NOVEL THIAZOLO[5,4-D]PYRIMIDINE DERIVATIVES AS INVERSE AGONISTS OF A2A ADENOSINE RECEPTORS

Abstract

The present invention refers to novel thiazolo[5,4-d]pyrimidine derivatives that are inverse agonists of the adenosine A.sub.2A receptor, to a process for their preparation, to the pharmaceutical compositions containing them and to their use in the medical field, in particular in the therapeutic treatment of diseases or disorders associated to an activity of the adenosine A.sub.2A receptor, and more in particular in the therapeutic treatment of neurological diseases, of pain, of cancer, and of dermal fibrosis and scarring.

Claims

1. Compounds of general formula (I) ##STR00005## wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl optionally substituted, (CH.sub.2)naryl optionally substituted and (CH.sub.2).sub.nheteroaryl optionally substituted, wherein n is an integer ranging from 0 to 4, and pharmaceutically acceptable salts, tautomers and enantiomers thereof.

2. The compounds according to the claim 1, wherein R.sub.3 is (CH.sub.2)naryl or (CH.sub.2).sub.nheteroaryl, optionally substituted, wherein n=1 or 2.

3. A compound according to claim 1, selected from the group consisting of: 2-(furan-2-yl)-N.sup.5-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(4-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(3-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 3-((7-amino-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5-yl)amino)methyl) phenol 2-(furan-2-yl)-N.sup.5-(furan-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(thiophen-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(pyridin-3-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(pyridin-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine N.sup.5-(3-fluorobenzyl)-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(2-(thiophen-2-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-propyl-[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine N.sup.5-butyl-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(thiophen-3-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(pyridin-4-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(pyrazin-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine 2-(furan-2-yl)-N.sup.5-(2-(furan-2-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine, and 2-(furan-2-yl)-N.sup.5-(2-(pyridin-3-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine.

4. A pharmaceutical composition comprising a compound of claim 1, in admixture with one or more excipients and/or diluents and/or pharmaceutically acceptable carriers.

5. The pharmaceutical composition according to claim 4, further comprising one or more further active principles.

6. (canceled)

7. A method for the therapeutic treatment of diseases or disorders associated with an activity of the adenosine A.sub.2A receptor, comprising administering a compound of claim 1 to a subject in need thereof.

8. The method according to claim 7, wherein said diseases or disorders are selected from the group consisting of neurological pathologies, pain, cancer, dermal fibrosis and scarring.

9. A process for the preparation of the compounds of general formula (I) as defined in claim 1, comprising the following steps according to the scheme illustrated below: ##STR00006## wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl optionally substituted, (CH.sub.2)naryl optionally substituted and (CH.sub.2).sub.nheteroaryl optionally substituted, wherein n is an integer ranging from 0 to 4: a) reacting compound A, 5-amino-6-sulphanylpyrimidin-2,4-diol, with 2-furoylchloride to form a compound B, which is the 2-(furan-2-yl)-thiazolo[5,4-d]pyrimidin-5,7-diol; b) chlorinating compound B obtained in step a) with substitution of the two hydroxyl groups and formation of a 5,7-dichloro derivative C; c) substituting of chloro at position 7 in the compound C obtained in step b) by reacting it with an aqueous solution of ammonia, to form a compound D 7-amino-5-chloro substituted; and d) substituting of chloro at position 5 in the compound D obtained in step c) with an amine R.sub.3NH.sub.2 appropriately selected in order to obtain compounds of formula (I) with the desired R.sub.3 group.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0024] The Figures from 1 to 6 here attached show some of the most significant results obtained in the experimental studies described in detail in the following in Example 6. In particular:

[0025] FIGS. 1A, 1B, 10, 1D, 1E, 1F and 1G represent the competition curves of [.sup.3H]-ZM 241385 at the human receptor A.sub.2A of adenosine for compound 1 (FIG. 1A), compound 3 (FIG. 1B), compound 5 (FIG. 10), compound 6 (FIG. 1D), compound 7 (FIG. 1E), and compound 10 (FIG. 1F) of the present invention, which show the presence of two binding sites while the reference compound ZM 241385 (FIG. 1G) is characterised by single-phase curve;

[0026] FIGS. 2A, 2B, 2C, 2D, 2E, 2F and 2G show the inhibition curves of the cAMP levels in CHO cells transfected with the human receptor A.sub.2A obtained for the compound 1 (FIG. 2A), for the compound 3 (FIG. 2B), for the compound 5 (FIG. 2C), for the compound 6 (FIG. 2D), for the compound 7 (FIG. 2E), and for the compound 10 (FIG. 2F) of the present invention, and for the reference compound ZM 241385 (FIG. 2G); in these figures the effects of the active compound are expressed as percentage with respect to the basal production of cAMP;

[0027] FIGS. 3A, 3B, 3C, 3D, 3E, 3F and 3G show the inhibition curves of the cAMP levels in CHO cells transfected with the human receptor A.sub.2A obtained for the compound 1 (FIG. 3A), for the compound 3 (FIG. 3B), for the compound 5 (FIG. 3C), for the compound 6 (FIG. 3D), for the compound 7 (FIG. 3E), and for the compound 10 (FIG. 3F) of the present invention, and for the reference compound ZM 241385 (FIG. 3G), wherein the effects of the compounds are expressed as percentage with respect to the production of cAMP in the presence of CGS 21680 (10 nM);

[0028] FIG. 4 shows, in the form of histograms, the percentages of cells viability with respect to a control for samples of breast cancer cells MRMT-1 treated with CGS 21680 and with the compounds 1, 5, 6, 7 and 10 of the present invention, according to what described in the experiments of cellular proliferation of the following Example 6;

[0029] FIGS. 5A, 5B, 5C, 5D show, in the form of histograms, the number of abdominal contractions induced by intraperitoneal administration of acetic acid in mice following treatment with carrier and with increasing doses of compound 1 (FIG. 5A) and of compound 3 (FIG. 5B) of the invention, and of the reference compounds ZM 241385 (FIG. 5C) and of morphine (FIG. 5D);

[0030] FIGS. 6A, 6B, 6C and 6D show, in the form of histograms, the latency time of the tail withdrawal after immersion in hot water measured on mice following administration of carrier and increasing doses of compound 1 (FIG. 6A) and of the compound 3 (FIG. 6B) of the invention, and of the reference compounds ZM 241385 (FIG. 6C) and of morphine (FIG. 6D).

