United States Patent
`
`[191
`
`[11] Patent Number:
`
`5,684,014
`
`
`Miiller et al.
`[45] Date of Patent:
`Nov. 4, 1997
`
`US005684014A
`
`[54] CYCLOALKANO-INDOLE AND
`-AZAINDOLE DERIVATIVES
`
`[55]
`
`References Cited
`U_S_ PATENT DOCUMENTS
`
`E25]
`
`Inventqrs:
`
`[]]1-ich Miner, Wmppcxtaj, Geamany;
`Richard Connell, Trumbull, Conn.;
`Siegfried Goldmann. Wuppertal; Rudi
`Grfimmum’ Solmgen’ both of
`Gamay; Mara” Back’ Ifmfmd’
`C°’1°'; mm“ BM“; D“k D°““*
`W“ °f W“PP“*31s GWWY? A111“
`Domdey-Bette» Hfickfiswasw: Stefln
`Wohlfeil, Hildcn. both of Gennany
`
`[73] Assigncc: BayerAktiengesellschaft,Lcverkuscn,
`Gcnnany
`
`[21]
`
`N0.: 535,693
`
`[22] Filed:
`
`Sep. 23, 1995
`
`.......................... 5463345
`H1972 Manner el :11.
`3,632,807
`.......................... 514£411
`4,'nS,680 1oz19ss Giuard et a1.
`FOREIGN PATENT DOCUMENTS
`0 234 703 A1
`9:193?
`E ropean Pat of
`o 300 676 A2
`H1939
`Euuropean Fat. 05' '
`o 310 179 A2
`#1939 European Pat. of '
`0 496 237 A2
`"mm European Pat: ctr: 1
`0509359 1ox1992 European Fat. 011 .
`o 617 035 A1
`9!1994 European Fat. 011 .
`UI'I-IER PUBI..ICA'I‘IONS
`
`Hctcrocyclcs, VOL 22, No. 10, 1934 (pp. 2277-2279).
`Prinmry E.mm.Ener—Ala.I1 L. Rounan
`Axioms); Agent, or Finn—SpIl1l1g Klalner Schaefer &
`Briscoc
`
`[57]
`
`ABSTRACT
`
`Foreign Application Priority Data
`[30]
`Oct. 4, 1994
`[DE]
`Genznany ........................ 44 35 477.0
`
`[51]
`
`Int. CL"
`
`AISIK 31144; Corn) 471/02;
`C07D 471104
`
`_.............. 514392‘, 54-6-2'86; 54-6f8':'
`[52] U.S. CL
`5461536,’ 8'7; 514-I292
`[58] Field of Search .-.-._.........
`
`Cycloalkano-indole and —az.a.1'.I1do1e delivatives are prepared
`by reaction of appropriately substituted carboxylic acids
`with amines. The cyc1oa]ka.no—indo1e and —azai.ndole dcriva-
`lives are suitable as active compounds for medicamcnls,
`preferably anliaiherosclerotic medicaments.
`
`.
`
`11 Claims, No Drawings
`
`Iof67
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`5,634,014
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`2
`
`1
`CYCLOALKANO-INI)OLE AND
`-AZAINDOLE DERIVATIVES
`
`The present invention relates to cycloalkano-indole and
`—azaindo.le derivatives, processes for their preparation and 5
`their use as medicarnents, in particular as antziatltcrosclerotic
`medicatnents.
`It is lcnown that increased blood levels of triglycerides
`(hypertriglyceridaernia)
`and
`cholesterol
`(hypercholesterolaemia) are associated with the genesis of
`atherosclerotic vessel wall changes and coronary heart dis-
`eases.
`
`10
`
`A distinctly increased risk of the development of coro-
`nary heart disease is moreover present if these two risk
`factors occur in combination, which is accompanied in turn,
`with an ove.rprcdt.1ct.ion of apolipoproteiu B-100. There is
`therefore, as before, a great need to make available eifective
`medicarnents for the control of atherosclerosis and coronary
`heart diseases.
