WO2013087954A1 - Compounds that inhibit beta amyloid peptide aggregation - Google Patents

Compounds that inhibit beta amyloid peptide aggregation Download PDF

Info

Publication number
WO2013087954A1
WO2013087954A1 PCT/ES2012/000299 ES2012000299W WO2013087954A1 WO 2013087954 A1 WO2013087954 A1 WO 2013087954A1 ES 2012000299 W ES2012000299 W ES 2012000299W WO 2013087954 A1 WO2013087954 A1 WO 2013087954A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
peptide
aggregation
use according
compounds
Prior art date
Application number
PCT/ES2012/000299
Other languages
Spanish (es)
French (fr)
Inventor
Javier Sancho Sanz
Laura Catalina LÓPEZ RODRÍGUEZ
José Alberto CARRODEGUAS VILLAR
Salvador Ventura Zamora
Original Assignee
Universidad De Zaragoza
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Zaragoza filed Critical Universidad De Zaragoza
Publication of WO2013087954A1 publication Critical patent/WO2013087954A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/416—2,5-Pyrrolidine-diones with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to other ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/64—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/30—Hetero atoms other than halogen
    • C07D333/34—Sulfur atoms

Definitions

  • the present invention relates to the use of a compound of general formula 5 (I) for the preparation of a pharmaceutical composition, more preferably for the treatment of Alzheimer's disease.
  • the invention relates to a pharmaceutical composition comprising said compound.
  • AD Alzheimer's disease 15
  • senile neurofibrillary plaques are composed of beta amyloid peptides ( ⁇ ),
  • ⁇ peptide and fragments thereof have been shown to be toxic in vitro and in vivo, indicating an important role of ⁇ in the pathogenesis of AD.
  • ADDLs diffuseusible ligands derived from ⁇
  • protofibrils or soluble oligomers Walsh DM, Selkoe DJ, Protein Pept Lett, 2004. 1 1 (3): p. 213-28.
  • the main targets for therapeutic intervention in the ⁇ cascade are classified as: (i) Inhibition of ⁇ production, (ii) Inhibition of ⁇ aggregation and fiber formation (iii) Inhibition of the inflammatory response caused by the ⁇ deposit (Grundman and Thal., Neurol Clin, 2000. 18 (4): p. 807-28).
  • AD symptoms include the use of acetylcholinesterase (AchE) inhibitors.
  • AchE acetylcholinesterase
  • Those available clinically to treat mild or moderate AD are galantamine, donepecyl hydrochloride and rivastigmine (Matharu et al., J Neurol Sci, 2009. 280 (1-2): p. 49-58).
  • DESCRIPTION OF THE INVENTION The present invention provides compounds that are useful for the preparation of a medicament and more preferably for use in the treatment of Alzheimer's disease.
  • a first aspect of the present invention relates to the use of a compound of general formula (I) for the preparation of a medicament (from now on compound of the invention):
  • Xi and X2 are independently selected from S, O or NH;
  • R1 is selected from -NR to R b , -OR c and -SRd, where R a and Rb are independently selected from a (C1-C5) or H alkyl group or are joined to form a heterocycloalkyl, and R c and Rd are, independently, an alkyl group (CC 5 );
  • R 2 is selected from a halogen or a hydrogen
  • n has a value of 1, 2 or 3, preferably 2.
  • alkyl refers, in the present invention, to hydrocarbon chains, linear or branched, having 1 to 5 carbon atoms, and which bind to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ero-butyl, sec-butyl, n-pentyl, etc., preferably the alkyl group has 1 to 3 carbon atoms and more preferably it is a methyl group.
  • the alkyl groups may be optionally substituted by one or more substituents such as aryl, halogen, (called haloalkyl), hydroxy, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto or thioalkyl.
  • substituents such as aryl, halogen, (called haloalkyl), hydroxy, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto or thioalkyl.
  • substituents such as aryl, halogen, (called haloalkyl), hydroxy, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto or thioalkyl.
  • heterocycloalkyl refers to a saturated carbocyclic chain, which has 3 to 12 carbon atom
  • the heterocycloalkyl is a monocyclic and more preferably has 3 to 6 carbon atoms.
  • the heterocycloalkyl may be opclonally substituted by one or more substituents such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, amino or nitro.
  • substituents such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, amino or nitro.
  • halogen is meant in the present invention a bromine, chlorine, iodine or fluorine atom.
  • it is chlorine.
  • X is NH. In another preferred embodiment, X2 is S.
  • R1 is -NR to Rb, more preferably R a and R b bind forming a heterocycloalkyl, and even more preferably the heterocycloalkyl is piperidinyla.
  • the compound of the invention is 2,5-dichloro-N- (4-piperidophenyl) -3-thiophenesulfonamide, of the formula:
  • the compounds of the invention may be presented in the form of pharmaceutically acceptable salts, solvates or stereoisomers.
  • pharmaceutically acceptable salts, solvates or stereoisomers refers to any pharmaceutical salt, ester, solvate or any other compound that, being administered to a receptor, is capable of providing (directly or indirectly) a compound described herein.
  • pharmaceutically unacceptable salts are also within the scope of the invention since the latter may be useful in the preparation of pharmaceutically acceptable salts.
  • the preparation of salts, stereoisomers and derivatives can be carried out by means of methods known in the art.
  • the present invention further provides pharmaceutical compositions comprising compounds of formula (I) together with an acceptable pharmaceutical carrier, adjuvant or vehicle for administration to a patient.
  • Said composition can be used together with other additional drugs to provide a combination therapy.
  • additional drugs may be part of the same pharmaceutical composition or, alternatively, be provided in a form of a separate composition for simultaneous or not administration, with the pharmaceutical composition comprising a compound of the invention. Therefore, the composition
  • the pharmaceutical invention can also comprise another active ingredient.
  • another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the invention, in addition to at least one pharmaceutically acceptable carrier.
  • compositions are the vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
  • compositions suitable for oral administration include any solid composition (tablets, pills, capsules, granulated forms, etc.) or liquid (solutions, suspensions, emulsions, syrups, etc.) and may contain conventional excipients known in the art.
  • parenteral administration can be carried out by intramuscular, intraarterial, intravenous, intradermal, subcutaneous, intrathecal or intraosseous administration but not limited to these types of parenteral administration routes. Administration may also be sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled.
  • treatment refers to eliminating, reducing or decreasing the cause or effects of the disease.
