WO2007112922A1 - Trimeric, water-soluble aminopyrazole compounds having an effect against neurodegenerative diseases - Google Patents

Abstract

The invention relates to trimeric, water-soluble aminopyrazole compounds of formula (I), wherein R<SUP>1</SUP> represents H, a straight-chain or branched alkyl group with up to 10 carbon atoms or an amino acid group or polyamino acid group, and R<SUP>2</SUP> represents an OH, OR<SUP>3</SUP> or NHR<SUP>3</SUP> group in which R<SUP>3</SUP> is a straight-chain or branched alkyl group with 1 to 10 carbon atoms or an amino acid group or polyamino acid group.

Description

Trimers, water-soluble aminopyrazole compounds with activity against neurodegenerative diseases

The invention relates to novel, non-peptide aminopyrazole-based active compounds and to processes for their preparation. These drugs are able to prevent the de novo aggregation of Alzheimer's peptide, prion protein, α-synuclein and huntingtin as well as to dissolve existing aggregates.

It is well-known that illnesses, which occur increasingly with old humans, in particular dementia illnesses due to the increased life expectancy are more common today than before. Of particular importance in this context is Alzheimer's disease, which is associated with a serious impairment of cognitive abilities. Neurobiological characteristics of Alzheimer's disease are the conversion of normal α-helical protein structures to abnormal β-sheet structures (β-amyloid patches, β-AP), excessive loading of cytoskeletal τ proteins with phosphate groups, and muscarinic degeneration, cholinergic neurons. ß-AP are deposited between the nerve cells, so that the oxygen and energy supply of the brain is impaired and the nerve cells eventually die. τ proteins are normal components of the cytoskeleton. Excessive loading of phosphate groups disturbs the stabilization and transport processes leading to cell death. The degeneration of the muscular, cholinergic neurons is due to a chronic deficiency of the messenger acetylcholine, which leads to a greatly reduced signal transmission between the nerve cells.

At present, drug therapy is mainly focused on acetylcholine esterase inhibitors, which slow down and prevent the breakdown of acetylcholine. Alternatively or additionally, phytotherapeutics (cat's claw, St. John's wort, etc., as well as some non-steroidal anti-inflammatory drugs such as I-buprofen used, the mode of action of these preparations is unknown. DE 102 21 052 A1 already describes heterocyclic active substances which bind to proteins or peptides having a β-sheet structure and are present at least as dimers. The active substances mentioned there also contain amino pyrazole groups and can be used for the treatment, diagnosis and prophylaxis of diseases that are associated with the formation of a β-sheet structure and subsequent abnormal protein aggregation. The interest in the active ingredients described there is considerable and has given rise to the search for further, pharmacologically interesting agents whose structures differ from those of DE 102 21 052 A1 and which are better than the previously known agents for dissolving pathogenic protein fibrils.

This object is achieved by trimeric, water-soluble aminopyrazole compounds of the general formula I ₁ which are able to form a perfect β-sheet with peptides in the extended conformation.

The invention therefore trimeric, water-soluble aminopyrazole compound of the formula I.

In which R ¹ for H _{1 is} a straight-chain or branched alkyl group having up to 10 carbon atoms or an amino acid or polyamino acid residue, and R ^{2 is} an OH, OR ³ or NHR ³ group in which R ^{3 is} a straight-chain or branched alkyl group 1 to 10 carbon atoms or an amino acid or polyamino acid residue. Very particular preference is given to compounds of the formula II

and compounds of the formula III

The abovementioned aminopyrazole derivatives according to the invention can be prepared by a surprisingly simple preparation process: the iterative coupling of Λ / -PMB-protected nitropyrazolecarboxylic esters with the aid of the coupling reagent PyClop followed by reduction of the nitro group with H ₂ / Pd-C leads directly to oligomers, possibly after attachment of further solubility or affinity-mediating groups such as lysine building blocks be transferred by acidic deprotection in the finished active ingredients.

