ITMI981620A1 - PROPHARMACIES OF COMPOUNDS ACTIVE ON THE CENTRAL NERVOUS SYSTEM - Google Patents

Description

Description of the industrial invention entitled: "PROFARMACI OF COMPOUNDS ACTIVE ON THE CENTRAL NERVOUS SYSTEM"

The present invention relates to the preparation of drugs of compounds active on the Central Nervous System, to a process for their preparation and to the pharmaceutical compositions which contain them.

Notoriously, one of the major difficulties encountered in the design of drugs intended for the Central Nervous System (CNS) is represented by the difficulty of obtaining compounds which, in addition to possessing a high intrinsic activity towards a given receptor system, are able to overcome the blood-brain barrier is sufficient to ensure appropriate drug concentrations in the CNS or brain and thus ensure appropriate pharmacological activity.

To date, several compounds are known which are endowed with considerable in vitro activity, and therefore potentially useful as drugs for the CNS, but which do not exhibit any pharmacological activity when administered in vivo.

Several strategies are used to improve the ability of a drug to penetrate the blood-brain barrier and among these the design of prodrugs is certainly one of the most promising.

The design of a prodrug useful for this purpose involves the conjugation of the active drug with a pro-moiety capable of giving the conjugate chemical-physical characteristics such that it allows greater penetration of the blood-brain barrier. Furthermore, for the success of the prodrug it is appropriate that the link between the drug and the promoiety is such as to allow, once the blood brain barrier has been overcome, the reconversion in the brain of the prodrug into a starting drug and pro-moiety, obviously with the latter devoid of toxicity. cerebral. At the present stage of the research, one of the main objectives, which most of the scientific works concerning the design of profanacis intended for the CNS, is represented by the increase in lipophilicity which seems to constitute a critical parameter for overcoming the blood-brain barrier ( 1) Unfortunately, the increase in lipophilicity does not always allow to obtain an improvement in the delivery of the drug to the brain and has rarely allowed the creation of prodrugs of therapeutic interest.

It has now been found that it is possible to obtain prodrugs of CNS active substances, which as such have little application in therapy due to their inability to cross the blood brain barrier, through a conjugation system that allows the active substance to be transported inside of the CNS by means of a facilitated mechanism based on specific transporters called GLUT-1, and to be subsequently released in order to exert its specific pharmacological action.

This conjugation system consists in the reversible covalent chemical bond between the active substance and a hydroxyl group of a monosaccharide. By reversible covalent bond we mean a bond that can be split in order to restore the active molecule to its initial form prior to conjugation, by the intervention of monovalent enzymes present in the brain.

Preferably, this bond will be of the ester type. The active substances that can be used for the preparation of the conjugates (prodrugs) of the invention are chosen from:

1) competitive antagonists of NMDA, AMPA, Kainic receptors, such as 7-chloroquinurenic acid; D-2-amino-5-phosphonopentanoic acid and others;

2) agonists and antagonists of metabotropic receptors, such as 1-aminoindane-1, 5 (RS) dicarboxylic acid and others;

3) non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, naproxen and others;

4) classic and recently introduced antiepileptic drugs, such as valproic acid and others.

The monosaccharides that participate in the formation of prodrugs are chosen from those that are actively transported across the blood-brain barrier.

Preferably the monosaccharides are glucose and galactose.

A preferred embodiment of the invention is represented by the conjugation of 7-chloro-kynurenic acid with a monosaccharide, preferably with glucose.

7-Chloro-kynurenic acid is a powerful antagonist of one of the excitatory amino acid receptors and exactly of the N-methyl-D-aspartate (NMDA) receptor (Kemp J.A. and Leeson P.D., The glycine site of the NMDA receptor-five years on Trends Pharmacol. Sci. 14: (1993) 20-25), whose activation is involved in the etiology of several brain dysfunctions such as epilepsy, cerebral ischemias, Parkinson's, chronic degenerative disorders, anxiety, movement disorders etc.

