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Número de publicaciónWO2003087109 A1
Tipo de publicaciónSolicitud
Número de solicitudPCT/IB2002/001198
Fecha de publicación23 Oct 2003
Fecha de presentación12 Abr 2002
Fecha de prioridad12 Abr 2002
Número de publicaciónPCT/2002/1198, PCT/IB/2/001198, PCT/IB/2/01198, PCT/IB/2002/001198, PCT/IB/2002/01198, PCT/IB2/001198, PCT/IB2/01198, PCT/IB2001198, PCT/IB2002/001198, PCT/IB2002/01198, PCT/IB2002001198, PCT/IB200201198, PCT/IB201198, WO 03087109 A1, WO 03087109A1, WO 2003/087109 A1, WO 2003087109 A1, WO 2003087109A1, WO-A1-03087109, WO-A1-2003087109, WO03087109 A1, WO03087109A1, WO2003/087109A1, WO2003087109 A1, WO2003087109A1
InventoresCesare Biagi
SolicitanteSilbiotec Due S.A.
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  Patentscope, Espacenet
Medicaments containing glycerophosphoinositol-4-phosphate derivatives
WO 2003087109 A1
Resumen
Compounds of general formula (I): their enantiomers, diastereoisomers, racemes, their mixtures, their hydrates e solvates, wherein R1, R2, R3, R4, R5, R6 and M have the meaning described, and compositions containing them for use as medicaments.
Reclamaciones  (El texto procesado por OCR puede contener errores)
CLAIMS 1. Compounds of general formula (I):
their enantiomers, diastereoisomers, racemes, their mixtures, their hydrates and solvates, wherein:
II) Rl, R2, R3, R4, R5, R6 can be equal or different among each other being a) H or .
- a group C(0)A, acylic residue of mono-carboxylic acid or emiacylic residue of di-carboxylic acid, where A can be: an alkyl radical saturated or unsaturated having from 1 to 4 double bond, linear or branched, or an aryl, arylalkyl or heterocyclic group having one or more heteroatoms or an alkyl or alkenyl group mono- or poly-cyclic; these groups can be optionally substituted with one or more groups selected among keto, hydroxy, acylamido, halogen, mercapto, alkylthio or alkyldithio, -COOH and where these -COOH can be optionally salified in equivalent ratioto form the related COOM salt wherein M has the same meaning described at point (II) ; (II) M is a cation H or a cation of a pharmacologically acceptable inorganic element, or a cation of a pharmacologically acceptable organic base, monovalent, divalent or trivalent; when M is monovalent, m=3 and n=l; when M is divalent m=3 and n=2"; when M is trivalent m=l and n=l,for use as medicaments.
2. Compounds according to claim 1 , wherein i) when A is an alkyl group it is preferably satured or mono-unsatured and preferably has from 1 to 8 carbon atoms, and preferably from 2 to 6. ii) when A is an alkyl or alkenyc group mono- or poly- cyclic it has preferably from 5 to 30 carbon atoms, more preferably from 6 to 24 carbon atoms; iii) when A is an aryl, arylalkyl or heterocycle group having one or more heteroatoms, it has preferably from 4 to 15 carbon atoms and more preferably from 4 to 8 carbon atoms and it has from 1 to 5 heteroatoms and preferably from 1 to 3 heteroatoms, selected among N, 0 or S, preferably N.
3. Compounds according to claim 2, wherein: i) when M is the cation of a pharmacologically acceptable inorganic element, it is preferably selected among sodium, lithium, potassium, magnesium, calcium, zinc, iron, copper, silver, manganese and chromium; ii) when M is the cation of a pharmacologically acceptable organic base, it is preferably a mono-, di-, tri- o tetra-alkylammonium, more preferably N- (2- hydroxyethyl) -dimethylammonium, a cation of choline or of an amino acid, preferably lysine' or arginine or a cation of mono-, di-, tri and tetra-peptides or cation of xanthine bases, preferably, caffeine.
4. Compounds according to any claim from 1 to 3, wherein compounds of formula (I) are selected among zinc salt of L-α-phosphoglycero-D-myo- inositol-4-phosphate , lysine salt of L-α~phosphoglycero-D~myo- inositol-4-phosphate,
- potassium salt of L-α-glycero-phospho-D-myo- inositol-4-monophosphate peracetylate, and compounds of formula (I) wherein
- R1=R2=R3=R4=R5=R6=CH3-C(0)-; M=Zn; n=l; m=l,
R1=CH3-(C0)- R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l,
R1=CH3- (CO) - R2=R3=R4=R5=R6=H; M=Zn; n=l; m=l,
R1=CH3- (CO) - R2=R3=R4=R5=R6=H; M=Lysine; n=l; m=2,
R1=C6H5- (CO) -; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l, - R1=CH3- (CH2)3- (CO) -; R2=R3=R4=R5=R6=-H; M=Ca; n=l; m=l,
- Rl=KO(CO) -CHa- (CO) -; R2=R3=R4=R5=R6=H; M=K; n=l; m=2 , - Rl=acylic radical of nicotinic acid; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l,
- Rl= acylic radical of cholic acid; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l,
- R1=H2N-CH2- (CO)-; R2=R3=R4=R5=R6=H; M=Na; n=l; m=l and
- Rl=acylic radical of lipoic acid; R2=R3=R4=R5=R6=H; M=K; n=l; m=2.
5. Compounds according to any claim from 1 to 4, for use in treating pathologies mediated by the activation or over-stimulation of enzymatic and metabolic G protein associated pathways.
6. Use of the compounds according to any claim from 1 to 4 and of there non-salified derivatives for the preparation of a drug for the treatment of the pathologies mediated by the activation or over- stimulation of enzymatic and metabolic G protein associated pathways.
7. Use according to claim 6 , for the treatment of articular pathologies such as arthritis and arthrosis including rheumatoid arthritis and psoriatic arthritis.
8. Use according to claim 6 , for the treatment of ophthalmic pathologies.
9. Use according to claim 6 , for the treatment of skin disease such as psoriasis, sebofrhoea, atopic dermatitis, UVB damage and more in general cuteneous dis-reactivity.
10. Use according to claim 6, for the treatment of septical shock and viral and bacterial infections, gengival tissue damage also due to bacterial infection.
11. Use according to claim 6, for the treatment of pathologies of the respiratory apparatus, such as acute pulmonary damage including newborn pathologies, chronic obstructive bronchopathies BCPO including asthma.
12. Use according to claim 6 , for the treatment of intestinal ischemia.
13. Use according to claim 6, for the treatment of cardio-vascular pathologies associated with vascular remodelling, contractile strength and platelet aggregation.
14. Use according to claim 6, for the treatment of kidney pathologies and any pathology associated to the over-stimulation of PLA2 enzyme even mediated by the activation of different pathways such as thrombin and specific receptors and growth factors such as EGF, NGF or of kinins such as the NK and Bl receptors, or of purines such as the P2Y receptor, or of neuropeptides such as bombesin or other G protein coupled receptor activating CPLA2.
15. Use according to claim 6, for the treatment of tumor pathologies .
16. Use according to claim 6 , for the treatment of headaches and migraine, pain and hyperalgesia, pathologies of Central Nervous System and Peripheral Nervous System for instance pathologies associated with damage to the barrier between vessel and nervous system such as the perinerium of the nerves including peripheral neuropathy, and pathology associated with blood brain barrier damage, for example neuronal edema, stroke, cerebral hemorrhage, cerebral edema and, TIA (Transient Ischaemic Attack) .
17. Use according to claim 6, for the treatment of Alzheimer's disease or behavioural disorders such as schizophrenia and depression.
18. Use according to claim 6, for the treatment of pathologies related to food intake disorders such as bulimia, anorexia, cachessia, obesity; dis-metabolic pathologies such as diabetes; pancreatic disorders.
19. Compounds of general formula (I) according to any claims from 1 to 4, on condition that Rl, R2, R3 , R4 ,
R5 , R6 are not on contemporaneousness hydrogen.
20. Compositions containing as active principle at least one of the derivatives according to any of the claim from 1 a 4 in association with pharmaceutically acceptable excipients.
21. Compositions according to claim 20, suitable for systemic, parenteral, oral and rectal, inhalational, topic, transdermic intravenous, intramuscular, subcutaneous administration.
22. Compositions according to claim 21, wherein the oral form of administration are tablets, sugar coated tablets and, capsules, powder, solutions, suspensions or nebulizated form.
23 Compositions according, to claim 21, wherein the inhalational formulation are powders, solutions, suspensions .
24 Composition according to claim 21, wherein the topic formulation are emulsions, gels, and solutions compatible with skin and mucosal use, eye drops
25. Use according to any claim from 6 to 18, wherein the drug is for human therapy.
26. Use according to any claim from 6 to 18, wherein the drug is for veterinary therapy.
27. Use according to any claim from 6 to 18 and from 25 to 26, wherein such treatment involves dosages of active principle including between 0.1 and 100 mg/Kg for 1-100 -days for each therapy cycle of, in relation to the type, stage, severity and apparatus of manifestation of the pathology or alteration or possible application in human or veterinarian health care.
28. Use according to any claim from 6 to 18 and from 25 to 26, wherein, the medicament is formulated as supplements for oral intake for preventive or coadiuvant treatment .
Descripción  (El texto procesado por OCR puede contener errores)

