WO2008031888A2

WO2008031888A2 - Condensed pyrimidinones active on glutamatergic receptors

Info

Publication number: WO2008031888A2
Application number: PCT/EP2007/059721
Authority: WO
Inventors: Giuseppe Campiani; Stefania Butini; Caterina Fattorusso; Sandra Gemma; Salvatore Sanna Coccone; Marianna Borriello
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-09-15
Filing date: 2007-09-14
Publication date: 2008-03-20
Anticipated expiration: 2009-03-15
Also published as: WO2008031888A3; ITRM20060491A1

Abstract

The present invention relates to a compound of Formula (I) process for its preparation and uses thereof.

Description

COMPOUNDS ACTIVE ON GLUTAMATERGIC RECEPTORS

TECHNICAL FIELD

The present invention relates to the synthesis of novel compounds represented by Formula (I) as selective agonists and/or antagonists of glutamatergic ionotropic receptors, preferably the AMPA and kainate GluR5 receptor subtypes. These compounds show to be useful for the treatment of pain, including neuropathic pain and neurodegenerative diseases.

BACKGROUND Z-Glutamate (GIu) is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and it modulates the functions of most neuronal circuits in the CNS. The physiological and pathological actions of GIu are mediated by activation of a range of excitatory amino acid (EAA) receptors: i) ionotropic glutamate receptors, (iGluRs, ligand gated ion channels) and ii) the metabotropic glutamate receptors (mGluRs, G-protein coupled receptors). The GIu receptor ion channels are abundantly expressed in the brain and binding of GIu to iGluRs is a key step in the mechanism of rapid excitatory synaptic transmission in the CNS. Even if the complex roles of the iGluRs are far from being understood in detail, these receptors are implicated in learning and memory functions and are associated to a number of psychiatric and neurological disorders such as Alzheimer's, Parkinson's and Huntington's disease, ALS and epilepsy (Ozawa, S. et AL; Prog. Neurobiol. 1998, 54, 581-618). iGluRs in the mammalian brain are encoded by a family of 18 genes that co-assemble to form the kainate, NMDA and AMPA receptors, classified on the basis of the binding of selective agonists: JV-methyl-d-aspartate (NMDA), (5)-2-amino- 3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid (AMPA) and kainate receptors (KA). Co-assembly of these ion channels within families generates several receptor subtypes. iGluRs form tetramer ligand-gated ion channels and one copy of the ligand-binding core, as a discrete domain, is present in each subunit.

AMPA receptors consist of different subunits (GIuRl -GluR4). The ion channel activity can be regulated by the interaction of the agonist with the main binding site or with the allosteric binding site. AMPA receptors are widely distributed, often co-localized with the NMDA receptors, and in particular their concentration increases in telencephalic area. KA receptors are also associated to ion channels, and consist of different subunits (GluR5- GluR7; KAl; KA2). They are widely distributed with high levels in proencephalic areas (Guo, W. Et AL; Eur. J. Pharmacol, 2002, 452, 309-318).

Since glutamate receptors are involved in a great number of functions, it is necessary to find molecules that selectively interact with specific glutamate subtypes to lower possible side effects (Furuyama, T. et AL; MoI. Brain Res., 1993, 18, 141-151).

Evidence from the last decades indicates that the excitatory amino acid GIu plays a significant role in nociceptive processing. GIu and glutamate receptors are located in areas (brain, spinal cord and periphery) that are involved in pain sensation and transmission. KA receptors are widely distributed in the CNS, including the dorsal horn, and are also found in small and medium- sized dorsal root ganglion neurons. The dorsal horn contains abundant mRNA for KA2 subunits, but mRNAs for GluR5-7 are also present. Dorsal root ganglion cells are rich in GluR5 mRNA (Huettner, J. E. et AL; Neuron, 1990, 5, 255-266; Guo, W. Et AL; Eur. J. Pharmacol, 2002, 452, 309-318; Partin, K. M. et AL; Neuron, 1993, 11, 1069-1082). Transfer of nociceptive information from the periphery to the spinal cord occurs via C- fiber primary afferents. Within dorsal roots, dorsal root ganglion (DRG) cell bodies are associated with primary afferent neurons. It has been demonstrated that C-fiber afferents possess KA receptors and that high intensity single shock stimulation of primary afferent sensory fibres produces a fast, KA receptor-mediated excitatory postsynaptic current in the superficial dorsal horn of the spinal cord.

More recently, biophysical and pharmacological studies aimed to clarify the profile of KA receptors in DRG neurons suggested that they likely comprise GluR5 homomers. Moreover, studies in spinal cord slices have implicated KA receptors, specifically the GluR5 receptor subtype, in pain transmission (Jensen, T. S. et AL; Eur. J. Pharmacol, 2001, 429, 1-11).

Throughout history, pain relief has been a major focus of medical science. In normal conditions pain is a natural defence mechanism but extreme pain and chronic pain may become debilitating. Opioid analgesics have been the most commonly used agents for the treatment of pain. However, repeated use of opioids is often associated with the development of analgesic tolerance, as well as physical and psychological dependence.

Pain associated with disease or injury of the peripheral or central nervous system is defined as neuropathic pain, a challenging pain category which is considered to be particularly difficult to treat and which resists to conventional analgesics. Hyperalgesia (the lowering of pain threshold and the increased response to noxious stimuli) and allodynia (the evocation of pain by non-noxious stimuli) are typical elements of neuropathic pain. NMDA and non-NMDA receptors play a crucial role in spinal cord nociceptive transmission, under both normal and neuropathic conditions. Adverse side effects limit the clinical value of NMDA receptor antagonists. Conversely, KA receptor antagonists could be useful in neuropathic pain, because of their role in chronic pain states, and drugs with a relatively selective action on these receptors are becoming available.

Potential drugs which interact with the orthosteric or allosteric GIu binding sites of specific receptor subtypes could therefore represent a new therapeutic option for the treatment of severe CNS diseases such as schizophrenia, neuropathic pain, epilepsy, ALS.

For these reasons the authors focused their work on the synthesis and pharmacological evaluation of chemical structures which can modulate GIu receptor subtypes.

DESCRIPTION OF THE INVENTION Recently the authors reported the synthesis of (S)-CPW399 (Campiani, G et AL, J. Med. Chem. 2001, 44, 4501-4504; Kastrup, J., Campiani, G. et AL, MoL Pharmacol. 2005, 67, 703-713) a compound structurally related to the naturally occurring ligand (S)-Willardiine. This derivative was found to be a weakly desensitizing, partial agonist of GluRl/GluR2

2+ subunits that, unlike (5)-Willardiine, stimulated an increase in [Ca ] in mouse cerebellar granule cells and induced neuronal cell death. The present invention is based on the activity of (S)-CPW399 on kainate GluR5 receptors. With the aim to provide potential selective drugs able to interact with AMPA and KA receptor subtypes, a new set of (S)- CPW399 related analogues was rationally designed. These compounds represent new potential pharmacological tools for a better understanding of the physiological role of AMPA and KA-GluR5 subtypes and for the subsequent development of drugs for the treatment and/or prevention of pain and neurodegenerative diseases. Therefore the object of the present invention is a compound of Formula (I):

I wherein n=l or 2; A=O or S; B=sp2 carbon; D=sp2 carbon; X, Y and Z =S, N, O, C or CH;

- when Z-B and Y-D are single bond, then B-D is carbon-carbon double bond;

- when Z=N, C or CH, then Ri= H, Me, Et or is not present;

- when X=S and Z-B and Y-D are double bonds, then B-D is a single bond;

- when X=C or N, then R₂=H, Me, Et or is not present and R₃=H, Me, Et or is not present; - when X=S or O, then R₂ and R₃ are not present;

- when Y=N or C or CH, then R₄=H, Me, Et;

- when R₂ and R₃ are H, and when Z=S, O and when Ri is not present or when X=NH or CH and when Z=CH and when Ri=H, then X-Z=single bond;

- when X=C, R₂=H, and R₃ is not present, or when X=N and Z=N or CH and Ri is not present, then X-Z=double bond;

- when X=C and R₂ and R₃ are H, or when X=N and R₂=H and R₃ is not present, then X-Y is a single bond;

