WO2001010832A1 - Composes de pyrrolidine et leur utilisation dans la therapie de maladies hyperproliferatives et de maladies tumorales - Google Patents

Composes de pyrrolidine et leur utilisation dans la therapie de maladies hyperproliferatives et de maladies tumorales Download PDF

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WO2001010832A1
WO2001010832A1 PCT/EP2000/007740 EP0007740W WO0110832A1 WO 2001010832 A1 WO2001010832 A1 WO 2001010832A1 EP 0007740 W EP0007740 W EP 0007740W WO 0110832 A1 WO0110832 A1 WO 0110832A1
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cio
alkenyl
alkyl
aryl
cycloalkenyl
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PCT/EP2000/007740
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German (de)
English (en)
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Harm Brummerhop
Tatjana Achenbach
Rolf Müller
Thorsten Bach
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Vectron Therapeutics Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the present invention relates to new pyrrolidine compounds and their use as medicaments, in particular for the therapy of diseases which are characterized by disorders in the induction of cell death or the regulation of the cell cycle.
  • Apoptosis also called programmed cell death, is the controlled sequence of a series of molecular processes that lead to biochemical and morphological changes and ultimately to the death of a cell. It is a means of selective cell killing, which is of crucial importance both in the selection of autoantigenic T cells in the spleen during the formation of the immune system and in normal embryonic development. In accordance with the importance of apoptosis, a disruption in the molecular processes associated with apoptosis is responsible for the development of many diseases (L. Aravind et al., TIBS 1999, 24, 47).
  • Apoptosis is caused by changes such as a reduction in cell volume, chromatin condensation, DNA fragmentation, cell surface changes, the formation of membrane-bound, apoptotic bodies and the activation of the so-called caspases (ES Alnemri et al, Cell 1996, 87, 171; DW Nicholson et al ., N ⁇ twre 1996, 376, 37; C. Widmann et al., J. Biol. Chem. 1998, 273, 7141; SM Srinivasula et al., J. Biol. Chem. 1998, 273, 10107). These morphological and biochemical changes can be detected by a number of methods, for example by a DNA ladder (M.
  • Pyrrolidine compounds in particular pyrrolidinol (+) - Preussin, induce apoptosis and are specifically active against tumor cells. While for not transformed cells resulted in only a concentration of 47 uM (IC 5 o) value for the cell cytotoxicity induced (+) - Prussian already at concentrations between 1 -6 uM (IC 50 - values) Tumorzellzytotoxiztician. Thus, tumor cells are 9 to 47 times more sensitive to the pyrrolidine compounds according to the invention than non-transformed cells.
  • the pyrrolidine compounds according to the invention in low concentrations lead to an accumulation of cells in the G] phase of the cell cycle and are therefore also suitable for the treatment of diseases which are characterized by disorders in the regulation of the cell cycle ,
  • the present invention accordingly relates to pyrrolidine compounds of the general formula 1
  • C 1 to C 20 alkyl preferably C 1 to C 5 alkyl, particularly preferably C 1 to C 10 alkyl, especially C 1 alkyl, C 2 to C 1Q alkenyl, preferably C 2 to C 8 Alkenyl, particularly preferably C 2 to C 5 alkenyl, in particular C 2 alkenyl, C to C 0 cycloalkyl, preferably C 3 to C 6 cycloalkyl, C 3 to Cio-cycloalkenyl, preferably C 3 to C 6 -
  • Ci- to C ⁇ alkoxy preferably C 5 - to Cio-alkoxy, particularly preferably C 8 - to Cio-alkoxy, in particular C 9 -alkoxy, optionally substituted; m 0 to 10, preferably 0 to 5, particularly preferably 0 to 2, in particular 0 to 1; R 3 H, Cp to C 2 o-alkyl, preferably Cp to C 5 alkyl, particularly preferably C 1 to C 10 alkyl, in particular C 2 alkyl, C 2 to Cio alkenyl, preferably C 2 to C 8 Alkenyl, particularly preferably C to C 5 alkenyl, in particular C alkenyl, C to Cio cycloalkyl, preferably C 3 to C 6 cycloalkyl, C 3 to Cio cycloalkenyl, preferably C 3 to C 6 -
  • Cycloalkenyl Ci- to C 20 -acyl, preferably Ci- to C ⁇ 5 -acyl, particularly preferably Cp to Cio-acyl, especially d-acyl, (CH 2 ) n -aryl or (CH 2 ) n- heteroaryl, preferably with one or more N, S and / or O heteroatoms, optionally substituted; n 0 to 10, preferably 0 to 5, particularly preferably 0 to 2, in particular
  • R 4 (CH 2 ) aryl or (CH 2 ) heteroaryl, preferably with one or more N, S and / or O heteroatoms, optionally substituted.
