Title of Invention	SUBSTITUTED IMIDAZOPYRIMIDINES FOR THE PREVENTION AND TREATMENT OF CANCER
Abstract	Compounds of general formula (I), wherein from A1 to A5, and from B1 to B5 are H, alkyl, alkoxyl, halogen, carboxylic derivatives or sulfur derivatives, among others; and from P1 to P3 are H, halogen, alkyl or alkoxyl, among others. Said compounds may be used for the chemoprevention and treatment of both precancerous lesions and cancer.

Full Text

Substituted imidazopyrimidines for the prevention and treatment of cancer The present invention relates to new compounds, and the use thereof for the chemoprevention and treatment of both precancerous lesions [e.g. familial adenomatous polyposis (FAP), and actinic keratoses (AKs)] and cancer (e.g. colorectal, prostate, breast, bladder or skin cancer). BACKGROUND ART Colorectal cancer (CRC) is one of the most common cancers in the world, with an overall mortality exceeding 40%. About 15% of patients with CRC report a family history of the disease. Hereditary CRC generally develops from two syndromes: familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). FAP is caused by germline mutations in the tumour suppressor gene adenomatous polyposis coli (APC) and is characterized by the early development of multiple adenomas in the large intestine. If these lesions are not removed, they may progress to carcinomas. Approximately 9 0% of all CRC cases and deaths are thought to be .preventable. The objective of chemopreventive strategies is to avoid the formation of adenomatous polyps and their subsequent progression to CRC. Chemopreventive agents can act at various levels, by increasing apoptosis, reducing cell proliferation, and/or decreasing carcinogen-induced DNA damage- A number of pharmacological agents have been studied for the prevention of CRC, including nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors. The inhibition of COX-2 enzymatic activity underlies part of the preventative action of these compounds (there is an over expression of COX-2 in. tumour cells), although COX-2-independent mechanisms have also been described- (Hsu A.L. J. Biol. Chem. 2000, 275, 11397-403). Some NSAID derivatives that do not inhibit COX, retain their chemopreventive activity on precancerous and cancerous processes, thereby acting through a mechanism independent of COX inhibition (Piazza G.A. Cancer Res. 1997, 57, 2909-15). Actinic keratosis (AK) and skin cancer are increasingly frequent dermatological diseases in the population.. They appear in regions of chronic sun exposure such as the face and the back of” the hands. Although the exact incidence of AK is unknown, 40-50% of Australians over 40 years of age harbour AK, and the incidence increases with age. There is strong evidence that AK can progress to squamous cell carcinoma (SCC), in fact, approximately 60% of SCC arise from pre-existing AK, The incidence of skin cancer is increasing due to many factors, including greater life expectancy of the population and increases in ambiental ultraviolet radiation. UV light induces molecular signalling pathways and results in specific genetic alterations (i.e. mutation of p53) that are likely critical to skin cancer development. It has been shown that celecoxib, a COX-2 inhibitor, reduces the development of murine UVB-induced skin tumours, although its precise mechanism of action has not been fully elucidated. Additionally, recent reports on cyclooxygenase knockout-fibroblasts confirm that some of the antiproliferative and antineoplastic effects of NSAIDs are independent .of the inhibition of either COX-1 or COX-2 (Zhang X. J. Exp. Med. 1999, 190, 451-459). The development of safe and effective NSAIDs for chemoprevention is complicated by the fact that severe toxicity may counteract the benefits of treatment with these drugs when administered to healthy individuals who have a low probability of developing the disease. Moreover, there is’ increasing concern due to the risk of serious gastrointestinal reactions, cardiovascular safety, and the potential for serious skin reactions and