United States Patent m
`Miiller et al.
`
`• • • ( • • • • • • ( • • • I ll
`5,684,014
`Nov. 4, 1997
`
`US005684014A
`[ii] Patent Number:
`[45] Date of Patent:
`
`[54] CYCLOALKANO-INDOLEAND
`-AZAINDOLE DERIVATIVES
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`[75]
`
`Inventors: Ulrich Mullen Wuppertal, Germany;
`Richard Connell, Trumbull, Conn.;
`Siegfried Goldmann, Wuppertal; Rudi
`Grutzmann, Solingen, both of
`Germany; Martin Beuck, Nilford,
`Conn.; Hilmar Bischoff; Dirk Denzer,
`both of Wuppertal, Germany; Anke
`Domdey-Bette, Hiickeswagen; Stefan
`Woblfdl, Hilden, both of Germany
`
`[73] Assignee: Bayer Aktiengesellschaft, Leverkusen,
`Germany
`
`[21] Appl. No.: 535,698
`
`[22] Filed:
`
`Sep. 28,1995
`
`Maureretal
`1/1972
`3,632,807
`10/1988
`4,775,680
`GUlaidetal
`FOREIGN PATENT DOCUMENTS
`
`546/345
`514/411
`
`0 234 708 Al
`0 300 676 A2
`0 310179 A2
`0 496 237 A2
`0509359
`0 617 035 Al
`
`European Fat. Off.
`9/1987
`European Pat. Off.
`1/1989
`European Pat. Off.
`4/1989
`European Pat. Off.
`7/1992
`European Pat. Off.
`10/1992
`European Pat. Off.
`9/1994
`OTHER PUBUCAnONS
`Heterocycles, vol. 22, No. 10, 1984 (pp. 2277-2279).
`Primary Examiner—Man L. Rotman
`Attorney, Agent, or Firm—Sprung Kramer Schaefer &
`Briscoe
`[57]
`
`ABSTRACT
`
`[30]
`
`Foreign Application Priority Data
`
`44 35 477.0
`Oct. 4, 1994 [DE] Germany
`[51] Int CI.6
`A61K 31/44; C07D 471/02;
`C07D 471/04
`514/292; 546/86; 546/87
`546/86, 87; 514/292
`
`[52] U.S. CI
`[58] Field of Search
`
`Cycloalkano-indole and -azaindole derivatives are prepared
`by reaction of appropriately substituted carboxylic acids
`with amines. The cycloalkano-indole and -azaindole deriva(cid:173)
`tives are suitable as active compounds for medicaments,
`preferably antiatherosclerotic medicaments.
`
`11 Claims, No Drawings
`
`1 of 67
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`PENN EX. 2203
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`5,684,014
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`CYCLOALKANO-INDOLE AND
`-AZAENDOLE DERIVATIVES
`
`The present invention relates to cycloalkano-indole and
`-azaindole derivatives, processes for their preparation and 5
`their use as medicaments, in particular as antiatherosclerotic
`medicaments.
`It is known that increased blood levels of triglycerides
`( h y p e r t r i g l y c e r i d a e m i a)
`and
`c h o l e s t e r ol
`(hypercholesterolaemia) are associated with the genesis of
`atherosclerotic vessel wall changes and coronary heart dis(cid:173)
`eases.
`A distinctly increased risk of the development of coro(cid:173)
`nary heart disease is moreover present if these two risk
`factors occur in combination, which is accompanied, in turn,
`with an overproduction of apolipoprotein B-100. There is 15
`therefore, as before, a great need to make available effective
`medicaments for the control of atherosclerosis and coronary
`heart diseases.
