GB1598126A - Lipogenesis-inhibiting compositions - Google Patents

Description

(54) LIPOGENESIS-INHIBITING COMPOSITIONS (71) We, SHELL INTERNATIONALE RESEARCH MAATSCHAPPIJ B.V., a company organised under the laws- of The Netherlands, of 30 Carel van Bylandtlaan, The Hague, The Netherlands, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to pharmaceutical or veterinary compositions for the inhibition of lipogenesis in mammals.

The invention provides a lipogenesis-inhibiting composition which comprises a pharmaceutically- or veterinarily-acceptable carrier and as active ingredient, in a pharmaceutically- or veterinarily-acceptable state of purity, a compound of the general formula:

in which X is

Y is

R is halogen; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms; cycloalkyl or cycloakyloxy of from three to six carbon atoms; phenyl, phenoxy, benzyl or 2-phenethyl, optionally-substituted by one or two of one or more substituents selected from alkyl groups of from one to six carbon atoms, halogen atoms and nitro groups; n is zero, one or two; each of R' and R2 is hydrogen or alkyl of from one to four carbon atoms, at least one of R' and R2 being hydrogen; R3 is hydrogen, phenylalkyl or alkyl of from one to four carbon atoms; and R4 is phenylalkyl, alkyl of from one to four carbon atoms, alkenyl, alkynyl, or cycloalkyl of from three to six carbon atoms; with the proviso that when X is -NR3-, Y is a substituent in the 2-position of the oxygen-containing ring, and when X is -0-, -CH2 or -CHOH, Y is a substituent in either the 2- or the 3position of the oxygen-containing ring; or, when X rePresents an -NR3- roup, an acid addition salt thereof.

The compounds of the general formula I I may exist as optical and/or geometric isomers. The general formula I should be understood to include all such isomers and mixtures thereof.

The compounds of the general formula I inhibit the formation of lipids, especially cholesterol and triglycerides, in mammals. The invention therefore also provides a method of inhibiting lipogenesis in a non-human mammal, which comprises administering to the mammal a compound of the general formula I or an acid addition salt of such a compound wherein X represents -NR3-, or a lipogenesis-inhibiting composition of this invention.

The process according to the invention may be applied to livestock, furbearing animals and domestic animals, including for example dogs, cats, mink, sheep, goats, swine, cattle, horses, mules and donkeys. The active compound may be administered orally or Parenterally to the animal. For example, the active compound may be orally administered as a drench, by intubation, in the animal's food or water, in a food supplement or as a formulation expressly designed for administration of the compound. Suitable formulations include solution, suspensions, dispersions, emulsions, tablets, boluses, powders, granules, capsules, syrups and elixirs. For parenteral administration, the active compounds may be in the form of a solution, suspension, dispersion or emulsion, or in the form of an implant or other controlled, sustained release formulation. Inert carriers, such as one or more of water, edible oil, gelatin, lactose, starch, magnesium stearate, talc or vegetable gum can be used. Tie dosage of the compound needed will depend upon the particular compound used, and the particular animal being treated.

However, in general, satisfactory results are obtained when the compounds are administered in a dosage of from about 1 to about 500 milligrams per kilogram of the animal's body weight. The compound can be administered in a single dose or in a series of doses in the same day, or over a period of days.

Certain of the compounds of the general formula I are novel compounds per se. and are described and claimed in our co-pending Applications 31589/78 (Serial No. 1598127) and 31590/78 (Serial No. 1598128).

Useful compounds for inhibiting lipogenesis include 2,3 - dihydro - N - (2 propenyl) - 1,4 - benzodioxin - 2 carboxamides, of the formula:

wherein R' and R2 have the meanings given for the formula I; n is zero or one; and R is halogen; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms; cycloalkyl or cycloalkyloxy of from three to six carbon atoms; or phenyl or phenoxy optionally-substituted by one or two of the same or different substituents selected from alkyl of from one to six carbon atoms, halogen atoms and nitro groups. Unless otherwise stated, any halogen atoms is preferably a chlorine or bromine atom.

Preferred compounds of the general formula II because of their activity in inhibiting lipogenesis, are those wherein n is zero, or n is one and R is a chlorine or bromine atom in the 6-position of the ring structure; and R' and R2 each is hydrogen.

In compounds of the general formula II wherein R2 is alkyl, the compounds can exist in the form of a cis- and trans-isomers, depending on the spatial relationship of the carboxamide and alkyl moieties. It has been found that, generally, the cis-form when R2 is alkyl inhibits lipogenesis more than the corresponding trans-form.

Further, chirality exists in the compounds of formula II due to the asymmetric carbon atom at the 2-position and, when R2 is alkyl, at the 3-position, of the 2,3dihydro-1,4-benzodioxin ring.

For illustration, preparation of certain compounds of formula II are described in the Examples hereinafter. Other compounds of the formula II include those listed in the following Table I: TABLE I R n R' R2 6-Br 1 H H 6-trifluoromethyl 1 H H 6-methoxy 1 H H 6-cyclohexyl 1 H H 6-cyclohexyloxy I H H 6-phenyl 1 H H 6-phenoxy 1 H H 6-nitro 1 H H 6-amino 1 H H 6-methylsulphonylamino 1 H H 6-(4-chlorophenyl) 1 H H 6-(4-chlorophenoxy) 1 H H 5-methoxy 1 H H 8-methoxy 1 H H 5chloro 1 H methyl 8-chloro 1 H methyl O H propyl 0 O H n-butyl 0 O H isopropyl Some of the compounds of the general formula II are known: 2,3 - dihydro N - (2 - propenyl) - 1,4 - benzodioxin - 2 - carboxamide is disclosed by J. Koo, et al., J. Am. Chem. Soc., 77, 5373 (1955). Compounds of the general formula II can be prepared by treating an alkyl, suitably methyl or ethyl, ester of the corresponding carboxylic acid, in solution in a suitable solvent such as ethanol, with 2-propenamine. The reaction will proceed at room temperature; however, higher temperatures-for example, the mixture can be refluxed-may be employed to reduce the reaction time. This procedure is described by Koo et al. Preferably, about a four-to-six fold excess of the amine is used. The desired product can be recovered by evaporating the solvent and excess amine, then employing conventional techniques, such as selective extraction, recrystallization and/or drycolumn chromatography, to isolate the desired product.

Alternatively, the compounds of the general formula II can be prepared by treating the corresponding carboxylic acid with thionyl chloride, to form the corresponding acid chloride, then treating the acid chloride with 2-propenamine.

This procedure is also described in the article by Koo et al. An excess of thionyl chloride is used, in part acting as solvent. Conveniently, the treatment is conducted by refluxing the mixute. The excess thionyl chlorine is then evaporated and the acid chloride is isolated. Alternatively, the crude acid chloride can be treated with an excess of the amine, a solvent such as methylene chloride being added if needed to moderate the reaction and/or to ensure a liquid reaction mixture. The desired product can be recovered from the reaction mixture as indicated above.

Further useful compounds for inhibiting lipogenesis are those of the formula

or an acid addition salt thereof, wherein n, R2 and R4 have the meanings given for formula I; R is bromine, chlorine or fluorine; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms: or cycloalkyl of from three to six carbon atoms; and R3 is hydrogen or phenalkyl. If R represents a halogen atom this is preferably a chlorine atom.

In a phenalkyl group represented by R3 or R4, the alkyl moiety may for example contain up to four carbon atoms, and preferably contains one or two carbon atoms linking the phenyl moiety with the indicated ring nitrogen atom.

Preferred compounds of formula III, because of their activity in inhibiting lipogenesis, are those wherein n is zero, or n is one and R is a substituent bonded to the carbon atom at the 6-position in the ring structure, R4 is ethyl, R2 is hydrogen and R3 is hydrogen or benzyl.

Compounds of formula III are basic in character and can form salts with acids, for example the hydrohalic acids, which when the salt is physiologically tolerable are also effective inhibitors of lipogenesis in mammals. The use of such salts and compositions containing them are therefore included in this invention.

For illustration preparation of certain compounds of the formula III are described in the Examples hereinafter. Other include the following: R4 is ethyl, R2 is hydrogen, R3 is hydrogen, n is one, R is: 6-bromo; 5-chloro 6-chloro; 7-nitro; 8-chloro.

R4 is ethyl, R2 is methyl, R3 is hydrogen, n is zero.

R4 is ethyl, R2 is hydrogen, R3 is hydrogen, n is two, R is: 6,7-dichloro; 6-nitro, 8-chloro; 6-chloro, 8-methyl; 6-nitro, 8-methyl; 6,8-dichloro.

