United States Patent [w]
`Miiller et al.
`
`[54] PYRIMIDO[l,2-A]INDOLES
`
`[75]
`
`Inventors: Ulrich Miiller, Wuppertal; Peter
`Eckenberg, Erkrath; Rudi Griitzmann,
`Solingen; Hilmar Bischoff; Dirk
`Denzer, both of Wuppertal; Stefan
`Wohlfeil, Hilden, all of Germany;
`Stefan Lohmer, Milan, Italy; Ulrich
`Nielsch; Peter Kolkhof, both of
`Wuppertal, Germany
`
`[73] Assignee: Bayer Aktiengesellschaft, Leverkusen,
`Germany
`
`[ * ] Notice:
`
`This patent issued on a continued pros(cid:173)
`ecution application filed under 37 CFR
`1.53(d), and is subject to the twenty year
`patent
`term provisions of 35 U.S.C.
`154(a)(2).
`
`[21] Appl. No.: 08/829,015
`
`[22] Filed:
`
`Mar. 31, 1997
`
`[30]
`
`Foreign Application Priority Data
`
`Apr. 4, 1996 [DE] Germany
`[51] Int. CI.7
`[52] U.S. CI
`[58] Field of Search
`
`A01N 43/54; C07D 239/00
`514/267; 544/252
`514/267; 544/252
`
`196 13 550
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,850,957 11/1974 White
`4,783,455 11/1988 Cliffe
`5,306,820 4/1994 Decker et al
`5,352,687 10/1994 Muller et al
`
`260/309.6
`514/220
`546/153
`514/341
`
`US006114341A
`[ii] Patent Number:
`[45] Date of Patent:
`
`6,114,341
`*Sep. 5,2000
`
`5,420,149 5/1995 Muller et al
`5,521,206
`5/1996 Muller et al
`5,527,809 6/1996 Muller-Gliemann et al
`5,576,342 11/1996 Muller
`5,705,498 1/1998 Gaster et al
`
`514/399
`514/400
`514/303
`514/399
`514/214
`
`FOREIGN PATENT DOCUMENTS
`
`509359
`0513533 A2
`513533
`560163
`0622358 Al
`2200584
`4302956
`4309968
`
`10/1992
`11/1992
`11/1992
`9/1993
`11/1994
`7/1972
`8/1994
`9/1994
`
`European Pat. Off.
`European Pat. Off.
`European Pat. Off.
`European Pat. Off.
`European Pat. Off.
`Germany .
`Germany .
`Germany .
`
`OTHER PUBLICATIONS
`
`R.A. Glennon und M. von Stradtmann, J. Heterocycl. Chem.
`vol. 12, pp. 135-138, (1975).
`C.A. Grob und O. Weissbach, Helv. Chim. Acta 44, pp.
`1748-1753, (1961).
`A.N. Kost, R.S. Sagitullin, V.I. Gorbunov und N. N. Mody-
`anov, Khim. Geterosikl. Soedin vol. 6, 359-363, (1970);
`English translation pp. 334-337.
`
`Primary Examiner—Mukund J. Shah
`Assistant Examiner—Tamthom T. Ngo
`Attorney, Agent, or Firm—Norris, McLaughlin & Marcus,
`PA.
`
`[57]
`
`ABSTRACT
`
`The pyrimido[l,2-a]indoles according to the invention are
`prepared by reacting appropriately substituted phenylacetic
`acid derivatives with phenylglycinols. The pyrimido[l,2-a]
`indoles can be used as active compounds in medicaments, in
`particular in medicaments with antiatherosclerotic activity.
`
`18 Claims, No Drawings
`
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`PYRIMIDO[l,2-A]INDOLES
`
`The present invention relates to pyrimido[l,2-a]indoles,
`to processes for
`their preparation and to their use as
`medicaments, in particular as antiatherosclerotic medica- 5
`ments.
`It is known that elevated blood levels of triglycerides
`( h y p e i t r i g l y c e r i d a e m i a)
`a nd
`c h o l e s t e r ol
`(hypercholesterolaemia) are associated with the develop(cid:173)
`ment of atherosclerotic changes in vessel walls and coronary 10
`heart disease.
`There is, furthermore, a distinctly increased risk of
`developing coronary heart disease when these two risk
`factors occur in combination, which is in turn associated
`with an overproduction of apolipoprotein B-100. Hence 15
`there is a continuing pressing need to provide effective
`medicaments for controlling atherosclerosis and coronary
`heart disease.
