Methods of Treating Atherosclerosis With Dialkylureas and Dialkylthioureas

This invention relates to a method for treating atherosclerosis in a manner designed to prevent, arrest, or reverse the course of the disease; using dialkylureas and dialkylthioureas. The compounds of the present invention are antiatherosclerotic agents capable of ameliorating atherosclerosis by counteracting the formation or development of atheromatous lesions in the arterial wall of mammals. The invention also relates to the chemical synthesis of the compounds disclosed herein. In addition, the invention pertains to novel pharmaceutical compositions for the utilization of these compounds in the treatment of disease in mammals.

A variety of urea and thiourea compounds can be found in the literature, for example, in J. Med. Chem. 18, 1024 (1975); U.S. Pat. Nos. 2,688,039; 3,335,142; 3,856,952; 3,903,130; and in West German Offenlegungsschrift No. 29 28 485. The compounds found in the literature are disclosed as being useful, herbicides, plant growth regulators, bactericides, pesticides, fungicides, algecides, photographic sensitizers, antihelmintics, sympatholytics and antivirals. Those urea compounds found in Offenlegungsschrift 29 28 485 are disclosed as useful in inhibiting lipid absorption. There are, however, no literature references disclosing the use of the dialkylureas and dialkylthioureas of this invention in the treatment of atherosclerosis or hyperlipidemia.

Atherosclerosis is a form of arteriosclerosis characterized by lipid accumulation in and thickening of the arterial walls of both medium and large-sized arteries. Arterial walls are thereby weakened and the elasticity and effective internal size of the artery is decreased. Atherosclerosis is the most common cause of ischemic heart disease and is of great medical importance since the occlusion of medium and large-sized arteries diminishes the supply of blood to vital organs such as the heart muscles and the brain. The sequelae to atherosclerosis include ischemic heart disease, heart failure, lift-threatening arrythmias, senility, and stroke.

The fact that cholesterol is a major component of atherosclerotic lesions or plaques has been known for more than 100 years. Various researchers have studied the role of cholesterol in lesion formation and development and also, more importantly, whether lesion formation can be prevented or lesion development arrested or reversed. Atheromatous lesions have now been shown [Adams, et al., Atherosclerosis, 13, 429 (1974)] to contain a greater quantity of esterified as opposed to unesterified cholesterol than the surrounding undiseased arterial wall. The intracellular esterification of cholesterol with fatty acids is catalyzed by the enzyme Fatty acyl CoA:cholesterol acyl transferase or ACAT and the accumulation and storage of cholesteryl esters in the arterial wall is associated with increased activity of this enzyme [Hashimoto and Dayton, Atherosclerosis. 28, 447 (1977)]. In addition, cholesteryl esters are removed from cells at a slower rate than unesterified cholesterol [Bondjers and Bjorkerud, Atherosclerosis, 15, 273 (1972) and 22, 379 (1975)]. Thus, inhibition of the ACAT enzyme would diminish the rate of cholesterol esterification, decrease the accumulation and storage of cholesteryl esters in the arterial wall, and prevent or inhibit the formation and development of atheromatous lesions. The compounds of the present invention are very potent inhibitors of the ACAT enzyme. Thus, these compounds are useful for controlling and reducing the cholesteryl ester content of mammalian arterial walls, and decreasing the accumulation and storage of cholesterol in the arterial walls of mammals. Further, the compounds of this invention inhibit the formation or development of atherosclerotic lesions in mammals.

The evidence that hyperlipidemia is one of the factors involved in coronary heart disease is very impressive. A most important study carried out in Framingham, Mass. (Gordon and Verter, 1969) in over 5,000 persons for more than 12 years established a correlation between high concentrations of blood cholesterol and increased risk of heart attack. Although the causes of coronary artery disease are multiple, one of the most constant factors has been the elevated concentration of lipids in the blood plasma. A combined elevation of choelsterol and triglycerides has been shown (Carlson and Bottiger, 1972) to carry the highest risk of coronary heart disease. The majority of patients with ischemic heart disease or peripheral vascular disease were found to have hyperlipoproteinemia, involving very low-density and/or low-density lipoproteins (Lewis, et al., 1974).

