Biphenyl amides as dual-targeting SOCE / dhodh inhibitors

Abstract

Compound of formula (I) able to simultaneously modulate Store Operated Calcium Entry (SOCE) and human dihydroorotate dehydrogenase (hDHODH). The disclosure also relates to compositions and uses of compounds of formula (I) for treatment of autoimmune, inflammatory, and chronic degenerative diseases, as well as neurodegenerative and multisystem inflammatory diseases associated with SOCE overactivation and DHODH pathway alterations, while avoiding immunosuppression, the main side effect of the currently available therapies.

Description

[0001] “Biphenyl amides as dual-targeting SOCE / DHODH inhibitors”

[0002] Field of the invention

[0003] The disclosure concerns new biphenyl amide derivatives able to simultaneously modulate SOCE and DHODH, compositions, and uses thereof.

[0004] Background art

[0005] Calcium (Ca2+) represents a ubiquitous messenger serving as universal signal molecule to codify information between and inside the cells, regulating a wide range of cellular functions ranging from short-term responses, such as contraction and secretion, to longer-term control of transcription, cell division and cell death (Smyth J. T. et al. J. Cell. Mol. Med. 2010;14(10):2337-2349. Lewis R. S. Cold Spring Harb Perspect Biol. 201 l;3:a003970). The huge gradient across the cell plasma membrane together with the very low concentration of Ca2+in the cytosol (Ca2+is 105times more abundant in organelles and in the extracellular medium) became thereafter a great opportunity to use this ion as a specific second messenger. A Ca2+-signal codifies a message through the exact spatial localization in the cell, the amplitude, the duration and the frequency of its rise. High concentrations of calcium ions are present in intracellular organelles (in particular in the endoplasmic reticulum (ER) and / or sarcoplasmic reticulum (SR)) and the opening of Ca2+-channels (e.g. RyR, IP3R) located on these membranes allows this ion to flux out of the deposit and elicit cellular signals. Given that Ca2+- pumps and exchangers are located on both membranes to extrude Ca2+from the cytosol, it would be expected that the intracellular organelle pool would be soon depleted. Among these pathways, Store-Operated-Ca2+Entry (SOCE), so named for its regulation by the free Ca2+concentration ([Ca2+]) of the ER Ca2+stores, is a widespread Ca2+entry mechanism in animal cells that delivers Ca2+to refill ER stores and evoke cellular Ca2+signals (Putney J. W. Front. Biosci. (Schol. Ed.) 2011;3(3):980-984).

[0006] SOCE is associated with the electrophysiological current ICRAC, firstly described by Hoth and Penner (Hoth M., Penner R. Nature 1992;355(6358):353-356). The exact molecular mechanism behind this phenomenon has been elucidated between 2005 and 2006, when the principal components of the SOCE machinery, the Ca2+-Release Activated-Ca2+(CRAC) channels, have been discovered. CRAC channels are assembled from two fundamental protein complexes: ORAI proteins that form the ion channel pore on the plasma membrane, and the Stromal Interaction Molecule (STIM) proteins, which act as calcium sensors on the ER. (Kodakandla G., etal. Front Physiol. 2023; 14: 1330259. Berna-Erro A. etal. Adv. Exp. Med. Biol.

[0007] 2012;740:349-382. Soboloff J. et al. Nat. Rev. Mol. Cell Biol. 2012;13:549-565. Lacruz R. S., Feske S. Ann. N. Y. Acad. Sci. 2015;1356:45-79). Besides STIM and ORAI, it should be underlined that other crucial proteins participate in the SOCE mechanism, including Transient Receptor Potential Channels (TRPCs) (Manning D. et al. Front. Physiol. 2023; 14: 1141006. Ong H. L. and Ambudkar I. S. Cell Calcium 2015; 58:376-386).

[0008] STIM proteins are single-span membrane proteins, highly conserved across species. Two members of the family have been described, STIM1 and STIM2, of which the former appears more expressed (Grabmayr H. et al. Int. J. Mol. Sci.

[0009] 2020;22(l):378. Roos J. et al. J. Cell Biol. 2005;169(3):435-45). STIM1 was identified as a Ca2+sensor for SOCE since it is specialized for responding to significant changes in ER Ca2+signals. STIM1 localization is crucial to the role of SOCE: when Ca2+stores are full, STIM1 is localized in tubular structures throughout the ER membrane, but when stores are depleted, it moves to punctate structures at sites where the ER is in contact with the plasma membrane. This relocalization of STIM1 within the ER towards the plasma membrane allows the direct or indirect interaction and activation of ORAI channels. ORAI channels reside on the plasma membrane and three members of the family (Orail, Orai2, and Orai3) have been described, with Orail being the most abundant and closely connected to the ICRAC (Ong H. L. et al. Biochim Biophys Acta Mol Cell Res.

[0010] 2019;1866(7):1037-1045. Smyth J. T. etal. J. Cell. Mol. Med. 2010;14(10):2337-2349. Lewis R. S. Cold Spring Harb Perspect Biol. 201 l;3:a003970. Feske S. etal. J. Exp. Med. 2005;202(5):651-62. Feske S. etal. Nature 2006; 441(7090): 179-185).

[0011] The key experiment to exemplify SOCE is depicted in Figure 1. Briefly, emptying of the ER / SR store leads to opening of a channel located on the plasma membrane through which Ca2+can flow back in the cell and these two phenomena can be dissected by adding Ca2+to the extracellular solution after the intracellular stores are depleted. This simple, yet powerful, in vitro experimental approach remains valid to unmask the phenomenon in screenings.

[0012] CRAC currents were initially identified in lymphocytes and mast cells, and simultaneously characterized in different cell lines such as DT40 B cells, hepatocytes, dendritic, megakaryotic and Madin-Darby canine kidney cells.

[0013] In lymphocytes and mast cells, the activation through T-cell receptor or Fc receptor initiates the release of Ca2+ion from intracellular stores caused by the second messenger inositol (l,4,5)-triphosphate (IP3), that leads to Ca2+ion influx through CRAC channels in the plasma membrane.

[0014] CRAC channels also mediate crucial functions from secretion to gene expression and cell growth and form a process essential for the activation of adaptive immune response. It has been demonstrated that Ca2+oscillations triggered through stimulation of the T-Cell antigen Receptor (TCR) involved only the influx pathway of the store operated CRAC channel. Therefore, Ca2+ion influx mediated by the store operated CRAC channel is fundamental in lymphocyte activation (Parekh A. B. and Putney Jr. J. W. Physiol. Rev. 2005;85:757-810. Hogan G. P. et al. Annu. Rev. Immunol. 2010;28:491-533; Hogan P. G. and Rao A. Biochem. Biophys. Res. Commun. 2015;24,460(l):40-49. Feske S. et al. Biochem. Biophys. Res. Commun. 2003;311(4): 1117-32). Accordingly, it has been extensively reported the SOCE involvement in Ca2+- calcineurin - Nuclear Factor of Activated T cell (NF AT) pathway, of which alterations are responsible for triggering pro-inflammatory cytokines release and autoimmune reaction initiation in different autoimmune disorders. In fact, Ca2+- calcineurin - NF AT pathway plays a key role in: (i) the transcriptional regulation in T lymphocytes, (ii) the production of pro-inflammatory cytokines, (ii) the differentiation of CD4+T cells into helper and regulatory T cells (Park Y. J. et al. Front. Immunol. 2020;10:111-195). In this regard, different evidence shows that T and B cells in Multiple Sclerosis (MS), a chronic autoimmune and neurodegenerative disease, present SOCE over-activation with consequent overload of cytosolic Ca2+and Ca2+- calcineurin - NF AT pathway alteration (Kaufmann U. et al. J. Immunol. 2016;196(2):573-85). Thus, different approaches aiming at silencing or inhibiting SOCE were able to drastically ameliorate disease progression both in ex-vivo and in vivo model of MS (Ma J. et al. Eur. J. Immunol. 2010;40(l l):3028-3042. Kaufmann U. et al. J. Immunol.

[0015] 2016;196(2):573-585). Interestingly, as reported in literature, by properly interfering with SOCE it is possible to attenuate pro-inflammatory cytokines production and the consequent autoimmune response without altering the ability of CD8+T cells to provide immunity against infections or regulatory T cells to maintain immune tolerance (Vaeth M. et al. Trends Immunol. 2020;41(10):878- 901).

[0016] These findings suggest that modulators of SOCE would be useful for the treatment of diseases, such as autoimmune disease, where abnormal SOCE has a predominant role.

[0017] Several SOCE modulators have been postulated as being of therapeutic interest in disorders characterized by SOCE overactivation. L-651582 (CAI) was the first inhibitor to enter clinical trials for cancer. Although its development was initially discontinued because of lack of promising efficacy in tumours, it is now gaining novel impulse in gliobastoma (Omuro, A. et al. J. Clin. Oncol.

[0018] 2018;36(17): 1702-1709). The most recent phase lb trial shows that the drug is tolerable with no dose-limiting toxicities. SOCE inhibitors are being tested also for other conditions, including acute pancreatitis, given the importance that Ca2+has in triggering exocytosis of zymogen granules (Gerasimenko, J. V. et al. Proc. Natl. Acad. Sci. USA 2013; 110(32): 13186— 13191). In this respect, CM4620 (also known as Auxora26) has completed phase 1 trials and is now in phase 2 (NCT03401190) for acute pancreatitis as well as in phase VII trials for asparaginase-associated acute pancreatitis, a rare condition triggered by asparaginase treatment (NCT04195347). The same molecule is also in phase 2 for severe COVID-19 pneumonia (NCT04345614) for its potential to reduce pulmonary inflammation (Miller J. et al. Crit. Care 2020;24(l):l-9). A small, phase 2 study has been published on this compound, showing that at a dose of 1.0 mg / kg to 1.4 mg / kg for 4 days it is well tolerated (Bruen, C. et al. Pancreas 2021;50(4):537-543). A third compound, RP3128, developed for autoimmune disorders, has published data on its first-inhuman trial again showing that the drug was well tolerated with no limiting toxicities (Barde P. et al. J. Clin. Pharm. Ther. 2021;46(3):677-687). Therefore, these emerging data suggest that safety associated with SOCE inhibition is manageable.

[0019] Cellular metabolism is a pivotal set of biochemical reactions taking place within a cell to maintain life, either consuming or producing energy. These metabolic pathways can be categorized into three main groups: those that create uncomplicated molecules or combine them into more intricate macromolecules (anabolism); those that break down molecules to liberate energy (catabolism); and those that assist in the removal of toxic waste generated by the other two categories (waste disposal, DeBerardinis R. J. and Thompson C. B., Cell 2012; 148(6): 1132- 1144). Among the macromolecules that are essential for cell growth, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) represent the core of cellular nucleus as the first one carries genetic information, while the second one carries genetic code from DNA to the ribosomes, launching proteins synthesis (Minchin S. and Lodge J. Essays Biochem. 2019;63(4):433-456). Both DNA and RNA synthesis require nucleosides that can be obtained through a salvage pathway starting from preformed pyrimidine bases (uracil, cytosine, and thymine) or nucleosides (uridine, thymidine, and cytidine) or by de novo synthesis (starting from glutamine). The latter is the main mechanism leveraged by rapidly proliferating cells, such as cancer cells, T-lymphocytes, and viruses. One of the key players that regulates this process is represented by dihydroorotate dehydrogenase (DHODH), a flavin mononucleotide-containing enzyme located within the cell (Evans D. R. and Guy H. I. J. Biol. Chem. 2004;279(32):33035-33038). Here, DHODH catalyses the transformation of dihydroorotate (DHO) into orotate (ORO), the fourth rate-limiting step of the pyrimidine de novo synthetic process. The initial half-reaction involves the reduction of DHO to ORO, with electron transfer occurring to the flavine mononucleotide component (FMN), leading to its oxidation into dihydroflavin mononucleotide (FMNH2). Once ORO dissociates from the enzyme, FMNH2 is regenerated through interaction with a ubiquinone molecule, which is sourced from the inner mitochondrial membrane (Baumgartner R. et al. J. Med. Chem. 2006;49(4): 1239-47).

[0020] There are more than 3000 genes encoding for different DHODHs, and according to their subcellular localization, oligomeric state, and cofactor they can be divided into two classes: Class 1 and Class 2, sharing about 20% of sequence identity. Class 1, which in turn can be divided into 1A, IB, and IS, includes soluble DHODHs located in the cytoplasm. The first type consists of homodimeric proteins primarily found in gram-positive bacteria, while the second comprises heterotetrameric proteins, composed of two different proteins, also prevalent in gram-positive bacteria. Finally, class IS is a newly discovered subtype that cannot utilize any of the natural electron acceptors. Instead, it employs serine as a catalytic base, which is unique among cytosolic DHODHs. On the other hand, Class 2 includes monomeric proteins that anchor themselves to the inner mitochondrial membrane in eukaryotes and certain prokaryotes (Reis R. et al. Arch. Biochem. Biophys. 2017;632:175-91). Human DHODH belongs to Class 2 and its structure relies on a large C-terminal domain, connected through a long loop to a smaller N-terminal domain. The larger domain is composed of a central barrel comprising eight parallel P strands encircled by eight a helices and contains the redox site, which is formed by the substrate binding pocket and the FMN cofactor binding pocket. The TV-terminal comprises two a helices (al and a2), linked by a short loop and contains the binding site for the ubiquinone cofactor (Zhou Y. et al. Cancer Metab. 2021;9(1): 22). As the process involving this enzyme is crucial for rapidly proliferating cells, it has been reported that DHODH inhibitors exhibit advantageous effects in anticancer therapy (Madak J. T. et al. Pharmacol. Ther.

[0021] 2019;195:111-131) and demonstrate immunosuppressive and anti -proliferative effects, with the most significant impact observed on T cells, providing a positive outcome in autoimmune disorders (Fairbanks L. D. et al. J. Biol. Chem.

[0022] 1995;270(50):29682-29689).

[0023] Considering their substantial therapeutic potential, several DHODH inhibitors have been developed over the years, such as brequinar, leflunomide, teriflunomide, 195 vidofludimus, farudodstat (also named as ALASN003) and BAY 2402234 (Vyas V. K. and Ghate M. Mini Rev. Med. Chem. 2011; 11(12)11039-1055).

[0024] Summary of the invention

[0025] The object of this disclosure is to provide new compounds able to modulate SOCE and DHODH.

[0026] According to the invention, the above object is achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure.

[0027] The present invention provides a class of compounds as novel SOCE / DHODH modulators and their use in therapy. More particularly, the invention provides a family of biphenyl amide-based compounds.

[0028] The present disclosure provides compounds of formula (I):

[0029]

[0030] wherein

[0031] Ai, A2, A3, A4 and A5 are identical or different from each other and independently selected from H, CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN, a 5-6 membered O-heterocyclic group, an F containing group;

[0032] Ai and A2, or A?, and A3, or A3 and A4, or A4 and As can form together a 5-6 membered O-heterocyclic group fused to the phenyl ring to which they are attached;

[0033] Bi, B?, B3 and B4 are identical or different from each other and independently selected from H or F, Cl, Br, I, an F containing group;

[0034] at least one of Bi, B2, B3 and B4 is F;

[0035] C is selected from H or unsubstituted or substituted Ci-s alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;

[0036] Di, D2, D3 and D4 are identical or different from each other and independently selected from H or CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN;

[0037] E is selected from H or unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;

[0038] Ri and R2 are identical or different from each other and independently selected from unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4, CF3; pharmaceutically acceptable hydrates and / or solvates and / or salts thereof. The present disclosure relates to biphenyl amide compounds of formula (I) with a dual activity on SOCE and DHODH.