- when X=C, R₂=H and R₃ is not present, or when X=N and Y=N or CH and R₄ is not present, then X-Y=double bond; - when Ri-R₂=-(CH₂)₄-, -(CH=CH)₂-, cycloalkyl, cycloheteroalkyl (N, S, O), aromatic or heteroaromatic (N, S) systems, then X=C and R₃ is not present and Z=C and Y=O or S and R₄ is not present, or Y=N and R₄ is H, Me, Et, Alkyl (C₃-C₅) or Y=CH and R₄=H;

- when R₃-R₄=-(CH₂)₄-, -(CH=CH)₂-, cycloalkyl, cycloheteroalkyl (N, S, O), aromatic or heteroaromatic (N, S) systems, then X=C and R₂ is not present and Y=C and Z=O, S and Ri is not present or Z=N and Ri is H, Me, Et, Alkyl (C₃-C₅) or Z=CH and Ri=H; R₅=H, alkyl (Ci-C₈) or cycloalkyl (C₅-C₆);

R₆=H, (2-carboxythien-3-yl)methyl, (5-carboxythien-2-yl)methyl, (5-carboxythien-3- yl)methyl, (4-carboxythien-3-yl)methyl, (4-carboxythien-2-yl)methyl, (3-carboxythien-2- yl)methyl, 2-carboxybenzyl, 3-carboxybenzyl, 4-carboxybenzyl, (2-carboxyfuran-3- yl)methyl, (5-carboxyfuran-2-yl)methyl, (5-carboxyfuran-3-yl)methyl, (4-carboxyfuran-3- yl)methyl, (4-carboxyfuran-2-yl)methyl, (3-carboxyfuran-2-yl)methyl, (2-carboxypyrrol-3- yl)methyl, (5-carboxypyrrol-2-yl)methyl, (5-carboxypyrrol-3-yl)methyl, (4-carboxypyrrol- 3-yl)methyl, (4-carboxypyrrol-2-yl)methyl, (3-carboxypyrrol-2-yl)methyl, (2- phosphonothien-3 -yl)methyl, (5 -phosphonothien-2-yl)methyl, (5 -phosphonothien-3 - yl)methyl, (4-phosphonothien-3-yl)methyl, (4-phosphonothien-2-yl)methyl, (3- phosphonothien-2-yl)methyl, 2-phosphonobenzyl, 3-phosphonobenzyl, 4- phosphonobenzyl, (2-phosphonofuran-3-yl)methyl, (5-phosphonofuran-2-yl)methyl, (5- phosphono furan-3 -yl)methyl, (4-phosphono furan-3 -yl)methyl, (4-phosphono furan-2- yl)methyl, (3-phosphonofuran-2-yl)methyl, (2-phosphonopyrrol-3-yl)methyl, (5- phosphonopyrrol-2-yl)methyl, (5-phosphonopyrrol-3-yl)methyl, (4-phosphonopyrrol-3- yl)methyl, (4-phosphonopyrrol-2-yl)methyl, (3-phosphonopyrrol-2-yl)methyl, and the corresponding methyl and ethyl esters, (2-(lH-tetrazol-5-yl)thien-3-yl)methyl, (5-(1H- tetrazol-5 -yl)thien-2-yl)methyl, (5 -( lH-tetrazol-5 -yl)thien-3 -yl)methyl, (4-( lH-tetrazol-5 - yl)thien-3-yl)methyl, (4-(lH-tetrazol-5-yl)thien-2-yl)methyl, (3-(lH-tetrazol-5-yl)thien-2- yl)methyl, 2-(lH-tetrazol-5-yl)benzyl, 3-(lH-tetrazol-5-yl)benzyl, 4-(lH-tetrazol-5- yl)benzyl, (2-(lH-tetrazol-5-yl)furan-3-yl)methyl, (5-(lH-tetrazol-5-yl)furan-2-yl)methyl, (5-(lH-tetrazol-5-yl)furan-3-yl)methyl, (4-(lH-tetrazol-5-yl)furan-3-yl)methyl, (4-(1H- tetrazol-5-yl)furan-2-yl)methyl, (3-(lH-tetrazol-5-yl)furan-2-yl)methyl, (2-(lH-tetrazol-5- yl)pyrrol-3 -yl)methyl, (5 -( lH-tetrazol-5 -yl)pyrro l-2-yl)methyl, (5 -( lH-tetrazol-5 -yl)pyrrol- 3-yl)methyl, (4-(lH-tetrazol-5-yl)pyrrol-3-yl)methyl, (4-(lH-tetrazol-5-yl)pyrrol-2- yl)methyl, (3 -( lH-tetrazol-5 -yl)pyrro l-2-yl)methyl; a salt, tautomer, geometric isomer, enantiomer, diastereoisomer or racemate thereof able to bind at least one glutamatergic system receptor for medical use. Prefered compounds are: (5)-l-(2'-Amino-2'-carboxyethyl)-thieno[3,4-<i]pyrimidine- 2,4(lH,3H,5H,7H)-dione (5a), (5)-l-(2'-Amino-2'-carboxyethyl)-6,6-dimethyl-

2,3,4,5,6,7-hexahydrocyclopentapyrimidin-2,4-dione (5b), (5)-l-(2'-Amino-2'- carboxyethyl)thieno[3,2-J]pyrimidin-2,4-dione (8a), (5)-l-(2'-Amino-2'- carboxyethyl)thieno[2,3-J]pyrimidin-2,4-dione (8b), (S)- 1 -(2 '-Amino -2 '- carboxyethyl)thieno[3,4-(i]pyrimidin-2,4-dione (12), (S)-l-(2'-Amino-2'-carboxyethyl)- 6,7,8,9-tetrahydrobenzo[6]thieno[3,2-<i]pyrimidine-2,4-dione (16), (5)-l-(2'-Amino-2'- carboxyethyl)-3-[(2-carboxythien-3-yl)methyl]thieno[3,2-(i]pyrimidin-2,4-dione (21a) and (S)- 1 -(2 ' -Amino-2 ' -carboxyethyl)-3 - [(2-carboxythien-3 -yl)methyl]thieno [3 ,4-<i]pyrimidin- 2,4-dione (21b).

It is another object of the invention a compound having the general formula I wherein the compound having A=O, n=l, Z=CH, X=C, Y=CH, Ri=R₂=R₃=R₄=R₅=H is not included. It is a further object of the invention a pharmaceutical compositions comprising a pharmacologically effective and acceptable amount of at least one compound of the invention.

It is another object of the invention the use of the compound of the invention for the preparation of a medicament for pain therapy and/or prevention. Preferably for neuropathic pain.

It is another object of the invention the use of the compound of the invention for the preparation of a medicament for therapy and/or prevention of a CNS disease. Preferably the CNS disease is comprised in the group of: epilepsy, ALS, neurodegenerative disease or schizophrenia. More preferably the neurodegenerative disease is Alzheimer's disease. It is a further object of the invention a process for the preparation of a compound of the invention comprising the phases reported in Schemes 1-5. Compounds of Formula (I) also comprise tautomers, geometrical isomers, optically active forms as enantiomers, diastereoisomers and racemate forms, as well as pharmaceutically acceptable salts of the compounds of Formula (I).

Depending on the meanings of the radicals in the compounds of Formula (I) one or more chiral centres may be present. For the purposes of the present invention it is pointed out that each of the products of Formula (I) can exist both as a racemic mixture R/S, and in the separate isomeric forms R and S.

Preferred pharmaceutically acceptable salts of the Formula (I) are acid addition salts formed with pharmaceutically acceptable acids like hydrobromide, hydrochloride, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, Αnάpara- toluenesulfonate salts.

Suitable pharmaceutically acceptable base addition salts for the compound of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N₅N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (/V-methylglucamine) and procaine. Sodium salts are particularly preferred. The compounds of Formula (I) may be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred experimental conditions (i.e. reaction temperatures, time, moles of reagents, solvents, etc.) are given, other experimental conditions can also be used, unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimisation procedures. Specific reference is made to the methods described in the non limiting examples and Schemes 1-5.