hypersensitivity reactions, related to the use of COX-2 inhibitors, that currently limits their clinical application to the prevention • and/or treatment of precancerous lesions and cancer. Thus, the development of new, less toxic, and more efficient therapeutic agents’for these pathologies is essential. Among the many antiproliferative compounds proposed, there are some that are structurally similar to the present invention, such as those of the following formulae, described in documents WO 99/51590 and US 5700826, that are still in the development phase. WO 00/08024 discloses some compounds that are ‘structurally similar to those of the present invention. Such compounds are selective COX-2 inhibitors, and as such, are useful for the treatment of inflammation and cancer. Wang and collaborators (J\ Med. Chem. 2002, 45, 1697-1711) have described 4,5-diphenylimidazo derivatives having potent antitubulin and cytotoxic activity. For the aforementioned reasons, ■ it is necessary to provide new compounds for chemoprevention and treatment of both precancerous lesions and cancer. SUMMARY OF THE INVENTION The compounds of the present invention inhibit the proliferation of, and induce apoptosis in cancer cell lines, through a COX-2 independent pathway, thereby minimizing the toxicity associated with COX-2 inhibition. The present invention relates to a compound of general formula (I): wherein: Ai / A2, A3, A4, A5, Bi, B2, B3, B4 y B5 are radi cal s independently selected from the group consisting of H, (C1-C4) -alkyl, (C3-C7) -cycloalkyl, CF3, OCF3/ CN, (CH2)nORl, (CH2)nNRlR2, C0NR1R2, F, Cl”, Br, I, NR1R2, NR2C0R1, 0R1, COR1, COOR1, COSR1, OCOR1, SRI, SOR1, S (0)OH, SO2R1, SO2NR2R3, SO2NHCOR1/ and SCOR1; wherein n is an integer from 1 to 3; Rl is a radical selected from H, CH20C0R2, CF3, (C1-C4) -alkyl, and (C3-C7) -cycloalkylmethyl and (C3-C7) -cycloalkyl; R2 is a radical selected from H, and Some of the compounds of formula (I) of the present invention may have one or more chiral centres. The present invention includes each one of the possible stereoisomers and mixtures thereof, particularly racemic mixtures thereof. A single enantiomer may be prepared by any of the commonly used processes/ for example, by chromatographic separation of the racemic mixture on a stationary chiral phase, by resolution of ‘ the racemic mixture by fractional crystallization techniques of the diastereomeric salts, by•chiral synthesis, by enzymatic resolution or by biotransformation. Pharmaceutically acceptable salts include, among others, addition salts of inorganic acids such as hydrochloric, hydrobromic, nitric, sulphuric and phosphoric, as well as addition salts - of organic acids ‘ such as acetic, benzenesulphonic, benzole, camphorsulphonic, mandelic, methanesulphonic, oxalic, succinic, fumaric, tartaric, and maleic. Likewise, an acid proton in compounds of formula (I)-may be substituted by a metallic ion, for example, ■ an alkaline metal ion, an alkaline-earth metal ion or an aluminium ion; or may be coordinated with an organic or inorganic base. An acceptable organic base includes diethylamine and triethylamine. An acceptable inorganic base includes aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. There may be more than one cation or anion depending on the number of functions with charge and on the valence of cations and anions. Some of the compounds of formula (I) of the present invention may exist in unsolvated as well as solvated forms such as, for example, hydrates.. The present invention encompasses all such above-mentioned forms that are pharmaceutically active. Some of the compounds of general formula (I) may exhibit polymorphism, encompassing the present invention all the possible polymorphic forms, and mixtures thereof. Compounds of the present invention may be synthesized using the methods described below, as well as other processes known in the field of organic synthesis. Preferred methods include, but are not limited to, the general processes shown in the attached schemes. According to Scheme 1, ct-bromodesoxybenzoine (III) may