Compound for the treatment of transthyretin amyloidosis

Abstract

A benzophenone compound of formula (I) and the pharmaceutically salts thereof are provided, which are useful in the treatment of a transthyretin amyloidosis. The compound presents higher affinity to transthyretin, higher bioavailability and reduced toxicity. A process for preparing the compound and a pharmaceutical composition comprising the compound are also provided.

Description

[0001] TITLE: Compound for the treatment of transthyretin amyloidosis

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of small molecules for their use in the treatment of transthyretin amyloidosis.

[0004] BACKGROUND ART

[0005] STATEMENT REGARDING PRIOR ART DISCLOSURES BY THE INVENTOR OF A JOINT INVENTOR UNDER UNITED STATES 37 C.F.R. 1.77(b)(6)

[0006] At the filing date of the present application, the article F. Pinheiro, et al., 2022 published on 28th October 2022 described the compound (3,4-dihydroxy-5-nitrophenyl)-(3-fluoro-5-hydroxyphenyl)- methanone, therein referred to as M-23 (chemical structure shown below). M-23 is a transthyretin (TTR) kinetic stabilizer that displays affinity for both binding sites and binds to TTR in human plasma with higher TTR stabilizing activity in comparison with tafamidis and tolcapone molecules, two therapeutically relevant molecules for the treatment of transthyretin amyloidosis.

[0007] All the inventors of the present application have assigned all herein relevant rights to applicant of the present application, said in other words, the discussed article publication of present applicant may be considered as so-called "inventor originated disclosure", i.e., the subject matter in the public disclosure must be attributable to the inventor, one or more co-inventors, or another who obtained the subject matter directly or indirectly from the inventor or a co-inventor.

[0008] Amyloid diseases encompass a diverse range of disorders characterized by the accumulation of amyloid fibrils, highly organized structures that arise from the misfolding and subsequent aggregation of proteins. Amyloids build up in specific organs and tissues, interfering with their normal function.

[0009] Transthyretin amyloidosis (ATTR) is a group of life-threatening systemic disorders caused by the extracellular deposition of TTR amyloid fibrils. ATTR amyloidosis can be either hereditary or sporadic (non-hereditary).

[0010] Received at EPO via Web-Form on Jul 18, 2023 Native TTR is a 55 kDa homotetrameric protein, which is mainly synthesized in the liver, choroid plexus and retinal pigment epithelium. Besides its well-known role as a transporter of thyroxine (T4) and retinol binding protein-vitamin A complex, TTR is increasingly recognized as having a neuropro- tective activity in the central nervous system.

[0011] Individuals with mutations in the TTR gene produce amyloidogenic TTR variants throughout their lives. In general, TTR variants are more amyloidogenic than the wild-type protein.

[0012] The most common forms of ATTR are related with the aggregation of TTR mutants and inherited in an autosomal dominant manner. Most of the genetic variants causes familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC), e.g., V30M-TTR and V122I-TTR mutations / vari- ants. In some rare cases, TTR mutations can induce leptomeningeal amyloidosis. ATTR can also be caused by the wild-type (WT) protein, known as senile systemic amyloidosis (SSA).

[0013] Amyloid deposits start to form and then build up until they cause clinical disease, mainly affecting the nerves and / or heart, and sometimes the kidneys, eyes and synovial tissues (tendons and ligaments). Symptoms may appear at any time from early adult life onwards. This condition runs in families.

[0014] There is no cure for transthyretin-associated amyloidosis; liver or combined liver and heart transplantation are the default palliative therapeutic strategies for these disorders. However, it has been shown that treatment with small molecules can help to manage signs and symptoms and limit further accumulation of amyloid protein.

[0015] These small molecules stabilize the TTR tetramer acting as kinetic stabilizers, thus stalling TTR aggregation. Since pathogenic mutations in TTR decrease its thermodynamic stability and / or lower the kinetic barrier for tetramer dissociation, which is thought to be the rate-limiting step in amyloid formation, the occupancy of the T^binding sites by specific small molecules is known to stabilize TTR, inhibiting its dissociation and, thus, preventing ATTR onset. These molecules may include tolcapone, tafamidis, AGI O, and diflunisal, which have the following chemical structures:

[0016] Received at EPO via Web-Form on Jul 18, 2023

[0017] Tafamidis is the only drug approved for ATTR, and is the INN of the compound named 2-(3,5-dichlo- rophenyl)-1 ,3-benzoxazole-6-carboxylic acid. The so-called kinetic stabilization strategy is the mode of action of the drug tafamidis, the only pharmacological treatment in the market for both TTR-related polyneuropathy and cardiomyopathy. However, the evidence that 30% of patients do not respond to tafamidis, together with its high cost, has prompted the development of alternative molecules to treat ATTR.

[0018] Tolcapone is the INN of the compound named 5-(4-methylbenzoyl)-3-nitrobenzene-1 ,2-diol. It was first disclosed in European patent application EP-A-0237929, and it is an inhibitor of central and peripheral catechol-O-methyltransferase. It is used in medicine in the treatment of Parkinson’s disease, but has been repositioned for the treatment of familial amyloid polyneuropathy. The results of a Phase Ila clinical trial demonstrated that tolcapone stabilizes TTR in patients with FAP, supporting its further development as a drug to treat ATTR. Noteworthy, tolcapone can cross the blood-brain barrier, unlike tafamidis, and it was shown to bind and inhibit the aggregation of the highly destabilized TTR variants associated with leptomeningeal amyloidosis.

[0019] The higher stabilizing activity of tolcapone compared to tafamidis was proposed to be related with its similar high affinity for both T^binding sites, which contrasts with the strong negative cooperativity displayed by tafamidis. Despite tolcapone binds better than tafamidis to the second binding cavity, tafamidis affinity for the first cavity is > 3-fold higher than the one of tolcapone. The high-resolution structure of TTR in complex with tolcapone revealed that its 4-methyl-phenyl ring does not establish any enthalpic interaction with the binding pocket.

[0020] Moreover, the use of tolcapone has been linked to cases of severe liver injury, including acute liver failure, which can be life-threatening. Therefore, tolcapone is generally reserved for patients with Parkinson's disease who have not adequately responded to or cannot tolerate other medications.

[0021] Received at EPO via Web-Form on Jul 18, 2023 AGI O did not show the expected results in clinical trials for FAP / SSA. On the other hand, AGI O binds to V122I-TTR variant (associated with FAC) with negative cooperativity.

[0022] Diflunisal is the INN of the compound named 5-(2,4-difluorophenyl)-2-hydroxybenzoic acid. It is a nonsteroidal anti-inflammatory drug, which has been shown to be inhibitor of transthyretin-related hereditary amyloidosis. However, it displays a low affinity for TTR and, since it is an inhibitor of cyclooxygenase enzymes, it has gastrointestinal and cardiovascular side effects.

[0023] Considering that transthyretin amyloidosis, in particular FAP, is a disease that currently has few treatment options, there is still a need to provide further active compounds for said treatment.

[0024] SUMMARY OF THE INVENTION

[0025] The present invention provides a new small molecule compound which exhibits higher affinity to transthyretin, higher bioavailability and reduced toxicity compared to compounds known in the art, and has shown to be effective in the treatment of transthyretin amyloidosis, and more particularly FAP.

[0026] Particularly, inventors have developed the compound of formula (I), (3-fluoro-5-hydroxyphenyl)(4- hydroxy-3-methoxy-5-nitrophenyl)methanone, hereinafter also referred to as PITB (Pharmacokinet- ically Improved TTR Binder), and the pharmaceutically salts thereof.

[0027] This compound has shown to be a kinetic stabilizer of TTR, exhibiting one of the highest affinities reported for TTR so far. In addition, pharmacokinetic (PK) studies in mice have confirmed that PITB has an excellent PK profile. TTR / PITB crystal structures have demonstrated the establishment of protein-ligand contacts at the dimer-dimer interface that are lacking in tolcapone. These interactions underly the higher binding selectivity and stabilizing activity showed by PITB in serum from both control individuals and patients with FAP, when compared to tolcapone. Overall, the obtained results confirm the potential of PITB as a novel molecule for ATTR therapeutic intervention.

[0028] The working examples herein provide evidence of the compound of formula (I) to exhibit higher

[0029] Received at EPO via Web-Form on Jul 18, 2023 affinity to TTR, higher bioavailability and reduced toxicity compared to tolcapone, a compound with a similar chemical structure.

[0030] Inventors have shown that PITB strongly binds to the T^binding sites of WT-TTR and the two most

[0031] 5 clinically relevant TTR variants V30M and V122I. As a result, PITB greatly increases the stability of TTR tetramers in vitro, inhibiting their aggregation. Remarkably, compared with tolcapone, PITB presents a higher binding selectivity and stabilization potency in plasma. The high-resolution crystal structures of TTR:PITB complexes have confirmed that PITB keeps critical interactions which might underlie its increased efficacy in plasma relative to tolcapone. See EXAMPLE 2 to EXAMPLE 6 (FIG.

[0032] 10 1 to FIG. 4).

[0033] Additionally, cytotoxicity assays suggest a low risk associated with PITB administration (see EXAMPLE 7, FIG. 5), thereby being a drug potentially without side effects such severe liver injury. Since tolcapone is contraindicated for patients with liver-related diseases, PITB would be a perfect candidate for these patients.