`The present invention relates to pyrimido[l,2-a]indoles of
`the general formula (I)
`
`20
`
`(I)
`
`—L
`
`CO NH
`
`; ^<
`
`R2
`
`in which
`A, D, E, G, L and M are identical or different and
`represent hydrogen, halogen, trifluoromethyl, carboxyl,
`hydroxyl, straight-chain or branched alkoxy or alkoxy-
`carbonyl with, in each case, up to 6 carbon atoms or
`straight-chain or branched alkyl with up to 6 carbon
`atoms, which in turn can be substituted by hydroxyl or
`by straight-chain or branched alkoxy with up to 4
`carbon atoms,
`R1 and R2 are identical or different and
`represent
`hydrogen, cycloalkyl with 3 to 8 carbon atoms or
`straight-chain or branched alkyl with up to 10 carbon
`atoms, which is optionally substituted by cycloalkyl
`with 3 to 6 carbon atoms, or represent phenyl which is
`optionally substituted by halogen or trifluoromethyl, or
`R1 and R2 form, together with the carbon atom, a 4—8-
`membered cycloalkyl ring,
`
`30
`
`35
`
`40
`
`45
`
`50
`
`and
`R3 represents phenyl which is optionally substituted up to
`3 times, identically or differently, by nitro, carboxyl,
`halogen, cyano or by straight-chain or branched alkenyl 55
`or alkoxycarbonyl with, in each case, up to 6 carbon
`atoms or by straight-chain or branched alkyl with up to
`6 carbon atoms, which is optionally substituted by
`hydroxyl, carboxyl or by straight-chain or branched
`alkoxy or alkoxycarbonyl with, in each case, up to 6
`carbon atoms, and/or is optionally substituted by a
`group of the formula —OR4 or —NR5R6,
`in which
`R4 is hydrogen or straight-chain or branched alkyl or
`alkenyl with, in each case, up to 6 carbon atoms,
`R5 and R6 are identical or different and denote phenyl,
`hydrogen or straight-chain or branched alkyl with up to
`
`60
`
`65
`
`6 carbon atoms, or denote straight-chain or branched
`acyl with up to 8 carbon atoms, which is optionally
`substituted by a group of the formula —NR7R8,
`in which
`R7 and R8 are identical or different and denote hydrogen
`or straight-chain or branched acyl with up to 8 carbon
`atoms;
`where appropriate in an isomeric form and the salts thereof.
`The pyrimido[l,2-a]indoles according to the invention
`can also be in the form of their salts. Salts which may be
`generally mentioned here are those with organic or inorganic
`bases or acids.
`the
`Physiologically acceptable salts are preferred for
`purpose of the present invention. Physiologically acceptable
`salts of the compounds according to the invention may be
`salts of the substances according to the invention with
`mineral acids, carboxylic acids or sulphonic acids. Particu(cid:173)
`larly preferred examples are salts with hydrochloric acid,
`hydrobromic acid, sulphuric acid, phosphoric acid, meth-
`anesulphonic acid, ethanesulphonic acid, toluenesulphonic
`acid, benzenesulphonic acid, naphthalenedisulphonic acid,
`acetic acid, propionic acid, lactic acid, tartaric acid, citric
`acid, fumaric acid, maleic acid or benzoic acid.
`Physiologically acceptable salts may likewise be metal or
`ammonium salts of the compounds according to the inven(cid:173)
`tion which have a free carboxyl group. Particularly preferred
`examples are sodium, potassium, magnesium or calcium
`salts, and ammonium salts which are derived from ammonia
`or organic amines such as, for example, ethylamine, di- or
`triethylamine, di- or triethanolamine, dicyclohexylamine,
`dimethylaminoethanol, arginine, lysine, ethylenediamine or
`2-phenylethylamine.
`The compounds according to the invention can exist in
`stereoisomeric forms which either are related as image and
`mirror image (enantiomers) or are not related as image and
`mirror image (diastereomers). The invention relates to the
`enantiomers or diastereomers or mixtures thereof in each
`case. These mixtures of enantiomers and diastereomers can
`be separated into the stereoisomerically pure components in
`a manner known per se.