[0039] The present disclosure also relates to the compounds of formula (I) for use in in vivo treatment of pathological conditions linked to SOCE overactivation and DHODH pathway alterations, autoimmune, inflammatory, and chronic degenerative diseases, as well as neurodegenerative and multisystem inflammatory diseases, in which the simultaneous modulation of SOCE and DHODH is beneficial.

[0040] The present disclosure also provides pharmaceutical compositions comprising at least one compound of formula (I) and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more additional therapeutic agents.

[0041] Brief description of the drawings

[0042] The invention will now be described in detail, purely by way of illustrative and non-limiting example, with reference to the attached figures, wherein:

[0043] - Figure 1: Representative trace of Store Operated Calcium Entry (SOCE).

[0044] Experiments were carried out prior to and during exposure of the cells to the Ca2+-free solution. In the absence of Ca2+, the intracellular Ca2+stores were depleted by 2,5-t-butylhydroquinone ( / BHQ, 50 mM; Sigma-Aldrich, Italy), a SERCA poison, and then Ca2+2 mM was re-added to the extracellular solution.

[0045] - Figure 2: Effect of the proposed dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid) on Ca2+response.

[0046] A. Jurkat cells were with 2 pM Fluo4 AM and placed in an extracellular solution containing 0 mM Ca2+. Stores were depleted with 50 pM / BHQ and Ca2+influx was stimulated by the addition of 2 mM Ca2+alone (Ctrl), or in combination with: 1 or 3 pM compound 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid, a proposed dual SOCE / DHODH modulator that negatively regulate SOCE. Ca2+responses are expressed as changes in fluorescence intensity before and after the addition of the selected modulator. The compound negatively modulates SOCE at both concentrations evaluated.

[0047] B. Jurkat cells were assayed for a Ca2+response to different concentrations of 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (0.3; 1; 3; 10; 30 pM) using Fluo-4 fluorimetric assay. Concentration-response curves represent the AUC % of the compound as compared to positive control. The compound inhibits SOCE with an IC50 of 2.91 ± 0.63 pM.

[0048] - Figure 3: Effect of the proposed dual SOCE / DHODH inhibitors on cell viability in presence or absence of uridine.

[0049] Viability assays were performed in Jurkat cells that were plated in 96-well plates at the density of 25,000 cells per well. Cells were treated for 72 h with the dual SOCE / DHODH inhibitors indicated in the Figure legend, in comparison wi. th the compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R in graph), previously described as SOCE negative modulator with off-target activity on DHODH (Serafini M. et al. J. Med. Chem. 2020;63(23): 14761-14779). The involvement of the de novo pyrimidine synthesis pathway was evaluated by supplementing the medium with an excess of uridine that should counter-balance the effect of DHODH inhibition by triggering the de novo pathway. Therefore, to evaluate the effects on the DHODH enzyme, Jurkat cells were treated with the compounds depicted in the Figure in the absence or presence of 100 pM uridine for 72 h. The graph shows average ± SEM of cell viability. A Student t-test was performed on compound vs co-treatment (compound + uridine).

[0050] Based on cut-off values cell viability percentage (> 45%) and percentage increase in viability (> 10.5%) following uridine co-treatments, the SOCE / DHODH inhibitors (i.e., Compounds A, H, I, J, L, N in graph) slightly reduce Jurkat cells viability (percentage of viability reduction from 20% to 50 %), while the SOCE inhibitor with off-target activity on DHODH, (Serafini M. et al. J. Med. Chem.

[0051] 2020;63(23): 14761-14779, Compound R in graph) significantly impairs cell survival (reduction higher than 55%). Furthermore, their effects are counterbalanced by uridine co-treatment (percentage increase in viability from 10.5 % to 36.3 %), demonstrating that all compounds act on DHODH and dual SOCE / DHODH inhibitors are endowed with a better safety profile.

[0052] - Figure 4: The proposed dual SOCE / DHODH modulator, 3-((2, 3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid, does not impair cell viability as compared to 3-((4-(2,3-dihydrobenzo[ / >][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid.

[0053] Viability assays were performed in Jurkat cells that were plated in 96-well plates at the density of 25,000 cells per well. Cells were treated for 24, 48 and 72 h with increasing concentrations (0.3, 1, 3, 10 and 30 pM) of selected dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid or compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid. Values are means from three independent experiments ± S. E. M. A Student t-test was performed comparing the two compounds.

[0054] The data obtained from these viability assays strongly support that the dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid is well tolerated and safer compared to 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid, a SOCE inhibitor with an off-target activity on DHODH.

[0055] - Figure 5: The proposed dual SOCE / DHODH modulator, 3-((2, 3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid, does not alter cell cycle as compared to 3-((4-(2,3-dihydrobenzo[ / >][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid.

[0056] Jurkat cells were treated for 72 h with the selected dual SOCE / DHODH modulator, 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid, and compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (10 and 30 pM). Cell cycle distribution was assessed in Jurkat by flow cytometry after propidium iodide staining. Graphs show the % of cells ± SEM in the different phases of cell cycle. A Student t-test was performed on compounds vs control. The data obtained from cell cycle distribution assays demonstrates that dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid does not impair cell cycle in Jurkat cells, while 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid induces significant alterations both in Phase G1 and S at all concentrations tested.

[0057] - Figure 6: SOCE alterations and effects of dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid in PBMCs derived from patients affected by MS.

[0058] SOCE induced by / BHQ was evaluated in PBMCs derived from healthy volunteer (HV) or patient affected by MS, in presence or absence of compound 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid at 3 and 10 pM. 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4- yl)carbamoyl)benzoic acid impairs SOCE in PBMCs derived from MS patient. In detail, the compound is able to revert the over-activation of SOCE, restoring Ca2+to physiological level.

[0059] - Figure 7: The proposed dual SOCE / DHODH modulator 3-((2, 3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid does not affect MS PBMCs proliferation as compared to teriflunomide.

[0060] PBMCs proliferation was evaluated by performing a proliferation assay with the fluorescent dye 5(6)-carboxyfluorescein diacetate N-succinimidil ester (CFSE) using flow cytometry.

[0061] (A) PBMCs were treated with increasing concentrations (0.1, 0.3, 1, 3, 10 and 30 pM) of selected dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid or teriflunomide, a DHODH inhibitor approved for the treatment of MS. Values are means from three patients ± S. E. M. A Student t-test was performed comparing the two compounds. The data obtained from this proliferation assay demonstrates that dual SOCE-DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid does not alter cell division, thus proving to be well tolerated and safer than the DHODH inhibitor teriflunomide.

[0062] (B) The proliferation rate of CD4+T cells was assessed by treating with 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20), or 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R), or teriflunomide at 10 pM for 72 h. Values are means from three patients ± S. E. M. A Student t-test was performed comparing the three compounds.

[0063] The data obtained from this proliferation assay demonstrates that dual SOCE-DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) does not significantly impair cell proliferation in each generation analysed compared to both 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) and teriflunomide.

[0064] - Figure 8: The proposed dual SOCE-DHODH modulator 3-((2, 3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid significantly reduces the gene expression of Thl and Thl7 related proinflammatory cytokines as compare to teriflunomide.

[0065] PBMCs were treated for 72 h with 10 pM (3-((2,3,5,6-tetrafluoro-3',4',5'- trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) or teriflunomide.

[0066] (A) mRNA levels of Thl effector cytokines (IL-2 and TFNy). Data reported represent the average ± SEM of two patients. Anova test followed by Tukey’s multiple comparison test.

[0067] (B) mRNA levels of Thl7 effector cytokines (IL-17A and GM-CSF). Data reported represent the average ± SEM of 9 replicates from 2 MS patients. Anova test followed by Tukey’s multiple comparison test.

[0068] Data obtained by qRT-PCR, demonstrated that 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) significantly downregulates both Thl and Th 17 pro-inflammatory cytokines, while teriflunomide only moderates GM-CSF mRNA expression. Therefore, it can be assumed that 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) efficacy in reducing inflammatory-mediated autoimmune reaction is higher than teriflunomide used at the same treatment condition (10 pM, 72 h of treatment).

[0069] Detailed description of the invention

[0070] In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0071] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0072] The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0073] The term “alkyl” as used herein refers to a monovalent straight or branched chain group derived from an unsaturated hydrocarbon of one to eight carbons. The alkyl groups of this invention can be optionally substituted.

[0074] The term “alkenyl” as used herein refers to a monovalent straight or branched chain group derived from a hydrocarbon of two to eight carbons having at least one carbon-carbon double bond. The alkenyl groups of this invention can be optionally substituted.

[0075] The term “alkynyl” as used herein refers to a monovalent straight or branched chain group derived from a hydrocarbon of two to eight carbons having at least one carbon-carbon triple bond. The alkynyl groups of this invention can be optionally substituted.

[0076] The term “aryl” as used herein refers to a mono- or bicyclic carbocyclic ring system having at least one aromatic ring that can be optionally substituted. The aryl group can be fused to a cyclohexane, cyclohexene, cyclopentane or cyclopentene ring in which case the aryl group can be attached through the ring to which it is attached or through the aromatic ring itself. The aryl groups of this invention can be optionally substituted.

[0077] The term “heteroaryl” as used herein refers to an aryl group as defined above containing one, two, three or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. Preferably, the heteroaryl group is represented by benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl, isothiazolidinyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolidinyl, oxazolyl, pyrazolidinyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinolinyl, quinoxaloyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, triazolyl, and the like. More preferably, the heteroaryl group is represented by nitrogen-containing heterocycles such as pyridyl, triazolyl and the like.

[0078] The term “cycloalkyl” as used herein refers to a monovalent saturated cyclic or bicyclic hydrocarbon of three to six carbons. The cycloalkyl groups of this invention can be optionally substituted.

[0079] The term “halogen” as used herein refers to F, Cl, Br, or I.

[0080] The term “heterocyclic” as used herein refers to a 4-, 5-, 6- or 7-membered ring containing one, two or three heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The 4- and 5-membered rings have 0, 1, or 2 double bonds and the 6- and 7-membrered rings have 0, 1, 2, or 3 double bonds. The nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen atom can be optionally quaternized. The term “heterocyclic” also includes bicyclic, tricyclic, and tetracyclic groups in which a heterocyclic ring is fused to one or two rings selected from an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring or another monocyclic heterocyclic ring. Heterocycles of this type can be attached through the ring to which they are fused or through the heterocyclic ring itself. Heterocycles include, but are not limited to, acridinyl, benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, dioxanyl, dioxolanyl, furyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl, isothiazolidinyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinolinyl, quinoxaloyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, thiomorpholinyl, triazolyl, and the like. The heterocycle groups of this invention can be optionally substituted. Preferably, the heterocyclic group is selected from nitrogen-containing heterocycles such as pyridyl, triazolyl, and oxygen-containing heterocycles, such as dioxanyl and dioxolanyl.

[0081] The term “pharmaceutically acceptable salt” as used herein refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio.

[0082] The term “pharmaceutically acceptable hydrate and / or solvate” as used herein refers to a crystal form of a substance which contains one or more water and / or solvent molecules.

[0083] In an embodiment the present disclosure provides a compound of formula (I):

[0084]

[0085] wherein

[0086] Ai, A2, A3, A4 and A5 are identical or different from each other and independently selected from H, CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN, a 5-6 membered O-heterocyclic group, an F containing group;

[0087] Ai and A2, or A? and A3, or A and A, or A4 and As can form together a 5-6 membered O-heterocyclic group fused to the phenyl ring to which they are attached;

[0088] Bi, B2, B3 and B4 are identical or different from each other and independently selected from H or F, Cl, Br, I, an F containing group;

[0089] at least one of Bi, B?., B3 and B4 is F;

[0090] C is selected from H or unsubstituted or substituted Ci-s alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;

[0091] Di, D2, D3 and D4 are identical or different from each other and independently selected from H or CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN;

[0092] E is selected from H or unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;

[0093] Ri and R2 are identical or different from each other and independently selected from unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4, CF3;

[0094] pharmaceutically acceptable hydrates and / or solvates and / or salts thereof. In one or more embodiments, when Ri and R2, if present, are independently selected from substituted Ci-8 alkyl group, substituted C2-8 alkenyl group, substituted C2-8 alkynyl group, substituted C3-6 cycloalkyl, substituted aryl, substituted heterocyclic group, the one or more substituents are independently selected from halogen, CH3, CH2F, CHF2, CF3, OR2, CN, COOR4, CONR4R5, NR4R5, NHCOR4, NHSO2R4, S(O)R4, S(O)2R4, and SO2NHR4,

[0095] wherein R4 and R5 are the same or different and independently selected from H, Ci-Cs alkyl group unsubstituted or substituted with one or more halogen atoms, and C3-C6 cycloalkyl group unsubstituted or substituted with one or more halogen atoms.

[0096] In one or more embodiments, R4 and R5 are independently selected from H, methyl, ethyl, tert-butyl, iso-propyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0097] In one or more embodiments, Ri and R2 are selected from Ci-Cs alkyl group unsubstituted or substituted with one or more halogen atoms, C3-C6 cycloalkyl group unsubstituted or substituted with one or more halogen atoms, unsubstituted or substituted aryl, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

[0098] In one or more embodiments, Ri and R2 are selected from unsubstituted methyl, ethyl, tert-butyl, iso-propyl, phenyl and benzyl.

[0099] In one or more embodiments, Ai, A2, A3, A4 and A5 are identical or different from each other and independently selected from H, F, a F containing group, O-Ci-salkyl, O-heterocyclic group.

[0100] In one or more embodiments, Ai is selected from H, F, OCH3.

[0101] In one or more embodiments, A2 is selected from H, F, OCH3, OCH2CH2CH3, OCF3, CF3, a 5-6 membered O-heterocyclic group. In one or more embodiments, A3 is selected from H, F, OCH3, a 5-6 membered O-heterocyclic group.

[0102] In one or more embodiments, A4 is selected from H, F, OCH3, OCH2CH2CH3, OH, OCF3, CF3, a 5-6 membered O-heterocyclic group.

[0103] In one or more embodiments, A5 is selected from H, F, OCH3.

[0104] In one or more embodiments, when A2, A3, and A4 are independently selected from a 5-6 membered O-heterocyclic group, the 5-6 membered O-heterocyclic group is selected from oxolanyl, oxanyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, dihydrofuranyl, dihydropyranyl, morpholinyl, furanyl, pyranyl, oxazolyl, isoxazolyl, oxadiazinyl.

[0105] In one or more embodiments, when two adjacent groups at position Ai, A2, A3, A4 or A5 form together a 5-6 member O-heterocyclic group fused with the phenyl ring to which they are attached, the heterocyclic group fused with the phenyl ring is selected from dihydrobenzodioxinyl or benzodi oxolyl.