DESCRIPTION

MEDICAMENTS CONTAINING GLYCEROPHOSPHOINOSITOL-4-PHOSPHATE DERIVATIVES

The present invention relates to the use of glycerophosphoinositols for the preparation of a drug for the treatment of pathologies mediated by an activation or over-stimulation of the G protein associated enzymatic and metabolic pathways, new derivatives having the glycerophosphoinositol structure and compositions containing them.

It is known that the G protein associated receptors are a plurality of trans-membrane molecules capable of interaction with a different type of ligands. This interaction is associated with the cascade of second messenger "signaling" mechanisms via G protein, in particular through heterotrimeric G proteins.

Such G proteins have been suggested to mediate, at least in part, also the activation of phospholipase C, adenylate cyclase and phospholipase A2 (PLA2) .

Furthermore it is known that the receptor-mediated G protein activation is blocked by pertuxis toxin capable to abolish the G protein modulating effect on PLA2 enzyme activity. The use of gene knockout techniques has shown the biological relevance of the cPLA2 involvement into specific cellular systems or cellular events including cytotoxicity and apoptosis further to inflammation and tissue damage. Through these techniques it has been shown that the arachidonic acid release is mediated overall by cPLA2 as an answer to a variety of stimuli such as lypopolysaccharide, thrombin o cytokins (for instance Interleuchina-lβ) , Tumor Necrosis Factor α, but also neuropeptides such as takykinin, bradykinin or neurotransmitters such as purines, in particular ATP, and serotonergic , adrenergiσ and muscarinic agonists, which activate the enzyme leading to intracellular Ca2+ increase and to a rapid enzyme phosphorilation by protein Kynase C and subsequent translocation to plasmatic membrane where it bind to phospholipidic substrate. These mediators activate • also the phospholipid de novo synthesis. (Sapirstein, A. and Bonventre, J.V. , "Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts" , Biochim. Biophys . Acta, 1488, 139-48, 2000).

