Urea derivatives and salts thereof in method for inhibiting the ACAT enzyme

Urea derivatives of the general formula (I) ##STR1## and salts thereof, pharmaceutical compositions containing the same, and methods for producing the same are disclosed. The urea derivatives of the general formula (I) and salts thereof are novel compounds having the acyl-CoA cholesterol acyltransferase (ACAT) inhibiting activity.

FIELD OF THE INVENTION 
The present invention relates to urea derivatives of the following general 
formula (I) and salts thereof, which are of value as drugs for the 
treatment and prevention of various diseases related, particularly to 
atherosclerosis. 
##STR2## 
wherein R.sup.1 represents a condensed carbocyclic group containing at 
least 11 carbon atoms; R.sup.2 represents a cycloalkyl group which may 
optionally have bridgeheads; R.sup.3 represents a phenyl group which may 
optionally be substituted by one or more substituents selected from the 
group consisting of halogens, lower alkyl groups, amino, and mono- or 
di-lower alkylamino groups or a tetrahydronaphthyl group; A represents a 
single bond or a straight-chain or branched alkylene group containing 1 to 
6 carbon atoms. The present invention further relates to pharmaceutical 
compositions containing the compound (I) or a pharmaceutically acceptable 
salt thereof, and processes for producing the compound (I) or a salt 
thereof. 
BACKGROUND OF THE INVENTION 
It is known that accumulation of cholesterol in the vascular system is an 
etiologic factor in various diseases such as coronary heart disease. 
Atherosclerosis, among them, is a form of arteriosclerosis which is 
characterized by the deposition of lipids, particularly cholesterol 
esters, on the walls, and the resulting thickening, of medium- and 
large-sized arteries. 
It has recently been made clear that the production of such cholesterol 
ester is catalyzed by acyl-CoA cholesterol acyltransferase (ACAT). Thus, 
the excessive accumulation of cholesterol ester on the arterial wall is 
related to an increase in the ACAT enzyme level. Therefore, it is thought 
that if the ACAT enzyme is successfully inhibited, the esterification 
reaction of cholesterol will be retarded and the development and 
progression of atheromatous lesions due to excessive accumulation of 
cholesterol ester on the arterial wall be successfully prevented. 
On the other hand, cholesterol in diets is absorbed as unesterified 
cholesterol, esterified by the action of ACAT in the body and released 
into the bloodstream in the form of chylomicrons. Therefore, inhibition of 
ACAT would suppress not only absorption of dietary cholesterol from the 
intestinal tract but also reabsorption of the cholesterol released into 
the intestine. 
GB-A-2 113 684 discloses a series of antiatherosclerotic agents which are 
certain substituted urea and thiourea compounds having ACAT-inhibiting 
activity. 
EP-A-0 335 375 also discloses a series of antihyperlipidemic and 
antiatherosclerotic agents which are certain substituted urea compounds 
having ACAT-inhibiting activity. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide urea derivatives of the 
above general formula (I) and salts thereof. 
Another object of the invention is to provide processes for producing the 
urea derivatives of the general formula (I) and salts thereof. 
Still another object of the invention is to provide pharmaceutical 
compositions containing a urea derivative of the general formula (I) or a 
pharmaceutically acceptable salt thereof. 
The compounds (I) according to the present invention are structurally 
different from the known compounds mentioned above and as has been fully 
demonstrated in the comparative pharmacological investigations described 
hereinafter, have markedly superior pharmacologic activity than any of the 
known compounds. 
The compounds (I) according to the present invention is structurally 
characterized in that a urea derivative is directly attached to a 
condensed carbocyclic nucleus with or without interposition of an alkylene 
group. 
DETAILED DESCRIPTION OF THE INVENTION 
Referring to the definitions of the general formula (I), the "condensed 
carbocyclic group containing at least 11 carbon atoms" includes, among 
others, fluorenyl 
##STR3## 
and the like. 
The "cycloalkyl group which may optionally have bridgeheads" is a 
cycloalkyl group containing 3 to 18 carbon atoms, such as cyclopropyl, 
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, 
cyclodecyl, cyclododecyl, cyclotridecyl, cyclopentadecyl, adamantyl, 
norbornyl and so on, and more preferably includes cycloalkyl groups 
containing 6 to 10 carbon atoms. 
Referring to the "phenyl group which may optionally be substituted by one 
or more substituents selected from the group consisting of halogens, lower 
alkyl groups, amino, and mono- or di-lower alkylamino groups", the halogen 
may be chlorine, fluorine, bromine or iodine; the lower alkyl group is a 
straight-chain or branched alkyl group containing 1 to 5 carbon atoms such 
as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, 
tert-butyl, pentyl (amyl), isopentyl, tert-pentyl, neopentyl, 
1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, etc.; and the mono- or 
di-lower alkylamino group may be an amino group substituted by one or two 
lower alkyl groups mentioned above. 
