used which forms the 3-hydroxy- quinuclidinyl compounds in one step is as follows:

Reaction of 3-hydroxy-3-(4-bromophenyl)quinuclidine with sodium hydride to deprotonate the alcohol forms the corresponding sodium alkoxide which in tum is treated with t-butyl lithium which replaces the bromide to form the lithium compound. The later is treated with zinc chloride to form the zinc chloride which in turn is reacted with 6-quinoyltrifluoromethanesulfonate in the presence of palladium catalyst to form the desired compound.

The reaction can be carried out in an inert solvent such as tetrahydrofuran. Other deprotonating agents may be used such as KH. This reaction is carried out at raised temperature, usually at the boiling point of the solvent used. After alkoxide formation, the reaction mixture is cooled followed by Li/Br exchange and replacement with the zinc chloride. After formation of the zinc chloride, introduction of the 6-quinoyltrifiuoromethanesulfonate is carried out at room temperature at which time a catalyst is added (tetrakis(triphenylphosphine)-palladium is preferred) and the reaction mixture again heated as before. Workup is in the normal manner by cooling, quenching (methanol) and extracting.

It is convient for purification to convert the reaction product to a pharmaceutically acceptable salt in the normal manner.

iVteOH

In a similar manner the 3-quinolinyl compounds may also be prepared.

The starting materials are either known or may be prepared by the following reactions.

The quinolinylstannanes may be prepared from the corresponding halide (preferably bromide or iodide) by conversion to the quinolinyllithium (by reaction with t-butyllithium at decreased temperatutes (preferably -78°C) followed by reaction with a halotrialkylstannane.

The triflates may be prepared from the corresponding alcohol with triflic anhydride (trifluoromethanesulfonic anhydride) in pyridine.

The 3-hydroxy-3-(4-bromophenyl)quinuclidine is prepared from dihalobenzene and 3-quinuclidinone. The dihalobenzene is reacted with n- butyllithium in an inert solvent at decreased temperatures (-78°C) followed by addition of the 3-quinuclidinone, warming slightly to complete the reaction, quenching and extracting.

In the case where B or D is SO or Sθ2, then treatment of the thio compound with m-chlorobenzoic acid or sodium periodate results in the sulfinyl compound. Preparation of the sulfonyl compound may be accomplished by known procedures such as dissolving the sulfinyl compound in acetic acid and treating hydrogen peroxide, preferably about 30% aqueous H2O2.

Condensation of the ketone with an appropriate hydroxylamine results in the formation of the oxime, while Wittig condensation of the ketone using Ph ₃ P=CH2 results in the methylene compounds; Wittig condensation also may take place at the D position of the molecule of Formula I as follows:

-C-R'

This may be carried out using normal Witting reaction conditions. When the appropriate aldehyde or ketone is reacted with a Wittig reagent then condensation

results in formation of the double bond. This may then be reduced catalytically by known procedures such as Pd/C or any other suitable hydrogenating condition. The Wittig reagent is prepared by known art recognized procedures such as reaction of triphenylphosphine or diethylphosphone, with a substituted alkyl bromide followed by treatment with a strong organometallic or alkoxide base, such as n-BuLi or NaOH, results in the desired ylide. Of course this Witting condensation may also take place when the Wittig reagent is formed on the quinuclidine position of the molecule which is then condensed with the appropriate aldehyde.

Halogenation with Br in CCU on the double bond followed by double dehydration with NaNH / liq NH3 results in the triple bond compounds.

Of course preparation of the compounds of this invention are not limited to the above procedures and one skilled in the art can prepare compounds of this invention by known processes found in the literature. Thus for example, 3- hydroxy-3-[4-(quinolin-2-yl)phenoxymethyl]-1 -azabicyclo[2.2.2]octane may be prepared by the following scheme.

Conversion of the alcohol to the lithium methoxide may be carried out by the procedures of W. Clark Still in J. Am. Chem. Soc 1978, 100,1481.

A further example is the following preparation of 3-hydroxy-3-[4-(quinolin-3- yl)styryl]-1-azabicyclo[2.2.2]octane where the final step of the scheme may be carried out by the procedure of Frank, W. O; Kim, Y. C; and Heck, R. F. J. Org. Chem. 1978, 43, 2947.

When 3-bromoquinoline is replaced by 6-bromoquinoline, 3-bromo-7- methoxyquinoline or 7-bromoquinoline then the corresponding products are prepared.

