WO2002008220A1 - 2-thenoic acid (thiophene) derivatives having glutamate receptor antagonistic activity - Google Patents

Abstract

Derivatives of 2-thenoic acid of formula (I) with antagonist activity on the glutamate receptors, are described.

Description

-THEONIC ACID (THIOPHENE) DERIVATIVES HAVING GLUTAMATE RECEPTOR ANTAGONISTIC ACTIVITY

The present invention relates to derivatives of 5 2-thenoic acid with antagonist activity on glutamate receptors. BACKGROUND OP THE INVENTION

Glutamate can activate both channel receptors and receptors associated .with G proteins. The first are

10 commonly called ionotropic receptors (iGlu) whereas the second are called metabotropic receptors (mGlu) . The mGlu receptors are selectively stimulated by 1-amino- cyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and their structure is similar to that of other members of

15 a complex family of receptors associated with G proteins that also comprises the receptors ^' for GABAb, for pheromones and a ■ sensor for Ca²⁺. Eight cDNAs have been identified (from mGluRl to mGluR8) which code for them and on the basis of their structure and their

20 association with the effectors are classified into three subgroups :

1st) Group; Comprises the mGlu receptors that activate the inositol cycle (mGlul and mGlu5) and are selectively stimulated . by DHPG. Other agonists

25 stimulate these receptors in the following order of potency: quisgualate » 1S,3R-ACPD > CCG1 > L-AP-4. A selective antagonist for these receptors has been described recently (see: Pellicciari et al., J. Med. Chem. 38: 3717-3719; 1995 and Moroni et al., J.

Pharmacol. Exp . Tere. 281: 721-729; 1997).

2nd) mGlu receptors that inhibit adenylate cyclase and are stimulated preferentially by DCG-IV and by CCG1 (mGlu2, mGlu3) . The order of potency of the agonists is as follows: L-CCG1 > 1S,3R-ACPD > QUIS »»> L-AP4. Good antagonists for this subgroup are also available now (see Pellicciari et al . , J. Med. Chem. 39: 2259- 2269; 1996 and Cozzi et al . , Eur. J. Neurosci. 9: 1350- 1355; 1997) .

3rd) mGlu receptors stimulated preferentially by L-AP4 (mGlu4, mGluβ, mGlu7, mGlu8) and also associated with inhibition of adenyl cyclase but in this order of potency: L-AP4 » 1S,3R-ACPD »» L-CCG1. Their . distribution in the CNS is now well characterized and it is known that several receptor subtypes can be expressed by the same neuron. The end effects of their activation depend on the types of receptors present and therefore, when they come into contact with the agonist, there may be both inhibitory effects and excitatory effects. For example, at the cerebellar level, stimulation of Glul leads to activation of calcium-dependent potassium channels (production of IP3 causes an increase in the intracellular concentration of calcium) and hence to hyperpolarization; it also leads to phosphorylation of the NR2C subunit of the NMDA receptor and to diminished functioning of this channel receptor; at the level of the hippocampus, activation of the mGlu 1 receptors increases neuronal excitability through inhibition of the potassium channels operated by the voltage and in other structures of the central nervous system, such as the spinal cord, activation of the mGlu receptors of the first group causes a notable amplification of the synaptic responses mediated both by the NMDA and AMPA receptors. Then there are mGlu receptors localized at presynaptic level that are able to regulate the release of transmitter with particularly interesting mechanisms. Thus, the stimulation of mGlu4 or mGlu7 can reduce the ingress of Ca²⁺ into the nerve terminals, directly inhibiting the voltage-dependent channels and thus reducing the synaptic release of transmitter. A similar result can be obtained by stimulating the mGlu2 receptors that may even be located at quite a distance from the active site of the synapse, but which, having a high affinity for glutamate, seem to be able to be activated, even tonically, by the amino acid present in the extracellular spaces. These receptors inhibit the formation of cAMP and in some way reduce the effects of depolarization on the release of transmitter. On the other hand, the stimulation of other subtypes of mGlu receptors (perhaps mGlul) amplifies the depolarization- transmitter release coupling.

