ITMI20011483A1 - USE OF COMPOUNDS AS FUNCTIONAL ANTAGONISTS TO CENTRAL DEICANNABINOID RECEPTORS - Google Patents

Description

Description of industrial invention entitled: "Use of compounds as functional antagonists to central cannabinoid receptors"

FIELD OF THE INVENTION

The present invention relates to the use of acylamides as functional antagonists of the central cannabinoid receptors.

STATE OF THE TECHNIQUE

Cannabis sativa, in addition to being one of the most popular recreational drugs in the world, has been used for medical purposes for centuries due to its multiple pharmacological effects, a use that has been limited in the past century due to its effects on the CNS. The main active component identified is Δ <9> -tetrahydrocannabinol (Δ <9> -THC), which in fact, in addition to possessing psychotropic activity, produces an innumerable series of pharmacalogical effects in both humans and animals, thus demonstrating a great therapeutic potential, however limited by the central effects mentioned.

In the last fifteen years, research on Δ <9> -THC has led to the discovery of cannabinoid receptors and, more recently, of lipid substances, such as anandamide (ANA) and 2-arachidonylglycerol (2- Arach), considered as the natural endogenous ligands for these receptors (Martin B.R. et al. 1999 Life Sci. 65, 573-595). These receptors together with the ligands just described constitute the so-called “endocannbinoid system” (Piomelli D. et al. 2000 TIPS 21, 218-224).

In the "endocannbinoid system" two receptors have been identified to date: i) the so-called central receptor, as initially identified in areas of the CNS and considered the molecular transducer of the central effects of cannabinoids and ii) the so-called peripheral receptor initially identified in tissues peripheral and in turn considered the molecular transducer of peripheral effects. The central receptors are in fact present in high concentrations in the CNS, and more precisely in the cerebral cortex, hippocampus, nuclei caudatoputamen, substantia nigra, pars reticulata, globus pallidus, endopedicular nucleus, cerebellum and spinal cord. Although to a lesser extent, the central receptors are also present at the peripheral level mainly at nerve endings such as in the intestine, as well as on the endothelium and immunocompetent cells. Peripheral receptors have instead been identified mainly at the peripheral level and especially in immunocompetent cells such as T lymphocytes, mast cells, macrophages, etc. There is no evidence to support their expression in the CNS of adult animals, although it has been seen to be expressed in vitro in newborn mouse granular cells (Skaper S.D. et al. 1996 Proc. Natl.Sci.

96, 3984-3989; Ameri A. 1999 Progr. Neurobiol. 58, 315-348).

Regarding the effects, the central receptors are considered to mediate not only the "unwanted" effects of cannabinoids (such as psychoactive effects, decreased memory and attention, loss of motor coordination and echo.) But also some of their "desired" effects under the therapeutic profile (analgesic, anti-hyperalgic effect, stimulation of appetite, ocular hypotensive, etc.). The peripheral receptors, as well as the central ones present in immunocompetent cells, have been associated with the more peripheral effects of cannabinoids such as the anti-inflammatory effect (Ameri A. 1999 ref. Cit.).

All these discoveries have given impetus to the development of a wide range of cannabinomimetic molecules, such as classical (e.g. Δ <8> -THC, Δ <6> -THC etc.) and non-classical (e.g. e.g. CP 55940, HU-210 etc.), endocannabinoid derivatives (e.g. methanandamide, etc.), aminoalkylindoles (e.g. Win 55-212) and molecules capable of interfering with the uptake and inactivation of endocannabinoids (eg AM 404, trifluoromethylketone, etc.), of potential therapeutic interest whose applications range from the control of pain and inflammation, to the control of nausea and appetite and in the reduction of intraocular pressure. Synthetic derivatives, pyrrole derivatives (eg SR141716, SR144528), with antagonistic activity on cannabinoid receptors, have also been characterized, considered useful in the control of eating disorders, in improving memory and motor activities and in weaning addiction in tobacco and cannabis smokers (Ameri A. 1999 ref. cit.).

Although it is potentially possible to distinguish the effects mediated by central receptors from peripheral ones, to date most of the synthesized cannabinoidomimetic molecules do not have such selectivity and receptor specificity that they can be used without incurring the unwanted effects of cannabinoids. In fact, although with different characteristics of binding to receptors, both the classical cannabinoids and their derivatives, as well as the aminoalkylindoles as well as the endocannabinoids themselves and their derivatives, possess activity on both central and peripheral receptors (Martin B.R. et al. 1999 ref. cit; Khanolkar A.D. et al.

2000 Chem. Phys. of Lipids 108, 37-52). To date, only a few molecules have been identified to possess a sensitive receptor selectivity and among these we find: synthetic derivatives of pyrazole to which specific antagonistic activity has been attributed for the central receptors (SR 141716) and for the peripheral ones (SR 144258), as well as the CP55940, HU210 etc. with agonist activity mainly on central receptors and HU-308, JTE-907 and palmitoylethanolamide (PEA) and analogues with agonist activity on peripheral receptors. The latter molecules, therefore not possessing activity referable to the central receptors, are devoid of the central effects of cannabinoids and have been indicated above all as molecules essentially with anti-inflammatory activity, as they are able to inhibit the pro-inflammatory activation of mast cells through the specific interaction with the peripheral receptor present on those cells (WO 9618600 and WO 9618391).