Furthermore, it is thought that excessive activation of NMDA receptors may mediate calcium-dependent neurotoxicity associated with cerebral ischemia, trauma, epilepsy and other autonomic disorders.

Unfortunately, the "therapeutic use of 7-chloró-kynurenic acid for the treatment of acute and chronic autonomic disorders is severely limited by the inability of this compound to overcome the blood-brain barrier and therefore to reach adequate concentrations in the brain tissue (Kemp J.A. and Leeson P.D., The glycine site of the NMDA receptor-five years on Trends Pharmacol. Sci. 14: (1993) 20-25).

The conjugation of 7-chloro-kynurenic acid, according to the invention, allows these drawbacks to be overcome.

The synthesis scheme of two types of 7-chloro-kynurenic acid / glucose conjugates and the in vitro and in vivo study of the pharmacological activity of the conjugation products will be described below.

The latter were obtained by conjugating 7-chloroquinurenic acid (4-hydroxy-7-chloro-2-carboxy-quinolinic) with D-glucose in position 6 (compound 1) and in position 3 (compound 2).

For the preparation of the esters of compounds 1 and 2, 0-benzyl-7-chloro-kynurenic acid is initially synthesized according to scheme 1:

DIAGRAM 1

a) Benzyl chloride-NaH-DMF; b) Na0H-H2O-Me0H.

Subsequently, the O-benzi1-7-chynurenic acid is reacted with 1,2,3,4-O-tetramethylcarbonate-D-glucose and, by subsequent basic and acid deprotection, the 6-0-D ester is obtained -glucosyl-chloroquinurenate, according to scheme 2:

DIAGRAM 2

Alternatively, the O-benzi * 1-7-chloroquinurenic acid is reacted with diacetone-D-glucose and by subsequent acid deprotection the 3-0-D-glucosii-7-chloro-kynurenate ester is obtained, according to the scheme 3:

DIAGRAM 3

The pharmacological study was conducted on mice in which compounds 1 and 2 and subsequently NMDA were injected, evaluating the degree of induced seizures compared to the control.

In control mice, NMDA injection (200 mg / kg, i.p.) induced generalized tonic-clonic seizures, causing death in 13 of 21 animals (see Table 1).

Only two animals experienced only partial seizures, while only one animal showed no particular response after administration of the NMDA. The extent of NMDA-induced seizures was not attenuated in mice treated with 200 mg / kg of 7-chloro-kynurenic acid (see Table 1). On the contrary, pretreatment with both compounds 1 and 2 (both at a dose of 200 mg / kg, i.p.) significantly protected the tested animals from NMDA-induced seizures. Of the two prodrugs, compound 1 was more effective than compound 2.

Of the 19 animals pretreated with compound 1, only four experienced generalized seizures and only one died. Five of these animals showed no response to NMDA. In animals pretreated with compound 1 and responding to NMDA, the latency to both seizures (partial and generalized) was twice that observed in animals pretreated with the vehicle alone ( Tab.l).

All animals (n = 7) pretreated with compound 2 were sensitive to NMDA, but only one developed generalized seizures and none died. In these animals, however, the latency to partial seizures was comparable to that observed in the controls (Table 1).

To verify whether prodrugs 1 and 2 are hydrolyzed at the "target" site, protection from NMDA-induced toxicity in mixed cultures of mouse cortical cells was evaluated. This culture system constitutes a good model capable of reproducing the cell medium encountered by prodrugs after crossing the blood brain barrier. Compounds 1 and 2 were applied to cultures by incubating them in the absence of fetal bovine serum to avoid any interference with enzymes or enzyme inhibitors present in the serum. Initially only 7-chloro-kynurenic acid (173 ± 18 μΜ, n = 3) was incubated and it was observed that its concentration did not change in the extracellular medium three hours after incubation indicating that this drug alone is not captured by the neurons or astrocytes and is not metabolized on the cell surface. Conversely, the application of compound 1 was followed by a rapid decline in its concentration in the extracellular medium, with a half-life of 10 min. (see Fig.1A). This effect (rapid disappearance of compound 1) was accompanied by a parallel increase in the concentration of 7-chloro-kynurenic acid in the extracellular medium. The disappearance of compound 2 in the extracellular medium was much slower with an apparent half-life of about 40 min. In cultures treated with compound 2, the extracellular concentration of 7-chloro-kynurenic acid shows a modest increase, which a plateau already after 10 min (Fig 1B).