[0034] Most importantly, the PK evaluation of PITB in mice has shown that it has a favorable PK profile, with a 10.1 h half-life and 85.1 % oral bioavailability. Remarkably, PITB presents improved PK parameters compared to tolcapone, an FDA-approved drug (see EXAMPLE 8, FIG. 6, Table 2).

[0035] Considering that tolcapone has already demonstrated effectiveness in a phase Ila clinical trial in FAP caused by V30M-TTR, and given that PITB outperforms tolcapone in every analyzed aspect, evidence presented herein underscores the strong potential of PITB as a therapeutic agent for ATTR, particularly for FAP caused by V30M-TTR.

[0036] 25

[0037] Furthermore, PITB proved to be a better molecule for the treatment of ATTR compared to M-23. PITB has a better PK profile (10.1 h vs <1 h) and better affinity for V30M-TTR (36 vs 64 nM, respectively) and V122I-TTR mutants (14 vs 28 nM, respectively).

[0038] 30 Accordingly, a first aspect of the invention relates to a compound defined by formula (I) or a pharmaceutically acceptable salt thereof.

[0039] Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in the first aspect of the invention and at least a pharmaceutically acceptable excipient or vehicle.

[0040] Another aspect of the invention relates to a compound as defined in the first aspect of the invention, for use as a medicament.

[0041] 40 Another aspect relates to a compound as defined in the first aspect of the invention or a

[0042] Received at EPO via Web-Form on Jul 18, 2023 pharmaceutical composition as defined herein, for use in the treatment of a transthyretin amyloidosis in a subject. Alternatively, the invention relates to a method of treatment of a transthyretin amyloidosis comprising administering a therapeutically effective amount of a compound as defined in the first aspect or a pharmaceutical composition as defined herein to a subject in need thereof. Alternatively, the invention relates to the use of a compound as defined in the first aspect for the manufacture of a medicament for the treatment of a transthyretin amyloidosis.

[0043] Another aspect relates to a process for preparing a compound of formula (I) as defined in the first aspect of the invention, comprising the removal of two of the methyl groups of compound of formula (l)-1 : to yield compound of formula (I).

[0044] Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and without intending to be limiting to the present invention.

[0045] DESCRIPTION OF DRAWINGS

[0046] FIG. 1 shows the PITB effect over the kinetic stability and aggregation of WT-, V30M- and V122I- transthyretin (TTR). (A) Unfolding time course at 6 M urea in the absence or presence of two molar equivalents of PITB and tolcapone, as monitored by Trp fluorescence. The values correspond to mean ± SEM (n = 3). (B) Percentage of TTR aggregation (represented as fibril formation (%)) as a function of PITB and tolcapone concentration as measured by turbidity at 340 nm. The values were normalized to 100%, which was assigned to the turbidity of samples aggregated in the absence of any compound. Data represents mean ± SEM (n = 3).

[0047] FIG. 2 shows the interaction of PITB or tolcapone with WT-, V30M- and V122I-TTR proteins. The top panels correspond to the raw data after baseline correction (dQ / dt in pcal / s between time in minutes), while the lower panels represent the integrated data as a function of the molar ratio (Q in kcal / mol of injection) between the concentration of PITB or tolcapone and protein ([PITB or

[0048] Received at EPO via Web-Form on Jul 18, 2023 tolcapone]i7[protein]T). The solid lines in the bottom panels represent the best fit to the data to acquire the binding parameters.

[0049] FIG. 3 shows the crystal structures of WT-TTR (A), V30M-TTR (B) and V122I-TTR (C) in complex

[0050] 5 with PITB. On the left, the global view of TTR bound to PITB (cartoon representation). The dashed line corresponds to the twofold symmetry axis of the dimer-dimer interface. The insets show the detailed view of one the TTR binding sites for PITB and for tolcapone. PITB, tolcapone and some of the TTR interacting residues are shown as sticks. The dashed lines show key interactions between each ligand and TTR. The structural data for Tolcapone:WT and Tolcapone:V122l was obtained from

[0051] 10 PDB files 4D7B and 5A6I, respectively. (D) PITB binding at the WT-, V30M- and V122I-TTR dimerdimer interface. PITB and some of the TTR residues interacting with the ligand are depicted by sticks. Dashed lines indicate relevant contacts between PITB and TTR and between the hydroxyl groups of S117 / S117’.

[0052] FIG. 4 (A-B) shows the PITB binding and stabilizing activity in human plasma from control individuals (WT-TTR) or patients with FAP (V30M-TTR). (A) Fraction of [125I]-T4 bound to TTR determined by densitometry analysis of the native gel electrophoresis, as explained in the experimental section. The values were normalized to the negative control and correspond to mean ± SEM (n = 3). (B) Percentage of TTR tetramer stabilization calculated from the densitometry analysis of the isoelectric focusing (IEF) gels, as detailed in the experimental section. Error bars represent SEM of mean values (n = 2 for WT plasma; n = 3 for V30M plasma). (C-D) Representative films and IEF gels for the analysis of the binding and stabilizing activity of PITB in human plasma from control individuals (WT-TTR) and patients with FAP (V30M-TTR). (C) Representative native gel electrophoresis of plasma samples of WT-TTR and V30M-TTR carriers incubated with [125I]-T4, in the absence or presence of PITB / tolca-

[0053] 25 pone. A plasma sample incubated with the same amount of DMSO was used as a negative control. The main T4-binding proteins are indicated: T4-binding globulin (TBG), albumin (ALB) and transthyretin (TTR). The gels are cropped to avoid showing the results of other compounds that were tested in parallel but are not part of this study. (D) IEF analysis under partially denaturing conditions of plasma samples from WT-TTR and V30M-TTR carriers treated with PITB, tolcapone, or DMSO (as

[0054] 30 control). The distinct molecular species observed in the IEF gels are depicted (monomers, oxidized monomers and tetramers). These gels are representative of others run in parallel. Cropped images of the gels are shown since compounds that are not included in this work were run alongside with PITB and tolcapone.

[0055] FIG. 5 shows the toxicity of PITB to two human cell lines. It shows cell viability (%) of HeLa (A) and HepG2 (B) cells between PITB or tolcapone concentration (pM) after 72 h exposure to PITB or tolcapone as assessed by the PrestoBlue assay. Data represents mean ± SEM (n = 3).

[0056] FIG. 6 shows the plasma concentration-time profiles for PITB and tolcapone (ng / mL) over time

[0057] 40 (hours) after administration in CD-1 mice. (A) Pharmacokinetics (PK) profiles for PITB over 24 h after

[0058] Received at EPO via Web-Form on Jul 18, 2023 intravenous (IV) (1 mg / Kg) and oral (PO) (10 mg / Kg) administration. Data represents mean ± SEM (n = 3). (B) Mean plasmatic levels of tolcapone (± SEM) over 24 h after oral (PO) administration (10 mg / Kg) (n = 3). Noteworthy, no tolcapone was detected after 24 h.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] Throughout the present description and in the claims, the expressions in singular preceded by the articles “a” or “the” are understood to also include, in a broad manner, the reference to the plural, unless the context clearly indicates the contrary.

[0061] In the context of the present invention, it is understood that the term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). Particularly, the term referred to a determined value indicates that a certain variation for said value is accepted, generally of + / - 5 %.

[0062] The ranges disclosed in this description include both the lower and the upper limit thereof. and salts thereof

[0063] As said, a first aspect of the present invention relates to a compound defined by formula (I) or a pharmaceutically acceptable salt thereof. Particularly, the compound is as defined in formula (I).

[0064] The compound of the present invention is a benzophenone compound. Particularly, compound of formula (I) is herein named PITB.

[0065] In an embodiment, the compound of formula (I) can also be converted into a pharmaceutically acceptable salt. Particularly, suitable salts are salts with pharmaceutically acceptable bases. Examples of such salts are the salts of alkali metals, such as sodium and potassium salts. These salts can be prepared by methods which are known per se and are familiar to any person skilled in the art.

[0066] Received at EPO via Web-Form on Jul 18, 2023 Process of preparing the compound of formula (I)

[0067] In general, the compound according to the first aspect of the present invention can be synthesized by methods known in the art. One synthetic route for preparing the compound of formula (I) is represented in Scheme I:

[0068] SCHEME I

[0069] 1) / -PrMgBr, THF anh., -40 °C,1 h

[0070] In an embodiment, the synthesis leading to PITB (I) can start with the coupling reaction of the freshly prepared Grignard reagent of iodoarene compound (l)-2 (step 1 of Scheme I) to the methyl diprotected nitro-catechol, 3,4-dimethoxy-5-nitrobenzaldehyde (step 2 of Scheme I) followed by a Dess- Martin periodinane oxidation reaction (step 3 of Scheme I) to furnish ketone (l)-1 in about 45-80% yield for the two steps, particularly 58% yield. The Grignard reagent of iodoarene compound (l)-2 is particularly freshly prepared by an iodine-magnesium exchange re-action with i-PrMgBr at about -40 °C in an organic solvent, such as, e.g., tetrahydrofuran (THF). In an embodiment, selective removal of two methyl protecting groups on (l)-1 can be carried out using boron tribromide (BBrs) in an organic solvent, such as dichloromethane (DCM, CH2CI2), to furnish monoprotected catechol PITB (I) in excellent yield, particularly in about 70-99%, and more particularly about 88%.

