Selective phosphodiesterase 9a inhibitors as medicaments for improving cognitive processes

Translation of description of equivalent WO 2004026286 A2

Selective phosphodiesterase 9A inhibitors as drugs to improve cognitive processes

The invention relates to the use of selective phosphodiesterase 9A (PDE 9A) -lhhibitoren for the preparation of medicaments for improving perception, concentration, learning and / or memory.

The cellular activation of adenylate or guanylate cyclases effects the cyclicality capitalization of ATP or GTP to 5'-3 'cyclic adenosine monophosphate (cAMP) and 5'-3' cyclic guanosine monophosphate (cGMP). These cyclic nucleotides (cAMP and cGMP) are important second messengers and therefore play a central role in cellular signal transduction. Each of them reactivates inter alia, but not exclusively, protein kinases. The protein kinase activated by cAMP is called protein kinase A (PKA), activated by cGMP protein kinase called protein kinase G (PKG). Activated PKA and PKG are able in turn a number of cellular effector phosphoryheren (eg ion channels, G-protein coupled receptors, Strulcturproteine). In this way for the second messengers cAMP and cGMP to control a wide variety of physiological processes in a variety of organs. However, the cyclic nucleotides can also act directly on effector. For example, it is known that cGMP may act on ion channels directly and thus is able to influence the cellular ion concentration (review in: Wei et al, Prog Neurobiol, 1998, 56:... 37-64). A control mechanism for controlling the activity of cAMP and cGMP and thus in turn these physiological processes, the phosphodiesterases (PDE). PDEs hydrolyze the cyclic monophosphates to the inactive monophosphates AMP and GMP. There are now described at least 21 PDE genes (Exp. Opin. Investig. Drugs 2000, 9, 1354-3784). These 21 PDE genes can be due to their sequence homology into 11 PDE families divided (for proposed nomenclature, see http://depts.washmgton.edu/pde/Nomenclatare.html.). are Individual PDE genes within a family distinguished by letters (eg PDE1A and PDE1B). If there occur different splice variants within a gene, this is then indicated by an additional numbering after the letters (eg PDE1 AI).

pictures:

Fig. 1: Intracellular cGMP concentration in primary rat (E18) -Kortex- kulruren after treatment with Example 2. The cells were incubated for 20 min with Example 2 in the indicated concentrations. The medium control indicates the cGMP value in untreated cells. The DMSO content was in the Medium- checks as in the substance-treated cells 0.2%. The cGMP concentration is plotted in fmol / 150,000 cells.

Fig. 2: effect of Example 2 (10 microns) on the potentiation of the focal excitatory postsynaptic potential (fEPSP) after a weak tetanus. Example 2 enhanced the increase in fEPSPs significant when it 10 minutes prior to

was washed in 30 minutes after a weak tetanus (closed symbols). The control was treated with 0.01% DMSO (open symbols). *: P <0.05; Analysis of variance with the factors dosage (0 and 10 uM) and test time point by a factor of measuring time.

Fig. 3: Effect of example 2 on the percentage reduction (Trial 2 / Trial 1) the interaction time in the social recognition test (mean ± SEM). The animals were vehicle (10% ethanol, 20% Solutol, 70% physiological saline), or 0.1 mg / kg, 0.3 mg / kg, 1.0 mg kg or 3.0 mg / kg of Example 2 intra - injected intraperitoneally immediately after the first Zusarnmentreffen (Trial 1). Statistical

Evaluation: * p <0.05.

PDE9A - discovery, iron properties, distribution

The Humane PDE9A (GenBank / EMBL accession number NM_002606, cDNA sequence, see Sequence Listing, SEQ LD NO: l) was cloned and sequenced in 1998.. The amino acid identity with other PDEs does not exceed 34% (PDE8A) and minimal 28% (PDE5A). With a Km of 170 nM PDE9A hochaffm for cGMP. Moreover PDE9A is selective for cGMP (Km for cAMP = 230 .mu.M). PDE9A has no cGMP binding domain on (GAF domain) which would suggest allosteric enzyme regulation by cGMP. In a Western blot analysis it was shown that the PDE9A in human testis, brain,

