WO2004073705A1

WO2004073705A1 - 1-phenyl-2- monoalkyl carboxylic acid derivatives for the treatment of neurodegenerative diseases

Info

Publication number: WO2004073705A1
Application number: PCT/EP2004/001595
Authority: WO
Inventors: Luca Raveglia; Ilaria Peretto; Stefano Radaelli; Bruno Pietro Imbimbo; Andrea Rizzi; Gino Villetti
Original assignee: Chiesi Farmaceutici S.P.A.
Priority date: 2003-02-21
Filing date: 2004-02-19
Publication date: 2004-09-02
Also published as: ITMI20030312A1

Abstract

Novel 1-phenyl-2-monoalkylcarboxylic derivatives, the process for the preparation thereof and the use thereof in the treatment and/or prevention of neurodegenerative diseases such as Alzheimer’s disease.

Description

l-PHENYL-2- MONOALKYL CARBOXYLIC ACID DERIVATIVES FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES

The present invention concerns novel l-phenyl-2-monoalkylcarboxylic acids, pro-drugs and bioisosters on the carboxylic moiety thereof. The invention is also directed to the process for their preparation and the use thereof in the preventive or therapeutical treatment of neurodegenerative diseases, in particular Alzheimer's disease. INTRODUCTION

Alzheimer's disease is a neurodegenerative disorder characterized by atrophy of the cerebral cortex and by a massive loss of cortical neurons and cholinergic projections of the nucleus basalis towards the cortex. From a histopathologic point of view there is a diffuse presence of extracellular and perivascular neuritic plaques and intracellular neurofibrillary tangles in the cerebral parenchyma of Alzheimer patients.

Neuritic plaques are mainly composed of aggregates of a protein with 39-43 amino acid residues known as β-amyloid (βA), and, depending on the numbers of aminoacids, Aβ₃₉, Aβ₄₀, Aβ₄₂ and Aβ₄₃.

In addition to these histopathologic lesions, there is lack in some neurotransmitters, particularly acetylcholine, serotonin, noradrenalin, dopamine, glutamate and substance P. The pharmacological approaches aimed at increasing acetylcholine cerebral levels, mainly through acetylcholine- esterase inhibitors, attained poor results from the clinical standpoint, or anyhow results which cannot significantly prevent the progress of the disease. For this reason, in recent years interest has been focused on the mechanisms of formation of the main pathologic lesions in the brain of the patients, namely both neuritic plaques and neurofibrillary tangles, and more effective therapeutical approaches have been looked for. PRIOR ART

Epidemiological studies evidenced that chronic administration of non steroid anti-inflammatory drugs (NSAIDs) significantly decreases the onset of Alzheimer's disease in the population regularly taking these drugs. The mechanism underlying such NSAID preventive action has not been fully elucidated yet, but is apparently connected with their ability of inhibiting cyclooxygenase enzymes. More recently, a novel pharmacological action of some NSAIDs has been described: indomethacin, sulindac, ibuprofen and flurbiprofen can selectively reduce the production of the most neurotoxic isoform of β-amyloid peptide in cell cultures, namely the form containing 42 amino acids (Aβ₄₂), thus favouring the release of a less harmful isoform, Aβ₃₈ (Weggen et al., Nature 2001; 414 (6860): 212-6). However, the inhibition of the production of Aβ ₂, which can be ascribed to the interaction of these drugs with γ-secretase (a macromolecular/multiprotein enzymatic complex with aspartyl-protease activity) has been observed in vitro at very high concentrations. Plasma and cerebral levels corresponding to the dosages used in the in vitro experimentation could significantly increase in treated patients the risk of side effects typical of COX inhibitors, such as gastrointestinal bleeding and perforating ulcers.

