ITMI20010450A1 - PROPHARMACIES DERIVED FROM ASCORBIC ACID AT THE PASSAGE OF THE BLOOD-ENCEPHALIC BARRIER - Google Patents

Description

"Ascorbic acid derivative prodrugs suitable for the passage of the blood-brain barrier"

Field of the invention

The present invention relates to prodrugs consisting of ascorbic acid from a substance active on the central nervous system, linked by covalent bond.

State of the art

It is known that one of the important problems encountered in the treatment of diseases of the central nervous system (CNS) is the ability of drugs to cross the blood-brain barrier. In fact, several compounds are known which are endowed with remarkable activity in vitro, and therefore potentially useful as drugs for the CNS which, however, do not exhibit any pharmacological activity when administered in vivo due to their inability to cross the blood-brain barrier. .

Several strategies have been proposed to improve the ability of a drug to pass the blood-brain barrier and among these the design of prodrugs is certainly one of the most promising.

The design of prodrugs useful for this purpose mainly involves the conjugation of an active drug with a substance capable of giving the conjugate characteristics such as to allow greater penetration of the blood-brain barrier. Furthermore, for the success of the prodrug, the link between the drug and said substance should be such as to allow, once the blood brain barrier has been overcome, the reconversion of the prodrug into the starting drug. To date, most of the research efforts have been directed towards prodrugs that show an increase in lipophilicity that seems to be a critical parameter for overcoming the blood brain barrier (Hansch C, Bjorkroth JP, Leo A Hydrophobicity and center nervous System agents: on the principle of minimal hydrophobicity in drug design. J Pharm Sci 1987 Sep; 76 (9): 663-87)

However, this strategy does not allow specific tissue accumulation and the achievement of the optimal effective concentration and also causes side effects on other organs and tissues.

According to another approach, starting from active substances on the CNS which as such have little application in therapy due to their inability to cross the blood-brain barrier, prodrugs with amino acids or glucose have been prepared that allow the active substance to be transported to the of the CNS by means of a facilitated mechanism based on specific transporters, and to be subsequently released in order to exert its specific pharmacological action. (Bonina F.P., Arenare, L., Palagiano, F., Saija, A., Nava, F., Trombetta, D., De Capraris, P. Synthesis, stability and pharmacological evaluation of nipecotic acid prodrugs. J Pharm ,. Sci ., 88, 561-567, 1999).

The pharmacological application of these transporters is limited by some problems. In the specific case of GLUT, a limit is represented by the blood glucose levels, which can influence the transport efficiency as the transporter is completely saturated in the case of hyperglycemia (Battaglia G., La Russa M., Bruno V., Arenare L ., Ippolito R., Copani A., Bonina F., Nicoletti F., Systematically administered D-glucose conjugates of 7-chlorokynurenic acid are centrally available and exert anticonvulsivant activity in rodents. Brain research 860, 149-156, 2000). Another limitation is represented by the fact that this transporter has not proved effective for some drugs such as in the case of nipecotic acid. (Bonina Fp., Arenare L., Palagiano F., Saija A., Nava F., Trombetta D, de Caprariis P., Synthesis, stability, and pharmacological evaluation of nipecotic acid prodrugs. J. Pharm Sci 19999 May; 88 ( 5): 561-7).

Summary

The Applicant has surprisingly found that compounds potentially active for the treatment of CNS pathologies, unable to cross the blood-brain barrier as such, take on the ability to cross the blood-brain barrier when they are transformed into prodrugs by conjugation with covalent bond. with ascorbic acid, directly or through a linker, in positions 5 and / or 6. Said covalent bond is split by enzymes present in the brain so as to restore the active compound to its initial form. The prodrugs according to the present invention have the following general formula (I)

where W is a formula linker

m is zero or 1,

n is zero or a number from 1 to 10,

and when m is zero, R is a hydrogen atom or an active substance having a carboxylic group which ester-bonds to the OH group of ascorbic acid in position 5 and / or 6 and when m is 1, R is an atom hydrogen or an active substance having a hydroxyl, thiol or amino group which binds to the carboxy group of W with ester, thioester or amide bond, in position 5 and / or 6, with the proviso that R cannot be simultaneously an atom of hydrogen in both positions 5 and 6.

The active substances to be used for the preparation of the prodrugs according to the present invention are selected from the group comprising antioxidants, non-steroidal anti-inflammatory drugs, adenosine agonists, competitive antagonists of NMDA, AMPA and Kainic receptors, agonists and antagonists of metabotropic receptors, inhibitors of the uptake of GABA, antiviral, anticancer and antiepileptic drugs.