  • the pyrrolidine compounds of the present invention accordingly do not comprise any compounds which fall under the formula 1 and in which the substituents have the meanings given above.
  • the present invention further provides a pyrrolidine compound of the general formula 1, the substituent P having the general formula Ia
  • Ci to C 2 o-alkyl preferably C 1 -C 5 -alkyl, particularly preferably d to Cio-alkyl, in particular Cp to C 4 alkyl, C 2 to Cio Alkenyl, preferably C 2 to C 8 alkenyl, particularly preferably C 2 to C 4 alkenyl, C 3 to Cio-cycloalkyl, preferably C to C 6 cycloalkyl, C 3 to Cio-cycloalkenyl, preferably C - to C 6 -cycloalkenyl, Cp to C ⁇ 2 -alkoxy, preferably Cp to C 8 -alkoxy, particularly preferably C ⁇ - to C -alkoxy, especially C 2 -alkoxy, Cp to C 20 -alkoxycarbonyl, preferably Cp to Cio- Alkoxycarbonyl, particularly preferably Cp to C 6 -alkoxycarbonyl, especially Cp to C 3 -alkoxy
  • Another object of the present invention is the use of one or more of the pyrrolidine compound described above as a medicament.
  • the pyrrolidine compounds according to the invention are particularly suitable for the therapy of diseases which are characterized by disorders in the induction of cell death or the regulation of the cell cycle.
  • Another subject is accordingly a medicament which contains one or more of the pyrrolidine compounds described above and optionally suitable auxiliaries and / or additives.
  • Another object of the present invention is the use of one or more pyrrolidine compounds according to general formula 1
  • C ⁇ - to C 2 o-alkyl preferably Cp to C ⁇ 5 -alkyl, particularly preferably Cp to Cio-alkyl, in particular CpAlkyl, C 2 - to Cio-alkenyl, preferably C 2 - to C 8 -alkenyl, particularly preferably C 2 to C 5 alkenyl, in particular C 2 alkenyl, C 3 to Cio-cycloalkyl, preferably C 3 to C 6 cycloalkyl, C 3 to Cio-cycloalkenyl, preferably C 3 to C 6 -
  • R 2 H Cp to C 20 alkyl, preferably C 5 - to -CC alkyl, particularly preferred
  • R 3 is H, Cp to C 2 o-alkyl, preferably Cp to C 5 -alkyl, particularly preferably d- to Cio-alkyl, in particular C 2 -alkyl, C 2 - to Cio-alkenyl, preferably C 2 - to C 8 Alkenyl, particularly preferably to C 5 alkenyl, in particular C 2 alkenyl, C 3 to Cio-cycloalkyl, preferably C 3 to C 6 cycloalkyl, C 3 to Cio-cycloalkenyl, preferably C 3 to C 6 - Cycloalkenyl, Cp to C 2 o-acyl, preferably C ⁇
  • auxiliaries and / or additives for the therapy of diseases which are characterized by disorders in the induction of cell death or the regulation of the cell cycle.
  • Auxiliaries and additives are understood to mean substances which change the solubility, stability, release kinetics and / or the biological half-life of the pyrrolidine compound, such as DMSO or buffer solutions.
  • disorders in the induction of cell death are understood to mean disorders in mechanisms that lead to cell death. These mechanisms primarily include necrosis and apoptosis. A disturbance in the sense of the present invention is present especially when cells are no longer able to initiate and / or carry out apoptosis.