[0071] The structural identity and purity of the compounds can be ascertained by NMR spectroscopy and high-resolution mass spectrometry (HRMS).

[0072] Accordingly, another aspect of the invention is a process for preparing the compound of formula (I),

[0073] Received at EPO via Web-Form on Jul 18, 2023 which comprises the removal of two of the methyl groups of the compound of formula (l)-1 : to yield compound of formula (I).

[0074] In a particular embodiment, the (selective) removal of two of the methyl groups of compound (l)-1 is carried out using boron tribromide (BBrs) in an organic solvent, particularly methylene dichloride (DCM, dichloromethane). In a particular embodiment, the compound (I) is obtained in a yield of about 70-99%, and more particularly of about 88%.

[0075] In an embodiment, the process for preparing the compound of formula (I), further comprises the coupling reaction of a Grignard reagent of compound (l)-2: with 3,4-dimethoxy-5-nitrobenzaldehyde, to obtain compound of formula (l)-1 :

[0076] In a particular embodiment, the Grignard reagent of the iodoarene compound (l)-2 is prepared by an iodine-magnesium exchange reaction with i-PrMgBr at about -40 °C in an organic solvent, such as, e.g., tetrahydrofuran (THF), e.g., for about 1 hour.

[0077] In a particular embodiment, the synthesis leading to compound of formula (l)-1 comprises the coupling reaction of the prepared Grignard reagent of iodoarene compound (l)-2 to the 3,4-dimethoxy- 5-nitrobenzaldehyde e.g., in THF, overnight.

[0078] In a particular embodiment, the synthesis leading to compound of formula (l)-1 further comprises an oxidation of compound (l)-2, particularly a Dess-Martin periodinane oxidation reaction using e.g., DMPI (Dess-Martin periodinane or DMP) and methylene dichloride (DCM, dichloromethane) anhydrous, overnight.

[0079] Received at EPO via Web-Form on Jul 18, 2023 In a particular embodiment, the compound of formula (l)-1 is obtained in a yield of about 45-80%, particularly about 58%.

[0080] 5 In a particular embodiment, the process for preparing the compound of formula (l)-1 is performed as detailed in EXAMPLE 1.

[0081] In an embodiment, the process comprises the following steps: a) compound (l)-1 is obtained by a coupling reaction of a Grignard reagent of compound (l)-2 and

[0082] 10 3,4-dimethoxy-5-nitrobenzaldehyde; b) compound (l)-1 is obtained by oxidation of compound (l)-2, particularly using a Dess-Martin peri- odinane oxidation; and c) compound (I) is obtained by demethylation of compound (l)-1 , particularly using boron tribromide in an organic solvent, more particularly the organic solvent is methylene dichloride.

[0083] Pharmaceutical composition

[0084] Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in the first aspect of the present invention and at least a pharmaceutically acceptable excipient or vehicle.

[0085] Alternatively, the compound as defined in the first aspect is in the form of a pharmaceutical composition comprising appropriate amounts of acceptable excipients.

[0086] 25 The term "acceptable excipient or vehicle" as used herein pertains to compounds, materials, compositions and / or administration forms which are suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, vehicle, etc. must also be “acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0087] 30 Suitable carriers, excipients, vehicles etc. can be found in standard pharmaceutical texts.

[0088] Pharmaceutically acceptable excipients or vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, fillers / diluents / bulking agents, binders, antiadherents, disintegrants, coatings, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colours, tensides, stabilizers, control-release agents, antimicrobial preservatives, acidifying agents, gelling agents, suspending agents, cosolvents, emulsifying agents and other classes of acceptable excipients.

[0089] The compound of the present invention is formulated into pharmaceutical compositions that can be

[0090] 40 administered e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally,

[0091] Received at EPO via Web-Form on Jul 18, 2023 vaginally or via an implanted reservoir. The term "parenteral" as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, in- tralesional and intracranial injection or infusion techniques.

[0092] 5 Therefore, in some embodiments, the pharmaceutical composition is formulated as an oral, parenteral (e.g., intravenous or intramuscular), inhalant, topical, rectal, nasal, buccal, vaginal or implanted reservoir composition. In a particular embodiment, the pharmaceutical composition is formulated as an oral or a parenteral composition.

[0093] 10 In a particular embodiment, the pharmaceutical composition is formulated as an oral composition. In another particular embodiment, e.g., in cases of severe ATTR, the pharmaceutical composition is formulated as an injectable composition for e.g., intravenous or intraperitoneal administration.

[0094] In each case, the presentation of the composition will be adapted to the type of administration used. Thus, the composition can be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount. The pharmaceutical composition can be thus formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders (such as those derived from lyophilization (freeze-drying) or air-drying), granules, solutions, suppositories, gels or microspheres. In a particular embodiment, the pharmaceutical composition is formulated for administration in liquid form or in solid form.

[0095] In a particular embodiment, the pharmaceutical composition is in solid form such as tablets, lozenges, sweets, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, pills, troches, gastro-resistant tablets and capsules, dispersible strips and

[0096] 25 films. More particularly, the pharmaceutical composition is in form of a capsule, a powder, a tablet, a pill, lozenges, sachets, sticks, or granules. In a more particular embodiment, the composition is in form of a pill, a tablet or a capsule.

[0097] In another embodiment, the pharmaceutical composition is in liquid form such as oral solutions,

[0098] 30 drops, suspensions, emulsions and syrups.

[0099] Both inorganic and organic excipient materials are suitable as acceptable excipients or vehicles. For example, lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof can be used as carrier materials for tablets, coated tablets, coated tablets and hard gelatin capsules. Suitable carriers for soft gelatin capsules are, e.g., vegetable oils, waxes, fats and semi-solid and liquid polyols. Suitable carrier materials for the production of solutions and syrups are, e.g., water, polyols, sucrose, invert sugar and glucose. Suitable carrier materials for injection solutions are, e.g., water, alcohols, polyols, glycerin and vegetable oils. Examples of suitable carrier materials for suppositories are natural or hardened oils, waxes, fats and semi-liquid or liquid polyols.

[0100] 40

[0101] Received at EPO via Web-Form on Jul 18, 2023 In an embodiment, the pharmaceutical composition can be in the form of a sterile injectable preparation, e.g., a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspen¬

[0102] 5 sion in a non-toxic parenterally-acceptable diluent or solvent, e.g., as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful

[0103] 10 in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant.

[0104] In a particular embodiment, the pharmaceutical composition is intravenous and comprises about 5% of nonionic surfactants (e.g., Cremophor®), 5% of mannitol, and / or 5% of dimethyl sulfoxide (DMSO). Particularly, the pharmaceutical composition is administered at about 1 mg / kg. Particularly, the administration volume is of about 5 ml / kg.

[0105] In an embodiment, the pharmaceutical composition can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents.

[0106] 25 If desired, certain sweetening, flavouring or colouring agents can also be added.

[0107] In a particular embodiment, the pharmaceutical composition is oral and comprises about 0.5% of carboxymethyl cellulose (CMC) and / or about 0.1 % of polysorbate 80 (Tween 80). Particularly, the pharmaceutical composition is administered at about 10 mg / kg. Particularly, the administration vol¬

[0108] 30 ume is of about 10 ml / kg.

[0109] In another particular embodiment, the pharmaceutical composition is a tablet (e.g., a film-coated table). Tablet core excipients can be, without limitation, calcium hydrogen phosphate, microcrystalline cellulose, povidone K30, sodium starch glycolate, lactose monohydrate, talc, and magnesium stearate; and film-coat excipients can be, e.g., hydroxypropyl methylcellulose, talc, yellow iron oxide, ethyl cellulose, titanium dioxide (E171), triacetin and sodium lauryl sulfate. In a particular embodiment, the tablet comprises about 100 mg of the compound of the present invention, and about 7.5 mg of lactose monohydrate.

[0110] 40 Alternatively, the pharmaceutical composition can be administered in the form of suppositories for

[0111] Received at EPO via Web-Form on Jul 18, 2023 rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0112] 5

[0113] The pharmaceutical composition can also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0114] 10

[0115] For topical applications, the pharmaceutical composition can be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compound of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0116] For ophthalmic use, the pharmaceutical composition can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, particularly, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions can be formulated in an ointment such as petrolatum.

[0117] 25

[0118] The pharmaceutical composition can also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters

[0119] 30 to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0120] The amount of compound as defined in the first aspect of the present invention that can be combined with the excipient or vehicle to produce a single dosage form will vary depending upon the subject treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular subject will depend upon a variety of factors, including the activity of the compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of compound can also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co-administered.

[0121] 40

[0122] Received at EPO via Web-Form on Jul 18, 2023 A therapeutically effective amount of a pharmaceutical composition is the amount which is required to confer a therapeutic effect on the treated patient, and will depend on a variety of factors, such as the size of the subject, the goal of the treatment, the nature of the pathology to be treated, the pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between

[0123] 5 about 0.001 and about 100 mg / kg body weight per day, particularly between about 0.1 and about 10 mg / kg body weight per day of the compound as defined in the first aspect of the present invention can be useful.

[0124] In an embodiment, the pharmaceutical composition comprises about 25 mg to about 300 mg, partic¬

[0125] 10 ularly about 50 mg to about 150 mg, of the compound as defined in the first aspect of the present invention.

[0126] Depending on the dosage, it is expedient to administer the daily dose in several dosage units.

