WO2013178545A1

WO2013178545A1 - Acid ceramidase inhibitors and their use as medicaments

Info

Publication number: WO2013178545A1
Application number: PCT/EP2013/060729
Authority: WO
Inventors: Daniele Piomelli; Natalia REALINI; Marco Mor; Chiara PAGLIUCA; Daniela PIZZIRANI; Rita Scarpelli; Tiziano Bandiera
Original assignee: Fondazione Istituto Italiano Di Tecnologia; The Regents Of The University Of California; Universita' Degli Studi Di Parma
Priority date: 2012-05-28
Filing date: 2013-05-24
Publication date: 2013-12-05
Also published as: ITMI20120923A1

Abstract

The present invention concerns, in a first aspect, compounds of Formula I as defined herein, pharmaceutically acceptable salts thereof and pharmaceutical compositions containing such compounds. The present invention also relates to compounds of Formula I for use as acid ceramidase inhibitors, and in the treatment of cancer and other disorders in which modulation of the levels of ceramide is clinically relevant.

Description

Acid ceramidase inhibitors and their use as medicaments

CROSS-REFERENCES TO RELATED APPLICATIONS

Not applicable.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was made, in part, with government support under NIH Grant R01 DA1 241 3 awarded by the National Institutes of Health ; the United States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to acid ceramidase inhibitors and their use as medicaments.

In particular, the present invention concerns acid ceramidase inhibitors, pharmaceutical compositions containing them and methods for preparing these inhibitors.

The present invention also provides methods of inhibiting acid ceramidase for the treatment of cancer and other disorders in which modulation of the levels of ceramide is clinically relevant.

BACKGROUND OF THE INVENTION

The sphigolipids are a family of membrane lipids derived from the aliphatic amino alcohol sphigosine and its related sphigoid bases. They are present in eukaryote membranes, where they exert important structural roles in the regulation of fluidity and subdomain structure of the lipid bilayer. In addition to that, they have emerged as key effectors in many aspects of cell biology including inflammation, cell proliferation and migration, senescence and apoptosis [see, for instance, Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 2008, 9, 139-150].

Ceramide is considered a central molecule in sphingolipid signaling. The generic term "ceramide" comprises a family of several distinct molecular species deriving from the /V-acylation of sphingosine with fatty acids of different chain length, typically from 14 to 26 carbon atoms. Ceramide can be synthesized de novofrom condensation of serine with palmitate, catalyzed by serine palmitoyltransferase, to form 3-keto-dihydrosphingosine. In turn, 3-keto-dihydrosphingosine is reduced to dihydrosphingosine, followed by acylation by a (dihydro)-ceramide synthase. Ceramide is formed by the desaturation of dihydroceramide. Alternatively, ceramide can be obtained by hydrolysis of sphingomyelin by sphingomyelinases. Ceramide is metabolized by ceramidases to yield sphingosine and fatty acid [Hannun YA, Obeid LM Nat. Rev. Mol. Cell Biol. 2008, 9, 139-150].

Ceramide plays important roles in a varietyof cellular processes. Ceramide concentrations increase in response to cellular stress, such as DNA damage, exposure to cancer chemotherapeutic agents and ionizing radiation, and increased ceramide levels can trigger senescence and apoptosis in normal cells. [Wymann MP, Schneiter R Lipid signalling in disease. Nat. Rev. Mol. Cell. Biol. 2008, 9, 162- 176]. Ceramide is involved in the regulation of cancer cell growth, differentiation, senescence and apoptosis [Ogretmen B and Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer 2004, 4, 604-616]. Many anticancer drugs increase ceramide levels in cells by stimulating \tsde novo synthesis and/or the hydrolysis of sphingomyelin. For example, daunorubicin elicits ceramide production through the de novo pathway [Bose R et al. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995, 82, 405-414]. De novo ceramide induction was observed in various human cancer cells after treatment with camptothecin and fludarabine [Chauvier D et al. Ceramide involvement in homocamptothecin- and camptothecin induced cytotoxicity and apoptosis in colon HT29 cells. Int. J. Oncol. 2002, 20, 855-863; Biswal SS et al., Changes in ceramide and sphingomyelin following fludarabine treatment of human chroni chronic B-cell leukemia cells. Toxicology 2000, 154, 45-53], and with gemcitabine [Chalfant CE et al., De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase- 1. J. Biol. Chem. 2002, 277, 12587-12595]. In many of these studies, inhibition of de novo ceramide synthesis was found to prevent, at least in part, the cytotoxic responses to these agents, thus indicating that the de novo pathway might function as a common mediator of cell death. Therefore, increasing or sustaining the levels of ceramide in cancer cells could be envisaged as a novel therapeutic strategy to induce cell death.

An approach to increasing or sustaining the levels of ceramide in cells consists in the inhibition of enzymes responsible for ceramide clearance. Enzymes that contribute to decreasing the intracellular levels of ceramide are glucosylceramide synthase, which incorporates ceramide into glucosylceramide, sphingomyelin synthase, which synthesizes shpinghomyelin, and ceramidases, which hydrolyze ceramide to sphingosine and fatty acid. Currently, there are five known human ceramidases: acid ceramidase, neutral ceramidase, alkaline ceramidase 1 , alkaline ceramidase 2, and alkaline ceramidase 3 [Mao C, Obeid LM Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1 -phosphate. Biochim. Biophys. Acta 2008, 1781, 424-434\. Among them, acid ceramidase is emerging as an important enzyme in the progression of cancer and in the response to tumor therapy [Gangoiti P et al. Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease. Prog. Lipid Res. 2010, 49, 316-34]. Messenger RNA and protein levels of acid ceramidase are heightened in a wide variety of cancers including prostate cancer [Seelan RS et al. Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000, 29, 137- 146], head and neck cancer [Norris JS et al. Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report. Cancer Gene Ther. 2006, 13, 1045-1051; Elojeimy S et al. Role of acid ceramidase in resistance to FasL: therapeutic approaches based on acid ceramidase inhibitors and FasL gene therapy. Mol. Ther. 2007, 15, 1259-1263], and melanoma [Musumarra G et al. A bioinformatic approach to the identification of candidate genes for the development of new cancer diagnostics. Biol. Chem. 2003, 384, 321-327]. In prostate cancer, acid ceramidase expression correlates with the malignant stage of the disease [Seelan RS et al. Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000, 29, 137- 146]. Up-regulation of acid ceramidase has also been observed in prostate cancer cells in response to radiotherapy, and this mechanism desensitizes cells to both chemotherapy and radiotherapy. Restoration of acid ceramidase levels in radio-resistant cells by either gene silencing or inhibition of acid ceramidase activity confers radiation sensitivity to prostate cancer cells. Improvement of tumor sensitivity to ionizing radiation by inhibition of acid ceramidase has been shown in vivo in a PPC-1 xenograft model [Mahdy AE et al. Acid ceramidase upregulation in prostate cancer cells confers resistance to radiation: AC inhibition, a potential radiosensitizer. Mol. Ther. 2009, 17, 430-438\. Together, the data outlined above indicate that acid ceramidase provides a growth advantage to cancer cells and contributes to the altered balance between proliferation and death eventually leading to tumor progression. The data further suggest that up-regulation of acid ceramidase expression and activity renders cancer cellsresistant to chemotherapy and radiotherapy. The resistance to chemotherapy and radiotherapy is a major obstacle to the successful eradication of cancer in many patients. The inhibition of acid ceramidase represents, therefore, a promising strategy both to enhance thepotency and effectiveness of current anti-tumoral treatments and to reduce the adverse eventsassociated with administration of high dosages of these compounds. Thus, acid ceramidase inhibitors represent a novel class of chemosensitizing agents that may find broad applications to anti-tumoral therapy in combination with chemotherapeutic agents and/or radiation therapy.

Certain methods for inhibiting ceramidase activity by compound containing a sphingoid base, a derivative of a sphingoid base, or a salt of a sphingoid base are described in the European patent application EP1287815. Other methods for inhibiting ceramidase activity using cyclopropenyl-sphingosine derivatives are described in the patent application WO2005/051891 . Still other methods of inhibiting ceramidase activity in cells using cationic ceramide derivatives are reported in the patent application WO2006/050264. Further methods for inhibiting or modulating acid ceramidase activity are disclosed in the patent application WO2007/136635 and in the patent application WO2010/054223.

Acid ceramidase inhibitors disclosed in the scientific and patent literature, such as B13 [Selzner M et al. Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res.2001, 61, 1233- 1240], LCL 204 [Holman DH et al. Lysosomotropic acid ceramidase inhibitor induces apoptosis in prostate cancer cells. Cancer Chemother. Pharmacol.2008, 61, 231-242,] or E-tb [Bedia C et al. Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides. Chem. Phys. Lipids 2008, 156, 33-40], are ceramide analogs that inhibit acid ceramidase activity in cell-free assays and proliferation of cancer cell lines only at high micromolar concentrations.

There is therefore still a substantial need for novel acid ceramidase inhibitors with improved potency and drug-likeness.

Accordingly one of the aims of the present invention resides in the provision of novel compounds which are acid ceramidase inhibitors for use in the prevention or treatment of disorders or diseases in which the modulation of the levels of ceramide is clinically relevant.

SUMMARY OF THE INVENTION

The inventors have discovered that selected compounds bearing a 5-fluoro uracil moiety inhibit acid ceramidase effectively and are therefore useful as pharmacological agents in the treatment of cancer, in particular in combination with chemotherapeutic agents and/or radiation therapy.

In a first aspect, the present invention provides a compound of Formula I or pharmaceutically acceptable salts thereof

Formula I wherein

Y represents a bond, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyl, or a group -(CR_aRb)_n-Q-(CR_cRd)m-; W represents hydrogen, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, a group -(CR_eR_f)p-CR_gR_hRi or a group Z-R₃; Ri represents an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyl, or a group C(=0)R₄;

Q represents O, NR₅;

Z represents O, NR₅;

R₃ represents hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocyclyl;

R₄ represents an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl selected from pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzofuranyl, benzothiophenyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazolinyl, an optionally substituted heterocyclyl, or a group 0-R₆;

Rs represents hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl;

R₆ represents an optionally substituted C-1 -C3 alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or an optionally substituted heterocyclyl;

R_a, Rb, Rc, Rd, R_e, Rf, R_g, Rh, and R, are independently selected from the group consisting of hydrogen, halogen, an optionally substituted lower alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, alkoxy, cycloalkyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, or trifluoromethoxy;

n is an integer from 1 to 12;

m is an integer from 0 to 12;

p is an integer from 0 to 6.

In a second aspect the invention concerns a compound of Formula I as a medicament, in particular it concerns compounds of Formula I for use in the treatment of pathologies where modulation or inhibition of acid ceramidase is needed, such as in the treatment of cancer and other disorders where modulation of ceramide levels is clinically relevant. In a third aspect the invention concerns pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier and/or excipient.

In a fourth aspect, the present invention provides a method for modulating the levels of ceramides in a subject by administering a compound of Formula I or a pharmaceutical composition containing such compound.