DETAILED DESCRIPTION OF THE INVENTION

[0031] In the present invention by the term aryl a monovalent aromatic hydrocarbon group is meant preferably having a single ring (for instance phenyl). Unless defined otherwise, these aryl groups typically contain from 6 to 10 carbon atoms in the ring. Preferred aryl groups comprise phenyl and benzyl.

[0032] As used herein, the term heteroaryl refers to heteroaromatic groups, formed by a minimum of 5 to a maximum of 10 terms and containing from 1 to 3 heteroatoms, selected for instance from the group consisting of N, O, S, and oxidised derivatives; preferred heteroaryl groups comprise thienyl, furyl, and pyridyl.

[0033] As used herein, the term alkyl refers to a monovalent saturated hydrocarbon, which may be linear or branched. Representative alkyl groups include, as a non-limitative example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, n-pentyl, n-hexyl, n-eptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-hexadecyl, and n-octadecyl. Methyl is a preferred alkyl group according to the invention.

[0034] By optionally substituted group a non-substituted group is meant or a group substituted with one or more substituents including nitro, cyano, halogen, amino, amido, oxo, carboxy, hydroxy, alkoxy, sulphoxy, or aliphatic chains, for example alkyl, alkenyl or alkynyl.

[0035] As used herein, the term alkoxy represents a monovalent group of formula (alkyl)-O, wherein the term alkyl is defined as above and typically is methoxy, whereas the terms alkenyl and alkynyl refer to unsaturated hydrocarbon radicals, linear or branched, respectively with double or triple bonds.

[0036] As used herein, by the term halogen is meant fluoro, chloro, bromo or iodio. Preferred compounds according to the present invention are the compounds of general formula (I) wherein R.sub.3 is (CH.sub.2).sub.naryl or (CH.sub.2).sub.nheteroaryl, optionally substituted, wherein n=1 or 2.

[0037] The compounds of formula (I) as defined above can be prepared according to the synthetic scheme illustrated in the Scheme 1 illustrated below, and exemplified in the preparations of the compounds 1-17 of the following Examples 1-5:

##STR00002##

[0038] wherein R.sub.3 is defined as above.

[0039] The preparation process of the Scheme 1 comprises therefore the following steps:

[0040] a) reaction of the starting compound A, 5-amino-6-sulphanylpyrimidin-2,4-diol, with 2-furoylchloride to yield a compound B that is the 2-(furan-2-yl)-thiazolo[5,4-d]pyrimidin-5,7-diol;

[0041] b) chlorination of the compound B obtained in step a) with substitution of the two hydroxyl groups and formation of a 5,7-dichloro derivatives C;

[0042] c) substitution of chlorine at position 7 in the compound C obtained in step b) by its reaction with an aqueous solution of ammonia, to yield a compound D 7-amino-5-chloro substituted; and

[0043] d) substitution of chlorine at position 5 in the compound D obtained in step c) with an amine R.sub.3NH.sub.2 appropriately selected to obtain the compounds of formula (I) with the desired group R.sub.3.

[0044] According to a preferred embodiment of the present invention, the process of Scheme 1 may be so accomplished:

##STR00003##

[0045] The process can therefore include heating of the starting compound A (G. P. Hager et al. J. Am. Pharm. As. 1955, 44, 193-196) easily synthesisable starting from commercial products, with 2-furoylchloride in N-methyl-2-pyrrolidone (NMP), so as to obtain with high yields the desired compound B, i.e. the 2-(furan-2-yl)-thiazolo[5,4-d]pyrimidin-5,7-diol. The so obtained compound B is treated with phosphoryl chloride to yield the 5,7-dichloro derivative C, which is then made to react with an aqueous solution of ammonia to yield the compound 7-amino-5-chloro-substituted (compound D). The reaction of this latter, carried out by microwaves irradiation with a (hetero)arylalkyl or alkylamine, appropriately selected depending on the group R.sub.3 to be introduced. The so obtained compounds bearing methoxy groups can then be transformed in the corresponding phenols by means of procedures known to any person skilled in the art, for instance by treatment with BBr.sub.3 in CH.sub.2Cl.sub.2. By this process, as described in the Examples 1-5 reported below, are obtained the compounds 1-17 wherein R.sub.3 is defined as follows:

##STR00004##

[0046] If in the compounds of general formula (I) one or more asymmetric carbon atoms are present, the present invention comprises not only the respective pure enantiomeric forms, but also their scalemic or racemic mixtures. Moreover, if the compounds of general formula (I) exist in tautomeric forms, the present invention comprises any possible tautomeric forms.

[0047] In the present invention the term pharmaceutically acceptable salt refers to derivatives of the compound of formula (I) wherein the compound was appropriately modified by conversion of any acidic or basic group, if present, into the corresponding addition salt with any base or acid conventionally considered as acceptable for pharmaceutical use.

[0048] Suitable examples of these salts may include addition salts with organic or mineral acids of basic residues such as amine groups, or addition salts of acidic residues, such as carboxylic acids with bases such as those containing alkaline and alkaline earth metals (sodium, potassium, magnesium and calcium) or appropriate organic amines. Possibly, the compounds of general formula (I) described in the present invention can form salts with aminoacids too.

[0049] The compounds of general formula (I) defined above according to the invention are useful in the treatment of diseases or disorders that are responsive to the blockade of adenosine A.sub.2A receptors, and they can be used, alone or in combinations of two or more compounds, in pharmaceutical compositions with pharmaceutically acceptable carriers, excipients and/or diluents, and with possible further active principles. The present compounds can be present in the compositions as such or in the form of pharmaceutically acceptable salts.

[0050] The present pharmaceutical compositions can be formulated in several pharmaceutical forms, for different administration routes, for example as oral compositions or injectable solutions. They can moreover find application in the treatment of diseases or disorders associated to the activity of adenosine A.sub.2A receptors, which can be treated therefore therapeutically thanks to the blockade of activity of the adenosine A.sub.2A receptors. In other words, in the present invention, by diseases or disorders associated or related to an activity of the adenosine A.sub.2A receptor are meant diseases or disorders that are responsive to the inhibition of an activity of the adenosine A.sub.2A receptors, such as in particular neurologic diseases, pain, dermal fibrosis and scarring, and cancer.

