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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

• This invention relates to 5,7-disubstituted 3-isopropylpyrazolo[4,3-d]pyrimidine derivatives for inhibiting proliferation and inducing apoptosis in mammalian cells, in particular for preventing, inhibiting and/or treating abnormal vascular smooth muscle cell (VSMC) proliferation and specifically in preventing, inhibiting or treating of restenosis.
• VSMC vascular smooth muscle cell
• Restenosis after coronary angioplasty is due to a proliferation of smooth muscle cells growing in the vascular lumen, beneath the residual fragments of the atherosclerotic plaque, as seen in necropsy studies and examination of the specimens removed by atherectomy. At the histological analysis thrombi or their fibrocellular organization are usually not detectable. Smooth muscle cell proliferation leading to restenosis is very similar to the one observed in the experimental models of response-to-injury, so that these models are used to investigate into the pathogenetic mechanisms of restenosis.
• the main stimulus to the loss of the contractile phenotype and to the start of the smooth muscle cell proliferation is represented by the growth factors delivered by platelets adhered to the disendothelialized wall and by the smooth muscle cells themselves, stretched during the dilatation.
• Other stimuli can be growth factors delivered by monocytes and fibroblasts, by thrombin, endothelin, angiotensin and interleukin 1.
• the elastic recoil of the vessel wall, the plaque debris and the regional wall shear stress can also contribute to restenosis.
• the restenosis tissue is different from the atheromatous plaque in that it is almost only constituted by smooth muscle cells and intercellular matrix, while atheroma is much more complex due to the presence of various kinds of cells, of necrotic debris and lipid substances.
• the smooth muscle cells proliferation also contributes to the pathogenesis of atherosclerosis, but the stimuli starting this process have not been clarified yet; moreover this process is much slower than restenosis, interacting with several factors. Encouraging results have been achieved in the prevention of restenosis after angioplasty in experimental models, but not in humans. In order to reduce the incidence of restenosis one should improve the results of angioplasty, even by the use of atherectomy and intracoronary stents. Among pharmacologic approaches anticoagulants, heparin, antiplatelet agents, calcium-channel blockers, corticosteroids all proved ineffective.
• vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis.
• drugs interfering with cell cycle progression in VSMC are promising candidates for an anti-restenotic therapy.
• Roscovitine (ROSC) has been characterised as a selective inhibitor of cyclin-dependent kinases (CDK) 1 , 2, and 5 in enzyme-based assays (Meijer et al. 1997, J. Biochem. 243: 527-536) inhibiting proliferation of various cell types ranging from numerous cancer cell lines to keratinocytes and fibroblasts.
• CDK cyclin-dependent kinases
• the aim of this invention was to provide more effective compounds in terms of their potency to inhibit PDGF-induced VSMC proliferation. Additionally, without being bound by a theory, an explanation of the mode of action of the compounds in comparison to ROSC itself is provided. Therefore, it is an object of this invention to provide new anti-restenosis compounds having improved selectivity and efficiency index, i.e. that are less toxic yet more efficacious than the analogues known heretofore.
• R7 is R7'-NH-;
• R7' is selected from aryl and arylalkyl, wherein each of the groups can optionally be substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, and amino groups;
• R5 is R5'-NH-;
• R5' is selected from branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkyl alkyl, wherein each of the groups can optionally be substituted with one or more substituents selected from halogen, amino, alkoxy and hydroxy groups, for use as medicaments.
• This invention relates more specifically to said derivatives for use for inhibiting proliferation and inducing apoptosis in mammalian cells, in particular for preventing, inhibiting and/or treating abnormal vascular smooth muscle cell (VSMC) proliferation and specifically in preventing, inhibiting and/or treating of restenosis.
• VSMC vascular smooth muscle cell
• Halogen is selected from fluorine, bromine, chlorine and iodine atoms.
• Haldroxy refers to the group -OH.
• Amino refers to the group -NH 2 .