The present invention also provides methods for treating conditions associated with over-expression or over-activity of acid ceramidase, including various cancers and hyperproliferative diseases, by administering a therapeutically effective amount of a compound of Formula I, in particular in combination with chemotherapeutic agents and/or radiation therapy.

In a fifth aspect, the present invention provides methods for preparing compounds of Formula I, as defined above, through a process consisting of suitable synthetic transformations.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a bar graph illustrating the effect of compound 1 , compound 2, 5- fluorouracil (5FU), or vehicle (0.1 % DMSO in DMEM)on acid ceramidase activity in the human colon adenocarcinoma cell line SW403. Levels of acid ceramidase activity were measured after 3hr exposure to different concentrations (1 and 10μΜ) of compound 1 , compound 2, 5FU or vehicle. Results are expressed as mean ± SEM (n=3). ^***p<0.0001 vs vehicle, one-way ANOVA followed by Tukey's. Figure 2 shows a bar graph illustrating the effect of compound 1 , compound 2, 5- fluorouracil (5FU), or vehicle (0.1 % DMSO in DMEM)on ceramide levels in the human colon adenocarcinoma cell line SW403. Levels of the most abundant endogenous ceramide species (C14, C16 and C18:0) were measured after 3hr exposure to compound 1 (10μΜ), compound 2 (10μΜ), 5FU (300nM) or vehicle. Results are expressed as mean ± SEM (n=3). ^***p<0.0001 , vs vehicle, one-way ANOVA followed by Tukey's.

Figure 3 shows four graphs illustrating the effect of compound 1 and compound 2 on SW403 cell viability, and synergism with 5-fluorouracil (5FU) measured with the trypan blue assay. The graph A shows the concentration-response curve of multiple treatments(72 hrs) with compound 1 on SW403 cell viability. The graph B shows the concentration-response curve of multiple treatments(72 hrs) with compound 2 on SW403 cell viability.

The graph C shows the isobolographic analysis of data obtained after multiple treatments of SW403 cells with compound 1 and 5FU.

The graph D shows the isobolographic analysis of data obtained after multiple treatments of SW403 cells with compound 2 and 5FU. DETAILED DESCRIPTION OF THE INVENTION

The present invention origins from the finding that compounds having a 5- fluorouracil moiety with selected substituent groups as represented by Formula I effectively inhibit or modulate acid ceramidase activity. Accordingly, such compounds can advantageously be used for the treatment of diseases or disorders associated with overexpression or overactivity of acid ceramidase in an organ or body compartment.

Thus, in accordance with the first aspect of the present invention, a compound of Formula I or pharmaceutically acceptable salts thereof are provided

Formula I wherein

Y represents a bond, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyl, or a group -(CR_aRb)_n-Q-(CR_cRd)m-; W represents hydrogen, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, a group -(CR_eR_f)p-CR_gR_hRi or a group Z-R₃;

Ri represents an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyl, or a group C(=0)R₄;

Q represents O, NR₅;

Z represents O, NR₅;

F¾5 represents hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl;

R_a, Rt>, Rc, Rd, Re, Rf, R_g, Rh, and R, are independently selected from the group consisting of hydrogen, halogen, an optionally substituted lower alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, alkoxy, cycloalkyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, or trifluoromethoxy;

n is an integer from 1 to 12;

m is an integer from 0 to 12;

p is an integer from 0 to 6.

Compounds of Formula I containing a carbon-carbon double bond can exist as E and Z geometric isomers. Geometric isomers of compounds of Formula I containing one or more carbon-carbon double bonds are within the scope of the present invention.

Compounds of Formula I may contain one or more chiral centers. Compounds containing one chiral center can occur as single enantiomers or mixtures of the two enantiomers. Such mixtures occur as racemates or racemic mixtures. Compounds containing more than one chiral center can occur as single enantiomers and pairs of enantiomers, and as stereoisomers which are not enantiomers, referred to as diastereoisomers. Compounds of Formula I are meant to encompass all possible stereoisomers and mixtures thereof.

Some of the compounds described herein may exist with different points of attachment of a hydrogen atom, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed by the Formula I.

The compounds of Formula I may have unnatural ratios of atomic isotopes at one or more of their atoms. For example, the compounds may be radiolabeled with isotopes such as tritium or carbon-14. All isotopic variations of the compounds of the present invention, whether radioactive or not, are within the scope of the present invention

Compounds of Formula I may be isolated in the form of their pharmaceutically acceptable acid addition salts, such as the salts derived from inorganic and organic acids. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable, non-toxic acids including inorganic or organic acids. Such acids include hydrochloric, sulfuric, phosphoric, glycolic, malic, maleic, tartaric, succinic, citric, malonic acid and the like.

The present invention also encompasses active metabolites of compounds of Formula I.

In certain embodiments of the compounds of Formula I,

R-i represents an optionally substituted alkyl, an optionally substituted cycloalkyl selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane, or a group C(=0)R₄,

R₄ represents an optionally substituted lower alkyl, typically a CrC₃ alkyl, an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, biphenyl, an optionally substituted heteroaryl selected from pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl, pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl, or an optionally substituted heterocyclyl selected from oxetane, tetrahydrofuran, tetrahydropyran, azetidine, pyrrolidine, piperidine, or morpholine. Y represents an optionally substituted alkyi, or an optionally substituted cycloalkyl selected from cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

W represents hydrogen, an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, biphenyl, an optionally substituted heteroaryl selected from oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and quinazolinyl, or an optionally substituted cycloalkyl selected from cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

In certain embodiments of the compounds of Formula I, R-i represents an optionally substituted CrC₆ alkyi, preferably a C₁-C₃ alkyi.

In certain embodiments, Ri represents an optionally substituted (CrC₆)-alkyl- cycloalkyl, preferably a (C-i-C₃)-alkyl-cycloalkyl.

In certain embodiments of the compounds of Formula I, R₄ represents an optionally substituted lower alkyi, typically a C₁ -C3 alkyi, an optionally substituted aryl, or a heteroaryl which is 5-membered aromatic ring containing N as heteroatom or containing two or three heteroatoms selected from N, O, S.

Suitable 5-membered aromatic rings containing two or three heteroatoms selected from N, O or S include oxazolyl, thiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl.

Suitable 6-membered aromatic rings containing one to three heteroatoms include pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl.

In certain embodiments R₄ represents an optionally substituted C₁-C₃ alkyi, selected from methyl, ethyl, n-propyl and /^'sopropyl, or an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, and biphenyl.

In certain preferred embodiments of the compounds of Formula I, R₄ represents a group 0-R₆, wherein R₆ represents an optionally substituted C₁-C₃ alkyi preferably selected from methyl, ethyl, n-propyl, and /sopropyl.

In certain embodiments R₆ represents an optionally substituted cycloalkyl preferably selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, or an optionally substituted heterocyclyl preferably selected from oxetane, tetrahydrofuran, tetrahydropyran, azetidine, pyrrolidine, piperidine, or morpholine. In certain embodiments R₆ is an optionally substituted cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In certain embodiments of the first aspect of the invention, compounds of Formula I are selected from the group comprising:

5-fluoro-N-hexyl-3-methyl-2,4-dioxo-pyrimidine-1 -carboxamide

3-(cyclopropylmethyl)-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

5-fluoro-N-hexyl-3-(2-methylpropanoyl)-2,4-dioxo-pyrimidine-1 -carboxamide methyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-benzoyl-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

Ethyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-Ethyl-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

Methyl 5-fluoro-3-(octylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-Benzoyl-5-fluoro-N-octyl-2,4-dioxo-pyrimidine-1 -carboxamide.

III. Definitions

All technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art, unless otherwise defined.

The following terms, used in the specification and claims of this application, have the meaning specified hereunder, unless otherwise defined.

The term "alkyi", as used herein, indicates a saturated aliphatic hydrocarbon radical, including straight chain and branched chain radicals of 1 to 1 6 carbon atoms. In certain embodiments, alkyi refers to straight chain and branched chain radicals of 1 to 1 2 carbon atoms. The term "lower alkyi", as used herein, refers to straight chain and branched chain radicals of 1 to 6 carbon atoms, preferably of 1 to 3 carbon atoms. Non-limiting examples of alkyi are, for instance, methyl, ethyl, propyl, isopropyl, n-butyl, /^'sobutyl, terf-butyl, n-amyl, iso-amyl, n-hexyl, n-heptyl, n-octyl and the like.

Any alkyi group may be unsubstituted or substituted by one or more substituents. Thus, the term "substituted alkyi" comprises alkyi groups as defined hereinabove in which one or more atoms or functional groups of the alkyi moiety are replaced with another atom or functional group including, by way of example, alkyi, cycloalkyl, halogen, aryl, substituted aryl, hydroxyl, amino, alkoxyl, alkylamino, sulfate. In certain embodiments alkyi is substituted by one or more substituents independently selected from the group consisting of halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

The term "alkenyl", as used herein, indicates an alkyl group, as defined herein, consisting of at least two carbon atoms and containing at least one carbon-carbon double bond. In certain embodiments, alkenyl refers to alkyl radicals of 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. Examples include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, 1 - or 2-butenyl, and the like. Any alkenyl group may be unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

The term "cycloalkyi", as used herein, indicates a 3- to 7-membered all-carbon monocyclic ring, which may contain one or more double bonds but does not have a completely conjugated pi-electron system.

Examples of cycloalkyi groups include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, and cycloheptane.

In certain embodiments a suitable cycloalkyi group is selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane.

A cycloalkyi group may be unsubstituted or substituted by one to three substituents independently selected from the group comprising lower alkyl, halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

The term "aryl", as used herein, indicates a hydrocarbon consisting of a mono-, bi- or tricyclic ring system, wherein the rings are fused together or linked to each other covalently and at least one of the carbocyclic rings is aromatic. The term "aryl" specifically encompasses heterocyclic aromatic compounds. In particular embodiments, the term "aryl" means a cyclic aromatic group comprising 3 to 7 carbon atoms, specifically including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

Not limiting examples of aryl groups include, but are not limited to, phenyl, alpha- or beta-naphthyl, 9,10-dihydroanthracenyl, indanyl, fluorenyl, biphenyl and the like. An aryl group may be unsubstituted or substituted by one to three substituents independently selected from the group consisting of lower alkyl, halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

In certain embodiments a suitable aryl group is selected from phenyl, alpha- or beta-naphthyl, indanyl, and biphenyl.

The aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents, which can be the same or different, wherein "aryl group substituent" includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NK'K", wherein K' and K" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or aralkyl.

In certain embodiments the substituted aryl includes by one to three substituents independently selected from the group consisting of lower alkyl, halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

The term "heteroaryl", as used herein, indicates a mono-, bi- or tricyclic ring system containing from one to three heteroatoms selected from nitrogen, oxygen and sulfur, wherein the rings are fused together or linked to each other covalently and at least one of the rings is aromatic. Not limiting examples of heteroaryl groups include pyrrolyl, thiophenyl, furoyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, indolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzopyrazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl and the like.