WO 01/78721 claims a method of preventing, delaying or reversing the progression of Alzheimer's disease by administering an Aβ₄₂ lowering agent, under conditions in which levels of Aβ₃₈ are increased and levels of Aβ₄₂ are left unchanged. Furthermore, methods and materials for identifying and developing Aβ₄₂ lowering agents and methods for identifying agents that increase the risk of developing, or hasten progression of, Alzheimer's disease, are disclosed. The examples concern indomethacin and flufenamic acid derivatives, but no examples concerning flurbiprofen derivatives are reported.

Jantzen et al, J Neurosci 2002; 22: 2246-2254, described a flurbiprofen derivative capable of releasing nitric oxide. The paper generically states that flurbiprofen derivatives are apparently more efficacious than other NSAIDs in clearing β-amyloid deposits but no mention concerning any A ₄2 lowering selective activity is made.

In this therapeutical scenario, and in the light of the potential problems of conventional NSAIDs, novel derivatives having more selective and more potent inhibitory activity on the peptide Aβ₄₂ release while inhibiting to a lesser extent, or not inhibiting at all, cyclooxygenase would be a significant improvement in therapies aimed at preventing the onset of Alzheimer's disease and/or at delaying the cognitive decline that represent an early stage disease.

WO 99/41224 claims novel biaryl-acetic acid derivatives with anti- inflammatory activity as cyclooxygenase-2 (COX-2) inhibitors, useful for the treatment of a number of diseases, including Alzheimer's disease. However it has been highlighted in the literature {vide supra) that an inhibitory effect on the COX-2 is associated to side effects such as gastrointestinal bleeding and perforating ulcers.

GB 1,396,726, GB 1,382,996, GB 1,359,987 disclose 2-biphen-4-yl propionic acid derivatives substituted in 2' and 4' as anti-inflammatory agents. SUMMARY OF THE INVENTION

The present invention concerns l-phenyl-2-monoalkyl carboxylic acids derivatives having a more selective inhibitory activity on the release of Aβ₄₂ peptide, thereby being able to modulate gamma-secretase activity without affecting other important metabolic processes.

The invention is also directed to a process for their preparation, to pro- drugs and bioisosters on the carboxylic moiety thereof, to pharmaceutical compositions containing them and the use thereof in the prevention or therapeutical treatment of neurodegenerative diseases, in particular _. Alzheimer's disease. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of general formula (ϊ):

(i) wherein:

R is linear or branched C₁-C₄ alkyl; G is:

- a COOR" group wherein R" is H, linear or branched C₁-C₄ alkyl, C3-C₆ cycloalkyl or ascorbyl;

- a tetrazolyl residue;

Ri is CF₃₎ OCF₃ or a halogen selected from the group of F, CI, Br, I, preferably fluorine.

Ar is a group of formula

wherein R_2, R₃ and R₄ are independently selected from the group of:

- H;

- CF₃;

- C₃-C₈ cycloalkyl optionally substituted;

- phenyl optionally substituted; with the proviso that R₂, R₃ and R₄ cannot simultaneously be H.

A group of preferred compounds is that in which: R is CH₃; Ri is fluorine; G is COOH; Ar is phenyl as defined above. Particularly preferred are the following compounds: 2-(2-fluoro-4'-trifluoromethylbiρhen-4-yl)ρroρionic acid (CHF 4732); 2-(2-fluoro-3'-trifluoromethylbiρhen-4-yl)ρropionic acid (CHF 4729); 2-(2-fluoro-3',5'-bis(trifluoromethyl)biphen-4-yl)ρroρionic acid (CHF 4801); 2-(4'-cyclohexyl-2-fluorobiphen-4-yl)propionic acid (CHF 4865).

The invention also relates to the enantiomers, pharmaceutically acceptable salts and esters prepared in order to increase the crossing of the hemato-encephalic barrier.

A further object of the present invention are the compounds of formula (I) as medicaments, in particular the use thereof in the preparation of pharmaceutical compositions for the treatment and/or the prevention of neurodegenerative diseases such as Alzheimer's disease.

Still a further object of the invention are solid or liquid pharmaceutical compositions, preferably for the oral use, comprising at least one compound of formula (I) in admixture with pharmaceutically acceptable excipients and/or carriers, for example those described in Remington's Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., N.Y., U.S.A..