To confirm that 7-chloro-kynurenic acid was generated by the hydrolysis of prodrugs in the extracellular medium, NMDA toxicity was evaluated in cultures in the presence and absence of compound 1. Toxicity was induced by 10 minutes of application of the NMDA to cultures: this exposure generally results in the death of 75-80% of the neuron population. Application of both 7-chloro-kynurenic acid and compound 1 was found to comparably protect cells from NMDA-induced toxicity. This evidence indicates that after the application of compound 1 in cell culture, free and pharmacologically active 7-chloro-kynurenic acid is released during the 10 minutes of application (see Fig. 2).

Although the two compounds examined exhibit different hydrolysis kinetics; overall, the results obtained in the in vitro and in vivo pharmacological tests allow us to conclude that:

the conjugation of 7-chloro-kynurenic acid with glucose allows to obtain prodrugs able to cross the blood-brain membrane,

prodrugs, after passing the blood-brain barrier, hydrolyze in the brain,

the drugs released by the prodrugs are able to exert their typical pharmacological action at the target site.

The conjugates of 7-chloro-kynurenic acid can be used for the preparation of medicaments useful in the treatment of acute and chronic cerebral ischemic degeneration, status epilepticus, degeneration from status epilepticus, hypoglycemic coma, head trauma and / or hemorrhage, subarachnoid, Alzheimer's disease, multi-infarct dementia, amyotrophic lateral sclerosis, parkinsonism, Huntington's chorea, dementia associated with AIDS, other pathologies in which anomalies in the functioning of the excitatory neurotransmission of NMDA receptors are involved.

In addition to the prodrugs described above, the invention provides pharmaceutical compositions comprising the prodrugs themselves along with pharmaceutically acceptable carriers and excipients. These compositions can be for example in the form of tablets, capsules, solutions, emulsions or suspensions (administered orally or parenterally), syrups, pharmaceutical forms with controlled release, transdermal therapeutic systems. the patient's age, sex, weight and general health conditions.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A - Enzymatic hydrolysis of compound 1 in mixed cultures of mouse cortical cells.

Figure 1B - Enzymatic hydrolysis of compound 2 in mixed cultures of mouse cortical cells

Figure 2 - NDMA-induced cell toxicity after pretreatment with compound 1 or 7-chloro-kynurenic acid

The following examples illustrate the invention in greater detail.

Example 1

Synthesis of O-benzyl-7-Cl-kynurenic acid (compound 3).

Sodium hydride (NaH) (23.27 mmol) and subsequently benzyl chloride was added dropwise to a well-stirred solution of 7-chloroquinurenic acid methyl ester (3.23 g, 23.27 mmol) in dimethylformamide (DMF). (3.23 g, 28.01 mmol) at 0 "C and the mixture was stirred for 3 hours at room temperature. Subsequently the solvent (DMF) was removed under vacuum and a mixture was added, stirring for 2 h. solution of NaOH (4 g, 100 mmol) in water-methanol (H2O / Me0H) (1: 1, 100 ml). The solution was concentrated under vacuum and the residue taken up with 100 ml of H2O and washed with 20 ml of Ethyl acetate (EtOAc). The aqueous phase was then neutralized with HC16N and the product, taken up with EtOAc (3x50 ml), was then washed with 20 ml of H2O 20 ml of H2O and ice and finally dried. The solvent was removed under vacuum and the residue purified by flash chromatography eluting with a mixture of chloroform-methanol (CHCl3 / MeOH (9: 1) to obtain 9.6 g of compound 3 as a yellow solid with a yield of 64.5% (mp 135-136'C).