In certain embodiments a suitable heteroaryl group is selected from oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzofuranyl, benzothiophenyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazolinyl. A heteroaryl group may be unsubstituted or substituted by one to three substituents independently selected from the group consisting of lower alkyl, halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino

The terms "heterocyclyl" or "heterocyclic ring", as used herein, mean a 3- to 7- membered, saturated or partially unsaturated carbocyclic ring wherein one or more carbon atoms are independently replaced by nitrogen, oxygen and sulfur. The heteroatom nitrogen and sulfur are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Not limiting examples of heterocyclyl groups include, for instance, radicals derived from oxirane, aziridine, oxetane, azetidine, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrolidine, dihydropyrrole, pyran, dihydropyran, tetrahydropyran, tetrahydrothiopyran, piperidine, pyrazoline, oxazoline, isoxazolidine, isoxazoline, thiazolidine, thiazoline, isothiazoline, dioxane, piperazine, morpholine, thiomorpholine, examethyleneimine, homopiperazine, and the like. A heterocyclyl group or a heterocyclic ring may be unsubstituted or substituted by one to three substituents independently selected from the group consisting of lower alkyl, halogen, trifluoromethyl, hydroxy, alkoxy, trifluoromethoxy, amino, monoalkylamino, or dialkylamino.

In certain embodiments a suitable heterocyclyl group is selected from oxetane, tetrahydrofuran, pyran, dihydropyran, tetrahydropyran, dioxane, oxazoline, azetidine, pyrrolidine, piperidine, piperazine, and morpholine.

The term "aromatic" refers to a moiety wherein the constituent atoms make up an unsaturated ring system, all atoms in the ring system are sp2 hybridized and the total number of pi electrons is equal to 4n+2, wherein n is an integer.

The term "alkoxy", as used herein, means an unsubstituted or substituted alkyl chain linked to the remainder of the molecule through an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propyloxy, isopropyloxy, benzyloxy and the like.

The term "amino" means a -NH₂ radical.

The term "aryloxy", as used herein, means an unsubstituted or substituted aryl group linked to the remainder of the molecule through an oxygen atom. Examples of aryloxy include, but are not limited to, phenoxy, alpha- or beta-naphthyloxy, biphenyloxy and the like.

The term "cycloalkyloxy", as used therein, means an unsubstituted or substituted cycloalkyl group linked to the remainder of the molecule through an oxygen atom. Examples of cycloalkyloxy include, but are not limited, to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclopentenyloxy, cyclohexyloxy, cyclohexenyloxy, cyclohexadienyloxy, cycloheptanyloxy and the like.

The term "heteroaryloxy", as used therein, means an unsubstituted or substituted heteroaryl group linked to the remainder of the molecule through an oxygen atom. The term "heterocyclyloxy", used therein, means an unsubstituted or substituted heterocyclyl group linked to the remainder of the molecule through an oxygen atom.

The term "halogen", as used herein, indicates fluorine (F), chlorine (CI), bromine (Br) or iodine (I).

The term "hydroxyl" means a -OH radical.

The term "monoalkylamino", as used herein, represents an amino group wherein one of the hydrogen atoms is substituted by an alkyl chain. Not limiting examples of monoalkylamino include methylamino, ethylamino, propylamino, butylamino and the like.

The term "dialkylamino", as used herein, represents an amino group wherein both hydrogen atoms are substituted by an alkyl chain. The two alkyl chains can be the same or different. Not limiting examples of dialkylamino include dimethylamino, diethylamino, dipropylamino, methylethylamino, methylisopropylamino and the like.

The term "trifluoromethyl" means a -CF₃ radical.

The term "trifluoromethoxy" means a -OCF₃ radical.

IV. Pharmaceutically acceptable salts

It will be understood that, as used herein, references to the compounds of Formula I are meant to include also the pharmaceutically acceptable salts or derivatives thereof.

Furthermore, the compound of the Formula I may form an acid addition salt or a salt with a base, depending on the kind of the substituents, and these salts are included in the present invention, as long as they are pharmaceutically acceptable salts.

The terms "the compound of the invention" and "the compounds of the present invention" and "the compounds of Formula I" refer to each of the compounds of Formula I and are meant to include their pharmaceutically acceptable salts, hydrates, solvates, and crystalline forms and also any suitable forms as illustrated hereinafter.

As used herein, the term "salt" refers to any salt of a compound according to the present invention prepared from an inorganic or organic acid or base and internally formed salts. Typically, such salts have a physiologically acceptable anion or cation.

Suitably physiologically or pharmaceutically acceptable salts of the compounds of the present invention include the hydrochloride, acetate, citrate, gluconate, lactate, tartrate, phosphate, borate, maleate, sulphate and nitrate, the hydrochloride being preferred.

The salts of compounds of Formula I may be prepared by reacting a basic compound with the desired acid in solution.

Physiologically or pharmaceutically acceptable salts are particularly suitable for medical applications because of their greater aqueous solubility relative to the parent compounds.

Pharmaceutical acceptable salts may also be prepared from other salts, including other pharmaceutically acceptable salts, of the compounds of Formula I, using conventional methods.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of the invention are within the scope of the invention. The compounds of Formula I may readily be isolated in association with solvent molecules by crystallization or evaporation of an appropriate solvent to give the corresponding solvates. The compounds of Formula I of the invention may be in crystalline forms. In certain embodiments, the crystalline forms of the compounds of Formula I are polymorphs.

The subject invention also includes isotopically-labelled compounds, which are identical to those recited in Formula I and following, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, iodine, and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ³¹ P, ³²P, ³⁵S, ¹⁸F, ³⁶CI, ¹²³l and ¹²⁵l.

Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as ³H, ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. ¹¹C and ¹⁸F isotopes are particularly useful in PET (positron emission tomography), and ¹²⁵l isotopes are particularly useful in SPECT (single photon emission computerized tomography), all useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of Formula I and following of this invention can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

Certain groups/substituents included in the present invention may be present as isomers or in one or more tautomeric forms. Accordingly, in certain embodiments, the compound of the Formula I may exist in the form of other tautomers or geometrical isomers in some cases, depending on the kinds of the substituents. In the present specification, the compound may be described in only one form of such isomers, but the present invention includes such isomers, isolated forms of the isomers, or a mixture thereof. Furthermore, the compound of the Formula I may have asymmetric carbon atoms or axial asymmetries in some cases, and correspondingly, it may exist in the form of optical isomers such as an (F?)-form, an (S)-form, and the like. The present invention includes all such isomers, including racemates, enantiomers and mixtures thereof.

In particular, within the scope of the present invention are included all stereoisomeric forms, including enantiomers, diastereoisomers, and mixtures thereof, including racemates and the general reference to the compounds of Formula I include all the stereoisomeric forms, unless otherwise indicated.

In general, the compounds or salts of the invention should be interpreted as excluding those compounds (if any) which are so chemically unstable, either per se or in water, that they are clearly unsuitable for pharmaceutical use through all administration routes, whether oral, parenteral or otherwise. Such compounds are known to the skilled chemist. Prodrugs or compounds which are stable ex vivo and which are convertable in the mammalian (e.g. human) body to the inventive compounds are however included.

V. Methods for preparing compounds of Formula I

In a further aspect the present invention provides methods for preparing compounds of Formula I.

In general, the compounds of Formula I can be prepared through a process including synthetic transformations reported, for instance, in Michael Smith, Jerry March - March's Advanced Organic Chemistry: reactions mechanisms and structure - 6th Edition, John Wiley & Sons Inc., 2007, which is herein incorporated as reference. It is well known to one of ordinary skill in the art that transformation of a chemical function into another may require that one or more reactive centers in the compound containing this function be protected in order to avoid undesired side reactions. Protection of such reactive centers, and subsequent de-protection at the end of the synthetic transformations, can be accomplished following standard procedures described, for instance, in Theodora W. Green and Peter G.M. Wuts - Protective Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons Inc., 2006, which is herein incorporated as reference.

In certain embodiments, a compound of Formula I can be obtained by reaction of compound of Formula II, or a salt thereof, with an isoc anate of Formula III,

II

WhereinY, and W are as defined above, and Ri represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl.

Compounds of Formula II are either commercially available or can be obtained according to standard synthetic methods as reported, for instance, in Michael Smith, Jerry March - March's Advanced Organic Chemistry: reaction mechanisms and structure - 6^th Edition, John Wiley & Sons Inc., 2007, and references cited therein, which is incorporated herein as reference.

A compound of Formula II can be obtained by treating another compound represented by the same Formula II with suitable reagents in order to remove one or more protective groups introduced in one of the synthetic steps, as known to those skilled in the art. Protection of reactive centers, and subsequent de- protection, can be accomplished following standard procedures described, for instance, in Theodora W. Greene and Peter G. M. Wuts - Protective Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons Inc., 2006, which is herein incorporated as reference.

A compound of Formula II can be obtained by treating another compound represented by the same Formula II, with suitable reagents in order to transform one or more functional groups into one or more new functional groups. An isocyanate of Formula I I I is either commercially available or can be prepared by synthetic methods as reported, for instance, in Molina P., Tarraga A., Arques A. in Katritzky A. ft, Taylor ft J. k., Comprehensive Organic Functional Group Trasformations II, Elsevier, 2004, Vol. 5, Pag. 949-973; or in Michael Smith, Jerry March - March's Advanced Organic Chemistry: reaction mechanisms and structure - 6^th Edition, John Wiley & Sons Inc., 2007, and references cited therein, which are incorporated herein as reference.

A compound of Formula I I, wherein R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl, can be obtained by base-mediated cleavage of the terf-butyloxycarbonyl group of a compound of Formula IV

wherein R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl. A compound of Formula IV, as defined above, can be obtained by reaction of a compound of Formula V with an halide of Formula VI,

VI

V wherein Hal represents bromine or iodine, and Ri is an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyi.

A compound of Formula V, as defined above, can be obtained by reaction of 5- fluorouracil VII

with di-terf-butyl dicarbonate, according to standard synthetic procedures, as known to those skilled in the art. A halide of Formula VI is either commercially available or can be prepared from suitable precursors, as known to a person skilled in the art, according to standard synthetic methods as reported, for instance, in Michael Smith, Jerry March - March's Advanced Organic Chemistry: reaction mechanisms and structure - 6^th Edition, John Wiley & Sons Inc., 2007, and references cited therein, which is incorporated herein as reference.

In another embodiment, a compound of Formula I, wherein Y and W are as defined above, and Ri represents a group C(=0)R₄, wherein R₄ represents an optionally substituted lower alkyl, an optionally substituted cycloalkyi, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocyclyl, or a group 0-R₆, can be obtained by reaction of compound of Formula VIII, wherein Y and W are as defined above, with a chloride of Formula IX,

wherein R₄ is as defined above. A chloride of Formula IX is either commercially available or can be prepared from suitable precursors, as known to a person skilled in the art, according to standard synthetic methods as reported, for instance, in Michael Smith, Jerry March - March's Advanced Organic Chemistry: reaction mechanisms and structure - 6^th Edition, John Wiley & Sons Inc., 2007, and references cited therein, which is incorporated herein as reference.