The compounds of general formula (I) wherein G is COOH can be prepared according to methods of literature by palladium-catalyzed reaction between an aryl halide of formula (II)

in which R and Ri are as defined above and X is bromine or iodine, preferably iodine, with a boronic acid or ester ArB(OL)₂ in which L is an alkyl chain, under the conditions reported in Scheme 1.

DME = 1,2-dimethoxyethane TTP = triphenylphosphine

Scheme 1

The compounds of formula (II) are commercially available, or can be prepared according to the following synthetic route.

The process comprises the alkylation of a phenylacetic acid (III) with an alkyl halide RX', wherein R is as defined above and X' is halogen selected from CI, Br and I, according to what reported in Scheme 2:

MeOH

Scheme 2 An alternative synthetic route that also affords phenylacetic acids (III) is reported in Scheme 3:

(II) or (III) for R = H

Scheme 3

Boronic acids or the corresponding boronates are either commercially available or can be prepared from the corresponding halide according to methods known in literature.

The compounds of formula (I) wherein G is COOR", where R" is linear or branched C₁-C₄ alkyl, C₃-C₆ cycloalkyl or ascorbyl, can be prepared by esterifying the compounds of formula (I) in which G is COOH.

The compounds of formula (I) in which G is tetrazolyl can be prepared from compounds of formula (I) according to known methods, for example transforming the carboxylic acid into amide, dehydrating the amide to nitrile and reacting the latter with tributyltin azide. EXAMPLES

EXAMPLES OF CHEMICAL PREPARATION

Example 1 - Preparation of 2-(2-fluoro-4'-trifluoromethyIbiphen-4-vI) propionic acid (CHF 4732) Preparation of ethyl r2-methyl 2-(3-fluoro-4-nitrophenyl)1 malonate

Sodium hydride (60% dispersion in vaseline oil, 6.12 g, 153 mmoles) under nitrogen atmosphere is added with dry dimethylsulfoxide (100 ml), under stirring and during 30 minutes. The resulting suspension, kept at a temperature of 15°C, is dropped, during 30 minutes, into a solution of diethyl- methylmalonate (24 g, 138 mmoles) in dry dimethylsulfoxide (100 ml). Stirring is continued for 30 minutes, then a solution of 2,4- difluoronitrobenzene (20.3 g, 128 mmoles) in dry dimethylsulfoxide (200 ml) is dropped, during 90 minutes, at a temperature of 15°C, to obtain a purple mixture. After stirring for 30 minutes, the reaction mixture is diluted in water (5.000 ml) at 0°C, then extracted with ethyl acetate (4 x 150 ml). The organic phase is washed with a NaCl saturated solution (50 ml), dried over Na₂S0₄, and concentrated under vacuum to give an orange oil (40 g). Purification by chromatography on 200 g of silica gel (eluting first with hexane, then with hexane / ethyl acetate 9 / 1 v/v), affords a yellow oil (35 g, 87%). HPLC - UV purity (215 nm): 90%.

1H NMR (CDC1₃): 8.04 (dd, 1H); 7.39 (dd, 1H); 7.33 (m, 1H); 4.26 (q, 2H); 4.25 (q, 2H); 1.88 (s, 3H); 1.27 (t, 6H).

Legenda: s = singlet; d = doublet; dd = double doublets; t = triplet; q = quartet; m = multiplet.

MS (El): 313 m/z (M+.); 268; 241; 213; 195; 184; 166. Preparation of ethyl [2-methyl-2-(3-fluoro-4-aminophenyl ]malonate

A solution of ethyl [2-methyl-2-(3-fluoro-4-nitrophenyl)]malonate (35 g, 112 mmoles) in ethanol (500 ml) is added with 2 g of wet 10%Pd/C (50%) H₂0). The mixture is hydrogenated for 1 h at room temperature under 10 psi pressure, the catalyst is filtered off, and the solution is concentrated under vacuum to give a colourless oil (30.2 g, 95%). HPLC - UV purity (215 nm): 95%.

