EP2035426A1 - Pyrazolopyrimidones - Google Patents

Pyrazolopyrimidones

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Publication number
EP2035426A1
EP2035426A1 EP07765401A EP07765401A EP2035426A1 EP 2035426 A1 EP2035426 A1 EP 2035426A1 EP 07765401 A EP07765401 A EP 07765401A EP 07765401 A EP07765401 A EP 07765401A EP 2035426 A1 EP2035426 A1 EP 2035426A1
Authority
EP
European Patent Office
Prior art keywords
methyl
propyl
benzyl
pyrazolo
pyrimidin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07765401A
Other languages
German (de)
French (fr)
Inventor
Thomas Maier
Thomas Bär
Andreas Lindenmaier
Matthias Vennemann
Jürgen BRAUNGER
Markus Boehm
Volker Gekeler
Astrid Zimmermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
4SC AG
Original Assignee
4SC AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 4SC AG filed Critical 4SC AG
Priority to EP07765401A priority Critical patent/EP2035426A1/en
Publication of EP2035426A1 publication Critical patent/EP2035426A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to pyrazolopyrimidone derivatives, which can be used in the pharmaceutical industry for the production of pharmaceutical compositions.
  • US application US2004/0242596 contains pyrazolo[3,4-d]pyrimidones which are said to be useful as anti-cancer agents and to induce mitotic arrest.
  • the compounds according to this invention can act as inhibitors of Eg5 kinesin.
  • these derivatives are potent and highly efficacious inhibitors of cellular (hyper)proliferation and/or cell-cycle specific inducers of apoptosis in cancer cells. Therefore, these compounds can be particular useful for treating (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, notably cancer.
  • these derivatives should have a higher therapeutic index compared to standard chemotherapeutic drugs targeting basic cellular processes like DNA replication or interfering with basic cellular molecules like DNA.
  • the compounds according to this invention are expected to be useful in targeted cancer therapy.
  • R2 is 1-4C-alkyl
  • A is Aryl-1-4C-alkyl, in which
  • Aryl is phenyl, or R3- and R31-substituted phenyl, in which
  • R3 is 1-4C-alkyl, trifluoromethyl, 1-4C-alkoxy, hydroxyl, halogen, or completely or predominantly fluorine-substituted 1-4C-alkoxy
  • R31 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy
  • R4 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl or 3-7C-cycloalkyl-1-4C-alkyl
  • R5 is hydrogen or 1-4C-alkyl
  • R6 is hydrogen or 1-4C-alkyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which R7 is 1-4C-alkyl, trifluoromethyl, cyano, 1-4C-alkoxy, halogen, carboxyl, 1-4C-alkylcarbonyl, methylenedioxy, ethylenedioxy, 1-4C-alkoxy-1-4C-alkyl, hydroxy-1-4C-alkyl, or completely or predominantly fluorine-substituted 1-4C-alkoxy, R71 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy, n is 2, 3, 4, 5 or 6, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • alkyl alone or as part of another group refers to both branched and straight chain saturated aliphatic hydrocarbon groups having the specified numbers of carbon atoms, such as for example:
  • 1-4C-Alkyl is a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.
  • Examples are the butyl, isobutyl, sec-butyl, tert-butyl, propyl, isopropyl, ethyl and methyl radicals, of which propyl, isopropyl, ethyl and methyl are more worthy to be mentioned.
  • Halogen within the meaning of the present invention is iodine or, in particular, bromine, chlorine or fluorine.
  • 1-4C-Alkoxy represents radicals which, in addition to the oxygen atom, contain a straight-chain or branched alkyl radical having 1 to 4 carbon atoms. Examples which may be mentioned are the butoxy, isobutoxy, sec-butoxy, tert-butoxy, propoxy, isopropoxy, ethoxy and methoxy radicals, of which propoxy, isopropoxy, and, particularly, ethoxy and methoxy are more worthy to be mentioned.
  • cycloalkyl alone or as part of another group refers to a monocyclic saturated aliphatic hydrocarbon group having the specified numbers of carbon atoms, such as for example: 3-7C-Cycloalkyl stands for cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, of which cyclopentyl, cyclobutyl and, in particular, cyclopropyl are more worthy to be mentioned.
  • 3-7C-Cycloalkyl-1-4C-alkyl stands for one of the abovementioned 1-4C-alkyl radicals, which is substituted by one of the abovementioned 3-7C-cycloalkyl radicals.
  • Examples which may be mentioned are the S-ZC-cycloalkylmethyl radicals, of which cyclopentylmethyl, cyclobutyl methyl and, in particular, cyclopropylmethyl are more worthy to be mentioned.
  • 1-4C-Alkoxy-1-4C-alkyl represents one of the abovementioned 1-4C-alkyl radicals, which is substituted by one of the abovementioned 1-4C-alkoxy radicals.
  • Examples which may be mentioned are the 2-isopropoxyethyl, 2-ethoxyethyl, 2-methoxyethyl and, in particular, isopropoxymethyl, ethoxymethyl and, in more particular, methoxymethyl radical.
  • Hydroxy-1-4C-alkyl represents one of the abovementioned 1-4C-alkyl radicals, which is substituted by a hydroxyl radical. Examples which may be mentioned are the 3-hydroxypropoxy, 2-hydroxyethyl and, in particular, hydroxymethyl radical.
  • Completely or predominantly fluorine-substituted 1-4C-alkoxy is, for example, the 2,2,3,3,3- pentafluoropropoxy, the perfluoroethoxy, the 1 ,2,2-trifluoroethoxy and in particular the 1 ,1 ,2,2- tetrafluoroethoxy, the 2,2,2-trifluoroethoxy, the trifluoromethoxy and the difluoromethoxy radical, of which the trifluoromethoxy and the difluoromethoxy radicals are preferred.
  • "Predominantly" in this connection means that more than half of the hydrogen atoms of the 1-4C-alkoxy groups are replaced by fluorine atoms.
  • 1-4C-Alkylcarbonyl is a carbonyl group, to which one of the abovementioned 1-4C-alkyl radicals is bonded.
  • An example is the acetyl radical (CH 3 CO-).
  • Aryl-1-4C-alkyl stands for one of the abovementioned 1-4C-alkyl radicals, which is substituted by an Aryl radical as defined below, such as e.g. the Aryl-methyl or 2-Aryl-ethyl radical, of which the Aryl- methyl radical is to be emphasized.
  • Aryl stands for phenyl, or R3- and R31-substituted phenyl.
  • Aryl-1-4C-alkyl radical examples include the phenethyl and, particularly, the benzyl radical, each of which is optionally substituted by R3 and R31 on the phenyl moiety.
  • Suitable salts for compounds according to this invention - depending on substitution - are all acid addition salts or all salts with bases. Particular mention may be made of the pharmacologically and/or pharmaceutically tolerable inorganic and organic acids and bases customarily used in pharmacy.
  • Those suitable include, but are not limited to, water-insoluble and, particularly, water-soluble acid addition salts with acids such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulphuric acid, acetic acid, citric acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)- benzoic acid, butyric acid, sulphosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid, stearic acid, toluenesulphonic acid, phenylsulphonic acid, methanesulphonic acid or 3-hydroxy-2-naphthoic acid, the acids being employed in salt preparation - depending on whether a mono- or polybasic acid is concerned and depending on which salt is desired - in an equimolar quantitative ratio or one differing therefrom.
  • acids being employed in
  • salts with bases are - depending on substitution - also suitable.
  • salts with bases are mentioned the lithium, sodium, potassium, calcium, aluminium, magnesium, titanium, ammonium, meglumine or guanidinium salts, here, too, the bases being employed in salt preparation in an equimolar quantitative ratio or one differing therefrom.
  • Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds of formula I or their pharmaceutically acceptable salts, are also included.
  • the compounds of formula I according to this invention as well as their salts may contain, e.g. when isolated in crystalline form, varying amounts of solvents. Included within the scope of the invention are therefore all solvates and in particular all hydrates of the compounds of formula I according to this invention as well as all solvates and in particular all hydrates of the salts of the compounds of formula I according to this invention.
  • salts of compounds of formula I include a salt of a compound of formula I with hydrochloric acid (hydrochloride).
  • R3 and R31 of compounds of formula I can be attached in the ortho, meta or para position with respect to the binding position in which the Aryl ring is bonded to the 1-4C-alkyl group, whereby emphasis is given to the attachment of R3 in the meta or para position.
  • R31 is hydrogen.
  • R7 and R71 of compounds of formula I can be attached in the ortho, meta or para position with respect to the binding position in which the phenyl ring is bonded to the carbonyl group, whereby emphasis is given to the attachment of R7 in the meta or para position.
  • R71 is hydrogen.
  • hyperproliferation and analogous terms are used to describe aberrant / dysregulated cellular growth, a hallmark of diseases like cancer.
  • This hyperproliferation might be caused by single or multiple cellular / molecular alterations in respective cells and can be, in context of a whole organism, of benign or malignant behaviour.
  • Inhibition of cell proliferation and analogous terms is used herein to denote an ability of the compound to retard the growth of and/or kill a cell contacted with that compound as compared to cells not contacted with that compound. Most preferable this inhibition of cell proliferation is 100%, meaning that proliferation of all cells is stopped and/or cells undergo programmed cell death.
  • the contacted cell is a neoplastic cell.
  • a neoplastic cell is defined as a cell with aberrant cell proliferation and/or the potential to metastasize to different tissues or organs.
  • a benign neoplasia is described by hyperproliferation of cells, incapable of forming an aggressive, metastasizing tumor in-vivo.
  • a malignant neoplasia is described by cells with different cellular and biochemical abnormalities, e.g. capable of forming tumor metastasis.
  • the aquired functional abnormalities of malignant neoplastic cells are replicative potential ("hyperproliferation"), self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion from apoptosis, sustained angiogenesis and tissue invasion and metastasis.
  • Inducer of apoptosis and analogous terms are used herein to identify a compound which executes programmed cell death in cells contacted with that compound.
  • Apoptosis is defined by complex biochemical events within the contacted cell, such as the activation of cystein specific proteinases ("caspases") and the fragmentation of chromatin.
  • caspases cystein specific proteinases
  • Induction of apoptosis in cells contacted with the compound might not necessarily be coupled with inhibition of cell proliferation.
  • the inhibition of cell proliferation and/or induction of apoptosis is specific to cells with aberrant cell growth (hyperproliferation).
  • cytotoxic is used in a more general sense to identify compounds which kill cells by various mechanisms, including the induction of apoptosis / programmed cell death in a cell cycle dependent or cell-cycle independent manner.
  • Cell cycle specific and analogous terms are used herein to identify a compound as inducing apoptosis only in continously proliferating cells actively passing a specific phase of the cell cycle, but not in resting, non-dividing cells.
  • Continously proliferating cells are typical for diseases like cancer and characterized by cells in all phases of the cell division cycle, namely in the G (“gap”) 1 , S (“DNA synthesis”), G2 and M (“mitosis”) phase.
  • R1 is hydrogen or halogen
  • R2 is 1-2C-alkyl
  • A is Aryl-1-2C-alkyl, in which
  • Aryl is phenyl, or R3- and R31-substituted phenyl, in which
  • R3 is 1-2C-alkyl, trifluoromethyl, 1-2C-alkoxy, hydroxyl, halogen, or completely or predominantly fluorine-substituted 1-2C-alkoxy
  • R31 is hydrogen, halogen, 1-2C-alkyl or 1-2C-alkoxy
  • R4 is hydrogen, 1-4C-alkyl, cyclopropyl or cyclopropyl-I ⁇ C-alkyl,
  • R5 is hydrogen or 1-3C-alkyl
  • R6 is hydrogen or 1-3C-alkyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which
  • R7 is 1-2C-alkyl, trifluoromethyl, cyano, 1-2C-alkoxy, halogen, carboxyl, 1-2C-alkylcarbonyl, methylenedioxy, ethylenedioxy, 1-2C-alkoxy-1-2C-alkyl, hydroxy-1-2C-alkyl, or completely or predominantly fluorine-substituted 1-2C-alkoxy
  • R71 is hydrogen, halogen, 1-2C-alkyl or 1-2C-alkoxy
  • n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is hydrogen or halogen
  • R2 is methyl or ethyl
  • A is Arylmethyl, in which
  • Aryl is phenyl, or R3- and R31-substituted phenyl, in which
  • R3 is methyl, ethyl, trifluoromethyl, methoxy, ethoxy, hydroxyl, halogen, difluoromethoxy or trifluoromethoxy,
  • R31 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy
  • R4 is methyl, ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl
  • R5 is hydrogen, methyl, ethyl, propyl or isopropyl
  • R6 is hydrogen, methyl, ethyl, propyl or isopropyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, acetyl, methylenedioxy, ethylenedioxy, methoxymethyl, hydroxymethyl, difluoromethoxy or trifluoromethoxy,
  • R71 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is hydrogen, fluorine, chlorine or bromine
  • R2 is methyl or ethyl
  • A is Arylmethyl, in which
  • Aryl is phenyl, or R3- and R31-substituted phenyl, in which
  • R3 is methyl, ethyl, trifluoromethyl, methoxy, ethoxy, hydroxyl or halogen,
  • R31 is hydrogen, halogen, methyl or methoxy
  • R4 is ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl
  • R5 is hydrogen, methyl, ethyl, propyl or isopropyl
  • R6 is hydrogen, methyl, ethyl, propyl or isopropyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which
  • R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl
  • R71 is hydrogen, halogen, methyl, ethyl or methoxy
  • n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is hydrogen, chlorine or bromine
  • R2 is methyl or ethyl
  • A is benzyl, halobenzyl (e.g. fluorobenzyl, chlorobenzyl or bromobenzyl), methylbenzyl, methylhalobenzyl, methoxybenzyl, hydroxybenzyl, dihalobenzyl (e.g. dichlorobenzyl), or dimethoxybenzyl,
  • R4 is ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl
  • R5 is hydrogen, methyl, ethyl, propyl or isopropyl
  • R6 is hydrogen, methyl, ethyl, propyl or isopropyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl
  • R71 is hydrogen, halogen, methyl or ethyl, such as, for example, B is phenyl, methylphenyl, ethylphenyl, halophenyl, methylhalophenyl, (hydroxymethyl)phenyl, methoxyphenyl
  • R1 is hydrogen, chlorine or bromine
  • R2 is methyl or ethyl
  • A is benzyl, halobenzyl (e.g. fluorobenzyl, chlorobenzyl or bromobenzyl), methylbenzyl, methoxybenzyl or hydroxybenzyl, R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either R5 and R6 are both hydrogen, or
  • R5 is methyl
  • R6 is hydrogen, or
  • R5 is ethyl
  • R6 is hydrogen, or
  • R5 is propyl
  • R6 is hydrogen, or
  • R5 is isopropyl
  • R6 is hydrogen, or
  • R5 and R6 are both methyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which
  • R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl
  • R71 is hydrogen, halogen, methyl or ethyl, such as, for example, B is phenyl, methylphenyl, ethylphenyl, halophenyl, dihalophenyl, methylhalophenyl,
  • B is phenyl, 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3- fluoro-4-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 3,4- dichlorophenyl or 2,3-dichlorophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is hydrogen, chlorine or bromine, particularly
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl, fluorobenzyl, chlorobenzyl, bromobenzyl, methylbenzyl, methoxybenzyl or hydroxybenzyl, in particular
  • A is benzyl or fluorobenzyl (e.g. 3-fluorobenzyl or 4-fluorobenzyl), R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 is methyl
  • R6 is hydrogen, or
  • R5 is ethyl
  • R6 is hydrogen, or
  • R5 and R6 are both methyl
  • B is phenyl, or R7- and R71-substituted phenyl, in which
  • R7 is methyl, ethyl, trifluoromethyl, fluorine, chlorine or bromine,
  • R71 is hydrogen, methyl, fluorine, chlorine or bromine, such as, for example,
  • B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, such as, for more detailed example, B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, fluorophenyl, chlorophenyl, bromophenyl, dichlorophenyl, difluorophenyl, methylfluorophenyl, methylchlorophenyl or methylbromophenyl, such as, for yet more detailed example, B is phenyl, 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3- fluoro-4-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl or 3,4-dichlorophenyl, n is 2 or 3, and the salts, stereo
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl or fluorobenzyl (e.g. 3-fluorobenzyl or 4-fluorobenzyl),
  • R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, such as, for example, B is methylphenyl, trifluoromethylphenyl, methoxyphenyl, fluorophenyl, chlorophenyl, bromophenyl, dichlorophenyl, difluorophenyl, methylfluorophenyl, methylchlorophenyl or methylbromophenyl, such as, for more detailed example,
  • B is 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3-fluoro-4- methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl or 3,4-dichlorophenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl, 3-fluorobenzyl or 4-fluorobenzyl
  • R4 is ethyl, propyl, isopropyl or cyclopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is methylphenyl, trifluoromethylphenyl, methoxyphenyl, chlorophenyl, dichlorophenyl, bromophenyl, fluorophenyl or methylfluorophenyl, such as, for example, B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-chlorophenyl,
  • n 2 or 3
  • the salts, stereoisomers and the salts of the stereoisomers of these compounds are 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • the invention relates to compounds of formula I according to the invention, in which
  • R1 is hydrogen, chlorine or bromine
  • R2 is methyl
  • A is benzyl or fluorobenzyl
  • R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl
  • R4 is ethyl or isopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is methylphenyl, chlorophenyl, trifluoromethylphenyl, methoxyphenyl, bromophenyl or fluorophenyl, such as, for example, B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-chlorophenyl,
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl
  • R4 is ethyl or isopropyl
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- chlorophenyl, 4-bromophenyl or 3-fluoro-4-methylphenyl; and n is 3; and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • R5 and R6 are both hydrogen
  • B is methylphenyl, chlorophenyl, trifluoromethylphenyl, methoxyphenyl, bromophenyl or fluorophenyl, such as, for example,
  • B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl, n is 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • the invention also relates to compounds of formula I according to the invention comprising one or more of the following:
  • R1 is chlorine or bromine
  • R2 is methyl
  • A is benzyl
  • R4 is isopropyl
  • R5 and R6 are both hydrogen
  • B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl; and n is 3; and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • the invention relates to compounds of formula I in which
  • R1 is hydrogen, chlorine, bromine or fluorine
  • R2 is methyl
  • A is benzyl
  • R4 is ethyl, isopropyl, sec-butyl, cyclopropyl or cyclobutyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, 4- methoxyphenyl, 4-trifluoromethylphenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methylphenyl, 3,4- dichlorophenyl or 2,3-dichlorophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • the invention relates to compounds of formula I according to the invention, in which
  • R1 is hydrogen, chlorine or bromine
  • R2 is methyl
  • A is benzyl
  • R4 is ethyl or isopropyl, either
  • R5 and R6 are both hydrogen, or
  • R5 and R6 are both methyl
  • B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, n is 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • the invention relates to compounds of formula I in which
  • R1 is chlorine, bromine or fluorine
  • R2 is methyl
  • A is benzyl
  • R4 is ethyl, isopropyl, sec-butyl or cyclopropyl
  • R5 and R6 are both hydrogen
  • B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methylphenyl or 3,4-dichlorophenyl, n is 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
  • a special interest in the compounds according to this invention refers to those compounds of formula I which are included -within the scope of this invention- by one or, when possible, by a combination of more of the following special embodiments:
  • Another special embodiment (embodiment 1 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is hydrogen.
  • Another special embodiment (embodiment 2) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine.
  • Another special embodiment (embodiment 3) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is bromine.
  • Another special embodiment (embodiment 3a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is fluorine.
  • Another special embodiment (embodiment 4) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R2 is methyl.
  • Another special embodiment (embodiment 5) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine, and R2 is methyl.
  • Another special embodiment (embodiment 6) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is bromine, and R2 is methyl.
  • a special embodiment (embodiment 7) of the compounds of formula I according to this invention refers to those compounds of formula I, in which A is benzyl.
  • A is fluorobenzyl, such as e.g. 3-fluorobenzyl or 4-fluorobenzyl.
  • A is benzyl, halobenzyl (such as e.g. chlorobenzyl or bromobenzyl), methylbenzyl, hydroxybenzyl or methoxybenzyl.
  • Another special embodiment (embodiment 10) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is ethyl.
  • Another special embodiment (embodiment 1 1 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is isopropyl.
  • Another special embodiment (embodiment 11 a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine, and R2 is methyl, and R4 is isopropyl.
  • Another special embodiment (embodiment 11 b) of the compounds of formula I according to this invention refers to those compounds of formula I, in which A is benzyl, and
  • R4 is isopropyl
  • R4 is isopropyl
  • Another special embodiment (embodiment 12) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is propyl.
  • Another special embodiment (embodiment 13) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is cyclopropyl.
  • Another special embodiment (embodiment 13a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is cyclobutyl.
  • Another special embodiment (embodiment 14) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is sec-butyl.
  • Another special embodiment (embodiment 15) of the compounds of formula I according to this invention refers to those compounds of formula I, in which n is 2.
  • Another special embodiment (embodiment 16) of the compounds of formula I according to this invention refers to those compounds of formula I, in which n is 3.
  • R5 and R6 are the same and selected from hydrogen, methyl and ethyl.
  • Another special embodiment (embodiment 18) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
  • R5 is methyl, ethyl, propyl or isopropyl
  • R6 is hydrogen
  • Another special embodiment (embodiment 19) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 is methyl or ethyl, and R6 is hydrogen.
  • Another special embodiment (embodiment 20) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both hydrogen, and n is 2.
  • Another special embodiment (embodiment 21 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both methyl, and n is 2.
  • Another special embodiment (embodiment 22) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both hydrogen, and n is 3.
  • Another special embodiment (embodiment 23) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both methyl, and n is 3.
  • B is phenyl, or R7- and R71-substituted phenyl, in which
  • R7 is fluorine, chlorine, bromine, methyl, trifluoromethyl or ethyl
  • R71 is hydrogen, fluorine, chlorine or methyl.
  • B is phenyl, halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl, (hydroxymethyl)phenyl, (methoxymethyl)phenyl, methoxyphenyl, ethoxyphenyl, carboxyphenyl, methylphenyl, ethylphenyl, methylenedioxyphenyl, ethylenedioxyphenyl, methoxychlorophenyl, methylhalophenyl or (trifluoromethyl)phenyl.
  • Another special embodiment (embodiment 26) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is phenyl, methylphenyl, halophenyl (e.g. chlorophenyl or bromophenyl), dihalophenyl (e.g. dichlorophenyl), methylhalophenyl (e.g. methylfluorophenyl), (hydroxymethyl)phenyl, halo(trifluoromethyl)phenyl, methylenedioxyphenyl, (trifluoromethyl)phenyl, methoxyphenyl or cyanophenyl.
  • B is phenyl, methylphenyl, halophenyl (e.g. chlorophenyl or bromophenyl), dihalophenyl (e.g. dichlorophenyl), methylhalophenyl (e.g. methylfluorophenyl), (hydroxymethyl)phenyl, halo(
  • Another special embodiment (embodiment 27) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is methylphenyl, chlorophenyl, dichlorophenyl, bromophenyl, fluorophenyl, (trifluoromethyl)phenyl, methoxyphenyl or methylfluorophenyl.
  • B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- chlorophenyl, 4-bromophenyl or 3-fluoro-4-methylphenyl.
  • Another special embodiment (embodiment 29) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl.
  • Another special embodiment (embodiment 30) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is 4-methylphenyl.
  • Another special embodiment (embodiment 32) of the compounds of formula I according to this invention refers to those compounds which are from formula I** as shown below.
  • a respective another special embodiment (embodiment 33 to 92) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
  • R1 is bromine
  • R2 is methyl
  • R4, n, R5 and R6 have the respective meanings indicated in Table 1 given below.
  • a respective another special embodiment (embodiment 93 to 152) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
  • R1 is chlorine
  • R2 is methyl
  • R4, n, R5 and R6 have the respective meanings indicated in Table 2 given below.
  • the compounds of formula I, in which R4 is different from hydrogen, are chiral compounds having a chiral center in position 2'.
  • the invention includes all conceivable stereoisomers of the compounds of this invention, like e.g. diastereomers and enantiomers, in substantially pure form as well as in any mixing ratio, including the racemates, as well as the salts thereof.
  • the invention therefore relates to compounds of formula I as described in this application, wherein said compounds have the configuration as shown in formula I*, and the salts thereof.
  • R4 has the meaning methyl, ethyl, propyl, isopropyl, sec- butyl or cyclopropyl
  • the configuration - according to the rules of Cahn, lngold and Prelog - is R in the 2' position.
  • R4 has the meaning methyl, ethyl, propyl, isopropyl, sec- butyl or cyclopropyl
  • the configuration - according to the rules of Cahn, lngold and Prelog - is S in the position 2'.
  • enantiomerically pure compounds of this invention may be prepared according to art- known processes, such as e.g. via asymmetric syntheses, for example by preparation and separation of appropriate diastereoisomeric compounds/intermediates, which can be separated by known methods (e.g. by chromatographic separation or (fractional) crystallization from a suitable solvent), or by using chiral synthons or chiral reagents; by chromatographic separation of the corresponding racemate on chiral separating columns; by means of diastereomeric salt formation of the racemic compounds with optically active acids (such as e.g.
  • one possible alternative for enatiomer separation may be carried out at the stage of the compounds of formula I or of the starting compounds having a protonatable group, e.g. a free amino group, such as starting compounds of formula Il as defined later.
  • separation of the enantiomers may be carried out, for example, by means of salt formation of the racemic compounds of formula Il with optically active acids, preferably carboxylic acids, subsequent resolution of the salts and release of the desired compound from the salt.
  • optically active carboxylic acids which may be mentioned in this connection, without being restricted thereto, are the enantiomeric forms of mandelic acid, tartaric acid, O,O'-dibenzoyltartaric acid, camphoric acid, quinic acid, glutamic acid, pyroglutamic acid, malic acid, camphorsulfonic acid, 3-bromocamphorsulfonic acid, ⁇ -methoxyphenylacetic acid, ⁇ -methoxy- ⁇ -trifluoromethylphenylacetic acid or 2-phenylpropionic acid or the like.
  • Another possible alternative for enantiomer separation may be carried out by chromatographic separation of a racemic mixture of compounds of formula I or of starting compounds thereof on a chiral separating column, such as e.g. described in the following examples or analogously or similarly thereto, using the appropriate separation conditions.
  • the enantiomers having the formula I* and the salts thereof are part of the invention.
  • the enantiomers having the formula I** and the salts thereof are also part of the invention.
  • R1 is chlorine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
  • R1 is hydrogen, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
  • R1 is bromine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
  • R1 is chlorine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
  • R1 is hydrogen, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
  • the compounds according to the invention can be prepared e.g. as described as follows and according to the following specified reaction steps, or, particularly, in a manner as described by way of example in the following examples, or analogously or similarly thereto according to preparation procedures or synthesis strategies known to the person skilled in the art.
  • the trione compound of formula IX in which R2 has the meanings given above (particularly, R2 is methyl), is condensed and cyclized with semicarbazide (H 2 N-C(O)-NHNH 2 ⁇ CI) to give the corresponding pyrazolopyrimidone compounds of formula VIII.
  • Said cyclocondensation reaction can be carried out as it is habitual for the skilled person or as described in the following examples or analogously or similarly thereto, in the presence of a suitable base (e.g. sodium carbonate) in a suitable solvent, for example water, at elevated temperature.
  • a suitable base e.g. sodium carbonate
  • chlorination or bromination reaction can be carried out using an appropriate electrophilic chlorinating or brominating reagent, e.g. N-chlorosuccinimide (NCS) or N-bromosuccinimide (e.g. NBS / AIBN), respectively, under conditions customary for the skilled person or as described in the following examples.
  • an appropriate electrophilic chlorinating or brominating reagent e.g. N-chlorosuccinimide (NCS) or N-bromosuccinimide (e.g. NBS / AIBN)
  • R1 H
  • optionally halogenation e.g. bromination
  • Fluorination reaction may be carried out using a suitable electrophilic fluorinating reagent, such as e.g. 1-chloromethyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) (Selectfluor I) or 1- methyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) (Selectfluor II) or the like, under appropriate conditions.
  • a suitable electrophilic fluorinating reagent such as e.g. 1-chloromethyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) (Selectfluor I) or 1- methyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate
  • said halogenation reaction is carried out largely regioselectively to obtain halogenation on the pyrazole moiety of the pyrazolopyrimidone scaffold, i.e. the 3-position of the pyrazolopyrimidone scaffold.
  • methyl lithium forming in situ more reactive organomagnesium reagents, e.g. lithium trialkyl magnesiates such as R4Me 2 Mgl_i from 2 equiv. LiMe and 1 equiv. R4-Mg-X.
  • the nucleophilic addition reaction can be carried out in a manner customary for the skilled person or as described in the following examples.
  • Alcohols of formula IV in which R1 is hydrogen, fluorine or chlorine and A, R2 and R4 have the meanings given above, are converted into the corresponding ketones of formula III by oxidation reaction using an appropriate oxidation reagent, such as e.g. NMO (N-methylmorpholine-N-oxide) / TPAP (tetrapropylammonium perruthenate) or the like, under conditions known for the skilled person or as described in the following examples.
  • an appropriate oxidation reagent such as e.g. NMO (N-methylmorpholine-N-oxide) / TPAP (tetrapropylammonium perruthenate) or the like, under conditions known for the skilled person or as described in the following examples.
