Inhibition of lipogenesis

Lipogenesis in mammals is inhibited by benzofurancarboxylic acids substituted on the aromatic ring by one of certain moieties, and the 2,3-dihydro counterparts thereof.

DESCRIPTION OF THE INVENTION 
It has been found that lipogenesis in mammals is inhibited by 
benzofurancarboxylic acids substituted on the aromatic ring by one of 
certain moieties, and the 2,3-dihydro counterparts thereof, all of these 
compounds being described by the formula: 
##STR1## 
wherein R is cycloalkyl of from three to six carbon atoms; alkyl or alkoxy 
of from one to eight carbon atoms; or is phenyl, phenoxy, benzyl or 
benzoyl or any of these substituted by one of alkyl or alkoxy of from one 
to six carbon atoms, fluorine, chlorine, bromine, nitro, acetyl, acetoxy 
and hydroxy, with the proviso that R also can be chlorine substituted at 
the 7-position of the molecule. In these compounds, each aliphatic moiety 
may be of straight-chain or branched-chain configuration. 
The dotted line between the carbon atoms at the 2- and 3-positions in the 
ring structure indicates that these carbon atoms may be linked by a double 
bond (i.e., the benzofuran subgenus) or by a single bond (i.e., the 
2,3-dihydrobenzofuran subgenus). 
Chirality exists in the 2,3-dihydro members, hence they can exist in two 
optical isomeric forms. None of the isomers has been separated, nor has 
the lipogenesis inhibition activity of any of the individual isomers been 
determined. The 2,3-dihydro species that have been prepared inhibit 
lipogenesis. Under the circumstances, the invention contemplates the furan 
subgenus, the 2,3-dihydrofuran subgenus, including the individual isomers 
of the latter subgenus, as well as mixtures thereof. 
Generally speaking, acids of the furan subgenus can be prepared by 
hydrolysis of the corresponding ethyl esters, by the method of Kurkudar 
and Rao, Indian Acad. Sci., Section A, 58, 336 (1963), while acids of the 
2,3-dihydrofuran subgenus can be prepared by reduction of the 
corresponding acids of the furan subgenus, through use of sodium/-mercury 
amalgam, according to the method of Fredga, Acta Chem. Scand., 9, 719 
(1955). 
The precursor ethyl esters of the furan subgenus can be prepared by 
condensation of the appropriate R-substituted salicylaldehyde with diethyl 
bromomalonate in the presence of anhydrous potassium carbonate, according 
to the methods of Kurkudar and Rao, and of Witiak, D.T., et al., Lipids, 
11 (5), 384-391 (1976) and the references mentioned therein. 
The precursor R-substituted salicylaldehydes can be prepared by treating 
the appropriate phenol with chloroform under strongly basic conditions, 
according to the Reimer-Tieman Reaction. (References cited in the Merck 
Index, 9th edition, page ONR-74; also, Russell, A. and Lockhart, L.B., 
Organic Synthesis, 22, 63 (1942)). 
Many of the precursor phenols are known compounds; others can be prepared 
by conventional procedures. 
Preparation of compounds of Formula I is illustrated in the following 
examples. In each case, the identities of the product, and of the 
precursor(s) involved, were confirmed by appropriate chemical and spectral 
analyses.

EXAMPLE 1 
5-phenyl-2-benzofurancarboxylic acid (1) 
22 g of phenylphenol was dissolved in 95% ethanol; a solution of 40 g of 
sodium hydroxide in 80 ml of water was rapidly added. The resulting 
solution was heated to 75.degree.-80.degree. C. and 20 ml of chloroform 
was added over a one-hour period, the mixture being gently refluxed. The 
mixture then was stirred for three hours, cooled and the ethanol and 
excess chloroform were evaporated under reduced pressure. The resulting 
residue was cooled and poured into cold water. The resulting mixture was 
acidified by slow addition of hydrochloric acid and extracted with ether. 
The solvent was evaporated from the extract under reduced pressure, the 
residue was poured in twice its volume of saturated sodium metabisulfite 
solution and the mixture was shaken vigorously for forty-five minutes. The 
resulting semisolid bisulfite addition compound was allowed to stand for 
one hour, filtered in the dark and washed with small portions of ethanol 
and ether to remove the phenol. The bisulfite addition compound was 
decomposed with dilute sulfuric acid, the mixture being warmed on a 
water-bath for thirty minutes. The cooled mixture was extracted with 
ether, dried (Na.sub.2 SO.sub.4), and the solvent was evaporated under 
reduced pressure. The residue was treated with activated charcoal and 
recrystallized from ethanol/water to give 5-phenylsalicylaldehyde (1A), as 
yellow crystals, mp: 98.degree.-99.degree. C. 
