Covalent inhibitors of pfkfb3

Abstract

The present invention relates to compounds of formula (I) salts and derivatives thereof. Further object of the present invention are compositions, which comprise at least a compound of formula (I) or its pharmaceutically acceptable salts and derivatives, and at least one excipient. Further object of the present invention are compounds of formula (I) or compositions which comprise them, for use in the treatment or prevention of a cancer and / or a metabolic disease and / or an immunological disease. Further object of the present invention is the in vitro use of the compounds as chemical probes.

Description

[0001] “Covalent inhibitors of PFKFB3”

[0002] *******

[0003] DESCRIPTION FIELD OF THE INVENTION

[0004] The present invention relates to covalent inhibitors of the protein PFKFB3, and their use in the treatment or prevention of a cancer and / or a metabolic disease and / or an immunological disease. A further object of the present invention is the in vitro use thereof as chemical probes to investigate PFKFB3 activity.

[0005] STATE OF ART

[0006] Enzymatic reactions are fundamental to carry out a wide variety of cellular mechanisms and functions. Kinases are enzymes that transfer phosphate groups from ATP to other substrates, such as proteins or metabolites. In response to extracellular mediators or molecules such as nutrients, growth factors, mitogens or cytokines, kinase enzymes participate in transducing cellular signaling, contributing to cell growth, proliferation, differentiation, survival, and migration. A non-physiological increase in kinases expression and activity can lead to a range of diseases including neoplastic, immunological, neurodegenerative, or metabolic diseases. Such increase can be caused for instance by somatic mutations or defects in the control of expression of these enzymes. [1, 2]

[0007] The enzyme 6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase (PFK-2 / FBPase - PFKFB) is a bifunctional glycolytic enzyme constituted by two subunits. The kinase subunit, named PFK-2, catalyzes the reaction of phosphorylation of the fructose-6-phosphate (F6P) in fructose-2,6-bisphosphate (F2,6P2), while the phosphatase FBPase domain dephosphorylates the F2,6P2 to obtain F6P. The main PFK-2 product, fructose-2,6-bisphosphate (F2,6P2), is a key regulator of the glycolytic flux since it is the main allosteric activator of the glycolytic rate-limiting enzyme phosphofructokinase 1 (PFK1), which catalyzes the phosphorylation of fructose-6-phosphate into fructose-1,6-bisphosphate. Among all isoforms, PFKFB3 has the highest kinase / phosphatase ratio, and its activity greatly increases the glycolytic flux. PFKFB3 is almost ubiquitously expressed, and it is upregulated upon mitogen, inflammatory or hypoxic stimuli. Indeed, PFKFB3 is overexpressed in several human cancers that are associated with poorprognosis, such as breast, colon, pancreatic, gastric, liver and lung cancer, glioblastoma, nasopharyngeal carcinoma and many other neoplasms. [3] By increasing the glycolysis flux, PFKFB3 overexpression promotes the Warburg effect, wherein cancer cells preferentially rely on anaerobic glycolysis for energy production even in the presence of oxygen, facilitating their rapid growth and survival. Thus, PFKFB3 inhibitors have potential applications in the treatment of different types of cancer. [4-8]

[0008] In addition to cancer, PFKFB3 inhibitors could find application in the treatment of autoimmune diseases (e.g rheumatoid arthritis, chronic inflammatory diseases) [9, 10], multiple sclerosis

[0010] , cardiovascular disorders (atherosclerosis, heart failure) [11, 12], neurodegenerative diseases (Alzheimer's and Parkinson's diseases) [13-15], fibrosis

[0016] , endometriosis

[0017] and other disorders.

[0009] Despite reversible PFKFB3 inhibitors belonging to different chemical classes were discovered [18-21], no covalent inhibitor has been reported to date. Recently, there has been a renewed interest in covalent inhibitors

[0022] , which present a chemical reactive moiety, named “warhead”, able to form an irreversible covalent bond with the targeted protein. Covalent drugs have proven to be beneficial for the treatment of various diseases since they can display unmatched potency, selectivity, and duration of action. [22, 23] Indeed, several covalent kinase inhibitors have been approved for oncology applications to date (e.g. Afatinib, Ibrutinib, Osimertinib, Neratinib, Acalabrutinib, Dacomitinib). [24-29]

[0010] Beside the therapeutic approaches, thanks to their high selectivity and specificity, covalent inhibitors find application as chemical probes in vitro and in vivo to investigate the biological functions of their target protein in the contexts of basic and applied research.

[0030] High-quality chemical probes have served as powerful research tools and as seeds to spur the development of new medicines. [31, 32] In light of the above, it is essential to develop novel and potent inhibitors, preferably covalent inhibitors, due to their enhanced potency and selectivity, to target PFKFB3, an enzyme often dysregulated across a broad spectrum of diseases.

[0011] BRIEF DESCRIPTION OF THE FIGURESFigure 1: Cell viability of human PDAC cell lines treated with compounds 2 and 3 of the present invention.

[0012] PANC-1 (A, C) and MIA PaCa-2 (B, D) cells were treated with PFKFB3 inhibitors (reversible compound as control and compounds 2 and 3 of the present invention) at four different concentrations (1 pM, 10 pM, 25 pM and 50 pM) for 48 h. The effect of the inhibitors was analyzed using the Crystal Violet assay (A, B) and MTT assay (C, D). Two-way ANOVA (Tukey's multiple comparisons test) was performed for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001.

[0013] Figure 2: Cell viability of human pancreatic cancer cell lines treated with compounds 2 and 3 of the present invention.

[0014] Suit2 (A, E), AsPc1 (B, F) Hs776t (C, G), PaCa3 (D, H) cells were treated with PFKFB3 inhibitors (reversible compound as control and compounds 2 and 3 of the present invention) at 25 pM concentrations for 48 h. The effect of the inhibitors was analysed using the Crystal Violet assay (A-D) and MTT assay (E-H). Ordinary one-way ANOVA (Dunnett's multiple comparisons test) was performed for statistical analysis * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001.

[0015] Figure 3: Cell viability of human PDAC cell lines treated with compound 3 of the present invention and washed out after determined timepoints.

[0016] PANC-1 (A) and MIA PaCa-2 (B) cells were treated with DMSO or Compound 3 (25 pM) for 15 minutes, 1 h, 2 h, 3 h or 4 h. Subsequently, the free inhibitor was washed out and media was replaced. The effect of the inhibitor was analysed using the Crystal Violet assay after 72 h. Two-way ANOVA (Sidak's multiple comparisons test) was performed for statistical analysis, *** p < 0.001, **** p< 0.0001.

[0017] - Figure 4: In vivo efficacy of covalent PFKFB3 inhibition in zebrafish xenografts.

[0018] (A-B) Representative images and fluorescence quantification of MIA PaCa-2 (A) and PANC-1 (B) xenografts in zebrafish larvae treated with compound 3 (7 pM) or vehicle (CTRL). Fluorescent signals were measured at 24 (T1) and 48 hours (T2) post-treatment. The fluorescent signal has been circled in the representative zebrafish pictures. Statistical analysis was performed using Dunn’s multiple comparisons test (* p < 0.05, *** p < 0.001 ).- Figure 5: Covalent PFKFB3 inhibition disrupts glycolytic flux in PDAC cells.

[0019] (A) PFK activity measured in MIA PaCa-2 cell lysates after 2 h treatment with compound 3 (25 pM). Cells were washed prior to lysis to remove non-covalently bound material. (B-C) ATP rate assay performed 1 h and 2 h after treatment with 25 pM of compound 3. (D-E) Glycolysis stress tests in PANC-1 (D) and MIA PaCa-2 (E) cells following 2 h and 6 h treatment with compound 3, indicating significant decreases in glycolytic rate, glycolytic capacity, and glycolytic reserve. Statistical analysis was conducted using Tukey's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0020] - Figure 6: (A-D) Dose-response curves showing cell viability of MIA PaCa-2 (A, B) and PANC-1 (C, D) cells treated with increasing concentrations of compound 3, chemotherapeutic agents alone, or their combinations.

[0021] (E-H) Combination index (Cl) analysis using the Chou-Talalay method. In MIA PaCa-2 cells (E, F), compound 3 + FOI or gemcitabine is additive at low fractional effects (Fa < 0.4) and synergistic at higher Fa (Fa > 0.5). In PANC-1 cells (G, H), compound 3 + FOI shows strong synergy at low to intermediate Fa (0.1 -0.6; Cl 0.3-0.5), while compound 3 + gemcitabine maintains robust synergy over a broader range (Fa = 0.1-0.8; Cl 0.3-0.6), becoming additive only at very high Fa (>0.9).

[0022] DEFINITIONS

[0023] The term “PFKFB3” refers to the glycolytic enzyme 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 3, one of the isoforms of the PFKFB enzyme.

[0024] The term “inhibitor” refers to a compound that by binding its target protein inhibits its catalytic activity. A “covalent inhibitor” is a compound that irreversibly inhibits its target protein through a covalent bond.

[0025] The term “irreversible” or “irreversible inhibitor” refers to a molecule that inhibits its molecular target in a non-reversible manner, generally forming a covalent bond. As “reversible inhibitor” is intended a molecule that is able to bind its target not irreversibly and usually forming bonds other than covalent bonds.

[0026] The term “warhead” or “warhead group” refers to electrophilic functional groupspresent on covalent inhibitors that are able to form covalent bonds with nucleophilic residues (i.e. the thiol group of cysteines) of the target protein.