More specifically, PEA, like the endocannabinoid ANA, belongs to the class of N-acylethanolamides (NAE). This family includes ethanolamine derivatives condensed with both saturated (PEA) and unsaturated (ANA) acid radicals. Both ANA and PEA can be produced in the brain, as well as in other tissues, following the hydrolysis of N-acylated phosphatidylethanolamine (Piomelli et al. 2000 ref. Cit .; Schmid H.H.O. 2000, Chem. Phys. Lipids 108, 71- 87), suggesting their possible role as neuromodulators belonging to the endocannabinoid system. However, while acute administration of anandamide in vivo causes cannabinoid-like effects such as hypothermia, hypomotility, catalepsy, etc., these effects have never been reported after administration of PEA in vivo, given this is compatible with the absence

of affinity of this molecule for the central receptor. Although PEA and

ANA are both able to exert anti-inflammatory effects in vivo, there are discrepancies on the ability of PEA, as opposed to ANA, to interact with cannabinoid receptors in the periphery (Sugiura T. et al.

2000 J. Biol. Chem. 275, 605-612; Let us L. et al. 1995 Proc. Nati. Ski.

USA 92, 3376-3380). In fact, although it has been shown that the PEA

is able to significantly reduce in vitro the release of proinflammatory factors from stimulated mast cells, through an action on the peripheral receptors expressed by these cells (Facci L. et al. 1995 ref. cit.), works

more recent facts on cell lines transfected overexpressing the peripheral receptor have shown that PEA is not able to displace the

binding of cannabinoidomimetic molecules to this receptor (Sugiura

T. et al. 2000 ref. cit.). On the other hand, in vivo studies show that the anti-inflammatory / anti-pain activity of PEA is reduced following co-administration of the antagonist to the peripheral receptor SR1 44528 {Calignano A. et al. 2001 Eur. J. Pharm. 419, 191-198). Also it was

since the simultaneous administration of PEA and ANA induces a

synergistic anti-inflammatory / anti-pain effect (WO99 / 60987) suggesting that the molecules act on two different systems. In fact, while the ANA effect was antagonized by the central receptor agonist SR141716, the effect of PEA was sensitive to the effect of the peripheral receptor antagonist (Calignano A. 2001 ref. Cit.).

It should also be noted that PEA and analogs have been shown to possess neuroprotective activity in vitro and therefore useful in pathological conditions associated with neuronal death (WO 9525509 and WO 9618600). This effect was observed in vitro on granular cells of the cerebellum from newborn mice expressing the peripheral receptor or a CB2-like receptor (Skaper S.D. et al. 1996 ref.cit.). Furthermore, a patent application has recently been filed for the use of PEA as cardioprotective (WO01 / 28588), an effect also in this case probably mediated by peripheral receptors, as it is antagonized by SR 144528, an antagonist of the peripheral receptor, and not from SR 141716, central receptor antagonist. Finally, it should be noted that the only central effects reported for PEA are antispasmodic and anticonvulsant, transient effects obtained following acute parenteral administration (Baker D. et al. 2000 Nature 404, 84-87; Lambert D. et al. 2001 Epilepsia 42 , 321-327).

In conclusion, there is no doubt that the evidence available to date indicates that PEA and its analogues have no effects on the central receptor and that these compounds exert their effects (anti-inflammatory, painkiller, neuro-protective, etc.) through the peripheral receptor. or in any case a CB-like receptor (non-CB1 and non-CB2) present on immunocompetent cells (eg mast cells) or other cells.

SUMMARY

The Applicant has now surprisingly found that following a chronic administration the acylamides of saturated acids of general formula (I)

where is it:

R1 - CO-- can be an acyl residue of a linear or branched saturated organic acid comprising from 10 to 20 C atoms

- N - R2 can be:

- a residue of a linear or branched aminohydroxyalkyl comprising from 2 to 6 C atoms, optionally substituted with one or more aromatic groups on the alkyl chain

- a residue of an amino acid of the α, β or γ series

R3 can be H or CH3

or where -N - R2, R3 form with the N atom a cyclic aminoether comprising from 5 to 7 C atoms possibly substituted with linear or branched alkyl groups

they behave as functional antagonists of central cannabinoid receptors. They can therefore be usefully used as drugs in pathological states that can be controlled by reducing the functionality of these receptors or by reducing the effect of the endocannabinoids themselves caused by a lower availability or affinity of the receptors.

Therefore, the subject of the present invention is the use of said saturated acylamides, or esters or salts thereof, for the preparation of pharmaceutical compositions for the treatment of pathological states connected with an altered functionality and / or "abusive" activation of the central cannabinoid receptors .

DETAILED DESCRIPTION OF THE INVENTION

The purposes and advantages of the therapeutic use of saturated acylamides as functional antagonists to central cannabinoid receptors in pathological states that can be controlled by reducing the functionality of these receptors or by preventing the activity of the endocannabinoids, object of the present invention, will be better understood in the course of the following detailed description.