R2 is hydrogen, R3 is hydrogen, n is zero, R4 is methyl or butyl.

R4 is butyl, R2 is hydrogen, R3 is benzyl, n is zero.

R4 is ethyl, R2 is ethyl, R3 is hydrogen, n is one, R is 6-nitro.

R4 is propyl, R2 is hydrogen, R3 is hydrogen, n is one, R is 6methylsulphonylamino.

R4 is ethyl, R2 is hydrogen, R3 is benzyl, n is one, R is 6-nitro.

Preferably n is zero or, where n is other than zero, the substituent or substituents R are selected from chlorine, methyl, nitro, amino, methylsulphonylamino, trifluoromethyl, methoxy and cyclohexyl.

The ethyl ester of 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid is a known compound: British patent 1,057,568. Esters wherein the R4 moiety is other than ethyl can be prepared by the Fischer-Speier esterification of the acids, i.e., treating the corresponding acid with the appropriate alcohol in a solvent, such as toluene, in the presence of a catalytic amount of an acid, such as sulphuric acid, hydrochloric acid, or para-toluene-sulphonic acid. The precursor acids can be prepared by hydrolysis of the ethyl ester, using conventional techniques. Precursor esters, wherein R2 is alkyl can be prepared by condensing a methyl or ethyl ester of the appropriate 2,3-dibromobutyric, -pentanoic or -hexanoic acid with the appropriate 4-R-2-aminophenol, in the presence of a base, such as potassium carbonate, in a solvent, such as acetone, at or somewhat above room temperature.

Some of the precursor phenols (n=0 or R=chloro, methyl, methoxy, nitro) are known. Methods for preparation of other phenols are shown in Examples 13 and 15 hereinafter, and by Katz et al., J. Org. Chem., 19, 758 (1954).

Further useful compounds of the invention are 3,4 - dihydro - N - (2 propenyl) - 2H - 1,4 - benzoxazine - 2 - carboxamides of the formula:

and acid addition salts thereof, wherein n and R2 have the meanings given for formula I; R is chlorine, fluorine or bromine, preferably chlorine; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms: or cycloalkyl of from three to six carbon atoms; and R3 is hydrogen or alkyl of from one to four carbon atoms.

Preferred compounds of formula IV, because of their activity in inhibiting lipgenesis, are those wherein n is zero, or n is one and R is chlorine, alkyl or trifluoromethyl, R2 is hydrogen and R3 is hydrogen or methyl.

Compounds of formula IV are basic in character, and form salts with acids, for example the hydrohalic acids which, when the salt is physiologically tolerable, are also effective inhibitors of lipogenesis in mammals. Such salts accordingly may be used in the performance of this invention.

For illustration, preparation of certain compounds of formula IV are described in the Examples hereinafter. Other such compounds are as follows: R2 is hydrogen, R3 is hydrogen, n is one, R is: 6-methoxy; 6-cyclohexyl; 6-amino; 6-bromo; 7-methyl; 5-chloro; 8-chloro.

R2 is methyl, R3 is hydrogen, n is zero.

R2 is methyl, R3 is methyl, n is zero.

R2 is hydrogen, R3 is butyl, n is zero.

R2 is hydrogen, R3 is isopropyl, n is 1, R is 6-chloro.

R2 is hydrogen, R3 is ethyl, n is 1, R is 6-bromo.

R2 is hydrogen, R3 is isobutyl, n is 1, R is 6-methyl.

R2 is propyl, R3 is hydrogen, n is 1, R is 6-chloro.

R2 is hydrogen, R3 is hydrogen, n is 2, R is: 6,7-dichloro; 6,8-dichloro; 6-chloro, 8-methyl; 6-nitro, 8-methyl; 6-nitro, 8-chloro.

Preferred groups R are substituents in the 6-position selected from chloro, methyl, nitro, amino, methylsulphonylamino, trifluoromethyl, methoxy, cyclohexyl or hydrogen.

Compounds of formula IV can be prepared by treating an alkyl, suitably methyl or ethyl, ester of the corresponding carboxylic acid, in solution in a suitable solvent such as ethanol, with 2-propenamine. The reaction will proceed at room temperature; however, higher temperatures-for example, the mixture can be refluxed-may be employed to reduce the reaction time. Preferably, about a fourto-six fold excess of the amine is used. The desired product can be recovered by evaporating the solvent and excess amine, then employing conventional techoiques, such as selective extraction, recrystallization and/or dry column chromatography, to isolate the desired product. Use of these procedures in particular instances is illustrated in the working examples included hereinafter.

A fourth group of lipogenesis inhibiting compounds are those of the formula:

wherein X is -CH2- or -CHOH-; n is zero or one; and R is halogen; nitro; amino; trifluoromethyl; methylsulphonyl-amino; alkyl or alkoxy of from one to six carbon atoms; cycloalkyl of from three to six carbon atoms; or phenyl, phenoxy, benzyl or 2-phenethyl, optionally-substituted on the ring by one or two of the same or different substituents selected from alkyl of from one to six carbon atoms, halogen and nitro; with the proviso that when X is -CHOH, the compound is the cis-isomeric configuration. Any halogen atom is preferably a bromine or chlorine atom.

Preferred compounds of the formula V because of their activity in inhibiting lipogenesis, are those wherein X is -CH2- and either n is zero, or n is one and R is chlorine, phenyl or cyclohexyl. When X is -CH2OH- the preferred substituents R are chlorine, phenyl or cyclohexyl. Preferably R is a substituent in the 6-position.

Preparation of certain compounds of formula V are described in the Examples included hereinafter. Other compounds of this type are those wherein: n=l, the aminocarbonyl moiety is at the 2-position of the ring, and R=fluoro trifluoromethyl methyl methoxy nitro amino methylsulphonylamino phenoxy.

n=l, the aminocarbonyl moiety is at the 3-position of the ring, and R=methoxy amino methylsulphonylamino.

Compounds of the formula V wherein X is CH2 can be prepared by treating the appropriate corresponding carboxylic acid chloride with 2-propenamine.

The acid chloride precursors can be prepared by treating the corresponding carboxylic acids with thionyl chloride. An excess of the thionyl chloride is used, part acting as solvent.

Conveniently, the treatment is conducted by refluxing the mixture. The excess thionyl chloride then is evaporated, leaving the acid chloride crude product, from which the acid chloride can be isolated by conventional means.

The acid chloride-either the crude or isolated products then treated with an excess of the amine, a solvent, such as methylene chloride, being added, if needed, to moderate the reaction and/or to ensure a liquid reaction medium. The amide product can be recovered and isolated from the reaction mixture by conventional means.

The carboxylic acid and ester precursors are in part a known class of compounds: Witiak et al., J. Med. Chem., 14, 758-66 (1971); Witiak et al., J. Med.

Chem., 18, 93442 (1975); Taylor et al., J. Chem. Soc. London, 1950, 27245.

Compounds of the formula V can also be prepared by heating an alkyl, suitably methyl or ethyl, ester of the corresponding carboxylic acid, in solution in a suitable solvent such as ethanol, with 2-propenamine. While the reaction can be effected at somewhat lower temperatures, suitably it can be conducted efficiently by refluxing the mixture. Preferably, about a four-to-six fold excess of the amine is used. The desired product can be recovered by evaporating the solvent and excess amine, then employing conventional techniques, such as selective extraction, recrystallization and/or dry-column chromatography, to isolate the desired product.

The following Examples illustrate the invention.

EXAMPLE 1 2,3-dihydro-N-(2-propenyl)-l ,4-benzodioxin-2-carboxamide (I) A solution of 5.2 g of the ethyl ester of 2,3 - dihydro - 1,4 - benzodioxin - 2 carboxylic acid (Koo et al., supra) and 5.7 g of 2 - propenamine in 50 ml of ethanol was refluxed for 20 hours. The solvent was stripped and the residue was treated with charcoal and crystallized from ether-hexane to give 1, as white needles, m.p.: 55.5--57"C. (Koo et al.: 59--61"C).

EXAMPLE 2 2,3-dihydro-N-(2-propenyl)-2-methyl- 1 ,4-benzodioxin-2-carboxamide (2) A mixture of 27.5 g of catechol and 28.3 g of 1 - chloro - 2,3 - epoxy - 2 methylpropane (U.S. patent 3,150,154) in 100 ml of 10% aqueous sodium hydroxide was stirred and refluxed for 4 hours. The mixture then was cooled and extracted with ether. The combined extracts were washed with 5% aqueous sodium hydroxide, dried (MgSO4) and stripped of solvent. The residue was vacuum distilled to give a mixture of 70% 2,3-dihydro-2-methyl- 1 ,4-benzodioxin-2-methanol and 30% 3,4-dihydro-3-methyl-2H- 1 ,5-benzodioxepin-3-ol.