The present invention also includes a method for the preparation of said prodrugs and the pharmaceutical compositions containing them.

Detailed description of the invention

The characteristics and advantages of the prodrugs derived from ascorbic acid suitable for the passage of the blood-brain barrier according to the present invention, will be better illustrated in the course of the following detailed description.

The prodrug having formula (I) as defined in the summary, where m is zero, is prepared according to the following synthetic scheme.

where R represents an acyl group of an active substance, R3 and R4 represent protective groups selected from benzyl, benzoyl, dimethylterbutylsilyl, trimethylsilyl or acetyl and X represents a hydroxyl or alkoxy group or a halogen.

The protecting groups, R3 and R4, are able to easily liberate the hydroxyl groups. Preferably benzyl is used as the protecting group, which allows easy purification and therefore higher overall yields. More specifically, the ascorbic acid or a derivative thereof protected in position 2 and 3 or a salt thereof is reacted with (4) which is a biologically active substance containing a carboxy group, a salt or ester thereof or the corresponding acyl chloride to give the corresponding derivative of ascorbic acid or a salt thereof. This reaction is carried out in the presence of a condensing agent such as dicyclohexylcarbodiimide or highly concentrated sulfuric acid. For example, when sulfuric acid is used as a condensing agent, sulfuric acid and ascorbic acid or 2,3-dibenzylated ascorbic acid or 2-phosphate and an active substance having a carboxylic group or an ester thereof or salt are mixed and reacted. The reaction times vary from 1 to 120 hours, preferably between 4 and 50 hours, and the temperature is generally between 5 and 80 ° C and preferably between 20 and 35 ° C. The quantities of reactants are preferably equimolar and the reactants have a moisture content lower than 5% and preferably they are anhydrous. The solvents used for the reaction can be polar organic solvents, preferably dimethylformamide. If the condensing agent is sulfuric acid, this can also be used as a solvent. Alternatively, the halide of the corresponding carboxylic acid can be used. Purification can be carried out by traditional techniques such as extraction or column chromatography and subsequent crystallization. When using ascorbic acid, its extraction is difficult, therefore it may be advantageous, to increase yields, to use the derivative of 2,3-dibenzylated ascorbic acid which is easily extractable from the aqueous medium that retains the reaction impurities. However, this alternative requires the subsequent unblocking with hydrogen and catalysts or BCI3 and subsequent purification, after filtration on celite and evaporation of the solvent by crystallization.

In the case in which the use of a linker (spacer) is envisaged, i.e. in the case in which in the formula (I) m = 1, the reaction can be carried out as in the following synthetic scheme, similar to the previous one, which provides for the reaction of ascorbic acid or one of its protected derivatives, with a compound (7) suitable to give the spacer group (linker) and the subsequent condensation with the alcoholic, thiol or amino group of an active substance RX-i. The R3 and R4 groups are as defined above while RX1 represents a biologically active substance containing at least one alcoholic, thiol or amino residue.

Said compound (7) suitable for giving the linker is preferably succinic acid or tartaric acid.

The prodrugs derived from ascorbic acid of the present invention can be used in compositions for medical use in all those CNS pathologies (cerebral and myocardial ischemias, neoplasms, inflammation, bacterial and viral infections) where there are difficulties in specifically conveying the substances biologically active at the CNS. CNS active substances suitable for the preparation of prodrugs according to the present invention preferably belong to the following pharmaceutical groups.

a) Antioxidants. In recent years, the implication of the oxidative component in acute and chronic neurodegeneration, and in the damage associated with post reperfusion has been widely confirmed.

ischemic. Particularly useful is a compound developed and patented by us, Fe-FI-21 or its analogue Trolox, as they are water-soluble derivatives of alpha-tocopherol.

b) Non-steroidal anti-inflammatory drugs. No alternative approach to the treatment of neurodegenerative diseases is suggested by numerous experimental evidences that show an inverse correlation between the use of anti-inflammatory drugs and the incidence of the aforementioned diseases. Among the anti-inflammatories, diclofenac, meclophenomic acid, indomethacin, naproxen, etc. can be mentioned. C) Adenosine agonists. It is known that adenosine is able to regulate various functions at the CNS and cardiovascular level. The activation of the A1 adenosine receptors has shown a number of effects, including a depression of neuronal excitability. On the basis of these effects, the A1 adenosine receptors have been proposed as targets for the development of drugs against disorders caused by ischemic states in the brain. The N6 substituted derivatives of adenosine have been reported to be potent A1 agonists, and in particular N6-cyclopentyladenosine (CPA), which is currently considered a prototype of the A1 agonists.

d) Competitive receptor antagonists. NMDA, AMPA, Kainico, eg.