The Applicant has in fact found that after repeated administration of acylamides of saturated acids there is surprisingly a marked reduction in the receptor density of the central cannabinoid receptors at the level of the spinal cord and a reduction in the affinity constant (Kd) always of these receptors in different brain areas (cerebellum, cortex, hippocampus). A similar effect is known to occur following repeated administration of agonists to the central receptor endowed with psychotropic activity, such as Δ <9> -THC or CP55940 which in fact leads to a change in receptor density or to a desensitization of the central receptor. in the areas where this is expressed (Ameri A.1999 ref. cit.). However, an effect on central cannabinoid receptors has never been reported with molecules of the saturated acylamide class, such as PEA, as described below. In the acylamides of saturated acids defined by the general formula (I)

where is it:

R1 - CO-- can be an acyl residue of a linear or branched saturated organic acid comprising from 10 to 20 C atoms

--N-R2 can be:

- a residue of a linear or branched aminohydroxyalkyl comprising from 2 to 6 C atoms, optionally substituted with one or more aromatic groups on the alkyl chain, and the hydroxyl can optionally be esterified with a pharmaceutically acceptable acid group such as acetic, tartaric and succinic and equivalents

- a residue of an amino acid of the α, β or γ series in which the carboxylic group can be esterified with pharmaceutically acceptable groups, such as for example methyl, ethyl, propyl, or salified with pharmaceutically acceptable counterions such as for example sodium, potassium, magnesium and equivalents

R3 can be H or CH3

or where -N-R2, R3 form with the N atom a cyclic aminoether comprising from 5 to 7 C atoms possibly substituted with linear or branched alkyl groups

R1 - CO-- can be preferentially selected from the group consisting of saturated aliphatic monocarboxylic acids such as decenoic, lauric, myristic, paimitic, stearic or arachidic acid and

- N - R2, when it is a residue of an aminohydroxyalkyl, can be preferentially chosen for example from the group consisting of monoethanolamine, 2-hydroxypropylamine, while when it is a residue of an α, β or γ amino acid it can be chosen from the group consisting of serine , gliein, β-alanine, γ-aminobutyric, phenylalanine and tyrosine when instead -N - R2, R3 form a cyclic aminoether with the N atom, this is preferentially morpholine.

The saturated acylamides described in the present invention can be prepared according to various methods and preferably by fusion of the alkylamine salt with the carboxylic acid and formation of the corresponding alkylamide, or by acylation of the alkylamine nitrogen with suitable activated carboxylic derivatives.

Some examples of saturated monocarboxylic acid starches are reported below for illustrative and non-limiting purposes.

Example 1: Preparation of N- (2-hydroxyethyl) -palmitoylamide 100 mmoles of ethanolamine dissolved in 120 ml of dichloromethane were placed in a 250 ml flask. To this solution, 40 mmoles of palmitoyl chloride dissolved in anhydrous dichloromethane were added drop by drop by means of a loading funnel. The reaction was maintained under continuous stirring at the temperature of 0-4 ° C and at the appropriate time, stopped by adding a 10% aqueous solution of citric acid. The organic phase was then dried with anhydrous sodium sulfate and filtered. By means of a rotary evaporator at reduced pressure, the solvent and the solid residue were removed and crystallized with a suitable solvent.

The reaction yield was approximately 75%.

Physico-chemical properties of N- (2-hydroxyethyl) -palmitoylamide: appearance: white crystalline powder

formula: C18H37NO2

elemental analysis: C = 72.15%; H = 12.35; N = 4.73; O = 10.88

solubility in organic solvents: DMSO, CHCI3, ethanol

solubility in water: insoluble

melting temperature: 93-95 ° C

TLC: eluent toluene / CHCl3, 9/1; Rf = 0.42

Example 2: Preparation of N- (3-hydroxypropyl) -palmitoylamide 80 mmoles of 3-amino-1-propanol were dissolved in 100 ml of dichloromethane in a 250 ml flask. To this solution, 40 mmoles of palmitoyl chloride dissolved in anhydrous dichloromethane were added drop by drop by means of a loading funnel and under constant stirring. The reaction was maintained under continuous stirring at the temperature of 0-4 ° C and at the appropriate time, stopped by adding a 10% aqueous solution of citric acid. The organic phase was then dried with anhydrous sodium sulfate and filtered. By means of a rotary evaporator at reduced pressure, the solvent and the solid residue were removed and crystallized with a suitable solvent.

The reaction yield was approximately 88%.

Physico-chemical properties of N- (2-hydroxypropyl) -palmitoylamide:

appearance: white crystalline powder

formula: C19H39NO2

molecular weight: 313.53

elemental analysis: C = 72.51%; H = 12.38; N = 4.43; 0 = 10.78

solubility in organic solvents: about 1 mg / ml in DMSO

solubility in water: very slightly soluble

melting temperature: 90-91 ° C

TLC: eluent CH3OH / CHCI395 / 5; Rf = 0.38

Example 3: Preparation of N- (3-hydroxypropyl) -lauroylamide

In a bubble refrigerant flask, they were reacted for 5 h a

160 ° C, 1.73 g of lauric acid (8.65 mmol) and 0.8 g of ethanolamine (13

mmol). The reaction mixture was then crystallized from ethanol

80%. The crystalline body was separated by filtration on buchner,

washed three times with cold 80% ethanol and finally dried underneath

empty.

The reaction yield was approximately 88%.