`The present invention relates to cycloalkano-indole and
`-azaindole derivatives of the general formula (I)
`
`20
`
`0)
`
`CH2 -fy^-E-
`
`D
`
`in which
`R1 and R2, including the double bond connecting them,
`together form a phenyl or pyridyl ring or a ring of the
`formula
`
`NR»
`
`wherein
`R8 denotes hydrogen or straight-chain or branched
`alkyl having up to 4 carbon atoms,
`R3 and R4, including the double bond connecting them,
`together form a phenyl ring or a 4- to 8-membered
`cycloalkene at oxocycloalkene radical,
`all ring systems mentioned under R1^2 and R3/R4
`optionally being substituted up to 3 times by iden(cid:173)
`tical or different halogen, trifluoromethyl, carboxyl
`or hydroxyl substituents, by straight-chain or
`branched alkoxy or alkoxycarbonyl each having up
`to 6 carbon atoms or by straight-chain or branched
`alkyl having up to 6 carbon atoms, which, for its part,
`can be substituted by hydroxyl or by straight-chain
`or branched alkoxy having up to 4 carbon atoms,
`D represents hydrogen, cycloalkyl having 4 to 12 carbon
`atoms or straight-chain or branched alkyl having up to
`12 carbon atoms,
`E represents the —CO— or —CS— group,
`L represents an oxygen or sulphur atom or a group of the
`formula —NR9,
`wherein
`R9 denotes hydrogen or straight-chain or branched
`alkyl having up to 6 carbon atoms, which is option(cid:173)
`ally substituted by hydroxyl or phenyl,
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`R5 represents phenyl or a 5- to 7-membered saturated or
`unsaturated heterocycle having up to 3 heteroatoms
`from the series consisting of S, N and/or O, the cycles
`optionally being substituted up to 3 times by identical
`or different nitro, carboxyl, halogen or cyano substitu(cid:173)
`ents or by straight-chain or branched alkenyl or alkoxy(cid:173)
`carbonyl each having up to 6 carbon atoms or by
`straight-chain or branched alkyl having up to 6 carbon
`atoms, which is optionally substituted by hydroxyl,
`carboxyl or by straight-chain or branched alkoxy or
`alkoxycarbonyl each having up to 6 carbon atoms,
`and/or the cycles optionally being substituted by a group
`of the formula —OR10 or —NR1^1 2,
`wherein
`R10 denotes hydrogen or straight-chain or branched
`alkyl or alkenyl each having up to 6 carbon atoms,
`R11 and R12 are identical or different and denote
`phenyl, hydrogen or straight-chain or branched alkyl
`having up to 6 carbon atoms or straight-chain or
`branched acyl having up to 8 carbon atoms, which is
`optionally substituted by a group of the formula
`—NR13R14,
`wherein
`R13 and R14 are identical or different and denote
`hydrogen or straight-chain or branched acyl hav(cid:173)
`ing up to 8 carbon atoms,
`R6 represents hydrogen, carboxyl or straight-chain or
`branched alkoxycarbonyl having up to 5 carbon atoms,
`or represents straight-chain or branched alkyl having up
`to 6 carbon atoms, which is optionally substituted by
`hydroxyl or by a group of the formula —O—CO—R15,
`wherein
`Rls denotes phenyl which is optionally substituted up
`to 3 times by identical or different halogen or
`hydroxyl substituents or by straight-chain or
`branched alkyl having up to 5 carbon atoms,
`eg- straight-chain or branched alkyl or alkenyl each
`having up to 22 carbon atoms, each of which is
`optionally substituted by a group of the formula
`—OR16,
`wherein
`R16 is hydrogen, benzyl, triphenylmethyl or straight-
`chain or branched acyl having up to 6 carbon
`atoms,
`R7 represents hydrogen or
`R6 and R7 together represent the group of the formula
`= 0,
`if appropriate in an isomeric form, and their salts.
`The cycloalkano-indole and -azaindole derivatives
`according to the invention can also be present in the form of
`their salts. In general, salts with organic or inorganic bases
`or adds may be mentioned here.
`In the context of the present invemion, physiologically
`acceptable salts are preferred. Physiologically acceptable
`salts of the compounds according to the invention can be
`salts of the substances according to the invention with
`mineral acids, carboxylic acids or sulphonic adds. Particu(cid:173)
`larly preferred salts are, for example, those with hydrochlo(cid:173)
`ric add, hydrobromic add, sulphuric add, phosphoric acid,
`methanesulphonic add, ethanesulphonic add, toluenesul-
`phonic add, benzenesulphonic add, naphthalenedisul-
`phonic acid, acetic add, propionic acid, lactic acid, tartaric
`acid, citric add, fumaric add, maleic add or benzoic add.
`Physiologically acceptable salts can also be metal or
`ammonium salts of the compounds according to the inven(cid:173)
`tion which have a flee carboxyl group. Particularly preferred
`
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`5,684,014
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`salts are, for example, sodium, potassium, magnesium or
`calcium salts, and also ammonium salts which are derived
`from ammonia, or organic amines, such as, for example
`ethylamine, di- or triethylamine, di- or ttiethanolamine,
`dicyclohexylamine, dimethylaminoethanol, arginine, lysine, 5
`ethylenediamine or 2-phenylethylainine.