  • a suitable titanium reagent like titanium(IV)isopropoxide to give the corresponding imine compounds, which are reduced in a second step, such as with the aid of a suitable reducing reagent, e.g. a suitable hydride like a borohydride (e.g. NaCNBH 3 , NaBH 4 or Na(OAc) 3 BH) or the like, Raney- Ni or in the presence of hydrogen/transition metal catalyst (e.g. H 2 /Pd).
  • a suitable reducing reagent e.g. a suitable hydride like a borohydride (e.g. NaCNBH 3 , NaBH 4 or Na(OAc) 3 BH) or the like, Raney- Ni or in the presence of hydrogen/transition metal catalyst (e.g. H 2 /Pd).
  • PG1 stands for a suitable temporary protective group for the amino group, for example tert- butoxycarbonyl, benzyloxycarbonyl or 4-methoxy-benzyloxycarbonyl or one of those art-known protective groups mentioned in “Protective Groups in Organic Synthesis” by T. Greene and P. Wuts (John Wiley & Sons, Inc. 1999, 3 rd Ed.) or in “Protecting Groups (Thieme Foundations Organic Chemistry Series N Group” by P. Kocienski (Thieme Medical Publishers, 2000).
  • methyl and in which R1 , A, n, B, R2 and R4 have the meanings given above, can be obtained by alkylation, e.g. methylation, of compounds of formula I, in which R5 and R6 are hydrogen.
  • these compounds of formula I, in which R5 and R6 are 1-4C-alkyl, e.g. methyl can be obtained by any other method that is known to the person skilled in the art.
  • the invention therefore relates to a process for preparing a compound as described in this application, comprising at least one of the steps
  • compounds of the formula I can be converted into their salts, or, optionally, salts of the compounds of the formula I can be converted into the free compounds.
  • Corresponding processes are customary for the skilled person.
  • the compounds of formula I may be obtained - depending on their individual chemical nature and the individual nature of the acid used - as free base or containing said acid in an stoechiometric or non-stoechiometric quantity.
  • the amount of the acid contained can be determined according to art-known procedures, e.g. by titration.
  • the substances according to the invention are isolated and purified in a manner known per se, for example by distilling off the solvent under reduced pressure and recrystallizing the residue obtained from a suitable solvent or subjecting it to one of the customary purification methods, such as, for example, column chromatography on a suitable support material.
  • Salts can be obtained by dissolving the free compound in a suitable solvent (e.g. a ketone, such as acetone, methyl ethyl ketone or methyl isobutyl ketone, an ether, such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon, such as methylene chloride or chloroform, or a low-molecular-weight aliphatic alcohol, such as methanol, ethanol or isopropanol) which contains the desired acid or base, or to which the desired acid or base is then added.
  • a suitable solvent e.g. a ketone, such as acetone, methyl ethyl ketone or methyl isobutyl ketone, an ether, such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon, such as methylene chloride or chloroform, or a low-
  • Salts obtained can be converted into the free compounds, which can in turn be converted into salts, by alkalization or by acidification. In this manner, pharmacologically and/or pharmaceutically unacceptable salts can be converted into pharmacologically and/or pharmaceutically acceptable salts.
  • the present invention also relates to intermediates (including their salts, stereosiomers as well as salts of these stereoisomers), methods and processes useful in synthesizing compounds according to this invention.
  • m.p. stands for melting point, h for hour(s), min for minutes, cone, for concentrated, calc. for calculated, fnd. for found, EF for elemental formula, MS for mass spectrometry, M for molecular ion in mass spectrometry, TFA for trifluoroacetic acid, and other abbreviations have their meanings customary per se to the skilled person.
  • (RS) characterizes a racemate comprising the one enantiomer having the configuration R and the other enantiomer having the configuration S; each of these enantiomers and their salts in pure form as well as their mixtures including the racemic mixtures is part of this invention.
  • the enantiomers elute at 7.49 min and 13.95 min. Both isomers are isolated by solving in dichloromethane and treatment with 4M solution of HCI in dioxane. After stirring for 6 hours at ambient temperature, the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo.
  • (+)-N-(3-Amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7- dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride (8a) is tentatively assigned to the enantiomer with the R-configu ration (+)-N-(3-Amino-propyl)-N-[(R)-1-(6- benzyl-S-chloro ⁇ -methyl ⁇ -oxo- ⁇ y-dihydro-pyrazoloIi . ⁇ -clpyrimidin- ⁇ -yl ⁇ -methyl-propyll ⁇ -methyl- benzamide hydrochloride. Consequently, the S-configu ration is assigned to the (-)-enantiomer.
  • the enantiomers elute at 6.59 and 15.38 min. Both enantiomers are isolated by mixing with 1 N HCI in diethylether and stirring in this mixture. The mixture is evaporated and the residue is dissolved in a mixture of water and acetonitrile and lyophilized.
  • Some of the compounds in this section have an optionally substituted benzoyl group, which may be designated in the systematic name as phenyl-carbonyl group or alternatively, as a phenyl-methanoyl group.
  • a special substituted phenyl group in this connection is the m-tolyl group (3-methyl-phenyl) or the p-tolyl group (4-methyl-phenyl).
  • the following compounds may be prepared by reductive amination reaction of the appropriate compound C1 to C6 with N 1 ,N 1 -dimethyl-propane-1 ,3-diamine (Me 2 N-(CH 2 ) 3 -NH 2 ) analogously or similarly as described for compounds B1 to B3.
  • the title compound may be prepared by reductive amination reaction of compound C4 with ⁇ /-Boc-1 ,3- diaminopropane analogously or similarly as described for compounds B1 to B3.
  • the mixture of the crude imine is dissolved in 12 ml_ acetic acid. 0.37 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 16 hours at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO 3 -solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.34 g (34%) of the title compound as colorless foam.
  • the mixture of the crude imine is dissolved in 11 ml_ acetic acid. 0.33 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 1 hour at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO 3 -solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.21 g (25%) of the title compound as colorless foam.
  • the mixture of the crude imine is dissolved in 12 mL acetic acid. 0.33 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 90 minutes at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO 3 -solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a cyclohexane ethyl acetate in a 2:1 mixture. This yielded 0.16 g (19%) of the title compound as colorless solid.
  • the title compound may be prepared by oxidation of compound D4 analogously or similarly as described for compounds C1 to C3.
  • the title compound may be prepared from compound E1 analogously or similarly as described for compound D1.
  • the compounds of formula I, I* and I**, and their pharmacologically and/or pharmaceutically acceptable salts have valuable pharmacological and/or pharmaceutical properties which can make them commercially applicable.
  • the compounds according to this invention can act as inhibitors of the mitotic kinesin Eg5 and these compounds are expected to be commercially applicable in the therapy of diseases responsive to the inhibition of this kinesin, such as e.g. those diseases mentioned below.
  • the compounds according to this invention can display cell-cycle dependent, anti-proliferative and/or apoptosis inducing activity.
  • the mitotic kinesin Eg5 is an enzyme essential for the assembly and function of the bipolar mitotic spindle. Eg5 plays essential roles during all phases of mitosis. Drugs that perturb mitosis have proven clinically effective in the treatment of many cancers. Despite the diverse array of essential spindle proteins that could be exploited as targets for the discovery of novel cancer therapies, all spindle- targeted therapeutics in clinical use today act on only one protein, tubulin. Surprisingly, kinesin Eg5 expression is most abundant in proliferating human tissues, whereas it is absent from most postmitotic cells, such as e.g. human central nervous system neurons, consistent with an exclusive or almost confined role for Eg5 in cell proliferation.
  • Eg5 kinesin inhibitors are expected not to disrupt microtubule-based cellular processes, e.g. neuronal transport, that are unrelated to proliferation.
  • Eg5 is essentially involved in organizing microtubules into a bipolar structure that forms the mitotic spindle.
  • Experimental perturbation of Eg5 function causes a characteristic malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest and cell death.
  • the compounds according to this invention can be used to modulate mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis, which is frequently followed by apoptosis.
  • modulate herein is meant altering mitotic spindle formation, including increasing and decreasing spindle formation.
  • mitotic spindle formation herein is meant organization of microtubules into bipolar structures by mitotic kinesins.
  • disfunction of the mitotic spindle herein is meant mitotic arrest and monopolar spindle formation.
  • “Malformation of the mitotic spindle” encompasses the splaying of mitotic spindle poles, or otherwise causing morphological perturbation of the mitotic spindle.
  • a "neoplasia” is defined by cells displaying aberrant cell proliferation and/or survival and/or a block in differentiation.
  • a "benign neoplasia” is described by hyperproliferation of cells, incapable of forming an aggressive, metastasizing tumor in-vivo.
  • a "malignant neoplasia” is described by cells with multiple cellular and biochemical abnormalities, capable of forming a systemic disease, for example forming tumor metastasis in distant organs.
  • Various diseases are caused aberrant cell proliferation ("hyperproliferation") as well as evasion from apoptosis. These diseases include e.g.
  • Malignant neoplasia include solid and hematological tumors. Solid tumors are exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g.
  • malignant neoplasia include inherited cancers exemplified by retinoblastoma and Wilms tumor. In addition, malignant neoplasia include primary tumors in said organs and corresponding secondary tumors in distant organs ("tumor metastases").
  • Hematological tumors are exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers of unknown primary site as well as AIDS related malignancies.
  • the invention therefore relates to a use of the compounds according to the invention in the manufacture of pharmaceutical compositions, a method of treatment or a combination according to the invention, in which the cancer to be treated is selected from the group consisting of cancer of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands, esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva; inherited cancers, retinomblastoma and Wilms tumor; leukemia, lymphoma, non-Hodgkins disease, chronic and acute myeloid leukaemia, acute lymphoblastic leukemia, Hodgkins disease, multiple myeloma and T-cell lymphoma; myelody
  • a cancer disease as well as a malignant neoplasia does not necessarily require the formation of metastases in distant organs. Certain tumors exert devastating effects on the primary organ itself through their aggressive growth properties. These can lead to the destruction of the tissue and organ structure finally resulting in failure of the assigned organ function.
  • Neoplastic cell proliferation might affect normal cell behaviour and organ function. For example the formation of new blood vessels, a process described as neovascularization, is induced by tumors or tumor metastases.
  • Compounds according to this invention can be commercially applicable for the treatment of pathophysiological relevant processes caused by benign or neoplastic cell proliferation, such as but not limited to neovascularization by unphysiological proliferation of vascular endothelial cells.
  • Drug resistance is of particular importance for the frequent failure of standard cancer therapeutics. This drug resistance is caused by various cellular and molelcular mechanisms like overexpression of drug efflux pumps or mutation within the cellular target protein.
  • the commercial applicability of the compounds according to this invention is not limited to 1 st line treatment of patients. Patients with resistance to defined cancer chemotherapeutics or target specific anti-cancer drugs (2 nd or 3 rd line treatment) can be also amenable for treatment with the compounds according to this invention.
  • compounds according to the present invention may be also commercially usable for treatment of diseases associated with cell cycle and cell proliferation, such as, besides cancer discussed above, for example, fibroproliferative and differentiative disorders, psoriasis, rheumatoid arthritis, atherosclerosis, hyperplasia, restenosis, cardiac hypertrophy, (auto)immune disorders, fungal disorders, bone diseases, or acute or chronic inflammation.
  • diseases associated with cell cycle and cell proliferation such as, besides cancer discussed above, for example, fibroproliferative and differentiative disorders, psoriasis, rheumatoid arthritis, atherosclerosis, hyperplasia, restenosis, cardiac hypertrophy, (auto)immune disorders, fungal disorders, bone diseases, or acute or chronic inflammation.
  • diseases associated with cell cycle and cell proliferation such as, besides cancer discussed above, for example, fibroproliferative and differentiative disorders, psoriasis, rheumatoid arthritis, atheros
  • Compounds according to the present invention can be commercially applicable for treatment, prevention or amelioration of the diseases of benign and malignant behavior as described before, such as e.g. benign or malignant neoplasia, particularly cancer (such as e.g. any of those cancer diseases described above), especially a cancer that is susceptible to Eg5 inhibition.
  • diseases of benign and malignant behavior such as e.g. benign or malignant neoplasia, particularly cancer (such as e.g. any of those cancer diseases described above), especially a cancer that is susceptible to Eg5 inhibition.
  • the compounds according to the present invention are expected to be distinguished by valuable and desirable effects related therewith, such as e.g. by low toxicity, superior bioavailability in general (such as e.g. good enteral absorption), superior therapeutic window, absence of significant side effects, and/or further beneficial effects related with their therapeutic and pharmaceutical suitability.
  • the invention further includes a method for treating (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, particularly those diseases, disorders, conditions or illnesses mentioned above, in mammals, including humans, suffering therefrom comprising administering to said mammals in need thereof a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention.
  • the present invention further includes a method useful to modulate apoptosis and/or aberrant cell growth in the therapy of benign or malignant neoplastic diseases, such as e.g. cancer, comprising administering to a subject in need of such therapy a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention.
  • the invention further includes a method for modulating, particularly inhibiting, Eg5 activity in cells comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such modulation, particularly inhibition.
  • the present invention further includes a method to modulate the mitotic spindle, i.e., for example, altering mitotic spindle formation, including decreasing spindle formation, or increasing or decreasing spindle pole separation causing malformation of the mitotic spindle poles, comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such modulation.
  • the present invention further includes a method to inhibit mitosis in cells comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such inhibition.
  • the present invention further includes a method for treating, preventing or ameliorating diseases and/or disorders associated with Eg5 kinesin activity, such as, for example, (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, for example, benign neoplasia or malignant neoplasia, e.g. cancer, in a mammal comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more compounds according to the present invention to said mammal in need thereof.
  • diseases and/or disorders associated with Eg5 kinesin activity such as, for example, (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, for example, benign neoplasia or malignant neoplasia, e.g. cancer
  • the present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which are employed for the treatment, prophylaxis and/or amelioration of one or more of the illnesses mentioned.
  • the present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which can be used in the treatment, prevention or amelioration of (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis in a mammal, such as, for example, benign or malignant neoplasia, e.g. cancer.
  • the present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which can be used use in the treatment, prevention or amelioration of disorders responsive to arresting of aberrant cell growth and/or induction of apoptosis.
  • the present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
  • the present invention further relates to pharmaceutical compositions comprising one or more of the compounds according to this invention and a pharmaceutically acceptable carrier or diluent.
  • the present invention further relates to pharmaceutical compositions made by combining one or more of the compounds according to this invention and a pharmaceutically acceptable carrier or diluent.
  • the present invention further relates to pharmaceutical compositions comprising one or more of the compounds according to this invention and pharmaceutically acceptable auxiliaries and/or excipients.
  • the present invention also relates to pharmaceutical compositions for treating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, which include benign neoplasia and malignant neoplasia, including cancer, comprising a compound according to this invention.
  • the present invention further relates to combinations comprising one or more of the compounds according to this invention and pharmaceutically acceptable auxiliaries, excipients and/or vehicles, e.g. for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
  • the present invention further relates to a combination comprising a compound according to this invention and a pharmaceutically acceptable excipient, carrier and/or diluent, e.g. for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
  • a pharmaceutically acceptable excipient, carrier and/or diluent e.g. for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
  • the present invention further relates to a composition consisting essentially of a therapeutically effective and tolerable amount of one or more compounds according to this invention together with the usual pharmaceutically acceptable vehicles, diluents and/or excipients for use in therapy, e.g. for treating, preventing or ameliorating hyperproliferative diseases, such as e.g. cancer, and/or disorders responsive to induction of apoptosis.
  • a composition consisting essentially of a therapeutically effective and tolerable amount of one or more compounds according to this invention together with the usual pharmaceutically acceptable vehicles, diluents and/or excipients for use in therapy, e.g. for treating, preventing or ameliorating hyperproliferative diseases, such as e.g. cancer, and/or disorders responsive to induction of apoptosis.
  • the present invention further relates to compounds according to this invention for use in therapy, such as, for example, in the treatment, prevention or amelioration of (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis, such as e.g. those diseases mentioned herein, particularly cancer.
  • the present invention further relates to compounds according to this invention having anti-proliferative and/or apoptosis inducing activity.
  • the present invention further relates to compounds according to this invention having Eg5 inhibiting properties.
  • the present invention further relates to pharmaceutical compositions according to this invention having Eg5 inhibiting properties.
  • the present invention further relates to pharmaceutical compositions according to this invention having anti-proliferative activity.
  • the present invention further relates to pharmaceutical compositions according to this invention having apoptosis inducing activity.
  • the invention further relates to the use of a pharmaceutical composition comprising one or more of the compounds according to this invention as sole active ingredient(s) and a pharmaceutically acceptable carrier or diluent in the manufacture of pharmaceutical products for the treatment and/or prophylaxis of the illnesses mentioned above.
  • the invention relates to an article of manufacture, which comprises packaging material and a pharmaceutical agent contained within said packaging material, wherein the pharmaceutical agent is therapeutically effective inhibiting Eg5 and/or inhibiting cellular (hyper)proliferation and/or inducing apoptosis, ameliorating the symptoms of a Eg5 mediated disease and/or a (hyper)proliferative disease and/or a disorder responsive to the induction of apoptosis, and wherein the packaging material comprises a label or package insert which indicates that the pharmaceutical agent is useful for preventing or treating a Eg5 mediated disease and/or a (hyper)proliferative disease and/or a disorder responsive to the induction of apoptosis, and wherein said pharmaceutical agent comprises one or more compounds according to the invention.
  • the packaging material, label and package insert otherwise parallel or resemble what is generally regarded as standard packaging material, labels and package inserts for pharmaceuticals having related utilities.
  • compositions according to this invention are prepared by processes which are known per se and familiar to the person skilled in the art.
  • auxiliaries and/or excipients e.g. a delayed release form or an enteric form
  • auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge.
  • gel formers In addition to solvents, gel formers, ointment bases and other active compound excipients, for example antioxidants, dispersants, emulsifiers, preservatives, solubilizers (such as e.g. polyoxyethylenglyceroltriricinoleat 35, PEG 400, Tween 80, Solutol HS15 or the like), colorants, complexing agents, permeation promoters, stabilizers, fillers, binders, thickeners, disintegrating agents, buffers, pH regulators (e.g. to obtain neutral, alkaline or acidic formulations), polymers, lubricants, coating agents, propellants, tonicity adjusting agents, surfactants, flavorings, sweeteners or dyes, can be used.
  • active compound excipients for example antioxidants, dispersants, emulsifiers, preservatives, solubilizers (such as e.g. polyoxyethylenglyceroltriricino
  • auxiliaries and/or excipients of a type appropriate to the desired formulation and the desired mode of administration are used.
  • the administration of the compounds, pharmaceutical compositions or combinations according to the invention may be performed in any of the generally accepted modes of administration available in the art.
  • suitable modes of administration include intravenous, oral, nasal, parenteral, topical, transdermal and rectal delivery. Oral and intravenous delivery are preferred.
  • the compounds of the invention can be in particular administered in the form of those pharmaceutical compositions which are suitable for topical application.
  • suitable pharmaceutical formulations are, for example, powders, emulsions, suspensions, sprays, oils, ointments, fatty ointments, creams, lotions, pastes, gels or solutions.
  • the pharmaceutical compositions according to the invention can be prepared by processes known per se.
  • Topical application forms (such as ointments) for the treatment of dermatoses thus contain the active compounds in a concentration of, for example, 0.1-99%.
  • the customary dose in the case of systemic therapy (p.o.) may be between 0.03 and 60 mg/kg per day, (i. v.) may be between 0.03 and 60 mg/kg/h.
  • the customary dose in the case of systemic therapy (p.o.) is between 0.3 and 30 mg/kg per day, (i. v.) is between 0.3 and 30 mg/kg/h.
  • additional therapeutic active agents which are normally administered to treat or prevent that disease, may optionally be coadministered with the compounds according to this invention.
  • additional therapeutic agents that are normally administered to treat or prevent a particular disease are known as appropriate for the disease being treated.
  • compounds according to this invention may be combined with one or more standard therapeutic agents used for treatment of the diseases as mentioned before.
  • compounds according to this invention may be combined with one or more art-known anti-cancer agents, such as e.g. with one or more chemotherapeutic and/or target specific anti-cancer agents as described below.
  • chemotherapeutic anti-cancer agents frequently used in combination therapy include, but not are limited to (i) alkylating/carbamylating agents such as Cyclophosphamid (Endoxan®), lfosfamid (Holoxan®), Thiotepa (Thiotepa Lederle®), Melphalan (Alkeran®), or chloroethylnitrosourea (BCNU); (ii) platinum derivatives like cis-platin (Platinex® BMS), oxaliplatin, satraplatin or carboplatin (Cabroplat® BMS); (iii) antimitotic agents / tubulin inhibitors such as vinca alkaloids (vincristine, vinblastine, vinorelbine), taxanes such as Paclitaxel (Taxol®), Docetaxel (Taxotere®) and analogs as well as new formulations and conjugates thereof, epothilones such as Epothilone
  • target specific anti-cancer drug classes used in experimental or standard cancer therapy include but are not limited to (i) kinase inhibitors such as e.g. lmatinib (Glivec®), ZD-1839 / Gefitinib (Iressa®), Bay43-9006 (Sorafenib), SU11248 / Sunitinib (Sutent®) or OSI-774 / Erlotinib (Tarceva®); (ii) proteasome inhibitors such as PS-341 / Bortezumib (Velcade®); (iii) histone deacetylase inhibitors like SAHA, PXD101 , MS275, MGCD0103, Depsipeptide / FK228, NVP-LBH589, NVP-LAQ824, Valproic acid (VPA) and butyrates (iv) heat shock protein 90 inhibitors like 17-allylaminogeldanamycin (17-AAG); (v) vascular targeting agents (
  • Gemtuzumab ozogamicin Mylotarg®
  • lbritumomab tiuxetan Zevalin®
  • antibody fragments oligonucleotide based therapeutics like G-3139 / Oblimersen (Genasense®)
  • Toll-like receptor / TLR 9 agonists like Promune®, TLR 7 agonists like Imiquimod (Aldara®) or lsatoribine and analogues thereof, or TLR 7/8 agonists like Resiquimod as well as immunostimulatory RNA as TLR 7/8 agonists
  • protease inhibitors x
  • hormonal therapeutics such as anti-estrogens (e.g.
  • Tamoxifen or Raloxifen include Tamoxifen or Raloxifen, anti-androgens (e.g. Flutamide or Casodex), LHRH analogs (e.g. Leuprolide, Goserelin or Triptorelin) and aromatase inhibitors.
  • anti-androgens e.g. Flutamide or Casodex
  • LHRH analogs e.g. Leuprolide, Goserelin or Triptorelin
  • aromatase inhibitors include
  • target specific anti-cancer agents which may be used for combination therapy include bleomycin, retinoids such as all-trans retinoic acid (ATRA), DNA methyltransferase inhibitors such as the 2-deoxycytidine derivative Decitabine (Dacogen®) and 5-azacytidine, alanosine, cytokines such as interleukin-2, interferons such as interferon ⁇ 2 or interferon- ⁇ , death receptor agonists, such as TRAIL, DR4/5 agonistic antibodies, FasL and TNF-R agonists.
  • ATRA all-trans retinoic acid
  • DNA methyltransferase inhibitors such as the 2-deoxycytidine derivative Decitabine (Dacogen®) and 5-azacytidine
  • alanosine alanosine
  • cytokines such as interleukin-2
  • interferons such as interferon ⁇ 2 or interferon- ⁇
  • death receptor agonists such as TRAIL,
  • any of the following drugs may be mentioned, without being restricted thereto, 5 FU, actinomycin D, ABARELIX, ABCIXIMAB, ACLARUBICIN, ADAPALENE, ALEMTUZUMAB,
  • ALTRETAMINE AMINOGLUTETHIMIDE, AMIPRILOSE, AMRUBICIN, ANASTROZOLE, ANCITABINE, ARTEMISININ, AZATHIOPRINE, BASILIXIMAB, BENDAMUSTINE, BEVACIZUMAB, BEXXAR, BICALUTAMIDE, BLEOMYCIN, BORTEZOMIB, BROXURIDINE, BUSULFAN, CAMPATH, CAPECITABINE, CARBOPLATIN, CARBOQUONE, CARMUSTINE, CETRORELIX, CHLORAMBUCIL, CHLORMETHINE, CISPLATIN, CLADRIBINE, CLOMIFENE, CYCLOPHOSPHAMIDE, DACARBAZINE, DACLIZUMAB, DACTINOMYCIN, DAUNORUBICIN, DECITABINE, DESLORELIN, DEXRAZOXANE, DOCETAXEL, DOXIFLURIDINE, DOXORUBICIN, D
  • anti-cancer agents mentioned herein above as combination partners of the compounds according to this invention are meant to include pharmaceutically acceptable derivatives thereof, such as e.g. their pharmaceutically acceptable salts.
  • total daily dosage(s) and administration form(s) of the additional therapeutic agent(s) coadministered can vary within a wide range.
  • the compounds according to this invention may be administered in combination therapy separately, sequentially, simultaneously, concurrently or chronologically staggered (such as e.g. as combined unit dosage forms, as separate unit dosage forms, as adjacent discrete unit dosage forms, as fixed or non-fixed combinations, as kit-of-parts or as admixtures) with one or more standard therapeutics (chemotherapeutic and/or target specific anti-cancer agents), in particular art-known anti-cancer agents, such as any of e.g. those mentioned above.
  • standard therapeutics chemotherapeutic and/or target specific anti-cancer agents
  • anti-cancer agents such as any of e.g. those mentioned above.
  • the present invention further relates to a combination comprising a first active ingredient, which is at least one compound according to this invention, and a second active ingredient, which is at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy, such as e.g. in therapy of any of those diseases mentioned herein.
  • a first active ingredient which is at least one compound according to this invention
  • a second active ingredient which is at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy, such as e.g. in therapy of any of those diseases mentioned herein.
  • combination may be present as a fixed combination, a non-fixed combination or a kit-of-parts.
  • a “fixed combination” is defined as a combination wherein the said first active ingredient and the said second active ingredient are present together in one unit dosage or in a single entity.
  • a “fixed combination” is a pharmaceutical composition wherein the said first active ingredient and the said second active ingredient are present in admixture for simultaneous administration, such as in a formulation.
  • Another example of a "fixed combination” is a pharmaceutical combination wherein the said first active ingredient and the said second active ingredient are present in one unit without being in admixture.
  • a “kit-of-parts” is defined as a combination wherein the said first active ingredient and the said second active ingredient are present in more than one unit.
  • One example of a “kit-of-parts” is a combination wherein the said first active ingredient and the said second active ingredient are present separately.
  • the components of the may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a first active ingredient, which is at least one compound according to this invention, and a second active ingredient, which is at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, and, optionally, a pharmaceutically acceptable carrier or diluent, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy.
  • the present invention further relates to a combination product comprising a.) at least one compound according to this invention formulated with a pharmaceutically acceptable carrier or diluent, and b.) at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, formulated with a pharmaceutically acceptable carrier or diluent.
  • the present invention further relates to a kit-of-parts comprising a preparation of a first active ingredient, which is a compound according to this invention, and a pharmaceutically acceptable carrier or diluent; a preparation of a second active ingredient, which is an art-known anti-cancer agent, such as one of those mentioned above, and a pharmaceutically acceptable carrier or diluent; for simultaneous, concurrent, sequential, separate or chronologically staggered use in therapy.
  • said kit comprises instructions for its use in therapy, e.g. to treat (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, such as e.g. cancer, more precisely, any of those cancer diseases described above.
  • the present invention further relates to a combined preparation comprising at least one compound according to this invention and at least one art-known anti-cancer agent for simultaneous, concurrent, sequential or separate administration.
  • the present invention further relates to combinations, compositions, formulations, preparations or kits according to the present invention having Eg5 inhibitory activity and/or anti-proliferative and/or apoptosis inducing properties.
  • the present invention further relates to a method for treating in combination therapy (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, such as e.g. cancer, in a patient comprising administering a combination, composition, formulation, preparation or kit as described herein to said patient in need thereof.
  • the present invention further relates to a method for treating (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis, such as e.g.
  • a pharmaceutical composition which comprises a compound according to this invention and a pharmaceutically acceptable carrier or diluent, and a pharmaceutically active and therapeutically effective and tolerable amount of one or more art-known anti-cancer agents, such as e.g. one or more of those mentioned herein, to said patient in need thereof.
  • the present invention relates to a method for treating, preventing or ameliorating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as, for example, benign or malignant neoplasia, e.g. cancer, particularly any of those cancer diseases mentioned herein, in a patient comprising administering separately, simultaneously, concurrently, sequentially or chronologically staggered to said patient in need thereof an amount of a first active compound, which is a compound according to the present invention, and an amount of at least one second active compound, said at least one second active compound being a standard therapeutic agent, particularly at least one art-known anti-cancer agent, such as e.g. one or more of those chemotherapeutic and target-specific anti-cancer agents mentioned herein, wherein the amounts of the first active compound and said second active compound result in a therapeutic effect.
  • a first active compound which is a compound according to the present invention
  • at least one second active compound being a standard therapeutic agent, particularly at least one art-known
  • the present invention relates to a method for treating, preventing or ameliorating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as e.g. benign or malignant neoplasia, e.g. cancer, particularly any of those cancer diseases mentioned herein, in a patient comprising administering a combination according to the present invention.