7-chloroquinurenic acid; D-2-amino-phosphonopentanoic acid etc .; e) Agonists and antagonists of metabotropic receptors, eg. 1-aminoindane-1,5 (RS) dicarboxylic acid etc.

f) GABA uptake inhibitors. An alternative approach to the treatment of pathologies such as eg. Parkinson's, Huntington's and epilepsy predict an enhancement of central GABAergic activity. Among the compounds potentially very effective in vitro, nipecotic acid was selected, which is one of the most potent in vitro inhibitors of neuronal and glial uptake of GABA.

g) Antivirals. Zidovudine (AZT) is one of the most effective drugs for the treatment of AIDS, but it has a high toxicity that limits its use.

Active substances particularly suitable for use for the preparation of prodrugs according to the present invention are selected from the group comprising 7-chloro-kynurenic acid, D-2-amino-5-phosphonopentanoic acid, 1-aminoindane-1,5 (RS) acid dicarboxylic acid, meclophenamic acid, indomethacin, naproxen, valproic acid, nipecotic acid, trolox, AZT, aciclovir and CPA (N6-cyclopentyl-adenosine).

The preferred prodrugs of the present invention are:

• the prodrug consisting of 7-chloro-kynurenic acid or nipecotic acid or trolox bound with an ester bond to ascorbic acid in position 6; • the prodrug consisting of 7-chloro-kynurenic acid or nipecotic acid or trolox linked with an ester bond to ascorbic acid in position 5; • the prodrug consisting of 7-chloro-kynurenic acid or nipecotic acid or trolox linked with ester bonds in positions 5 and 6 of the ascorbic acid.

A biological experiment was carried out with the prodrugs according to the present invention which demonstrated that they are able to easily cross the blood-brain barrier.

In particular, a biological experiment was performed to determine the effects of 6-O-nipecotyl-ascorbate in the prevention of seizures induced by pentylenetetrazole (PTZ).

Swiss albino mice with a body weight of 25-30 g were used for the experimentation. In each experimental session a maximum number of 15 animals were injected i.p. respectively with saline solution (control), nipecotic acid (0.75 mmol / kg), 6-O-nipecotyl-ascorbate (0.75 mmol / kg).

25 minutes after this treatment, all mice were injected subcutaneously with pentylenetetrazole (80 mg / kg) and were observed for the next 30 minutes by an observer who was unaware of the treatment. In order to evaluate the effects induced by treatment with pentylenetetrazole, the latency (sec) to the onset of generalized tonic seizures and mortality were measured.

Mortality was defined as the percentage of animals that died within 60 minutes of injection with pentylenetetrazole.

Statistical analysis of the results was performed by the ANOVA method followed by the Newmann-Keuls test for multiple comparisons.

The following results were obtained.

Mice treated with 6-O-nipecotyl-ascorbate and with nipecotic acid showed no apparent abnormality in general behavior except for a slight defecation which appeared 40 minutes after injection only in animals treated with 6-O-nipecotyl-ascorbate. The results relating to the latency (seconds) of the onset of pentylenetetrazole-induced tonic seizures (PTZ) are shown in Table 1 Table 1

As can be seen from the table, the injection of 6-O-nipecotyl-ascorbate produced a significant increase in the latency of the onset of tonic convulsions induced by pentylenetetrazole, thus demonstrating an effect on the CNS that involves the passage through the blood brain barrier.

The prodrugs according to the present invention can be prepared in conventional pharmaceutical forms such as tablets, capsules, liquid preparations or injections in admixture with pharmaceutically acceptable diluents or excipients. The dosage depends on the subject to be treated, the pathology in question and the route of administration and can vary between 0.05 and 100 mg / kg of body weight, preferably between 0.5 and 25 mg / kg of body weight per day. for oral administration and preferably between about 1 and 10 mg / kg of body weight for parenteral administration, preferably between 0.05 and 10 mg / kg of body weight when administered by injection.

The ascorbic acid derivatives described in the present invention can be used in pharmaceutical compositions as active components in quantities that can vary between 0.01 and 100%.

Examples of compositions for oral administration include tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and nebulization preparations. These compositions can be obtained by known methods using excipients such as lactose, starch, sucrose or magnesium stearate.

For parenteral administration, an injection composition, suppositor, poultice, ophthalmic solution or preparation for external application can be prepared. Suppositories include endorectal and vaginal suppositories. Preparations for external applications include ointments, agents for nasal administration and agents for oral administration.