The mutual modulation of the G proteins, PKC, cPLA2 and adenylate cyclase pathways, lipid and lysolipid mediators including arachidonic acid, configures G proteins and the enzyme cascades under G protein control as an important pharmacological target .

Among lipid mediators, it is known the importance of the inositol phospholipid "signaling" pathway. Glycerophosphoinositols originate from membrane phosphoinositides following the phospholipase A2 activation wherein the starting main products of this reaction are . arachidonic acid and lysophosphatidylinositols. These latter are the substrate for a lysophospholipase A (LysoPLA) leading to the release of fatty acids bound at sn-1 position and to the glycerophosphoinositols formation.

Glycerophosphoinositols , including glycerophosphoinositol-4-phosphate, increase in relation to the RAS-induced cell mutation and anyhow in relation to RAS pathway activation. Furthermore glycerophosphoinositol-4-phosphate has been observed to be increased following α^adrenoceptor stimulation in mammalian heart (Debetto G, Cargnelli G, Antolini M, Bova S., Travisi L, Varotto R, Luciani S, wαl- Adrenorecptor -Mediated _ Formation of

Glycerophosphoinisitol -4 -phosphate in Rat Heart" , Possible Role in the Positive Inotropic Response Biochemical Pharmacology, 58, 1437-1446, 1999) and hence has been associated with positive inotropic effect. Glycerophosphoinositol-4-phosphate and its hydrolisis product glycerophosphoinositol as well as its precursor glycerophosphoinositol-4 , 5-diphosphate, can increase following a variety of stimuli, not necessarily according to the same rate (Valitutti, S., Cucchi, P., Colletta, G., Di Filippo, C. and Corda, D. , " Trans format ion by the k-RAS oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinosi tol production and phosphoinositide synthesis in thyroid cells" , Cell. Signal. 3, 321-32, 1991; Berrie, C.P., Iurisci, C. and Corda, D., "Membrane transport and in vitro metabolism of the RAS cascade messenger, glycerophosphoinositol 4 -phosphate" , Eur. J. Biochem. 266, 413-9, 1999; Corda, D. e Falasca, M. , "Glycerophosphoinositols as potential markers of RAS- induced transformation and novel second messengers" , Cancer Res. 16, 1341-50, 1996).

Studies carried out to clarify the biological role of glycerophosphoinositols have been shown that as in non- odified-cell cultures, glycerophosphoinositol-4- phosphate inhibits in a dose-dependent manner the cAMP accumulation, inhibits iodide-uptake and timidine incorporation which are cAMP regulated functions.

No biological activity has been associated with the precursor glycerophosphoinositol-4, 5-diphosphate. The applicant has now surprisingly found that L-α- glycero-phospho-D-myo-inositol-4-monophosphate (GPI-4-P) and its derivatives obtained by semi synthesis and characterised by the general formula (I) here following described, are endowed with inhibitory activity, both in vitro and in vivo, toward G proteins and biological signals mediated by G proteins, showing an high in vivo bioavailability.

The above described activities allow the use of L- α-glycero-phospho-D-myo-inositol-4-monophosphate (GPI-4- P) and its derivatives for the treatment of pathologies mediated by PLA2 activation, as following described, as well as for the alterations where the activation and/or deactivation path is coupled with G protein, for example, platelet aggregation via thrombin and heart contractile strength.

Object of the present invention are the new salts and covalent derivatives of L-α-glycero-phospho-D-myo- inositol-4-monophosphate (GPI-4-P) , characterized by the following general formula:

where Rl, R2, R3, R4, R5, R6, equal or different between each other, can be either H, or -C(0)A, where the meaning of A and M will be subsequently detailed The present invention relates also to the use of L- α-glycero-phospho-D-myo-inositol-4-monophosphate (GPI-4- P) , and its salts, in particular with metal alkaline or earth alkaline, in particular zinc and with organic ions in particular aminoacids and in particular lysine, and also of the new covalent derivatives according to the formula (I) for the preparation of a drug suitable for the treatment of pathologies mediated by the activation or over-stimulation of G protein-associated enzymatic and metabolic pathways, such as, for example, thrombin and in particular PLA2 activation.

