WO1994013686A1

WO1994013686A1 - Glycosyl phosphotriester derivatives and their utilization for the central nervous system

Info

Publication number: WO1994013686A1
Application number: PCT/FR1993/001253
Authority: WO
Inventors: Tam Huynh-Dinh; Gilles Fillion
Original assignee: Institut Pasteur
Priority date: 1992-12-16
Filing date: 1993-12-15
Publication date: 1994-06-23

Abstract

The invention relates to compounds having a pharmacological activity in the central nervous system, having the capacity of traversing the blood-brain barrier after peripheral administration of said compound, and having the general formula (I) wherein A is O, N or S; R1 is (1) either: [sugar or desoxy sugar in C5 or C6, substituted or unsubstituted ]n in series D or L: n = 1 to 6, and the junction between the sugar and the phosphorous being on one of the hydroxyls at the apex 2, 3, 4 or 6; (2) or: (Ose) X - O - [(CH2)y - O]z - wherein Ose is a sugar in C5 or C6, D or L, the substitution of the chain 0 - [(CH2)y - O]z bearing on the hydroxyl of apex 1 of the sugar; x is an integer from 1 to 12; y is an integer from 1 to 6; z is an integer from 1 to 4; R2 is an alkyl group having from 6 to 30 carbon atoms, saturated or unsaturated, which may carry branches or cycles; N is a molecule susceptible of acting on the central nervous system.

Description

GLYCOSYL PHOSPHOTRIESTERS DERIVATIVES AND THEIR USE AT THE CENTRAL NERVOUS SYSTEM

The subject of the invention is glycosyl phosphotriester derivatives of substances capable of passing the blood-brain barrier (BBB) and having pharmacological activity in the central nervous system (CNS).

An important problem posed in pharmacology is that of the availability of drugs at the level of their therapeutic target, and in this area, the passage of the BBB. Among these. substances include psychotropics (antidepressants, neuroleptics or anxiolytics), analgesics, or even various regulators of the reticuloendothelial system or the cardiovascular system. A bad passage of the BBB constitutes a major drawback to the administration of substances which must act on the level of the central nervous system.

These substances are generally administered by peripheral route (oral, intravenous, intramuscular or intraperitoneal) in massive doses with the objective that a minimal fraction reaches the cerebral target. The result is most often the induction of significant and disabling side effects.

Different strategies have been developed to allow targeting of substances with pharmacological effect in the central nervous system. One of these, developed by N. Bodor et al. since the early 1980s, the redox-dihydropyridine / pyridiniu system has been used (cf. in particular the Bodor patents: US n ° 4,479,932, n ° 4,540,564, n ° 4,617,298, n "4,727,079, n" 4.824.850, n ° 4.829.070, n "4.863.911, n ° 4.880.816, n" 4.880.921, n '4.900.837, as well as PCT patent applications WO83 / 03968 and WO86 / 00897 in the name of University of Florida.

This system, if it has given encouraging results in the laboratory, encounters major drawbacks during the transposition to the conception of a drug: derivatives containing dihydropyridine are unstable, and extremely sensitive to oxidation. In addition, this type of compound is insoluble in an aqueous solution, which makes its systemic administration problematic.

Other approaches have been developed, also by Bodor in PCT patent application 092/00988. In this approach, phosphonate derivatives have been developed. These compounds have a residue composed of a pharmacological substance having a functional reactive group, which group is coupled, directly or by a bridging group, and via an -O- link, to the phosphorus atom of the compound; these compounds are hydrolyzed in situ, allowing the release of the pharmacological substance and its active site.

The present invention aims to develop a new approach and new compounds in order to allow the targeting of molecules at the central level, by a means usable as a drug in 1 • man or animal.

A pharmacologically active substance which, originally or badly passes the blood-brain barrier at all, is modified by the addition of a phosphate group esterified by a sugar and a lipid chain which allow it to cross this barrier; at the level of the central nervous system (CNS), metabolic reactions make it possible to release the active molecule again in its initial state and this, in the vicinity of its targets.

The present invention is a new family of compounds exhibiting pharmacological activity central, having the capacity to cross the BBB after administration of said compound by the peripheral route, and having the general formula I: 0

II

R., 0 - P - AM

I

0R ₂ in which: A = O, N or S

- R-, represents

1) either: [C ₅ to C ₆ sugar or deoxy sugar, substituted or not] _n , in series D or L; n = 1 to 6, and the junction between sugar and phosphorus being on one of the hydroxyls of vertices 2, 3, 4, 6, or

2) either: (Ose) x - 0 - [(CH ₂ ) _y - 0] _z - in which Ose represents a C ₅ or C ₆ , D or L sugar, the substitution of the chain 0 - [(CH ₂ ) _y - 0] ₂ being done on the top 1 of the sugar.

- x is an integer from 1 to 12;

- y is an integer from 1 to 6;

- z is an integer from 1 to 4;

- R ₂ represents an alkyl group of 6 to 30 carbon atoms, saturated or not, which can carry ramifications or rings;

- M represents a molecule capable of acting at the central level. Mention may in particular be made of psychotropics, analgesics, regulators of the immune or cardiovascular systems; it can also represent a precursor of said molecules capable of being transformed in the CNS into the active molecule.

Said molecule can be, for example, a peptide or polypeptide or a glycosylated protein or not, a cyclic or heterocyclic molecule chosen for example from the different families of psychotropics. This list is not exhaustive and may include any active substance having a hydroxyl, thiol or amino radical capable of being esterified or amidified on a phosphate function.