In general terms, the manufacturing process used comprises the following steps:

1. Preparation of the PMB-Protected Nitropyrazolecarboxylic Acid (Building Block A)

2. Esterification of the Carboxylic Acid and Reduction (H ₂ / Pd-C) of the Nitro Group to the Amine (Building Block B)

3. Coupling of building block A and B using PyClop / DIEA

4. Reduction of the nitro group (H ₂ / Pd-C)

5. renewed coupling with component A

6. renewed reduction of the nitro group (H ₂ / Pd-C)

7. Λ / -terminal attachment of amino acids or peptides with suitable coupling reagents (CI-HOBT, HCTU ₁ lutidine)

8. possibly C-terminal attachment of amino acids or peptides after esterase saponification (LiOH) with suitable coupling reagents (CI-HOBT, HCTU, lutidine)

9. Complete cleavage of all acid-labile protecting groups with TFA at 60-70 ⁰ C.

To 1.-5. "Aminopyrazole Oligomers for β-Sheet Stabilization of Peptides", P. Rzepecki, M. Wehner, O. MoIt, R. Zadmard, K. Harms, T. Schrader *, Synthesis 2003, 1815-1826. To 6. The nitro compound is dissolved in a 3: 1 mixture of methanol and tetrahydrofuran. As catalyst, Pd (5% on C) is added in catalytic amount. Under a stationary hydrogen atmosphere, it is stirred for about 1 d at room temperature. The course of the reaction is monitored by TLC. The catalyst is removed by filtration through kieselguhr, washed with a 3: 1 mixture of methanol and tetrahydrofuran and the solvent removed under reduced pressure. The crude product is purified by flash chromatography on SiO ₂ .

To 7. Under argon, the C-terminal, side-chain protected amino acid (or a corresponding C-terminal, side-chain protected peptide) is dissolved in a 3: 1 mixture of dichloromethane and dimethylformamide. At 0 ° C CI-HOBt (2.75 moleq.), HCTU (1.1 moleq.) And 2,6-lutidine (3.3 moleq.) Are added. It is warmed to room temperature and stirred for 10 min. After addition of the N-terminal, protected Pyrazolbausteins 1-2 d is stirred at room temperature. The solvent is removed on a rotary evaporator under reduced pressure and the crude product is purified by flash chromatography on SiO ₂ .

To 8. In methanol / THF at RT with 2 eq. LiOH and then precipitated by acidification. Continue as at 7.

To 9. The PMB-protected pyrazole component (with or without peptidically linked amino acid units) is treated with trifluoroacetic acid (about 1 mL per 0.01 g of compound to be deprotected). Optionally, it is largely dissolved in the ultrasonic bath. After 6-24 h at 60-70 ⁰ C, the reaction mixture is cooled in an ice bath and mixed with the 5-10-fold volume of diethyl ether. The precipitate is collected by centrifugation, washed with a little diethyl ether and dried in an oil pump vacuum. The active compounds according to the invention can be used for the treatment of Parkinson's disease, Alzheimer's disease and prion diseases and show their superior pharmacological properties in the following experiments:

In vitro experiments with trimeric aminopyrazoles via FCS (fluorescence correlation spectroscopy) and above all thioflavin T tests prove the efficient prevention of the aggregation of isolated Aβ, as well as its dose- and time-dependent aggregate dissolution.

The dose dependence: the aggregate dissolution is shown in FIG.

The time dependence of the aggregate resolution is shown in FIG.

Ultracentrifugation experiments also show an altered sedimentation profile, which particularly suppresses the small and medium aggregates A and B, which pose a particular neurotoxicity. This is shown by FIG.

Schädigung am 8. Tag):/ n-wVo experiments with trimeric aminopyrazole derivatives on living telencephalon neurons (8-9 day old chicken embryos - Lohman Brown hybrids) prove their neuroprotective (2% cell stress assay) and antiaggregatory capacity (5% cell culture assay with

Damage on the 8th day):

The neuroprotective capacity of trimeric aminopyrazole derivatives is demonstrated by the in vivo experiments shown in Figure 4. Shown is a 2% cell stress assay (100% control).