The present invention is also related to pharmaceutical compositions containing as active principle at least one of the derivatives according to the formula (I) in association with suitable excipients for the treatment of pathologies mediated by the activation or over-stimulation of G protein-associated and activated enzymatic and metabolic pathway, for instance PLA2 such as articular pathologies i.e. arthritis and arthrosis including rheumatoid arthritis and psoriatic arthritis; eye pathologies; skin disease such as psoriasis, seborrhoea, atopic dermatitis, UVB damage and more in general cuteneous dis-reactivity; septical shock and viral and bacterial infection, gengival tissue damage also due to bacterial infection; pathologies of the respiratory apparatus, such as acute pulmonary damage including new-born pathologies, chronic obstructive bronchopathies BCPO including asthma; intestinal ischemia; cardio-vascular pathologies associated with vascular remodelling, contractile strength and platelet aggregation; kidney pathologies and any pathology associated to over-stimulation of PLA2 enzyme even mediated by the activation of different pathways such as thrombin and specific receptors and growth factors such as EGF, NGF or of kinins such as the NK and Bl receptors, or of purines (for example ATP) such as the P2Y, or of neuropeptides such as bombesin or other G proteins coupled receptor activating cPLA2 ; tumor pathologies;' headaches and migraine, pain and hyperalgesia, pathologies of Central Nervous System and Peripheral Nervous System for instance pathologies associated with damage to the barrier between vessel and nervous system such as the perinerium of the nerves including peripheral neuropathy, and pathology associated with blood brain barrier damage, for example neuronal edema, stroke, cerebral hemorrhage, cerebral edema and, TIA (Transient Ischaemic Attack); Alzheimer's disease or behavioural disorders such as schizophrenia and depression; pathologies related to food intake disorders such as bulimia, anorexia, cachessia, obesity; dis-metabolic pathologies such as diabetes; pancreatic disorder .

The characterization and the advantages of the L-α- glycero-phospho-D-myo-inositol-4-monophosphate derivatives according to the formula (I) as agents capable to negatively modulate G protein activation and to influence the G protein-associated activities for

' example phospholipase A2 activation, in particular of the cytosolic isoform (cPLA2) , resulting in the inhibition of arachidonic acid release and its metabolite formation, will be described in detail in the following sections.

The present invention refers to compounds according to the general formula (I)

their enantiomers, diastereoisomers, racemes, their mixtures, their hydrates e solvates, wherein:

I) Rl, R2, R3, R4, R5, R6 can be equal or different among each other being a) H or

- a group C(0)A, acylic residue of mono-carboxylic acid or emiacylic residue of di-carboxylic acid, where A can be: an alkyl radical saturated or unsaturated having from 1 to 4 double bond, linear or branched, or an aryl, arylalkyl or heterocyclic group having one or more heteroatoms or an alkyl or alkenyl group mono- or poly-cyclic; these groups can be optionally substituted with one or more groups selected among keto, hydroxy, acylamido, halogen, mercapto, alkylthio or alkyldithio, -COOH and where these -COOH can be optionally salified in equivalent ratio to form the related -COOM salt wherein M has the same meaning described at point (II) ;

•(II) M is a cation H or a cation of a pharmacologically acceptable inorganic element, or a pharmacologically acceptable cation of an organic base, monovalent, divalent or trivalent; when M is monovalent it is m=3 and n=l; when M is divalent it is m=3 and n=2 ; when M is trivalent it is m=l and n=l. More detailed:

Description of group A: i) when A is an alkyl group it is preferably satured or mono-unsatured and preferably has from 1 to 8 carbon atoms, and preferably from 2 to 6. ii) when A is an alkyl or alkenyc group mono- or poly- cyclic it has preferably from 5 to 30 carbon atoms, more preferably from 6 to 24 carbon atoms; iii) when A is an aryl, arylalkyl or heterocycle group having one or more heteroatoms, it has preferably from 4 to 15 carbon atoms and more preferably from 4 to 8. The heteroatoms are from 1 to 5 and preferably from 1 to 3. They are N, 0 or S, preferably N. These heteroatoms N can be optionally salified with a pharmacologically acceptable organic or inorganic acid. Description of H: i) when M is the cation of a pharmacologically acceptable inorganic element, it is preferably selected among sodium, lithium, potassium, magnesium, calcium, zinc, iron, copper, silver, manganese and chromium; ii) when M is the cation of a pharmacologically acceptable organic base, it is preferably a mono-, di-, tri- o tetra-alkylammonium, more preferably N- (2- hydroxyethyl) -dimethylammonium, a cation of choline or of an amino acid, preferably lysine or arginine or a cation of mono-, di-, tri and tetra-peptides or cation of xanthine bases, preferably, caffeine.

The term acylamino means preferably an acetylammino group .

The term alkoxy means preferably methoxy- , ethoxy or allyloxy groups.

The term halogen means preferably chloride, fluoride, bromide or iodide .

The term arylalkyl radical means preferably a C7-C9- arylalkyl group and more preferably a benzyl . The term aryl radical means preferably a C6-C12-aryl, preferably a phenyl .

The term heterocycle radical means preferably the radical of a saturated, unsaturated or aromatic heterocycle, with a 5 or 6 member ring. The term cycloalkyl or cicloalkylenic radical means preferably a ring mono- or poly- cyclic having preferably from 5 to 30 carbon atoms, more preferably from 6 to 24.

A further object of the present invention is the use of the compounds of formula (I) for the treatment of pathologies mediated by G Protein-associated metabolic and/or enzymatic pathways for example phospholipase A2 activation, in particular of its cytosolic isoform (cPLA2) , and the consequent inhibition of arachidonic acid release and the formation of its metabolites, will be described in detail in the following sections . Preparation of the compounds of the invention The preparation of the compounds of the present invention is carried our preferably, but not limited to1, starting from L-α-glycero-phospho-D-myo-inositol-4- monophosphate (GPI-4-P) ; GPI-4-P is a known substance, commercially available as potassium salt.

L-α-glycero-phospho-D-myo-inositol-4-phosphate can be obtained starting from L-α-phosphatydylinositol-4- monophosphate, extracted from crude or partially purified preparation of phospholipids preferably of plant origin, through alcoholysis and deacylation of the glycerol at 1 and 2 positions. The reaction has been carried out in alcoholic solvent and catalized by an alcoxyde.