Regulation of the functioning of the neuronal circuits is particularly interesting in this respect. In the hippocampus, stimulation of the mGlu4 and mGlu7 receptors reduces transmission at the level of the glutamatergic synapses, whereas stimulation of mGlu5 can increase the excitability of the circuit perhaps also because it amplifies the responses of ionotropic type. The result of the reduction in transmission and increase in excitability is that stimuli of low intensity are blocked, whereas potent stimuli, capable of surmounting the presynaptic inhibition, are amplified. In this way, the strategic localization of the mGlu receptors can lead to the formation of filtering systems that are able to increase the signal/noise ratio of the stimuli that converge on this neuronal circuit. Systems of this kind, in which other types of receptors are also involved, seem to operate at the level of various sensory signals and might operate in the regulation of pain afferents both at a spinal and a thalamic level. The pharmacology of the mGlu receptors seems to promise vast areas for therapeutic application. The fact that stimulation of the mGlu receptors can cause an increase in the sensitivity of the ionotropic receptors for the same transmitter makes these receptors an ideal target for the modulation of synaptic excitatory function.

Numerous molecules capable of modifying glutamatergic excitatory neurotransmission are available (Lubeluzol, Ketanin, Cerestat, Eliprodil, Aniraceta , LI-314582, ACEA 1021, GV-150526A) and have had a clinical use proposed for them. Unfortunately, clinical testing of many of the ligands of the ionotropic glutamate receptors proposed as anti- epileptic molecules or able to reduce ischaemic brain damage has shown that the ratio between the therapeutic results and the side-effects is not favourable. Numerous research groups are trying to identify molecules that have acceptable side effects and are active selectively on subtypes of the NMDA or AMPA receptors . Their identification might alter the prognosis of diseases for which modern medicine is still devoid of acceptable therapies (for example ictus/stroke, degenerative diseases of the CNS) . DESCRIPTION OF THE INVENTION

Compounds have now been found that are characterized by the presence of a thiophene carboxylate ring and exhibit selective antagonist activity with respect to mGlul and mGlu5 receptors

(Group 1) .

The compounds of the invention have the following formula (I)

in which:

Ri is hydrogen, methyl, cyclopropyl or a group of formula

in which X is sulphur or oxygen;

R₂ is hydrogen, methyl, methoxy, hydroxy, halogen, cyano; R₃ is carboxy.

The invention also relates to the salts of compounds (I) with pharmaceutically acceptable acids or bases, and physiologically equivalent derivatives such as esters or amides and the individual enantiomers of compounds (I) .

In a preferred class of compounds, Ri is hydrogen and R₂ is hydrogen or methyl.

In a further preferred class of compounds, Ri is a group of formula

in which X is sulphur or oxygen; R₂ is hydrogen or methyl, R₃ is carboxy. According to one aspect of the invention, X is oxygen.

The configuration of the asymmetric carbon atom is preferably R. The compounds of formula (I) in which Ri is hydrogen can be prepared by reaction of a compound of formula (II)

where R₂ is as defined above, R'₃ is a protected carboxy group, for example such as alkyl ester, with alpha-phenylglycinol, preferably with

(R) -alpha-phenylglycinol, in the presence of trimethylsilylcyanide, followed by hydrolysis of the

N-substituted alpha-amino-nitriles obtained, for example by means of lead tetra-acetate and removal of any protective groups on the carboxyl.

The reaction with alpha-phenylglycinol is stereoselective and is carried out in the presence of anhydrous polar solvents such as alcohols, ethers, esters, or ketones at temperatures between -10 and +10°C.

Hydrolysis with lead tetra-acetate is normally carried out in the presence of anhydrous solvents, for example alcohols, ketones, esters, halogenated hydrocarbons or their mixtures at a temperature of approx. 0°C. Final hydrolysis in acids gives the desired compounds.

The compounds of formula (I) in which Ri is methyl or cyclopropyl can be prepared by reaction of a compound of formula (III)

where R₂ and R'₃ are as defined above and Ri ' is methyl or cyclopropyl, by reaction with alkaline cyanides in the presence of ammonium carbonate in the conditions of the Buchere-Berg reaction, followed by hydrolysis with alkaline hydroxides.

The compounds of formula (II) and (III) are known or can be prepared by known methods.