2 g of the mixture were dissolved in acetone and at 100C were added dropwise over a 20-minute period 10 ml of 2.7 molar Jones reagent. The resulting mixture was stirred at 10"C for 1 hour and overnight at room temperature. The mixture then was quenched with water and extracted with ether. The combined ether extracts were washed with water, then extracted with aqueous sodium bicarbonate solution.

The combined sodium bicarbonate extracts were acidified with concentrated hydrochloric acid and extracted with ether. The combined ether extracts were washed with water, dried (MgSO4) and stripped of solvent. Recrystallization of the residue from ether-hexane gave 2,3 - dihydro - 2 - methyl - 1,4 - benzodioxin 2 - carboxylic acid (2A).

3.0 g of 2A were dissolved in 15 ml of thionyl chloride and the solution was refluxed for 45 minutes. The excess thionyl chloride was stripped, the residual liquid was cooled with ice, and 20 ml of 2-propenamine were cautiously added. The resulting solution was stirred for 4 hours at room temperature, then the excess amine was stripped off. The residue was partitioned between methylene chloride and water. The organic layer was separated, dried (MgSO4) and stripped of solvent.

The residue was dry column chromatographed through silica gel using Solvent No.

3 (a 4:30:66 by volume mixture of tetrahydrofuran, ethyl acetate and hexane) as eluent, and the product was crystallized from ether-hexane to give 2, as a white solid, m.p.: 67--68"C.

EXAMPLE 3 6-chloro-2,3-dihydro-N-(2-propenyl)- 1 ,4-benzodioxin-2-carboxamide (3A) and the 7-chloro-isomer (3B) 4 - chlorocatechol was condensed with ethyl 2,3 - dibromopropionate by the method shown in F. DeMarchi, et al., Gaze. Chim. Ital., 95, 1447-54 (1965). The product was a mixture of isomers: approximately 70% of the 7-chloro-isomer and 30% of the 6 - chloro - isomer of the ethyl ester of 2,3 - dihydro - 1,4 benzodioxin - 2 - carboxylic acid. A solution of 10 g of the product mixture and 9.1 g of 2-propenamine in 50 ml of ethanol was refluxed for 20 hours. The solvent then was stripped and the resulting gum was dry column chromatographed through silica gel using Solvent No. 3 as eluent. A series of ultraviolet-fluorescent bands in the column were obtained. The bands were isolated by cutting the column at appropriate points. Each separated portion of the column then was extracted with a solvent such as methylene chloride or acetone, each extract was filtered and stripped of solvent. On repeated recrystallization from ether-hexane, one of the residues gave 3A, as a white crystalline solid, m.p.: 72--73"C, while another gave 3B, as a white crystalline solid, m.p.: 60.5--61.5"C.

EXAMPLE 4 2,3-dihydro-3-methyl-N-(2-propenyl)- 1 ,4-benzodioxin- 2-carboxamide cis-isomer (4) A mixture of diastereoisomeric ethyl esters of 2,3 - dihydro - 3 - methyl 1,4 - benzodioxin - 2 - carboxylic acid, consisting of approximately 67% cis- and 33% trans-isomers, was prepared from catechol and ethyl 2,3-dibromobutyrate according to the procedure of Koo et al., supra.

A solution of 7.0 g of the mixture of isomers and 14.0 g of 2-propenamine in 100 ml of ethanol was refluxed for 24 hours. Then the solvent was stripped off and the residue was dry column chromatographed through silica gel, using Solvent No.

3 as eluent. Work-up by the technique described in Example 3 gave, as one fraction, a yellow liquid, which on repeated recrystallization from ether-hexane gave 4, a white crystalline solid, m.p.: 70.5--72.5"C, identified as the cis-isomer by nuclear magnetic resonance spectrum analysis.

EXAMPLE 5 6-( 1,1 -dimethylethyl)-2,3-dihydro-N-(2-propenyl)- 1,4 benzodioxin-2-carboxamide (5) 40 g of ethyl 2,3 - dibromopropionate were added slowly to a stirred mixture of 23 g of 4-tertiary-butylcatechol, 56 g of potassium carbonate and 200 ml of acetone at 250C. The mixture was warmed, then was heated and refluxed for 4 hours. It was then filtered. The solid material was dissolved in water, the solution was extracted with toluene; the toluene extract was combined with the acetone filtrate; then the solvents were evaporated under vacuum and the residue was vacuum-distilled, to give a mixture of the ethyl esters of 6- and 7 - (1,1 dimethylethyl) - 2,3 - dihydro - 1,4 - benzodioxin - 2 - carboxylic acids (5A), a boiling point: 141--1420C, 0.1 Torr.

A mixture of 13.2 g of 5A and 8.5 g of 2-propenamine was held at room temperature for 19 hours. It then was stripped of excess amine under vacuum. The residue was extracted with pentane. On standing overnight, crystals precipitated from the extract. The crystals were collected and recrystallized from hexane to give 5, as a solid, m.p.: 95--96"C.

EXAMPLE 6 2, 3-dihydro-6-metyl-N-(2-propenyl)- 1 ,4-benzodioxin-2-carbox am ide (6) and its 7-methyl counterpart (7) 15 g of ethyl 2,3 - dibromopropionate were added slowly to a stirred refluxing mixture of 24.8 g of 4 - methylcatechol, 23 g of potassium carbonate and 250 ml of acetone. In three additional increments were added over a one-hour period: 20 g of potassium carbonate and 27.2 g of ethyl 2,3-dibromopropionate. The mixture then was refluxed for 6 hours and allowed to stand over a week-end. The mixture then was filtered, the solids were washed with acetone, the solvent was stripped from the combined filtrate and washings and the residue was vacuum-distilled to give a mixture of the ethyl esters of 2,3 - dihydro - 6 - (and 7 - )methyl - 1,4 benzodioxin - 2 - carboxylic acids (6A), b.p.: 124--126.5"C, 0.045 Torr.

A mixture of 11.1 g of 6A, 20 g of 2-propenamine and 50 ml of ethanol was stirred at room temperature for 24 hours. The solvent and excess amine were stripped under vacuum. The residue was dry column chromatographed over silica, using a 4:30:50 v/v/v mixture of tetrahydrofuran, ethyl acetate and hexane as eluent.

The liquid product was crystallized from ether-hexane. The product was taken up in ether, the solution was cooled and filtered, the solid being product 6B. Upon standing, crystals formed in the mother liquor, and were separated, being product 6C, m.p.: 69-710C; 6B was taken up in ether and the solution was cooled, giving crystals, which were separated and recrystallized from ether. The product was recrystallized from pentane-methylene dichloride to give white needles (6D), m.p.: 7679.5 C. 6D was designated as the 6-methyl isomer and 6C was designated as the 7-methyl isomer on the basis that the less symmetrical isomer would have the lower melting point.

EXAMPLE 7 7-fluoro-2,3-dihydro-N-(2-propenyl)- 1 ,4-benzodioxin-2-carboxamides (8) 26 g of 4 - fluorocatechol (prepared by the procedure of Corse, J. et. al., J.

Org. Chem., 16, 1345 (1951)) were mixed with 57 g of ethyl 2,3-dibromopropionate at 150C, the temperature of the mixture being allowed to rise over a 2-hour period.

The mixture then was refluxed for 3.5 hours, cooled and filtered. The filtrate was stripped of solvent to give a paste, which was filtered. The filtrate was treated with water and then with methylene chloride. The methylene chloride phase was separated and stripped of solvent, and the residue was vacuum-distilled to give a mixture of the ethyl esters of 2,3 - dihydro - 6 - (and 7 - ) - fluoro - 1,4 benzodioxin - 2 - carboxylic acids (8A), b.p.: 116--120"C, 0.1 Torr.

A mixture of 7 g of 8A and 10 g of 2-propenamine was held overnight at room temperature. Then it was stripped of solvent under reduced pressure and the residue was extracted with pentane. The residue then was extracted with cyclohexane, the solvent was stripped under reduced pressure. The cyclohexanesoluble residue was recrystallized from pentane to give 8, as a solid, m.p.: 54- 56aC.

EXAMPLE 8 The carboxamides of formula II have been found to inhibit lipogenesis in mammalian tissues. Their effectiveness for this purpose has been ascertained by immersing samples of mammalian liver or 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 uptake of the radioactive carbon by means of scintillation counting techniques. These tests were conducted with both liver and adipose tissues, because in some animals the primary site of lipogenesis appears to be liver tissue, while in others it appears to be adipose tissue. The test animals were pigs, sheep, rabbits, cats and dogs.