`Preferred compounds of the general formula (I) are those
`in which
`A, D, E, G, L and M are identical or different and
`represent hydrogen, fluorine, chlorine, bromine,
`trifluoromethyl, carboxyl, hydroxyl, straight-chain or
`branched alkoxy or alkoxycarbonyl with, in each case,
`up to 4 carbon atoms or straight-chain or branched
`alkyl with up to 4 carbon atoms, which can in turn be
`substituted by hydroxyl or by straight-chain or
`branched alkoxy with up to 3 carbon atoms,
`R1 and R2 are identical or different and
`represent
`hydrogen, cyclopropyl, cyclobutyl, cyclopentyl,
`cyclohexyl, cycloheptyl, cyclooctyl or straight-chain or
`branched alkyl with up to 8 carbon atoms, which is
`optionally substituted by cyclopropyl, cyclopentyl or
`cyclohexyl, or represent phenyl which is optionally
`substituted by fluorine, chlorine or bromine, or
`R1 and R2 form, together with the carbon atom, a 4-7-
`membered cycloalkyl ring,
`
`and
`R3 represents phenyl which is optionally substituted up to
`3 times, identically or differently, by nitro, carboxyl,
`fluorine, chlorine, bromine, cyano, by straight-chain or
`branched alkenyl or alkoxycarbonyl with, in each case,
`up to 4 carbon atoms or by straight-chain or branched
`alkyl with up to 5 carbon atoms, which is optionally
`
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`6,114,341
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`substituted by hydroxyl, carboxyl or by straight-chain
`or branched alkoxy or alkoxycarbonyl with, in each
`case, up to 5 carbon atoms, and/or is optionally sub(cid:173)
`stituted by a group of the formula —OR4 or —NR5R6,
`in which
`R4 denotes hydrogen or straight-chain or branched alkyl
`or alkenyl with, in each case, up to 4 carbon atoms,
`R5 and R6 are identical or different and denote phenyl,
`hydrogen or straight-chain or branched alkyl with up to
`5 carbon atoms, or straight-chain or branched acyl with
`up to 6 carbon atoms, which is optionally substituted by
`a group of the formula —NR7R8,
`in which
`R7 and R8 are identical or different and denote hydrogen
`or straight-chain or branched acyl with up to 6 carbon
`atoms,
`where appropriate in an isomeric form and the salts thereof.
`Particularly preferred compounds of the general formula
`(I) are those in which
`A, D, E, G, L and M are identical or different and
`represent hydrogen, fluorine, chlorine, bromine,
`trifluoromethyl, carboxyl, hydroxyl, straight-chain or
`branched alkoxy or alkoxycarbonyl with, in each case,
`up to 3 carbon atoms or represents straight-chain or
`branched alkyl with up to 3 carbon atoms,
`R1 and R2 are identical or different and represent
`hydrogen, cyclopropyl, cyclobutyl, cyclopentyl,
`cyclohexyl, cycloheptyl, cyclooctyl or represent
`straight-chain or branched alkyl with up to 6 carbon
`atoms, which is optionally substituted by cyclopentyl
`or cyclohexyl, or represent phenyl which is optionally
`substituted by fluorine, chlorine or bromine, or
`R1 and R2 form, together with the carbon atom, a 5-7-
`membered cycloalkyl ring,
`
`and
`R3 represents phenyl which is optionally substituted up to
`3 times, identically or differently, by hydroxyl,
`trifluoromethyl, trifluoromethoxy, carboxyl, or by
`straight-chain or branched alkoxy, alkyl or alkoxycar(cid:173)
`bonyl with, in each case, up to 3 carbon atoms,
`
`30
`
`35
`
`40
`
`where appropriate in an isomeric form and the salts thereof.
`A process for the preparation of the compounds of the
`general formula (I) according to the invention has also been
`found and is characterized in that racemic or else already
`enantiomerically pure carboxylic acids or their activated
`derivatives of the general formula (II)
`
`(II)
`
`racemic or enantiomerically pure in which
`A, D, E, G, L, M, R1 and R2 have the indicated meaning,
`and
`R9 represents hydroxyl or represents an activating radical,
`preferably chlorine,
`are amidated with phenylglycinols of the general formula
`(III)
`
`(in)
`
`HoN'
`
`in which
`R3 has the indicated meaning,
`in inert solvents, where appropriate in the presence of
`bases and/or ancillary substances.
