Integrin-linked kinase modulating ligands and use of same for treating diseases

Abstract

The present invention provides a series of novel sulphonamides that act as integrin-linked kinase (ILK) ligands, which are capable of activating the actin cytoskeleton and promoting cell receptor traffic. Furthermore, the invention relates to use in the treatment or prevention of ILK-mediated diseases, such as type 2 diabetes mellitus or nephrogenic diabetes insipidus, as well as pathologies involving reduced transmembrane transport.

Description

[0001] Integrin-linked kinase modulating ligands and their use in the treatment of diseases: Description and Technical Sector

[0002] The present invention relates to novel compounds that act as ligands of integrin-linked kinase (ILK), facilitating the polymerization of the actin cytoskeleton and thus promoting the trafficking of cell receptors from intracellular compartments to the cell surface. For this reason, they can be used in diseases such as type 2 diabetes mellitus or nephrogenic diabetes insipidus and, by extension, in any disease in which the pathophysiological alterations are due, wholly or partially, to decreased transmembrane transport, such as renal tubulopathies caused by a loss of transporters.

[0003] STATE OF THE ART

[0004] The human proteome consists of approximately 550 kinases, proteins capable of covalently incorporating a phosphate group derived from ATP hydrolysis into various substrates, including proteins, lipids, and sugars (Mace, PD; Murphy, JM, J. Biol. Chem., 2021, 296, 100705). Thus, protein kinases play an essential role in intracellular signaling processes in all living organisms, controlling numerous events related to cell growth, differentiation, proliferation, and communication.

[0005] Despite the importance of this group of proteins, it is estimated that approximately 10% of kinases have lost all or part of their catalytic capacity over the course of evolution, due to mutations and / or deletions of key amino acids in their active site. This group of proteins, called pseudokinases, are capable of decisively influencing the cell cycle, either through allostehic modulation, as activators of other proteins through their protein-protein interactions, or as scaffolds for protein macroassemblies (Goldberg, T; Sreelatha, A., Biochem. J., 2023, 480, 715-728).

[0006] Integrin-linked kinase (ILK), a protein discovered towards the end of the last century, is a pseudokinase that retains its ability to bind ATP to its active site. For this reason, it has been extensively studied as a potential therapeutic target for the treatment of neoplasms, primarily due to its ability to phosphorylate GSK3-P and Akt at sennas 9 and 473, respectively (Hu, HF et al., Am. J. Cancer. Res., 2019, 9, 186-197). ILK forms a bidirectional signal transduction bridge mediated by the cytoskeleton, between the extracellular matrix and intracellular components. In this way, ILK regulates various aspects such as adhesion, migration, proliferation, differentiation, and changes in gene expression. The role of ILK as a scaffolding protein has therefore gained interest in the last decade (Górska A, Cell Mol Life Sci. 2022;79(2):100).Transgenic deletion of ILK dysregulates both transcriptional and post-transcriptional levels of several metabolite transporters in insulin-sensitive tissues (muscle, heart, white fat, and liver; Hatem-Vaquero M., J. Endocrinol. 2017;234(2):115-128; Hatem-Vaquero M., Cell Physiol Biochem. 2020; 54(1 ):71-87.), as well as the tubular water channel AQP2 in the kidney (Hatem-Vaquero M., Biochim. Biophys. Acta Gene Regul. Meeh. 2017;1860(9):922-935; Mamuya FA, Am. J. Physiol. Renal. Physiol. 2016;311(6):F1346-F1357; Cano-Peñalver JL, FASEB J.

[0007] 2014;28(8):3645-59). Therefore, it has been demonstrated that the absence or inhibition of ILK, both in cell culture models and in transgenic rodents, is compatible with an increase in obesity, type 2 diabetes and diseases related to insulin resistance, as well as with nephrogenic diabetes insipidus.