For example, 2-formyl-thiophenes can be converted to the corresponding protected imidazolidine derivatives by reaction with N,N- dimethylethylenediamine and then submitted to reactions of carboxylation in position 5 (in the presence of strong bases and C0₂) , or halogenation followed by carboxylation .

Some synthesis schemes that can be used for preparing compounds of formula (I) are shown below, by way of examples. Scheme 1

Scheme 2

H₃C

ol

ATIDA

Scheme 3

Compounds in which X = 0 and/or R₂ is different from H can be prepared using procedures similar to Scheme 3.

The compounds of formula (I) have antagonist activity on the glutamate receptors and can be used both for scientific purposes (for the characterization of glutamate receptors) and for therapeutic purposes for the treatment of diseases in which there is excessive stimulation of the mGlu receptors. Among the clinical situations in which excessive stimulation of this kind has been demonstrated, mention may be made of: focal cerebral ischaemia (stroke) and global ischaemia (cardiac arrest ^' or fibrillation) , CNS traumas, cerebral and subarachnoid haemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, dementia in the course of AIDS, convulsive disorders, pain and hyperalgesic syndromes, muscle spasms and myoclonus, schizophrenia, psychiatric syndromes of anxiety and depression, drug dependence, vomiting. For the therapeutic uses envisaged, compounds (I) will be formulated in suitable pharmaceutical compositions, employing conventional techniques and excipients .

Compounds (I) can be administered by the oral, parenteral, rectal or transdermal route, at dosages that will depend on several factors (weight, sex, age of the patient, seriousness and type of pathology) and on the pharmacokinetic and toxicological characteristics of each individual compound.

A pharmaceutical composition of the invention, which can be prepared by mixing, conveniently at room temperature and at atmospheric pressure, is generally suitable for oral, parenteral, rectal or transdermal administration and, as such, can be in the form of tablets, capsules, liquid oral preparations, powders, granules, pastilles, powders that can be reconstituted, solutions or suspensions that are injectable or can be administered by infusion, or suppositories.

The tablets and the capsules for oral administration can be in the form of a unit dose, and can contain conventional excipients, such as acceptable binders, fillers, lubricants for tableting, disintegrating agents and wetting agents. The tablets can be coated according to methods that are well known in usual pharmaceutical practice.

The liquid oral preparations can be in the form of, for example, suspensions in water or oil, solutions, emulsions, syrups or elixirs, or in the form of a dry product for reconstituting with water or some other suitable vehicle before use. It will be possible for these liquid preparations to contain conventional additives such as suspending agents, emulsifiers, non- aqueous vehicles (which can include food-grade oils) , preservatives and, if desired, conventional flavourings and colouring matter. For parenteral administration, fluid forms of unit dose are prepared using a compound of the invention or one of its pharmaceutically acceptable salts and a sterile vehicle. The compound, depending on the vehicle and on the concentration employed, can be suspended or dissolved in a vehicle. For preparing the solutions, the compound can be dissolved for injection and submitted to sterile filtration prior to distribution in suitable vials or ampoules, and sealing. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents are dissolved in the vehicle. To increase stability, after distribution in vials the composition can be frozen and the water eliminated under vacuum. The parenteral suspensions are prepared substantially in the same way, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be effected by filtration. The compound can be sterilized by treatment with ethylene oxide prior to suspension in a sterile vehicle. Advantageously, the composition includes a surfactant or wetting agent to facilitate uniform distribution of the compound.

It will be possible for the composition to contain from 0.1% to 99% by weight, preferably from 10 to 60% by weight, of the active ingredient, depending on the method of administration.

Examples of formulations include capsules, tablets, vials, granules containing single doses from 10 to 500 mg of compounds (I) . These unit doses can be administered once or more per day, for example two or three times per day, and the therapy can be spread over many weeks or months. The following examples illustrate the invention in greater detail.