  • the cell cycle is the regulated sequence of a large number of molecular processes, which are also manifested in morphological changes as the cell progresses through the cell cycle and which culminate in the division of the cell.
  • the cell moves through four phases characterized by microscopic observation, the Gp, S, G 2 and M phase.
  • Proteins that play a key role in the course of the cell cycle are the so-called cyclins.
  • the cyclin / cyclin dependent kinase pair cyclin E / cdk2 (cyclin dependent kinase) is active in the GpPhase.
  • S phase also activates Cyclin A / cdk2.
  • Cyclin B which is also associated with cdc2, is activated in the late M phase.
  • the activity of the various cyclin-cyclin-dependent kinase pairs is regulated both by association with other proteins, such as p21 CIP , p27 ⁇ p , pl6 rNK or p57 INK2, and by phosphorylation or dephosphorylation or ubiquitinilation (J. Zwicker and R. Müller, 91; C. Desdouets et al., 115; K. Sauer and CF Lehner, 125; A. Koff and K. Polyak, 141; L.
  • a cell is either caused to advance and divide through the cell cycle or is prevented from dividing and then enters a fifth cell cycle phase, the Go phase or resting phase, or also apoptosis.
  • Disruption of the regulation of the cell cycle in the sense of the present invention is therefore understood to mean all the changes which lead to a cell not performing the physiologically normal regulation of the cell cycle, in particular that a cell enters the cell cycle or remains in the cell cycle, although it does should be in the G 0 phase or should enter the Go phase.
  • the present invention furthermore relates to the use of one or more pyrrolidine compounds of the formula 1, characterized in that the substituent P has the general formula Ia,
  • Cp to C 2 o-alkyl preferably Cp to Cis-alkyl, particularly preferably Cp to Cio-alkyl, in particular Cp to C 4 -alkyl, C 2 - to Cio-alkenyl, preferably C 2 to C 8 alkenyl, particularly preferably C 2 to C 4 alkenyl, C 3 to Cio-cycloalkyl, preferably C 3 to C 6 cycloalkyl, C 3 to Cio-cycloalkenyl, preferably C 3 to C 6 -cycloalkenyl, Cp to C ⁇ 2 alkoxy, preferably Cp to C 8 alkoxy, particularly preferably Cp to C 3 alkoxy, especially C 2 alkoxy, Cp to C o-alkoxycarbonyl, preferably Cp to Cio-alkoxycarbonyl, particularly preferred Cp to C 6 alkoxycarbonyl, in particular Cp to C 3 alkoxycarbonyl, Cp to C 2 o-alkyl, preferably C
  • Another object of the present invention is the use of one or more pyrrolidine compound according to formula 1, characterized in that the substituent P has the general formula Ib,
  • a preferred use of the pyrrolidine compounds described above is characterized in that one or more of the pyrrolidine compounds, optionally together with auxiliaries and / or additives, are used to induce apoptosis.
  • pyrrolidine compounds described above are characterized in that one or more of the pyrrolidine compounds, optionally together with auxiliaries and / or additives, are used for the therapy of hyperproliferative diseases.
  • Hyperproliferative diseases in the sense of the present invention are all diseases which are characterized by cell division, since it is stronger than it would be physiologically normal.
  • Another preferred use of the pyrrolidine compounds described above is characterized in that one or more of the pyrrolidine compounds, optionally together with auxiliaries and / or additives, are used for the therapy of tumors, autoimmune diseases and psoriasis.
  • the pyrrolidine compounds of the present invention induce apoptosis in a number of tumor cell lines.
  • (+) - Preussin induced apoptosis in 8 different tumor cell lines at a concentration of 1 - 5.8 ⁇ M (IC o -values) whereas in non-transformed control cells (NLH3T3) cell cytotoxicity was only observed at a concentration of 47 ⁇ M (Ido-value) .