`Including the double bond of parent structure, the
`cycloalkene radical (R3/R4) in the context of the invention in
`general represents a 4- to 8-membered hydrocarbon radical,
`preferably a 5- to S-membered hydrocarbon radical, for 10
`example a cyclobutene, cyclopentene, cyclohexene, cyclo-
`heptene or cyclooctene radical. The cyclopentene,
`cyclohexene, cyclooctene or cycloheptene radicals are pre(cid:173)
`ferred
`Heterocycle (R5) in the context of the invention in general is
`represents a saturated or unsaturated 5- to 7-menibered
`heterocycle, preferably a 5- to 6-membered heterocycle,
`which can contain up to 3 heteroatoms from the series
`consisting of S, N and/or 0. Examples which may be
`mentioned are: pyridyl, thienyl, furyl, pyrrolyl, thiazolyl, 20
`oxazolyl, imidamlyl, morpholinyl or piperidyl. Pyridyl and
`thienyl are preferred.
`The compounds according to the invention can exist in
`stereoisomeric forms which either behave as image and
`mirror image (enantiomers), or do which do not behave as 25
`image and mirror image (diastereomers). The invention
`relates both to the enantiomers and diastereomers and their
`respective mixtures. These mixtures of the enantiomers and
`diastereomers can be separated in a known manner into the
`stereoisomerically uniform constituents.
`Preferred compounds of the general formula (I) are those
`in which
`R1 and R2, including the double bond connecting them,
`together form a phenyl or pyridyl ring or a ring of the
`formula
`
`30
`
`35
`
`NR!,
`
`40
`
`wherein
`R9 denotes hydrogen or straight-chain or branched
`alkyl having up to 5 carbon atoms, which is option(cid:173)
`ally substituted by hydroxyl or phenyl,
`R5 represents phenyl, pyridyl, furyl, thienyl or imidazolyl,
`each of which is optionally substituted up to 2 times by
`identical or different nitro, carboxyl, fluorine, chlorine,
`bromine or cyano substituents, by straight-chain or
`branched alkenyl or alkoxy carbonyl each having up to
`4 carbon atoms or by straight-chain or branched alkyl
`having up to 5 carbon atoms, which is optionally
`substituted by hydroxyl, carboxyl or by straight-chain
`or branched alkoxy or alkoxycarbonyl each having up
`to 5 carbon atoms,
`and/car the cycles are optionally substituted by a group
`of the formula —OR10 or —NR1^1 2,
`wherein
`R10 denotes hydrogen or straight-chain or branched
`alkyl or alkenyl each having up to 4 carbon atoms,
`R11 and R2 are identical or different and denote phenyl,
`hydrogen or straight-chain or branched alkyl having
`up to 5 carbon atoms
`or denote straight-chain or branched acyl having up
`to 6 carbon atoms, which is optionally substituted by
`a group of the formula —NR13R14,
`wherein
`R13 and R14 are identical or different and denote
`hydrogen or straight-chain or branched acyl hav(cid:173)
`ing up to 6 carbon atoms,
`R6 represents hydrogen, carboxyl or straight-chain or
`branched alkoxycarbonyl having up to 4 carbon atoms,
`or represents straight-chain or branched alkyl having up
`to 5 carbon atoms, which is optionally substituted by
`hydroxyl or by a group of the formula —O—CO—R15,
`wherein
`R15 denotes phenyl which is optionally substituted up
`to 3 times by identical or different fluorine, chlorine,
`bromine or hydroxyl substituents or by straight-
`chain or branched alkyl having up to 4 carbon atoms,
`or straight-chain or branched alkyl or alkenyl each
`having up to 20 carbon atoms, each of which is
`optionally substituted by a group of the formula
`—OR16,
`wherein
`Rlfiis hydrogen, benzyl, triphenylmethyl or straight-
`chain or branched acyl having up to 5 carbon
`atoms,
`R7 represents hydrogen or
`R6 and R7 together represent the group of the formula
`= 0,
`if appropriate in an isomeric form, and their salts.