To demonstrate the effectiveness of this methodology, 5-hydroxy N-acetyltryptamine was chosen as a model. This compound is a derivative of serotonin having only a chemically reactive OH function; it is a metabolite of serotonin which is transformed in the brain by an O-methyl transferase into melatonin (CT15850). Melatonin is a hormone secreted by the pineal gland; its role is quite complex: it can be considered as a mediator involved in photoperiodicity, thermoregulation, reproduction and locomotor activity in a certain number of animals (L. Tamar in, CJ Baird and OFX Almeida, Science (1985) ), 227: 714-720; SM Reppert, DR Weaver, SA Rivkees and EG Stopa, Science (1988), 242.78-81 and VM Cassone, TINS (1990), 1_3: 457-467; DN Kranse and ML Dubocovitch, TINS (1990), 2 ^ 3: 464-470. In humans, melatonin has been used to resynchronize circadian rhythms and to combat the physiological and psychological effects of jet lag (jet lag); Recent studies seem to indicate in addition an immunomodulatory and anti-stress role (A. Conti and G. Maestroni, Neuroimmunomodulation in pharmacology, 2nd Course of the Federation of the European Pharmacological Societies, 5-7 February 1992, PARIS). When administered by intraperitoneal (IP) melatonin at high c oncentration (30 mg / kg) has no effect on the locomotion of the mouse (Ducobovitch et al., 1990, Eur. J. Pharmacol. 182: 313-325), while a low dose intracerebral injection (0.1 to 100 ng) directly into the nucleus accumbens decreases the locomotor activity (Gaffori and Van Ree, 1985, Neuropharmacology, 24: 237-244). These observations strongly suggest a very weak passage of melatonin across the blood-brain barrier after peripheral administration.

The present invention illustrates the capacity of glycosylphosphotriester derivatives to cross the blood-brain barrier by measuring the functional effect of the brain on locomotion in mice. This effect has been compared to that of melatonin alone after central or peripheral administration. Hydroxy-N-acetyltryptamine (CT15166) has been tested in the same way.

A group of glycosylated phosphotriester derivatives of the invention corresponds to formula II below:

in which :

- R ₂ has the same definition as in formula I above and / or

- R has the following meanings:

. R = H: the sugar is then a deoxyglucose and the general formula II is coded under the n ° CT15165,

. R = OH: the sugar is a mannose and the general formula II above is coded n ° CT16729,

. R = OH: the sugar is a glucose and the general formula II is coded n ° CT16731,

- M is N-acetyltryptamine. The three derivatives CT15165, CT16729 and CT16731 will be used in the experiments below as glycosylated phosphotriester derivatives of melatonin.

However, any other sweet derivative in which the sugar is a C ₅ or C ₆ D or L sugar also meets the characteristics of the invention. Among these, mention may be made of fructose, fucose, amino hexoses for hexoses, or arabinose, xylose, ribose, deoxyribose for pentoses.

Another compound of the invention can be represented by formula III.

in which R, R ₂ and M have the same meaning as in formula II.

The invention also relates to the process for the preparation of compounds of formula I. This process is a three-step process.

The first step is to phosphorylate a suitably protected sugar with a phosphite which is in situ coupled with N-acetyl hydroxy-5 tryptamine and oxidized to the corresponding phosphotriester.

The second step consists in removing the protective groups from the sugar and the phosphate to phosphόdiester, isolated in the form of tetrabutyl ammonium salt. The third step consists of a nucleophilic substitution of an alkyl halide with the phosphodiester salt to give the phosphotriester.

It is understood that any variant of this reaction scheme is also possible, for example phosphorylation of 5-hydroxy trypta ine and of a long chain aliphatic alcohol and final alkylation with a halogen-sugar derivative. Similarly, the chemistry described here with a derivative of trivalent phosphorus (chlorophosphora idite) can be carried out with other phosphoric derivatives of phosphotriester type (cf. oligonucleotide synthesis), phosphonate or phosphorothioate.

A particular example of the synthesis of the compounds CT15165, CT16729 and CT16731 will be given in Example I below.

All the compounds obtained as well as the intermediate compounds have spectral characteristics (nuclear magnetic resonance, ultraviolet, SM) in agreement with the formulas proposed. The purity was tested by HPLC chromatography (Table I).

The following examples are intended to show the particularly advantageous effects of the compounds of the invention; a series of hexadecylphosphates glycosyls of hydroxy-5N-acetyltryptamine (CT15165, CT16729 and CT16731) were synthesized. Their effect on locomotor 1'activité mice was measured and compared with the effects of melatonin alone ^(CT15850) and the precursor of the hydroxy-5 melatonin N-acetyltryptamine (CT15166).

- EXAMPLE I: Synthesis of CT15165, CT16729 and CT16731 derivatives:

To this end, we start with a sugar (deoxyglucose, mannose or glucose) which is coupled to a hydroxy-5-N-acetyl tryptamine by a phosphate. Chain hexadecyl is then condensed on this phosphodiester structure via a nucleophilic substitution reaction. ,. ,

The chemistry of trivalent phosphorus (phosphoramidite) has been used to allow much faster reaction times than that of pentavalent phosphorus.

The different steps are as follows and illustrated in Figure 1.