[0027] In this context, as “chemical probe” is intended a compound which, by binding its target protein in vitro or in vivo, allows the measurement of the enzymatic activity of said target protein for research or diagnostic purposes.

[0028] The term “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of the compound. The compound of the present invention is a base, and the pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

[0029] The term “isomers” refers to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric centre, for example a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric centre, which can be defined as R- or S-according to the sequencing rules of Cahn-lngold-Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomer respectively). A chiral compound can exist as either individual enantiomer or as a mixture of two enantiomers. A mixture containing equal percentages of the two enantiomers is called a “racemic mixture”. The methods for the determination of stereochemistryand the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of “Advanced Organic Chemistry”, 4thedition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic mixture.

[0030] The compounds of the present invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including mixtures of such isomers form part of the present invention.

[0031] The term “tautomer” or “tautomeric form” refers to structural isomers of chemical compounds that can easily transform into one another through a process called tautomerization, which involves the relocation of a proton. Examples of this isomerization are keto-enol and imine-enamine proton tautomers.

[0032] For “alpha-haloacetamides” or “acrylamides” are intended amides with general chemical formula XCH2C(O)NH2 and CH2=CHC(O)NH2 respectively, that in the context of the present invention form covalent bonds with the side chain of a cysteine.

[0033] The term “protecting group” refers to a substituent that is commonly employed to block or protect a particular functionality during the reaction of other functional groups on the compound. For example, an “amino-protecting group” is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, fe / Y-butoxycarbonyl (BOC), benzyloxycarbonyl and 9-fluorenylmethylenoxycarbonyl (Fmoc).

[0034] For the purposes of the present invention, the term “treatment” refers to any act intended to ameliorate the health status of a patient, such as therapy, prevention, prophylaxis and retardation of a disease.

[0035] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, and even more preferably to a human, including adult, child, newborn or at the prenatal stage. However, the term “subject” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others. The terms “quantity” and “dose” are used interchangeably herein and may refer to an absolute quantification of a molecule.The terms “active principle”, “active ingredient” and “ingredient pharmaceutically active” are equivalent and refer to a component of a pharmaceutical composition having a therapeutic effect.

[0036] The term “excipient” refers to any ingredient, except active ingredients, that is present in a pharmaceutical composition. Its addition may be aimed to confer a particular consistency or other physical properties to the final product. An excipient must not be responsible for any interaction, in particular chemical, with the active ingredients.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] A first aspect of the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof,

[0039]

[0040] wherein L is a moiety selected from the group consisting of:

[0041] R’A. / <

[0042]

[0043] R3,RI,R4; and wherein

[0044] R1 is selected from the group consisting of:

[0045] H;

[0046] Linear or branched, saturated or unsaturated C1 -C6 alkyl;

[0047] R2 is selected from the group consisting of:

[0048] H;

[0049] Linear or branched, saturated or unsaturated C1 -C6 alkyl;

[0050] R3 is selected from the group consisting of:

[0051] H;

[0052] Linear or branched, saturated or unsaturated C1 -C6 alkyl;

[0053] -CH2-N-R’R” and wherein R’ and R” are selected independently between H and CH3;

[0054]

[0055] wherein

[0056] R5 is selected from halogen, H, a C1-C6 alkyl;

[0057] z is 0, or 1 or 2;

[0058] R6 is H or a linear or branched, saturated or unsaturated C1-C6 alkyl; and / or R4 is a halogen.

[0059] In a preferred embodiment of the invention,

[0060] R1 is selected from the group consisting of:

[0061] H;

[0062] Linear or branched, saturated or unsaturated C1-C4 alkyl; and / or R2 is selected from the group consisting of:

[0063] H;

[0064] Linear or branched, saturated or unsaturated C1-C4 alkyl; and / or

[0065] R3 is selected from the group consisting of:

[0066] H;

[0067] Linear or branched, saturated or unsaturated C1 -C4 alkyl;

[0068] -CH2-N-R’R”, R’ and R” are selected independently between H and CH3;

[0069] <•? > k ' '■J ■

[0070] :i f ' r r

[0071]

[0072] andR / I'd > >

[0073] wherein R5 is a halogen, or H, or a C1 -C4 alkyl;

[0074] z is 0, or 1 or 2; and / or

[0075] R6 is H or a linear or branched, saturated or unsaturated C1-C4 alkyl; and / or R4 is a halogen.

[0076] In another preferred embodiment,

[0077] R1 is H or a saturated or unsaturated C1-C2 alkyl; and / or

[0078] R2 is H or a saturated or unsaturated C1-C2 alkyl; and / or

[0079] R3 is selected from the group consisting of:

[0080] H;saturated or unsaturated C1-C2 alkyl;

[0081] -CH2-N-R’R” and wherein R’ and R” are selected independently between H and CH3;

[0082]

[0083] wherein

[0084] R5 is a halogen, or H, or a C1 -C2 alkyl;

[0085] z is 0, or 1 or 2; and / or

[0086] R6 is H or a linear or branched, saturated or unsaturated C1-C2 alkyl; and / or R4 is a halogen.

[0087] In another preferred embodiment of the invention,

[0088] R1 is H or CH3; and / or

[0089] R2 is H or CH3; and / or

[0090] R3 is selected from the group consisting of:

[0091] H;

[0092] CH3;

[0093] -CH2-N-R’R” and wherein R’ and R” are H;

[0094] ' " N " ' ".-x A J

[0095] ; / N:. I ' x-v. / f' ”N' V

[0096] v J?J, i ’ wherein (

[0097]

[0098] RS)z,, Ox>andRg.

[0099] R5 is a halogen, or H, or an alkyl chain C1-C2; and / or

[0100] z is 0 or 1; and / or

[0101] R6 is H or CH3; and / or

[0102] R4 is a halogen.

[0103] In another preferred embodiment,

[0104] R1 is H or CH3; and / or

[0105] R2 is H or CH3; and / or

[0106] R3 is selected from the group consisting of:H;

[0107] CH3;

[0108] -CH2-N-R’R” and wherein R’ and R” are H;

[0109] f\ x f IN I -

[0110]

[0111] (•w..,«4>;,ANDR«'N- wherein R5 is a halogen, or H, or CH3;

[0112] z is 0 or 1 or 2; and / or

[0113] R6 is H or CH3; and / or

[0114] R4 is a halogen.

[0115] In another preferred embodiment of the invention,

[0116] R1 is H or CH3; and / or

[0117] R2 is H or CH3; and / or

[0118] R3 is selected from the group consisting of:

[0119] H;

[0120] CH3;

[0121] -CH2-N-R’R” and wherein R’ and R” are H;

[0122]

[0123] , wherein

[0124] R5 is F, or H, or CH3;

[0125] z is 0 or 1 or 2; and / or

[0126] R4 is a halogen.

[0127] In another preferred embodiment of the invention,

[0128] R1 is H or CH3; and / or

[0129] R2 is H or CH3; and / or

[0130] R3 is selected from the group consisting of:

[0131] H,

[0132] CH3,

[0133] -CH2-N-R’R” and wherein R’ and R” are H;

[0134]

[0135] 1 5,z, wherein

[0136] R5 is H;

[0137] z is 1; and / or

[0138] R4 is a Cl.

[0139] In a particularly preferred embodiment,

[0140] R1 is H;

[0141] R2 is H;

[0142] R3 is H or CH₃; and / or

[0143] R4 is a Cl.

[0144] In a further embodiment of the invention, said compound is preferably selected from the group consisting of:

[0145] (S)-N-(4-((1 -acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0146] (S)-N-(4-((1 -(2-chloroacetyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0147] (S, E)-N-(4-((1 -(but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0148] (S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 -(3-methylbut-2-enoyl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0149] (S)-N-(4-((1 -methacryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0150] (S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 -propioloy 1-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0151] (S)-N-(4-((1 -(but-2-ynoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0152] (S, E)-N-(4-((1-(4-(dimethylamino)but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)- 1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2-carboxamide,

[0153] (S, E)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1-(4-(piperidin-1-yl)but-2-enoyl)- 1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2-carboxamide.

[0154] The formulas of the above-mentioned compounds are represented in Table 1.In a particularly preferred embodiment, said compound is preferably selected from the group consisting of:

[0155] (S)-N-(4-((1 -acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0156] (S)-N-(4-((1 -(2-chloroacetyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,

[0157] (S, E)-N-(4-((1 -(but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide.

[0158] The compounds of the present invention may possess one or more asymmetric centres; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or nomenclature of a particular compound in the specification is intended to include both individual enantiomers and mixtures thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of " Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic mixture. Some of the compounds of the invention may have geometric isomeric centres (E / Z-isomers). It is to be understood that the present invention encompasses all optical, diastereomeric and geometric isomers and mixtures thereof.

[0159] It is also to be understood that certain compounds of formula (I) may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the present invention encompasses all said solvated forms. It is also to be understood that certain compounds of formula (I) may exhibit tautomerism, and that the present invention encompasses all the tautomers of said compounds.

[0160] Without intending to be bound to any specific theory, the compound of the present invention is capable of targeting and binding PFKFB3. In an embodiment, said compound preferably inhibits PFKFB3 by binding a cysteine of PFKFB3 with a covalent bond, more preferably, said cysteine is cysteine 154 (Cys154). In aparticularly preferred embodiment, the compound of the present invention is a covalent inhibitor of PFKFB3 and binds irreversibly the Cys154 of PFKFB3.

[0161] In particular, the functional group of the compound of formula (I) that is able to form the covalent bond with the target protein is named “warhead” or “warhead group”.