be obtained by bromination of desoxybenzoine (II) by using Br2”/HBr/AcOH, Br2/CCl4, or CuBr2 in ethyl acetate (EtOAc) . The reaction of compound (III) with 2-aminopyrimidine (IV), in the presence of a base such as potassium carbonate or an excess of aminopyrimidine, gives a mixture of compounds (la) and (Ib). Scheme 1 As shown in Scheme 2, the starting desoxybenzoine (II) may be prepared at least by one of these four different routes: route 1 consists in a Friedel-Crafts reaction of an aromatic substrate (VI) with a substituted phenacetyl chloride (V) ; route 2 consists in a Perkin condensation between a benzaldehyde (VIII) and a phenylacetic acid derivative (VII) to yield a 2,3-diphenylacrilic acid, followed by Curtius rearrangement, and subsequent hydrolytic treatment; route 3 consists in the addition of a benzylmagnesiane (IX) over benzaldehyde (VIII), and subsequent oxidation of the compound obtained; and route 4 consists in the addition of benzonitrile (X) to the-benzylmagnesiane (IX) . When in compound (I) either one of Ai to A5 or one of Bi to B5 (being the rest of them as defined above) is a methylsulfide, this may be transformed in the corresponding sulfonamide as shown in Scheme 4 (where only the sulfonamide substituent on ring A is represented, although the same applies for ring B7 and where the rest of the positions on A and B ring may be substituted as defined above) . Thus, the methylsulfide (XV) is oxidized with metachloroperbenzoic acid (jnCPBA) to obtain the sulfoxide (XVI), Pummerer reaction of (XVI) affords the acetoxymethylthio (XVII), which can be oxidized with monoperoxyphthalic acid magnesium salt hexahydrate (MMPP) to give compound (XVIII) . The treatment of (XVIII) with NaOH (IN) in MeOH allows to obtain the sulf inate (XIX) . This is first treated with sulfuryl chloride in dichloromethane (DCM) r and then with aqueous ammonium hydroxide in tetrahydrofurane (THF) to yield the sulfonamide (XX). Compound (XVIII) may be also converted in the sulfonamide (XX) by treatment- with NaOAc and K2CO3 followed by reaction with HOSA. (hydroxylamina-O-sulfonic acid) . For purposes of simplicity, the possible substitituents in A and B rings in the scheme 4 above are not shown. Alternatively, sulfonamide (XX) may be obtained starting from sulfoxide (XVI) by successive reactions with: a) TFAA (trifluoroacetic anhydride); b) triethylamine in MeOH; c) chlorine in acetic acid; and, finally, d) ammonium hydroxide. N-acetylation of sulfamoyl group in (XX) with .acetic anhydride in presence of triethylamine allows to obtain its corresponding acetyl derivative (XXI) . On the other hand, the compound (XX) where B3 is a sulfonamide may also be obtained starting from compound (II) where B3 is H by chlorosulfonation and subsequent amination. As shown in scheme 5, some substitutions on the pyrimidine ring (i.e. PI, P2 and P3), may be obtained by treatment of hydroxyimidazopyrimidine (XXII) with phosphorous oxychloride to yield the chloroderivative (XXIII), which then can be reacted either with thiourea to give the corresponding mercaptan (XXIV), or with an alcohol or amine to give the corresponding ether (XXV) or aminoderivative (XXVI), respectively. The compounds of the present invention may induce apoptosis of cancerous and/or precancerous cells. Thus, an aspect of the present invention relates to the use of said compounds for the manufacture of a medicament for the chemoprevention and treatment of both a precancerous lesion (such as, familial adenomatous polyposis, and actinic keratoses) and a cancer (particularly, colorectal, prostate, breast, bladder, or skin cancer) . Therefore, this aspect of the invention is related to a method for the prophylactic and/or curative treatment of an animal, including a human, suffering from the above-mentioned pathologies, which comprises administering a therapeutically effective amount of a compound of formula (I) - Another aspect of the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of the compound (I), as an active ingredient, together with appropiate amounts of