For any compound, the therapeutically effective dose can be estimated initially either in cellular culture or in animal, usually mice, monkeys, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination , reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones. The medicament may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co. , N. J.1991). Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be Formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Once Formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated. The medicament of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual. Dosage treatment may be a single dose schedule or a multiple dose schedule. A further object of the present invention are pharmaceutical compositions containing one or more of the compounds of Formula (I) described earlier, in combination with excipients and/or pharmacologically acceptable diluents.

The compositions in question may, together with the compounds of Formula (I), contain known active principles. A further embodiment of the invention is a process for the preparation of pharmaceutical compositions characterised by mixing one or more compounds of Formula (I) with suitable excipients, stabilizers and/or pharmaceutically acceptable diluents.

The invention will now be illustrated in greater detail by means of non- limiting examples, and in particular with reference to the following figures: Figure IA: Compounds 5a and 8a activate human 1GIuRl₁ expressed in CHO-Kl cells. a) Dose-response curves for the activation of 1GIuR₁ by 5a (filled squares) and 8a (filled circles) in the presence of 30 μM cyclothiazide. Values were normalized to a maximal current induced by 10 mM L-glutamate in the presence of 30 μM cyclothiazide and represent the mean ± SEM of 3-4 separate experiments, b) Representative current responses induced by 8a in an 1GIuRl₁ expressing cell, which were voltage-clamped at -60 mV in the whole-cell configuration. The stimulations were conducted in the presence of 30 μM cyclothiazide. The cell was initially subjected to Is pulses of 10 mM L-glutamate. After the washout of glutamate, increasing concentrations of 8a were applied to the cell in 1 s pulses.

Figure IB: Representative current responses induced by 8a (a) or 5a (b) in iGluR5(Q)ib expressing cells, which were voltage-clamped at -60 mV in the whole-cell configuration. The cells were initially subjected to 100 ms pulses of 10 mM L-glutamate. After the washout of glutamate (NaR), a 100 ms pulse of either 8a or 5a was applied to the cell. Figure 2: Compounds 5a and 8a desensitize both human 1GIuRl₁ and iGluR5(Q)ib receptors expressed in CHO-Kl cells. a) Dose-response curve for the inactivation of iGluR5(Q)ib by 5a. The cells were stimulated with 100 ms control pulses of 3 mM L-glutamate in 30 s intervals. When a stable response level was obtained, the cells were pretreated with 5a, before the next pulses with 3 mM L-glutamate. Increasing the concentration of 5a caused a reduction of the L- glutamate induced responses. Values were normalized to the control L-glutamate currents in the absence of 5a and represent the mean ± SEM of 4-10 separate experiments, b) Representative L-Glutamate (3 mM) induced current responses at iGluR5(Q)ib receptors without (left) and in the presence of 3 nM 5a (right), c) Dose-response curve for the inactivation of 1GIuRl₁ (open circles) and iGluR5(Q)ib (filled circles) by 8a. The cells were stimulated with 100 ms control pulses of 3 mM L-glutamate (iGluR5(Q)ib) or 1 mM L- glutamate (1GIURI₁) in 30 S intervals. When a stable response level was obtained, the cells were pretreated with 8a, before the next pulses with L-glutamate. Values were normalized to the control L-glutamate currents in the absence of 8a and represent the mean ± SEM of 4-10 separate experiments, d) Representative L-Glutamate (3 mM) induced current responses at iGluR5(Q)ib receptors without (left) and in the presence of 3 nM 8a (right), e) Representative L-Glutamate (1 mM) induced current responses at 1GIuRl₁ receptors without (left) and in the presence of 3 nM 8a (right).

EXAMPLES Experimental Procedure

Example 1 - Synthesis of Derivatives 5a,b according to Scheme 1

Ia X=S 2a X=S 3a X=S

1b X= (CH₃)₂C 2b X= (CH₃)₂C 3b X= (CH₃)₂C

4a X=S, R=Boc

- 5a X=S, R=H

4b X= (CH₃)₂C, R=Boc

- 5b X= (CH₃)₂C, R=H

Scheme 1. (a)S-Ethyl-isothiouroniumbromide, Na₂CO₃, H₂O; b) HCI, AcOH, H₂O; c) NaH, DMF, (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one; d) TFA, CH₂CI₂.

2-(Ethylthio)thieno[3,4-</]pyrimidin-4(3H,5H,7H)-one (2a).

To a solution of S-ethyl isothiouronium bromide (2.3 g, 12.5 mmol) in water (10.0 rnL) kept in the dark, sodium carbonate (Na₂COs) (1.3 g, 12.5 mmol) and Ia (2.0 g, 12.5 mmol) were added portionwise. The resulting mixture was stirred for 18 hours at room temperature in the dark. The suspension was filtered and the solid was washed with water, diethyl ether, methanol and acetone. Then the collected solid was dried in an oven at 50 ⁰C under vacuum. Crystallization of title compound from methanol afforded derivative 2a as white prisms in 84.6% yield (mp >300°C); ¹H NMR (DMSO-J₆) δ 7.90 (bs, 1Η), 4.06 (m, 2Η), 3.87 (m, 2H), 3.06 (q, 2H, J = 7.2 Hz), 1.25 (t, 3H, J = 7.4 Hz); ¹³C NMR (DMSO-J₆) δ 165.27, 161.01, 118.33, 114.54, 34.66, 32.98, 24.92, 15.02; ESI-MS m/z 237 (M+Na)⁺; Anal. (C₈H₁₀N₂OS₂): C,H,N.

Thieno[3,4-</]pyrimidine-2,4(lH,3H,5H,7H)-dione (3a). To a water solution (15.0 mL) of 2a (2.0 g, 9.34 mmol), HCl cone. (1.45 mL) and glacial acetic acid (2.90 mL) were added and the mixture was heated to reflux for 5 hours. The resulting suspension was filtered and the solid was washed with water and methanol. The collected solid was dried in an oven at 50 ⁰C under vacuum. Crystallization of the crude solid from methanol afforded derivative 3a as white prisms in 87.0% yield, (mp >300°C); ¹H NMR (DMSO-J₆) δ 11.16 (bs, IH), 11.00 (bs, IH), 3.93 (t, 2H, J= 3.3 Hz), 3.72 (t, 2H, J= 3.2 Hz); ¹³C NMR (DMSO-J₆) δ 161.88, 152.80, 152.34, 109.58, 36.00, 32.77; ESI-MS m/z 361 (2M-2H+Na)^", 339 (2M-H)^", 169 (100) (M-H)^"; Anal. (C₆H₆N₂O₂S): C,H,N.

6,6-Dimethyl- 1 ,5,6,7-tetr ahydrocyclopentapyrimidin-2,4-dione (3b).

To a solution of S-ethyl isothiouronium bromide (1.15 g, 6.3 mmol) in water (5.0 mL) kept in the dark, sodium carbonate (Na₂COs) (0.7 g, 6.3 mmol) and then Ib (810 mg, 4.4 mmol) were added portionwise. The resulting mixture was stirred for 18 hours at room temperature in the dark. The suspension was filtered and the solid was washed with water, diethyl ether, methanol and acetone. Then the collected solid was dried in an oven at 50 ⁰C under vacuum. Crystallization of the crude solid from methanol afforded derivative 2b as white prisms in 84.6% which was used in the following step without further purification. To a water solution (8 mL) of 2b (0.6 g, 3.3 mmol), HCl cone. (0.5 mL) and glacial acetic acid (1.0 mL) were added and the mixture was heated under reflux for 5 hours. The resulting suspension was filtered and the solid was washed with water and methanol. The collected solid was dried in an oven at 50 ⁰C under vacuum. Crystallization of the crude solid from methanol afforded derivative 3b as white prisms in 23.0% yield (mp 290-295 ⁰C). ¹H-NMR (DMSO-J₆) δ 10.94 (br, IH), 10.67 (br, IH), 2.40 (s, 2H), 2.22 (s, 2H), 1.08 (s, 3H), 1.07 (s, 3H); ESI-MS m/z 179 (M-H)^"; Anal. (C₉Hi₂N₂O₂): C,H,N.

(S)-l-(2'-(tert-Butoxycarbonyl)amino-2'-carboxyethyl)-thieno[3,4-</]pyrimidine- 2,4(lH,3H,5H,7H)-dione (4a).