The synthesis of a compound of Formula I, according to the synthetic processes described above, can be conducted in a stepwise manner, whereby each intermediate is isolated and purified by standard purification techniques, like, for example, column chromatography, before carrying out the subsequent reaction. Alternatively, two or more steps of the synthetic sequence can be carried out in a so-called "one-pot" procedure, as known in the art, whereby only the compound resulting from the two or more steps is isolated and purified.

The compounds described above can be prepared as exemplified in the following procedures.

A compound of Formula I, wherein Y and W are as defined above, and R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl, can be obtained by reaction of compound of Formula II, as defined above, with an isocyanate of Formula III, as defined above. The reaction is preferably conducted in anhydrous, polar aprotic solvents, such as pyridine, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), dichloromethane, and the like, at a temperature ranging from room temperature to 100°C, and for a period of time from 10 minutes to 18 hours. The reaction can be conducted in the presence of tertiary amines such as 4-dimethylaminopyridine, di-isopropyl ethyl amine and the like.

A compound of Formula II, wherein R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl, can be obtained by base-mediated cleavage of the terf-butyloxycarbonyl group of a compound of Formula IV, as defined above. The reaction is conducted in a polar protic solvent such as water, methanol, ethanol, and the like, and in the presence of a base such as potassium carbonate (K₂C0₃), potassium terf-butoxide, and the like. The reaction is carried out at a temperature ranging from 0°C to 100°C and for a period of time from 15 minutes to 18 hours.

A compound of Formula IV, wherein R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyl, can be obtained by reaction of a compound of Formula V, as defined above, with an halide of Formula VI, as defined above. The reaction is carried out in a suitable solvent such as Λ/,/V-dimethylformamide (DMF), acetonitrile, tetrahydrofuran (THF), and the like, and in the presence of an inorganic base such as sodium hydride (NaH), sodium carbonate (Na₂C0₃), potassium carbonate (K₂C0₃), cesium carbonate (Cs₂C0₃), and the like, at a temperature ranging from 0°C to 40 °C and for a period of time from 1 hour to 18 hours.

A compound of Formula V, can be obtained by reaction of 5-fluorouracil VII with di- terf-butyl dicarbonate. The reaction is conducted in a suitable solvent such as acetronitrile, tetrahydrofuran (THF), Λ/,/V-dimethylformamide (DMF), dichloromethane, dimethoxyethane (DME), and the like, at a temperature ranging from 0°C to 50 °C and for a period of time from 1 hours to 18 hours. Occasionally, the reaction can be conducted in the presence of tertiary amines such as 4- dimethylaminopyridine, di-isopropyl ethyl amine, imidazole, triethylamine, and the like.

A compound of Formula I, wherein Y, and W are as defined above, and R-i represents a group C(=O)R₄, wherein R₄ represents an optionally substituted lower alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocyclyl, or a group O-R₆,can be obtained by reaction of compound of Formula VIII with a chloride of Formula IX. The reaction is conducted in a suitable solvent such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran, pyridine, or mixtures thereof, and in the presence of a suitable base such as triethylamine, di- isopropylethylamine, or pyridine, at a temperature ranging from -10°C to 40 °C, and for a period of time from 1 hour to 72 hours.

VI. Medical uses of compounds of Formula I

In accordance with a second aspect of the present invention compounds of Formula I are provided for use as a medicament. In accordance with an additional aspect, the present invention provides the compounds of Formula I for use in treating diseases or disorders associated with increased (relative to physiological or desired) levels of acid ceramidase protein or function, for example in subjects where acid ceramidase protein is overactive or over-expressed.

In accordance to an aspect, a method of treatment of diseases or disorders associated with increased (relative to physiological or desired) levels of acid ceramidase protein or function, for example in subjects where acid ceramidase protein is overactive or over-expressed, is also provided.

In some embodiments, the compounds of Formula I and their pharmaceutical compositions and methods of administering them, are useful in treating diseases or disorders involving cell overproliferation and/or dysfunctional sphingolipid signal transduction.

These diseases and disorders include, but are not limited to, primary and metastatic neoplastic diseases. Diseases and disorders involving cell overproliferation include, but are not limited to, pre-malignant conditions, for example hyperplasia, metaplasia or dysplasia, cancers, cancer metastasis, benign tumors, hyperproliferative disorders and benign dysproliferative disorders.

The treatment may be prophylactic or therapeutic.

The subject to be treated may be an animal (e.g., mouse, rat, non-human primate, and non-human mammal) or human.

Primary and metastatic neoplastic diseases and related disorders that can be treated and/or prevented by the methods, compounds and compositions of the presently disclosed subject matter include, but are not limited to, prostate cancers, colorectal cancers, liver cancer, head and neck cancer, breast cancer, melanoma, metastatic melanoma, precancerous skin conditions such as actinic keratosis, skin cancers such as squamous cell carcinoma and basal cell carcinoma, and hematological malignancies such as chronic myelogeneous leukemia.

In accordance with certain aspects the present invention provides a method for the treatment or prevention of cancer, cancer metastasis, inflammation, neuropathic pain, asthma, atherosclerosis, stenosis, psoriasis or atopic dermatitis, comprising the administration of a therapeutically effective compound of Formula I according to one or more of the embodiments described above, in a subject in need of treatment.

Cancers and related disorders that can be treated and/or prevented by the methods and compositions of the presently disclosed subject matter include, but are not limited to acute and chronic leukemia; polycythemia vera; lymphomas such as Hodgkin's disease, non-Hodgkin's disease; multiple myelomas, plasmacytoma; Waldenstrom's acroglobulinemia; gammopathy; heavy chain disease; bone and connective tissue sarcomas; brain tumors; breast cancer; adrenal cancer; thyroid cancer; pancreatic cancer; pituitary cancers; eye cancers; vaginal cancers; vulvar cancer; cervical cancers; uterine cancers; ovarian cancers; head and neck squamous cell cancers (HNSCCs), esophageal cancers; stomach cancers; colon cancers; rectal cancers; liver cancers; cholangiocarcinomas; testicular cancers, prostate cancers; penal cancers; oral cancers; basal cancers; salivary gland cancers; pharynx cancers; skin cancers; kidney cancers; Wilms' tumor; bladder cancers, myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas. In certain embodiments, the present invention provides compounds of Formula I for the use in the treatment and/or prevention of breast cancer, prostate cancer, melanoma, alveolar cancer, or head and neck cancer. In some embodiments, the compounds of Formula I, and their pharmaceutical compositions and methods of administering them, are useful in treating or preventing a disease or disorder when administered in combination with other treatments.

In an additional aspect, the present invention also concerns combination therapies or treatment with a compound of Formula I or pharmaceutical composition containing them.

In some embodiments, the compounds of Formula I, and their pharmaceutical compositions and methods of administering them, are useful in treating various cancers when administered in combination with other pharmacological agents or active ingredients. In certain embodiments these pharmacological agents are chemotherapeutic agents including, but not limited to, doxorubicin, daunorubicin, etoposide, cisplatin, oxaliplatin, carboplatin, gemcitabine, 5-fluorouracil, capecitabine, tegafur-uracil (UFT), dacarbazine, fenretinide, camptothecin, irinotecan, fludarabine, vinblastine, taxol, mitomycin C.

In accordance with certain aspects, the compounds of Formula (I) of the invention are chemosensitizer agents.

A chemosensitizer agent is a medicament that is administered to make tumor cells more sensitive to chemotherapy.

In certain aspects the present invention provides for a chemosensitizing therapy. One of the aim of the chemosensitizing therapy of the invention is to make tumor cells more sensitive to chemotherapy so that lower doses of antitumoral agent is administered and a reduction of side effects is achieved.

In another aspect the present invention provides a method of treating a subject suspected of having or having developed chemoresistance, i.e. resistance to chemotherapy, the method comprising administering to the subject a therapeutically effective amount of at least one chemotherapeutic agent and an effective amount of at least one chemosensitizing compound of Formula I.

In some embodiments, the compounds of Formula I, and their pharmaceutical compositions, are administered before, during or after patient's treatment with one or more chemotherapeutic agents.

In a further aspect the present invention provides for a product or kit containing a compound of Formula I and a chemotherapeutic agent as a combined preparation for simultaneous, separate or sequential use in antitumoral therapy.

As used herein, the terms chemotherapeutic agent, antitumoral agent, anticancer agent, anticancer are interchangeable and can be considered to have the same meanings.

In some embodiments, the compounds of Formula I, and their pharmaceutical compositions and methods of administering them, are useful in treating various cancers when administered before, during or after patient's treatment with radiation therapy. In accordance with an additional aspect, the present invention provides a method of inhibiting ceramidase-related activity by contacting a biological sample with a compound of Formula I as described hereinabove.

In certain embodiments the biological sample is an in vitro cell sample or an in vivo cell sample. The biological sample includes cells in culture media or lysed cells containing acid ceramidase. The biological sample includes cells present in plasma, urine, a tissue or organ sample or present in a subject. According to certain aspects of the invention, the methods can be used in medical or scientific research related to acid ceramidase and ceramidase-related activity.

VIII. Pharmaceutical Compositions

In a third aspect, the invention provides pharmaceutical compositions of compounds of Formula I.

The pharmaceutical compositions of the present invention encompass any compositions made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. Such compositions are suitable for pharmaceutical use in an animal or human.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more compounds of Formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A pharmaceutical composition may optionally contain other active ingredients.

The term "carrier" refers to a vehicle, excipient, diluent, or adjuvant with which the therapeutic or active ingredient is administered. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated for use with the compounds disclosed herein.

The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

In certain embodiments, the compounds of the present invention can be combined as the active ingredient in intimate admixture with a suitable pharmaceutical carrier and/or excipient according to conventional pharmaceutical compounding techniques.

The compositions include compositions suitable for, parenteral including subcutaneous, intramuscular, and intravenous, pulmonary, nasal, rectal, topical or oral administration. Suitable route of administration in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient. An exemplary route of administration is the oral route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

The preferred compositions include compositions suitable for oral, parenteral, topical, subcutaneous, or pulmonary, in the form of nasal or buccal inhalation, administration. The compositions may be prepared by any of the methods well- known in the art of pharmacy.

The pharmaceutical compositions may be in the form of tablets, pills, capsules, solutions, suspensions, emulsion, powders, suppository and as sustained release formulations.

If desired, tablets may be coated by standard aqueous or non-aqueous techniques. In certain embodiments such compositions and preparations can contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 1 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a therapeutically effective dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor. To prevent breakdown during transit through the upper portion of the gastrointestinal tract, the composition may be an enteric coated formulation.

Compositions for topical administration include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, sticks, liposomes, nanoparticles, patches, bandages and wound dressings. In certain embodiments, the topical formulation comprises a penetration enhancer.

Compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of the powder of a compound of Formula I or a salt thereof, and the powder of a suitable carrier and/or lubricant. The compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to a person skilled in the art.

Administration of the compositions is performed under a protocol and at a dosage sufficient to reduce the inflammation and pain in the subject.

In some embodiments, in the pharmaceutical compositions of the present invention the active principle or active principles are generally formulated in dosage units. The dosage unit may contain from 0.1 to 1000 mg of a compound of Formula I per dosage unit for daily administration.

In some embodiments, the amounts effective for topical formulation will depend on the severity of the disease, disorder or condition, previous therapy, the individual's health status and response to the drug. In some embodiments, the dose is in the range from 0.001 % by weight to about 60% by weight of the formulation.

When used in combination with one or more other active ingredients, the compound of the present invention and the other active ingredients may be used in lower doses than when each is used singly.

With respect to formulations with respect to any variety of routes of administration, methods and formulations for the administration of drugs are disclosed in Remington's Pharmaceutical Sciences, 17^th Edition, Gennaro et al. Eds., Mack Publishing Co., 1985, and Remington's Pharmaceutical Sciences, Gennaro AR ed. 20^th edition, 2000, Williams & Wilkins PA, USA, and Remington: The Science and Practice of Pharmacy, 21^st Edition, Lippincott Williams & Wilkins Eds., 2005; and in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8^th Edition. Lippincott Williams & Wilkins Eds., 2005, which are herein incorporated as reference.

The present invention claims the priority of the Italian application MI2012A000923 filed on 28 May 2012, whose content is fully incorporated hereby.

IX. Methods for testing compounds on Acid Ceramidase

Recombinant rat and human AC expression

Rat Acid Ceramidase (rAC) was cloned from a brain cDNA library using primers based on the sequence obtained from the National Center for Biotechnology Information (NCBI) database: 5'rAC (5'-GACCATGCTGGGCCGTAGT-3') and 3'rAC (5'-CCAGCCTATACAAGGGTCT-3'). The PCR (High Fidelity PCR Master, Roche) product was subcloned into a pEF6-V5/His vector (Invitrogen) to construct a mammalian expression vector encoding V5/His-tagged rat AC. HEK293 cells were transfected with pEF6-rAC-V5/His using Super-Feet reagent (Qiagen) and screened with G418 (0.3 mg/mL).

Human Acid Ceramidase (hAC) cDNA was purchased from Open Biosystem (clone ID 3923451 ) and subcloned in the mammalian expression vector pCDNA3.1 , containing the neomycin resistance gene. The cell line HEK293 was transfected with hAC-pCDNA3.1 construct using JetPEI reagent (Polypus transfectionTM, lllkirch, FR) following the manufacturer instructions. A stable cell line of hAC-overexpressing HEK293 was generated by selection with G418 (1 mg/ml) and cell clones were derived by limited dilution plating. hAC-expressing clones were analyzed by western blot.

Protein preparation Hek293 cells overexpressing rAC or hAC were suspended in 20 mM Tris HCI (pH 7.5) containing 0.32M sucrose, sonicated and centrifuged at 800xg for 15 min at 4°C. The supernatants were centrifuged again at 12,000xg for 30 min at 4°C. The pellets were suspended in phosphate-buffered saline (PBS) and subjected to 2 freeze-thaw cycles at -80 °C. The suspensions were centrifuged at 105,000xg for 1 h at 4 °C. The supernatants containing hAC were kept at -80 °C until use. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce). Acid Ceramidase activity

A rAC or hAC protein preparation (25 or 10 μg, respectively) was preincubated with inhibitors (final DMSO concentration 1 %) in assay buffer (100 mM sodium phosphate, 0.1 % Nonidet P-40, 150 mM NaCI, 3 mM DTT, 100 mM sodium citrate, pH 4.5) for 30 min at 37 °C. Reactions were started by the addition of 50 μΜ N- lauroyl ceramide (Nu-Chek Prep, Elysian, MN) and carried on for 30 min at 37 °C. Reactions were stopped by addition of a mixture of chloroform/methanol (2:1 , vol/vol) containing 1 nmol 1 1 -lauroleic acid (NuChek Prep). The organic phases were collected, dried under nitrogen and analyzed by UPLC/MS (Acquity, Waters) in the negative-ion mode monitoring the reaction product (lauric acid, m/z=199) using 1 1 -lauroleic acid as internal standard.

Lipids were eluted on an Acquity UPLC BEH C18 column (50mm length, 2.1 mm i.d., 1 .7 μιη pore size, Waters) column at 0.5 mL-min^"1 for 1 .5 min with a gradient of acetonitrile (CH₃CN) and water, both containing 0.25% acetic acid and 5 mM ammonium acetate (70% to 100% CH₃CN in 0.5 min, 100% CH₃CN for 0.5 min, 70% CH₃CN for 0.4 min). The column temperature was 40 °C. Electrospray ionization (ESI) was in the negative mode, capillary voltage was 1 kV and cone voltage was 50 V. Nitrogen was used as drying gas at a flow rate of 500 L/h and at a temperature of 400 °C.

The [M-H]^" ion was monitored in the selected-ion monitoring mode (m/z values: lauric acid 199, 1 1 -lauroleic acid 197.35). Calibration curves were generated with authentic lauric acid (Nu Check Prep). Inhibition of AC activity was calculated as reduction of lauric acid in the samples compared to vehicle controls. IC₅o values were calculated by non-linear regression analysis of log[concentration]/inhibition curves using GraphPad Prism 5 (GraphPad Software Inc., CA, USA) applying a standard slope curve fitting.

The IC₅o value of the compounds described in the Examples are reported in Table 1 .

Table 1. IC50 value on rat acid ceramidase (rAC) and human acid ceramidase (hAC) of selected compounds of the invention.

Compound 1 and compound 2, but not 5-fluorouracil (5FU), inhibited acid ceramidase activity in SW403 cells, as shown in Figure 1 . Acid ceramidase activity in SW403 cells was measured, using a method analogous to that reported above, after three hours exposure to different concentrations (1 and 10μΜ) of compound 1 and compound 2, 300 nM 5-fluorouracil, or vehicle. Results are expressed as mean ± SEM (n=3). ^***p<0.0001 vs vehicle, one-way ANOVA followed by Tukey's. Methods for screening compounds for a therapeutic activity

Lipid Extraction and ceramide analysis. Lipids were extracted using a chloroform/methanol mixture (2:1 vol/vol, 3 ml_) containing internal standards. The organic phases were collected, dried under nitrogen, and dissolved in methanol/chloroform (3:1 vol/vol) for LC/MS analyses.

Ceramides were analyzed by LC/MSⁿ, using a 1 100-LC system (Agilent Technologies) equipped with an Ion Trap XCT and interfaced with ESI (Agilent Technologies). They were separated on a Poroshell 300 SB C18 column (2.1 x 75 mm i.d., 5 μιη; Agilent Technologies) maintained at 30°C. A linear gradient of methanol in water containing 5 mM ammonium acetate and 0.25% acetic acid (from 80% to 100% of methanol in 3 min) was applied at a flow rate of 1 mL/min. Detection was in the positive mode, capillary voltage was 4.5 kV, skimmer voltage at -40 V, and capillary exit -151 V. Nitrogen was used as drying gas at a flow rate of 10 L/min, temperature was 350 °C, and nebulizer pressure of 80 psi. Helium was used as collision gas.

Tissue-derived ceramides were identified by comparison of their LC retention times and MSⁿ fragmentation patterns with those of authentic standards (Avanti Polar Lipids). Extracted ion chromatograms were used to quantify myristoyl ceramide (C14:0, m/z 510.5 > 492.5 > 264.3), palmitoyi ceramide (C16:0, m/z 538.5 > 520.3 > 264.3), stearoyl ceramide (C18:0 m/z 566.5 > 548.3 > 264.3), lignoceroylceramide (C24:0 m/z 650.5 > 632.3 > 264.3), nervonoylceramide (C24:1 m/z 648.5 > 630.3 > 264.3) and using lauroyi ceramide standard (m/z 482.5 > 464.5 > 264.3). Detection and analysis were controlled by Agilent/Bruker Daltonics software version 5.2. MS spectra were processed using MS Processor from Advanced Chemistry Development.

Compound 1 and compound 2, but not 5-fluorouracil (5FU), increase ceramide levels in the human colon adenocarcinoma cell line SW403, consistent with their ability to inhibit acid ceramidase.

Exposure of SW403 cells to 10μΜ compound 1 or 10μΜ compound 2, for three hours, caused an increase in the levels of the most abundant endogenous ceramide species (C14, C16 and C18:0), as shown in Figure 2. The levels of ceramide species did not increase after three hours exposure to 300 nM 5- fluorouracil or vehicle. Results are expressed as mean ± SEM (n=3). ^***p<0.0001 , vs vehicle, one-way ANOVA followed by Tukey's.

Cell viability and proliferation assays.

Cell viability can be defined as the number of living cells in a sample. There are many well described and widely used methods to evaluate cell viability such as trypan blue dye exclusion, MTT reduction or crystal violet [for a review, see Stoddart MJ, Cell viability assays: introduction. Methods in Molecular Biology, 201 1, Vol. 740].

Cells are seeded in 12- or 96- well plates in complete medium 24 hrs before treatment and then incubated for 24 hrs (single treatment) or 72 hrs (multiple treatments) with different drug concentrations in media without serum. Cell viability is then evaluated.

The trypan blue exclusion assay is based on the principle that viable cells have intact cell membrane and can therefore exclude the trypan blue dye. Cell viability is measured after 24 hrs incubation with the drug. Cells are harvested, centrifuged at 1200 rpm for 10min and pellets re-suspended in PBS. To evaluate viability, cells are diluted 1 :1 with 0.4% trypan blue dye (Sigma), incubated for 1 min and white (viable) cells are counted with a hemacytometer.

Alternatively, cell viability can be assessed measuring mitochondrial functionality by the MTT assay, which is based on the reduction of the soluble tetrazolium salt MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into insoluble formazan by mitochondria. Briefly, after treatment, cells are washed with PBS and incubated with 0.5 mg/mL MTT for 2 hrs at room temperature. MTT reduction is quantified by absorbance at 570 nm using a UV-visible plate reader.

A method to evaluate cells morphology and proliferation is the crystal violet assay. At specific time points (every 24 hrs for 7 days) media are removed, cells are washed once with PBS and fixed with 4% formaldehyde for 10 min. Cells are stained with 0.4% crystal violet in 50% MeOH for 20 min and extensively washed with water to remove excess dye. Crystal violet is dissolved in DMSO. The absorbance of the dissolved dye, corresponding to the number of viable cells, is measured in a UV-visible plate reader at 570 nm.

Isobolographic analysis.