Example 1 a) 1,3-Dime hyl-2- (3-methyl-2- thienyl) imidazolidine (2) Add N, ' -dimethylethylenediamine (17.5 g, 198.5 mmol) to a solution of 3-methyl-2-thiophene- carboxaldehyde (1) (25 g, 198.1 mmol) in anhydrous benzene (280 ml) , with magnetic stirring, in an inert atmosphere. The mixture obtained is reacted under reflux for 12 hours, from time to time removing the water that is formed by the reaction, using Dean-Stark apparatus. The solvent is evaporated under vacuum and the residue is distilled under vacuum, obtaining derivative 2 (33.8 g, 172.2 mmol, yield 87%) as oil (end point 75 °C at 0.37 mmHg) . ^XH-NMR (CDC1₃) δ: 2.20 (9H, bs, NCH₃ and CH₃-Ar) , 2.49 (2H, m, CH₂) , 3.30 (2H, , CH₂) , 3.74 (1 H, s, CH) , 6.68 (1 H, d, J=5.48, H Ar) , 7.16 (1 H, d, J=5.48, H Ar) . b) 5-Formyl-4-methyl-thiophene-2-carboxylic acid (3) A solution of 1, 3-dimethyl-2- (3-methyl-

2-thienyl) imidazolidine (2) (11.30 g, 57.7 mmol) in anhydrous THF (610 ml), while stirring by magnetic stirring in an inert atmosphere, is cooled to -78 °C and treated with N,N, ' , ' -tetramethyl-ethylenediamine

(TMEDA) (9.52 ml, 63.05 mmol) and nBuLi (42 ml, 1.5 M solution in hexane) . The mixture is reacted at -78°C for 2 hours. The reaction mixture is then poured into a flask containing a pasty mixture of diethylether

(200 ml) and solid C0₂ (sufficient for partial solidification of the mixture) . The mixture thus obtained is left to return to room temperature over night, with stirring. The mixture is evaporated to dryness under vacuum and treated, while stirring at room temperature, with an aqueous solution of H₂S0 at 10% (250 ml) . The mixture is extracted with ethyl acetate (2 x 200 ml) and the organic phase is extracted with a saturated solution of NaHC0₃ (200 ml) . The aqueous phase is acidified with HCl (3N) and extracted with ethyl acetate (4 x 100 ml) . The organic phases are combined, dried over anhydrous Na₂S0₄, and evaporated under vacuum, obtaining acid 3 (8.3 g, 48.7 mmol, yield 84%) as a grey solid (end point 177-180°C) . ^XH-NMR (CDC1₃) δ: 2.53 (3H, s, CH₃) , 7.54 (1 H, s, Ar) , 10.02 (1 H, s, CHO) . c) 5-Formyl-4-methyl-thiophene-2-carboxylic acid methyl ester (4)

Gaseous HCl is bubbled several times through a solution of 5-formyl-4-methyl-thiophene-2-carboxylic acid (3) (8.20 g, 48.2 mmol) in methanol (200 ml) and the mixture is reacted at room temperature for 36 hours. The solvent is removed under vacuum and the residue is distributed between water (200 ml) and ethyl acetate (200 ml) , the organic phases are washed with a saturated solution of NaHC0₃ (2 x 100 ml) , dried over anhydrous Na₂S0₄ and evaporated under vacuum, obtaining ester 4 (7.1 g, 38.6 mmol, yield 80%) as a white solid (end point 75-77°C), which is used "as is" for the subsequent reaction.

¹H-NMR (CDC1 ) δ: 2.48 (3H, s, CH₃) , 3.81 (3H, s, C0₂CH₃), 7.51 (1 H, s, Ar) , 9.96 (1 H, s, CHO) . d) (2R) -N- [ (R) -α-Phenylglycinyl] -2- (5 ' - carbomethoxy-3 ' -methyl-2 ' -thienyl) -glycinonitrile (5)

and (2S) -N- [ (R) -α-phenylglycinyl] -2- (5 ' -carbomethoxy- 3 ' -methyl-2 ' -thienyl) -glycinonitrile (6)

1 ' (R) -α-phenylglycinol (2.17 g, 15.8 mmol) is added to a solution of 5-formyl-4-methyl-thiophene-

2-carboxylic acid methyl ester (4) (1.98 g, 10.8 mmol) in anhydrous methanol (100 ml), with magnetic stirring, in an inert atmosphere and at room temperature, and it is left to react for 2 hours. The mixture is cooled to 0°C (ice bath), TMSCN is added (2.9 ml, 21.44 mmol) and it is left to react for 12 hours, removing the ice bath. The solvent is evaporated under vacuum and the residue is purified by flash chromatography, using petroleum ether/ethyl acetate as eluent mixture (gradient 90/10 down to 75/25) . The two aminonitriles 5