In this diagram, a) if R = H:

the starting compound (1) is deoxy-2-D-glucose,

- the compound (6) is CT15164 (6a),

- the compound (7) is CT15165 (7a), b) if R = OH, located "inside" the cycle:

- compound (1) is mannose,

- the compound (6) is CT16728 (6b),

- the compound (7) is CT16729 (7b), c) if R = OH, located "outside" the cycle:

- compound (1) is glucose,

- the compound (6) is CT16730 (6c),

- the compound (7) is CT16731 (7c).

First step: Protection of sugar, and phosphorylation with a phosphyte coupled with N-acetyl hydroxy-5 tryptamine and oxidation to phosphotriester: a) Synthesis of Tetra-O-benzoyl-1,3,4,6 deoxy-2-D- glucose (2a):

10 g (61 mmol) of deoxy-2-D-glucose co-evaporated with pyridine are redissolved in 100 ml of anhydrous pyridine, 58 ml (70.24 g; 500 mmol) of benzoyl chloride are added dropwise now the temperature at 0 ° C. The medium is left overnight at 4 ° C., then methanol is added at 0 ° C. until the precipitate formed is completely dissolved. The solvent is evaporated to dryness and the residue is taken up in dichloromethane, washed twice with saturated NaHCO _{3 solution} , then with water. The organic phase is dried over Na ₂ SO _[ and evaporated to dryness to give a residue (32.46 g, 92%) which is washed several times with petroleum ether. Rf = 0.27 (AcOEt-hexane, 1-2). Mp 131 ° C. b) Synthesis of Tri-O-benzoyl-3,4,6-benzyloxy-2 ethyl deoxy-2-D-glucopyranoside (3a):

A release of HCl gas is made to arrive in a suspension of 32.26 g of 2a in 12 ml (84.5 mmol) of benzyloxyethanol at room temperature for 2 h 30 min until complete dissolution. The mixture is diluted in dichloromethane, washed twice with saturated NaHCO _{3 solution} , once with water. The residue is chromatographed on a Merck 9385 silica column eluted with dichloromethane to give 78% 3a (oil). Rf = 0.07 (CH ₂ C1 ₂ ).

NMR (CDC1 ₃ , ppm) = ¹ H: 5.05 (H-,), 2.0 and 2.60 (H ₂ , H ₂ '), 5.80 (H ₃ ), 5.6 (H ₄ ), 3.9 (H ₅ ), 4.45 (H ₆ , H ₆ '), 3.6-3.75 (CH ₂ ), 4.55 (CH ₂ <>). c) Synthesis of Tri-O-benzoyl-3,4,6-hydroxy-2 ethyl deoxy-2-D glucopyranoside (4a):

18.5 g (30 mmol) of 3a dissolved in 250 ml of ethyl acetate are catalytically hydrogenated in the presence of Pd-C (10%) for 5 h at 30 psi.

After filtration through Celite, the solvent is evaporated to dryness and the residue chromatographed on a column of silica eluted with dichloromethane to give 19.4 g (86%) of 4a. Rf = 0.49 (MeOH-CH ₂ Cl ₂ , 5-95). M = 42-46 ° C. MS (IC, NH ₃ ) = 538 (M + 18). NMR (CDCl ₃ , ppm): ¹ H: 5.1 (H _T ), 2.05 and 2.55 (H ₂ , H ₂ '), 5.75 (H ₃ ), 5.6 (H ₄ ) , 4.4 (H ₆ , H ₆ '), 3.6 (CH ₂ CH ₂ OH), 3.8 (CH ₂ CH ₂ OH). d) Synthesis of 5-cyanoethyl (tri-O-benzoyl 3,4,6 deoxy-2-D-glucopyranosidyl) ethyl-2 (N-acetγl tryptarainyl) -5 phosphate (5a):

To a solution of 2.29 g (4.4 mmol) of 4a in 20 ml of anhydrous acetonitrile and 3.3 ml (2.44 g, 19 mmol) of diisopropyl ethylamine, 975 μl (1.034 g, 4.4 mmol) of β-cyanoethyl N, N diisopropyl chlorophosphora idite are added under a nitrogen atmosphere. The mixture is left for 15 min at room temperature and then a solution of 1.284 g (18.3 mmol) of tetrazole and 800 g (3.67 mmol) of hydrox-5-N-acetyltrypta ine dissolved in 15 ml of acetonitrile are added. anhydrous. After 1 h at room temperature, the mixture is oxidized with 30 ml of a 0.1 M iodine solution (H ₂ O-pyridine-THF, 1-10-40) diluted with dichloromethane and washed twice with a solution of NaHS0 ₃ (5%) and once in water. After evaporation to dryness, the residue is precipitated with petroleum ether and chromatographed on a column of Merck 7734 silica eluted with dichloromethane enriched in methanol (0-5%). A second chromatography on a Sephadex LH-20 column eluted with a THF-MeOH mixture (95-5) gives 1.78 g 57% of 5a. Rf 0.40 (MeOH-CH ₂ Cl ₂ , 75-92.5).

. Second step: Synthesis of CT15164, CT16728 and

CT16730 a) Synthesis of (deoxy-2-D-glucopyranosidyl) ethyl-2

(N-acetyl trypta inyl) -5 phosphate (6a) (CT15164):

900 mg (1.06 mmol) of 5a are treated with 50 ml of sodium methylate (1%) for 10 min at room temperature. After neutralization with a Do ex AG-50 X8 resin, the solvent is filtered and evaporated to dryness to give a residue which is purified on a column of Lichroprep RP-18 silica (Merck 9303) eluted with water gradually enriched in methanol ( 0-100%) to give 457 mg (89%) of 6a as the ammonium salt. Mp 139 ° C. Rf = 0.62 (isopropanol-NH ₄ OH-H ₂ 0, 7-1-2).