[0008] The development of small molecules capable of binding to ILK that promote its activity by modulating the cell cytoskeleton is therefore an interesting and novel strategy in the treatment of these or other disorders (García-Marín, J. et al., ACS Med. Chem. Lett., 2021 , 12, 1656-1662). There are small molecules that inhibit ILK activity, such as the pyrazole Cpd22 (Lee, SL et al., J. Med. Chem., 2011, 54, 6364-6374.), the diaminopyrazoles QLT0267 and QLT0254 (Edwards, LA et al., Mol. Cancer. Ther., 2008, 7, 59-70), the dihydroquinoline alkaloid chelidonine (Kim, O. et al., Mol. Med. Rep., 2015, 12, 2161-2168) or miscellaneous (Liu, J. et al., J. Oncol., 2022, 3658334). Cpd22 and QLT0267 have been extensively studied, but have not demonstrated their physical interaction with the protein and their direct modulation, so they can only be considered as inhibitors of the ILK signaling pathway without having clarified their mechanism of action.

[0009] International application W02003045379 describes hydrazonodiaminopyrazole derivatives as ILK inhibitor antiproliferative agents. International application WO2018046666 describes the use of fibronectin and ILK inhibitors for the treatment of leukemia.

[0010] International application WO2012071310 relates to pyrazole-derived compounds, integrin-linked kinase inhibitors, and their use for cancer treatment.

[0011] International application WO2019147552 describes quinoxaline and azaquinoxaline derivatives, ILK ligands, and their use for the treatment of ILK-related diseases, such as cancer.

[0012] Finally, international application WO 2022023612 describes alpha-parvine-based peptide compounds that bind to ILK and modify actin polymerization.

[0013] DESCRIPTION OF THE INVENTION

[0014] The invention relates, in a first aspect, to at least one bisulfonamide of Formula I:

[0015]

[0016] where R is a cyclic radical preferably selected from a group consisting of:

[0017]

[0018] where n = 2, 3 or 4; where each of the rings is optionally substituted by at least one substituent selected from a group consisting of: H, an alkyl group (preferably of 1 to 4 carbon atoms), an alkoxy group (preferably of 1 to 4 carbon atoms), F, Cl, CF3, OH, NO2, NH2, CN and COOH, as well as any combinations thereof;

[0019] and where Ar is a carbocycle or heterocycle of one or two fused aromatic rings selected from a group consisting of:

[0020]

[0021] where Ri and R2 are selected independently from a group preferably consisting of: F, Cl, Br, H, methyl, ethyl, butyl, propyl, OH, NH2, NO2, NHCH3, NHCH2CH3, NHCH2CH2CH3, CH2CH3OH, CH2CH2CH3OH and CH3OH, as well as any combinations thereof.

[0022] For the purposes of this patent, "alkyl" means any saturated hydrocarbon chain, linear or branched, having from 1 to 6 carbon atoms, such as, for example, the methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, or cyclohexyl groups. In particular embodiments of the invention, the alkyl groups may be optionally substituted by one or more substituents, preferably selected from a group consisting of alkynyl, alkenyl, halogen, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, and mercapto, as well as any combinations thereof. The bisulfonamide of Formula I is characterized as a ligand of integrin-linked kinase (ILK). Specifically, it is a compound capable of acting as an ILK activator at the level of the actin cell cytoskeleton.Therefore, another object of the present invention is a use of the bisulfonamide described herein, in any of its forms, as a ligand of the ILK-linked kinase.

[0023] Furthermore, the claimed Formula I bisulfonamide has been shown to be effective in treating any disease in which the pathophysiological alterations are wholly or partially due to (i.e., associated to a greater or lesser extent with) ILK dysregulation, resulting in decreased transmembrane transport, such as renal tubulopathies caused by a loss of transporters. Preferably, the claimed Formula I bisulfonamide has been shown to be particularly effective in an in vitro model of type 2 diabetes mellitus or other diseases associated with insulin resistance.

[0024] Thus, the invention also relates to a bisulfonamide of Formula I or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, for use in medicine.

[0025] For the purposes of this patent, "pharmaceutically acceptable salt" means any salt that can be used in a pharmaceutical form and that is capable of providing, directly or indirectly, in vivo a compound of formula (I). The nature of the salt is not essential, as long as it is pharmaceutically acceptable.

[0026] Additionally, the invention relates to a Formula I bisulfonamide or a pharmaceutically acceptable salt, isomer, solvate, or prodrug thereof, for use in the treatment and / or prevention of pathologies where the membrane transporters of metabolites and water have been altered, such as type 2 diabetes mellitus and other diseases associated with insulin resistance, nephrogenic diabetes insipidus, and other pathologies associated with ILK dysregulation.