The compounds of this invention have at least one asymmetric carbon atom at the 3-hydroxyquinuclidinyl position. Further, certain compounds of this invention may exist in their cjs or trans configuration such as those compounds where B or D is CR'=CR'. As a result, those compounds of this invention may be obtained either as racemic mixtures, diastereoisomeric mixtures or as individual enantiomers. When two or three asymmetric centers are present the product may exist as mixtures of two or four diastereomers. Of course it is understood that certain other compounds within the scope of this invention could have a number of stereocenters. In general, a compound with x stereocenters can have a maximum of 2 ^X stereoisomers. Therefore, a compound having three such centers gives rise to a maximum of eight stereoisomers, while one having four produces sixteen, etc.

The product may be synthesized as a mixture of the isomers and then the desired isomer separated by conventional techniques such as chromatography or fractional crystallization from which each diastereomer may be resolved. On the other hand, synthesis may be carried out by known stereospecific processes using the desired form of the intermediate which would result in obtaining the desired stereospecificity.

Reference to the separation of cjs and trans isomers by chromatography may be found in W.K. Chan, el aj, J. Am. Chem. Soc. 9J5, 3642, 1974.

It is to be understood that the scope of this invention encompasses not only the various isomers which may exist but also the various mixture of isomers which may be formed.

The resolution of the compounds of this invention and their starting materials may be carried out by known procedures. Incorporation by reference is hereby made to the four volume compendium Optical Resolution Procedures for Chemical Compounds: Optical Resolution Information Center, Manhattan College, Riverdale, New York. Such procedures are useful in the practice of this invention. A further useful reference is Enantiomers. Racemates and Resolutions: Jean Jacques, Andre Collet and Samuel H. Wilen; John Wiley & Sons, Inc., New York, 1981. Basically, the resolution of the compounds is based on the differences in the physical properties of diastereomer salts. Conversion of the racemates into a mixture of diastereomer salts, by crystallization with a chiral acid results in forms that are separable by fractional crystallization.

The present compounds form salts with acids when a basic amino function is present and salts with bases when an acid function, i.e., carboxyl, is present. All such salts are useful in the isolation and/or purification of the new products. Of particular value are the pharmaceutically acceptable salts with both acids and bases. Suitable acids include, for example, hydrochloric, sulfuric, nitric, benzenesulfonic, toluenesulfonic, acetic, maleic, tartaric and the like which are pharmaceutically acceptable. Basic salts for pharmaceutical use are the Na, K, Ca and Mg salts.

Various substituents on the present new compounds can be present in the starting compounds, added to any one of the intermediates or added after formation of the final products by known methods of substitution or conversion reactions. If the substituents themselves are reactive, then the substituents can

themselves be protected according to the techniques known in the art. A variety of protecting groups known in the art, may be employed. Examples of many of these possible groups may be found in "Protective Groups in Organic Synthesis" by T.W. Green, John Wiley and Sons, 1981. For example, nitro groups can be added by nitration and the nitro group converted to other groups, such as amino by reduction, and halo by diazotization of the amino group and replacement of the diazo group. Amino groups can be alkylated to form mono- and di-alkylamino groups. Thus, substitution or alteration reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product.

The compounds of the present invention may be prepared by the following representative examples.

EXAMPLE 1

3-hvdroxy-3-f4-(αuinolin-6-vπphenvn-1-azabicvclor2.2.21 octane dihvdrochloride

Step A 6-quinolyl-trifluoromethanesulfonate

6-Hydroxyquinoline (10.2 g, 0.0703mol) is partially dissolved in a mixture of dichloromethane (200 mL) and pyridine (17 mL, 0.211 mol) and cooled to 0 °C. Trifluoromethanesulfonic anhydride (12.4 mL, 0.074 mol) is added dropwise over 15 minutes with stirring. The reaction is diluted with dichloromethane (300 mL) and washed with 0.1 M HCI (3 X 400 mL) followed by saturated NaHC0 ₃ (1 X 300 mL). The solution is dried (MgS04), filtered and concentrated to yield a low melting solid, 6-quinoyltrifluoromethanesulfonate, which is used directly in the next step.