Small intestine, skeletal muscle, heart, lung, and spleen Thyrnus is expressed. The highest expression was found in the brain, small intestine, heart and spleen (Fisher et al, J Biol Chem, 1998, 273 (25): 15559-15564..). The gene for human PDE9A is located on chromosome 21q22.3 and comprises 20 exons. To date, 20 alternative splice variants of PDE9A have been identified (Guipponi et al, Hum Genet, 1998, 103rd:

386-392; Rentero et al., Biochem. Biophys. Res. Commun., 2003, 301: 686-692). Classical PDE inhibitors do not inhibit human PDE9A. To view LBMX, dipyridamole, SKF94120, rolipram and vinpocetine in concentrations up to 100 uM no inhibition on the isolated enzyme. For Zaprinast, an IC₅₀Value of 35 microns detected (Fisher et al, J. Biol Chem, 1998, 273 (25): 15559-15564.).

The mouse was PDE9A 1998 by Soderling et al. (J. Biol Chem, 1998, 273 (19):. 15553-15558) cloned and sequenced. This is like the human form, high affinity for cGMP with a Km of 70 nM. In the mouse, a particularly high expression was found in the kidney, brain, lung and heart. Murine PDE9A is not inhibited by IBMX in concentrations below 200 uM; the IC50 for Zaprinast is 29 microns (Soderling et al, J. Biol Chem, 1998, 273 (19): 15553-15558..). In the rat brain has been shown that PDE9A is strongly expressed in some regions of the brain. These include olfactory bulb, hippocampus, cortex, basal ganglia and basal forebrain (Andreeva et al, J. Neurosci, 2001, 21 (22): 9068-9076..).

The hippocampus, cortex and basal forebrain in particular play an important role in learning and memory processes.

PDE9A is characterized by a particularly high affinity for cGMP. The Km for cGMP is 170 nM (Fisher et al, J. Biol Chem, 1998, 273 (25)...:

15559-15564). Therefore PDE9A is 10 uM unlike PDE2A (Km =; ... Martins et al, J. Biol Chem, 1982, 257: 1973-1979), PDE5A (Km = 4 microns; Francis et al, J. Biol Chem, 1980, 255: 620-626), PDE6A... (Km = 17 uM; Gillespie and Beavo, J. Biol Chem, 1988, 263 (17):.. 8133-8141). and PDE11A (Km = 0.52 uM; Fawceft et al, PNAS, 2000, 97 (7 ): 3702 - 3707) is active even at low physiological concentrations, fm Unlike PDE2A (Murashima et al.

Biochemistry, 1990, 29: 5285-5292) is not increased, the catalytic activity of PDE9A by cGMP because it has no GAF domain (Beavo et al, Current Opinion in Cell Biology, 2000, 12: 174 -179).. therefore PDE9A Inlήbitoren lead to an increase in the baseline cGMP concentration (see Figure 1). This increase in the baseline cGMP concentration surprisingly led to an improvement in learning and memory in the social recognition test.

Surprisingly, it has been demonstrated that PDE9A inhibitors have an effect on the function of the central nervous system. In particular, now GE was round, that selective PDE9A lhhibitoren for the preparation of medicaments for the

are improving perception, concentration, learning or memory suitable.

A PDE9A inhibitor for the purposes of the invention is a compound which inhibits human PDE 9A under the conditions indicated below with an IC50 of less than

10 .mu.M, preferably less than lμM inhibits.

A selective PDE9A -] _ nhibitor within the meaning of the invention is a Verbmdung, human PDE9A under the conditions indicated below more strongly inhibits the human PDE1C, PDE2A, PDE3B, PDE4B, PDE5A, PDE7B, PDE8A, PDE10A and

PDE11A. It is preferred IC50 (PDE9A) / IC₅₀ (PDE1C, PDE2A, PDE3B, PDE4B, PDE5A, PDE7B, PDE8A, PDE10A undPDEHA) less than 0.2.