[0162] Examples of warhead groups are acetamides and acrylamides that for instance, can form irreversible covalent bonds with cysteine thiols via the SN2 mechanism (Nucleophilic Substitution).

[0163] Methods to identify if a compound is an irreversible inhibitor are known to one of ordinary skill in the art. Examples of said methods may include enzymatic kinetic analysis upon compound supplementation, the analysis of the modifications on the target protein by the compound with mass spectrometry or the discontinuous exposure of the protein to the compound, also known as “wash out” experiments. A second aspect of the present invention relates to a pharmaceutical composition comprising at least one compound previously described in detail and / or at least a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0164] Preferably, said at least one pharmaceutically acceptable salt is selected from the group consisting of: acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate.

[0165] The skilled person is aware that said at least one pharmaceutically acceptable excipient is adapted according to the necessary formulation and route of administration. The selection of the at least one pharmaceutically acceptable excipient is a standard procedure well known to those of ordinary skills in the art. In this context, as pharmaceutically acceptable excipient is intended any component of the pharmaceutical composition that is not the compound or an active principle.

[0166] In an embodiment of the invention, the pharmaceutical composition further comprises at least one anticancer agent chosen from the group consisting of: achemotherapeutic agent, an immunotherapeutic agent, a targeted therapeutic agent.

[0167] In another embodiment, the chemotherapeutic agent is chosen from the group consisting of: gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan and combination thereof.

[0168] In another embodiment, the composition comprises said at least one compound according to the invention and gemcitabine; or the composition comprises said at least one compound according to the invention and a mixture of at least one or a mixture of fluorouracil, oxaliplatin or irinotecan.

[0169] Unexpectedly, and without being bound to any specific theory, the combination of the compound of the invention with at least an anticancer agent, preferably when the anticancer agent is a chemotherapeutic agent, exhibits a synergistic effect, meaning that their combined efficacy exceeds the sum of their individual effects. The compound and / or the pharmaceutical composition described in the present specification may be administered by any conventional route of administration. In particular, said compound and / or said pharmaceutical can be administered by a topical, enteral, oral, parenteral, intranasal, intravenous, intra-arterial, intramuscular, intravesical, intraurethral, subcutaneous or intraocular administration and the like.

[0170] In certain embodiments of the invention, the compound and / or the pharmaceutical composition is formulated into pharmaceutical dosage forms to provide an easily controllable dosage of said compounds.

[0171] In some preferred embodiments the compound and / or the pharmaceutical composition is formulated in a way that is suitable for topical administration such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like, comprising the active ingredient together with one or more of solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those skilled in the art.

[0172] In some other embodiments the compound and / or the pharmaceutical composition can be formulated for enteral administration, particularly oral administration or rectal administration. In addition, the compound and / or the pharmaceutical composition of the present invention can be made up in a solid form (includingwithout limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). In some other embodiments the compound and / or the pharmaceutical composition can be formulated for parenteral administration, for example by i.v. infusion, intradermal, subcutaneous or intramuscular administration.

[0173] The characteristics of the pharmaceutical composition, the route and the dose of administration of the compound and / or of the pharmaceutical composition described herein, can be adjusted by the man skilled in the art according to the type and seventy of the disease, and to the state of the patient, in particular his / her / its age, weight, sex, or general physical condition. The quantity of the compound or of the pharmaceutical composition according to the present invention to be administered has to be determined by standard procedure well known by those of ordinary skills in the art.

[0174] In some preferred embodiments, the pharmaceutical compositions according to the present invention may be formulated to release the active ingredient either substantially immediately upon administration or at any predetermined time or time period after administration. The compounds or the pharmaceutical composition according to the present specification may be administered as a single dose or in multiple doses. Preferably, the treatment is administered regularly, preferably between daily and monthly, or preferably between daily and weekly.

[0175] A third aspect of the present invention relates to the compound and / or the composition for use as a medicament.

[0176] In a preferred embodiment, the use as a medicament relates to the prevention or treatment of a disease in a subject, preferably selected from the group consisting of: a tumor, a neurodegenerative disease, an autoimmune disease, an inflammatory disease, a multiple sclerosis, a metabolic disease.

[0177] Without intending to bind to any specific theory, such applications derive from the almost ubiquitous expression of the protein PFKFB3, that when expressed over physiological levels might exert aberrant effects on cellular proliferation, growth, migration, and metabolism.

[0178] In a preferred embodiment, said tumor to be prevented or treated derives from a tissue preferably chosen from the group consisting of: mesenchyme; oral cavity; pharynx; larynx; head and neck; lung; bronchus; esophagus; stomach; intestine;thyroid; liver; prostate; breast; pancreas; adrenal gland; brain; endometrium; skin; kidney; urinary bladder; uterus, cervix; vagina; ovary; bone marrow; lymph node, thymus.

[0179] In another preferred embodiment, said tumor is a blood tumor chosen from the group consisting of: myeloproliferative neoplasm; chronic or acute leukemia; lymphocytic or myeloid leukemia.

[0180] In another preferred embodiment, said tumor is a lymphoma.

[0181] In a further preferred embodiment of the invention, said compound and / or said composition is administered in combination with at least an anticancer treatment, preferably chemotherapy and / or immunotherapy and / or targeted therapy.

[0182] In an embodiment of the invention, the anticancer treatment comprises the administration of a chemoterapeutic agent chosen from the group consisting of: gemcitabine, 5-fluorouracil, oxaliplatin and irinotecan.

[0183] In a particularly preferred embodiment, said compound and / or said composition is administered in combination with gemcitabine or in combination with at least one or a mixture of 5-fluorouracil, oxaliplatin and irinotecan.

[0184] Without being bound to any specific theory, the compound and / or composition in combination with an anticancer treatment, especially when the anticancer treatment is a chemotherapy, exhibits a synergistic effect, meaning that their combined efficacy exceeds the sum of their individual effects.

[0185] A fourth aspect of the present invention relates to a method of treating a patient comprising at least one step of administering said compound and / or said composition to a patient in need thereof. Said patient has preferably been diagnosed for a disease selected from the group consisting of: tumor, neurodegenerative disease, autoimmune disease, inflammatory disease, multiple sclerosis, metabolic disease. As previously disclosed in the second aspect of the present invention, the route and the dose of administration of the compound and / or of the pharmaceutical composition can be adjusted by the man skilled in the art according to the type and severity of the disease, and to the state of the patient, in particular his / her / its age, weight, sex, or general physical condition. A fifth aspect of the present invention relates to the in vitro use of the described compound as a chemical probe. In the context of the present invention, for chemical probe is intended a molecule that by binding its target protein allows themeasurement of the enzymatic activity of said target protein. The measurement of enzymatic activity of proteins are kinetic analysis that involve various types of assays, including spectrophotometric, fluorometric, and colorimetric methods, which are commonly employed in routine experiments to monitor reaction rates, substrate conversion, or product formation under controlled conditions. The choice and optimization of said kinetic assays are activities well known to those skilled in the art.

[0186] In a preferred embodiment of the invention, the compound allows to measure the enzymatic activity of PFKFB3 in a sample. Preferably, said sample is a biological sample isolated from a patient. In an embodiment, said sample is chosen from the group consisting of: blood, plasma, serum, urine, milk or a tissue biopsy.

[0187] In another embodiment of the invention, the in vitro use of the compound as chemical probe, allows to detect alterations in PFKFB3 expression and / or activity. Said alterations refer to increases or decreases in the expression and / or activity of PFKFB3 compared to physiological levels. Said alterations are detected when the expression and / or activity of PFKFB3 in a sample derived from a patient is compared to the physiological levels of expression and / or activity of PFKFB3 in a sample derived from a healthy subject.

[0188] In a preferred embodiment, the in vitro use of the compound as a chemical probe allows to diagnose alterations in PFKFB3 expression and / or activity in a biological sample isolated from a patient, when compared to the expression and / or activity of PFKFB3 in a sample derived from a healthy subject. Preferably, said alterations are increases in PFKFB3 expression and / or activity in the biological sample isolated from the patient.

[0189] In another preferred embodiment the ex vivo use of the compound as chemical probe, allows the detection of alterations in PFKFB3 expression and / or activity in samples derived from an animal, preferably an animal for research purposes.

[0190] The compounds and relative formulas disclosed in the first aspect of the present invention are depicted in Table 1.