Interaction between drugs was assessed by isobolographic analysis, based on the concept of dose-equivalence which follows from the dose effect curves of the individual drugs {Tallarida RJ, Interactions between drugs and occupied receptors. Pharmacol. Ther. 2007, 1 13, 197-209; Tallarida RJ, Raff a RB, The application of drug dose equivalence in the quantitative analysis of receptor occupation and drug combinations. Pharmacol. Ther. 2010, 127, 165-174). Specifically, the individual drugs' potency and efficacy allow calculating the expected effect of a combination of the two drugs.

In the experiment of combined treatment, isobolograms were constructed by plotting on vertical and horixontal axes the ED₅₀ data of the single drugs measured by trypan blue assay after subcronic treatment for 72 hrs. The straight line with axial intercepts represents the isobole of additivity and allows calculating the theoretical additive dose. Synergism is indicated by an observed pair (x, y) that plots below the isobole for the specified effect, whereas sub-additivity is indicated when an observed pair (x, y) plots above the isobole.

Compound 1 and compound 2 caused a reduction of the viability of the human colon adenocarcinoma cell line SW403, as measured by the trypan blue assay (Figure 3A and 3B). Compound 1 and compound 2 also showed a synergistic effect with the anti-neoplastic agent 5-fluorouracil (5FU, Figure 3C and 3D) in reducing the viability of SW403 cells. Isobolograms were constructed with ED₅o data measured by trypan blue assay. The theoretical doses (T_h) of compound 1 or compound 2 that must be used with 300nM 5FU to obtain an additive effect are respectively 1 1 μΜ and 20.3 μΜ, higher than the experimental values (E_x). The effect is synergistic. Results are expressed as mean ± SEM (n=3).

Statistics.

GraphPad Prism software (GraphPad Software, Inc., USA) was used for statistical analysis. Data were analyzed using the Student's t-test or one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. Two-way ANOVA was used to compare the means of data with two independent variables. Differences between groups were considered statistically significant at values of p < 0.05. Results are expressed as mean ± SEM.

General purification and analytical methods

UPLC-MS analyses were run on a Waters ACQUITY UPLC-MS instrument consisting of a SQD Single Quadrupole Mass Spectrometer equipped with an electrospray ionization interface and a photodiode array detector. The analyses were performed on an ACQUITY UPLC BEH C18 column (50x2.1 mmID, particle size 1 .7μιη) with a VanGuard BEH C18 pre-column (5x2.1 mmID, particle size 1 .7μιη). The mobile phases were 10 mM ammonium acetate at pH 5 adjusted with acetic acid (A) and 10mM ammonium acetate in acetonitrile-water (95:5) at pH 5 (B). Electrospray ionization in positive and negative mode was used in the mass scan range 100-500Da.

Automated column chromatography purification was done using a Teledyne ISCO apparatus (CombiFlash^® Rf) with normal phase pre-packed silica gel columns of different sizes (from 4g to 40g). Typical silica gel column chromatography is intended as a purification performed using normal glass columns filled with Merck silica gel 60 (230-400 mesh) as stationary phase. In both cases, mixtures of increasing polarity of cyclohexane or petroleum ether and ethyl acetate were used as eluents.

Flash column chromatography was performed manually on pre-packed silica cartridges (2g or 5g) from Biotage or on glass columns using Merck silica gel 60 (230-400 mesh) as stationary phase.

Purifications by preparative HPLC-MS were run on a Waters Autopurification system consisting of a 3100 Single Quadrupole Mass Spectrometer equipped with an electrospray ionization interface and a 2998 Photodiode Array Detector. The HPLC system included a 2747 Sample Manager, 2545 Binary Gradient Module, System Fluidic Organizer and 515 HPLC Pump. The purifications were performed on a XBridge™ Prep d₈ OBD column (100x19mmlD, particle size 5μιτι) with a XBridge™ Prep C-|₈ (10x19mmlD, particle size 5μιτι) Guard Cartridge. The mobile phases were either 1 ) water and acetonitrile (B) or 2) 10 mM ammonium acetate at pH 5 adjusted with acetic acid (A) and 10 mM ammonium acetate in acetonitrile- water (95:5) at pH 5 (B). Electrospray ionization in positive and negative mode was used in the mass scan range 100-500Da.

Flow hydrogenation reactions were performed on the H-Cube apparatus from Thalesnano Nanotechnology Inc. using cartridges of various commercial prepacked heterogeneous catalysts. Small format (s-cart) cartridges were adopted (30 x 4 mm, containing -140 mg catalyst).

Microwave heating was performed using Explorer^®-48 positions instrument (CEM). NMR experiments were run on a Bruker Avance III 400 system (400.13 MHz for 1 H, and 100.62 MHz for 13C), equipped with a BBI inverse probe and Z-gradients. Unless indicated, spectra were acquired at 300 K, using deuterated dimethylsulfoxyde (DMSO-d₆) and deuterated chloroform (CDCI₃) as solvents. With the aim of better illustrating the present invention, the examples reported in Table 2 are provided.

Table 2. Examples ofcompounds of the invention

C2oH2₄FNs0₄ 3-benzoyl-5-fluoro-N- octyl-2,4-dioxo- pyrimidine-1 - carboxamide

The compounds reported in Table 2 were synthesized as described below.

The following Examples provide illustrative embodiments of the present invention. EXAMPLES

Materials and Methods

In the procedure that follows, after the starting materials, reference to a description is typically provided. The starting materials may not necessarily have been prepared from the description referred to.

Solvents and reagents used in the following examples were commercially available from various suppliers and were used without further purification. For simplicity, solvents and reagents were indicated as follows.

Acetonitrile (MeCN), ammonium chloride (NH₄CI), cesium carbonate (Cs₂C0₃), 4- dimethylaminopyridine(DMAP), ethyl acetate (EtOAc), sodium bicarbonate (NaHC0₃), sodium hydride (NaH), sodium sulfate (Na₂S0₄), tetrahydrofuran (THF).

Example 1. 5-Fluoro-/\/-hexyl-3-methyl-2,4-dioxo-pyrimidine-1 -carboxamide

Step 1 . Preparation of terf-butyl 5-fluoro-2,4-dioxo-pyrimidine-1 -carboxylate

The title compound was obtained according to the procedure reported in the literature {Synthetic Comm.2001 , 37(24), 3739-3746) starting from 5- fluorouracil(0.16 g, 0.45 mmol). The crude product (0.26 g) was obtained as white powder, and it was used in the next step without further purification. Analytical data were consistent with those reported in the literature {Bioorg. Med. Chem. Lett.2009, 79(4), 1261 -1263). ¹H NMR (400 MHz, DMSO-d₆): δ 1 .52 (s, 9H), 8.13 (d, J = 7.2 Hz, 1 H), 1 1 .91 (s, 1 H). MS (ESI) m/z: 229 [M - H]\

Step 2. Preparation of terf-butyl 5-fluoro-3-methyl-2,4-dioxo-pyrimidine-1 - carboxylate

Terf-butyl 5-fluoro-2,4-dioxo-pyrimidine-1 -carboxylate (0.26 g, 1 .15 mmol) was dissolved in dry THF (5.8 ml_). The resulting solution was cooled to 0°C and NaH (95%, 0.03 g, 1 .21 mmol) was added. The reaction was stirred for 1 hr under nitrogen atmosphere. Then, the mixture was cooled to 0°C and iodomethane (0.1 1 ml_, 1 .72 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred under nitrogen atmosphere for 20 hrs. Water was added and the aqueous phase was extracted with EtOAc (3 x 15 ml_). The combined organic phases were washed with brine, dried over Na₂S0₄ and the solvent was removed under reduced pressure. The crude was purified by column chromatography using a Teledyne ISCO apparatus (petroleum ethenEtOAc from 70:30 to 60:40) to afford the title compound (0.08 g, 27%) as white powder. ¹ H NMR (400 MHz, CDCI₃): δ 1 .62 (s, 9H), 3.37 (s, 3H), 7.93 (d, J = 6.1 Hz, 1 H). MS (ESI) m/z: 243 [M - H]\

Step 3. Preparation of 5-fluoro-3-methyl-1 /-/-pyrimidine-2,4-dione

The title compound was obtained according to the procedure reported in the literature {Synthetic Comm.2001 , 37(24), 3739-3746) starting from terf-butyl 5- fluoro-3-methyl-2,4-dioxo-pyrimidine-1 -carboxylate (0.08 g, 0.32 mmol). The crude product (0.05 g) was obtained as white powder, and it was used in the next step without further purification. ¹ H NMR (400 MHz, CDCI₃) δ 3.37 (s, 3H), 7.24 (d, J = 5.1 Hz, 1 H), 9.29 (s, 1 H). MS (ESI) m/z: 143 [M - H]\

Step 4. Preparation of5-fluoro-/V-hexyl-3-methyl-2,4-dioxo-pyrimidine-1 - carboxamide

5-Fluoro-3-methyluracil (0.04 g, 0.31 mmol) was dissolved in dry pyridine (1 .6 ml_). DMAP (0.04 g, 0.34 mmol) was added and the reaction mixture was stirred under nitrogen atmosphere at room temperature for 30 min. Hexyl isocyanate (0.08 ml_, 0.56 mmol) was then added and the resulting mixture was stirred for 12 hrs. The solvent was evaporated under reduced pressure and the crude was purified by silica gel column chromatography (cyclohexane:EtOAc 70:30) to afford the title compound (0.06 g, 68%) as colorless oil. ¹H NMR (400 MHz, CDCI₃): δ 0.90 (t, J = 6.4 Hz, 3H), 1 .21 -1 .44 (m, 6H), 1 .56-1 .69 (m, 2H), 3.34-3.44 (m, 2H), 3.40 (s, 3H), 8.47 (d, J = 6.6 Hz, 1 H), 9.23 (s, 1 H). ¹³C NMR (101 MHz, CDCI₃): δ 14.12 , 22.65 , 26.64 , 28.71 , 29.28 , 31 .51 , 41 .61 , 121 .1 1 (d, J = 37.4 Hz), 140.62 (d, J = 238.2 Hz), 149.61 , 151 .01 , 156.69 (d, J = 26.4 Hz). MS (ESI) m/z: 272 [M - H]⁺. Example 2. 3-(Cyclopropylmethyl)-5-f luoro-/\/-hexyl-2,4-dioxo-pyrimidine-1 - carboxamide

Step 1 . Preparation of terf-butyl 3-(cyclopropylmethyl)-5-fluoro-2,4-dioxo- pyrimidine-1 -carboxylate

Terf-butyl 5-fluoro-2,4-dioxo-pyrimidine-1 -carboxylate (0.10 g, 0.43 mmol) was dissolved in dry MeCN (2.2 mL) and Cs₂C0₃ was added (0.23 g, 0.65 mmol), followed by (bromomethyl)cyclopropane (0.12 mL, 1 .29 mmol). The reaction mixture was stirred under nitrogen atmosphere at room temperature for 18 hrs. The reaction was then diluted with EtOAc and saturated aqueous NH₄CI solution was added. The aqueous layer was extracted with EtOAc (3 x 15 mL), the combined organic phases were washed with brine, dried over Na₂SO₄ and the solvent was removed under reduced pressure. The crude was purified by column chromatography using a Teledyne ISCO apparatus (petroleum ether:EtOAc 80:20) to afford the title compound (0.08 g, 64%) as white solid. ¹ H NMR (400 MHz, CDCIs): δ 0.38-0.45 (m, 2H), 0.44-0.54 (m, 2H), 1 .20-1 .32 (m, 1 H), 1 .62 (s, 9H), 3.86 (d, J = 7.2 Hz, 2H), 7.91 (d, J = 6.1 Hz, 1 H). MS (ESI) m/z: 307 [M-Na]⁺.