[0051] Experimental Part

[0052] Chemistry.

[0053] All reagents and solvents available on the market were purchased from Sigma Aldrich (Italy), and have been used without further purification. The microwave assisted synthesis were performed using an initiator EXP Microwave Biotage equipment (irradiation frequency: 2.45 GHz). Analytical silica gel plates (0.20 mm, F254, Merck, Germany), preparative silica gel plates (2 mm, F254, Merck, Germany) and silica gel 60 (70-230 mesh, Merck, Germany) were used for analytical and preparative TLC, and for column chromatography, respectively. Melting points were determined in glass capillary tubes on a Gallenkamp melting point apparatus. Compounds were named according to the IUPAC rules as applied by ACD/ChemSketch. Elemental analyses were performed with an elemental analyser for C, H and N Flash E1112 Thermofinnigan. IR spectra were recorded with a Perkin-Elmer Spectrum RX I spectrometer in Nujol dispersions and the data were expressed in cm.sup.1. Nuclear Magnetic Resonance experiments (NMR) were conducted on a Bruker Avance 400 (400 MHz for .sup.1H and 100 MHz for .sup.13C NMR). The spectra were recorded at 300 K using DMSO-d6 as a solvent. Spectrum chemical shifts for .sup.1H and .sup.13C were recorded in parts per million using residual non-deuterated solvent as internal standard. The following abbreviations are used: s=singlet, d=doublet, t=triplet, m=multiplet, br=broad, ar=aromatic protons, exch=exchangeable proton.

Example 1

Preparation of 2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diol (Compound B of the Scheme 1)

[0054] To a suspension of 5-ammino-6-sulphanylpyrimidin-2,4-diol (Compound A) (10 mmol) in anhydrous NMP, furan-2-carbonyl chloride (10 mmol) was slowly added. The resulting mixture was heated to 150 C. under N.sub.2 atmosphere for 14 hours. The reaction mixture was then cooled down to room temperature and diluted with cold water (100 ml) obtaining a precipitate that was then collected by filtration. Yield 82%. Pf: >300 C. (DMSO). .sup.1H NMR: 6.73-6.74 (m, 1H, ar), 7.18-7.19 (m, 1H, ar), 7.93 (s, 1H, ar), 11.39 (br s, 1H, exch), 12.07 (br s, 1H, exch). .sup.13C NMR: 110.75, 113.37, 130.71, 147.37, 147.95, 149.49, 150.47, 157.85. IR: 1673, 1703. Anal. calc. for C.sub.9H.sub.5N.sub.3O.sub.3S.

Example 2

Preparation of 5,7-dichloro-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidine (Compound C of the Scheme 1)

[0055] To a suspension in POCl.sub.3 (20 ml) of the 5,7-diol (5 mmol) prepared as described above in the Example 1, was added at room temperature N,N-dimethylaniline (1.15 mL, 10 mmol). The resulting mixture was heated at 100 C. for 6 hours. The organic phase was concentrated under vacuum, then the raw material was re-dissolved twice with cyclohexane (20 ml) and the organic portions evaporated under vacuum. The obtained residue was added with a mixture of water and ice (100 g), yielding a precipitate that was collected by filtration and used in the subsequent step without further purification. Yield 73%. .sup.1H NMR: 6.88-6.89 (m, 1H, ar), 7.65-7.66 (m, 1H, ar), 8.17 (s, 1H, ar). .sup.13C NMR: 114.38, 116.23, 142.58, 147.02, 152.80, 152.84, 158.78, 167.66.

Example 3

Preparation of 5-chloro-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-7-amine (Compound D of the Scheme 1)

[0056] A suspension of the 5,7-dichloro derivative (4 mmol) prepared as described above in the Example 2, in a mixture of 33% ammonia aqueous solution (15 ml) and ethanol (10 ml) was heated at 85 C. for 6 hours. The reaction mixture was then cooled down to room temperature, obtaining a solid product, which was collected by filtration. Yield 75%. Pf: 296-300 C. dec. (2-metoxyethanol/H.sub.2O). .sup.1H NMR: 6.80-6.81 (m, 1H, ar), 7.29-7.30 (m, 1H, ar), 8.03 (s, 1H, ar), 8.27 (br s, 2H, exch). .sup.13C NMR: 112.73, 113.62, 130.33, 147.70, 152.92, 155.35, 158.12, 163.05. IR: 3136, 3298. Anal. calc. for C.sub.9H.sub.5ClN.sub.4OS.

[0057] ESEMPIO 4

[0058] General procedure for the preparation of the compounds 1-3 and 5-17

[0059] A (hetero)arylalkylamine or an alkylamine (3 mmol), appropriately selectable by any person with ordinary skills in the art depending on the final product to be obtained, was added to a solution in n-BuOH (2 ml) of the 5-chloro-7-amino derivative (1 mmol) prepared as described above in the Example 3. The reaction mixture was then irradiated with microwaves at 200 C. for 20 minutes, then cooled down to room temperature and rendered basic with an aqueous solution of KOH (50%, 2 ml). The addition of water (approximately 100 ml) yielded a solid that was collected by filtration and washed with diethyl ether. The raw material was purified by crystallisation with organic solvents or by chromatography. The following compounds were so prepared and characterised:

Compound 1 2-(furan-2-yl)-N.SUP.5.-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0060] Yield 81%. Pf: 254-256 C. (acetic acid). .sup.1H NMR: 3.81 (s, 3H, OCH.sub.3), 4.47 (d, 2H, CH.sub.2, J=6.2 Hz), 6.71-6.72 (m, 1H, ar), 6.87 (t, 1H, J=7.3), 6.97 (d, 1H, ar, J=7.8 Hz), 7.04-7.05 (m, 1H, ar), 7.14-7.24 (m, 5H, 2 ar+3 exch), 7.89 (s, 1H, ar). .sup.13C NMR: 55.69, 110.04, 110.67, 113.14, 120.51, 127.46, 128.00, 128.43, 148.62, 157.05, 157.56, 160.61. Anal. calc. for C.sub.17H.sub.15N.sub.5O.sub.2S.

