WO1996033210A1

WO1996033210A1 - Amines exhibiting analgesic action, their preparation and use

Info

Publication number: WO1996033210A1
Application number: PCT/EP1996/001598
Authority: WO
Inventors: Giorgio Adembri; Lia Fantetti; Alberto Giotti; Ilaria Masini; Gabrio Roncucci
Original assignee: L. Molteni & C. Dei Fratelli Alitti - Societa' Di Esercizio Societa' Per Azioni
Priority date: 1995-04-19
Filing date: 1996-04-17
Publication date: 1996-10-24
Also published as: ITFI950073A1; AU5688996A; IT1277841B1; ITFI950073A0; EP0825999A1

Abstract

The present invention relates to amines of general formula (I), exhibiting analgesic action, the methods of preparation and use thereof.

Description

AMINES EXHIBITING ANALGESIC ACTION, THEIR PREPARATION AND USE

Field of the invention

The present invention relates to compounds of general formula (I)

where :

R₁, R₂, R₃, R₄, identical or different, are each selected from the group consisting of H, C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, an amine protective group;

R₅ and R₆, identical or different, are each selected from the group consisting of H, C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, or R₅ and R₆ with the carbon atom whereto they are bound form a CO group;

R₇ is selected from the group consisting of: halogen; OR₈; NR₁₁R₁₂; proteic amino acid, optionally protected, bound to Y by a nitrogen atom, where :

R₈ is selected from the group consisting of H, C_1-4 alkyl, aryl, aryl- C_1-4 alkyl-, carboxyl-C_1-4 alkyl- and

R₁₁ and R_12' identical or different, are each selected from the group consisting of H, C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, carboxyl-C_1-4 alkyl- or an ester thereof;

x is a whole number from 1 to 10;

n is a whole number from 1 to 5;

Y is selected from the group consisting of: CO, CR₉R₁₀, where R₉ and R₁₀ , identical or different, are each selected from the group consisting of H, C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, taking into account that when Y = CO, CR₅R₆ is different from CO; and pharmaceutically acceptable salts thereof.

Prior art

The need for highly therapeutically active compounds capable of acting as pain-killers through a mechanism differing from that of the traditional compounds used to this end (opioids, cyclooxygenase inhibitors) that cause the well known untoward side effects, is acknowledged. In fact, said compounds would be extremely useful for many curative indications in all the sectors in which an excessive release of endogenous aminoacidergic-peptidergic factors causes pain, ischaemia and in the degenerative conditions of organs and systems mediated by same.

It is known, in this regard, that the tripeptide, (L)lysine- (L)proline-(L)threonine exhibits hyperalgesic action, whereas the corresponding tripeptide, (L)lysine-(D)proline-(L)threonine exhibits an opposite action (analgesic) against the effects caused by IL-1β [cf. Nature, 334 , 698 (1988)].

It has surprisingly been found that compounds of formula (I) not having the aforesaid tripeptide structure show excellent analgesic properties.

Detailed description of the invention

It is an object of the present invention to solve the aforesaid problem and, therefore, to provide compounds - based on the products of general formula (I) above - that, at low dosage, exert a pain- killing action without causing the adverse side effects arising from the administration of the common pain-killers. According to the present invention, C_1-4 alkyl means: methyl-, ethyl-, n-isopropyl-, n-iso-tert-butyl- group. Aryl means: phenyl group. Aryl- C_1-4 alkyl- means: benzyl-, phenyl-ethyl-, phenyl-propyl-, phenyl-butyl group. C_1-4 alcoxy means: methoxy-, ethoxy- propoxy-, butoxy group. Amine protective group preferably means: Boc, Cbz, CH₃CO-.

Halogen means: F, Cl, Br, I.

n is preferably 3 or 4; x is 4, 5. 10.

Particularly preferred are the compounds wherein x = 4 and n = 4.

Among the compounds of formula (I), particularly preferred are the following:

N,N'-diCbz-LysΨ[CH₂N]DPro

LysΨ[CH₂N]DPro

N,N'-diCbz-LysΨ[CH₂N]Pro-Gly-OEt

N,N'-diCbz-LysΨ[CH₂N]Pro-Gly

LysΨ[CH₂N]Pro-Gly

LysΨ[CH₂N]Pro-Thr

N,N'-diCbz-LysΨ[CH₂N]DPro-Thr(Bz)

LysΨ[CH₂N]DPro-Thr

N,N'-diCbz-Lysy[CH₂N]DPro-Thr(tBu)-OCH₃

Cbz-Lys(Boc)Ψ[CH₂N]DPro

N,N'-diCbz-LysΨ[CH₂N]Pro-Val

N,N^,-diCbz-LysΨ[CH₂N]Pro-Thr(Bz)