  • apoptosis such as e.g. benign or malignant neoplasia, e.g. cancer, particularly any of those cancer diseases mentioned herein
  • the present invention further relates to the use of a composition, combination, formulation, preparation or kit according to this invention in the manufacture of a pharmaceutical product, such as e.g. a commercial package or a medicament, for treating, preventing or ameliorating (hyper)proliferative diseases, such as e.g. cancer, and/or disorders responsive to the induction of apoptosis, particularly those diseases mentioned herein, such as e.g. malignant or benign neoplasia.
  • a pharmaceutical product such as e.g. a commercial package or a medicament
  • the present invention further relates to a commercial package comprising one or more compounds of the present invention together with instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein.
  • the present invention further relates to a commercial package consisting essentially of one or more compounds of the present invention as sole active ingredient together with instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein.
  • the present invention further relates to a commercial package comprising one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein, together with instructions for simultaneous, concurrent, sequential or separate use with one or more compounds according to the present invention.
  • chemotherapeutic and/or target specific anti-cancer agents such as e.g. any of those mentioned herein, together with instructions for simultaneous, concurrent, sequential or separate use with one or more compounds according to the present invention.
  • compositions, combinations, preparations, formulations, kits or packages mentioned in the context of the combination therapy according to this invention may also include more than one of the compounds according to this invention and/or more than one of the art-known anti-cancer agents mentioned.
  • the first and second active ingredient of a combination or kit-of-parts according to this invention may be provided as separate formulations (i.e. independently of one another), which are subsequently brought together for simultaneous, concurrent, sequential, separate or chronologically staggered use in combination therapy; or packaged and presented together as separate components of a combination pack for simultaneous, concurrent, sequential, separate or chronologically staggered use in combination therapy.
  • the type of pharmaceutical formulation of the first and second active ingredient of a combination or kit-of-parts according to this invention can be similar, i.e. both ingredients are formulated in separate tablets or capsules, or can be different, i.e. suited for different administration forms, such as e.g. one active ingredient is formulated as tablet or capsule and the other is formulated for e.g. intravenous administration.
  • the amounts of the first and second active ingredients of the combinations, compositions or kits according to this invention may together comprise a therapeutically effective amount for the treatment, prophylaxis or amelioration of a (hyper)proliferative diseases and/or a disorder responsive to the induction of apoptosis, particularly one of those diseases mentioned herein, such as e.g. malignant or benign neoplasia, especially cancer, like any of those cancer diseases mentioned herein.
  • compounds according to the present invention can be used in the pre- or post-surgical treatment of cancer.
  • compounds of the present invention can be used in combination with radiation therapy.
  • a combination according to this invention can refer to a composition comprising both the compound(s) according to this invention and the other active anti-cancer agent(s) in a fixed combination (fixed unit dosage form), or a medicament pack comprising the two or more active ingredients as discrete separate dosage forms (non-fixed combination).
  • a medicament pack comprising the two or more active ingredients
  • the active ingredients are preferably packed into blister cards which are suited for improving compliance.
  • Each blister card preferably contains the medicaments to be taken on one day of treatment. If the medicaments are to be taken at different times of day, the medicaments can be disposed in different sections on the blister card according to the different ranges of times of day at which the medicaments are to be taken (for example morning and evening or morning, midday and evening).
  • the blister cavities for the medicaments to be taken together at a particular time of day are accommodated in the respective range of times of day.
  • the various times of day are, of course, also put on the blister in a clearly visible way. It is also possible, of course, for example to indicate a period in which the medicaments are to be taken, for example stating the times.
  • the daily sections may represent one line of the blister card, and the times of day are then identified in chronological sequence in this column.
  • Medicaments which must be taken together at a particular time of day are placed together at the appropriate time on the blister card, preferably a narrow distance apart, allowing them to be pushed out of the blister easily, and having the effect that removal of the dosage form from the blister is not forgotten.
  • the ATPase activity of Eg5 kinesin motor domains can be used to monitor the effects of modulating agents.
  • the test compounds are dissolved as 10 mM solutions in dimethylsulfoxide (DMSO). 2 ⁇ l of appropriate DMSO dilutions of the test compounds are added to each well of a 96 well flat bottom plate. Each compound dilution is tested as triplicates.
  • DMSO dimethylsulfoxide
  • the reagents are added and the final reaction of the standard assay contains 15 mM Pipes, pH 6.8, 5.0 mM MgCI 2 , 0.5 mM KCI, 1 mM EGTA, 0.1 mg/ml BSA, 1 ⁇ M Paclitaxel, 250 nM preformed microtubules (Cytoskeleton, cat. No. MT001 ), 300 ⁇ M ATP, and Eg5 protein (50 ng) in a reaction volume of 100 ⁇ l.
  • the controls include buffer wells with ATP and 2% DMSO.
  • Reactions are started by the addition of ATP, incubated at room temperature for 30 min., and terminated by removing 20 ⁇ l of the reaction volume and adding it to 80 ⁇ l of 1 M perchloric acid, followed by the addition of 80 ⁇ l Malachite green reagent.
  • Malachite green reagent is prepared by mixing a solution of 4.2 g ammonium molybdate in 100 ml 4 N HCI with a solution of 0.135 g Malachite green in 300 ml H 2 O. The reactions are incubated for a further 20 min. and then read at 615 nm.
  • the corresponding IC 50 values of the compounds for Eg5 inhibition are determined from the concentration-effect curves.
  • the anti-proliferative / cytotoxic activity of the compounds described herein can be tested on subclones of RKO human colon adenocarcinoma cells (Schmidt et al., Oncogene 19, 2423-2429; 2000) using the Alamar Blue cell viability assay (described in O ' Brien et al. Eur J Biochem 267, 5421- 5426, 2000).
  • the compounds are dissolved as 10 mM solutions in DMSO and subsequently diluted in semi-logarithmic steps.
  • DMSO dilutions are further diluted 1 :100 into Dulbecco ' s modified Eagle ' s medium (DMEM) containing 10% fetal calf serum to a final concentration twice as much as the final concentration in the test.
  • DMEM Dulbecco ' s modified Eagle ' s medium
  • RKO subclones are seeded into 96 well flat bottom plates at a density of 4000 cells per well in a volume of 50 ⁇ l per well. 24 hours after seeding the 50 ⁇ l each of the compound dilutions in DMEM medium are added into each well of the 96 well plate. Each compound dilution is tested as triplicates.
  • Wells containing untreated control cells are filled with 50 ⁇ l DMEM medium containing 1 % DMSO.
  • the cells are then incubated with the substances for 72 hours at 37 0 C in a humidified atmosphere containing 5% carbon dioxide.
  • 10 ⁇ l of an Alamar Blue solution (Biosource) are added and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm.
  • the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values.
  • the Graphpad Prism program is used for the calculation of EC 50 values for anti-proliferative / cytotoxic activity out of the obtained dose- response curves.
  • RKO colon adenocarcinoma cells (RKOp27 as described by Schmidt et al. in Oncogene 19, 2423-2429; 2000) are seeded into 96 well flat bottom plates at a density of 16000 cells per well in a volume of 50 ⁇ l per well in DMEM growth medium with 10% FCS containing 10 ⁇ M Ponasterone A. 24 hours after seeding the 50 ⁇ l each of the compound dilutions in DMEM medium are added into each well of the 96-well plate. Each compound dilution is tested as triplicates. Wells containing untreated control cells are filled with 50 ⁇ l DMEM medium containing 1% DMSO.
  • the cells are then incubated with the substances for 72 hours at 37 0 C in a humidified atmosphere containing 5% carbon dioxide.
  • 10 ⁇ l of an Alamar Blue solution (Biosource) are added and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm.
  • the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values.
  • the Graphpad Prism program is used for the calculation of EC 50 values out of the obtained dose-response curves. Viability is compared of proliferating cells grown in the absence of the inducer Ponasterone A, versus viability of cells arrested by the expression of ectopic p27Kip1 induced by Ponasterone A.
  • the induction of apoptosis can be measured by using a Cell death detection ELISA (Roche Biochemicals, Mannheim, Germany).
  • NCI-H460 non-small cell lung cancer cells are seeded into 96 well flat bottom plates at a density of 10000 cells per well in a volume of 50 ⁇ l RPMI medium (containing 10% fetal calf serum) per well.
  • the compounds are dissolved as 10 mM solutions in DMSO and subsequently diluted in semi-logarithmic steps.
  • DMSO dilutions are further diluted 1 :100 into RPMI medium (containing 10% fetal calf serum) to a final concentration twice as much as the final concentration in the test.
  • the degree of apoptosis is calculated as follows: The absorbance at 405 nm obtained with lysates from cells treated with 50 ⁇ M cisplatin is set as 100 cpu (cisplatin units), while an absorbance at 405 nm of 0.0 is set as 0.0 cpu. The degree of apoptosis is expressed as cpu in relation to the value of 100 cpu reached with the lysates obtained from cells treated with 50 ⁇ M cisplatin.
  • Eg5 function causes a characteristic malformation of the mitotic spindle, which can be examined by confocal laser scanning microscopy.
  • HeLa cervical cancer cells are grown overnight on glass cover slips (NuncTM Lab-TekTM Chamber Slides) in 1800 ⁇ l DMEM medium containing 10% fetal calf serum.
  • the test compounds are dissolved as 10 mM solutions in DMSO.
  • Appropriate DMSO dilutions of the test compounds are further diluted 1 :20 into DMEM medium containing 10% fetal calf serum to a final concentration ten times as much as the final concentration in the test. 24 hours after seeding, 200 ⁇ l of the compound dilutions in DMEM medium are added into each well of the cover slip.
  • DMEM medium containing 5% DMSO 200 ⁇ l DMEM medium containing 5% DMSO are added. 24 hours after incubation with the test compounds, the cells are washed with PBS, and fixed with 3.7% formaldehyde in H 2 O for 20 min. at 37 0 C. Subsequently, cells are washed with PBS and incubated with 0,1 % Triton X-100 in a buffer containing 1.471 mM KH 2 PO 4, 8.504 mM Na 2 HPO 4 , 137 mM NaCI, 1.325 mM CaCI 2 , 2.685 mM KCI, 0.542 mM MgCI 2 , pH 7.2 for 15 min. at room temperature.
  • Some of the compounds according to this invention may be efficacious against p-glycoprotein mediated multidrug-resistent tumour cell lines (e.g. HCT-15), that can be measured as follows: All cell lines used are cultured at standard conditions in a tissue culture incubator at 37 0 C, 5% CO 2 and 95% humidity. At day 1 , cells are detached with Trypsin / EDTA and pelleted by centrifugation. Cells are resuspended at the appropriate density in culture medium, seeded into 96well microtiter plates and incubated over night in a tissue culture incubator at 37 0 C, 5% CO 2 and 95% humidity.
  • p-glycoprotein mediated multidrug-resistent tumour cell lines e.g. HCT-15
  • All cell lines used are cultured at standard conditions in a tissue culture incubator at 37 0 C, 5% CO 2 and 95% humidity. At day 1 , cells are detached with Trypsin / EDTA and pelleted by centrifug
  • the cells are incubated in a tissue culture incubator at 37 0 C, 5% CO 2 and 95% humidity for additional 1-6 hours and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm.
  • the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values.
  • the Graphpad Prism program is used for the calculation of EC 50 values out of the obtained dose- response curves.

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Abstract

Compounds of a certain formula (I), in which R1, R2, A, R4, B, R5 and R6 have the meanings indicated in the description, are effective compounds with antiproliferative and/or apoptosis inducing activity.

Description

Pyrazolopyrimidones
Field of application of the invention
The invention relates to pyrazolopyrimidone derivatives, which can be used in the pharmaceutical industry for the production of pharmaceutical compositions.
Known technical background
The US application US2004/0242596 contains pyrazolo[3,4-d]pyrimidones which are said to be useful as anti-cancer agents and to induce mitotic arrest.
Description of the invention
It has now been found, that the pyrazolo[1 ,5-c]pyrimidone derivatives, which are described in greater details below, differ from prior art compounds by unanticipated structural features and have surprising and particularly advantageous properties.
Thus, for example, the compounds according to this invention can act as inhibitors of Eg5 kinesin. In more detail, it has been unexpectedly found that these derivatives are potent and highly efficacious inhibitors of cellular (hyper)proliferation and/or cell-cycle specific inducers of apoptosis in cancer cells. Therefore, these compounds can be particular useful for treating (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, notably cancer. By having a cell-cycle specific mode of action, these derivatives should have a higher therapeutic index compared to standard chemotherapeutic drugs targeting basic cellular processes like DNA replication or interfering with basic cellular molecules like DNA.
Thus, for example, the compounds according to this invention are expected to be useful in targeted cancer therapy.
The invention thus relates to compounds of formula I
(I) in which R1 is hydrogen or halogen,
R2 is 1-4C-alkyl,
A is Aryl-1-4C-alkyl, in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is 1-4C-alkyl, trifluoromethyl, 1-4C-alkoxy, hydroxyl, halogen, or completely or predominantly fluorine-substituted 1-4C-alkoxy, R31 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy, R4 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl or 3-7C-cycloalkyl-1-4C-alkyl, R5 is hydrogen or 1-4C-alkyl, R6 is hydrogen or 1-4C-alkyl,
B is phenyl, or R7- and R71-substituted phenyl, in which R7 is 1-4C-alkyl, trifluoromethyl, cyano, 1-4C-alkoxy, halogen, carboxyl, 1-4C-alkylcarbonyl, methylenedioxy, ethylenedioxy, 1-4C-alkoxy-1-4C-alkyl, hydroxy-1-4C-alkyl, or completely or predominantly fluorine-substituted 1-4C-alkoxy, R71 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy, n is 2, 3, 4, 5 or 6, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
As used herein, "alkyl" alone or as part of another group refers to both branched and straight chain saturated aliphatic hydrocarbon groups having the specified numbers of carbon atoms, such as for example:
1-4C-Alkyl is a straight-chain or branched alkyl radical having 1 to 4 carbon atoms. Examples are the butyl, isobutyl, sec-butyl, tert-butyl, propyl, isopropyl, ethyl and methyl radicals, of which propyl, isopropyl, ethyl and methyl are more worthy to be mentioned.
Halogen within the meaning of the present invention is iodine or, in particular, bromine, chlorine or fluorine.
1-4C-Alkoxy represents radicals which, in addition to the oxygen atom, contain a straight-chain or branched alkyl radical having 1 to 4 carbon atoms. Examples which may be mentioned are the butoxy, isobutoxy, sec-butoxy, tert-butoxy, propoxy, isopropoxy, ethoxy and methoxy radicals, of which propoxy, isopropoxy, and, particularly, ethoxy and methoxy are more worthy to be mentioned.
The term "cycloalkyl" alone or as part of another group refers to a monocyclic saturated aliphatic hydrocarbon group having the specified numbers of carbon atoms, such as for example: 3-7C-Cycloalkyl stands for cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, of which cyclopentyl, cyclobutyl and, in particular, cyclopropyl are more worthy to be mentioned.
3-7C-Cycloalkyl-1-4C-alkyl stands for one of the abovementioned 1-4C-alkyl radicals, which is substituted by one of the abovementioned 3-7C-cycloalkyl radicals. Examples which may be mentioned are the S-ZC-cycloalkylmethyl radicals, of which cyclopentylmethyl, cyclobutyl methyl and, in particular, cyclopropylmethyl are more worthy to be mentioned.
1-4C-Alkoxy-1-4C-alkyl represents one of the abovementioned 1-4C-alkyl radicals, which is substituted by one of the abovementioned 1-4C-alkoxy radicals. Examples which may be mentioned are the 2-isopropoxyethyl, 2-ethoxyethyl, 2-methoxyethyl and, in particular, isopropoxymethyl, ethoxymethyl and, in more particular, methoxymethyl radical.
Hydroxy-1-4C-alkyl represents one of the abovementioned 1-4C-alkyl radicals, which is substituted by a hydroxyl radical. Examples which may be mentioned are the 3-hydroxypropoxy, 2-hydroxyethyl and, in particular, hydroxymethyl radical.
Completely or predominantly fluorine-substituted 1-4C-alkoxy is, for example, the 2,2,3,3,3- pentafluoropropoxy, the perfluoroethoxy, the 1 ,2,2-trifluoroethoxy and in particular the 1 ,1 ,2,2- tetrafluoroethoxy, the 2,2,2-trifluoroethoxy, the trifluoromethoxy and the difluoromethoxy radical, of which the trifluoromethoxy and the difluoromethoxy radicals are preferred. "Predominantly" in this connection means that more than half of the hydrogen atoms of the 1-4C-alkoxy groups are replaced by fluorine atoms.
1-4C-Alkylcarbonyl is a carbonyl group, to which one of the abovementioned 1-4C-alkyl radicals is bonded. An example is the acetyl radical (CH3CO-).
Aryl-1-4C-alkyl stands for one of the abovementioned 1-4C-alkyl radicals, which is substituted by an Aryl radical as defined below, such as e.g. the Aryl-methyl or 2-Aryl-ethyl radical, of which the Aryl- methyl radical is to be emphasized.
Aryl stands for phenyl, or R3- and R31-substituted phenyl.
Examples of the Aryl-1-4C-alkyl radical include the phenethyl and, particularly, the benzyl radical, each of which is optionally substituted by R3 and R31 on the phenyl moiety.
Suitable salts for compounds according to this invention - depending on substitution - are all acid addition salts or all salts with bases. Particular mention may be made of the pharmacologically and/or pharmaceutically tolerable inorganic and organic acids and bases customarily used in pharmacy. Those suitable include, but are not limited to, water-insoluble and, particularly, water-soluble acid addition salts with acids such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulphuric acid, acetic acid, citric acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)- benzoic acid, butyric acid, sulphosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid, stearic acid, toluenesulphonic acid, phenylsulphonic acid, methanesulphonic acid or 3-hydroxy-2-naphthoic acid, the acids being employed in salt preparation - depending on whether a mono- or polybasic acid is concerned and depending on which salt is desired - in an equimolar quantitative ratio or one differing therefrom. Preferred are the salts selected from hydrochlorides, mesylates, tartrates, citrates, fumarates or sulfates.
On the other hand, salts with bases are - depending on substitution - also suitable. As examples of salts with bases are mentioned the lithium, sodium, potassium, calcium, aluminium, magnesium, titanium, ammonium, meglumine or guanidinium salts, here, too, the bases being employed in salt preparation in an equimolar quantitative ratio or one differing therefrom.
Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds of formula I or their pharmaceutically acceptable salts, are also included.
Pharmaceutically unacceptable salts, which can be obtained, for example, as process products during the preparation of the compounds according to this invention on an industrial scale, are converted into pharmaceutically acceptable salts by processes known to the person skilled in the art.
According to expert's knowledge the compounds of formula I according to this invention as well as their salts may contain, e.g. when isolated in crystalline form, varying amounts of solvents. Included within the scope of the invention are therefore all solvates and in particular all hydrates of the compounds of formula I according to this invention as well as all solvates and in particular all hydrates of the salts of the compounds of formula I according to this invention.
In one embodiment of this invention, salts of compounds of formula I include a salt of a compound of formula I with hydrochloric acid (hydrochloride).
The substituents R3 and R31 of compounds of formula I can be attached in the ortho, meta or para position with respect to the binding position in which the Aryl ring is bonded to the 1-4C-alkyl group, whereby emphasis is given to the attachment of R3 in the meta or para position. In one embodiment R31 is hydrogen.
The substituents R7 and R71 of compounds of formula I can be attached in the ortho, meta or para position with respect to the binding position in which the phenyl ring is bonded to the carbonyl group, whereby emphasis is given to the attachment of R7 in the meta or para position. In one embodiment R71 is hydrogen.
In the context of this invention, hyperproliferation and analogous terms are used to describe aberrant / dysregulated cellular growth, a hallmark of diseases like cancer. This hyperproliferation might be caused by single or multiple cellular / molecular alterations in respective cells and can be, in context of a whole organism, of benign or malignant behaviour. Inhibition of cell proliferation and analogous terms is used herein to denote an ability of the compound to retard the growth of and/or kill a cell contacted with that compound as compared to cells not contacted with that compound. Most preferable this inhibition of cell proliferation is 100%, meaning that proliferation of all cells is stopped and/or cells undergo programmed cell death. In some preferred embodiments the contacted cell is a neoplastic cell. A neoplastic cell is defined as a cell with aberrant cell proliferation and/or the potential to metastasize to different tissues or organs. A benign neoplasia is described by hyperproliferation of cells, incapable of forming an aggressive, metastasizing tumor in-vivo. In contrast, a malignant neoplasia is described by cells with different cellular and biochemical abnormalities, e.g. capable of forming tumor metastasis. The aquired functional abnormalities of malignant neoplastic cells (also defined as "hallmarks of cancer") are replicative potential ("hyperproliferation"), self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion from apoptosis, sustained angiogenesis and tissue invasion and metastasis.
Inducer of apoptosis and analogous terms are used herein to identify a compound which executes programmed cell death in cells contacted with that compound. Apoptosis is defined by complex biochemical events within the contacted cell, such as the activation of cystein specific proteinases ("caspases") and the fragmentation of chromatin. Induction of apoptosis in cells contacted with the compound might not necessarily be coupled with inhibition of cell proliferation. Preferably, the inhibition of cell proliferation and/or induction of apoptosis is specific to cells with aberrant cell growth (hyperproliferation). Thus, compared to cells with aberrant cell growth, normal proliferating or arrested cells are less sensitive or even insensitive to the proliferation inhibiting or apoptosis inducing activity of the compound. Finally, cytotoxic is used in a more general sense to identify compounds which kill cells by various mechanisms, including the induction of apoptosis / programmed cell death in a cell cycle dependent or cell-cycle independent manner.
Cell cycle specific and analogous terms are used herein to identify a compound as inducing apoptosis only in continously proliferating cells actively passing a specific phase of the cell cycle, but not in resting, non-dividing cells. Continously proliferating cells are typical for diseases like cancer and characterized by cells in all phases of the cell division cycle, namely in the G ("gap") 1 , S ("DNA synthesis"), G2 and M ("mitosis") phase.
Compounds according to this invention worthy to be mentioned are those compounds of formula I, in which
R1 is hydrogen or halogen, R2 is 1-2C-alkyl, A is Aryl-1-2C-alkyl, in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is 1-2C-alkyl, trifluoromethyl, 1-2C-alkoxy, hydroxyl, halogen, or completely or predominantly fluorine-substituted 1-2C-alkoxy, R31 is hydrogen, halogen, 1-2C-alkyl or 1-2C-alkoxy,
R4 is hydrogen, 1-4C-alkyl, cyclopropyl or cyclopropyl-I^C-alkyl,
R5 is hydrogen or 1-3C-alkyl,
R6 is hydrogen or 1-3C-alkyl,
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is 1-2C-alkyl, trifluoromethyl, cyano, 1-2C-alkoxy, halogen, carboxyl, 1-2C-alkylcarbonyl, methylenedioxy, ethylenedioxy, 1-2C-alkoxy-1-2C-alkyl, hydroxy-1-2C-alkyl, or completely or predominantly fluorine-substituted 1-2C-alkoxy, R71 is hydrogen, halogen, 1-2C-alkyl or 1-2C-alkoxy, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention more worthy to be mentioned are those compounds of formula
I, in which
R1 is hydrogen or halogen,
R2 is methyl or ethyl,
A is Arylmethyl, in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is methyl, ethyl, trifluoromethyl, methoxy, ethoxy, hydroxyl, halogen, difluoromethoxy or trifluoromethoxy,
R31 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy, R4 is methyl, ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl, R5 is hydrogen, methyl, ethyl, propyl or isopropyl, R6 is hydrogen, methyl, ethyl, propyl or isopropyl, B is phenyl, or R7- and R71-substituted phenyl, in which R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, acetyl, methylenedioxy, ethylenedioxy, methoxymethyl, hydroxymethyl, difluoromethoxy or trifluoromethoxy,
R71 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention in particular worthy to be mentioned are those compounds of formula I, in which
R1 is hydrogen, fluorine, chlorine or bromine,
R2 is methyl or ethyl,
A is Arylmethyl, in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is methyl, ethyl, trifluoromethyl, methoxy, ethoxy, hydroxyl or halogen,
R31 is hydrogen, halogen, methyl or methoxy, R4 is ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl,
R5 is hydrogen, methyl, ethyl, propyl or isopropyl,
R6 is hydrogen, methyl, ethyl, propyl or isopropyl,
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl, R71 is hydrogen, halogen, methyl, ethyl or methoxy, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention in more particular worthy to be mentioned are those compounds of formula I, in which
R1 is hydrogen, chlorine or bromine,
R2 is methyl or ethyl,
A is benzyl, halobenzyl (e.g. fluorobenzyl, chlorobenzyl or bromobenzyl), methylbenzyl, methylhalobenzyl, methoxybenzyl, hydroxybenzyl, dihalobenzyl (e.g. dichlorobenzyl), or dimethoxybenzyl,
R4 is ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropylmethyl, R5 is hydrogen, methyl, ethyl, propyl or isopropyl, R6 is hydrogen, methyl, ethyl, propyl or isopropyl, B is phenyl, or R7- and R71-substituted phenyl, in which R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl, R71 is hydrogen, halogen, methyl or ethyl, such as, for example, B is phenyl, methylphenyl, ethylphenyl, halophenyl, methylhalophenyl, (hydroxymethyl)phenyl, methoxyphenyl, ethoxyphenyl, trifluoromethylphenyl, halo(trifluoromethyl)phenyl, dihalophenyl, methylenedioxyphenyl, ethylenedioxyphenyl, (methoxymethyl)phenyl, methoxychlorophenyl, carboxyphenyl or cyanophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention in further more particular worthy to be mentioned are those compounds of formula I, in which
R1 is hydrogen, chlorine or bromine,
R2 is methyl or ethyl,
A is benzyl, halobenzyl (e.g. fluorobenzyl, chlorobenzyl or bromobenzyl), methylbenzyl, methoxybenzyl or hydroxybenzyl, R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either R5 and R6 are both hydrogen, or
R5 is methyl, and
R6 is hydrogen, or
R5 is ethyl, and
R6 is hydrogen, or
R5 is propyl, and
R6 is hydrogen, or
R5 is isopropyl, and
R6 is hydrogen, or
R5 and R6 are both methyl,
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl, R71 is hydrogen, halogen, methyl or ethyl, such as, for example, B is phenyl, methylphenyl, ethylphenyl, halophenyl, dihalophenyl, methylhalophenyl,
(hydroxymethyl)phenyl, halo(trifluoromethyl)phenyl, trifluoromethylphenyl, methoxyphenyl, methylenedioxyphenyl or cyanophenyl, such as, for more detailed example, B is phenyl, 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3- fluoro-4-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 3,4- dichlorophenyl or 2,3-dichlorophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be emphasized are those compounds of formula I, in which
R1 is hydrogen, chlorine or bromine, particularly
R1 is chlorine or bromine,
R2 is methyl,
A is benzyl, fluorobenzyl, chlorobenzyl, bromobenzyl, methylbenzyl, methoxybenzyl or hydroxybenzyl, in particular
A is benzyl or fluorobenzyl (e.g. 3-fluorobenzyl or 4-fluorobenzyl), R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
R5 and R6 are both hydrogen, or
R5 is methyl, and
R6 is hydrogen, or
R5 is ethyl, and
R6 is hydrogen, or
R5 and R6 are both methyl,
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is methyl, ethyl, trifluoromethyl, fluorine, chlorine or bromine,
R71 is hydrogen, methyl, fluorine, chlorine or bromine, such as, for example,
B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, such as, for more detailed example, B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, fluorophenyl, chlorophenyl, bromophenyl, dichlorophenyl, difluorophenyl, methylfluorophenyl, methylchlorophenyl or methylbromophenyl, such as, for yet more detailed example, B is phenyl, 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3- fluoro-4-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl or 3,4-dichlorophenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be more emphasized are those compounds of formula I, in which
R1 is chlorine or bromine,
R2 is methyl,
A is benzyl or fluorobenzyl (e.g. 3-fluorobenzyl or 4-fluorobenzyl),
R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, such as, for example, B is methylphenyl, trifluoromethylphenyl, methoxyphenyl, fluorophenyl, chlorophenyl, bromophenyl, dichlorophenyl, difluorophenyl, methylfluorophenyl, methylchlorophenyl or methylbromophenyl, such as, for more detailed example,
B is 4-methylphenyl, 3-methylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 3-fluoro-4- methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl or 3,4-dichlorophenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be in particular emphasized are those compounds of formula I, in which
R1 is chlorine or bromine,
R2 is methyl,
A is benzyl, 3-fluorobenzyl or 4-fluorobenzyl,
R4 is ethyl, propyl, isopropyl or cyclopropyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is methylphenyl, trifluoromethylphenyl, methoxyphenyl, chlorophenyl, dichlorophenyl, bromophenyl, fluorophenyl or methylfluorophenyl, such as, for example, B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-chlorophenyl,
4-bromophenyl, 4-fluorophenyl or 3-fluoro-4-methylphenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
In a preferred embodiment, the invention relates to compounds of formula I according to the invention, in which
R1 is hydrogen, chlorine or bromine,
R2 is methyl,
A is benzyl or fluorobenzyl,
R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is phenyl, methylphenyl, trifluoromethylphenyl, methoxyphenyl, halophenyl, dihalophenyl or methylhalophenyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be in more particular emphasized are those compounds of formula I, in which
R1 is chlorine or bromine,
R2 is methyl,
A is benzyl,
R4 is ethyl or isopropyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is methylphenyl, chlorophenyl, trifluoromethylphenyl, methoxyphenyl, bromophenyl or fluorophenyl, such as, for example, B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-chlorophenyl,
4-bromophenyl or 4-fluorophenyl, n is 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be particularly emphasized are those compounds of formula
I comprising one or more of the following:
R1 is chlorine or bromine;
R2 is methyl;
A is benzyl;
R4 is ethyl or isopropyl; either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl;
B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- chlorophenyl, 4-bromophenyl or 3-fluoro-4-methylphenyl; and n is 3; and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
Compounds according to this invention to be in further more particular emphasized are those compounds of formula I, in which R1 is chlorine or bromine, R2 is methyl, A is benzyl, R4 is isopropyl,
R5 and R6 are both hydrogen,
B is methylphenyl, chlorophenyl, trifluoromethylphenyl, methoxyphenyl, bromophenyl or fluorophenyl, such as, for example,
B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl, n is 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
The invention also relates to compounds of formula I according to the invention comprising one or more of the following:
R1 is chlorine or bromine;
R2 is methyl;
A is benzyl;
R4 is isopropyl;
R5 and R6 are both hydrogen;
B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl; and n is 3; and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
In a further preferred embodiment, the invention relates to compounds of formula I in which
R1 is hydrogen, chlorine, bromine or fluorine,
R2 is methyl,
A is benzyl,
R4 is ethyl, isopropyl, sec-butyl, cyclopropyl or cyclobutyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, 4- methoxyphenyl, 4-trifluoromethylphenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methylphenyl, 3,4- dichlorophenyl or 2,3-dichlorophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
In a further preferred embodiment, the invention relates to compounds of formula I according to the invention, in which
R1 is hydrogen, chlorine or bromine,
R2 is methyl,
A is benzyl, R4 is ethyl or isopropyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, n is 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
In a further preferred embodiment, the invention relates to compounds of formula I in which
R1 is chlorine, bromine or fluorine,
R2 is methyl,
A is benzyl,
R4 is ethyl, isopropyl, sec-butyl or cyclopropyl,
R5 and R6 are both hydrogen,
B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methylphenyl or 3,4-dichlorophenyl, n is 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
A special interest in the compounds according to this invention refers to those compounds of formula I which are included -within the scope of this invention- by one or, when possible, by a combination of more of the following special embodiments:
Another special embodiment (embodiment 1 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is hydrogen.