`Particularly preferred compounds of the general formula
`(I) are those in which
`R1 and R2, including the double bond connecting them,
`together form a phenyl or pyridyl ring or a ring of the
`formula
`
`NR8,
`
`wherein
`R8 denotes hydrogen or methyl,
`
`45
`
`50
`
`wherein
`R8 denotes hydrogen or straight-chain or branched
`alkyl having up to 3 carbon atoms,
`R3 and R4, including the double bond connecting them,
`together form a phenyl ring or a cyclopentene,
`c y c l o h e x e n e, c y c l o h e p t e n e, c y c l o o c t e n e,
`oxocyclopentene, oxocyclohexene, oxocycloheptene or
`oxocyclooctene radical,
`all ring systems mentioned under RVR2 and R3/R4
`optionally being substituted up to 2 times by iden(cid:173)
`tical or different fluorine, chlorine, bromine,
`trifluoromethyl, carboxyl or hydroxyl substituents, 55
`by straight-chain or branched alkoxy or alkoxycar-
`bonyl each having up to 4 carbon atoms or by
`straight-chain or branched alkyl having up to 4
`carbon atoms, which, in turn, can be substituted by
`hydroxyl or by straight-chain or branched alkoxy
`having up to 3 carbon atoms,
`D represents hydrogen, cyclobutyl, cyclopentyl,
`cyclohexyl, cycloheptyl, cyclooctyl or straight-chain or
`branched alkyl having up to 10 carbon atoms,
`E represents the —CO— or —CS— group,
`L represents an oxygen or sulphur atom or represents a
`group of the formula —NR9,
`
`60
`
`65
`
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`5,684,014
`
`R3 and R4, including the double bond connecting them,
`together form a phenyl ring or a cyclopentene,
`c y c l o h e x e n e, c y c l o h e p t e n e,
`c y c l o o c t e n e,
`oxocyclopentene, oxocyclohexene, oxocycloheptene or
`oxocyclooctene radical,
`all ring systems mentioned under R'/R2 and R3/R4
`optionally being substituted up to 2 times by iden(cid:173)
`tical or different fluorine, chlorine, bromine,
`trifluoromethyl, carboxyl or hydroxyl substituents,
`by straight-chain or branched alkoxy or alkoxycar-
`bonyl each having up to 3 carbon atoms or by
`straight-chain or branched alkyl having up to 3
`carbon atoms, which, for its part, can be substituted
`by hydroxyl, methoxy or ethoxy,
`D represents hydrogen, cyclopentyl, cyclohexyl,
`cycloheptyl, cyclooctyl or straight-chain or branched
`alkyl having up to 6 carbon atoms,
`E represents the —CO— or —CS— group,
`L represents an oxygen or sulphur atom or represents a
`group of the formula —NR ,
`wherein
`R9 denotes hydrogen or straight-chain or branched
`alkyl having up to 4 carbon atoms, which is option(cid:173)
`ally substituted by hydroxyl or phenyl,
`R5 represents phenyl, pyridyl or thienyl, each of which is
`optionally substituted up to 2 times by identical or
`different nitro, carboxyl, fluorine, chlorine, bromine or
`cyano substituents, by straight-chain or branched alk-
`enyl or aikoxycarbonyl each having up to 3 carbon
`atoms or by straight-chain or branched alkyl having, up
`to 4 carbon atoms, which is optionally substituted by
`hydroxyl, carboxyl or by straight-chain or branched
`alkoxy or aikoxycarbonyl each having up to 4 carbon
`atoms, and/or the cycles are optionally substituted by a
`group of the formula —ORld or —NR^R12,
`wherein
`R10 denotes hydrogen or straight-chain or branched
`alkyl or alkenyl each having up to 3 carbon atoms,
`R11 and R12 are identical or different and denote
`phenyl, hydrogen or straight-chain or branched alkyl
`having up to 4 carbon atoms
`or denote straight-chain or branched acyl having up
`to 5 carbon atoms, which is optionally substituted by
`a group of the formula —-NR13R14,
`wherein
`R13 and R14 are identical or different and denote
`hydrogen or straight-chain or branched acyl hav(cid:173)
`ing up to 5 carbon atoms,
`R6 represents hydrogen, carboxyl or straight-chain or
`branched aikoxycarbonyl having up to 3 carbon atoms,
`or represents straight-chain or branched alkyl having up
`to 4 carbon atoms, which is optionally substituted by
`hydroxyl or by a group of the formula —CO—R15,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`wherein
`R15 denotes phenyl which is optionally substituted up
`to 3 times by identical or different straight-chain or
`branched alkyl having up to 3 carbon atoms,
`or denotes straight-chain or branched alkyl or alk-
`egyl each having up to 19 carbon atoms, each of
`which is optionally substituted by a group of the
`formula —OR16,
`wherein
`R16 denotes hydrogen, benzyl, triphenylmethyl or
`straight-chain or branched acyl having up to 4
`carbon atoms,
`R7 represents hydrogen or
`R6 and R7 together represent the group of the formula
`= 0,
`if appropriate in an isomeric form, and their salts.