To suspension of 3a (2.0 g, 11.6 mmol) in dry DMF (100.0 mL) sodium hydride (NaH) (253.0 mg, 10.54 mmol) was added portionwise and the resulting mixture was stirred 2 hours at room temperature. Then the mixture was cooled to -65°C and a solution of (S)-3- [(te/t-butoxycarbonyl)amino]oxetan-2-one (1.97 g, 10.54 mmol) in dry DMF (30.0 mL) was added during 1 hour. When the addition was completed the mixture was allowed to room temperature and left 18 hours stirring. The solvent was removed under reduced pressure, and the crude product taken up in water (30.0 mL). The aqueous phase acidified until pH = 2 and then extracted with ethyl acetate (EtOAc) (3 x 35 mL). The collected organic layers were dried on sodium sulphate (Na₂SO₄) and then the solvent removed under vacuum. The crude product was purified by means of flash chromatography using a gradient of elution (from CH₂Cl₂/Me0H/Ac0H 9:0.3:0.01 v/v to CH₂Cl₂/Me0H/Ac0H 9:2:0.1 v/v) affording compound 4a as amorphous white solid (18.3% yield). ¹H NMR (DMSO-J₆) δ 11.22 (bs, IH), 6.66 (bs, IH), 4.21 (m, 4H), 3.75 (m, 2H), 3.43 (m, 2H), 1.28 (s, 9H); ¹³C NMR (DMSO-J₆) δ 171.70, 160.91, 155.95, 154.22 152.29, 110.77, 79.06, 51.84, 48.84, 36.60, 33.37, 28.57; ESI-MS m/z 356 (M-H)^", 282; ESI-MS/MS of (M-H)^" m/z 282 (100), 238; Anal. (Ci₄Hi₉N₃O₆S): C,H,N.

(^-l-Cl'-^rt-ButoxycarbonylJamino-l'-carboxyethylJ-o^-dimethyH^^^^,?- hexahydrocyclopentapyrimidin-2,4-dione (4b).

Derivative 4b was synthesized starting from 3b (90 mg, 0.5 mmol) following the same synthetic strategy previously reported for compound 4a. Title compound was obtained as amorphous solid in 40% yield. ¹H-NMR (CD₃OD) δ 4.60-4.58 (m, 2H), 4.30-4.23 (m, IH), 3.82-3.70 (m, IH), 2.76 (s, IH), 2.74 (s, IH), 2.42 (s, 2H), 1.36 (s, 9H), 1.19 (s, 3H), 1.18 (s, 3H); ESI-MS m/z 366 (M-H)^", 292; [α]²⁰ _D = -87° (c = 0.57 in HCl IN); Anal.

(^-l-Cl'-Amino-l'-carboxyethyO-thienoIS^-^pyrimidine-l^ClH^H^H^HJ-dione (5a).

A solution of 4a (0.70 g, 1.96 mmol) in dichloromethane (5.0 mL) and trifluoroacetic acid (TFA) (5.0 mL) was stirred for 16 hours at room temperature. Then the solvent was removed under reduced pressure and the crude product was purified by means of ion exchange resin (Dowex 50 WX 8-400), using first a mixture water/ethanol 1 :1 v/v as eluent and then pyridine IM in water to recover displaced 5a from the resin. Compound 5a was obtained as white amorphous solid in 74.8% yield. ¹H NMR (CD₃OD:DC1 9:1) δ 4.51 (m, IH), 4.35 (m, 4H), 3.92 (t, 2H, J = 3.1 Hz); ¹³C NMR (CD₃OD:DC1 9:1) δ 168.36, 162.54, 155.14, 154.20, 113.81, 53.19, 46.62, 37.35, 33.43; ESI-MS m/z 513 (100) (2M-H)^", 256 (M-H)^"; [α]²⁰D = -14.1° (c = 0.21 in HCl IN); Anal. (C₉HnN₃O₄S) C,H,N. (S)-l-(2'-Amino-2'-carboxyethyl)-6,6-dimethyl-2,3,4,5,6,7-hexahydrocyclopenta- pyrimidin-2,4-dione (5b). The title compound was prepared starting from 4b (75 mg, 0.2 mmol) following the same procedure used for the synthesis of compound 5a. Compound 5b was obtained as white amorphous solid in 64% yield. ¹H-NMR (D₂O:DC1 9:1) δ 4.33-4.24 (m, IH), ), 4.16-4.11 (m, 2H), 2.62 (s, 2H), 2.28 (s, 2H), 0.98 (s, 6H); ESI-MS m/z 266 (M- H)^"; [Ci]²⁰D = -46° (c = 0.52 in HCl IN); Anal. (Ci₂Hi₇N₃O₄): C,H,N.

Example 2 - Synthesis of Derivatives 8a,b according to Scheme 2 NH₂

6a Z=CH, Y=S 7a Z=CH, Y=S 8a Z=CH, Y=S 6b Z= S, Y=CH 7b Z= S, Y=CH 8b Z= S, Y=CH

Scheme 2. (a) NaOCN, H₂O, AcOH; (b) NaH, (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one, DMF;(c) TFA, CH₂CI₂

Thieno [3,2-d\ pyrimidine-2,4-dione (7a).

A solution of sodium cyanate (5.0 g, 77.0 mmol) in water (15.0 rnL) was added dropwise to a mixture of 6a (6.05 g, 38.4 mmol) in a solution 50% of glacial acetic acid in water (90.0 mL) and the resulting mixture was stirred for 5 hours at room temperature. The resulting precipitate was collected by filtration and then dissolved in NaOH 2N (90.0 mL). The solution was cooled to 0 ⁰C, acidified with acetic acid and the solid filtered and dried in an oven at 60 ⁰C to give 7a (86.7% yield) as white solid (mp >300°C); ¹H NMR (DMSO-J₆) δ 9.17 (bs, IH), 7.91 (d, IH, J= 5.2 Hz), 7.72 (d, IH, J= 5.2 Hz), 6.70 (bs, IH); ESI-MS m/z

191 (M+Na)⁺; Anal. (C₆H₄N₂O₂S): C,H,N .

Thieno [2,3-</| pyrimidine-2,4-dione (7b).

Title compound was prepared following the same procedure used for the synthesis of compound 7a starting from 6b (1 g, 6.3 mmol). The compound was obtained as a white solid in 13% yield. ¹H-NMR (DMSO- d₆) δ 11.88 (bs, IH), 11.11 (bs, IH), 7.11-7.05 (m, 2H), ESI-MS m/s 191 [M+Na]⁺; Anal. (C₆H₄N₂O₂S): C,H,N.

(S)-l-(2'-Amino-2'-carboxyethyl)thieno[3,2-</]pyrimidin-2,4-dione (8a).

Title compound was synthesized starting from 7a (1 g, 6.0 mmol) following the same procedure described for synthesis of 5a. Compound 8a was obtained as amorphous solid in 10.0% overall yield. ¹H NMR (CD₃OD:DC1 9:1) δ 8.09 (d, IH, J= 5.5 Hz), 7.30 (d, IH, J = 5.5 Hz), 4.61 (m, 2H), 4.45 (m, IH); ESI-MS m/z 508 (100) (2M-H)^", 254 (M-H)^", 167; [(X]²⁰D = -9.7° (c = 0.17 in HCl IN); Anal. (C₉H₉N₃O₄S): C,H,N. (S)- 1-(2 '- Amino-2 '-carboxyethyl)thieno [2,3-d] pyrimidin-2,4-dione (8b).

Title compound was prepared sterting from 7b (139 mg, 0.82 mmol) following the same procedure used for the synthesis of compound 5a .Compound 8b was obtained as a white amorphous solid in 19.1% overall yield. ¹H-NMR (DCl 20%wt in D₂O) δ 6.98 (d, IH, J= 5.5 Hz), 6.90 (d, IH, J = 5.5 Hz), 4.38-4.15 (m, 3H); ESI-MS m/s 254 [M-H]^"; Anal. (C₉H₉N₃O₄S): C,H,N.

Example 3 - Synthesis of Derivative 12, according to Scheme 3

10 11 c,d

NH₂ OH

Scheme 3. (a) NaOCN, H₂O, AcOH; (b) NaOMe, methanol; (c) NaH, (S)-3-[(tert- butoxycarbonyl)amino]oxetan-2-one, DMF; (d) TFA, CH₂CI₂

Methyl 4-ureidothiophene-3-carboxylate (10).