`
`The present invention relates to cycloalkano-indole and
`azaindole derivatives of the general formula (I)
`
`R3
`
`114:
`
`inwhich
`
`R1
`
`(1)
`
`: N:
`I
`ctn
`
`:22
` E L Rik’
`
`“’
`_ ’i\ ««
`
`D
`
`R1-and R2, including the double bond connecting them,
`together form a phenyl or pyridyl ring or at ring of the
`formula
`
`15
`
`25
`
`30
`
`35
`
`NR‘,
`
`wherein
`
`R’ represents phenyl or a 5- to 7-membered saturated or
`unsaturated heterocycle having up to 3 heteroatoms
`from the series consisting of S, N andfor 0, the cycles
`optionally being substituted up to 3 times by identical
`or different nitro, carbonyl, halogen or cyano substitu-
`ents or by straight-chain or branched alkenyl or alkoxy-
`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,
`andfor the cycles optionally being substituted by a group
`of the formula —oR1° or —NR”R”,
`wherein
`R1” denotes hydrogen or straight-chain or branched
`alltyl or alkenyl each having up to 6 carbon atoms,
`R” and R13 are identical or difierent and denote
`phenyl, hydrogen or straight—chain or branched alkyl
`having up to 6 carbon atoms or straight-chain or
`branched acyl having upto 8 carbon atoms, which is
`optionally substituted by a group of the formula
`__NR13R14!
`wherein
`R13 and R“ are identical or diiferent and denote
`hydrogen or st:ra.ight-chain or branched acyl hav-
`ing up to 8 carbon atoms,
`R5 represents hydrogen, carboxyl or straight-chain or
`branched alkoxycarhonyl having up to 5 carbon atoms,
`or represenm sira.ight—chain or branched alkyl having up
`to 6 carbon atoms, which is optionally substituted by
`hydroxyl or by a group of the formula —0——C0—-R”,
`wherein
`
`R” denotes phenyl which is optionally substituted up
`to 3 times by identical or diiferent halogen or
`hydroxyl substituents or by straight-chain or
`branched alkyl having up to 5 carbon atoms,
`or straight-chain or branched alkyl or alkyl each
`having up to 22 carbon atoms, each of which is
`optionally substituted by a group of the formula
`4Rl61
`wherein
`
`R1‘ is hydrogen, benzyl, triphenylrnethyl or straight-
`chain or branched acyl having up to 6 carbon
`atoms,
`
`R7 represents hydrogen or
`R‘ 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 acids may be mentioned here.
`In the context of the present invernion, pbysiologitztlly
`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 acids. Particu-
`larly preferred salts are, for example, those with hydrochlo-
`ric acid, hydrobtornic acid, sulphuric acid, phosphoric acid,
`methanesulphonic acid, ethanesulphonic acid, toluenesul-
`phonic acid, benzenesulphonic acid. naphthalcnedisul-
`phonic acid, acetic acid, propionic acid, lactic acid, tartaric
`acid, citric acid, fumaric acid, rnaleic acid or benzoic acid.
`Physiologically acceptable salts can also be metal or
`arnmonium salts of the compounds according to the inven-
`tion which have a flee carbonyl group. Particularly prefened
`
`45
`
`50
`
`R3 denotes hydrogen or straight-chain or branched
`alkyl having up to 4 carbon atoms,
`R“ and R‘, including the double bond connecting them,
`together form a phenyl ring or a 4- to 8-membened
`cycloalkene or oxocycloallrene radical,
`all ring systems mentioned under RVR’ and Ram‘
`optionally being substituted up to 3 times by iden-
`tical or diiferent halogen, trifluorornetltyl, carboxyl
`or hydroxyl substituents, by straight-chain or
`branched alkoxy or alkoxycarbonyl each having up
`to 6 carbon atoms or by stl‘a.igl1t-chain D1’ branched
`alkyl having up to 6 carbon atoms, which, forits 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,
`Lrepresents an oxygen or sulphur atom or a group of the
`formula —NR9,
`wherein
`R9 denotes hydrogen or straight-chain or branched 65
`alkyl having up to 6 carbon atoms, which is option-
`ally substituted by hydroxyl or phenyl,
`
`55
`
`60
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`3
`salts are, for example. sodium, potassitun, magnesium or
`calcium salts, and also ammonium salts which are derived
`from ammonia, or organic amines, such as, for example
`ethylamine, d.i~ or l:riethyla.mine, di- or lriethanolarnine,
`dicyclohexylamine, dimefliylarninoethanol. arginine. lysine.