`The process according to the invention can be illustrated
`by way of example by the following formula diagram:
`
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`6,114,341
`
`-continued
`
`OH
`
`Suitable solvents for the amidation in this case are inert
`organic solvents which are not changed under the reaction
`conditions. These include ethers such as diethyl ether or
`tetrahydrofuiran, halogenated hydrocarbons such as
`dichloromethane, trichloromethane, tetrachloromethane,
`1,2- dichloroethane, trichloroethane, tetrachloroethane, 1,2-
`dichloroethylene or trichloroethylene, hydrocarbons such as
`benzene, xylene, toluene, hexane, cyclohexane, or petro(cid:173)
`leum fractions, nitromethane, dimethylformamide, acetone,
`acetonitrile or hexamethylphosphoric triamide. It is likewise
`possible
`to employ mixtures of
`the solvents.
`Dichloromethane, tetrahydrofuran, acetone or dimethylfor(cid:173)
`mamide are particularly preferred.
`Bases which can be employed for the process according
`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
`or potassium carbonate, alkaline earth metal carbonates such
`as calcium carbonate or alkali metal or alkaline earth metal
`alcoholates such as sodium or potassium methanolate,
`sodium or potassium ethanolate or potassium tert-butoxide,
`or organic amines (trialkyl (C1-C6)amines) such as
`triethylamine, or heterocycles such as 1,4-diazabicyclo
`[2.2.2]octane (DABCO),
`l,8-diazabicyclo[5.4.0]undec-7-
`ene (DBU), pyridine, dimethylaminopyridine, methylpiperi-
`dine or morpholine. It is also possible to employ as bases
`alkali metals such as sodium and hydrides thereof such as
`sodium hydride. Sodium and potassium carbonates and
`triethylamine are preferred.
`The base is employed in an amount of from 1 mol to 5
`mol, preferably from 1 mol to 3 mol, based on 1 mol of the
`compound of the general formula (II).
`The reaction is generally carried out at a temperature in
`the range from 0° C. to 150° C, preferably from +20° C. to
`+110° C.
`The reaction can be carried out under atmospheric,
`elevated or reduced pressure (for example 0.5 to 5 bar).
`Atmospheric pressure is generally employed.
`The reaction can, where appropriate, also take a course
`via the activated stage of the acid halides which can be
`prepared from the corresponding acids by reaction with
`thionyl chloride, phosphorus trichloride, phosphorus
`pentachloride, phosphorus tribromide or oxalyl chloride.
`The bases listed above may also be employed as acid-
`binding aids for the amidation.
`Likewise suitable as ancillary substances are dehydrating
`reagents. These include, for example, carbodiimides such as
`diisopropylcarbodiimide, dicyclohexylcarbodiimideorN-(3-
`
`dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
`or carbonyl compounds such as carbonyldiimidazole or
`1,2-oxazolium compounds such as 2-ethyl-5-phenyl-l,2-
`oxazolium-3'-sulphonate or propanephosphonic anhydride
`or
`isobutyl chloroformate or benzotriazolyloxy
`(dimethylamino)phosphonium hexafluorophosphate or
`diphenylphosphoryl azide or methanesulphonyl chloride,
`where appropriate in the presence of bases such as triethy(cid:173)
`lamine or N-ethylmorpholine or N-methylpiperidine or
`dicyclohexylcarbodiimide and N-hydroxysuccinimide.
`The ancillary substances are generally employed in an
`amount of from 0.5 to 3 mol, preferably from 1 to 1.5 mol,
`based on 1 mol of the appropriate carboxylic acids.
`The carboxylic acids of the general formula (II) are novel
`and can be prepared by initially preparing, by reacting
`compounds of the general formula (IV)
`
`T-CH2
`
`COjR11
`
`(IV)
`
`30
`
`35
`
`40
`
`45
`
`in which
`
`R1 and R2 have the indicated meaning,
`T represents a typical leaving group such as, for example,
`chlorine, bromine, iodine, tosylate or mesylate, and
`preferably represents bromine,
`
`50
`
`and
`R10 represents (Cj-CJ-alkyl,
`55 with compounds of the general formula (V)
`
`(V)
`
`60
`
`65 in which
`
`A, D, E, G, L and M have the indicated meaning,
`
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`the compounds of the general formula (VI)
`
`6,114,341
`
`8
`
`(VI)
`
`and
`Z represents halogen, preferably bromine,
`the enantiomerically pure menthyl esters of the general
`formula (IXa) or (IXb)
`
`CO2R11
`
`R1
`
`R2
`
`in which
`A, D, E, G, L, M, R1, R2 and R10 have the abovemen-
`tioned meaning,
`in inert solvents, where appropriate in the presence of
`bases,
`and subsequently hydrolysing the esters by conventional
`methods.