A solution of sodium cyanate (511 mg, 7.8 mmol) in water (5.0 mL) was added dropwise to a mixture of 9 (620 mg, 3.9 mmol) in a solution 50% of glacial acetic acid in water (15 mL) and the resulting mixture was stirred for 18 hours at room temperature. Then the solvent was removed under reduced pressure and the residue was suspended in HCl IM and the aqueous phase was extracted with ethyl acetate (4 x 50 mL), the collected organic layers were dried over anhydrous sodium sulphate (Na₂SO₄), filtered and the solvent evaporated. The crude product was purified by means of flash chromatography (50% n-hexane in EtOAc) to give compound 10 as a white solid in 82 % yield; ¹U NMR (DMSO-J₆) δ 8.88 (bs, IH), 8.26 (d, IH, J= 3.6 Hz), 7.63 (d, IH, J= 3.6 Hz), 6.51 (bs, 2H), 3.82 (s, 3H), ESI- MS m/z 199 [M-H]^"; Anal. (C₇H₈N₂O₃S): C,H,N.

Thieno [3,4-</| pyrimidine-2,4-dione (11). 650 mg (3.2 mmol) of 10 were added to a solution of sodium methoxide (440 mg, 8.1 mmol) in methanol and the resulting mixture was stirred at room temperature for 4 hours. Then the solvent was removed under reduced pressure and the residue was dissolved in water. The solution was then cooled to 0 ⁰C, acidified with HCl 4N and the precipitate was filtered and dried in oven at 50 ⁰C to give compound 11 as a white solid in 89 % yield. ¹H NMR (DMSO-J₆) δ 10.9 (bs, 2H), 8.32 (d, IH, J= 3.2 Hz), 6.78 (d, IH, J = 3.2 Hz). ¹³C- NMR (DMSO-J₆) δ 159.20, 151.32, 138.34, 130.89, 122.63, 103.40, ESI-MS m/z 191 (M+Na)⁺ ; Anal. (C₆H₄N₂O₂S): C,H,N.

(SH-^'-Amino-l'-carboxyethyOthienoβ^-^pyrimidin-l^-dione Cll). Title compound was prepared following the same procedure described for the synthesis of compound 5a starting from 11 (487 mg, 2.8 mmol). The compound was obtained as a white amorphous solid in 7.7% overall yield. ¹H-NMR (DCl 20%wt in D₂O) δ 8.19 (d, IH, J = 3.0 Hz), 6.91 (d, IH, J = 3.0 Hz), 4.45-4.25 (m, 3H).¹³C-NMR (DCl 20%wt in D₂O) δ 169.15, 160.04, 152.44, 137.81, 132.71, 121.39, 105.12, 51.32, 44.36. ESI-MS m/s 256 [M+H]⁺ ; Anal. (C₉H₉N₃O₄S): C,H,N.

Example 4- Synthesis of Derivative 16, according to Scheme 4

13 14 15 c,d

Scheme 4. (a) Triphosgene, toluene; EtOH, NH₃; (b) MeONa, methanol; (c) NaH, (S)-3-[(tert- butoxycarbonyl)amino]oxetan-2-one, DMF; (d) TFA, CH₂CI₂

Ethyl 3-ureido-4,5,6,7-tetrahydrobenzo[ό]thiophene-2-carboxylate (14). To a solution of 13 (3 g, 13.3 mmol) in 40 niL of dry toluene, triphosgene (1.3 g, 4.4 mmol) was added and the solution was heated under reflux for 3 hours. Then the solvent was removed under reduced pressure, the residue was dissolved in 20 mL of ethanol and 40 mL of saturated solution of ammonia in ethanol was added and the mixture was stirred at room temperature for 18 hours. Then the solvent was evaporated and the crude product was purified by means of flash cromatography (n-hexane/EtOAc 1 :1) to provide derivative 14 as a white solid in 32.5% yield. ¹H-NMR (DMSO-J₆) δ 10.21 (bs, IH), 6.93 (bs, 2H), 4.23 (q, 2H, J= 7.0 Hz), 2.64 (m, 2H), 2.48 (m, 2H), 1.67 (m, 4H), 1.27(t, 3H, J= 7.0 Hz), ESI-MS m/z 267 [M-H]^" ; Anal. (Ci₂Hi₆N₂O₃S): C,H,N. 6,7,8,9-Tetrahydrobenzo [b] thieno [3,2-d\ pyrimidine-2,4-dione (15).

The title compound was prepared starting from 14 (1.16 g, 4.3 mmol) following the same procedure described for the synthesis of compound 11. Compound 15 was obtained as a white solid in 85% yield. ¹H-NMR (DMSO-J₆) δ 11.73 (bs, IH), 10.93 (bs, IH), 2.71 (m, 2H), 2.57 (m, 2H), 1.75-1.66 (m, 4H); ESI-MS m/z 221 [M-H]^" ; Anal. (C₁₀H₁₀N₂O₂S): C,H,N.

(S)- 1-(2 '- Amino-2 '-carboxyethyl)-6,7,8,9-tetrahydrobenzo [b] thieno [3,2-d] pyrimidine- 2,4-dione (16).

The title compound was prepared following the same procedure described for the synthesis of compound 5a starting from 15 (500 mg, 2.2 mmol). Compound 16 was obtained as a white amorphous solid in 30 % overall yield. ¹H-NMR (DCl 20%wt in D₂O) δ 4.25-4.15 (m, 2H), 4.06-3.99 (m, IH), 2.30 (m, 4H), 1.42-1.38 (m, 4H). ¹³C-NMR (DCl 20%wt in D₂O) δ 168.55, 160.17, 152.87, 151.49, 132.71, 129.20, 114.49, 51.14, 47.05, 24.99, 23.97, 22.36, 21.32; ESI-MS m/s 310 [M+H]⁺ ; Anal. (Ci₃Hi₅N₃O₄S): C,H,N.

Example 5- Synthesis of Derivatives 21a,b, according to Scheme 5

6a X=CH, Y=S 18a X=CH, Y=S 19a X=CH, Y=S 9 X=S, Y=CH 18b X= S, Y=CH 19b X=S, Y=CH

C

20a X=CH, Y=S, R=Boc -21a X=CH, Y=S, R=H

20b X= S, Y=CH, R=Boc -21b X= S, Y=CH, R=H

Scheme 5. (a) 1) triphosgene, toluene; 2) terf-butyl 3-(aminomethyl)thiophene-2-carboxylate, toluene; (b)1) TFA, CH₂CI₂; 2) NaOH, MeOH; (c) NaH, (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one, DMF; (d) TFA, CH₂CI₂

*-Butyl 3-{ 3- [(2-(methoxycarbonyl)thiophen-3-yl)ureido] methyl} thiophene-2- carboxylate (18a). To a solution of 6a (252 mg, 1.6 mmol) in 15 niL of dry toluene triphosgene (160 mg, 0.52 mmol) was added and the solution was stirred under reflux for 3 hours. Then the volatiles were removed under reduced pressure and the residue was dissolved in 10 mL of dry toluene. Subsequently, a solution of tert-butyl-3-(aminomethyl)thiophene-2- carboxylate (341 mg, 1.6 mmol) in 5 mL of toluene was added and the mixture was heated under reflux for 3 hours. Then the solvent was evaporated and the crude product was purified by means of flash chromatography (n-hexane/EtOAc 5:1) to provide derivative 18a as a white solid in 96.3 % yield. ¹H-NMR (DMSO-J₆) δ 9.34 (bs, IH), 8.15 (t, IH, J = 5.8 Hz), 7.91(d, IH, J= 5.5 Hz), 7.78-7.73 (m, 2H), 7.09 (d, IH, J= 5.2Hz), 4.54 (d, 2H, J = 5.8 Hz), 3.79 (s, 3H), 1.52 (s,9H); ESI-MS m/z 419 [M+Na]⁺; Anal. (Ci₇H₂₀N₂O₅S₂): C,H,N.