Described in more detail, the tests were conducted according to the following general procedure: Tissue slices (200 milligrams for liver; 150 milligrams for adipose tissue) were incubated, at 37"C for 2 hours with shaking in 3 millilitres of Krebs-Ringer bicarbonate solution containing one-half the normal calcium ion concentration, 60 micromoles of glucose, 0.5 micro-Curie of glucose-U-14C, 300 micro-units of insulin, and 5% dimethyl sulphoxide (DMSO). The test compounds were added as a solution or suspension in DMSO and were present at a concentration of 100 micrograms per millilitre of the incubation mixture.

The incubation was terminated by addition of 0.25 millilitre of 1 N sulphuric acid. The resulting mixture was extracted with a total of 25 millilitres 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 millilitres of counting fluid (two parts toluene containing 0.4% w/v New England Nuclear Omnifluor: 1 part Triton X-100) ("Triton" is a registered Trade Mark). The tests were conducted in triplicate and were accompanied by control tests in which all ingredients, proport administration, after 7 days of drug administration, and 6 days after withdrawal of drug. Adipose tissue slices were prepared from the biopsy sample, and in vitro lipogenesis was determined with radio-active glucose as substrate. The incubation was similar to that previously described except there was no DMSO or test compound in the flasks. Compared to the control animals, it was found that the test compound had not significantly affected lipogenesis at day 7, but has significantly reduced the lipogenic rate at day 13.

EXAMPLE 9 Ethyl 3,4-dihydro-4-benzyl-2H- 1 ,4-benzoxazine-2-carboxylate (9) 3,4 - dihydro - 2H - 1,4 - benzoxazine - 2 - carboxylic acid ethyl ester hydrochloride (9A) was prepared as white crystals, m.p.: 186--1880C (British patent 1,057,528: m.p.: 181--185"C) by the potassium carbonate mediated condensation of o-aminophenol and ethyl 2,3-dibromopropionate in dry acetone, according to the procedure shown in British patent 1,057,528.

9A was treated with sodium bicarbonate to prepare the free base (9B). A mixture of 5.3 g of 9B and 8.23 g of thallium ethoxide in 50 ml of dimethylformamide was stirred at room temperature for 16 hours. 5.64 g of benzyl bromide then was added and the resulting mixture was stirred at room temperature for 5 hours. Thallium bromide precipitated and was filtered. The filtrate was stripped of solvent under reduced pressure and the residue was dry column chromatographed through silica gel using a 4:16:80 by volume mixture of tetrahydrofuran, ethyl acetate and hexane as eluent. The product was recrystallized from ether/hexane to give 9, as a solid, m.p.: 83.5--84.5"C.

EXAMPLE 10 Ethyl 6-chloro-3 ,4-dihydro-2H- 1 ,4-benzoxazine-2-carboxylate (10) 43.0 g of 2 - amino - 4 - chlorophenol were dissolved in 500 ml of anhydrous acetone containing 42.0 g of anhydrous potassium carbonate. That mixture was heated to reflux temperature and 23.0 g of ethyl 2,3-dibromopropionate were added slowly. In three additional portions each, additional ester and carbonate were added to the refluxing mixture until a total of 124 g of carbonate and 85.8 g of ester had been added. The mixture was refluxed for 21 hours. Solids were filtered from the mixture and the filtrate was stripped of solvent. The residue was taken up in water and the solution was extracted with ether. The ether extract was dried (MgSO4) and the solvent was stripped. The residue was recrystallized from ethanol, eluted through a silica gel column using a 4:30:66 by volume mixture of tetrahydrofuran, ethyl acetate and hexane as eluent, and the product was recrystallized from ethanol, and then from ether, to give 10 as white crystals, m.p.: 85.5--86.5"C.

EXAMPLE 11 Ethyl 6-methyl-3,4-dihydro-2H- 1 ,4-benzoxazine-2-carboxylate hydrochloride (1 I) 29.3 g of ethyl 2,3-dibromopropionate were added over a ten minute-period to a refluxing mixture of 19.0 g of anhydrous potassium carbonate, 56.6 g of 2 amino - p - cresol and 500 ml of dry acetone. The procedure was repeated thrice as in Example 10. The mixture then was refluxed for 17 hours and filtered, and the filtrate stripped of solvent under reduced pressure. The liquid residue was diluted with 300 ml of 1N sodium hydroxide solution at 5--10"C, then extracted four times with 300 ml portions of ether. The extracts were combined, dried (MgSO4) and treated with hydrogen chloride gas at 5--10"C. A solid which formed was filtered and washed with acetone. The residue was recrystallized from ethanol to give 11, as white crystals, m.p.: 158--160"C.

EXAMPLE 12 Ethyl 6-nitro-3,4-dihydro-2H-1 ,4-benzoxazine-2-carboxylate (12) 29.2 g of ethyl 2,3 - dibromopropionate were added dropwise to a refluxing mixture of 19 g of anhydrous potassium carbonate, 70.9 g of 4 - nitro - 2 aminophenol and 500 ml of dry acetone. This procedure was repeated thrice. The reaction mixtue was refluxed for 17 hours, then filtered. The solvent was evaporated from the filtrate under reduced pressure. The residue was washed with dilute sodium hydroxide solution, extracted with ether, then with methylene chloride. The solvent was evaporated separately from each extract. Thin layer chromatographic analysis indicated that the products from each of the extracts were the same. The products were combined and recrystallized from ether to give 12, as a solid, m.p.: 88-900C.

EXAMPLE 13 Ethyl 6-amino-3,4-dihydro-2H- ,4-benzoxazine-2-carboxylate hydrochloride (13) Two l-gram portions of 10% palladium-on-carbon catalyst were added to 10 g of 12 in 700 ml of ethanol. The mixture was hydrogenated at 50 psig for 2 hours.

Fresh catalyst was added and the mixture was again hydrogenated. This procedure was repeated four times. Thin layer chromatographic analysis indicated that the nitro moiety had been completely converted to the amino moiety. The mixture was filtered and the filtrate was evaporated. Part of the resulting liquid (13A) was dissolved in ethanol, then ether was added. The solution was cooled with an icebath and hydrogen chloride gas was bubbled into the solution until precipitation ceased. The precipitate was filtered, washed with ether and recrystallized from ethanol to give 13, as a solid, m.p.: 222"C (with decomposition).

EXAMPLE 14 Ethyl 3,4-dihydro-6-((methylsulphonyl)amino)-2H-l ,4- benzoxazine-2-carboxylate (14) A mixture of 14.7 g of 13A and 7.3 g of triethylamine in 200 ml of methylene chloride was treated with 8.3 g of methanesulphonyl chloride at Q--5"C. The mixture was then stirred for 2 hours, washed with water, dried, filtered, and the solvent was evaporated. The residue was mixed with 100 ml of ethanol and filtered.

Recrystallization from ethanol gave 14 as a solid, m.p.: 149--1510C.

EXAMPLE 15 Ethyl 6-(trifluoromethyl)-3,4-dihydro-2H- 1,4- benzoxazine-2-carboxylate (15) 87.6 g of finely powdered sodium hydroxide were added in portions over an 8hour period to a stirred solution of 164.0 g of 2-nitro-4 (trifluoromethyl)chlorobenzene in 200 ml of dimethyl sulphoxide at room temperature. The mixture was allowed to stand overnight, then poured into 1.5 litres of cold water. The resulting mixture was acidified to pH 1 with concentrated hydrochloric acid. An oil formed; it was separated and dissolved in ether. The solution was dried (MgSO4) and stripped of solvent under reduced pressure. The residue was mixed with cold sodium hydroxide solution and the mixture was extracted with petroleum ether. The water layer was acidified with concentrated hydrochloric acid and the resulting oil was separated and dissolved in ether. The solution was dried (MgSO4) and stripped of solvent to give 2-nitro-4 (trifluoromethyl)phenol (15A).

82.2 g of 15A were dissolved in 300 ml of ethanol. 0.5 g of platinum oxide catalyst was added and the mixture was hydrogenated at 50 psig. Fresh portions of catalyst were added periodically. The resulting mixture was filtered, and the solvent was evaporated from the filtrate. The residue was crystallized from water to give 2 - amino - 4 - trifluoromethylphenol (15B).