• Alkyl is a branched or unbranched C 1 -C 6 alkyl chain, preferably selected from the group comprising methyl, propyl, isopropyl, tert-butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl.
• Alkenyl is a branched or unbranched C 2 -C 6 alkenyl chain, containing at least one double bond, preferably selected from the group comprising allyl, vinyl, 1-propenyl, 1-methylethenyl, but-1 to 3-enyl, pent-1 to 4-enyl, isopentenyl, hex-1 to 5-enyl, hept- 1 to 6-enyl, isopentenyl, 3,3-dimethylallyl groups.
• Alkynyl is is a branched or unbranched C 2 -C 6 alkenyl chain, containing at least one triple bond. Preferably, it may be represented by ethynyl or propargyl groups.
• Alkoxy denotes the group -OR 3 , wherein R 3 is alkyl, alkenyl, alkynyl, wherein each of the groups can optionally be substituted with one or more substituents selected from the group comprising halogen, hydroxy and amino groups; R 3 is preferably methyl.
• Aryl or “Ar” is an aromatic carbocyclic group having at least one aromatic ring or multiple condensed rings, preferably having 6 to 14 carbon atoms.
• Aryl is preferably selected from the group comprising phenyl, biphenyl, naphthyl, tetrahydronaphtyl, fluorenyl, indenyl and phenanthrenyl.
• Arylalkyl is the group -R-Ar wherein Ar is an aryl group and R is a branched or unbranched CpC 6 bridging hydrocarbon chain which is saturated or unsaturated.
• the arylalkyl is benzyl.
• Cycloalkyl is C 3 -Ci 5 monocyclic or polycyclic alkyl group.
• C 3 -Ci 5 cycloalkyl is preferably selected from the group comprising cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and adamantyl.
• Cycloalkyl alkyl is the group -R-cycloalkyl wherein cycloalkyl is the cycloalkyl group as defined above and R is the C 1 -C 6 bridging hydrocarbon group which is saturated or unsaturated.
• R7 is arylalkyl-NH-, which can be optionally substituted by one or more substituents selected from the group consisting of halogen, alkoxy, alkyl, hydroxy and amino, in particular R7 is selected from the group consisting of benzylamino and benzylamino substituted by one or more substituents selected from the group consisting of halogen, alkoxy, alkyl, hydroxy and amino.
• the arylalkyl is substituted by one to three substituents.
• R7 is more preferably selected from the group consisting of benzylamino and benzylamino substituted with one to three substituents selected from halogen, methoxy, methyl, hydroxy and amino; in particular R7 is selected from the group consisting of benzylamino, 2-, 3-, 4-fluorobenzylamino, 2-, 3-, 4-chlorobenzylamino, 2,3-difluorobenzylamino, 3,4-difluorobenzylamino, 2,3,4-trifluorobenzylamino, 3- methoxybenzylamino, 2-hydroxy-3-methoxybenzylamino, 2-hydroxy-3- methylbenzylamino, 2-hydroxy-5-fluorobenzylamino, 2-hydroxy-5- chlorobenzylamino, 3-hydroxy-5-chlorobenzylamino, 2- and 4-aminobenzylamino.
• R5 is selected from the group consisting of alkyl-NH-, alkenyl-NH-, alkynyl-NH-, cycloalkyl-NH-, which is substituted by at least one amino and/or hydroxy substituent, more preferably, R5 is substituted by one amino and/or one hydroxy substituent.
• R5 is more preferably selected from 2-aminoethylamino, 3-aminopropylamino, 4- aminobutylamino, 2-aminocyclohexylamino, 3-aminocyclohexylamino, 4- aminocyclohexylamino, (3-amino-2-hydroxypropyl)amino, [2-(2- aminoethoxy)ethyl]amino, and (2-amino-2-methylpropyl)amino.
• One object of this invention are 5,7-disubstituted 3-isopropylpyrazolo[4,3- d]pyrimidines of formula I for use as medicaments.