  • the pyrrolidine compounds described above, in particular Preussin are not only able to efficiently kill tumor cells, they also show a remarkable tumor cell specificity. This differential effect on tumor cells and "normal" cells is usually a prerequisite for the successful use of a new chemotherapy drug.
  • a starting point for the synthesis of the pyrrolidine compounds of the present invention is, for example, commercially available (S) - or (R) - pyroglutamic acid, which is converted into enantiomerically pure 2,3-dihydropyrroles of type 3 in 8 steps (T. Bach and H. Brummerhop, Angew Chem. 1998, 110, 3577; T. Bach and H. Brummerhop, Angew. Chem. Int. Ed. Engl. 1998, 37, 3400; T. Bach and H. Brummerhop, J. Prakt. Chem. 1999, 341 , 312).
  • Type 3 2,3-dihydropyrroles can be synthesized as follows. Starting from pyroglutamic acid, the toluenesulfonyl compound 6 can be prepared in 3 stages (E. Hardegger, H. Ott, He / v. Chim. Acta 1955, 38, 312) and both reduced to the methyl-substituted pyrrolidinone 5a (B. Ringdahl et al., J. Med. Chem. 1985, 28, 1760) as well as in the sense of a Corey-House reaction with cuprates (J. Ackermann et al. He / v. Chim. Acta 1990, 73, 122; M Matthes and C. Tamm, He / v. Chim. Acta 1991, 74, 1585) thereby 6 is converted into pyrrolidinones of type 5b.
  • R 2 C 2 -C 20 alkyl, CH 2 - (C 3 -C 10 cycloalkyl), CH 2 - (C 3 -C 10 cycloalkenyl), CH 2 aryl, CH 2 heteroaryl
  • R 2 C 2 -C 20 alkenyl
  • Scheme 3 Synthesis of substituted pyrrolidinones
  • the substituted pyrrolidinones of type 5 can be converted into the dihydropyrroles of type 3 in a 4-step sequence (T. Bach and H. Brummerhop, 1999, supra).
  • R 2 hydrogen, CC 20 alkyl, CH 2 - (C 3 -C 10 cycloalkyl), CH 2 - (C 3 -C 10 cycloalkenyl), CH 2 aryl, CH 2 -O- (CpC 20 - Alkyl), CH 2 -0- (C 3 -C-, 0- cycloalkyl), CH 2 -0- (C 3 -C 1 o-cycloalkenyl), CH 2 -0- (C 1 -C 2 o-acyl ), CH 2 -0-aryl, CH 2 -0-heteroaryl, C 2 -C 2 o-alkylene
  • the cyclic enamides of type 3 can be converted in a [2 + 2] photocycloaddition with aromatic aldehydes to give diastereomeric, bicyclic compounds of types 9a and 9b (T. Bach and H. Brummerhop, Angew. Chem. 1998, 110, 3577; T. Bach and H. Brummerhop, Angew. Chem. Int. Ed. Engl. 1998, 37, 3400).
  • Subsequent opening of the hydrogenolytic oxetane ring leads to pyrrolidinols of type 10a and 10b, which are converted into the diastereomeric pyrrolidinols 10c and lOd are transferred (Scheme 5).
  • R 2 hydrogen, CC 20 alkyl, (MeOH)
  • Ar aryl, heteroaryl, 2.K 2 C0 3 , (MeOH) optionally substituted
  • Another starting point for the synthesis of the pyrrolidine compounds of the present invention is, for example, an ⁇ -amino acid (M. Overhand and SM Hecht, J Chem. 1994, 59, 4721).
  • ⁇ -amino acid M. Overhand and SM Hecht, J Chem. 1994, 59, 4721.
  • enantiomers and non-natural amino acids which can easily be obtained using a variety of synthetic methods (RM Williams, Synthesis of Optically Active ⁇ -Amino Acids, Pergamon Press, Oxford 1989; H.-J. Altenbach in Organic Synthesis Highlights, editor: J. Mulzer, H.-J. Altenbach, M. Braun, K. Krohn, VCH, Weinheim 1991, 300), as the Schöllkopf see bislactimethersynthesis (U. Schöllkopf, Top.