  • treatment includes, but is not limited to, alleviating, reducing or eliminating one or more symptoms of the disease; reduce the degree of disease, stabilize (i.e. no worsen) the state of the disease, delay or slow the progression of the disease, alleviate or improve the state of the disease and remit (either total or partial).
  • the compound of the invention in particular the compound 2,5-dichloro-N- (4- piperidinophenyl) -3-thiophenesulfonamide (hereinafter "compound") strongly inhibits aggregation of the human ⁇ peptide, so one of its applications is the treatment of Alzheimer's disease.
  • compound 2,5-dichloro-N- (4- piperidinophenyl) -3-thiophenesulfonamide
  • another aspect of the present invention relates to the use of the compound of general formula (I) for the inhibition of aggregation of the ⁇ peptide, preferably of the human ⁇ peptide, and even more preferably for the preparation of a medicament for the treatment of Alzheimer's disease.
  • Fig. 1 It shows the fluorescence of thioflavin associated with aggregation of the peptide in the presence and absence of the compound, which is arbitrary units.
  • Fig. 2. Represents the increase in light scattering in solution associated with aggregation of the peptide in the presence of the compound.
  • Fig. 3. Shows the images by electron microscopy of the formation of amyloid fibers under different conditions: negative control: in the absence of ⁇ peptide, positive control: ⁇ peptide (17-40) at 50 ⁇ concentration, compound: ⁇ peptide (17 -40) at concentration 50 ⁇ + compound at a concentration 100 ⁇ .
  • Fig. 4. Shows the cell viability of human neuroblastoma cells and HeLa cells by an XTT assay. MCC is minimum cytotoxic concentration.
  • Fig. 5 It shows the cellular survival in yeast related to the inhibition of aggregation of the ⁇ peptide (1-42).
  • the bars refer to equivalent tests carried out in the presence of 10 ⁇ (black column) and 20 ⁇ (white column) of MTX (methotrexate), the dihydrofolate reductase (DHFR) enzyme inhibitor.
  • MTX metalhotrexate
  • DHFR dihydrofolate reductase
  • Example 1 The first test was carried out as described in LeVine, Protein Sci, 1993. 2 (3): p. 404-10. This assay monitors the fluorescence of thioflavin associated with the aggregation of the ⁇ peptide (fragment 17-40) and compares the aggregation of said peptide that occurs in the absence and in the presence of the compound (Fig. 1).
  • the compound was purchased lyophilized and was resuspended in 100% DIVISE at a 4 mM stock concentration and subsequently diluted in PBS to a final concentration of 100 ⁇ .
  • the second test monitors the increase in light scattering in solution associated with aggregation of the ⁇ peptide (fragment 17-40) in the presence of the compound (Fig. 2).
  • the presence of the compound prevents the formation of amyloid fibers.
  • the ⁇ 17-40 peptide was resuspended in a 0.02% NH 4 OH solution until a 500 ⁇ stock solution was obtained, subsequently centrifuged at 10,000 rpm for 30 minutes at 4 s C, and diluted in a phosphate buffer solution ( PBS) to a final concentration 50 ⁇ . To the solution of the ⁇ 17-40 peptide the compound was added to a final concentration of 100 ⁇ , which had previously been resuspended in 100% DMSO (stock concentration of the compound of 20 mM).
  • PBS phosphate buffer solution
  • the absorbance value at 360 nM was analyzed every 30 minutes for 8 hours and compared with the negative control, which is a sample of 0.002% NH 4 OH, 0.5% DMSO and PBS, and the positive control, the which is a sample of the ⁇ 17-40 50 ⁇ peptide in PBS and 0.5% DMSO.
  • the test was carried out using a Varian Cary 100 Bio spectrophotometer, and Hellma 104QS quartz cuvettes with a 10 mm light path. This test allows quantifying and confirming the inhibitory effect of the formation of amyloid fibers that the compound presents, since in a sample where the aggregation process occurs (positive control), the absorbance values increase proportionally to the formation of amyloid aggregates in the weather.
  • the third test allows to visualize directly the formation of amyloid fibers (or the absence of formation in the presence of the compound) by electron microscopy (Fig. 3). As Figure 3 shows, the compound significantly reduced the formation of amyloid fibers.
  • ⁇ (17-40) 50 ⁇ , PBS and 0.5% DMSO peptide was used as a positive control.
  • the ⁇ peptide was initially resuspended in a 0.02% NH 4 OH solution at an initial concentration of 500 ⁇ , centrifuged at 10,000 rpm for 30 minutes at 4 Q C and subsequently diluted in PBS solution until a 50 ⁇ concentration was obtained, which It was incubated for 24 hours at 37 s C in the presence of the compound, which was at a final concentration of 100 ⁇ (and which had previously been resuspended in 100% DMSO, 20 mM stock solution).
  • the toxicity of the compound to human SH-SY5Y neuroblastoma cells and HeLa cells was determined by assay with compound XTT.
  • the compound shows a reasonably low toxicity judging by its Minimum Cytotoxic Concentration (MCC) (Fig. 4).
  • the cells were cultured in DMEM with phenol red, supplemented with 100 U / ml penicillin, 100 g / ml streptomycin and 10% fetal bovine serum. The culture was maintained at 37 Q C in an atmosphere of 5% C0 2. The cells were grown in 25 ml culture bottles and subcultured every 3 days. To assess the toxicity of the compound in HeLa cells, the cells were cultured in 96-well plates. 3 x 10 4 cells were seeded per well in 100 ⁇ of medium for 24 hours. Subsequently, the cells were incubated with the compound at different concentrations, as shown in Figure 4.
  • cell viability was determined using the compound 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5- [(phenyl-amino) carbonyl] -2H- Tetrazolium hydroxide (XTT) (Cell Proliferation Kit II, Roche), following the manufacturer's instructions.
  • XTT Tetrazolium hydroxide
  • the culture medium is replaced by DMEM medium without phenol red and 50 ⁇ of the XTT reagent is added to each well.
  • the culture plates are incubated again for 4 hours at 37 9 C at an atmosphere of 5% C0 2 .
  • Example 5 The minimum cytotoxic concentration (MCC) was calculated by adjustment of the viability values at each concentration of the compound evaluated, using a dose-response function with a variable slope, using the Origin Pro ® 8 software (Northampton, USA).
  • MCC cytotoxic concentration
  • the ability of the compound to inhibit aggregation of the ⁇ (1-42) peptide expressed in yeast cells has been determined using an assay that relates to cell survival with inhibition of ⁇ aggregation as described in orell, et al., Mol. BioSyst, 201 1, 7, 1 121-1 128.
  • the compound increases the survival of Saccharomyces Cerevisiae yeast cells, in a dose-dependent manner, inhibiting intracellular aggregation of the ⁇ peptide (1-42) (Fig. 5).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to the use of a compound of formula (I) for treating Alzheimer's disease, and in particular relates to a pharmaceutical composition that comprises said compound and to the use thereof for the treatment of Alzheimer's disease.