[0191] Table 1.Compound 1:

[0192] (S)-N-(4-((1 -acryloyl-3-(1 -methyl-1 H- / / N'NX

[0193] pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2- Z'f^N''^'^

[0194] VNHHZ^O carboxamide

[0195] \\

[0196] Compound 2:

[0197] (S)-N-(4-((1 -(2-chloroacetyl)-3-(1 - / / N'NX

[0198] methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2- Z'f 1XN- £A'^y TX >

[0199] V~NHHZ*O carboxamide

[0200] Cl

[0201] Compound 3:

[0202] (S, E)-N-(4-((1 -(but-2-enoyl)-3-(1 - / ZN'NX

[0203] methyl-1 H-pyrazol-4-yl)-1 H-indol-5- jQ XX > yl)oxy)phenyl)pyrrolidine-2- Z'-f^'N'^^

[0204] ^NHHZ^O carboxamide

[0205] J\

[0206] Compound 4:

[0207] / / N'NX(S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4- yl)-1 -(3-methylbut-2-enoyl)-1 H-indol-5- ^ ja°^0z yl)oxy)phenyl)pyrrolidine-2- ^NHHZ^O carboxamide

[0208] Compound 5:

[0209] / / N'NX(S)-N-(4-((1 -methacryloyl-3-(1 -methyl- 1 H-pyrazol-4-yl)-1 H-indol-5- ^

[0210] Hja°Dz yl)oxy)phenyl)pyrrolidine-2- VNH carboxamide

[0211]

[0212] Compound 6:

[0213] (S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4- / / N'NX

[0214] yl)-1 -propioloy 1-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2- \ / V 1 W LI W

[0215] V-NH carboxamide

[0216] Compound 7:

[0217] < / N-N / (S)-N-(4-((1 -(but-2-ynoyl)-3-(1 -methyl- 1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2- \ | u \

[0218] VNH carboxamide

[0219] Compound 8:

[0220] (S, E)-N-(4-((1-(4-(dimethylamino)but- 2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)- JL XACA

[0221] 1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2- ANHHAO

[0222] carboxamide

[0223] z fN"

[0224] Compound 9:

[0225] / / N'NX

[0226] (S, E)-N-(4-((3-(1 -methyl-1 H-pyrazol-4- yl)-1 -(4-(piperid in-1 -yl)but-2-enoyl)-1 H- Z^A ANrAAy°r Wo

[0227] ANHHAO indol-5-yl)oxy)phenyl)pyrrolidine-2- f carboxamide

[0228] 0

[0229]

[0230] EXAMPLESThe present invention will now be described in more detail by the following examples, which are included to disclose some embodiments of the invention, but not in any way to limit the scope of the invention.

[0231] The compounds of the invention may be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, particularly considering the detailed description contained herein. The starting materials are generally available from commercial sources or are readily prepared using methods well known to those skilled in the art.

[0232] For illustrative purposes, Scheme 1 shows a general method for preparing the compounds of the present invention. Refer to the Methods and Examples below for a more detailed description of the individual reaction steps. However, other synthetic routes may be employed to synthesize the compounds of the present invention using techniques well known to those skilled in the art. Although specific starting materials and reagents are specified in the Examples below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and / or reaction conditions. In addition, the compounds disclosed herein can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0233] In preparing the compounds of the present invention, protection and deprotection of chemical groups (e.g., primary or secondary amine) of intermediates may be necessary. The need for such protection will vary depending on the nature of the chemical groups and the conditions of the preparation methods. Suitable aminoprotecting groups include tert-butyloxycarbonyl (BOC) or N, N-dimethylformamidinyl. The need for such protection is readily determined by one skilled in the art.

[0234] In the context of the present invention, reaction products and intermediates are separated and purified with techniques common in the art and the selection of appropriate methods of separation depends on the physical-chemical properties of the materials involved. Typically, such separations involve extraction, crystallization from a solvent or solvent mixture, or chromatography.

[0235] Materials and Methods.Reagents were purchased at the highest commercial quality from Sigma-Aldrich or Fluorochem and used without further purification. 1H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 (300 MHz) spectrometer or with an AvanceTM NEO 400 MHz (Bruker) spectrometer. NMR spectra were obtained in deuterated solvents, such as CDCl3, Methanol-d4 or DMSO-d6. The chemical shift (5 values) is reported in ppm and corrected to the signal of the deuterated solvents [7.26 ppm (1H NMR) and 77.16 ppm (13C NMR) for CDCl3; 2.50 ppm (1H NMR) and 39.52 ppm (13C NMR) for DMSO-d6; and 3.31 ppm (1 H NMR) and 49.00 ppm (13C NMR) for methanol-d4]. Peak multiplicities are reported as: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), m (multiplet), br (broadened). Chemical shifts (5) are expressed in ppm and coupling constants (J) in Hertz (Hz). Thin layer chromatography (TLC) plates were purchased from Sigma-Aldrich (silica gel 60 F254 aluminum sheets, with fluorescence indicator 254 nm) and UV light (254 nm) was used to visualize the compounds. Flash chromatography was performed using silica gel, pore size 60 A, 230-400 mesh particle size. High-resolution mass spectrometry (HRMS) analyses were performed using the Ultimate 3000 HPLC (Dionex), set to automatically inject into the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, USA), operating in full scan mode. Analyses were carried in positive ion mode.

[0236] The chromatographic purity of final compounds was determined by high-performance liquid chromatography (HPLC) analyses on a Waters 1525 Binary HPLC Pump, equipped with a Waters 2489 UV-vis detector (Waters, Milford, MA), using a Symmetry C18 Column (4.6 x 75 mm2, 3.5 pm particle size). Solvents A and B (A = Millipore water with 0.1% TFA, B = ACN with 0.1% TFA) and three different methods were used. Method A: gradient elution (85:15 for 0.2 min, 85:15 → 5:95 over 14 min) of the mobile phase consisting of solvents A and B at a flow rate of 1 mL / min at room temperature. Method B: gradient elution (70:30 for 0.2 min, 70:30 → 5:95 over 14 min) of the mobile phase consisting of solvents A and B at a flow rate of 1 mL / min at room temperature. Method C: gradient elution (60:40 for 0.2 min, 60:40 → 5:95 over 14 min) of the mobile phase consisting of solvents A and B at a flow rate of 1 mL / min at room temperature. The purity of all final compounds was >95%.The following abbreviations are used hereinafter: DMSO (dimethyl sulfoxide), HCI (hydrochloric acid), M (molar), HRMS (High resolution mass spectra), MS (mass spectrometry), PBS (phosphate buffered saline), TLC (thin layer chromatography). Chromatography can involve any number of methods including, for example: reverse-phase and normal phase; high, medium and low-pressure liquid chromatography methods and apparatus; small scale analytical; and preparative thin or thick layer chromatography, as well as techniques of small-scale thin layer and flash chromatography.

[0237] Chemical synthesis

[0238] Scheme 1 represents a general method for the synthesis of the compounds 1-3 of the present invention. The methods of synthesis of the intermediates are named from i) to vii) and described in detail below.

[0239]

[0240] Scheme 1. Reagents and conditions: (i) 1-fluoro-4-nitrobenzene, Cs2CO3, dry DMF, r.t., o / n; (ii) NBS, dry DMF, r.t., 2 hrs; (iii) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole, Pd(PPh3)4, 1 M K3PO4, 1,4-dioxane, 80°C, o / n; (iv) 10% Pd / C, H2, MeOH, r.t., 2 hrs; (v) Boc-Pro-OH, HATU, DIPEA, dry DMF, r.t., 4 hrs; (vi) acryloyl chloride, Cs2CO3, dry DMF, r.t. o / n (for 1); or bromoacetyl bromide, DBU, dry DCM, r.t. 2 hrs (for 2); or (2E)-2-butenoyl chloride, NaH, dryTHF, r.t. o / n (for 3); (vii) acetyl chloride, dry MeOH, r.t., 2 hrs. (r.t. = room temperature, o / n = overnight).

[0241] Detailed chemical synthesis of the intermediates reported in Scheme 1.

[0242] Method i): 5-(4-Nitrophenoxy)-1 H-indole

[0243]

[0244] To a stirred solution of 1-H-Indol-5-ol (1.00 g, 7.51 mmol, 1.0 eq.) in dry DMF (13 mL) under nitrogen atmosphere, 1-fluoro-4-nitro-benzene (1.10 g, 7.51 mmol, 1.0 eq.), and Cs2CO3 (2.90 g, 9.01 mmol, 1.2 eq.) were added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction monitored by TLC, EtOAc was added, and the organic phase was washed with water (3x) and brine (1x). The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane / EtOAc 8:2) afforded 5-(4-nitrophenoxy)-1 H-indole as yellow solid (1.70 g, 6.81 mmol, 75% yield).

[0245] 1H NMR (300 MHz, DMSO-d6) 5 11.26 (br, 1H), 8.24-8.16 (m, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.43 (dd, J = 2.4, 2.4 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.06-6.99 (m, 2H), 6.88 (dd, J = 8.7, 2.3 Hz, 1H), 6.46-6.42 (m, 1H).

[0246] Method ii): 3-Bromo-5-(4-nitrophenoxy)-1 H-indole

[0247] Br

[0248]

[0249] To a stirred solution of 5-(4-nitrophenoxy)-1 H-indole (1.70 g, 6.81 mmol, 1.0 eq.) in dry DMF (20 mL) under nitrogen atmosphere, N-bromosuccinimide (1.21 g, 6.81 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction monitored by TLC, EtOAc was added, and the reaction mixture was washed with water (3x) and brine (1x). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reducedpressure. Purification by column chromatography on silica gel (cyclohexane / EtOAc 9:1) afforded 3-bromo-5-(4-nitrophenoxy)-1 H-indole as a brown solid (2.26 g, 6.81 mmol, quantitative yield).

[0250] 1H NMR (300 MHz, DMSO-d6) 5 11.66 (br, 1H), 8.24-8.17 (m, 2H), 7.65 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 2.4, 1H), 7.09-7.03 (m, 2H), 7.00 (dd, J = 8.7, 2.4 Hz, 1H).

[0251] 13C NMR (75 MHz, DMSO-d6) 5 164.74 (s, 1 C), 148.36 (s, 1 C), 142.21 (s, 1 C), 133.55 (s, 1C), 127.36 (s, 1 C), 127.25 (s, 1 C), 126.59 (s, 2C), 117.10 (s, 2C), 116.53 (s, 1C), 114.47 (1 C), 109.71 (s, 1 C), 89.12 (s, 1C).