Another special embodiment (embodiment 2) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine.
Another special embodiment (embodiment 3) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is bromine.
Another special embodiment (embodiment 3a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is fluorine. Another special embodiment (embodiment 4) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R2 is methyl.
Another special embodiment (embodiment 5) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine, and R2 is methyl.
Another special embodiment (embodiment 6) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is bromine, and R2 is methyl.
A special embodiment (embodiment 7) of the compounds of formula I according to this invention refers to those compounds of formula I, in which A is benzyl.
Another special embodiment (embodiment 8) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
A is fluorobenzyl, such as e.g. 3-fluorobenzyl or 4-fluorobenzyl.
Another special embodiment (embodiment 9) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
A is benzyl, halobenzyl (such as e.g. chlorobenzyl or bromobenzyl), methylbenzyl, hydroxybenzyl or methoxybenzyl.
Another special embodiment (embodiment 10) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is ethyl.
Another special embodiment (embodiment 1 1 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is isopropyl.
Another special embodiment (embodiment 11 a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine, and R2 is methyl, and R4 is isopropyl. Another special embodiment (embodiment 11 b) of the compounds of formula I according to this invention refers to those compounds of formula I, in which A is benzyl, and
R4 is isopropyl.
Another special embodiment (embodiment 1 1c) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R1 is chlorine, and R2 is methyl, and A is benzyl, and
R4 is isopropyl.
Another special embodiment (embodiment 12) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is propyl.
Another special embodiment (embodiment 13) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is cyclopropyl.
Another special embodiment (embodiment 13a) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is cyclobutyl.
Another special embodiment (embodiment 14) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R4 is sec-butyl.
Another special embodiment (embodiment 15) of the compounds of formula I according to this invention refers to those compounds of formula I, in which n is 2.
Another special embodiment (embodiment 16) of the compounds of formula I according to this invention refers to those compounds of formula I, in which n is 3.
Another special embodiment (embodiment 17) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
R5 and R6 are the same and selected from hydrogen, methyl and ethyl. Another special embodiment (embodiment 18) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
R5 is methyl, ethyl, propyl or isopropyl, and R6 is hydrogen.
Another special embodiment (embodiment 19) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 is methyl or ethyl, and R6 is hydrogen.
Another special embodiment (embodiment 20) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both hydrogen, and n is 2.
Another special embodiment (embodiment 21 ) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both methyl, and n is 2.
Another special embodiment (embodiment 22) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both hydrogen, and n is 3.
Another special embodiment (embodiment 23) of the compounds of formula I according to this invention refers to those compounds of formula I, in which R5 and R6 are both methyl, and n is 3.
Another special embodiment (embodiment 24) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is fluorine, chlorine, bromine, methyl, trifluoromethyl or ethyl,
R71 is hydrogen, fluorine, chlorine or methyl.
Another special embodiment (embodiment 25) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
B is phenyl, halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl, (hydroxymethyl)phenyl, (methoxymethyl)phenyl, methoxyphenyl, ethoxyphenyl, carboxyphenyl, methylphenyl, ethylphenyl, methylenedioxyphenyl, ethylenedioxyphenyl, methoxychlorophenyl, methylhalophenyl or (trifluoromethyl)phenyl.
Another special embodiment (embodiment 26) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is phenyl, methylphenyl, halophenyl (e.g. chlorophenyl or bromophenyl), dihalophenyl (e.g. dichlorophenyl), methylhalophenyl (e.g. methylfluorophenyl), (hydroxymethyl)phenyl, halo(trifluoromethyl)phenyl, methylenedioxyphenyl, (trifluoromethyl)phenyl, methoxyphenyl or cyanophenyl.
Another special embodiment (embodiment 27) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is methylphenyl, chlorophenyl, dichlorophenyl, bromophenyl, fluorophenyl, (trifluoromethyl)phenyl, methoxyphenyl or methylfluorophenyl.
Another special embodiment (embodiment 28) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- chlorophenyl, 4-bromophenyl or 3-fluoro-4-methylphenyl.
Another special embodiment (embodiment 29) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is 4-methylphenyl, 4-chlorophenyl or 4-bromophenyl.
Another special embodiment (embodiment 30) of the compounds of formula I according to this invention refers to those compounds of formula I, in which B is 4-methylphenyl.
Another special embodiment (embodiment 31 ) of the compounds of formula I according to this invention refers to those compounds which are from formula I* as shown below.
Another special embodiment (embodiment 32) of the compounds of formula I according to this invention refers to those compounds which are from formula I** as shown below.
A respective another special embodiment (embodiment 33 to 92) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
R1 is bromine, R2 is methyl, and
A, R4, n, R5 and R6 have the respective meanings indicated in Table 1 given below.
Table 1 :
A respective another special embodiment (embodiment 93 to 152) of the compounds of formula I according to this invention refers to those compounds of formula I, in which
R1 is chlorine, R2 is methyl, and
A, R4, n, R5 and R6 have the respective meanings indicated in Table 2 given below.
Table 2:
It is to be understood that the present invention includes any or all possible combinations and subsets of the special embodiments defined hereinabove.
The compounds of formula I, in which R4 is different from hydrogen, are chiral compounds having a chiral center in position 2'.
Numbering:
The invention includes all conceivable stereoisomers of the compounds of this invention, like e.g. diastereomers and enantiomers, in substantially pure form as well as in any mixing ratio, including the racemates, as well as the salts thereof.
In this connection, compounds of formula I worthy to be mentioned are those which have, with respect to the position 2', the same configuration as shown in formula I*:
In a preferred embodiment, the invention therefore relates to compounds of formula I as described in this application, wherein said compounds have the configuration as shown in formula I*, and the salts thereof.
If, for example, in compounds of formula I* R4 has the meaning methyl, ethyl, propyl, isopropyl, sec- butyl or cyclopropyl, then the configuration - according to the rules of Cahn, lngold and Prelog - is R in the 2' position.
Further on, compounds of the formula I also worthy to be mentioned are those which have, with respect to the position 2', the same configuration as shown in formula I**:
If, for example, in compounds of formula I** R4 has the meaning methyl, ethyl, propyl, isopropyl, sec- butyl or cyclopropyl, then the configuration - according to the rules of Cahn, lngold and Prelog - is S in the position 2'.
In general, enantiomerically pure compounds of this invention may be prepared according to art- known processes, such as e.g. via asymmetric syntheses, for example by preparation and separation of appropriate diastereoisomeric compounds/intermediates, which can be separated by known methods (e.g. by chromatographic separation or (fractional) crystallization from a suitable solvent), or by using chiral synthons or chiral reagents; by chromatographic separation of the corresponding racemate on chiral separating columns; by means of diastereomeric salt formation of the racemic compounds with optically active acids (such as e.g. those mentioned below) or bases, subsequent resolution of the salts and release of the desired compound from the salt; by derivatization of the racemic compounds with chiral auxiliary reagents, subsequent diastereomer separation and removal of the chiral auxiliary group; by resolution via diastereomeric inclusion compounds (e.g. complexes or clathrates); by kinetic resolution of a racemate (e.g. by enzymatic resolution); by enantioselective (preferential) crystallization (or crystallization by entrainment) from a conglomerate of enantiomorphous crystals under suitable conditions; or by (fractional) crystallization from a suitable solvent in the presence of a chiral auxiliary.
Thus, e.g. one possible alternative for enatiomer separation may be carried out at the stage of the compounds of formula I or of the starting compounds having a protonatable group, e.g. a free amino group, such as starting compounds of formula Il as defined later. Hereby, separation of the enantiomers may be carried out, for example, by means of salt formation of the racemic compounds of formula Il with optically active acids, preferably carboxylic acids, subsequent resolution of the salts and release of the desired compound from the salt. Examples of optically active carboxylic acids which may be mentioned in this connection, without being restricted thereto, are the enantiomeric forms of mandelic acid, tartaric acid, O,O'-dibenzoyltartaric acid, camphoric acid, quinic acid, glutamic acid, pyroglutamic acid, malic acid, camphorsulfonic acid, 3-bromocamphorsulfonic acid, α-methoxyphenylacetic acid, α-methoxy-α-trifluoromethylphenylacetic acid or 2-phenylpropionic acid or the like.
Another possible alternative for enantiomer separation may be carried out by chromatographic separation of a racemic mixture of compounds of formula I or of starting compounds thereof on a chiral separating column, such as e.g. described in the following examples or analogously or similarly thereto, using the appropriate separation conditions.
The enantiomers having the formula I* and the salts thereof are part of the invention. The enantiomers having the formula I** and the salts thereof are also part of the invention.
Preference is given in this connection to those compounds of formula I which have with respect to the asymmetric -C(R4)H- atom (position 2') the same absolute configuration as the compound (+)-N-(3- amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-methyl-benzamide hydrochloride having the specific optical rotation [α]D 20 = + 231 ° (c=0.4535, CHCI3), as well as the salts thereof.
The invention therefore relates to compounds of formula I according to the invention, in which R4 is different from hydrogen, and which have with respect to the asymmetric -C(R4)H- atom the same absolute configuration as the compound (+)-N-(3-amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7- oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide hydrochloride having the specific optical rotation [α]D 20= + 231 ° (c=0.4535, CHCI3), as well as the salts thereof.
As exemplary compounds according to this invention the following compounds of formula I*, in which R1 is bromine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
As further exemplary compounds according to this invention the following compounds of formula I*, in which
R1 is chlorine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
As further exemplary compounds according to this invention the following compounds of formula I*, in which
R1 is hydrogen, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
As further exemplary compounds according to this invention the following compounds of formula I**, in which
R1 is bromine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
As further exemplary compounds according to this invention the following compounds of formula I**, in which
R1 is chlorine, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
As further exemplary compounds according to this invention the following compounds of formula I**, in which
R1 is hydrogen, and R2 is methyl, and the salts thereof, may be mentioned by means of the substituent meanings for A, R4, n, R5, R6 and B in the Table 3 given below.
The compounds according to the invention can be prepared e.g. as described as follows and according to the following specified reaction steps, or, particularly, in a manner as described by way of example in the following examples, or analogously or similarly thereto according to preparation procedures or synthesis strategies known to the person skilled in the art.
As shown in the synthesis route outlined in scheme 1 below, the trione compound of formula IX, in which R2 has the meanings given above (particularly, R2 is methyl), is condensed and cyclized with semicarbazide (H2N-C(O)-NHNH2ΗCI) to give the corresponding pyrazolopyrimidone compounds of formula VIII. Said cyclocondensation reaction can be carried out as it is habitual for the skilled person or as described in the following examples or analogously or similarly thereto, in the presence of a suitable base (e.g. sodium carbonate) in a suitable solvent, for example water, at elevated temperature.
Compounds of formula IX, in which R2 has the meanings given above, particularly methyl, are known or can be prepared according to known procedures or as described in the following examples, or analogously or similarly thereto. Thus, e.g. compounds of formula IX can be obtained from corresponding compounds of formula X by alkaline hydrolysis
Compounds of formula X are known or can be obtained according to known procedures.
Compounds of formula VIII are benzylated with compounds of formula A-X1 , in which A has the meanings given above and X1 is a suitable leaving group (e.g. halogen or the like), in a standard manner or as described exemplarily in the following examples to give corresponding compounds of formula VII.
Compounds of formula VII, in which R2 and A have the meanings given above, are halogenated using a suitable halogenating reagent under appropriate conditions to obtain corresponding compounds of formula Vl, in which R2 and A have the meanings given above and R1 is halogen, in particular fluorine or, in more particular, chlorine or bromine. Said halogenation reaction can be carried out according to customary procedures or as described in the following examples, or analogously or similarly thereto.
Thus, chlorination or bromination reaction can be carried out using an appropriate electrophilic chlorinating or brominating reagent, e.g. N-chlorosuccinimide (NCS) or N-bromosuccinimide (e.g. NBS / AIBN), respectively, under conditions customary for the skilled person or as described in the following examples. Reaction scheme 1 :
n
oxidation
(V) (IV)
1. B-C(O)-X2, benzoylation
2. When R1 = H, optionally halogenation (e.g. bromination)
3. If necessary, removal of temporary ' protecting groups
(I) Fluorination reaction may be carried out using a suitable electrophilic fluorinating reagent, such as e.g. 1-chloromethyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) (Selectfluor I) or 1- methyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) (Selectfluor II) or the like, under appropriate conditions.
Using appropriate conditions, said halogenation reaction is carried out largely regioselectively to obtain halogenation on the pyrazole moiety of the pyrazolopyrimidone scaffold, i.e. the 3-position of the pyrazolopyrimidone scaffold.
The methyl group attached to the pyrimidone moiety of the pyrazolopyrimidone of compounds of formula Vl, in which R1 is halogen and A and R2 have the meanings given above, is oxidized under suitable conditions to obtain corresponding compounds of formula V. Said oxidation is carried out in a manner habitual for the skilled person or as described in the following examples using a suitable oxidation reagent, e.g. selenium dioxide, to give largely regioselectively the 5-formyl derivative of formula V.
Compounds of formula V, in which R1 is halogen and A and R2 have the meanings given above, are reacted in a nucleophilic addition reaction with compounds of formula R4-M, in which R4 is different from hydrogen and has the meanings given above and M is a suitable metal or metal complex, to give corresponding compounds of formula IV. Said nucleophilic addition reaction can be obtained using organomagnesium halides (Grignard reagents) of formula R4-Mg-X (X = Halogen) with or without further additives (e.g. LiCI or alkyl lithium reagents). Advantageously said reaction is carried out in the presence of alkyl lithium reagents, e.g. methyl lithium, forming in situ more reactive organomagnesium reagents, e.g. lithium trialkyl magnesiates such as R4Me2Mgl_i from 2 equiv. LiMe and 1 equiv. R4-Mg-X. The nucleophilic addition reaction can be carried out in a manner customary for the skilled person or as described in the following examples.
When compounds of formula V, in which R1 is bromine, are reacted with Grignard reagents in said nucleophilic addition reaction, debromination of the 3-bromo substituent can be obtained depending on the reaction conditions to give corresponding compounds of formula IV, in which R1 is hydrogen. Said debromination is performed as described in the following examples.
Alcohols of formula IV, in which R1 is hydrogen, fluorine or chlorine and A, R2 and R4 have the meanings given above, are converted into the corresponding ketones of formula III by oxidation reaction using an appropriate oxidation reagent, such as e.g. NMO (N-methylmorpholine-N-oxide) / TPAP (tetrapropylammonium perruthenate) or the like, under conditions known for the skilled person or as described in the following examples. Compounds of formula III, in which R1 is hydrogen, fluorine or chlorine and A, R2 and R4 have the meanings given above, are subjected to a reductive amination reaction with amines of formula H2N- (CH2)n-N(R5)R6, in which n has the meanings indicated above and R5 and R6 stand for a suitable temporary protective group PG 1 or for the meanings given above with the exception that R5 and R6 are not hydrogen, to afford corresponding compounds of formula Il in a manner which is habitual for the skilled person or which is described in the following examples. In more detail, in a first step of the reductive amination reaction the reaction partners are reacted in the presence of a suitable Lewis acid, e.g. a suitable titanium reagent like titanium(IV)isopropoxide, to give the corresponding imine compounds, which are reduced in a second step, such as with the aid of a suitable reducing reagent, e.g. a suitable hydride like a borohydride (e.g. NaCNBH3, NaBH4 or Na(OAc)3BH) or the like, Raney- Ni or in the presence of hydrogen/transition metal catalyst (e.g. H2/Pd).
PG1 stands for a suitable temporary protective group for the amino group, for example tert- butoxycarbonyl, benzyloxycarbonyl or 4-methoxy-benzyloxycarbonyl or one of those art-known protective groups mentioned in "Protective Groups in Organic Synthesis" by T. Greene and P. Wuts (John Wiley & Sons, Inc. 1999, 3rd Ed.) or in "Protecting Groups (Thieme Foundations Organic Chemistry Series N Group" by P. Kocienski (Thieme Medical Publishers, 2000).
Compounds of formula II, in which R1 is hydrogen, fluorine or chlorine and A, n, R2 and R4 have the meanings given above and R5 and R6 stand for a suitable temporary protective group PG1 as defined above or for the meanings given above with the exception that R5 and R6 are not hydrogen, are benzoylated with compounds of formula B-C(O)-X2, in which B has the meanings given above and X2 is a suitable leaving group (e.g. chlorine), to give corresponding compounds of formula I.
Compounds of formula I, in which R1 is hydrogen and A, n, B, R2 and R4 have the meanings given above and R5 and R6 stand for a suitable temporary protective group PG1 as defined above or for the meanings given above with the exception that R5 and R6 are not hydrogen, can be halogenated, particularly brominated, using a suitable halogenating reagent (for bromination e.g. NBS) under appropriate conditions to obtain corresponding further compounds of formula I, in which R1 is halogen. Said halogenation reaction can be carried out according to customary procedures, such as those mentioned above, or as described in the following examples, or analogously or similarly thereto.
Compounds of formula I, in which R1 , A, n, B, R2 and R4 have the meanings given above and R5 and R6 stand for a suitable temporary protective group PG1 as defined above, are deprotected by removal of said protecting group PG1 in a manner known to the person skilled in the art or as described in the following examples to give corresponding deprotected compounds of formula I.
Compounds of formula I, in which R5 and R6 are 1-4C-alkyl, e.g. methyl, and in which R1 , A, n, B, R2 and R4 have the meanings given above, can be obtained by the methods described above, starting from compounds of formula I, in which R5 and R6 are 1-4C-alkyl, e.g. methyl, and in which R1 , A, n, B, R2 and R4 have the meanings given above. Alternatively, compounds of formula I, in which R5 and R6 are 1-4C-alkyl, e.g. methyl, and in which R1 , A, n, B, R2 and R4 have the meanings given above, can be obtained by alkylation, e.g. methylation, of compounds of formula I, in which R5 and R6 are hydrogen. Alternatively, these compounds of formula I, in which R5 and R6 are 1-4C-alkyl, e.g. methyl, can be obtained by any other method that is known to the person skilled in the art.
The invention therefore relates to a process for preparing a compound as described in this application, comprising at least one of the steps
(i) benzoylation of a compound of formula II, with the meanings of R1 , R2, A, R4, R5 and R6 as indicated for the compounds according to the invention, or with at least one of R5 and R6 being part of a protective group,
(H) (I)
(ii) halogenation of a compound of formula I wherein R1 = H to give a compound of formula I wherein R1 = halogen, or
(I) R1 = H (I) R1 = halogen
(iii) optionally the removal of protecting groups represented by at least one of R5 and R6 as indicated under (i).
Optionally, compounds of the formula I can be converted into their salts, or, optionally, salts of the compounds of the formula I can be converted into the free compounds. Corresponding processes are customary for the skilled person.
When one of the final steps or purification is carried out under the presence of an inorganic or organic acid (e.g. hydrochloric, trifluoroacetic, acetic or formic acid or the like), the compounds of formula I may be obtained - depending on their individual chemical nature and the individual nature of the acid used - as free base or containing said acid in an stoechiometric or non-stoechiometric quantity. The amount of the acid contained can be determined according to art-known procedures, e.g. by titration. It is moreover known to the person skilled in the art that if there are a number of reactive centers on a starting or intermediate compound it may be necessary to block one or more reactive centers temporarily by protective groups in order to allow a reaction to proceed specifically at the desired reaction center. A detailed description for the use of a large number of proven protective groups is found, for example, in "Protective Groups in Organic Synthesis" by T. Greene and P. Wuts (John Wiley & Sons, Inc. 1999, 3rd Ed.) or in "Protecting Groups (Thieme Foundations Organic Chemistry Series N Group" by P. Kocienski (Thieme Medical Publishers, 2000).
The substances according to the invention are isolated and purified in a manner known per se, for example by distilling off the solvent under reduced pressure and recrystallizing the residue obtained from a suitable solvent or subjecting it to one of the customary purification methods, such as, for example, column chromatography on a suitable support material.
Salts can be obtained by dissolving the free compound in a suitable solvent (e.g. a ketone, such as acetone, methyl ethyl ketone or methyl isobutyl ketone, an ether, such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon, such as methylene chloride or chloroform, or a low-molecular-weight aliphatic alcohol, such as methanol, ethanol or isopropanol) which contains the desired acid or base, or to which the desired acid or base is then added. The salts can be obtained by filtering, reprecipitating, precipitating with a nonsolvent for the addition salt or by evaporating the solvent. Salts obtained can be converted into the free compounds, which can in turn be converted into salts, by alkalization or by acidification. In this manner, pharmacologically and/or pharmaceutically unacceptable salts can be converted into pharmacologically and/or pharmaceutically acceptable salts.
Suitably, the conversions mentioned in this invention can be carried out analogously or similarly to methods which are familiar per se to the person skilled in the art.
The person skilled in the art may know on the basis of his/her knowledge and on the basis of those synthesis routes, which are shown and described within the description of this invention, how to find other possible synthesis routes for compounds according to this invention. All these other possible synthesis routes are also part of this invention.
The present invention also relates to intermediates (including their salts, stereosiomers as well as salts of these stereoisomers), methods and processes useful in synthesizing compounds according to this invention.
Having described the invention in detail, the scope of the present invention is not limited only to those described characteristics or embodiments. As will be apparent to persons skilled in the art, modifications, analogies, variations, derivatizations, homologisations and adaptations to the described invention can be made on the base of art-known knowledge and/or, particularly, on the base of the disclosure (e.g. the explicite, implicite or inherent disclosure) of the present invention without departing from the spirit and scope of this invention as defined by the appended claims.
The following examples illustrate the invention in greater detail without restricting it. Likewise, further compounds according to this invention, of which the preparation is not explicitly described, can be prepared in an analogous or similar manner or in a manner familiar per se to the person skilled in the art using customary process techniques.
Any or all of the compounds of formula I according to the present invention which are mentioned as final compounds in the following examples, as well as the salts, stereoisomers and salts of the stereoisomers thereof, are a preferred subject of the present invention.
In the examples, m.p. stands for melting point, h for hour(s), min for minutes, cone, for concentrated, calc. for calculated, fnd. for found, EF for elemental formula, MS for mass spectrometry, M for molecular ion in mass spectrometry, TFA for trifluoroacetic acid, and other abbreviations have their meanings customary per se to the skilled person.
Further on, according to common practice in stereochemistry, the term "(RS)" characterizes a racemate comprising the one enantiomer having the configuration R and the other enantiomer having the configuration S; each of these enantiomers and their salts in pure form as well as their mixtures including the racemic mixtures is part of this invention.
Examples
Final compounds
1. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide hydrochloride
150 mg {3-[[1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(1-p-tolyl- carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (0.26 mmol) are solved in 1 ml diethylether. 10 ml HCI in diethylether (ca. 4 N) are added and the suspension is stirred 1 h at ambient temperature. Afterwards the suspension is evaporated, the colorless solid is washed twice with diethylether and dried in vacuo. By this method 120 mg (91%) of a colorless solid are obtained. M. p.: 235-250 0C (decomposition). MS: m/z (MH+) = 472.1.
2. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide hydrochloride
150 mg {3-[[1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(1- p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert butyl ester (0.26 mmol) are solved in 1 ml diethylether, 4 ml HCI in diethylether (ca. 4N) are added and the mixture is stirred 1 h at ambient temperature. Afterwards the suspension is evaporated, the colorless solid is washed twice with diethylether and dried in vacuo. By this method 135 mg (88%) of a colorless solid are obtained. M. p.: > 190 0C (decomposition). MS: m/z (MH+) = 550.0, 552.0.
3. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-3-methyl-benzamide; isolated as trifluoroacetate salt
0.144 g (1.0 eq) {S-tti^e-Benzyl-S-chloro^-methyl^-oxo-e^-dihydro-pyrazoloti .δ-clpyrimidin-δ-yl)^- methyl-propyl]-(1-m-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester is dissolved in 8 ml_ dichloromethane and treated with 2 ml_ trifluoro acetic acid. After stirring 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and yields after drying 0.108 g (73%) of the title compound as a trifluoroacetate. M.p. 162 0C. MS: m/z (MH+) = 520.1 , 522.1. 4. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-fluoro-benzamide; isolated as trifluoroacetate salt
0.090 g (1.0 eq) (3-{[1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(4-fluoro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester is dissolved in 6 ml_ dichloromethane and treated with 1.5 ml_ trifluoro acetic acid. After stirring 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and yields after drying 0.068 g (75%) of the title compound as a trifluoroacetate. M.p. 182 0C. MS: m/z (MH+) = 524.1 , 526.1.
5. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methoxy-benzamide; isolated as trifluoroacetate salt
0.014 g (1.0 eq) (S-Ki^e-Benzyl-S-chloro^-methyl^-oxo-e^-dihydro-pyrazoloti .δ-clpyrimidin-δ-yl)^- methyl-propyl]-[1-(4-methoxy-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester is dissolved in 0.8 ml_ dichloromethane and treated with 0.2 ml_ trifluoro acetic acid. After stirring 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and yields after drying 0.007 g (48%) of the title compound as a trifluoroacetate. M.p. 149 0C. MS: m/z (MH+) = 536.1 , 538.1.
6. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-bromo-benzamide; isolated as trifluoroacetate salt
0.153 g (1.0 eq) (S-Ki^e-Benzyl-S-chloro^-methyl^-oxo-e^-dihydro-pyrazoloti .δ-clpyrimidin-δ-yl)^- methyl-propyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester is dissolved in 10 ml_ dichloromethane and treated with 2.5 ml_ trifluoro acetic acid. After stirring 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and yields after drying 0.086 g (δδ%) of the title compound as a trifluoroacetate. M.p. 204-20δ 0C (decomposition). MS: m/z (MH+) = δ84.0, δ8δ.9, δ87.9. 7. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-chloro-benzamide; isolated as trifluoroacetate salt
0.130 g (1.0 eq) (3-{[1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(4-chloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester is dissolved in 8 ml_ dichloromethane and treated with 2 ml_ trifluoro acetic acid. After stirring 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and yields after drying 0.075 g (56%) of the title compound as a trifluoroacetate. M. p. 199-201 0C (decomposition). MS: m/z (MH+) = 540.0, 542.0, 544.0.
8. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride
0.129 g (1.0 eq) {S-tti^e-Benzyl-S-chloro^-methyl^-oxo-e^-dihydro-pyrazoloti .δ-clpyrimidin-δ-yl)^- methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A8) is dissolved in 20 ml_ of a 1 M solution of hydrochloric acid in diethylether at ambient temperature. After stirring for 16 hours the precipitate is filtered off and washed with diethylether. This yields after drying
0.083 g (71%) of the title compound as a hydrochloride salt.