[0106] In one or more embodiments, Bi, B2, B3 and B4 are identical or different from each other and independently selected H and F.

[0107] In one or more embodiments, C is selected from H or unsubstituted or substituted Ci-s alkyl group, unsubstituted or substituted C3-6 cycloalkyl, (CH2)n-Ci-s alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

[0108] In one or more embodiments, C is H.

[0109] In one or more embodiments, Di, D2, D3 and D4 are identical or different from each other and independently selected from H, CF3, Br, I, Cl, F, OH, ORi, SRi, NO2, CN.

[0110] In one or more embodiments, Di, D2, D3 and D4 are identical or different from each other and independently selected from H and F.

[0111] In one or more embodiments, E is selected from H or unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

[0112] In one or more embodiments, E is selected from H and CH3.

[0113] In one or more embodiment, the F containing group is selected from CF3, CHF2, CH2F, 0CF3, 0CHF2, 0CH2F.

[0114] In one or more embodiment, when C is selected from substituted Ci-8 alkyl group, substituted C2-8 alkenyl group, substituted C2-8 alkynyl group, substituted C3-6 cycloalkyl, substituted aryl, substituted heterocyclic group, the one or more substituents are independently selected from halogen, CF3, OCH3, OCF3, OH, CH2OH.

[0115] In one or more embodiment, when E is selected from substituted Ci-8 alkyl group, substituted C2-8 alkenyl group, substituted C2-8 alkynyl group, substituted C3-6 cycloalkyl, substituted aryl, substituted heterocyclic group, the one or more substituents are independently selected from halogen, CF3, OCH3, OCF3, OH, CH2OH.

[0116] In one or more embodiment, the solvate of a compound of formula (I) is selected from hydrates, alcoholates and the like, preferably hydrates or alcoholates.

[0117] Hydrates and solvates of the compounds of formulae (I) are also provided by the invention and may be formed according to techniques known to one having ordinary skill in the pharmaceutical arts. As an example, solvates of any embodiments encompassing the compounds represented by formula (I) may be made according to known techniques. Suitable solvents for use in providing the solvates are known in the art and may vary according to the particular embodiment. Exemplary solvents include alcohols, such as, without limitation, methanol, ethanol, and the like.

[0118] In one or more embodiment, the salt of a compound of formula (I) is selected from “pharmaceutically acceptable salts” including sodium, potassium, ammonium, succinate, acetate, maleate, tartrate, fumarate, and succinate.

[0119] To establish the dual target activity versus SOCE and DHODH of the claimed compounds, four specific parameters have been considered. These parameters are closely associated to simultaneous inhibition of SOCE and DHODH, and have been defined as specified below: (i) cell viability percentage; (ii) percentage increase in viability; (iii) SOCE inhibition; (iv) DHODH residual activity. Preferably the claimed compounds must satisfy at least two of the above parameters (i)-(iv) for exerting the dual target activity. For each parameter (i)-(iv) the present inventors also identified their cut-off values: (i) cell viability percentage > 45 % following 72h treatments with biphenyl amide compounds at 50 pM; (ii) percentage increase in viability > 10.5 % following 72h co-treatments with biphenyl amide compounds at 50 pM and uridine at 100 pM; (iii) percentage of SOCE inhibition > 50 % following treatments with biphenyl amide compounds at 10 pM; (iv) percentage of DHODH residual activity < 50 % following treatments with biphenyl amide compounds at 10 pM.

[0120] Compared to the SOCE inhibitor with off-target activity on DHODH previously disclosed 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (identified in the following as "Compound R"), all the dual SOCE-DHODH inhibitors reported in Table 2 fulfill at least two of the four established cut-off values: (i) cell viability, (ii) reversal capability in presence of uridine, (iii) SOCE inhibition and (iv) residual DHODH activity.

[0121] In detail, according to Table 2, compounds D, E, K, M, O and P meet reversal capability in presence of uridine (> 10.5% at 50 pM with uridine 100 pM) and residual DHODH activity (< 50% at 10 pM). Compounds I and J satisfy cell viability (> 45% at 50 pM) and reversal capability in presence of uridine (> 10.5% at 50 pM with uridine 100 pM). Compound Q fulfills cell viability (> 45% at 50 pM) and SOCE inhibition (> 50% at 10 pM). Compound F meets cell viability (> 45% at 50 pM) and residual DHODH activity (< 50% at 10 pM). Compounds B, C and G satisfy cell viability (> 45% at 50 pM), residual DHODH activity (< 50% at 10 pM) and SOCE inhibition (> 50% at 10 pM). Compounds A, L and N fulfill cell viability (> 45% at 50 pM), reversal capability in presence of uridine (> 10.5% at 50 pM with uridine 100 pM) and residual DHODH activity (< 50% at 10 pM). Compound H (SP20) satisfies all the four established cut-off values.

[0122] Lastly, it should be noted that Compound H (SP20) does not alter cell division in PBMCs derived from patients affected by MS, thus proving to be well tolerated and safer than the DHODH inhibitor teriflunomide, a drug currently approved for the treatment of this disorder (Figure 7A, B).

[0123] In one or more embodiments, the compounds of formula (I) herein described are for use in the treatment or prevention of a disease condition depending on SOCE overactivation and DHODH pathway alteration(s).

[0124] In one or more embodiments, the compounds of formula (I) herein described are for use in the treatment of autoimmune, inflammatory, and chronic degenerative diseases, as well as neurodegenerative and multisystem inflammatory diseases associated with SOCE overactivation and DHODH pathway alterations, while avoiding immunosuppression, the main side effect of the currently available therapies.

[0125] In one or more embodiments, the disease condition depending on SOCE overactivation and DHODH pathway alteration(s) is selected from:

[0126] - Autoimmune and inflammatory disorders including, but not limited to, Multiple Sclerosis (in all its forms of onset and progression, Multiple Sclerosis-related diseases such as Neuromyelitis Optica and Myelin oligodendrocyte glycoprotein antibody disease (MOGAD), Systemic Lupus Erythematosus, Rheumatoid Arthritis (Ma J. et al. Eur. J. Immunol. 2010;40(l l):3028-3042. Kaufmann U. et al. J. Immunol.

[0127] 2016;196(2):573-585. Park Y. J. et al. Front. Immunol. 2020;10:lll- 195. VaethM. et al. Trends Immunol. 2020;41(10):878-901. Rahman S, and Rahman T. Set Rep. 2017;(l): 12881. Muehler A. et al. Mult. Scler. Relat. Disord. 2020;43: 102129);

[0128] - Neurodegenerative diseases (Secondo A. et al. Front. Mol. Neurosci.

[0129] 2018;l 1:87; Gottle P. et al. J. Neuroinflammation. 2018; 15(1):76); - Inflammatory diseases, including Psoriasis in its different types such as Plaque Psoriasis and Psoriatic Arthritis (Stauderman K. A. Cell Calcium 2018;74:147-159. Abdel-Magid A. F. ACS Med. Chem. Lett.

[0130] 2020; 11(11):2072-2074);

[0131] - Inflammatory bowel diseases and Crohn’s diseases (Kappel S. and Peinelt C. EMBO Mol. Med. 2022;14(9):el6489. Glauben R. et al. Cell Mol. Gastroenterol. Hepatol. 2022;14(l):243-244. Vaeth M. et al. Trends Immunol. 2020;41(10):878-901. Zhou X. et al. J. Med. Chem.

[0132] 2023;66(21): 14755-14786).

[0133] - fibrotic diseases, such as fibrosis affecting liver, kidney, heart, and lung, including, but not limited to, idiopathic pulmonary fibrosis and cystic fibrosis; graft-versus-host disease (GVDH); lymphoproliferative disease (Balghi H. et al. FASEB J. 2011;25(12):4274-91. Mohis M. et al. Am. J. Physiol. Heart. Circ. Physiol. 2018;315(l): H83-H91. Vaeth M. et al. Trends Immunol. 2020;41(10):878-901. Lu K. et al. Bioorg. Chem. 2019;86:44-51. Sexauer A. N. Blood Adv. 2023;7(21):6685- 6701).

[0134] In one or more embodiment, the present invention concerns pharmaceutical compositions comprising one or more compounds of formula (I).

[0135] In line with the data reported in the brief description of the drawings section, the dual SOCE / DHODH inhibitors described in this application display a different safety and efficacy profile compared to both single-target DHODH inhibitors (e.g., teriflunomide) and the prior art SOCE negative modulator with off-target activity on DHODH (i.e., 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6- yl)phenyl)carbamoyl)benzoic acid, Compound R).

[0136] In detail, compound SP20 (z.e., 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)carbamoyl)benzoic acid, also named Compound H) shows an IC50of 2.9 pM on SOCE (Figure 2A, B) and a residual hDHODH activity at 10 pM of 45.3% (Table 1). Furthermore, as reported in Figure 4, compound H, compared to the SOCE negative modulator with off-target activity on DHODH (z.e., 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid, Compound R), exhibits a better safety profile, reducing Jurkat cells viability by approx. 30% (vs approx. 60% for 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) and demonstrates a higher percentage reversal of this effect (16.5% vs 10.3%) in presence of uridine (100 pM, Figure 3, Table 2). Accordingly, 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) does not alter Jurkat cell cycle at 10 and 30 pM, while 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) induces significant alterations both in Phase G1 and S at all concentrations tested (Figure 5).

[0137] Of note, 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) contrasts the over-activation of SOCE, restoring Ca2+to physiological level in PBMCs derived from MS patients (Figure 6). As a corroboration of its safety profile, compound H (SP20) does not affect both MS PBMCs proliferation (Figure 7A) and CD4+T cells proliferation rate (Figure 7B) as compared to compound R and teriflunomide (DHODH inhibitor approved for MS treatment).

[0138] From an efficacy perspective, the data obtained by qRT-PCR reported in Figure 8A, B, demonstrate that 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) significantly downregulates both Thl (IL-2 and IFNy) and Thl7 (IL-17A and GM-CSF) pro-inflammatory cytokines, while teriflunomide only moderates GM-CSF mRNA expression at the same treatment condition (10 pM, 72 h of treatment).

[0139] Compounds of formula (I) can be administered in various routes appropriate to the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intra-arterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal.

[0140] Compounds of formula (I) can be formulated as a pharmaceutical composition in the form of tablet, capsule, aqueous solution, granule, powder, suspension, cream, syrup, gel, emulsion, and the like.

[0141] The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Although daily dosage can vary from one individual to another, the compound / s will be administered to an adult human in a range of 0.0001-50 mg / kg of body weight as daily single dose or 0.01 to 1 mg / kg as daily repeated doses.

[0142] Tablets contain the compound / s of formula (I) in a mixture with non-toxic pharmaceutically excipients suitable for the manufacture of tablets. Exemplary excipients could be: inert diluents, such as sodium carbonate, lactose, dextrose, cellulose etc.; granulating and disintegrating agents as maize starch, glycolate, alginic acid; binding agents as gelatin or acacia; lubricating agents, for example silica magnesium or calcium stearate, stearic acid or talc. For preparing suppositories, a mixture of for example fatty acid glycerides or cocoa butter is first melted and the compound / s of formula (I) is / are dissolved homogenously by stirring. The homogenous mixture is then cooled into convenient sized molds. Liquid preparations, which include solutions, suspensions and emulsions, contain the formula (I) compound / s in a mixture of excipients suitable for the manufacture of aqueous suspension such as sodium carboxymethylcellulose, methylcellulose, resin, sodium alginate and natural or synthetic gums. Eventually the liquid preparation may contain suitable colorants, flavors, stabilizers, preservatives and thickening agents as desired.

[0143] Compounds of the present invention may also be co-administered with one or more additional therapeutic agents. In a preferred embodiment, said additional therapeutic agents, include, but are not limited to, non-steroidal anti-inflammatory drugs (such as indomethacin) and steroidal anti-inflammatory drugs.

[0144] Moreover, more than one compound according to formula (I) can be coadministered.

[0145] Compounds of formula (I) include, but are not limited to, the compounds shown in Table 1.

[0146] Table 1

[0147]

[0148]

[0149]

[0150]

[0151] General Synthesis of Compounds of Formula (I)

[0152] The following schemes show a method for preparing the compounds of formula (I) of the present description. For a more detailed description of the individual reaction steps, see the Examples herein below. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of the invention. Although specific starting materials and reagents are depicted in the Schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of the instant disclosure using conventional chemistry well known to those skilled in the art.

[0153] In detail, compounds of formula (II)

[0154]

[0155] wherein Ari is selected from 2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl, phenyl, 3- methoxyphenyl, 2-fluoro-5 -methoxyphenyl, 3 -propoxyphenyl, 3,4- dimethoxyphenyl, 2,3-dimethoxyphenyl, and 3,4,5-trimethoxyphenyl, can be prepared as outlined in Scheme 1 below:

[0156]

[0157] Compounds of Formula II are synthesized via Suzuki cross-coupling reaction using commercially available boronic acids and bromoanilines, affording biarylamines of formula 1 (intermediates la-m), which are activated with A1(CH3)3 and acylated using methyl 3-(chlorocarbonyl)benzoate, in turn prepared by reacting the corresponding carboxylic acid with thionyl chloride. The resulting biarylamides of formula 2 (intermediates 2a-m) are finally hydrolyzed under basic conditions, yielding the title compounds. Compounds of formula (III)

[0158]

[0159] wherein An is selected from 2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl, phenyl, 3 -methoxyphenyl, 3 -propoxyphenyl, and 3,4,5-trimethoxyphenyl, can be prepared as outlined in Scheme 2 below:

[0160] Scheme 2

[0161]

[0162] Compounds of Formula II are synthesized via Suzuki cross-coupling reaction using commercially available boronic acids and bromoanilines, affording biarylamines of formula 3 (intermediates 3a-e), which are coupled with monomethyl isophthalate to yield biarylamides of formula 4 (intermediates 4a-e). A final hydrolysis of the methyl ester function under basic condition gives the title compounds.

[0163] The chemical reactions described in the Examples below may be readily adapted to prepare a number of other SOCE / DHODH modulators of the present invention, and alternative methods for preparing the compounds of formula (I) belong to the common general knowledge of the skilled man.

[0164] For example, the synthesis of non-exemplified compounds according to the invention may be successfully performed by modifications apparent to those skilled in the art, by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art other than those described, and / or by making routine modifications of reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the invention.

[0165] General procedure A: Suzuki cross-coupling reaction for the synthesis of intermediates la, Ic-g, li, Ik, 11, 3a, 3c and 3d

[0166] Pd(PPh3)4 (0.2 equiv) and K2CO3 (3.0-4.0 equiv) were sequentially added to a degassed mixture of the appropriate boronic acid (1.5-2.0 equiv) and 4-bromoaniline (1 mmol) in a 1,4-dioxane / water mixture (9:1, 0.4 M) or in a DMF / water mixture (4:1, 0.1 M) under a nitrogen atmosphere. The resulting mixture was heated at 90 °C under an inert atmosphere overnight. Upon completion, the resulting mixture was cooled down to room temperature and was partitioned between EtOAc and water. The separated aqueous phase was extracted with EtOAc (x2). The combined organic phase was washed with brine, separated, dried over Na2SO4, concentrated under reduced pressure, and purified by normal phase silica gel flash chromatography.