[0252] Method iii): 3-(1-Methyl-1H-pyrazol-4-yl)-5-(4-nitrophenoxy)-1H-indole

[0253] N

[0254] N N

[0255]

[0256] H

[0257] To a stirred solution of 3-bromo-5-(4-nitrophenoxy)-1 H-indole (2.00 g, 6.00 mmol, 1.0 eq.) in dry dioxane (30 mL) under nitrogen atmosphere, 1 -methyl-4-(4, 4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyrazole (2.50 g, 12.00 mmol, 2.0 eq.), palladium tetrakistriphenylphosphine (556 mg, 0.48 mmol, 0.08 eq.) and a 1 M K3PO4 degassed solution (12 mL, 12.00 mmol, 2.0 eq.) were added. The reaction mixture was stirred at 80°C overnight. After completion of the reaction monitored by TLC, the aqueous phase was extracted with EtOAc (3x). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane / EtOAc from 7:3 to 4:6) afforded 3-(1 -methyl-1 H-pyrazol-4-yl)-5-(4-nitrophenoxy)-1H-indole as a yellow solid (863 mg, 2.58 mmol, 43% yield).

[0258] 1H NMR (300 MHz, DMSO-d6) 5 11.32 (br, 1H), 8.24-8.17 (m, 2H), 8.08 (s, 1H), 7.74 (s, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.08-7.01 (m, 2H), 6.94 (dd, J = 8.7, 2.3 Hz, 1H), 3.83 (s, 3H).

[0259] 13C NMR (75 MHz, DMSO-d6) 5 165.24 (s, 1 C), 147.47 (s, 1 C), 141.89 (s, 1 C), 136.44 (s, 1C), 134.65 (s, 1 C), 127.17 (s, 1 C), 126.54 (s, 2C), 126.18 (s, 1 C), 123.92 (s, 1C), 116.69 (s, 2C), 115.82 (s, 1 C), 115.38 (s, 1 C), 113.65 (s, 1 C), 111.32 (s, 1 C), 108.50 (s, 1 C), 38.83 (s, 1 C).Method iv): 4-((3-( 1 -Methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)aniline.

[0260]

[0261] To a stirred solution of 3-(1 -methyl-1 H-pyrazol-4-yl)-5-(4-nitrophenoxy)-1 H-indole (863 mg, 2.58 mmol, 1.0 eq.) in MeOH (18 mL) under nitrogen atmosphere, 10% palladium on charcoal (200 mg) was added. The reaction flask was then evacuated and flushed with hydrogen. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction monitored by TLC, the suspension was filtered on Celite. The filtrate was evaporated under reduced pressure to afford 4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)aniline as a brown solid (785 mg, 2.58 mmol, quantitative yield).

[0262] 1H NMR (300 MHz, DMSO-d6) 5 11.15 (br, 1H), 7.99 (s, 1H), 7.67 (s, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz, 1H), 3.84 (s, 3H), 2.49 (br, 2H).

[0263] 13C NMR (75 MHz, DMSO-d6) 5 151.78 (s, 1 C), 149.22 (s, 1 C), 144.54 (s, 1 C), 136.35 (s, 1C), 133.21 (s, 1 C), 127.01 (s, 1 C), 126.07 (s, 1 C), 123.29 (s, 1 C), 119.40 (s, 2C), 116.22 (s, 1 C), 115.25 (s, 2C), 114.11 (s, 1 C), 112.82 (s, 1 C), 108.24 (s, 1 C), 107.85 (s, 1 C), 38.86 (s, 1C).

[0264] Method y): Tert-butyl 2-((4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate

[0265]

[0266] To a stirred suspension of 4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)aniline (785 mg, 2.58 mmol, 1.0 eq.) in dry DCM (25 mL) under nitrogenatmosphere, (2S)-1-tert-butoxycarbonylpyrrolidine-2-carboxylic acid (722 mg, 3.35 mmol, 1.3 eq.), N, N, N', N'-tetramethyl-O-(1 H-benzotriazol-1-yl)uronium hexafluorophosphate (1.50 g, 3.87 mmol, 1.5 eq.), and diisopropylethylamine (667 mg, 0.9 mL, 5.16 mmol, 2.0 eq.) were added. The reaction mixture was stirred at room temperature for 4 h. After completion of the reaction monitored by TLC, the organic solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane / EtOAc from 3:7 to 2:8) afforded tertbutyl 2-((4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate as a yellow solid (1.29 g, 2.58 mmol, quantitative yield).

[0267] 1H NMR (300 MHz, DMSO-d6) 5 9.35 (s, 1H), 8.31 (s, 1H), 7.69 (s, 1H), 7.58 (s, 1 H), 7.44-7.39 (m, 3H), 7.36 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 6.97 (d, J = 8.7, 2.4 Hz, 1H), 6.95-6.89 (m, 2H), 4.51-4.36 (m, 1H), 3.94 (s, 3H), 3.57-3.30 (m, 2H), 2.01-1.87 (m, 2H), 1.67-1.58 (m, 2H), 1.48 (s, 9H).

[0268] Method vi) for compound 1: Tert-butyl (S)-2-((4-((1-acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate)

[0269]

[0270] To a stirred solution of tert-butyl 2-((4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate (250 mg, 0.50 mmol, 1.0 eq.) in dry DMF (4 mL) under nitrogen atmosphere, acryloyl chloride (68 mg, 60 pL, 0.75 mmol, 1.5 eq.) and Cs2CO3 (487 mg, 1.50 mmol, 3.0 eq.) were added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction monitored by TLC, EtOAc was added and the reaction mixture was washed with water (3x) and brine (1x). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Purification by preparative TLC (DCM / MeOH 98:2) afforded tert-butyl (S)-2-((4-((1-acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 - carboxylate) as a yellow solid (222 mg, 0.25 mmol, 50% yield).

[0271] 1H NMR (300 MHz, CDCI3) 59.44 (br, 1 H), 8.50 (d, J = 9.0 Hz, 1 H), 7.72 (s, 1 H), 7.63 (s, 1 H), 7.57 (s, 1 H), 7.50-7.42 (m, 2H), 7.31 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 9.0, 2.4 Hz, 1H), 7.01-6.90 (m, 3H), 6.68 (dd, J = 16.7, 1.5 Hz, 1H), 6.05 (dd, J = 10.4, 1.5 Hz, 1H), 4.51-4.36 (m, 1H), 3.96 (s, 3H), 3.56-3.32 (m, 2H), 2.56-2.42 (m, 1H), 1.98-1.83 (m, 3H), 1.48 (s, 9H). HPLC (Method B): tR = 7.53 min (98.9% purity).

[0272] Alternative method vi) for compound 1: Tert-butyl (S)-2-((4-((1-acryloyl-3-(1- methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 - carboxylate)

[0273]

[0274] To a stirred solution of tert-butyl 2-((4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate (300 mg, 0.60 mmol, 1.0 eq.) in dry THF (10 mL) under nitrogen atmosphere, at 0°C acryloyl chloride (432 mg, 389 pL, 4.78 mmol, 8.0 eq.) was added. After 15 minutes at 0°C NaH 60% (210 mg, 5.26 mmol, 8.8 eq.) was added. The reaction mixture was stirred at 0°C for 30 minutes then allowed to reach room temperature and reacted overnight. Then, water was added, and the solvent was evaporated under reduced pressure. The reaction mixture was extracted with EtOAc (3x). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Purification by preparative TLC (DCM / MeOH 99:1) afforded tert-butyl 2-((4-((3-(1 -methyl-1 H- pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate as a yellow solid (89 mg, 0.16 mmol, 27% yield).

[0275] 1H NMR (300 MHz, CDCI3) 59.44 (br, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.72 (s, 1H), 7.63 (s, 1H), 7.57 (s, 1H), 7.50-7.42 (m, 2H), 7.31 (d, J = 2.4 Hz, 1H), 7.07 (dd, J =9.0, 2.4 Hz, 1H), 7.01-6.90 (m, 3H), 6.68 (dd, J = 16.7, 1.5 Hz, 1H), 6.05 (dd, J = 10.4, 1.5 Hz, 1H), 4.51-4.36 (m, 1H), 3.96 (s, 3H), 3.56-3.32 (m, 2H), 2.56-2.42 (m, 1H), 1.98-1.83 (m, 3H), 1.48 (s, 9H).

[0276] Alternative method vi) for compound 2: Tert-butyl (S)-2-((4-((1-(3-bromopropanoyl)-3-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate

[0277]

[0278] To a stirred solution of (S)-2-((4-((3-(1-methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate (328 mg, 0.65 mmol, 1.0 eq.) in dry DCM (15 mL) under nitrogen atmosphere, 8-diazabiciclo[5.4.0]undec-7-ene (496 mg, 490 pL, 3.27 mmol, 5.0 eq.) was added. The reaction mixture was cooled at 0 °C and bromo acetyl bromide (396 mg, 170 pL, 1.96 mmol, 3.0 eq.) was added. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction monitored by TLC, the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane / EtOAc from 4:6 to 1:9) afforded compound tert-butyl (S)-2-((4-((1-(3-bromopropanoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate as a yellow solid (190 mg, 0.31 mmol, 47% yield).