Compound 2 2-(furan-2-yl)-N.SUP.5.-(4-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0061] Yield 68%. Pf: 189-192 C. (EtOAc). .sup.1H NMR: 3.72 (s, 3H, OCH.sub.3), 4.42 (d, 2H, CH.sub.2, J=5.8 Hz), 7.02-7.03 (m, 1H, ar), 6.86 (d, 2H, ar, J=7.3 Hz), 7.04-7.05 (m, 1H, ar), 7.17 (br s, 2H, exch), 7.24-7.31 (m, 3H, 2 ar+1 exch), 7.89 (s, 1H, ar). .sup.13C NMR: 44.08, 55.47, 110.04, 113.16, 114.01, 128.83, 133.03, 148.61, 157.49, 158.46, 160.39. Anal. calc. for C.sub.17H.sub.15N.sub.5O.sub.2S.

Compound 3 2-(furan-2-yl)-N.SUP.5.-(3-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0062] Yield 70%. Pf: 199-201 C. (EtOAc). .sup.1H NMR: 3.72 (s, 3H, OCH3), 4.46 (d, 2H, CH.sub.2, J=6.2 Hz), 6.71-6.72 (m, 1H, ar), 6.76-6.78 (m, 1H, ar), 6.88-6.89 (m, 2H, ar), 7.04-7.05 (m, 1H, ar), 7.19-7.23 (m, 3H, 1 ar+2 exch), 7.32-7.38 (m, 1H, exch), 7.90 (s, 1H, ar). .sup.13C NMR: 44.59, 55.39, 110.06, 112.24, 113.17, 119.70, 129.64, 142.83, 148.60, 157.51, 159.69, 160.42. Anal. calc. for C.sub.17H.sub.15N.sub.5O.sub.2S.

Compound 5 2-(furan-2-yl)-N.SUP.5.-(furan-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0063] Yield 73% Pf 220-224 C. (chromatographic column with cyclohexane/ethyl acetate 3/7 as eluent).sup.1H NMR: 4.47 (d, 2H, CH.sub.2, J=6.0 Hz), 6.24-6.25 (m, 1H, ar), 6.35-6.37 (m, 1H, ar), 6.71-6.72 (m, 1H, ar), 7.05-7.06 (m, 1H, ar), 7.23-7.27 (m, 3H, exch), 7.55 (s, 1H, ar), 7.90 (s, 1H, ar). Anal. calc. for C.sub.14H11 N.sub.5O.sub.2S.

Compound 6 2-(furan-2-yl)-N.SUP.5.-(thiophen-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0064] Yield 89% Pf 192-196 C. (isopropanol).sup.1H NMR: 4.64 (d, 2H, CH.sub.2, J=6.2 Hz), 6.71-6.73 (m, 1H, ar), 6.93-6.95 (m, 1H, ar), 6.99-7.00 (m, 1H, ar), 7.06-7.07 (m, 1H, ar), 7.23 (s, 2H, exch), 7.32-7.33 (m, 1H, ar), 7.40 (t, 1H, exch, J=6.2 Hz), 7.90 (s, 1H, ar). Anal. calc. for C.sub.14H.sub.11N.sub.5OS.sub.2.

Compound 7 2-(furan-2-yl)-N.SUP.5.-(pyridin-3-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0065] Yield 91% Pf 212-215 C. (EtOH).sup.1H NMR: 4.60 (d, 2H, CH.sub.2, J=6.1 Hz), 6.71-6.72 (m, 1H, ar), 7.04-7.05 (m, 1H, ar), 7.22-7.25 (m, 3H, 1 ar+2 exch), 7.31 (d, 1H, ar, J=7.9 Hz), 7.37 (br s, 1H, exch), 7.73 (t, 1H, ar, J=7.6 Hz), 7.89 (s, 1H, ar), 7.19-7.23 (m, 3H, ar), 8.49-8.50 (m, 1H, ar). Anal. calc. for C.sub.15H.sub.12N.sub.6OS.

Compound 8 2-(furan-2-yl)-N.SUP.5.-(pyridin-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0066] Yield 94% Pf 213-217 C. (EtOH) 1H NMR: 4.62 (d, 2H, CH.sub.2, J=5.9 Hz), 6.71-6.74 (m, 1H, ar), 7.05-7.09 (m, 1H, ar), 7.25-7.40 (m, 5H, 2ar+3 exch), 7.77-7.81 (m, 1H, ar), 7.87-7.90 (m, 1H, ar), 8.50-8.52 (m, 1H, ar). Anal. calc. for C.sub.15H.sub.12N.sub.6OS.

Compound 9 N.SUP.5.-(3-fluorobenzyl)-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0067] Yield 64% Pf 217-221 C. (nitromethane).sup.1H NMR: 4.51 (d, 2H, CH.sub.2, J=6.0 Hz), 6.71-6.72 (m, 1H, ar), 7.01-7.22 (m, 6H, 4ar+2exch), 7.32-7.37 (m, 1H, ar), 7.40-7.42 (m, 1H, exch), 7.89-7.91 (s, 1H, ar). Anal. calc. for C.sub.16H.sub.12FN.sub.5OS.

Compound 10 2-(furan-2-yl)-N.SUP.5.-(2-(thiophen-2-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0068] Yield 78% Pf 216-218 C. (nitromethane).sup.1H NMR: 3.06 (t, 2H, CH.sub.2, J=7.2 Hz), 3.51 (dd, 2H, CH.sub.2, J=13.4, 7.0 Hz), 6.71-6.72 (m, 1H, ar), 6.92-6.96 (m, 3H, ar), 7.04-7.05 (m, 1H, ar), 7.18 (br s, 2H, exch), 7.32-7.34 (m, 1H, exch), 7.89-7.90 (m, 1H, ar). Anal. calc. for C.sub.15H.sub.13N.sub.5OS.sub.2.

Compound 11 2-(furan-2-yl)-N.SUP.5.-propyl-[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0069] Yield 65% Pf 204-207 C. (EtOAc).sup.1H NMR: 0.87-0.91 (m, 3H, CH.sub.3), 1.50-1.55 (m, 2H, CH.sub.2), 3.18-3.22 (m, 2H, CH.sub.2), 6.71-6.72 (m, 1H, ar), 6.83 (br s, 1H, exch), 7.03-7.04 (m, 1H, ar), 7.10 (br s, 2H, exch), 7.89-7.90 (m, 1H, ar). Anal. calc. for C.sub.12H.sub.13N.sub.5OS.