Analysis C ₂₀ H ₃₂ O ₁₀ N ₃ P, H ₂ 0 (C, H, N): Wedge. 45.89, 6.50, 8.03; ïr. 45.66, 6.53, 7.92. HPLC: R _t 11.07 min.

MS (FAB +): 527.3 (M + Na +), 506.2 (M + H) +. UV (MeOH): 222 (27000), 277 (6000). NMR (table). b) Synthesis of (D-Mannopyranosidyl) ethyl-2 (N-acetyl tryptaminyl) -5 phosphate (6b) (CT16728):

The same sequence of reactions is carried out from 900 mg (1.406 mmol) 2-hydroxy-ethyl-D-α-mannopyranoside 4b (Rf = 0.62, CH ₂ Cl ₂ -MeOH, 95-5) (Y. Hénin and al., J. Med. Chem. 1191, 3-4: 1830-1837; C. Gouyette et al. Tetrahedron Lett. 1989, 30: 6019-6022) to give 40% 5b. (Rf = 0.57, CH ₂ Cl ₂ -MeOH, 90-10) which is deprotected by methylate in 6b (95%). This phosphate is exchanged in N (Bu) _{4+ form} for the rest of the reactions. (Rf = 0.78, isopropranol-NH ₄ 0H-H ₂ 0, 7-1-2) MS (FAB +): 987.4 (M + N (Bu) ₄ ) ⁺ . UV (MeOH): 223 (31000), 276 (6800).

Analysis 0 ₃ ^ ₄ 0 ^ ₃ ?, 1/2 H ₂ 0. (C, H, N, P): Wedge. 57.29, 8.62, 5.57, 4.11; Tr. 57.27, 8.67, 5.35, 4.28. HPLC: R _t 11.07 min. NMR (Table I). c) Synthesis of (D-glucopyranosidyl) ethyl-2 (N-acetyl tryptaminyl) -5 phosphate (6c) (CT16730):

Penta-0-benzoyl-1,2,3,4,6 glucose is transformed into bromo-1 tetra-0-benzoyl-2,3,4,6 glucose according to R.K. Ness et al. (JACS, 1950, 7 ^: 2200-2205).

Is allowed to rotate at room temperature for 1 h 30 min a suspension of 4.6 g of bro o-glucose (7 mmol) with 2.14 g of Hg (CN) _2, 3.05 g of Hg (Br) _2, 670 mg of 4Â molecular sieve and 1 ml of benzyloxyethanol in 40 ml of a toluene-nitromethane mixture (1/1). After filtration, the solution is diluted with dichloromethane, washed 3 times with H ₂ 0, NaHC0 ₃ , H ₂ 0. After chromatography on a silk column Merck 9385 with dichloromethane, 60% of 3c is obtained. (Rf = 0.35, AcEt-Hexane, 1-2) (oil) which is hydrogenated on Pd / C to give 85% of 4c. (Rf = 0.76, CH ₂ Cl ₂ -Me0H, 95-5).

The series of similar reactions as for the previous series gives 5c (9%) Rf = 0.42 (CHC1 ₂ - MeOH, 10-90) which is deprotected in 6c (78%). (Rf = 0.40, isopropranol-NH ₄ OH-H ₂ 0, 7-1-2)

Analysis C ₃₆ H ₆₄ 0 ₁₁ N ₃ P, 1/2 H ₂ 0 (C, H, N): Wedge. 57.29, 8.62, 5.57; Tr. 57.20, 8.78, 5.60. HPLC: R _t : 7.05 min. MS (FAB +): 987.5 (M + N (Bu) 4) +. NMR (table).

Third step: nucleophilic substitution of an alkyl halogen; synthesis of CT15165, CT16729, and CT16731. a) Hexadecyl (deoxy-2-D-glucopyranosidyl) ethyl-2 (N-acetyl tryptaminyl) -5 phosphate (7a) (CT15165):

The phosphodiester 6a is passed through a column of Dowex AG-50 X8, N (Butyl) + to obtain the tetrabutylammonium salt. 260 mg (0.537 mmol) of the phosphodiester in 17 ml of anhydrous CH ₃ CN and 1.7 ml (1.9 g, 5.4 mmol) of 16-iodo hexadecane are heated at 80 ° C overnight. After evaporating the solvent, the residue is precipitated with petroleum ether and chromatographed on a Merck 9385 silica column eluted with dichloromethane gradually enriched in methanol to give 86% of 7a. (Rf = 0.44, CH ₂ Cl ₂ -Me0H, 80-20).

Analysis C ₃₆ H ₆₁ O ₁₀ N ₂ P, 1/2 H ₂ 0; (C, H, N, F): Wedge. 59.92, 8.60, 3.88, 4.30; Tr. 60.06, 8.66, 3.89, 3.97. HPLC: R _t 15.35 min. MS (FAB +): 735.7 (M + Na +). NMR (table). b) Synthesis of hexadecyl (α-D-mannopyranosidyl) ethyl-2 (N-acetyl tryptaminyl) -5 phosphate (7b) (CT16729):

The same protocol as for 7a on 250 mg of 6b (0.336 mmol) gives 74% of 7b. (Rf = 0.32, CH ₂ C1 ₂ - MeOH, 80-20).