11.4 g of potassium carbonate were added to 48.7 g of 15B in 320 ml of acetone. Then 18.2 g of ethyl 2,3-dibromopropionate were added dropwise to the refluxing mixture. This procedure was repeated thrice. The mixture was refluxed for 17 hours and filtered, and the filtrate was stripped of solvent under reduced pressure. The residue was dissolved in ether; the solution was washed with dilute sodium hydroxide solution, then dried (MgSO4) and stripped of solvent. The residue was washed with petroleum ether and dried. The residue was mixed with ether and filtered. Part of the solvent was evaporated from the filtrate; the remaining solution was cooled. Crystals which formed were filtered and recrystallized from ether to give 15, m.p.: 105--1070C.

EXAMPLE 16 Ethyl 6-methoxy-2,3-dihydro-2H- 1 ,4-benzoxazine-2-carboxylate (16) 336.4 g of 2 - nitro - 4 - methoxyaniline were refluxed for 20 hours with 200-g of sodium hydroxide and 10 g of arsenic trioxide, in 6500 ml of water. The resulting solution was cooled on an ice-bath, then acidified to pH 1 with concentrated hydrochloric acid. The solid which formed was filtered and washed with water and dried under reduced pressure over P205 to give 2 - nitro - 4 - methoxyphenol, 16A, m.p.: 78-800C.

193.1 g of 16A were mixed with 1400 ml of water and 513 ml of ammonium hydroxide were added. Then 595 g of powdered sodium dithionite were added in portions over a 50 minute-period. The mixture then was stirred for 2 hours, the solid which formed was filtered and dried over P205 under reduced pressure to give 4 - methoxy - 2 - aminophenol, 16B, m.p.: 134--1360C.

A mixture of 56 g of 16B and 58.4 g of potassium carbonate in 640 ml of dry dimethylformamide was heated to 60"C. Then 104 g of ethyl 2,3dibromopropionate were added, and the mixture was held at 600C for 48 hours.

The mixture then was poured into 4.5 litres of ice-water and extracted with ether.

The extract was washed with water, then with 0.5 N sodium hydroxide solution, then was dried (MgSO4) and the solvent evaporated under reduced pressure. The residue was wet column chromatographed over silica gel, eluents being ether/hexane (1:9 by volume), ether/hexane (1:4 by volume), ether/hexane (1:1 by volume). The last fraction obtained was dissolved in ether. The solution was chilled to give a solid, which was filtered and recrystallized from ether/petroleum ether (30--60"C) to give 16, as off-white crystals, m.p.: 59--61"C.

EXAMPLE 17 Ethyl 6-cyclohexyl-2,3-dihydro-2H-1 ,4-benzoxazine-2-carboxylate (17) 90 g of cyclohexanol were added slowly over a period of one hour to a stirred solution of o-nitrophenol in 1500 ml of 85% sulphuric acid, at room temperature.

The mixture was heated at 700C for 4 hours, diluted with 2000 ml of ice-water, and extracted with 6000 ml of benzene. Two-thirds of the solvent were evaporated from the extract under reduced pressure, the concentrated solution was washed with water, dried (MgSO4) and the remainder of the solution was evaporated under reduced pressure. The residue was mixed with 300 g of silica gel, then purified with 2300 g of silica gel, then was dry column chromatographed with petroleum ether.

Four fractions were separated and extracted with ether. Evaporation of the ether from the extract of the second fraction gave 4-cyclohexyl-2-nitrophenol (I 7A), as a yellow liquid.

25.5 g of 17A were dissolved in 200 ml of methanol, 200 ml of methylene chloride and 400 ml of water. 86 ml of ammonium hydroxide were added, and then 82 g of sodium dithionite were added, in portions. The mixture then was refluxed for 5 hours, 300 ml of methylene chloride were added, the methylene chloride phase which formed was separated, dried (MgSO4) and the methylene chloride evaporated. The residue was washed with petroleum ether, and the insoluble white solid was collected, dried and identified as 4-cyclohexyl-2-aminophenol (17B).

m.p.: 103-l050C.

6.9 g of anhydrous potassium carbonate were added to 15.9 g of 1 7B in 90 ml of dry acetone. Then 11.6 g of 2,3-dibromopropionate were added slowly to the refluxing mixture. This procedure was repeated. The mixture then was refluxed for 24 hours and filtered. The filtrate was concentrated and the residue was taken up in ether. The ether layer that formed was separated, dried (MgSO4) and the solvent were evaporated. The residue was mixed with petroleum ether, the mixture was filtered and the solvent was evaporated from the filtrate. The residue was recrystallized from hexane to give 17, m.p.: 119--121"C.

EXAMPLE 18 150 milligrams of slices of swine adipose tissue were incubated at 370C for 2 hours with shaking in 3 millilitres of Krebs-Ringer bicarbonate solution containing one-half the normal calcium ion concentration, 60 micromoles of glucose, 0.5 micro-Curie of glucose-U'4C, and 300 micro-units of insulin, and 5% dimethyl sulphoxide (DMSO). The test compounds were added as a solution or suspension in DMSO and were present at a concentration of 100 micrograms per millilitre of the incubation mixture.

The incubation was terminated by addition of 0.25 millilitre of IN sulphuric acid. The resulting mixture was extracted with a total of 25 millilitres 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 millilitres of counting fluid (two parts toluene containing 0.4% w/v New England Nuclear Omnifluor: 1 part 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 percent inhibition of lipid synthesis by the test compound in each case. The data obtained from the tests were set out in Table 2 as the percent inhibition of lipogenesis compared to the results obtained in the control tests wherein only the test compound was omitted.

TABLE 2 Compound Percent No. inhibition 9A 62 9 53 10 28 11 18 12 74 13 43 14 76 15 31 EXAMPLE 19 3 ,4-dihydro-N-(2-propenyl)-2H- 1 ,4-benzoxazine-2-carboxamide (18) 3,4 - dihydro - 2H -- 1,4 - benzoxazine - 2 - carboxylic acid ethyl ester, hydrochloride (18A) was prepared as white crystals, m.p.: 186--188"C (British patent 1,057,528: m.p.: 181--1850C) by the potassium carbonate mediated condensation of o-aminophenol and ethyl 2,3-dibromopropionate in dry acetone according to the procedure shown in British patent 1,057,528. 18A was treated with sodium bicarbonate to prepare the free base (18B).

A solution of 7.3 g of (18B) and 6.84 g of 2-propenamine in 50 ml of ethanol was stirred at room temperature for 3 days. Then solvent and excess amine were stripped off and the residue was partitioned between ether and water. The ether layer was separated and the aqueous layer was extracted with ether. The ether solutions were combined, washed with water, dried (MgSO4), and concentrated to give 18, as white crystals, m.p.: 90--91"C.

EXAMPLE 20 3 ,4-dihydro-4-methyl-N-(2-propenyl)-2H- 1 ,4-benzoxazine 2-carboxamide (19) To a stirred solution of 2.6 g of (18B) in 50 ml of ethanol was added dropwise at room temperature a solution of 3.76 g of thallium ethoxide in 50 ml of ethanol. The resulting mixture was stirred at room temperature for 24 hours. Then a solution of 1.8 g of methyl iodide in 50 ml of dimethylformamide was added and the resulting mixture was stirred at room temperature for 72 hours. The mixture then was filtered and the solvent was stripped from the filtrate to leave a gum. This product was refluxed overnight in ethanol containing a small amount of sulphuric acid as catalyst. The resulting mixture was cooled and neutralized with sodium bicarbonate; the solvent was stripped off and the residue was partitioned between water and methylene chloride. The methylene chloride layer was separated, the aqueous layer was extracted with methylene chloride; the methylene chloride solutions were combined, washed with water, dried (MgSO4) and the solvent was stripped off to give a liquid residue. The residue was dry column chromatographed through silica gel, using Solvent No. 3 (a 4:30:66 by volume mixture of tetrahydrofuran, ethyl acetate and hexane) as eluent. On work-up, the fastermoving, major component was separated, dissolved in ether and treated with hydrogen chloride gas, to form the hydrochloride salt, as as gum. The gum was separated and triturated with ethanol and the resulting solution was cooled to give the hydrochloride salt of the ethyl ester of 3,4-dihydro-4-methyl-2H- 1,4benzoxazine-2-carboxylic acid (19A) as white crystals, m.p.: 89--93"C.

A solution of 1.1 g of 19A, and 5 ml of 2-propenamine in 25 ml of ethanol was refluxed for 16 hours. The solvent and excess amine were stripped off and the residue was passed through a silica gel column using Solvent No. 3 as eluent. The solvent was stripped off to give 19, as light yellow liquid, boiling point not determined.