1H NMR (CDCl3) d 8.20 (m, 2H, 6-C H 8-CH); 7.70 (m, 1H, 5-CH); 7.40 (m, 6H, 3-CH and phenyl group); 5.40 (s, 2H, CH2Ph).

Example 2

Synthesis of 1,2,3,4-0-tetramethylcarbonate-6-0-D-glucosyl-0-benzyl-7-chloro-kynurenate (compound 4).

To a solution of 0-benzyl kynurenic acid (compound 3), (1 g, 3.18 mmol) in CH2Cl2-DMF (9: 1, 30 ml), 1-1 <1> -carbonyldiimidazole (CDI) was added (0.516 g, 3.18 mmol) and the mixture was stirred for 1 h at room temperature (Scheme 2). A solution of 1,2,3,4-tetramethylcarbonate-D-glucose (1.45 g, 3.50 mmol) in dichloromethanodimethylformamide (CH2CL2-DMF) (1: 5, 30ml) was then added drop by drop. continuing to stir for 24 hours at room temperature. Subsequently, most of the DMF solvent was removed under vacuum and the reaction mixture obtained was taken up with 30 ml of EtOAc. The solvent was then removed under vacuum and the crude purified by flash chromatography eluting with hexane / EtOAc (4: 6) to obtain 1.46 g of compound 4 as a gray solid with a yield of 64.5% (m.p.

140-141 ° C).

Example 3

Synthesis of 6-0-D-glucosyl-7-chloro-kynurenate (compound 1).

K2CO3 (200 mg) was added to a solution of compound 4 (1.46 g, 2.05 mmoles) in 20 ml of MeOH and the mixture was then stirred for 12 h at room temperature. The evaporation of the solvent then led to the formation of a residue which was dissolved in 25 ml of EtOAc and then washed with 20 ml of H2O. The organic phase was dehydrated on anhydrous Na2SO4, filtered and concentrated under vacuum. The residue obtained was dissolved in 30 ml of CH2Cl2 (30ml) and 3.05 ml of CF3COOH and 1.89 ml of thioanisole were added to this solution and it was stirred for 72 hours at room temperature. The solution obtained was then concentrated under vacuum and the residue taken up with 100 ml of H2O and subsequently washed with 20 ml of EtOAc. The aqueous phase was neutralized with 10% NH4OH and subsequently concentrated under vacuum. The residue was taken up with 100 ml of H2O and washed with 20 ml of EtOAc. The aqueous phase was concentrated under vacuum and the subsequent crystallization of the residue from H2O led to the formation of 0.615 g of compound 1 as a yellowish solid with a yield of 893⁄4 (m.p. 170-171 ° C).

Example 4

Synthesis of diacetone-3-O-D-glycosyl-0-fcenzyl-7-chloro kynurenate (compound 5).

1.1 < 1> -carbons Idimidazole (516 mg, 3.18 mmol) and the mixture was stirred for 1 hour at room temperature (Scheme 3). Subsequently, a diacetone-D-glucose solution (0.911 g, 3.50 mmol) in CH2Cl2-DMF (9: 1, 30 ml) by continuing to stir for 24 hours at room temperature, then most of the DMF solvent was vacuumed and the reaction mixture was taken up with 30 ml of EtOAc. The solution obtained was washed with 20 ml of H2O, 20 ml of water and ice and finally dried with anhydrous Na2SO4. After filtering and removing the solvent under vacuum, the product was purified by flash chromatography eluting with a 1: 1 hexane / EtOAc mixture thus obtaining 1.44 g of compound 5 in the form of a gray solid with a yield of 81% (m.p. 119-120 ° C).

Example 5

Synthesis of 3-0-D-glucosyl-7-cyoro-kynurenate (compound 2).