Boc-Lys-Cbz Ψ [CH₂N]DPro

Lys (Boc ) Ψ [CH₂N]DPro

Boc-Lys Ψ [CH₂N]DPro

N,N'-diCbz-Lys-prolinol

N,N'-diCbz-Lys-2-chloromethylpyrrolidine The products according to the invention may be prepared by reactions already known, e.g.:

a) by reaction of an aldehyde of general formula (II) with a cyclic amine of general formula (III)

in the presence of sodium cyanoboron hydride; where the various substituents are as defined above;

b) by peptide synthesis in the liquid and/or solid phase between amino acids of general formula (IV)

and an amine of general formula (III) as defined above, where the substituents are as defined above;

c) by reaction of compounds of general formula (V)

where W = CHO, CH₂X and X = halogen

with compounds of general formula HNR₁₁R₁₂ or proteic amino acids, where R₁₁ and R₁₂ are as defined above, according to synthesis procedures already known.

Examples of reactions of the aforesaid type are described in Peptide

Research, Vol. 5. No. 1, p. 30 (1992).

The following examples are intended as illustrative of the present invention but not limiting.

EXAMPLE 1

N,N'-diCbz-LysΨ[CH₂N]DPro.2H₂O

[Compound of formula (I), where:

R_1' R₃, R₅, R₆ = H; R₂, R₄, = C₆H₅-CH₂-O-CO-; n = 3; x = 4;

Y = CO; R₇ = OH]

The aldehyde, N,N'-diCbz-2,6-diaminohexanal, (3-75 g; 9.41 mmol) was solubilized in a 99:1 methanol-acetic acid mixture (40 ml). The solution obtained was added with 1.5 equivalent of D-proline at a time. The reaction mixture was added portionwise with NaBH₃CN (1.77 g) over a period of 30 min. The reaction was maintained at room temperature for 30', the reaction solvent was partially evaporated and the residue was added with a sodium sulphate saturated solution (200 ml), acidified with sulphuric acid (1M in H₂SO₄). A few minutes later, the reaction mixture was extracted at room temperature with ethyl acetate (300 ml) in 3 portions. The extracts were combined, dehydrated with anhydrous sodium sulphate and evaporated yielding a colourless oil which was initially purified by solubilization in ethyl acetate and two precipitations with n-hexane followed by solubilization in ethyl acetate and reprecipitation with ethyl ether.

The product obtained was an oil, which then solidified.

Elem. Anal. Found: C, 62.53; H, 7.07; N, 8.05

Calcd: C, 62.90; H, 7.10; N, 8.40 EXAMPLE 2

LysΨ[CH₂N]Pro.2CF₃COOH

[Compound of formula (I), where:

R_1' R₂, R₃, R₄, R₅, R₆ = H; n = 3; x = 4; Y = CO; R₇ = OH]

N,N'-diCbz-LysΨ[CH₂N]Pro (207 mg) was solubilized in methanol (5 ml) and subjected to catalytic hydrogenation at atmospheric pressure in the presence of 102% Pd-C.

The reaction was discontinued after 3 hrs, once the removal of the benzyloxycarbonyl group had been ascertained. The catalyst was then eliminated and the reaction solvent was evaporated to give a white solid which was triturated twice in ethyl ether, solubilized in H₂O-

0.1% TFA and purified by HPLC (reversed phase, gradient H₂O-0.1% TFA- methanol).

Freeze-drying of the eluted fractions, concentrated at reduced pressure, yielded a white solid consistent with the assigned product.

Elem. Anal. Found: C, 39.74; H, 5.67; N, 9.92

Calcd: C, 39.38; H, 5.50; N, 9.18

EXAMPLE 3

N,N' -diCbz-LysΨ[CH₂N]Pro-Gly-OEt

[Compound of formula (I), where:

R₁, R₃, R₅, R₆ = H: R₂, R₄ = C₆H₅-CH₂-O-CO-; n = 3; x = 4; Y = CO; R₇

= -NH-CH₂-COOC₂H₅]

N,N'-diCbz-LysΨ[CH₂N]Pro (200 mg; 0.40 mol) was solubilized in anhydrous THF (15 ml), added, in order, with N-ethylmorpholine (0.40 mmol) and ethyl chloroformate (0.40 mmol). The resulting turbid solution was allowed to stir at room temperature for a period of 30 min and, after ascertaining the formation of mixed anhydride by TLC, added with 5 equivalents of glycine ethyl ester hydrochloride, solubilized in a mixture of THF (10 ml) and DMF (1 ml) containing 5 equivalents of N-ethylmorpholine.

The reaction mixture was stirred at room temperature for additional 4 hrs and, after partial evaporation of the reaction solvent, added with a sodium bicarbonate saturated solution (200 ml). The suspension obtained was extracted portionwise with ethyl acetate (100 ml). The extracts were combined, washed with water, dehydrated, and evaporated to yield an oil (190 mg) which was purified by silica column chromatography (1.2 x 10 cm) eluting with chloroform-methanol, 98/2.

The chromatographic fractions were evaporated yielding 180 mg of the assigned compound.