`ethylenediamine or 2-phenylethylamine.
`the
`Including the double bond of parent structure,
`cycloallrene radical (R3;'R"‘) in the context of the invention in
`general represents a 4- to 8-rnernbered hydrocarbon radical,
`preferably a S~ to 8-membered hydrocarbon radical, for
`example a cyclobutene, cyclopentene, cyclone:-rene, cyclo-
`heptene or cyclooctene radical. The cyclopentene,
`cyclohexene, cyclooctene or cycloheptene radicals are pre-
`ferred
`
`Heterocycle (R5) in the context ofthe invention in general
`represents a saturated or unsaturated 5- to 7—rnembered
`hetcrocycle, preferably a 5- to 6-Inernbered heterocycle,
`which can contain up to 3 heteroatoms from the series
`consisting of S, N andfor 0. Examples which may be
`mentioned are: pyridyl, thienyl, fury], pyrrolyl, thiazlolyl.
`oxazolyl, imidarulyl, rnorpholinyl or piperidyl. Pyridyl and
`thienyl are preferred.
`The compounds according to the invention can exist in
`stereoisorneric forms which either behave as image and
`mirror image (enantiomers). or do which do not behave as
`image and mirror image (diastereomers). The invention
`relates both to the enanliorners and diastereorners and their
`respective mixtures. These mixtures of the enantiorners and
`diastereorners can be separated in a known manner into tl1e
`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
`
`III]
`
`'25
`
`NR‘,
`
`wherein
`
`R5 denotes hydrogen or straight—chain or branched
`alkyl having up to 3 carbon atoms,
`R3 and R‘, including the double bond connecting them.
`together form a phenyl ring or a cyclopentenc,
`cyclohexene. cycloheptene, cyclooctenc,
`oxocyclopentene, oxocyclohexene, oxocycloheptene or
`oxocyclooctene radical,
`all ring systems mentioned under RIIRZ and R3fR“
`optionally being substituted up to 2 times by iden-
`tical or different fluorine, chlorine, bromine,
`trifluorornethyl, carboxyl or hydroxyl substituents,
`by st:rzu'ght—chain or branched alkoxy or a1koxycar-
`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
`hychoxyl or by straight-chain or branched alkoxy
`having up to 3 carbon atoms,
`D represents hydrogen, cyclobutyl, cyclope ntyl,
`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 sulghur atom or represents a
`group of the formula -—i
`,
`
`wherein
`
`4
`
`R9 denotes hydrogen or straight-chain or branched
`alkyl having up to 5 carbon atoms. which is option-
`ally substituted by hydroxyl or phenyl,
`R5 represents phcnyl, pyridyl, fiiryl, thienyl or irnidazolyl,
`each of which is optionally substituted up to 2 times by
`identical or different nitro, carboxyl. fluorine, chlorine,
`bromine or cyano subslituents, by straighl—chai.n or
`branched alkenyl or alkotty carbonyl each having up to
`4 carbon atoms or by straight-chain or branched allryl
`having up to 5 carbon atoms, which is qationally
`substituted by hydroxyl, carbonyl or by straight-cltain
`or branched allroxy ctr allroxycarbonyl each having up
`to 5 carbon atoms,
`
`a.nd:‘or the cycles are raationally substituted by a group
`of the formula -—-OR‘ or ——NR“R",
`wherein
`
`R” denotes hydrogen or straight~chain or branched
`alkyl or alkeuyl each having up to 4 carbon atoms,
`R“ and R2 are identical or diiferent 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 —NR”R"‘,
`wherein
`R” and R“ are identical or different and denote
`hydrogen or straight-chain or branched acyl hav-
`ing up to 6 carbon atoms,
`R6 represents hydrogen, carboxyl or straight-chain or
`branched allroxymrbonyl having up to 4 carbon atoms,
`orrepresents straight-chain or branched alkyl having up
`to 5 carbon atoms, which is optionally substituted by
`hydroxyl or by a group ofthe formula ——O-—CO——R‘5,
`wherein
`
`R” denotes phenyl which is optionally substituted up
`to 3 times by identical or dilferent fluorine, chlorine,
`bromine or hydroxyl substittucnts 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
`ZORIE,
`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
`109
`if appropriate in an isomeric form, and their salts.