One or more, whether the same or different, of these halogens and lower 
alkyl, amino, and mono- or di-lower alkylamino groups may be present on 
the phenyl group. 
Such substituted phenyl group includes, among others, 
2,4,6-trifluorophenyl, 2,6-dimethylphenyl, 2,6-diethylphenyl, 
2,4,6-trimethylphenyl, 2,4,6-triethylphenyl, 4-propylphenyl, 
2,6-diisopropylphenyl, 4-t-butylphenyl, 4-dimethylaminophenyl and the 
like. 
The compound of the general formula (I) may form salts, and such salts are 
also included within the scope of the present invention. Among such salts 
are acid addition salts with inorganic acids, such as hydrochloric acid, 
hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric 
acid, etc., and acid addition salts with organic acids, such as formic 
acid, acetic acid, oxalic acid, citric acid, succinic acid, fumaric acid, 
maleic acid, malic acid, tartaric acid, methanesulfonic acid, 
ethanesulfonic acid and so on. 
As mentioned hereinbefore, the present invention also provides processes 
for producing the compounds (I) and salts thereof. Some representative 
processes are described below. 
##STR4## 
In the above reaction formula, R.sup.1, R.sup.2, R.sup.3 and A are as 
defined above, X represents a halogen atom and R.sup.4 represents a phenyl 
group or a lower alkyl group. 
The compound (I) can be prepared by reacting the compounds of the general 
formulae (II), (III) and (IV) either concurrently or in an optional order. 
Preferably, the compound (I) can be produced by reacting an amino compound 
of the general formula (IV) with a haloformic acid ester of the general 
formula (III) and reacting the resulting carbamic ester with a compound of 
the general formula (II). 
The haloformic acid ester of the general formula (III) includes isobutyl 
chloroformate, methyl chloroformate, methyl bromocarbonate, phenyl 
chloroformate and the like. There are cases in which the reaction can be 
advantageously hastened using a base such as potassium carbonate, sodium 
carbonate, sodium hydroxide, potassium hydroxide, triethylamine, 
N,N-dimethylaniline and the like. 
The reaction solvent may be virtually any inert solvent, such as 
N,N-dimethylformamide, chloroform, benzene, toluene, xylene, dioxane, 
ether, tetrahydrofuran, chloroform, dichloromethane, dichloroethane and so 
on. In regard of reaction temperature, the reaction between amino compound 
(IV) and haloformic acid ester (III) is conducted under cooling or at room 
temperature and the reaction between the resulting carbamic acid ester and 
compound (II) is conducted at room temperature or under warming. 
##STR5## 
In the above reaction formula, R.sup.1, R.sup.2, R.sup.3 and A are as 
defined above. 
The compound (I) according to the present invention can also be produced by 
reacting an amino compound of the general formula (II) with an isocyanate 
compound of the general formula (V). The isocyanate compound (V) is used 
generally in an equimolar amount with respect to the compound (II). 
This reaction is conducted in an inert solvent, such as 
N,N-dimethylformamide, pyridine, benzene, toluene, dioxane, 
tetrahydrofuran, ether, chloroform, dichloromethane, dichloroethane, 
n-hexane, etc., at room temperature or with heating. 
##STR6## 
In the above reaction formula, R.sup.1, R.sup.2, R.sup.3 and A are as 
defined above, and X represents a halogen atom. 
The compound (I) of the present invention can also be produced by reacting 
an amino compound of the general formula (II) with a halogen compound of 
the general formula (VI). 
This reaction is conducted by reacting amino compound (II) with halogen 
compound (VI) in equimolar proportions in an inert solvent such as 
N,N-dimethylformamide, benzene, toluene, dioxane, tetrahydrofuran, ether, 
chloroform, dichloromethane, dichloroethane, n-hexane and so on. The 
reaction temperature is suitably decided depending on the starting 
compounds and the solvent used in the reaction, but the reaction is 
generally carried out at room temperature or under warming. 
The resulting compound (I) of the present invention can be isolated and 
purified in the free form or in the form of a salt thereof by salt-forming 
or desalting in a conventional manner. The isolation and purification 
procedure may involve extraction, crystallization, recrystallization, 
chromatography and/or other chemical processes which are commonly 
employed. 
The compounds (I) of the present invention and salts thereof inhibit ACAT 
to thereby inhibit the accumulation of cholesterol ester in the smooth 
muscle cells of the arterial wall. It also inhibits the absorption of 
cholesterol from the intestinal tract and facilitates the catabolism and 
excretion of cholesterol in the liver to thereby lower blood cholesterol 
levels and reduce the accumulation and storage of cholesterol ester in the 
arterial wall, which in turn inhibits the formation or progression of 
atherosclerotic lesions. These actions are not seen in the conventional 
lipid-lowering agents. 