*-Butyl 3-{ 3- [(4-(methoxycarbonyl)thiophen-3-yl)ureido] methyl} thiophene-2- carboxylate (18b). Title compound was prepared following the same procedure described for the synthesis of 18a starting from 9 (400 mg, 2.54 mmol). The derivative 18b was obtained as a white solid in 77.7% yield. ¹H-NMR (DMSO-J₆) δ 9.03 (bs, IH), 8.27 (d, IH, J= 3.5 Hz), 7.92 (t, IH, J = 5.8 Hz), 7.73 (d, IH, J = 5.2 Hz), 7.64 (d, IH, J = 3.5 Hz), 7.09 (d, IH, J = 5.2 Hz), 4.53 (d, 2H, J = 5.8 Hz), 3.81 (s, 3H), 1.52 (s, 9H); ESI-MS m/z 419 [M+Na]⁺ ; Anal. (C₁₇H₂₀N₂O₅S₂): C,H,N.

3- [(2-Carboxythien-3-yl)methyl] thieno [3,2-</| pyrimidin-2,4-dione (19a). A mixture of 18a (470 mg, 1.2 mmol) in 15 mL of dichloromethane (CH₂Cl₂) and trifluoroacetic acid was stirred for 3 hours at room temperature. Then the solvent was removed under reduced pressure and the residue was suspended in 10 mL of MeOH and 8 mL of NaOH 4 M were added and the mixture was refluxed for 1 hour. Then methanol was evaporated under reduced pressure, water (5 mL) was added and the solution was cooled to 0 ⁰C , acidified with HCl 4N and the solid filtered and dried in oven at 50 ⁰C to give the derivative 19a as a white solid in 69 % yield. ¹H-NMR (DMSO-J₆) δ 13.16 (bs, IH), 11.99 (bs, IH), 8.09 (d, IH, J= 4.9 Hz), 7.67 (d, IH, J = 4.9 Hz), 6.95 (d, IH, J = 5.2 Hz), 6.74 (d, IH, J= 5.2 Hz), 5.31 (s, 2H); ESI-MS m/z 307 [M-H]^" ; Anal. (Ci₂H₈N₂O₄S₂): C,H,N. 3- [(2-Carboxythien-3-yl)methyl] thieno [3,4-</| pyrimidin-2,4-dione (19b). The title compound was prepared following the same procedure described for the synthesis of 19a starting from 18b (782 mg, 1.97 mmol). Derivative 19b was obtained as a white solid in 76.1% yield. ¹H-NMR (DMSO-J₆) δ 13.16 (bs, IH), 11.34 (bs, IH), 8.40 (d, IH, J = 3.2 Hz), 7.44 (d, IH, J= 5.2 Hz), 6.87 (d, IH, J= 3.2 Hz), 6.61 (d, IH, J= 5.2 Hz), 5.31 (m, 2H); ESI-MS m/z 307 [M-H]^"; Anal. (Ci₂H₈N₂O₄S₂): C,H,N. (S)-I- [2 '-(*-Butoxycarbonylamino)-2 '-carboxy ethyl] -3- [(2-carboxythien-3- yl)methyl] thieno [3,2-</] pyrimidin-2,4-dione (20a). To a solution of 19a (195 mg, 0.63 mmol) in dry DMF (10 mL) NaH (30 mg, 1.26 mmol) was added portionwise and the resulting mixture was stirred for 2 hours at room temperature. Then a solution of (5)-3-[(t-butoxycarbonyl)amino]oxetan-2-one (118 mg, 0.63 mmol) in DMF (2 mL) was added and the mixture was stirred at room temperature for 18 hours. Then the solvent was removed under reduced pressure and water (15 mL) was added to the residue. The pH was adjusted to 2 by the addition of HCl 2M. The aqueous phase was extracted with ethyl acetate (4 x 30 mL), the collected organic layers were dried over anhydrous sodium sulphate, filtered and the solvent was evaporated. The crude product was purified by means of flash chromatography using a gradient elution (from CH₂Cl₂/Me0H/Ac0H 97:3:0.1 v/v to CH₂Cl₂/Me0H/Ac0H 90:10:1 v/v) to give the derivative 19a as white amorphous solid in 38.4 % yield; ¹H-NMR (CD₃OD) δ 7.98 (d, IH, J = 5.2 Hz), 7.46 (d, IH, J = 5.2 Hz), 7.34 (d, IH, J = 5.2 Hz), 7.01 (d,lH, J = 5.2 Hz), 5.65-5.46 (m, 2H), 4.71-4.65 (m, 2H), 4.16 (m, IH), 1.22 (s, 9H); ESI-MS m/z 494 [M-H]^" ; Anal. (C₂₀H₂₁N₃O₈S₂): C,H,N.

(S)-l-[2'-(^-Butoxycarbonylamino)-2'-carboxyethyl]-3-[(2-carboxythien-3- yl)methyl] thieno [3,4-</] pyrimidin-2,4-dione (20b).

Title compound was prepared following the same procedure described for the synthesis of 20a starting from 19b (465 mg, 1.5 mmol). Derivative 20b was obtained as a white amorphous solid in 19.2% yield. ¹H-NMR (CD₃OD) δ 8.34 (d, IH, J = 3.2 Hz), 7.47 (d, IH, J = 5.2 Hz), 7.19 (d, IH, J = 3.2 Hz), 6.96 (d, IH, J = 5.2 Hz), 5.60-5.43 (m, 2H), 4.69-4.12 (m, 3H), 1.22 (s, 9H); ESI-MS m/z 494 [M-H]^" ; Anal. (C₂₀H_2IN₃O₈S₂): C,H,N. (S)-l-(2'-Amino-2'-carboxyethyl)-3-[(2-carboxythien-3-yl)methyl]thieno[3,2- </]pyrimidin-2,4-dione (21a). The title compound was prepared starting from 20a (120 mg, 0.24 mmol) following the same procedure described for the synthesis of compound 5a. Compound 21a was obtained as a white amorphous solid in 95% yield.

¹H-NMR (DCl 20%wt in D₂O) δ 7.55 (d,lH, J= 5.2 Hz), 7.05 (d, IH, J= 5.2 Hz), 6.73 (d, IH, J= 5.2 Hz), 6.30 (d,lH, J= 5.2), 4.92-4.78.(m,2H), 4.14-4.02 (m, 3H); ¹³C-NMR (DCl 20%wt in D₂O) δ 168.57, 165.19, 159.52, 152.81, 145.61, 144.24, 137.98, 132.68, 127.61, 126.80, 116.56, 113.07, 51.33, 46.00, 41.39; ESI-MS m/z 394 [M-H]^"; Anal. (Ci₅H₁₃N₃O₆S₂): C,H,N.

(S)-l-(2'-Amino-2'-carboxyethyl)-3-[(2-carboxythien-3-yl)methyl]thieno[3,4- </]pyrimidin-2,4-dione (21b). The title compound was prepared starting from 20b (143 mg, 0.28 mmol) following the same procedure used for the synthesis of compound 5a .Compound 21b was obtained as a white amorphous solid in 98% yield. ¹H-NMR (DCl 20% wt in D₂O) δ 8.27 (d, IH, J= 3.2 Hz), 7.22 (d, IH, J = 5.2 Hz), 7.01 (d, IH, J = 3.2 Hz), 6.65 (d, IH, J = 5.2 Hz), 5.31 (m, 2H), 4.46-4.26 (m, 2H), 4.04 (m, IH); ESI-MS m/s 394 [M-H]^" ; Anal. (Ci₅Hi₃N₃O₆S₂): C,H,N.

Example 6-Bio logical activity Cell culture and transfections The CHO-Kl cell line was used for transfections of expression vectors containing human iGluR5(Q)ib or 1GIuRl₁. The cells were cultured and maintained in Dulbecco's modified Eagle's medium DMEM) supplemented with 10 % (V /V) fetal calf serum and 2 mM L- proline in polystyrene culture flasks (12.5 or 25 cm²) and in a humidified atmosphere of 5 % CO₂ / 95 5 air, at 37°C. The cells were transiently transfected using the LipofectAMINE PLUS™ (Life Technologies) transfection kit as described by the manufacturer. Cells were incubated over night and used the day after transfection. The cells were seeded onto glass cover slips (3.5 mm) pre-coated with poly-D-lysine. 2.5 mL of cells suspension (0.1 x 10⁶ cells/mL) was added and the cells were allowed to attach to the glass cover slips for 1 h before us.