`Particularly preftrred compounds of the general formula
`(I) are those in which
`R’ and R’, including the double bond connecting them,
`togetha form a phenyl or pyridyl ring or a ring of the
`formula
`
`wherein
`
`R“ denotes hydrogen or methyl,
`
`3uf67
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`5,684,014
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`5
`R3 and R4, including the double bond connecting them,
`together form a phenyl ring or a Cyclopentene,
`cycloheiiene, cycloheptene, cyclooctene,
`oxocyclopentene, oxocyclohexene, oxocycloheptene or
`oxocyclooctene radical,
`all ring systems mentioned under aim’ and R3}R“
`optionally being substituted up to 2 times by iden-
`tical or different
`fluorine, chlorine, bromine,
`u-ifluoromethyl, carbonyl or hydroxyl substituents,
`by straight-chain or branched alkoxy or a1koxycar-
`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, rnethoxy or ethoxy,
`D represents hydrogen, cyclopentyl, cyclohexyl,
`cyclohcptyl, cycloociyl ccr so-aight-chain or branched
`allryl having up to 6 carbon atoms,
`E represents the —C0— or —CS— group,
`L represents an oxygen or sulphur atom or represents a
`group of the formula — ,
`wherein
`
`R9 denotes hydrogen or straight-chain or branched
`alkyl having up to 4 carbon atoms, which is option-
`ally substituted by bydroxyl 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 substitnents, by straight-chain or branched alk-
`enyl or alkoxycarbonyl 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 allcoxycar-bony] each having up to 4 carbon
`atoms, andfor the cycles are téptionally substituted by a
`group of the fomiula -011‘ or —NR“R",
`wherein
`R10 denotes hydrogen or straight—chain or branched
`alkyl or alkenyl each having up to 3 carbon atoms,
`R“ and R” are identical or different and denote
`phenyl, hydrogen or straight-chain or branched alkyl
`having up to 4 carbon atoms
`or denote slraightachain or branched acyl having up
`to 5 carbon atoms, which is optionally substituted by
`a group of the formula —NR‘3R1"',
`wherein
`R” and R“ are identical or dilferent and denote
`hydrogen or straight-chain or branched acyl hav-
`ing up to 5 carbon atoms,
`R“ represents hydrogen, carboxyl or straight-chain or
`branched alkoxyarrbonyl having up to 3 carbon atoms,
`or represents sIra.ight—cha.in orbranched allryl having up
`to 4 carbon atoms, which is optionally substituted by
`hydroxyl or by a group of the formula —CO——-R”,
`
`wherein
`
`6
`
`R” dencles phenyl which is optionally substiurted. up
`to 3 times by identical or dilferent straight-chain or
`branched alkyl having up to 3 carbon stores,
`or denotes straight-chain or branched allryl or talk.
`egyl each having up to 19 carbon atoms, each of
`which is optionally substituted by a group of the
`formula _o121‘,
`wherein
`
`R” denotes hydrogen, bcnzyl, triphenylrnmhyl or
`straight—chain or branched acyl having up to 4
`carbon atoms,
`
`R7 represents hydrogen or
`R‘ and R’ together represent the group of die formula
`=0,
`if appropriate in an isomeric form, and their salts.