We have now found that certain members of this class of compounds can safely and effectively lower serum lipids in warm-blooded animals. Such action on serum lipids is considered to be very useful in the treatment of atherosclerosis. For some time it has been considered desirable to lower serum-lipid levels and to correct lipoprotein imbalance in mammals as a preventive measure against atherosclerosis. The compounds of the present invention do not act by blocking late stages of cholesterol biosynthesis and thus do not produce accumulation of intermediates such as desmosterol, as equally undesirable as cholesterol itself. Compounds with the combination of therapeutically favorable characteristics possessed by those of the present invention can be safely administered to warm-blooded mammals for the treatment of hyperlipidemic and atherosclerotic states found in patients with or prone to heart attacks, to peripheral or cerebral vascular disease and to stroke.

The compounds of this invention exhibit antiatherosclerotic activity and the invention should not be construed as limited to any particular mechanism of antiatherosclerotic action.

This invention relates to a method for treating atherosclerosis, employing ureas and thioureas which may be represented by formula I: ##STR1## wherein X represents at least one substituent selected from the group consisting of hydrogen, C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4 alkenyl, C.sub.1 -C.sub.4 alkynyl, hydroxy, C.sub.1 -C.sub.4 alkoxy, phenoxy, mercapto, C.sub.1 -C.sub.4 alkylthio, amino, C.sub.1 -C.sub.4 alkylamino, di-(C.sub.1 -C.sub.4 alkyl)amino, halo, trihalomethyl, C.sub.1 -C.sub.4 alkanoyl, benzoyl, C.sub.1 -C.sub.4 alkanamido, C.sub.1 -C.sub.4 alkanesulfonyl, C.sub.1 -C.sub.4 alkanesulfinyl, benzenesulfonyl, toluenesulfonyl, nitro, cyano, carboxy, C.sub.1 -C.sub.4 carboalkoxy, carbamoyl, sulfamyl, methylenedioxy, phenyl, ortho-phenylene, tolyl, benzyl, halobenzyl, methylbenzyl and the group ##STR2## wherein Y is selected from the group consisting of oxygen and sulfur; R is selected from the group consisting of C.sub.4 -C.sub.12 alkyl, C.sub.4 -C.sub.12 alkenyl, C.sub.4 -C.sub.12 alkynyl, C.sub.4 -C.sub.12 cycloalkyl and C.sub.4 -C.sub.12 cycloalkylalkyl.

Preferred embodiments of the invention are those in which Y is oxygen. More preferred are those in which X represents at least one C.sub.1 -C.sub.4 alkyl or halo substituent, and R is C.sub.4 -C.sub.12 alkyl. The most preferred are those in which X represents at least one methyl or chloro substituent.

Preferred specific embodiments involve 1,1-di-(n-butyl)ureas, for example:

1,1-di-(n-butyl)-3-(3-methylphenyl)urea,

1,1-di-(n-butyl)-3-(3-trifluoromethylphenyl)urea,

1,1-di-(n-butyl)-3-(3-chlorophenyl)urea,

1,1-di-(n-butyl)-3-(2,4-dichlorophenyl)urea,

1,1-di-(n-butyl)-3-(3,5-dichlorophenyl)urea,

1,1-di-(n-butyl)-3-(3-chloro-4-methylphenyl)urea,

1,1-di-(n-butyl)-3-(2,4-dimethylphenyl)urea,

1,1-di-(n-butyl)-3-(2-methylphenyl)urea,

1,1-di-(n-butyl)-3-(4-methylphenyl)urea,

1,1-di-(n-butyl)-3-(2,3-dimethylphenyl)urea,

1,1-di-(n-butyl)-3-(2,5-dimethylphenyl)urea,

1,1-di-(n-butyl)-3-(2,6-dimethylphenyl)urea,

1,1-di-(n-butyl)-3-(3,5-dimethylphenyl)urea,

1,1-di-(n-butyl)-3-(3,4-dimethylphenyl)urea,

1,1-di-(n-pentyl)-3-(2,4-dimethylphenyl)urea,

1,1-di-(n-hexyl)-3-(2,4-dimethylphenyl)urea,

1,1-di-(n-heptyl)-3-(2,4-dimethylphenyl)urea,

1,1-di-(n-octyl)-3-(2,4-dimethylphenyl)urea,

1,1-di-(n-decyl)-3-(2,4-dimethylphenyl)urea,

This invention further relates to a method of inhibiting atherosclerotic lesion development in mammals which comprises administering to said mammal an effective amount of a compound as recited above.

This invention also relates to a method of treating hyperlipidemia in mammals which comprises administering to said mammal an effective amount of a compound as recited above.