Step B 3-(4-bromophenyl.-3-hvdroxyαuinuclidine

1 ,4-Dibromobenzene (29.5 g, 0.125 mol) is dissolved in dry THF (600 mL) and cooled to -78 °C. A solution of n-butyllithium (50.0 mL, 0.125 mol, 2.5 M in ether) is added slowly over 15 minutes. A solution of 3-quinuclidinone (15.6 g, 0.125 mol) in dry THF (50 mL) is added over 5 minutes and the reaction is allowed to warm to -50 °C. The reaction is then poured into water (300 mL) and extracted with ether (250 mL). The ether is washed with water (1 X 100 mL) then dried (MgS04) and filtered. The filtrate is cooled in ice to yield 3-(4-bromc- phenyl)-3-hydroxyquinuclidine (m.p. 191-193 °C).

Step C 3-hvdroxy-3- ^r ι4-ιquinolin-6-vnphenyl1-1-azabicvclor2.2.21octane

3-(4-Bromophenyl)-3-hydroxyquinuclidine (5.14 g, 0.0182 mol) is dissolved in dry tetrahydrofuran (150 mL) and sodium hydride (1.02 g, 0.026 mol, 60% in oil) is added. Imidazole (40 mg, .00058 mol) is added as a catalyst and the reaction is heated at 60 °C for 5 h. The reaction is cooled to -78 °C and t- butyllithium (24 mL, 0.041 mol, 1.7 M in pentane) is added slowly over 15 minutes. After 30 minutes, a solution of zinc chloride (47 mL, 0.047 mol, 1.0 M in ether) is added slowly over 10 minutes. After 10 minutes the reaction is warmed to room temperature and stirred 20 minutes. 6-Quinoyltrifluoromethanesulfonate (5.73 g, 0.0207 mol) is added, followed by tetrakis(triphenylphosphine)-palladium(0) (0.67 g, 0.58 mmol), and the reaction is heated at 60 °C for 14 h. The reaction is cooled to 0 °C and carefully quenched by the addition of methanol (20 mL). After the reaction is concentrated, the residue is dissolved in 10% MeOH/CH2Cl2 (750 mL) and washed with a saturated solution of Na2Cθ3 (150 mL). The aqueous layer is back-extracted with 10% MeOH/CH2Cl2 (2 X 150 mL). The organic solutions are combined, dried (MgSθ4) and concentrated to yield an oil which is chromatographed on basic alumina using 5% MeOH/CH2Cl2 as the eluant. The isolated product is recrystallized from chloroform to yield 2.2 g of 3-hydroxy-3-[4- (quinolin-6-yl)phenyl]-1-azabicyclo[2.2.2]octane as a white solid.

Step D 3-hvdroxy-3-f4-ιαuinolin-6-yπphenyl1-1-azabicvclor2.2.2lo ctane dihvdrochloride

3-Hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo[2.2.2]octane (2.73 g, 8.26 mmol) is added to a stirring solution of methanol (100 mL) followed by the slow addition of a solution of HCI in ether (17.3 mL, 17.3 mmol). After 5 minutes the reaction is concentrated in vacυo, dissolved in de-ionized water (40 mL) and lyophylized to yield 3.3 g of 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1- azabicyclo[2.2.2]octane dihydrochloride (m.p. dec. 274 °C) as a pale yellow solid.

EXAMPLE 2 3-hvdroxy-[4-f2-methoxyαuinolin-6-vπphenvn-1-azabicvclor2. 2.21octane

Step A 6-bromo-2-hydroxyαuinoline

To a solution of 16 g (110.3 mmol) of 2-hydroxyquinoline in 225 ml of glacial acetic acid is added 5.69 ml (110.3 mmol) of bromine dropwise over 3 minutes. The thick mixture is stirred at room temperature for 30 minutes and is then diluted with 250 ml of water slowly. The mixture is stirred for 15 minutes and is then filtered. The filter cake is air dried and recrystallized from 150 ml of ethanol to give 6-bromo-2-hydroxyquinoline (m.p. 262-265°C).

Step B 6-bromo-2-chloroαuinoline

A mixture of 12.5 g (55.8 mmol) of 6-bromo-2-hydroxyquinoline in 40 ml of phosphorus oxychloride is heated in an oil bath of 125°C for 1 hour. The temperature is then raised and the phosphorus oxychloride is distilled. The residue is cooled and dissolved in chloroform. The solution is slowly added to cold aqueous sodium hydroxide. The organic layer is washed with water and is dried over magnesium sulfate and is then filtered. The filtrate is evaporated to give 6- bromo-2-chloroquinoline (m.p. 156-159°C).