Step 2. Preparation of 3-(cyclopropylmethyl)-5-fluoro-1 /-/-pyrimidine-2,4-dione The title compound was obtained according to the procedure described in the literature {Synthetic Comm.2001 , 37(24), 3739-3746) starting from terf-butyl 3- (cyclopropylmethyl)-5-fluoro-2,4-dioxo-pyrimidine-1 -carboxylate (0.08 g, 0.27 mmol). The crude product (0.05 g) was obtained as white powder, and it was used in the next step without further purification. ¹H NMR (400 MHz, CDCI₃) δ 0.39-0.45 (m, 2H), 0.46-0.55 (m, 2H), 1 .20-1 .32 (m, 1 H), 3.85 (d, J = 7.2 Hz, 2H), 7.19-7.25 (m, 1 H), 9.01 (s, 1 H). MS (ESI) m/z: 185 [M-H]⁺.

Step 3. Preparation of 3-(cyclopropylmethyl)-5-fluoro-/V-hexyl-2,4-dioxo- pyrimidine-1 -carboxamide

The title compound was obtained according to the procedure described for the synthesis of Example 1 (Step 4), starting from 3-(cyclopropylmethyl)-5-fluoro-1 H- pyrimidine-2,4-dione (0.05 g, 0.27 mmol). The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 85:15) to afford the title compound (0.06 g, 66%) as colorless oil.¹H NMR (400 MHz, CDCI₃): δ 0.39-0.45 (m, 2H), 0.47-0.56 (m, 2H), 0.89 (t, J = 6.9 Hz, 3H), 1 .20-1 .27 (m, 1 H), 1 .27-1 .43 (m, 6H), 1 .53-1 .70 (m, 2H), 3.39 (dt, J = 7.2, 5.6 Hz, 2H), 3.88 (d, J = 7.3 Hz, 2H), 8.46 (d, J = 6.6 Hz, 1 H), 9.26 (s, 1 H). ¹³C NMR (101 MHz, CDCI₃): δ 4.07, 9.63, 14.12, 22.65, 26.65, 29.28, 31 .51 , 41 .60, 46.96, 121 .16 (d, J = 37.5 Hz), 140.76 (d, J = 238.3 Hz), 149.76, 151 .1 1 , 156.82 (d, J = 26.5 Hz). MS (ESI) m/z: 312 [M - H]⁺, 334 [M - Na]⁺.

Example 3. 5-Fluoro-/V-hexyl-3-(2-methylpropanoyl)-2,4-dioxo-pyrimidine-1 - carboxamide

5-Fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide (0.04 g, 0.15 mmol) was dissolved in dry dichloromethane (0.7 mL). To the resulting solution, cooled at 0°C, triethylamine (0.09 mL, 0.62 mmol) was added, followed by isobutyryl chloride (0.05 mL, 0.05 mmol). The reaction was allowed to warm to room temperature and stirred under nitrogen atmosphere for 45 min. Water was added and the aqueous phase extracted with dichloromethane (3 x 15 mL). The combined organic layers were washed with saturated aqueous NaHC0₃ solution and brine, dried over Na₂S0₄ and the solvent was removed under reduced pressure.The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 85:15) to afford the title compound (0.01 g, 19%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 0.89 (t, J = 7.0 Hz, 3H), 1 .24-1 .40 (m, 6H), 1 .31 (d, J = 7.0 Hz, 6H), 1 .57-1 .64 (m, 2H), 3.05 (hept, J = 7.0 Hz, 1 H), 3.38 (td, J = 5.5, 7.2 Hz, 2H), 8.48 (d, J = 6.7 Hz, 1 H), 8.86 (t, J = 5.6 Hz, 1 H). ¹³C NMR (101 MHz, CDCI₃) δ 14.1 1 , 17.94, 22.63, 26.60 , 29.21 , 31 .48 , 40.02 , 41 .74, 122.78 (d, J = 37.4 Hz), 140.55 (d, J = 243.3 Hz), 148.92, 149.40, 155.64 (d, J = 28.5 Hz), 177.21 . MS (ESI) m/z: 256 [M - COC(CH₃)₂] .

Example 4. Methyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1- carboxylate

5-Fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide (0.05 g, 0.20 mmol) was dissolved in dry dichloromethane (4 mL) and pyridine (0.04 mL, 0.43 mmol) was added, followed by methyl chloroformate (0.02 mL, 0.23 mmol) at 0 °C. The reaction was stirred at room temperature for 18 h, and then the solvent was removed under reduced pressure. The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 90:10) to afford the title compound (0.02 g, 32%) as colorless oil. ¹ H NMR (400 MHz, CDCI₃) δ 0.86-0.92 (m, 3H), 1 .22-1 .43 (m, 6H), 1 .55-1 .65 (m, 2H), 3.33-3.43 (m, 2H), 4.09 (s, 3H), 8.48 (d, J = 6.7 Hz, 1H), 8.79 (t, J = 5.5 Hz, 1H).¹³C NMR (101 MHz, CDCI₃) δ 14.33, 22.35, 26.52, 28.98, 31.50, 41.69, 56.85, 122.71 (d, J= 37.5 Hz), 140.17 (d, J= 242.8 Hz), 148.38, 148.46, 148.51, 153.88 (d, J= 29.3 Hz). MS (ESI) m/z: 316 [M-H]⁺, 333 [M-NH₄]⁺.

Example 5.3-Benzoyl-5-fluoro-/\/-hexyl-2,4-dioxo-pyrimidine-1-carboxamide

5-Fluoro-N-hexyl-2,4-dioxo-pyrimidine-1-carboxamide (0.05 g, 0.20 mmol) was dissolved in dry pyridine (4 mL). To the resulting solution, benzoyl chloride (0.03 mL, 0.25 mmol) was added. The reaction was stirred at room temperature under nitrogen for 2 hrs then concentrated under reduced pressure. The crude was purified by column chromatography using silica gel (5 g) cartridge (cyclohexane:EtOAc 90:10) to afford the title compound (0.03 g, 45%) as colorless oil.¹H NMR (400 MHz, CDCI₃): δ 0.87 (t, J= 6.9 Hz, 3H), 1.24-1.39 (m, 6H), 1.49- 1.65 (m, 2H), 3.36 (dt, J =5.7, 7.0 Hz, 2H), 7.47-7.61 (m, 2H), 7.65-7.79 (m, 1H), 7.89-8.01 (m, 2H), 8.58 (d, J= 6.7 Hz, 1H) 8.82 (t, J= 5.7 Hz, 1H).¹³C NMR (101 MHz, CDCIs): δ 13.95, 22.45, 26.43, 29.02, 31.31, 41.62, 122.89 (d, J= 31.7 Hz), 129.48, 130.50, 130.61, 135.87, 148.71, 166.03, 186.23. MS (ESI) m/z: 378 [M- Na]⁺.

Example 6. Ethyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1- carboxylate

The title compound was obtained according to the procedure described for the synthesis of Example 4, starting from 5-fluoro-/V-hexyl-2,4-dioxo-pyrimidine-1- carboxamide (0.05 g, 0.20 mmol); 2.8 equivalents of ethyl chloroformate were used therein. The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 80:20) to afford the title compound (0.03 g, 45%) as colorless oil.¹H NMR (400 MHz, CDCI₃) δ 0.89 (t, J= 7.5 Hz, 3H), 1.23-1.41 (m, 6H), 1.44 (t, J= 7.1 Hz, 3H), 1.55-1.65 (m, 2H), 3.38 (td, J= 7.1, 5.6 Hz, 2H), 4.53 (q, J = 7.1 Hz, 2H), 8.48 (d, J= 6.8 Hz, 1H), 8.82 (t, J= 5.7 Hz, 1H).¹³C NMR (101 MHz, CDCI₃) δ 13.87, 14.13, 22.64, 26.60, 29.21, 31.49, 41.78, 67.17, 122.77 (d, J = 37.4 Hz), 140.38 (d, J= 242.7 Hz), 147.92, 148.57, 148.70, 155.16 (d, J = 31.5 Hz). MS (ESI) m/z: 347 [M-NH₄]⁺.

Example 7.3-Ethyl-5-f luoro-/V-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

Step 1. Preparation of terf-butyl 3-(ethyl)-5-fluoro-2,4-dioxo-pyrimidine-1- carboxylate

The title compoundwas obtained according to the procedure described for the Example 2 (Step 1), employing terf-butyl 5-fluoro-2,4-dioxo-pyrimidine-1- carboxylate (0.10 g, 0.43 mmol); 3.0 equivalents of iodo ethane were used therein. The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 80:20) to afford the title compound (0.06 g, 54 %) as white powder.¹H NMR (400 MHz, CDCIs) δ 1.26 (t, J= 7.1 Hz, 3H), 4.02 (q, J= 7.1 Hz, 2H), 7.21 (dd, J = 4.3, 6.3 Hz, 1H), 8.56 (s, 1H). MS (ESI) m/z: 157 [M-H]\

Step 2. Preparation of 3-ethyl-5-fluoro-1 /-/-pyrimidine-2,4-dione

The title compound was obtained according to the procedure described in the literature {Synthetic Comm.2001, 37(24), 3739-3746) starting from terf-butyl 3- (ethyl)-5-fluoro-2,4-dioxo-pyrimidine-1 -carboxylate (0.06 g, 0.23 mmol). The crude product (0.03 g) was obtained as white powder, and it was used in the next step without further purification. ¹H NMR (400 MHz, CDCI₃) δ 1.26 (t, J= 7.1 Hz, 3H), 4.02 (q, J =7.1 Hz, 2H), 7.21 (dd, J =4.3, 6.3 Hz, 1H), 8.56 (s, 1H). MS (ESI) m/z: 157 [M-H]\

Step 3. Preparation of 3-ethyl-5-fluoro-/V-hexyl-2,4-dioxo-pyrimidine-1- carboxamide

The title compoundwas obtained according to the procedure described for the synthesis of Example 1(Step 4), starting from 3-ethyl-5-fluoro-1 /-/-pyrimidine-2,4- dione (0.04 g, 0.25 mmol). The crude was purified by silica gel column chromatography (petroleum ethenEtOAc 85:15) to afford the title compound (0.02 g, 25%) as colorless oil.¹H NMR (400 MHz, CDCI₃) δ 0.90 (t, J= 6.8 Hz, 3H), 1.26 (t, J= 7.1 Hz, 3H), 1.2-1.43 (m, 6H), 1.5-1.68 (m, 2H), 3.28-3.50 (m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 8.45 (d, J = 6.6 Hz, 1 H), 9.22-9.29 (m, 1 H).¹³C NMR (101 MHz, CDCI₃) δ 12.74, 14.14, 22.65, 26.66, 29.29, 31.52, 37.74, 41.61, 121.11 (d, J = 37.4 Hz), 140.72 (d, J= 238.6 Hz), 149.73, 150.69, 156.40 (d, J= 29.7 Hz). MS (ESI) m/z: 157 [M-CONH(CH₂)₅CH₃]^".