Chemical shifts selected from ¹H NMR (200 MHz) CDCl₃: 7.33 (s,2x5H, Ph); 5-0 (s,2x2H, 0-CH₂-Ph); 4.05 (q,2H, 0-CH₂); 1.12 (t,3H,CH₃).

EXAMPLE 4

N,N' -diCbz-Lys ψ[CH₂N]Pro-Gly

[Compound of formula ( I ) , where:

R₂ , R ₄ , R₅ , F ₆ = H; R₁ , R₃ = C₆H₅-CH₂O-CO- ; n = 3 ; x = 4 ; Y = CO; R₇ = -NH-CH₂-COOH]

The ester prepared as described in Example 3 (180 mg) was treated with a mixture of 1N NaOH (10 ml) and methanol (3 ml). The mixture was allowed to stir at room temperature overnight to obtain complete solubilization of the starting product. After methanol elimination, the aqueous solution was acidified with a few drops of 1N HCl and extracted portionwise with ethyl acetate (50 ml). The organic solution was washed, dehydrated and evaporated to give a colourless oil, which then solidified. EXAMPLE 5

LysΨ[CH₂N]Pro-Gly

[Compound of formula (I), where:

R₁, R₂, R₃, R₄, R₅, R₆ = H; n = 3; x = 4; Y = CO; R₇ = -NH-CH₂-COOH] The product prepared as described in Example 4 (100 mg) was solubilized in methanol (10 ml) and hydrogenated at atmospheric pressure in the presence of 10% Pd-C catalyst. After 2 hrs, the reaction was discontinued and the solvent evaporated completely. The resulting colourless vitreous solid was taken up with little water- 0.1% TFA and purified by HPLC (reversed-phase, gradient H₂O-0.1% TFA- methanol).

EXAMPLE 6

Lys Ψ [CH₂N]Pro-Thr.3CF₃COOH.2H₂O

[Compound of formula (I), where:

R₁, R₂, R₃, R₄, R₅, R₆ = H; n = 3; x = 4; Y = CO; R₇ = -NH-CH-(COOH)- CH(CH₃)OH]

The product prepared as described in Example 12 (150 mg) was hydrogenated in a methanol solution at atmospheric pressure in the presence of 10% Pd-C catalyst for a period of 18 hrs. Once the catalyst had been removed, methanol was evaporated to give a colourless vitreous solid which was taken up with little H₂O-0.1% TFA and purified by HPLC (reversed-phase C18; gradient H₂O-0.1% TFA- methanol).

Elem. Anal. Found: C, 35-38; H, 4.71; N, 7.69

Calcd: C, 35-59; H, 4.69; N, 7.90

EXAMPLE 7

N,N'-diCbz-Lysψ[CH₂N]DPro-Thr(Bz).HCl .2H₂O [Compound of formula (I), where:

R₁, R₃, R₅, R₆ = H; R₂, R₄, = C₆H₅-CH₂-O-CO- ; n = 3; x = 4; Y = CO; R₇

= -NH-CH(COOH)-CH(CH₃)-O-CH₂-C₆H₅]

The compound prepared as described in Example 1 (900 mg) was suspended in anhydrous THF (70 ml). The resulting suspension was added with

N-ethylmorpholine (480 μl) and, after cooling to 0°C, slowly added with ethyl chloroformate (180 μl), while the reaction mixture was stirred for 4 hrs.

Separately, Boc-threonine(Bz) (1.070 g) was added at room temperature with TFA (3 ml). The solution was caused to react for 30', then the excess of TFA was distilled off under vacuum. The residue obtained was taken up with anhydrous THF (30 ml) containing 1 equivalent of N- ethylmorpholine and the solution was added to the above reaction mixture under stirring at room temperature.

The reaction was continued for 18 hrs. The reaction solvent was then evaporated, the residue was taken up with a sodium sulphate saturated solution acidified with sulphuric acid, and the suspension was extracted portionwise with ethyl acetate (300 ml). Once the organic extracts had been combined, the solution was washed with water, dehydrated and evaporated to dryness yielding an oil which was purified by silica column chromatography (1.2 x 30 cm) eluting with chloroform-methanol, 90/10.

The product obtained (200 mg) was salified with HCl.

Elem. Anal. Found: C, 60.40; H, 6.55; N, 7.49

Calcd: C, 59-94; H, 6.75; N, 7-36

EXAMPLE 8

LysΨ [CH₂N]DPro-Thr.3CF₃COOH.2H₂O [Compound of formula (I), where:

R_1' R₂, R₃, R₄, R₅, R₆ = H; n = 3; x = 4; Y = CO; R₇ = -NH-CH-(COOH)-

CH(CH₃)OH]

The product prepared as described in Example 7 (460 mg) was solubilized in methanol (20 ml) and hydrogenated at room temperature in the presence of 10% Pd-C catalyst for a period of 18 hrs. The solvent was evaporated and the resulting reaction product solution yielded a vitreous white solid, which was triturated in anhydrous ethyl ether, taken up with little H₂O-0.1% TFA and purified by HPLC (isocratic, H₂O-0.1% TFA) .