`A process for the preparation of the compounds of the
`general formula (I) according to the invention has addition(cid:173)
`ally been found, characterized in that
`carboxylic acids of the general formula (IT)
`
`Rl
`
`.R1
`
`(H)
`
`N - ^ R*
`
`CO2H,
`
`in which
`R1, R2, R3, R4 and D have the meaning indicated,
`are amidated using compounds of the general formula
`(HI)
`
`R5
`
`H2rr^R«
`
`(HI)
`
`in which
`Rs has the meaning indicated and
`R17 has the indicated meaning of R6, but does not
`represent carboxyl,
`in an inert solvent and in the presence of bases and/or
`auxiliaries,
`and, if appropriate, functional groups are varied by
`hydrolysis, esterification or reduction.
`The process according to the invention can be illustrated
`by the following reaction scheme:
`
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`CHa
`
`5,684,014
`
`8
`
`Dichloromethane/triethylamiig
`1-hydroxy-lH-benzotriazole and
`N1 -{3 -dimcthylamiBopropyl)-
`N-ethykarbodiimide hydrochloride
`
`-^
`^
`
`CO—NH
`
`OH
`
`Suitable solvents for the amidation are in this case inert
`organic solvents which do not change under the reaction
`conditions. These include ethers, such as diethyl ether or 50
`tetrahydrofuran, halogenohydrocarbons
`such as
`dichloromethane, trichloromethane, tetrachloromethane,
`trichloroethane, tetrachloroethane, 1,2-dichloroethane or
`trichloroethylene, hydrocarbons such as benzene, xylene,
`toluene, hexane, cyclohexane or petroleum fractions, 55
`nilromethane, dimethylfonnamide, acetone, acetonilrile or
`hexamethylphosphoramide. It is also possible to employ
`mixtures of the solvents. Dichloromethane, tetrahydrofuran,
`acetone and dimethylformamide are particularly preferred.
`Bases which can, be employed for the process according 60
`to the invention are in general inorganic or organic bases.
`These preferably include alkali metal hydroxides such as, for
`example, sodium hydroxide or potassium hydroxide, alka(cid:173)
`line earth metal hydroxides, such as, for example, barium
`hydroxide, alkali metal carbonates such as sodium carbonate 65
`or potassium carbonate, alkaline earth metal carbonates such
`as calcium carbonate, or alkali metal alkoxides such as
`
`sodium or potassium methoxide, sodium or potassium
`ethoxide or potassium tert-butoxide, or organic amines
`(trialkyl(C1-C)5)aimnes) such as triethylamine, or hetero-
`cycles such as l,4-diazabicyclo[2.2.2]octane (DABCO),
`l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine,
`diaminopyridine, methylpiperidine or morpholine. It is also
`possible to employ alkali metals such as sodium and their
`hydrides such as sodium hydride as bases. Sodium and
`potassium carbonate and triethylamine are preferred.
`The base is employed in an amount from 1 mol to 5 mol,
`preferably from 1 mol to 3 mol, relative to 1 mol of the
`compound of the general formula (IT).
`The reaction is in general carried out in a temperature
`range from 0° C. to 150° C, preferably from +20° C. to
`+110° C.
`The reaction can be carried out at normal, increased or
`reduced pressure (e.g. 0.5 to 5 bar). In general, the reaction
`is carried out at normal pressure.
`The amidation can optionally proceed via the activated
`stage of the acid halides, which can be prepared from the
`
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`T—ifcc ^ / ^ ^^
`
`ff^
`
`c c, Ris
`
`3^
`
`^
`
`10
`9
`The bases employed for the process according to the
`corresponding adds by reaction with thionyl chloride, phos-
`invention can in general be inorganic or organic bases. These
`phorus trichloride, phosphorus pentachloride, phosphorus
`preferably include alkali metal hydroxides, for example,
`tribromide or oxalyl chloride
`socUum hydroxide or potassium hycfroxide, alkaline earth
`The abovementioned bases can optionally also be
`5 m e^ hydroxides, for example, banum hydroxide alkali
`employed for the amidation as acid-bindkg auxiliaries.