[0167] General procedure B: Suzuki cross-coupling reaction for the synthesis of intermediates Ih, Im and 3e

[0168] Pd(PPh3)4 (0.2-0.3 equiv) and a 2M solution of Na2CC>3 (3.0-4.0 equiv) were sequentially added to a degassed mixture of the appropriate boronic acid (1.2-1.5 equiv) and 4-bromoaniline (1 mmol) in DMF (0.1 M) under a nitrogen atmosphere. The resulting mixture was heated at 70 °C under an inert atmosphere overnight. Upon completion, the resulting mixture was cooled down to room temperature and was partitioned between EtOAc and water. The separated aqueous phase was extracted with EtOAc (x2). The combined organic phase was washed with brine, separated, dried over Na2SO4, concentrated under reduced pressure, and purified by normal phase silica gel flash chromatography.

[0169] General procedure C: Suzuki cross-coupling reaction for the preparation of intermediates lb, Ij and 3b

[0170] Pd(OAc)2 (0.01-0.1 equiv) and K2CO3 (2.6-6.0 equiv) were sequentially added to a degassed mixture of the appropriate boronic acid (1.3-3.0 equiv) and the proper 4-bromoaniline (1 mmol) in a DMF / EtOH mixture (1:1, 0.5 M) under a nitrogen atmosphere. The resulting mixture was heated at 80 °C overnight. Upon completion, the resulting mixture was cooled down to room temperature and was partitioned between EtOAc and water. The separated aqueous phase was extracted with EtOAc (x2). The combined organic phase was washed with brine, separated, dried over Na2SO4, concentrated under reduced pressure, and purified by normal phase silica gel flash chromatography.

[0171] Example la (intermediate la): Synthesis of 4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)-2, 3,5, 6-tetrajluoroaniline

[0172] Intermediate la was synthesised following general procedure A using 1,4-benzodioxane-6-boronic acid (2.0 equiv), K2CO3 (4.0 equiv) and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography with Cy / CEbCh 9:1 as eluent, affording a white powder (570.7 mg, 1.91 mmol, 93%).

[0173] Characterization: 'H-NMR (400 MHz, CDCh): b 6.95-6.93 (m, 1H), 6.93 (s, 1H), (dq, Js= 8.4 and 1.5 Hz, 1H), 4.30-4.28 (m, 4H), 4.00 (br s, 2H). HRMS (ESI) m / z calculated for C14H10F4NO2 (M+H)+: 300.0642; found: 300.0646.

[0174] Example lb (intermediate lb): Synthesis of 2, 3,5, 6-tetrafluoro-[ 1,1 '-biphenyl] -4-amine

[0175] Intermediate lb was prepared following general procedure C using phenyl boronic acid (3.0 equiv), Pd(OAc)2 (0.06 equiv) and K2CO3 (6.0 equiv). The crude material was purified by column chromatography with PEZEtOAc 98:2 as eluent, affording a white powder (114.4 mg, 0.47 mmol, 58%).

[0176] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.52-7.40 (m, 5H), 5.49 (br s, 2H). HRMS (ESI) m / z calculated for Ci2H8F4N (M+H)+: 242.0442; found: 240.0444.

[0177] Example 1c (intermediate 1c): Synthesis of 2,3,5,6-tetrafluoro-3'-methoxy-[l,l'-biphenyl -4-amine

[0178] Intermediate 1c was prepared following general procedure A using 3-methoxyphenylboronic acid (2.0 equiv), K2CO3 (4.0 equiv) and a mixture of 1,4.-dioxane / water as solvent. The crude material was purified by column chromatography using PEZEtOAc 98:2 as eluent, affording a white powder (188.1 mg, 0.69 mmol, 85%).

[0179] Characterization: 'H-NMR (400 MHz, CDCh): b 7.36 (t, J= 7.9 Hz, 1H), 7.01 (dt, Js= 7.9 and 1.3 Hz, 1H), 6.97-6.92 (m, 2H), 4.02 (br s, 2H), 3.84 (s, 3H). HRMS (ESI) m / z calcd for C13H9F4NO (M+H)+: 272.0693; found: 272.0694.

[0180] Example Id (intermediate Id): Synthesis of 2,2',3,5,6-pentafluoro-5'-methoxy-[1,1 '-biphenyl -4-amine

[0181] Intermediate Id was prepared following general procedure A using 2-fluoro-5-methoxyphenylboronic acid (2.0 equiv), K2CO3 (4.0 equiv) and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PEZEtOAc 99:1 as eluent, affording a white powder (169.2 mg, 0.59 mmol, 71%).

[0182] Characterization: 'H-NMR (400 MHz, CDCh): b 7.21 (t, J= 9.1 Hz, 1H), 7.07-7.03 (m, 1H), 7.01 (dd, Js= 5.7 and 3.2 Hz, 1H), 5.60 (br s, 2H). HRMS (ESI) m / z calcd for C13H9F5NO (M+H)+: 290.0599; found: 290.0599.

[0183] Example le (intermediate le): Synthesis of 2,3,5,6-tetrafluoro-3'-propoxy-[l,l'-biphenyl -4-amine

[0184] Intermediate le was prepared following general procedure A using 3-propoxyphenylboronic acid (2.0 equiv), K2CO3 (4.0 equiv) and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PEZEtOAc 96:4 as eluent, affording a white powder (256.2 mg, 0.85 mmol, 60%).

[0185] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.38 (t, J = 8.20 Hz, 1H), 7.05-6.95 (m, 3H), 5.48 (br s, 2H), 3.99 (t, J= 6.5 Hz, 2H), 1.80 (dtd, Js= 13.9, 7.4 and 6.4 Hz, 2H), 1.03 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calcd for C15H14F4NO (M+H)+: 300.1006; found: 300.1006.

[0186] Example If (intermediate If): Synthesis of 2,3,5,6-tetrafluoro-3',4'-dimethoxy-[1,1 '-biphenyl ]-4-amine

[0187] Intermediate If was prepared following general procedure A using 3,4-dimethoxyphenylboronic acid (2.0 equiv), K2CO3 (4.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / CH2CI270:30 as eluent, affording a white powder (352.5 mg, 1.17 mmol, 82%).

[0188] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.05 (d, J= 8.3 Hz, 1H), 7.02 (t, J= 1.5 Hz, 1H), 7.00-6.96 (m, 1H), 5.42 (br s, 2H), 3.86 (s, 3H), 3.84 (s, 3H). HRMS (ESI) m / z calcd for C14H12F4NO2 (M+H)+: 302.0799; found: 302.0800. Example 1g (intermediate 1g): Synthesis of 2,3,5,6-tetrafluoro-2',3'-dimethoxy-[1,1 '-biphenyl -4-amine

[0189] Intermediate 1g was prepared following general procedure A using 2,3-dimethoxyphenylboronic acid (2.0 equiv), K2CO3 (3.0 equiv), and 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / EtOAc 95:5 as eluent, affording a yellow powder (222.9 mg, 0.74 mmol, 92%).

[0190] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 5 7.15-7.14 (m, 2H), 6.88 (t, J = 4.6 Hz, 1H), 5.42 (br s, 2H), 3.91 (s, 3H), 3.68 (s, 3H). HRMS (ESI) m / z (calcd for C14H12F4NO2 (M+H)+: 302.0799; found: 302.0796.

[0191] Example Ih (intermediate Ih): Synthesis of 2,3,5, 6-tetrafluoro-3', 4', 5'-trimethoxy-[1,1 '-biphenyl -4-amine

[0192] Intermediate Ih was prepared following general procedure B using 3,4,5-trimethoxyphenylboronic acid (1.5 equiv), Pd(PPh3)4 (0.2 equiv) and 2M Na2CO3(3.0 equiv). The crude material was purified by column chromatography using PE / EtOAc 94:6 as eluent, affording a light-yellow powder (344.60 mg, 1.04 mmol, 63%).

[0193] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 6.74 (t, J= 1.2 Hz, 2H), 5.42 (br s, 2H), 3.85 (s, 6H), 3.78 (s, 3H). HRMS (ESI) m / z (M+H)+calcd for C15H14F4NO3: 332.0904, found: 332.0901.

[0194] Example li (intermediate li): Synthesis of 4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)-2, 6-difluoroaniline

[0195] Intermediate li was prepared following general procedure A using 1,4-benzodioxane-6-boronic acid (1.5 equiv), K2CO3(3.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / EtOAc 96:4 as eluent, affording a yellow oil (413.8 mg, 1.57 mmol, 82%).

[0196] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.13 (dd, Js= 8.0 and 2.3 Hz, IH), 7.09-7.02 (m, 2H), 6.88-6.83 (m, IH), 4.72 (br s, IH), 4.28 (s, 4H). HRMS (ESI) m / z calcd for C14H12F2NO2 (M+H)+: 264.0831; found: 264.0828.

[0197] Example Ij (intermediate Ij): Synthesis of 3, 5 -difluor o-[ 1,1 '-biphenyl] -4 -amine Intermediate Ij was prepared following general procedure C using phenylboronic acid (3 equiv), Pd(OAc)2 (0.1 equiv) and K2CO3 (5.0 equiv). The crude material was purified by column chromatography using PE / EtOAc 99: 1 as eluent, affording a white powder (339.4 mg, 1.65 mmol, 76%).

[0198] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 37.64-7.58 (m, 2H), 7.44-7.39 (m, 2H), 7.33-7.27 (m, 1H), 7.27-7.15 (m, 2H), 4.79 (br s, 2H). HRMS (ESI) m / z calcd for C12H10F2N (M+H)+: 206.0776; found: 206.0776.

[0199] Example Ik (intermediate Ik): Synthesis of 3,5-difluoro-3'-methoxy-[l,l'-biphenyl -4-amine

[0200] Intermediate Ik was prepared following general procedure A using 3-methoxyphenylboronic acid (2 equiv), K2CO3 (4.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / EtOAc 99:1 as eluent, affording a white powder (257.9 mg, 1.09 mmol, 57%).

[0201] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 37.32 (t, J= 7.9 Hz, 1H), 7.24-7.20 (m, 2H), 7.17 (ddd,.4 = 7.6, 1.8 and 1.0 Hz, 1H), 7.16-7.14 (m, 1H), 6.88 (ddd, J= 8.2, 2.5 and 1.0 Hz, 1H), 4.80 (br s, 1H), 3.86 (s, 3H). HRMS (ESI) m / z calcd for C13H12F2NO (M+H)+: 236.0881; found: 236.0878.

[0202] Example 11 (intermediate 11): Synthesis of 3,5-difluoro-3'-propoxy-[l,l'-biphenyl -4-amine

[0203] Intermediate 11 was prepared following general procedure A using 3-methoxyphenylboronic acid (2 equiv), K2CO3 (4.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / EtOAc 98:2 as eluent, affording an orange oil (311.0 mg, 1.18 mmol, 82%).

[0204] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 37.31 (ddd, J= 8.1, 7.3 and 0.8 Hz, 1H), 7.22 (dd, J= 8.0 and 2.3 Hz, 2H), 7.17-7.12 (m, 2H), 6.87 (ddd, J= 8.2, 2.4 and 1.1 Hz, 1H), 4.79 (s, 2H), 4.02 (t, J = 6.5 Hz, 2H), 1.80 (dtd, J = 13.8, 7.4 and 6.5 Hz, 2H), 1.04 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calcd for C15H16F2NO (M+H)+: 264.1194; found: 264.1193.

[0205] Example 1 m (intermediate 1 m): Synthesis of 3, 5-difluoro-3 ',4', 5 ' -trimethoxy- [ 1, 1 '-biphenyl -4-amine

[0206] Intermediate Im was prepared following general procedure C using 3,4,5- trimethoxyphenylboronic acid (1.5 equiv), Pd(PPh3)4 (0.2 equiv), 2M solution of Na2CC>3 (3.0 equiv). The crude material was purified by column chromatography using PE / EtOAc 95:5 as eluent, affording a white powder (310.0 mg, 1.05 mmol, 73%).

[0207] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 7.30-7.16 (m, 2H), 6.89 (s, 2H), 4.75 (br s, 2H), 3.91 (s, 6H), 3.74 (s, 3H). HRMS (ESI) m / z calcd for C15H16F2NO3 (M+H)+: 296.1093; found: 296.1089.

[0208] Example 3a (intermediate 3a): Synthesis of 4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)-3, 5-dijluoroaniline

[0209] Intermediate 3a was prepared following general procedure A using 1,4-benzodioxane-6-boronic acid (1.5 equiv), K2CO3 (3.0 equiv), and a mixture of 1,4-dioxane as solvent. The crude material was purified by column chromatography using cyclohexane / EtOAc 97:3 as eluent, affording a light-yellow oil (413.8 mg, 1.57 mmol, 82%).

[0210] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 36.33 (d, J= 1.7 Hz, 1H), 6.35 (d, J= 1.7 Hz, 1H), 6.33 (d, J= 1.7 Hz, 1H), 5.28 (s, 2H). HRMS (ESI) m / z calcd for C14H12F2NO2 (M+H)+: 264.0831; found: 264.0828.

[0211] Example 3b (intermediate 3b): Synthesis of 2, 6-dijluoro-[ 1,1 '-biphenyl] -4-amine Intermediate 3b was prepared following general procedure C using phenylboronic acid (3 equiv), Pd(OAc)2(0.1 equiv) and K2CO3 (5.0 equiv). The crude material was purified by column chromatography using PE / EtOAc 99: 1 as eluent, affording an orange powder (150.3 mg, 0.73 mmol, 34%).

[0212] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 37.45-7.36 (m, 4H), 7.35-7.29 (m, 1H), 6.39-6.35 (m, 2H), 5.34 (s, 1H). HRMS (ESI) m / z calcd for C12H10F2N (M+H)+: 206.0776; found: 206.0775.

[0213]

[0214] Intermediate 3c was prepared following general procedure A using 3-methoxyphenylboronic acid (2 equiv), K2CO3 (4.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PE / EtOAc 95:5 as eluent, affording a light-pink oil (301.0 mg, 1.28 mmol, 66%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.32 (ddd, Js= 8.1, 7.5 and 0.7 Hz, 1H), 6.97-6.92 (m, 2H), 6.90 (ddd, Js= 8.2, 2.6 and 1.0 Hz, 1H), 6.44-6.27 (m, 2H), 5.34 (br s, 1H), 3.81 (s, 4H). HRMS (ESI) m / z calcd for C13H12F2NO (M+H)+: 236.0881; found: 236.0878.

[0215] Example 3d (intermediate 3d): Synthesis of 2,6-dijluor o-3 '-propoxy- [1,1'-biphenyl -4-amine

[0216] Intermediate 3d was prepared following general procedure A using 3-propoxyphenylboronic acid (2 equiv), Pd(PPh3)4 (0.2 equiv), K2CO3(4.0 equiv), and a mixture of 1,4-dioxane / water as solvent. The crude material was purified by column chromatography using PEZEtOAc 97:3 as eluent, affording a brown oil (165.0 mg, 0.63 mmol, 90%).