90 mg of a hygroscopic white solid was obtained from the chromatographic fractions concentrated under vacuum and freeze-dried.

Elem. Anal. Found: C, 35.59; H, 4.69; N, 7-90

Calcd: C, 35.68; H, 4.90; N, 7-77

EXAMPLE 9

N,N'-diCbz-LysΨ[CH₂N]DPro-Thr(tBu)-OCH₃

[Compound of formula (I), where:

R₁, R₃, R₅, R₆ = H; R₂, R₄ = C₆H₅-CH₂-O-CO-; n = 3: x = 4; Y = CO; R₇

= -NH-CH(COOCH₃)-CH(CH₃)-O-C(CH₃)₃]

The compound prepared as described in Example 1 (350 mg; 0.6 mmol) was added with N-ethylmorpholine (180 μl; 1.4 mmol). The solution was cooled to 0ºC and slowly added with ethyl chloroformate (70 μl; 0.7 mmol).

The solution was then stirred for 3 hrs and added with threonine(tBU) methyl ester hydrochloride (400 mg; 1.77 mmol) at a time and then slowly under stirring with N-ethylmorpholine (270 μl).

Stirring of the reaction mixture was continued overnight. Then the solvent was evaporated completely and the residue was taken up with water and extracted with ethyl acetate (200 ml) in 3 portions.

The extracts were washed with water, dehydrated with anhydrous sodium sulphate and evaporated. The resulting residue was purified by silica column chromatography eluting with chloroform yielding 350 mg of a colourless oil.

Elem. Anal. Found: C, 64.11; H, 7.82; N, 8.29

Calcd: C, 64.65; H, 7.84; N. 8.38

EXAMPLE 10

Cbz-Lys(Boc)Ψ[CH₂N]DPro.H₂O

[Compound of formula (I), where:

R₁, R₃, R₆, R₆ = H; R₂ = (CH₃)₃C-O-CO-; R₄= C₆H₅-CH₂-O-CO-; n = 3; x = 4; Y = CO; R₇ = OH]

2-NCbz-6NBoc-diaminohexanal (1.85 g; 4.8 mmol) was solubilized in the reaction solvent (25 ml) consisting of 99:1 methanol-acetic acid. The solution was added with 1.5 equivalent of D-proline.

The reaction mixture was then added portionwise with sodium cyanoboron hydride (904 mg; 14.4 mmol) and stirred at room temperature for 4 hrs. The reaction solvent was evaporated, the residue was added with a sodium sulphate solution (250 ml) acidified with sulphuric acid and the suspension was extracted with ethyl acetate (200 ml) in 3 portions.

The extracts were combined, washed with water, dehydrated, and finally evaporated to dryness yielding an oil which was taken up with hexane. The two phases were maintained in an ultrasound bath for 1 hr, the solvent was decanted and the product was purified by repeated eolubilizations in ethyl acetate followed by reprecipitation with hexane .

1.1 g of a pure liquid was isolated.

Elem. Anal. Found: C, 59.46; H, 7-95; N, 8.42

Calcd: C. 59.70; H, 8.09; N, 8.72

EXAMPLE 11

N,N'-diCbz-LysΨ[CH₂N]Pro-Val.2.5 H₂O

[Compound of formula (I), where:

R₁, R₃, R₅ , R₆ = H; R₂, R₄ = C₆H₅-CH₂-O-CO-; n = 3; x = 4; Y = CO; R₇

= -NH-CH(COOH)-CH(CH₃)₂]

N,N'-diCbz-LysΨ [CH₂N]Pro (1 g; 2 mmol) was suspended in anhydrous

THF, added with N-ethylmorpholine (550 ml). The resulting suspension was cooled to 0ºC and slowly added with ethyl chloroformate (200 μl).

The reaction mixture was stirred for 3 hrs and, after ascertaining the formation of mixed anhydride, added with valine (703 mg; 6 mmol). The reaction was continued at room temperature for 18 hrs.

The solvent was evaporated yielding an oil which was taken up with a sodium sulphate saturated solution (300 ml) acidified with sulphuric acid. The suspension was extracted with ethyl acetate (300 ml) in 3 portions.

The extracts were washed with water, dehydrated with anhydrous sodium sulphate, and evaporated to dryness yielding an oil (500 mg) which was purified 3 times by silica column chromatography (1.2 x 25 cm) eluting with chloroform-methanol, 90/10.

200 mg of a colourless viscous liquid was thus obtained.