`Enantiomerically pure acids of the formula (Ha) or (lib):
`
`(IXa)
`
`(IXb)
`
`H,C.
`
`H,c
`
`K-
`
`CH
`
`R1
`
`C O o — R1
`
`K CH
`
`C O ; — R1
`
`>*N
`V*
`
`A3
`
`CH2-
`
`(Ha)
`
`in which
`R1, R2 and R11 have the indicated meaning,
`converting the latter in a next step by a halogenation into
`the compounds of the general formula (Xa) or (Xb)
`
`25
`
`- C H — C O —B?
`
`\
`
`/
`
`R1
`
`(lib)
`
`30
`
`35
`
`C H — C O — R9
`
`40
`
`^^
`
`J^CH
`
`C O 2 — R1
`
`T - C H j. XJ, CH
`
`R2
`
`C O j — R1
`
`(Xa)
`
`(Xb)
`
`„
`_
`.
`in which R and R are not hydrogen and R represents
`,
`j
`,
`,. •
`, ,
`.,
`r .,
`r
`hydroxyl, are furthermore obtained by preparing, from the
`D- or L-menthyl esters of the general formula (VII)
`
`45
`
`in which
`R1, R2, and R11 have the indicated meaning, and T
`represents halogen,
`,
`,
`. ,
`.
`.
`subsequently preparing, by reaction with the compounds
`^
`if
`1 /-in u
`• 11
`of the general formula (V), the enantiomerically pure
`compounds of the general formula (XIa) or (Xlb)
`
`(VII)
`
`(XIa)
`
`^
`
`7 ? ~ - C H 2 — C O j — R1
`
`in which
`R11 represents D- or L-menthyl,
`by reaction with compounds of the general formula
`(Villa) or (VHIb) R1—Z (Villa) or R2—Z (VHIb)
`in which
`R1 and R2 have the indicated meaning,
`
`50
`
`55
`
`- CH
`
`CO2R1
`
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`10
`
`H3C.
`
`X)
`x>
`
`' C H-
`
`R1
`
`' C H-
`
`R2
`
`- C 0 2 t Bu
`
`(XlVa)
`
`(XlVb)
`
`9
`-continued
`
`6,114,341
`
`(xib)
`
`- CH
`
`CO2R1
`
`in which
`A, D, E, G. L, M, R1, R2 and R11 have the indicated
`meaning,
`or then converting the latter by hydrolysis into the enan(cid:173)
`tiomerically pure acids of the general formula (Ha) and
`(lib).
`It is additionally possible to prepare enantiomerically pure
`acids of the formula (Ha) or (lib) by initially converting
`racemic carboxylic acids of the general formula (Xlla) or
`(Xllb)
`
`converting the latter as described above by a halogenation
`into the enantiomerically pure compounds of the general
`formula (XVa) or (XVb)
`
`TV
`
`JJ CH
`
`k^
`
`TV
`
`Jl CH
`
`k^
`
`(XVa)
`
`(XVb)
`
`COjtBu
`
`COjtBu
`
`(Xlla)
`
`(Xllb)
`
`30
`
`-COoH
`
`X)-• C H-
`H3C. X)—
`
`R1
`
`- C H-
`
`R2
`
`in which R1 and R2 have the indicated meaning,
`by reaction with (R)- or (S)-phenylethylamine in inert
`solvents and subsequent crystallization of the phen-
`ethylammonium salts and subsequent hydrolysis of the
`salts, into the enantiomerically pure compounds of the
`general formula (XHIa) or (XHIb)
`
`(XHIa)
`
`H3C
`
`k^Y"
`
`35 wherein R1 and R2 have the indicated meaning and T'
`represents halogen and converting the latter by reaction with
`the compounds of the general formula (V) into the enantio(cid:173)
`merically pure esters of the general formula (XVIa) or
`(XVIb):
`
`40
`
`45
`
`50
`
`(XVIa)
`
`(XVIb)
`
`(XHIb)
`
`55
`
`^ 3 ^ CH
`
`C02H
`
`60
`
`- C 0 2 t Bu
`
`in which R1 and R2 have the indicated meaning,
`preparing from the latter in another step with isobutene, in
`inert solvents and in the presence of acids, the enan(cid:173)
`tiomerically pure esters of the general formula (XlVa)
`or (XlVb)
`
`in which A, D, E, G, L, M, R1 and R2 have the indicated
`65 meaning and in the final steps as described above, preparing
`the corresponding enantiomerically pure acids of the for(cid:173)
`mula (Ha) or (lib) and activated derivatives.