M. p. 183 0C.
MS: m/z (MH+) = 520.0, 522.1.
Separation of enantiomers:
8a. (+)-N-(3-Amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride (assumed (R)-isomer) and
8b. (-J-N-β-Amino-propylJ-N-ti-fθ-benzyl-S-chloro^-methyl^-oxo-θ^-dihydro-pyrazolotijS- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride (assumed (S)-isomer)
1.79 g of racemic N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride are separated into its enantiomers by preparative HPLC (column: CHIRALPAK AD 10 μm, 250 x 20 mm; mobile phase: n-Heptane / ethanol / diethylamine 80:20:0.1 (v/v/v); flow rate: 30 ml/min; detection: UV 230 nm; temperature: 25 0C).
The enantiomers elute at 7.49 min and 13.95 min. Both isomers are isolated by solving in dichloromethane and treatment with 4M solution of HCI in dioxane. After stirring for 6 hours at ambient temperature, the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. The HCI-salt of the first eluting enantiomer (τ = 7.49 min, > 99.5 % ee, 94 % purity, 556 mg, example
8a) has an optical rotation of [α]D 20 = +225 ° (c = 1.02, dichloromethane), a melting point of 203-263
0C (decomposition) and MS: m/z (MH+) = 519.8, 521.9.
The HCI-salt of the second eluting enantiomer (τ = 13.95 min, > 98 % ee, > 99 % purity, 870 mg, example 8b) has an optical rotation of [α]D 20 = -205 ° (c = 0.725, dichloromethane), a melting point of
206-233 0C (decomposition) and MS: m/z (MH+) = 520.1 , 522.0.
The absolute stereochemistry of (+)-N-(3-Amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7- dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride (8a) is tentatively assigned to the enantiomer with the R-configu ration (+)-N-(3-Amino-propyl)-N-[(R)-1-(6- benzyl-S-chloro^-methyl^-oxo-εy-dihydro-pyrazoloIi .δ-clpyrimidin-δ-yl^-methyl-propyll^-methyl- benzamide hydrochloride. Consequently, the S-configu ration is assigned to the (-)-enantiomer.
Furthermore, a variety of salts of compound 8, 8a and 8b is prepared:
8c. N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide phosphate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) are suspended in 1 ml_ ethanol and heated at reflux until the amine is completely dissolved. 13.1 μl_ phosphoric acid (85% in water, 1.0 eq) are added and the mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded (after 30 min ultrasonification) in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 63 mg (53%) of the title compound. There are slight impurities of diethylether and diethylammonium phosphate.
M. p.: foaming at 123 0C, melting 160-205 0C (decomposition). MS: m/z (MH+) = 519.9, 522.1.
8d. N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide semi fumarate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) and 22.3 mg of fumaric acid (1.0 eq) are suspended in 1 ml_ ethanol and heated at reflux for 2 minutes. The mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 61 mg (55%) of the title compound. There are slight impurities of diethylether and diethylammonium fumarate. The semi fumarate is elucidated by NMR spectroscopy. M. p.: sinter from 140 0C on, melting 195-215 0C (decomposition). MS: m/z (MH+) = 519.9, 522.1. 8e. N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide sulfate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) are suspended in 1 ml_ ethanol and heated at reflux until the amine is completely dissolved. 10.2 μl_ sulfuric acid (1.0 eq) are added and the mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry (after 15 min ultrasonification) that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 73 mg (62%) of the title compound. There are slight impurities of diethylether and diethylammonium sulfate.
M. p.: foaming at 130 0C, melting 198-209 0C (decomposition). MS: m/z (MH+) = 519.9, 522.1.
8f. N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide sesqui citrate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) and 37 mg of citric acid (1.0 eq) are suspended in 1 ml_ ethanol and heated at reflux for 2 minutes. The mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded (after 15 min ultrasonification) in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 85 mg (55%) of the title compound. There are slight impurities of diethylether and diethylammonium citrate. The sesqui citrate is elucidated by NMR spectroscopy.
M. p.: sinter from 110 0C on, melting 160-191 0C (decomposition). MS: m/z (MH+) = 520.0, 522.0.
8g. N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (D)-tartrate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) and 29 mg of D-tartaric acid (1.0 eq) are suspended in 1 ml_ ethanol and heated at reflux for 2 minutes. The mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded (after 20 min ultrasonification) in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 98 mg (76%) of the title compound. There are slight impurities of diethylether and diethylammonium tartrate. The mono tartrate is elucidated by NMR spectroscopy.
M. p.: foaming at 100 0C, melting 162-176 0C (decomposition). MS: m/z (MH+) = 519.9, 522.0. 8h. N^S-Amino-propyO-N-^SJ-i^θ-benzyl-S-chloro^-methyl-Z-oxo-ΘJ-dihydro-pyrazoloti.S- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide mesylate
100 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) are suspended in 1 ml_ ethanol and heated at reflux until the amine is completely dissolved. 12.5 μl_ methane sulfonic acid (1.0 eq) are added and the mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry (after 15 min ultrasonification) that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 63 mg (53%) of the title compound. There are slight impurities of diethylether and diethylammonium mesylate. The mono mesylate is elucidated by NMR spectroscopy. M. p.: melting 150-214 0C (decomposition). MS: m/z (MH+) = 520.0, 522.0.
8i. N^S-Amino-propyO-N-^SJ-i^e-benzyl-S-chloro^-methyl-Z-oxo-ej-dihydro-pyrazoloti.S- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide benzoate
50 mg of (-)-N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8b) are suspended in 1 ml_ ethanol and heated at reflux until the amine is completely dissolved. 11.8 mg benzoic acid (1.0 eq) are added and the mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry (after 10 min ultrasonification) that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 29 mg (47%) of the title compound. There are slight impurities of diethylether and diethylammonium benzoate. The mono benzoate is elucidated by NMR spectroscopy. M. p.: sinter from 145 0C, melting 160-175 0C (decomposition). MS: m/z (MH+) = 520.0, 522.0.
8j. N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide mesylate
60 mg of (-)-N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8a) are suspended in 1 ml_ ethanol and heated at reflux until the amine is completely dissolved. 7.5 μl_ methane sulfonic acid (1.0 eq) are added and the mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry (after 10 min ultrasonification) that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 63 mg (89%) of the title compound. The mono mesylate is elucidated by NMR spectroscopy. M. p.: foaming at 125 0C, melting 190-198 0C (decomposition). MS: m/z (MH+) = 519.9, 521.9. 8k. N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide fumarate
59 mg of (-)-N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8a) and 13.2 mg of fumaric acid (1.0 eq) are suspended in 1 ml_ ethanol and heated at reflux for 2 minutes. The mixture is stirred for 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 73 mg (quant.) of the title compound. The mono fumarate is elucidated by NMR spectroscopy.
M. p.: sinter from 165 0C on, melting 190-200 0C (decomposition). MS: m/z (MH+) = 520.0, 522.0.
8I. N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide tosylate
58 mg of (-)-N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (example 8a) and 21.2 mg of p-toluenesulfonic acid monohydrate (1.0 eq) are suspended in 1 ml_ ethanol and heated at reflux for 2 minutes. The mixture is stirred for another 5 minutes without heating anymore. Evaporation of the solvent and subsequent addition of diethylether yielded in a white slurry that is filtered and washed with several portions of diethylether. The colorless solid is dried in vacuo to yield 65 mg (85%) of the title compound. The mono tosylate is elucidated by NMR spectroscopy. M. p.: sinter from 160 0C on, melting 170-175 0C (decomposition). MS: m/z (MH+) = 519.9, 522.0.
9. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide hydrochloride
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester (5.00 g, 8.2 mmol) is treated with HCI/diethylether (4N, 100 ml_), stirred for 1 h at ambient temperature, the solvents are evaporated and the residue is washed with diethylether. 3.90 g (87 %) of the title compuond are obtained as colorless solid with m.p. 201-203 0C. MS: m/z (MH+) = 506.0, 508.1.
Separation of enantiomers: N-(3-Amino-propyl)-N-[(R)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-propyl]-4-methyl-benzamide hydrochloride and
N-(3-Amino-propyl)-N-[(S)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-propyl]-4-methyl-benzamide hydrochloride
The separation of the enantiomers of racemic N-(3-amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2- methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide hydrochloride is performed by preparative chromatography: 250 x 20 mm CHIRALPAK AD-H 5μM, 0.7 ml/min of ethanol + 0.1% diethylamine at 40 0C:
The enantiomers elute at 6.59 and 15.38 min. Both enantiomers are isolated by mixing with 1 N HCI in diethylether and stirring in this mixture. The mixture is evaporated and the residue is dissolved in a mixture of water and acetonitrile and lyophilized.
The first eluting enantiomer (6.59 min, example 9a) has a MH+ of 506.1 (ee = 97.9%) and at Na
589nm at 2O0C in CHCI3 (0.4535 g/100ml) [α]D 20 = + 226 °. Thus, enantiomerically pure (+)-N-(3-
Amino-propyl)-N-[1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-methyl-benzamide hydrochloride has [α]D 20 = + 231 ° (c=0.4535, CHCI3).
The second eluting enantiomer (15.38 min, example 9b) has a MH+ of 506.0 (ee = 99.7%).
The absolute stereochemistry of the (+)-isomer (9a) is tentatively assigned to the enantiomer with the
R-config u ration. Consequently, the S-configu ration is assigned to the (-)-enantiomer (9b).
10. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-trifluoromethyl-benzamide trifluoroacetate
81 mg of (3-{[(RS)-1-6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(4-trifluoromethyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A10) are dissolved in 4 ml_ dichloromethane and 1.5 ml_ trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 64 mg (78%) of the title compound are obtained as colorless solid. M.p.: 218 °C. MS: m/z (MH+) = 574.0, 575.9.
Using similar procedures to those described herein above but with suitable choice of the starting materials, which are mentioned below, the following compounds of formula I (especially in form of their hydrochloride salts) may be prepared:
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-fluoro-benzamide N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- propyl]-4-trifluoromethyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-fluoro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-trifluoromethyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-fluoro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-propyl]-4-trifluoromethyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-fluoro-benzamide N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-4-trifluoromethyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-fluoro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-propyl]-4-trifluoromethyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-3-methyl-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-fluoro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-methoxy-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-bromo-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-chloro-benzamide
N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-
5-yl)-2-methyl-butyl]-4-trifluoromethyl-benzamide 11. N-[(RS)-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)- methyl-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide
A solution of 89 mg (1.0 eq) 6-Benzyl-3-chloro-5-[(RS)-1-(3-dimethylamino-propylamino)-2-methyl- propyl]-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (compound A11 ), 87 μL (3.0 eq) triethylamine and 33 μL (1.2 eq) 4-methylbenzoyl chloride in 2 ml_ dichloromethane is stirred for 16 hours at ambient temperature. The product is extracted between dichloromethane and a saturated NaHCO3-solution, the organic phase is dried over magnesium sulfate and the crude product is purified by preparative HPLC. 20 mg (17%) of the title compound is isolated by means of lyophilization. M. p.: sinter at 71-72 0C and melting at 88-91 0C. MS: m/z (MH+) = 548.1 , 550.2.
Using similar procedures to those described for Example 1 1 but with suitable choice of the starting materials, which are mentioned below, and the appropriate benzoic acid derivatives the following compounds of formula I may be prepared:
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-3-methyl-benzamide
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-4-fluoro-benzamide
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-4-methoxy-benzamide
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-4-bromo-benzamide
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-4-chloro-benzamide
N-[(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl-propyl]-N-(3- dimethylamino-propyl)-4-trifluoromethyl-benzamide
12. N^-Amino-butyO-N-^RSJ-i^θ-benzyl-S-chloro^-methyl^-oxo-ΘJ-dihydro-pyrazoloti.S- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide trifluoroacetate
126 mg of {4-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-butyl}-carbamic acid tert-butyl ester (compound A12) are dissolved in 4 mL dichloromethane and 1 mL trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 85 mg (66%) of the title compound are obtained as colorless foam. M. p.: 179-182 0C. MS: m/z (MH+) = 534.1 , 536.1. 13. N-(2-Amino-ethyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide
164 mg of {2-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-ethyl}-carbamic acid tert-butyl ester (compound A13) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 1 hour at ambient temperature the solvent is removed. Addition of dichloromethane and basification with saturated aqueous NaHCO3-solution followed by extraction of the aqueous phase with dichloromethane results after drying over magnesium sulfate and evaporation of the solvent in 145 mg (quant.) of the title compound. M. p.: 1 19-12O 0C. MS: m/z (MH+) = 506.1 , 507.9.
14. N-(2-Amino-ethyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide trifluoroacetate
125 mg of {2-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-ethyl}-carbamic acid tert-butyl ester (compound A13) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 70 mg (55%) of the title compound are obtained as colorless solid. M. p.: 173-174 0C. MS: m/z (MH+) = 506.0, 508.0.
15. N-^RSJ^Θ-Benzyl-S-chloro^-methyl^-oxo-ΘJ-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- methyl-propyl]-N-(2-dimethylamino-ethyl)-4-methyl-benzamide
A mixture of 70 mg N-(2-Amino-ethyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide (compound 13), 33 μl_ formalin and 52 μl_ formic acid is heated for 8 hours at 100 0C. After cooling, the crude product is purified by silica gel flash chromatography using a gradient of dichloromethane and methanol from 100:0 to
90:10. This yielded 35 mg (46%) of the title compound as colorless solid.
M. p.: 94-95 0C.
MS: m/z (MH+) = 534.0, 536.0.
16. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo [i^-cjpyrimidin-S-yO-i-cyclobutyl-methylH-methyl-benzamide trifluoroacetate
159 mg of {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-1- cyclobutyl-methyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A14) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 1 hour at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 140 mg (87%) of the title compound are obtained as colorless solid. M. p.: sinter at 158-179 0C and melting at 180-185 0C. MS: m/z (MH+) = 532.0, 534.1.
17. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-butyl]-4-methyl-benzamide trifluoroacetate
60 mg of {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-butyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A15) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 1.5 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 43 mg (70%) of the title compound are obtained as colorless solid. M. p.: 179-182 0C. MS: m/z (MH+) = 534.0, 535.8.
18. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazoloti^-cjpyrimidin-S-ylJ-i-cyclopropyl-methylj^-methyl-benzamide hydrochloride
80 mg of {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-1- cyclopropyl-methyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A16) dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. The residue is dissolved in dichloromethane and treated with saturated aqueous NaHCO3-solution. Extraction of the aqueous layer followed by drying over MgSO4 results in the free base of the title compound. The solvent is evaporated and the residue is treated with 1 M HCI in diethylether. The precipitate is filtered, washed with diethylether and dried in vacuo. This afforded 18 mg (26%) of the title compound as colorless solid. M. p.: 156-158 0C. MS: m/z (MH+) = 518.0, 520.1.
19. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazoloti.S-clpyrimidin-S-yO-i-cyclopropyl-methyll^-bromo-benzamide trifluoroacetate
34 mg of (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-1- cyclopropyl-methyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A17) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 21 mg (60%) of the title compound are obtained as colorless solid. M. p.: 178-180 0C (decomposition). MS: m/z (MH+) = 581.9, and also MH++2 and MH++4. 20. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide trifluoroacetate
104 mg of (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(3-fluoro-4-methyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A18) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 1.5 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 61 mg (57%) of the title compound are obtained as pale yellow solid. M. p.: 140-158 0C (decomposition). MS: m/z (MH+) = 537.9, 540.0.
21. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-2-fluoro-4-methyl-benzamide trifluoroacetate
83 mg of (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(2-fluoro-4-methyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A19) are dissolved in 4 ml_ dichloromethane and 1 ml_ trifluoroacetic acid. After stirring this mixture for 2 hours at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 39 mg (46%) of the title compound are obtained as colorless powder. M. p.: sinter 178-181 0C and melting at 182-192 0C. MS: m/z (MH+) = 538.0, 539.9.
22. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-3,4-dichloro-benzamide trifluoroacetate
88 mg of (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(3,4-dichloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A20) are dissolved in 2 ml_ dichloromethane and 0.5 ml_ trifluoroacetic acid. After stirring this mixture for 1 hour at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 37 mg (41 %) of the title compound are obtained as colorless solid. M. p.: 184-185 0C. MS: m/z (MH+) = 573.8, 575.9, 577.8.
23. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-2,3-dichloro-benzamide trifluoroacetate
67 mg of (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-[1-(2,3-dichloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound A21 ) are dissolved in 2 ml_ dichloromethane and 0.5 ml_ trifluoroacetic acid. After stirring this mixture for 1 hour at ambient temperature the solvent is removed. Addition of diethylether results in a white slurry that is filtered. The precipitate is washed with diethylether and dried in vacuo. By this method 31 mg (46%) of the title compound are obtained as colorless solid.
M. p.: 187-188 0C.
MS: m/z (MH+) = 573.9, 576.0, 577.8.
24. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride
87 mg of {3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A22) are dissolved in 4 M HCI in dioxane. After stirring this mixture for 3 hour at ambient temperature the solvent is removed. By this method 78 mg (quant.) of the title compound are obtained as colorless solid.
MS: m/z (MH+) = 563.9, 566.0.
25. N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide hydrochloride
155 mg of {3-[[(RS)-1-(6-Benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (compound A24) are dissolved in 2 ml_ dioxane and treated with 4 M HCI in dioxane (3 ml_). After stirring this mixture for 3 hour at ambient temperature the solvent is removed. By this method 80 mg (57%) of the title compound are obtained as colorless solid. MS: m/z (MH+) = 504.0.
Starting materials:
Some of the compounds in this section have an optionally substituted benzoyl group, which may be designated in the systematic name as phenyl-carbonyl group or alternatively, as a phenyl-methanoyl group. A special substituted phenyl group in this connection is the m-tolyl group (3-methyl-phenyl) or the p-tolyl group (4-methyl-phenyl).
A1. {S-t^RSJ-i^θ-Benzyl^-methyl-Z-oxo-ΘJ-dihydro-pyrazoloti.S-clpyrimidin-S-yO-propyl]- (4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
393 mg {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propylamino]- propyl}-carbamic acid tert-butyl ester (0.87 mmol) (compound B1 ) are solved in 5 ml dichlormethane, afterwards 0.37 ml triethylamine (2.61 mmol) and 161 mg 4-Methyl-benzoyl chloride (1.04 mmol) are added and the reaction mixture is stirred 4 h at ambient temperature. The product is extracted between CH2CI2 and saturated NaHCO3-solution, the organic phase is dried over sodium sulphate and the crude product is purified by silica gel flash chromatography. By this method 377 mg (77%) of a colorless solid are obtained.
A2. {3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
220 mg {3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(1-p- tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (0.38 mmol) (compound A1 ) are solved in 2ml dichloromethane. 70 mg N-Bromo-succinimide (0.39 mmol) are added and the reaction mixture is stirred 4 h at ambient temperature. The product is extracted between CH2CI2 and saturated NaHCO3- solution. The organic layer is dried over sodium sulfate. The solvents are evaporated and the crude product is purified by silica gel flash chromatography. By this method 170 mg ( 69 %) of a colorless solid are obtained. M. p.: 95-97 0C.
A3. {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-(1-m-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
0.300 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.095 ml_ (1.2 eq) m- toluoyl chloride and 0.248 ml_ (3.0 eq) triethylamine are dissolved in 3 ml_ dichloromethane and stirred for 48 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using n-hexane and ethyl acetate in a mixture of 3:2 povides a mixture of starting material and the title compound. A second silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yields 0.151 g (41%) of the title compound as a white solid.
Furthermore 0.105 g (35%) of the starting material is recovered.
M. p. 83.9 0C.
MS: m/z (MH+) = 619.9, 621.9.
A4- (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(4-fluoro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
0.300 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.085 ml_ (1.2 eq) 4- fluorobenzoyl chloride and 0.248 ml_ (3.0 eq) triethylamine are dissolved in 3 ml_ dichloromethane and stirred for 48 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using gradient of n-hexane, ethyl acetate and acetic acid from 59:39:2 to 0:100:0 provides the title compound with some impurities and the pure starting material (0.190 g, 63%). A second silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yields 0.096 g (26%) of the title compound as a white solid. M. p. 143.0 0C (foam). MS: m/z (MH+) = 623.9, 625.9.
A5. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(4-methoxy-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
0.300 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.122 g (1.2 eq) p- anisoyl chloride and 0.248 ml_ (3.0 eq) triethylamine are dissolved in 3 ml_ dichloromethane and stirred for 48 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using of n-hexane, ethyl acetate and acetic acid in a mixture of 39:59:2 yields 0.018 g (5%) of the title compound as a white solid. M. p. 1 15.7 0C (decomposition). MS: m/z (MH+) = 635.9, 637.9.
A6. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
0.300 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.158 mg (1.2 eq) p- bromobenzoyl chloride and 0.248 ml_ (3.0 eq) triethylamine are dissolved in 3 ml_ dichloromethane and stirred for 48 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using n-hexane and ethyl acetate in a mixture of 3:2 povides a mixture of starting material and the title compound. A second silica gel flash chromatography using a gradient of n- hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yields 0.159 g (39%) of the title compound as a white solid. Furthermore 0.18O g (60%) of the starting material are recovered. M. p. 96 0C (foam). MS: m/z (MH+) = 683.8, 685.8, 687.8.
A7- (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(4-chloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
0.300 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.092 ml_ (1.2 eq) A- chlorobenzoyl chloride and 0.248 ml_ (3.0 eq) triethylamine are dissolved in 3 ml_ dichloromethane and stirred for 48 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using n-hexane and ethyl acetate in a mixture of 3:2 povides a mixture of starting material and the title compound. A second silica gel flash chromatography using a gradient of n- hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yields 0.160 g (42%) of the title compound as a white solid. Furthermore 0.17O g (57%) of the starting material is recovered. M. p. 93 0C (foam). MS: m/z (MH+) = 639.9, 641.8, MH++4.
A8. {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
0.405 g (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 0.336 ml_ (1.2 eq) A- methylbenzoyl chloride and 0.128 ml_ (3.0 eq) triethylamine are dissolved in 4 ml_ dichloromethane and stirred for 16 hours at ambient temperature. After addition of 10 ml_ dichloromethane and 10 ml_ saturated aqueous sodium hydrogen carbonate solution the phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yield 0.145 g (29%) of the title compound as a white solid. Furthermore 0.259 g (64%) of the starting material are recovered. M. p. 92-94 0C, sinter 82 0C. MS: m/z (MH+) = 619.9, MH++2.
A9. {3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propylamino]- propyl}-carbamic acid ferf-butyl ester (4.00 g, 8.2 mmol) (compound B2) is dissolved in dichlormethane (40 ml_), treated with triethylamine (3.6 ml_, 24.6 mmol) and 4-methyl benzoyl chloride (1.30 ml_, 9.8 mmol) and stirred 15 h at ambient temperature. It is diluted with dichlormethane, washed with aqueous NaHCO3-solution and the organic phase is dried. After evaporation, the residue is purified by chromatography. 4.5 g (90 %) of a colorless solid of m.p. 80-82 0C is obtained.
A10. (S^^RSJ-i^e-Benzyl-S-chloro^-methyl^-oxo-ej-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 2-methyl-propyl]-[1-(4-trifluoromethyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert- butyl ester
A solution of 200 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-propyll-carbamic acid tert-butyl ester (compound B3), 88 μl_ (1.5 eq) 4-(trifluoromethyl)-benzoyl chloride with catalytic amount of DMAP in 10 ml_ pyridine is heated for 16 hours at 70 0C. Evaporation of the pyridine followed by two times coevaporation with toluene results in the crude product. This material is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. By this method 102 mg (38%) of the title compound are obtained as a colorless solid. M.p.: 106-108 0C. MS: m/z (MH+) = 673.9, 675.8.
The following compounds may be prepared by benzoylation reaction of compound B1 with the appropriate benzoic acid derivative analogously or similarly as described for compounds A1 and A3 to A9:
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(3-methyl- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4-fluoro- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4-methoxy- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4-bromo- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4-chloro- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- trifluoromethyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester The following compounds may be prepared by bromination of the appropriate compounds mentioned afore analogously or similarly as described for compound A2:
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(3- methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- fluoro-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- methoxy-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- bromo-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- chloro-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- trifluoromethyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
The following compounds may be prepared by benzoylation reaction of compound B2 with the appropriate benzoic acid derivative analogously or similarly as described for compounds A1 and A3 to A9:
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(3- methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- fluoro-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- methoxy-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- bromo-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- chloro-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-propyl]-(4- trifluoromethyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
The following compounds may be prepared by benzoylation reaction of compound B4 with the appropriate benzoic acid derivative analogously or similarly as described for compounds A1 and A3 to A9:
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-(1-p- tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-(1-m- tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-[1-(4- fluoro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-[1-(4- methoxy-phenyO-carbonyll-aminol-propyO-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-[1-(4- bromo-phenylj-carbonyll-aminol-propylj-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-[1-(4- chloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl-propyl]-[1-(4- trifluoromethyl-phenylj-carbonyll-aminol-propylj-carbamic acid tert-butyl ester
The following compounds may be prepared by bromination of the appropriate compounds mentioned afore analogously or similarly as described for compound A2:
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-(1-m-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-[1-(4-fluoro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-[1-(4-methoxy-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-[1-(4-chloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propyl]-[1-(4-trifluoromethyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
The following compounds may be prepared by benzoylation reaction of compound B5 with the appropriate benzoic acid derivative analogously or similarly as described for compounds A1 and A3 to A9:
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
{3-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-(1-m-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
(3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-[1-(4-fluoro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-[1-(4-methoxy-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-[1-(4-chloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- butyl]-[1-(4-trifluoromethyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
A11. θ-Benzyl-S-chloro-S-^RSJ-I^S-dimethylamino-propylaminoJ^-methyl-propyll^-methyl- 6H-pyrazolo[1,5-c]pyrimidin-7-one
A suspension of 544 mg (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-propanoyl)-6H- pyrazolo[1 ,5-c]pyrimidin-7-one (compound C3) and 742 μL N1,N1-dimethylpropane-1 ,3-diamine (2.5 eq) in 1.5 ml_ Ti(O1Pr)4 is heated for 16 hours at 50 0C. This crude mixture with the imine as main product is dissolved in 5 ml_ glacial acetic acid and treated with 400 mg (6.6 eq) NaBH4. This mixture is stirred for 16 hours at ambient temperature to give the crude mixture containing the title compound. The solution is treated with 2 M aqueous NaOH-solution to an pH of 12 and extracted with 1-butanol. Purification by silica gel flash chromatography using a gradient of dichloromethane, methanol and triethylamine from 89:9:2 to 79:19:2 resulted in 105 mg (15%) of the title compound as a colorless oil. MS: m/z (MH+) = 430.1 , 432.2.
The following compounds may be prepared by reductive amination reaction of the appropriate compound C1 to C6 with N1,N1-dimethyl-propane-1 ,3-diamine (Me2N-(CH2)3-NH2) analogously or similarly as described for compounds B1 to B3.
6-Benzyl-5-[(RS)-1-(3-dimethylamino-propylamino)-propyl]-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin-7- one
6-Benzyl-3-chloro-5-[(RS)-1-(3-dimethylamino-propylamino)-propyl]-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one
6-Benzyl-5-[(RS)-1-(3-dimethylamino-propylamino)-2-methyl-propyl]-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one
6-Benzyl-5-[(RS)-1-(3-dimethylamino-propylamino)-2-methyl-butyl]-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one
6-Benzyl-3-chloro-5-[(RS)-1-(3-dimethylamino-propylamino)-2-methyl-butyl]-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one
A12. {^[[(RSJ-i^e-Benzyl-S-chloro^-methyl-Z-oxo-ej-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 2-methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-butyl}-carbamic acid tert-butyl ester
A solution of 180 mg (1.0 eq) {4-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-butyll-carbamic acid tert-butyl ester (compound B6), 175 μL (1.5 eq) 4-methyl benzoyl chloride and 145 μL (3.7 eq) triethylamine in 3.5 mL dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 mL dichloromethane and 10 mL saturated aqueous NaHCO3-solution, the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 127 mg (71%) of the title compound as a colorless solid.
M. p.: 82-84 0C.
MS: m/z (MNa+) = 656.2, and also MNa++2.
A13. {2-[[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-ethyl}-carbamic acid tert-butyl ester
A solution of 200 mg (1.0 eq) {2-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propylamino]-ethyl}-carbamic acid tert-butyl ester (compound B7), 205 μL (3.8 eq) 4-methyl benzoyl chloride and 171 μl_ (3.0 eq) triethylamine in 4 ml_ dichloromethane is stirred for 62 hours at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 mL saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yielded 125 mg (50%) of the title compound as a colorless solid. M. p.: 1 19-12O 0C. MS: m/z (MH+) = 605.9, 608.0.
A14. {S-t^RSJ-i^e-Benzyl-S-chloro^-methyl^-oxo-ej-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 1-cyclobutyl-methyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
A solution of 300 mg (1.0 eq) (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-1-cyclobutyl-methyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound B8), 115 μL (1.5 eq) 4-methyl benzoyl chloride and 241 μl_ (3.0 eq) triethylamine in 6 ml_ dichloromethane is stirred for 2 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 mL saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 194 mg (53%) of the title compound as a colorless foam. Furthermore 76 mg (25%) of the starting material is recovered. M. p.: 108-111 0C. MS: m/z (MH+) = 631.8, 633.8.