Step C 6-bromo-2-methoxyquinoline

To a suspension of 12 g (49.6 mmol) of 6-bromo-2-chloroquinoline in 350 ml of methanol is added 19.2 ml (84 mmol) of 25% sodium methoxide (Aldrich). The mixture is heated under reflux for 18 hours. The mixture is evaporated and the residue is dissolved in dichloromethane and is then washed with water. The organic layer is dried over magnesium sulfate and is filtered. The filtrate is evaporated to give 10.6 g of product (m.p. 96-98°C).

Step D 6-(4-bromophenvπ-2-methoxyαuinoline

To a solution of 10.6 g (44.5 mmol) of 6-bromo-2-methoxyquinoline in 250 ml of anhydrous THF cooled to -78°C is added 17.8 ml (44.5 mmol) of 2.5M n- butylithium hexane solution dropwise over 3 minutes. The mixture is then stirred for 5 minutes and 44.5 ml (44.5 mmol) of 1M zinc chloride is added. The cooling bath is removed and the mixture, is stirred for 1.5 hours warming to room temperature. This solution is added to a mixture of 12.5 g (44.5 mmol) of 1-bromo-4- iodobenzene and 2.5 g (2.2 mmol) of tetrakis(triphenylphosphine)palladium (0) in 100 ml of THF. The mixture is stirred at room temperature for 2 hours and is then added to 250 ml of water. The mixture is extracted with ether. The ether is dried

over magnesium sulfate and is filtered. The filtrate is evaporated and the residue recrystallized from hexane to give 6-(4-bromophenyl)-2-methoxyquinoline (m.p. 134-136°C).

Step E 3-hvdroxy-f4-(2-methoxyquinolin-6-yl.phenvM-1- azabicvclor2.2.21octane

To a solution of 7.9 g (25.15 mmol) of 6-(4-bromophenyl)-2- methoxyquinoline in 125 ml of anhydrous THF cooled to -78°C is added 10 ml (25 mmol) of 2.5M n-butyllithium hexane solution dropwise over 3 minutes. The mixture is stirred for 5 minutes and a solution of 3.15 g (25.15 mmol) of 3- quinuclidinone (prepared from the hydrochloride salt) in 25 ml of THF dropwise over 3 minutes. The cooling bath is removed and the mixture is stirred for 30 minutes warming to about 0°C. The mixture is poured into water and is extracted with ether. The ether is dried over magnesium sulfate and is then filtered. The filtrate is evaporated and the residue is triturated with 50 ml of cold ethanol. The insoluble material is collected and is recrystallized from ethyl acetate to give 3- hydroxy-[4-(2-methoxyquinolin-6-yl)phenyl]-1-azabicyclo-[2.2 .2]octane (m.p. 202-204°C (dec)).

Analysis Calc for C2 ₃ H24N2O2 - 1/4 H 0

C: 75.69 Found C: 75.85

H: 6.77 H: 6.62

N: 7.68 N: 7.59

EXAMPLE 3 3-hvdroxy-3-[4-ι ^/ αuinolin-3-vπphenyl1-1-azabicvclof2.2.2]octane dihvdrochloride

Step A 3-trimethylstannylquinoline

To a solution of 1.51 g (7.27 mmol) of 3-bromoquinoline in 30 ml of Et2θ, cooled to -78°C, is added 9.40 ml (16.0 mmol 220 M%) t-BuLi 1.7 M/pentane dropwise and then stirred under N2 at -78°C for 15 minutes (yellow-orange suspension). To this is injected 2.17 g (10.90 mmol) of MesSnCI dropwise and when the addition is complete 10 ml of E.2O. During the addition the solution becomes more homogenous and is orange-red in color while toward the end it becomes a heterogeneous light yellow suspension. The dry ice/acetone both is

removed and the reaction is allowed to come to R.T. then quenched with 5 ml MeOH. The solution is partitioned between water and EtOAc and then washed with brine, dried (MgSθ4) and chromatographed with 10% EtOAc/hexane to obtain 3-trimethylstannylquinoline which is used directly in the next step.