[0127] Particularly for the compound according to the first aspect of the present invention, the dosage units can be once daily, twice daily, or three times daily. In an embodiment, the compound is administered in one dosage (e.g., 100 or 200 mg per dosage unit). In another embodiment, the compound is administered in two dosages (e.g., 100 mg or 200 mg per dosage unit). In another embodiment, the compound is administered in three dosages (e.g., 100 mg or 200 mg per dosage unit).

[0128] In an embodiment, the compound is administered in a dose of about: 100 mg / day, 200 mg / day, 300 mg / day, 400 mg / day, 500 mg / day, or 600 mg / day.

[0129] Medical uses

[0130] 25

[0131] As described, the examples of the present invention provide evidence of the TRR kinetic stabilizing activity of the compound described herein and its potential use for the treatment of transthyretin amyloidosis. It is observed that compound of formula (I) increases the stability of TTR tetramers in vitro and consequently inhibits their aggregation. Remarkably, the compound performed better than

[0132] 30 tolcapone, inhibiting up to 89.2% and 92% the aggregation of WT-TTR and V30M-TTR, respectively. The compound strongly interacts with the T4 binding sites of the tetramer, with an affinity of 16, 36 and 14 nM for WT-, V30M- and V122I-TTR, respectively, which is significantly better than that of tolcapone. Furthermore, the compound shows a low risk of cytotoxicity and a favorable PK profile in vivo, with a 10.1 h half-life and 85.1 % oral bioavailability.

[0133] Accordingly, another aspect of the invention relates to a compound as defined in the first aspect of the invention for use as a medicament.

[0134] In another aspect, the invention refers to a compound as defined in the first aspect of the invention

[0135] 40 or a pharmaceutical composition provided herein for use in the treatment of a transthyretin

[0136] Received at EPO via Web-Form on Jul 18, 2023 amyloidosis (ATTR) (also known as a TTR-associated amyloidosis) in a subject.

[0137] Alternatively, an aspect of the invention refers to a method for the treatment of a transthyretin amyloidosis comprising administering a therapeutically effective amount of a compound as defined in the

[0138] 5 first aspect of the present invention or a pharmaceutical composition provided herein to a subject in need thereof.

[0139] Alternatively, an aspect of the invention refers to the use of a compound as defined in the first aspect of the present invention for the manufacture of a medicament for the treatment of a transthyretin

[0140] 10 amyloidosis.

[0141] The most common forms of ATTR are related with the aggregation of TTR mutants and inherited in an autosomal dominant manner. To date, ATTR onset has been associated with more than 130 genetic variants, most of them causing peripheral neuropathy (known as familial amyloid polyneuropathy - FAP) or cardiomyopathy (known as familial amyloid cardiomyopathy - FAC). In some rare cases, TTR mutations can induce leptomeningeal amyloidosis, which is often accompanied by ocular involvement.

[0142] The most common amyloidogenic variant worldwide is the V30M-TTR mutation, which is associated with FAP. It has been identified as the predominant mutation in several populations affected by transthyretin amyloidosis, including Portugal, Sweden, and some Japanese populations. V122I-TTR mutation is also relatively common, particularly in the United States, especially in individuals of African descent or West Africa, and is associated with FAC.

[0143] 25 Familial amyloid polyneuropathy is disclosed e.g., in the review article of V. Pante-Bordeneuve et al., 2011 . Familial amyloid cardiomyopathy is disclosed e.g., in C. Rapezzi et al., 2010. Leptomeningeal amyloidosis is disclosed e.g., in H. Goren and MC. Steinberg, 1980.

[0144] ATTR can also be caused by the wild-type (WT) protein. The deposition of WT-TTR, especially in

[0145] 30 the heart, underlies the development of senile systemic amyloidosis, a disease that mainly affects elderly man. Senile systemic amyloidosis is increasingly recognized as an important cause of heart failure, affecting up to 25% of individuals over 80 years of age. Senile systemic amyloidosis is disclosed e.g., in P. Westermark et al., 1990.

[0146] Accordingly, in an embodiment, the transthyretin amyloidosis is selected from the group consisting of familial amyloid polyneuropathy (FAP), senile systemic amyloidosis, familial amyloid cardiomyopathy (FAC), and leptomeningeal amyloidosis.

[0147] In an embodiment, the transthyretin amyloidosis is an hereditary amyloidosis, particularly, familial

[0148] 40 amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) and leptomeningeal

[0149] Received at EPO via Web-Form on Jul 18, 2023 amyloidosis.

[0150] In a particular embodiment, the transthyretin amyloidosis is familial amyloid polyneuropathy (FAP). In another embodiment, the transthyretin amyloidosis is senile systemic amyloidosis. In another em¬

[0151] 5 bodiment, the transthyretin amyloidosis is familial amyloid cardiomyopathy (FAC). In yet another embodiment, the transthyretin amyloidosis is leptomeningeal amyloidosis.

[0152] In some embodiments, the ATTR is associated with a mutation in transthyretin (TTR) protein / gene (i.e. , amyloidogenic TTR variants). Particularly, the TRR mutation is selected from mutations listed

[0153] 10 in http: / / amyloidosismutations.com / mut-attr.php

[0154] In an embodiment, the familial amyloid polyneuropathy is associated with V30M, V28M, L55P, L58H and / or S77Y transthyretin mutations / variants. In a particular embodiment, the familial amyloid polyneuropathy is associated with V30M transthyretin variant.

[0155] In another embodiment, the familial amyloid cardiomyopathy is associate with V122I, T60A, I68L and / or L1 11 M transthyretin mutations / variants. In a particular embodiment, the familial amyloid cardiomyopathy is associated with V122I transthyretin variant.

[0156] S77Y mutation is one of the most frequent mutations in France (apart from V30M); L55P is the most aggressive TTR variant; I68L is the most frequent mutation in Italy; and L111 M is the most frequent in Denmark.

[0157] The terms "treat", "treatment", "therapy", as used herein refer to a clinical intervention to prevent

[0158] 25 (e.g., suppress or inhibit) a disease or condition; cure the disease or condition; delay the onset of the disease or condition; reduce the seriousness or severity of the disease or condition; ameliorate or eliminate one or more symptoms or sequelae associated with a disease or condition; or the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.

[0159] 30

[0160] In some embodiments, the term refers to a clinical intervention to improve one or more symptoms; improve one or more sequelae; prevent (e.g., suppress, inhibit or delay) one or more symptoms; prevent (e.g., suppress, inhibit or delay) one or more sequelae; delay one or more symptoms; delay one or more sequelae; ameliorate one or more symptoms; ameliorate one or more sequelae; shorten the duration of one or more symptoms; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more sequelae; improve the quality of life; increase survival; prevent (e.g., suppress, inhibit or delay) a recurrence of the disease or condition; delay a recurrence of the disease or condition; reduce the severity of the disease; or any

[0161] 40 combination thereof, e.g., with respect to what is expected in the absence of the treatment with the

[0162] Received at EPO via Web-Form on Jul 18, 2023 compound as defined in the first aspect of the present invention.

[0163] The term "treatment" also includes prophylaxis or prevention (e.g., suppression, inhibition or delay) of a disease or condition or its symptoms or sequelae thereof. Prophylaxis refers to a therapeutic or

[0164] 5 course of action used to prevent, inhibit, suppress, reduce the risk, reduce the occurrence or delay the onset of a disease or condition, or to prevent, inhibit, suppress, or delay a symptom associated with a disease or condition.

[0165] The terms "subject", "patient", and "individual", and variants thereof are used interchangeably herein

[0166] 10 and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The uses and methods described herein are applicable to both human therapy and veterinary applications.

[0167] In an embodiment, the subject is a mammal, including a human. In a further particular embodiment, the subject is a human subject.

[0168] In some embodiments, the subject presents a mutation in the transthyretin (TTR) protein / gene. Particularly, the TRR mutation is selected from mutations listed in http: / / amyloidosismutations.com / mut- attr.php.

[0169] In another embodiment, the subject presents:

[0170] (i) a mutant TTR comprising at least one point mutation selected from the group consisting of V30M,

[0171] 25 V28M, L55P, L58H, S77Y, V122I, T60A, I68L and L1 11 M, wherein the numbering of said amino acid positions is relative to the full-length sequence of TTR (Uniprot P02766, version 1 , date June 28, 2023), or

[0172] (ii) a mutant TTR comprising at least one amino acid substitution at a position in the sequence of TTR (Uniprot P02766, version 1 , date June 28, 2023) selected from the group consisting of positions

[0173] 30 30, 28, 55, 58, 77, 122, 60, 68, 111 , or a combination thereof.