Patch-clamp electrophysiology in cell cultures

Electrophysiological measurements were performed in the voltage-clamp mode using conventional whole-cell patch-clamp techniques (Hamill et al., 1981). All data were obtained with an EPC-9 amplifier (HEKA-electronics, Lambrect, Germany) run by a Machintosh G3 computer. Experimental conditions and data acquisition were set and obtained using the PULSE-software accompanying the amplifier. Data was low-pass filtered and sampled directly to the hard disk. Pipettes were pulled from borosilicate glass using a horizontal electrode puller (Zeitz Instrumente, Augsburg, Germany), and the final pipette resistance was approximately 2 MΩ when filled with internal solution (in mM: 120 KCl, 31 KOH, 10 EGTA, 1.8 MgCl₂, 10 HEPES, pH 7.2) and submerged into the external solution (in mM: 140 NaCl, 5 KCl, 10 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4) used in the experiments.

Cover slips with cultured cells were transferred to a perfusion chamber mounted on the stage of an inverted microscope, and cells were continuously superfused with external solution at a rate of 2.5 mL/min. Compounds were dissolved in external solution and applied to the patched cell through double-barreled application pipettes fabricated from theta glass tubes (1.5 mm outer diameter, World Precision Instruments, Sarasota, FL= and mounted on a piezoelectric device (PZS-100HS, Burleigh Instruments, Quebec, Canada), which was controlled by the data-acquisition software. One barrel of the application pipette contained external solution and is used for preincubation and was, whereas the other barrel contained the agonist dissolved in external solution. In the resting phase the cell was only subjected to the fluid coming out of the first barrel and stimulation was performed by moving the application pipette sideward and thereby subjecting the cell to the solution coming out of the agonist containing barrel.

One minute after the onset of the flow in the application pipette a PULSE protocol was initiated and the current was recorded 3 times separated by 30 s waiting periods. For the agonist recordings at iGluR5(Q)ib the maximal efficacy was first evaluated by applying 10 mM L-glutamate to the cell in 100 ms pulses. After the establishment of a constant response level the cells were washed with external solution for several minutes (>10 min) until all L-glutamate had been washed out. Subsequently, 300 μM of either 5a or 8a was applied, but due to strong desensitization of these compounds only one response could be obtained at each cell.

For the agonist recordings at 1GIuRl₁, 30 μM cyclothiazide was included in all solutions. The maximal efficacy was evaluated by applying 10 mM L-glutamate to the cell in 1 s pulses. After the establishment of a constant response level the cells were washed with external solution for several minutes (>10 min) to eliminate all trace of L-glutamate. Subsequently, increasing concentrations (1-300 μM) of 5a or 8a was applied to the cell. Because of good reproducibility and a constant low series resistance (< 5 MΩ), several concentrations of 5a or 8a could be tested at each cell.

Antagonist effects: For eah cell, a control response induced by 3 mM L-glutamate (iGluR5(Q)ib) or 1 mM L-glutamate (IGIuRl₁) was recorded followed by recordings in the presence of increasing concentrations of 8a or 5a. Because of good reproducibility and a constant low series resistance (< 5 MΩ), several concentrations of 5a or 8a could be tested at each cell.

Table 1 shows the binding affinities for recombinant rat AMPA receptor subtypes and for kainate GluR5 and kainate GluR6 subunits in Sf9 and HEK293 cells.

Table 1: The binding affinities for recombinant rat AMPA receptor subtypes and for kainate GluR5 and kainate GluR6 subunits in Sf9 and HEK293 cells.

GluR5 Compd GIuRl₀ GluR2₀(7?) GluR3₀ GluR4₀ GluR6(V,C,R)

(R,S)- 21.9 ± 16.8 ± 2.9 20.6 ± 40.0 ± 1150 ± NA

AMPA 4.4 2.6 19.9 114

L-GIu 169 ± 27 282 ± 90 249 ± 12 354 ± 140 ± 7 332 ± 21

178

Kainate 477 ± 76 3700 ± 980 1980 ± 3565 ± 76 ± 27 12.7 ± 3.5

340 1,018

(5)-CPW399 109 ± 5 223 ± 24 1890 ± 2088 ± 44.0 ± NA

584 495^a 6.8

(R,S)- 410 ± 55 1200 ± 305 19000 ± 14200 ± 155 ± 22 NA

Willardiine 477 3560

(S)Sa. 58.2 ± 79.7 ± 29.6 311 ± 22 288 ± 5.2 ± 0.5 NA

18.3 124

(S)Sb 102 ± 30 126 ± 8 1500 ± 1590 ± 976 ± NA

570 588 470

(S)-Sa 49.9 ± 112 ± 26 936 ± 804 ± 38 5.3 ± 1.3 NA

6.7 114 a Pooled values from GluR4₀ and GluR4c₀, ,NA not active at Ki > 1 mM

Table 1 demonstrates that 5a and 8a derivatives obtained from CPW399 structure modifications are two of the most potent iGluR5 kainate receptor ligands with a significant selectivity. Compound 5a has a high affinity for AMPA receptor subtype GIuRi in comparison to the reference compound CPW399 (K; = 44 nM) but its affinity is 20 times lower on GIuRs receptors.

Table 2 shows Willardine, CPW399 and compounds 5a, 8a EC50S on recombinant AMPA receptors expressed in Xenopus lαevis oocytes Table 2. Potencies (EC50) at recombinant AMPA receptors expressed in Xenopus lcevis oocytes.

EC₅₀ (μM)

Compd GIuRl GluR2(0 GluR3 GluR4

(5)-Willardiine 11.5 ± 1.8^a'^* -

(5)-CPW399^*b 24.9 ± 3.6^d 13.9 ± 1.4^d 224 ± 20^d 34.3 ± 2.1^#'^d

(S)-(5a)^**b 11.8 ± 1.6 (8) 12.6 ± 0.6 (6) 33.9 ± 3.4 (8) 24.2 ± 2.4 (10)

(S)-(8a)^**b 7.2 ± 0.8 (6) 11.8 ± 1.1 (5) 120 ± 14 (9) 90 ± 15 (14)

EC50 at flop receptor isoforms, EC50 at flip receptor isoforms, EC50 at GluR4c₀ ^aKKiizzeellsszztteeiinn eett aall.. ((22000000)),, ^bbVVaalluueess aarree means ± S.E.M. (N value, number of oocytes, in parentheses), ^c Campiani et al. (2001).

5a, 8a affinity and potency on recombinant homomeric AMPA receptors are expressed in Xenopus lαevis oocytes. EC50 values demonstrate that tested compounds present better affinity for recombinant receptors GIuRi and GIuR₂ than GIUR3 and GIuR₄ even if compound 5a is more potent than 8a on GIUR3 and GIuR₄. receptors. Compound 8a shows higher selectivity than CPW399.

In addition, the pharmacology of 5a and 8a was evaluated at human 1GIuRl₁ and iGluR5(Q)lb expressed in the CHO-Kl cell line using whole-cell patch clamp electrophysiology. The compounds were both partial agonists at 1GIuRl₁ and responses could only be detected in the presence of 30 μM cyclothiazide, which inhibits the receptor desensitization (figure IA). 5a and 8a had EC50-values of 20.3 μM and 4.35 μM, respectively, whereas their maximal efficacies relative to 10 mM L-glutamate responses were 17.1% and 27.5%. Both of the compounds were also partial agonists at iGluR5(Q)ib, exhibiting rapid desensitization kinetics (figure IB). The two compounds displayed extremely slow wash off rates and their EC50- values could therefore not be determined. The low efficacies of 5a and 8a at iGluR5(Q)ib and slow recovery from desensitization suggested that 5a and 8a could have antagonistic properties at this receptor. Indeed, both compounds were able to completely inhibit the responses induced by 3 mM L-glutamate (figure 2) with IC50 values of 3.8 nM and 1.9 nM for 5a and 8a, respectively. L-glutamate responses (1 mM) at 1GIuRl₁ were also completely antagonized by higher concentrations of 8a due to its desensitization properties. The IC50 value for this inhibition was 0.37 μM.