EXAMPLE 21 6-chloro-3 ,4-dihydro-N-(2-propenyl)-2H- 1 ,4-benzoxazine- 2-carboxamide (20) 43.0 g of 2-amino-4-chlorophenol were dissolved in 500 ml of anhydrous acetone containing 42.0 g of anhydrous potassium carbonate. That mixture was heated to reflux temperature and 23.0 g of ethyl 2,3-dibromopropionate were added dropwise. In three additional portions each, additional ester and carbonate were added to the refluxing mixture until a total of 124 g of carbonate and 85.8 g of ester had been added. The mixture was refluxed for 21 hours. Solids were filtered from the mixture and washed with acetone. The filtrate was stripped of solvent and the residue was taken up in water. The water solution was extracted with ether.

The ether extract was dried (MgSO4) and the solvent was stripped. The residue was eluted through a silica gel column using Solvent No. 3, the solvent was stripped and the residue was recrystallized from ethanol, then from ether to give ethyl 6-chloro 3,4-dihydro-2H-1 ,4-benzoxazine-2-carboxylate (20A), as white crystals, m.p.: 85.5--86.5"C.

20 was prepared as white platelets, m.p.: 1 14--1 14.5"C, by treating 20A with 2propenamine by the procedure of Example 1.

EXAMPLE 22 6-methyl-3,4-dihydro-N-(2-propenyl)-2H- 1 ,4-benzoxazine- 2-carboxamide (21) 29.3 g of ethyl 2,3-dibromopropionate were added over a ten minute-period to a refluxing mixture of 19.0 g of anhydrous potassium carbonate, 56.6 g of 2-aminop-cresol and 500 ml of dry acetone. The ethyl 2,3-dibromopropionate and potassium carbonate addition was repeated thrice to give a total of 76 g of potassium carbonate and 118 g of ethyl 2,3-dibromopropionate. The mixture then was refluxed for 17 hours, filtered and the filtrate was stripped of solvent under reduced pressure. The liquid residue was diluted with 300 ml of IN sodium hydroxide solution at 5--100C, then extracted four times with 300 ml portions of ether. The extracts were combined, dried (MgSO4) and treated with hydrogen chloride gas at 5--100C. A solid which formed was filtered and extracted with acetone. The residue was recrystallized from ethanol to give the ethyl ester of 6 methyl-3,4-dihyro-2H-I ,4-benzoxazine-2-carboxylic acid hydrochloride (21A), as white crystals, m.p.: 158--160"C.

42.4 g of 21A and 38 g of 2-propenamine were mixed in 165 ml of ethanol and the mixture was stirred at room temperature for 90 hours. The solvent was removed under reduced pressure. The residue was partitioned between ether and water. The ether solution was dried (MgSO4), about half the solvent was evaporated, and solid which formed was filtered, then dried under reduced pressure to give 21, m.p.: 113--1150C.

EXAMPLE 23 6-nitro-3,4-dihydro-N-(2-propenyl)-2H- 1,4-benzoxazine 2-carboxamide (22) 29.2 g of ethyl 2,3-dibromopropionate were added dropwise to a refluxing mixture of 19 g of anhydrous potassium carbonate, 70.9 g of 4-nitro-2-aminophenol and 500 ml of dry acetone. The ethyl 2,3-dibromopropionate and potassium carbonate addition was repeated thrice to give a total of 76 g of potassium carbonate and 118 g of ethyl 2,3-dibromopropionate. The reaction mixture was refluxed for 17 hours, then filtered. The filtrate was concentrated under reduced pressure. The residue was washed with dilute sodium hydroxide solution, then was extracted with ether and with methylene chloride. The solvents were evaporated.

Thin layer chromatographic analyses indicated that both products were the same.

They were combined and recrystallized from ether to give the ethyl ester of 6-nitro 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid (22A), as a solid, m.p.: 88 90"C.

A mixture of 10.0 g of 22A, 9.1 g of 2-propenamine and 35 ml of ethanol was stirred at room temperature for 3 days. The solvent was stripped under reduced pressure. The residue was washed with ether, dried, and then recrystallized from ethanol to give 22, as orange crystals, m.p.: 142--144"C.

EXAMPLE 24 3,4-dihydro-6-((methylsulphonyl)amino)-N-(2-propenyl)- 2H-1 ,4-benzoxazine-2-carboxamide (23) Two I-gram portions of 10% palladium-on-carbon catalyst were added to 10 g of 22A in 700 ml of ethanol. The mixture was hydrogenated at 50 psig for 2 hours.

Fresh catalyst was added and the mixture again was hydrogenated. This procedure was repeated four times, when thin layer chromatographic analysis indicated that the nitro moiety had been completely converted to the amino moiety. The reaction mixture was filtered and the filtrate was concentrated to give the amino derivative (23A) as a brown liquid.

A mixture of 14.7 g of 23A and 7.3 g of triethylamine in 200 ml of methylene chloride was treated with 8.3 g of methanesulphonyl chloride at 0--50C. The mixture then was stirred for 2 hours, washed with water, dried, filtered, and the solvent was evaporated. The residue was washed with a small amount of ethanol, filtered, and recrystallized from ethanol to give the methylsulphonylamino derivative (23B), as a solid, m.p.: 149--1510C A mixture of 7.3 g of 23B, 20 ml of 2-propenamine and 10 ml of ethanol was stirred at room temperature for 20 hours. The solid product was filtered, dried and recrystallized from ethanol/acetone (6/1 v/v) to give 23, as white crystals, m.p.: 178--1800C.

EXAMPLE 25 6-trifluoromethyl-3,4-dihydro-N-(2-propenyl)-2H-1 ,4 benzoxazine-2-carboxamide (24) 87.6 g of finely powdered sodium hydroxide were added in portions over an 8hour period to a stirred solution of 164.0 g of 2-nitro-4 (trifluoromethyl)chlorobenzene in 220 ml of dimethyl sulphoxide at room temperature. The mixture was allowed to stand overnight, then poured into 1.5 litres of cold water. The resulting mixture was acidified to pH 1 with concentrated hydrochloric acid. An oil formed; it was separated and dissolved in ether. The solution was dried (MgSO4) and stripped of solvent under reduced pressure. The residue was mixed with cold sodium hydroxide solution and the mixture was extracted with petroleum ether. The water layer was acidified with concentrated hydrochloric acid. The resulting oil was separated and dissolved in ether. The solution was dried (MgSO4) and stripped of solvent to give 2-nitro-4 (trifluoromethyl)phenol (24A).

82.2 g of 24A were dissolved in 300 ml of ethanol. 0.5 g of platinum oxide catalyst was added and the mixture was hydrogenated at 50 psig. Fresh portions of catalyst were added periodically. The reaction mixture was filtered, and the filtrate was concentrated. The residue was crystallized from water to give 2-amino-4 (trifluoromethyl)phenol (24B).

11.4 g of potassium carbonate were added to 48.7 g of 24B in 320 ml of acetone. Then 18.2 g of ethyl 2,3-dibromopropionate were added dropwise to the refluxing mixture. The ethyl 2,3-dibromopropionate and potassium carbonate addition was repeated thrice, to give a total of 45.6 g of potassium carbonate and 72.8 g of ethyl 2,3-dibromopropionate. The reaction mixture then was refluxed for 17 hours and filtered, and the filtrate was stripped of solvent under reduced pressure. The residue was dissolved in ether; the solution was washed with dilute sodium hydroxide solution, then dried (MgSO4) and stripped of solvent. The residue was washed with petroleum ether, dried and dissolved in ether. The ether solution was partially concentrated and cooled. The resulting crystals were filtered and recrystallized from ether to give the ethyl ester of 6 - trifluoromethyl - 3,4 dihydro - 2H - 1,4 - benzoxazine - 2 - carboxylic acid (24C), m.p.: 105--107"C.

A mixture of 20.6 g of 24C, 25.7 g of 2-propenamine and 34 ml of ethanol was stirred at room temperature for 36 hours. The excess amine and solvent were evaporated under reduced pressure. The solid residue was mixed with 150 ml of ether and the remaining solid material was filtered. The filtrate was added to petroleum ether and cooled; the resulting viscous material was filtered. The filtrate was stripped and the residue was triturated with petroleum ether to give an offwhite powder which was purified by dry-column chromatography (silica gel), using ether as eluent. The higher Rf band was collected and extracted with ether, the solvent was stripped and the solid residue was recrystallized from ether/hexane (4/5 v/v). Thin-layer chromatography indicated that two components were present. The product was purified by wet-column chromatography (silica gel) using petroleum ether/ethyl ether (1:4 v/v) as eluent. The solvent was stripped and the residue was recrystallized from ether/hexane (75:100 v/v) to give 24, as a solid, m.p.: 95--97"C.