[0174] The symptoms and sequelae of ATTR can vary depending on the type and progression of the disease. Some common symptoms and sequelae associated with ATTR are:

[0175] - peripheral neuropathy, e.g., numbness, tingling, and weakness in the extremities, starting in the lower limbs and progressing upwards. Loss of sensation and motor function may lead to difficulty in walking or performing fine motor tasks;

[0176] - autonomic dysfunction: impaired regulation of involuntary bodily functions, such as blood pressure, heart rate, digestion, and sweating. Symptoms can include orthostatic hypotension (low blood pressure upon standing), gastrointestinal disturbances, sexual dysfunction, and abnormal sweating;

[0177] 40 - cardiac involvement: cardiomyopathy, which is characterized by the stiffening and thickening of the

[0178] Received at EPO via Web-Form on Jul 18, 2023 heart muscle, leading to impaired pumping function. Symptoms can include shortness of breath, fatigue, chest pain, palpitations, and eventually heart failure;

[0179] - gastrointestinal involvement: in some cases of ATTR, the gastrointestinal tract can be affected, leading to symptoms such as abdominal pain, diarrhea, constipation, and weight loss;

[0180] 5 - ocular manifestations: deposition of amyloid fibrils in the eye can cause various ocular abnormalities, including dry eyes, blurred vision, and vitreous opacities;

[0181] - renal involvement: ATTRwt, in particular, can result in the deposition of amyloid fibrils in the kidneys, leading to renal impairment and potentially progressing to end-stage renal disease;

[0182] - central nervous system involvement: in rare instances, ATTR can involve the central nervous sys¬

[0183] 10 tem, leading to cognitive impairment, dementia, and movement disorders; and

[0184] - other organ involvement: amyloid deposition can affect other organs, such as the liver, lungs, and spleen, although the severity and impact vary.

[0185] The main symptoms and sequelae of ATTR for each type of ATTR can be:

[0186] - familial amyloid polyneuropathy (FAP): peripheral neuropathy and autonomic dysfunction;

[0187] - senile systemic amyloidosis: cardiac involvement and other organ involvement;

[0188] - familial amyloid cardiomyopathy (FAC): cardiac involvement; and

[0189] - leptomeningeal amyloidosis: central nervous system involvement.

[0190] Accordingly, in some embodiments, the administration of the compound as defined in the first aspect of the invention results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of:

[0191] - stabilization of transthyretin (TTR) tetramers;

[0192] - preservation of nerve function;

[0193] 25 - slowed disease progression;

[0194] - prolonged survival;

[0195] - reduction in amyloid deposition;

[0196] - improved cardiac function (e.g., slowing or halting the progression of cardiomyopathy, or improved heart muscle function);

[0197] 30 - enhanced mobility;

[0198] - improved sensory function (e.g., reduction or alleviation of numbness or tingling);

[0199] - enhanced motor function (e.g., slowing or halting the progression of muscle weakness);

[0200] - improved blood pressure regulation;

[0201] - enhanced gastrointestinal function (e.g., alleviation of diarrhea or constipation, or improvement of digestion);

[0202] - better control of sweating;

[0203] - reduced risk of heart failure (e.g., prevention or delay of the onset of heart failure);

[0204] - slowed progression of renal impairment (e.g., delay or prevention end-stage renal disease); and

[0205] - cognitive preservation (e.g., slowing the progression of cognitive impairment and dementia).

[0206] 40

[0207] Received at EPO via Web-Form on Jul 18, 2023 The invention also refers to a compound as defined in the first aspect of the invention in combination with one or more therapeutic agents, particularly for the treatment of a transthyretin amyloidosis.

[0208] In a particular embodiment, the additional therapeutic agent is selected from the group consisting of

[0209] 5 tolcapone, entacapone, tafamidis, AGI O, iododiflunisal, diflunisal, and benzbromarone or pharmaceutically acceptable salts thereof; and more particularly, in combination with tolcapone, tafamidis, AGI O, and diflunisal, or pharmaceutically acceptable salt thereof.

[0210] The combination of the compound as defined in the first aspect and the other therapeutic agent(s)

[0211] 10 can be administered in a single pharmaceutical composition, or in two different pharmaceutical compositions, either administered simultaneously, sequentially or separately after a certain period of time. Particularly, if the administration is not simultaneous, the compounds are administered in a relatively close time proximity to each other. Furthermore, compounds are administered in the same or different dosage form or by the same or different administration route, e.g., one compound can be administered intravenously and the other compound can be administered orally.

[0212] Therefore, the term "combination" relates herein to the various combinations of the compound as defined in the first aspect and other therapeutic agent(s), e.g., in a single pharmaceutical composition, in a combined mixture composed from separate pharmaceutical formulations / compositions of the single active compounds, such as a "tank-mix", and in a combined use of the single active ingredients when applied in a sequential manner, i.e. one after the other with a reasonably short period, such as a few hours or days or in simultaneous administration. The order of applying the compounds is not essential.

[0213] 25 For instance, the compound as defined in the first aspect is independently administered from the other therapeutic agent(s) (i.e., in two units) but at the same time. In another suitable example, the compound of the present invention is administered first and then the other therapeutic agent(s) is separately or sequentially administered; alternatively, the other therapeutic agent(s) is administered first and then the compound of the present is separately or sequentially administered.

[0214] 30

[0215] EXAMPLES

[0216] EXAMPLE 1 : MATERIALS AND METHODS

[0217] 1.1

[0218] Tolcapone was purchased from Fisher. PITB (I) was chemically synthesized as shown in Scheme I. The detailed synthesis procedure is described below.

[0219] Chemical synthesis, general. Commercially available reagents were used as received. Solvents were

[0220] 40 dried by distillation over the appropriate drying agents. All reactions were monitored by analytical

[0221] Received at EPO via Web-Form on Jul 18, 2023 thin-layer chromatography (TLC) using silica gel 60 precoated aluminum plates (0.20 mm thickness). Flash column chromatography was performed using silica gel Geduran® SI 60 (40-63 pm).1H NMR, 19F NMR and13C NMR spectra were recorded at 250, 400 MHz and 90, 100 MHz, respectively.1H NMR spectra were referenced to the residual proton signals of CDCI3, 7.26 ppm, and acetone-ck

[0222] 5 2.05 ppm.13C NMR spectra were referenced to the residual13C signal of CDCh, 77.16 ppm, and acetone-ck, 29.84 ppm. The purity of the PITB was determined to be over 95% by HPLC analysis. Melting points were determined on hot stage and are uncorrected. HRMS were recorded using electrospray ionization (ESI).

[0223] 10 (3,4-dimethoxy-5-nitrophenyl)(3-fluoro-5-methoxyphenyl)methanone, (l)-1. To a solution of (l)-2 (2.50 g, 9.93 mmol) in dry tetrahydrofuran (THF) (25 mL) at -40 °C, / PrMgBr (1 M in THF, 15.9 mL,

[0224] I I .92 mmol) was added dropwise under an Ar atmosphere and the reaction mixture was stirred for 1 h. Then, a solution of 3,4-dimethoxy-5-nitrobenzaldehyde (2.53 g, 12.00 mmol) in dry THF (20 mL) was added and the mixture was warmed to room temperature (rt) and stirred overnight. The reaction was quenched by the slow addition of brine (20 mL) and the aqueous layer was extracted with EtOAc (3 x 35 mL). The resulting organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to furnish the corresponding alcohol as a yellow oil (1 .98 g) which was used in the next step without further purification. To a solution of the alcohol (1.98 g, 5.87 mmol) in dry CH2CI2 (90 mL) under an Ar atmosphere, a solution of Dess-Martin periodinane (DMPI) (5.08 g, 12.03 mmol) in dry CH2CI2 (30 mL) was added dropwise and the mixture was stirred overnight at rt. The reaction was quenched with the addition of 12 mL of a prepared solution of Na2S20s (17.0 g) in a saturated aqueous solution of NaHCOs (80 mL) and the mixture was stirred for 15 min. The aqueous phase was extracted with CH2CI2 (3 x 50 mL) and the combined organic extracts were dried over anhydrous Na2SO4 and concentrated under vacuum. Purification by flash column chromatography (hex-

[0225] 25 anes / EtOAc, 5:2) of the resulting residue provided the ketone (l)-1 as a white solid (1.92 g, 5.72 mmol, 58% yield for the two-steps). (I)-1 : Mp 96 - 94 °C (from CHCb).1H NMR (400 MHz, CDCh) 6 7.67 (d, J = 2.0 Hz, 1 H), 7.62 (d, J = 2.0 Hz, 1 H), 7.06 (m, 1 H), 7.02 (ddd, J = 8.4 Hz, J = 2.3 Hz, J = 1.4 Hz, 1 H), 6.86 (dt, J = 10.2 Hz, J = 2.3 Hz, 1 H), 4.06 (s, 3H), 3.97 (s, 3H), 3.84 (s, 3H);19F NMR (250 MHz, CDCh) 6 -110.1 (s);13C NMR (90.5 MHz, CDCh) 6 192.4 (d, J =2.8 Hz), 163.3 (d, J =

[0226] 30 248.3 Hz), 161.1 (d, J = 10.8 Hz), 154.4, 146.7, 144.2, 139.0 (d, J = 8.4 Hz), 132.0, 118.7, 116.3,

[0227] I I I .2 (d, J = 2.8 Hz), 109.1 (d, J = 23.1 Hz), 106.4 (d, = 25.0 Hz), 62.4, 56.8, 56.1. HRMS (ESI+) calcd. for [Ci6Hi4FNO6+Na]+358.0703; found [M+Na]+358.0705.