[0217] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 7.33-7.28 (m, 1H), 6.95-6.92 (m, 2H), 6.89 (ddd, Js= 8.2, 2.4 and 1.2 Hz, 1H), 6.39-6.33 (m, 2H), 5.33 (br s, 1H), (3.97 (t, J= 6.5 Hz, 2H), 1.83-1.74 (m, 2H), 1.03 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z C15H16F2NO calcd for (M+H)+: 264.1194; found: 264.1192.

[0218] Example 3e (intermediate 3e): Synthesis of 2,6-difluoro-3',4',5'-trimethoxy-[l,l'-biphenyl -4-amine

[0219] Intermediate 3e was prepared following general procedure B using 3,4,5-trimethoxyphenylboronic acid (1.2 equiv), Pd(PPh3)4 (0.3 equiv), 2M solution of Na2CO3(4.0 equiv). The crude material was purified by column chromatography using PE / CH2CI230:70 as eluent, affording a white powder (162.7 mg, 0.55 mmol, 38%).

[0220] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 6.66 (t, J= 1.2 Hz, 2H), 6.43-6.32 (m, 2H), 5.33 (br s, 2H), 3.85 (s, 6H), 3.77 (s, 3H). HRMS (ESI) m / z calcd for C15H16F2NO3(M+H)+: 296.1093; found: 296.1090.

[0221] General procedure D: Amide coupling for the synthesis of intermediates 2a-m A mixture of methyl isophtalic acid (3 equiv) and SOCh (6.9 equiv) was heated at 65°C for 1 h under nitrogen atmosphere and was then concentrated to dryness under reduced pressure. In parallel, in a sealed vial tube, a 2.0 M solution of trimethylaluminum in hexane (1.2 equiv) was added dropwise to a solution of aniline (1 equiv) in dry toluene (0.2 M) at 0 °C under a nitrogen atmosphere. The resulting mixture was warmed to room temperature and was stirred for 2-3 h at the same temperature. To this solution, a solution of the freshly prepared acyl chloride in dry toluene (0.6 M) was added dropwise at 0 °C and under an inert atmosphere. The resulting mixture was heated at 85 °C for 2-3 h and was then allowed to stir at room temperature overnight. The reaction was quenched by adding MeOH and the resulting mixture was concentrated under reduce pressure, obtaining a residue that was dissolved in CH2CI2 and washed with H2O and brine (x2). The separated organic layer was dried over Na2SO4, concentrated under reduced pressure and purified by normal phase silica gel flash chromatography.

[0222] General procedure E: Amide coupling to prepare intermediates 4a-e

[0223] To a solution of 3 -(methoxy carbonyl )benzoic acid (1.5 equiv) in dry CH2CI2 (0.2 M), EDC-HC1 (3 equiv), DMAP (0.3 equiv) and DIPEA (5 equiv) were added and the resulting mixture was stirred under a nitrogen atmosphere for 30 min before adding the proper biarylamine. The resulting mixture was stirred at room temperature overnight. Upon completion, the reaction mixture was diluted with CH2Q2 and washed with H2O and brine. The separated organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was then purified by normal phase silica gel flash column chromatography.

[0224] Example 2a: Synthesis of methyl 3-((4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)~ 2, 3,5, 6- tetrajluorophenylcarbamoyl)benzoate

[0225] Example 2a was prepared following general procedure D, starting from biarylamine la. The crude material was purified by column chromatography using PEZEtOAc 85:15 as eluent, affording a white powder (94.0 mg, 0.20 mmol, 61%).

[0226] Characterization: 'H-NMR (400 MHz, CDCh): 88.70-8.60 (m, 2H), 8.21-8.16 (m, 1H), 7.54 (t, J= 7.8 Hz, 1H), 6.97-6.90 (m, 3H), 4.32-4.26 (m, 4H), 3.90 (s, 3H). HRMS (ESI) m / z calcd for C23H16F4NO5 (M+H)+: 462.0959; found: 462.0956.

[0227] Example 2b (intermediate 2b): Synthesis of methyl 3f (2,3,5, 6-tetrafluoro-[l, 1'-biphenyl ]-4-yl) carbamoyl) benzoate

[0228] Intermediate 2b was prepared following the general procedure D, starting from biarylaniline lb. The crude material was purified by column chromatography using PEZEtOAc 9:1 as eluent, affording a white powder (191.4 mg, 0.47 mmol, 76%). Characterization: 'H-NMR (400 MHz, CDCh): d 9.99 (br s, 1H), 8.68 (t, J= 1.8 Hz, 1H), 8.33 (dt, Js= 7.8, 1.4 Hz, 1H), 8.27 (dt, Js= 7.8, 1.4 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.63-7.52 (m, 5H), 3.95 (s, 3H). HRMS (ESI) m / z calcd for C21H14F4NO3 (M+H)+404.0904; found: 404.0901.

[0229] Example 2c (intermediate 2c): Synthesis of methyl 3-((2,3,5,6-tetrafluoro-3'-methoxy-[ 1, 1 '-biphenyl] -4-yl)carbamoyl)benzoate

[0230] Intermediate 2c was prepared following the general procedure D, starting from biarylamine 1c. The crude material was purified by column chromatography using PEZEtOAc 20:80 as eluent, affording a white powder (67.00 mg, 0.14 mmol, 28%). Characterization: 'H-NMR (400 MHz, CDCh): b 9.99 (br s, 1H), 8.68 (t, J= 1.8 Hz, 1H), 8.33 (ddd, = 7.8, 1.8 and 1.3 Hz, 1H), 8.27 (dt, Js= 7.8 and 1.3 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.51-7.46 (m, 1H), 7.20-7.11 (m, 2H), 7.12-7.08 (m, 1H) 3.95 (s, 3H), 3.88 (s, 3H). HRMS (ESI) m / z calcd for C22H16F4NO4 (M+H)+: 434.1010; found: 434.1009.

[0231] Example 2d (intermediate 2d): Synthesis of methyl 3-((2,2',3,5,6-pentafluoro-5'-methoxy-[ 1, 1 '-biphenyl] -4-yl)carbamoyl)benzoate

[0232] Intermediate 2d was prepared following the general procedure D, starting from biarylamine Id. The crude material was purified by column chromatography using PEZEtOAc 90:10 as eluent, affording a white powder (35.0 mg, 0.08 mmol, 15%). Characterization: 'H-NMR (400 MHz, CDCh): b 10.08 (br s, 1H), 8.69 (td, J= 1.9 and 0.6 Hz, 1H), 8.41-8.32 (m, 1H), 8.27 (dt, J= 7.8, 1.4 Hz, 1H)

[0233] 7.74 (td, J= 7.8 and 0.6 Hz, 1H), 7.31 (t, J= 9.1 Hz, 1H), 7.22-7.13 (m, 2H), 3.95 (s, 3H), 3.87 (s, 3H). HRMS (ESI) m / z calcd for C22H13F5NO4 (M+H)+: 450.0770; found: 450.0774.

[0234] Example 2e (intermediate 2e): Synthesis of methyl 3-((2,3,5,6-tetrafluoro-3'-propoxy-[ 1, 1 '-biphenyl] -4-yl)carbamoyl)benzoate

[0235] Intermediate 2e was prepared following the general procedure D, starting from biarylamine le. The crude material was purified by column chromatography using PEZEtOAc 93:7 as eluent, affording a white powder (110.3 mg, 0.25 mmol, 41%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.99 (br s, 1H), 8.68 (t, J = 1.8 Hz, 1H), 8.33 (ddd, Js= 7.8, 1.9 and 1.2 Hz, 1H), 8.27 (dt, Js= 7.8 and 1.4 Hz, 1H), 7.74 (td, Js= 7.8 and 0.6 Hz, 1H), 7.52-7.44 (m, 1H), 7.18-7.11 (m, 2H), 7.09 (ddd, Js= 8.4, 2.6 and 1.0 Hz, 1H), 4.04 (t, J= 6.47 Hz, 2H), 3.95 (s, 3H), 1.82 (dtd, Js= 13.9, 7.4 and 6.5 Hz, 2H), 1.04 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calcd for (M-H)- C24H18F4NO4: 460.1177; found: 460.1177.

[0236] Example 2f (intermediate 2j): Synthesis of methyl 3-((2,3,5,6-tetrafluoro-3',4'-dimethoxy-[ 1, 1 '-biphenyl / -4-yl) carbamoyl) benzoate

[0237] Intermediate 2f was prepared following the general procedure D, starting from biarylamine If. The crude material was purified by column chromatography using PE / EtOAc 96:4 as eluent, affording a yellow powder (240.0 mg, 0.52 mmol, 48%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 8.84 (t, J= 1.8 Hz, 1H), 8.52 (ddd, Js= 7.9, 2.0 and 1.2 Hz, 1H), 8.36 (dt, Js= 7.8 and 1.4 Hz, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.18 (t, J= 1.4 Hz, 1H), 7.14 (d, J= 1.0 Hz, 2H), 3.97 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H). HRMS (ESI) m / z calcd for C23H16F4NO5(M-H)-: 462.0970; found: 462.0976.

[0238] Example 2g: Synthesis of methyl 3-((2,3f,6-tetrafhioro-2',3'-dimethoxy-[l,l'-biphenyl ]-4- yl)carbamoyl)benzoate

[0239] Example 2g was prepared following the general procedure D, starting from biarylamine 1g. The crude material was purified by column chromatography using PE / EtOAc 92:8 as eluent, affording a white powder (90.8 mg, 0.20 mmol, 58%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.95 (s, 1H), 8.68 (t, J= 1.8 Hz, 1H), 8.33 (dt, Js= 7.7 and 1.5 Hz, 1H), 8.27 (dd, Js= 7.9 and 1.4 Hz, 1H), 7.75 (t, J= 7.8 Hz, 1H), 7.31-7.14 (m, 2H), 7.01 (dd, Js= 5.8 and 3.4 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.76 (s, 3H). HRMS (ESI) m / z calcd for C23H18F4NO5(M+H)+464.1116; found 464.1114.

[0240] Example 2h: Synthesis of methyl 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl ]-4-yl) carbamoyl) benzoate

[0241] Example 2h was prepared following the general procedure D, starting from biarylamine Ih. The crude material was purified by column chromatography using PE / EtOAc 95:5 as eluent, affording a white powder (85.6 mg, 0.17 mmol, 52%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.95 (s, IH), 8.67 (t, J= 1.9 Hz, IH), 8.33 (dt, Js= 7.9 and 1.5 Hz, IH), 8.27 (dt, Js= 7.8 and 1.5 Hz, IH), 7.73 (d, J= 7.8 Hz, IH), 6.91 (d, J= 1.3 Hz, 2H), 3.95 (s, 3H), 3.90 (s, 6H), 3.82 (s, 3H). HRMS (ESI) m / z calcd for C24H20F4NO6 (M+H)+494.1221; found 494.1220.

[0242] Example 2i (intermediate 2i): Synthesis of methyl 3-((4-(2,3- dihydrobenzo [b ][ 1, 4 ]dioxin-6-yl)-2, 6-difluorophenyl)carbamoyl)benzoate Example 2i was prepared following the general procedure D, starting from biarylamine li. The crude material was purified by column chromatography using PEZEtOAc 83: 17 as eluent, affording a yellow powder (208.4 mg, 0.49 mmol, 53%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.56 (br s, 1H), 8.68 (t, J = 1.8 Hz, 1H), 8.33-8.30 (m, 1H), 8.23 (dt, Js= 7.8 and 1.5 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.40-7.32 (m, 2H), 7.24-7.20 (m, 2H), 6.96-6.92 (m, 1H), 4.32 (s, 4H), 3.94 (s, 3H). HRMS (ESI) m / z calcd for C23H18F2NO5 (M+H)+426.1148; found 426.1147.

[0243] Example 2j (intermediate 2j): Synthesis of methyl 3-((3,5-difluoro-[l,l'-biphenyl]-4-yl)carbamoyl) benzoate

[0244] Example 2j was prepared following the general procedure D, starting from biarylamine Ij. The crude material was purified by column chromatography using PEZEtOAc 90:10 as eluent, affording a white powder (57.0 mg, 0.16 mmol, 16%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.62 (s, 1H), 8.68 (t, J= 1.8 Hz, 1H), 8.32 (dt, Js= 7.8 and 1.4 Hz, 1H), 8.25 (dt, Js= 7.8 and 1.4 Hz, 1H), 7.79-7.74 (m, 2H), 7.72 (t, J= 7.8 Hz, 1H), 7.56-7.48 (m, 2H), 7.48-7.41 (m, 3H), 3.95 (s, 3H). HRMS (ESI) m / z calcd for C21H16F2NO3(M+H)+: 368.1093; found 368.1090.

[0245] Example 2k (intermediate 2k): Synthesis of methyl 3-((3,5-difluoro-3'-methoxy-[1,1 '-biphenyl ]-4-yl) carbamoyl) benzoate

[0246] Example 2k was prepared following the general procedure D, starting from biarylamine Ik. The crude material was purified by column chromatography using PEZEtOAc 92:8 as eluent, affording a white powder (58.0 mg, 0.15 mmol, 21%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.64 (s, 1H), 8.68 (t, J= 1.8 Hz, 1H), 8.32 (ddd, J= 7.8, 1.9 and 1.2 Hz, 1H), 8.25 (dt, J= 7.8 and 1.3 Hz, 1H), 7.72 (td, J= 7.8 and 0.6 Hz, 1H), 7.49 / 7.43 (m, 2H), 7.43 / 7.39 (m, 1H), 7.33-7.31 (m, 1H), 7.31-7.30 (m, 1H), 3.94 (s, 3H), 3.90 (s, 3H). HRMS (ESI) m / z calcd for C22H18F2NO4(M+H)+: 398.1198; found 398.1196.

[0247] Example 21 (intermediate 21): Synthesis of methyl 3-((3,5-difhioro-3'-propoxy-[1,1 '-biphenyl ]-4-yl) carbamoyl) benzoate

[0248] Example 21 was prepared following the general procedure D, starting from biarylamine 11. The crude material was purified by column chromatography using PEZEtOAc 93:7 as eluent, affording a white powder (149.4 mg, 0.35 mmol, 32%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 9.65 (br s, 1H), 8.68 (t, J = 1.7 Hz, 1H), 8.32 (dt, J= 7.8 and 1.5 Hz, 1H), 8.24 (dt, J= 7.7 and 1.5 Hz, 1H), 7.71 (td, J= 7.7 and 3.2 Hz, 1H), 7.56-7.38 (m, 3H), 7.31-7.28 (m, 2H), 7.00 (ddd, J= 8.2, 2.4 and 1.1 Hz, 1H), 4.07 (t, J= 6.5 Hz, 2H), 3.94 (d, J= 2.0 Hz, 3H), 1.90- 1.76 (m, 2H), 1.05 (t, J= 7.5 Hz, 3H). HRMS (ESI) m / z calcd for C24H20F2NO4 (M-H)': 424.1366; found 424.1368.