*H NMR (CDCI₃): 7.06 (dd, 1H); 6.95 (ddd, 1H); 6.72 (dd, 1H); 4.22 (q, 2H); 4.21 (q, 2H); 1.80 (s, 3H); 1.25(t, 6H). Preparation of ethyl [^"2-methyl-2-(3-fluoro-4-iodophenyl)1malonate

A suspension of ethyl [2-methyl-2-(3-fluoro-4-aminophenyl)]malonate (30 g, 106 mmoles) in 6N HC1 (180 ml), kept under stirring at 0°C, is added drop by drop with a solution of NaN0₂ (7.3 g, 106 mmoles) in water (40 ml). The resulting mixture, kept at 0°C, is dropped under stirring into a solution of KI (73 g, 440 mmoles) in water (75 ml) cooled at 0°C. The mixture is stirred at room temperature for 3 h, then extracted with ethyl acetate (2 x 200 ml). The organic phase is washed with a 10% Na₂S₂0 aqueous solution until negative response with aminoiodinated strip, then with a NaCl saturated solution, finally dried over Na₂S0₄ and concentrated under vacuum to give a dark oil (37 g, 88%) which is used without further purification in the subsequent preparation. HPLC - UV purity (215 nm): 75%.

1H NMR (CDC1₃): 7.70 (dd, 1H); 7.14 (dd, 1H); 6.94 (dd, 1H); 4.23 (q, 2H); 4.22 (q, 2H); 1.83 (s, 3H); 1.26(t, 6H). MS (El): 394 m/z (M+.); 321; 293; 276; 247; 166. Preparation of 2-(3-fluoro-4-iodophenyl)propionic acid

A solution of ethyl [2-methyl-2-(3-fluoro-4-iodophenyl)]malonate (titre 75%, 37 g, 70 mmoles) in ethanol (130 ml) is added with a 2M NaOH aqueous solution (130 ml). The mixture is heated to 100°C for 8 h, then concentrated under vacuum to remove the ethanol. The residue is taken up with water (300 ml), washed with ethyl ether (2 x 100 ml), and the exhausted organic phase is discarded. The aqueous phase is acidified to pH 2 with 10% HC1, then extracted with ethyl acetate (3 x 150 ml). The organic phase is washed with a NaCl saturated solution, dried over Na₂S0₄, and concentrated under vacuum to give a dark oil (27 g), which is subjected to chromatography on 150 g of silica gel, eluting with dichloromethane. The resulting product is a pale yellow solid (18 g), that is purified by cold crystallization with methanol / water 1 / 3 v/v, to afford a white solid (16 g, 77%).

HPLC - UV purity (215 nm): 94%.

'H NM (CDC1₃): 7.69 (dd, 1H); 7.05 (dd, 1H); 6.87 (dd, 1H); 3.71 (q, 1H);

1.50 (d, 3H).

Preparation of 2-(2-fluoro-4'-trifluoromethylbiphen-4-vQpropionic acid

4-Trifuoromethylphenylboronic acid (190 mg, 1 mmol), under nitrogen atmosphere, is added, under stirring, with a 2M Na₂C0₃ solution (0.75 ml) previously degassed with nitrogen. After 5 minutes, a solution of 2-(3-fluoro- 4-iodophenyl)propionic acid (196 mg, 0.667 mmoles) and tetrakis(triphenylphosphine)palladium(0) (38.5 mg, 0.033 mmoles) in dimethoxyethane, previously degassed with nitrogen (3 ml), is added. The mixture is heated at 80°C for 24 hours, then cooled at room temperature, added with 2M HC1 (1.5 ml) and diluted with ethyl acetate (5 ml). The phases are separated, and the organic phase is concentrated under vacuum to give a dark oil (0.4 g), which is purified by preparative HPLC. The fractions containing the product are concentrated under vacuum to give a pale brown solid (87 mg, 42%). HPLC - UV purity (215 nm): 99% MS (EI): 312 m/z (M+.); 267.