  • the amino acid 12 is provided with a Boc protective group and into the corresponding Weinreb amide 13 (S. Nahm and SM Weinreb, Tetrahedron Lett. 1981, 22, 3815; J.-A. Fehrentz and B. Castro, Synthesis 1983, 676) transferred (Scheme 7).
  • Type 16 compounds can be reduced diastereoselectively to type 17a pyrrolidinols and transformed into type 17b pyrrolidinols by Mitsunobu inversion (O. Mitsunobu, supra).
  • By choosing the starting material all enantiomers of all diastereomers of skeleton 1 are accessible (Scheme 9).
  • (+) - Preussin was synthesized according to the synthesis described by T. Bach and H. Brummerhop (Angew. Chem. 1998, 110, 3577; Angew. Chem. Int. Ed. Engl. 1998, 37, 3400).
  • S commercially available (S) -pyroglutaminol 19 (see Scheme 11)
  • the w-nonyl side chain was built up by nucleophilic suspension with a di - «- octyl cuprate.
  • the endocyclic double bond was formed after acylation of pyrrolidinone 21 with methyl chloroformate.
  • the pyrrolidinone 22 was first reduced to the half-aminal with LiBEt 3 H, which was not isolated, but instead was transacetalated directly to the N, O-acetal 23 using dimethoxypropanone in the presence of camphorsulfonic acid. This was converted into dihydropyrrole 24 using an Et ⁇ zPr 2 / TMSOTf elimination method.
  • (+) - Preussin was completed by converting the main diastereomer 25a into pyrrohdinol 26 by hydrogenolysis.
  • the reduction of the methoxycarbonyl group with LiAlH 4 to the methyl group went smoothly and (+) - Preussin 27 could be isolated as a light yellow oil.
  • the overall yield over nine stages was 10%.
  • the purity of the compound obtained as a light yellow wax was> 97% (GC).
  • the analytical data agree with the data reported for the natural product (JH Johnson et al., J. Antibiot. 1989, 42, 1184) (mass spectrum, ⁇ -NMR, 13 C-NMR, NOESY, rotation value).
  • the Annexin-V kit was developed by Nexins Research BN (Holland), ATP by Pharmacia Biotech (USA), the PARP monoclonal mouse antibody by Pharmingen, Signal Transduction, Dianova (Hamburg, Germany) and the caspase inhibitor zVAD-fmk by Biomol (Hamburg, Germany) received.
  • Flavopiridol (H.H. Sedlacek et al., Int. J. Oncol. 1996, 9, 1143) was used as the control substance. Cdk2 kinase was overexpressed and purified in SF9 insect cells infected with baculovirus.
  • HeLa, MeWo, MCF-7 (with 0.9 mg / 1 insulin), ⁇ IH3T3 and A549 were cultivated in DMEM, PC-3, LNCaP, DU-145 and HL60 in RPMI 1640 medium, each with 10% fetal-bovine- Serum, 100 units / ml penecillin and 100 ⁇ g / ml streptomycin.
  • (+) - Preussin, Cam, Anisomycin and FP were dissolved in DMSO (ethanol) and diluted in various concentrations in the cell culture medium.
  • concentration of DMSO (ethanol) in the medium was always less than 1% (v / v).
  • the cells were incubated at 37 ° C for different times.
  • Treatment of cells with the caspase inhibitor zVAD-fmk The cells were preincubated with the inhibitor (zVAD-fmk; 50 ⁇ M final concentration) in full medium for 1 hour and then treated with the chemotherapeutic agents as described above.
  • the cells from 10 cm plates were harvested and washed twice in phosphate salt buffer (PBS). After the last washing step, the pellets were placed in an equal volume of buffer with 20 mM HEPES, pH 7.8; 450 mM NaCl; 0.2 mM EDTA; 25% glycerol; 5 ⁇ M DTT (dithiothreitol), 5 ⁇ M PMSF; 0.5 ⁇ g / ml leupeptin and 5 ⁇ g / ml aprotinin resuspended. The cells were kept for 5 min. Incubated on ice and then lysed by freezing three times in liquid nitrogen and thawing in a 30 ° C water bath. The lysates were at 13000 x g for 10 min. Centrifuged at 4 ° C and transferred to a new Eppendorf tube. The protein concentration was determined and the extracts at -80 ° C. were kept.
