3,239,417 United States Patent Office Patented Mar. 8, 1966

2 advantageously be employed on a b.i.d. or t.i.d. basis. 3,239,417 quite surprisingly not only do such compositions reduce METHODS AND COMPOSTEONS FOR INHIBITING STEROL BIOSYNTHESIS lipid biosynthesis but in addition they appear to actually Nicholas W. Di Tullio, Holmes, and William L. Holmes, reduce absorption of ingested cholesterol from the gas Rosemont, Pa., assignors to Smith Kline & French trointestinal tract. Laboratories, Philadelphia, Pa., a corporation of The pharamaceutical compositions of this invention Pennsylvania thus embrace quaternary salts of the above formula. No Drawing. Filed Dec. 7, 1962, Ser. No. 242,932 The anionic halide moiety is of relatively minor im 5 Claims. (CI. 167-65) portance since the properties of the compounds result 10 from the cationic form of the steroidal compound. Thus Our invention is concerned with methods and compo the anion need only be a pharmaceutically acceptable sitions for reducing the levels of certain sterol materials nontoxic anion and may thus be for example iodide, from the plasma and organs of animal organisms. chloride, bromide and the like. In fact, the highly singul Among the etiological factors for arteriosclerosis is an lar properties of these compounds appear to be a function abnormally high level of various steroidal lipids, notably 5 of the structure found in the A and B rings. Thus while cholesterol, in the plasma and organs of the particular the cholestane side chain at C-17 is preferred for eco subject and it has accordingly been an object of metabolic nomic reasons, other compounds containing the various research to discover agents which would inhibit the bio pregnane or testosterone C-17 groups also demonstrate synthesis of cholesterol. To a large extent such an ob the properties herein described. jective has been realized with several chemical agents 20 The preparation of the compounds utilized in this in which successfully prevent the formation of cholesterol. vention is more fully described hereafter. It involves It appears however that mere inhibition of cholesterol the preparation of a 4-aza-5a-cholestane by treatment of synthesis not infrequently results in a concomitant and a 3,5-seco-4-norcholestan-5-one-3-oic acid with a lower unduly large accumulation in the body of the cholesterol alkyl amine and subsequent hydrogenation. Introduction precursor sterols, such as lanosterol, Zymosterol and par 25 of the 3-benzyl group is next accomplished by treatment ticularly desmosterol. with benzylmagnesium bromide followed by hydrogena We have now discovered a method for inhibiting bio tion. Formation of the quaternary salt is then effected synthesis of squalene. Since this substance serves direct by standard procedures, such as treatment with a lower liy as a precursor in animal organisms for lanosterol, and alkyl halide. therefore demosterol and cholesterol, the objective of in 30 The following examples will further typify the nature hibiting cholesterol biosynthesis without substantial ac of our invention but should not be construed as a limi cumulation of squalene, lanosterol, Zymosterol and/or tation thereof. desmosterol is thus realized. In short, by virtue of inhibit Example I ing the synthesis at an early stage, the precursor thereby not utilized in cholesterol synthesis can be readily utilized 35 3,5-seco-4-norcholestan-5-on-3-oic acid (20 g., 0.05 by the body in other metabolic pathways. Since the site mole) is dissolved in 100 ml. of absolute alcohol which of inhibition is prior to the synthesis of any steroidal lipid, has been previously saturated with methylamine. The e.g., as squalene biosynthesis, the desired result of sterol solution is heated in a pressure vessel at 150 for five inhibition is effected without such side effects as fatty in hours and then concentrated to about 50 ml. and allowed 40 to cool overnight. The crystals which form are collected filtration, liver damage, hypertrophy of the adrenals and to yield 4-methyl-4-aza-5-cholesten-3-one, M.P. 98-100°. the like. Recrystallization from ethanol produces white needles, Our method involves the administration of a compound M.P. 101-102. of the formula: A solution of 10.5 g. of 4-methyl-4-aza-5-cholesten CH 45 3-one in 150 ml. of glacial acetic acid is hydrogenated C817 for 8 hours at 60° and 60 lb./in. pressure in the pres CH ence of 100 mg. of platinum catalyst. The solution is then filtered and the solvent distilled. The residue is dissolved in ether and washed with sodium bicarbonate Solution and water. After drying the solution over an / 50 hydrous sodium sulfate, the ether is removed and the res = Xe idue crystallized from aqueous alcohol. There is thus { 2-CH S./ obtained 4-methyl-4-aza-5o-cholestan-3-one, M.P. 118 - / N 120°. The melting point is raised at 121-122 by fur lower alkyl . . . lower alkyl ther crystallization. To a Grignard reagent prepared from 960 mg. (0.04 wherein X is a pharamaceutically acceptable nontoxic mole) of magnesium, 20 ml. of