A15. {S-t^RSJ-i^e-Benzyl-S-chloro^-methyl^-oxo-ej-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 2-methyl-butyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
A solution of 260 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazololi .δclpyrimidin-δ-yl^-methyl-butylaminol-propyll-carbamic acid tert-butyl ester (compound B5), 100 μL (1.5 eq) 4-methyl benzoyl chloride and 208 μL (3.0 eq) triethylamine in 5 mL dichloromethane is stirred for 16 hours at ambient temperature. After addition of 10 mL of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yielded 81 mg (26%) of the title compound as a pale yellow foam. Furthermore 147 mg (57%) of the starting material is recovered. M. p.: 67-68 0C. MS: m/z (MH+) = 633.9, 635.8.
A16. {S-t^RSJ-i^e-Benzyl-S-chloro^-methyl-Z-oxo-ej-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 1-cyclopropyl-methyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
A solution of 257 mg (1.0 eq) (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-1-cyclopropyl-methyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound B9), 135 μl_ (2.0 eq) 4-methyl benzoyl chloride and 212 μl_ (3.0 eq) triethylamine in 5 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yielded 90 mg (29%) of the title compound as a colorless foam. Furthermore 73 mg (29%) of the starting material is recovered. M. p.: 85-87 0C. MS: m/z (MH+) = 617.9, 619.9.
A17. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 1-cyclopropyl-methyl]-[1-(4-bromo-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
A solution of 196 mg (1.0 eq) (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-1-cyclopropyl-methyl]-amino}-propyl)-carbamic acid tert-butyl ester (compound B9), 105 mg (1.2 eq) 4-bromo benzoyl chloride and 166 μl_ (3.0 eq) triethylamine in 4 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 50 mg (18%) of the title compound as a colorless foam. M. p.: 120-122 0C. MS: m/z (MH+) = 681.8, 683.6, 685.4. A18. (S^^RSJ-i^θ-Benzyl-S-chloro^-methyl-Z-oxo-ΘJ-dihydro-pyrazoloti.S-clpyrimidin-S-yl)- 2-methyl-propyl]-[1-(3-fluoro-4-methyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert- butyl ester
A solution of 330 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester (compound B3), 171 mg (1.5 eq) 3-fluoro-4-methyl benzoyl chloride and 274 μl_ (3.0 eq) triethylamine in 8 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 59:39:2 to 0:100:0 yielded 115 mg (27%) of the title compound as a pale yellow foam. Furthermore 122 mg (37%) of the starting material is recovered. M. p.: 88-91 0C. MS: m/z (MH+) = 637.8, 639.8.
A19. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(2-fluoro-4-methyl-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert- butyl ester
A solution of 277 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-propyll-carbamic acid tert-butyl ester (compound B3), 142 mg (1.5 eq) 2-fluoro-4-methyl benzoyl chloride and 229 μl_ (3.0 eq) triethylamine in 7 ml_ dichloromethane is stirred for 2 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 59:39:2 to 0:100:0 yielded 100 mg (28%) of the title compound as a colorless foam. Furthermore 100 mg (36%) of the starting material is recovered.
M. p.: sinter at 100 0C and melting at 110-112 0C. MS: m/z (MH+) = 637.9, 639.9.
A20. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(3,4-dichloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
A solution of 259 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-propyll-carbamic acid tert-butyl ester (compound B3), 129 mg (1.2 eq) 3,4-dichloro benzoyl chloride and 214 μl_ (3.0 eq) triethylamine in 6 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 88 mg (22%) of the title compound as a colorless foam.
M. p.: 104-107 0C.
MS: m/z (MH+) = 673.6, 675.7, 677.8.
A21. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-[1-(2,3-dichloro-phenyl)-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester
A solution of 300 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-propyll-carbamic acid tert-butyl ester (compound B3), 143 mg (1.2 eq) 2,3-dichloro benzoyl chloride and 249 μl_ (3.0 eq) triethylamine in 7 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a gradient of n-hexane, ethyl acetate and acetic acid from 39:59:2 to 0:100:0 yielded 67 mg (17%) of the title compound as a colorless solid. Furthermore 164 mg (55%) of the starting material is recovered. M. p.: 109-110 0C. MS: m/z (MH+) = 673.8, 676.0, 677.9.
A22. {3-[[(RS)-1-(6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester
0.10 g of {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2-methyl- propylamino]-propyl}-carbamic acid tert-butyl ester (compound A23) are dissolved in 3 ml_ DMF. After adding 36.3 mg (1.2 eq) NBS at 0 0C the mixture is stirred for 16 hours at ambient temperature. Addition of water followed by extraction of the aqueous phase with dichloromethane yielded after drying over magnesium sulfate and evaporation of the solvent the crude product. The residue is purified by flash chromatography using n-hexane / ethylacetate in a ratio of 2:1 and yielded 90 mg (80%) of the title compound as colorless oil.
A23. {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl- propylamino]-propyl}-carbamic acid tert-butyl ester
A solution of 151 mg {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-2- methyl-propylamino]-propyl)-carbamic acid tert butyl ester (1.0 eq) (compound B4), 55 mg (1.5 eq) p- methyl benzoyl chloride and 132 μl_ (3.0 eq) triethylamine in 5 ml_ dichloromethane is stirred for 1 days at ambient temperature and for 1 day at 40 0C. After addition of 10 ml_ of dichloromethane and 10 ml_ saturated aqueous NaHCO3-solution the phases are separated and the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a ratio of n-hexane, ethyl acetate and acetic acid 66:32:2 yielded 105 mg (80%) of the title compound with impurities of acetic acid.
A24. {3-[[(RS)-1-(6-Benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propyl]-(1-p-tolyl-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester
A solution of 155 mg (1.0 eq) {3-[(RS)-1-(6-Benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yl^-methyl-propylaminol-propyll-carbamic acid tert-butyl ester (compound B10), 62 μl_ (1.5 eq) p-tolyl benzoyl chloride and 137 μl_ (3.0 eq) triethylamine in 6 ml_ tetrahydrofurane is stirred for 3 days at ambient temperature. The solvent is evaporated in vacuo. Purification of the crude product by silica gel flash chromatography using a ratio of n-hexane, ethyl acetate and acetic acid 66:32:2 yielded 150 mg (78%) of the title compound with impurities of acetic acid.
B1. {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- propylamino]-propyl}-carbamic acid tert-butyl ester
437 mg 6-Benzyl-2-methyl-5-propionyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (1.48 mmol) (compound C1 ) and 516 mg (3-Amino-propyl)-carbamic acid tert-butyl ester (2.96 mmol) are dissolved in 4 ml THF. 1.9 ml Ti(O1Pr)4 (4.44 mmol) are added and the reaction mixture is stirred 14 h at ambient temperature. Afterwards 4 ml ethanol and 412 mg NaCNBH3 (5.92 mmol) are added and the mixture is stirred 2 h at ambient temperature. After adding water the inorganic salts are filtrated, washed with ethanole and the filtrate is evaporated. The residue is suspended in dichloromethane, dried over sodium sulphate and filtrated. After removing the solvents the crude product is purified by silica gel flash chromatography. By this method 540 mg (80 %) of a colorless solid is obtained. M. p.: 72-74 0C.
B2. {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- propylamino]-propyl}-carbamic acid tert-butyl ester
6-Benzyl-3-chloro-2-methyl-5-propionyl-6/-/-pyrazolo[1 ,5-c]pyrimidin-7-one (3.70 g, 11.2 mmol) (compound C2) and Λ/-Boc-1 ,3-diaminopropane (2.60 g, 14.6 mmol) are dissolved in THF (28 ml_), treated with Ti(O1Pr)4 (6.40 g, 22.4 mmol) and stirred for 14h at ambient temperature. Then ethanol (28 ml_) and NaCNBH3 (1.40 g, 22.4 mmol) are added and it is stirred for 2 h at ambient temperature. After adding of water it is filtered and the filtrate is evaporated. The residue is purified by silica gel flash chromatography. A colorless solid of m.p. 73-75 0C is obtained. B3. {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester
1.10 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1 ,5-c]pyrimidin-7- one (compound C3) are dissolved in 15 ml tetrahydrofurane. 1.39 g (2.5 eq) tert-butyl-N-(3- aminopropyl)carbamate and 2.86 ml_ (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred for 72 hours at reflux. After cooling the mixture is treated with an aqueous citric acid solution and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yield a two-phase system. The two phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yields the crude mixture of the corresponding imine. This mixture of the crude imine is dissolved in 20 ml_ acetic acid. 0.61 g (5.0 eq) sodium borohydride are added. The reaction mixture is stirred for 1 hour at ambient temperature. After addition of water the mixture is extracted with etyhl acetate. The combined organic phase is once washed with saturated aqueous sodium hydrogencarbonate solution. The organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yields 0.62 g (39%) of the title compound as a white foam. M. p. 51-53 0C (foam). MS: m/z (MH+) = 501.9, 504.0.
B4. {3-[(RS)-1-(6-Benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)-2-methyl- propylamino]-propyl}-carbamic acid tert-butyl ester
The title compound may be prepared by reductive amination reaction of compound C4 with Λ/-Boc-1 ,3- diaminopropane analogously or similarly as described for compounds B1 to B3.
B5. {3-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-butylamino]-propyl}-carbamic acid tert-butyl ester
0.70 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-butanoyl)-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (compound C5) are dissolved in 40 ml_ tetrahydrofurane. 1.36 g (4.0 eq) tert-butyl-N-(3- aminopropyl)carbamate and 3.50 ml_ (6.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 72 hours at reflux. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine.
The mixture of the crude imine is dissolved in 12 ml_ acetic acid. 0.37 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 16 hours at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.34 g (34%) of the title compound as colorless foam.
M. p.: 64-65 0C.
MS: m/z (MH+) = 516.0, 518.0.
B6. {4-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propylamino]-butyl}-carbamic acid tert-butyl ester
1.87 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1 ,5-c]pyrimidin-7- one (compound C3) are dissolved in 23 ml_ tetrahydrofurane. 2.56 g (2.5 eq) tert-butyl-N-(3- aminobutyl)carbamate and 4.86 ml_ (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 72 hours at 80 0C. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine.
The mixture of the crude imine is dissolved in 35 ml_ acetic acid. 1.05 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 1 hour at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-solution and dichloromethane. After phase separation the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.44 g (16%) of the title compound as colorless foam. M. p.: 63-65 0C. MS: m/z (MH+) = 516.0, 518.0.
B7. {2-[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propylamino]-ethyl}-carbamic acid tert-butyl ester
0.61 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1 ,5-c]pyrimidin-7- one (compound C3) are dissolved in 8 ml_ tetrahydrofurane. 0.71 g (2.5 eq) tert-butyl-N-(3- aminoethyl)carbamate and 1.58 ml_ (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 72 hours at 80 0C. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine.
The mixture of the crude imine is dissolved in 11 ml_ acetic acid. 0.33 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 1 hour at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.21 g (25%) of the title compound as colorless foam.
M. p.: 73-75 0C.
MS: m/z (MH+) = 487.9, 490.0.
B8. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 1-cyclobutyl-methyl]-amino}-propyl)-carbamic acid tert-butyl ester
0.63 g (1.0 eq) of 6-Benzyl-3-chloro-5-(1-cyclobutyl-carbonyl)-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin-7- one (compound C6) are dissolved in 4 ml_ tetrahydrofurane. 0.54 g (2.5 eq) tert-butyl-N-(3- aminopropyl)carbamate and 1.10 ml_ (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 72 hours at 80 0C. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine.
This mixture of the crude imine is dissolved in 8 ml_ acetic acid. 0.23 g (5.0 eq) sodium borohydride is added and the mixture is stirred for 1 hour at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.50 g (79%) of the title compound as colorless foam. M. p.: 73-75 0C. MS: m/z (MH+) = 514.0, 516.0.
B9. (3-{[(RS)-1-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolot1,5-c]pyrimidin-5-yl)- 1-cyclopropyl-methyl]-amino}-propyl)-carbamic acid tert-butyl ester
1.81 g (1.0 eq) of 6-Benzyl-3-chloro-5-(1-cyclopropyl-carbonyl)-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin- 7-one (compound C7) are dissolved in 20 ml_ tetrahydrofurane. 2.31 g (2.5 eq) tert-butyl-N-(3- aminopropyl)carbamate and 4.70 ml_ (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 5 days at 80 0C. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine. This mixture of the crude imine is dissolved in 30 ml_ acetic acid. 1.0 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 2 hours at ambient temperature. The sovent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-SoI ution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50. This yielded 0.26 g of a mixture containing the title compound. This mixture was used for the further synthetic pathway. M. p.: 74-76 0C.
MS: m/z (MH+) = 500.0, 502.0 (analytical data measured with recovered pure starting material from A16).
B10. {3-[(RS)-1-(6-Benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidin-5-yl)- 2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester
0.55 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1 ,5-c]pyrimidin-7- one (compound C8) are dissolved in 7 ml_ tetrahydrofurane. 0.74 g (2.5 eq) tert-butyl-N-(3- aminoetyl)carbamate and 1.51 mL (3.0 eq) titanium(IV)isopropoxide are added and this mixture is stirred in a sealed tube for 72 hours at 80 0C. After cooling, the mixture is treated with an aqueous solution of citric acid and stirred for another 15 min at ambient temperature. Addition of dichloromethane and filtration of the mixture yielded a two-phase system. The phases are separated and the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is removed in vacuo. This yielded the crude mixture of the corresponding imine.
The mixture of the crude imine is dissolved in 12 mL acetic acid. 0.33 g (5.0 eq) sodium borohydride are added and the mixture is stirred for 90 minutes at ambient temperature. The solvent is evaporated and the residue is partitioned between saturated aqueous NaHCO3-solution and dichloromethane. After phase separation, the aqueous phase is extracted three times with dichloromethane. The combined organic phase is dried over magnesium sulfate and the solvent is evaporated in vacuo. The crude product is purified by silica gel flash chromatography using a cyclohexane ethyl acetate in a 2:1 mixture. This yielded 0.16 g (19%) of the title compound as colorless solid.
C1. 6-Benzyl-2-methyl-5-propionyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one
770 mg 6-Benzyl-5-((RS)-1-hydroxy-propyl)-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (2.59 mmol) (compound D1 ) are dissolved in 15 ml dichloromethane, 1.5 g molecular sieve (4 A, 500 mg/mmol Substrat) and 1060 mg NMO ( 7.8 mmol) are added and the reaction mixture is stirred 2 h at ambient temperature. Afterwards TPAP ( 100 mg, 0.26 mmol) are added and the mixture is stirred 14 h at ambient temperature. The reaction mixture is filtrated over silica gel, washed with etylacetate and evaporated. The residue is purified by silica gel flash chromatography. By this method 452 mg ( 59 %) of a yellow solid are obtained. M. p.: 128-130 0C. C2. θ-Benzyl-S-chloro^-methyl-S-propionyl-ΘH-pyrazolotijS-clpyrimidin-Z-one
6-Benzyl-3-chloro-5-((RS)-1-hydroxy-propyl)-2-methyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (6.0O g, 18.0 mmol) (compound D2) is dissolved in CH2CI2 (90 ml_) and treated with molecular sieve (4λ, 9.00 g) and NMO (7.30 g, 54.0 mmol) and stirred for 2h at ambient temperature. To the mixture is added TPAP (633 mg, 1.8 mmol) and it is stirred for 14 h at ambient temperature. The mixture is filtered and evaporated. The residue is purified by silica gel flash chromatography. The title compound is obtained as 3.80 g of a yellow solid with m.p. 128 0C.
C3. θ-Benzyl-S-chloro^-methyl-S^-methyl-propanoyO-ΘH-pyrazoloti.S-clpyrimidin-Z-one
1.61 g (1.0 eq) of 6-Benzyl-3-chloro-5-((RS)-1-hydroxy-2-methyl-propyl)-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one (compound D3) are dissolved in 60 ml_ of dichloromethane at room temperature. To this mixture are added 7 g (1 ,5 g/mmol) of mol sieves 4 A and 2.53 g (4.0 eq) N-methylmorpholine-N- oxide. After 30 min stirring at ambient temperature 0.33 g (0.2 eq) tetrapropylammonium perruthenate is added. After 16 hours the reaction mixture is filtered through silica gel and washed out with ethyl acetate. The solvent is removed in vacuo. Flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yields 1.09 g (68%) of the title compound as a white solid. M.p. 146.7 0C. MS: m/z (MH+) = 344.1 , 346.1.
C4. 6-Benzyl-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1,5-c]pyrimidin-7-one
The title compound may be prepared by oxidation of compound D4 analogously or similarly as described for compounds C1 to C3.
C5. θ-Benzyl-S-chloro^-methyl-S^-methyl-butanoyO-ΘH-pyrazoloti.S-clpyrimidin-Z-one
1.12 g (1.0 eq) of 6-Benzyl-3-chloro-5-((RS)-1-hydroxy-2-methyl-butyl)-2-methyl-6H-pyrazolo[1 ,5- c]pyrimidin-7-one (compound D5) are dissolved in 50 ml_ of dichloromethane at ambient temperature. 4.6 g (1.5 g/mmol) of mol sieves 4 A and 1.68 g (4.0 eq) N-methylmorpholine-N-oxide are added to this mixture. After 30 min stirring at ambient temperature 0.22 g (0.2 eq) tetrapropylammonium perruthenate are added. After 16 hours the reaction mixture is filtered through silica gel using ethyl acetate as eluent. The solvent is removed in vacuo. Flash chromatography using a gradient of n- hexane and ethyl acetate from 100:0 to 50:50 yielded 0.78 g (70%) of the title compound as a colorless solid. M.p.: 122-123 0C. MS: m/z (MH+) = 358.1 , 360.1.
C6. 6-Benzyl-3-chloro-5-(1-cyclobutyl-carbonyl)-2-methyl-6H-pyrazolo[1,5-c]pyrimidin-7-one
A suspension of 0.63 g (1.0 eq) of 6-Benzyl-3-chloro-5-((RS)-1-cyclobutyl-1-hydroxy-methyl)-2-methyl- 6H-pyrazolo[1 ,5-c]pyrimidin-7-one (compound D6), 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and 1.29 g iodobenzene diacetate in 4 ml_ dichloromethane is stirred for 2 days at ambient temperature. This mixture is treated with aqueous Na2S2O3-solution (1 M) and dichloromethane. Phase separation and extraction of the aqueous layer with dichloromethane resulted after drying over magnesium sulfate and evaporation of the solvent in vacuo in the crude product. Silica gel flash chromatography with a ratio of n-hexane and ethyl acetate 3:7 yielded 0.63 g (quant.) of the title compound as a colorless solid.
M. p.: 96-97 0C.
MS: m/z (MH+) = 356.1 , 358.1.
C7. 6-Benzyl-3-chloro-5-(1-cyclopropyl-carbonyl)-2-methyl-6H-pyrazolo[1,5-c]pyrimidin-7- one
0.33 g (1.0 eq) of 6-Benzyl-3-chloro-5-(1-cyclopropyl-(RS)-1-hydroxy-methyl)-2-methyl-6H- pyrazolo[1 ,5-c]pyrimidin-7-one (compound D7) are dissolved in 15 ml_ of dichloromethane at ambient temperature. 1.43 g (1.5 g/mmol) of mol sieves 4 A and 0.51 g (4.0 eq) N-methylmorpholine-N-oxide are added to this mixture. After 30 min stirring at ambient temperature 67 mg (0.2 eq) tetrapropylammonium perruthenate are added. After 16 hours the reaction mixture is filtered through silica gel with ethyl acetate as eluent. The solvent is removed in vacuo. Flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 30:70 yielded 0.23 g (68%) of the title compound as a colorless solid. M. p.: 151-152 0C. MS: m/z (MH+) = 342.2, 344.2.
C8. 6-Benzyl-3-fluoro-2-methyl-5-(2-methyl-propanoyl)-6H-pyrazolo[1,5-c]pyrimidin-7-one
A suspension of 0.55 g (1.0 eq) of 6-Benzyl-3-fluoro-5-(1-hydroxy-2-methyl-propyl)-2-methyl-6H- pyrazolo[1 ,5-c]pyrimidin-7-one (compound D8), 55 mg (0.22 eq) 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and 1.21 g (2.2 eq) iodobenzene diacetate in 10 ml_ dichloromethane is stirred for 3 days at ambient temperature. After addition of 25 mg (0.1 eq) TEMPO the mixture is stirred for another 2 days at ambient temperature. This mixture is treated with aqueous Na2S2O3-solution (1 M) and dichloromethane. Phase separation and extraction of the aqueous layer with dichloromethane resulted after drying over sodium sulfate and evaporation of the solvent in vacuo in the crude product. Silica gel flash chromatography with a ratio of cydohexane and ethyl acetate of 3:2 yielded 0.55 g (quant.) of the title compound as a yellow solid.
D1. 6-Benzyl-5-((RS)-1-hydroxy-propyl)-2-methyl-6H-pyrazolo[1,5-c]pyrimidin-7-one
To a solution of 7.2 ml MeLi (11.5 mmol, 1.6 M in Et2O) in THF (20 ml), 2.3 ml EtMgBr (6.9 mmol, 3 M in Et2O) are added at - 78 0C. The solution is stirred 1 h at - 780C.
1 g ε-Benzyl-S-bromo^-methyl^-oxo-εy-dihydro-pyrazoloIi .δ-clpyrimidine-δ-carbaldehyde (2.89 mmol) (compound E1 ) diluted in 4 ml DMPU and 10 ml THF are added dropwise under argon atmosphere. Afterwards the reaction mixture is stirred 2 h at - 780C and 3 h at -30 0C. The reaction mixture is quenched with aqueous NH4CI-solution and then warmed up to ambient temperature. The product is extracted between ethylacetate and water. The organic phase is dried over sodium sulphate. After evaporating the solvents the crude product is purified by silica gel flash chromatography. By this method 352 mg (42%) of the title compound are obtained. M. p.: 190-191 0C.
D2. 6-Benzyl-3-chloro-5-((RS)-1-hydroxy-propyl)-2-methyl-6H-pyrazolo[1,5-c]pyrimidin-7-one
To a mixture of THF (4 ml.) and MeLi (1.25 ml_, 2.0 mmol, 1.6 M in Et2O) are added at -78 0C EtMgBr (0.48 ml_, 1.2 mmol, 3.0 M in Et2O), the solution is stirred 1 h at this temperature and 6-benzyl-3- chloro^-methyl^-oxo-e^-dihydro-pyrazoloti .δ-clpyrimidine-δ-carbaldehyde (compound E2) is added. The mixture is stirred for 6h at - 70 0C and then the mixture is hydrolyzed with aqueous NH4CI solution and stirred at ambient temperature. After adding water the mixture is extracted with ethylacetate, the organic phase is dried and evaporated. The residue is purified by silica gel flash chromatography. 201 mg are obtained with a melting point of 80-82 0C.
D3. 6-Benzyl-3-chloro-5-((RS)-1-hydroxy-2-methyl-propyl)-2-methyl-6H-pyrazolo[1,5- c]pyrimidin-7-one
25 ml_ of methyllithium (2.0 eq, 1.6 M solution in diethylether) and 24 ml_ of isopropylmagnesium bromide (1.2 eq, ~1 M solution in tetrahydrofurane) are added to 200 ml_ dry tetrahydrofurane at - 780C. After one hour at -780C 6g (1.0 eq) of 6-Benzyl-3-chloro-2-ιmethyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidine-5-carbaldehyde (compound E2) is added. After stirring this mixture for an additional 2 hours, a saturated aqueous solution of ammonium chloride is added. At ambient temperature the mixture is extracted with ethyl acetate and the combined organic phase is dried over magnesium sulfate. After filtration the solvent is evaporated under vacuo. The crude product is crystallized from ethyl acetate. This yields 2.83 g of the title compound. Flash chromatography of the mother liquor using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yields another 1.15 g of the title compound (together 58% yield) as a white solid. M. p. 72.9 0C. MS: m/z (MH+) = 345.9, 347.9.
D4. 6-Benzyl-5-((RS)-1-hydroxy-2-methyl-propyl)-2-methyl-6H-pyrazolo[1,5-c]pyrimidin-7- one
The title compound may be prepared from compound E1 analogously or similarly as described for compound D1.
D5. 6-Benzyl-3-chloro-5-((RS)-1-hydroxy-2-methyl-butyl)-2-methyl-6H-pyrazolo[1,5- c]pyrimidin-7-one
12.5 ml_ of methyllithium (2.0 eq, 1.6 M solution in diethylether) and 12 mL of sec-butylmagnesium bromide (1.2 eq, ~1 M solution in tetrahydrofurane) are added to 100 mL dry tetrahydrofurane at - 780C. After one hour at -780C 3.0 g (1.0 eq) of 6-Benzyl-3-chloro-2-ιmethyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidine-5-carbaldehyde (compound E2) are added. After stirring this mixture for an additional 2.5 hours, a saturated aqueous solution of ammonium chloride is added. The mixture is extracted with ethyl acetate and the combined organic phase is dried over magnesium sulfate. After filtration, the solvent is evaporated in vacuo. Silica gel flash chromatography using a gradient of n- hexane and ethyl acetate from 100:0 to 50:50 yielded 1.2 g (33%) of the title compound as colorless foam.
M. p.: 72-73 0C.
MS: m/z (MH+) = 360.2, 362.2.
D6. 6-Benzyl-3-chloro-5-((RS)-1-cyclobutyl-1-hydroxy-methyl)-2-methyl-6H-pyrazolo[1,5- c]pyrimidin-7-one
1.0 ml_ (3.0 eq) Cyclobutylbromide and 0.24 g (3.0 eq) magnesium (Grignard) are dissolved in 20 ml_ diethylether. The grignard reaction is started with iodine and heating of the suspension to reflux. After 90 minutes at reflux a solution of 1.0 g (1.0 eq) of 6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro- pyrazolo[1 ,5-c]pyrimidine-5-carbaldehyde (compound E2) in 15 ml_ THF is added dropwise. After stirring for 16 hours at ambient temperature the reaction is stopped with ammonium chloride. Extraction of the aqueous phase with ethyl acetate resulted after drying over magnesium sulfate and evaporation of the solvent in vacuo in the crude product. Silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 0.20 g (17%) of the title compound as a colorless solid. M. p.: 188-193 0C (decomposition). MS: m/z (MH+) = 358.1 , 360.0.
D7. e-Benzyl-S-chloro-S^I-cyclopropyl^RSJ-i-hydroxy-methyO^-methyl-eH-pyrazoloti.S- c]pyrimidin-7-one
1.0 g (1.0 eq) of ε-Benzyl-S-chloro^-methyl^-oxo-εy-dihydro-pyrazoloIi .δ-clpyrimidine-δ- carbaldehyde (compound E2) are dissolved in 30 ml. THF and at 0 0C 16.5 ml. (2.5 eq, 0.5 M in THF) cyclopropylmagnesiumbromide are added dropwise. After stirring for 3 days at ambient temperature the mixture is treated with a saturated aqueous solution of ammonium chloride. Extraction of the water phase with dichloromethane resulted after drying over magnesium sulfate and evaporation of the solvent in vacuo the crude product. Silica gel flash chromatography using a gradient of n-hexane and ethyl acetate from 100:0 to 50:50 yielded 0.56 g (49%) of the title compound as a colorless solid. M. p.: 1 19-12O 0C. MS: m/z (MH+) = 344.1 , 346.1.
D8. θ-Benzyl-S-fluoro-S^I-hydroxy^-methyl-propylJ^-methyl-ΘH-pyrazolotijS-clpyrimidin- 7-one
3.2 ml_ of methyllithium (2.0 eq, 1.6 M solution in diethylether) and 3 ml_ of isopropylmagnesium bromide (1.2 eq, ~1 M solution in tetrahydrofurane) are added to 25 ml_ dry tetrahydrofurane at -780C. After one hour at -780C 0.6 g (1.0 eq) of 6-Benzyl-3-fluoro-2-ιmethyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidine-5-carbaldehyde (compound E3) are added. After stirring this mixture for an additional 3 hours the reaction is quenched by treatment with a 10% aqueous ammonium chloride solution. The mixture is extracted with diethylether and the combined organic phase is dried over magnesium sulfate. After filtration, the solvent is evaporated in vacuo. Silica gel flash chromatography using a ratio of n-hexane and ethyl acetate of 1 :1 yielded 0.23 g (27%) of the title compound as slightly yellow solid.
E1. 6-Benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidine-5-carbaldehyde
6-Benzyl-3-bromo-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (24.0 g, 72.2 mmol) (compound F1 ) are dissolved in 350 ml dioxane. 24 g selendioxide are added and the reaction mixture is heated to reflux for 12 h. Afterwards the reaction mixture is filtered, the residue is washed with diethylether and the filtrate is evaporated. By this method 22.7 g (90 %) of the title compound are obtained. M. p.: 187- 189 0C.