Initially a solution containing 1.44 g (2.59 mmoles) of compound 5, 4.46 ml of CF3COOH and 2.77 ml of thioanisole in 20 ml of CH2Cl 2 was stirred at room temperature for 72 hours. The solution was then concentrated under vacuum and the residue taken up with 100 ml of H2O and washed with 20 ml of EtOAc. The aqueous phase was neutralized with a 10% NH4OH solution and then concentrated under vacuum. The residue was taken up with a further 100 ml of H2O and washed with 20 ml of EtOAc. The aqueous phase was concentrated and from the crude, by crystallization from water, 0.85 g of compound 2 were obtained with yields of 85% as a yellowish solid (mp 175-176 “C).

Example 6

Evaluation of protection of HMDA-induced seizures in tapi Swiss Webster mice (30-35 g) were used for this experiment. In each experimental session, a maximum of 12 mice were injected intraperitoneally and at a dose of 200 mg / kg (in saline solution-pH 7.4) one of the following drugs: 7-chloroquinurenic acid, compound 1, compound 2 The control consisted of the saline solution used as vehicle. 15 minutes later, all mice were administered NMDA (200 mg / kg, i.p., dissolved in saline solution pH 7.4). Then all the animals were examined for 60 minutes by an observer not aware of the treatment performed.

In control animals, seizures developed through a sequence of paroxysmal "strecthing", hypermotility. "circling", tonic-clonic convulsions and occasionally death. The following scale was used to assess the intensity of the seizures (SS = seizures severity): 0 = no response; 1 = excessive "grooming" + paroxysmal "stretching"; 2 = moderate hypermotility and "circling"; 4 = clony of the forelimbs hypertonia of the tail; 5 = generalized tonic-clinical convulsions; 6 = "status epilepticus" and death. The evaluation of epileptic seizures (SS) results from the numerical sum of the registered effects. In addition, the latency in minutes for partial and generalized seizures was also determined. Example 7

Evaluation of NMDA-induced toxicity protection in mixed cultures of mouse cortical cells.

Mixed cortical cell cultures, containing both neurons and astrocytes, were prepared from mouse fetuses at day 14-16 of gestation, as described in the literature (Rose K., Goldberg M.P. and Choi D.D. Cytotoxicity in murine neocortical celi cultures. In: Methods in Toxicology, Vol.l, 1993, pp. 46-60; Tyson C.A. and Frazier J.M. Eds., Academic Press, San Diego).

Briefly, dissociated cortical cells were placed in 15 mm culture plates (Falcon Primaria, Lincon Park, NY) on a state of confluent astrocytes, using a "MEM-Eagle's salts" medium (glutamine-free) spiked with horse serum. heat inactivated, 5% bovine fetal serum, glutamine (2 mM), glucose (21 mM) and NaHCC> 3 (25 mM) .After 3-5 days of in vitro growth (DIV), non-cell division neuronal was obtained by exposure for 1-3 days to cytosine arbinoside (10 μΜ), and the cultures were placed in a maintenance broth identical to that of the plates, but without bovine serum. Subsequently, a partial change of the maintenance broth was performed twice in a week. Cell cultures at 13-14 DIV were used for the experiments.

For the evaluation of NMDA-induced toxicity, we exposed the cultures to NMDA in the presence or absence of compound 1 for 10 minutes at room temperature in a buffered solution (HEPES) containing in mM: 120 NaCl, 5.4 KCl, 0 , 8 MgCL21.8 CaCl2 / 20 HEPES, 15 glucose. Subsequently, the cultures were washed abundantly and incubated in a MEM-Eagle's medium (added with 25 mM of NaHC03 and 21 mM of glucose) at 37 ° C. The neuronal damage was estimated by examining the cultures with a phase contrast microscope. after 24 insult (NMDA addition), when the cell death process was largely completed.Neurone damage was quantitatively determined by trypan blue staining.Stained neurons were randomly counted from three fields per well.

Example 8

Evaluation of the enzymatic hydrolysis of compounds 1 and 2 in the extracellular medium of cortical cell cultures.