[0027] The invention also relates to a pharmaceutical composition or medicament comprising bisulfonamide of Formula I as previously described, or a pharmaceutically acceptable salt, isomer, solvate, or prodrug thereof, as the active ingredient. Finally, the invention relates to a pharmaceutical composition comprising bisulfonamide of Formula I as previously described, or a pharmaceutically acceptable salt, isomer, solvate, or prodrug thereof, for use in medicine, preferably in the treatment and / or prevention of pathologies where membrane transporters of metabolites and water have been altered, such as type 2 diabetes mellitus and other diseases associated with insulin resistance, nephrogenic diabetes insipidus, and other pathologies associated with ILK dysregulation.The claimed medical use of the product also covers the treatment and / or prevention of pathologies where membrane transporters of metabolites and water are altered, such as type 2 diabetes mellitus and other diseases associated with insulin resistance, nephrogenic diabetes insipidus, and other pathologies associated with ILK dysregulation. This includes administering bisulfonamide of Formula I, as described above, or a pharmaceutical composition comprising it, to a patient. The invention also relates to the use of bisulfonamide of Formula I, as described herein, for the manufacture of a pharmaceutical composition for the treatment and / or prevention of the pathologies mentioned in this paragraph.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] To complement the description being made, and in order to help a better understanding of the characteristics of the invention, a series of figures are included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0030] Figure 1.

[0031] Figure 1A shows the polymerization of F-Actin, detected by phalidinine fluorescent probe in confocal microscopy on C2C12 differentiated myotubes treated with vehicle (a) or 12.5, 25, 50, 75, 100 pM of compound 1 (b, c, d, e, f, respectively) for 1 h. Bar 100 pm.

[0032] Figure 1B shows the histogram of phalloidin quantification with respect to the total number of cells after treatment with vehicle (a) or 50 pM compound 1 (d) for 1 h, as shown in Figure 1A. Values ​​shown are the means + SEM. * = p>0.05 vs (a). Figure 1C shows intracellular glucose uptake, using the fluorescent probe 2-NBDG, on differentiated C2C12 myotubes treated with vehicle (a), 50 pM compound 1 (d), or 100 mM insulin (INS) as a positive control for glucose uptake for 1 h. The histogram represents intracellular 2-NBDG quantification with respect to total protein content. Values ​​shown are the means + SEM. * = p>0.05 vs (a).

[0033] Figure 2.

[0034] Figure 2A shows the polymerization of F-Actin, detected by phalloidin fluorescent probe in confocal microscopy on C2C12 myoblasts treated with vehicle (a), 50 pM compound 1 (b) or compound 2 (c) for 1 h. Bar 50 pM.

[0035] Figure 2B represents the histogram of phalloidin quantification with respect to total number of cells after treatment with vehicle (a), 50 pM compound 1 (b) or compound 2 (c) for 1 h shown in Figure 2A. The values ​​represented are the means + SEM. * = p>0.05 vs (a).

[0036] Figure 3.

[0037] Figure 3 represents sensograms of compounds 1 and 2 with ILK at different concentrations.

[0038] Figure 3A shows the binding signal of compound 1 to recombinant ILK, detected by SPR at different concentrations: 5, 10, 25, 50, 75, 100, 200, 300, 400 pM (a, b, c, d, e, f, g, h, i respectively) in phosphate buffer.

[0039] Figure 3B shows the binding signal of compound 2 to recombinant ILK, detected by SPR at different concentrations: 2, 5, 10, 20, 30, 40, 60, 80 pM (a, b, c, d, e, f, g, h respectively) in phosphate buffer.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The invention is then described in detail by means of various tests that have been carried out by the inventors, which demonstrate the activity and effectiveness of the claimed compounds.

[0042] Example 1. Synthesis of the compounds of the invention. The compounds whose biological activity is the subject of the present invention were synthesized by organic synthesis procedures, as shown in Scheme 1. In a first step (“General Procedure 1”), sulfonyl chloride was reacted with various aminosulfonamides in a suitable organic solvent (such as, for example, dimethylformamide or dichloromethane), yielding reaction products I. Subsequently, these were reacted in a second step (“General Procedure 2”) with the corresponding nucleophile in aqueous or methanol solution, yielding final compounds II.