[0279] Method vi) for compound 3: Tert-butyl (S, E)-2-((4-((1-(but-2-enoyl)-3-(1-methyl-1l-l-pyrazol-4-yl)-1H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate

[0280]

[0281] To a stirred solution of tert-butyl 2-((4-((3-(1-methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate (300 mg, 0.60 mmol, 1 eq.) in dry DMF (4 mL) under nitrogen atmosphere, crotonyl chloride (94 mg, 86 pL, 0.90 mmol, 1.5 eq.) and Cs2CO3 (585 mg, 1.70 mmol, 3.0 eq.) were added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction monitored by TLC, EtOAc was added, and the reaction mixture was washed with water (3x) and brine (1x). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Purification by preparative TLC (DCM / MeOH 98:2) afforded tert-butyl (S, E)-2-((4-((1-(but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate as a yellow solid (45 mg, 0.080 mmol, 13% yield).

[0282] 1H NMR (300 MHz, CDCI3) 5 9.43 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.73 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.50-7.42 (m, 2H), 7.34-7.25 (m, 2H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 7.00-6.92 (m, 2H), 6.71 (dq, J = 15.0, 1.7 Hz, 1H), 4.51-4.40 (m, 1H), 3.97 (s, 3H), 3.54-3.30 (m, 2H), 2.60-2.46 (m, 1H), 2.07 (dd, J = 6.9, 1.7 Hz, 3H), 2.01-1.86 (m, 3H), 1.49 (s, 9H).

[0283] 13C NMR (75 MHz, CDCI3) 5 169.78 (s, 1C), 163.64 (s, 1 C), 153.73 (s, 1 C), 146.88 (s, 1C), 137.59 (s, 1 C), 132.69 (s, 1 C), 132.15 (s, 1 C), 132.01 (s, 1 C), 130.50 (s, 1C), 128.59 (s, 1 C), 128.42 (s, 1 C), 127.67 (s, 1 C), 122.04 (s, 1 C), 121.22 (s, 2C), 118.51 (s, 2C), 118.14 (s, 1 C), 117.38 (s, 1 C), 114.66 (s, 1 C), 113.91 (s, 1C), 109.59 (s, 1 C), 80.92 (s, 1 C), 77.24 (s, 1 C), 60.34 (s, 1 C), 47.23 (s, 1 C), 39.13 (s,1C), 29.69 (s, 1 C), 28.40 (s, 3C), 18.65 (s, 1C). HPLC (Method C): tR = 5.10 min (98.0% purity).

[0284] Method vii) for compounds 1-3, general procedure for Boc deprotection.To a stirred solution of MeOH dry (2.5 mL) under nitrogen atmosphere at 0°C, acetyl chloride (0.4 mL) was added dropwise, and the mixture was allowed to warm up to room temperature. After 5 minutes, the previously prepared solution (6 eq.) was added to the Boc-protected intermediate (1 eq.). The reaction mixture was stirred at room temperature under nitrogen atmosphere for one hour. After completion of the reaction, monitored by TLC, the reaction mixture was evaporated under reduced pressure to afford the desired final compound.

[0285] Method vii) for compound 1: (S)-N-(4-((1-acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1H-indol-5-yl)oxy)phenyl)pyrrolidine-2-carboxamide hydrochloride (Compound 1)

[0286]

[0287] (S)-N-(4-((1 -Acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide hydrochloride (Compound 1) was prepared following general procedure 1 from (S)-2-((4-((1 -acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate) (70 mg, 0.13 mmol, 1.0 eq.), using 362 pL of the solution dry methanol / acetyl chloride. After one hour the reaction mixture was evaporated under reduced pressure to afford the desired compound as a yellow solid (48 mg, 0.09 mmol, 70% yield).

[0288] 1H NMR (300 MHz, Methanol-d4) 58.52 (d, J = 9.0 Hz, 1H), 8.08 (s, 1H), 8.04 (s, 1H), 7.86 (s, 1H), 7.60-7.54 (m, 2H), 7.41 (d, J = 2.3 Hz, 1H), 7.29 (dd, J = 16.6, 10.5 Hz, 1H), 7.05 (dd, J = 9.0, 2.4 Hz, 1H), 7.02-6.97 (m, 2H), 6.65 (dd, J = 16.6, 1.5 Hz, 1H), 6.09 (dd, J = 10.5, 1.5 Hz, 1H), 4.43-4.31 (m, 1H), 3.95 (s, 3H), 2.60- 2.42 (m, 2H), 2.21-2.01 (m, 4H).

[0289] 13C NMR (75 MHz, Methanol-d4) 5 166.27 (1 C), 163.74 (1 C), 154.88 (1 C), 153.94 (1C), 134.63 (1C), 132.87 (1C), 132.46 (1 C), 131.66 (1 C), 131.15 (1C), 129.84 (1C), 127.77 (1C), 123.41 (1C), 121.58 (2C), 118.21 (2C), 117.79 (1C), 116.86 (1C), 114.97 (1C), 112.62 (1 C), 109.34 (1 C), 60.36 (1C), 46.25 (1C), 37.87 (1 C),29.89 (1 C), 23.85 (1C). HRMS (m / z): [M + H]+ calcd for C26H26N5O3 456.2036; found, 456.2027. HPLC (Method B): tR = 6.17 min (97.36% purity).

[0290] Method vii) for compound 2: (S)-N-(4-((1-(3-chloropropanoyl)-3-(1-methyl-1H-pyrazol-4-yl)-1H-indol-5-yl)oxy)phenyl)pyrrolidine-2-carboxamide hydrochloride (Compound 2)

[0291]

[0292] (S)-N-(4-((1 -(3-chloropropanoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide hydrochloride was prepared following general procedure 1 from tert-butyl (S)-2-((4-((1-(3-bromopropanoyl)-3-(1-methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate as (50 mg, 0.08 mmol, 1.0 eq), using 230 pL (0.48 mmol, 6 eq.) of the solution dry methanol / acetyl chloride. After one hour the reaction mixture was evaporated under reduced pressure to afford the desired compound as a yellow solid (27 mg, 0.05 mmol, 61% yield).

[0293] 1H NMR (400 MHz, Methanol-d4) 58.42 (d, J = 9.0 Hz, 1H), 8.22 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.61-7.55 (m, 2H), 7.42 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 9.0, 2.4 Hz, 1H), 7.01-6.95 (m, 2H), 4.90 (s, 2H), 4.47-4.38 (m, 1H), 4.02 (s, 3H), 3.49-3.37 (m, 2H), 2.18-2.05 (m, 4H).

[0294] 13C NMR (101 MHz, Methanol-d4) 5 166.36 (s, 1 C), 165.03 (s, 1 C), 154.94 (s, 1 C), 154.19 (s, 1C), 135.52 (s, 1 C), 132.92 (s, 1 C), 132.46 (s, 1 C), 130.18 (s, 1 C), 129.82 (s, 1C), 122.57 (s, 1 C), 121.57 (s, 2C), 118.27 (s, 2C), 117.45 (s, 1 C), 117.06 (s, 1C), 114.40 (s, 1C), 113.98 (s, 1 C), 109.47 (s, 1 C), 60.32 (s, 1 C), 46.12 (s, 1 C), 42.25 (s, 1 C), 37.56 (s, 1 C), 29.74 (s, 1 C), 23.74 (s, 1C). HRMS (m / z): [M + H]+ calcd for C25H25CIN5O3478.1646; found, 478.1649.

[0295] Method vii) for compound 3:(S, E)-1-(3-(1 -methyl- 1 H-pyrazol-4-yl)-5-( 4-( ( pyrrolidin-2-ylmethyl)amino)phenoxy)- 1 H-indol-1 -yl)but-2-en-1 -onehydrochloride (Compound 3)

[0296]

[0297] (S, E)-1-(3-(1-methyl-1H-pyrazol-4-yl)-5-(4-((pyrrolidin-2-ylmethyl)amino)phenoxy)- 1 H-indol-1 -yl)but-2-en-1 -onehydrochloride (Compound 3) was prepared following general procedure 1 from (S)-2-((4-((1-acryloyl-3-(1-methyl-1 H-pyrazol-4-yl)-1 H-indol-5-yl)oxy)phenyl)carbamoyl)pyrrolidine-1 -carboxylate) (45 mg, 0.08 mmol, 1 eq.), using 230 pL (0.48 mmol, 6 eq.) of the solution dry methanol / acetyl chloride. After one hour the reaction mixture was evaporated under reduced pressure to afford the desired compound as a yellow solid (27 mg, 0.5 mmol, 62% yield).

[0298] 1H NMR (300 MHz, Methanol-d4) 58.46 (d, J = 9.0 Hz, 1H), 8.04 (s, 1H), 8.01 (s, 1 H), 7.85 (s, 1 H), 7.60-7.54 (m, 2H), 7.36 (d, J = 2.4 Hz, 1H), 7.31-7.18 (m, 1H), 7.04-6.92 (m, 4H), 4.46-4.37 (m, 1H), 3.94 (s, 3H), 3.51-3.33 (m, 2H), 2.60-2.47 (m, 1H), 2.16-2.08 (m, 3H), 2.05 (dd, J = 6.9, 1.3 Hz, 3H).

[0299] 13C NMR (75 MHz, Methanol-d4) 5 166.22 (s, 1 C), 164.11 (s, 1 C), 155.06 (s, 1 C), 153.72 (s, 1C), 146.84 (s, 1 C), 136.62 (s, 1C), 132.76 (s, 1C), 132.63 (s, 1 C), 130.32 (s, 1C), 128.72 (s, 1 C), 122.21 (s, 1C), 121.84 (s, 1C), 121.54 (s, 2C), 118.18 (s, 2C), 117.70 (s, 1 C), 116.53 (s, 1C), 114.15 (s, 1C), 113.95 (s, 1 C), 109.27 (s, 1 C), 60.31 (s, 1 C), 46.12 (s, 1 C), 37.57 (s, 1 C), 29.72 (s, 1 C), 23.74 (s, 1C), 17.17 (s, 1C). HRMS (m / z): [M + H]+ calcd for C27H28N5O3 470.2192; found, 470.2193. HPLC (Method A): tR = 6.16 min (96.9% purity).