  • PBS phosphate salt buffer
  • Soluble proteins were applied to an SDS-polyacrylamide gel and by
  • the proteins were then blotted onto a nitrocellulose by electrophoresis and identified by immunoblot analysis with the specific antibodies in each case.
  • the nitrocellulose membrane was incubated for 2 hours with the antibodies (1: 1000 diluted in 1 x PBS with 5% milk powder), washed 5 times with 1 x PBS and diluted with horseradish peroxidase-conjugated IgG (1: 2000 diluted) and enhanced chemiluminescence (ECL ) Immunoblot system visualized.
  • the cells were harvested from a 10 cm plate, washed twice with PBS, fixed with 75% ethanol for 1 hour and stained with Hoechst 33258.
  • the flow cytometry analyzes were carried out on a FACStarPlus (Becton Dickenson) carried out.
  • the cell cycle DNA distribution was determined by the cell-fit program or manual classification.
  • Fluorescence microscopy cells were washed with Hoechst 33342 (10 ⁇ M) and propidium iodide (10 ⁇ M) for 10 min. colored and analyzed under the fluorescence microscope at a wavelength of 360 nm. Since Hoechst 33342 stains all nuclei and propidium iodide nuclei with destroyed plasma membrane, nuclei from living, necrotic and apoptotic cells could be analyzed as blue round, pink round and fragmented blue or pink nuclei.
  • the cells were plated in 96 microtiter plates (20,000 cells / well). 16
  • the IC 5 o-value was defined as the concentration of chemotherapeutic agents was needed in each test, to achieve strength at a 50%> reduction of absorption.
  • the IC 50 value was determined graphically by a "dose-response" curve.
  • the cell line to be examined was plated out and treated with FP or (+) - Preussin in various concentrations.
  • the cells were harvested with the supernatant and washed twice in PBS. After the pellet had been taken up in 440 ⁇ l annexin-V binding buffer, 10 ⁇ l annexin FITC (1:10 diluted with annexin binding buffer) and 50 ⁇ l propidium iodide (100 ⁇ g / ml) were added and incubated on ice for 10 min FACStar measured to avoid non-specific bindings.
  • HL-60 cells were cultured in RPMI medium and treated with FP (control) or (+) - Preussin in various concentrations. All cells were collected by centrifugation and the pellet was taken up in 200 ⁇ l ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.2% Triton X-100). After extraction of the DNA with phenol and phenol chloroform / isoamyl alcohol (25: 24: 1), the aqueous solution was precipitated with ethanol.
  • the pellet was taken up in 20 ⁇ l TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), applied to 5-10 ⁇ l on a 2% agarose gel and visualized by ethidium bromide and UV radiation.
  • TE buffer 10 mM Tris-HCl, pH 7.5, 1 mM EDTA
  • the proteins were radiolabelled in cells that had previously been treated with defined concentrations of (+) - Preussin, Anisomycin and FP.
  • the installation was carried out in L-methionine-free RPMI 1640 medium with 7.5 ⁇ Ci [ 35 S] -methionine per 200 ⁇ l medium for the last two hours of chemotherapy induction.
  • the cells were washed in methionine-free medium and worked up as previously described under protein extraction. Trichloroacetic acid was added to the samples (10%> final concentration) and for 10 min. incubated on ice to get the proteins precipitate. After centrifugation, the pellets were resuspended in 50 ⁇ l 0.1 M NaOH and provided with 3 ml scintillation mix. The samples were measured on a scintillation machine (Beckman, USA).
  • (+) - Preussin induces apoptosis in HL-60 cells
  • the molecular mechanisms of apoptosis involve
  • Table 1 Induction of apoptosis by (+) - Preussin in HL-60 cells
  • HL-60 cells show a condensed nucleus after 18 hours. Generally, cells show a dotted staining characteristic of chromatin condensation that occurs during apoptosis.