[0228] (3-fluoro-5-hydroxyphenyl)(4-hydroxy-3-methoxy-5-nitrophenyl)methanone, PITB (I). BBra (1 M in CH2Cl2, 9.50 mL, 9.50 mmol) was added dropwise to a solution of (l)-1 (400 mg, 1 .19 mmol) in DCM (CH2CI2) (6.0 mL) at -10 °C. The reaction was allowed to proceed at rt for 1 .5 h (TLC, CH2Cl2 / MeOH, 10:1). The reaction was quenched with water (8 mL) and the resulting aqueous layer was extracted with EtOAc (2 x 20 mL). The organic extracts were dried with anhydrous Na2SO4 and the solvent removed under vacuum to give the crude product which was purified by flash column chromatog¬

[0229] 40 raphy (CH2Cl2 / MeOH, 20:1) to afford PITB (I) as a yellow solid (31 1 mg, 1 .01 mmol, 85% yield). Mp

[0230] Received at EPO via Web-Form on Jul 18, 2023 157 - 155 °C (from CHCb).1H NMR (300 MHz, acetone-cfe) 5 8.06 (br s, 1 H), 7.61 (br s, 1 H), 7.07 (br s, 1 H), 6.97 (br d, J = 9.0 Hz, 1 H), 6.87 (dt, J = 10.4 Hz, 1 H), 4.03 (s, 3H);19F NMR (250 MHz, CDCb) 6 -110.01 ;13C NMR (125 MHz, acetone-cfe) 6 192.4, 164.0 (d, J = 244.3 Hz), 162.4, 159.7 (d, J= 11.6 Hz), 155.1 , 142.1 (d, J = 9.2 Hz), 135.1 , 125.5, 120.3, 113.1 , 112.3, 107.6 (d, J = 23.2 Hz), 106.4 (d, J = 24.2 Hz), 57.2. HRMS (ESI-) calcd. for [C14H10FNO6-H]- 306.0414; found [M-H]- 306.0424.

[0231] 1.2 Recombinant TTR expression and purification

[0232] The cDNA encoding for WT-TTR was cloned into a pET28a vector (Novagen). The vectors encoding for V30M- and V122I-TTR were prepared by classical site-directed mutagenesis protocols using the WT-TTR vector as a template. WT-, V30M- and V122I-TTR were expressed in Escherichia coli BL21 (DE3) and purified as previously described (F. Pinheiro et al., 2021). The purest fractions eluting from the gel filtration chromatography were combined and kept at -20 °C until use. The concentration of protein was determined spectrophotometrically at 280 nm, using a molar extinction coefficient of 77 600 M-1cm1.

[0233] 1.3 Urea-induced TTR tetramer dissociation measured by Trp fluorescence

[0234] Samples containing TTR (1 .8 pM in PBS) were incubated with 3.6 pM PITB or tolcapone for 30 min at RT, and 6 M urea was added to trigger denaturation. Since the stock solutions of both compounds were prepared in dimethyl sulfoxide (DMSO), a control sample containing the same percentage of DMSO instead of compound was prepared. Trp fluorescence spectra were recorded over time (Aex = 295 nm; Aem = 310-410 nm) using an FP-8200 Spectrofluorometer (Jasco). Upon denaturation, the Trp residues become more exposed to the polar solvent, which is accompanied by a change in the emission maximum from ~ 335 to ~ 355 nm. The fluorescence intensity ratio 355 / 335 nm was calculated for each time point and the values normalized from minimum (folded state) to maximum (unfolded state), with the maximum being the one of the control sample after 96 h incubation.

[0235] 1.4 TTR in vitro aggregation inhibition

[0236] To assess the effect of PITB and tolcapone in the aggregation of WT-, V30M- and V122I-TTR, a well- established TTR aggregation assay was employed. Briefly, TTR solutions (7.2 pM in 10 mM sodium phosphate, 100 mM KCI, 1 mM EDTA, 1 mM DTT, pH 7.0) were incubated with varying concentrations of test compound for 30 min at 37 °C. The percentage of DMSO was the same in all samples (5%). Following incubation, the pH of the solutions was dropped to 4.2 via addition of an equal volume of acidification buffer (100 mM sodium acetate, 100 mM KCI, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), pH 4.2) and the samples were kept at 37 °C for more 72 h. The aggregation was assessed by measuring turbidity at 340 nm using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). Since PITB and tolcapone display dose-dependent absorbance at 340 nm, the turbidity of a sample containing the same concentration of compound, but lacking TTR, was subtracted to every measurement. The percentage of TTR aggregation for

[0237] Received at EPO via Web-Form on Jul 18, 2023 each inhibitor concentration was calculated by dividing the turbidity of the test sample by that of a sample aggregated in the absence of compound, and multiply by 100.

[0238] 1.5 Isothermal titration calorimetry (ITC)

[0239] 5 All ITC measurements were carried out with protein and compounds dissolved in the same buffer (in a PBS buffer pH 7.0 containing 100 mM KCI, 1 mM EDTA and 2.5% DMSO), and degassed. The ITC measurements were performed at 25 °C using a Nano ITC calorimeter (TA Instruments). Titrations were performed as a set of 20 injections of 100 pM compound into protein (5 pM), programmed as a 2.02 pl injection with 150 s of equilibration period. The NanoAnalyze software (TA Instruments)

[0240] 10 was used to integrate the peaks and find their areas. The fittings were performed with a general model for a protein with two-ligand binding sites implemented in Origin 7.0 (OriginLab), which allows to establish if the binding is cooperative or non-cooperative.

[0241] 1.6 Crystallization and structure determination

[0242] Cocrystals of WT-TTR / PITB, V30M-TTR / PITB, V122I-TTR / PITB and V30M-TTR / tolcapone were obtained as explained previously (F. Pinheiro et al., 2021). Briefly, purified proteins (140 pM) were mixed with 1 .4 mM of PITB / tolcapone and cocrystallized at 18 °C by hanging-drop vapor diffusion methods. The reservoir solution contained 20-30% PEG 400, 200 mM CaCh, 100 mM HEPES, pH 7.0-8.0. The crystals were flash-frozen in liquid nitrogen (100 K) and diffraction data were collected at the BL13-XALOC beamline from the ALBA Synchrotron in Barcelona. Data were integrated and merged using XDS and scaled, reduced, and further analyzed using CCP4. The structures of TTR / PITB and V30M-TTR / tolcapone complexes were determined from the X-ray data by molecular replacement with Phenix (version 1.19.2-4158) using a previous TTR structure (PDB 1 F41) as a model. Model refinement and building were done with Phenix and Coot, respectively. The structural

[0243] 25 representations were prepared using Pymol (The PyMOL Molecular Graphics System, Version 2.0, Schrodinger, LLC).

[0244] 1.7 T4 binding competition assays

[0245] The ability of PITB and tolcapone to displace T4 from plasmatic TTR was evaluated by incubation of

[0246] 30 whole plasma (5 pl) with 1 pl of [125I]-T4 (specific radioactivity 1250 pCi / pg; concentration 320 pCi / mL; Perkin Elmer) in the presence of the test compounds (5 molar excess relative to TTR tetramer). A negative control containing the same percentage of DMSO than the samples was also prepared. Following 1 h incubation at room temperature, plasma proteins were separated by native PAGE, the gels were dried, and subjected to phosphor imaging using a Typhoon 8600 variable mode imager (Molecular Diagnostics, Amersham Biosciences). Then, the autoradiography films were scanned, and the bands quantified using Image Lab software version 5.2.1 (Bio-Rad). The amount of T4 bound to TTR in comparison to total T4 (T4-TBG (globulin) + T4-ALB (albumin) + T4-TTR) was determined for each sample and normalized to the maximum value, which corresponds to the negative control. Plasma from three V30M-TTR carriers was analyzed.

[0247] 40

[0248] Received at EPO via Web-Form on Jul 18, 2023 1.8 TTR stabilization in human plasma assessed by isoelectric focusing

[0249] To assess the effect of PITB and tolcapone on the stability of TTR in plasma, isoelectric focusing (IEF) under semi-denaturing conditions was performed. To carry out this assay, 30 pl of human plasma from V30M-TTR carriers (n = 3) were incubated overnight at 4 °C in the presence of 19.1 pM

[0250] 5 compounds. Additionally, control samples containing the same amount of DMSO rather than compound were prepared. After incubation, the samples were subjected to native PAGE, and the TTR gel band was excised and applied to an IEF gel. IEF was performed in the presence of 4 M urea (semi-denaturing conditions) and 5% (v / v) ampholytes (pH 4-6.5; Sigma-Merck), at 1200 V for 5 h. Proteins were then fixed with 20% trichloroacetic acid and stained with Coomassie blue. The gels

[0251] 10 were scanned using a GS-900 calibrated densitometer (Bio-Rad) and the bands analyzed by densitometry with the Image Lab software version 5.2.1 (Bio-Rad). The ratio of the TTR tetramer over total TTR (TTR tetramer + monomer) was determined for each sample, and the percentage of tetramer stabilization was calculated as ((ratio treated sample - ratio control sample) / ratio control sample) x 100.

[0252] 1.9 Cytotoxicity studies

[0253] The potential chemical toxicity of PITB was evaluated in vitro in HeLa (ATCC CCL-2) and HepG2 (ATCC HB-8065) human cell lines. Tolcapone was tested in parallel for comparison. HeLa and HepG2 cells were cultured in MEM ALPHA medium (Gibco) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 humidified atmosphere. HeLa cells were seeded at 3500 cells / well and HepG2 cells at 4500 cells / well in 96-well plates and incubated with increasing concentrations of compound (2-100 pM) for 72 h at 37°C. Controls were prepared with the equivalent amount of DMSO relative to each concentration of compound. Then, 10 pl of PrestoBlue® reagent (Thermo Fisher Scientific) were added to each well, and after incubating for 15 min at 37°C, the fluorescence emis¬

[0254] 25 sion was recorded using a 590 / 20 filter with an excitation wavelength of 535 nm in a Victor3 Multilabel Reader (PerkinElmer). The assays were performed in triplicate and the percentage of cell viability for each well was calculated as (intensity sample - mean intensity blank) / (mean intensity control - mean intensity blank) x 100, where "mean intensity blank" corresponds to the mean intensity of wells with PrestoBlue® alone and "mean intensity control" is the mean intensity of wells that contain the

[0255] 30 corresponding percentage of DMSO.