(1.88 g, 5.7 mmol, yield 53%) and 6 (0.39 g, 1.18 mmol, yield 11%) are obtained as amorphous solids. 5: ²H-NMR (CDC1₃) δ: 2.15 (3H, s, CH₃) , 3.65 (1 H, dd, J= 10.8, 9.4, CH₂OH) , 3.72-3.90 (4H, m, CHzOH and

C0₂CH₃), 4.20 (1 H, dd, J = 9.4, 4.0, CH-F) , 4.51 (1 H, s, CHCN) , 7.20-7.40 (5H, m, Ar) , 7.45 (1 H, s, H-Ar) .

¹³C-NMR (CDCI₃) δ: 14.49, 21.48, 46.34, 52.72,

60.88, 63.58, 67.43, 68.14, 117.67, 127.75, 128.13,

129.11, 129.48, 131.92, 136.68, 137.16, 137.97, 138.70,

164.57, 162.81.

6: ^-H-NMR (CDCI₃) δ: 2.11 (3H, s, CH₃) , 3.65-4.00 (5H, m, CH₂OH and C0₂CH₃) , 4.45 (1 H, dd, J= 9.4, 4.0_r CH-Ar) , 4.87 (1 H, s, CHCN), 7.20-7.40 (5H, m, Ar) , 7.49 (1 H, s, H-Ar) .

¹³C-NMR (CDCI₃ δ: 14.49, 45.60, 52.76, 62.46, 67.30, 76.63, 118.10, 127.94, 128.95, 129.05, 131.60, 136.77, 137.61, 138.05, 142.23, 163.03. e) (2S) -2- (5 ' -Carboxy-3 ' -methyl-2 ' -thienyl) - glycine (7) (3-MATIDA)

Lead tetra-acetate (2.54 g, 5.52 mmol) is added to a solution of (2R) -N- [ (R) -α-phenylglycinyl] -2- (5 ' -carbomethoxy-3 ' -methyl-2 ' -thienyl) -glycinonitrile

(5) (0.90 g, 2.72 mmol) in a mixture (1:1) of anhydrous methanol (30 ml) and anhydrous dichloromethane (30 ml) cooled to 0°C, with magnetic stirring and in an inert atmosphere. The mixture obtained is left to react at 0°C for a further 30 minutes and is then treated with 50 ml of water, allowing the reaction mixture to return to room temperature. The mixture is filtered on Celite and the solvent recovered is evaporated under vacuum.

The residue obtained is dissolved in HCl (6N, 40 ml) and is reacted under reflux for 16 hours. The mixture is left to return to room temperature, washed with dichloromethane (3 x 10 ml) and the aqueous phase is evaporated under vacuum. The residue obtained is purified by ion-exchange chromatography with Dowex

50 X2-200 resin, eluting with 10% pyridine. The final α-amino acid (7) is obtained as a grey solid (0.32 g, 1.49 mmol, yield 55%) (end point 245-247°C).

^XH-NMR (D₂0) δ: 2.15 (3H, s, CH₃) , 5.01 (1 H, s, CH) , 7.35 (1 H, s, H-Ar) .

¹³C-NMR (D₂0+Pyr d6) δ: 16.83, 55.72, 136.47, 137.37, 142.13, 144.81, 171.47, 174.99. Example 2

Similarly to Example 1, the following compound was obtained:

(2S) -N-2- (5 ' -carboxy-2 ' -thienyl) -glycine (ATIDA)

^XH-NMR (D₂0) δ: 5.45 1H, s, CH) , 7.34 (1 H, d, J=3.8, 4-H Th) , 7.77 (1 H, d, J=3.8, 3-H Th) .

¹³C-NMR (D₂0+Py dβ) δ: 53.84, 127.81, 128.90, 137.61, 141.45, 166.82, 171.58.