A mixture of 0.99 g of 1A, 0.96 g of diethyl bromomalonate and 1.25 g of 
anhydrous potassium carbonate in 20 ml of 2-butanone was refluxed for 10 
hours. The solvent was evaporated under reduced pressure. The residue was 
cooled, poured into 100 ml of water and extracted with ether. The extract 
was washed with cold 5% sodium hydroxide solution and water and then 
concentrated under reduced pressure. The residue was recrystallized from 
ethanol to give ethyl 5-phenylbenzofuran-2-carboxylate (1B), as white 
crystals, mp: 109.degree.-110.degree. C. 
A mixture of 1.3 g of Compound 1B and 50 ml of 10% alcoholic potassium 
hydroxide was refluxed for 4 hours. The solvent was evaporated under 
reduced pressure and the residue was washed with ether and dissolved in 
water. The basic solution was acidified with dilute hydrochloric acid and 
extracted with ether. The ether layer was extracted with dilute sodium 
bicarbonate solution. The aqueous extract was re-acidified with dilute 
hydrochloric acid and extracted with ether. The ether extract was dried 
(Na.sub.2 SO.sub.4) and concentrated under reduced pressure. The residue 
was crystallized from ethanol to give 1, as white solid, mp: 
220.degree.-221.degree. C. 
EXAMPLE 2 
2,3-dihydro-5-phenyl-2-benzofurancarboxylic acid (2) 
5.0 g of 1 was mixed with 90 ml of 10% sodium hydoxide solution. Sodium 
amalgam (prepared from 1.5 g of sodium and 50 l g of mercury) was added to 
the stirred mixture over a period of one hour. The mixture was then 
stirred for 24 hours and allowed to stand at room temperature for an 
additional 24 hours. The mercury was separated, and the solution was 
neutralized with dilute hydrochloric acid and extracted with ether. The 
extract was dried (Na.sub.2 SO.sub.4) and concentrated under reduced 
pressure, and the residue was recrystallized from ethanol to give 2, as 
white crystals, mp: 186.degree.-187.degree. C. 
EXAMPLES 3-8 
By procedures similar to those described in Example 1, the following 
additional individual species of the benzofuran subclass of the class of 
compounds defined in Formula I were prepared from known phenols: 
______________________________________ 
R 
Example 
Compound (number indicating 
Melting Point 
No. No. position on ring) 
(.degree.C.) 
______________________________________ 
3 3 5-(1-methylpropyl) 
124-126 
4 4 5-cyclohexyl 203.5-206 
5 5 6-phenoxy 215-217 
6 6 5-benzoyl 195-198 
7 7 5-hexyloxy 147-149 
8 8 5-benzyl 229-231 
______________________________________ 
EXAMPLE 9 
4-phenoxy-2-benzofurancarboxylic acid (9) 
9, mp: 215-217, was prepared by procedures similar to those described in 
Example 1, from the precursor salicyaldehyde prepared as follows: 
14.0 g of meta-phenoxyphenol was dissolved in 700 ml of 95% ethanol. 468 g 
of sodium hydroxide was then added rapidly. The resulting suspension was 
heated to 70.degree.-80.degree. C. Then 558.7 g of chloroform was added, 
at such a rate that gentle reflux was maintained; the addition required 10 
hours. The mixture then was stirred for 2 hours at 75.degree.-80.degree. 
C., held at room temperature overnight and filtered. The solid product was 
dissolved in 1000 ml of water. The solution was acidified to pH=2 with 
concentrated hydrochloric acid, then was extracted with ether. The ether 
layer was dried (MgSO.sub.4) and concentrated. The residue was extracted 
with hot petroleum ether. The extract was dried (Na.sub.2 SO.sub.4) and 
concentrated to give an oil, which was wet column chromatographed over 
silica gel, using first a 9/1 v/v mixture, then a 4/1 v/v mixture of 
petroleum ether and ether as eluent. The fourth fraction obtained, after 
removal of the solvents, was identified as 
2-hydroxy-6-phenoxybenzaldehyde. 
EXAMPLE 10 
5-(4-acetylphenyl)-2-benzofurancarboxylic acid (10) 
12.2 g of acetyl chloride was added to a mixture of 10.3 g of 1B, and 120 
ml of carbon disulfide, then 21.8 g of anhydrous aluminum chloride was 
added in portions to the stirred mixture. The mixture then was stirred at 
room temperature for 2.5 hours, the temperature rising to 32.degree. C. 