Analysis C _j ^^ O ^^ P, H ₂ 0 (C, H, N, P). Wedge: 57.91, 8.44, 3.75, 4.16; Tr. 58.51, 8.45, 3.82, 4.59. UV (MeOH): 224 (34000), 286 (5700).

HPLC: R _t 13.19 min. MS (FAB +): 751.6 (M + Na +), 728 (M +). NMR (Table 1). c) Synthesis of hexadecyl (D-glucopyranosidyl-ethyl-2 (N-acetyl tryptaminyl) -5 phosphate (7c) (CT16731):

The reaction with iodohexadecane on 6c gives 69% of 7c. (Rf = 0.35, CH ₂ C1 ₂ - MeOH, 80-20).

Analysis C ^ H ^ N ^ E, H ₂ 0 (C, H, N, PJ. Calc: 57.91, 8.44, 3.75, 4.16; Tr .: 58.45, 8.5, 3.83, 4.12. HPLC: R _t : 13.11 min. MS (FAB +): 751.8 (M + Na +), 729.7 (MH +), 728 (M +). NMR (Table 1). HPLC chromatographic conditions:

TEAA buffer: triethylam onium acetate 10 ^{* 2} M pH 6.5.

Acetonitrile Merck (ACN).

Duration of the gradient: 20 minutes.

Nucleosil 5-C18 column (Macherey-Nagel) (15 cm x 0.46 cm).

Flow 1 ml / min.

- EXAMPLE II: Measurement of the pharmacological activity of the five products CT15165, CT16729, CT16731, CT15850 and CT15166 on the locomotor activity of mice:

The animals used are Swiss mice weighing 15 to 18 g (Iffa Credo). The mice used are kept in a pet store in a light-dark cycle from 12 pm to 12 pm at 22 ° C., and having food and drink ad libitum.

The intraperitoneal (IP) injections are carried out in a volume of 0.5 ml per 20 g of animal, corresponding to 1.10 or 30 mg / kg for melatonin or the equivalent doses for the glycosylated phosphotriester derivatives. The animals then remain 15 minutes at rest in their cage before starting the measurement of their motor activity in an activometer, according to a process described below.

Intracerebroventricular (ICV) injections are performed by freehand in the third ventricle of the right cerebral hemisphere in a maximum volume of 5 μl. The injection time is 30 seconds. The doses used vary from 5 to 50 ng / kg of melatonin or their equivalence for glycosylated phosphotriester derivatives.

The animals are observed and left to rest for 15 minutes in their cage before starting to measure their motor activity.

The locomotor activity of animals is measured in a six-compartment activometer in which the activity of animals is measured by their passage through two light beams arranged at 90 ° and directed towards two photoelectric cells. The only horizontal locomotion is recorded for 15, 30, 45 and 60 minutes.

The results are expressed as a percentage of locomotion measured relative to control mice having received NaCl in an equivalent volume and measured 15, 30, 45 and 60 min after the injection, which results are obtained at variable doses of compound injected.

EXAMPLE III Comparison of the Effects of Melatonin, of N-acetyltryptamine and of the Glycosyl Phosphotriester Derivative of N-Acetyltryptamine of the Following Formulas:

. phosphotriester derivatives: CT15165 a) R = H deoxyglucose CT16729 b) R = OH annose CT16731 c) R = OH glucose

CT15850 melatonin:

CT15166 5-hydroxy-acetyltryptamine

3.1. Intracerebroventricular injection (ICV):

The injection is carried out under the conditions described above.

Two doses were used 5 or 50 ng / kg of animal weight, in melatonin equivalent.

Figure 2 shows the results obtained for each of these doses with CT15850, CT15165 and CT15166.

Each of the three compounds has an effect on locomotion.

The ICV administration of the products studied made it possible to observe at the dose of 5 ng / kg an inhibiting effect on locomotion progressing over time for the CT15850, as well as the CT15165. The melatonin precursor did not show significant effects under the same conditions.

At 50 ng / kg, melatonin itself showed a marked inhibitory effect after 60 minutes, its precursor CT15166 also induced an effect inhibitor progressing over time; it was the same with the ester derivative.

3.2. Administration on the outskirts:

Intraperitoneal administration was carried out under the conditions described above.

Four doses were used: 0.01, 1, 10 and 30 mg / kg.

The results of the locomotion tests obtained are shown in Figure 3a.

Melatonin itself and its precursor, N-acetyltryptamine, had no significant effect in increasing or inhibiting animal locomotion during the four time periods analyzed after injection. A slight reduction in activity was observed after 45 and 60 minutes at 10 mg / kg of N-acetyltrypta ine, but this effect was not significant and it was not confirmed at the higher dose of 30 mg / kg.

On the contrary, the ester derivative CT15165 reduced the activity at the three doses studied, moderately to 1 mg / kg and 10 mg / kg and very markedly, to 30 mg / kg.

Figure 3b indicates that 1 • intraperitoneal administration of the two derivatives CT16729 and CT16731 are as effective, injected at a dose of 30 mg of melatonin equivalent per kg of mouse as CT15165.

The results obtained in this experimental series showed that by the intraperitoneal route, the only ester derivative was capable of inhibiting locomotor activity in mice at the 3 doses studied (1, 10, 30 mg / kg), while in the same conditions, no significant effect was observed for melatonin itself or its precursor N-acetyltryptamine. These results strongly suggest that the only ester derivative was able to cross the barrier. hematoencephalic to induce locomotor effects. This hypothesis is supported by the fact that the three substances studied reduce the locomotor activity when administered directly at the central level.