• the invention also provides a pharmaceutical composition, which comprises at least one 5, 7-disubstituted 3 -isopropylpyrazolo[4,3 -djpyrimidine and at least one pharmaceutically acceptable carrier.
• This invention further constitutes a method for inducing apoptosis and inhibiting proliferation in mammalian cells, in particular for preventing, inhibiting or treating abnormal vascular smooth muscle cell (VSMC) proliferation and specifically in preventing, inhibiting or treating of restenosis, in a mammal in need of such treatment by administering an effective amount 5,7-disubstituted-3- isopropylpyrazolo[4,3-d]pyrimidine to the mammal.
• VSMC vascular smooth muscle cell
• this invention is a pharmaceutical composition of matter comprising the composition in an admixture with one or more pharmaceutical excipients.
• Another aspect of the invention are 5, 7-disubstituted 3-isopropylpyrazolo[4,3- d]pyrimidine of the general formula Ia
• R5 is selected from the group consisting of alkyl-NH-, alkenyl-NH-, alkynyl- NH-, cycloalkyl-NH-, wherein each of these groups may be substituted by at least one substituent selected from amino and hydroxy;
• R7 is selected from the group consisting of 2-fluorobenzylamino, 3- fiuorobenzylamino, 4-fluorobenzylamino, 2-chlorobenzylamino, 3- chlorobenzylamino, 4-chlorobenzylamino, 2,3-difluorobenzylamino, 3,4- difiuorobenzylamino, 2,3,4-trifluorobenzylamino, 3-methoxybenzylamino, 2- hydroxy-5-fluorobenzylamino, 2-hydroxy-5-chlorobenzylamino, 3-hydroxy-5- chlorobenzylamino, 4-aminobenzylamino.
• R5 is selected from the group consisting of 2-aminoethylamino, 3- aminopropylamino, 4-aminobutylamino, 2-aminocyclohexylamino, 3- aminocyclohexylamino, 4-aminocyclohexylamino, (3-amino-2-hydroxypropyl)amino, [2-(2-aminoethoxy)ethyl]amino, and (2-amino-2-methylpropyl)amino.
• the pharmaceutical composition preferably comprises about 1 % to about 95 % of the active ingredient, single-dose forms of administration preferably comprising about 20 % to about 90 % of the active ingredient and administration forms, which are not single-dose preferably comprising about 5 % to about 20 % of the active ingredient.
• Unit dose forms may be, for example, coated tablets, tablets, ampoules, vials, suppositories or capsules.
• Other forms of administration are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions and the like. Examples are capsules containing from about 0.05 g to about 1.0 g of the active ingredient.
• compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.
• solutions of the active ingredient, and in addition also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions are used, if being possible for these to be prepared before use, for example in the case of lyophilised compositions which comprise the active substance by itself or together with a carrier, for example mannitol.
• the pharmaceutical compositions can be sterilised and/or comprise excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilizing agents, salts for regulating the osmotic pressure and/or buffers, and they are prepared in a manner known per se, for example by means of conventional dissolving or lyophilising processes.
• solutions or suspensions mentioned can comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatine.
• Suspensions in oil comprise, as the oily component, the vegetable, synthetic or semisynthetic oils customary for injection purposes.
• Oils which may be mentioned are, in particular, liquid fatty acid esters which contain, as the acid component, a long-chain fatty acid having 8 - 22, in particular 12-22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidonic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, euric acid, brasidic acid or linoleic acid, if appropriate with the addition of antioxidants, for example vitamin E, ⁇ -carotene or 3,5-di-tert-butyl-4- hydroxytoluene.
• a long-chain fatty acid having 8 - 22, in particular 12-22, carbon atoms for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic
• the alcohol component of these fatty acid esters has not more than 6 carbon atoms and is mono- or polyhydric, for example mono-, di- or trihydric alcohol, for example methanol, ethanol, propanol, butanol, or pentanol, or isomers thereof, but in particular glycol and glycerol.