Step B S-i -bromophenvπαuinoline

A mixture of 2.0 g (6.85 mmol) of 3-trimethylstannylquinoline, 0.39 g (.34 mmol) of tetrakis(triphenylphosphine)palladium (0) and 1.9 g (6.85 mmol) of 1- bromo-4-iodobenzene in 20 ml of anhydrous DMF is heated under an atmosphere of nitrogen in an oil bath at 135°C for 15 minutes. The black mixture is poured into 150 ml of 50% aqueous ammonium hydroxide and 200 ml of ether is added. The mixture is stirred for 1 hour. The ether layer is dried over magnesium sulfate and filtered. The filtrate is evaporated and the residue is recrystallized from hexane to give 3-(4-bromophenyl)quinoline (m.p. 125-129°C).

Step C 3-hvdroxy-3-f4-fquinolin-3-vπphenyl1-1-azabicvclo[2.2.2]oct ane dihydrochloride

To a solution of 0.7 g (2.46 mmol) of 3-(4-bromophenyl)quinoline in 20 ml of anhydrous THF cooled to -78°C is added 1 ml (2.5 mmol) of 2.5M n-butyllithium hexane solution dropwise over 2 minutes. The mixture is stirred for 5 minutes and a solution of 0.3 g (2.46 mmol) of 3-quinuclidinone (prepared from the hydrochloride salt) in 5 ml of THF is added dropwise over 1 minute. The cooling bath is removed and the mixture is stirred for 45 minutes warming to room temperature. The mixture is then added to water and extracted with ether. The ether is dried over magnesium sulfate and filtered. The filtrate is evaporated and the residue recrystallized from ethyl acetate. The solid is dissolved in ethanol and is acidified with ethanolic HCI. A few ml of ether is added to give 3-hydroxy-3-[4- (quinolin-3-yl)phenyl]-1-azabicyclo[2.2.2]octane dihydrochloride (m.p. 222-225°C (dec)).

Analysis Calc for C22H24CI2N2O H ₂ 0

C: 62.71 Found C: 62.83

H: 6.22 H: 6.28

N: 6.65 N: 6.43

EXAMPLE 4 3-hydroxy-3-f4-fquinolin-6-vhphenvn-1-azabicyclof2.2.2loctan e

Step A 6-ι4-bromophenyπquinoline

A mixture of 2.50 g (8.56 mmol) 6-trimethylstannylquinoline and 2.67 g (9.42 mmol) 4-iodobromobenzene in 30 mL of DMF and 4.95 mg (0.43 mmol) of Pd(PPh ₃ )4 is heated to 100°C under N2 for 9 hours. The reaction mixture is evaporated to dryness and partitioned between CH2CI2 and 15% NH4OH and stirred vigorously for 15 minutes, separated, dried (MgSθ4), evaporated and chromatographed (CH2CI2) to obtain 6-(4-bromophenyl)quinoline which is used directly in the next step.

Step B 3-hvdroxy-3-r4-ιαuinolin-6-vπphenvn-1-azabicvclor2.2.2loc tane

To a solution of 6-(4-bromophenyl)quinoline 5.00 g (17.6 mmol) in 25 ml THF and 25 ml anhydrous ether in an inert atmosphere and cooled to about -120°C using pentane/liquid N2 bath is injected slowly 8.8 ml (19.4 mmol, 1.1 eq) 2.2 N n- BuLi and stirred at -105°C for about 1 hour. To this is injected 2.42 g (19.4 mmol, 1.1 eq) of quinuclidinone in 10 ml THF, warmed until the internal temperature reaches about -20°C, quenched with 200 ml 1 N HCI and stirred with 100 ml ether for a few minutes. The reaction mixture is separated from the aqueous and organic layers and the aqueous is neutralized with a previously cooled solution of 8 g NaOH in 50 ml H2O. This is then extracted 2x with CH CI ₂ , dried (Na S04) and evaporated to dryness. The residue is chromatographed using 10% CH ₃ OH/CH2CI2 until all less polar products come over and then 10% CH ₃ θH/10% (Me3)2EtN/CH2Cl2. The latter fractions are evaporated, dissolved in CH2CI2, washed 1x with 1N NaOH, dried (Na2S04) and evaporated to dryness to obtain 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo[2.2.2]octane.