This last observation also indicates that the locomotor effects observed are indeed of central origin since melatonin and its precursor had no effect after their administration at the peripheral level.

The large difference that can be observed between the active doses of the ester derivative when administered ICV (5 to 50 ng / kg) and PI (1 to 30 mg / kg) is probably due to the fact that this lipophilic product is sequestered extremely important in a large number of peripheral compartments; despite this inactivation by its sequestration or by a rapid metabolism which denatures it in the form of native melatonin, a small but functionally very significant percentage is capable of reaching brain targets. Under the same conditions, melatonin itself does not show any activity at these same doses.

These results therefore indicate that a molecule active at the central level but whose physicochemical properties prevent the passage of the BBB can receive a chemical group giving it the property of passing this obstacle.

On the basis of these results, it has not been investigated whether the ester could act as such or as melatonin generated by the degradation of this molecule. However, there is every reason to believe that the molecule is metabolized since the brain probably contains in large numbers the enzymes necessary for this operation. This example is not limiting of the importance of the invention in terms of new means for targeting an effector molecule at the level of specific CNS receptors.

- EXAMPLE IV: Comparison of the effects of an analgesic peptide and its glycosylphosphoesters derivatives on the manifestations of pain:

This example relates to the measurement of the intracerebral transport capacity of the derivatives of the invention for a peptide exhibiting an analgesic effect.

The tests undertaken consisted in seeking in mice, for example the EOPS mouse, the properties of natural enkephalin (TyrGlyGlyPheLeu) and of various derivatives after administration directly into the brain (ICV injection) or after peripheral administration (IP injection).

The measurement of the analgesic activity of these substances is measured by the tail-flick test. Briefly, the animals are placed in an individual cage where they cannot move. Their tail is then exposed for a short time (maximum 7 seconds) under a light beam whose intensity is controlled (1 = 8OmA) and corresponds to a significant contribution of heat. The time taken by the mouse to withdraw the tail outside the beam by a short movement (tail-flick) is measured (two or three seconds in a normal animal).

The animal which receives an analgesic reacts in a longer time: the maximum exposure time ("eut off") is fixed at 7 seconds, considered to be sufficient to indicate a state of analgesia.

In this study, the effect of IP or ICV injections of [D-Ala ² -D-Leu ⁵ ] enkephalin amide YAGFL-NH ₂ and the derivative 2-deoxy-glycoside phosphodiester were compared. The peptide YAGFL-NH ₂ is close to the natural enkephalin TyrGlyGlyPheLeu (YGGFL).

Method for obtaining diesters and triesters derivatives of YAGFL-NH ₂ :

It is done by the different stages described below.

First stage :

H-Tyr-D-Ala-Gly-Phe-D-Leu-NH ₂ > Z-Tyr-D-Ala-Gly-

Phe-D-Leu-NH ₂ (1)

95 mg of (D-Ala-D-Leu ⁵ ) enkephalin amide (0.31015 mmol) are dissolved in 5.5 ml of water and 43 μl of triethylamine (1 eq). The reaction is followed by HPLC: CH ₃ CN / CH ₃ COO ^" NH ₄ ⁺ 0.05 M; λ = 230 nm: column C _18. 1 μl of the reaction mixture is injected into a gradient I5 ²⁰ⁿ > 100% t _r = 9, 55 mins).

78 mg (0.31015 mmol) of N- (benzyloxycarbonyoxy) succinimide are added in 5.5 ml of methanol. Agitation for 1 h 30 min. Injection 1 μl of the reaction mixture t _r = 1.88 min (hydroxysuccinimide released) t _r = 12.4 min Z (D-Ala ² -D-Leu ⁵ ) enkephalin amide. The reaction mixture is evaporated to dryness and purified on a Sephadex LH 20 column (THF 80 / MeOH 20). 198 mg of (1) (84%) are obtained. ccm: 10% MeOH / CH ₂ Cl ₂ ; Rf: 0.25, H- NMR, (DMSO-d ₆ ): <5 (ppm), CH ₃ L: 0.5 and 0.7 (doublet), CH ₃ A: 1 (doublet), CH ₇ L : 1.1, CH _{2 /?} L: 1.2, CH _{2; 5} Y: 2.5 and 2.7, CH2 F: 2.8, CH ₂ G: 3.5, CH _ff L: 3.9, CH.-A: 4, 1, CH _β Y: 4.2, CH _α F: 4.3, CH ₂ Benzyl: 4.8, Aromatics Y: 6.4 and 6.9, Aromatics F: 7 to 7.2, Aromatics Benzyl, NH Y: 7.3, NH F-NH G-NH L: 7.9 to 8.1, NH A-OH Y: 9

Second step : Z-Tyr-vD-Ala-Gly-Phe-D-Leu-NH ₂

AGF -NH.

OBz (2)

272 mg of (2-hydroxyethyl) -deoxy-2-D-perbenzoylated glucopyrannoside (0.523 mmol) are dissolved in 2.3 ml of anhydrous acetonitrile and 387 μl of diisopropylethylamine. 120 μl (0.541 mmol) of 2 cyanoethyl N, N diisopropylchlorophosphoramidite are added using a syringe and stirred at room temperature 30 min (TLC: MeOH 05 / CH ₂ Cl ₂ 95 Rf: 0.8).