Compound 12 N.SUP.5.-butyl-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0070] Yield 70% Pf 201-205 C. (EtOAc).sup.1H NMR: 0.85-0.90 (m, 3H, CH.sub.3), 1.32-1.35 (m, 2H, CH.sub.2) 1.48-1.52 (m, 2H, CH.sub.2), 3.24-3.26 (m, 2H, CH.sub.2), 6.71-6.72 (m, 1H, ar), 6.81 (br s, 1H, exch), 7.03-7.04 (m, 1H, ar), 7.10 (br s, 2H, exch), 7.88-7.89 (m, 1H, ar). Anal. calc. for C.sub.13H.sub.15N.sub.5OS.

Compound 13 2-(furan-2-yl)-N.SUP.5.-(thiophen-3-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0071] Yield 74% Pf: 209-212 C. (EtOAc). .sup.1H NMR: 4.47 (d, 2H, CH.sub.2, J=6.1 Hz), 6.72 (dd, 1H, ar, J=3.3 Hz, 1.7 Hz), 7.05-7.06 (m, 1H, ar), 7.10 (broad s, 1H, exch), 7.22 (broad s, 2H, exch), 7.29-7.32 (m, 2H, ar), 7.45 (dd, 1H, ar, J=4.8 Hz, 3.0 Hz), 7.89-7.90 (m, 1H, ar). Anal calc. for C.sub.14H.sub.11N.sub.5OS.sub.2.

Compound 14 2-(furan-2-yl)-N.SUP.5.-(pyridin-4-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0072] Yield 72% Pf: 223-225 C. (preparative plate with ethyl acetate/methanol 9/2.5 as eluent). .sup.1H NMR: 4.51 (d, 2H, CH.sub.2, J=6 Hz) 6.71-6.72 (m, 1H, ar), 7.04-7.05 (m, 1H, ar), 7.23 (s, 2H, exch), 7.30-7.31 (m, 2H, ar), 7.47 (broad s, 1H, exch), 7.89-7.90 (m, 1H, ar), 8.47-8.48 (m, 2H, ar) Anal calc. for C.sub.15H.sub.12N.sub.6OS.

Compound 15 2-(furan-2-yl)-N.SUP.5.-(pyrazin-2-ylmethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0073] Yield 68% Pf: 236-238 C. (acetic acid/EtOH). .sup.1H NMR: 4.63 (d, 2H, CH.sub.2, J=6.1 Hz), 6.72 (dd, 1H, ar, J=3.4 Hz, 1.7 Hz), 7.05-7.06 (m, 1H, ar), 7.27 (s, 2H, exch), 7.48 (broad s, 1H, exch), 7.89-7.90 (m, 1H, ar), 8.50-8.51 (m, 1H, ar), 8.57-8.58 (m, 1H, ar), 8.62 (s, 1H, ar). Anal calc. for C.sub.14H.sub.11N.sub.7OS

Compound 16 2-(furan-2-yl)-N.SUP.5.-(2-(furan-2-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0074] Yield 86% Pf: 198-200 C. (chromatographic column with ethyl acetate/cyclohexane 1/1 as eluent). .sup.1H NMR: 2.87 (t, 2H, CH.sub.2, J=7.0 Hz), 3.51-3.53 (m, 2H, CH.sub.2), 6.17-6.18 (m, 1H, ar), 6.35-6.36 (m, 1H, ar), 6.71-6.72 (m, 1H, ar), 6.90 (broad s, 1H, exch), 7.04-7.05 (m, 1H, ar), 7.17 (broad s, 2H, exch), 7.51-7.52 (m, 1H, ar), 7.88-7.89 (m, 1H, ar). Anal calc. for C.sub.15H.sub.13N.sub.5O.sub.2S

Compound 17 2-(furan-2-yl)-N.SUP.5.-(2-(pyridin-3-yl)ethyl)[1,3]thiazolo[5,4-d]pyrimidin-5,7-diamine

[0075] Yield 59% Pf: 176-178 C. (preparative plate with ethyl acetate/cyclohexane/methanol 8/1/1 as eluent). .sup.1H NMR: 2.87 (t, 2H, CH.sub.2, J=7.0 Hz), 3.50 (dd, 2H, CH.sub.2, J=12.9 Hz, 6.7 Hz), 6.72-6.73 (m, 1H, ar), 6.95 (broad s, 1H, exch), 7.04-7.05 (m, 1H, ar), 7.18 (slargato s, 2H, exch), 7.30 (dd, 1H, ar, J=7.6 Hz, 4.7 Hz), 7.68 (d, 1H, ar, J=6.6 Hz), 7.89-7.90 (m, 1H, ar), 8.40 (d, 1H, ar, J=4.7 Hz), 8.47 (s, 1H, ar). Anal calc. for C.sub.16H.sub.14N.sub.6OS

Example 5

Preparation of 3-(((7-amino-2-(furan-2-yl)[1,3]thiazolo[5,4-d]pyrimidin-5-yl)amino)methyl) phenol (Compound 4)

[0076] A solution of BBr.sub.3 in CH.sub.2Cl.sub.2 (1 M, 1.5 ml) was added drop by drop to a suspension in anhydrous dichloromethane (40 ml) of the Compound 3 (0.5 mmol) prepared as described above in the Example 4. Upon completion of the addition, the suspension was maintained under stirring at 50 C. for 1 day. The solution was diluted with water and ice (50 g) and maintained under stirring for 4 hours, then a saturated aqueous solution of NaHCO.sub.3 (6 ml) was added. The resulting precipitate was collected by filtration and washed with water. Yield 80%. Pf: 207-209 C. (EtOAc). .sup.1H NMR: 4.44 (d, 2H, CH.sub.2, J=5.3 Hz), 6.58 (d, 1H, ar, J=8.9 Hz), 6.68-6.71 (m, 3H, ar), 7.04-7.09 (m, 2H, ar), 7.23 (br s, 2H, exch), 7.35 (br s, 1H, exch), 7.89 (s, 1H, ar), 9.25 (br s, 1H, exch). .sup.13C NMR: 44.45, 110.06, 113.17, 113.81, 114.13, 117.97, 129.54, 142.65, 145.52, 148.59, 157.50, 157.76, 160.35, 164.86. Anal. calc. for C.sub.16H.sub.13N.sub.5O.sub.2S.