The antiaggregatory capacity of trimeric aminopyrazole derivatives is demonstrated by the in vivo experiments shown in Figure 5. Shown is a 5% cell culture assay with ß-amyloidi _-4 2 damage on the 8th day (control = 40% surviving cells). The preparation of the active compounds according to the invention is documented by the following examples:

5-Amino-1- (p-methoxybenzyl) pyrazole-3-carbonyl- [5'-amino- [1 '- (p-methoxybenzyl) pyrazole-3'-carbonyl] -5 "-amino-1" - ( p-Methoxybenzyl) pyrazole-3 "-carboxylic acid methyl ester = (1) = H ₂ N-Pz (PMB) -Pz (PMB) -Pz (PMB) -OMe:

H _2, Pd / C, RT, d 1, MeOH / THF 3: 1

There are dissolved 0.720 g of the nitro compound (0.96 mmol) in a 3: 1 mixture of methanol (30 ml_) and tetrahydrofuran (10 ml_). To this solution are added 0.10 g of Pd (5% on C) as a catalyst. Under a stationary hydrogen atmosphere, it is stirred for about 1 d at room temperature. The reaction is monitored by TLC (SiO ₂ maps, dichloromethane / methanol 10: 1, R f = 0.54). The catalyst is purified by filtration through diatomaceous earth removed, washed with a 3: 1 mixture of methanol and tetrahydrofuran and the solvent removed under reduced pressure. After flash chromatography (SiO 2, dichloromethane / tetrahydrofuran 1: 1, R _f = 0.40), 1 is isolated as a white solid. Yield: 0.522 g, 0.73 mmol (76%). C ₃₇ H ₃₇ N ₉ O ₇ (719.746). MS: (HR-ESI in MeOHfTHF) 742.2696 C ₃₇ H ₃₇ N ₉ O ₇ Na. ¹ H-NMR (300 MHz, CDCl ₃ ): δ [ppm] = 3.56 (s, 3H), 3.73 (s, 3H), 3.76 (s, 3H), 3.88 (s, 3H), 5.45-5.70 (m , 6H), 6.57-6.67 (m, 6H), 7.11-7.45 (m, 9H).

5-Amino-1- (p-methoxybenzyl) pyrazole-3-carbonyl [5'-amino-1 '- (p-methoxybenzyl) pyrazole-3'-carbonyl] -5 "-amino] -1 ^M - ( p-methoxybenzyl) pyrazole-3 "-carboxylic acid = (2) = H ₂ N-Pz (PMB) -Pz (PMB) -Pz (PMB) -OH:

eq LiOH, MeOH / THF / H ₂ O 4: 4: 1, 55 ° C, 20 h

Es werden 0.150 g des Esters 1 (0.208 mmol) in einem Gemisch aus Methanol (2.0 ml_), Tetrahydrofuran (2.0 ml_) und H₂O (0.5 ml_) gelöst. Nach Zugabe von 0.010 g Lithiumhydroxid (0.42 mmol) wird ca. 20 h bei 55°C gerührt. Durch DC- Kontrolle (SiO₂-Karten, Dichlormethan / Tetrahydrofuran 1:1) wird der Reaktionsverlauf verfolgt. Nach Beendigung der Reaktion wird das Lösungsmittel am Rotationsverdampfer unter vermindertem Druck entfernt und der Rückstand in H₂O aufgenommen. Mit ca. 1.5 mL Salzsäure (c = 1.0 mol/L) wird bis auf pH = 1 angesäuert, der ausfallende Niederschlag abzentrifugiert, mit ca. 2 mL Salzsäure (c = 0.2 mol/L) gewaschen und im Ölpumpenvakuum getrocknet. Es wird 2 als blassgelber Feststoff erhalten. Ausbeute: 0.083 g, 0.118 mmol (57%). C₃₆H₃₅N₉O₇ (705.720). MS: (HR-ESI) 706.2732 C₃₆H₃₆N₉O₇. ¹H-NMR (200 MHz, CD₃OD): δ [ppmj: 3.74 (s, 6H), 3.75 (s, 3H), 5.65 (m, 6H), 6.83 (m, 6H)₁ 7.18-7.39 (m, 9H).