After addition of 180 mg of tetrazole (2.535 mmol), 198 mg of (1) (0.2816 mmol) and 3.4 ml of anhydrous acetonitrile, the medium is returned to nitrogen and allowed to stir for 1 h 30 min (TLC : MeOH 05 / CH ₂ Cl ₂ 95 Rf: 0.2).

Oxidation is carried out with a 0.1 M iodine solution in pyridine / water (addition until iodine color persists). After extraction with CH C1 ₂ , the crude product is chromatographed on a MERCK 7734 silica column (CH ₂ Cl ₂ / MeOH).

(TLC: MeOH 10 / CH ₂ C1 ₂ 90 Rf: 0.3). 227 mg of (2) (60%) are obtained.

The characteristics are as follows: SM (FAB ⁺ ): 1337, NMR H. (DMS0-d ₆ ): δ (ppm), CH ₃ L: 0.7 and 0.8, CH ₃ A: 1.15, CH _y L>: 1.2, CH / 3L: 1.4, H ' ₂ H " ₂ : 2.1 and 2.45, CH ₂₃ Y: 2.7 and 2.9, CH ₂₃ F: 2, 9, CH ₂ G: 3.7, [C ₂ ] chain: 3.7; 3.9 and 4.4, CH _α L: 4.1, CH _α A-CH _α Y: 4.3, CH _α F: 4.5, H ' ₃ : 5.2, H'-,: 5.6, Cyanoethyl: 4.4 and 5.5, CH ₂ Benzyl: 4.9, Aromatics Y: 6.4 and 6.9, Aromatics F-Aromatics Benzyl-Aromatics Benzoyl: 6.9 to 8, NH Y: 7.3, NH F: 7 , 6, NH G: 8.1, NH L: 8.15, NH A: 8.25.

Third step :

227 mg (0.1698 mmol) of (2) are dissolved in 9.2 ml of methanol and 1.65 ml of a 1% sodium methylate solution (0.3054 mmol). The mixture is stirred for 2 h (TLC: Isopropanol 70 / ammonia 10 / water 20 Rf: 0.7) and then neutralized with an H ⁺ AG50 -X8 resin. Purification on Sephadex G10, then HPLC (CH ₃ CN / CH ₃ COO ^" NH ₄ ⁺ 0.05 M, C ₁₈ : λ = 230 nm; gradient 0 20 E2> 60% t _r = 13.5 min) gives 112 mg (67%) of (3) Fourth step:

70 mg of (3) (0.07078 mmol) are dissolved in 8 ml of methanol and hydrogenated on palladium-C for 1 h (TLC: Isopropanol 70 / ammonia 10 / water 20 Rf: 0.6).

After filtration and evaporation of the solvent,

56 mg of product (92%) of (4), the characteristics of which are as follows:

MS (FAB ⁺ ): 838, NMR E ^ (DMSO-d ₆ ): δ (ppm), CH ₃ L: 0.7 and 0.8, CH ₃ A: 1.15, CH ₇ L: 1.25 , CH _2/3 L: 1,4, H ' ₂ H " ₂ :

1.4 and 1.9, CH _{/ 3} Y-CH _2/3 F 2.8 to 3, H ' ₄ : 3, H' ₃ : 3.6, CH ₂ G: 3.6, CH _α L 4 , 1, CH _j A: 4.3, CH _a Y: 4, CH _α F: 4.5, H ',: 4.8, OH _c , ₄ 4.9, Aromatics Y-Aromatics F: 6.9 at 7.4, NH F 8.1, NH G: 8.35, NH L: 8.2, NH A: 8.75, NH ₂ Y: 7.9 at 8.3 Fifth step:

112 mg of (3) (0.11132 mmol) are exchanged on Dowex resin (tetrabutylammonium). After lyophilization, 137 mg of salt is obtained which is dissolved in 3.5 ml anhydrous CH ₃ CN. 355 μl of iodohexadecane (1.13 mmol) are added and the mixture is stirred at 80 ° C. overnight. (TLC: 15 MeOH / 85 CH C1 ₂ Rf: 0.5). After evaporation and trituration with petroleum ether, the product is purified on Sephadex LH 20 (THF 80 / MeOH 20) and 62 mg of product (45%) is obtained, the characteristics of which are as follows:

MS (FAB ⁺ ): 1197, NMR H-, (DMSO-d ₆ ): δ (ppm), CH ₃ L: 0.5 and 0.6 (doublet), CH ₃ [C ₁₆ ]: 0.65 ( triplet), CH ₃ A: 0.95 (doublet), CH ₂ [C ₁₆ ]: 1 to 1.15; 1.4; 3.9, CH ₂₃ L: 1.2, H ' ₂ H " ₂ : 1.3 and 1.7, CH _2/3 Y: 2.55 to 2.75, CH _{2 / J} F: 3.3 and

3.45, CH ₂ G: 3.5, CH _a L: 3.9, CH _α Y and A: 4.1, CH _α F:

4.3, H ¹ ,: 4.65, OH: 4.6, OH: 4.75, CH ₂ benzyl: 4.8,

Aromatic Y-Aromatic F-Aromatic Benzyl: 6.7 to

7.2, NH Y: 7.35, NH F: 7.9, NH G: 8, NH L: 7.95, NH

A: 8.1.