Example 6Pharmacologic Tests

[0077] In Vitro Pharmacological Tests

[0078] Cell Culture and Membrane Preparation

[0079] Chinese Hamster Ovary (CHO) cells transfected with human (h) adenosine receptors A.sub.1, A.sub.2A, A.sub.2B and A.sub.3 were cultured and maintained in Dulbecco's modified with a mixture of nutrients F12, containing the 10% of bovine foetal serum, penicillin (100 U/ml), streptomycin (100 g/ml), 1-glutamine (2 mM), geneticin (G418; 0.2 mg/ml) at 37 C. in 5% CO.sub.2 and 95% of air until use in cAMP assays. For the membranes preparation the culture medium was removed, and the cells were washed with a saline phosphate-buffered solution collected with hypotonic buffer (5 mM Tris HCl, 1 mM EDTA, pH 7.4). The cell suspension was homogenised with a Polytron, centrifuged for 30 minutes at 40000 g at 4 C. and the resulting membrane pellet was used in competition binding experiments (Varani K, et al., Mol. Pharmacol. 2000; 57:968-975).

[0080] Competition Binding Experiments

[0081] The compounds of the present invention and the known compound ZM 241385 as reference were tested for their affinity to the following human receptors of adenosine: hA.sub.1, hA.sub.2A and hA.sub.3. Competition binding experiments for the receptor A.sub.1 were carried out incubating 1 nM [.sup.3H]-DPCPX with the membrane suspension (50 g of protein/100 l) and different concentrations of the compounds evaluated at 25 C. for 90 minutes in 50 mM Tris HCl, pH 7.4. Non-specific binding was defined as binding in the presence of 1 M DPCPX and was always <10% of the total binding. Inhibition experiments to A.sub.2A receptors were carried out by incubating the radioligand [.sup.3H]-ZM 241385 (1 nM) with the membrane suspension (50 g of protein/100 l) and at least 12 different concentrations of the tested compounds for 60 minutes at 4 C. in 50 mM Tris HCl (pH 7.4), 10 mM MgCl.sub.2. Non-specific binding was determined in the presence of ZM 241385 (1 M) and was approximately the 20% of the total binding.

[0082] Competition binding experiments for the binding to the A.sub.3 receptor were carried out by incubating the membrane suspension (50 g of protein/100 l) with 0.5 nM [.sup.125I]-ABMECA in the presence of the compounds under evaluation at various concentrations for an incubation time of 120 minutes at 4 C. in 50 mM Tris HCl (pH 7.4), 10 mM MgCl.sub.2, 1 mM EDTA. The non-specific binding was defined as binding in the presence of 1 M ABMECA and was always <10% of the total binding.

[0083] Bound radioactivity and free radioactivity were separated by filtering the solution through Whatman GF/B glass fibre filters using a Brandel cell harvester (Brandel Instruments, Unterfhring, Germany). The filter-bound radioactivity was counted by a scintillation counter Packard Tri Carb 2810 TR (Perkin Elmer) (Varani K. et al., Mol. Pharmacol. 2000; 57:968-975).

[0084] Cyclic AMP Assays

[0085] CHO cells transfected with the adenosine receptors were washed with a phosphate-buffered saline solution, detached with trypsin and centrifuged for 10 minutes at 200 g. The pellet containing CHO cells (110.sup.6 cells/sample) was suspended in 0.5 ml of the incubation mixture (mM): NaCl 15, KCl 0.27, NaH.sub.2PO.sub.4 0.037, MgSO.sub.4 0.1, CaCl.sub.2 0.1, Hepes 0.01, MgCl.sub.2 1, glucose 0.5, pH 7.4 at 37 C., 2 IU/ml adenosine deaminase and 4-(3-butoxy-4-metoxybenzyl)-2-imidazolidinone (Ro 20-1724) as phosphodiesterase inhibitor and pre-incubated for 10 minutes in a thermostated bath under stirring at 37 C. The potencies of the evaluated compounds for the hA.sub.2B receptors have been determined by evaluating their ability to inhibit the cAMP levels stimulated by NECA (100 nM). In order to better investigate the behaviour of inverse antagonism/agonism, the potency towards the hA.sub.2A receptors of the compounds tested at 12 different concentrations was determined by studying their ability to inhibit the production of cAMP both under basal conditions and in the presence of the known agonist CGS 21680 (10 nM). Additional experiments have been carried out by evaluating the studied compounds at the concentration of 10 M in hA.sub.1, hA.sub.2B or hA.sub.3CHO cells in order to verify their effect on the cAMP production under basal conditions. The reaction was terminated by addition of cold 6% trichloroacetic acid (TCA). The TCA suspension was centrifuged at 2000 g for 10 minutes at 4 C. and the supernatant was extracted four times with diethyl ether with water. The final aqueous solution was tested for the cAMP levels by a competition protein binding assay with a cAMP-binding protein. The standard samples of cAMP (0-10 pmoli) were added to each test tube containing the incubation buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2 mercaptoethanol 6.0 mM, pH 7.4) and [.sup.3H]-cAMP. The binding protein previously prepared from beef adrenals, was added to the samples previously incubated at 4 C. for 150 minutes, and after the addition of charcoal were centrifuged at 2000 g for 10 min. The transparent surnatant was counted in a 2810-TR Packard scintillation counter (Varani K et al., Biochem Pharmacol 2005; 70:1601-1612).

[0086] Cell Proliferation Assay

[0087] For the experiments of cellular proliferation, the DELFIA kit was used and a multimode plate reader Ensight from PerkinElmer. The DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay) assay is based on Time-Resolved Fluorescence (TRF) and on the incorporation of the 5-bromo-2-deoxyuridine (BrdU) in the DNA filaments recently synthesised by the proliferating cells seeded on microplates. The incorporated BrdU is detected by using a monoclonal antibody conjugated with europium, a long-lived chelated lanthanide, and the fluorescence measured is proportional to the synthesis of DNA in the cells population present in each well. The MRMT-1 cells, breast cancer cells, were pre-treated with some of the antagonists under evaluation at the concentration of 100 nM (compounds 1, 5, 6, 7, 10 of the present invention) for 30 minutes, then stimulated with CGS 21680 100 nM and after 30 minutes the solution of BrdU (10 l/well) was added. At the end of the incubation period of 48 hours 100 l/well of Anti-BrdU-Eu (0.5 g/ml) were added and the cells were incubated for 120 minutes at room temperature. After 4 washings, 200 l of DELFIA stimulator were added at room temperature for 15 minutes and the Eu-fluorescence was detected by means of the Ensight reader from Perkin Elmer (Perkin Elmer, Milan, Italy).