The mixture was poured into 1 liter of ice water, and stirred for 30 
minutes, and the solution was extracted with ether. The extract was dried 
(MgSO.sub.4) and concentrated. The residue was washed with ether and 
dissolved in 100 ml of chloroform. The solution was treated with activated 
charcoal, and 100 ml of hexane was added. The resulting solution was 
concentrated to about 100 ml and cooled to give ethyl 
5-(4-acetylphenyl)-benzofuran-2-carboxylate (10A), mp: 
105.degree.-107.degree. l C. 
10A was treated with potassium hydroxide in ethanol to give 10, mp: 
264.degree.-266.degree. C. 
EXAMPLE 11 
5-(4-(acetyloxy)phenyl)-2-benzofurancarboxylic acid (11) 
2 ml of trifluoroacetic acid and 92 ml of 30% hydrogen peroxide solution 
were added to a mixture of 13 g of 10 in 300 ml of acetic acid. The 
mixture was heated at 70.degree.-75.degree. C. for 10 hours, then was 
cooled in a refrigerator. The solid that formed was collected and dried. 
The acetic acid solution was poured into 3.6 liters of ice water. The 
solid that formed was collected and dried (P.sub.2 O.sub.5). The two 
solids were combined and dissolved in 150 ml of tetrahydrofuran. The 
solution was filtered, concentrated to 20 ml and chilled. The solid that 
formed was collected, dried under reduced pressure, and recrystallized 
from tetrahydrofuran to give 11, mp: 251.degree.-254.degree. C. 
EXAMPLE 12 
5-(4-chlorophenyl)-2-benzofurancarboxylic acid (12) 
A mixture of 6.2 g of 10A, 100 ml of ethanol and 200 ml of tetrahydrofuran 
was heated to 60.degree.-70.degree. C. A solution of 1.53 g of 
hydroxylamine hydrochloride and 1.16 g of sodium carbonate in 20 ml of 
water was added. The mixture was heated for 3 hours at 
60.degree.-70.degree. C. The resulting solid was collected, washed with 
water, then ethanol, and extracted with methylene chloride. The solvent 
was evaporated from the extract under reduced pressure to give ethyl 
5-(4-(1-(hydroxyimino)ethyl)phenyl)-2-benzofurancarboxylate (12A), mp: 
220.degree.-222.degree. C. 
56.5 g of phosphorus pentachloride was added in portions to a solution of 
55.0 g of 12A in 1 liter of chloroform, and the mixture was stirred at 
room temperature for 16 hours. The solvent was evaporated under reduced 
pressure. The residue was suspended in 4 liters of water, the mixture was 
stirred vigorously for 30 minutes and filtered. The solid was extracted 
with chloroform. The extract was washed, successively, with water, 
saturated sodium bicarbonate solution, and water, then was filtered 
through celite (to break the emulsion). The filtrate was dried 
(MgSO.sub.4) and the solvent was evaporated under reduced pressure. The 
residue was triturated with ether. The ether phase was separated, then 
concentrated to about half its volume under reduced pressure and allowed 
to stand over a weekend. The solid which formed was collected and 
dissolved in 400 ml of chloroform. The solution was filtered over 
charcoal, and diluted with 400 ml of hexane. The resulting solid was 
collected and dry column chromatographed over silica gel, using a 1/9 v/v 
mixture of tetrahydrofuran and chloroform as eluent. After removal of the 
solvents, the appropriate fractions were combined and extracted with 
tetrahydrofuran. The solvent was evaporated under reduced pressure and the 
residue was triturated with ether. The resulting solid was collected and 
dried under reduced pressure to give ethyl 
5-(4-(acetylamino)phenyl)-2-benzofurancarboxylate (12B), mp: 
174.degree.-176.degree. C. 
A mixture of 3.0 g of 12B, 50 ml of 6 N hydrochloric acid and 50 ml of 
ethanol was stirred and refluxed for 5 hours. The solution was 
concentrated to about half its volume, the solid that formed was 
collected, and dissolved in chloroform containing some triethylamine. The 
solution was washed with water, dried (MgSO.sub.4) and filtered. The 
solvent was evaporated from the filtrate under reduced pressure. The 
residue was dry column chromatographed over silica gel, using a 1/9 v/v 
mixture of tetrahydrofuran and chloroform as eluent. The solvents were 
evaporated, the appropriate sections were combined and extracted with 
chloroform. The extract was filtered and the solvent was evaporated under 
reduced pressure. The residue was refluxed in petroleum ether for 8 hours. 