Sixth step:

62 mg of (5) (0.0518 mmol) are dissolved in 4 ml of methanol and hydrogenated on palladium-C for 1 hour. (CCM: 15 MeOH / 85 CH ₂ C1 ₂ Rf: 0.15). Purification on Sephadex LH 20 (THF 80 / MeOH 20) gives 38 mg of (6) (70%), the characteristics of which are as follows:

MS (FAB ⁺ ): 1064, NMR H, (DMSO-d ₆ ): δ (ppm), CH ₃ L: 0.6 and 0.7 _r CH ₃ [C ₁₆ ]: 0.75, CH ₃ A: 1.05, CH ₂ [C ₁₆ ]: 1 to 1.15; 1.5; 4, CH ₇ L: 1.15, CH _{2 /?} L: 1.3, H ' ₂ H " ₂ : 1.3 and 1.7, H' ₃ : 3.5, H ¹ ,: 3.5, OH: 4.80, H ' ₄ : 2.95 CH _{2 /?} Y: 3.45 to 3.65, CH _{2 / J} F: 3.5, CH ₂ G: 3.55, CH _a L: 4.1, CH _α A: 4.1, CH _a Y: 4.1, CH _a F: 4.35, Aromatic Y-Aromatic F: 6.6 to 7.2, NH F: 8, NH G: 8.15, NH L: 8.02, NH A: 8.1. 24

Treatment of animals: a) with the peptide YAGFL-NH ₂ :

The mice receive an ICV injection of 3 μg of the peptide, of 50 mg / kg by IP injection. b) with the derivative (4):

The following table indicates that the analgesic activity of the phosphodiester derivative is observed, whether the injection is carried out in ICV, with a maximum effect at 25 μg per mouse, or in PI at a dose of 30 mg / kg.

ANALGESIC EFFECT OF ^"enkephalin OR DIESTER

c) with derivative 6:

The first results obtained with the phosphotriester derivative (6) confirm the analgesic effect of an icv injection.

These results strongly suggest that the analgesic effect observed after administration of the phosphoesters derivatives of enkephalin is due to the passage through the BBB of the esterification complex and the release of the peptide in the brain from these "prodrugs".

Claims

1. Compounds having pharmacological activity at the level of the CNS, having the capacity to cross the BBB after administration of said compound by the peripheral route, and having the general formula: O

R., 0 - P - AM

OR ₂ in which: A = 0, N, or S

- R-, represents

1) either: [C ₅ or C sugar or deoxy sugar, substituted or not] _n in series D or L: n = 1 to 6, and the junction between the sugar and the phosphorus being on one of the hydroxyls of the vertices 2, 3, 4 or 6 or

2) either: (Ose) x - 0 - [(CH ₂ ) _y - 0] _z - in which Ose represents a C ₅ or C ₆ , D or L sugar, the substitution of the chain 0 - [(CH ₂ ) _y - 0] _z being done on the top 1 of the sugar,

- x is an integer from 1 to 12;

- y is an integer from 1 to 6;

- z is an integer from 1 to 4;

- R ₂ represents an alkyl group of 6 to 30 carbon atoms, saturated or not, which can carry ramifications or rings,

M represents a molecule capable of acting at the central level, mention may in particular be made of psychotropics, analgesics, cerebral regulators of the immune or cardiovascular systems; it can also represent a precursor of said molecules capable of being transformed in the CNS into the active molecule, said molecule being able to be, for example, a peptide or polypeptide or a protein glycosylated or not, a cyclic or heterocyclic molecule chosen for example from the different families of psychotropics, this list not being limiting and can include any active substance having a hydroxyl, thiol or amino radical capable of being esterified or amidified on a function phosphate.

2. Compound according to claim 1, characterized in that the dare motif is chosen from mannose, deoxyglucose, glucose, galactose, fructose and fucose.

3. Compound according to Claims 1 and 2, characterized in that x is equal to 1, y is equal to 2 and z is equal to 1.

4. Compound according to one of claims 1 to 3, characterized in that it is glycosylated phosphotriesters of formula II:

in which :

- R ₂ has the same definition as in formula I above and can for example be a cholestryl, phytyl and / or

- R has the following meanings:

. R = OH: the sugar is then a mannose and the general formula II above is coded n ° CT16729, . R = OH: the sugar is then a glucose and the general formula II is coded n ° CT16731, - M is N-acetyltryptamine.

5. Compound according to one of claims 1 to 3, characterized in that it is glycosylated phosphotriesters of formula III:

in which R, R ₂ and M have the same s gn f cat on as in formula II.

6. Compound according to one of claims 1 to 4, characterized in that the group R ₂ represents an alkyl chain containing from 2 to 30 carbon atoms, where appropriate, substituted by a group NH ₂ , -NHR ¹ or - NR'R "in which R ¹ and R" represent an alkyl group of 1 to 4 carbon atoms.

7. A method of preparing a compound according to claims 1 to 6 characterized by the following steps: a) phosphorylation of a sugar suitably protected with a phosphite which is in situ coupled with N-acetyl hydroxy-5 tryptamine and oxidized to corresponding phosphotriester; b) removal of the protective groups of the sugar and of the phosphate in phosphodiester, isolated in the form of tetrabutyl ammonium salt; c) nucleophilic substitution of an alkyl halide with the salt of the phosphodiester to give the phosphqtriester; it being understood that the different variants allowing the derivatives of formula I, II or III to be obtained using the conventional chemistry of trivalent phosphorus, also form part of the invention.

8. Pharmaceutical composition for the prevention or treatment of disorders having their origin in the central nervous system, comprising as active ingredient a compound according to one of claims 1 to 5 in association with a pharmaceutically acceptable carrier.