`
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`11
`Solvents suitable for the processes are conventional
`organic solvents which are not changed under the reaction
`conditions. These preferably include ethers such as diethyl
`ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or
`hydrocarbons such as benzene, toluene, xylene, hexane, 5
`cyclohexane or petroleum fractions, or halogenated hydro(cid:173)
`carbons such as dichloromethane,
`trichloromethane,
`tetrachloromethane, dichloroethylene, trichloroethylene or
`chlorobenzene, or ethyl acetate, triethylamine, pyridine,
`dimethyl sulphoxide, dimethylformamide, hexamethylphos- 10
`phoric triamide, acetonitrile, acetone or nitromethane. It is
`likewise possible to use mixtures of the said solvents.
`Dimethylformamide, toluene and tetrahydrofuran are pre(cid:173)
`ferred.
`Bases which can be employed for the processes according 15
`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 and bicarbonates such as 20
`sodium carbonate, sodium bicarbonate or potassium
`carbonate, alkaline earth metal carbonates such as calcium
`carbonate, or alkali metal or alkaline earth metal alcoholates
`such as sodium or potassium methanolate, sodium or potas(cid:173)
`sium ethanolate or potassium tert-butoxide, or organic 25
`amines (trialkyl(C1-C6)-amines) such as triethylamine, or
`heterocycles such as
`l,4-diazabicyclo[2.2.2]octane
`(DABCO), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
`pyridine, dimethylaminopyridine, methylpiperidine or mor-
`pholine. It is also possible to employ as bases alkali metals 30
`such as sodium or hydrides thereof such as sodium, hydride.
`Sodium bicarbonate, potassium carbonate and potassium
`tert-butoxide, DBU or DABCO are preferred.
`Solvents suitable for the hydrolysis are water or the
`organic solvents customary for a hydrolysis. These prefer- 35
`ably include alcohols such as methanol, ethanol, propanol,
`isopropanol or butanol, or ethers such as tetrahydrofuran or
`dioxane, or dimethylformamide, or dimethyl sulphoxide.
`Alcohols such as methanol, ethanol, propanol or isopropanol
`are particularly preferably used. It is likewise possible to 40
`employ mixtures of the said solvents.
`The hydrolysis can, where appropriate, also be carried out
`with acids such as, for example, trifluoroacetic acid, acetic
`acid, hydrochloric acid, hydrobromic acid, methanesul-
`phonic acid, sulphuric acid or perchloric acid, preferably 45
`with trifluoroacetic acid.
`The hydrolysis is generally carried out at a temperature in
`the range from 0° C. to +100° C, preferably from +20° C.
`to +80° C.
`The hydrolysis is generally carried out under atmospheric 50
`pressure. However, it is also possible to employ reduced
`pressure or elevated pressure (for example from 0.5 to 5
`bar).
`When carrying out the hydrolysis, the base is generally
`employed in an amount of from 1 to 3 mol, preferably from 55
`1 to 1.5 mol, based on 1 mol of the ester. Equimolar amounts
`of the reactants are particularly preferably used.
`The hydrolysis of tert-butyl esters is generally carried out
`with acids such as, for example, hydrochloric acid or trif(cid:173)
`luoroacetic acid, in the presence of one of the abovemen- 60
`tioned solvents and/or water or mixtures thereon preferably
`with dioxane or tetrahydrofuran.
`General process [A] according to the invention is gener(cid:173)
`ally carried out at a temperature in the range from -30° C.
`to +150° C, preferably from 80° C. to 150° C.
`Suitable and preferred conditions for the individual steps
`to prepare enantiomerically pure acids are the following:
`
`65
`
`12
`Compounds of the general formula (IXa) and (IXb) are
`preferably prepared in dimethylformamide and potassium
`tert-butanolate at a temperature in the range from -10° C. to
`+10° C.
`Halogenation to give the compounds of the general for(cid:173)
`mula (Xa) and (Xb) is carried out in chlorobenzene with
`l,3-dibromo-5,5-dimethylhydantoin in the presence of azo-
`bisisobutyronitrile at a temperature in the range from 0° C.
`to 110° C.