`metal carbonates such as sodium carbonate or potassium
`"^ .•'77
`",.
`.
`,
`, .
`J
`.
`x r~
`e a r th m e t al c a l b o n ates such as calcium
`Suitable auxiliaries are also dehydrating reagents. These
`c a r b o l l a t e5 aikaiille
`such as sodium or
`c a r b o n a t e? or a ] ]^ m e t al ^xifes
`include, for example, carbodnmides such as
`potassium methoxide, sodium or potassium ethoxide or
`diisopropylcarbodiimide, dicyclohexylcarbodumide and
`potassium tert-butoxide, or organic amines (trialkyKd-Ce)
`N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydro-
`chloride or carbonyl compounds such as carbonyldiimida- 10 amines) s u ch as triethylamine, or heterocycles such as
`zole or 1^-oxazolium compounds such as 2-ethyl-5-phenyl-
`l,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo
`l,2-oxazolium-3-sulphonate or propanephosphonic
`[5.4.0]undec-7-ene (DBU), pyridine, diaminopyridine,
`anhydride or iso-butyl chlorofonnate or benzotriazolyloxy-
`methylpiperidine . or moipholine. It is also possible to
`tris-(dimethylamino)phosphonium hexafluorophosphate or
`employ alkali metals such as sodium or their hydrides such
`diphenyl phosphoramidate or methanesulphonyl chloride, if 15 as sodium hydride as bases. Sodium hydride, potassium
`appropriate in the presence of bases such as triethylamine or
`carbonate, triethylamine, pyridine and potassium tert-
`N-ethylmorpholine or N-methylpiperidine or dicyclohexyl-
`butoxide, DBU or DABCO are preferred,
`carbodiimide and N-hydroxysuccinimide.
`In general, the base is employed in an amount from 0.05
`The acid-binding agents and dehydrating reagents are in
`m o1 to 10 mol> preferably from 1 mol to 2 mol, relative to
`general employed in an mount from 0.5 to 3 mol, preferably 20 * m o1 of ^ compound of the formula (IV).
`T*16 Pr o c e ss according to the invention is in general
`from 1 to 1.5 mol, relative to 1 mol of the corresponding
`carbo
`lie
`-^
`carried out in a temperature range from -30° C. to +100° C,
`The variation of functional groups, for example
`preferably from-10° C. to+60° C.
`,
`,
`.
`.
`.
`The process according to the invention is in general
`.„
`..
`,
`. ^
`,i ,
`£
`. ,r
`,
`e
`hydrolysis, esterification and reduction, and also separation
`.
`.
`„
`.. .
`,
`-.,
`J.
`'
`'
`,
`, „
`^,
`earned out at normal pressure. However, it is also possible
`,.
`.
`. .
`,
`of isomers and salt formation is earned out by customary 25 to c a ny o ut ^
`p[oc£a
`at e l e v a t ed p r e s s l l re or ^reduced
`mS^
`j.
`,.
`.,
`**
`, ™
`pressure (e.g. in a range from 0.5 to 5 bar).
`* ^
`The carboxyhc acids of the general formula (II) are new
`j^ compounds of the general formula (EI) are known
`and can be prepared by reacting
`pg,. se>
`compounds of the general formula (IV)
`The compounds of the general formula (IV) are known or
`30 can be prepared in analogy to known methods.
`The compounds of the general formula (V) are known or
`can be prepared in analogy to known methods.
`The compounds of the general formula (I) according to
`the invention have an unforeseeable spectrum of pharma-
`35 cological action.
`They can be used as active compounds in medicamnents
`for the reduction of changes to vessel walls and for the
`in which
`treatment of coronary heart disorders, cardiac insufficiency,
`D has the meaning indicated,
`brain power disorders, ischaemic brain disorders, apoplexy,
`T represents a typical leaving group, for example
`chlorine, bromine, iodine, tosylate or mesylate, prefer- 40 circulatory disorders, disorders of the microcirculation and
`thromboses.
`ably bromine, and
`i (C -C \ alkvl
`Ri8
`Furthermore, the proliferation of smooth muscle cells
`j, it 4^~^^, .
`, __
`^
`.„.
`j
`plays a decisive part in the occlusion of vessels. The
`with compounds of the general formula (V)
`compounds accortfng to the invention are suitable for
`_
`inhibiting this proliferation and thus preventing atheroscle-
`( ) 45 rotic processes.