`A process for the preparation of the compounds of the
`general formula (1) according to the inveniion has addition-
`ally been found, characterined in that
`carboxylic acids of the general formula (II)
`
`10
`
`15
`
`210
`
`R’
`
`R‘
`
`N
`
`RI
`
`R:
`
`(:1)
`
`C0211.
`
`D
`
`in which
`
`35
`
`R‘, R2, R3, R‘ and D have the meaning indicated,
`are alnidated using compounds of the general l'urml.1l.a
`(111)
`
`.
`
`R5
`M2".
`
`(E0
`
`in which
`
`45
`
`R5 has the meaning indicated and
`R” has the indicated meaning of R5, but does not
`represent carboxyl,
`in an inert solvent and in the presence of bases andfor
`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 reac1ion scheme:
`
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`7
`
`CH3
`
`CW1!
`
`5 ,684,014
`
`8
`
`+ (D Dicliltzzomedaanefn-§e%1amine E
`
`.1 _
`-
`N'-{3-diInefl:|ylam.i.tDp!Upyl)—
`OH N¢&y iiyrhoehloricb
`
`:-
`3
`H2N/\\/
`
`
`
`Co -11}! /‘\x 0H
`
`Separation of
`diastereomers
`
`
`
`
`
`also
`
`Suitable solvents for the arnidation are in this case inert
`organic solvents which do not change under the reaction
`conditions. These include ethers. such as diethyl ether or 5'”
`tetrahydrofuran, halogenohydrocarbons such as
`dichloromethane,
`trichloromethane,
`tetrachloromethane,
`lrichloroethane,
`tetrachloroethane,
`l,2-dicltloroethane or
`n-ichloroethylene, hydrocarbons such as benzene, xylene,
`toluene, hexane, cyclohexane or petroleum fractions, 55
`nilromethane, dimethylformamide, acetone, aoetonflrfle or
`hexarnethylphosphoramide. It is also possible to employ
`mixtures of the solvents. Dichloromethane, tetrahyclrofiiran,
`acetone and diinethylforruarnide 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-
`line earth metal hydroxides, such as, for example, barium
`hydroxide, alkalirnetal carbonates such as sodium carbonate 55
`or potassium carbonate. alkaline earth metal. carbonates such
`as calcium carbonate, or alkali metal alkoxides such as
`
`sodium or potassium methoxide, sodium or potassium
`elhoxide or potassium tert-buloxide, or organic amines
`(tn'alkyl{C1—C5)arnines) such as triethylamine, or hetero-
`cycles such as 1,4-—diaz.abicyclo[2.2.2]octane (DABCO),
`1,8-diazabicyclo[5.4.0]under:-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 n-iethylamiue are preferred.
`The base is employed in an amount from 1 mol to 5 mol,
`preferably from 1 mo] to 3 mol, relative to 1 mol of the
`compound of the general formula (11).
`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 opfionally proceed via the activated
`stage of the acid halides, whichcan be prepared from the
`
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`10
`The bases employed for the process according to the
`invention can in general be inorganic or organic bases. These
`preferably include alkali metal hydroxides, for example,
`sodium h droxide or potassium hydroxide, alkaline earth
`metal hy oxides, for example, barium hydroxide, alkali
`metal carbonates such as sodium carbonate 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 (tr1'alkyl(C1—C5)
`amines) such as triethyiarninc, or hetcrocycles such as
`1,4-diazabicyclo[2.2.2]octa.ne (DABCO), 1,8—diazabicyclo
`[S.4.0]undec-7-ene (DB1 I), pyridine, diarninopyridine,
`methylpiperidine . or morpholine. It is also possible to
`employ alkali metals such as sodium or their hydrides such
`as sodium hydride as bases. Sodium hydride, potassium
`carbonate,
`triethylamine, pyridine and potassium tert-
`butoxide, DBU or DABCO are preferred.
`In general, the base is employed in an amount from 0.05
`incl to 10 mol, preferably from 1 ml to 2 mol, relative to
`1 mol of the compound of the formula (IV).