The compounds (I) and salts thereof according to the present invention have 
been demonstrated by animal experiments to have excellent blood total 
cholesterol and low-density lipoprotein (LDL) lowering effects and is 
useful in lowering lipids as well as in the prevention and treatment of 
various diseases related to arteriosclerosis, such as cerebral infarction, 
transient ischemic attack, angina pectoris, peripheral thrombus and 
alteriosclerotic obliterans. 
The effects of the compound of the present invention have been confirmed in 
the following manner. 
i) ACAT enzyme inhibiting activity: 
Inhibitory action against acyl-CoA cholesterol acyltransferase (ACAT) 
activity in rabbit liver microsome 
The rabbit liver microsome was prepared as an enzyme fraction according to 
the method of Heider (J. G. Heider et al., J. of Lipid Res., Vol. 24, 
1127-34 (1983)). 
To the mixture of 0.154M phosphate buffer solution (pH 7.4), 2 mM 
dithiothreitol, 36 .mu.M bovine serum albumin and 10-100 .mu.g of 
microsome fraction was added liposome prepared by the method of Suckling 
(K. E. Suckling et al., FEBS Letters, Vol. 151, No. 1, 111-116 (1983)) so 
that the proposition of liposome became 20% v/v. To the mixture was added 
2% v/v of each concentration of test compound solution in dimethyl 
sulfoxide and the mixture was heated at 37.degree. C. for 5 minutes. Then 
36 .mu.M oleoyl CoA containing 1-.sup.14 C-oleoyl CoA was added and the 
resultant mixture was heated at 37.degree. C. for 10 minutes. The reaction 
was stopped by adding chloroform/methanol (=2/1). After stirring, 
cholesterol oleate extracted into the chloroform layer was separated by 
thin layer chromatography and the radioactivity was determined as ACAT 
activity. The results obtained are shown in Table 1. 
TABLE 1 
______________________________________ 
ACAT Inhibiting Activity 
Test Compound IC.sub.50 * 
______________________________________ 
Compound of Example 1 
7.3 .times. 10.sup.-8 M 
Compound of Example 10 
6.1 .times. 10.sup.-8 M 
______________________________________ 
*IC.sub.50 : 50% Inhibition Concentration 
ii) Lipid-lowering activity: 
Male Sprague-Dawley rats, 5 weeks of age, were fed with a diet containing 
1.5% cholesterol and 0.5% bile acid for 7 days and during the last 5 days, 
the compound (I) of this invention suspended in a 0.5% aqueous solution of 
methylcellulose was orally administered via sonde once a day. Two hours 
after the last administration, blood samples were collected under ether 
anesthesia for determination of serum total cholesterol level and 
HDL-cholesterol level. The cholesterol level was determined by the method 
of Siedel et al. (Siedel, J. et al., J. Clin. Chem. Clin. Biochem., 19, 
838 (1981)) and the HDL-cholesterol level was determined by the method of 
Ishikawa et al. (Ishikawa, T. et al., Lipids, 11, 628 (1976)). The results 
obtained are shown in Table 2. 
TABLE 2 
______________________________________ 
% Reduction in Serum 
Total Cholesterol 
Test Compound ED.sub.50 
______________________________________ 
Compound of Example 1 
3.8 mg/kg 
Compound of Example 10 
1.7 mg/kg 
Compound of Example 212 
249 mg/kg 
in GB-A-2,113,684 
Compound of Example 1 
514 mg/kg 
in EP-A-0,335,375 
______________________________________ 
As mentioned hereinbefore, another object of the present invention is to 
provide a pharmaceutical composition containing the compound (I) or a salt 
thereof. 
Such pharmaceutical composition can be manufactured by formulating the 
compound (I) or a salt thereof with the pharmaceutically acceptable 
carrier, vehicle or excipient which is commonly employed in the art in 
accordance with the established pharmaceutical manufacturing practice. 
The pharmaceutical composition of the present invention can be administered 
orally in such dosage forms as tablets, pills, capsules, granules, 
powders, solutions, etc., parenterally in the form of an injectable 
preparation, or otherwise, for example in the form of suppositories. The 
dosage depends on the symptom, the age and sex of the patient and other 
factors but generally the daily oral dose per adult human, for instance, 
is about 50 mg to about 500 mg, which can be administered in a single dose 
or in 2 to 4 divided doses. 
The following examples are further illustrative of the present invention 
and should by no means be construed as defining the metes and bounds of 
the invention. In the examples, .sup.1 H-NMR stands for proton nuclear 
magnetic resonance spectrum, Mass for mass spectrum, and IR for infrared 
absorption spectrum. 
Reference Examples are also given hereinafter for describing the processes 
for production of the starting compounds used in the Examples.