EXAMPLE 5 M 3-(4-bromophenvfl-3-hvdroquinuclidine 3-(4-brornophenvfl-3-hvdroquinuclidine

Step A

To a hot mixture of 20 ml of acetone and 20 ml of ethyl acetate is dissolved in 1.1 g (3.9 mmol) of 3-hydroxy-3-(4-bromophenyl)quinuclidine. To this solution is added a solution of 1.39 g (3.9 mmol) of dibenzoyl-L-tartaric acid in

2 ml of acetone in one portion. The mixture is allowed to stand at room temperature for 5 minutes and is filtered. The clear solution is allowed to stand at room temperature for 24 hours. The precipitate which forms is collected to give 0.3 g. This solid is dissolved in hot ethanol and the solution is diluted with petroleum ether to the cloudy point and is allowed to stand at room temperature for 6 hours. The solid which forms is collected to give 154°C (dec). This compound is dissolved in 50% aqueous ethanol and the solution is basified with a sodium carbonate solution to give 216-218°C; >99.5% by analytical HPLC.

Step B

The original acetone-ethyl acetate filtrate is evaporated and the residue is treated with aqueous sodium hydroxide and extracted with chloroform. The chloroform is dried and evaporated to give 0.3 g. This solid (1.06 mmol) is dissolved in a hot mixture of 10 ml of acetone and 10 ml of ethyl acetate and a solution of 0.38 g (1.06 mmol) of dibenzoyl-d-tartaric acid in 1 ml of acetone is added. The solution is allowed to stand at room temperature for 24 hours. The precipitate which forms is collected to give 0.4 g. This solid is dissolved in ethanol and the solution is diluted with petroleum ether to the cloudy point and is allowed to stand at room temperature for 6 hours. The solid is collected to give 154°C (dec). This compound is dissolved in 50% aqueous ethanol and the solution is basified with a sodium carbonate solution to give 217-217°C; .99.5% by analytical HPLC.

Step C Preparative Chromatography Conditions:

Column: Chiralcel OD, 10 urn particle size, 250 mm X 20 mm

ID,

Daicel Inc.

Mobile Phase: Heptane:Ethanol (92:8)

Flow rate: 15 mLmin.

Detection: UV absorbance at 230 nm

Injection

Volume: 2 mL

Amount Injected: 40 mg

Retention Enantiomer l: 17 minutes Times: Enantiomer II: 23 minutes

EXAMPLE 6

(+) 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo[2.2.2]- octane dihydrochloride ^*

(-) 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo[2.2.2]- octane dihydrochloride .

When the procedure of Example 1 , Steps C and D are followed, and 3-(4- bromophenyl)-3-hydroxyquinuclidine is replaced by the enantiomers of Example 5, then the compounds prepared are (+) 3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1- azabicyclo[2.2.2]octane dihydrochloride and (-) 3-hydroxy-3-[4-(quinolin-6- yl)phenyl]-1 -azabicyclo[2.2.2]octane dihydrochloride.

Preparative Chromatography Conditions:

Column: Chiralcel OD, 10 urn particle size, 250 mm x 20 mm ID, Daicel, Inc.

Mobile Phase: Ethanol containing 0.1% DEA Flow rate: 7 mlJmin. Detection: UV absorbance at 320 nm Injection Volume: 2 mL Amount Injected: 50 mg Retention Times: Enantiomer l: 12 minutes Enantiomer II: 20 minutes

Representative compounds which may be prepared according to the above examples are as follows:

Various tests have been carried out to show the ability of the compounds of the present invention to exhibit pharmacological responses that can be correlated with activity in animals, rapticularly in humans. These tests involve such factors as the effect of the compounds of Formula I to inhibit squalene synthesis. It has been found that compounds within the scope of this invention when tested using the following procedures show a marked activity for the inhibition of squalene synthase and hence are believed to be useful in the treatment of cholesterol-related disorders in humans and other animals.

Squalene Synthase Inhibition Assay

The squalene synthase assay used is described by Amin et al.in

"Bisphosphonates Used for the Treatment of Bone Disorders Inhibit Squalene Synthase and Cholesterol Biosynthesis," Journal of Lipid Research, 33: 1657- 1663 (1992).

I. Preparation of Assay Substances:

A) Test Solutions: Test solutions are prepared fresh in 100% DMSO or dH ₂ θ. Subsequent dilutions are made in the same solvent. Compounds are tested initially at 1 μM (final concentrations).

B) Assay Buffers: Potassium Phosphate (50 mM,) pH 7.4, and HEPES (4-(2-hydroxyethyl)-

1-piperazineethanesulfonic acid, 50 mM) pH 7.4 stock buffers are prepared and stored at 4°C until use.