L-α-phosphatydylinositol-4-monophosphate is suspended into a pure alcoholic solvent or in a mixture of alcohols or a mixture formed by an alcohol and a different organic solvent. The concentration of L-α-phosphatydylinositol-4- monophosphate will be between 10 and 500mg/ml, preferably between 30 e 300mg/ml and more preferably between 50 and 250mg/ml. The alcoholic solvent used for the reaction will be preferably, but not limitatively an aliphatic satured alcohol, selected among methanol, ethanol, n-propanol, n-butanol, n-octanol, isopropanol, isobutanol, isopentanol, terbutanol, ethylene glycol, propandiol, glycerol or an arylaliphatic alcohol and preferably benzilic alcohol.

The organic solvent eventually used in mixture with the selected alcohol can be an aliphatic hydrocarbon or a mixture of aliphatic hydrocarbons and preferably, pentane, hexane, cyclohexane or petrol ether, or an aromatic hydrocarbon and preferably toluene or xylene, or an ether preferably ethylether, isopropylic ether, tert-buthylmethylic ether, or an ester and preferably methyl acetate, ethyl acetate, butyl acetate, ethyl propionate, or a ketone and preferably acetone, methylethylketone or methylisobutylketone, or a nitrile and preferably acetonitrile.

The alcoxyde used as catalyst will be preferably the alcoxyde of an alkaline or alkaline earth metal, and more preferably sodium methylate, sodium ethylate, potassium terbutylate, magnesium ethylate or calcium ethylate .

The reaction is carried out under anhydrous condition and preferably under inert gas atmosphere , 5 preferably nitrogen.

The reaction is carried out at temperature between -10°C and +60°C and preferably between +5°C and +40°C.

The reaction is carried out for a time between 10 minutes and 24 hours and preferably between 30 minutes 10. and 6 hours.

At the end of the reaction the alcoxyde can be optionally neutralized with an organic acid selected preferably among formic, acetic, benzoic, maleic, methansulfonic, para-toluensulfonic or inorganic 15 selected among hydrochloric, sulfuric, phosphoric acid or a salt of phosphoric acid such as sodium or potassium monobasic phosphate.

GPI-4-P can be isolated from the reaction mixture by centrifugation or filtration or by solvent / water 20 partitioning. The partition can be performed directly between reaction-solvent and water if the case of. non- miscibility of the solvent with water, whereas the reaction-solvent can be dried and the residue submitted to partition between a non-water-miscible solvent and 25 water. GPI-4-P can be recovered by evaporation under vacuum of the aqueous phase, lyophilization or spray- drier. The compound can be utilized as such or further purified by normally-used techniques.

General method for the preparation of the GPI-4-P derivatives covalently acilated on alcoholic -OH groups

The preparation of these derivatives is performed generally starting from a GPI-4-P salt, in general potassium, pyridinium or quaternary ammonium, and among these it is preferable tetrabutylammonium, in a pure organic solvent or in organic solvent mixtures or mixtures of organic solvents with water at a concentration between 10 and 500 mg/ml and preferably between 50 and 200 mg/ml. The preferred organic solvents are: acetone, 2-butanone, methylisobutylketone, dimethylsulfoxide, sulfolane, dimethylformamide, dimethylacetamide, diethylacetamide, n-methyl-2- pyrrolidone, pyridine, tetrahydrofuran, methyltetrahydrofuran, acetonitrile, dimethoxyethane, diethylether, terbutylmethylether, ethyl acetate, chloroform, dichloromethane, 1, 1, 1-trichloroethane, methanol, ethanol, 2-propanol, butanol . The reactions are carried out at a temperature between -30°C ' and 120°C, preferably between 5°C and 40°C along a time between 15 minutes and 48 hours, preferably between 1 hour and 15 hours. In order to insert -C(0)A groups, monocarboxylic acid acylic residues or dicarboxylic acid emiacylic residues as defined at point (a) of the detailed description of the invention, GPI-4-P is submitted' to a reaction with activated derivatives of these acids among them are preferred chlorides and bromides, anhydrides, mixed anhydrides, cyclic anhydrides, active esters such as p-nitrophenylesters, succinimydylesters, acylimydazoles, O-acylisoureas preferably, but not limited to, in presence of a inorganic or organic base. Among these bases are preferred bicarbonates, carbonates, oxydes, hydroxydes, hydrides and alkaline metals or alkaline earth metals alkoholates and preferably litium, sodium, potassium, magnesium or calcium, trimethylamine, triethylamine, tributhylamine, tetramethylammonium hydroxyde, tetrabutylammonium hydroxyde, pyridine or picolina.

Example 1 Preparation of Zinc salt of L-α-glycero- phospho-D-myo-inositol-4-monophosphate 5,3g of GPI-4-P potassium salt (10 moles) are dissolved in 35 ml of distilled water. The solution is cooled at 4°C and eluted in a column containing 35 ml of sulfonic cation exchange resin generated in H+ form and thermostated at 4°C. The column is then eluted with 15 ml of distilled water and the eluate is collected at 4°C, flowed with nitrogen and neutralised with l,08g of basic zinc carbonate. The resulting solution is filtered, frozen and lyophilized. The reaction yields 99%. The chemical-physical characteristics of the product L- α-glycero-phospho-D-myo-inositol-4 -monophosphate zinc salt are: formula : (C374P2) 2Zn3 molecular formula : Cι8H340P4Zn3 formula weight : 1018.5 elemental composition : C=21.23%; H=3.36%; 0=43.99%; P=12.16%; Zn=19.26% solubility : > lOmg/ml in water, slightly soluble in organic solvents TLC identification : lOOmcg of compound applied to silica gel plate, eluents with chloroform/methanol/water 2:2:1 and acetonitrile/water 3:1, spray KMn04 basic - - complies with the standard of GPI-4P, no secondary degradation products are observed. Example 2 Preparation of Lysine salt of L-α- glycero-phospho-D-myo-inositol-4-monophosphate.