• Fatty acid esters are, for example: ethyl oleate, isopropyl myristate, isopropyl palmitate, "Labrafil M 2375” (polyoxyethylene glycerol trioleate from Gattefosee, Paris), "Labrafil M 1944 CS” (unsaturated polyglycolated glycerides prepared by an alcoholysis of apricot kernel oil and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris), “Labrasol” (saturated polyglycolated glycerides prepared by an alcoholysis of TCM and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris) and/or "Miglyol 812" (triglyceride of saturated fatty acids of chain length C 8 to Ci 2 from HuIs AG, Germany), and in particular vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil
• compositions for oral use can be obtained by combining the active ingredient with one or more solid carriers, if appropriate granulating the resulting mixture, and, if desired, processing the mixture or granules to tablets or coated tablet cores, if appropriate by addition of additional excipients.
• Suitable carriers are, in particular, fillers, such as sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium diphosphate, or calcium hydrogen phosphate, and furthermore binders, such as starches, for example maize, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and/or, if desired, desintegrators, such as the above mentioned starches, and furthermore carboxymethyl-starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
• fillers such as sugars, for example lactose, sucrose, mannitol or sorbitol
• cellulose preparations and/or calcium phosphates for example tricalcium diphosphate, or calcium hydrogen phosphate
• binders such as starches, for example maize
• Additional excipients are, in particular, flow regulators and lubricants, for example salicylic acid, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
• flow regulators and lubricants for example salicylic acid, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
• Coated tablet cores can be provided with suitable coatings which, if appropriate, are resistant to gastric juice, the coatings used being, inter alia, concentrated sugar solutions, which, if appropriate, comprise gum arabic, talc, polyvinylpyrrolidine, polyethylene glycol and/or titanium dioxide, coating solutions in suitable organic solvents or solvent mixtures or, for the preparation of coatings which are resistant to gastric juice, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be admixed to the tablets or coated tablet coatings, for example for identification or characterisation of different doses of active ingredient.
• suitable coatings which, if appropriate, are resistant to gastric juice
• the coatings used being, inter alia, concentrated sugar solutions, which, if appropriate, comprise gum arabic, talc, polyvinylpyrrolidine, polyethylene glycol and/or titanium dioxide, coating solutions in suitable organic solvents or solvent mixtures or, for the preparation of coatings which are resistant
• compositions which can be used orally, are also hard capsules of gelatine and soft, closed capsules of gelatine and a plasticiser, such as glycerol or sorbitol.
• the hard capsules can contain the active ingredient in the form of granules, mixed for example with fillers, such as maize starch, binders and/or lubricants, such as talc or magnesium stearate, and stabilisers if appropriate.
• the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as greasy oils, paraffin oil or liquid polyethylene glycol or fatty acid esters of ethylene glycol or propylene glycol, it being likewise possible to add stabilisers and detergents, for example of the polyethylene sorbitan fatty acid ester type.
• suitable liquid excipients such as greasy oils, paraffin oil or liquid polyethylene glycol or fatty acid esters of ethylene glycol or propylene glycol, it being likewise possible to add stabilisers and detergents, for example of the polyethylene sorbitan fatty acid ester type.
• suitable liquid excipients such as greasy oils, paraffin oil or liquid polyethylene glycol or fatty acid esters of ethylene glycol or propylene glycol, it being likewise possible to add stabilisers and detergents, for example of the polyethylene sorbitan fatty acid ester type.
• Other oral forms of administration are, for example, syrups prepared in the customary manner, which comprise the active ingredient, for
• compositions which can be used rectally, are, for example, suppositories that comprise a combination of the active ingredient with a suppository base.
• Suitable suppository bases are, for example, naturally occurring or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
• Compositions which are suitable for parental administration are aqueous solutions of an active ingredient in water-soluble form, for example of water-soluble salt, or aqueous injection suspensions, which comprise viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if appropriate, stabilizers.