pharmaceutically acceptable excipients. Preferably, the compound is administered orally, parenterally or topically. Throughout th: description and claims the word “comprise” and variations of the word, such as “comprising”, is not intended to exclude other additives, components, elements or steps. The disclosures in the abstract accompanying this application and in the application from which priority is claimed, are incorporated herein as reference. Throughout the description and claims, the terms “alkyl” and “alkoxyl” shall be construed as straight or branched. Additional aspects, advantages and novel features of the invention will be set forth in part in the description, and in part will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The present invention will be further illustrated by the following examples. The examples are given by way of illustration only and are not to be construed as limiting. EXAMPLES The structure of the different compounds of the present invention is confirmed either by ^-NMR (in CDC13/ unless otherwise stated; and using a VARIAN UNITY-3 0 0 MHz equipment; wherein chemical shifts are expressed as ppm (5) from the internal reference TMS) or by mass spectrometry obtaining the molecular ions by a electrospray probe of an Agilent 1100 VL. All of the reactions under microwave irradiation were conducted in heavy-walled Pyrex tubes. Microwave heating was carried out with a single mode cavity Discover Microwave Synthesizer (CEM corporation) . The nomenclature used in the present document is based on the software AUTONOM (Automatic Nomenclature) from the Beilstein Institute, which uses the IUPAC systematic nomenclature. The following examples are given by way of illustration only and are not to be construed as limiting. General methods for bromination of desoxybenzoine a) To a mixture of a desoxybenzoine derivative (50 inmol) in chloroform (210 mL) and CC14 (826 mL), bromine (50 mmol) dissolved in CC14 was added dropwise. When decolouration was complete, the organic layer was washed with 5% sodium bicarbonate and brine. Then, it was dried over anhydrous sodiuin sulfate and the solvent was evaporated to give the ct-bromodesoxibenzoine of interest. b) To a solution of a desoxybenzoine derivative (50 mmol) in EtOAc (210 mL), cooper (II) bromide (24, 5 g, 110 mmol) was added, and then the mixture was stirred at 60°C for 3 h. The reaction mixture was allowed to cool down and filtered through Celite, and the solvent was evaporated to give the a -bromodesoxibenzoine of- interest. General method for 2-Phenylimidazo [1, 2-a3pyrimidine derivatives To a solution of a bromoacetofenone derivative (7.37 mmol) in dimethylformamide (DMF, 75 mL), a 2-aminopyriinidine derivative (18.4 mmol) was added. The mixture was stirred at 70°C for 12 h. Then, it was allowed to cool down and diluted with EtOAc. The solution was washed first with 5% sodium bicarbonate and then with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash). General method for 3-bromo~2-phenylimidazo [1,2-a]pyrimidines To a suspension of a 2-phenylimidazo [1,2-a]pyrimidine derivative (4.60 mmol) in acetonitrile (50 mL), NBS (750 mg, 4.20 mmol) was added in one portion at Q°C. The resulting solution was stirred at the same temperature for Ih, whereupon the reaction mixture was concentrated under reduced pressure. The obtained residue was purified by column chromatography over silica gel (flash). Examples 1 and 2: 2-(4-Ethoxyphenyl)-3-(4- methylsulfanylphenyl)imidazo [1,2-a]pyrimidine and .3- (4- ethoxyphenyl) -2- (4-methylsulf anylphenyl) imidazo [1,2- a]pyrimidine, respectively To. a solution of 1.1 g (2.9 mmol) of 2-bromo-2-(4- ethoxyphenyl) -1- (4-methylsulf anylphenyl) ethanone in 3 0 mL of DMF, 0.7 g (7.4 ‘mmol) of 2 - aminopyrimidine were added. The mixture was heated at 70°C with, stirring for 12 tu Then, it was allowed to cool down and diluted with EtOAc. The solution was washed first with 5% sodium bicarbonate and then with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash), using EtOAc as eluent, to give 300 mg of Example 1, and 70 