`The compunds according to the invention are distin-
`guished by a lowering of the ApoB-100-associated lipopro-
`teins (VLDL and its degradation products, e.g. LDL), of
`ApoB-100, of triglycerides and of cholesterol. They thus
`50 have useful, superior pharmacological properties in com-
`parison with the prior art.
`in which
`R1, R2, R3 and R4 have the meaning indicated,
`Surprisingly, the action of the compounds according to the
`invention consists first in a decrease or complete inhibition
`in inert solvents, if appropriate in the presence of a base.
`of the formation and/or the release of ApoB-100-associated
`Suitable solvents for the process are the customary
`organic solvents which do change under the reaction con- 55 lipoproteins from liver cells, which results in a lowering of
`ditions. These preferably include ethers such as diethyl
`the VLDL plasma level. This lowering of VLDL must be
`ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or
`accompanied by a lowering of the plasma level of ApoB-
`hydrocarbons such as benzene, toluene, xylene, hexane,
`1 0 0' L D L' triglycerides and cholesterol; a number of the
`cyclohexane or petroleum fractions, or halogenohydrocar-
`abovementioned risk factors which are involved in vessel
`bons such as dichloromethane, trichloromethane, 60 waU changes are thus simultaneously decreased,
`. rbc compounds according to the invention can therefore
`tetrachloromethane, dichloroethylene, trichloroethylene or
`. .J ^.\,
`,
`J
`.,
`,
`'
`. ,
`...
`be employed, for the prevention and treatment of
`chlorobenzene, or ethyl acetate, tnefliylamme, pyndme,
`o b e s i t y, p ^ e a t i t is and constipation,
`^
`^^
`d i m e t h yl
`s u l p h o x i d e,
`d i m e t h y l f o r m a m i d e,
`hexamethylphosphoramide, acetonitrile, acetone or
`nitromethane. It is also possible to use mixtures of the 65
`solvents mentioned. Dimethylformainide and tetrahydrofu-
`ran are preferred.
`
`I- Inhibition of the Release of ApoB-lOO-assodated
`Lipoproteins
`The test fen detecting the inhibition of the release of
`ApoB-100-associated lipoproteins from liver cells was car-
`
`XX
`
`^ „,
`
`R
`
`^
`
`,,
`R'
`
`?
`g
`
`6 of 67
`
`PENN EX. 2203
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`5,684,014
`
`11
`ried out in vitro using cultured liver cells, preferably using
`cells of the human line HepG2. These cells are cultured
`under standard conditions in medium for the culture of
`eukaryotic cells, preferably in RPMI1640 with 10% foetal
`calf serum. HepG2 cells synthesize and secrete into the
`culture supenatant ApoB-lOO-assodated lipoprotein par(cid:173)
`ticles which in principle are built up in a similar manner to
`the VLDL and LDL particles which are to be found in the
`plasma.
`These particles can be detected using an immunoassay for
`human LDL. This immunoassay is carried out using anti(cid:173)
`bodies which have been induced against human LDL in
`rabbits under standard conditions. The anti-LDL antibodies
`(rabbit anti-LDL Ab) were purified by affinity chromat-
`agraphy on an immunosorbent using human LDL. These
`purified rabbit anti-LDL Ab are adsorbed on the surface of
`plastic. Expediently, this adsorption is carried out on the
`plastic surface of microtitre plates having 96 wells, prefer(cid:173)
`ably on MaxiSorp plates. If ApoB-100-associated particles
`are present in the supernatant of Hep-G2 cells, they can be
`bound to the insolubilized rabbit anti-LDL Ab, and an
`immune complex results which is bound to the plastic
`surface. Unbound proteins are removed by washing. The
`immune complex located on the plastic surface is detected
`using monoclonal antibodies which have been induced
`against human LDL and purified according to standard
`conditions. These antibodies were conjugated with the
`enzyme peroxidase. Peroxidase converts the colourless sub(cid:173)
`strate TMB into a coloured product in the presence of HJOJ.
`After acidification of the reaction mixture with H2SO4, the
`specific light absorption at 450 nm is determined, which is
`a measure of the amount of ApoB-100-associated particles
`which have been secreted into the culture supernatant by the
`HepG2 cells.
`Surprisingly, the compounds according to the invention
`inhibit the release of the ApoB-100-associated particles. The
`IC50 value indicates at which substance concentration the
`light absorption is inhibited by 50% in comparison with the
`control (solvent control without substance).