For external applications, the compositions of the present invention can be prepared in a solid, semisolid or liquid solvent according to known methods. For example, in the case of a solid composition, the compounds of the present invention are added in the form of powder to an excipient (e.g. glycol, mannitol, starch, cellulose) or to a thickener (e.g. gum arabic, cellulose derivatives, acrylic polymers ).

In the case of an injection, the composition can be prepared as an oil or aqueous suspension. In the case of a semi-solid composition, an aqueous or oily gel or an ointment is preferred.

A pH correcting agent (e.g. carbonic acid, phosphoric acid, hydrochloric acid, sodium hydroxide) and an antiseptic (e.g. para-hydroxy benzoic esters, benzalkonium chloride) can be added. To obtain suppositories, the compositions of the present invention can be placed in a solid, semisolid or liquid suppositor of aqueous or oily nature according to known methods.

Example 1

Piperidin-, 3-carboxylic acid-3- [2- (3,4-bis-benzyloxy-5-oxo-2,5dihydrofuran2-yl) -2-hydroxy-ethyl] 1-tert-butyl ester

To a solution of nipecotic N-BOC acid (1 g, 4.36 mmol), DMAP (42 mg, 0.165 mmol) and 2,3 di-O-benzyl ascorbic acid (1.55 g, 4.36 mmol) in Anhydrous CH3CN (62 ml) at 0 ° C, a solution composed of N, N'-Dicyclohexylcarbodiimide (DCC) (525 mg, 2.85 mmol) in anhydrous CH3CN (9 ml) is slowly added.

The reaction mixture is stirred at room temperature for 3 hours, in an inert argon atmosphere. After evaporation of the reaction solvent under vacuum, the product was dissolved in Et20, washed with an aqueous solution of 1N HCl (20 ml), then Brine (20 ml), NaHC03 (20 ml) and dried with Na2SO4. The organic phase is evaporated to dryness, and the residual product purified by chromatography on silica gel EtOAc / hexane (2/8) to give 1.06g of the desired conjugation product, as yellow oil (Yield 43%)

1H NMR (CDCI3): δ 1.2 (m, 9H, Boc), 1, 3-1, 4 (m, 2H, C5-H), 1, 8-2.1 (m, 2H, C4-H ), 2.5 (m, 1H, C3-H), 2, 7-3, 6 (m, 2H, C6-H), 3.85-4.0 (m, 2H, C2-H) 4, 05-4.48 (m, 3H, C6'-H, C5'-H); 4.8 (d, 1H, J = 2 Hz, C4'-H); 5.2 (m, 4H, CH2-Ph); 7.25-7.4 (m, 10H, Ph).

Maldi MS: 590.7 Da (M Na) +; 606.7 (M + K) +

3-carboxylic acid piperidin-2- (3,4-bis-benzyloxy-5-oxo-2,5-dihydrofuran-2-yl) -2-hydroxy-ethyl ester

Trifluoroacetic acid (3.8 ml) is slowly added to a solution of the previously obtained conjugate (1.21 g, 2.1 mmol) in anhydrous CH2CI2, and the mixture is stirred at room temperature for 2 hours, in an inert argon atmosphere. . The reaction solvent is removed under vacuum, the residue is taken up with CH2CI2 (20 ml) and subsequently washed with a saturated NaHC03 solution (2 x 20 ml). The organic phase is dried with Na2SO4 and evaporated to dryness. The residual product is purified by silica gel chromatography with CH2CI2 / MeOH (9/1) to give 630 mg (63% of yield) of the desired product, as yellow oil.

1H NMR (CDCI3): δ 1.3-1.4 (m, 2H, C5-H), 1, 8-2.1 (m, 2H, C4-H), 2.5 (m, 1H, C3 -H), 2.7-3.6 (m, 2H, C6-H), 3.85-4.0 (m, 2H, C2-H) 4.05-4.48 (m, 3H, C6 '-H, C5'-H); 4.8 (d, 1H, J = 2 Hz, C4'-H), 5.2 (m, 4H, CH2-Ph); 7.25-7.4 (m, 10H, Ph).

Maldi MS: 469.0 Da (M + H) +; 490.7 (M + Na) +; 506.6 (M + K) +

Piperidin-3-carboxylic acid-2- (3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl) -2-hydroxy-ethyl ester

To a solution of the previously obtained product (1 g, 2.14 mmol) in MeOH (50 ml), 10% Pd / C (200 mg) is added, and the mixture is kept stirring for 5 hours, at room temperature, in an inert atmosphere of argon. The reaction mixture is filtered over celite, and the solvent is removed in vacuo. The residual product is purified by silica gel chromatography, with CH2CI2 / MeOH (8/2) to give 510 mg of the final deprotected product as yellow oil (yield 83%).