Physico-chemical properties of N- (2-hydroxyethyl) -lauroylamide:

appearance: white crystalline powder

formula: C14H29NO2

molecular weight: 243.39

s) elemental analysis: C = 68.98%; H = 11.95; N = 5.73; 0 = 13.35

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in CHCI3

solubility in water: very slightly soluble

melting temperature: 86-88 ° C

TLC: eluent CHCl3-CH3OH-H2O-NH3, 80/17/2/1; Rf = 0.82

Example 4: Preparation of N- (2-hydroxyethyl) -stearoylamide

In a bubble refrigerant flask, they were reacted for 5 h a

160 ° C, 2.42 g of stearic acid (8.65 mmol) and 0.8 g of ethanolamine (13 mmol). The reaction mixture was then crystallized from ethanol

95%. The crystalline body was separated by filtration on buchner,

washed three times with cold 95% ethanol and finally dried underneath

empty.

The reaction yield was approximately 85%.

Physico-chemical properties of N- (2-hydroxyethyl) -stearoylamide:

appearance: white crystalline powder

formula: C20H41NO2

molecular weight: 243.39

elemental analysis: C = 73.18%; H = 12.45; N = 4.13; 0 = 10.14

solubility in organic solvents: about 1 mg / ml in CHCI3

solubility in water: insoluble

melting temperature: 100-101 ° C

TLC: eluent CHCI3-CH3OH-H2O-NH3, 80/17/2/1; Rf = 0.82

Example 5: Preparation of N-palmitoyl-morpholinamide

0.75 g of morpholine (8.6 mmol) e were dissolved in a flask at 0 ° C

0.92 g of triethylamine (9 mmol) in 50 ml of dimethylformamide. At the ; solution, 2.35 g of palmitoyl chloride (8.5 mmol) dissolved in dimethylformamide were added drop by drop and reacted for 1 h at 0 ° C and for 5 h at room temperature. The suspension obtained was dried in an evaporator at reduced pressure. The solid residue was then washed with ethyl ether and crystallized from 70% ethanol. The crystalline body was separated by filtration on buchner, washed three times with cold 70% ethanol and finally dried under vacuum.

The reaction yield was approximately 90%.

Physico-chemical properties of N-palmitoyl-morpholinamide:

appearance: white crystalline powder

formula: C20H39NO2

molecular weight: 325.53

elemental analysis: C = 73.52%; H = 11.95; N = 4.18; 0 = 10.35

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in boiling ethanol solubility in water: insoluble

melting temperature: 42-44 ° C

TLC: eluent CH3OH / CHCI395 / 5; Rf = 0.90

Example 6: Preparation of N- (2-methoxy-ethyl) -palmitoylamide In a flask, 0.75 g of 2-methoxy-ethyl-amine (10 mmol) and 1.1 g of triethylamine (11 mmol ) in tetrahydrofuran. To the solution were added, drop by drop and under continuous stirring, 2.75 g of palmitoyl chloride (10 mmol) dissolved in tetrahydrofuran and reacted for 8 h at 0 ° C. A double volume of water was added to the reaction mixture and the whole was extracted three times with ethyl acetate. The organic phase, after collection, was washed twice with 1N HCI and two more times with water. After anhydrification with sodium sulphate, the solvent was removed in an evaporator under reduced pressure and the residue crystallized from terbutyl-methyl ether and dried under vacuum.

The reaction yield was approximately 70%.

Physico-chemical properties of N- (2-methoxy-ethyl) -palmitoylamide:

appearance: white crystalline powder

formula: C19H39NO2

molecular weight: 313.52

elemental analysis: C = 72.92%; H = 12.22; N = 4.69; 0 = 10.17

solubility in organic solvents:> 10 mg / ml in ethanol

solubility in water: insoluble

melting temperature: 75-78 ° C

TLC: eluent toluene / ethanol / acetic acid 65/30/5; Rf = 0.70

Example 7: Preparation of N-lauroyl-morpholinamide

0.75 g of morpholine (8.6 mmol) and 0.92 g of triethylamine (9 mmol) in tetrahydrofuran were dissolved in a flask at 0 ° C. 1.86 g of lauroyl chloride (8.5 mmol) dissolved in tetrahydrofuran and reacted for 1 h at 0 ° C and for 5 h at room temperature were added drop by drop to the solution. An equal volume of water was added to the reaction mixture and the whole was extracted three times with terbutyl-methyl ether. The organic phase, after collection, was washed twice with 1N HCI and two more times with water. After anhydrification with sodium sulphate, the solvent was removed in an evaporator under reduced pressure and the residue purified by chromatography on silica gel flowed with the mixture hexane / ethyl acetate / acetic acid (79.5 / 20 / 0.5). The fractions containing the product were evaporated in an evaporator at reduced pressure and the residue brought to dryness under vacuum.

The reaction yield was approximately 85%.

Physico-chemical properties of N-lauroyl-morpholinamide:

appearance: white crystalline powder

formula: C16H31N02

molecular weight: 369.43

elemental analysis: C = 71.52%; H = 11.85; N = 5.11; 0 = 11.52

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in ethanol

solubility in water: insoluble

melting temperature: 20-23 ° C

TLC: eluent hexane / ethyl acetate / acetic acid, 75/24/1; Rf = 0.25 Example 8: Preparation of N-stearoyl-morpholinamide

0.75 g of morpholine (8.6 mmol) and 0.92 g of triethylamine (9 mmol) in anhydrous dimethylformamide were dissolved in a flask at 0 ° C. 2.56 g of stearoyl chloride (8.5 mmol) dissolved in anhydrous dimethylformamide and reacted for 1 h at 0 ° C and for 16 h at room temperature were added dropwise to the solution. The reaction mixture was in an evaporator under reduced pressure and the residue, after washing with water, was crystallized first from ethanol and subsequently from terbutyl-methyl ether. The crystalline residue was washed three times with terbutyl methyl ether and dried under vacuum.