Description

COMPUESTOS INHIBIDORES DE LA AGREGACION DEL PEPTIDO  INHIBITING COMPOUNDS OF THE AGGREGATION OF THE PEPTIDE
BETA AMILOIDE  AMILOID BETA
La presente invención se refiere al uso de un compuesto de fórmula general 5 (I) para la elaboración de una composición farmacéutica, más preferiblemente para el tratamiento de la enfermedad de Alzheimer. Además, la invención se refiere a una composición farmacéutica que comprende dicho compuesto. The present invention relates to the use of a compound of general formula 5 (I) for the preparation of a pharmaceutical composition, more preferably for the treatment of Alzheimer's disease. In addition, the invention relates to a pharmaceutical composition comprising said compound.
Figure imgf000002_0001
Figure imgf000002_0001
I O (0  I O (0
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
15 La enfermedad de Alzheimer (AD) es la causa más común de declive progresivo de la función cognitiva en ancianos. Se caracteriza por la presencia de numerosas placas seniles y ovillos neurofibrilares, y se ve acompañada por pérdida de neuronas colinérgicas. Las placas seniles neurofibrilares están compuestas por péptidos beta amiloides (Αβ),15 Alzheimer's disease (AD) is the most common cause of progressive decline in cognitive function in the elderly. It is characterized by the presence of numerous senile plaques and neurofibrillar clews, and is accompanied by loss of cholinergic neurons. Senile neurofibrillary plaques are composed of beta amyloid peptides (Αβ),
20 fragmentos peptídicos de 40-42 residuos de una proteína precursora (APP) codificada en el cromosoma 21 . Se ha demostrado que el péptido Αβ y fragmentos del mismo son tóxicos in vitro e in vivo, lo que indica un papel importante de Αβ en la patogénesis de AD. 20 peptide fragments of 40-42 residues of a precursor protein (APP) encoded on chromosome 21. The Αβ peptide and fragments thereof have been shown to be toxic in vitro and in vivo, indicating an important role of Αβ in the pathogenesis of AD.
25 La naturaleza precisa de la forma tóxica de Αβ no ha sido establecida por completo pero, recientemente, la toxicidad se ha relacionado con formas tempranas de agregados peptídicos llamadas ADDLs (ligandos difundibles derivados de Αβ), protofibrillas u oligómeros solubles (Walsh D.M., Selkoe D.J., Protein Pept Lett, 2004. 1 1 (3): p. 213-28). Las dianas principales para intervención terapéutica en la cascada Αβ se clasifican en: (i) Inhibición de la producción de Αβ, (ii) Inhibición de la agregación de Αβ y de la formación de fibras (iii) Inhibición de la respuesta inflamatoria causada por el depósito de Αβ (Grundman y Thal., Neurol Clin, 2000. 18(4): p. 807-28). La inhibición de formación de fibras y, mejor aún, de agregación de Αβ es de interés terapéutico en el tratamiento de AD (Findeis y Molineaux., Methods Enzymol, 1999. 309: p. 476-88) porque los agregados de bajo peso molecular de Αβ son extremadamente tóxicos para neuronas (Pike et al., J Neurosci, 1993. 13(4): p. 1676-87). Aunque se ha hecho mucho esfuerzo para desarrollar agentes terapéuticos tales como inmunoterapia (Thatte., Curr Opin Investig Drugs, 2001 . 2(5): p. 663-7) así como compuestos que rompan láminas β (Findeis y Molineaux., Methods Enzymol, 1999. 309: p. 476-88), la terapia específica disponible para combatir AD está limitada a la basada en la mejora de la función colinérgica y el efecto clínico no es suficiente (Grundman y Thal., Neurol Clin, 2000. 18(4): p. 807-28). 25 The precise nature of the toxic form of Αβ has not been fully established but, recently, toxicity has been related to forms Early peptide aggregates called ADDLs (diffusible ligands derived from Αβ), protofibrils or soluble oligomers (Walsh DM, Selkoe DJ, Protein Pept Lett, 2004. 1 1 (3): p. 213-28). The main targets for therapeutic intervention in the Αβ cascade are classified as: (i) Inhibition of Αβ production, (ii) Inhibition of Αβ aggregation and fiber formation (iii) Inhibition of the inflammatory response caused by the Αβ deposit (Grundman and Thal., Neurol Clin, 2000. 18 (4): p. 807-28). The inhibition of fiber formation and, better still, of Αβ aggregation is of therapeutic interest in the treatment of AD (Findeis and Molineaux., Methods Enzymol, 1999. 309: p. 476-88) because low molecular weight aggregates of Αβ are extremely toxic to neurons (Pike et al., J Neurosci, 1993. 13 (4): p. 1676-87). Although much effort has been made to develop therapeutic agents such as immunotherapy (Thatte., Curr Opin Investig Drugs, 2001. 2 (5): p. 663-7) as well as compounds that break β sheets (Findeis and Molineaux., Methods Enzymol , 1999. 309: p. 476-88), the specific therapy available to combat AD is limited to that based on the improvement of cholinergic function and the clinical effect is not sufficient (Grundman and Thal., Neurol Clin, 2000. 18 (4): p. 807-28).
Por tanto, la necesidad de intervención terapéutica es imperiosa. La utilidad de los compuestos que reducen la agregación de Αβ o su neurotoxicidad in vitro se ve a menudo comprometida por su toxicidad in vivo. Los tratamientos de los síntomas de AD incluyen el uso de inhibidores de acetilcolinesterasa (AchE). Los disponibles clínicamente para tratar AD suave o moderado son galantamina, donepecil-hidrocloruro y rivastigmina (Matharu et al., J Neurol Sci, 2009. 280(1 -2): p. 49-58). DESCRIPCIÓN DE LA INVENCIÓN La presente invención proporciona unos compuestos que son útiles para la elaboración de un medicamento y más preferiblemente para su uso en el tratamiento de la enfermedad de Alzheimer. Therefore, the need for therapeutic intervention is imperative. The usefulness of compounds that reduce the aggregation of oβ or its in vitro neurotoxicity is often compromised by its toxicity in vivo. Treatments for AD symptoms include the use of acetylcholinesterase (AchE) inhibitors. Those available clinically to treat mild or moderate AD are galantamine, donepecyl hydrochloride and rivastigmine (Matharu et al., J Neurol Sci, 2009. 280 (1-2): p. 49-58). DESCRIPTION OF THE INVENTION The present invention provides compounds that are useful for the preparation of a medicament and more preferably for use in the treatment of Alzheimer's disease.
Un primer aspecto de la presente invención se refiere al uso de u compuesto de fórmula general (I) para la elaboración de un medicamento ( partir de ahora compuesto de la invención): A first aspect of the present invention relates to the use of a compound of general formula (I) for the preparation of a medicament (from now on compound of the invention):
Figure imgf000004_0001
Figure imgf000004_0001
( donde:  ( where:
Xi y X2 se seleccionan, de manera independiente, de entre S, O ó NH;  Xi and X2 are independently selected from S, O or NH;
R1 se selecciona de entre -NRaRb, -ORc y -SRd¡ donde Ra y Rb se selecciona, de manera independiente, de entre un grupo alquilo (C1-C5) o H o se unen formando un heterocicloalquilo, y Rc y Rd son, de manera independiente, un grupo alquilo (C C5); R1 is selected from -NR to R b , -OR c and -SRd, where R a and Rb are independently selected from a (C1-C5) or H alkyl group or are joined to form a heterocycloalkyl, and R c and Rd are, independently, an alkyl group (CC 5 );
R2 se selecciona entre un halógeno o un hidrógeno; y R 2 is selected from a halogen or a hydrogen; Y
n tiene un valor de 1 , 2 o 3, preferiblemente es 2. El término "alquilo" se refiere, en la presente invención, a cadenas hidrocarbonadas, lineales o ramificadas, que tienen de 1 a 5 átomos de carbono, y que se unen al resto de la molécula mediante un enlace sencillo, por ejemplo, metilo, etilo, n-propilo, /-propilo, n-butilo, íero-butilo, sec-butilo, n-pentilo, etc., preferiblemente el grupo alquilo tiene de 1 a 3 átomos de carbono y más preferiblemente es un grupo metilo. Los grupos alquilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como arilo, halógeno, (denominándose haloalquilo), hidroxilo, alcoxilo, carboxilo, carbonilo, ciano, acilo, alcoxicarbonilo, amino, nitro, mercapto o tioalquilo. Preferiblemente el grupo alquilo no esta sustituido. Por "heterocicloalquilo" se refiere a una cadena carbocíclica saturada, que tiene de 3 a 12 átomos de carbono y al menos uno de esos carbonos está sustituido por un nitrógeno y en concreto el que se une al anillo aromático (N-cicloalquilo), pudiendo ser monocíclíco o bicíclico, en este último caso los anillos pueden estar separados o condensados. Preferiblemente el heterocicloalquilo es un monocíclico y más preferiblemente tiene de 3 a 6 átomos de carbono. El heterocicloalquilo puede estar opclonalmente sustituido por uno o más sustituyentes tales como alquilo, halógeno, hidroxilo, alcoxilo, carboxilo, carbonilo, ciano, acilo, amino o nitro. Por "halógeno" se entiende en la presente invención a un átomo de bromo, cloro, yodo o flúor. Preferiblemente es cloro. n has a value of 1, 2 or 3, preferably 2. The term "alkyl" refers, in the present invention, to hydrocarbon chains, linear or branched, having 1 to 5 carbon atoms, and which bind to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ero-butyl, sec-butyl, n-pentyl, etc., preferably the alkyl group has 1 to 3 carbon atoms and more preferably it is a methyl group. The alkyl groups may be optionally substituted by one or more substituents such as aryl, halogen, (called haloalkyl), hydroxy, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto or thioalkyl. Preferably the alkyl group is not substituted. By "heterocycloalkyl" refers to a saturated carbocyclic chain, which has 3 to 12 carbon atoms and at least one of those carbons is substituted by a nitrogen and in particular the one that joins the aromatic ring (N-cycloalkyl), being able be monocyclic or bicyclic, in the latter case the rings can be separated or condensed. Preferably the heterocycloalkyl is a monocyclic and more preferably has 3 to 6 carbon atoms. The heterocycloalkyl may be opclonally substituted by one or more substituents such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, amino or nitro. By "halogen" is meant in the present invention a bromine, chlorine, iodine or fluorine atom. Preferably it is chlorine.
En una realización preferida, X es NH. En otra realización preferida, X2 es S. In a preferred embodiment, X is NH. In another preferred embodiment, X2 is S.
En otra realización preferida, R1 es -NRaRb, más preferiblemente Ra y Rb se unen formando un heterocicloalquilo, y aún más preferiblemente el heterocicloalquilo es piperidinila. In another preferred embodiment, R1 is -NR to Rb, more preferably R a and R b bind forming a heterocycloalkyl, and even more preferably the heterocycloalkyl is piperidinyla.
En una realización más preferida, el compuesto de la invención es el 2,5- dicloro-N-(4-piperid¡nofenil)-3-tiofenesulfonamida, de fórmula: In a more preferred embodiment, the compound of the invention is 2,5-dichloro-N- (4-piperidophenyl) -3-thiophenesulfonamide, of the formula:
Figure imgf000006_0001
Figure imgf000006_0001
Los compuestos de la invención pueden presentarse en forma de sales farmacéuticamente aceptables, solvatos o esteroisómeros. The compounds of the invention may be presented in the form of pharmaceutically acceptable salts, solvates or stereoisomers.
El término "sales farmacéuticamente aceptables, solvatos o estereoisómeros" se refiere a cualquier sal farmacéutica, éster, solvato o cualquier otro compuesto que, siendo administrado a un receptor, es capaz de proporcionar (directa o indirectamente) un compuesto descrito en el presente documento. Sin embargo se observará que las sales farmacéuticamente inaceptables están también en el ámbito de la invención ya que estas últimas pueden ser útiles en la preparación de sales farmacéuticamente aceptables. La preparación de sales, estereoisómeros y derivados pueden ser llevadas a cabo por medio de métodos conocidos en la materia. The term "pharmaceutically acceptable salts, solvates or stereoisomers" refers to any pharmaceutical salt, ester, solvate or any other compound that, being administered to a receptor, is capable of providing (directly or indirectly) a compound described herein. However, it will be noted that pharmaceutically unacceptable salts are also within the scope of the invention since the latter may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, stereoisomers and derivatives can be carried out by means of methods known in the art.
La presente invención proporciona además composiciones farmacéuticas que comprenden compuestos de fórmula (I) junto con un transportador farmacéutico aceptable, adyuvante o vehículo para la administración a un paciente. Dicha composición se pueden utilizar junto con otros fármacos adicionales para proporcionar una terapia de combinación. Dichos fármacos adicionales pueden formar parte de la misma composición farmacéutica o, alternativamente, ser provistos en una forma de una composición separada para su administración simultanea o no, con la composición farmacéutica que comprende un compuesto de de la invención. Por tanto, la composición farmacéutica de la invención puede comprender además otro principio activo. The present invention further provides pharmaceutical compositions comprising compounds of formula (I) together with an acceptable pharmaceutical carrier, adjuvant or vehicle for administration to a patient. Said composition can be used together with other additional drugs to provide a combination therapy. Such additional drugs may be part of the same pharmaceutical composition or, alternatively, be provided in a form of a separate composition for simultaneous or not administration, with the pharmaceutical composition comprising a compound of the invention. Therefore, the composition The pharmaceutical invention can also comprise another active ingredient.