[0043]

[0044] SCHEME 1

[0045] General Procedure 1

[0046] In one particular embodiment, under an inert atmosphere, the corresponding 4-aminobenzenesulfonamide (1.0 eq) is added to a solution of 4-chloro, 3-nitrobenzenesulfonyl chloride (1.0 eq). The mixture is stirred at room temperature for 45 minutes. The reaction product is neutralized with a solution of HCl (20 mL). The aqueous phase is extracted with ethyl acetate (AcOEt) (5 x 10 mL), and the organic phase is subsequently washed with HCl (5 x 10 mL) and brine (3 x 10 mL). Finally, it is dried with anhydrous MgSC, filtered, and the solvent is removed using a rotary evaporator, yielding the corresponding reaction product I.

[0047] General Procedure 2

[0048] The corresponding reaction product I is reacted with 7N NH3 in methanol (20 mL / mmol) for 180 minutes at 1600 kPa in a microwave reactor. The solvent is removed from the resulting solution, and 20 mL of EtAc are added. The organic phase is washed with distilled water (2 x 10 mL), dried with anhydrous MgSO4, filtered, and concentrated to dryness using a rotary evaporator.

[0049] The reaction products I (compounds 1 to 9) obtained in the tests carried out are defined below: Compound 1: 4-amino-3-nitro- / V-(4-( / V-(thiazol-2- l)sulfamoyl)phenyl)benzenesulfonamide

[0050]

[0051] 1 H-NMR (400 MHz, DMSO) 58.39 (d, J= 2.3 Hz, 1H), 8.03 (bs, 2H), 7.67 (m, 2H), 7.65 (dd, J= 2.3 Hz, J= 9.0 Hz, 1H), 7.23 (d, J= 4.6 Hz, 1H), 7.21 (m, 2H), 7.07 (d, J= 9.0 Hz, 1H), 6.80 (d, J= 4.6 Hz, 1 H)

[0052] 13C-RMN (101 MHz, DMSO-d6): 5 168.4, 148.3, 140.8, 136.9, 131.9, 128.7, 127.2, 126.00, 124.5, 124.2, 120.2, 118.4, 108.0

[0053] Compuesto 2: 4-amino- / V-(4-( / V-(4-fluorofenil)sulfamoil)fenil)-3-nitrobencenosulfonamida

[0054]

[0055] 1 H-RMN (500 MHz, Acetona) 58.86 (bs, 1 H), 8.53 (d, J= 2.3 Hz, 1 H), 7.70 (dd, J= 9.0, 2.3 Hz, 1 H), 7.66 (bs, 2H) 7.66 - 7.61 (m, 2H), 7.39 - 7.31 (m, 2H), 7.16 (d, J = 9.0 Hz, 1 H), 7.16 - 7.11 (m, 2H), 7.05 - 6.96 (m, 2H)

[0056] 13 C-RMN (126 MHz, Acetona) 5 160.1 (d, J = 242.7 Hz), 148.6, 142.1, 134.6, 133.9 (d, J = 2.9 Hz), 132.5, 130.0, 128.7, 126.7, 125.7, 124.0 (d, J = 8.5 Hz), 120.0, 119.1, 115.6 (d, J = 22.9 Hz)

[0057] Compuesto 3: 4-amino- / V-(4-( / V-(5-metilisoxazol-3-il)sulfamoil)fenil)-3-nitrobencenosulfonamida

[0058]

[0059] 1H-RMN (400 MHz, DMSO) 511,05 (bs, 2H), 8,40 (d, J= 2,3 Hz, 1H), 8,04 (bs, 2H), 7,76-7,70 (m, 2H), 7,67 (dd, J = 9,0, 2,3 Hz, 1H), 7,27 - 7,22 (m, 2H), 7,07 (d, J = 9,1 Hz, 1H), 6,05 (s, 1H), 2,27 (s, 3H)

[0060] 13 C-RMN (101 MHz, DMSO) 5 170,6, 158,3, 149,0, 142,7, 134,4, 132,5, 129,4, 128,9, 126,7, 125,1, 120,8, 118,9, 95,9, 12,5

[0061] Compuesto 4: 4-amino- / V-(4-( / V-(4,6-dimetilpirimidin-2-il)sulfamoil)fenil)-3-nitrobencenosulfonamida

[0062]