[0088] In Vivo Pharmacological Tests

[0089] Animals

[0090] Female CD1 mice (22-24 g) were obtained from Charles River (Milano, Italia). The animals were kept under standard environmental temperature conditions (222 C.) and under moisture-controlled conditions with 12 hours light/dark cycle and food and water ad libitum. The animals were acclimated to the laboratory settings for at least 1 hour before testing and were used only once throughout the experiments. All the procedures used in the present study were carried out in accordance with the European Communities Council Directives (86/609/EEC) and the National Laws and Policies (D.L.116/92) after authorization from the Italian Ministry for Health (4/2014-B). In addition, the experimental procedures were in agreement with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (Couto M. Methods Mol Biol 2011; 770:579-599).

[0091] Writhing Test

[0092] The acetic acid-induced writhing response was performed after intraperitoneal injection of 10 ml/Kg of 0.6% acetic acid solution. The response to the abdominal constrictions induced by acetic acid was evaluated after the intraperitoneal injection of 10 ml/kg of a 0.6% solution of acetic acid. The compounds under evaluation were dissolved in DMSO and then diluted in a saline solution. The carrier consists of saline solution and 5% of DMSO. A writhe is indicated by stretching of the abdomen followed by the extension of the hind limbs. The animals (8 mice per group) were placed singly in a glass cylinder and the number of writhing episodes of abdominal contractions was counted in a 30 minutes period. The compounds were administered intraperitoneally 15 minutes before injection of acetic acid solution. As expected, no abdominal constrictions were observed in mice treated with saline solution instead of with acetic acid solution (Vincenzi F. et al., Pain 2013; 154:864-873). The values of ED.sub.50 were calculated by a linear regression analysis converting the data to percentage of maximum possible effect (MPE) using the following equation: 100(post drug responseresponse to carrier)/(response to carrier).

[0093] Tail Immersion Test

[0094] The warm-water tail immersion assay was performed using a water thermostated bath at a temperature maintained at 52 C. The compounds under evaluation were dissolved in DMSO and then diluted in saline solution. The carrier consists of saline solution and 5% of DMSO. Before intraperitoneally injecting the compound, the natural time of response of the mice was determined and the distal part of the tail was then immersed in the thermostated bath. The latency in responding to the heat stimulus with a vigorous flexion of the tail was measured by means of a manual stopwatch. A 20 seconds maximum cut-off time was imposed to prevent tissue damage. The latency of the tail withdrawal was then tested 15 minutes after injection of the compound. The values of ED.sub.50 were calculated by a linear regression analysis converting the data to % MPE using the following equation: 100(post-drug latencybasal latency)/(cut-off latencybasal latency).

[0095] Statistical Analysis of the Data

[0096] The statistical analysis of the data was performed using ANOVA followed by Dunnett's test. The inhibitory binding constants, Ki, will be calculated from the IC50 values according to the Cheng e Prusoff equation: Ki=IC.sub.50/(1+[C*]/KD*), wherein [C*] is the radioligand concentration and KD* its dissociation constant. KH and KL were obtained by using a two sites binding model and Graph PAD Prism (San Diego, Calif., USA). The values of IC.sub.50 obtained in the cAMP assays were calculated by non-linear regression analysis using the equation for a sigmoid concentration-response curve. All data are expressed as the meanSEM of four independent experiments each performed in duplicate for in vitro assays and n=8-10 mice/group for in vivo assays.

[0097] Results of the In Vitro Tests

[0098] Evaluation of affinity at human adenosine A.sub.1, A.sub.2A and A.sub.3 receptors Affinity at human adenosine A.sub.1, A.sub.2A and A.sub.3 receptors of the tested compounds of the present invention expressed as Ki values are listed in the Table 1 below, together with those of ZM 241385 as the reference compound.

TABLE-US-00001 TABLE 1 Receptor hA.sub.2A.sup.[b] Receptor Ki (nM) or Receptor Receptor hA.sub.1.sup.[a] KH* (fM) and hA.sub.3.sup.[c] hA.sub.2B.sup.[d] Compound Ki (nM) KL** (nM) Ki (nM) IC.sub.50 (nM) 1 3.54 0.32 3.55 0.42* 36 3 313 29 6.45 0.57** 2 163 12 171 16 381 37 283 27 3 8.16 0.72 5.31 0.52* 92 8 452 42 26 2** 4 27 3 20 2 55 4 24 3 5 38 4 39 4* 4.72 0.38 82 9 1.73 0.15** 6 12.5 1.1 10.7 1.0* 6.43 8 75 8 3.82 0.31** 7 7.12 0.65 217 19* 18.2 1.7 109 11 0.68 0.05** 8 28 3 0.42 0.04 59 5 95 9 9 8.51 0.76 0.82 0.07 35 4 103 10 10 4.92 0.37 10.6 0.9* 65 6 112 11 18 2** 11 64 10 17 2 35 4 323 28 12 41 5 8.21 0.78 23 3 185 17 13 8.12 0.71 0.25 0.02 3.14 0.29 8.96 0.82 14 47 4 12 1 827 48 33 2 15 25 4 5.14 0.48 157 14 13.2 1.2 16 5.24 0.46 2.15 0.19 23 2 14 1 17 2.61 0.22 0.24 0.01 174 11 4.21 0.32 ZM 241385 185 14 0.91 0.08 683 64 48 5 Affinity values obtained from competition binding experiments using [.sup.3H]-DPCPX.sup.[a], [.sup.3H]-ZM 241385.sup.[b] or [.sup.125I]-ABMECA.sup.[c] binding to human adenosine A.sub.1, A.sub.2A, A.sub.3 receptors respectively (n = 3-6). .sup.[d]Potency (IC.sub.50) in cAMP assays to human adenosine A.sub.2B receptor. Data are expressed as mean SEM.