Dissolve 0.150 g of ester 1 (0.208 mmol) in a mixture of methanol (2.0 mL), tetrahydrofuran (2.0 mL), and H ₂ O (0.5 mL). After addition of 0.010 g of lithium hydroxide (0.42 mmol) is stirred at 55 ° C for about 20 h. The course of the reaction is monitored by TLC monitoring (SiO ₂ maps, dichloromethane / tetrahydrofuran 1: 1). After completion of the reaction, the solvent is removed on a rotary evaporator under reduced pressure and the residue taken up in H ₂ O. With about 1.5 mL hydrochloric acid (c = 1.0 mol / L) is acidified to pH = 1, the precipitate precipitated centrifuged off, washed with about 2 mL hydrochloric acid (c = 0.2 mol / L) and dried in an oil pump vacuum. 2 is obtained as a pale yellow solid. Yield: 0.083 g, 0.118 mmol (57%). C ₃₆ H ₃₅ N ₉ O ₇ (705.720). MS: (HR-ESI) 706.2732 C ₃₆ H ₃₆ N ₉ O ₇ . ¹ H-NMR (200 MHz, CD ₃ OD): δ [ppmj: 3.74 (s, 6H), 3.75 (s, 3H), 5.65 (m, 6H), 6.83 (m, 6H) ₁ 7.18-7.39 (m , 9H).

5-aminopyrazole-3-carbonyl-5'-aminopyrazole-3'-carbonyl-5'-aminopyrazole-3'-carboxylic acid = (3) = H ₂ N-Pz ₃ -OH:

Add 0.020 g of the protected compound 2 (0.028 mmol) with 1.5 mL of trifluoroacetic acid and dissolve in an ultrasound bath. Thereafter, it is heated to 60 ° C for 17 h. The red-brown, slightly turbid reaction mixture is cooled in an ice bath and treated with 7.5 mL diethyl ether. The precipitate is collected by centrifugation, washed with 3 mL diethyl ether and dried in an oil pump vacuum. 3 is obtained as a brown solid. Yield: 0.007 g. C ₁₂ H ₁₁ N ₉ O ₄ (345,274) (excluding TFA). ¹ H-NMR (500 MHz, DMSO): δ [ppm]: 7.00 (s, 1H), 7.42 (s, 1H), 7.49 (s, 1H), 11.03 (s, 1H), 11.15 ( s, 1H), 12.17 (s, 1H), 13.68 (s, broad, 1H). 5-aminopyrazole-3-carbonyl-5'-aminopyrazole-3'-carbonyl-5 "-aminopyrazole-3" -carboxylic acid methyl ester = (4) = H ₂ N-Pz ₃ -OMe:

0.010 g of the protected compound 1 (0.014 mmol) are mixed with 0.8 ml of trifluoroacetic acid. It is then heated to 6O ⁰ C for 6 h. It is cooled to RT, displaced TFA largely in the argon stream and treated with 4.0 mL diethyl ether. The precipitate is collected by centrifugation, washed with 3 mL diethyl ether and dried in an oil pump vacuum. 4 is obtained as a brown solid. Yield: 0.005 g.

Ci ₃ Hi ₃ N ₉ O ₄ (359.301) (without consideration of TFA). ¹ H-NMR (400 MHz ₁ DMSO): δ [ppm]: 3.84 (s, 3H), 6.75 (s, 1H), 6.98 (s, 1H), 7.47 (s, 1H), 11.14 (s , 2H), 12.14 (s, 1H), 13.6 (s, 2H).

Fmoc-octa (ε-Boc-lysyl) -5-amino-1- (p -methoxybenzyl) pyrazole-3-carbonyl [5'-amino- [1 '- (p-methoxybenzyl) -pyrazole-3' carbonyl] -5 "-amino-1" - (p-methoxybenzyl) pyrazole-3 "-carboxylic acid methyl ester = (5) = Fmoc- [K (Boc)] ₈ -Pz (PMB) -Pz (PMB) -Pz (PMB) -OMe:

Under argon, 0.050 g of Fmoc- [K (Boc)] ₈ -OH (0.0242 mmol) are dissolved in a mixture of 1.5 ml of dichloromethane and 0.5 ml of dimethylformamide. At ⁰ ° C 0.011 g CI-HOBt (0.0666 mmol), 0.011 g HCTU (0.0266 mmol) and 0.009 mL 2,6-lutidine (0.0799 mmol) was added. It is warmed to room temperature and stirred for 10 min. After addition of 0.031 g of 1 (0.0436 mmol) is stirred at room temperature for 16 h and the resulting viscous reaction mixture diluted with a further 2 mL of dichloromethane. After 42 h, the solvent is removed on a rotary evaporator under reduced pressure and the crude product is purified by flash chromatography (SiO ₂ , dichloromethane / methanol 30: 1, R _f = 0.41). Yield 5: 0.038 g, 0.014 mmol (57%). C ₁₄₀ H ₂ OTN ₂₅ O ₃₃ (2768.290). MS (ESI and HR-ESI): 1406.7553 (10%, C ₄₀ H _2O rN ₂₅ O ₃₃ Na _2). ¹ H-NMR (300 MHz, CDCl ₃ ): δ [ppm]: 1.25 (s, 72H), 1.26- 1.76 (m, 48H), 2.71-3.22 (m, 16H), 3.55-3.95 (m, 9H) ₁ 4.09 (m, 14H), 5.46 (m, 6H), 6.68 (m, 6H), 7.01-7.67 (m, 17H).

Fmoc-octa- (ε-Boc-lysyl) -5-aminopyrazole-3-carbonyl-5 ¹ -aminopyrazole-3 ¹ -carbonyl-5 "-aminopyrazole-3" -carboxylic acid methyl ester = (6) = Fmoc- [K (Boc )] ₈ - Pz ₃ -OMe:

TFA, 60 ^° C, 17 h

Add 0.025 g of the protected compound 5 (0.009 mmol) with 1.5 mL of trifluoroacetic acid. Thereafter, it is heated to 6O ⁰ C for 17 h. It is cooled to RT, the trifluoroacetic acid largely displaced in the argon stream and treated with 8.0 mL diethyl ether. The precipitate is centrifuged off, washed with 4 mL diethyl ether and dried in an oil pump vacuum. It is obtained as a beige 6 solid. Yield: 0.017 g. C ₇₆ Hi ₁₉ N ₂₅ O ₁₄ (1606.918) (excluding TFA). MS (ESI): 803 (100%, M ²⁺ ). ¹ H-NMR (400 MHz, D ₂ O): δ [ppm]: 1.20-1.95 (m, 48H), 2.85-3.10 (m, 16H), 3.39 (t, 1H), 3.70 (dd, 2H) , 3.98 (s, 3H), 4.20-4.45 (m, 8H), 6.91-6.99 (m, 3H), 7.35-8.02 (m, 8H).

Claims

claims 1. Trimere, water-soluble aminopyrazole compound of the formula I.

In which R ^{1 is} H, a straight-chain or branched alkyl group having up to 10 carbon atoms or an amino acid or polyamino acid residue and R ^{2 is} an OH, OR ³ or NHR ³ group, in the R ^{3 is} a straight-chain or branched alkyl group with 1 -10 carbon atoms or an amino acid or polyamino acid residue. 2. Trimere, water-soluble aminopyrazole compounds according to claim 1, characterized in that they carry solubility or affinity-promoting groups. 3. A compound according to claim 1, characterized in that it of the formula II

equivalent. 4. A compound according to claim 1, characterized in that it has the formula IM

equivalent. 5. A process for the preparation of compounds of claims 1 to 4, characterized by the steps: a. Masking of the amino acids, b. Reaction with a peptide coupling reagent, c. Removal of the protecting groups and d. Cleaning the products. 6. The method according to claim 5, characterized in that oligomeric end products are formed. 7. Use of compounds of claims 1 to 4 for the preparation of medicaments associated with a β-sheet structure followed by abnormal protein aggregation. 8. Use of compounds of claims 1 to 4, characterized in that they are used for the treatment of Parkinson's disease, Alzheimer's disease and prion diseases. 9. Use of compounds of claims 1 to 4, characterized in that they are used intravenously, subcutaneously, intraperitoneally, intrathecally, intravesically, topically or as an aerosol. 10. Pharmaceutical preparation, characterized in that it contains a compound of claims 1-4. 11. Pharmaceutical preparation according to claim 10 for the dissolution or prevention of pathological aggregations of peptides and / or proteins which lead to neurodegenerative diseases.