The reaction yield was approximately 85%.

in Physico-chemical properties of N-stearoyl-morpholinamide:

appearance: white crystalline powder

formula: C22H43NO2

molecular weight: 325.59

elemental analysis: C = 74.12%; H = 11.98; N = 4.11; 0 = 10.79

solubility in organic solvents:> 10 mg / ml in boiling ethanol

solubility in water: insoluble

melting temperature: 58-60 ° C

TLC: eluent hexane / ethyl acetate / acetic acid, 65/30/5; Rf = 0.63

Example 9: Preparation of N-palmitoyl-L-serine

1.02 g of L-serine (10 mmol) were dissolved at 4 ° C in 1 M potassium carbonate. 2.75 g of palmitoyl chloride (10 mmol) were added dropwise and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and then acidified with 6N HCl. The precipitate was separated by buchner filtration and brought to dryness under vacuum. The residue was crystallized first from terbutyl-methyl ether and subsequently from methanol. The crystalline residue was washed three times with methanol and dried under vacuum.

The reaction yield was approximately 80%.

Physico-chemical properties of N-palmitoyl-L-serine:

appearance: white crystalline powder

formula: C19H37NO4

molecular weight: 343.51

elemental analysis: C = 66.23%; H = 11.08; N = 4.19; 0 = 19.50

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in ethanol

solubility in water: insoluble

melting temperature: 95-96 ° C

TLC: eluent chloroform / methanol / water / ammonia, 78/25/2/1; Rf = 0.13

Example 10: Preparation of N-lauroyl-L-serine

1.02 g of L-serine (10 mmol) were dissolved at 4 ° C in 1 M potassium hydroxide. 2.20 g of lauroyl chloride (10 mmol) were added dropwise and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and then acidified with 6N HCl and then extracted with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated in an evaporator under reduced pressure. The residue was crystallized from acetonitrile. The crystalline residue, separated by filtration, was washed three times with cold acetonitrile and dried under vacuum.

The reaction yield was approximately 78%.

Physico-chemical properties of N-lauroyl-L-serine:

appearance: white crystalline powder

formula: C15H29NO4

molecular weight: 287.40

elemental analysis: C = 63.02%; H = 10.38; N = 4.79; 0 = 22.81

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in ethanol

solubility in water: insoluble (soluble> 10 mg / ml as sodium salt) melting temperature: 121 ° C

TLC: eluent toluene / ethanol / acetic acid, 65/30/5; Rf = 0.50 Example 11: Preparation of N-lauroyl-L-glycine

1.2 g of gliein (16 mmol) were dissolved at 4 ° C in 1 M potassium hydroxide. 2.20 g of lauroyl chloride (10 mmol) were added drop by drop and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and, after this time, acidified with 6N HCl and then extracted with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated in an evaporator under reduced pressure. The residue was crystallized from acetonitrile. The crystalline residue, separated by filtration on buchner, was washed three times with cold acetonitrile and dried under vacuum.

The reaction yield was approximately 80%.

Physico-chemical properties of N-lauroyl-glycine:

appearance: white crystalline powder

formula: C14H27NO3

molecular weight: 257.37

elemental analysis: C = 65.02%; H = 10.78; N = 5.89; 0 = 18.31

solubility in organic solvents:> 10 mg / ml in DMSO

> 10 mg / ml in ethanol

solubility in water: insoluble (soluble> 10 mg / ml at pH 7.5) melting temperature: 120 ° C

TLC: eluent toluene / ethanol / acetic acid, 65/30/5; Rf = 0.60 Example 12: Preparation of N-palmitoyl-L-glycine

1.2 g of gliein (16 mmol) were dissolved at 4 ° C in 1 M potassium hydroxide. 2.75 g of palmitoyl chloride (10 mmol) were added drop by drop and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and, after this time, acidified with 6N HCl and then extracted with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated in an evaporator under reduced pressure. The residue was crystallized from ethanol. The crystalline residue, separated by filtration on buchner, was washed three times with cold ethanol and dried under vacuum.

The reaction yield was approximately 70%.

Physico-chemical properties of N-palmitoyl-glycine:

appearance: white crystalline powder

formula: C18H35NO3

molecular weight: 313.48

elemental analysis: C = 69.12%; H = 11.01; N = 4.86; 0 = 15.01

solubility in organic solvents:> 10 mg / ml in DMSO

solubility in water: very slightly soluble

melting temperature: 120 ° C

TLC: eluent chloroform / methanol / water / ammonia, 77/25/2/1; Rf = 0.15

Example 13: Preparation of N-palmitoyl-β-alanine

1.5 g of β-alanine (17 mmol) were dissolved at 4 ° C in 1 M potassium hydroxide. 2.75 g of palmitoyl chloride (10 mmol) were added dropwise and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and, after this time, acidified with 6N HCl and then extracted with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated in an evaporator under reduced pressure. The residue was crystallized from terbutyl methyl ether. The crystalline residue, separated by filtration on buchner, was washed three times with cold terbutyl-methyl ether and dried under vacuum.

The reaction yield was approximately 85%.