[0256] 1.10 In vivo pharmacokinetic studies in mice

[0257] Inventors conducted PK studies of PITB (intravenous, IV and oral, PO) and tolcapone (oral, PO) in CD-1 mice. For each study, 24 male CD-1 mice were weighted and identified by a distinct number at the base of the tail.

[0258] For the IV studies, PITB was dissolved in 5% Cremophor / 5% Mannitol / 5% DMSO. The compound was administered at 1 mg / Kg, with an administration volume of 5 mL / Kg. At different timepoints after administration (0.0833, 0.5, 1 , 2, 4, 6 and 24 h), three animals were anesthetized with isoflurane,

[0259] 40 and their blood collected by cava vein puncture in tubes containing K2-EDTA 5%. Three animals

[0260] Received at EPO via Web-Form on Jul 18, 2023 were not administered and referred as t = 0 h (predose). Blood samples were centrifuged at 10,000 rpm for 5 min to obtain the plasmas, which were stored at -80 °C until analysis.

[0261] For the PO studies, PITB and tolcapone were formulated in 0.5% CMC / 0.1 % tween 80 and admin¬

[0262] 5 istered at 10 mg / Kg. The administered volume was 10 mL / Kg. Plasma samples were obtained as described above at 0.25, 0.5, 1 , 2, 4, 6 and 24 h post-dosing (three animals for time point). Three animals were used as blank with no administration (predose). All plasma samples were analyzed using the API 3200 LC-MS / MS system (Sciex) coupled to an UPLC-Acquity (Waters). A calibration curve was done for each compound in plasma. The lower limit of quantification (LLOQ) for PITB and

[0263] 10 tolcapone were 7.68 ng / mL and 2.73 ng / mL, respectively. PK parameters were determined with a non-compartmental model using Phoenix 64 8.3 (WinNolin) software.

[0264] RESULTS

[0265] The following examples describe experiments wherein the techniques and the specific exemplified materials and devices described in EXAMPLE 1 were used.

[0266] EXAMPLE 2: PITB kinetically stabilizes TTR in vitro

[0267] Inventors tested whether PITB could protect TTR from urea-induced tetramer dissociation. In urea, TTR subunits do not unfold until the tetramer dissociates. Since monomer unfolding is much faster than tetramer dissociation, it is possible to evaluate the rate of tetramer dissociation by irreversibly linking these two processes. This can be achieved by using urea concentrations in the post-transition zone for tertiary structural changes, which assures that monomers unfold extremely fast and cannot

[0268] 25 refold. Accordingly, WT-TTR and the two most clinically important TTR variants, V30M- and V122I- TTR (1.8 pM) were incubated in the absence or presence of PITB (3.6 pM) and TTR denaturation was initiated by adding urea to a final concentration of 6 M. Tryptophan (Trp) intrinsic fluorescence was measured each 24 h and used to calculate the fraction of unfolded protein (FIG. 1 (A)). The effect of PITB was compared to the one of tolcapone.

[0269] 30

[0270] In the presence of equimolar levels of PITB relative to T^binding sites, 94.1 ± 0.5%, 79.2 ± 0.2% and 92.5 ± 1.6% of WT-, V30M- and V122I-TTR, respectively, tetramers were protected from urea-induced dissociation, indicating that PITB exerts a higher stabilizing effect over these proteins. Importantly, PITB stabilizing activity was equivalent to the one exerted by tolcapone for each of the analyzed proteins.

[0271] EXAMPLE 3: PITB protects TTR from aggregation in vitro

[0272] After confirming that PITB can prevent tetramer dissociation, it was assessed whether this effect is

[0273] 40 translated into an inhibition of TTR aggregation. For this purpose, TTR samples were mixed with

[0274] Received at EPO via Web-Form on Jul 18, 2023 increasing concentrations of PITB and aggregation was induced by acidification. After an incubation of 72 h at 37 °C, aggregation was quantified by recording turbidity at 340 nm and normalized to the maximum turbidity value, which was assigned to the TTR samples incubated in the absence of compound (FIG. 1 (B)).

[0275] The data indicate that PITB is highly effective at inhibiting the aggregation of WT-TTR and, most importantly, of V30M- and V122I-TTR. In all cases, there was a decrease in TTR aggregation >60% at a 1 :1 TTR / PITB ratio and >87.5% when PITB concentration is higher than or equal to the one of T4-binding sites. Remarkably, PITB performed better than tolcapone, inhibiting up to 89.2% and 92% the aggregation of WT-TTR and V30M-TTR, respectively. In the case of V122I-TTR, PITB equaled the potency of tolcapone, reaching up to 90% inhibition at 20 pM.

[0276] EXAMPLE 4: PITB binds with high affinity to TTR

[0277] After confirming the high stabilization and anti-aggregation effect of PITB on WT-, V30M- and V122I- TTR, its binding affinity to these proteins was assessed. To determine the binding parameters of PITB to TTR, isothermal titration calorimetry (ITC) was used. Most ligands bind to TTR with negative cooperativity, which means that when the first binding site is occupied, there is a loss of affinity for the second one. To account for this possibility, ITC data was analyzed using two models: one that considers independent binding to both sites, and other that assumes cooperative binding.

[0278] The obtained data indicated that PITB binds with high affinity and no cooperativity to both WT and TTR variants V30M and V1 121 (FIG. 2 and Table 1). Moreover, PITB binds to WT- and V122I-TTR with an affinity > 3-fold than that of tolcapone, and to V30M-TTR with an affinity > 12-fold. Remarkably, PITB presents a significantly higher affinity for V30M- and V122I-TTR than tafamidis (available data for tafamidis: V30M-TTR: Kd1 = 153.8 nM and Kd2 = 625 nM; V122I-TTR: Kd1 = 8.1 nM and Kd2 ~ 1 pM), the only kinetic stabilizer approved for FAP and FAC. Independently of the tested protein, the binding of PITB was entirely enthalpically driven (AH < 0; -TAS > 0), suggesting the establishment of noncovalent interactions (e.g., hydrogen bonds or ionic interactions) at the binding interface.

[0279] Table 1. Thermodynamic parameters for the interaction of PITB with WT-, V30M- and V122I-TTR determined by ITC.

[0280] Received at EPO via Web-Form on Jul 18, 2023

[0281] Of note, the much higher binding affinity of PITB for the FAP-related variant V30M might be translated into an improved biological activity for this variant.

[0282] EXAMPLE 5: PITB stabilizes the weaker TTR dimer-dimer interface

[0283] To elucidate the interactions that drive the binding of PITB to WT-TTR and TTR variants V30M and V122I, the X-ray crystal structures of TTR:PITB complexes were determined at 1 .85 (WT-TTR:PITB), 1.20 (V30M-TTR:PITB) and 1.42 (V122I-TTR:PITB) A resolution (FIG. 3). Additionally, inventors obtained, for the first time, the cocrystal structure of tolcapone with V30M-TTR, at a resolution of 1 .57 A.

[0284] PITB binds to TTR T^binding sites at the weaker AB / CD dimer-dimer interface. Because of the twofold symmetry axis that runs through the binding pockets, two symmetry-related binding conformations appear for PITB. In all structures, PITB was found in the forward binding mode, with the 3- methoxy-4-hydroxy-5-nitrophenyl pointing to the outer binding cavity, where it makes hydrophobic interactions with the residues from halogen binding pockets (HBPs) 2 / 2’ and 1 / 1 ’ (FIG. 3). In this orientation, the 4-hydroxy group of PITB forms hydrogen bonds with the K15 / K15’ residues of TTR, which in turn stabilize their electrostatic interactions with E54 / E54’. These interactions by K15 are also observed in TTR:tolcapone complexes and were proposed to close the binding cavity, protecting the protein-ligand complex from the solvent.

[0285] On the other hand, PITB establishes important interactions in the inner binding cavity, which are not present in the TTR:tolcapone structures. In particular, the fluorine atom of the 3-fluoro-5-hydroxy- phenyl ring interacts with A108, while the hydroxy group forms hydrogen bonds with the S117 residues from adjacent subunits. These additional contacts by PITB might justify its increased affinity for WT-, V30M- and V122I-TTR in comparison to tolcapone.

[0286] Remarkably, the hydrogen bonds between PITB and S117 help to bridge the dimers at the weaker dimer-dimer interface, increasing the kinetic barrier for tetramer dissociation. Additionally, in one of the S1 17 side-chain conformations, a short hydrogen bond can be formed between the S117 residues from adjacent monomers in the same dimer (FIG. 3(D)). This inter-subunit interaction was also observed in the structure of the kinetically stabilized TTR variant T119M and could further contribute to the high stabilizing effect of PITB.