Por tanto, otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos un compuesto de la invención, además de al menos un vehículo farmacéuticamente aceptable. Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of the invention, in addition to at least one pharmaceutically acceptable carrier.
Los "vehículos farmacéuticamente aceptables" que pueden ser utilizados en dichas composiciones son los vehículos conocidos por los técnicos en la materia y utilizados habitualmente en la elaboración de composiciones terapéuticas. The "pharmaceutically acceptable vehicles" that can be used in said compositions are the vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
Las formas farmacéuticas adecuadas para la administración oral incluyen cualquier composición sólida (tabletas, pastillas, cápsulas, formas granuladas, etc.) o líquida (soluciones, suspensiones, emulsiones, jarabes, etc.) y pueden contener excipientes convencionales conocidos en la materia. Pharmaceutical forms suitable for oral administration include any solid composition (tablets, pills, capsules, granulated forms, etc.) or liquid (solutions, suspensions, emulsions, syrups, etc.) and may contain conventional excipients known in the art.
Los compuestos descritos en esta invención, sus sales farmacéuticamente aceptables, estereoisómeros y/o solvatos, así como las composiciones farmacéuticas que los contienen, pueden ser administrados de forma oral o parenteral. La administración parenteral se puede llevar a cabo por vía de administración intramuscular, intraarterial, intravenosa, intradérmica, subcutánea, intratecal o intraósea pero sin limitarse únicamente a estos tipos de vías de administración parenteral. La administración puede ser también sublingual, nasal, intracatecal, bronquial, linfática, rectal, transdérmica o inhalada. The compounds described in this invention, their pharmaceutically acceptable salts, stereoisomers and / or solvates, as well as the pharmaceutical compositions containing them, can be administered orally or parenterally. Parenteral administration can be carried out by intramuscular, intraarterial, intravenous, intradermal, subcutaneous, intrathecal or intraosseous administration but not limited to these types of parenteral administration routes. Administration may also be sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled.
A lo largo de la presente descripción, el término "tratamiento" se refiere a eliminar, reducir o disminuir la causa o efectos de la enfermedad. Para los propósitos de esta invención, tratamiento incluye, aunque sin quedar limitados a los mismos, aliviar, disminuir o eliminar uno o más síntomas de la enfermedad; reducir el grado de enfermedad, estabilizar (es decir, no empeorar) el estado de la enfermedad, retrasar o ralentizar la progresión de la enfermedad, aliviar o mejorar el estado de la enfermedad y remitir (ya sea total o parcial). Los autores de la presente invención han demostrado en los ejemplos el compuesto de la invención, en concreto el compuesto 2,5-dicloro-N-(4- piperidinofenil)-3-tiofenesulfonamida (en adelante, "compuesto") inhibe fuertemente la agregación del péptido Αβ humano, por lo que una de sus aplicaciones es el tratamiento de la enfermedad de Alzheimer. Throughout the present description, the term "treatment" refers to eliminating, reducing or decreasing the cause or effects of the disease. For the purposes of this invention, treatment includes, but is not limited to, alleviating, reducing or eliminating one or more symptoms of the disease; reduce the degree of disease, stabilize (i.e. no worsen) the state of the disease, delay or slow the progression of the disease, alleviate or improve the state of the disease and remit (either total or partial). The authors of the present invention have demonstrated in the examples the compound of the invention, in particular the compound 2,5-dichloro-N- (4- piperidinophenyl) -3-thiophenesulfonamide (hereinafter "compound") strongly inhibits aggregation of the human Αβ peptide, so one of its applications is the treatment of Alzheimer's disease.
Por tanto, otro aspecto de la presente invención se refiere al uso del compuesto de fórmula general (I) para la inhibición de la agregación del péptido Αβ, preferiblemente del péptido Αβ humano, y aún más preferiblemente para la elaboración de un medicamento para el tratamiento de la enfermedad de Alzheimer. Therefore, another aspect of the present invention relates to the use of the compound of general formula (I) for the inhibition of aggregation of the Αβ peptide, preferably of the human Αβ peptide, and even more preferably for the preparation of a medicament for the treatment of Alzheimer's disease.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. BREVE DESCRIPCION DE LA FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES
Fig. 1. Muestra la fluorescencia de tioflavina asociada a la agregación del péptido en presencia y ausencia del compuesto, u.a. es unidades arbitrarias. Fig. 2. Representa el incremento de dispersión de luz en solución asociado a agregación del péptido en presencia del compuesto. Fig. 3. Muestra las imágenes por microscopía electrónica de la formación de fibras amiloides en distintas condiciones: control negativo: en ausencia de péptido Αβ, control positivo: péptido Αβ (17-40) a concentración 50 μΜ, compuesto: péptido Αβ (17-40) a concentración 50 μΜ + compuesto a una concentración 100 μ . Fig. 1. It shows the fluorescence of thioflavin associated with aggregation of the peptide in the presence and absence of the compound, which is arbitrary units. Fig. 2. Represents the increase in light scattering in solution associated with aggregation of the peptide in the presence of the compound. Fig. 3. Shows the images by electron microscopy of the formation of amyloid fibers under different conditions: negative control: in the absence of Αβ peptide, positive control: Αβ peptide (17-40) at 50 μΜ concentration, compound: Αβ peptide (17 -40) at concentration 50 μΜ + compound at a concentration 100 μ.
Fig. 4. Muestra la viabilidad celular de células humanas de neuroblastoma y células HeLa mediante un ensayo con XTT. MCC es concentración mínima citotóxica. Fig. 4. Shows the cell viability of human neuroblastoma cells and HeLa cells by an XTT assay. MCC is minimum cytotoxic concentration.