Physico-chemical properties of N-palmitoyl-β-alanine:

appearance: white crystalline powder

formula: C19H37NO3

molecular weight: 327.51

elemental analysis: C = 70.11%; H = 11.72; N = 4.68; 0 = 14.49

solubility in organic solvents:> 5 mg / ml in DMSO

> 5 mg / ml in ethanol

solubility in water: very slightly soluble

melting temperature: 122 ° C

TLC: eluent toluene / ethanol / acetic acid, 65/30/5; Rf = 0.60 Example 14: Preparation of N-palmitoyl-aminobutyrate

2.0 g of y-aminobutyric acid (19 mmol) were dissolved at 4 ° C in 1 M potassium hydroxide. 2.75 g of palmitoyl chloride (10 mmol) were added drop by drop and under continuous stirring to the solution. The reaction mixture was kept at 0 ° C for 16 h and, after this time, acidified with 6N HCl and then extracted with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated in an evaporator under reduced pressure. The residue was crystallized from terbutyl methyl ether. The crystalline residue, separated by filtration on buchner, was washed three times with cold terbutyl-methyl ether and dried under vacuum.

The reaction yield was approximately 80%.

Physico-chemical properties of N-palmitoyl-y-aminobutyrate:

appearance: white crystalline powder

formula: C20H39NO3

molecular weight: 341.54

elemental analysis: C = 69.81%; H = 11.02; N = 4.85; 0 = 14.32

solubility in organic solvents:> 5 mg / ml in DMSO

> 5 mg / ml in ethanol

solubility in water: very slightly soluble

melting temperature: 102 ° C

TLC: eluent toluene / ethanol / acetic acid, 65/30/5; Rf = 0.70.

The starch derivatives of saturated fatty acids described here, as previously briefly mentioned, have surprisingly been shown to act, after repeated administration in normal adult rats, as functional antagonists of central cannabinoid receptors, as a reduction in the number of receptors or their affinity leads to a reduction in their activity, without determining the cannabinomimetic effects known to be mediated by the central receptor (eg hypothermia, motor deficits, etc.). These effects of drastic reduction in the expression of the central receptor in the spinal cord and a significant decrease in the affinity of this receptor in other areas of the CNS are described in detail below.

A) Characterization of central cannabinoid receptors in different areas of the CNS

Cannabinode receptors were characterized using receptor binding techniques. These determinations were carried out on preparations of different CNS areas of animals repeatedly treated with the compounds described here and compared with animals treated with the vehicle alone.

a) Treatment of animals

Adult male Sprague Dawley rats weighing 200-300 grams were used (Harlan-Italy, San Pietro al Natisone, UD, Italy). The animals, maintained on a "normal" diet, received, per os 10mg / kg for 10 days, two daily administrations of the compounds described herein.

b) Preparation enriched with membranes of rat nervous tissue for the analysis of central cannabinoid receptors.

Nervous tissue membranes were prepared by taking and rapidly freezing the following brain areas at -80 ° C: spinal cord, cortex, hippocampus and cerebellum. The tissue, weighed and suspended in 30 volumes of cold Tris-HCI 50 mM pH 7.4 buffer containing 0.25% of Trypsin inhibitor Soybean Type II, 1 mM EDTA and 4mM MgCI, was homogenized with 10 strokes in Potter Teflon / Glass. After subsequent centrifugations and membrane washes, the pellet was taken up in a small volume (approximately 1.5ml per 1 gram of initial wet tissue) of 50mM pH 7.4 Tris-HCL buffer containing 1mM EDTA, 4mM MgCI2 and 0.05% BSA Fatty Acid Free. The protein concentration was determined on these samples with the bicinquinonic acid method.

c) Determination of Kd and Bmax of the central receptor. The binding to the central cannabinoid receptors was performed in polystyrene tubes in the presence of PMSF, as a protease inhibitor, and of DMSO using as the labeled ligand <3> H-WIN55,212-2 known to bind to cannabinoid receptors. After addition of the membranes, prepared as described above, the tubes were incubated for one hour at 30 ° C. The <3> H-WIN55,212-2, possessing a high affinity for the central cannabinoid receptors, was used at concentrations around 1-2 nM, while the final cold WIN55.212-2 concentration used to obtain the total displacement of the label from its receptors was 1 or 10μΜ. For a typical binding experiment, the final volume was 1ml, consisting of: 870 μΙ of binding buffer, 10 μl of DMSO, 10 μl of <3> H-WIN55, 212-2 100nM, 10 μΙ of PMSF 1mM in 1% DMSO and 100 μl of preparation enriched with synaptosomal membranes from nervous tissue (concentration of the homogenate around 1-2 μg of protein per μl). At the end of the incubation the samples were filtered on Watman GF / C filters using a celi harvester. The filters were then washed with buffer (3X5 ml) and dehumidified with a hot air dryer. Once dry they were placed in vials containing three milliliters of scintillation liquid and the radioactivity measured with a liquid scintillation Beta Counter. The values obtained from the samples used for non-specific binding were subtracted from those of total binding thus obtaining the specific binding values.

The results obtained with respect to the KD and Bmax of the central receptor with the compounds described are reported in table 1.