[0300] Testing methods of the compounds of the present invention

[0301] Example 1: recombinant PFKFB3 expression and purification

[0302] The gene encoding human 6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase 3 isoform 1 (PFKFB3, EC:3.1.3.46) was codon-optimized for expression in E. coli, fused with an N-terminal 6xHis-tag and subcloned into a pET28 expression vector, which confers kanamycin resistance. E. coli BL21 cells were transformed with thecloned vector, and the expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside, followed by overnight incubation at 20°C. Protein purification was performed using conventional IMAC chromatography.

[0303] The purified human recombinant PFKFB3 protein was then used for the subsequent assays.

[0304] Example 2: Measurement of PFKFB3 Activity

[0305] PFKFB3 enzymatic activity was measured using three methods:

[0306] 1. A continuous coupled assay measuring the conversion of ATP to ADP in the presence of the co-substrate fructose 6-phosphate. Once produced by PFKFB3, ADP is converted back to ATP by pyruvate kinase in the presence of phosphoenolpyruvate, with concomitant production of pyruvate. Pyruvate is then reduced to lactate by the enzyme lactate dehydrogenase in the presence of NADH. NADH oxidation is monitored by real-time absorbance at 340 nm. The assay was performed in the absence and presence of inhibitors at various concentrations and timepoints. Using this assay, full inhibition was observed when PFKFB3 at 2.5 pM was incubated for 5 minutes with the inhibitors at 10 pM concentration.

[0307] 2. A discontinuous enzyme assay based on the same reaction described above, carried out on aliquots of the reaction mixture containing PFKFB3 and its substrates fructose 6-phosphate and ATP, collected at different timepoints. In each aliquot, the concentration of ADP was measured by endpoint readings at 340 nm using a plate reader. This assay allows PFKFB3 to react in the absence of phosphoenolpyruvate, an allosteric inhibitor that could potentially influence the reaction rate in the continuous coupled assay. This assay was used to confirm that phosphoenolpyruvate had no significant effect on the primary assay. Residual activity following incubation with the covalent inhibitors was negligible.

[0308] 3. A continuous1H NMR-based assay. The activity of PFKFB3 was monitored by measuring ATP consumption and ADP formation using1H NMR spectra performed at different time points. The H-8 peak of adenine in ATP shifts to slightly higher ppm values when ATP is converted to ADP

[0033] . Spectra were acquired using a JEOL 600 MHz spectrometer at 25°C. Initially, a reference spectrum of 200 pM ATP and 1 mM fructose-6P in 10 mM Na phosphate, 10 mM MgCI2, 50mM NaCI, pH 7.5 in D20 was obtained. After adding 2.5 pM PFKFB3 to the NMR tube, spectra were recorded every 5 minutes for 3 h. Data were analyzed using the MestReNova software (Mestrelab Research). The accumulation of ADP and the corresponding decrease of ATP were observed. No enzyme activity was detected when PFKFB3 was pre-incubated with compounds 1, 2, or 3. The loss of activity was correlated with the rapid precipitation of the enzyme upon interaction with compounds 1, 2, and 3, as confirmed by UV-vis spectra of the enzyme solution.

[0309] Example 3: Identification of Reactive Cysteines of PFKFB3

[0310] To demonstrate that the inhibitors covalently bind to cysteine in the enzyme's active site (Cys154), mass spectrometry experiments were conducted on trypsin-digested PFKFB3 that was incubated with the compounds. PFKFB3 incubated with compound 1 was precipitated in acetone overnight at 20°C. The pellet was resuspended in a buffer solution containing 50 mM Tris, 8 M urea, pH 8.0. Tris(2-carboxyethyl)phosphine (TCEP) was added, and the mixture was incubated for 2 h at 30°C. lodoacetamide was added, and the mixture was incubated for 30 minutes at room temperature in the dark. The solution was diluted with 50 mM Tris, pH 8.0 to reach a final urea concentration of 2 M. The protein was then digested with trypsin overnight at 30°C, and the reaction was stopped with 1% TFA. A reference enzyme sample, not incubated with compound 1, was subjected to the same protocol. The resulting peptides were analyzed using an LTQ ORBITRAP XL (Thermo) mass spectrometer, equipped with a Phenomenex AERIS Peptide 3.6u XB-C18 150x2.1 mm column. Fragmentation was carried out in CID mode in the instrument’s linear trap with a normalized collision energy of 35. Data were analyzed using PEAKS Studio 11, with two possible cysteine modifications considered: derivatization with iodoacetamide (+57.02 Da) and adduct formation with compound 1 (+455.20 Da). Coverage was 80%. The only post-translational modification identified with high confidence was the derivatization of Cys160 (Cys154 in the native sequence) in the peptide AFFIESVC(+455.20)DDPTVVASNIMEVK with compound 1, but not in its absence. Similar results were obtained with compounds 2 and 3.Example 4: Targeting PFKFB3 inhibits PDAC cell viability

[0311] The efficacy of the compounds to affect cell growth and cell viability was tested across a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines with varying degrees of genetic complexity.

[0312] Panel, MIA PaCa-2, Suit2, AsPc1, Hs776t and PaCa3 epithelial cell lines that were established from human pancreatic tumor, were employed for the following experiments. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented (v / v) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 50 pg / mL Gentamicin sulfate (BioWhittaker, Lonza) and maintained at 37 °C in a humidified atmosphere with 5% CO2. The effect of the compounds was assessed by treating the cells for 48 h. Cells were seeded in 96-well plate (5 x 103cells / well), and after 24 h they were either left untreated or treated with some of the compounds of the invention and with a non-covalent PFKFB3 inhibitory 9] as control, at the concentrations of 1 pM, 10 pM, 25 pM and 50 pM. After 48 h, cell proliferation and cell viability were measured by Crystal Violet assay and MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay.

[0313] The Crystal Violet assay is based on staining cells that remain attached to cell culture plates after the 48 h of treatment with the compounds. Before staining the cells with the Crystal Violet solution, the wells are washed once with PBS 1x. Then, the Crystal violet dye is solubilized and measured by absorbance at 595 nm. The amount of Crystal Violet staining in the assay is directly proportional to the cell biomass that is attached to the plate. The cell biomass can be used to infer levels of cell viability I cytotoxicity. For the MTT assay, MTT solution (at final concentration 5.5 mg / mL) was added for 2 h to the cells. The medium was then replaced with 25 pL of medium and 50 pL of DMSO to each well to lyse the cells and the absorbance of the formazan product released was measured at 540 nm. The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. Both for the Crystal violet assays and MTT assays, absorbance was measured by spectrophotometric analysis (GENios Pro, Tecan) and the values reported in the figures are expressed as percentage of the viability of treated cells compared with DMSO treated control (100% viability). As shown in Figure 1, the two PFKFB3 covalent inhibitors (Compound 2 and Compound 3) significantly affect cell viability of both Panel and MIA PaCa-2 cellsin a dose-dependent manner, compared to the non-covalent inhibitor (Compound 43 from paper

[0019] ). The percentage of cell viability inhibition was calculated using the Crystal violet (Figure 1A-B) and the MTT assays (Figure 1C-D).

[0314] In addition, the inhibitory activity of the compounds on PDAC cell proliferation was also confirmed using 4 different human PDAC cell lines: Suit2 (Figure 2A and E), AsPc1 (Figure 2B and F), Hs776t (Figure 3C and G) and PaCa3 (Figure 2D and H) cells. For this experiment the cells were treated with 25pM concentration of the inhibitors (DMSO treatment was used as control). Both the Crystal Violet (Figure 2A-D) and MTT assays (Figure 3E-H) showed a significant impairment in cell viability, with Compound 3 being the most effective.

[0315] To demonstrate the extended duration of action of the covalent compounds of the present invention, washout experiments on Panel and MIA PaCa-2 cells were performed. Cells were seeded in 48-well plate (5 x 103 cells / well), and after 24 h were treated with DMSO or 25 pM of Compound 3. The cells were incubated for 15 minutes, 1 h, 2 h, 3 h or 4 h before the free compound was washed out and the media was replaced. The effect of the inhibitor was then analyzed using the Crystal Violet assay after 72 h. As shown in Figure 3, a long-term effect on cell viability was achieved also after the compound washout: 1 h of incubation with Compound 3 is sufficient to significantly compromise cell viability highlighting the advantage of the covalent inhibitor.

[0316] Example 5: Zebrafish xenografts reveal in vivo efficacy of covalent PFKFB3 inhibition

[0317] To further evaluate the antitumor efficacy of covalent compound 3 in vivo, a zebrafish xenograft model was used. MIA PaCa-2 and PANC-1 cells were fluorescently labeled with a lipophilic membrane dye to enable visualization after injection into 2 days post-fertilization (dpf) embryos. Prior to treatment, the maximum tolerated dose of the compound was determined by LD50analysis in zebrafish larvae, which indicated that larvae tolerated doses below 10 pM without observable toxicity. Based on these results, a concentration of 7 pM of covalent compound 3, well below the tolerated threshold, was selected for subsequent experiments. At 24 hours post-injection (hpi), fluorescence imaging was performedto assess initial tumor cell engraftment, and larvae were treated with 7 pM of compound 3 in water. Fluorescence intensity was monitored at 24 (T1) and 48 hours (T2) post-treatment. For both MIA PaCa-2 and PANC-1 xenografts, control larvae showed a decrease in fluorescence over time, likely reflecting normal developmental clearance or minimal cell loss in this model. In contrast, larvae treated with compound 3 exhibited a pronounced and more statistically significant reduction in fluorescence intensity, especially at T2 (Figure 4 A-B). The strong efficacy observed at 7 pM reflects the high sensitivity of these pancreatic cancer cells to the compound in the zebrafish xenograft system, where direct exposure in a small aquatic volume allows efficient drug uptake and rapid pharmacological effects.