[0063] 1 H-RMN (500 MHz, DMSO) 511,10 (bs, 2H), 8,33 (d, J= 2,2 Hz, 1H), 8,02 (bs, 2H), 7,80 -7,75 (m, 2H), 7,63 (dd, J = 9,0, 2,3 Hz, 1H), 7,23 - 7,17 (m, 2H), 7,04 (d, J = 9,1 Hz, 1H), 6,68 (s, 1H), 2,17 (s, 6H)

[0064] 13 C-RMN (126 MHz, DMSO) 5 156,5, 148,9, 142,1, 135,6, 132,6, 130,1, 129,2, 126,6, 125,0, 120,7, 118,5, 113,8, 110,0, 23,1

[0065] Compuesto 5: 4-amino- / V-(4-( / V-ciclopropilsulfamoil)fenil)-3-nitrobencenosulfonamida

[0066]

[0067] 1 H-RMN (400 MHz, Acetona) 58.52 (d, J= 2.2 Hz, 1 H), 7.79 - 7.76 (m, 2H), 7.74 (dd, J = 9.1, 2.3 Hz, 1H), 7.64 (bs, 2H), 7.46-7.40 (m, 2H), 7.16 (d, J= 9.0 Hz, 1H), 6.67 (bs, 1H), 2.19 - 2.08 (m, 1H), 0.52 - 0.45 (m, 2H), 0.44 - 0.39 (m, 2H)

[0068] 13 C-RMN (126 MHz, Acetona) 5 148.6, 141.7, 135.9, 132.6, 130.0, 128.8, 126.7, 125.8, 120.1, 119.5, 24.1, 5.0

[0069] Compuesto 6: 4-amino- / V-(4-( / V, / V-dietilsulfamoil)fenil)-3-nitrobencenosulfonamida

[0070]

[0071] 1 H-RMN (400 MHz, Acetona) 59.49 (bs, 1 H), 8.51 (d, J= 2.2 Hz, 1 H), 7.77 - 7.69 (m, 3H), 7.63 (bs, 2H), 7.47 - 7.38 (m, 2H), 7.16 (d, J = 9.0 Hz, 1H), 3.17 (q, J = 7.1 Hz, 4H), 1.03 (t, J = 7.1 Hz, 6H)

[0072] 13C-RMN (101 MHz, Acetona) 5 149.6, 142.5, 136.9, 133.5, 130.9, 129.4, 127.6, 126.7, 121.0, 120.6, 42.8, 14.4

[0073] Compuesto 7: 4-chloro-3-nitro- / V-(4-(N-(tiazol-2-¡l)sulfamoil)fen¡l)bencenosulfonamida

[0074]

[0075] 1 - 7.74 (m, 2H), 7.42 - 7.33 (m, 2H), 7.22 (d, J= 4.7 Hz, 1 H), 6.77 (d, J= 4.7 Hz, 1 H)

[0076] 13 C-RMN (101 MHz, Acetona) 5 170.1, 149.0, 141.0, 140.7, 140.2, 134.2, 132.5, 131.9, 128.7, 125.4, 124.6, 120.7, 108.7

[0077] Compuesto 8: 5-(dimetilamino)-M(4-(M(tiazol-2-il)sulfamoil)fenil)naftaleno-1-sulfonamida

[0078]

[0079] 1H-RMN (400 MHz, DMSO) 512,65 (bs, 1H), 11,29 (bs, 1H), 8,60 (d, J= 8,6 Hz, 1H), 8,47 (d, J = 8,7 Hz, 1 H), 8,32 (d, J = 7,4, 1 H), 7,74 - 7,64 (m, 2H), 7,64 - 7,56 (m, 2H), 7,49 (d, = 7,3 Hz, 1H), 7,21 (d, J= 4,6 Hz, 1 H), 7,20 - 7,16 (m, 2H), 6,78 (d, J= 4,6 Hz, 1 H), 2,93 (s, 6H)

[0080] 13 C-RMN (101 MHz, DMSO) 5 168,6, 147,5, 140,9, 136,6, 134,6, 130,3, 129,8, 128,7, 128,3, 127,9, 127,3, 124,5, 124,4, 121,0, 117,6, 117,2, 108,1, 45,6

[0081] Compuesto 9: M(4-(K-(tiazol-2-il)sulfamoil)fenil)benzo[c][1 ,2,5]tiadiazol-5-sulfonamida