mg of Example 2. Example 3: 2-(4-Methoxyphenyl)-5,7-dimethyl-3-(4-methylsulfanylphenyl) imidazo{l, 2-a]pyrimidine To a solution of 750 mg of 2-bromo-l- (4-methoxyphenyl) -2- (4-methylsulf anylphenyl) ethanone (2.1 mmol) in 21 mL of acetonitrile, 660 mg of 2-amino-4, 6-dimethylpyrimidine (5.3 mmol) were added. The reaction mixture was refluxed with stirring for 72 h, then, it was allowed to cool down and diluted with EtOAc. The solution was washed, first with 5% sodium- bicarbonate and then with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash), using EtOAc as eluent, to give 3 0 mg of the title compound. Example 4: 31(4-Fluorophenyl)-2-p-tolylimidazo[1,2- a]pyrimidine A solution of 150 mg of 3-bromo-2-p-tolylimidazo [1,2-a]pyrimidine (0.55 mmol), 92 mg of 4-fluoroboronic acid (0.66 mmol), 120 mg of Na2CO3 (2.10 mmol), and 0,5 mg of tetrakis (triphenylphosphine) (0.005 mmol) in 2 mL of THP and 2 mL of water was exposed to microwave irradiation (60 W) at a temperature of 170°C for 20 min. The pressure in the closed reaction vessel was comprised between 140-150 psi. After irradiation, the solution was diluted with EtOAc, and washed with water. The organic layer was dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash), using EtOAc:DCM (1:1) as eluent, to give 10 0 mg of the title compound. Example 73: 7-Chloro-3-(4-methoxyphenyl)-2-(4-methylsulf any lphenyl) imidazo [1, 2-a]pyrimidine A solution of 200 mg of Example 72 (0.55 mmol) in 5 mL of POC13 was refluxed with stirring for 3 h and then allowed to cool down. The solvent was evaporated, the residue obtained was diluted with water, and ammonia was added to basic pH. The solution was extracted with EtOAc, and the organic extracts were dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash), using EtOAc as eluent, to obtain 80 mg of the title compound. Example 102 : 5~Methoxy-2-(4-methoxyphenyl)-3-(4-methylsulf anylphenyl) imidazo [1, 2-a] pyrimidin-7-ol To a solution of 750 mg (2.1 mmol) of 2-bromo-l- (4-methoxyphenyl)-2-(4-methylsulf anylphenyl) ethanone in 21 mL of acetonitrile, 826 mg (5.3 mmol) of 2-amino-4,6-dimethoxypyrimidine were added. The mixture was refluxed for 72 h, and allowed to cool down. Then, it was diluted with EtOAc, and 5% sodium bicarbonate was added. The organic layer was dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatogi-aphy over silica gel (flash), using EtOAc as eluent, to give 40 mg of the title compound. Example 22 0: Acetic acid 4-(7-Methyl-3-phenylimidazo [1,2-a] pyrimidin-2-yl) phenylsulf anylmethyl ester . To a solution of 550 mg of Example 166 (1.58 mmol) in 7 mL of acetic anhydride, 700 mg of potassium acetate (7.12 mmol) were added. The mixture was refluxed for 10 ‘h, and then allowed to cool down. The solvent was evaporated, the residue obtained was diluted with EtOAc, and the solution was washed with a saturated solution of NH4C1 and brine. The organic layer was dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 610 mg of the title compound. Example 221: Acetic acid 4- (7-Methyl-3-phenylimidazo[1,2-a3pyrimidin-2-yl)benzenesulfonylmethyl ester To a solution of 610 mg of Example 22 0 (1.56 mmol) in 20 mL of DCM:MeOH (3:1), 1,06 g of MMPP (1.72 mmol) were added. The mixture was .stirred at room temperature for 10 h. The reaction, mixture was neutralized with saturated bicarbonate and the solvent was evaporated. The residue obtained was diluted with DCM and the solution was washed with 5% sodium bicarbonate; The organic layer was dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was purified by column chromatography over silica gel (flash), using EtOAc as eluent, to give 260 mg of the title compound. Example 216: 4-(7-Methyl-3-phenylimidazo[l,2-a]pyrimidin-2-yl)benzenesulfonamide To a solution of 250 mg of Example 221 (0.60 mmol) in 6 mL of MeOH, 460 mg of potassium acetate (4.74 mmol) were added. The mixture was stirred at room temperature for 10 min. Then, 170 mg of potassium carbonate (1.18 mmol) were added and the reaction mixture was stirred for 1.5 h. Then, 270 mg of HOSA (2.37 mmol) were added