Particularly, the selective PDE9A] are hhibitoren for improving perception, concentration, learning, or memory after

Cognitive impairments, as defined in particular in SitaationenKrankheiten / syndromes occur as "mild cognitive impairment", age-associated learning and memory impairments, age-associated memory losses, vascular dementia, craniocerebral trauma, stroke, dementia occurring after strokes ( "post stroke dementia"), post-traumatic dementia, general concentration impairments, concentration impairments in children with learning and memory problems,

Alzheimer's disease, dementia with Lewy bodies, dementia with degeneration of the frontal lobes emschließlich Pick's syndrome, Parkinson's disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob- dementia, HIV dementia, schizophrenia with dementia or Korsakoff's psychosis.

The invention relates to the use of selective PDE9A inhibitors for producing medicaments for improving perception, concentration performance, cognitive processes, learning and / or memory.

The invention further relates to the use of selective hihibitoren PDE9A for the prophylaxis and / or treatment of disorders of perception, concentration, cognitive processes, learning power and / or mnemonic performance.

It may be partly as a result of a disease selected from the group of dementia, stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, depression, or dementia with frontal lobe degeneration disorder.

The invention further relates to the use of PDE9A inhibitors for the treatment of diseases of the central nervous system that can be herapiert by influencing the cGMP level. Thus, the invention relates to, for example, the treatment of dementia, stroke traumatic brain injury, Alzheimer's

Disease, dementia with frontal lobe degeneration, Lewy body dementia, vascular Dementia, Attention Deficit syndromes, attention and concentration problems, Parkinson's disease, schizophrenia, depression, affective disorders, psychosis, neurosis, anxiety, mania or manic-depressive disorders, Pick's disease, pain and epilepsy.

The invention preferably relates to the novel use of inhibitors of formulas PDE9A

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The compounds of formulas (I) and (II) may also exist in form of their salts, solvates or solvates of its salts. In the context of the invention physiologically acceptable salts are preferred.

Physiologically acceptable salts can be salts of the compounds of the invention with inorganic or organic acids. Preference is given to salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or salts with organic carboxylic or sulphonic acids such as acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulphonic acid,

Ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid or Naphthalindisulfon-.

Physiologically acceptable salts can also be metal or ammonium salts of the compounds of the invention. Particularly preferred are Alkalirnetallsalze

(Eg sodium or potassium salts) (for example, magnesium or calcium salts), and ammonium salts derived from ammonia or organic amines arnin such as Emylamin, di- or triethylamine, di- or triethanolamine, dicyclohexylamine ylamine, Dimelnylaminoefhanol, arginine, lysine, ethylenediamine or 2-phenyls.

WO 98/40384 discloses pyrazolopyrimidine which vascular as PDE1-, 2- and 5- inhibitors are distinguished and for the treatment of cardiovascular, cerebrovascular ankungen Erl and disorders of the urogenital can be used.

In CH 396 924, CH 396 925, CH 396 926, CH 396 927, DE 1,147,234, DE 1,149,013, GB 937,726 describe pyrazolopyrimidines which a coronary effects which can be used to treat circulatory disorders of the heart muscle.

In US 3,732,225 describes pyrazolopyrimidines have an antiinflammatory and blood glucose-lowering effect.

In DE 2408906 Styrolpyrazolpyrimidme describes the bielle as antimicro- and anti-inflammatory agents for the treatment of, for example,

Edema can be used.

The compounds of the invention can be prepared by the synthetic scheme described below: <img id="imgf000009_0001" he="20" wi="95" file="imgf000009_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

H₂O₂/ NH₃

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An optionally subsequent reaction of the compounds of formula (I) and (H) with the appropriate (i) solvents and / or (ii) bases or acids leads to the corresponding salts, solvates and / or solvates of the salts.

The active compound can act systemically and / or locally. For this purpose it can be administered in a suitable manner, such as oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, transdermal, conjunctival, otic or as an implant.

For these administration routes, the active ingredient can be administered in suitable Apphkationsformen.

For oral administration known, the active ingredient rapidly and / or in modified form are suitable, such as tablets (uncoated and coated tablets, eg enteric coatings), capsules, granules, pellets, powders, emulsions, suspensions and solutions.