Elem. Anal. Found: C, 59.82; H, 7.45; N, 8.77

Calcd: C, 59.88; H, 7.63; N. 8.72 EXAMPLE 12

N,N' -diCbz-Lys ψ[CH₂N]Pro-Thr(Bz ) , H₂O

[Compound of formula ( I ) , where :

R₁ , R₃ , R₅ , R₆ = H; R₂ , R₄ = C₆H₅-CH₂-O-CO- ; n = 3 ; x = 4 ; Y = CO ; R₇ = -NH-CH (COOH) -CH(CH₃) -O-CH₂-C₆H₅]

N,N'-diCbz-Lys V[CH₂N]Pro (1.2 g) was solubilized in anhydrous THF (100 ml). The resulting solution was added with N-ethylmorpholine (314 μl), cooled to 0ºC and slowly added with ethyl chloroformate (186 μl), while the reaction mixture was allowed to stir for 1 hr. The mixture was then added with threonine(Bz) (1.088 g) solubilized in anhydrous THF (30 ml) containing 1 equivalent of N-ethylmorpholine. The solution was added to the above reaction mixture under stirring at room temperature.

The reaction was continued for 18 hrs, the reaction solvent was removed by evaporation, the residue was taken up with a sodium sulphate saturated solution acidified with sulphuric acid. The suspension was extracted portionwise with ethyl acetate (300 ml).

Once the organic extracts had been combined, the solution was washed with water, dehydrated and evaporated to dryness yielding an oil (900 mg) which was purified by silica column chromatography (1.2 x 30 cm) eluting with chloroform-methanol, 90/10.

200 mg of a colourless viscous oil was thus obtained.

Elem. Anal. Found: C, 64.22; H, 7.01; N, 7-90

Calcd: C, 64.50; H, 7.13; N, 7-92

EXAMPLE 13

Boc-Lys(Cbz)Ψ[CH₂N]DPro.0.5 H₂O

[Compound of formula (I), where: R₁, R₃, R₅, R₆ = H; R₄ = (CH₃)₃C-O-CO-; R₂= C₆H₅-CH₂-O-CO-; n = 3; x =

4; Y = CO; R₇ = OH]

2N-Boc-6N-Cbz-diaminohexanal (2.3 g; 5-98 mmol) was solubilized in a

99-1 methanol-acetic acid mixture and added with 1.5 equivalent of D- Proline under stirring. After solubilization completion, the solution was added portionwise with sodium cyanoboron hydride (1.03 g) at room temperature, over 1 hr approx.

The reaction mixture was stirred at room temperature for 3 hrs, then added with a sodium sulphate solution (200 ml) acidified with sulphuric acid. The resulting suspension was extracted with ethyl acetate (300 ml) in 3 portions.

The extracts were combined, washed with water to neutrality, dehydrated with anhydrous sodium sulphate and evaporated under vacuum.

The resulting oily residue solidified by treatment with cyclohexane. 2 g of product was recovered and purified by silica column chromatography eluting with chloroform-methanol, 95/5.

800 g of the assigned product was thus obtained.

Elem. Anal. Found: C, 60.88; H, 7-91; N, 8.63

Calcd: C, 60.99; H, 7-89; N, 8.89

EXAMPLE 14

Lys(Boc)Ψ[CH₂N]DPro.2 CF₃COOH

[Compound of formula (I), where:

R₁, R₃, R₄, R₅, R₆ = H; R₂ = C(CH₃)₃-O-CO-; R₇= OH; n = 3; x = 4; Y =

CO]

The compound prepared as described in Example 10 ( 800 mg) was solubilized in methanol ( 15 ml ) . The resulting solution was subjected to catalytic hydrogenation at atmospheric pressure in the presence of 10% Pd-C .

Once the reaction had been completed, the solvent was eliminated and the vitreous transparent solid was purified by HPLC (reversed phase,

H₂O-0.1% TFA-methanol). The assigned product - a white hygroscopic solid - was recovered from the chromatographic fractions by partial evaporation at reduced pressure and freeze-drying.

Elem. Anal. Found: C, 42.60; H, 5.92; N, 7.64

Calcd: C, 43.08; H, 5.96; N, 7.53

EXAMPLE 15

Boc-Lys Ψ[CH₂N]DPro.2 CF₃COOH.H₂O

[Compound of formula (I), where:

R_1' R₂, R₃, R₅, R₆ = H; = R₄ = (CH₃)₃ -C-O-CO-; n = 3; x = 4; Y = CO; R₇ = OH]

The compound prepared as described in Example 13 (500 mg) was solubilized in methanol (15 ml) and hydrogenated at atmospheric pressure in the presence of 10% Pd-C as catalyst. The reaction was discontinued after 24 hrs, the solvent was evaporated and the vitreous solid residue was purified by HPLC (reversed phase, gradient H₂O-0.1%

TFA-methanol).

The assigned product was recovered from the chromatographic fractions by partial evaporation followed by freeze-drying.