This invention still further relates to a pharmaceutical composition which comprises an effective antiatherosclerotic amount of a compound as recited above in association with a pharmaceutically acceptable carrier.

Finally, this invention relates to processes for preparing compounds as recited above. One process especially useful for the preparation of trisubstituted ureas and thioureas involves reacting an arylisocyanate or arylisothiocyanate of formula II with a secondary amine of formula III, where X, Y and R are as defined above. ##STR3##

A second process for the preparation of ureas and thioureas involves reacting a compound of formula IV; wherein A and B are leaving groups, which may be the same or different, selected from the group consisting of halo, C.sub.1 -C.sub.4 alkoxy, C.sub.1 -C.sub.4 alkylthio phenoxy, 4-chlorophenoxy and 4-nitrophenoxy; with a secondary amine of formula III to yield an intermediate of formula V and then reacting the intermediate with an arylamine of formula VI, wherein X, Y and R are as defined above. ##STR4##

A third process for the preparation of ureas and thioureas involves reacting a compound of formula IV with an arylamine of formula V to yield an intermediate of formula VI, wherein X, Y and B are as defined above and then reacting this intermediate with a secondary amine of formula III. ##STR5##

Many of the ureas and thioureas of this invention are prepared by reacting arylisocyanates are arylisothiocyanates with secondary amines. These reactions may be preformed in aprotic solvents such as hexane, diethyl ether, toluene, tetrahydrofuran and the like at temperatures from room temperature or below up to the boiling point of the solvent used. The ureas and thioureas are isolated by filtration or by evaporating the solvent and they may be purified by recrystallization, absorbtion chromatography, or distillation under reduced pressure. An example of this process is the reaction of 2,4-dimethylphenylisocyanate with di-(n-butyl)amine to yield 1,1-di-(n-butyl)-3-(2,4-dimethylphenyl)urea.

Certain of the ureas and thioureas of this invention are prepared by reacting activated derivatives of carbonic acid such as phosgene, thiophosgene, or phenyl chloroformate with secondary amines to yield an intermediate, for instance, a disubstituted carbamyl chloride. This intermediate is in turn reacted with an arylamine to yield the urea or thiourea. The preparation of the intermediate is conducted in an aprotic solvent such as tetrahydrofuran, toluene, xylene, or the like at temperatures from about room temperature up to the boiling point of the solvent. The intermediate may be isolated by evaporation and purified by distillation if necessary. The intermediate is then reacted with an arylamine in an aprotic solvent such as dimethylacetamide in the presence of a base such as sodium hydride at temperatures from about room temperature up to the boiling point of the solvent used. An example of this process is the reaction of phosgene with di-(n-butyl)amine in toluene to yield the intermediate N,N-di-(n-butyl)carbamyl chloride, which is then reacted with 3-bromoaniline in N,N-dimethylacetamide in the presence of sodium hydride to yield 1,1-di-(n-butyl)-3-(3-bromophenyl)urea.

Other of the ureas and thioureas of this invention are prepared by reacting arylamines with activated derivatives of carbonic acid such as phosgene or thiophosgene to yield an intermediate, for instance, an arylcarbamyl chloride. This intermediate is then reacted with a secondary amine to yield the urea or thiourea. The preparation of this intermediate is conducted in an aprotic solvent such as toluene or xylene at temperatures from about room temperature up to the boiling point of the solvent in the presence of a base, for example, N,N-dimethylaniline. The intermediate is then reacted with a secondary amine in an aprotic solvent such as toluene at temperatures from room temperature or below up to the boiling point of the solvent. An example of this process is the reaction of phosgene with 3-chloroaniline to yield the intermediate N-(3-chlorophenyl)carbamyl chloride which is then reacted with di-(n-butyl)amine to yield 1,1-di-(n-butyl)-3-(3-chlorophenyl)urea.

The ureas and thioureas of this invention which contain carboxy groups are prepared by alkaline hydrolysis of the corresponding carboalkoxy ureas and thioureas, prepared by the synthetic methods described above. Likewise, those which contain hydroxy, mercapto, or amino groups are prepared by alkaline hydrolysis of the corresponding O-acetyl, S-acetyl and N-acetyl ureas and thioureas, respectively, the latter also having been obtained by the urea and thiourea syntheses described above. Alternatively, ureas and thioureas containing hydroxy groups are prepared by cleavage of the corresponding methoxy compounds using Lewis acids such as boron tribromide.