Example 3 a) Methoxycarbonyl-2-thiophenecarboxylic acid (9) Jones reagent 8N was added to a solution of (8) (4.5 g, 26.5 mmol) in acetone (225 ml) cooled with ice, under magnetic stirring, until a persistent orange colour was obtained. The reaction mixture was stirred at room temperature for 1 hour. 2-Propanol (5 ml) was then added, and after 10 min the mixture was filtered on Celite, washing with methanol. The solvent was eliminated under vacuum and the residue was absorbed in ethyl acetate, dried (Na₂S0₄) and concentrated to reduced volume (approx. 10 ml) . The precipitated compound was filtered to give (9) (3.76 g, 76.4%).

¹H-NMR (CDC1₃) δ: 3.90 (3H, s, C0₂CH₃) , 7.70 (1 H, d, J = 2.5 Hz; 3-CH) , 7.80 (1 H, d, J = 2.5 Hz, 4-CH) . b) Ethyl 3- [5 ' - (methoxycarbonyl) -2 ' -thienyl] - 3-oxopropanoate (10)

S0C1₂ (1.17 g, 9.8 mmol) was added to a solution of (9) (1.30 g, 7.3 mmol) in anhydrous benzene (25 ml) with magnetic stirring in an argon atmosphere and the mixture was kept under reflux for 2 hours. The excess S0C1₂ was eliminated under vacuum to obtain the raw chloride. n-BuLi (19 ml of a 1.6 M solution in hexane) was added dropwise to a solution of monoethyl malonate (1.93 g, 14.6 mmol) and 2, 2 ' -bipyridyl (5 mg) in anhydrous THF (35 ml) cooled to -78 °C, stirred mechanically in an argon atmosphere until a pink colour persisted for several minutes, while the internal temperature was allowed to rise to approx. -10 °C. The reaction mixture was cooled again to -65 °C and a solution of raw acyl chloride (1.50 g, 7.30 mmol) in anhydrous THF (10 ml) was added dropwise in 10 min. After 10 min, the reaction mixture was poured into a separating funnel containing 130 ml of diethyl ether and 80 ml of cold IN HCl. The organic phase was separated and washed with saturated NaHC0₃ (2 x 40 ml) , water (2 x 40 ml) , and dried (Na₂S0₄) . The solvents were eliminated under vacuum to give (10) (1.30 g, 68%) .

²H-NMR (CDC1₃) δ: 1.20 (3H, t, J = 7.0 Hz, CH₂CH₃) ,

3.80 (4H, m, CH₂CH₃ and C0CH₂) , 4.20 (2H, m, CH₂CH₃) ,

7.60 (1 H, d, J = 3.3 Hz, 3'-CH), 7.70 (1 H, d, J = 3.3

Hz, 4'-CH);

¹³C-NMR (CDCI₃) δ: 14.39, 46.74, 53.06, 62.09, 132.94, 133.81, 140.90, 147.74, 162.18, 166.89, 185.77. c) Ethyl 3- [5 ' - (methoxycarbonyl) -2 ' -thienyl] - 3-OXO-2- (9H-9-thioxanthenyl) -propanoate (11)

(10) (1.20 g, 4.70 mmol) was added to a solution of 9H-thioxanthen-9-ol (1.07 g, 4.70 mmol) in glacial AcOH-EtOH (50 ml, 1:1), stirred magnetically, and the resulting mixture was held under reflux for 3 h. The solvents were evaporated under vacuum and the residue was diluted with water (100 ml) and extracted with ethyl acetate (4 x 50 ml) . The organic phases were combined, washed with saturated NaHC0₃ and dried (Na₂S0₄) . The solvent was eliminated under vacuum to give a residue that was purified by flash chromatography. Elution with petroleum ether-ethyl acetate (70:30) gave (11) (1.93 g; 91%).

^XH-NMR (CDCI3) δ: 1.30 ( , 3H, CH₂-CH₃) , 3.90 (m 2H, (CH₂-CH₃), 4.00 (s, 3H, CH₃-O-CO) , 7.10-7.7 (10H, m aromatics) . d) 5- [3 ' - (9H-thioxanthenyl) propanoyl] -2-thiophene- carboxylic acid (12)

A solution of NaOH (0.176 g, 4.42 mmol) in water

(1.8 ml) was added to a solution of (11) (1.0 g, 2.21 mmol) in absolute EtOH (20 ml) and the resulting mixture was held under reflux for 96 h. The solution was cooled, acidified with 2N HCl and extracted with ethyl acetate (4 x 20 ml) . The organic phases were combined and dried (Na₂S0₄) and the solvent was evaporated under vacuum to give (12) (0.720 g, 86%) as a white solid: end point 190-193°C.