Example 8. Methyl 5-fluoro-3-(octylcarbamoyl)-2,6-dioxo-pyrimidine-1- carboxylate

Step 1.Preparation of5-fluoro-/V-octyl-2,4-dioxo-pyrimidine-1-carboxamide

The title compound was obtained according to the procedure described for the synthesis of Example 1 (Step 4), starting from 5-fluorouracil (0.50 g, 3.85 mmol); 1.5 equivalents of octyl isocyanate were used therein. The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 80:20) to afford the title compound (0.05 g, 46%) as white powder.¹H NMR (400 MHz, CDCI₃) δ 0.88 (t, J = 7.1 Hz, 3H), 1.21-1.39 (m, 10H), 1.51-1.67 (m, 2H), 3.39 (td, J= 7.1, 5.5 Hz, 2H), 8.48 (d, J= 6.8 Hz, 1H), 8.93-9.05 (m, 1H), 9.10 (s, 1H).¹³C NMR (101 MHz, CDCIs) δ 14.20, 22.75, 26.94, 29.27, 31.89, 41.63, 123.40 (d, J= 37.3 Hz), 140.89 (d, J = 241.3 Hz), 149.13, 150.03, 156.156.43. MS (ESI) m/z: 284 [M - H]\

Step 2. Preparation ofmethyl 5-fluoro-3-(octylcarbamoyl)-2,6-dioxo-pyrimidine-1- carboxylate

The title compound was obtained according to the procedure described for the synthesis of Example 4, starting from 5-fluoro-/V-octyl-2,4-dioxo-pyrimidine-1- carboxamide (0.20 g, 0.70 mmol). The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 85:15) to afford compound the title compound (0.16 g, 66%) as colorless oil.¹H NMR (400 MHz, CDCI₃) δ 0.74-1.00 (m, 3H), 1.11-1.43 (m, 11H), 1.47-1.69 (m, 2H), 1.55-1.65 (m, 2H), 3.37 (td, J = 5.6, 7.1 Hz, 2H), 4.08 (q, J= 7.1 Hz, 2H), 8.47 (d, J= 6.7 Hz, 1H), 8.78 (t, J= 5.6 Hz, 1H).¹³C NMR (101 MHz, CDCI₃) δ 14.17, 22.73, 26.88, 29.18, 29.23, 31.87, 41.76, 56.96, 122.85 (d, J= 37.5 Hz), 140.30 (d, J= 242.6 Hz), 148.52, 148.60, 148.65, 154.01 (d, J = 29.3 Hz). MS (ESI) m/z: 344 [M-H]⁺.

Example 9.3-benzoyl-5-f luoro-N-octyl-2,4-dioxo-pyrimidine-1 -carboxamide Step 1.Preparation of5-fluoro-/V-octyl-2,4-dioxo-pyrimidine-1 -carboxamide

The title compound was obtained according to the procedure described for the synthesis of Example 1 (Step 4), starting from 5-fluorouracil (0.50 g, 3.85 mmol); 1.5 equivalents of octyl isocyanate were used therein. The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 80:20) to afford the title compound (0.05 g, 46%) as white powder.¹H NMR (400 MHz, CDCI₃) δ 0.88 (t, J = 7.1 Hz, 3H), 1.21-1.39 (m, 10H), 1.51-1.67 (m, 2H), 3.39 (td, J= 7.1, 5.5 Hz, 2H), 8.48 (d, J= 6.8 Hz, 1H), 8.93-9.05 (m, 1H), 9.10 (s, 1H).¹³C NMR (101 MHz, CDCI₃) δ 14.20, 22.75, 26.94, 29.27, 31.89, 41.63, 123.40 (d, J= 37.3 Hz), 140.89 (d, J = 241.3 Hz), 149.13, 150.03, 156.156.43. MS (ESI) m/z: 284 [M-H]\

Step 2. Preparation of3-benzoyl-5-fluoro-/V-octyl-2,4-dioxo-pyrimidine-1- carboxamide

The title compound was obtained according to the procedure described for the synthesis of Example 5, starting from 5-fluoro-/V-octyl-2,4-dioxo-pyrimidine-1- carboxamide (0.15 g, 0.53 mmol). The crude was purified by silica gel column chromatography (cyclohexane:EtOAc 85:15) to afford compound the title compound (0.15 g, 74%) as colorless oil.¹H NMR (400 MHz, CDCI₃) δ 0.86 (t, J = 6.79 Hz, 3H), 1.16-1.39 (m, 8H), 1.45-1.64 (m, 4H), 3.31-3.43 (m, 2H), 7.55 (t, J = 7.87 Hz, 2H), 7.68-7.78 (m, 1H), 7.89-8.02 (m, 2H), 8.58 (d, J = 6.70 Hz, 1H), 8.74-8.87 (m, 1H). MS (ESI) m/z: 407 [M-NH₄]⁺.

Claims

1 . A compound of Formula I or pharmaceutically acceptable salts thereof

Formula I wherein

Y represents a bond, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyi, or a group -(CR_aR_b)n-Q-(CR_cRd)m-; W represents hydrogen, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyi, a group -(CR_eRf)_p-CR_gRh Ri or a group Z-R₃;

R-i represents an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted cycloalkyi, or a group C(=0) R₄;

Q represents O, N R₅;

Z represents O, N R₅;

R₃ represents hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyi, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocyclyl;

R₄ represents an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl which is 5-membered aromatic ring containing N as heteroatom or containing two or three heteroatoms selected from N, O, S, an optionally substituted heterocyclyl, or a group 0-R₆;

Rs represents hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyi, an optionally substituted aryl;

R₆ represents an optionally substituted C-1 -C3 alkyl, an optionally substituted cycloalkyi, an optionally substituted aryl, an optionally substituted heteroaryl, or an optionally substituted heterocyclyl; R_a, Rt>, Rc, Rd, Re, Rf, Rg, Rh, and R, are independently selected from the group consisting of hydrogen, halogen, an optionally substituted lower alkyl, an optionally substituted cycloalkyi, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, alkoxy, cycloalkyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, or trifluoromethoxy;

n is an integer from 1 to 12;

m is an integer from 0 to 12;

p is an integer from 0 to 6.

2. A compound of Formula I according to claim 1 wherein

Y represents an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted cycloalkyi selected from cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

W represents hydrogen, an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, biphenyl, or an optionally substituted heteroaryl selected from oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and quinazolinyl, or an optionally substituted cycloalkyi selected from cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

3. A compound of Formula I according to claim 1 or 2 wherein R-i is a C-rC₆ alkyl or a cycloalkyi with 3 to 6 carbon atoms.

4. A compound of Formula I according to anyone of claims 1 to 3 wherein Rirepresents a CrC₃ alkyl or an optionally substituted cycloalkyi selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane.

5. A compound of Formula I according to claim 1 wherein R-i is a group C(=0)R₄ wherein R₄represents

- an optionally substituted CrC₃ alkyl;

- an optionally substituted heterocyclyl selected from oxetane, tetrahydrofuran, tetrahydropyran, azetidine, pyrrolidine, piperidine, or morpholine,

- an optionally substituted cycloalkyi selected from cyclopropane, cyclobutane,

cyclopentane, and cyclohexane, - an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, biphenyl, or

- an optionally substituted heteroaryl selected from pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzofuranyl, benzothiophenyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazolinyl.

6. A compound of Formula I according to claim 1 or 5 wherein R-i is a group C(=0)R₄ wherein R₄represents

an optionally substituted CrC₃ alkyl, selected from methyl, ethyl, n-propyl and iso- propyl, or

an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, and

biphenyl.

7. A compound of Formula I according to claim 1 wherein R-i is a group C(=0)R₄ wherein R₄ is a group 0-R₆ wherein R₆ is an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or an optionally substituted heterocyclyl.

8. A compound of Formula I according to claims 1 or 7 wherein R-i is group C(=0)R wherein R₄represents a group 0-R₆, wherein R₆ represents

an optionally substituted C1 -C3 alkyl;

an optionally substituted heterocyclyl selected from oxetane, tetrahydrofuran, tetrahydropyran, azetidine, pyrrolidine, piperidine, or morpholine,

an optionally substituted cycloalkyl selected from cyclopropane, cyclobutane, cyclopentane, and cyclohexane,

an optionally substituted aryl selected from phenyl, alpha- or beta-naphthyl, biphenyl, or

an optionally substituted heteroaryl selected from oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzofuranyl, benzothiophenyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazolinyl.

9. A compound according to claim 1 selected from the group comprising 5-Fluoro-N-hexyl-3-methyl-2,4-dioxo-pyrimidine-1 -carboxamide

3-(Cyclopropylmethyl)-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide 5-Fluoro-N-hexyl-3-(2-methylpropanoyl)-2,4-dioxo-pyrimidine-1 -carboxamide Methyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-Benzoyl-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

Ethyl 5-fluoro-3-(hexylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-Ethyl-5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1 -carboxamide

Methyl 5-fluoro-3-(octylcarbamoyl)-2,6-dioxo-pyrimidine-1 -carboxylate

3-Benzoyl-5-fluoro-N-octyl-2,4-dioxo-pyrimidine-1 -carboxamide

10. A pharmaceutical composition comprising an effective amount of a compound of Formula I according to anyone of claims 1 -9 or a pharmaceutically salt thereof and a pharmaceutically acceptable carrier.

1 1 . A compound of Formula I according to anyone of claims 1 -9 for use as a medicament.

12. A compound of Formula I according to anyone of claims 1 -9 or a pharmaceutical composition according to claim 10 for use in the treatment or prevention of a disease or disorder associated with acid ceramidase overexpression or overactivity in a mammal.

13. A compound of Formula I, according to anyone of claims 1 -9 or a pharmaceutical composition according to claim 10 for use in the treatment or prevention of a pathological condition selected from cancer, cancer metastasis, hyperplasia, metaplasia, dysplasia, hyper-proliferative disorders, benign dysproliferative disorders, and psoriasis.

14. A product or kit containing a compound of Formula I according to anyone of claims 1 to 9 and a chemotherapeutic agent as a combined preparation for simultaneous, separate or sequential use in antitumoral therapy.

15. A compound of Formula I, according to anyone of claims 1 -9 or a pharmaceutical composition according to claim 10 for use in combination with an active ingredient, preferably an anti-cancer agent or in combination with radiotherapy.