• the active ingredient can also be present here in the form of a lyophilisate, if appropriate, together with excipients, and be dissolved before, parenteral administration by addition of suitable solvents. Solutions such as are used, for example, for parental administration can also be used as infusion solutions. Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic or benzoic acid.
• Tinctures and solutions usually comprise an aqueous-ethanolic base to which, humectants for reducing evaporation, such as polyalcohols, for example glycerol, glycols and/or polyethylene glycol, and re-oiling substances, such as fatty acid esters with lower polyethylene glycols, i.e. lipophilic substances soluble in the aqueous mixture to substitute the fatty substances removed from the skin with ethanol, and, if necessary, other excipients and additives, are admixed.
• humectants for reducing evaporation such as polyalcohols, for example glycerol, glycols and/or polyethylene glycol
• re-oiling substances such as fatty acid esters with lower polyethylene glycols, i.e. lipophilic substances soluble in the aqueous mixture to substitute the fatty substances removed from the skin with ethanol, and, if necessary, other excipients and additives, are admix
• Fig. 1 displays dose-response curves obtained for Compound 1 (5-(2- aminocyclohexylamino)-7-benzylamino-3-isopropylpyrazolo[4,3-d]pyrimidine) and ROSC (roscovitine) measuring DNA synthesis in PDGF-activated VSMC.
• Serum- starved VSMC were pretreated with rising concentrations of Compound 1 (0.1-15 ⁇ M) or ROSC (1-100 ⁇ M) for 30 min following PDGF (20 ng/ml) treatment. After 4 h, cells were incubated with BrdU and PDGF for additional 20 h. BrdU incoorporation was determined 24 h after cell activation with PDGF-BB.
• IC 50 values were calculated by fitting a sigmoidal dose-response curve (variable slope, GraphPad Prism).
• Fig. 2 shows that Compound 1 potently induces Gl -phase arrest in PDGF-BB- activated vascular smooth muscle cells.
• Fig. 3 shows potently induced Gl -phase arrest in PDGF-BB-activated vascular smooth muscle cells by Compound 1.
• Fig. 4 shows the effect of Compound 1 and ROSC on PDGF-induced S-phase entry of VSMC. Serum-starved VSMC were pretreated for 30 min with Compound 1 (5 ⁇ M), ROSC (20 ⁇ M) or vehicle (DMSO 1%) and cultured in the presence or absence of PDGF (20 ng /ml). After the indicated periods of time the percentage of cells in S- phase was determined by flow cytometric analysis of Pi-stained nuclei. Columns show the mean of three independent experiments ⁇ SEM. n.s., not significant, **p ⁇ 0.0 ⁇ , ***p ⁇ 0.00 ⁇ (two-way ANOVA followed Bonferroni post-test versus PDGF-treatment).
• Fig. 5 shows the potent induction of Gl -phase arrest in PDGF-BB-activated vascular smooth muscle cells by Compound 1.
• Fig. 6 shows the effect of Compound 1 and ROSC on early signalling events downstream of the PDGF receptor. Using western blot analyses we excluded any inhibitory influence of ROSC or Compound 1 on MAPK phosphorylation (p38, ERK, JNK) or the activation of Akt and STAT3.
• Fig. 7 shows the effect of Compound 1 and ROSC on cyclin levels.
• Serum-starved VSMCs were pretreated for 30 min with Compound 1 (5 ⁇ M), ROSC (20 ⁇ M) or vehicle (DMSO 1%) prior to stimulation with PDGF-BB (20 ng/ml) for the indicated periods of time.
• Total cell lysates were then subjected to western blot analysis for cyclin Dl, A, E and ⁇ -tubulin. Representative western blots out of at least three independent experiments with consistent results are shown for cyclin Dl (a), cyclin A (b) and cyclin E (c).