Elem. Anal. Found: C, 41.82; H, 5.99; N, 7.35

Calcd: C, 41.73; H, 6.12; N, 7.30

EXAMPLE 16

N,N'-diCbz-Lys-Propinol

[Compound of formula (I), where:

R₁, R₃ = H; CR₅R₆ = 0; R₂, R₄ = -CO-O-CH₂-C₆H₅; Y = CH₂; R₇ = OH; n = 3; x = 4]

N,N'-diCbz-Lys (1.5 g) was solubilized in a 10% DMF-DCM mixture containing N-hydroxysuccinimide (417 mg). The stirred solution was added with a 0.5M solution of N,N'-dicyclohexyl carbodiimide in DCM (7-5 ml). The resulting solution was allowed to stand at room temperature for 18 hrs.

The dicyclohexylurea separated from the solution in the course of the reaction was filtered off. The protected lysine N-hydroxysuccinimide ester solution was evaporated to dryness to obtain complete DMF removal. The residue was taken up with DCM (10 ml) to give a turbid suspension. After solid removal by filtration, the active ester solution was slowly added to a prolinol solution (406 mg) under stirring at room temperature for 48 hrs.

Once the reaction had been completed, the solvent was partially evaporated, the oily residue was taken up with water (100 ml approx.), and the resulting sospension was extracted portionwise with ethyl acetate (200 ml). The organic phase was dehydrated with anhydrous sodium sulphate and evaporated to dryness to give a colourless oil which may be purified by silica gel column chromatography, eluting with CHCl ₃-CH₃OH, 90/10.

¹H NMR (200 MHz) (CDCl₃) : 7-35 (2x5H,Ph); 5.80 and 5.83 (s,H,N-H); 5.1 (s,2x2H,0-CH₂-Ph); 4.47 (m,2H,CH₂-CO); 4.65 (s,b,exc.,H,OH); 4.42 (m,2H,Lys); 3.72-3.0 (6H,Pro-Lys); 2.2-1.6 (m,8H,Lys-Pro).

By procedures analogous to the aforementioned ones, the following products were also obtained:

N,N'-diCbz-Lys-2-chloromethylpyrrolidine

N,N'-diCbz-LysΨ[CH₂N]DProΨ[CH₂NH]Thr N,N' -diCbz-LysΨ[CH₂N]Pro-V[CH₂NH]Thr Cbz-Lys(Boc) Ψ[CH₂N]DProΨ [CH₂NH]Thr Boc-Lys(Cbz)Ψ[CH₂N]ProΨ[CH₂NH]Thr Cbz-Lys(Boc)Ψ[CH₂N]ProΨ[CH₂NH]Thr Boc-Lys(Cbz)Ψ[CH₂N]DProΨ[CH₂ NH]Thr N,N'-diAcLysΨ[CH₂N]DProV[CH₂NH]Thr N,N' -diAcLysΨ[CH₂N]ProΨ[CH₂NH]Thr LysΨ[CH₂N]DProΨ[CH₂NH]Thr

Boc-LysΨ[CH₂N]DProΨ[CH₂NH]Thr N,N'-diCbz-LysΨ[CH₂N]ProΨ[CH₂NH]Val N,N'-diCbz-LysΨ[CH₂N]DProΨ[CH₂NH]Val Cbz-Lys(Boc)Ψ [CH₂N]DProΨ[CH₂NH]Val Boc-Lys(Cbz)Ψ[CH₂N]DProΨ[CH₂NH]Val N,N'-diAcLysΨ[CH₂N]DProΨ[CH₂NH]Val Lys Ψ[CH₂N]DProΨ[CH₂NH]Val

Boc-LysΨ[CH₂N]DProΨ[CH₂NH]Val N,N'-diCbz-Lys-DProψ[CH₂NH]Thr N,N'-diAcLys-DProψ[CH₂NH]Thr