EXAMPLE 26 The effectiveness of the compounds prepared in Examples 19-25 has been ascertained by immersing samples 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 uptake of the radioactive carbon by means of scintillation counting techniques. These tests were conducted in swine adipose tissue 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 370C for 2 hours with shaking in 3 millilitres of Krebs-Ringer bicarbonate solution containing one-half the normal calcium ion concentration, 60 micromoles of glucose, 0.5 micro-Curie of glucose-Ul4C, and 300 micro-unites of insulin, and 5% dimethyl sulphoxide (DMSO). The test compounds were added as a solution or suspension in DMSO and were present at a concentration of 100 micrograms per millilitre of the incubation mixture.

The incubation was terminated by addition of 0.25 millilitre of IN sulphuric acid. The resulting mixture was extracted with a total of 25 millilitres 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 scintiallation counter with 15 millilitres of counting fluid (two parts toluene containing 0.4% w/v New England Nuclear Omnifluor:l part 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 percent inhibition of lipid synthesis by the test compounds in each case. The data obtained from the tests are set out in Table 3, as the percent inhibition of lipogenesis compared to the results obtained in the control tests wherein only the test compound w chroman-2-carboxylic acid (Witiak et al. (1971)) and 2-propenamine, by the procedure described in the last paragraph of Example 28.

EXAMPLE 30 3,4-dihydro-6-phenyl-N-(2-propenyl)-2H- I -benzopyran-2-carboxamide (28) 28 was prepared as a white solid, m.p.: 123--124"C from the ethyl ester of 3,4 dihydro - 6 - phenyl - 2H - 1 - benzopyran - 2 - carboxylic acid (Witiak et al.

(1975)) and 2 - propenamine by the procedure described in the last paragraph of Example 28.

EXAMPLE 31 6-cyclohexyl-3 ,4-dihydro-N-(2-propenyl)-2H- 1 -benzopyran 2-carboxamide (29) 29 also was prepared, as white crystals, m.p.: 93--940C, by the procedure described in the last paragraph of Example 28, from the ethyl ester of 6 cyclohexyl - 3,4 - dihydro - 2H - I - benzopyran - 2 - carboxylic acid (Witiak et al. (1975)) and 2-propenamine.

EXAMPLE 32 6-chloro-3 ,4-dihydro-N-(2-propenyl)-2H- I -benzopyran 3-carboxamide (30) To a stirred, refluxing mixture of 50 g of 3 - chloro - 6 - hydroxybenzaldehyde and 62 ml of acrylonitrile in 50 ml of water was added dropwise over a three-hour period a solution of 12.8 g of sodium hydroxide in 120 ml of water. Then an additional 62 ml of acrylonitrile was added and the stirred mixture was refluxed for 2 hours, then allowed to stand at room temperature overnight. The crystals which formed were filtered off, washed with water and dried to give a solid, which was recrystallized from ethanol. The solution was filtered and cooled to give 6 chloro - 3,4 - dihydro - 4 - hydroxy - (2H) - 1 - benzopyran - 3 - carbonitrile (30A), as a solid. A mixture of 30A with 250 ml of methanol containing 2 ml of sulphuric acid was refluxed for 4 days and stripped of solvent. The resulting residue was dry column chromatographed over silica gel, using Solvent No. 3 as eluent. The product obtained on work-up was rechromatographed and recrystallized from ether to give methyl 6-chloro-2H-l-benzopyran-3-carboxylate (30B), as light yellow needles, m.p.: 106--109"C.

700 mg of 30B were dissolved in 75 ml of ethyl acetate and the solution was treated with hydrogen (initial pressure of 30 psig), in the presence of a 10% palladium-on-carbon catalyst, for 8 hours. The reaction mixture then was filtered and stripped of solvent to give methyl 6 - chloro - 3,4 - dihydro - 2H - 1 benzopyran - 3 - carboxylate (30C), as a yellow liquid, boiling point not determined.

A mixture of 500 mg of 30C, 5 ml of 2-propenamine and 25 ml of ethanol was refluxed for 18 hours. The solvent was then stripped off and the residue was taken up in methylene chloride, triturated with hexane and cooled to give a solid. The solid was redissolved in ethanol, the solution was passed through a short silica gel column and the eluent was stripped of solvent. The residue was recrystallized from methylene chloride/hexane to give 30, as white needles, m.p.: 171--171.5"C.

EXAMPLE 33 6-chloro-4-hydroxy-N-(2-propenyl)-2H- 1 -benzopyran- 2-carboxamide (cis) (31) A solution of 512 mg of the ethyl ester of 6 - chloro - 3,4 - dihydro - 4 hydroxy - 2H - I - benzopyran - 2 - carboxylic acid (prepared by treating the ethyl ester of 6 - chloro - 3,4 - dihydro - 4 - oxo - 2H - 1 - benzopyran - 2 carboxylic acid (Witiak et al., supra) with sodium borohydride), 570 mg of 2propenamine and 20 ml of ethanol was stirred at room temperature for 72 hours, then stirred for 3 hours while heated by a steam bath. The solvent was stripped off and the residue was crystallized from methylene chloride/hexane to give 31, as white crystals, m.p.: 129--130.5"C.

EXAMPLE 34 Tests to determine lipogenesis inhibition of compounds prepared in samples 27-33 were conducted according to the following general procedure: Tissue slices (200 milligrams for liver; 150 milligrams for adipose tissue) were incubated, at 370C for 2 hours with shaking in 3 millilitres of Krebs-Ringer bicarbonate solution containing one-half the normal calcium ion concentration, 60 micromoles of glucose, 0.5 micro-Curie of glucose-l4C, 300 micro-units of insulin, and 5% dimethyl sulphoxide (DMSO). The test compounds were added as a solution or a suspension in DMSO and were present at a concentration of 100 micrograms per millilitre of the incubation mixture.

The incubation was terminated by addition of 0.25 millilitre of IN sulphuric acid. The incubation mixture was extracted with a total of 25 millilitres 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 millilitres of counting fluid (two parts toluene containing 0.4 /n w/v New England Nuclear Omnifluor: 1 part 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 was calculated the percent inhibition of lipid synthesis by the test compound in each case.

Compound 26 was tested with respect to all of the animals. The other six compounds were tested only with respect to the pig.

From these and other tests, it was established that in pigs there is little lipogenic activity in the liver tissue. From these and other tests, it also has been established that swine adipose tissue utilizes glucose for lipogenesis, and is the major site of fatty acid synthesis. The data obtained from the tests using adipose tissue and glucose are set out in Table 4, as the percent inhibition of lipogenesis compared to the results obtained in the control tests wherein only the test compound was omitted.

TABLE 4 Compound Percent No. inhibition 25 59 26 83 27 72 28 76 29 40 30 67 31 55 EXAMPLE 35 The effects of some of the compounds prepared in Examples 1, 27 and 28 on the levels of cholesterol and triglycerides in the blood of a mammal were established as follows: The procedure of Schurr et al., Lipids, 7, 68-74 (1972) was followed. In this procedure, hyperlipemia was induced in rats by intraperitoneal injection of Triton WR-1339 ("Triton" is a registered Trade Mark) (oxyethylated tertiary actylphenoUformaldehyde polymer, Ruger Chemical Co.), employing four groups of ten Sprague-Dawley strain male albino rats, each rat weighing 260280 grams.

After a 2-week stabilizing period, two groups (III and IV) were fasted for 24 hours. Then each rat was injected with a solution of the polymer in a saline vehicle (0.15 M sodium chloride solution; (62.5 milligrams bf polymer per millilitre solution)), to give a dosage of 225 milligrams of polymer per kilogram of rat body weight. Two control groups (I and II) were also fasted and each rat received 2 millilitres of the saline vehicle. Groups II and IV received the test compounds in the vehicle, while Groups I and III received the vehicle only. The concentration of test compound in the vehicle was 8.22x10-3 millimoles/millilitre.

A total dose of 0.124 millimoles/kilogram of body weight was administered to the rats. Each rat received two 2-millilitre doses by gastric intubation, the first immediately after the polymer injection, and the other 20 hours later. Fasting was continued following injection of the polymer. 43 hours after the polymer was injected, the rats were anesthetized and blood was drawn from the abdominal aorta and centrifuged. Triglyceride content of the plasma was determined by the method of Eggstein, Klin. Wochenschr., 44, 267 (1966). Cholesterol content of the plasma was determined by the method of Holub et al., Clin. Chem., 18, 239 (1972). The results are shown in Tables 5 and 6.