`
`Ex. No.
`
`IC50 [109 msM]
`
`1
`5
`31
`50
`
`28
`1.1
`170
`29
`
`2. Determination of the VLDL Secretion in vivo in
`the Hamster
`
`The effect of the test substances on VLDL secretion in
`vivo is investigated in the hamster. To do this, golden
`hamsters are anaesthetized with Ketaset (83 mg/kg s.c.) and
`Nembutal (50 mg/kg i.p.) after premedication with atriopine
`(83 mg/kg s.c). When the animals have become reflex-free,
`the jugular vein is exposed and cannulated. 0.25 ml/kg of a
`20% strength solution of Triton WR-1339 in physiological
`saline solution is then administered. This detergent inhibits
`the lipoprotein lipase and thus leads to a rise in the triglyc(cid:173)
`eride level as a result of a lack of catabolism of secreted
`VLDL particles. This triglyceride rise can be used as a
`measure of the VLDL secretion rate.
`Blood is taken from the animals before and also one and
`two hours after administration of the detergent by puncture
`of the retroorbital venous plexus. The blood is incubated for
`two hours at room temperature, and then overnight at 4° C,
`
`12
`in order to end clotting completely. It is then centrifuged at
`10,000 g for 5 minutes. The triglyceride concentration in the
`serum thus obtained is determined with the aid of a modified
`commercially available enzyme test (Merckotest® triglyc(cid:173)
`eride No. 14354). 100 ^ of serum are treated with 100 pi of
`test reagent in 96-hole plates and incubated at room tem(cid:173)
`perature for 10 minutes. The optical density is then deter(cid:173)
`mined at a wavelength of 492 nM in an automatic plate-
`reading apparatus (SLT Spectra). Serum samples having an
`excessively high triglyceride concentration are diluted with
`10
`physiological saline solution. The triglyceride concentration
`contained in the samples is determined with the aid of a
`standard curve measured in parallel. In this model, test
`substances are administered intravenously either immedi(cid:173)
`ately before administration of the detergent or orally or
`15
`subcutaneously before initiation of anaesthesia.
`
`20
`
`EicNo.
`
`ED51, [mg/kg] p.o.
`
`10-15
`3-6
`10-20
`
`25
`
`3. Inhibition of Intestinal Triglyceride Absorption
`in vivo (Rats)
`The substances which are to be investigated for their
`triglyceride absorption-inhibiting action in vivo are admin(cid:173)
`istered orally to male Wistar rats having a body weight of
`between 170 and 230 g. For this purpose, the animals are
`30
`divided into groups of 6 animals 18 hours before substance
`administration and food is then withdrawn from them.
`Drinking water is available to the animals ad libitum. The
`animals of the control groups receive an aqueous tragacanth
`suspension or a tragacanth suspension which contains olive
`35
`oil. The tragacanth-olive oil suspension is prepared using an
`Ultra-Turrax. The substances to be investigated are sus(cid:173)
`pended in an appropriate tragacanth-olive oil suspension
`likewise using the Ultra-Turrax, directly before substance
`administration.
`40
`To determine the basal serum triglyceride content, blood
`is taken from each rat by puncture of the retroorbital venous
`plexus before stomach tube application. The tragacanth
`suspension, the tragacanth-olive oil suspensions without
`substance (control animals) or the substances suspended in
`45
`an appropriate tragacanth-olive oil suspension are then
`administered to the fasting animals using a stomach tube.
`Further taking of blood to determine the postprandial serum
`triglyceride rise is caxried out, as a rule, 1, 2 and 3 hours
`after stomach tube application.
`50
`The blood samples are centrifuged and, after recovering
`the serum, the triglycerides are determined photometrically
`using an EPOS analyzer 5060 (Eppendorf Geratebau,
`Netheler & Hinz GmbH, Hamburg). The determination of
`the triglycerides is carried out completely enzymatically
`55
`using a standard commercial UV test
`The postprandial serum triglyceride rise is determined by
`subtraction of the triglyceride preliminary value of each
`animals from its corresponding postprandial triglyceride
`concentrations (1, 2 and 3 hours after administration).
`60
`The differences (in mmol/1) at each point in time (1,2 and
`3 hours) are averaged in the groups, and the mean values of
`the serum triglyceride rise (ATG) of the substance-treated
`animals is compared with the animals which only received
`the tragac