[0082]

[0083] 1 H-RMN (400 MHz, DMSO) 512,60 (bs, 1H), 11,19 (bs, 1H), 8,60 (d, J= 1,8, 1H), 8,30 (d, J = 9,2, 1 H), 7,97 (dd, J = 9,2, 1 ,8 Hz, 1 H), 7,72 - 7,62 (m, 2H), 7,33 - 7,25 (m, 2H), 7,21 (d, J= 4,6 Hz, 1 H), 6,78 (d, J= 4,6 Hz, 1 H)

[0084] 13C-NMR (101 MHz, DMSO) 5 168.7, 155.1, 152.7, 140.5, 140.0, 137.4, 127.4, 125.4,124.4, 123.4, 121.6, 118.8, 108.2

[0085] Example 2. Effect of compounds on insulin-sensitive cell culture

[0086] Example 2.1 Induction of actin polymerization in myotubes

[0087] Cytoskeletal activity can be determined by observing actin polymerization (F-actin). The effect of compounds on actin polymerization (F-actin levels) was analyzed using confocal microscopy techniques, as well as actin polymerization in mouse myoblasts (C2C12 cell line, CRL-1772 from ATCC distributed by LGC Standards, Barcelona, ​​Spain) or in myotubes differentiated from myoblasts under culture conditions, following standardized protocols in the literature (Alcalde-Estévez E., Sci Rep. 2021, 12;11(1):512).

[0088] To this end, cells were cultured on sterile circular glass coverslips (Microscope Cover Glass 12 mm; Thermo Scientific; Chicago, IL, USA) at a density of 100,000 cells / well in 10% FBS medium. The following day, the cells were dehydrated from serum and treated with the compounds at the indicated concentrations for 1 hour. After the treatment time, the cells were washed twice with 1X PBS and fixed with 4% paraformaldehyde in 1X PBS for 20 minutes at room temperature. Subsequently, the cells were washed twice with PBS and the coverslips were treated for 10 minutes with PBS / 0.1% Triton X-100 at room temperature. The coverslips were washed twice with PBS and blocked for 30 minutes with a blocking solution (5% BSA) in PBS / 0.1% Triton X-100 at room temperature.The coverslips were incubated with the fluorescent F-actin marker phalloidin-FITC (Merck, Darmstadt, Germany), diluted in PBS with 0.5% BSA, for 1 hour at room temperature. Subsequently, the samples were washed and mounted with ProLong Gold antifade Reagent mounting fluid (Invitrogen, Paisley, UK), which incorporates cell nucleus staining with 4',6-diamidino-2-phenylindole (DAPI). The samples were observed using a LEICA TCS-SP5 confocal microscope (Leica Microsystems; Wetzlar, Germany) with a 560 nm excitation laser to detect the fluorescent product and a 405 nm diode to detect DAPI. Images were captured, and fluorescence intensity was measured by densitometry using ImageJ software. (The values ​​obtained per image were normalized with respect to the number of cells present (DAPI-labeled nuclei count).Figures 1A and 1B show an increase in F-Actin polymerization in differentiated myotubes after 1 h of treatment using various concentrations (12.5, 25, 50, 75, 100 pM) of compound 1. The quantification of the mean data + / - the standard mean error when using 50 pM of compound 1 was statistically significant (p>0.05 by ANOVA analysis), compared to a control treatment (with vehicle).