Following the same procedure and using the suitable reagents, compounds CHF 4729, CHF 4801, and CHF 4865 were prepared. Example 2 - Preparation of 5-ri-(2-fluoro-4^,-trifluoromethylbiphen-4--yl)- ethvn-2H-tetrazole (CHF 4871 Preparation of 2-(2-fluoro-4 ' -trifluoromethylbiphen-4-y Ppropionamide

A solution of 2-(2-fluoro-4'-trifluoromethylbiphen-4-yl)propionic acid (0.5 g, 1.53 mmoles) in dichloromethane (5 ml) is added with oxalyl chloride (0.5 ml, 5.73 mmoles) and kept under stirring for 20 hours at room temperature. The mixture is concentrated under vacuum to remove the oxalyl chloride excess, thereby obtaining an oil (0.5 g) which is redissolved in 10 ml of dichloromethane. The resulting solution is dropped into 50 ml of saturated ammonia solution in dichloromethane, obtained bubbling 6 g of gas ammonia in 50 ml of the solvent. The mixture is kept under stirring for 2 hours at room temperature, then concentrated under vacuum. The resulting solid is triturated with isopropyl ether, then with water, after that is filtered and dried under vacuum to afford the product as a white solid (0.4 g, 80%). m.p. 136-138°C.

HPLC-UV purity (215 nm): 98%

1H NMR (CDC1₃): 7.70 (d, 2H); 7.64 (d, 2H); 7.43 (dd, 1H); 7.20 (s, 1H); 9.19

(dd, 1H); 5.37 (s br, 2H); 3.64 (q, 1H); 1.57 (d, 3H).

MS (EI): 311 m/z (M+.); 267

Preparation of 2-(2-fluoro-4 ' -trifluoromethy lbiphen-4-yl propionitrile

A solution of 2-(2-fluoro-4' -trifluoromethy lbiphenyl-4-yl)propionamide (150 mg, 0.48 mmoles) in pyridine (1 ml) and 1,4-dioxane (1 ml), kept at 0°C, is added dropwise with trifluoroacetic anhydride (1 ml). The mixture is left to warm at room temperature for 2 hours under stirring, then diluted with water and extracted with ethyl acetate. The organic phase is washed in sequence with 2N hydrochloric acid, 10% sodium bicarbonate solution and a sodium chloride saturated solution, then dried over sodium sulfate and concentrated under vacuum to afford a white solid (100 mg, 71%) which is directly used in the subsequent transformation. Preparation of 5-[l-(2-fluoro-4'-trifluoromethylbiphen-4-yl ethyπ-2H-tetrazole

100 mg of 2-(2-fluoro-4' -trifluoromethy lbiphen-4-yl) propionitrile (0.34 mmoles) are suspended in 500 mg of tributyltin azide (1.5 mmoles). The mixture is heated at 100°C for 3 hours, then cooled at room temperature and added with 37% hydrochloric acid (1 ml) and methanol (1 ml). After stirring for 2 hours, the mixture is concentrated under vacuum. The resulting residue is treated with isopropyl ether to afford the product such as a white solid

(40 mg, 35%). m.p. 182-183°C

HPLC - UV purity (215 nm): 99% H NMR (CDC1₃): 15.48 (s br, 1H); 7.67 (d, 2H); 7.60 (d, 2H); 7.37 (dd, 1H);

7.20-7.13 (m, 2H); 4.42 (q, 1H); 1.80 (t, 3H).

MS (El): 336 m/z (M+.); 267

Example 3 - Pharmacological activity

Inhibition of Aβa? release in the supernatant of H4- 15x cells

H4-15x cells (human neuroglioma cells transfected with the human gene encoding for the precursor of β-amyloid protein APP695) were cultured in flasks (in incubator at 37°C, under aqueous vapour saturated atmosphere with 5% carbon dioxide), in the presence of hygromycin and blasticidin, which maintain the selective pressure for the cells continuously expressing the gene of interest.