BocLys(Cbz)-DProΨ[CH₂NH]Thr

CbzLys(Boc)-DProΨ[CH₂NH]Thr

Lys-DProΨ[CH₂NH]Thr

N,N'-diCbz-Lys-DProΨ[CH₂NH]Val N,N'-diAcLys-DProΨ[CH₂NH]Val

BocLys(Cbz)-DProΨ-[CH₂NH]Val

CbzLys(Boc)-DProΨ[CH₂NH]Val

Lys-DProΨ[CH₂NH]Val

N,N'-diCbz-Lys-DProΨ[CH₂NH]Gly N,N'-diAcLys-DProΨ[CH₂NH]Gly

BocLys(Cbz)-DProΨ[CH₂NH]Gly

Lys-DProΨ[CH₂NH]Gly

N,N'-diCbz-Lysyl-pipecoline acid

N,N'-diCbz-LysΨ[CH₂N]-pipecoline acid

N,N'-diAcLysΨ [CH₂N]-pipecoline acid

LysΨ[CH₂N]-pipecoline acid

N,N'-diCbz-LysΨ[CH₂N]DProΨ[CH₂NH]Gly

CbzLys(Boc)Ψ[CH₂N]-DProΨ[CH₂NH]Gly

BocLys(Cbz)Ψ[CH₂N]DProΨ[CH₂NH][ly

N,N'-diAc-LysΨ[CH₂N]DProΨ[CH₂NH]Gly

LysΨ[CH₂N]DProΨ[CH₂NH]Gly

BocLysΨ[CH₂N]DProΨ[CH₂NH]Gly

N,N'-diAc-Lys-DProlinol

BocLys(Cbz)-DProlinol

CbzLys(Boc)-DProlinol

N,N'-diCbz-Lys-DProlinol

N,N'-diCbz-Lys-DPro-CH₂-O-CH₂-COOH

Pharmacological activity

The compounds forming the object of the present invention, administered to test animals (mus rattus, mus musculus I) by the oral or parenteral (i.p., i.e. v.) route do not affect, within a wide interval of doses, the physiological functions and behaviour of same, also in activation conditions (e.g. rota rod).

Moreover, at the tolerated doses, said compounds exert potent selective antihyperalgesic. antiederaatous and antipyretic action, obtained by peripheral administrations in the order of μg/kg bodyweight. Said action is evident in the case of hyperalgesia from interleukin-1β (IL-1β) intraplantar administration and of edema from carrageenan intraplantar administration and is selective on inflamed tissue. In fact, the claimed compounds do not reduce the response to mechanical stimulation of a normal paw injected with physiological saline solution. Conversely, said response is deeply modified by morphine.

The anti-inflammatory activity does not depend on a direct antagonism of the drug on the receptor of IL-1β (used in this study as a phlogogenic agent) as proved by binding tests with labelled agonist. Furthermore, it is not connected, in case to a slight degree, in its antihyperalgesic component, with an increase (release) in endogenous endorphins, as proved by its persistent effect in the animals pretreated with naloxone doses capable of suppressing the action of morphine.

The action may therefore be related to a selective interference with the multifactorial route from IL-1β receptor activation to tissual damage causing edema and pain, in which the activation of inducible cyclooxygenase ( cyclooxygenase 2 ) is the final fundamental moment . Said sequence has been clearly demonstrated by experimental studies carried out with IL-1β and the "releasers" of same: prototypical is the LPS (lipopolysaccharide of bacterial origin).

The activity is quantitatively connected with the chemical structure and is obtained with doses that, except for unexpected selective compartmentalization phenomena during distribution, cause concentrations in the organism fluids in the submicromolar order. Therefore, it is likely that the molecule of the drug interferes selectively with key factors (e.g. neurokinins, substance P) and receptors thereof on the phlogistic pain route.

The selective action of the claimed compounds is reversible, but its duration is such that its therapeutic use in animal and human medicine may be envisaged.

The doses that have been found to be active in and tolerated by test animals range from microgram fractions/kg bodyweight to 10 mg/kg bodyweight and may be varied according to the type of application for phlogistic pain control (e.g. local application to mucous membranes, systemic oral or i.v. administration, etc.) and depending on the pharmacokinetic and pharmacometabolic characteristics of the various compounds described herein.

E

* p<0.05 vs IL-1β

TABLE 2

Anti-inflammatory activity of the product as per Example 1 and indomethacin

(experimental model: edema from carrageenan in the rat)

Compound Animals Dose Increase in paw volume (ml)

* p<0.05 cs control (500 μg carrageenan/rat, i.pl.)

Carrageenan was injected 30' after drug administration. The test was carried out 3 hrs after carrageenan intraplantar administration.

Claims

1. Compounds of general formula (I)

where :

R₇ is selected from the group consisting of: halogen; OR₈; NR₁₁R₁₂; proteic amino acid, optionally protected, bound to Y by an N atom, where:

R₁₁ and R₁₂, identical or different, are each selected from the group consisting of H, C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, carboxyl-C_1-4 alkyl- or an ester thereof;

x is a whole number from 1 to 10;

n is a whole number from 1 to 5;

Y is selected from the group consisting of: CO, CR₉R₁₀, where R₉ and R₁₀, identical or different, are each selected from the group consisting of H,C_1-4 alkyl, aryl, aryl-C_1-4 alkyl-, taking into account that when Y = CO, CR₅R₆ is different from CO; and pharmaceutically acceptable salts thereof.

2. The compounds according to claim 1, wherein n = 3. 4 and x = 4, 5, 10.

3. The compounds according to claim 2, wherein x = 4 and n = 4.

4. The compounds according to claim 1 of formula:

N,N'-diCbz-LysΨ[CH₂N]DPro

Lys Ψ[CH₂N] DPro

N,N'-diCbz-LysΨ[CH₂N]Pro-Gly-OEt

N,N'-diCbz-LysΨ[CH₂N]Pro-Gly

LysΨ[CH₂N]Pro-Gly

LysHΨ[CH₂N]Pro-Thr

N,N'-diCbz-LysΨ[CH₂N]DPro-Thr(Bz)