[0318] Example 6: Covalent PFKFB3 inhibition disrupts glycolytic flux in PDAC cells

[0319] To assess the cellular efficacy of compound 3 in the MIA PaCa-2 cell model, PFK activity was measured in cell lysates following a 2-hour incubation with covalent compound 3 at a final concentration of 25 pM. After incubation, cells were extensively washed to remove any non-covalently bound material prior to lysis. A statistically significant reduction in PFK activity was observed in cells treated with compound 3 (Figure 5A). These findings are consistent with the covalent mode of action of compound 3, leading to sustained enzyme inhibition. To further test the cellular efficacy of compound 3, an ATP rate assays at two time points, 1 h and 2 h post-treatment was conducted. The results revealed a rapid, time-dependent decrease in total ATP production, with glycolytic ATP production markedly reduced already at 1 h and completely abolished by 2 h in compound 3-treated cells (Figure 5 B, C). These data demonstrate that compound 3 rapidly and effectively shuts down glycolytic ATP rate in MIA PaCa2 cells, confirming its potent intracellular activity. Importantly, the ATP rate assay provided a dynamic readout of the compound’s impact on cellular bioenergetics, enabling precise temporal resolution of its inhibitory effects on glycolytic ATP generation.

[0320] Furthermore, glycolysis stress tests performed on MIA PaCa-2 and PANC-1 cells demonstrated a significant decrease in key glycolytic parameters, including glycolytic rate, capacity, and reserve, after only one hour of treatment (Figure 5 D-E). These findings suggest that compound 3 effectively impairs the glycolytic flux and the ability of cancer cells to respond to metabolic stress, further supporting its mechanism of action as a PFKFB3 inhibitor.

[0321] Example 7: Synergistic anticancer effects of compound 3 combined with chemotherapy in chemoresistant PDAC cell lines

[0322] The potential synergistic effects between the covalent PFKFB3 inhibitor, compound 3, and chemotherapeutic agents commonly used in PDAC treatment were investigated. The two most aggressive and chemoresistant PDAC cell lines, MIA PaCa-2 and PANC-1, were selected for this study. Compound 3 was tested in combination with gemcitabine at a fixed ratio of 1:10, as well as with a FOLFIRINOX-like cocktail (FOI) (excluding folinic acid), to evaluate whether the metabolic inhibition mediated by compound 3 could enhance chemotherapy efficacy. The FOLFIRINOX-like cocktail (FOI) is a combination of 5-fluorouracil, oxaliplatin and irinotecan.

[0323] Cell viability assays were performed after treatment with increasing concentrations of compound 3, chemotherapeutic agents alone, or their combinations. In both cell lines, single-agent treatments with either compound 3 or chemotherapy resulted in a dose-dependent reduction in cell viability. Notably, the combined treatments produced a more pronounced cytotoxic effect than the drug alone, particularly at intermediate and higher concentrations, suggesting a potentiating interaction between compound 3 and the chemotherapeutics (Figure 6 A-D). To quantitatively assess drug interactions, data were analyzed using CompuSyn software following the Chou-Talalay method. In MIA PaCa-2 cells, the combination of compound 3 with FOI or gemcitabine showed additive effects at low fractional effects (Fa < 0.4) and clear synergism at higher Fa values (Fa > 0.5), as confirmed by isobologram analysis (Figure 3 E-F).

[0324] In PANC-1 cells, compound 3 combined with FOI displayed strong synergy at low to intermediate effect levels (Fa < 0.6; Cl 0.3-0.5), becoming additive at higher Fa. Conversely, the compound 3 + gemcitabine combination maintained robust synergy over a wider range (Fa = 0.1 -0.8; Cl 0.3-0.6), turning additive only at very high Fa (>0.9) (Figure 6 g-h).Overall, the dose-response and combination index analyses demonstrate that compound 3 enhances the cytotoxic activity of both FOI and gemcitabine in PDAC cells, suggesting that metabolic inhibition via PFKFB3 targeting may sensitize tumor cells to chemotherapy-induced stress, thereby offering a promising combinatorial therapeutic approach for chemoresistant PDAC.

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Claims

CLAIMS1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,o, Hwherein L is a moiety selected from the group consisting of:R1, R2 are selected independently from the group consisting of:- H;- Linear or branched, saturated or unsaturated C1 -C6 alkyl;R3 is selected from the group consisting of:- H;- Linear or branched, saturated or unsaturated C1 -C6 alkyl;- -CH2-N-R’R” and wherein R’ and R” are selected independently between H and CH3;(R5)Zwherein R5 is selected from a halogen, H, a C1-C6 alkyl; z is 0, or 1 or 2;R4 is a halogen.

2. The compound according to claim 1, whereinR1, R2 are selected independently from the group consisting of:- H;- Linear or branched, saturated or unsaturated C1-C4 alkyl; and / orR3 is selected from the group consisting of:- H;- Linear or branched, saturated or unsaturated C1 -C4 alkyl;- -CH2-N-R’R”; R’ and R” are selected independently between H and CH3;(R5)Zwherein R5 is a halogen, or H, or a C1-C4 alkyl; z is 0, or 1 or 2; and / orR4 is a halogen.

3. The compound according to claim 1 or 2, whereinR1 is H or a saturated or unsaturated C1-C2 alkyl; and / orR2 is H or a saturated or unsaturated C1-C2 alkyl; and / orR3 is selected from the group consisting of:- H;- saturated or unsaturated C1-C2 alkyl;-CH2-N-R’R”, wherein R’ and R” are selected independently between H and CH3;(R5)Zwherein R5 is a halogen, or H, or a C1-C2 alkyl; z is 0, or 1 or 2; and / orR4 is a halogen.

4. The compound according to any one of claims 1-3, whereinR1 is H or CH3; and / orR2 is H or CH3; and / orR3 is selected from the group consisting of:- H;- CH3;- -CH2-N-R’R” wherein R’ and R” are H;(R5)Zwherein R5 is a halogen, or H, or an alkyl chain C1-C2; z is 0 or 1; and / orR4 is a halogen.

5. The compound according to any one of claims 1-4, whereinR3 is selected from the group consisting of:H;CH3;-CH2-N-R’R” wherein R’ and R” are H;(R5)Zwherein R5 is F, or H, or CH3; z is 0 or 1 or 2; and / orR4 is a halogen.

6. The compound according to any one of claims 1-5, whereinR1 is H or CH3; and / orR2 is H or CH3; and / orR3 is selected from the group consisting of:- H,- CH3,- -CH2-N-R’R” wherein R’ and R” are H;(R5)Zwherein R5 is H; z is 1; and / orR4 is a Cl.

7. The compound according to any one of claims 1-6, wherein:R1 is H;R2 is H;R3 is H or CH3; and / orR4 is a Cl.

8. The compound according to any one of claims 1-7, wherein said compound is selected from the group consisting of:- (S)-N-(4-((1 -acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((1 -(2-chloroacetyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide- (S, E)-N-(4-((1 -(but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 -(3-methylbut-2-enoyl)-1 H- indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((1 -methacryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 -propioloy 1-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((1 -(but-2-ynoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S, E)-N-(4-((1-(4-(dimethylamino)but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4- yl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S, E)-N-(4-((3-(1 -methyl-1 H-pyrazol-4-yl)-1 -(4-(piperid in-1 -yl)but-2- enoyl)-1 H-indol-5-yl)oxy)phenyl)pyrrolidine-2-carboxamide.

9. The compound according to any one of claims 1-8, wherein said compound is selected from the group consisting of:- (S)-N-(4-((1 -acryloyl-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide,- (S)-N-(4-((1 -(2-chloroacetyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide- (S, E)-N-(4-((1 -(but-2-enoyl)-3-(1 -methyl-1 H-pyrazol-4-yl)-1 H-indol-5- yl)oxy)phenyl)pyrrolidine-2 -carboxamide.

10. A pharmaceutical composition comprising the compound according to any one of claims 1-9, and / or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10, further comprising at least an anticancer agent chosen from the group consisting of: chemotherapeutic agent, immunotherapeutic agent, targeted therapeutic agent.

12. The pharmaceutical composition according to claim 11, wherein the chemotherapeutic agent is at least one compound chosen from the group consisting of: gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan.

13. The compound according to any one of claims 1-9, or the pharmaceutical composition according to any one of claims 10-12, for use as a medicament.

14. The compound according to any one of claims 1-9, or the pharmaceutical composition according to any one of claims 10-12, for use in the treatment of a cancer and / or a metabolic disease and / or an immunological disease.

15. The compound according to any one of claims 1-9, or the pharmaceutical composition according to any one of claims 10-12, for use according to claim 14, wherein the cancer is selected from the group consisting of: pancreatic, colorectal, gastrointestinal, hepatocellular, lymphoma or leukemia.

16. The in vitro use of the compound according to any one of claims 1-9, as a chemical probe for PFKFB3 activity.

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