Claims

1. A compound of Formula (I):

I wherein n=l or 2; A=O or S; B=sp2 carbon; D=sp2 carbon;

X, Y and Z =S, N, O, C or CH;

- when Z-B and Y-D are single bond, then B-D is carbon-carbon double bond; - when Z=N, C or CH, then Ri= H, Me, Et or is not present;

- when X=S and Z-B and Y-D are double bonds, then B-D is a single bond;

- when X=C or N, then R₂=H, Me, Et or is not present and R₃=H, Me, Et or is not present;

- when X=S or O, then R₂ and R₃ are not present;

- when Y=N or C or CH, then R₄=H, Me, Et; - when R₂ and R₃ are H, and when Z=S, O and when Ri is not present or when X=NH or CH and when Z=CH and when Ri=H, then X-Z=single bond;

- when X=C, R₂=H and R₃ is not present, or when X=N and Y=N or CH and R₄ is not present, then X-Y=double bond;

- when Ri-R₂=-(CH₂)₄-, -(CH=CH)₂-, cycloalkyl, cycloheteroalkyl (N, S, O), aromatic or heteroaromatic (N, S) systems, then X=C and R₃ is not present and Z=C and Y=O or S and R₄ is not present, or Y=N and R₄ is H, Me, Et, Alkyl (C₃-C₅) or Y=CH and R₄=H; - when R₃-R₄=-(CH₂)₄-, -(CH=CH)₂-, cycloalkyl, cycloheteroalkyl (N, S, O), aromatic or heteroaromatic (N, S) systems, then X=C and R₂ is not present and Y=C and Z=O, S and Ri is not present or Z=N and Ri is H, Me, Et, Alkyl (C₃-C₅) or Z=CH and Ri=H; R₅=H, alkyl (Ci-C₈) or cycloalkyl (C₅-C₆); R₆=H, (2-carboxythien-3-yl)methyl, (5-carboxythien-2-yl)methyl, (5-carboxythien-3- yl)methyl, (4-carboxythien-3-yl)methyl, (4-carboxythien-2-yl)methyl, (3-carboxythien-2- yl)methyl, 2-carboxybenzyl, 3-carboxybenzyl, 4-carboxybenzyl, (2-carboxyfuran-3- yl)methyl, (5-carboxyfuran-2-yl)methyl, (5-carboxyfuran-3-yl)methyl, (4-carboxyfuran-3- yl)methyl, (4-carboxyfuran-2-yl)methyl, (3-carboxyfuran-2-yl)methyl, (2-carboxypyrrol-3- yl)methyl, (5-carboxypyrrol-2-yl)methyl, (5-carboxypyrrol-3-yl)methyl, (4-carboxypyrrol- 3-yl)methyl, (4-carboxypyrrol-2-yl)methyl, (3-carboxypyrrol-2-yl)methyl, (2- phosphonothien-3 -yl)methyl, (5 -phosphonothien-2-yl)methyl, (5 -phosphonothien-3 - yl)methyl, (4-phosphonothien-3-yl)methyl, (4-phosphonothien-2-yl)methyl, (3- phosphonothien-2-yl)methyl, 2-phosphonobenzyl, 3-phosphonobenzyl, 4- phosphonobenzyl, (2 -phosphono furan-3 -yl)methyl, (5-phosphonofuran-2-yl)methyl, (5- phosphono furan-3 -yl)methyl, (4-phosphono furan-3 -yl)methyl, (4-phosphono furan-2- yl)methyl, (3-phosphonofuran-2-yl)methyl, (2-phosphonopyrrol-3-yl)methyl, (5- phosphonopyrrol-2-yl)methyl, (5-phosphonopyrrol-3-yl)methyl, (4-phosphonopyrrol-3- yl)methyl, (4-phosphonopyrrol-2-yl)methyl, (3-phosphonopyrrol-2-yl)methyl, and the corresponding methyl and ethyl esters, (2-(lH-tetrazol-5-yl)thien-3-yl)methyl, (5-(1H- tetrazol-5 -yl)thien-2-yl)methyl, (5 -( lH-tetrazol-5 -yl)thien-3 -yl)methyl, (4-( lH-tetrazol-5 - yl)thien-3-yl)methyl, (4-(lH-tetrazol-5-yl)thien-2-yl)methyl, (3-(lH-tetrazol-5-yl)thien-2- yl)methyl, 2-(lH-tetrazol-5-yl)benzyl, 3-(lH-tetrazol-5-yl)benzyl, 4-(lH-tetrazol-5- yl)benzyl, (2-(lH-tetrazol-5-yl)furan-3-yl)methyl, (5-(lH-tetrazol-5-yl)furan-2-yl)methyl, (5-(lH-tetrazol-5-yl)furan-3-yl)methyl, (4-(lH-tetrazol-5-yl)furan-3-yl)methyl, (4-(1H- tetrazol-5-yl)furan-2-yl)methyl, (3-(lH-tetrazol-5-yl)furan-2-yl)methyl, (2-(lH-tetrazol-5- yl)pyrrol-3 -yl)methyl, (5 -( lH-tetrazol-5 -yl)pyrro l-2-yl)methyl, (5 -( lH-tetrazol-5 -yl)pyrrol- 3 -yl)methyl, (4-( lH-tetrazol-5 -yl)pyrro 1-3 -yl)methyl, (4-( lH-tetrazol-5 -yl)pyrro 1-2- yl)methyl, (3 -( lH-tetrazol-5 -yl)pyrro l-2-yl)methyl; a salt, tautomer, geometric isomer, enantiomer, diastereoisomer or racemate thereof able to bind at least one glutamatergic system receptor for medical use.

2. The compound according to claim 1 being the (5)-l-(2'-Amino-2'-carboxyethyl)- thieno[3,4-ύT]pyri^midine-2,4(lH,3H,5H,7H)-dione (5a).

3. The compound according to claim 1 being the (S)-l-(2'-Amino-2'-carboxyethyl)-6,6- dimethyl-2,3,4,5,6,7-hexahydrocyclopentapyrimidin-2,4-dione (5b).

4. The compound according to claim 1 being the (5)-l-(2'-Amino-2'- carboxyethyl)thieno[3,2-<i]pyrimidin-2,4-dione (8a).

5. The compound according to claim 1 being the (5)-l-(2'-Amino-2'- carboxyethyl)thieno[2,3-J]pyrimidin-2,4-dione (8b).

6. The compound according to claim 1 being the (5)-l-(2'-Amino-2'- carboxyethyl)thieno[3,4-J]pyrimidin-2,4-dione (12).

7. The compound according to claim 1 being the (5)-l-(2'-Amino-2'-carboxyethyl)- 6,7,8,9-tetrahydrobenzo[δ]thieno[3,2-d]pyrimidine-2,4-dione (16).

8. The compound according to claim 1 being the (5)-l-(2'-Amino-2'-carboxyethyl)-3-[(2- carboxythien-3-yl)methyl]thieno[3,2-(i]pyrimidin-2,4-dione (21a).

9. The compound according to claim 1 being the (5)-l-(2'-Amino-2'-carboxyethyl)-3-[(2- carboxythien-3-yl)methyl]thieno[3,4-(i]pyrimidin-2,4-dione (21b).

10. A compound having the general formula as in claim 1 wherein the compound having A=O, n=l, Z=CH, X=C, Y=CH, Ri=R₂=R₃=R₄=R₅=H is not included.

11. A pharmaceutical compositions comprising a pharmacologically effective and acceptable amount of at least one compound according to any of previous claims.

12. Use of the compound according to claims 1-10 for the preparation of a medicament for pain therapy and/or prevention.

13. The use according to claim 12 wherein pain is neuropathic pain.

14. Use of the compound according to claims 1-10 for the preparation of a medicament for therapy and/or prevention of a CNS disease.

15. The use according to claim 14 wherein the CNS disease is comprised in the group of: epilepsy, ALS, neurodegenerative disease or schizophrenia.

16. The use according to claim 15 wherein the neurodegenerative disease is Alzheimer's disease.

17. Process for the preparation of a compound according to claim 1 comprising the phases reported in Schemes 1-5.