The mixture was filtered and the filtrate placed in a freezer overnight. 
The solid that formed was collected and dried to give ethyl 
5-(4-aminophenyl)benzofuran-2-carboxylate (12C), mp: 95.degree.-97.degree. 
C. 
A solution of 6.9 g of sodium nitrite in 40 ml of water was added 
drop-by-drop to a stirred mixture of 30 g of 12C and 160 ml of 6 N 
hydrochloric acid, the rate of addition being adjusted to maintain the 
temperature of the reaction mixture at 0.degree.-5.degree. C. The mixture 
was stirred for 1 hour at 0.degree.-5.degree. C., then added with vigorous 
stirring, to 50 ml of 20% aqueous cuprous chloride heated to 70.degree. C. 
The resulting mixture was heated to 70.degree. C., then allowed to cool. 
The solid was collected, washed with water, dried (P.sub.2 O.sub.5, 
reduced pressure) and dissolved in tetrahydrofuran. The solution was 
suspended on 75 g of silica gel and dry column chromatographed, using 
chloroform as eluent. The product was removed from the combined 
appropriate fractions with tetrahydrofuran. The solvent was evaporated ad 
the residue was dried (drying pistol, P.sub.2 O.sub.5, refluxing acetone) 
to give ethyl 5-(4-chlorophenyl)benzofuran-2-carboxylate (12 D), mp 
108.degree.-110.degree. C. 
12 D was treated with potassium hydroxide to give 12, mp: 
254.degree.-256.degree. C. 
EXAMPLES 13-15 
The following three individual species were purchased: 
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Example Compound 
No. No. R 
______________________________________ 
13 13 7-methoxy 
14 14 6,7-dimethoxy 
15 15 7-chloro 
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EXAMPLE 16 
5-(4-hydroxyphenyl)-2-benzofurancarboxylic acid (16) 
A mixture of 10.4 g of 11 and 7.2 g of potassium hydroxide in 170 ml of 
ethanol was refluxed for 2 hours. The solvent was evaporated under reduced 
pressure. The residue was mixed with 200 ml of water, and the mixture was 
acidified to pH=2 with concentrated hydrochloric acid, stirred for 30 
minutes and filtered. The solid was washed with water, dried under reduced 
pressure and mixed with tetrahydrofuran. The mixture was refluxed and 
filtered. The filtrate was concentrated, the residue was washed with ether 
and mixed with tetrahydrofuran. The mixture was filtered, the filtrate was 
concentrated to about one-fourth its volume and triturated with ether. The 
resulting solid was collected and dried under reduced pressure to give 16, 
mp: 297.degree.-300.degree. C. 
EXAMPLE 17 
5-(4-methoxyphenyl)-2-benzofurancarboxylic acid (17) 
16.6 g of methyl iodide and 17.8 g of potassium carbonate were added to a 
mixture of 20.0 g of 16, 400 ml of dimethyl sulfoxide and 150 ml of 
acetone. The mixture was heated at 65.degree.-70.degree. C. for 3 hours. A 
further 16.5 g of methyl iodide and 8.9 g of potassium carbonate were 
added, and the heating was continued for 4 hours. The mixture was poured 
into ice water. The resulting solid was collected, washed with water and 
dried (P.sub.2 O.sub.5, reduced pressure). The solid was mixed with 
tetrahydrofuran, the mixture was filtered, the filtrate was dried 
(NA.sub.2 SO.sub.4) and concentrated to a small volume. The resulting 
solid was separated and recrystallized from 2/1 v/v mixture of chloroform 
and hexane. The product was dry column chromatographed, using chloroform 
as eluent. The solvent was evaporated, the appropriate fractions were 
extracted with chloroform and the solvent was evaporated from the extract. 
The residue was washed with ether and dried under reduced pressure to give 
methyl 5-(4-methoxyphenyl)-2-benzofurancarboxylate, 17 A, mp: 
160.degree.-162.degree. C. 
17 A was treated with potassium hydroxide to give 17, mp: 
181.degree.-184.degree. C. 
EXAMPLES 18-24 
By the general procedure described in Example 2, the fllowing further 
individual species or the 2,3-dihydro subgenus of Formula I were prepared 
from the corresponding individual species of the furan subgenus: 
______________________________________ 
Example 
Compound Melting Point 
No. No. R (.degree.C.) 