C) Microsomal Enzyme Preparation: Fresh livers from male Sprague-Dawley rats (Taconic Farms, Germantown,

NY) weighing 150-200 g are collected after exsanguination. All subsequent procedures are performed at 4°C. The liver is homogenized in the assay buffer (50 mM, pH 7.4). Cellular fractions are separated as described by Popjak, G. in "Enzymes of sterol biosynthesis in liver and intermediates of sterol biosynthesis," Meth. Enzymol. 15: 393-454 (1969). Microsomes are prepared by centrifugation (100,000 g) and then resuspended in the assay buffer. Microsomes are rehomogenized with a motor-driven Teflon pestle to yield a uniform suspension (-30 mg protein/ml), aliquoted, and stored at -80°C until use.

II. Sαualene Svnthase Assay

The procedure is a modification of those described by Popjack (ibid) and Poulter et al. in "Squalene synthase Inhibition by ammonium analogues of carbocationic intermediates in the conversion of presqualene diphosphate to squalene" J. Am. Chem. Soc 111: 3734-3739 (1989). The assay is performed in 1 ml of 50 mM assay buffer, pH 7.4, containing 10 mM MgCl2, 0.5 mM NADPH, microsomes (30 μg protein), a bisphosphonate dissolved in distilled water, and substrate [ ³ H]FPP (0.5 μM, 0.27 Ci/mmol) in a 16x125 mm glass screw-cap tube. All components except [ ³ H]FPP are preincubated for 10 min. at 37°C. The reaction is initiated by the addition of pH]FPP. After 10 min at 37°C, the reaction is terminated by the addition of 1 ml 15% KOH in ethanol. The tubes are incubated at 65°C for 30 min. to solubilize proteins. The mixture is extracted with 5 ml petroleum ether for 10 min. After freezing the lower aqueous phase, the organic

phase is transferred to glass tubes containing 2 ml distilled water. After washing the lower aqueous phase is frozen and the petroleum ether phase is removed and counted with 10 ml Ready Safe liquid scintillation cocktail using a Beckman LS-9000 scintillation counter. DPM values are adjusted against a blank (no enzyme).

The difference in radioactivity in the presence and absence of the test compound is used to determine the level of inhibition. The IC50 values are calculated using a linear regression program of Tallarida and Murray (1987). Tallarida, R.J. and Murray, R.B. Manual of pharmacologic calculations with computer programs. Springer- Verlag, 1987.

The following table shows examples of representative compounds of this invention and their test results as determined in the squalene synthase inhibition assay.

COMPOUND IC ₅₀ (as % inhibition)

A further test which shows the hypolipidaemic activity of squalene synthase inhibitor in marmosets is carried out as follows:

Animals

Male Common Marmosets (Callithrix jacchus) housed in small groups (1-3) with access to food (CPDX primate diet) and tapwater ad libitum are used. Animals are aged 9 months to 5 years and bodyweight is about 300 g.

Blood Sampling

Animals are routinely bled (0.5 ml) from the femoral vein after an overnight fast. In order to reduce stress to the animals, marmosets are sedated with a neuroleptanalgesic (fentanyldroperidol, 0.05-0.1 ml Thalamonal, Janssen, intramuscularly), placed in a restrain device and 0.5 ml blood is obtained by venipuncture using a 25G hypodermic needle attached to a 1 ml syringe. Blood is placed in sample cups containing EDTA as anticoagulant and mixted well. Bleeding takes place between 10.00 and 12.00 h. The dosing period is 1 week only with blood samples taken on 0 and +7.

Assay of Plasma Cholesterol Concentration

Plasma is prepared by centrifugation at 2000 x g and aliquots are assayed for cholesterol concentration by means of a COBAS Bio centrifugal analyser using an enzymatic kit (Cholesterol C-system, Boehringer Mannheim GmbH). Apo A1 and Apo B levels are determined immunoturbidimetrically.

Plasma transaminase levels (ALAT, ASAT) are measured during the experimental phase.

Allocation to Treatment Groups

Plasma cholesterol levels range from about 2 to 6 mM in the animals. At the start of the dosing period, plasma cholesterol levels in 30 animals are used and are ranked and the rank is divided into 10 blocks of 3. One animal from each block is assigned randomly to each treatment group.