`‘The process according to the invention is in general
`carried out in a temperature range from —30° C. to +100° C.,
`preferably from —~10° C. to +6Cl° C.
`‘The process according to the invention is in geral
`carried out at normal pressure. However, it is also possible
`to carry out the process at elevated pressure or at reduced
`pressure (e.g. in a range from 0.5 to 5 bar).
`‘The compounds of the general formula (Ill) are known
`PG! 36.
`The compounds of the general formula (IV) are known or
`can be prepared in analogy to lcuown 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-
`cological action.
`They can be used as active compounds in medicamnents
`for the reduction of changes to vessel walls and for the
`treatment of coronary heart disorders, cardiac insuficicy,
`brain power disorders, ischaernic brain disorders, apcplexy,
`circulatory disorders, disorders of the mitrocircniation and
`thrornboses.
`
`Furthermore, the proliferation of smooth muscle cells
`plays a decisive part in the occlusion of vessels. The
`compounds according to the invention are suitable for
`inhibiting this proliferation and thus preventing atheroscle-
`rotic processes.
`‘The oompunds according to the invtion are distin—
`guished by a lowering of the ApoB-100-associated lipopro-
`teins (VLDL and its degradation products, e.g. LDL), of
`ApcB-100, of triglycerides and of cholesterol. They thus
`have useful, superior pharmacological properties in com-
`parison with the prior art.
`Slnprisingly, the action of the compounds according to the
`invention consists first in a decrease or complete inhibition
`of the formation andlor the release of ApcB-100-associated
`lipoproteins from liver cells, which results in a lowering of
`the VLDL plasma level. This lowering of VLDL must be
`accornpanicd by a lowering of the plasma level of Ape]!-
`I00, LDL, triglycerides and cholesterol; a number of the
`abovementioned risk factors which are involved in vessel
`wall changes are thus simultaneously decreased.
`The compounds according to the invention can therefore
`be employed, for
`the prevention and treatment of
`atherosclerosis, obesity, pancneatitis and constipation.
`I. Inhibition of the Release of ApoB-100-assotziatcd
`Lipoproteins
`The test for detecting the inhibition of the release of
`ApoB-100-associated lipoproteins from liver cells was car-
`
`9
`corresponding acids by reaction with thionyl chloride, phos-
`phorus trichlmide, phosphorus pentachloride, phosphorus
`tribrornide or oxalyl chloride.
`The abovementioned bases can optionally also be
`employed for the amidation as acid-binding auxiliaries.
`Suitable auxiliaries are also dehydrating reagents. ‘These
`include,
`for example, carbodiimides such as
`diisopropylcarbodiimide, dicyclohexylcarbodiirnide and
`N-(3-diiuethylaminopropyl]-N3ethylcarbodiirnide hydro-
`chloride or carbonyl compounds such as carbonyl.diimid.a—
`hole or 1,2-oztaholium compounds such as 2-ethyl-5-phenyl~
`l,2-oxazoliuIn-3-sulphonate or propanephosphonic
`anhydride or iso-butyl chlonoformate or benzotriazolyloxy—
`his-(dimethylamino)phosphonium hexailuorophosphate or
`diphenyl phosphcnamidate or methanesulphonyl chloride, if
`appropriate in the presence of bases such as triethylamine or
`N-ethylmorpholine or N—methylpiperi.d.ine or dicyclohexyl-
`carbodiimide and N-hydroxysuccinimide.
`The acid-binding agents and dehydrating reagents are in
`general employed in an mount from 0.5 to 3 mo], preferably
`from 1 to 1.5 mol, relative to 1 mol of the corresponding
`carboxylic acids.
`The variation of functional groups, for example
`hydrolysis, esterification and reduction, and also separation
`of isomers and salt formation is carried out by customary
`methods.
`The carboxylic acids of the general forrmila (II) are new
`and can he prepared by reacting
`compounds of the general formula (IV)
`
`'r—H,c
`
`C0211“.