Parenteral administration can take place with avoidance of an absorption step (intravenous, intraarterial, intracardiac, intraspinal or intralumbarly) or with inclusion of an absorption (intramuscular, subcutaneous, intracutaneous, percutaneous, or intraperitoneal). For parenteral administration, suitable administration forms, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilizates and sterile powders.

For the other administration routes, for example, pharmaceutical forms for inhalation (inter alia Pulveri are^ήhalatoren, nebulizers), nasal drops / solutions, sprays; lingual, sublingual or buccal administration, tablets or capsules, suppositories, ear and eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, milk, pastes, dusting powders or

Implants.

The active compounds can be converted in a known per se into the stated administration forms. This is done using inert, nontoxic, pharmaceutically suitable excipients. These include inter alia carriers (eg microcrystalline cellulose), solvents (eg liquid polyethylene glycols), emulsifiers (for example sodium dodecyl sulfate), dispersants (for example polyvinylpyrrolidone), synthetic and natural biopolymers (eg albumin), stabilizers (dantien eg antioxi- such as ascorbic acid), colors ( for example inorganic pigments such as iron oxides) or taste and / or odor.

It has generally proved advantageous to administer amounts of about 0.001 to 30 mg / kg, preferably about 0.01 to 10 mg / kg of body weight to achieve effective results in parenteral administration. In the case of oral administration the quantity is about 0.01 to 100 mg / kg, preferably about 0.1 to 30 mg / kg

Body weight. It may nevertheless be necessary where appropriate to deviate from the amounts mentioned, namely depending on body weight, administration route, individual behavior towards the active ingredient, type of preparation and time or interval at which administration takes place.

PDE Inhibition

Recombinant PDE1C (GenBank / EMBL accession number: NM_005020,

Loughney et al. . J. Biol. Chem 1996 271, 796-806), PDE2A (GenBank / EMBL Accession Number: NM_002599, Rosman et al 1997 Gene 191, 89-95), PDE3B.

(GenBank / EMBL accession number: NM_000922, Miki et al Genomics 1996 36, 476-485.), PDE4B (GenBank / EMBL accession number:.. NM_002600, Obernolte et al Gene 1993 129, 239-247), PDE5A (GenBank / EMBL Accession Number:. NM_001083, Loughney et al Gene 1998 216, 139-147), PDE7B (GenBank / EMBL accession number: NM_018945, Hetman et al Proc Natl Acad Sci USA 2000.....

97, 472-476), PDE8A (GenBank / EMBL accession number:..... AF_056490, Fisher et al Biochem Biophys Res Commun 1998 246, 570-577), PDE9A (GenBank / EMBL accession number NM_002606, cDNA sequence, see Sequence listing, SEQ ID NO: l, Fisher et al, J. Biol Chem, 1998, 273 (25):... 15559-15564), PDEIOA (GenBank / EMBL Accession Number: NM_06661, Fujishige et al J. Biol.. Chem.

. 1999 274, 18438-45), PDE11A (GenBank / EMBL accession number:..... NM_016953, Fawcett et al Proc Natl Acad Sci 2000, 97, 3702-3707) were using the pFASTBAC baculovirus expression system (GibcoBRL) in Sf9 cells expressing. 48 h after infection the cells are harvested and resuspended in 20 mL (IL per culture) lysis buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM MgCl₂, 1.5 mM EDTA,

10% glycerol plus 20 ul protease inhibitor cocktail Set HI [CalBiochem, La Jolla, CA USA]) suspended. The cells are treated at 4 ° C for 1 minute with ultrasound and then centrifuged for 30 minutes at 4 ° C at 10000 rpm. The supernatant (PDE preparation) was collected and stored at -20 ° C. The test substances are dissolved her to determine the in vitro effect on PDE 9A in 100% DMSO and serially diluted. Typically, serial dilutions are made from 200 uM to 1.6 uM (resulting final concentrations in the assay: 4 .mu.M to 0.032 uM). Each 2 ul of the diluted substance solutions are in the wells of microtiter plates (Isoplate; Wallac Inc., Atlanta,