[0249] Example 2m (intermediate 2m): Synthesis of methyl 3-((3,5-difluoro-3',4',5'~ trimethoxy-[ 1, 1 '-biphenyl ]-4-yl)carbamoyl)benzoate

[0250] Example 2m was prepared following the general procedure D, starting from biarylamine Im. The crude material was purified by column chromatography using CHzCb / EtOAc 99:1 as eluent, affording an orange powder (150.0 mg, 0.33 mmol, 32%).

[0251] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 9.57 (br s, 1H), 8.68 (t, J = 1.8 Hz, 1H), 8.32 (dt, Js= 7.8 and 1.6 Hz, 1H), 8.24 (dt, Js= 7.8 and 1.5 Hz, 1H), 7.72 (t, J= 7.8 Hz, 1H), 7.53-7.41 (m, 2H), 7.05 (s, 2H), 3.95 (d, J= 3.1 Hz, 9H), 3.79 (s, 3H). HRMS (ESI) m / z calcd for C24H20F2NO6 (M+H)+: 456.1264; found 456.1265.

[0252] Example 4a (intermediate 4a): methyl 3-((4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl) -3, 5-dijluorophenyl) carbamoyl) benzoate

[0253] Intermediate 4a was prepared following the general procedure E, starting from biarylamine 3a. The crude material was purified by column chromatography using PEZEtOAc 90: 10 as eluent, affording a white powder (102.8 mg, 0.24 mmol, 42%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.11 (s, 1H), 8.58 (t, J= 1.8 Hz, 1H), 8.26 (ddd, Js= 7.8, 1.9 and 1.2 Hz, 1H), 8.22 (dt, Js= 7.8 and 1.4 Hz, 1H), 7.77-7.57 (m, 3H), 6.97-6.92 (m, 3H), 4.32 (s, 4H). HRMS (ESI) m / z calcd for C23H18F2NO5(M+H)+: 426.1148; found 426.1147.

[0254] Example 4b (intermediate 4b): Synthesis of methyl 3-((2,6-difluoro-[l,l'-biphenyl ]-4-yl) carbamoyl) benzoate

[0255] Intermediate 4b was prepared following the general procedure E, starting from biarylamine 3b. The crude material was purified by column chromatography using PEZEtOAc 95:5 as eluent, affording a white powder (83.9 mg, 0.23 mmol, 39%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.11 (br s, 1H), 8.59 (t, J = 1.8 Hz, 1H), 8.31-8.24 (m, 1H), 8.26-8.19 (m, 1H), 7.77-7.65 (m, 3H), 7.55-7.48 (m, 4H), 7.46- 7.36 (m, 1H), 3.95 (s, 3H). HRMS (ESI) m / z calcd for C21H16F2NO3 (M+H)+: 368.1093; found 368.1092.

[0256] Example 4c (intermediate 4c): Synthesis of methyl 3-((2,6-difluoro-3'-methoxy-[1,1 '-biphenyl ]-4-yl) carbamoyl) benzoate

[0257] Intermediate 4c was prepared following the general procedure E, starting from biarylamine 3c. The crude material was purified by column chromatography using CH2Q2 / CH3OH 99:1 as eluent, affording a light pink powder (213.0 mg, 0.54 mmol, 72%).

[0258] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.10 (s, 1H), 8.58 (t, J= 1.9 Hz, 1H), 8.26 (dt, Js= 7.9 and 1.6 Hz, 1H), 8.22 (dt, Js= 7.7 and 1.4 Hz, 1H), 7.74-7.62 (m, 3H), 7.40 (t, J= 8.2 Hz, 1H), 7.09-6.90 (m, 3H), 3.94 (s, 3H), 3.81 (s, 3H). HRMS (ESI) m / z calcd for C22H18F2NO4(M+H)+: 398.1198; found 398.1196.

[0259] Example 4d (intermediate 4d): Synthesis of methyl 3-((2,6-difluoro-3'-propoxy-[1,1 '-biphenyl ]-4-yl) carbamoyl) benzoate

[0260] Intermediate 4d was prepared following the general procedure E, starting from biarylamine 3d. The crude material was purified by column chromatography using PEZEtOAc 95:5 as eluent, affording a white powder (78.4 mg, 0.18 mmol, 42%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.13 (s, 1H), 8.59 (t, J= 1.8 Hz, 1H), 8.27 (dt, Js= 6.7 and 1.1 Hz, 1H), 8.26-8.19 (m, 1H), 7.78-7.64 (m, 3H), 7.39 (t, J= 8.1 Hz, 1H), 7.09-7.01 (m, 2H), 7.03-6.96 (m, 1H), 4.01 (t, J= 6.5 Hz, 2H), 3.95 (s, 3H), 1.81 (dtd, Js= 13.9, 7.4 and 6.4 Hz, 2H), 1.04 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calcd for C24H22F2NO4 (M+H)+: 426.1511; found 426.1510.

[0261] Example 4e (intermediate 4e): Synthesis of methyl 3-((2,6-difluoro-3',4',5'-trimethoxy-[ 1, 1 '-biphenyl ]-4-yl)carbamoyl)benzoate

[0262] Intermediate 4e was prepared following the general procedure E, starting from biarylamine 3e. The crude material was purified by column chromatography using CH2Q2 / CH3OH 99:1 as eluent, affording a light-yellow powder (162.5 mg, 0.36 mmol, 74%).

[0263] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.13 (br s, 1H), 8.59 (t, J = 1.9 Hz, 1H), 8.27 (ddd, Js= 7.8, 1.9 and 1.2 Hz, 1H), 8.23 (dt, Js= 7.9 and 1.3 Hz, 1H), 7.74-7.63 (m, 3H), 6.77 (t, J= 1.2 Hz, 2H), 3.95 (s, 3H), 3.87 (s, 6H), 3.79 (s, 3H). HRMS (ESI) m / z calcd for C24H22F2NO6 (M+H)+: 458.1410; found 458.1407.

[0264] General procedure F: Methyl ester hydrolysis

[0265] LiOH (4 equiv) was added to the appropriate methyl ester (1 equiv) in a THF / H2O mixture (1:1, 0.1 M). The obtaining mixture was allowed to stir overnight at room temperature. Upon completion, the mixture was diluted with H2O and acidified with 3 N HC1 until pH reached approximately 3-4. The precipitated carboxylic acid was then filtrated under vacuum, dried, and treated with cold EtOAc. The obtained solid was filtered under vacuum and concentrated to dryness.

[0266] General procedure G: Methyl ester hydrolysis

[0267] LiOH (4 equiv) was added to the appropriate methyl ester (1 equiv) in a THF / H2O mixture (1:1, 0.1 M). The obtaining mixture was allowed to stir overnight at room temperature. Upon completion, the mixture was diluted with H2O and acidified with 3 N HC1 until pH reached approximately 3-4. The aqueous phase was extracted with EtOAc (x2) and the combined organic phase was dried over Na2SO3, concentrated under reduced pressure and purified by normal phase silica gel flash column chromatography.

[0268] Example A: Synthesis of 3-((4-(2,3-dihydrobenzo[b] [1, 4]dioxin-6-yl)-2, 3,5,6-tetrafluorophenyl) carbamoyl) benzoic acid

[0269] The title compound was synthesised following general procedure F starting from example 2a (80.0 mg, 0.17 mmol) to afford a white powder (52.2 mg, 0.12 mmol, 67%).

[0270] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.08 (br s, 1H), 8.70 (t, J = 1.7 Hz, 1H), 8.32 (ddd, Js= 7.8, 1.9 and 1.2 Hz, 1H), 8.28 (dt, Js= 7.8 and 1.4 Hz, 1H), 7.72 (t, J= 7.8 Hz, 1H), 7.10-7.02 (m, 2H), 7.00 (d, J= 8.3 Hz, 1H), 4.53-4.22 (m, 4H). HRMS (ESI) m / z calculated for C22H14F4NO5 (M+H)+: 448.0803; found: 448.0800.

[0271] Example B: Synthesis of 3-((2, 3,5, 6-tetrafluoro-[ 1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0272] The title compound was synthetised following general procedure G starting from intermediate 2b (100.0 mg, 0.25 mmol). The crude material was purified by column chromatography using PEZEtOAc 4:6 as eluent, affording a white powder (69.4 mg, 0.18 mmol, 72%).

[0273] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.00 (br s, 1H), 8.72 (s, 1H), 8.30 (t, J= 7.5 Hz, 2H), 7.72 (t, J= 7.8 Hz, 1H), 7.62-7.52 (m, 5H). HRMS (ESI) m / z calculated for C20H12F4NO3 (M+H)+: 390.0748; found: 390.0746.

[0274] Example C: Synthesis of 3-((2, 3,5, 6-tetrafluoro-3'-methoxy-[ 1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0275] The title compound was prepared following the general procedure G starting from intermediate 2c (60.0 mg, 0.14 mmol). The crude material was purified by column chromatography using PEZEtOAc 4:6 as eluent, affording a white powder (48.9 mg, 0.12 mmol, 84%).

[0276] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.00 (br s, 1H), 8.72 (s, 1H), 8.31 (t,.7= 7.6 Hz, 2H), 7.72 (t, J= 7.9 Hz, 1H), 7.48 (t, = 7.9 Hz, 1H), 7.18-7.13 (m, 2H), 7.10 (dd, Js= 8.3 and 2.5 Hz, 1H), 3.88 (s, 3H). HRMS (ESI) m / z calculated for C21H14F4NO4 (M+H)+: 420.0854; found: 420.0854.

[0277] Example D: Synthesis of 3-((2, 2’, 3,5, 6-pentafluoro-5' -methoxy- [1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0278] The title compound was prepared following the general procedure F starting from intermediate 2d (19.3 mg, 0.04 mmol) affording a white solid (19.4 mg, 0.04 mmol, quant, yield).

[0279] Characterization: 'H-NMR (400 MHz, (CD3)2CO): 3 10.05 (br s, 1H), 8.71 (t, J = 1.8 Hz, 1H), 8.33 (dt, J= 7.9 and 1.6 Hz, 1H), 8.30 (dt, J= 7.7 and 1.4 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.31 (t, J= 9.1 Hz, 1H), 7.25-7.10 (m, 2H), 3.87 (s, 3H). HRMS (ESI) m / z calculated for C21H13F5NO4(M+H)+: 438.0759; found: 438.0757.

[0280] Example E: Synthesis of 3-((2, 3,5, 6-tetrafluoro-3 '-propoxy- [1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0281] The title compound was prepared following the general procedure F starting from intermediate 2e (100.0 mg, 0.22 mmol) affording a white powder (53.0 mg, 0.12 mmol, 53%).

[0282] Characterization: 'H-NMR (400 MHz, (CD3)2CO): <5 11.59 (br s, 1H), 9.96 (s, 1H), 8.71 (t, J= 1.8 Hz, 1H), 8.34-8.29 (m, 2H), 7.74 (t, J= 7.8 Hz, 2H), 7.47 (t, J= 8.0 Hz, 1H), 7.50-6.96 (m, 4H), 4.04 (t, J= 6.5 Hz, 3H), 1.88-1.76 (m, 3H), 1.05 (t, J = 7.4 Hz, 4H). HRMS (ESI) m / z calculated for C23H16F4NO4(M-H)': 446.10209; found: 446.10217.

[0283] Example F: Synthesis of 3-((2,3,5,6-tetrafluoro-3',4'-dimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid

[0284] The title compound was prepared following the general procedure F starting from intermediate 2f (200.0 mg, 0.43 mmol) to yield a white powder (73.2 mg, 0.16 mmol, 38%).

[0285] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.97 (br s, 1H), 8.71 (t, J = 1.8 Hz, 1H), 8.34-8.29 (m, 2H), 7.74 (t, J= 7.8 Hz, 1H), 7.20 (s, 1H), 7.14 (s, 2H), 3.90 (s, 3H), 3.88 (s, 3H). HRMS (ESI) m / z calculated for C22H16F4NO5 (M+H)+: 450.0959; found: 450.0960.

[0286] Example G: Synthesis of 3-((2, 3, 5, 6-tetrafluoro-2 ', 3 '-dimethoxy- [ 1, 1 '-biphenyl ]-4-yl)carbamoyl)benzoic acid

[0287] The title compound was prepared following the general procedure G starting from example 2g (80.0 mg, 0.17 mmol). The crude material was purified by column chromatography using PEZEtOAc 6:4 as eluent, affording a white powder (31.4 mg, 0.07 mmol, 40%).

[0288] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.98 (br s, 1H), 8.72 (s, 1H), 8.32-8.28 (m, 2H), 7.74 (t, J = 7.8 Hz, 1H), 7.24-7.21 (m, 2H), 7.02-6.98 (m, 1H), 3.94 (s, 3H), 3.76 (s, 3H). HRMS (ESI) m / z calculated for C22H16F4NO5 (M+H)+: 450.0959; found: 450.0956.

[0289] Example H (SP20): Synthesis of 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl ]-4-yl) carbamoyl) benzoic acid

[0290] The title compound was prepared following the general procedure F starting from example 2h (85.0 mg, 0.17 mmol) to yield a beige powder (44.4 mg, 0.093 mmol, 54%).

[0291] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.96 (br s, 1H), 8.70 (t, J = 1.8 Hz, 1H), 8.34-8.28 (m, 2H), 7.74 (t, J= 7.8 Hz, 1H), 6.91 (s, 2H), 3.90 (s, 6H), 3.82 (s, 3H). HRMS (ESI) m / z calculated for C23H18F4NO6(M+H)+: 480.1065; found: 480.1067. Example I: Synthesis of 3-((4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)-3,5-dijluorophenyl) carbamoyl) benzoic acid

[0292] The title compound was prepared following the general procedure F starting from intermediate 4a (102.8 mg, 0.24 mmol) affording a white powder (58.0 mg, 0.14 mmol, 58%).

[0293] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.10 (br s, 1H), 8.61 (br s, 1H), 8.26 (d, J= 7.8 Hz, 2H), 7.78-7.63 (m, 3H), 7.02-6.89 (m, 3H), 4.34-4.30 (m, 4H). HRMS (ESI) m / z calculated for C22H14F2NO5 (M-H)': 410.0846; found: 410.0847.

[0294] Example J: Synthesis of 3-((4-(2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)-2,6-difluorophenyl) carbamoyl) benzoic acid

[0295] The title compound was prepared following the general procedure F starting from intermediate 2i (45.0 mg, 0.11 mmol) affording a white powder (25.0 mg, 0.06 mmol, 57 %).

[0296] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.61 (br s, 1H), 8.71 (t, J = 1.8 Hz, 1H), 8.35-8.31 (m, 1H), 8.27 (dt, J= 7.8, 1.4 Hz, 1H), 7.72 (t, J= 7.8 Hz, 1H), 7.42-7.34 (m, 2H), 7.25-7.22 (m, 2H), 6.98-6.92 (m, 1H), 4.33 (s, 4H). HRMS (ESI) m / z calculated for C22H16F2NO5 (M+H)+: 412.0991; found: 412.0989.

[0297] Example K: Synthesis of 3-((2,6-difluoro-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid

[0298] The title compound was prepared following the general procedure F starting from intermediate 4b (63.6 mg, 0.17 mmol) affording a white powder (36.0 mg, 0.10 mmol, 58%).