• Graphs depict compiled data of densitometrically evaluated cyclin/tubulin ratios for each timepoint normalised to the respective unstimulated vehicle control. Shown are means ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.0l, ***p ⁇ 0.00 ⁇ (two- way ANOVA followed Bonferroni post-test versus PDGF-treatment).
• Fig. 8 shows impact of Compound 1 and ROSC on pocket protein phosphorylation. Serum-starved VSMCs were pretreated for 30 min with Compound 1 (5 ⁇ M), ROSC (20 ⁇ M) or vehicle (DMSO 1%) prior to stimulation with PDGF-BB (20 ng/ml) for the indicated periods of time.
• 7,5-Dihydroxy-3-isopropylpyrazolo[4,3-d]pyrimidine precursor was prepared according to Krystof et al. (Eur. J. Med. Chem. 41,2006, 1405-1411). Unless noted otherwise all cell culture reagents and media were obtained from Lonza Group Ltd. (Basel, Switzerland) and all other reagents from Carl Roth (Karlsruhe, Germany). Trypsin, collagenase II and HAM's F- 12 medium were purchased from Invitrogen (Carlsbad, CA, USA) and DMSO from Fluka (Buchs, Switzerland).
• Roscovitine was obtained from Calbiochem (La Jolla, CA, USA) and recombinant human PDGF-BB from Bachem (Weil am Rhein, Germany).
• Anti-cyclin E, anti- cyclin A, anti- ⁇ -tubulin and anti-plO7 antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA), anti-phospho Ser 807+811 retinoblastoma protein, anti- cyclin Dl and rabbit IgG antibodies from New England Biolabs (Beverly, MA, USA).
• the mouse IgG antibody was obtained from Upstate (Charlottesville, VA, USA).
• the FITC-labeled anti-SM- ⁇ -actin antibody and luminol were purchased from Sigma Aldrich (St.Louis, MO, USA). CompleteTM protease inhibitor was obtained from Roche Diagnostics (Basel, Switzerland). Immuno-BlotTM PVDF membrane and precision plus protein standardTM were purchased from BIO-RAD (Hercules, CA, USA).
• the combined benzene extract was dried (over Na 2 SO 4 ) and than evaporated.
• the evaporated residue 0.5 mL of 2-fluorobenzylamine, 0.6 mL of ethyldiisopropylamine and 12 mL of N-methyl-2-pyrrolidone were stirred at 65 0 C for 3 hours.
• the reaction mixture was evaporated to drynes in vacuo.
• the residue was chromatographed on silica gel eluted with the following solvent system: CHCl 3 /MeOH (99/1), to give 5- chloro-7-(2-fiuorobenzyl)amino-3-isopropylpyrazolo[4,3-d]pyrimidine. Yield 28 %; amorphous.
• VSMC Vascular smooth muscle cell isolation and cultivation.
• VSMC employed in this study originate from two independent isolations from rat thoracic aortas of 3 sibling Sprague-Dawley rats each. Aortas were thoroughly cleaned from surrounding tissue, washed in PBS and incubated with digestion buffer for 3 h (HAM's Fl 2 medium supplemented with 253 U/ml collagenase II, 10 mM HEPES, 0.28 mM ascorbic acid, 0.1% BSA) for 15 min at 37 °C.
• VSMC vascular endothelial cells
• standard medium DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 10% calf serum
• Purity of VSMC cultures was checked by immunostaining of ⁇ -smooth muscle actin with a FITC-labeled antibody.
• VSMC were seeded at indicated density and after 24 h serum-starved (standard medium, 0.1% calf serum) for another 24 h prior to all experiments. Passages 6-14 were used.
• Relative light units were determined using a 96- well plate reader (TECAN GENios ProTM). Experiments were performed in triplicate. A panel of 26 new synthetic derivatives was screened for a general antiproliferative activity in VSMC using crystal violet staining (data not shown). This assay revealed the derivative No. 1 showing the highest activity, with a significantly higher potency than ROSC itself. We, therefore, compared the influence of both compounds on PDGF-BB (20 ng/ml)-induced DNA-synthesis using a BrdU incorporation assay. DNA-synthesis was dose-dependently reduced by both compounds with compound 1 again exerting a stronger antiproliferative activity on VSMC (IC 5 o: 3 ⁇ M) than ROSC (IC 50 : 17 ⁇ M) (Fig. 1).