GA) submitted. Then 50 L of a dilution of PDE9A preparation described above are added. The dilution of PDE9A preparation is selected so that during the subsequent incubation less than 70% of the substrate is reacted (typical dilution: 1: 10000; dilution buffer: 50 mM Tris / HCl pH 7.5, 8.3 mM MgCl₂, 1.7 mM EDTA, 0.2% BSA). The substrate,

[8- H] guanosine 3 ', 5'-cyclic phosphate (1 uCi / ul; Amersham Pharmacia Biotech, Piscataway, NJ.) Is 1: 2000 in assay buffer (50 mM Tris / HCl pH 7.5, 8.3 mM MgCl₂, 1.7 mM EDTA) to a concentration of 0.0005 uCi / ul. By adding 50 L (0.025 .mu.Ci) of the diluted substrate, the enzyme reaction is finally started. The test batches are incubated for 60 min at room temperature and the reaction is stopped by adding 25 ul of a dissolved in assay buffer PDE9A inhibitor (eg the inhibitor from preparation 2, 10 uM final concentration). Immediately after 25 .mu.l of a suspension containing 18 mg / mL yttrium scintillation proximity beads (Amersham Pharmacia Biotech., Piscataway, NJ.) Added. The microtiter plates are sealed with a film and left for 60 min at room temperature. Subsequently, the plates for 30 s per well in a Microbeta scintillation counter (Wallac Inc., Atlanta, GA) are measured. IC₅₀- Erte be determined from the graphical plot of the substance concentration versus the percentage inhibition.

The in vitro effect of test substances on recombinant PDE3B, PDE4B, PDE7B, PDE8A, PDEIOA and PDE11A is determined by the assay protocol described above for PDE 9A with the following adaptations: as the substrate [5 ', 8-³H] adenosine 3 ', 5'-cyclic phosphate (1 uCi / ul; Amersham Pharmacia Biotech, Piscataway, NJ.) Are used. The addition of a mhibitorlösung to stop the reaction is not necessary. Instead, in connection is the incubation of substrate and PDE described immediately by addition of the yttrium scintillation proximity beads as above proceeds and thus the reaction is stopped. To determine a corresponding effect on recombinant PDEIC, PDE2A and PDE5A the protocol is additionally adapted as follows: In addition PDEIC calmodulin 10^"7 M and CaCl₂ 3 mM added to the reaction mixture. PDE2A is stimulated 1 uM in the assay by adding cGMP and is assayed with a BSA concentration of 0.01%. For PDEIC and PDE2A is as a substrate [5 ', 8-³H] adenosine 3 ', 5'-cyclic phosphate (1 uCi / ul; Amersham Pharmacia Biotech, Piscataway, NJ.), For PDE5A [8- H] guanosine 3', 5'-cyclic phosphate (1 uCi / ul; Amersham Pharmacia Biotech., Piscataway, NJ) are used.

Inhibition of PDE isoenzymes by Example 2:

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The PDE9A inhibitory effect of Example 1, by an ICso value of

IC₅0 = 5 nM experimental proof. Increasing the intracellular cGMP concentration in neuronal cell cultures

PDE9A inhibitors increase the intracellular neuronal cGMP in cultivated primary cortical neurons.

Rat embryos (embryonic day 7 El - El 9) were decapitated, their heads in with preparation medium (DMEM, Pemcillin / streptomycin; both from Gibco) überfuhrt filled preparation dishes. The scalp and skull was removed, and the exposed brains were transferred into another Petri dish with Präparations- medium. Using a binocular microscope and two forceps, the cerebrum isolated (cortex) and cooled with ice at 4 ° C. This preparation and the separation of the cortical neurons were then following a standard protocol with the papain kit (Worthington Biochemical Corporation, Lakewood, New Jersey 08701, USA) (Huettner et al. J. Neurosci. 1986, 6, 3044-3060.) , The mechanically isolated cortical neurons were about 150,000 cells / well in