  • the induction of apoptosis in (+) - Preussin-treated HL-60 cells varies between 27% (500 nM) and 45% (2.5 ⁇ M) (Table 1).
  • Phosphatidylserine is a surface marker that is only presented on the surface during apoptosis and can be detected by Annexin-V.
  • the cells are treated with FITC-coupled Annexin-V and measured on the FACS device. The results correlate with the Propidium iodide-Hoechst staining (Table 1). DNA ladder
  • DNA from (+) - Preussin treated cells was isolated. The DNA was applied to an agarose gel and detected. A 100 bp ladder was used as the marker. DNA from cells was applied which were treated with various (+) - Preussin concentrations (0.5 - 5 ⁇ M). A characteristic DNA ladder is cells that have been treated with 2.5 and 5 ⁇ M.
  • caspase 3 is a protein that is important for many characteristic features of apoptosis
  • the activation of caspase 3 and the breakdown of PARP, an important repair enzyme and substrate of caspase 3 were followed by Western emblot analysis (see Table 1). Both activation of caspase 3 and the 85 kDa, characteristic cleavage product of PARP were detected in the protein extracts.
  • the uncleaved 116 kDa PARP protein was no longer detectable, which correlates with the observations from the maximum propidium iodide staining.
  • (+) - Preussin induces apoptosis in HL-60 cells. It was therefore tested whether (+) - Preussin also shows effects in other tumor cell lines. All rapidly growing cells should be susceptible to an effect of (+) - Preussin, since (+) - Preussin affects the cell cycle (see cell cycle analyzes).
  • Three different prostate carcinoma cell lines PC-3, DU-145 and LNCaP were tested and after incubating the cells for 48 hours with (+) - Preussin, the IC 50 values varied between 3 and 5.8 ⁇ M (Table 2). The immobilized but not transformed cell line NUT3T3 was used as a control.
  • the IC 5 o-value is for this Zelllmie at 47 uM (see Table 2) and 9- up to 47 times higher than in the tumor cell lines tested.
  • the induction of apoptosis is p53 independent because the cell lines PC-3 and DU-145 p53 are negative. Similar observations were made in the lung and breast carcinoma cell lines A549 and MCF-7 (Table 2).
  • Cell cycle analysis (+) - Preussin triggers cell cycle blocking in Gi at low concentrations (0.2 - 2 ⁇ M).
  • the cell cycle phase was determined using FACS analyzes. The number of cells in the Gi phase is higher in all tumor cell lines tested than in the control.
  • (+) - Preussin blocks cells in Gi
  • kinase assays were carried out with the G ⁇ specific kinase cdk2.
  • cdk2 kinase inhibitor FP In comparison with the cdk2 kinase inhibitor FP, a significant inhibition of the kinase activity was qualitatively evident after the addition of (+) - Preussin.
  • (+) - Preussin is a weaker competitive inhibitor than FP.
  • the analysis of the kinase activity indicates that (+) - Preussin is a selective cdk2 kinase inhibitor.

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Abstract

La présente invention concerne de nouveaux composés de pyrrolidine de formule générale (1), ainsi que leur utilisation en tant que médicament, notamment dans la thérapie de maladies qui se caractérisent par des troubles de l'induction de la mort cellulaire ou de la régulation du cycle cellulaire, telles que les maladies hyperprolifératives, les tumeurs et le psoriasis.
PCT/EP2000/007740 1999-08-09 2000-08-09 Composes de pyrrolidine et leur utilisation dans la therapie de maladies hyperproliferatives et de maladies tumorales WO2001010832A1 (fr)

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EP00958405A EP1202962A1 (fr) 1999-08-09 2000-08-09 Composes de pyrrolidine et leur utilisation dans la therapie de maladies hyperproliferatives et de maladies tumorales

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DE19936789A DE19936789A1 (de) 1999-08-09 1999-08-09 Neue Pyrrolidinole und ihre Verwendung für die Tumortherapie
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