5,3g of GPI-4-P potassium salt (10 mmoles) are solubilized in 35 ml of . distilled water. The solution is cooled at 4°C and eluted in a column containing 35 ml of sulfonic cationic exchange resin generated in H+ form and thermostated at 4°C. The column is then eluted with 15 ml of distilled water and the eluate is collected at 4°C, flowed with nitrogen and neutralised with 4.38 g of lysine. The resulting solution is filtered, frozen lyophilized.

The reaction yields 99%.

The characteristics of the product L-α-glycero- phospho-D-myo-inositol-4-monophosphate lysine salt are: formula : C9H20O14P2. (C6H14N202) 3 molecular formula : C27H62N602o 2 formula weight : 852.78 elemental composition :C=38.03%; H=7.33%;

N=9.86%; 0=37.52%; P=7.26% solubility : ^> lOmg/ml in water, slightly soluble in organic solvents

TLC identification : lOOmcg of compound applied to silica gel plate, eluents chloroform/methanol/water 2:2:1 and acetonitrile/water 3:1, spray KMn04 basic - complies with the standard of GPI-4P; no secondary degradation products are observed.

Example 3 Preparation of L-α-phosphoglycero-D-myo- inositol- -phosphate 500mg of pure L-α-phosphatydylinositol-4- monophosphato are suspended in 10ml of anhydrous methanol under nitrogen atmosphere. 50mg of potassium tert-butoxide are added and the mixture is kept under stirring at 20°C over 3 hours. 26.7mg of acetic acid are added, the mixture is centrifuged. The supernatant solution is discharged, the residue is washed with 2 ml of cold methanol, and then dried under high vacuum and used as such for further preparation. GPI-4-P aqueous solution at room temperature is stable over time only at pH value closed to neutrality. The methods described here after for the preparation of new GPI-4-P salts are effective in keeping the GPI-4-P structure stable. Example 4 Preparation of potassium salt of L-α- glycero-phospho-D-myo-inositol- -monophosphate peraαetylate

5,3g of GPI-4-P potassium salt (10 m oles) are solubilized in 35 ml of distilled water. The solution is cooled at 4°C and eluted in a column containing 35 ml of sulfonic cation exchange resin generated in H+ form and thermostated at 4°C. The column is then eluted with 15 ml of distilled water and the eluate is collected at 4°C, and lyophilized. The solid residue is suspended in 35 ml of anhydrous DMF. The mixture is cooled at 4°C and added under continuous stirring with 20 ml of anhydrous pyridine and then with 10 ml of acetic anhydride, slowly drop by drop over 30 minutes. The mixture is heated to room temperature and kept under continuous stirring over 48 hours in anhydrous conditions. The mixture is then exhaustively dried under high vacuum in presence of P205 and NaOH. The residue is solubilized in 30 ml of ethanol, eluted in a column containing 30 ml of sulfonic cationic exchange resin generated in potassium form; the eluate is evaporated under vacuum and the residue dried under high vacuum.

The reaction yields 90%.

The characteristics of the product L-α-glycero- phospho-D-myo-inositol-4-monophosphate potassium salt peracetilated are: molecular formula : C2ιH2902oP2K3 formula weight : 780.7 elemental composition : C= 32.31%; H= 3.74%; 0= 40.99%;. P= 7.93 %; K= 15.02% solubility : >10mg/ml in' ethanol,

>10mg/ml in DMF

Analogously have been prepared the salts of litium, sodium, magnesium, iron, silver, choline, dimethylaminoethanol , arginine and carnosine of GPI-4P and of the following compounds according to formula (I) where :

R1=R2=R3=R4=R5=R6=CH3-C(0) -; M=Zn; n=l; m=l;

R1=CH3- (CO) -; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l; R1=CH3- (CO) -; R2=R3=R4=R5=R6=H; M=Zn; n=l; m=l;

R1=CH3- (CO)-; R2=R3=R4=R5=R6=H; M=Lysine; n=l; m=2 ;

R1=C6H5- (CO) -; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l;

R1=CH3- (CH2)3- (CO) -; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l; Rl=KO(CO)-CH2-(CO)-; R2=R3=R4=R5=R6=H; M=K; n=l; m=2;

Rl=acylic radical of nicotinic acid;

R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l;

Rl= acylic radical of , cholic acid; R2=R3=R4=R5=R6=H; M=Ca; n=l; m=l;

R1=H2N-CH2- (CO) -; R2=R3=R4=R5=R6=H; M=Na; n=l; m=l; and

Rl= acylic radical of lipoic acid;

R2=R3=R4=R5=R6=H; M=K; n=l; m=2. BIOLOGICAL ACTIVITY

The tested compounds have been coded as examples and numbered as follows :

Example Chemical name

1 L-α-glycero-phospho-D-myo-inositol-4- monophosphate zinc salt. 2 L-α-glycero-phbspho-D-myo-inositol-4 - monophosphate lysine salt .