TABLE 1

The results obtained, in vivo, show that the repeated administration of amide derivatives of saturated acids are able to reduce the affinity, for WIN 212.55-2, of the central cannabinoid receptors in the cerebellum, hippocampus, cortex, without changing the number of receptors (Bmax). In this regard, it should be remembered that there is no experimental evidence of the presence of the peripheral cannabinoid receptor in the CNS of adult rats. In the marrow, where only the central cannabinoid receptors are known to be present, there is an increase in the affinity of these receptors for WIN 212.55-2. In this regard, it should be noted that the increase in the affinity of the central cannabinoid receptors corresponds to a marked reduction in the number of receptors expressed. This increase in receptor affinity is probably the result of an adaptation of the system induced by the strong reduction in the number of receptors, about 75%, induced by the repeated administration of starch derivatives of saturated acids. In any case, the strong reduction in the number of central receptors seen in the medulla and the decrease in the affinity of the central receptor in the cerebellum, hippocampus and cortex, indicate that these molecules act as functional antagonists to the central cannabinoid receptors. .

The present invention therefore describes an effect on the central cannabinoid receptors following repeated administration of N-acylamides of saturated acids which can be characterized as a functional antagonist-type effect at these receptors. These compounds can therefore be used, alone or in association with other compounds, in the treatment of symptoms associated with different disorders and pathological states in humans, which can be controlled by reducing the activity and / or functionality of the central cannabinoid receptors.

In order to make clear the therapeutic use object of the present invention, the following considerations should be made:

a) the role attributed to central cannabinoid receptors in the loss of both pyramidal and extrapyramidal control of motor movements and activities, in ataxias and hypoesthesia (Ameri A. 1999 ref.cit .; Patel S. and Hillard C.J. 2001 J. Pharmacol .Exp. Ther. 297, 629-637);

b) the role of central cannabinoid receptors in inducing deficits in memory, cognitive and attention capacities (Ameri A. 1999 ref.cit.)] c) the recent demonstration of a possible role of the endocannabinoid system in psychic functions and in schizophrenia (Piomelli

D. et al. 2000 ref. cit .; Ameri A. 1999 ref.cit.),

d) that it has recently been shown that chronic treatment with classical cannabinoids, known to be associated with a decrease in

receptor density or with a desensitization of central receptors

of cannabinoids, results in a lower appearance of opioid tolerance (Ameri A. 1999 ref.cit.);

e) the ability of central cannabinoid receptor antagonists to decrease pharmacological dependence on drugs and / or substances of abuse,

such as opioids, alcohol, marijuana, amphetamines and tobacco (Huestis M.A. et

to the. 2001 Arch. Gerì. Psychiatry 58, 322-328; Mas-Nieto M. et al. 2001

Brit. J. Pharmacol. 132, 1809-1816);

f) the role of central cannabinoid receptors in the control of appetite and hunger from which also derives the recent indication on the use of antagonists to the central cannabinoid receptor (SR

141716) as anorexizants (Di Marzo V. et al. 2001 Nature 410, 822-825; Kirkham T.C. and Williams C.M. 2001 Psycopharmacol. 153, 267-270)]

g) the presence of central cannabinoid receptors constitutively

expressed in peripheral organs and tissues such as the digestive system, the immune system and the vascular system with

vasodilative and hypotensive type (/ zzo A. A. et ai. 2000 Brit. J. Pharmacol.

129984-990; Salzet M. et al. 2000 Eur. J. Biochem 267, 4917-4927; / Hillard C. J. 2000 J. Pharmacol. Exp. Therap. 294, 27-32);

h) the role of central cannabinoid receptors in endotoxin-induced hypotension in advanced liver cirrhosis and the ability of central cannabinoid receptor antagonists (SR 141716) to induce a pressure effect associated with a decrease in mesenteric arterial flow and portal venous pressure (Baktai S. et al. 2001 Nature Med. 7, 827-832).

From the above considerations it is therefore clear that the effect of the acylamides obtained following repeated administration in vivo described in the present invention characterizes these compounds as functional antagonists of the central cannabinoid receptors which can therefore be usefully used for the preparation of pharmaceutical compositions for therapeutic treatment. , alone or in combination with other therapeutic agents of choice for the specific pathological state, such as antiepileptic drugs, neuroleptics, atypical neuroleptics, antidepressants, dopaminergics, dopamine agonists, gaba-agonists, against excess weight, for improvement of memory, anti-inflammatory / painkillers (e.g. opioids, salicylates, pyrazoles, indoles, arylantranilics, arylpropionics, arylacetics, oxicams, pyranocarboxyls, glucocorticoids), purgatives (emollients, osmotic, saline, irritants), of altered pathological states and / or "illegal" activation of central cannabinoid receptors, namely in:

- in the control of motor activities such as those induced by pyramidal deficits and extrapyramidal deficits;

- in hypoesthetic sensory nerve conduction disorders; - in neurological disorders characterized by deficits in memory, cognitive and attention skills;

- in the therapy of psychotic states such as schizophrenia and mood or affective disorders;

- in the reduction of opioid tolerance in analgesic therapy; - in the weaning of drug addiction to opioids, alcohol, marijuana, amphetamines and tobacco;

- in eating disorders to control the stimulus of hunger and appetite;

- in pathological states of immunosuppression, also induced by pharmacological means, in which an immunostimulating action is required;

- in the control of intestinal motility and blood pressure even in states of advanced cirrhosis.