and the solution was further stirred for 2 h. The solvent was evaporated and the residue obtained was diluted with EtOAc. The solution was washed with 5% sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, and evaporated to dryness. The obtained residue was washed with MeOH to give 90 mg of the title compound. The following examples were prepared using some of the methods previously described. Determination of apoptosis To stablish whether the antiproliferative activity of the compounds of the present invention is due to an apoptotic process, and not merely to a necrotic process, the generation of DNA fragments associated to histones (mono- and oligonucleosomes) was determined after incubation of the human colon cancer cell lines HCT-116 with the compounds of the present invention at different concentrations. DNA fragmentation into nucleosomes, which is an indicator of apoptosis phenomena, was quantified by a sandwich inmunoassay using monoclonal antibodies directed against DNA and histones (Cell Death Detection ELISAPLUS, Roche Diagnostics, cat #1920685). The quantity of fragmented DNA was expressed with the Enrichment Factor (EF) parameter, which is a coefficient of absorbance of nucleosomes liberated in the cytosol of the cells cultured in the presence of the products’compared with control cells. Determination of cell proliferation inhibition , _ . j The inhibitory capacity of cell proliferation of the compounds was determined in two human colon adenocarcinoma cell lines (HCT-116) obtained at the ATCC (American Type Collection). The cells were seeded in 96-well plates and kept at 3 7QC in a CO2 heater for 24 hours to allow cell-substrate-adhesion. Subsequently; cells were treated with the products under study at concentrations comprised between 1 and 10 0 /iM for 48 hours- After the treatment period, the medium was removed and cells were dyed with Sulforodamine B. Finally, decolouration of the dyed cells was carried out with Tris base 10 mM and the plates were read at 493-530 nm in a plate reader. IC50 was calculated as the product concentration inducing a growth inhibition of the 5 0% compared with control cells not treated. The compounds of the present invention are more potent than celecoxib as antiproliferative and pro-apoptotic agents, but much less potent than celecoxib as COX-2 inhibitory agents. These results and the fact that the human cancer cell line HCT-116 does not express COX-2 isoenzyme show that their mechanism of action is independent of their COX-2 inhibitory properties. 5. A pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the compound according to any one of the claims 1 to 4 together with• appropriate amounts of pharmaceutically acceptable excipients. 6. Use of the compound as defined in any one of claims 1 to 4 for the manufacture of a medicament for the chemopr event ion and/or treatment of a precancerous lesion. 7. Use according to claim 6, wherein the precancerous lesion is familial adenomatous polyposis or an actinic keratosis. S. Use of the compound as defined in any one of claims’ 1 to 4 for the manufacture of a medicament for the chemoprevention and/or treatment of a cancer. 9. Use according to claim 8, wherein the cancer is colorectal, prostate,, breast, bladders or skin cancer. 10. Use according to any one of the claims 6 to 9, wherein the medicament is administered orally, parenterally or topically. 11. A method for the prophylactic arid/or curative treatment of an animal, including a human, suffering from a precancerous lesion, comprising administering a therapeuticaly effective amount of a compound as defined in any one of claims 1 to 4 together with an appropriate amount of pharmaceutically acceptable excipients. 12. A method for the prophylactic and/or curative treatment of an animal, including a human, suffering from a cancer, comprising administering a therapeuticaly effective amount of a compound as defined in any one of claims 1 to 4 together with an appropriate amount of pharmaceutically acceptable excipients.

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