1 H NMR (DMSO-de): δ 1.3-1.4 (m, 2H, C5-H), 1.7-2.0 (m, 2H, C4-H), 2.4 (m, 1H, C3-H), 2.5-3.5 (m, 2H, C6-H), 3.65-3.8 (m, 2H, C2-H), 4.05-4.48 (m , 3H, C6'-H, C5'-H); 4.6 (d, 1H, J = 2 Hz, C4'-H).

Maldi MS: 288.4 Da (M + H) +.

Claims (13)

CLAIMS 1. Prodrugs derived from ascorbic acid suitable for the passage of the blood-brain barrier characterized by the general formula (I) where W is a formula linker m is zero or 1, n is zero or in number from 1 to 10, and when m is zero, R is a hydrogen atom or an active substance having an ester bonded carboxylic group to the OH group of ascorbic acid in position 5 and / or 6 and when m is 1, R is a hydrogen atom or an active substance having a hydroxyl, thiol or amino group bonded to the carboxylic group of W with an ester, thioester or amide bond, in position 5 and / or 6, with the proviso that R cannot be a hydrogen atom at the same time in both positions 5 and 6. 2. Prodrugs according to claim 1, characterized in that said active substance is selected from the group comprising non-steroidal anti-inflammatory drugs, adenosine agonists, competitive antagonists of NMDA, AMPA and Kainic receptors, agonists and antagonists of metabotropic receptors, inhibitors of GABA uptake , antiviral, antitumor and antiepileptic drugs. 3. Prodrugs according to claim 1, characterized in that said active substance is selected from the group comprising 7-chloro-kynurenic acid, D-2-amino-5-phosphonopentanoic acid, 1-aminoindane-1,5 (RS) dicarboxylic acid , meclophenamic acid, indomethacin, naproxen, valproic acid, nipecotic acid, trolox, AZT, aciclovir and N6-cyclopentyl-adenosine. 4. Prodrug according to claim 1, characterized by the fact that said active substance consists of 7-chloro-kynurenic acid or nipecotic acid or trolox bound with an ester bond to ascorbic acid in position 6. 5. Prodrug according to claim 1, characterized by the fact that said active substance consists of 7-chloro-kynurenic acid or nipecotic acid or trolox bonded with an ester bond to ascorbic acid in position 5. 6. Prodrug according to claim 1, characterized in that said active substance consists of 7-chloro-kynurenic acid or nipecotic acid or trolox linked with ester bonds in positions 5 and 6 of the ascorbic acid. 7. Use of prodrugs as defined in claim 1, for the preparation of pharmaceutical compositions suitable for the treatment of acute and chronic cerebral ischemic degenerations, status epilepticus, degenerations from status epilepticus, hypoglycemic coma, head trauma and / or subarachnoid haemorrhage, Alzheimer's, multi-infarct dementia, amyotrophic lateral sclerosis, parkinsonism, Huntington's chorea, dementia associated with AIDS, virosis, bacterial intentions, inflammatory-based pathologies and pathologies in which anomalies in the functioning of the excitatory neurotransmission of NMDA receptors are involved. 8. Pharmaceutical composition comprising at least one prodrug as defined in claim 1, in admixture with pharmaceutically acceptable diluents or excipients. 9. A process for the preparation of prodrugs having formula (I) in which m = O or their salts, as defined in claim 1, comprising the reaction of ascorbic acid or a derivative thereof with protecting groups in positions 2 and 3, with an active substance having a carboxyl group or with an ester, anhydride or chloride thereof in the presence of a condensing agent or catalyst. 10. A process for the preparation of prodrugs having formula (I) in which m = 1 or their salts, as defined in claim 1, comprising the reaction of ascorbic acid or a derivative thereof with protecting groups in positions 2 and 3, with a dicarboxylic acid suitable to give the linker W and subsequently the reaction with an active substance having a hydroxyl, thiol or amino group, said reactions being carried out in the presence of a condensing agent or a catalyst. 11. Process according to claim 10, characterized in that said dicarboxylic acid is selected from the group comprising succinic acid and tartaric acid. 12. Process according to claims 9 and 10, characterized in that said condensing agent is sulfuric acid. 13. Process according to claims 9 and 10, characterized in that said protecting groups are selected from the group comprising benzyl, benzoyl, dimethylterbutyl silyl, trimethyl silyl and acetyl.