[0089] Example 2.2 Increased glucose uptake in myotubes

[0090] Figure 1C shows how Compound 1 increases the intracellular glucose capacity of myotubes treated for 1 h with 50 pM of Compound 1. Differentiated C2C12 myotubes were treated with Compound 1 or vehicle under the same conditions as described in Example 1. As a positive control for glucose uptake under the stated conditions, other cells were treated with the canonical activator of intracellular trafficking of the GLUT4 sugar transporter, insulin (100 nM, Actrapid, Novo Nordisk A / S, Bagsvaerd, Denmark). Fifteen minutes later, 0.1 mM of the fluorescent probe analog D-glucose, 2-[N(7-nitrobenz-2-oxa-1,3-diazol-4-1)amino]-2-deoxy-glucose (2-NBDG, Sigma-Aldrich, St. Louis, MO, USA).After three washes with PBS, intracellular 2-NBDG fluorescence levels (excitation 485 nm, emission 535 nm) were determined using a wide-spectrum plate reader (VICTORX4, PerkinElmer, Waltham, MA, USA). These levels correspond to glucose uptake, and the values ​​were normalized to total protein content. Glucose uptake capacity, dependent on the insulin-sensitive membrane transporter GLUT4, is susceptible to modification by the presence or absence of ILK (Hatem-Vaquero M., Cell Physiol. Biochem. 2020; 54(1):71-87). The quantification of mean data (± standard error) using 50 pM of compound 1 or 100 mM insulin was statistically significant (p>0.05 by ANOVA) compared to a control treatment (vehicle). This demonstrates that glucose uptake capacity increased with compound 1 to the same level of efficiency as using insulin.

[0091]

[0092] 2 in the polymerization of actin in myoblasts

[0093] The effect of compounds 1 and 2 at a concentration of 50 pM for 1 h on actin polymerization (F-actin levels) was analyzed using confocal microscopy techniques in mouse myoblasts (C2C12 cell line) following the same protocol detailed in Example 2.1. Figure 2 shows an increase in F-actin polymerization in myoblasts after 1 h. The quantification of the mean data + / - the standard error when using 50 pM of compound 1 and 2 was statistically significant (p>0.05 by ANOVA analysis) with respect to the control treatment (vehicle), demonstrating that both compounds 1 and 2 are capable of increasing actin polymerization capacity in myoblasts to the same level.

[0094] Example 3. Surface plasmon resonance

[0095] For the SPR experiments, a Biacore X-100 instrument (Biacore, GE Healthcare Life Sciences) was used at room temperature with Tris buffer as the eluent (50 mM Tris, pH 7.5, 50 mM NaCl, 2 mM CaCh with 20 mM EDTA and 2% DMSO). Recombinant ILK (59.92 kDa) was obtained from Merck. The protein was immobilized on a CM5 sensor chip (Biacore, GE) using the standard amine docking method. The results indicate that compounds of formula (I) bind to ILK.

[0096] Affinity measurements were performed using a series of different concentrations injected at levels between 0.1 and 500 pM into the sensor chip at a flow rate of 90 pL / min for 1 minute, followed by a 1-minute dissociation time. Regeneration was not required. The sensogram data were double-referenced and solvent-corrected using BIAevaluation X-100 software (Biacore, GE Healthcare Life Sciences). The results are shown in Figure 3. Compound 2 exhibited the highest affinity for ILK, with an affinity constant (KD) of 9.1 pM, compared to Compound 1 (146 pM). The sensograms of Compounds 1 and 2 (Figure 3) indicate that both are ILK ligands, as they have the capacity to bind directly to the recombinant protein.

Claims

CLAIMS 1. Bisulfonamide of Formula I: Formula 1 where R is selected from a group consisting of: where n= 2, 3 or 4; and where Ar is a carbocycle or heterocycle of one or two fused aromatic rings selected from a group consisting of: where Ri and R2 are selected, independently, from a group consisting of F, Cl, Br, H, methyl, ethyl, butyl, propyl, OH, NH2, NO2, NHCH3, NHCH2CH3, NHCH2CH2CH3, CH2CH3OH, CH2CH2CH3OH and CH3OH, as well as any combinations thereof.

2. Bisulfonamide according to claim 1, wherein when R is a cyclic radical, said cyclic radical comprises a ring substituted by at least one substituent selected from a group consisting of H, an alkyl or alkoxy group consisting of between 1 and 4 carbon atoms, F, Cl, CF3, OH, NO2, NH2, CN and COOH, as well as any combinations thereof.

3. Bisulfonamide according to claim 1 or 2, wherein said bisulfonamide is characterized as an integrin-linked kinase ligand.

4. Pharmaceutical composition comprising a bisulfonamide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

5. Bisulfonamide according to any one of claims 1 to 3, or pharmaceutical composition according to claim 4, for use in medicine.

6. Bisulfonamide for use according to claim 5, in the treatment, prevention, or treatment and prevention of at least one pathology associated with ILK dysregulation.

7. Bisulfonamide according to claim 6, wherein said pathology consists of type 2 diabetes mellitus or nephrogenic diabetes insipidus.

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