Drug Preparation and Administration

The control sample is ground in a pestle and a mortar and then blended with sodium CMC (1%): Tween 80 (0.5%) with further grinding to produce a 25 mg/ml suspension. Suspension is prepared freshly every 3-4 days and stored at 4°C in

foil-wrapped tubes. The compound is dissolved in water at a concentration of 2.5 ^* mg base/ml, sufficient solution for 2 doses being prepared each afternoon. Animals receive 2 ml/kg of the appropriate suspension or solution via a stomach tube. The control group receives vehicle alone. Dosing takes place twice daily between 0.800 and 0.900 h and 20.00 and 21.00 h.

^* N.B. As the compound is a hydrochloride (90.8% base), 1.1 times the weight of base are required.

Statistical Analysis

The blocked design is used and permits analysis by:

(i) Two-way analysis of variance (ii) Covariate analysis

Covariate analysis is also particularly useful considering the wide range of predose plasma cholesterol concentrations in these animals.

Compounds within the scope of Formula I have been tested by the foregoing assay procedures and exhibit marked squalene synthase inhibition activity and are useful as hypocholesterolemic or hypolipidemic agents by virtue of their ability to inhibit the biosynthesis of cholesterol. Having such ability, the compounds are incorporated into pharmaceutically acceptable carriers and administered to a patient in need of such cholesterol biosynthesis inhibition. These pharmaceutical formulations contain at least one compound according to this invention.

Treatment with a combination of an HMG-CoA reductase inhibitor and a squalene synthase inhibitor would have a synergistic effect on inhibiting cholesterol biosynthesis. Inhibiting the squalene synthase enzyme and the HMG-CoA reductase enzyme at the same time would most closely resemble the physiological conditions of cholesterol homeostasis. A squalene synthase inhibitor could keep cellular concentrations of farnesyl diphosphate high enough for the synthesis of the small amounts of dolichol, ubiquinone, and the farnesylated proteins required by the cell. This would maintain some feedback regulation of the

HMG-CoA reductase enzyme and allow smaller amounts of the HMG-CoA reductase inhibitor to be used.

Other combinations with a squalene synthase inhibitor which could have a beneficial effect for controlling undesirable cholesterol levels in the body include niacin, antihyperlipoproteinemic agents such as gemfibrozil, cholesterol absorption inhibitors, bile acid sequestrants, antioxidants and lipoxygenase inhibitors.

Compounds of the present invention which inhibit squalene synthase may also be of use in combating fungal infections in animals and humans. They may be useful in the treatment of variety of systemic infections and treating tropical infections. They may be also useful as prophylactic agents to prevent systemic and tropical fungal infections. Prevention of fungal overgrowth during antibiotic treatment may also be desirable in some disease syndromes.

Compounds may be tested under a spectrum of activity against a panel of representative yeasts, filamentous fungi and bacteria. The ability of compounds of the invention to inhibit the enzyme squalene synthase in fungi and bacteria may be demonstrated in vitro using (14C)FPP as a substrate under assay conditions similar to those described by S. A. Biller et al. in J. Medicinal Chemistry 31 (10), 1869-1871 (1988), or Amin (ibid).

The in vitro evaluation of the anti-fungal activity of compounds of the invention can be performed by determining the minimum inhibitory concentration (MIC) which is the concentration of the test compound in a suitable medium at which growth of a particular microorganism fails to occur.

The compounds of the present invention can be administered to a mammalian host in a variety of forms adapted to the chosen route of administration, i.e., orally, or parenterally. Parenteral administration in this respect includes administration by the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelially including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation and aerosol and rectal systemic.

The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 6% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 50 and 300 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens a preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under

ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent of dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

The therapeutic compounds of this invention may be administered to a patient, and especially a mammalian patient such as a human in need of cholesterol lowering treatment, alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and

chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.

The physician will determine the dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment and it will vary with the form of administration and the particular compound chosen, and also, it will vary with the particular patient under treatment. He will generally wish to initiate treatment with small dosages by small increments until the optimum effect under the circumstances is reached. The therapeutic dosage will generally be from about 0.1 to about 1000 mg/day, and preferably from about 10 mg to about 100 mg/day, or from about 0.1 mg to about 50 mg/kg of body weight per day and preferably from about 0.1 to about 20 mg/kg of body weight per day and may be administered in several different dosage units. Higher dosages, on the order of about 2x to about 4x, may be required for oral administration.