[0099] It is worth noting that in the competition binding experiments for the binding with [.sup.3H]-ZM 241385, the compounds 1, 3, 5, 6, 7 and 10 of the invention showed two affinity values for the human adenosine A.sub.2A receptor, the first one having a high value of the affinity Ki (KH) of the femtomolar order and the second one having a nanomolar affinity value Ki (KL) (Table 1). It is also worth noting that the KH values of these compounds are approximately 10.sup.6 times lower than their corresponding KL values. On the contrary, in competition binding experiments the compounds 2, 4, 8, 9, 11-17 and ZM 241385, showed only an affinity value Ki in the nanomolar order. The competition binding curves of the compounds 1, 3, 5, 6, 7 and 10 showed a biphasic form that better match with a two sites binding model and can be interpreted as the interaction with two apparent binding sites whilst the competition binding curves of the reference compound ZM 241385 indicated the presence of a binding site recognition (FIG. 1).

[0100] Potency values at the human adenosine receptors Also studied was the in vitro activity of the compounds according to the present invention by evaluating their antagonist/inverse agonist potencies. In particular, the ability was tested for the compounds 1-8, 10, 13-17 and ZM 241385 to modulate the cAMP production in hA.sub.2A CHO cells in the absence or in presence of CGS 21680. The potency values and the efficacy values of the tested compounds in comparison with ZM 241385 are listed in the Table 2 below. According to their extremely high affinity for the human adenosine A.sub.2A receptor, the compounds 1, 3, 5, 6, 7 and 10 behaved as very potent inverse agonists, being able to inhibit the basal accumulation of cAMP at picomolar concentrations (IC.sub.50=1.9, 8.3, 1.6, 1.7, 11 and 6.4 M, respectively) (Table 2), and showing efficacy values of 63%, 41%, 64%, 61%, 61% and 62% respectively (FIG. 2A, 2B, 2C, 2D, 2E, 2F).

[0101] It is worth noting that the compounds 1, 3, 5, 6, 7 and 10 behaved as inverse agonists having higher potencies with respect to ZM 241385 (IC.sub.50=1.45 nM) (FIG. 2G). The compounds 2, 4, 8 and 13-17 significantly reduced the cAMP production in basal conditions with IC.sub.50 values comprised between 187 and 0.29 nM (Table 2). Moreover, the compounds 1, 3, 5, 6, 7 and 10 were also able to inhibit the cAMP production stimulated by CGS 21680 (10 nM) with high potency (IC.sub.50=51, 95, 40, 36, 59, 45 M, respectively) (FIG. 3A, 3B, 3C, 3D, 3E, 3F; Table 2). The reference compound ZM 241385 blocked the effect of the agonist with a lower potency (IC.sub.50=678 M, FIG. 3G) than those of the compounds 1, 3, 5, 6, 7 and 10.

[0102] As expected, all the tested compounds in the presence of an agonist showed an antagonist/inverse agonist profile. In particular, they reduced the cAMP accumulation, reaching lower values than those of the basal production as indicated in the Emax data (FIG. 3; Table 2). In functional assays performed in CHO cells transfected with human adenosine A.sub.1, A.sub.2B and A.sub.3 receptors, none of the compounds under evaluation were able to modulate the cAMP production in the absence of an agonist, suggesting that at these receptor subtypes the tested compounds do not behave as inverse agonists.

TABLE-US-00002 TABLE 2 Compounds IC.sub.50 (nM).sup.[a] Emax (%).sup.[b] IC.sub.50 (nM).sup.[c] Emax (%).sup.[d] 1 0.0019 0.0002 63 5 0.051 0.004 138 12 2 187 16 38 4 123 11 116 11 3 0.0083 0.0007 41 3 0.095 0.008 136 11 4 27 2 68 7 22 2 139 13 5 0.0016 0.0002 64 6 0.040 0.004 133 13 6 0.0017 0.0002 61 6 0.036 0.003 132 12 7 0.011 0.001 61 5 0.059 0.006 126 12 8 0.68 0.06 54 5 1.3 0.1 128 11 10 0.0064 0.0005 62 6 0.045 0.004 138 13 13 0.36 0.04 67 6 0.41 0.03 137 13 14 15.3 1.2 43 4 18.7 1.6 122 10 15 8.27 0.72 48 4 11.3 0.96 117 10 16 2.93 0.22 56 6 4.26 0.37 121 11 17 0.29 0.03 71 7 0.35 0.04 141 14 ZM 241385 1.45 0.42 46 2 0.678 0.061 123 10 Potency (IC.sub.50).sup.[a, c] and efficacy (Emax).sup.[b, d] of the tested compounds in cAMP assays in hA.sub.2A CHO cells in absence.sup.[a, b] or in presence.sup.[c, d] of CGS 21680 (10 nM), respectively. The data are expressed as mean SEM.

[0103] Results of Cellular Proliferation

[0104] The cellular proliferation assays performed on the breast cancer cells MRMT-1 expressing the adenosine receptors have showed that CGS 21680 is able to increase the proliferation of the cancer cells. The effect of the agonist CGS 21680 is blocked by the use of the compounds 1, 5, 6, 7 and 10 of the present invention that are able to reduce the proliferation of cancer cells induced by the activation of the adenosine A.sub.2A receptor (FIG. 4)

[0105] In Vivo Results

[0106] Analgesic Effects of the Novel Compounds of the Invention

[0107] To explore the anti-nociceptive activity of the novel inverse agonists of the human adenosine A.sub.2A receptor, the compounds 1 and 3 have been tested in comparison with ZM 241385 and with morphine in mice, in an evaluation test of the abdominal constrictions (Writhing Test) and in a tail immersion test. In the Writhing Test, the intraperitoneal administration of acetic acid induced 7512 abdominal constrictions in mice treated with a carrier. The dose-response curve of compounds 1, 3, ZM 241385 and morphine revealed a dose-dependent effect (P<0.001, one-way ANOVA). In particular, the compound 1 of the present invention proved to be more potent than the reference compounds. In fact, it shows an ED.sub.50 value of 0.03280.0021 mg/kg, which is 3.75 times lower than that obtained with morphine (0.1230.010 mg/kg) and approximately 42 times lower than that of ZM 241385 (1.3730.108 mg/kg) (FIG. 5A-D). The compound 3 of the present invention had an anti-nociceptive activity with potency similar to morphine. In the warm water tail immersion test, the compound 1 of the present invention shoed the highest analgesic activity with an ED.sub.50 value of 0.1340.011 mg/kg and a minimum effective dose of 0.01 mg/kg. The compound 3 of the present invention and morphine showed similar anti-nociceptive activity whereas ZM 241385 had no effect up to 10 mg/kg (FIG. 6A-D).