[0287] EXAMPLE 6: PITB selectively binds and stabilizes WT-TTR and V30M-TTR in human plasma

[0288] Received at EPO via Web-Form on Jul 18, 2023 To stabilize the native tetrameric TTR and thus prevent its aggregation in patients with ATTR, PITB must selectively bind to TTR in plasma over all other plasmatic proteins. To test this, inventors examined the capacity of PITB to compete with T4 for TTR binding in human plasma from control individuals (WT-TTR) and patients carrying V30M-TTR. Tolcapone was included as a control. In this

[0289] 5 assay, plasma samples were incubated with [125I]-T4, in the absence or presence of compounds, and subjected to native gel electrophoresis. T4-binding proteins were detected by autoradiography, as shown in FIG. 4(C). In the absence of any compound, three main plasma proteins bound to T4: the main binding protein was T4-binding globulin (TBG), followed by albumin (ALB) and TTR. The samples incubated with tolcapone and, especially, PITB, presented weaker T4-TTR bands, suggesting

[0290] 10 that these molecules can compete the binding of T4 to TTR in human plasma. Densitometry analysis of the bands revealed that PITB is a more potent competitor than tolcapone, presenting a displacement of T4 binding to plasmatic TTR of 97.3 ± 1 .2% and 94.5 ± 2.8% for normal plasma or plasma from patients, respectively (FIG. 4(A)).

[0291] Once confirmed that PITB selectively binds TTR in human plasma, inventors studied its effect on the stability of TTR in the plasma of normal individuals and FAP V30M patients. The ability of PITB to prevent tetramer dissociation was monitored ex vivo by isoelectric focusing (IEF) under partially dissociating conditions (4 M urea). Tolcapone was also used as a control. Briefly, plasma samples were incubated with or without compound overnight at 4 °C and, after IEF, the bands were compared. Under the conditions used, it is possible to distinguish two main sets of bands, one corresponding to monomers (normal and oxidized forms), and one including several bands of lower pl that represent different forms of tetramers (FIG. 4(D)). The tetramer / monomer ratios were used to calculate the degree of tetramer stabilization in each sample. The results showed that PITB has a higher stabilizing activity than tolcapone, especially in plasma from FAP patients, with an increase in tetramer stability

[0292] 25 of 17.4 ± 5.1 % (versus 4.9 ± 2.3% for tolcapone) (FIG. 4(B)). The increased binding selectivity and stabilization effect of PITB in human plasma, in comparison with tolcapone, is likely due to the higher number of interactions it establishes with the protein, as observed in the crystal structure.

[0293] EXAMPLE 7: PITB displays no toxicity for human cells

[0294] 30

[0295] Compound toxicity is an important concern in the development of new drugs. Therefore, the next step was to examine the potential chemical toxicity of PITB for human cervical carcinoma (HeLa) and human hepatoblastoma (HepG2) cells. These cell lines have been extensively studied and used in in vitro cytotoxicity studies. The HepG2 cell line in particular, is the most commonly used for detecting drug-induced hepatotoxicity. Liver injury is one of the major causes of drug withdrawal and, indeed, many medications require to monitor liver function during treatment, as it is the case of tolcapone.

[0296] In this work, HeLa and HepG2 cells were incubated with increasing concentrations of PITB or tolca¬

[0297] 40 pone and cell viability was assessed after 72 h using PrestoBlue®. For both cell lines, no significant

[0298] Received at EPO via Web-Form on Jul 18, 2023 toxicity was observed upon exposure to PITB, even at the high 100 pM concentration (FIG. 5). Importantly, PITB was substantially less toxic than tolcapone at concentrations higher than 10 pM, suggesting that PITB could be a safer molecule when administered at high doses than tolcapone.

[0299] EXAMPLE 8: Pharmacokinetic (PK) studies in mice confirm the bioavailability and stability of PITB

[0300] Altogether, the previous results support the potential of PITB to become a drug for the treatment of both hereditary and non-hereditary forms of ATTR. To further investigate this option, the PK parameters of PITB were determined from studies conducted in CD-1 mice.

[0301] The PK profiles for PITB following IV (1 mg / Kg) or PO (10 mg / Kg) administration of the compound are shown in FIG. 6(A), and the calculated parameters are depicted in Table 2. Following the IV injection of PITB, a Cmax value of 4043.3 ng / mL and a ti / 2 of 3.5 h were determined. Additionally, the results demonstrated that PITB is rapidly absorbed after PO administration, reaching a Cmax of 4176.7 ng / mL, at 0.5 h (Tmax). While the concentration achieved by the oral route was very similar to that observed for the IV infusion, the elimination of PITB was considerably slower in this case, with a ti / 2 of 10.1 h. In both cases, the concentration of PITB in plasma was still quantifiable 24 h post-dosing. The area under the plasma concentration-time curve (AUC) values were 3162.3 IT ng / mL and 26895.9 h ng / ml for the IV (1 mg / Kg) and PO (10 mg / Kg) administration of PITB, respectively. This yields an excellent oral bioavailability (%F) for PITB of around 85.1 %. Remarkably, the bioavailability of PITB is significantly higher than the one reported for AGI O (26.84%), a TTR kinetic stabilizer that is under clinical trials for ATTR- related cardiomyopathy.

[0302] For comparative purposes, the PK properties of tolcapone after PO administration (10 mg / Kg) in CD- 1 mice were also determined (FIG. 6(B) and Table 2). Following administration of equimolar doses of PITB and tolcapone, the last was more readily absorbed, achieving a Cmax of 7616.7 ng / mL in less than 15 min (Tmax). On the other hand, the elimination of PITB was significantly slower than the one of tolcapone (ti / 2 for PITB = 10.1 h; ti / 2 for tolcapone = 1.4 h), which is due to its higher volume of distribution (Vd) and decreased clearance (Cl). In agreement, no tolcapone was detected in plasma after 24 h, contrarily to PITB. As a result, the exposure to PITB was substantially greater than the one of tolcapone (AUCo-24 for PITB = 26895.9 h ng / mL; AUC0-24 for tolcapone = 11927.5 h ng / mL).

[0303] Table 2. PK parameters of PITB and tolcapone following administration in CD-1 mice.

[0304] Received at EPO via Web-Form on Jul 18, 2023 Overall, PITB seems to have better PK proper¬ ties than tolcapone, which might be translated into an improved pharmacologic activity in vivo.

[0305] Received at EPO via Web-Form on Jul 18, 2023 REFERENCES

[0306] Patent literature

[0307] EP-A-0237929

[0308] Non-patent literature

[0309] F. Pinheiro, et al., "Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation", J. Med. Chem.; 65, 21 , 14673-14691 , October 28, 2022.

[0310] V. Pante-Bordeneuve et al., "Familial amyloid polyneuropathy", Lancet Neurol., 2011 ,10(12), 1086- 97.

[0311] C. Rapezzi et al., "Transthyretin-related amyloidosis and the heart: a clinical overview", Nat. Rev, Cardiol., 2010, 7, 398-408.

[0312] H. Goren and MC. Steinberg, "Familial oculoleptomeningeal amyloidosis", Brain, 1980, 103, 473- 495.

[0313] P. Westermark et al., "Fibril in senile systemic amyloidosis is derived from normal transthyretin", Proc. Natl. acad. Sci. USA, 1990, 87, 2843-5. http: / / amyloidosismutations.com / mut-attr.php

[0314] F. Pinheiro et al., "Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis". The FEBS journal 288, 310-324 (2021).

[0315] Received at EPO via Web-Form on Jul 18, 2023

Claims

CLAIMS1. A compound defined by formula (I)or a pharmaceutically acceptable salt thereof.

2. A compound as defined in claim 1 , for use as a medicament.

3. A compound as defined in claim 1 , for use in the treatment of a transthyretin amyloidosis in a subject.

4. The compound for use according to claim 3, wherein the transthyretin amyloidosis is selected from the group consisting of familial amyloid polyneuropathy, senile systemic amyloidosis, familial amyloid cardiomyopathy, and leptomeningeal amyloidosis.

5. The compound for use according to claim 4, wherein the familial amyloid polyneuropathy is associated with V30M transthyretin variant.

6. The compound for use according to claim 4, wherein the familial amyloid cardiomyopathy is associated with V122I transthyretin variant.

7. The compound for use according to any one of claims 3 to 6, wherein the subject is a human subject.

8. The compound for use according to any one of claims 3 to 7, wherein the compound is in combination with one or more therapeutic agents for the treatment of transthyretin amyloidosis.

9. The compound for use according to claim 8, wherein the therapeutic agent is selected from the group consisting of tolcapone, entacapone, tafamidis, AGIO, iododiflunisal, diflunisal, and benzbro- marone or pharmaceutically acceptable salts thereof.

10. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in claim 1 and at least a pharmaceutically acceptable excipient or vehicle. ceived at EPO via Web-Form on Jul 18, 202311. The pharmaceutical composition according to claim 10, which is formulated as an oral or a parenteral composition.

12. A process for preparing the compound of formula (I) as defined in claim 1 , comprising the removal5 of two of the methyl groups of the compound of formula (l)-1 :to yield compound of formula (I).

13. The process according to claim 12, further comprising the coupling reaction of a Grignard reagent 10 of compound (l)-2:with 3,4-dimethoxy-5-nitrobenzaldehyde, to obtain compound of formula (l)-1 :ceived at EPO via Web-Form on Jul 18, 2023

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