Statistical analysis of the results show that: (1) Comparison of Groups I and II indicate the effect of the drug on the normal rat. Compound 25 significantly lowered serum cholesterol levels in the normal rat (Table 5).

(2) groups III and IV were hyperlipemic, Group III being the control and Group IV receiving the experimental drug. All three drugs lowered significantly the cholesterol level (III vs. IV). All three drugs also significantly lowered triglyceride levels in the hyperlipemic group (III vs. IV) (Table 6).

TABLE 5 Effect of Test Compounds on Plasma Cholesterol Drug-Treated Control Drug-Treated Triton Triton Group Control Hyperlipemic Hyperlipemic Compound (I) (Il) (III) (IV) 852il 1.97 87.3+16.08 148.9+40.25 126.6+32.74 25 71.4+ 8.04 62.8+ 8.77 144.9+72.88 102.6f17.38 26 64.9+12.04 63A+l2.18 167.1+72.76 109.6+22.14 TABLE 6 Effect of Test Compounds on Plasma Triglycerides Drug-Treated Control Drug-Treated Triton Triton Group Control Hyperlipemic Hyperlipemic Compound (I) (Il) (III) (IV) 25.7+9.46 42.6+ 7.26 115.2+28.77 83.0+33.54 25 43.8+6.52 44.7+10.87 142.5+64.52 39.2j4.80 26 33.1+9.09 39.6+ 8.24 136.8+60.8 48.1#16.8 WHAT WE CLAIM IS: 1. A lipogenesis-inhibiting composition which comprises a pharmaceutically or veterinarily-acceptable carrier and, as active ingredient, in pharmaceutically- or veterinarily-acceptable state of purity, a compound of the general formula

in which X is

R is halogen; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms; cycloalkyl or cycloalkyloxy of from three to six carbon atoms; phenyl, phenoxy, benzyl or 2-phenethyl, optionally-substituted by one or two of one or more substituents selected from alkyl groups of from one to six carbon atoms, halogen atoms and nitro groups; n is zero one or two; each of R1 and R2 is hydrogen or alkyl of from one to foulrcarbon atoms, at least one of Rl and R2 being hydrogen; R3 is hydrogen, phenylalkyl or alkyl of from one to four carbon atoms; and R4 is phenylalkyl, alkyl of from one to four carbon atoms, alkenyl, alkynyl, or cycloalkyl of from three to six carbon atoms; with the proviso that when X is -NR3-, Y is a substituent in the 2-position of the oxygen-containing ring, and when X is -O-, -CH2 or -CHOH, Y is a substituent in either the 2- or 3-position of the oxygen-containing ring; or, when X represents an -NR3- group, an acid addition salt thereof.

2. A composition as claimed in claim 1, which comprises as active ingredient a compound of the general formula

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (12)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   (2) groups III and IV were hyperlipemic, Group III being the control and Group IV receiving the experimental drug. All three drugs lowered significantly the cholesterol level (III vs. IV). All three drugs also significantly lowered triglyceride levels in the hyperlipemic group (III vs. IV) (Table 6).

   TABLE 5 Effect of Test Compounds on Plasma Cholesterol Drug-Treated Control Drug-Treated Triton Triton Group Control Hyperlipemic Hyperlipemic Compound (I) (Il) (III) (IV) 852il 1.97 87.3+16.08 148.9+40.25 126.6+32.74

   25 71.4+ 8.04 62.8+ 8.77 144.9+72.88 102.6f17.38

   26 64.9+12.04 63A+l2.18 167.1+72.76 109.6+22.14 TABLE 6 Effect of Test Compounds on Plasma Triglycerides Drug-Treated Control Drug-Treated Triton Triton Group Control Hyperlipemic Hyperlipemic Compound (I) (Il) (III) (IV) 25.7+9.46 42.6+ 7.26 115.2+28.77 83.0+33.54

   25 43.8+6.52 44.7+10.87 142.5+64.52 39.2j4.80

   26 33.1+9.09 39.6+ 8.24 136.8+60.8 48.1#16.8 WHAT WE CLAIM IS: 1. A lipogenesis-inhibiting composition which comprises a pharmaceutically or veterinarily-acceptable carrier and, as active ingredient, in pharmaceutically- or veterinarily-acceptable state of purity, a compound of the general formula

   in which X is

   R is halogen; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from one to six carbon atoms; cycloalkyl or cycloalkyloxy of from three to six carbon atoms; phenyl, phenoxy, benzyl or 2-phenethyl, optionally-substituted by one or two of one or more substituents selected from alkyl groups of from one to six carbon atoms, halogen atoms and nitro groups; n is zero one or two; each of R1 and R2 is hydrogen or alkyl of from one to foulrcarbon atoms, at least one of Rl and R2 being hydrogen; R3 is hydrogen, phenylalkyl or alkyl of from one to four carbon atoms; and R4 is phenylalkyl, alkyl of from one to four carbon atoms, alkenyl, alkynyl, or cycloalkyl of from three to six carbon atoms; with the proviso that when X is -NR3-, Y is a substituent in the 2-position of the oxygen-containing ring, and when X is -O-, -CH2 or -CHOH, Y is a substituent in either the 2- or 3-position of the oxygen-containing ring; or, when X represents an -NR3- group, an acid addition salt thereof.
2. 2. A composition as claimed in claim 1, which comprises as active ingredient a compound of the general formula

   wherein Rl and R2 have the meanings given in claim 1; n is0 or I; and R is halogen; nitro; amino; methylsulphonylamino; trifluoromethyl; alkyl or alkoxy of from 1 to 6 carbon atoms; cycloalkyl or cycloalkyloxy of from 3 to 6 carbon atoms; or phenyl or phenoxy optionally-substituted by 1 or 2 of the same or different subsituents selected from alkyl of from 1 to 6 carbon atoms, halogen atoms and nitro groups.
3. 3. A composition as claimed in claim 2, wherein n is 0, or n is 1 and R is a chlorine or bromine atom in the 6-position of the ring structure, and Rl and R2 each represents hydrogen.
4. 4. A composition as claimed in claim 1, which comprises as active ingredient a compound of the general formula

   or an acid addition salt thereof, wherein n, R2 and R4 have the meanings given in claim 1; R is bromine, chlorine, fluorine, nitro, amino, methylsulphonylamino, trifluoromethyl, alkyl or alkoxy of from 1 to 6 carbon atoms, or cycloalkyl of from 3 to 6 carbon atoms; and R3 is hydrogen or phenalkyl;
5. 5. A composition as claimed in claim 4, wherein n is 0, or n is 1 and R is a substituent bonded to the 6-position of the ring structure, R4 is ethyl, R2 is hydrogen and R3 is hydrogen or benzyl.
6. 6. A composition as claimed in claim 1 which comprises as active ingredient a compound of the general formula

   or an acid addition salt thereof, wherein n and R2 have the meanings given in claim 1; R is chlorine, fluorine or bromine; nitro; amino; methylsulphonylamino: trifluoromethyl; alkyl or alkoxy of from 1 to 6 carbon atoms; or cycloalkyl of from 3 to 6 carbon atoms; and R3 is hydrogen or alkyl of from 1 to 4 carbon atoms.
7. 7. A composition as claimed in claim 6, wherein n is 0, or n is I and R is chlorine, alkyl or trifluoromethyl, R2 is hydrogen and R3 is hydrogen or methyl.
8. 8. A composition as claimed in claim l,which comprises as active ingredient a compound of the general formula

   wherein X is CH2 or CH.OH; n is 0 or 1; and R is halogen, nitro, amino trifluoromethyl, methylsulphonylamino, alkyl or alkoxy of from I to 6 carbon atoms, cycloalkyl of from 3 to 6 carbon atoms, or phenyl, phenoxy, benzyl or 2 phenethyl optionally-subsituted on the ring by one or more of the same or different substituents selected from halogen atoms, nitro groups and alkyl groups having from I to 6 carbon atoms; with the proviso that when X is CH . OH, the compound is in the cis-configuration.
9. 9. A composition as claimed in claim 8, wherein X is CH2, and either n is 0 or n is 1 and R is chloro, phenyl or cyclohexyl
10. 10. A composition as claimed in claim 8 or 9, wherein R is a substituent in the 6-position.
11. I 1. A composition as claimed in claim 1, wherein the active ingredient is any one of the compounds of the general formula I named in the Examples herein.
12. 12. A method of inhibiting lipogenesis in a non-human mammal which comprises administering to the mammal a compound of the general formula I defined in claim 1 or a composition as claimed in any one of claims 1 to 11.