The ureas and thioureas of the present invention are obtained as crystalline solids or distillable liquids. They are characterized by distinct melting or boiling points and unique spectra. They are appreciably soluble in organic solvents but generally less soluble in water. Those compounds which contain carboxylic acid groups may be converted to their alkali metal and alkaline earth salts by treatment with the appropriate metal hydroxides and those which contain amino groups may be converted to their ammonium salts by treatment with organic or mineral acids. Both of these types of salts exhibit increased water solubility.

The preparation and properties of the compounds of this invention will be described in greater detail in conjunction with the specific examples shown below.

The compounds of the present invention were assayed for two types of biological activity related to their potential use as antiatherosclerotic agents. Compounds were tested in vitro for the ability to inhibit the enzyme fatty acyl CoA:cholesterol acyl transferase (ACAT) and in vivo for serum hypolipidemic activity as measured by their ability to inhibit lipid absorption in rats. The compounds were tested for their ability to inhibit ACAT according to the following procedure:

Rat adrenals were homogenized in 0.2 M monobasic potassium phosphate buffer, pH 7.4, and centrifuged at 1000 times gravity for 15 minutes at 5.degree. C. The supernatant, containing the microsomal fraction, served as the source of the cholesterol-esterifying enzyme, fatty acyl CoA:cholesterol acyl transferase (ACAT). A mixture comprising 50 parts of adrenal supernatant, 10 parts of albumin (BSA) (50 mg./ml.), 3 parts of test compound (final concentration 5.2 g./ml.) and 500 parts of buffer was preincubated at 37.degree. C. for 10 minutes. After treatment with 20 parts of oleoyl CoA(.sup.14 C-0.4 Ci) the mixture was incubated at 37.degree. C. for 10 minutes. A control mixture, omitting the test compound, was prepared and treated in the same manner. The lipids from the incubation mixture were extracted into an organic solvent and separated by thin-layer chromatography. The cholesteryl ester fraction was counted in a scintillation counter. This procedure is a modification of that described by Hashimoto, et al., Life Scie., 12 (Part II), 1-12 (1973).

The results of this test on representative compounds of this invention appear in Table I.

Inhibition of cholesterol absorption was determined by feeding male Sprague-Dawley rats, weighing 150-170 g., a 1% cholesterol:0.5% cholic acid diet for 2 weeks. The diet also contained compounds being tested at a dose of 0.03% of the diet. Control rats were fed the same diet without any compound. At the end of the test the rats were sacrificed by decapitation. Blood is collected, centrifuged at 1.5 kg times gravity for 10 minutes at 4.degree. C., and the serum is then analyzed for cholesterol and triglycerides enzymatically by the method of Trinder, P., Analyst, 77, 321(1952) on a Centrifichem 400 Analyzer. Livers are removed, a 0.4 g sample is taken from the center of the large lobe, and the sample is subjected to saponification using 25% saturated potassium hydroxide in ethanol. The resulting neutral sterols are extracted with petroleum ether and extract analyzed for cholesterol. The effectiveness of the compound in inhibiting cholesterol absorption is measured by the lowering of either serum cholesterol or liver cholesterol relative the values for control rats. The results of this test on representative compounds appear in Table II.

Inhibition of cholesterol absorption also was determined by feeding male Sprague-Dawley rats, weighing 150-170 g., a 1% cholesterol:0.5% cholic acid diet for 2 weeks. The diet also contained compounds being tested at doses of between 0.01% and 0.1% of the diet. After the rats had been on the test diet for 9 days each rat is given by gavage a sonicated mixture of [4-.sup.14 C] cholesterol (6.mu.Ci), 0.2 ml. triolein, 10 mg. cholic acid, 20 mg. cholesterol and 2 mg. of test compound in 0.8 ml. of 10% non-fat dry milk. Feces were collected for each 24 hour period for the remaining 5 days during which the rats were maintained on the 1% cholesterol:0.5% cholic acid plus test compound diet. Fecal .sup.14 C-neutral sterols were extracted with petroleum ether from saponified feces by the method of Grundy, S. M., et al., J. Lipid Res., 6, 397 (1965) and counted in a scintillation counter. Acidic sterols (bile acids) were extracted by acidifying the saponified feces and extracting in chloroform:methanol (2:1) and counting the chloroform phase in a scintillation counter. Total extraction of radioactivity (98-100%) from saponified feces is realized by this procedure.