¹H-NMR (CDC1₃ + CH₃OD) δ: 3.20 (2H, d, J = 6.40 Hz, 2'-CH₂), 4.70 (1 H, t, J = 6.40 Hz, 9-CH) , 7.0-7.60 (10H, m, aromatics) . e) 5-{2' ,5'-dioxy-4'-[2'-(9H-9-thioxanthenyl) ethyl-4 ' -i idazolidinyl} -2-thiophenecarboxylic acid (13)

KCN (0.360 g, 5.53 mmol) and ammonium carbonate

(1.05 g, 10.94 mmol) were added to a solution of (12)

(0.720 g, 1.97 mmol) in EtOH-water (10 ml, 1:1). The resulting reaction mixture was heated at 120 °C in a sealed test tube for 48 hours. The reaction mixture was cooled and acidified with 2N HCl. The solid precipitate was filtered under vacuum to give (13) (0.30 g, 35%) . ^XH-NMR (CO₃OD) δ: 2.30 (1 H, dd, J = 7.0 and 14.0 Hz 2'-CHa), 2.70 (1 H, dd, J = 7.0 and 14.0 Hz, 2'-CHb), 4.30 (1 H, t, J = 7.0 Hz, 9-CH), 6.80-7.60 (10H, m, aromatics) . f ) (±) -2- ( 9H-9-Thioxanthenylmethyl ) -2- ( 4 ' -carboxy-

2 ' -thienyl ) glycine ( 14 )

IN NaOH (15 ml, 1.70 mmol) was added to (13) (0.300 g, 0.69 mmol) and the resulting mixture was heated at 150°C in a sealed test tube for 20 h. The solution was then submitted to ion-exchange chromatography. Elution with 10% pyridine gave (14) (0.023 g, yield 8%) : end point 300°C. ^αH-NMR (Pi-d₅) δ: 1.95-2.05 (1 H, m, CHa) , 2.20- 2.30 (1 H, m, CHb) , 4.05 (1 H, t, J ^■= 5.1 Hz, 2-CH) , 6.70-7.00 (8H, m, aromatics).

¹³C-NMR (Pi-d₅) δ: 41.55, 47.77, 64.83, 125.34, 127.11, 127.20, 127.42, 127.61, 129.00, 129.13, 130.30, 132.19, 132.89, 138.27, 142.00, 169.41, 174.41. Example 4

The activity of the compounds of the invention was evaluated by means of the following tests:

1) Antagonism of the stimulating action on phospholipase C and formation of inositol phosphates by 1S,3R-ACPD (300 μM) in thin slices of rat cortex. Molecules that are active in this test are to be regarded as mGlul or mGlu5 antagonists (Group 1) .

2) Antagonism of the action of L-CCG1 (3 μM) on

the formation of cAMP induced by forskolin (30 μM) on thin slices of rat striatum. Molecules that are active in this test are to be regarded as mGlu2 or mGlu3 antagonists (Group 2) . 3) Antagonism of the action of L-AP4 (10 μM) on the formation of cAMP induced by forskolin (30 μM) in thin slices of rat cerebellum. Molecules that are active in this test are to be regarded as mGlu4, mGlu7 and mGluδ antagonists (Group 3) .

The molecules that are active on the mGlus of the 1st group were then tested for potentiation of transmitter release from thin slices of rat cortex and the molecules that are active on the mGlus of the 2nd group were tested for inhibition of the release of transmitter from thin slices of striatum. All the molecules investigated were also tested on mouse cortex preparations (cortical wedges: see Mannaioni et al . , Br. J. Pharmacol. 118: 1530-1536; 1996) to evaluate their possible selectivity and their action on ionotropic receptors. The methods employed for the above experiments are described in: Lombardi et al., British J. Pharmacol . 110: 1407-1412; 1993; and Lombardi et al . , British J. Pharmacol . Ill : 189-195, 1996 and Moroni et al . , Eur. J. Pharmacol. 347: 189- 195; 1998.