[0299] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.16 (br s, 1H), 8.62 (t, J = 1.8 Hz, 1H), 8.27 (ddt, Js= 9.2, 7.9 and 1.4 Hz, 2H), 7.76-7.65 (m, 3H), 7.50 (d, J = 4.7 Hz, 4H), 7.48-7.38 (m, 1H). HRMS (ESI) m / z calculated for C20H14F2NO3(M+H)+: 354.0936; found: 354.0934.

[0300] Example L: Synthesis of 3-((3,5-difluoro-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid

[0301] The title compound was prepared following the general procedure F starting from intermediate 2j (43.0 mg, 0.12 mmol) affording a white powder (21.0 mg, 0.059 mmol, 51%). Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.65 (br s, 1H), 8.72 (t, J = 1.8 Hz, 1H), 8.32 (dt, Js= 7.8 and 1.5 Hz, 1H), 8.28 (dt, Js= 7.7 and 1.4 Hz, 1H), 7.80-7.75 (m, 2H), 7.72 (t, J= 7.8 Hz, 1H), 7.56-7.49 (m, 2H), 7.49-7.42 (m, 3H). HRMS (ESI) m / z calculated for C20H14F2NO3 (M+H)+: 354.0936; found: 354.0934.

[0302] Example M: Synthesis of 3-((2, 6-difluoro-3'-methoxy-[ 1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0303] The title compound was prepared following the general procedure F starting from intermediate 4c (167 mg, 0.42 mmol) affording a white powder (150.0 mg, 0.39 mmol, 93%).

[0304] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.14 (br s, 1H), 8.61 (t, J = 1.8 Hz, 1H), 8.27 (tt, Js= 7.2 and 1.3 Hz, 2H), 7.75-7.65 (m, 3H), 7.44 -7.37 (m, 1H), 7.08-7.02 (m, 2H), 7.02-6.96 (m, 1H), 3.85 (s, 3H). HRMS (ESI) m / z calculated for C21H16F2NO4 (M+H)+: 384.1042; found: 384.1039.

[0305] Example N: Synthesis of 3-((3, 5-difluoro-3'-methoxy-[ 1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0306] The title compound was prepared following the general procedure F starting from intermediate 2k (24.0 mg, 0.06 mmol) affording a white powder (20 mg, 0.05 mmol, 87%).

[0307] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.66 (br s, 1H), 8.71 (t, J = 1.8 Hz, 1H), 8.32 (dd, J= 7.8 and 1.8 Hz, 1H), 8.27 (dt, J= 7.7 and 1.4 Hz, 1H), 7.72 (t, J= 7.8 Hz, 1H), 7.49-7.38 (m, 3H), 7.31 (dd, J= 7.4 and 1.5 Hz, 2H), 7.03-6.98 (m, 1H), 3.90 (s, 3H). HRMS (ESI) m / z calculated for C21H16F2NO4 (M+H)+: 384.1042; found: 384.1041.

[0308] Example O: Synthesis of 3 -((2, 6-difluoro-3' -propoxy- [1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0309] The title compound was prepared following the general procedure F starting from intermediate 4d (65.0 mg, 0.15 mmol) affording a white powder (27.0 mg, 0.07 mmol, 43%).

[0310] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.15 (br s, 1H), 8.61 (t, J = 1.8 Hz, 1H), 8.27 (tt, Js= 7.6 and 1.3 Hz, 2H), 7.77-7.64 (m, 3H), 7.39 (t, J= 8.1 Hz, 1H), 7.05-7.02 (m, 2H), 6.99 (ddd, Js= 8.2, 2.5 and 1.0 Hz, 1H), 4.01 (t, J = 6.5 Hz, 2H), 1.85-1.76 (m, 2H), 1.04 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calculated for C23H20F2NO4 (M+H)+: 412.1355; found: 412.1351.

[0311] Example P: Synthesis of 3-((3, 5-difluoro-3' -propoxy- [1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0312] The title compound was prepared following the general procedure F starting from intermediate 21 (65.0 mg, 0.15 mmol) affording a white powder (27.0 mg, 0.07 mmol, 43%).

[0313] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.15 (br s, 1H), 8.61 (t, J = 1.8 Hz, 1H), 8.27 (tt, Js= 7.6 and 1.3 Hz, 2H), 7.77-7.64 (m, 3H), 7.39 (t, J= 8.1 Hz, 1H), 7.05-7.02 (m, 2H), 6.99 (ddd, Js= 8.2, 2.5 and 1.0 Hz, 1H), 4.01 (t, J = 6.5 Hz, 2H), 1.85-1.76 (m, 2H), 1.04 (t, J= 7.4 Hz, 3H). HRMS (ESI) m / z calculated for C23H20F2NO4 (M+H)+: 412.1355; found: 412.1352.

[0314] Example Q: Synthesis of 3-((2,6-difluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid

[0315] The title compound was prepared following the general procedure F starting from intermediate 4e (132.5 mg, 0.31 mmol) affording a white powder (68.7 mg, 0.16 mmol, 50 %).

[0316] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 10.14 (s, 1H), 8.61 (t, J= 1.8 Hz, 1H), 8.27 (tt, Js= 7.5 and 1.4 Hz, 2H), 7.81-7.61 (m, 3H), 6.77 (t, J = 1.2 Hz, 2H), 3.87 (s, 6H), 3.79 (s, 3H). HRMS (ESI) m / z calculated C23H20F2NO6 (M+H)+: 444.1253; found: 444.1254.

[0317] Example S: Synthesis of 3-((3, 5-difluoro-3', 4', 5 '-trimethoxy-] 1,1 '-biphenyl] -4-yl)carbamoyl)benzoic acid

[0318] The title compound was prepared following the general procedure F starting from intermediate 2m (120.0 mg, 0.26 mmol) affording a white powder (66.2 mg, 0.15 mmol, 57%).

[0319] Characterization: 'H-NMR (400 MHz, (CD3)2CO): b 9.62 (s, 1H), 8.71 (t, J= 1.76 Hz 1H), 8.32 (dt, Js= 7.74 and 1.74 Hz, 1H), 8.28 (dt, Js= 7.74 and 1.40 Hz, 1H), 7.72 (t, J= 7.8 Hz, 1H), 7.53-7.42 (m, 2H), 7.05 (s, 2H), 3.95 (s, 6H), 3.78 (s, 3H). HRMS (ESI) m / z calculated for C23H20F2NO6 (M+H)+: 444.1253; found: 444.1253.

[0320] Biological Assays and Results

[0321] Cell cultures Jurkat cells (Jurkat, Clone E6-1, TIB-152, American Type Culture Collection - ATCC) were cultured in RPMI-1640 culture medium supplemented with bovine fetal serum (thermally decomplemented at 57°C for 30 minutes), L-glutamine 50 g / mL, penicillin 10 U / mL and streptomycin 100 g / mL. Cell density was adjusted every two to three days (0.5 x 106cells / mL). This cell line was used as an in vitro model of T cells, usually used to evaluate new molecular entities.

[0322] (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) MTT assay MTT assay was performed in the Jurkat cell line. Cells were treated at the initial density of 5 x 104and placed in 96-well plates (100 pL per well). At the end of the 72-hour treatment time (h), MTT (Sigma) was added to the culture medium at a concentration of 2.5 mg / mL. After 90 minutes of incubation (37°C, 5% CO2), the colorimetric reaction was stopped by adding a solution of 2-propanol added with triton 10% and hydrochloric acid 0.1%. The absorbance of each sample was evaluated at 570 nm wavelength by spectrophotometric analysis with 1420 Multilabel Counter Victor3 V.

[0323] Jurkat cells were treated (density 5 x 104) for 72 h with the compounds of interest at a concentration of 50 pM, in the presence or absence of uridine (100 pM), substrate of DHODH. The spectrophotometric analyses were carried out as described above.

[0324] The results obtained by this viability assay demonstrated that the dual SOCE / DHODH inhibitor SP20 (3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid, Compound H) displays a better safety profile as compared to the compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R, SOCE inhibitor with off-target activity on DHODH previously described, Serafini el al. J. Med. Chem.

[0325] 2020;63(23): 14761-14779). In particular, the data reported in Figure 4 demonstrates that compound H does not affect cell viability at all concentrations (0.3, 1, 3, 10 and 30 pM) and time points (24, 48 and 72 h) evaluated, while the prior art compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) significantly affects cell viability both at 10 and 30 pM at all time points of treatment (Figure 4).

[0326] Furthermore, the same viability assay was performed to discriminate the involvement of the de novo pyrimidine synthesis pathway by supplementing the medium with an excess of uridine (100 pM) that should counter-balance the effect of DHODH inhibition.

[0327] Based on cut-off values, cell viability percentage (> 45%) and percentage increase in viability (> 10.5%) following uridine co-treatments, the SOCE / DHODH inhibitors of the present invention (z.e., Compounds A, H, I, J, L, N in graph) slightly reduce Jurkat cells viability (percentage of viability reduction from 20% to 50 %), while prior art the SOCE inhibitor with off-target activity on DHODH, (Serafini M. et al. J. Med. Chem. 2020;63(23): 14761-14779, Compound R in graph) significantly impairs cell survival (reduction higher than 55%). Furthermore, their effects are counterbalanced by uridine co-treatment (percentage increase in viability from 10.5 % to 36 %), demonstrating that all compounds act on DHODH and dual SOCE / DHODH inhibitors are endowed with a better safety profile.

[0328] Analysis of Cell Cycle distribution by Flow Cytometry

[0329] At the end of the treatments, Jurkat cells were collected, washed once with Phosphate Buffer Saline (PBS) and resuspended in a 70% ethanol solution. Following to overnight incubation at -20°C, cells were washed again with PBS and incubated with RNAase (100 pg / mL, Sigma-Aldrich Inc., Milan, Italy). Cells were then incubated in PBS containing 5 mM EDTA and 50 pg / mL propidium iodide (Sigma-Aldrich Inc., Milan, Italy) for 30 minutes, prior to flow cytometric analysis with Accuri-C6 flow cytometer (BD Bioscience, Milan, Italy). FlowJo software was used for discrimination of different sub-populations along the cell-cycle.

[0330] Cell cycle distribution was analyzed in order to further corroborate the safety profile characterizing compound SP20 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid. In particular, the results reported in Figure 5 confirms that the treatment with 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (10 and 30 pM for 72 h, Compound H or SP20) does not impair cell cycle in Jurkat cells, while the prior art compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) induces significant alterations both in Phase G1 and S at all concentrations tested. Therefore, these data strongly support the improvements of the compounds of formula (I) compared to SOCE inhibitor with off-target activity on DHODH described in the prior art (3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R, Serafini M. et al. J. Med. Chem.

[0331] 2020;63(23): 14761-14779). Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Whole Blood The use of human whole blood samples refers to the clinical trial using biological samples entitled "Study of store-operated calcium entry inhibitors in immune cells from patients with Multiple Sclerosis" approved with code CE152 / 2024 by the Inter-Company Ethics Committee of the Hospital -University Major Charity of Novara in (code CE152 / 2024). Blood samples were collected at Hospital -University Major Charity of Novara, together with the express, free and informed consent to such collection and use of the patients.

[0332] Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of MS patients and healthy volunteers as per protocol using density gradient centrifugation with Ficoll-based solutions.

[0333] After collection, the whole blood samples were initially diluted with Roswell Park Memorial Institute medium (RPMI-1640, Sigma Aldrich) at a 1:1 ratio. A mixture containing two Ficoll Histopaque solutions, H-l 1191 and H-10771 (Sigma Aldrich), at a 1:1 ratio was then prepared, and an equivalent volume of the previously diluted blood samples in RPMI-1640 was added. At the end of this step, the samples were centrifuged at 300 g (RCF) (acceleration 3, deceleration 2) for 30 minutes at room temperature (RT). After centrifugation, a multi-phase system was obtained, with a specific separation ring containing the PBMCs.

[0334] These human derived cells were used as an in vitro model to evaluate safety and efficacy of compound SP20 (3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid, Compound H) in comparison with teriflunomide, a DHODH inhibitor approved for MS treatment.

[0335] Biological evaluation of compounds targeting SOCE by Fluo-4 Ca2+measurements

[0336] Jurkat cells or PBMCs were loaded with 2.5 pM Fluo-4 AM in the presence of 0.02% Pluronic-127 (both from Life Technologies, Italy) and 10 pM sulfinpyrazone (Sigma-Aldrich) in Krebs-Ringer buffer (KRB; 135 mM NaCl, 5 mM KC1, 0.4 mM KH2PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4) containing 2 mM CaCl2(30 min, RT). Then, cells were washed and incubated with KRB for 30 min to allow the de-esterification of Fluo-4 AM. To measure SOCE, cells were depleted of Ca2+in the ER with 2,5-t-butylhydroquinone (tBhQ, 50 pM; Sigma-Aldrich, Italy), a SERCA poison, in a Ca2+free solution, and then Ca2+was readded to the extracellular solution. During the experiments, cells were incubated in 96-well plates, and Fluo-4 AM fluorescence was monitored sequentially for a second in each well for 600s, generating time curves for each well.

[0337] The intracellular Ca2+evaluation performed using the probe Fluo-4 AM was performed to confirm SOCE over-activation in MS. As shown in Figure 6, SOCE induced by tBHQ is overactivated in PBMCs derived from patient affected by MS as compared to healthy volunteer (HV). Furthermore, the same graph demonstrates that the lead compound 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) at 3 and 10 pM significantly reduces intracellular Ca2+, reverting SOCE overactivation and restoring Ca2+to physiological level (closely to Ca2+level in HV, Figure 6).

[0338] Proliferation assay with the fluorescent dye 5(6)-carboxyfluorescein diacetate N-succinimidil ester (CFSE)

[0339] CFSE (CellTrace Thermo Fisher Scientific) is a marker used for cell proliferation tracking. PBMCs or CD19+B cells isolated from whole blood were labeled with CFSE in PBS and incubated at 37°C for 20 min according to manufacturer’s instructions. PBMCs were activated by pre-coating 24-well plates with 5 mg / mL human anti-CD3 (130-093-387, Miltenyi Biotec) and 2.5 mg / mL soluble anti-CD28 (130-093-386, Miltenyi Biotec) and stimulated for 6 days with 1 pg / ml anti-human CD40 (130-096-712, Miltenyi Biotec) antibody in the presence of interleukin 2 (IL-2 20 ng / ml, 130-097-743, Miltenyi Biotec), interleukin 4 (IL-4, 25 ng / ml, 130-093-917, Miltenyi Biotec) and interleukin 21 (IL-21, 50 ng / ml, 130-095-767, Miltenyi Biotec).

[0340] Cells were maintained in a humidified atmosphere at 37°C with 5% CO2. At endpoint of the experiment, cells were collected for FACS analysis.

[0341] For flow cytometry, PBMCs CFSE labelled cells were stained with APC-conjugated antihuman CD4 antibody (Invitrogen) for CD4+T cells subpopulation identification.

[0342] The proliferation assay with the fluorescent dye 5(6)-carboxyfluorescein diacetate A-succinimidil ester (CFSE) was conducted to analyse the proliferation of PBMCs derived from patients affected by MS treated or not with: (i) dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20); (ii) 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R); (iii) teriflunomide.