E2. 6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1,5-c]pyrimidine-5-carbaldehyde
2.29 g (1.0 eq) of 6-Benzyl-3-chloro-2,5-dimethyl-6/-/-pyrazolo[1 ,5-c]pyrimidin-7-one (compound F2) are dissolved in 40 ml_ dioxane and treated with 2.64 g (3.0 eq) selenium dioxide. This mixture is heated at reflux for 5 hours. After filtration off the inorganic salts, the filtrate is evaporated in vacuo. This residue is purified by silica gel flash chromatography using cyclohexane and ethyl acetate in a mixture of 2:1 to yield 2.10 g (88%) of the title compound. M. p. 187-188 0C.
E3. θ-Benzyl-S-fluoro^-methyl-Z-oxo-ΘJ-dihydro-pyrazoloti.S-clpyrimidine-S-carbaldehyde
A solution of 6.75 g (1.0 eq) of 6-Benzyl-3(4)-fluoro-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (compound F3) in 125 ml_ dioxane is treated with 10.55 g (3.0 eq) selenium dioxide. The mixture is heated for 7 days at reflux. This mixture is filtered, the solution is concentrated and washed three times with diethylether. The mother liquor is purified using flash chromatography (ethylacetate as eluent). This resulted in 0.60 g (8%) of the title compound.
F1. 6-Benzyl-3-bromo-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one
32 g (126 mmol) of the 6-Benzyl-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (compound G1 ) are dissolved in 300 ml CCI4. 22.5 g (126 mmol) NBS and 2.1 g (12.6 mmol) AIBN are added to the solution, it is stirred at ambient temperature for 18 h. Afterwards the solution is diluted with dichloromethane and washed with saturated aqueous NaHCO3 solution. The organic phase is dried over sodium sulphate and the solvents are evaporated. The crude product is purified by silica gel flash chromatography. By this method 24.1 g (58 %) of the title compound are obtained. F2. 6-Benzyl-3-chloro-2,5-dimethyl-6H-pyrazolo[1,5-c]pyrimidin-7-one
2.83 g (1.0 eq) 6-Benzyl-2,5-dimethyl-6/-/-pyrazolo[1 ,5-c]pyrimidin-7-one (compound G1 ) are dissolved in 35 ml_ tetrachloromethane and treated with 1.50 g (1.0 eq) N-chlorosuccinimide for 5 hours at reflux. The reaction mixture is diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution. Drying of the organic phase over sodium sulfate and evaporation of the solvent yields the crude product. The residue is purified by silica gel flash chromatography using cyclohexane and ethyl acetate in a mixture of 2:1 to yield 2.70 g (84%) of the title compound. M. p. 140-142 0C.
F3. 6-Benzyl-3(4)-fluoro-2,5-dimethyl-6H-pyrazolo[1,5-c]pyrimidin-7-one
14.7 g of 6-Benzyl-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one (1.0 eq, compound G1 ) are dissolved in 300 ml_ acetonitrile and treated at 0 0C with 22.6 g Selectfluor (1.1 eq). After stirring for 1 hour at this temperature, the solvent is removed in vacuo, the residue is dissolved in dichloromethane and neutralized with a saturated aqueous hydrogen carbonate solution. The aqueous phase is extracted with dichloromethane and the combined organic layer is dried over magnesium sulfate. After evaporation of the solvent, the oily residue is crystallized from diethylether. The crystals are further purified using flash chromatography (cyclohexane / ethylacetate = 1.5:1 ). This yielded 2.67 g (17%) of the title compound as a colorless solid.
G1. 6-Benzyl-2,5-dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one
2.30 g (1.0 eq) 2,5-dimethyl-6/-/-pyrazolo[1 ,5-c]pyrimidin-7-one (compound H1 ) are dissolved in 35 mL N,N-dimethylformamide (DMF). 2.20 g (1.1 eq) potassium carbonate and 1.9 ml_ benzyl bromide are added. The mixture is stirred at ambient temperature for 14 hours. After addition of water the aqueous phase is extracted with dichloromethane. The combined organic phase is dried over sodium sulfate and the solvent is evaporated in vacuo. The residue is washed with diethylether and yield 3.35 g (94%) of the title compound.
H1. 2,5-Dimethyl-6H-pyrazolo[1 ,5-c]pyrimidin-7-one
2.30 g (1.0 eq) semicarbazide hydrochloride are dissolved in 40 mL water. After addition of 1.10 g (0.5 eq) of a saturated aqueous solution of sodium carbonate and 2.90 g (1.0 eq) of heptane-2,4,6-trione (compound 11 ), the mixture is heated for 1 hour at 1000C. Extraction of the mixture with dichloromethane yields after evaporation of the solvent in vacuo 2.6 g (79%) of the title compound. M. p. 205-208 0C.
11. Heptane-2,4,6-trione
50.0 g 2,6-Dimethyl-γ-pyrone are dissolved in 250 mL ethanol and treated with 50 mL of a 16 M aqueous solution of sodium hydroxide. This mixture is heated for 5 hours at 6O0C and for another 1 hour at 1000C. The residue is filtered off and washed with diethylether. This residue is now dissolved in water and put in 500 mL of a 3 M aqueous solution of hydrochloric acid. The aqueous phase is extracted with diethylether. The combined organic phase is dried over sodium sulfate and the solvent is evaporated in vacuo. This yields 31.6 g (55%) of the title compound. M. p. 43-45 0C.
Commercial utility
The compounds of formula I, I* and I**, and their pharmacologically and/or pharmaceutically acceptable salts (= the compounds according to the present invention) have valuable pharmacological and/or pharmaceutical properties which can make them commercially applicable. Thus, for example, the compounds according to this invention can act as inhibitors of the mitotic kinesin Eg5 and these compounds are expected to be commercially applicable in the therapy of diseases responsive to the inhibition of this kinesin, such as e.g. those diseases mentioned below. Also, for example, the compounds according to this invention can display cell-cycle dependent, anti-proliferative and/or apoptosis inducing activity.
The mitotic kinesin Eg5 is an enzyme essential for the assembly and function of the bipolar mitotic spindle. Eg5 plays essential roles during all phases of mitosis. Drugs that perturb mitosis have proven clinically effective in the treatment of many cancers. Despite the diverse array of essential spindle proteins that could be exploited as targets for the discovery of novel cancer therapies, all spindle- targeted therapeutics in clinical use today act on only one protein, tubulin. Surprisingly, kinesin Eg5 expression is most abundant in proliferating human tissues, whereas it is absent from most postmitotic cells, such as e.g. human central nervous system neurons, consistent with an exclusive or almost confined role for Eg5 in cell proliferation. In contrary to drugs that directly interfere with microtubule dynamic instability, Eg5 kinesin inhibitors are expected not to disrupt microtubule-based cellular processes, e.g. neuronal transport, that are unrelated to proliferation. During mitosis, Eg5 is essentially involved in organizing microtubules into a bipolar structure that forms the mitotic spindle. Experimental perturbation of Eg5 function causes a characteristic malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest and cell death.
The compounds according to this invention can be used to modulate mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis, which is frequently followed by apoptosis. By "modulate" herein is meant altering mitotic spindle formation, including increasing and decreasing spindle formation. By "mitotic spindle formation" herein is meant organization of microtubules into bipolar structures by mitotic kinesins. By "dysfunction of the mitotic spindle" herein is meant mitotic arrest and monopolar spindle formation. "Malformation of the mitotic spindle" encompasses the splaying of mitotic spindle poles, or otherwise causing morphological perturbation of the mitotic spindle.
Further on, these compounds can be useful in the treatment of benign or malignant neoplasia. A "neoplasia" is defined by cells displaying aberrant cell proliferation and/or survival and/or a block in differentiation. A "benign neoplasia" is described by hyperproliferation of cells, incapable of forming an aggressive, metastasizing tumor in-vivo. In contrast, a "malignant neoplasia" is described by cells with multiple cellular and biochemical abnormalities, capable of forming a systemic disease, for example forming tumor metastasis in distant organs. Various diseases are caused aberrant cell proliferation ("hyperproliferation") as well as evasion from apoptosis. These diseases include e.g. benign hyperplasia like that of the prostate ("BPH") or colon epithelium, psoriasis, glomerulonephritis or osteoarthritis. Most importantly these diseases include malignant neoplasia commonly described as cancer and characterized by tumor cells finally metastasizing into distinct organs or tissues. Malignant neoplasia include solid and hematological tumors. Solid tumors are exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. Malignant neoplasia include inherited cancers exemplified by retinoblastoma and Wilms tumor. In addition, malignant neoplasia include primary tumors in said organs and corresponding secondary tumors in distant organs ("tumor metastases"). Hematological tumors are exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers of unknown primary site as well as AIDS related malignancies.
The invention therefore relates to a use of the compounds according to the invention in the manufacture of pharmaceutical compositions, a method of treatment or a combination according to the invention, in which the cancer to be treated is selected from the group consisting of cancer of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands, esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva; inherited cancers, retinomblastoma and Wilms tumor; leukemia, lymphoma, non-Hodgkins disease, chronic and acute myeloid leukaemia, acute lymphoblastic leukemia, Hodgkins disease, multiple myeloma and T-cell lymphoma; myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers of unknown primary site and AIDS related malignancies.
It is to be noted that a cancer disease as well as a malignant neoplasia does not necessarily require the formation of metastases in distant organs. Certain tumors exert devastating effects on the primary organ itself through their aggressive growth properties. These can lead to the destruction of the tissue and organ structure finally resulting in failure of the assigned organ function.
Neoplastic cell proliferation might affect normal cell behaviour and organ function. For example the formation of new blood vessels, a process described as neovascularization, is induced by tumors or tumor metastases. Compounds according to this invention can be commercially applicable for the treatment of pathophysiological relevant processes caused by benign or neoplastic cell proliferation, such as but not limited to neovascularization by unphysiological proliferation of vascular endothelial cells.
Drug resistance is of particular importance for the frequent failure of standard cancer therapeutics. This drug resistance is caused by various cellular and molelcular mechanisms like overexpression of drug efflux pumps or mutation within the cellular target protein. The commercial applicability of the compounds according to this invention is not limited to 1st line treatment of patients. Patients with resistance to defined cancer chemotherapeutics or target specific anti-cancer drugs (2nd or 3rd line treatment) can be also amenable for treatment with the compounds according to this invention.
Due to their cellular anti-proliferative properties, compounds according to the present invention may be also commercially usable for treatment of diseases associated with cell cycle and cell proliferation, such as, besides cancer discussed above, for example, fibroproliferative and differentiative disorders, psoriasis, rheumatoid arthritis, atherosclerosis, hyperplasia, restenosis, cardiac hypertrophy, (auto)immune disorders, fungal disorders, bone diseases, or acute or chronic inflammation. Thus, the invention relates to compounds according to the invention for use in the treatment of diseases.
Compounds according to the present invention can be commercially applicable for treatment, prevention or amelioration of the diseases of benign and malignant behavior as described before, such as e.g. benign or malignant neoplasia, particularly cancer (such as e.g. any of those cancer diseases described above), especially a cancer that is susceptible to Eg5 inhibition.
In the context of their properties, functions and usabilities mentioned herein, the compounds according to the present invention are expected to be distinguished by valuable and desirable effects related therewith, such as e.g. by low toxicity, superior bioavailability in general (such as e.g. good enteral absorption), superior therapeutic window, absence of significant side effects, and/or further beneficial effects related with their therapeutic and pharmaceutical suitability.
The invention further includes a method for treating (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, particularly those diseases, disorders, conditions or illnesses mentioned above, in mammals, including humans, suffering therefrom comprising administering to said mammals in need thereof a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention.
The present invention further includes a method useful to modulate apoptosis and/or aberrant cell growth in the therapy of benign or malignant neoplastic diseases, such as e.g. cancer, comprising administering to a subject in need of such therapy a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention. The invention further includes a method for modulating, particularly inhibiting, Eg5 activity in cells comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such modulation, particularly inhibition.
The present invention further includes a method to modulate the mitotic spindle, i.e., for example, altering mitotic spindle formation, including decreasing spindle formation, or increasing or decreasing spindle pole separation causing malformation of the mitotic spindle poles, comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such modulation.
The present invention further includes a method to inhibit mitosis in cells comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more of the compounds according to this invention to a patient in need of such inhibition.
The present invention further includes a method for treating, preventing or ameliorating diseases and/or disorders associated with Eg5 kinesin activity, such as, for example, (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, for example, benign neoplasia or malignant neoplasia, e.g. cancer, in a mammal comprising administering a pharmacologically and/or pharmaceutically active and therapeutically effective and tolerable amount of one or more compounds according to the present invention to said mammal in need thereof.
The present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which are employed for the treatment, prophylaxis and/or amelioration of one or more of the illnesses mentioned.
The present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which can be used in the treatment, prevention or amelioration of (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis in a mammal, such as, for example, benign or malignant neoplasia, e.g. cancer.
The present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions which can be used use in the treatment, prevention or amelioration of disorders responsive to arresting of aberrant cell growth and/or induction of apoptosis.
The present invention further relates to the use of the compounds according to this invention for the production of pharmaceutical compositions for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above. The present invention further relates to pharmaceutical compositions comprising one or more of the compounds according to this invention and a pharmaceutically acceptable carrier or diluent.
The present invention further relates to pharmaceutical compositions made by combining one or more of the compounds according to this invention and a pharmaceutically acceptable carrier or diluent.
The present invention further relates to pharmaceutical compositions comprising one or more of the compounds according to this invention and pharmaceutically acceptable auxiliaries and/or excipients.
The present invention also relates to pharmaceutical compositions for treating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, which include benign neoplasia and malignant neoplasia, including cancer, comprising a compound according to this invention.
The present invention further relates to combinations comprising one or more of the compounds according to this invention and pharmaceutically acceptable auxiliaries, excipients and/or vehicles, e.g. for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
The present invention further relates to a combination comprising a compound according to this invention and a pharmaceutically acceptable excipient, carrier and/or diluent, e.g. for treating, preventing or ameliorating benign or malignant neoplasia, particularly cancer, such as e.g. any of those cancer diseases described above.
The present invention further relates to a composition consisting essentially of a therapeutically effective and tolerable amount of one or more compounds according to this invention together with the usual pharmaceutically acceptable vehicles, diluents and/or excipients for use in therapy, e.g. for treating, preventing or ameliorating hyperproliferative diseases, such as e.g. cancer, and/or disorders responsive to induction of apoptosis.
The present invention further relates to compounds according to this invention for use in therapy, such as, for example, in the treatment, prevention or amelioration of (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis, such as e.g. those diseases mentioned herein, particularly cancer.
The present invention further relates to compounds according to this invention having anti-proliferative and/or apoptosis inducing activity.
The present invention further relates to compounds according to this invention having Eg5 inhibiting properties. The present invention further relates to pharmaceutical compositions according to this invention having Eg5 inhibiting properties.
The present invention further relates to pharmaceutical compositions according to this invention having anti-proliferative activity.
The present invention further relates to pharmaceutical compositions according to this invention having apoptosis inducing activity.
The invention further relates to the use of a pharmaceutical composition comprising one or more of the compounds according to this invention as sole active ingredient(s) and a pharmaceutically acceptable carrier or diluent in the manufacture of pharmaceutical products for the treatment and/or prophylaxis of the illnesses mentioned above.
Additionally, the invention relates to an article of manufacture, which comprises packaging material and a pharmaceutical agent contained within said packaging material, wherein the pharmaceutical agent is therapeutically effective inhibiting Eg5 and/or inhibiting cellular (hyper)proliferation and/or inducing apoptosis, ameliorating the symptoms of a Eg5 mediated disease and/or a (hyper)proliferative disease and/or a disorder responsive to the induction of apoptosis, and wherein the packaging material comprises a label or package insert which indicates that the pharmaceutical agent is useful for preventing or treating a Eg5 mediated disease and/or a (hyper)proliferative disease and/or a disorder responsive to the induction of apoptosis, and wherein said pharmaceutical agent comprises one or more compounds according to the invention. The packaging material, label and package insert otherwise parallel or resemble what is generally regarded as standard packaging material, labels and package inserts for pharmaceuticals having related utilities.
The pharmaceutical compositions according to this invention are prepared by processes which are known per se and familiar to the person skilled in the art. As pharmaceutical compositions, the compounds of the invention (= active compounds) are either employed as such, or preferably in combination with suitable pharmaceutical auxiliaries and/or excipients, e.g. in the form of tablets, coated tablets, dragees, pills, cachets, granules, capsules, caplets, suppositories, patches (e.g. as TTS), emulsions (such as e.g. micro-emulsions or lipid emulsions), suspensions (such as e.g. nano suspensions), gels, solubilisates or solutions (e.g. sterile solutions), or encapsuled in liposomes or as beta-cyclodextrine inclusion complexes or the like, the active compound content advantageously being between 0.1 and 95% and where, by the appropriate choice of the auxiliaries and/or excipients, a pharmaceutical administration form (e.g. a delayed release form or an enteric form) exactly suited to the active compound and/or to the desired onset of action can be achieved. The person skilled in the art is familiar with auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge. In addition to solvents, gel formers, ointment bases and other active compound excipients, for example antioxidants, dispersants, emulsifiers, preservatives, solubilizers (such as e.g. polyoxyethylenglyceroltriricinoleat 35, PEG 400, Tween 80, Solutol HS15 or the like), colorants, complexing agents, permeation promoters, stabilizers, fillers, binders, thickeners, disintegrating agents, buffers, pH regulators (e.g. to obtain neutral, alkaline or acidic formulations), polymers, lubricants, coating agents, propellants, tonicity adjusting agents, surfactants, flavorings, sweeteners or dyes, can be used.
In particular, auxiliaries and/or excipients of a type appropriate to the desired formulation and the desired mode of administration are used.
The administration of the compounds, pharmaceutical compositions or combinations according to the invention may be performed in any of the generally accepted modes of administration available in the art. Illustrative examples of suitable modes of administration include intravenous, oral, nasal, parenteral, topical, transdermal and rectal delivery. Oral and intravenous delivery are preferred.
For the treatment of dermatoses, the compounds of the invention can be in particular administered in the form of those pharmaceutical compositions which are suitable for topical application. For the production of the pharmaceutical compositions, the compounds of the invention (= active compounds) are preferably mixed with suitable pharmaceutical auxiliaries and further processed to give suitable pharmaceutical formulations. Suitable pharmaceutical formulations are, for example, powders, emulsions, suspensions, sprays, oils, ointments, fatty ointments, creams, lotions, pastes, gels or solutions.
The pharmaceutical compositions according to the invention can be prepared by processes known per se. The dosage of the compounds of the invention (= active compounds) is carried out in the order of magnitude customary for Eg5 inhibitors, inhibitors for cellular (hyper)proliferation or apoptosis inducers. Topical application forms (such as ointments) for the treatment of dermatoses thus contain the active compounds in a concentration of, for example, 0.1-99%. The customary dose in the case of systemic therapy (p.o.) may be between 0.03 and 60 mg/kg per day, (i. v.) may be between 0.03 and 60 mg/kg/h. In another embodiment, the customary dose in the case of systemic therapy (p.o.) is between 0.3 and 30 mg/kg per day, (i. v.) is between 0.3 and 30 mg/kg/h.
The choice of the optimal dosage regime and duration of medication, particularly the optimal dose and manner of administration of the active compounds necessary in each case can be determined by a person skilled in the art on the basis of his/her expert knowledge.
Depending upon the particular disease, to be treated or prevented, additional therapeutic active agents, which are normally administered to treat or prevent that disease, may optionally be coadministered with the compounds according to this invention. As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease are known as appropriate for the disease being treated.
For example, compounds according to this invention may be combined with one or more standard therapeutic agents used for treatment of the diseases as mentioned before.
In one particular embodiment, compounds according to this invention may be combined with one or more art-known anti-cancer agents, such as e.g. with one or more chemotherapeutic and/or target specific anti-cancer agents as described below.
Examples of known chemotherapeutic anti-cancer agents frequently used in combination therapy include, but not are limited to (i) alkylating/carbamylating agents such as Cyclophosphamid (Endoxan®), lfosfamid (Holoxan®), Thiotepa (Thiotepa Lederle®), Melphalan (Alkeran®), or chloroethylnitrosourea (BCNU); (ii) platinum derivatives like cis-platin (Platinex® BMS), oxaliplatin, satraplatin or carboplatin (Cabroplat® BMS); (iii) antimitotic agents / tubulin inhibitors such as vinca alkaloids (vincristine, vinblastine, vinorelbine), taxanes such as Paclitaxel (Taxol®), Docetaxel (Taxotere®) and analogs as well as new formulations and conjugates thereof, epothilones such as Epothilone B (Patupilone®), Azaepothilone (Ixabepilone®) or ZK-EPO, a fully synthetic epothilone B analog; (iv) topoisomerase inhibitors such as anthracyclines (exemplified by Doxorubicin / Adriblastin®), epipodophyllotoxines (examplified by Etoposide / Etopophos®) and camptothecin and camptothecin analogs (exemplified by lrinotecan / Camptosar® or Topotecan / Hycamtin®); (v) pyrimidine antagonists such as 5-fluorouracil (5-FU), Capecitabine (Xeloda®), Arabinosylcytosine / Cytarabin (Alexan®) or Gemcitabine (Gemzar®); (vi) purin antagonists such as 6-mercaptopurine (Puri-Nethol®), 6-thioguanine or fludarabine (Fludara®) and finally (vii) folic acid antagonists such as methotrexate (Farmitrexat®) or premetrexed (Alimta®).
Examples of target specific anti-cancer drug classes used in experimental or standard cancer therapy include but are not limited to (i) kinase inhibitors such as e.g. lmatinib (Glivec®), ZD-1839 / Gefitinib (Iressa®), Bay43-9006 (Sorafenib), SU11248 / Sunitinib (Sutent®) or OSI-774 / Erlotinib (Tarceva®); (ii) proteasome inhibitors such as PS-341 / Bortezumib (Velcade®); (iii) histone deacetylase inhibitors like SAHA, PXD101 , MS275, MGCD0103, Depsipeptide / FK228, NVP-LBH589, NVP-LAQ824, Valproic acid (VPA) and butyrates (iv) heat shock protein 90 inhibitors like 17-allylaminogeldanamycin (17-AAG); (v) vascular targeting agents (VTAs) like combretastin A4 phosphate or AVE8062 / AC7700 and anti-angiogenic drugs like the VEGF antibodies, such as Bevacizumab (Avastin®), or KDR tyrosine kinase inhibitors such as PTK787 / ZK222584 (Vatalanib); (vi) monoclonal antibodies such as Trastuzumab (Herceptin®) or Rituximab (MabThera / Rituxan®) or Alemtuzumab (Campath®) or Tositumab (Bexxar®) or C225/ Cetuximab (Erbitux®) or Avastin (see above) as well as mutants and conjugates of monoclonal antibodies, e.g. Gemtuzumab ozogamicin (Mylotarg®) or lbritumomab tiuxetan (Zevalin®), and antibody fragments; (vii) oligonucleotide based therapeutics like G-3139 / Oblimersen (Genasense®); (viii) Toll-like receptor / TLR 9 agonists like Promune®, TLR 7 agonists like Imiquimod (Aldara®) or lsatoribine and analogues thereof, or TLR 7/8 agonists like Resiquimod as well as immunostimulatory RNA as TLR 7/8 agonists; (ix) protease inhibitors (x) hormonal therapeutics such as anti-estrogens (e.g. Tamoxifen or Raloxifen), anti-androgens (e.g. Flutamide or Casodex), LHRH analogs (e.g. Leuprolide, Goserelin or Triptorelin) and aromatase inhibitors.
Other known target specific anti-cancer agents which may be used for combination therapy include bleomycin, retinoids such as all-trans retinoic acid (ATRA), DNA methyltransferase inhibitors such as the 2-deoxycytidine derivative Decitabine (Dacogen®) and 5-azacytidine, alanosine, cytokines such as interleukin-2, interferons such as interferon α2 or interferon-γ, death receptor agonists, such as TRAIL, DR4/5 agonistic antibodies, FasL and TNF-R agonists.
As exemplary anti-cancer agents, which may be useful in the combination therapy according to the present invention, any of the following drugs may be mentioned, without being restricted thereto, 5 FU, actinomycin D, ABARELIX, ABCIXIMAB, ACLARUBICIN, ADAPALENE, ALEMTUZUMAB,
ALTRETAMINE, AMINOGLUTETHIMIDE, AMIPRILOSE, AMRUBICIN, ANASTROZOLE, ANCITABINE, ARTEMISININ, AZATHIOPRINE, BASILIXIMAB, BENDAMUSTINE, BEVACIZUMAB, BEXXAR, BICALUTAMIDE, BLEOMYCIN, BORTEZOMIB, BROXURIDINE, BUSULFAN, CAMPATH, CAPECITABINE, CARBOPLATIN, CARBOQUONE, CARMUSTINE, CETRORELIX, CHLORAMBUCIL, CHLORMETHINE, CISPLATIN, CLADRIBINE, CLOMIFENE, CYCLOPHOSPHAMIDE, DACARBAZINE, DACLIZUMAB, DACTINOMYCIN, DAUNORUBICIN, DECITABINE, DESLORELIN, DEXRAZOXANE, DOCETAXEL, DOXIFLURIDINE, DOXORUBICIN, DROLOXIFENE, DROSTANOLONE, EDELFOSINE, EFLORNITHINE, EMITEFUR, EPIRUBICIN, EPITIOSTANOL, EPTAPLATIN, ERBITUX, ERLOTINIB, ESTRAMUSTINE, ETOPOSIDE, EXEMESTANE, FADROZOLE, FINASTERIDE, FLOXURIDINE, FLUCYTOSINE, FLUDARABINE, FLUOROURACIL, FLUTAMIDE, FORMESTANE, FOSCARNET, FOSFESTROL, FOTEMUSTINE, FULVESTRANT, GEFITINIB, GENASENSE, GEMCITABINE, GLIVEC, GOSERELIN, GUSPERIMUS, HERCEPTIN, IDARUBICIN, IDOXURIDINE, IFOSFAMIDE, IMATINIB, IMPROSULFAN, INFLIXIMAB, IRINOTECAN, IXABEPILONE, LANREOTIDE, LETROZOLE, LEUPRORELIN, LOBAPLATIN, LOMUSTINE, LUPROLIDE, MELPHALAN, MERCAPTOPURINE, METHOTREXATE, METUREDEPA, MIBOPLATIN, MIFEPRISTONE, MILTEFOSINE, MIRIMOSTIM, MITOGUAZONE, MITOLACTOL, MITOMYCIN, MITOXANTRONE, MIZORIBINE, MOTEXAFIN, MYLOTARG, NARTOGRASTIM, NEBAZUMAB, NEDAPLATIN, NILUTAMIDE, NIMUSTINE, OCTREOTIDE, ORMELOXIFENE, OXALI- PLATIN, PACLITAXEL, PALIVIZUMAB, PATUPILONE, PEGASPARGASE, PEGFILGRASTIM, PEMETREXED, PENTETREOTIDE, PENTOSTATIN, PERFOSFAMIDE, PIPOSULFAN, PIRARUBICIN, PLICAMYCIN, PREDNIMUSTINE, PROCARBAZINE, PROPAGERMANIUM, PROSPIDIUM CHLORIDE, RALOXIFEN, RALTITREXED, RANIMUSTINE, RANPIRNASE, RASBURICASE, RAZOXANE, RITUXIMAB, RIFAMPICIN, RITROSULFAN, ROMURTIDE, RUBOXISTAURIN, SARGRAMOSTIM, SATRAPLATIN, SIROLIMUS, SOBUZOXANE, SORAFENIB, SPIROMUSTINE, STREPTOZOCIN, SUNITINIB, TAMOXIFEN, TASONERMIN, TEGAFUR, TEMOPORFIN, TEMOZOLOMIDE, TENIPOSIDE, TESTOLACTONE, THIOTEPA, THYMALFASIN, TIAMIPRINE, TOPOTECAN, TOREMIFENE, TRAIL, TRASTUZUMAB, TREOSULFAN, TRIAZIQUONE, TRIMETREXATE, TRIPTORELIN, TROFOSFAMIDE, UREDEPA, VALRUBICIN, VATALANIB, VERTEPORFIN, VINBLASTINE, VINCRISTINE, VINDESINE, VINORELBINE, VOROZOLE and ZEVALIN.
The anti-cancer agents mentioned herein above as combination partners of the compounds according to this invention are meant to include pharmaceutically acceptable derivatives thereof, such as e.g. their pharmaceutically acceptable salts.
The person skilled in the art is aware on the base of his/her expert knowledge of the kind, total daily dosage(s) and administration form(s) of the additional therapeutic agent(s) coadministered. Said total daily dosage(s) can vary within a wide range.
In practicing the present invention, the compounds according to this invention may be administered in combination therapy separately, sequentially, simultaneously, concurrently or chronologically staggered (such as e.g. as combined unit dosage forms, as separate unit dosage forms, as adjacent discrete unit dosage forms, as fixed or non-fixed combinations, as kit-of-parts or as admixtures) with one or more standard therapeutics (chemotherapeutic and/or target specific anti-cancer agents), in particular art-known anti-cancer agents, such as any of e.g. those mentioned above.
In this context, the present invention further relates to a combination comprising a first active ingredient, which is at least one compound according to this invention, and a second active ingredient, which is at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy, such as e.g. in therapy of any of those diseases mentioned herein.
The term "combination" according to this invention may be present as a fixed combination, a non-fixed combination or a kit-of-parts.