200 ul Neurobasal hole (Neurobasal; B27 Supplement, 2 mM L-glutamine, in the presence of penicillin / streptomycin; all agents from Gibco) 7 days 96 well plates (poly-D lysine 100 ug / ml for 30 min pretreated) under standard conditions cultured (37 ° C, 5% CO₂). After 7 days, the medium removed to perform and the cells with HBSS buffer (Hank's balanced salt solution,

Gibco / BRL) washed. Subsequently, 100 .mu.l of test substance Example 2 dissolved in HBSS buffer (previously dissolved in 100% DMSO: 10 mM) added to the cells. 100 ul HBSS were then again added buffer, so that the final concentration of test substance Example 2 was as shown in Fig. 1 indicated and incubated at 37 ° C for 20 min. The assay buffer was then completely removed. Subsequently, the cells in 200 .mu.l of lysis buffer were (cGMP kit code RPN 226; from Amersham Pharmacia Biotech.) Lysed and the cGMP concentration according to the manufacturer's measured. All measurements were performed in triplicate. Statistical analysis was performed with Prism software version 2.0 (GraphPad Software Inc., San Diego, CA USA). Incubation of the primary neurons with Example 2 resulted in an increase of the cGMP content (FIG. 1).

LTP LTP is as a cellular correlate of learning and Gedächtnisvorg viewed ength. To determine whether PDE 9 inhibition has an influence on long-term potentiation, the following method was used:

Rat hippocampi were in an angle of about 70 degrees to the cutting blade placed (Chopper). At intervals of 400 microns of the hippocampus was cut. Sections were a soft using very thoroughly wetted

Brush (marten hair) taken from the blade and in a glass jar with carbo- genisierter cold nutrient solution (124 mM NaCl, 4.9 mM KC1, 1.3 mM MgSO_ι * 7H₂O, 2.5 mM CaCl²⁺ Water free, 1.2 mM BH ^ PO ^ 25.6 mM NaHCO_3y 10 mM glucose, pH 7.4) transferred. During the measurement, the sections were in a temperature-controlled chamber under a liquid level of 1-3 mm height. The

Flow rate was 2.5 ml / min. The preliminary gassing took place under a slightly elevated pressure (about 1 arm) and through a microneedle in the prechamber. The cutting chamber was connected to the antechamber that minicirculation could be maintained. When driving the minicirculation the cannula through the micro flowing carbogen was used. The freshly prepared hippocampal sections were adapted at least 1 hour at 33 ° C in the cutting chamber.

The stimulus intensity was chosen so that the focal excitatory postsynaptic potentials (fEPSP) 30% of the maximum excitatory postsynaptic potential (EPSP), respectively. With A monopolar stimulation electrode consisting of lacquered stainless steel, and a constant-current biphasic stimulus generator (AM Systems 2100), the Schaffer collaterals were locally energized (voltage: 1-5V, pulse width of one polarity 0.1 ms, total pulse 0.2 ms). With Glass electrodes (borosilicate glass with filament, 1-5 MOhm, diameter: 1.5 mm, tip diameter: 3-20 microns), which were filled with normal nutrient solution, were from the stratum radiatum the excitatory postsynaptic potentials (FEPSP). The measurement of field potentials were versus a chlorinated silver reference electrode, which was located on the edge of the slice chamber using a DC voltage amplifier. Filtering the field potentials through a low-pass filter (5 kHz). For statistical analysis of the experiments, the increase (slope) of fEPSPs (fEPSP slope) was determined. The

Recording, analysis and control of the experiment was carried out using a software program (RWLn)_&gt; which was developed in the Department of Neurophysiology of the Leibniz Institute for Neurobiology, Magdeburg. Averaging the fEPSP slope values ​​for each time point and the design stage the charts was performed using the EXCEL software, with an appropriate macro automated data recording.

basal synaptic transmission registers 120 minutes - In the control experiments was initially for 60 seconds. Then four two double pulses were with an interpulse interval of the double pulses of 10 ms and a width of the individual pulses of 0.2 ms (weak tetanus) administered (Zeitpu kt = 0, minutes Figure 2) at a distance of 200 ms. The resulting potentiation of EPSPs were recorded for at least 60 minutes. Example 2 was ngespült the illustrated Experiment 10 minutes before to 30 minutes after stimulation e.