______________________________________ 
18 18 5-(1-methylpropyl) 
91-94 
19 19 5-cyclohexyl 153-155 
20 20 5-benzyl 111-112 
21 21 5-hexyloxy 91-93 
22 22 6-phenoxy 131-133 
23 23 4-phenoxy 123-125 
24 24 5-(4-chlorophenyl) 
147-149 
______________________________________ 
Acids of Formula I have been found to inhibit lipogenesis in tissues of 
mammals. The manner in which they cause this effect is not known with 
certainty; it is believed that they interfere with the synthesis of fatty 
acids in the tissues. Their effectiveness for this purpose has been 
ascertained by immersing sales of swine adipose tissue in a liquid medium 
containing radioactive glucose and the test chemical for a period of time, 
then isolating the lipid from the treated tissue and determining the 
incorporation of the radioactive carbon into lipid by means of 
scintillation counting techniques. These tests were conducted in swine 
adipose tissues because in swine, the primary site of lipogenesis--i.e., 
fatty acid synthesis--appears to be adipose tissue. 
Described in more detail, the tests were conducted according to the 
following general procedure: 
150 milligrams of slices of swine adipose tissue were incubated at 
37.degree. C. for 2 hours with shaking in 3 milliliters of Krebs-Ringer 
bicarbonate solution containing one-half the normal calcium ion 
concentration, 60 micromoles of glucose, 0.5 micro-Curie of 
glucose-U.sup.14 C, and 300 microunits of insulin, and 5% 
dimethylsulfoxide (DMSO). The test compounds were added as a solution or 
suspension in DMSO and were present at a concentration of 100 micrograms 
per milliliter of the incubation mixture. 
The incubation was terminated by addition of 0.25 milliliter of 1 N 
sulfuric acid. The resulting mixture was extracted with a total of 25 
milliliters of chloroform/methanol (2:1 v/v). The extracts were washed 
according to Folch et al. (J. Biol. Chem. 226, 497-509, (1957)), air 
dried, and counted in a liquid scintillation counter with 15 milliliters 
of counting fluid (two parts toluene containing 0.4% w/v New England 
Nuclear Omnifluor/1part Triton X-100). The tests were conducted in 
triplicate and were accompanied by control tests in which all ingredients, 
proportions and conditions were the same except that no test compound was 
included. From the data obtained were calculated the precent inhibition of 
lipid synthesis by the test compounds in each case. The data obtained from 
the tests are set out in Table 1,as the percent inhibition of lipogenesis 
compared to the results obtained in the control tests wherein only the 
test compound was omitted. 
Table 1 
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Compound No. Percent Inhibition 
______________________________________ 
1 49 
2 50 
3 64 
4 91 
5 86 
6 44 
7 78 
8 87 
9 83 
10 41 
11 80 
12 55 
13 22 
14 24 
15 59 
16 30 
17 56 
18 35 
19 72 
20 40 
21 49 
22 62 
23 27 
24 56 
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The acids of Formula I can be used to control lipogenesis in mammals such 
as, for example, pets, animals in a zoo, livestock, furbearing animals and 
domestic animals, including, but not limited to dogs, cats, mink, sheep, 
goats, swine, cattle, horses, mules and donkeys. The effect is obtained by 
administering an effective amount of one or a mixture of two or more of 
the acids orally or parenterally to the animal. They may be administered 
as such, or as an active ingredient of a conventional pharmaceutical 
formulation. They may be administered orally by any convenient means. 
Thus, they may be orally administered as a drench, by intubation, in the 
animal's food and water, in a food supplement or in a formulation 
expressly designed for administration ofthe drug. Suitable formulations 
include solutions, suspensions, dispersions, emulsions, tablets, boluses, 
powders, granules, capsules, syrups and elixirs. For parental 
administration, they may be in the form of a solution, suspension, 
dispersion or emulsion. They can be administered in the form of an implant 
or other controlled sustained release formulation. Inert carriers, such as 
ne or more of water, edible oil, gelatin, lactose, starch, magnesium 
sterate, talc or vegetable gum can be used. The dosage of the acid needed 
to inhibit lipogenesis will depend upon the particular acid used, and the 
particular animal being treated. However, in general, satisfactory results 
are obtained when the acids are administered in a dosage of from about 1 
to about 400 milligrams per kilogram of the animal's body weight. The acid 
can be administered in a single dose or in a series of doses in the same 
day, or over a period of days. For any particular animal, a specific 
dosage regimen should be adjusted according to the individual need, the 
particular ester(s) used as the inhibitor, and the professional judgement 
of the person administering or supervising the administration of the 
inhibitor. It is to be understood that the dosages set forth herein are 
exemplary only, and that they do not, to any extent, limit the scope or 
practice of the invention.