The administration routes that can be used for the preventive or therapeutic treatment of the pathological states according to the present invention can be the oral, parenteral, intramuscular, subcutaneous or intravenous route, and the transdermal topical route, including the rectal, sublingual and intranasal route. The compounds, according to the therapeutic use as functional antagonists of the central cannabinoid receptors, can be administered in pharmaceutical compositions in combination with excipients, dispersants and diluents compatible with known or new pharmaceutical uses, in order to obtain a better delivery of the active ingredient. at the site of action and to obtain a rapid, sustained or delayed effect over time. For this purpose, fast, sustained or slow release pharmaceutical compositions can be used. Dosages depend on the severity of the pathology and the chosen route of administration, as well as on the state (age, body weight, general health conditions) of the patient and for illustrative but not limitative purposes of the present invention, they can be included between 1 mg / Kg and 50 mg / Kg in repeated daily administrations for a period between 2 and 16 weeks.

For oral administration, compositions in the form of dispersible granular powders, tablets, dragees, soft or hard gelatin capsules, suspensions, may be suitable; for intramuscular, subcutaneous, intravenous or epidural parenteral administration, compositions in the form of buffered aqueous solutions, oily suspensions or lyophilized powders dispersible in a suitable solvent at the time of administration may be suitable; compositions in suitable excipients or dispersants in the form of patches, suppositories, ovules, aerosols and sprays may be suitable for topical transdermal, rectal, intranasal or sublingual administration.

Claims (1)

Claims 1. use of an acylamide of saturated acids of formula (I) or pharmaceutically acceptable esters or salts thereof, where: R-i-CO-- is an acyl residue of a linear or branched saturated organic acid comprising from 10 to 20 C atoms, - N-- R2 is: - a residue of a linear or branched aminohydroxyalkyl comprising from 2 to 6 C atoms, optionally substituted with one or more aromatic groups on the alkyl chain, - a residue of an amino acid of the α, β or γ series, R3 can be H or CH3 or where -N-R2, R3 form with the N atom a cyclic aminoether comprising from 5 to 7 C atoms, possibly substituted with linear or branched alkyl groups, as functional antagonists of the central cannabinoid receptors for the preparation of pharmaceutical compositions for the preventive or therapeutic treatment of pathological states connected with an altered functionality and / or “abusive” activation of the central cannabinoid receptors; 2. use according to claim 1 characterized in that the acyl residue R-i-CO-- is selected from the group consisting of saturated aliphatic monocarboxylic acids; 3. use according to claim 2 wherein the acyl residue is selected from the group consisting of decenoic, lauric, myristic, paimitic, stearic or arachidic acid; 4. use according to claim 1 characterized in that, when -N - R2 is a residue of a linear or branched amino hydroxyalkyl, this is selected from the group consisting of monoethanolamine or 2 hydroxypropylamine; 5. use according to claims 1 and 4 characterized in that the hydroxyl of the aminohydroxyalkyl residue is esterified with a pharmaceutically acceptable acid group; 6. use according to claim 1 characterized in that, when --N - R2 is a residue of an amino acid of the α, β or γ series, this is selected from the group consisting of serine, glycine, β-alanine, γaminobutyric, phenylalanine or tyrosine; 7. use according to claims 1 and 6 characterized in that the carboxylic group of the amino acid residue is esterified or salified with pharmaceutically acceptable groups or counterions; 8. use according to claim 1 characterized in that, when -N - R2, R3 form a cyclic aminoether with the N atom, this is morpholine; 9. use according to claim 1 characterized in that said pathological states are selected from the group consisting of motor activity disorders deriving from pyramidal and extrapyramidal deficits; 10. use according to claim 1 characterized in that said pathological states are selected from the group consisting of disorders of the nervous sensitivity of the hypoaesthetic type; 11. use according to claim 1 characterized in that said pathological states are selected from the group consisting of neurological disorders characterized by deficits in memory, cognitive and attention capacities; 12. use according to claim 1 characterized in that said pathological states are selected from the group consisting of psychotic states and mood or affective disorders; 13. use according to claim 1 characterized in that said pathological states are selected from the group consisting of reduction of tolerance to opioids in analgesic therapy; 14. use according to claim 1 characterized in that said pathological states are selected from the group constituted in the disorders associated with the weaning of drug addiction to opioids, alcohol, marijuana, amphetamines and tobacco; 15. use according to claim 1 characterized in that said pathological states are selected from the group consisting of eating disorders; 16. use according to claim 1 characterized by the fact that said pathological states are selected from the group consisting of immunosuppressive conditions in which an immunostimulating action is required; 17. use according to claim 1 characterized in that said pathological states are selected from the group consisting of intestinal motility disorders; 18. use according to claim 1 characterized in that said pathological states are selected from the group consisting of blood pressure disorders including advanced states of liver cirrhosis; 19. use according to claims 9 to 18 characterized by the fact that in said pathological states the therapeutic compositions containing the acylamides of saturated acids, or esters or salts thereof, can be used alone or in association with drugs of choice for the same ; 20. use according to claim 1 characterized in that said pharmaceutical compositions are suitable for oral administration in the form of dispersible granular powders, tablets, dragees, soft or hard gelatin capsules, suspensions; 21. use according to claim 1 characterized in that said pharmaceutical compositions are suitable for intramuscular, subcutaneous, intravenous or epidural parenteral administration in the form of buffered aqueous solutions, oily suspensions or lyophilized powders dispersible in a suitable solvent at the time of administration; 22. use according to claim 1 characterized in that said pharmaceutical compositions are suitable for topical transdermal, rectal, intranasal, sublingual administration in suitable excipients or dispersants in the form of patches, suppositories, ovules, aerosols and sprays. (SL / lm)