Super of the hippocampus slices with a 10 .mu.M solution of Example 2 resulted in a significant increase in the LTP (Figure 2).

Social Recognition Test: The social recognition test is a learning and memory test. It measures the

Ability of rats to distinguish between known and unknown conspecifics. This test is therefore suitable for examining the learning- or memory-improving effect of test substances.

Adult rats were housed in groups, were 30 minutes ago

Test start individually in test cages. Four minutes before use was the brought test animal in an observation box. After this adaptation time, a juvenile animal was placed with the test animal and measured for 2 minutes, the absolute time, which the adult animal inspects the young (Trial 1). Measured were all clearly directed at the young animal behavior, ie anogenital inspection, pursuit and grooming, during which the old animal was at a distance of not more than 1 cm from the young animal. The juvenile was taken out, and the adult with Example 2 or vehicle treated and then returned to its home cage. After a retention time of 24 hours, the test was repeated (Trial 2). A diminished social interaction time compared with trial 1 would indicate that the adult rat remembers the young animal.

Adult animals were directly following trial 1 either with vehicle (10% Efhanol, 20% Solutol, 70% physiological saline) or 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg or 3.0 mg / kg of example 2 dissolved in 10% ethanol, 20% Solutol, 70% injected physiological saline solution intraperitoneally. Vehicle-treated rats showed no reduction in social interaction time in trial 2 compared with trial 1. They had consequently forgotten that they had contact with the young animal before. Surprisingly, the social interaction time was compared to vehicle-treated reduced significantly in the second run after treatment Example 2. FIG. This means that the substance-treated rats have remembered the juvenile animal and thus Example 2 exhibited an improving effect on learning and memory.

Used abbreviations:

DMSO dimethyl theory theory (in yield) equiv. Equivalent (s)

ESI electrospray ionization (in MS)

HPLC high pressure, high performance liquid chromatography

MS mass spectroscopy

NMR nuclear magnetic resonance spectroscopy

Mp. Melting point

Ausftihrungsb EXAMPLES:

Example 1 6- (Cyclohexylme yl) -l-cyclopentyl-l, 5-d ydro-4H-pyrazolo [3,4-d] pyrimidin-4-one

<img id="imgf000019_0001" he="39" wi="36" file="imgf000019_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

Step la) 5-amino-l-cyclopentyl-lH-pyrazole-4-carbonitrile

<img id="imgf000019_0002" he="29" wi="25" file="imgf000019_0002.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

A solution of ethoxymethylenemalononitrile (7.93 g, 64.9 mmol) in 100 ml of methanol is added at room temperature under argon with slow Cyclopentylhydrazin (6.5 g, 64.9 mmol), then heated under reflux for 3 h and then stirred overnight at room temperature. The solvent is removed on a rotary evaporator and the residue stirred Diefhylether. The solid is drained, washed with Diefhylether and dried under high vacuum. Yield: 7.24 g (63% of theory) MS (ESI): m / z = 177 (M + H)⁺ 1H-NMR (200 MHz, CDC1₃): Δ = 7.5 (s, 1H), 4.45 (br s, 2H), 4:35 (m, IE), 2.2-1.55 (m, 6H) ppm.. Step 1b) 5-amino-l-cyclopentyl-lH-pyrazole-4-carboxamide

<img id="imgf000020_0001" he="35" wi="30" file="imgf000020_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

A solution of 5-amino-l-cyclopentyl-lH-pyrazole-4-carbonitrile (6.74 g, 38.3 mmol) in a mixture of 300 ml Efhanol and 371 ml of concentrated aqueous ammonia solution is added at room temperature with 85 ml of 30% strength hydrogen peroxide solution and stirred overnight at room temperature. Subsequently, the non-aqueous solvents are removed on a rotary evaporator. From the remaining mixture, the product precipitates as a solid, which is suctioned off, washed with Diefhylether and dried under high vacuum. Yield: 5.31 g (71% of theory) MS (ESI): mz = 195 (M + H)⁺ 1H-NMR (200 MHz, CDC1₃): Δ = 7.5 (s, 1H), 5.6-4.8 (broad, 4H), 4:35 (m, 1H), 2.2-

1:55 (m, 8H) ρρm.