[0343] In Figure 7A is reported the cell division of PBMC following a 72 h treatment with increasing concentrations (0.1, 0.3, 1, 3, 10 and 30 pM) of 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) and the SOCE inhibitor with off-target activity on DHODH 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R). Dose-response curves in Figure 7 A demonstrate that 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) slightly reduces PBMCs proliferation at 10 and 30 pM (approx. 25%), while teriflunomide significantly compromises cell proliferation since the concentration of 0.3 pM.

[0344] To further confirm the safety profile of 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20), its effect at 10 pM was evaluated analysing the percentage of proliferative CD4+T cells from generation 1 to 6 in comparison with teriflunomide and 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R). As shown in Figure 7B, Compound H or SP20 slightly reduces cell proliferation (10% in generation 5 and 6), while teriflunomide and the prior art compound 3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid (Compound R) induce a significant impairment starting from generation 2.

[0345] Therefore, it can be stated that the compounds of formula (I) do not alter cell division, thus proving to be well tolerated and safer both than the DHODH inhibitor teriflunomide and the SOCE inhibitor with off-target activity on DHODH (3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)phenyl)carbamoyl)benzoic acid, Compound R).

[0346] Real-Time PCR

[0347] Total RNA was isolated using the TRIzol (Ambion-Life Technologies) reagent. The cDNA was obtained following the guidelines provided by the manufacturer SensiFASTTM cDNA Synthesis Kit (Meridian Bioscience). Real-Time PCR was performed on 96-well plates (CFX96 TM Real-Time PCR Detection Systems, Bio-Rad Inc.), in triplicate and with fluorescence intensity evaluated using the CFX96 TM Real-Time PCR Detection Systems (Bio-Rad Inc.).

[0348] The primers used are the following: human IFN-y: forward 5'-TGGAAAGAGGAGTGAC AGA-3' (SEQ ID No.: 1), reverse 5'- CACTCTTGGATGCTCTGGT-3' (SEQ ID No.: 2); human IL-2: 5'-CCAAGAAGGACACACTG-3' (SEQ ID No.: 3); reverse 5'-AGTCCCTCTGTCTTAAGTGA-3’ (SEQ ID No.: 4); human IL-17A: forward 5’-TACAACCGATCCACCACCTC-3' (SEQ ID No.: 5), reverse 5'-ACTTTGCCTCCCAGATCACA-3' (SEQ ID No.: 6); human GM-CSF: forward 5'-CCAGCCACTACAAGCAGCA-3' (SEQ ID No.: 7), reverse 5'-CAAAGGATGACAAGC AGA-3' (SEQ ID No.: 8).

[0349] Annealing temperature set at 60 °C. The transcripts were normalized to expression of ribosomal protein S18 mRNA levels (forward: 5'-TGCGAGTACTCAACCAACA-3' (SEQ ID No.: 9), reverse: 5'-CTGCTTTCCTCAACACCACA-3' (SEQ ID No.: 10)) and for each gene the threshold cycle (ACt) was calculated.

[0350] Real-Time PCR was carried out to examine pro-inflammatory cytokines mRNA expression levels in PBMCs derived from patients affected by MS treated or not with: dual SOCE / DHODH modulator 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) or teriflunomide. In detail, mRNA expression levels of Thl effector cytokines (IL-2 and IFNy) are shown in Figure 8A, whereas mRNA expression levels of Thl7 effector cytokines (IL-17A and GM-CSF) are reported in Figure 8B. Data obtained by Real-Time PCR demonstrates that (3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) significantly downregulates both Thl and Thl7 pro-inflammatory cytokines. On the other hand, teriflunomide only moderates GM-CSF and IFN-y mRNA expression levels. Therefore, it can be assumed that 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid (Compound H or SP20) has a higher efficacy than teriflunomide (used at the same treatment condition of 10 pM, 72 h of treatment) in reducing inflammatory -mediated autoimmune reaction.

[0351] Protein expression and purification

[0352] The open reading frame of hDHODH (Uniprot identifier: Q02127) was subcloned into pET-19b plasmid (GenScript, Piscataway, NJ, USA) using Ndel / BamHI restriction sites. The vector was designed to express the recombinant N-terminal His-tagged version of the protein exploiting E. coli BL21(DE3) (Novagen) as the host system. E. coli cells were transformed with the target construct and grown on a 2XYT-agar plate for 16 h at 37 °C. Transformed cells were inoculated into 1 L of sterile 2XYT liquid broth containing Ampicillin (50 g / mL) as a selective agent. The bacterial culture was grown in a 5 L shaking flask at 200 rpm and 37 °C until reaching an optical density at 600 nm (OD600) of 0.6.

[0353] The recombinant expression of hDHODH was induced using 0.2 mM isopropyl- 1- thio-D-galactopyranoside (IPTG), and the culture was incubated at 200 rpm and 16 °C for 20 h. The culture was then harvested by centrifugation at 6000 rpm and 4 °C for 10 min. The bacterial pellet was resuspended at a ratio of 1 g: 9 mL in lysis buffer (LB) [50 mM HEPES pH 7,8, 300 mM NaCl, 10% (v / v) Glycerol and 0.25% (w / v) nUndecyl-N, N-Dimethylamine-Oxide (UDAO)] supplemented with EDTA-free protease inhibitor cocktail (Merck) and DNAse.

[0354] All subsequent steps were carried out at 4 °C. Cells were lysed using a Sonics Vibra-Cell VC 130 Ultrasonic Homogenizer (Strokes: 10; Pulse: 30”; Stop: 60”; Amplitude: 45). The lysate was centrifuged at 17000 g at 4 °C for 45 min (Beckman Coulter Avanti Centrifuge J-26 XP), and the cleared lysate was applied to a Qiagen Ni-NTA agarose column pre-equilibrated with LB supplemented with 10 mM imidazole.

[0355] The resin was washed with 20 column volumes (CV) of wash buffer (LB supplemented with 80 mM imidazole), and the protein was eluted using an elution buffer (LB supplemented with 300 mM imidazole). Fractions containing hDHODH appeared yellow due to the presence of the FMN prosthetic group which is bound to the protein core. Positive fractions were verified by 12.5% SDS / PAGE and pooled, then concentrated using Amicon 15-30000 MWCO centrifugal concentrator (Sartorius).

[0356] Subsequently, the concentrated protein underwent a second chromatographic step and it was loaded onto a Hiload Superdex 200 16 / 600 (GE Healthcare, Chicago, IL, USA) column, pre-equilibrated with a size exclusion buffer [100 mM HEPES pH 7, 400 mM NaCl, 10% (v / v) Glycerol, 1 mM EDTA and 0.25% (w / v) UDAO], The size exclusion chromatography was monitored both at 280 nm, 350, and 442 nm wavelengths, corresponding to the FMN cofactor’s absorbance peaks. Protein quantification was performed via Nanodrop (MW=42566 Da; Ext. coefficient=15930).

[0357] Enzymatic assay

[0358] Michaelis-Menten kinetics The enzymatic activity was monitored using a Tecan Sunrise spectrophotometer in a transparent Greiner® microplate. The “ping-pong” reaction catalyzed by hDHODH was followed in a coupled reaction which involves the reduction of the DCIP reagent. In particular, the first substrate DHO is oxidized to the product ORO, while the cofactor FMN is reduced to FMNH2. Subsequently, the Q is reduced to regenerate the oxidized form of FMN. The re-oxidation of QH2 into Q is stoichiometrically equivalent to the reduction of DCIP (blue) to DCIPH2 (colorless).

[0359] The enzymatic assay was performed in a final 100 gL / well volume. The purified recombinant protein was tested at 0.1 gM in the reaction mixture [50 mM Tris- HC1 pH 8, 400 mM NaCl, 5% (v / v) Glycerol, 1 mM EDTA, 0.1% (v / v) triton X-100, 100 gM Q10 dissolved inDMSO, 50 / / MDCIP] up to a final volume of 100 / / L, the reaction was initiated by addition of DHO at increasing concentration from 0.49 to 500 / / M and the reduction was monitored at 600 nm for 10 min.

[0360] These experiments were performed to confirm the inhibitory activity of the compounds object of the present application on human DHODH. Data obtained by using this enzymatic assay are reported in Table 2. The properties evaluated include: (i) % of SOCE inhibition at 10 pM; (ii) IC50 for SOCE inhibition; (iii) % of viability at 50 pM; (iv) % of counterbalancing induced by uridine; (v) % of DHODH residual activity.

[0361] Table 2

[0362]

[0363]

[0364] 3-((4-(2.3- dihydrobcnzo[ / i|| 1.4|dioxin-6-x 1)- 3.5- ’ 46.2 ± 1.3 - 74.4 ± 4.0 10.5 71.5 ± 8.7 d ifl norophcny 1 )ca rbainoy 1 )bc nzo ic

[0365] l illlliiiiiiiiii

[0366]

[0367] dihx drobcnzo|b|| 1.4|dioxin-6-yl)- 2.6- ’ 14.4 ± 3.5 - 90.4 ± 8.8 12.1 77.3 ± 3.5 diflnorophcnx l)carbamox l)bcnzoic

[0368] acid (J)

[0369]

Claims

Claims1. Compound of formula (I):whereinAi, A2, A3, A4 and A5 are identical or different from each other and independently selected from H, CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN, a 5-6 membered O-heterocyclic group, an F containing group;Ai and A?, or A2 and A3, or A3 and A4, or 4 and A, can form together a 5-6 membered O-heterocyclic group fused to the phenyl ring to which they are attached;Bi, B2, B3 and B are identical or different from each other and independently selected from H or F, Cl, Br, I, an F containing group;at least one of Bi, B2, B3 and B4 is F;C is selected from H or unsubstituted or substituted Ci-s alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;Di, D2, D3 and D4 are identical or different from each other and independently selected from H or CF3, Br, I, Cl, F, OH, ORi, SRi, NH2, NHRi, NR1R2, S(O)Ri, S(O)2RI, NHCORI, NHSO2R1, CONHRi, CONR1R2, SO2NHR1, COOH, COORi, NO2, CN;E is selected from H or unsubstituted or substituted Ci-8 alkyl group,unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4;Ri and R2 are identical or different from each other and independently selected from unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C2-8 alkenyl group, unsubstituted or substituted C2-8 alkynyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic group, (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4, CF3;pharmaceutically acceptable hydrates and / or solvates and / or salts thereof.

2. Compound of formula (I) according to claim 1, wherein, when Ri and R2, if present, are independently selected from substituted Ci-8 alkyl group, substituted C2-8 alkenyl group, substituted C2-8 alkynyl group, substituted C3-6 cycloalkyl, substituted aryl, substituted heterocyclic group, the one or more substituents are independently selected from halogen, CH3, CH2F, CHF2, CF3, OR2, CN, COOR4, CONR4R5, NR4R5, NHCOR4, NHSO2R4, S(O)R4, S(O)2R4, and SO2NHR4, wherein R4and R5 are the same or different and independently selected from H, Ci-Cs alkyl group unsubstituted or substituted with one or more halogen atoms, and C3-C6 cycloalkyl group unsubstituted or substituted with one or more halogen atoms.

3. Compound of formula (I) according to any one of the preceding claims, wherein Ri and R2 are selected from Ci-Cs alkyl group unsubstituted or substituted with one or more halogen atoms, C3-C6 cycloalkyl group unsubstituted or substituted with one or more halogen atoms, unsubstituted or substituted aryl, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

4. Compound of formula (I) according to any one of the preceding claims, wherein Ai, A2, A3, A4and A5 are identical or different from each other and independently selected from H, F, a F containing group, O-Ci-salkyl, O-heterocyclic group.

5. Compound of formula (I) according to any one of the preceding claims, wherein C is selected from H, unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C3-6 cycloalkyl, (CH2)n-Ci-s alkyl, (CH2)n-C2-s alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

6. Compound of formula (I) according to any one of the preceding claims, wherein when C and E are independently selected from substituted Ci-s alkyl group, substituted C2-8 alkenyl group, substituted C2-8 alkynyl group, substituted C3-6 cycloalkyl, substituted aryl, substituted heterocyclic group, the one or more substituents are independently selected from halogen, CF3, OCH3, OCF3, OH, CH2OH.

7. Compound of formula (I) according to any one of the preceding claims, wherein Di, D2, D3 and D4 are identical or different from each other and independently selected from H, CF3, Br, I, Cl, F, OH, ORi, SRi, NO2, CN.

8. Compound of formula (I) according to any one of the preceding claims, wherein E is selected from H or unsubstituted or substituted Ci-8 alkyl group, unsubstituted or substituted C3-6 cycloalkyl,, unsubstituted or substituted (CH2)n-Ci-8 alkyl, (CH2)n-C2-8 alkenyl, (CH2)n-C2-8 alkynyl group, (CH2)n-cycloalkyl, (CH2)n-aryl and (CH2)n-heteroaryl, wherein n is an integer 1 to 4.

9. Compound of formula (I) according to any one of the preceding claims, wherein the F containing group is selected from CF3, CHF2, CH2F, OCF3, OCHF2, OCH2F.

10. Compound of formula (I) according to any one of the preceding claims, selected from:3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)-2,3,5,6-tetrafluorophenyl) carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-3'-methoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoicacid;3-((2,2',3,5,6-pentafluoro-5'-methoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-3'-propoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-3',4'-dimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-2',3'-dimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl) benzoic acid;3 -((4-(2,3 -dihy drobenzo[Z>] [ 1,4] dioxin-6-yl)-3, 5 -difluorophenyl)carbamoyl)benzoic acid;3-((4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)-2,6-difluorophenyl)carbamoyl)benzoic acid;3-((2,6-difluoro-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((3,5-difluoro-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((2,6-difluoro-3'-methoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid; 3-((3,5-difluoro-3'-methoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid; 3-((2,6-difluoro-3'-propoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid; 3-((3,5-difluoro-3'-propoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoic acid; 3-((2,6-difluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid;3-((3,5-difluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl)carbamoyl)benzoic acid;methyl 3-((4-(2, 3-dihy drobenzo[Z>][l, 4]dioxin-6-yl)-2, 3,5,6-tetrafluorophenyl)carbamoyl)benzoate;methyl 3-((2,3,5,6-tetrafluoro-2',3'-dimethoxy-[l,l'-biphenyl]-4-yl)carbamoyl)benzoate;methyl 3-((2,3,5,6-tetrafluoro-3',4',5'-trimethoxy-[l,r-biphenyl]-4-yl) carbamoyl)benzoate.

11. Compound of formula (I) according to any one of the preceding claims for use as a medicament.

12. Compound of formula (I) according to any one of the preceding claims for use in the treatment and / or prevention of a disease conditions depending on increased activity of SOCE and alterations of DHODH pathway,.

13. Pharmaceutical composition comprising at least one compound of formula (I) according to anyone of claims 1 to 10 and a pharmaceutically acceptable carrier and / or vehicle.

14. Pharmaceutical composition comprising at least one compound of formula (I) according to anyone of claims 1 to 10 and a pharmaceutically acceptable carrier and / or vehicle for use in the treatment and / or prevention of a disease condition depending on increased activity of SOCE and alterations of DHODH pathway.

Images