3 L-α-glycero-phospho-D-myo-inositol-4- monophosphate . 4 L- -glycero-phospho-D-myo-inositol-4- monophosphate peracetilate potassium salt.

Xn vi ro Experimental Models

Cell culture . A549 human lung adenocarcinoma cells are maintained in continuous growth phase in Dulbecco's Modified Eagles Medium DMEM/F-12 containing Phenol Red and 10% foetal calf serum (FCS) in T-150 flasks. The cells were not allowed to reach confluence because this diminishes their response to growth factors . Measurement of arachidonic acid release .

Subconfluent cells were seeded into 12-place multi-well plates at 3xl05 cell/mL/well in DMEM/F-12, 10% FCS and incubated overnight. 3H-AA in ethanol was evaporated to dryness under N2 and resuspended in an appropriate volume of DMEM/F-12 (w/o phenol red) , and after vortex mixing left for 1 hour at 37°C. After washing the cells with PBS, 9.25kBq 3H-AA in 0.5ml DMEM/F-12 (W/O phenol red) was added to each well and incubated overnight. The media containing free 3H-AA was then removed and the cells washed three times with 1 ml DMEM/F-12 containing lmg/ml BSA. The cells thus labelled with 3H-AA were exposed to the compounds under evaluation for different times and then exposed to the stimuli. After incubation, 0.4 ml of medium was removed from each well for scintillation counting.

Treatment. The compounds coded as Example 1, 2,

3 and 4 or the control vehicle suitably diluted in

. DMEM/F-12 (w/o phenol red) were incubated over 15, 30,

60, 90, 120 e 180 minutes at the fixed concentration of lOOμM or at the concentrations of O.OOSμM, O.OlμM,

O.lμM, l.OμM, lOμM, 50μM, lOOμM over 60 minutes.

Stimulation : EGF lOnm for 60 minutes increases the 3H-AA release above 70% compared to controls. EGF in fact activates cytosolic phospholipase A2 via a G protein - mediated and pertuxis toxin sensitive mechanism (lOng/ml for 3 hours). (Croxtall J.D. et al . ,

"lipocortin-1 and the control of CPLA2 in A549 cells .

Glucocorticoids block EGF stimulation of CPLA2

Phosphorilation" , Biochem Pharmacol., 52, 465-74, 1996). Results . All the tested compounds coded as Example

1, 2, 3 e 4 reduce in a dose-dependent (Table 1). and

time-dependent (Table 2) manner the arachidonic acid release induced by cPLA2 activation via EGF stimulation.

The inhibitory effect is 30% already at O.lμM and becomes maximum at lOOμM: this last concentration displays a partial effect which is already evident after a 15 minute pre-incubation.

TABLE 1 . The compounds of Examples 1 , 2 , 3 and 4 reduce in a dose -dependent manner the arachidonic acid release induced by EGF

Pre-labelled cells are incubated for 60 minutes at a concentration between 0.005 e 100 μm of compounds of

Examples 1 , 2, 3 and 4 and then stimulated with EGF (10 nm) for 60 minutes. EGF lOnM for 60 minutes increases the 3H-AA release over 70% in comparison with controls.

The results are expressed as the mean + SE of determinations in duplicate in 3 different experiments. TABLE 2. Examples 1, 2, 3 and 4 compounds reduce in time -dependent manner the arachidonic acid release induced by EGF.

Pre-labelled cells are incubated with the Example 1,2, 3 and 4 compounds at the concentration of lOOμM at the different time 15, 30, 60, 90, 120 and 180 minutes and then stimulated with EGF 10 nM for 60 minutes. EGF lOnM for 60 minutes increases the 3H-AA release over 70% in comparison with controls.

The results are expressed as the mean + SE of determinations in duplicate in 3 different experiments.

The evidences here reported indicate that the compounds of the invention reduce the arachidonic acid release induced by EGF in A549 human lung adenocarcinoma cell culture according to a dose- and time -dependent relationship being already partially active at 0,lμM concentration and, at lOOμM concentration, after 15 minutes exposure .

Furthermore, experimental tests have shown that the compounds of the invention above taken as example, when exogenously administered in vivo are endowed with inhibitory activity in relation to biological G protein mediated signals and have shown an high bioavailability. In vivo experimental models Phorbol - 12 -myrxstate- 13 -acetate induced inflammation

Method. Guinea Pig have been treated with 200 micrograms of phorbol-12-myristate-13-acetate (PMA) topical applied to the guinea pig ear. The testing compounds (Example 1 and 3) have been topically applied 15 minutes before inflammatory stimulus at the concentration of 0.1%, 0.5%, 1% and 5% as hydro-alcoholic solution.

Parameters . Edema has been selected as parameter and evaluated by measuring ear thickness .

RESULTS. Into the psoriatic lesion, an increase of pro-inflammatory metabolites derived from arachidonic acid has been shown.

The above-described model shows many characteristics of the human psoriasis; in fact the histological analysis suggests that the cutaneous inflammation is associated with acanthosis, edema, polymorphonucleated infiltration and ridge prominence as long as 96 hours after single application. Furthermore PMA induces an increase of the arachidonic acid via CPLA2 activation.

Both tested compounds (Examples 1 and 3) display a significant inhibition of edema according to a dose- effect relationship at concentrations between 0.5% and 5%.