`The reaction to give the compounds of the general for(cid:173)
`mula (XIa) and (Xlb) takes place under a protective gas
`atmosphere
`in dimethylformamide and potassium
`tert-
`butanolate at a temperature in the range from 0° C. to 30° C.
`Hydrolysis of the compounds of the general formula (XIa)
`and (Xlb) can be carried out as described above, with the
`HBr/formic acid system being particularly preferred. The
`hydrolysis is carried out at a temperature in the range from
`20° C. to 100° C.
`Suitable and preferred activating reagents are trifluo-
`romethanesulphonyl chloride, mesyl chloride, oxalyl chlo(cid:173)
`ride and thionyl chloride. Thionyl chloride is particularly
`preferred.
`The reaction to give the compounds of the general for(cid:173)
`mula (XHIa) and (XHIb) takes place in the first step pref(cid:173)
`erably in tetrahydrofuran and triethylamine, and in the
`second step in the water/hydrochloric acid system. The
`reaction is carried out at a temperature in the range from 30°
`C. to 70° C.
`Concentrated sulphuric acid is particularly preferably
`employed as acid for preparing the compounds of the
`general formula (XlVa) and (XlVb) according to the inven(cid:173)
`tion. The preparation is carried out with methylene chloride.
`In the further work-up step, potassium carbonate is
`employed as base. The reaction takes place at a temperature
`in the range from 0° C. to +20° C, particularly preferably at
`10° C.
`(XlVa) and
`The compounds of the general formula
`(XlVb) are halogenated with N-bromosuccinimide in meth(cid:173)
`ylene chloride in the presence of azobisisobutyronitrile.
`The base is generally employed in an amount of from 0.05
`mol to 10 mol, preferably from 1 mol to 2 mol, in each case
`based on 1 mol of the compounds of the general formulae
`(IV), (Villa) and (VHIb) and (XIa) and (Xlb).
`The processes according to the invention are generally
`carried out under atmospheric pressure. However, it is also
`possible to carry out the process under elevated pressure or
`under reduced pressure (for example in a range from 0.5 to
`5 bar).
`The compounds of the general formula (III) are known
`per se.
`The compounds of the general formula (IV), (Villa) and
`(VHIb) are known or can be prepared in analogy to known
`methods.
`The compounds of the general formula (V) are in some
`cases known or novel, but can then be prepared in analogy
`to published methods.
`The compounds of the general formula (VII) are novel as
`species and are prepared from the corresponding acid.
`The enantiomerically pure compounds of the general
`formula (IXa) and (IXb) are, with the exception of R1 or
`R2=isopropyl, novel and can be prepared as described
`above.
`The compounds of the general formulae (Xa), and (Xb),
`(XIa), (Xlb) are novel and can be prepared as described
`above.
`(XlVa) and
`The compounds of the general formula
`(XlVb) are in some cases known or can be prepared by
`customary methods.
`
`7 of 34
`
`PENN EX. 2217
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`6,114,341
`
`13
`The enantiomerically pure compounds of the general
`formula (XVa) and (XVb) and (XVIa) and (XVIb) are novel
`and can be prepared as described above.
`The compounds of the general formula (VI) are novel and
`can be prepared as described above.
`The compounds of the general formula (I) according to
`the invention have a spectrum of pharmacological effects
`which was unpredictable.
`They can be used as active compounds in medicaments
`for reducing changes in vessel walls and for treating coro(cid:173)
`nary heart disease, heart failure, brain dysfunction,
`ischaemic brain disease, stroke, disturbances of blood flow,
`microcirculation disturbances and thromboses.
`Furthermore, proliferation of smooth muscle cells plays a
`crucial part in the occlusion of vessels. The compounds
`according to the invention are suitable for inhibiting this
`proliferation and thus preventing atherosclerotic processes.
`The compounds according to the invention are distin(cid:173)
`guished by lowering the ApoB 100-associated lipoproteins
`(VLDL and its breakdown products such as, for example,
`LDL), and ApoB 100, the triglycerides and cholesterol.
`Hence they have valuable pharmacological properties which
`are superior by comparison with the prior art.
`Surprisingly, the effect of the compounds according to the
`invention initially comprises reducing or completely inhib(cid:173)
`iting the formation and/or the release of ApoB 100-
`associated lipoproteins from liver cells, which results in a
`lowering of the VLDL plasma level. This VLDL lowering
`must be associated with a lowering of the plasma levels of
`ApoB 100, LDL, triglycerides and cholesterol; thus a plu(cid:173)
`rality of the abovementioned risk factors involved in
`changes in vessel walls are reduced simultaneously.