`
`10
`
`35
`
`in which
`
`D has the meaning indicated,
`T represents a typical
`leaving group, for example
`chlorine, bromine, iodine, tosylate or mesylatc, prefer— 40
`ably bromine, and
`R13 represents (C1—C_,}alkyl
`with compounds of the general formula (V)
`
`CR-1
`
`R3,
`
`N
`
`I H
`
`R3:
`
`R‘
`
`in which
`
`_
`R‘, R2, R3 and R‘ have the meaning indicated,
`in inert solvents, if appropriate in the presence of a base.
`Suitable solvents for the process are the customary
`organic solvents which do change under the reaction con—
`ditions. These preferably include ethers such as diethyl
`ether, dioxane, ten-nhydrofur-an, glycol dimethyl ether, or
`hydrocarbons such as benzene,
`toluene, xylene, hexane,
`cyclohexane or petroleuin fractions, or l1a.iogenohydrocaI—
`bons such as dichloromethane,
`trichloromethane,
`tetrachloromethane, dichloroethylene, trichloroethylene or
`chlorobenzene, or ethyl acetate,
`trietllylaruine, pyridine,
`dimcthyl
`sulphoxide, dirnethylformamide,
`hexamethylphosphoramide, acetonitrile, acetone or
`nitromethane. It is also possible to use mixtures of the
`solvents mentioned. Dirnethylfozrmamide and tetraJ1ydrofu—
`ran are preferred.
`
`CV) 45
`
`50
`
`55
`
`65
`
`6of67
`
`PENN EX. 2203
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`5,634,014
`
`11
`ried out in vitro using cultured liver cells, preferably using
`cells of the human line I-lepG2. These cells are cultured
`under standard conditions in medium for the culture of
`eulraryolic cells, preferably in RPMI 1640 with 10% foetal
`calf serum. HepG2 cells synthesize and secrete into the
`culture supernatant ApoB—l00—associatcd lipoprotein par-
`ticles which in principle an: built up in a similar manner to
`the VLDL and LDL particles which are to be found in the
`plasma.
`'I‘hese particles can be detected using an immunoassay for
`human LDL. This immunoassay is carried out using anti-
`bodies which have been induced against human LDL in
`rabbits under standard conditions. The anti-LDL antibodies
`(rabbit anti-LDL Ab) were purified by aflinity chromat-
`agraphy on an immunosorbent using human LDL. ‘These
`purified rabbit anti-LDL Ab are adsorbed on the surface of
`plastic. Expedientiy, this adsorption is carried out on the
`plastic surface of microtitre plates having 96 wells, prefer-
`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 whidn have been induced
`against human LDL and ptlrifiod according to standard
`conditions. These antibodies were conjugated with the
`enzyme peroxidase. Peroxidase converts the colourless sulr
`strate TMB into a coloured product in the presence of H202.
`After acidification of the reaction mixture with H2804. the
`specific light absorption at 450 nm is determined, which is
`a measure of the amount of Apoli-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
`IC,0 value indicates at which substance concentration the
`light absorption is inhibited by 50% in comparison with the
`control (solvent control without substance).
`
`10
`
`25
`
`30
`
`35
`
`
`Ex. No.
`IC,., {1o° mum]
`1
`28
`5
`1.1
`31
`no
`so
`29
`
`
`45
`
`2. Determination of the VLDL Secretion in vivo in
`the Hamster
`
`The eifect of the test substances on VLDL secretion in
`vivo is investigated in the hamster. To do this, golden
`hamsters are anaesthetized with Ketaset (83 mgtkg s.c.) and
`Nembutal [50 mgjkg i.p.) after premedication with atricpine
`(83 111333 s.c.). When the animals have become reflex-free,
`the jugular vein is exposed and cannulated. 0.25 mlflrg of a
`20% strength solution of Triton WR—l339 in physiological
`saline solution is then administered. This detergent inhibits
`the lipoprotein lipase and thus leads to a rise in the triglyc-
`eride level as a result of a lack of cataboiism 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 retrooa'|:tital. venous plexus. The blood is incubated for
`two hours at room temperature, and then overnight at 4° C.,
`
`55
`
`65
`
`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-
`eride No. 14354). 100 pl of serum are treatedwith 100 pl of
`test reagent in 96-hole plates and incubated at room tem-
`perature for 10 minutes. The optical density is then deter-
`mined at a wavelength of 43.’. nM in an automatic plate-
`reading apparatus (SLT Spectra). Serum samples having an
`excessively high triglyceride concentration are diluted with
`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-
`ately before administration of the detergent or orally or
`subcutaneously before initiation of anaesthena.