Fig. 5. Muestra la supervivencia celular en levadura relacionada con la inhibición de la agregación del péptido Αβ (1 -42). Las barras se refieren a ensayos equivalentes realizados en presencia de 10 μΜ (columna negra) y 20 μΜ (columna blanca) de MTX (metotrexato), el inhibidor de la enzima dihidrofolato reductasa (DHFR). Fig. 5. It shows the cellular survival in yeast related to the inhibition of aggregation of the Αβ peptide (1-42). The bars refer to equivalent tests carried out in the presence of 10 μΜ (black column) and 20 μΜ (white column) of MTX (methotrexate), the dihydrofolate reductase (DHFR) enzyme inhibitor.
EJEMPLOS A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores. En todos los ejemplos se ha utilizado el compuesto 2,5- dicloro-N-(4-piperidinofenil)-3-tiofenesulfonamida (en adelante llamado compuesto), disponible comercialmente (Maybridge, Thermo Fisher Scientific). Como se demuestra en los ejemplos, el compuesto inhibe fuertemente la agregación del péptido Αβ humano. Se han realizado tres ensayos secuenciales in vitro que cuantifican el grado de agregación del péptido Αβ (fragmento 17-40). EXAMPLES The invention will now be illustrated by tests carried out by the inventors. In all the examples, the compound 2,5-dichloro-N- (4-piperidinophenyl) -3-thiophenesulfonamide (hereinafter called the compound), commercially available (Maybridge, Thermo Fisher Scientific) has been used. As demonstrated in the examples, the compound strongly inhibits the aggregation of the human Αβ peptide. Three sequential in vitro tests have been performed that quantify the degree of aggregation of the Αβ peptide (fragment 17-40).
Ejemplo 1 El primer ensayo se llevó a cabo como está descrito en LeVine, Protein Sci, 1993. 2(3): p. 404-10. Este ensayo monitoriza la fluorescencia de tioflavina asociada a la agregación del péptido Αβ (fragmento 17-40) y compara la agregación de dicho péptido que tiene lugar en ausencia y en presencia del compuesto (Fig. 1 ). El compuesto se adquirió liofilizado y fue resuspendido en DIVISO 100% a una concentración stock 4 mM y posteriormente diluido en PBS hasta una concentración final 100 μ . Example 1 The first test was carried out as described in LeVine, Protein Sci, 1993. 2 (3): p. 404-10. This assay monitors the fluorescence of thioflavin associated with the aggregation of the Αβ peptide (fragment 17-40) and compares the aggregation of said peptide that occurs in the absence and in the presence of the compound (Fig. 1). The compound was purchased lyophilized and was resuspended in 100% DIVISE at a 4 mM stock concentration and subsequently diluted in PBS to a final concentration of 100 μ.
Como muestra la figura 1 , el compuesto disminuye la fluorescencia de tioflavina y por tanto la agregación del péptido Αβ. As Figure 1 shows, the compound decreases the fluorescence of thioflavin and therefore the aggregation of the Αβ peptide.
Ejemplo 2 Example 2
El segundo ensayo monitoriza el incremento de dispersión de luz en solución asociado a agregación del péptido Αβ (fragmento 17-40) en presencia del compuesto (Fig. 2). La presencia del compuesto impide la formación de las fibras amiloides. The second test monitors the increase in light scattering in solution associated with aggregation of the Αβ peptide (fragment 17-40) in the presence of the compound (Fig. 2). The presence of the compound prevents the formation of amyloid fibers.
El péptido Αβ17-40 se resuspendió en una solución de NH4OH al 0,02% hasta obtener una solución stock 500 μΜ, posteriormente fue centrifugado a 10.000 r.p.m. durante 30 minutos a 4s C, y diluido en una solución de tampón fosfato (PBS) hasta una concentración final 50 μΜ. A la solución del péptido Αβ17-40 fue adicionado el compuesto a una concentración final 100 μΜ, el cual previamente había sido resuspendido en DMSO 100% (concentración stock del compuesto de 20 mM). The Αβ17-40 peptide was resuspended in a 0.02% NH 4 OH solution until a 500 μΜ stock solution was obtained, subsequently centrifuged at 10,000 rpm for 30 minutes at 4 s C, and diluted in a phosphate buffer solution ( PBS) to a final concentration 50 μΜ. To the solution of the Αβ17-40 peptide the compound was added to a final concentration of 100 μΜ, which had previously been resuspended in 100% DMSO (stock concentration of the compound of 20 mM).
El valor de absorbancia a 360 nM, fue analizado cada 30 minutos durante 8 horas y comparado con el control negativo, el cual es una muestra de NH4OH al 0,002%, DMSO 0,5% y PBS, y el control positivo, el cual es una muestra del péptido Αβ17-40 50 μΜ en PBS y DMSO 0,5%. El ensayo se llevo a cabo utilizando un espectrofotómetro Varían Cary 100 Bio, y cubetas de cuarzo Hellma 104QS de 10 mm de paso de luz. Este ensayo permite cuantificar y confirmar el efecto inhibidor de la formación de fibras amiloides que presenta el compuesto, ya que en una muestra donde ocurre el proceso de agregación (control positivo), los valores de absorbancia incrementan proporcionalmente a la formación de agregados amiloides en el tiempo. The absorbance value at 360 nM was analyzed every 30 minutes for 8 hours and compared with the negative control, which is a sample of 0.002% NH 4 OH, 0.5% DMSO and PBS, and the positive control, the which is a sample of the Αβ17-40 50 μΜ peptide in PBS and 0.5% DMSO. The test was carried out using a Varian Cary 100 Bio spectrophotometer, and Hellma 104QS quartz cuvettes with a 10 mm light path. This test allows quantifying and confirming the inhibitory effect of the formation of amyloid fibers that the compound presents, since in a sample where the aggregation process occurs (positive control), the absorbance values increase proportionally to the formation of amyloid aggregates in the weather.
Ejemplo 3 Example 3
El tercer ensayo permite visualizar directamente la formación de fibras amiloides (o la ausencia de formación en presencia del compuesto) por microscopía electrónica (Fig. 3). Como muestra la figura 3, el compuesto redujo significativamente la formación de fibras amiloides. The third test allows to visualize directly the formation of amyloid fibers (or the absence of formation in the presence of the compound) by electron microscopy (Fig. 3). As Figure 3 shows, the compound significantly reduced the formation of amyloid fibers.