A "fixed combination" is defined as a combination wherein the said first active ingredient and the said second active ingredient are present together in one unit dosage or in a single entity. One example of a "fixed combination" is a pharmaceutical composition wherein the said first active ingredient and the said second active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a "fixed combination" is a pharmaceutical combination wherein the said first active ingredient and the said second active ingredient are present in one unit without being in admixture.
A "kit-of-parts" is defined as a combination wherein the said first active ingredient and the said second active ingredient are present in more than one unit. One example of a "kit-of-parts" is a combination wherein the said first active ingredient and the said second active ingredient are present separately. The components of the may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.
The present invention further relates to a pharmaceutical composition comprising a first active ingredient, which is at least one compound according to this invention, and a second active ingredient, which is at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, and, optionally, a pharmaceutically acceptable carrier or diluent, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy.
The present invention further relates to a combination product comprising a.) at least one compound according to this invention formulated with a pharmaceutically acceptable carrier or diluent, and b.) at least one art-known anti-cancer agent, such as e.g. one or more of those mentioned herein above, formulated with a pharmaceutically acceptable carrier or diluent.
The present invention further relates to a kit-of-parts comprising a preparation of a first active ingredient, which is a compound according to this invention, and a pharmaceutically acceptable carrier or diluent; a preparation of a second active ingredient, which is an art-known anti-cancer agent, such as one of those mentioned above, and a pharmaceutically acceptable carrier or diluent; for simultaneous, concurrent, sequential, separate or chronologically staggered use in therapy. Optionally, said kit comprises instructions for its use in therapy, e.g. to treat (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, such as e.g. cancer, more precisely, any of those cancer diseases described above.
The present invention further relates to a combined preparation comprising at least one compound according to this invention and at least one art-known anti-cancer agent for simultaneous, concurrent, sequential or separate administration.
The present invention further relates to combinations, compositions, formulations, preparations or kits according to the present invention having Eg5 inhibitory activity and/or anti-proliferative and/or apoptosis inducing properties.
In addition, the present invention further relates to a method for treating in combination therapy (hyper)proliferative diseases and/or disorders responsive to the induction of apoptosis, such as e.g. cancer, in a patient comprising administering a combination, composition, formulation, preparation or kit as described herein to said patient in need thereof. In addition, the present invention further relates to a method for treating (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis, such as e.g. cancer, in a patient comprising administering in combination therapy separately, simultaneously, concurrently, sequentially or chronologically staggered a pharmaceutically active and therapeutically effective and tolerable amount of a pharmaceutical composition, which comprises a compound according to this invention and a pharmaceutically acceptable carrier or diluent, and a pharmaceutically active and therapeutically effective and tolerable amount of one or more art-known anti-cancer agents, such as e.g. one or more of those mentioned herein, to said patient in need thereof.
In further addition, the present invention relates to a method for treating, preventing or ameliorating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as, for example, benign or malignant neoplasia, e.g. cancer, particularly any of those cancer diseases mentioned herein, in a patient comprising administering separately, simultaneously, concurrently, sequentially or chronologically staggered to said patient in need thereof an amount of a first active compound, which is a compound according to the present invention, and an amount of at least one second active compound, said at least one second active compound being a standard therapeutic agent, particularly at least one art-known anti-cancer agent, such as e.g. one or more of those chemotherapeutic and target-specific anti-cancer agents mentioned herein, wherein the amounts of the first active compound and said second active compound result in a therapeutic effect.
In yet further addition, the present invention relates to a method for treating, preventing or ameliorating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as e.g. benign or malignant neoplasia, e.g. cancer, particularly any of those cancer diseases mentioned herein, in a patient comprising administering a combination according to the present invention.
In addition, the present invention further relates to the use of a composition, combination, formulation, preparation or kit according to this invention in the manufacture of a pharmaceutical product, such as e.g. a commercial package or a medicament, for treating, preventing or ameliorating (hyper)proliferative diseases, such as e.g. cancer, and/or disorders responsive to the induction of apoptosis, particularly those diseases mentioned herein, such as e.g. malignant or benign neoplasia.
The present invention further relates to a commercial package comprising one or more compounds of the present invention together with instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein.
The present invention further relates to a commercial package consisting essentially of one or more compounds of the present invention as sole active ingredient together with instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein.
The present invention further relates to a commercial package comprising one or more chemotherapeutic and/or target specific anti-cancer agents, such as e.g. any of those mentioned herein, together with instructions for simultaneous, concurrent, sequential or separate use with one or more compounds according to the present invention.
The compositions, combinations, preparations, formulations, kits or packages mentioned in the context of the combination therapy according to this invention may also include more than one of the compounds according to this invention and/or more than one of the art-known anti-cancer agents mentioned.
The first and second active ingredient of a combination or kit-of-parts according to this invention may be provided as separate formulations (i.e. independently of one another), which are subsequently brought together for simultaneous, concurrent, sequential, separate or chronologically staggered use in combination therapy; or packaged and presented together as separate components of a combination pack for simultaneous, concurrent, sequential, separate or chronologically staggered use in combination therapy.
The type of pharmaceutical formulation of the first and second active ingredient of a combination or kit-of-parts according to this invention can be similar, i.e. both ingredients are formulated in separate tablets or capsules, or can be different, i.e. suited for different administration forms, such as e.g. one active ingredient is formulated as tablet or capsule and the other is formulated for e.g. intravenous administration.
The amounts of the first and second active ingredients of the combinations, compositions or kits according to this invention may together comprise a therapeutically effective amount for the treatment, prophylaxis or amelioration of a (hyper)proliferative diseases and/or a disorder responsive to the induction of apoptosis, particularly one of those diseases mentioned herein, such as e.g. malignant or benign neoplasia, especially cancer, like any of those cancer diseases mentioned herein.
In addition, compounds according to the present invention can be used in the pre- or post-surgical treatment of cancer.
In further addition, compounds of the present invention can be used in combination with radiation therapy.
A combination according to this invention can refer to a composition comprising both the compound(s) according to this invention and the other active anti-cancer agent(s) in a fixed combination (fixed unit dosage form), or a medicament pack comprising the two or more active ingredients as discrete separate dosage forms (non-fixed combination). In case of a medicament pack comprising the two or more active ingredients, the active ingredients are preferably packed into blister cards which are suited for improving compliance.
Each blister card preferably contains the medicaments to be taken on one day of treatment. If the medicaments are to be taken at different times of day, the medicaments can be disposed in different sections on the blister card according to the different ranges of times of day at which the medicaments are to be taken (for example morning and evening or morning, midday and evening). The blister cavities for the medicaments to be taken together at a particular time of day are accommodated in the respective range of times of day. The various times of day are, of course, also put on the blister in a clearly visible way. It is also possible, of course, for example to indicate a period in which the medicaments are to be taken, for example stating the times.
The daily sections may represent one line of the blister card, and the times of day are then identified in chronological sequence in this column.
Medicaments which must be taken together at a particular time of day are placed together at the appropriate time on the blister card, preferably a narrow distance apart, allowing them to be pushed out of the blister easily, and having the effect that removal of the dosage form from the blister is not forgotten.
Bioloqical Investigations
The ATPase activity of Eg5 kinesin motor domains (Cytoskeleton, cat. No. EG01 ) can be used to monitor the effects of modulating agents. The test compounds are dissolved as 10 mM solutions in dimethylsulfoxide (DMSO). 2 μl of appropriate DMSO dilutions of the test compounds are added to each well of a 96 well flat bottom plate. Each compound dilution is tested as triplicates. The reagents are added and the final reaction of the standard assay contains 15 mM Pipes, pH 6.8, 5.0 mM MgCI2, 0.5 mM KCI, 1 mM EGTA, 0.1 mg/ml BSA, 1 μM Paclitaxel, 250 nM preformed microtubules (Cytoskeleton, cat. No. MT001 ), 300 μM ATP, and Eg5 protein (50 ng) in a reaction volume of 100 μl. The controls include buffer wells with ATP and 2% DMSO. Reactions are started by the addition of ATP, incubated at room temperature for 30 min., and terminated by removing 20 μl of the reaction volume and adding it to 80 μl of 1 M perchloric acid, followed by the addition of 80 μl Malachite green reagent. Malachite green reagent is prepared by mixing a solution of 4.2 g ammonium molybdate in 100 ml 4 N HCI with a solution of 0.135 g Malachite green in 300 ml H2O. The reactions are incubated for a further 20 min. and then read at 615 nm.
The corresponding IC50 values of the compounds for Eg5 inhibition are determined from the concentration-effect curves.
Representative inhibitory values [measured as -log IC50 (mol/l)] determined in the aforementioned assay follow from the following table A, in which the numbers of the compounds correspond to the numbers of the examples.
Table A
Inhibition of Eg5 activity
The anti-proliferative / cytotoxic activity of the compounds described herein can be tested on subclones of RKO human colon adenocarcinoma cells (Schmidt et al., Oncogene 19, 2423-2429; 2000) using the Alamar Blue cell viability assay (described in O'Brien et al. Eur J Biochem 267, 5421- 5426, 2000). The compounds are dissolved as 10 mM solutions in DMSO and subsequently diluted in semi-logarithmic steps. DMSO dilutions are further diluted 1 :100 into Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum to a final concentration twice as much as the final concentration in the test. RKO subclones are seeded into 96 well flat bottom plates at a density of 4000 cells per well in a volume of 50 μl per well. 24 hours after seeding the 50 μl each of the compound dilutions in DMEM medium are added into each well of the 96 well plate. Each compound dilution is tested as triplicates. Wells containing untreated control cells are filled with 50 μl DMEM medium containing 1 % DMSO. The cells are then incubated with the substances for 72 hours at 370C in a humidified atmosphere containing 5% carbon dioxide. To determine the viability of the cells, 10 μl of an Alamar Blue solution (Biosource) are added and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm. For the calculation of the cell viability the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values. The Graphpad Prism program is used for the calculation of EC50 values for anti-proliferative / cytotoxic activity out of the obtained dose- response curves.
To determine the cell cycle specific mode of action, subclones of RKO colon adenocarcinoma cells (RKOp27 as described by Schmidt et al. in Oncogene 19, 2423-2429; 2000) are seeded into 96 well flat bottom plates at a density of 16000 cells per well in a volume of 50 μl per well in DMEM growth medium with 10% FCS containing 10 μM Ponasterone A. 24 hours after seeding the 50 μl each of the compound dilutions in DMEM medium are added into each well of the 96-well plate. Each compound dilution is tested as triplicates. Wells containing untreated control cells are filled with 50 μl DMEM medium containing 1% DMSO. The cells are then incubated with the substances for 72 hours at 370C in a humidified atmosphere containing 5% carbon dioxide. To determine the viability of the cells, 10 μl of an Alamar Blue solution (Biosource) are added and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm. For the calculation of the cell viability the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values. The Graphpad Prism program is used for the calculation of EC50 values out of the obtained dose-response curves. Viability is compared of proliferating cells grown in the absence of the inducer Ponasterone A, versus viability of cells arrested by the expression of ectopic p27Kip1 induced by Ponasterone A.
Representative values for anti-proliferation / cytotoxicity [measured as -log EC50 (mol/l)] determined in the aforementioned assays follow from the following table B, in which the numbers of the compounds correspond to the numbers of the examples.
Table B
Anti-proliferative / cytotoxic activity on RKO colon cancer cells
The value of -log EC50 [mol/l] RKO p27 induced (arrested) was below the minimum determined by the assay specification (< 5.0, < 5.5 or < 6.0).
The induction of apoptosis can be measured by using a Cell death detection ELISA (Roche Biochemicals, Mannheim, Germany). NCI-H460 non-small cell lung cancer cells are seeded into 96 well flat bottom plates at a density of 10000 cells per well in a volume of 50 μl RPMI medium (containing 10% fetal calf serum) per well. The compounds are dissolved as 10 mM solutions in DMSO and subsequently diluted in semi-logarithmic steps. DMSO dilutions are further diluted 1 :100 into RPMI medium (containing 10% fetal calf serum) to a final concentration twice as much as the final concentration in the test. 24 hours after seeding the 50 μl each of the compound dilutions in RPMI medium are added into each well of the 96 Well plate. Each compound dilution is tested at least as duplicates. Wells containing untreated control cells are filled with 50 μl RPMI medium containing 1% DMSO. The cells are then incubated with the substances for 24 hours at 370C in a humidified atmosphere containing 5% carbon dioxide. As a positive control for the induction of apoptosis, cells are treated with 50 μM Cisplatin (Gry Pharmaceuticals, Kirchzarten, Germany). Medium is then removed and the cells are lysed in 200 μl lysis buffer. After centrifugation as described by the manufacturer, 10 μl of cell lysate is processed as described in the protocol. The degree of apoptosis is calculated as follows: The absorbance at 405 nm obtained with lysates from cells treated with 50 μM cisplatin is set as 100 cpu (cisplatin units), while an absorbance at 405 nm of 0.0 is set as 0.0 cpu. The degree of apoptosis is expressed as cpu in relation to the value of 100 cpu reached with the lysates obtained from cells treated with 50 μM cisplatin.
Experimental perturbation of Eg5 function causes a characteristic malformation of the mitotic spindle, which can be examined by confocal laser scanning microscopy. HeLa cervical cancer cells are grown overnight on glass cover slips (Nunc™ Lab-Tek™ Chamber Slides) in 1800 μl DMEM medium containing 10% fetal calf serum. The test compounds are dissolved as 10 mM solutions in DMSO. Appropriate DMSO dilutions of the test compounds are further diluted 1 :20 into DMEM medium containing 10% fetal calf serum to a final concentration ten times as much as the final concentration in the test. 24 hours after seeding, 200 μl of the compound dilutions in DMEM medium are added into each well of the cover slip. As a control, 200 μl DMEM medium containing 5% DMSO are added. 24 hours after incubation with the test compounds, the cells are washed with PBS, and fixed with 3.7% formaldehyde in H2O for 20 min. at 370C. Subsequently, cells are washed with PBS and incubated with 0,1 % Triton X-100 in a buffer containing 1.471 mM KH2PO4, 8.504 mM Na2HPO4, 137 mM NaCI, 1.325 mM CaCI2, 2.685 mM KCI, 0.542 mM MgCI2, pH 7.2 for 15 min. at room temperature. For saturation of non-specific binding, cells are incubated in 2% BSA/10% FCS in PBS (= blocking buffer) for 30 min. at room temperature prior to incubation with anti-alpha tubulin monoclonal antibodies (Sigma, #T5168; 1 :1000), followed by Cy3-conjugated rabbit anti-mouse IgG (H+L) antibody (Jackson lmmuno Research; 1 :1000). All antibody incubations are performed for one hour at 37 0C in blocking buffer, and cells are washed three times in PBS between different incubations. DNA is counterstained with Hoechst 33342 (0.1 μg/ml). Coverslips are mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined with a Leica TCS SP2 confocal laser scanning microscope fitted with appropriate filters (Leica Microsystems, Bensheim, Germany).
Some of the compounds according to this invention may be efficacious against p-glycoprotein mediated multidrug-resistent tumour cell lines (e.g. HCT-15), that can be measured as follows: All cell lines used are cultured at standard conditions in a tissue culture incubator at 370C, 5% CO2 and 95% humidity. At day 1 , cells are detached with Trypsin / EDTA and pelleted by centrifugation. Cells are resuspended at the appropriate density in culture medium, seeded into 96well microtiter plates and incubated over night in a tissue culture incubator at 370C, 5% CO2 and 95% humidity. Stock solution of all compounds to be tested are dissolved at 1 OmM in DMSO and at day 2 added to the microtiter plates in the desired dilutions. The final DMSO concentration in the microtiter plates is kept at 0.5 %. Control cells are treated with culture medium including a final concentration of 0.5% DMSO only. The microtiter plates are incubated with the compounds in a tissue culture incubator at 370C, 5% CO2 and 95% humidity for further 72 hours. To determine the viability of the cells at day 5, an Alamar Blue solution (Biosource) is added at 1/10 culture volume to the microtiter plates. The cells are incubated in a tissue culture incubator at 370C, 5% CO2 and 95% humidity for additional 1-6 hours and the fluorescence is measured at an extinction of 544 nm and an emission of 590 nm. For the calculation of the cell viability the emission value from untreated cells is set as 100% viability and the emission rates of treated cells are set in relation to the values of untreated cells. Viabilities are expressed as % values.
The Graphpad Prism program is used for the calculation of EC50 values out of the obtained dose- response curves.

Claims

Patent Claims
1. Compounds of formula I
(I) in which
R1 is hydrogen or halogen,
R2 is 1-4C-alkyl,
A is Aryl- 1 -4C-al kyl , in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is 1-4C-alkyl, trifluoromethyl, 1-4C-alkoxy, hydroxyl, halogen, or completely or predominantly fluorine-substituted 1-4C-alkoxy, R31 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy, R4 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl or 3-7C-cycloalkyl-1-4C-alkyl,
R5 is hydrogen or 1-4C-alkyl, R6 is hydrogen or 1-4C-alkyl, B is phenyl, or R7- and R71-substituted phenyl, in which R7 is 1-4C-alkyl, trifluoromethyl, cyano, 1-4C-alkoxy, halogen, carboxyl, 1-4C-alkylcarbonyl, methylenedioxy, ethylenedioxy, 1-4C-alkoxy-1-4C-alkyl, hydroxy-1-4C-alkyl, or completely or predominantly fluorine-substituted 1-4C-alkoxy, R71 is hydrogen, halogen, 1-4C-alkyl or 1-4C-alkoxy, n is 2, 3, 4, 5 or 6, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
2. Compounds of formula I according to claim 1 , in which
R1 is hydrogen or halogen,
R2 is methyl or ethyl,
A is Arylmethyl, in which
Aryl is phenyl, or R3- and R31-substituted phenyl, in which
R3 is methyl, ethyl, trifluoromethyl, methoxy, ethoxy, hydroxyl, halogen, difluoromethoxy or trifluoromethoxy,
R31 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy,
R4 is methyl, ethyl, propyl, isopropyl, sec-butyl, cyclopropyl or cyclopropyl methyl, R5 is hydrogen, methyl, ethyl, propyl or isopropyl,
R6 is hydrogen, methyl, ethyl, propyl or isopropyl,
B is phenyl, or R7- and R71-substituted phenyl, in which
R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, acetyl, methylenedioxy, ethylenedioxy, methoxymethyl, hydroxymethyl, difluoromethoxy or trifluoromethoxy,
R71 is hydrogen, halogen, methyl, ethyl, methoxy or ethoxy, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
3. Compounds of formula I according to claim 1 , in which
R1 is hydrogen, chlorine or bromine,
R2 is methyl or ethyl,
A is benzyl, halobenzyl (e.g. fluorobenzyl, chlorobenzyl or bromobenzyl), methylbenzyl, methoxybenzyl or hydroxybenzyl, R4 is ethyl, propyl, isopropyl, sec-butyl or cyclopropyl, either
R5 and R6 are both hydrogen, or
R5 is methyl, and R6 is hydrogen, or
R5 is ethyl, and R6 is hydrogen, or
R5 is propyl, and R6 is hydrogen, or
R5 is isopropyl, and R6 is hydrogen, or
R5 and R6 are both methyl,
B is phenyl, or R7- and R71-substituted phenyl, in which R7 is methyl, ethyl, trifluoromethyl, cyano, methoxy, ethoxy, halogen, carboxyl, methylenedioxy, ethylenedioxy, methoxymethyl or hydroxymethyl, R71 is hydrogen, halogen, methyl or ethyl, n is 2 or 3, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
4. Compounds of formula I according to any of the preceding claims comprising one or more of the following:
R1 is chlorine or bromine;
R2 is methyl;
A is benzyl;
R4 is ethyl or isopropyl; either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl;
B is 4-methylphenyl, 3-methylphenyl, 4-trifluoromethyl phenyl, 4-methoxyphenyl, 4-fluorophenyl, A- chlorophenyl, 4-bromophenyl or 3-fluoro-4-methyl phenyl; and n is 3; and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
5. Compounds of formula I according to claim 1 , in which R1 is hydrogen, chlorine, bromine or fluorine,
R2 is methyl,
A is benzyl,
R4 is ethyl, isopropyl, sec-butyl, cyclopropyl or cyclobutyl, either
R5 and R6 are both hydrogen, or
R5 and R6 are both methyl,
B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, 4-trifluoromethyl phenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methylphenyl, 3,4- dichlorophenyl or 2,3-dichlorophenyl, n is 2, 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
6. Compounds of formula I according to claim 1 , in which R1 is chlorine, bromine or fluorine,
R2 is methyl,
A is benzyl,
R4 is ethyl, isopropyl, sec-butyl or cyclopropyl,
R5 and R6 are both hydrogen,
B is 4-methylphenyl, 3-methylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, A- methoxyphenyl, 3-fluoro-4-methylphenyl, 2-fluoro-4-methyl phenyl or 3,4-dichlorophenyl n is 3 or 4, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
7. Compounds of formula I according to any of the preceding claims, wherein said compounds have the configuration as shown in formula I*
and the salts thereof.
8. Compounds of formula I, selected from:
(1 ) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5- yl)-propyl]-4-methyl-benzamide,
(2) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide,
(3) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-3-methyl-benzamide,
(4) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-fluoro-benzamide,
(5) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methoxy-benzamide,
(6) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-bromo-benzamide,
(7) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-chloro-benzamide,
(8) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide,
(9) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-propyl]-4-methyl-benzamide,
(10) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-trifluoromethyl-benzamide,
(11 ) N-[(RS)-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl- propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide,
(12) N-(4-Amino-butyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide,
(13) N-(2-Amino-ethyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide, (14) N-(2-Amino-ethyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide,
(15) N-[(RS)-(6-Benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5-c]pyrimidin-5-yl)-methyl- propyl]-N-(2-dimethylamino-ethyl)-4-methyl-benzamide,
(16) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo [1 ,5- c]pyrimidin-5-yl)-1-cyclobutyl-methyl]-4-methyl-benzamide,
(17) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-butyl]-4-methyl-benzamide,
(18) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- clpyrimidin-δ-yO-i-cyclopropyl-methyll^-methyl-benzamide,
(19) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-1-cyclopropyl-methyl]-4-bromo-benzamide,
(20) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide,
(21 ) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-2-fluoro-4-methyl-benzamide,
(22) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-3,4-dichloro-benzamide,
(23) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-chloro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-2,3-dichloro-benzamide,
(24) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-bromo-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide,
(25) N-(3-Amino-propyl)-N-[(RS)-1-(6-benzyl-3-fluoro-2-methyl-7-oxo-6,7-dihydro-pyrazolo[1 ,5- c]pyrimidin-5-yl)-2-methyl-propyl]-4-methyl-benzamide, and the salts, stereoisomers and the salts of the stereoisomers of these compounds.
9. Process for preparing a compound according to any of claims 1 to 8, comprising at least one of the steps
(i) benzoylation of a compound of formula II, with the meanings of R1 , R2, A, R4, R5 and R6 as indicated for the compounds according to any of claims 1 to 8, or with at least one of R5 and R6 being part of a protective group,
(H) (I)
(ii) halogenation of a compound of formula I wherein R1 = H to give a compound of formula I wherein R1 = halogen, or
(I) R1 = H (I) R1 = halogen
(iii) optionally the removal of protecting groups represented by at least one of R5 and R6 as indicated under (i).
10. Compounds according to any of the claims 1 to 8 for use in the treatment of diseases.
11. A pharmaceutical composition comprising one or more compounds according to any of the claims 1 to 8 together with customary pharmaceutical auxiliaries and/or excipients.
12. Use of the compounds according to any of the claims 1 to 8 in the manufacture of pharmaceutical compositions for treating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as, for example, benign and/or malignant neoplasia, e.g. cancer.
13. Pharmaceutical composition for treating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, which include benign neoplasia and malignant neoplasia, including cancer, comprising a compound according to any of claims 1 to 8.
14. A method for treating, preventing or ameliorating (hyper)proliferative diseases and/or disorders responsive to induction of apoptosis, such as, for example, benign or malignant neoplasia, e.g. cancer, in a mammal comprising administering a therapeutically effective and tolerable amount of one or more compounds according to any of the claims 1 to 8 to said mammal in need thereof.
15. A method for modulating Eg5 kinesin activity comprising administering a therapeutically effective and tolerable amount of one or more compounds according to any of the claims 1 to 8 to a mammal in need of said modulation.
16. A combination comprising a first active ingredient, which is at least one compound according to any of the claims 1 to 8, and a second active ingredient, which is at least one anti-cancer agent selected from the group consisting of chemotherapeutic anti-cancer agents and target-specific anti-cancer agents, for separate, sequential, simultaneous, concurrent or chronologically staggered use in therapy, such as e.g. therapy of (hyper)proliferative diseases of benign or malignant behaviour and/or disorders responsive to the induction of apoptosis, such as, for example, benign or malignant neoplasia, e.g. cancer.
17. A method for treating, preventing or ameliorating hyperproliferative diseases and/or disorders responsive to induction of apoptosis, such as, for example, benign or malignant neoplasia, e.g. cancer, in a patient comprising administering separately, simultaneously, concurrently, sequentially or chronologically staggered to said patient in need thereof an amount of a first active compound, which is a compound according to any of the claims 1 to 8, and an amount of at least one second active compound, said second active compound being an anticancer agent selected from the group consisting of chemotherapeutic anti-cancer agents and target- specific anti-cancer agents, wherein the amounts of the first active compound and said second active compound result in a therapeutic effect.
18. The combination or method according to claim 16 or 17, in which said chemotherapeutic anticancer agents are selected from (i) alkylating/carbamylating agents including Cyclophosphamid, Ifosfamid, Thiotepa, Melphalan and chloroethylnitrosourea; (ii) platinum derivatives including cis- platin, oxaliplatin, satraplatin and carboplatin; (iii) antimitotic agents / tubulin inhibitors including vinca alkaloids, such as e.g. vincristine, vinblastine or vinorelbine, taxanes, such as e.g. Paclitaxel, Docetaxel and analogs as well as formulations and conjugates thereof, and epothilones, such as e.g. Epothilone B, Azaepothilone or ZK-EPO; (iv) topoisomerase inhibitors including anthracyclines, such as e.g. Doxorubicin, epipodophyllotoxines, such as e.g. Etoposide, and camptothecin and camptothecin analogs, such as e.g. lrinotecan or Topotecan; (v) pyrimidine antagonists including 5- fluorouracil, Capecitabine, Arabinosylcytosine / Cytarabin and Gemcitabine; (vi) purin antagonists including 6-mercaptopurine, 6-thioguanine and fludarabine; and (vii) folic acid antagonists including methotrexate and pemetrexed.
19. The combination or method according to claim 16, 17 or 18, in which said target-specific anticancer agents are selected from (i) kinase inhibitors including Imatinib, ZD-1839 / Gefitinib, BAY43- 9006 / Sorafenib, SU11248 / Sunitinib and OSI-774 / Erlotinib; (ii) proteasome inhibitors including PS- 341 / Bortezomib; (iii) histone deacetylase inhibitors including SAHA, PXD101 , MS275, MGCD0103, Depsipeptide / FK228, NVP-LBH589, NVP-LAQ824, Valproic acid (VPA) and butyrates; (iv) heat shock protein 90 inhibitors including 17-allylaminogeldanamycin (17-AAG); (v) vascular targeting agents (VAT) including combretastatin A4 phosphate and AVE8062 / AC7700, and anti-angiogenic drugs including VEGF antibodies, such as e.g. Bevacizumab, and KDR tyrosine kinase inhibitors, such as e.g. PTK787 / ZK222584 (Vatalanib); (vi) monoclonal antibodies including Trastuzumab, Rituximab, Alemtuzumab, Tositumab, Cetuximab and Bevacizumab as well as mutants and conjugates of monoclonal antibodies, such as e.g. Gemtuzumab ozogamicin or lbritumomab tiuxetan, and antibody fragments; (vii) oligonucleotide based therapeutics including G-3139 / Oblimersen; (viii) Toll-like receptor / TLR 9 agonists including Promune®, TLR 7 agonists including Imiquimod and lsatoribine and analogues thereof, or TLR 7/8 agonists including Resiquimod as well as immunostimulatory RNA as TLR 7/8 agonists; (ix) protease inhibitors; (x) hormonal therapeutics including anti-estrogens, such as e.g. Tamoxifen or Raloxifen, anti-androgens, such as e.g. Flutamide or Casodex, LHRH analogs, such as e.g. Luprolide, Goserelin or Triptorelin, and aromatase inhibitors; bleomycin; retinoids including all-trans retinoic acid (ATRA); DNA methyltransferase inhibitors including the 2-deoxycytidine derivative Decitabine and 5-azacytidine; alanosine; cytokines including interleukin-2; interferons including interferon α2 and interferon-γ; and death receptor agonists including TRAIL, DR4/5 agonistic antibodies, FasL and TNF-R agonists.
20. The use, method or combination according to any of the claims 12, 14, 16 and 17, in which said cancer is selected from the group consisting of cancer of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands, esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva; inherited cancers, retinomblastoma and Wilms tumor; leukemia, lymphoma, non-Hodgkins disease, chronic and acute myeloid leukaemia, acute lymphoblastic leukemia, Hodgkins disease, multiple myeloma and T-cell lymphoma; myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers of unknown primary site and AIDS related malignancies.
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