To examine whether prodrugs 1 and 2 were hydrolyzed to 7-Chloro-kynurenic acid on the surface of neurons or astrocytes, these compounds were added to the previously described cell cultures.

Cultures were incubated for up to 180 minutes with HEPES buffered solution, and aliquots of the buffer were withdrawn at different time intervals. The analysis of 7-chloro-kynurenic acid and compounds 1 and 2 was conducted by HPLC using a fluorimetric detector. Cell cultures were collected at different times after application of prodrugs 1 and 2 and, after filtration, they were injected through a 20 μl loop. The chromatographic system used a Beckman 126 module (Beckman Instrumént, Ine-, Fulleton, CA) for programming the mobile phase, a 30 ° C C-18 reverse phase column (Ultrasphere ODS 3 um Spherical. 80 A pore, 4 , 6 mm x 75 mm, Beckman Instrumént, Ine., Fulleton CA), and a Shimadzu RF-551 spectrofluorimetric detector. The excitation and emission wavelengths were 344 and 398 nm, respectively. The mobile phase consisted of 50 mM sodium phosphate (pH 7.2) containing 10% methanol (phase A) and 50 mM sodium phosphate (pH 7.2) containing 70% methanol (phase B) , at a flow of 1 ml / min .. The elution gradient started with 98% of phase A and 2% of phase B, increasing to 98% of phase B in 15 minutes and then returning to the starting conditions in 5 min , before injecting the next sample.

From the peak area the concentration of 7-chloro-kynurenic acid and compounds 1 and 2 in the supernatant was determined by the use of external standards.

Claims (12)

CLAIMS 1. Prodrug of CNS active compounds characterized in that the active substance is linked by reversible covalent bond to a hydroxyl group of a monosaccharide which can be actively transported across the blood-brain barrier. 2. Prodrug according to claim 1, characterized in that said reversible covalent bond is an ester bond. 3. Prodrug according to claims 1-2, characterized in that the CNS-active compounds are selected from: a) conpetitive antagonists of NMDA, AMPA, Kainico receptors; b) agnists and antagonists of metabotropic receptors; c) non-steroidal anti-inflammatory compounds (NSAIDs); d) antiepileptic compounds. 4. Prodrug according to claim 3, characterized in that said compounds are selected from 7-chloro-kynurenic acid, D-2-amino-5-phosphonopentanoic acid, 1-aminoindane-1,5 (RS) dicarboxylic acid, indomethacin, naproxen, valproic acid. 5. Pre-drug according to claim 1, characterized in that the monosaccharide is selected from the monosaccharides actively transported across the blood-brain barrier by means of transport systems. 6. Pre-drug according to claim 5, characterized in that said monosaccharide is selected from glucose and galactose. 7. Prodrug according to any one of the preceding claims, comprising 7-chloro-kynurenic acid and a monosaccharide. 8. Prodrug according to claim 7, characterized in that said monosaccharide is glucose. 9. Pre-drug according to claim 8, characterized in that the 7-chloro-kynurenic acid is bound with an ester bond to the hydroxyl in position 6 of the D-glucose. 10. Pre-drug according to claim 8, characterized in that the 7-chloro-kynurenic acid is ester-bound to the hydroxyl in position 3 of the D-glucose. 11. Pharmaceutical compositions comprising a prodrug of claims 1-10 together with pharmaceutically acceptable carrier-excipients. 12. Use of the prodrugs of claims 7-10 for the preparation of medicaments useful in the treatment of acute and chronic cerebral ischemic degenerations, status epilepticus, status epilepticus degenerations, such as hypoglycemia, head trauma and / or subarachnoid hemorrhage, Alzheimer's disease, multi-infarct dementia, amyotrophic lateral sclerosis, parkinsonism, Huntington's chorea, dementia associated with AIDS, other pathologies in which anomalies in the functioning of the excitatory neurotransmission of NMDA receptors are involved.