LysΨ[CH₂N]DPro-Thr

N,N'-diCbz-LysΨ [CH₂N] DPro-Thr(tBu)-OCH₃

Cbz-Lys(Boc)Ψ[CH₂N]DPro

N,N'-diCbz-LysΨ[CH₂N]Pro-Val

N,N'-diCbz-LysΨ[ CH₂N]Pro-Thr(Bz)

Boc-Lys-CbzΨ[CH₂N]DPro

Lys(Boc)Ψ[CH₂N]DPro

Boc-LysΨ [CH₂N]DPro

N,N'-diCbz-Lys-prolinol

N,N'-diCbz-Lys-2-chloromethylpyrrolidine

N,N'-diCbz-Lysr[CH₂N]DProH-[CH₂NH]Thr

N,N'-diCbz-LysΨ[CH₂N]ProΨ[CH₂NH]Thr

Cbz-Lys(Boc)Ψ[CH₂N]DProΨ[CH₂NH]Thr

Boc-Lys(Cbz)Ψ[CH₂N]ProΨ[CH₂NH]Thr

Cbz-Lys(Boc)Ψ[CH₂N]ProΨ[CH₂NH]Thr Boc-Lys(Cbz)Ψ[CH₂N]DProΨ [CH₂NH]Thr N,N'-diAcLysΨ[CH₂N]DProΨ[CH₂NH]Thr N,N'-diAcLysΨ[CH₂N]ProΨ[CH₂NH]Thr LysΨ[CH₂N]DProΨ[CH₂NH]Thr

Boc-LysΨ[CH₂N]DProΨ[CH₂ΨH]Thr N,N'-diCbz-LysΨ[CH₂N]ProΨ[CH₂NH]Val N,N'-diCbz-LysΨ [CH₂N]DProΨ[CH₂NH]Val Cbz-Lys(Boc)Ψ[CH₂N]DProΨ[CH₂NH]Val boc-Lys(Cbz)Ψ[CH₂N]DProΨ [CH₂NH]Val N ,N'-diAcLysΨ[CH₂N]DProΨ[CH₂NH]Val LysΨ[CH₂N]DProΨ[CH₂NH]Val

BocLys(Cbz)-DProΨ[CH₂NH]Thr

CbzLys(Boc)-DProΨ[CH₂NH]Thr

Lys-DProΨ[CH₂NH]Thr

N,N'-diCbz-Lys-DProΨ[CH₂NH]Val N.N'-diAcLys-DProΨ [CH₂NH]Val

BocLys(Cbz)-DProΨ[CH₂NH]Val

CbzLys(Boc)-DProΨ[CH₂NH]Val

Lys-DProΨ[CH₂NH]Val

N,N'-diCbz-Lys-DProΨ[CH₂NH]Gly

N.N'-diAcLys-DProΨ[CH₂NH]Gly

BocLys(Cbz)-DProΨ[CH₂NH]Gly

Lys-DProΨ[CH₂NH]Gly

N,N'-diCbz-Lysyl-pipecoline acid N,N'-diCbz-LysΨ [CH₂N]-pipecoline acid

N,N' -diAcLysΨ[CH₂N]-pipecoline acid

LysΨ[CH₂N]-pipecoline acid

N,N' -diCbz-LysΨ[CH₂N]DProΨ[CH₂NH]Gly

CbzLys(Boc)Ψ[CH₂N]-DProΨ[CH₂NH]Gly

BocLys(Cbz)Ψ[CH₂N]DProΨ[CH₂NH]Gly

N,N'-diAc-LysΨ[CH₂N]DProΨ[CH₂NH]Gly

LysΨ[CH₂N]DProΨ[CH₂NH]Gly

BocLysΨ[CH₂N]DProΨ[CH₂NH]Gly

N,N'-diAc-Lys-DProlinol

BocLys(Cbz)-DProlinol

CbzLys(Boc)-DProlinol

N,N'-diCbz-Lys-DProlinol

N,N'-diCbz-Lys-DPro-CH₂-O-CH₂-COOH.

5. The process for the preparation of compounds of formula (I) according to claim 1, wherein an aldehyde of general formula (II) is caused to react with a cyclic amine of general formula (III)

in the presence of sodium cyanoboron hydride; where the various substituents are as defined above.

6. The process for the preparation of compounds of formula (I) according to claim 1, wherein a peptide synthesis of amino acids of general formula (IV)

where the substituents are as defined above, with an amine of general formula (III) as defined above, is carried out in the liquid and/or solid phase.

7. The process for the preparation of compounds of formula (I) according to claim 1, wherein compounds of general formula (V)

where W = CHO, CH₂X and X = halogen

are caused to react with compounds of general formula HNR₁₁R₁₂ or proteic amino acids, where R₁₁ and R₁₂ are as defined above, according to synthesis procedures already known.

8. Use of the compounds according to claim 1 for the preparation of pain-killing pharmaceutical compositions.

9. Pharmaceutical compositions containing, as active ingredient, a compound of formula (I) or mixtures thereof combined with suitable carriers.