• VSMC Cell cycle distribution of VSMC was determined after propidium iodide (PI) staining according to Riccardi and Nicoletti (Riccardi and Nicoletti, Nat Protoc 2006, 1(3):1458-1461). Briefly, VSMC were seeded into 12 well plates (1 x 10 5 cells per well), serum-starved, pretreated with test compounds or vehicle (DMSO 1%) for 30 min and subsequently stimulated with PDGF-BB (20 ng/ml).
• PI propidium iodide
• PDGF-BB led to a significantly elevated number of cells in S-phase as early as 10 h after addition of the stimulus indicating that the cells pass late Gl -phase after about 6-8 h and undergo S-phase transition after 8-10 h (black bars).
• Treatment with 5 ⁇ M Compound 1 completely abrogated the PDGF-BB-induced S-phase entry whereas 20 ⁇ M of the CDK inhibitor ROSC only slightly decreased but delayed cell cycle progression by about 8-10 h (grey bars). Consistently ROSC-treated cells also show a delayed entry into G2/M phase ( Fig.5). Further experiments using even up to 25 ⁇ M ROSC for up to 48 h showed no accumulation of VSMC in any phase of the cell cycle (data not shown).
• FIG. 7a shows that cyclin Dl levels are increased from 4 h on up to 24 h after PDGF-BB stimulation.
• ROSC and Compound 1 did not reduce but even markedly increased cyclin Dl levels compared to control cells, confirming that both compounds did not interfere with early signaling events leading to cell cycle entry and progression.
• Compound 1 inhibited expression of the S-phase cyclin A, whereas ROSC only delayed expression of this cyclin (Fig. 7b).
• the pocket proteins retinoblastoma protein (Rb) and pi 07, main CDK targets are hyper-phosphorylated from late Gl -phase (6-8 h) onwards (Fig.8).
• Compound 1 treatment completely abolished, whereas ROSC again only delayed the onset of PDGF-BB-induced pocket protein hyper-phosphory lation (Fig.8).
• Compound 1 and ROSC indeed directly interfere with CDK activity in PDGF- activated VSMC.
• compositions I as an active ingredient, are prepared as follows: Composition
• Preparation process The powdered substances mentioned are pressed through a sieve of mesh width 0.6 mm. Portions of 0.33 g of the mixture are transferred to gelatine capsules with the aid of a capsule-filling machine.
• Preparation process The powdered active ingredient is suspended in Lauroglykol ® (propylene glycol laurate, Gattefosse S. A., Saint Priest, France) and ground in a wet- pulveriser to a particle size of about 1 to 3 ⁇ m. Portions of in each case 0.419 g of the mixture are then transferred to soft gelatine capsules by means of a capsule-filling machine.
• Lauroglykol ® propylene glycol laurate, Gattefosse S. A., Saint Priest, France
• Preparation process The powdered active ingredient is suspended in PEG 400 (polyethylene glycol of Mr between 380 and 420, Sigma, Fluka, Aldrich, USA) and Tween ® 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Ind., Inc., USA, supplied by Sigma, Fluka, Aldrich, USA) and ground in a wet-pulveriser to a particle size of about 1 to 3 ⁇ m. Portions of in each case 0.419 g of the mixture are then transferred to soft gelatine capsules by means of a capsule-filling machine.
• PEG 400 polyethylene glycol of Mr between 380 and 420, Sigma, Fluka, Aldrich, USA
• Tween ® 80 polyoxyethylene sorbitan monolaurate, Atlas Chem. Ind., Inc., USA, supplied by Sigma, Fluka, Aldrich, USA

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