When the cells reached about 90% confluency, they were collected and re-seeded in 24 wells plates (2 x 10⁵ cells each), in 0.5 ml of complete culture medium. After approx. 24 hours, when the cells had adhered to the well surface and reached confluency, the medium of each well was replaced with 0.5 ml of fresh culture medium, supplemented with a compound (I) to 100 micromolar final concentration. Each tested concentration was repeated in triplicate. The molecules used for the treatment were previously dissolved in dimethylsulfoxide (DMSO) or in a dimethylsulfoxide/water mixture, the final concentration of DMSO in the wells not exceeding 1%. Thus, the prepared plates were incubated again overnight (14-16 hours); afterwards the cell supernatant was taken from each well and Aβ₄₂ and Aβ₄₀ proteins were quantitated. The assay was carried out with an instrumentation for microplates chemoluminescence analysis, which allows to separately quantify the two proteins and is based on the immobilization of an analyte-antibody complex on paramagnetic microbeads. One of the antibodies of this complex is marked with a ruthenium compound which, upon electrochemical excitement, gives a light signal, having intensity proportional to the amount of analyte present. Inhibition of cyclooxygenase- 1 (COX-1) in rat whole blood

Whole blood was taken from the rat abdominal aorta and immediately placed in heparinized tubes. Aliquots of heparinized blood (500 μl) were preincubated with 100 μM concentration of the tested compounds or with the only carrier (DMSO) for 1 h at 37°C. Eicosanoid production was induced by addition of calcium ionophore A23187 (final concentration 5x10^"5 M) and was interrupted after 30 minute incubation by quickly placing the samples in dry ice. Thereafter, samples were centrifuged (12.000 g x 3 minutes a 4°C) and the production of TxB₂ thromboxane B₂ was calculated by radioimmunoassay.

The results expressed as percent inhibition of Aβ₄₂ release at 100 μM and percent COX-1 inhibitory activity at the same concentration are reported in Table. Flurbiprofen used as comparison at the same concentration showed approx. 25% inhibition of Aβ₄₂ release and 100% COX-1 inhibitory activity.

Table: Percent inhibition of Aβ₄₂ release and percent COX-1 inhibitory activity of representative compounds of the invention at 100 μM concentration.

Claims

Compounds of general formula (I):

wherein:

R is linear or branched C₁-C₄ alkyl;

G is:

- a COOR" group wherein R" is H, linear or branched C1-C₄ alkyl, C₃-C₆ cycloalkyl or ascorbyl;

- a tetrazolyl residue;

Ri is CF_3] OCF₃ or a halogen selected from the group of F, CI, Br, I, preferably fluorine.

Ar is a group of formula

wherein R₂; R₃ and R₄ are independently selected from the group of:

- H;

•^■ CF₃;

- cycloalkyl C₃-C₈ optionally substituted:

- phenyl optionally substituted; with the proviso that R₂, R₃ and R₄ cannot be simultaneously H; enantiomers and pharmaceutically acceptable salts.

2. Compounds as claimed in claim 1, wherein R is CH₃; R₂ is fluorine, G is a COOH group; Ar is phenyl as defined above.

3. Compounds as claimed in claims .1 and 2 for use in medicine.

4. Pharmaceutical compositions containing a compound of formula (I) in mixture with pharmaceutically acceptable carriers and/or excipients.

5. Pharmaceutical compositions as claimed in claim 4, for the oral administration.

6. The use of the compounds of formula (I) as claimed in claims 1 and 2, for the preparation of a medicament for the treatment of diseases connected with an increased production of neurotoxic peptide Aβ₄₂.

7. The use of the compounds of formula (I) as claimed in claims 1 and 2, for the preparation of a medicament for the prevention or therapeutical treatment of neurodegenerative diseases.

8. The use of the compounds of formula (I) as claimed in claim 7, for the treatment of Alzheimer's disease.