dry ether, and 3.8 g. anion and “lower alkyl' embraces hydrocarbon chains (0.03 mole) of benzyl chloride is added under nitrogen of from one to four carbon atoms. This administration with stirring, 4 g. (0.01 mole) of 4-methyl-4-aza-5- to the particular animal organism is accomplished by 60 cholestan-3-one in 20 ml. of toluene. The ether is re utilization of a pharmaceutical composition such as a moved by warming and an additional 20 mi. of toluene tablet, capsule, solution, suspension or the like. Depend are added. The mixture is refluxed 12 hours and a satu ing on the particular animal organism and the degree rated aqueous solution of ammonium chloride is then add of inhibition desired, a dosage of from about 0.5 mg/ ed at 0'. The organic layer is separated and the aqueous kg. to about 50 mg/kg. is utilized. Such a dosage may 65 layer extracted with ether. The combined organic ex 3,239,417 3. 4. tracts are washed with sodium chloride solution and dried animal which comprises administering orally to said ani over anhydrous sulfate. Removal of the solvent yields a mal a compound of the formula: reddish oil which is very sensitive to air oxidation. CH, Without further purification, 4.5 g. of the oil is dissolved C817 in 150 ml. of 95% ethanol and one drop of 10% hydro CE3 chloric acid is added. The mixture is treated with hy drogen for 8 hours at 60 and 200 lb./in. pressure in the presence of 500 mg. of platinum catalyst. The catalyst is / removed by filtration and the solution allowed to cool overnight. The resulting precipitate is collected and re O XG crystallized from ethyl acetate to yield 3-benzyl-4-methyl 4-aza-5a-cholestane as white needles, M.P. 122-123. Two grams of 3-benzyl-4-methyl-4-aza-5a-cholestane is lower alkyl lower alkyl then treated with an excess of methyl iodide in ether. wherein X is a pharmaceutically acceptable, nontoxi isolation of the solid and washing with ether then yields 15 halide anion. : 2. The method of reducing sterol biosynthesis in an the quaternary salt, 3-benzyl-4-methyl-4-aza-5a-cholestane animal which comprises administering orally a pharmaceu methiodide,Alternatively M.P. by 259-260. using other lower alkyl halides, as for tical composition comprising a compound of the formula: example ethyl chloride, the corresponding quaternary Chs salts are obtained. 20 Cadi Similarly methylamine may be replaced by other lower CH alkyl amines in the initial step of this procedure. Example 2 Ingredients:3-benzyl-4-methyl-4-aza-5a-cholestane Amount, mg. 25 / methiodide ------10 s Xe Calcium Sulfate, dihydrate ------200 {X-chrs/ N Sucrose ------35 lower alkyl lower alkyl Starch ------men mana mals an assa as 18 30 Talc ------9 wherein X is a pharmaceutically acceptable nontoxic Stearic acid ------3 halide anion, to said animal, said compound being present The sucrose and calicum sulfate are thoroughly mixed in said composition in quantities sufficient to supply from and passed through a #40 mesh screen. Granulation is about 0.5 mg/kg. to about 50 mg. A kg. of body weight. then effected with a hot 10% gelatin solution and the Wet 35 3. The method according to claim 2 wherein the com granulation passed through a #4 mesh screen and dried pound is 3,3-benzyl-4-methyl-4-aza-50 - cholestane meth for three hours at 120 F. The dried granulation is passed iodide. through a #14 mesh screen and mixed with the starch, 4. A pharmaceutical composition having hypocholester talc, stearic acid and 3-benzyl-4-methyl-4-aza-5o-choles olemic activity in dosage unit form comprising a com 40 pound of the formula: tane methiodide (which have previously been passed CH3 through a #60 mesh screen). The granulation is then CSH17 compressed into tablets employing a 1%2' flat faced bevel CH3 edge single score punch and die. One tablet is administered theree times a day. 45 Example 3 r Ingredients:3-benzyl-4-ethyl-4-aza-5a-cholestane Amount, mg. Y ke ethochloride ------100 Magnesium Stearate ------10 50 Lactose ------400 lower alkyl >NC lower alkyl The above ingredients are passed through a #40 mesh wherein X is a pharmaceutically acceptable nontoxic screen, mixed well and introduced into a #40 hard gelatin halide anion and a pharmaceutical carrier. capsule. 5. A pharmaceutical composition having hypocholester One capsule is administered two to three times a day. olemic activity comprising from about 10 mg. to about . Example 4 100 mg. of 36-benzyl-4-methyl-4-aza-5a-cholestane meth Ingredients: Amount, mg. iodide and a pharmaceutical carrier. 3-benzyl-4-methyl-4-aza-5o-cholestane References Cited by the Examiner methiodide ------25 60. Magnesium Stearate ------a- 5 Doorenbos: Drug Trade News, Sept. 3, 1962, pp. 48, Lactose ------375 58,Kenna: 68. J. Chem. Soc. March 1960, pp. 945-952. - The above ingredients are mixed and introduced into a #1 hard gelatin capsule. 65 JULIAN S.LEVITT, Primary Examiner. One capsule is administered two times a day. FRANK CACCLAPAGLIA, JR., LEWIS GOTTS, What is claimed is: Examiners. 1. The method of reducing sterol biosynthesis in an