`
`
` E1. No. ED,“ [mylg] pp.
`
`2
`10-15
`5
`3-6
`7
`10-33
`
`
`3. Inhibition of Intestinal Triglyceride Absorption
`in vivo (Rats)
`
`The substances which are to be investigated for their
`triglyceride absorption-inlu'biting action in vivo are ad.rnin—
`istered orally to male Wistar rats having a body weight of
`between 170 and 230 g. For this purpose, the animals are
`divided into groups of 6 animals 18 hours before substance
`administration and food is then withdrawn from thorn.
`Drinking water is available to the an.imals ad libitum. The
`animals of the control groups receive an aqueous tragacanth
`suspension or a tragacanth suspension which contains olive
`oil. ‘The tragacanth-olive oil suspension is prepared using an
`Ultra-'I‘Lu'rax. The substances to be investigated are sus-
`pended in an appropriate tragacanth—olive oil suspension
`likewise using the Ult:ra-Turrax, directly before substance
`administration.
`
`To detennine the basal serum triglyceride content, blood
`is taken from each rat by puncture of the retroorbital venous
`plexus before stomach tube application. The tragacanth
`suwensicn,
`the tragacanthclive oil suspensions without
`substance (control animals) or the substances suspended in
`an appropriate tragacanth-olive oil suspension are t.hen
`administered to the fasting animals using a stomach tube.
`Further taking of blood to determine the fitprandial serum
`triglyceride rise is carried out, as a rule, 1, 2 and 3 hours
`after stomach tube application.
`The blood samples are centrifuged and, after recovering
`the serum. the triglycerides are determined photometrically
`using an EPOS analyzer 5060 (Eppendorf Geratebau,
`Netheler & Hinz Grub]-I, Hamburg). The determination of
`the triglycerides is carried out completely enzymatically
`using a standard commercial UV test.
`The postprandial serum triglyceride rise is determined by
`subtraction of the triglyceride 1:n‘el.iInina.ry value of each
`animals from its corresponding postprandial t:riglyceride
`concentrations (1, 2 and 3 hours after administration).
`The difierenoes (in mmolfl) at each point in time (1, 2 and
`3 hours) are averaged in the groups, and the mean values of
`the serum triglyceride rise (£rTG) of the substance-treated
`animals is compared with the animals which only received
`the tragacanth-oil suspension.
`The serum triglyceride course of the control animals
`which only received uagacanth is also calculated The
`
`7of67
`
`PENN EX. 2203
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`13
`
`5,684,014
`
`14
`
`and diabetes mellitus. Gluoosidase andlor amylase inhibitors
`in the context of the invention are, for exaruple, acarbose,
`adiposine. voglibase, Iniglitol, emiglitate. MDL 25637, cam-
`iglibase (MDL 73945),
`tendamistat, 131-3688,
`trestatin,
`pradimilin-Q and salbostatin.
`Combination of acarbose, miglitol, emiglitate or voglih—
`ase with one of the abovementioned compounds of the
`general fo1mula(I) according to the invention is preferred
`The new active compounds can be converted in a lmovm
`manner into the customary formulations, such as tablets,
`coated tablets, pills, granules, aerosols, syrups, emulsions,
`suspensions and solutions, using inert, non-toxic, pharma-
`ceutically suitable excipients or solvents. In this case, the
`therapeutically active compound should in each case be
`present in a concentration of approximately 0.5 to 90% by
`weight of the