Como control negativo se empleó NH40H al 0,002%, DMSO 0,5% y PBS. Como control positivo se empleó el péptido Αβ (17-40) 50 μΜ, PBS y DMSO 0,5%. El péptido Αβ inicialmente se resuspendió en una solución de NH4OH 0,02% a una concentración inicial 500 μΜ, centrifugado a 10.000 r.p.m. durante 30 minutos a 4Q C y posteriormente diluido en solución PBS hasta obtener una concentración 50 μΜ, el cual fue incubado durante 24 horas a 37s C en presencia del compuesto, el cual estaba a una concentración final 100 μΜ (y que había sido previamente resuspendido en DMSO 100%, solución stock 20 mM). As negative control, 0.002% NH 4 0H, 0.5% DMSO and PBS were used. The Αβ (17-40) 50 μΜ, PBS and 0.5% DMSO peptide was used as a positive control. The Αβ peptide was initially resuspended in a 0.02% NH 4 OH solution at an initial concentration of 500 μΜ, centrifuged at 10,000 rpm for 30 minutes at 4 Q C and subsequently diluted in PBS solution until a 50 μΜ concentration was obtained, which It was incubated for 24 hours at 37 s C in the presence of the compound, which was at a final concentration of 100 μΜ (and which had previously been resuspended in 100% DMSO, 20 mM stock solution).
Para la observación de las muestras (control positivo, control negativo y péptido Αβ incubado con el compuesto), se realizó el siguiente procedimiento: sobre una rejilla de cobre se adicionaron 10 μΙ de la muestra, que se dejó reposar 5 minutos, se retiró el exceso de muestra con ayuda de una tira de papel Watman, se adicionaron 10 μΙ del colorante Acetato de Uranilo, el cual se dejó actuar durante 1 minuto. Posteriormente, se retiró el exceso de colorante y las rejillas se dejaron secando a 37s C para su posterior observación en un microscopio electrónico HITACHI H-7000 a 75 kV. Ejemplo 4 For the observation of the samples (positive control, negative control and Αβ peptide incubated with the compound), the following procedure was performed: on a copper grid 10 μΙ of the sample was added, which was allowed to stand for 5 minutes, the excess sample with the help of a strip of Watman paper, 10 μΙ of the Uranyl Acetate dye was added, which was allowed to act for 1 minute. Subsequently, the Excess dye and gratings were allowed to dry at 37 s C for later observation in a HITACHI H-7000 electron microscope at 75 kV. Example 4
La toxicidad del compuesto hacia células humanas de neuroblastoma SH- SY5Y y células HeLa se determinó por ensayo con el compuesto XTT. El compuesto muestra una toxicidad razonablemente baja a juzgar por su Concentración Mínima Citotoxica (MCC) (Fig. 4). The toxicity of the compound to human SH-SY5Y neuroblastoma cells and HeLa cells was determined by assay with compound XTT. The compound shows a reasonably low toxicity judging by its Minimum Cytotoxic Concentration (MCC) (Fig. 4).
Las células se cultivaron en DMEM con rojo fenol, suplementado con 100 U/ml de penicilina, 100 g /mi de estreptomicina y 10% de suero bovino fetal. El cultivo se mantuvo a 37Q C en una atmósfera de 5% de C02. Las células fueron cultivadas en frascos de cultivo de 25 mi y subcultivadas cada 3 días. Para evaluar la toxicidad del compuesto en células HeLa, las células fueron cultivadas en placas de 96 pocilios. Se sembraron 3 x 104 células por pocilio en 100 μΙ de medio durante 24 horas. Posteriormente, las células fueron incubadas con el compuesto a diferentes concentraciones, como muestra la figura 4. The cells were cultured in DMEM with phenol red, supplemented with 100 U / ml penicillin, 100 g / ml streptomycin and 10% fetal bovine serum. The culture was maintained at 37 Q C in an atmosphere of 5% C0 2. The cells were grown in 25 ml culture bottles and subcultured every 3 days. To assess the toxicity of the compound in HeLa cells, the cells were cultured in 96-well plates. 3 x 10 4 cells were seeded per well in 100 μΙ of medium for 24 hours. Subsequently, the cells were incubated with the compound at different concentrations, as shown in Figure 4.
A las 24 horas de incubación con el compuesto, la viabilidad celular fue determinada utilizando el compuesto 2,3-bis(2-metoxi-4-nitro-5-sulfofenil)-5- [(fenil-amino)carbonil]- 2H-hidróxido de tetrazolio (XTT) (Cell Proliferation Kit II, Roche), siguiendo las instrucciones del fabricante. Después del periodo de incubación de 24 horas, el medio de cultivo es remplazado por medio DMEM sin rojo fenol y 50 μΙ del reactivo XTT se añaden a cada pocilio. Las placas de cultivo se incuban nuevamente durante 4 horas a 379 C a una atmosfera de 5% de C02. Posteriormente, la densidad óptica fue leída a 450 nm, con una longitud de onda de referencia de 650 nm, usando un lector de placas. Los valores obtenidos con controles no tratados se consideraron como 100% de viabilidad. La concentración mínima citotoxica (MCC) fue calculada por ajuste de los valores de viabilidad a cada concentración del compuesto evaluada, utilizando una función dosis-respuesta con una pendiente variable, utilizando el software Origin Pro ® 8 (Northampton, USA). Ejemplo 5 At 24 hours of incubation with the compound, cell viability was determined using the compound 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5- [(phenyl-amino) carbonyl] -2H- Tetrazolium hydroxide (XTT) (Cell Proliferation Kit II, Roche), following the manufacturer's instructions. After the 24 hour incubation period, the culture medium is replaced by DMEM medium without phenol red and 50 μΙ of the XTT reagent is added to each well. The culture plates are incubated again for 4 hours at 37 9 C at an atmosphere of 5% C0 2 . Subsequently, the optical density was read at 450 nm, with a reference wavelength of 650 nm, using a plate reader. The values obtained with untreated controls were considered as 100% feasibility. The minimum cytotoxic concentration (MCC) was calculated by adjustment of the viability values at each concentration of the compound evaluated, using a dose-response function with a variable slope, using the Origin Pro ® 8 software (Northampton, USA). Example 5
La capacidad del compuesto de inhibir la agregación del péptido Αβ (1 -42) expresado en células de levadura ha sido determinada usando un ensayo que relaciona supervivencia celular con inhibición de la agregación de Αβ como se describe en orell, et al., Mol. BioSyst, 201 1 , 7, 1 121 -1 128. El compuesto aumenta la supervivencia de las células de levadura Saccharomyces Cerevisiae, de manera dosis dependiente, inhibiendo la agregación intracelular del péptido Αβ (1 -42) (Fig. 5). The ability of the compound to inhibit aggregation of the Αβ (1-42) peptide expressed in yeast cells has been determined using an assay that relates to cell survival with inhibition of Αβ aggregation as described in orell, et al., Mol. BioSyst, 201 1, 7, 1 121-1 128. The compound increases the survival of Saccharomyces Cerevisiae yeast cells, in a dose-dependent manner, inhibiting intracellular aggregation of the Αβ peptide (1-42) (Fig. 5).