Step 1c)

6- (cyclohexylmethyl) -l -cyclopentyl- 1, 5-dihydro-4H-pyrazolo [3, 4-d] pyrimidin-4-one

<img id="imgf000020_0002" he="39" wi="36" file="imgf000020_0002.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="yes" /> Under argon, 75 mg (0:39 mmol) of 5-amino-l-cyclopentyl-lH-pyrazole-4-carboxamide and 183 mg (1.16 mmol, 3 equiv.) In 1.5 ml of absolute Cyclohexylessigsäuremethylester presented Efhanol. At 0 ° C 54 mg of sodium hydride (60% dispersion in mineral oil; 1:35 mmol, 3.5 equiv.) Was added in an argon countercurrent slowly to-. The resulting mixture is heated slowly and stirred for 18 h under reflux. For workup 20 mL of water are added and the mixture extracted several times with ethyl acetate. The combined organic phases are dried over sodium sulfate and concentrated in vacuo. The crude product is purified by preparative HPLC. Yield: 36 mg (31% of theory)

MS (ESI): m / z = 301 (M + H)⁺ M.p .: 147 ° C

1H-NMR (300 MHz, DMSO-d₆): Δ = 11.95 (s, 1H), 8.0 (s, 1H), 5.1 (m, 1H), 2.5 (d, 2H), 2.15-1.75 (m, 7H), 1.75-1.55 (m, 7H), 1.3-0.9 (m, 5H) ppm.

example 2

6- (cyclohexylmethyl) -l- (l-ethylpropyl) -l, 5-dihydro-4H-pyrazolo [3,4-d] - pyrimidin-4-one

<img id="imgf000021_0001" he="39" wi="42" file="imgf000021_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" /> Step 2a) 5-amino-l- (l-ethylρropyl) -lH-ρyrazol-4-carbonitrile

<img id="imgf000022_0001" he="31" wi="31" file="imgf000022_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

The production is similar to the method for Example 1 / stage la).

MS (ESI): mz = 179 (M + H)⁺

1H-NMR (300 MHz, DMSO-d₆): Δ = 7.55 (s, IH), 6.45 (s, 2H), 4.0 (m, IH), 1.8-1.55

(M, 4H), 0.65 (t, 6H) ppm.

Level 2b)

5-amino-1 - (1 -ethylpropyl) - lH-ρyrazol-4-carboxamide

<img id="imgf000022_0002" he="38" wi="36" file="imgf000022_0002.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

The production is similar to the method for Example 1 / step lb). S (ESI): mz = 197 (M + H)⁺

1H-NMR (300 MHz, DMSO-d₆). Δ = 7.65 (s, IH), 6.9 (br s, 2H), 6.1 (s, 2H), 3.9 (m, IH), 1.85-1.6 (m, 4H), 0.7 (t, 6H) ppm , Stage 2c)

6- (cyclohexylmethyl) - 1 - (1 -ethylpropyl) - 1, 5-dihydro-4H-pyrazolo [3, 4-d] pyrimidine

4-one

<img id="imgf000023_0001" he="40" wi="42" file="imgf000023_0001.tif" img-format="tif" img-content="drawing" orientation="portrait" inline="no" />

Analogously to Example 1 / Stafe lc) the product is obtained starting from 200 mg

(1:02 mmol) of 5-amino-l- (l-ethylρropyl) -lH-pyrazole-4-carboxamide and 482 mg

(3.06 mmol) Cyclohexylessigsäuremethylester obtained.

Yield: 146 mg (47% of theory)

MS (ESI): m / z = 303 (M + H)⁺

S p .: 122 ° C

1H-NMR (200 MHz, DMSO-d₆): Δ = 12.0 (s, IH), 8.0 (s, IH), 4.45 (m, IH), 2.5 (m,

2H), 2.0-1.5 (m, 10H), 1.4-0.9 (m, 5H), 0.6 (t, 6H, J = 7.5 Hz) ppm.