`The compounds according to the invention can therefore
`be employed for
`the prevention and treatment of
`atherosclerosis, of obesity, pancreatitis and of constipation.
`1. Inhibition of the Release of ApoB 100-associated Lipo(cid:173)
`proteins
`The test to detect inhibition of the release of ApoB
`100-associated lipoproteins from liver cells took place in
`vitro with cultivated liver cells, preferably with cells of the
`human line HepG2. These cells are cultured under standard
`conditions in medium for culturing eukaryotic cells, prefer(cid:173)
`ably in RPMI 1640 with 10% fetal calf serum. HepG2 cells
`synthesize, and secrete into the culture supernatant, ApoB
`100-associated lipoprotein particles which in principle have
`a similar structure to the VLDL and LDL particles to be
`found in the plasma.
`These particles can be detected using an immunoassay for
`human LDL. This immunoassay takes place with antibodies
`which had been induced against human LDL in rabbits under
`standard conditions. The anti-LDL antibodies (rab anti-LDL
`Abs) were purified by affinity chromatography on an immu(cid:173)
`nosorbent with human LDL. These purified rab anti-LDL
`Abs are adsorbed onto the surface of plastic. This adsorption
`expediently takes place onto the plastic surface of microtitre
`plates with 96 wells, preferably on MaxiSorp plates. If ApoB
`100-associated particles are present in the supernatant from
`Hep G2 cells, thesearticles can bind to the insolubilized rab
`anti-LDL Abs, resulting in an immune complex which is
`bound to the plastic surface. Unbound proteins are removed
`by washing. The immune complex present on the plastic
`surface is detected using monoclonal antibodies which had
`been induced against human LDL, and had been purified,
`under standard conditions. These antibodies were conju(cid:173)
`gated to the enzyme peroxidase. Peroxidase converts the
`colourless substrate TMB into a coloured product in the
`presence of H202. After acidification of the reaction mixture
`
`14
`with H2S04, the specific absorption of light at 450 nm is
`determined and is a measure of the amount of ApoB 100-
`associated particles secreted into the culture supernatant by
`the HepG2 cells.
`Surprisingly, the compounds according to the invention
`inhibit the release of ApoB 100-associated particles. The
`IC50 indicates the concentration of substance at which the
`absorption of light is inhibited by 50% compared with the
`control (solvent control without substance).
`
`Ex. No.
`
`2
`18
`24
`36
`54
`57
`
`Apo B
`1C50 [nM]
`
`1.3
`1.9
`0.6
`1.1
`0.7
`2.7
`
`2. Determination of VLDL Secretion in Vivo in Hamsters
`The effect of the test substances on VLDL secretion in
`vivo is investigated on hamsters. To do this, golden hamsters
`are premedicated with atropine (83 mg/kg s.c.) and then
`anaesthetized with Ketavet (83 mg/kg s.c.) and Nembutal
`(50 mg/kg i.p.). When the animals' reflexes have been lost,
`the jugular vein is exposed and cannulated. Subsequently,
`0.25 ml/kg of a 20% strength solution of Triton WR-1339 in
`physiological saline is administered. This detergent inhibits
`lipoprotein lipase and thus leads to an increase in the
`triglyceride level because there is no catabolism of secreted
`VLDL particles. This triglyceride increase can be used as a
`measure of the VLDL secretion rate. Blood is taken from the
`animals by puncture of the retroorbital venous plexus before
`and one and two hours after administration of the detergent.
`The blood is incubated at room temperature for two hours
`and then at 4° C. overnight in order to complete the
`coagulation. It is then centrifuged at 10,000 g for 5 minutes.
`The triglyceride concentration in the serum obtained in this
`way is determined using a modified commercially obtain(cid:173)
`able enzyme assay (Merckotest® Triglyceride No. 14354).
`100 fil of serum are mixed with 100 fil of assay reagent in
`96-well plates and incubated at room temperature for 10
`minutes. Subsequently, the optical density is determined at
`a wavelength of 492 nm in an automatic plate reader (SLT
`Spectra). Serum samples with a triglyceride concentration
`which is too high are diluted with physiological saline. The
`triglyceride concentration present in the samples is deter(cid:173)
`mined using a standard plot measured in parallel. In this
`model, test substances are administered either intravenously
`immediately before administration of the detergent or orally
`or su