Claims

REIVINDICACIONES
1 . Uso de un compuesto de fórmula general (I): one . Use of a compound of general formula (I):
Figure imgf000014_0001
Figure imgf000014_0001
(I) donde:  (I where:
Xi y X2 se seleccionan, de manera independiente, de entre S, O ó NH; Xi and X 2 are independently selected from S, O or NH;
R se selecciona de entre -NRaRb, -ORc y -SRd; donde Ra y Rb se selecciona, de manera independiente, de entre un grupo alquilo (CrC5) o H o se unen formando un heterocicloalquilo, y Rc y Rd son, de manera independiente, un grupo alquilo (CrC5); R is selected from -NR to R b , -OR c and -SR d ; where R a and Rb are independently selected from an alkyl group (CrC 5 ) or H or are joined to form a heterocycloalkyl, and R c and Rd are, independently, an alkyl group (CrC 5 );
R2 se selecciona entre un halógeno o un hidrógeno; y  R2 is selected from a halogen or a hydrogen; Y
n tiene un valor de 1 , 2 o 3, para la elaboración de un medicamento. n has a value of 1, 2 or 3, for the preparation of a medicine.
2. Uso según la reivindicación 1 , donde X1 es NH. 2. Use according to claim 1, wherein X1 is NH.
3. Uso según cualquiera de las reivindicaciones 1 o 2, donde X2 es S. 3. Use according to any of claims 1 or 2, wherein X2 is S.
4. Uso según cualquiera de las reivindicaciones 1 a 3, donde R1 es -NRaRb. 4. Use according to any of claims 1 to 3, wherein R1 is -NR to R b .
5. Uso según la reivindicación anterior, donde Ra y Rb se unen formando un heterocicloalquilo. 5. Use according to the preceding claim, wherein R a and Rb are joined forming a heterocycloalkyl.
6. Uso según la reivindicación anterior, donde el heterocicloalquilo es la piperidinila. 6. Use according to the preceding claim, wherein the heterocycloalkyl is piperidinyl.
7. Uso según cualquiera de las reivindicaciones 1 a 6, donde R2 es un halógeno. 7. Use according to any of claims 1 to 6, wherein R 2 is a halogen.
8. Uso según la reivindicación anterior, donde el halógeno es Cl. 8. Use according to the preceding claim, wherein the halogen is Cl.
9. Uso según cualquiera de las reivindicaciones 7 u 8, donde n es 2. 9. Use according to any of claims 7 or 8, wherein n is 2.
10. Uso según cualquiera de las reivindicaciones 1 a 9, donde el compuesto es de fórmula: 10. Use according to any of claims 1 to 9, wherein the compound is of the formula:
Figure imgf000015_0001
Figure imgf000015_0001
1 1. Uso del compuesto de fórmula general (I) según se describe en cualquiera de las reivindicaciones 1 a 10, para la elaboración de un medicamento para el tratamiento de la enfermedad de Alzheimer. 1 1. Use of the compound of general formula (I) as described in any of claims 1 to 10, for the preparation of a medicament for the treatment of Alzheimer's disease.
12. Composición farmacéutica que comprende un compuesto de fórmula general (I) según se describe en cualquiera de las reivindicaciones 1 a 10. 12. Pharmaceutical composition comprising a compound of general formula (I) as described in any one of claims 1 to 10.
13. Composición farmacéutica según la reivindicación anterior que además comprende un vehículo farmacéuticamente aceptable. 13. Pharmaceutical composition according to the preceding claim further comprising a pharmaceutically acceptable carrier.
PCT/ES2012/000299 2011-12-15 2012-11-30 Compounds that inhibit beta amyloid peptide aggregation WO2013087954A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP201132020 2011-12-15
ES201132020A ES2413911B1 (en) 2011-12-15 2011-12-15 INHIBITING AGENT OF THE AGGREGATION OF THE BETA AMILOID PEPTIDE.

Publications (1)

Publication Number Publication Date
WO2013087954A1 true WO2013087954A1 (en) 2013-06-20

Family

ID=48611885

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2012/000299 WO2013087954A1 (en) 2011-12-15 2012-11-30 Compounds that inhibit beta amyloid peptide aggregation

Country Status (2)

Country Link
ES (1) ES2413911B1 (en)
WO (1) WO2013087954A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998027081A1 (en) * 1996-12-19 1998-06-25 Smithkline Beecham Plc Sulphonamide derivatives, process for their preparation, and their use as medicaments
WO2002008179A1 (en) * 2000-07-21 2002-01-31 Biovitrum Ab Aryl sulfonamides as serotonin antagonist for the treatment of obesity
WO2004080882A1 (en) * 2003-03-07 2004-09-23 Mhe Technologies, Inc. Hoist apparatus rope sensing device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998027081A1 (en) * 1996-12-19 1998-06-25 Smithkline Beecham Plc Sulphonamide derivatives, process for their preparation, and their use as medicaments
WO2002008179A1 (en) * 2000-07-21 2002-01-31 Biovitrum Ab Aryl sulfonamides as serotonin antagonist for the treatment of obesity
WO2004080882A1 (en) * 2003-03-07 2004-09-23 Mhe Technologies, Inc. Hoist apparatus rope sensing device

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALLISTRERI, A. ET AL.: "The Acid Dissociation of Arene sulphonamides: 6Het Constants for Thia- and Oxa-substituents in Five-membered S-Linked Heterocycles and Effects of Substituents in the N-Linked Aromatic Ring", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS II, vol. 7, 1977, pages 984 - 987 *
COLE, D.C. ET AL.: "(S)-N-(5-Chlorothiophene-2-sulfonyl)-(3,(3- diethylalaninol: a Notch-1-sparing y-secretase inhibitor", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 19, 2009, pages 926 - 929 *
PU, J. ET AL.: "Synthesis and structure-activity relationship of a novel series of Heterocyclic sulfonamide y-secretase inhibitors", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 17, 2009, pages 4708 - 4717 *

Also Published As

Publication number Publication date
ES2413911B1 (en) 2014-05-14
ES2413911A1 (en) 2013-07-17

Similar Documents

Publication Publication Date Title
KR102561697B1 (en) Polymorphs of sepiapterin and salts thereof
Oakley et al. Tau filament self-assembly and structure: tau as a therapeutic target
US20140080780A1 (en) identification of compounds that disperse tdp-43 inclusions
AU2011302715B2 (en) Compounds as modulators of a mutant CFTR protein and their use for treating diseases associated with CFTR protein malfunction
ES2873510T3 (en) Water soluble derivatives of 3,5-diphenyl-diazole compounds
CA2906194A1 (en) Benzimidazole derivatives and uses thereof
US11364211B2 (en) Methods for selecting phosphatase selective and non-selective phosphatase inhibitors
WO2011134098A1 (en) Method for inhibiting tau protein aggregation and treatment of alzheimer's disease with a compound derived from quinoline
JP2019163314A (en) Novel formulations
JP6595345B2 (en) Chemical chaperonins as novel molecular modulators of beta protein aggregation in conformational diseases
JP2016536370A5 (en)
AU2015298491A1 (en) Modulators of caspase-6
US11278518B2 (en) Methods of treatment using 4-methylumbelliferone and derivatives thereof
WO2013087954A1 (en) Compounds that inhibit beta amyloid peptide aggregation
JP2015534990A (en) Compositions and methods for treating or preventing diseases or disorders associated with miscontrolled eIF4E
ES2449640B1 (en) COMPOUNDS FOR USE IN THE TREATMENT OF ALZHEIMER'S DISEASE
WO2013079745A1 (en) Compounds that inhibit aggregation of the beta amyloid peptide
ES2923022T3 (en) Agents that inhibit Ngly1 and methods of using them
WO2022114906A1 (en) Novel pharmaceutical composition for treating neurodegenerative diseases
ES2411804B1 (en) INHIBITING COMPOUNDS OF THE AGGREGATION OF THE BETA AMILOID PEPTIDE.
WO2018172587A1 (en) Cdc-7-inhibitor compounds and use thereof for the treatment of neurological conditions
RU2675794C1 (en) Multifunctional conjugates of tacrine and its analogues with derivatives of 1,2,4-tiadiazole, the way of their synthesis and application for the treatment of neurodegenerative diseases
WO2011095668A1 (en) Use of yessotoxin and analogues and derivatives thereof for treating and/or preventing neurodegenerative diseases linked to tau and beta amyloid
ES2924950T3 (en) Novel compounds for the treatment, alleviation or prevention of disorders associated with Tau aggregates
JP6970667B2 (en) Quinoline amide and its usage

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12857004

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12857004

Country of ref document: EP

Kind code of ref document: A1