Murine type II collagen- induced arthri tis (CIA)

Method

DBA/l mice were treated with bovine type II collagen diluted with 0.05 mol/1 acetic acid to a concentration of 2 mg/ml and emulsified in an equal volume of complete Freund's adjuvant (2 mg/ml MT H3 7Ra) . The mice were immunized intradermally at the base of the tail with 100 μl emulsion (100 μg di collagen) . At the day 21 the animals were stimulated with an intra- peritoneal injection of 100 μg type II collagen in phosphate-buffered saline, solution (pH 7.4). The animals were selected at day 28 in relation where the development of CIA occurred. Arthritic animals were divided into groups, each of 8 animals and treated with the compounds under evaluation (Examples 1 and 4) at concentrations of 3, 5, 10, mg/kg/die for 5 days by injection.

Parameter. The parameter taken into consideration was the arthritic score according to a severity score scale. The mice were considered arthritic when at significant occurrence of edema and/or redness of the paws and ankylosis for the most severe score. Clinical severity was graded on a scale of 0-2 for each paw, according to the changes in redness and edema: 0, no changes; 0.5 significant changes; 1.0 moderate; 1.5 marked; 2.0 maximal redness and edema and ankylosis.

Results . Arthritis is associated with chronic inflammation implying a cytokinin production increasing and increase of arachidonic acid pro-inflammatory metabolites. The collagen-induced arthritis is a well- standardised model capable of reproducing many characteristic of the human pathology.

The treatment with Examples 1 and 4 at all tested dosages, decrease the symptomatology at 3°day treatment. At the highest tested dosage an almost total remission of the symptoms occurred (6 animals at score 0; 4 animals at score 0.5) . None of the treated animals gains a score higher then 1. In conclusion, the compound of the invention here tested are capable to negatively modulate CPLA2 thus limiting the cascade of event associated with the arachidonic acid metabolism, according to a G protein regulated mechanism.

Therefore the compounds according to the formula (I) of the present invention are a valid therapeutic tool for the treatment of pathologies mediated by cPLA2 activation or over-stimulation such as the pathologies above listed and any how mediated by enzymatic and metabolic pathways whose activation/deactivation is G protein associated.

The dosages, timing and the route of administration will be chosen according to the type, stage, seriousness and district of manifestation of the pathology or alteration, or the possibility of application in human or veterinarian . health care and comprised between 0.1 and 100 mg/kg for 1-100 days for each therapy cycle in relation to the type, stage, severity and district of manifestation of the pathology or alteration or possible application in human or veterinarian health care .

For all the mentioned pathologies are indicated the systemic, parenteral, oral and rectal administration, but also inhalational, topic, transdermic and in any case such to achieve the highest availability of the active substance. For oral formulations are preferred administrations as tablets, sugar coated tablets and capsules, but also powders and solutions/suspensions also for nebulization. For the inhalational formulations prefer administration are powders and solutions/suspensions also as nebulization.

For topic treatment are preferred emulsions, gels, and solutions compatible with skin and mucosal use, including the gingival mucosa, together with eye drops for the administration into the conjunctival sac.

The injectable forms are formulated with solvents suitable for pharmaceutical use and for the endovenous, intramuscular and subcutaneous administration. The compounds of the formula (I) of the present invention can be, at the proper concentrations, formulated as supplements for oral intake for preventive or coadiuvant treatment of alterations related to disreactive conditions in human and veterinarian medicine .

The use of the above described compounds of formula (I) thus represents a further aspect of the present invention for the preparation of a medicament for the treatment of the pathologies mediated by an activation or over-stimulation of the enzymatic and metabolic G protein associated pathways in which the medicament is formulated as supplements for oral intake for the prevention or coadiuvant treatment .

Here after are some examples of pharmaceutical and cosmetic preparations which have only a descriptive, but not limitative purpose:

Example A - Tablet

Active principle Example 1 100 mg

Excipients Microcrystalline cellulose 160 mg

Starch 28 mg

Lactose 100 mg

Palmitic acid 6. 0 mg

Example B - injectable formulation

Active principle Example 1 50 mg Excipients Injectable Solution buffered to pH 7

To 4ml

Example C - emulsion for topic application mg Active principle Example 2 2. 0

Excipients Twin 60 0. 5

Pol wax 1 . 5

Akofine S 4. 0

P168-50 3 . 0 Cannabi s sativa 5.0

Dimethycone 0.7

Cet 868 7.0

Glycerin 3.0

Preservants 1.0 Water to 100 mg

Citas de patentes
Patente citada Fecha de presentación Fecha de publicación Solicitante Título
US5955453 *24 Abr 199821 Sep 1999The Regents Of The University Of CaliforniaMembrane-permeant phosphoinositides
US6258375 *5 Mar 199810 Jul 2001Abbott LaboratoriesAntibacterial phosphoinositides
Otras citas
Referencia
1 *BERRIE C P ET AL: "MEMBRANE TRANSPORT AND IN VITRO METABOLISM OF THE RAS CASCADE MESSENGER, GLYCEROPHOSPHOINOSITOL 4-PHOSPHATE", EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN, DE, vol. 266, no. 2, 1 December 1999 (1999-12-01), pages 413 - 419, XP001011370, ISSN: 0014-2956
Citada por
Patente citante Fecha de presentación Fecha de publicación Solicitante Título
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US81787042 Dic 200515 May 2012Cis Bio InternationalInositol-phosphate derivatives and method of detecting inositol-1-phosphate
US93519834 Oct 201331 May 2016Consiglio Nazionale Delle RicercheUse of glycerophosphoinositols for the treatment of septic shock
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