Radioactivity in liver and adrenals were determined by saponification and extraction into petroleum ether and counting by scintillation techniques. Total cholesterol in liver and adrenals was determined by the cholorimetric melthod of Zlatkis, A., et al., J. Lab. Clin. Med., 41, 486 (1953) on saponified-organic solvent extracted tissue prepared by the method of Trinder, P., Analyst, 77, 321 (1952). Serum cholesterol and triglycerides were assayed enzymaticaly by the method of Allain, C. C., et al., Clin. Chem., 20, 470 (1974) on a Centrifichem 400 Analyzer. .sup.14 C-Cholesterol in serum was determined by direct scintillation counting.

The effect of test compounds on cholesterol absorption was determined by:

1. increase in excreted .sup.14 C-neutral sterol

2. decrease in excreted .sup.14 C-acidic sterol

3. decrease in .sup.14 C-cholesterol or .sup.14 C-cholesteryl ester in the liver

4. decrease in .sup.14 C-cholesterol or .sup.14 C-cholesteryl ester in the serum.

A compound is considered active in inhibiting cholesterol absorption if it meets at least the first two criteria.

The results of this test on typical compounds of this invention appear in Table III.

The tests reported or shown in Tables I-III, inclusive, have been actually carried out and the results therein actually obtained or concluded therefrom.

When the compounds are employed for the above utility, they may be combined with one or more pharmaceutically acceptable carriers, e.g., solvents, dilutents and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, suspensions containing, for example, from about 0.5 to 5% of suspending agent, syrups containing, for example from about 10 to 50% of sugar, and elixirs containing, for example, from about 20 to 50% ethanol, and the like, or parenterally in the form of sterile injectable solutions or suspensions containing from about 0.5 to 5% suspending agent in an isotonic medium. These pharmaceutical preparations may contain, for example, from about 0.5% up to about 90% of the active ingredient in combination with the carrier, more usually between 5 and 60% by weight.

The antiatherosclerotic effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 2 mg. to about 500 mg./kg. of animal body weight, preferably given in divided doses two to four times a day, or in sustained release form. For most large mammals, the total daily dosage is from about 100 mg. to about 5,000 mg. preferably from about 100 mg. to 2,000 mg. Dosage forms suitable for internal use comprise from about 25 to 500 mg. of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A decided practical advantage is that these active compounds may be administered orally as well as by intravenous, intramuscular, or subcutaneous routes if necessary. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration desired. Adjuvants customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, e.g., vitamin E, ascorbic acid, BHT and BHA.

The preferred pharmaceutical compositions from the stand-point of ease of preparation and administration are solid compositions, particularly tablets and hardfilled or liquid-filled capsules. Oral administration of the compounds is preferred.

These active compounds may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof 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 must 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 or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

A solution of 4.89 g. of 2,4-dimethylphenylisocyanate in 100 ml. of hexane is added to a solution of 3.48 g. of di(n-butyl)amine in 150 ml. of hexane and the solution is stirred at room temperature for 2 hours and then evaporated. The residual solid is recrystallized from pentane to yield 1,1-di-(n-butyl)-3-(2,4-dimethylphenyl)urea, m.p. 48.degree.-50.degree. C.

The ureas and thioureas shown in Table IV were prepared from the appropriate arylisocyanates or arylisothiocyanates and secondary amines by the method of Example 1, or obtained commercially. The compound of Example 1 was actually prepared as shown.

A solution of 1.56 g. of phenyl chloroformate in 50 ml. of ether is added dropwise to a stirred solution of 3.44 g. of 3-bromoaniline in 35 ml. of ether and the mixture is stirred for one hour at room temperature and then filtered. The filtrate is evaporated and the residue is crystallized from hexane to yield phenyl N-(3-bromophenyl)carbamate, m.p. 88.degree.-90.degree. C.

A solution of 1.46 g. of phenyl N-(3-bromophenylcarbamate in 15 ml. of tetrahydrofuran is added to a solution of 1.52 g. of di-n-butylamine in 20 ml. of tetrahydrofuran and the mixture is stirred under reflux for 24 hours. The mixture is diluted with hexane and the precipitate collected by filtration. Recrystallization from pentane affords 1,1-di-(n-butyl)-3-(3-bromophenyl)urea, m.p. 80.degree.-81.degree. C.

The compound of Example 129 was actually prepared as shown.