Results

Of the molecules tested, compound (7) of Example 1, a selective antagonist of the mGluRs receptors of the first group, seems especially interesting. This molecule antagonizes the effect of 1S,3R-ACPD (300 μM) on the formation of inositol phosphates in slices of rat cortex with an IC₅₀ of 10 μM. It has no effect up to

300 μM on the other subgroups of mGlu receptors nor on ionotropic receptors for glutamate. It has an interesting ability to inhibit neuron death in cell cultures submitted to deprivation of oxygen and glucose as previously observed for other mGlu 1 antagonists (see: Pellegrini-Giampietro Neuropharmacology 38: 1607- 1619; 1999) .

The compound of Example 2 (ATIDA) antagonizes the effect of 1S,3R-ACPD (300 μM) on the formation of inositol phosphates in slices of rat cortex with an IC₅₀ of 70 μM. It has no agonistic or antagonistic effect (up to 300 μM) , in cortical or cerebral slices, on the cAMP accumulation test effected by forskolin. Moreover, it did not reduce the responses induced by NMDA (10 μM) or AMPA (5 μM) in cortical preparations ("cortical wedges" ) .

The compound of Example 3 also antagonizes the effect of 1S,3R-ACPD (300 μM) on the formation of inositol phosphates in slices of rat cortex with an IC₅₀ of 20 μM.

Example 5

Stroke model: The middle cerebral artery was occluded in anaesthetized Sprague-Dawley rats by inserting a silicone-coated nylon filament (0.28 mm) into the internal carotid artery as far as the Willis circle. Forty-eight hours after the procedure, the rats were anaesthetized again, the thorax was opened and a solution containing 2, 3, 5-triphenyltetrazolium chloride in physiological solution was slowly injected into the left cardiac ventricle in order to reveal necrotic tissue. The brain was removed, frontal ("coronal") sections were prepared, with thickness of 1 mm, the infarcted areas were measured and the volume of the infarct was calculated by the methods described previously. Results

Occlusion of the middle cerebral artery caused an extensive infarct in the hypsilateral hemisphere with a significant portion of frontal and parietal cortex, putamen caudatum, diencephalon and hippocampus seriously affected. Administration of 3-MATIDA (3-10 mg/kg i.p. 5 min after occlusion) reduced the necrotic area and the volumes of the infarct by 30 and 55% respectively. There was neuroprotection both in the cortical regions and in the subcortical regions and its level was comparable to that observed previously after treatment with glycine antagonists or with kynurenine- hydroxylase inhibitors (Chen et al . , JPET, 267: 937- 943; 1993 and Cozzi et al . , J. Cereb. Blood Flow Metab., 19: 771-777; 1999).

Claims

Compounds of formula (I)

NH,

in which: Ri is hydrogen, methyl, cyclopropyl or a group of formula

where X is oxygen or sulphur;

R₂ is hydrogen, methyl, methoxy, hydroxy, halogen, cyano;

R3 is carboxy; their salts with pharmaceutically acceptable acids or bases, physiologically equivalent derivatives and individual enantiomers .

2. Compounds according to Claim 1 in which Rα is hydrogen and R₂ is hydrogen or methyl.

3. Compounds according to Claim 1, in which R_x is a group of formula:

in which X is sulphur or oxygen; R₂ is hydrogen or methyl; R₃ is carboxy.

4. Compounds according to Claims 1-3 in which the configuration of the asymmetric carbon atom is R.

5. As compound according to Claim 1, (2S) -2- (5 ' -carboxy-3 ' -methyl-2 ' -thienyl) -glycine, (2S) -N-2- (5 ' -carboxy-2 ' -thienyl) -glycine, or (±) -2- (9H-9-thioxanthenylmethyl) -2- (4 ' -carboxy- 2 ' -thienyl) -glycine .

6. Pharmaceutical compositions containing as active principle a compound of Claims 1-5 mixed with suitable excipients .

7. Compounds of claims 1-5 as antagonists of the glutamate receptor.

8. Method for the treatment of diseases characterized by excessive stimulation of the mGlu receptors, which comprises the administration of a compound of Claims 1- 5.

9. Use of a compound of Claims 1-5 in the preparation of a drug for the treatment of diseases characterized by excessive stimulation of the mGlu receptors.