Novel compounds including a new class of transthyretin ligands for the treatment of common age-related comorbidities

Abstract

The invention provides a pharmaceutical composition comprising a compound of the invention and a pharma- ceutical acceptable carrier.The invention provides a method of stabilizing the TTR tetramer in a mammal, comprising administering to the mammal an amount of a compound of the invention or a composition of the invention effective to stabilize the TTR tetramer.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 149124 (filed February 12, 2021), the contents of which are incorporated herein by reference.

[0002] Throughout this application, references are referenced in parentheses. A full bibliography of these references is provided at the end of the specification. The disclosures of these references in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0003] This invention was made with Government support under R01EY028549 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. [Background technology]

[0004] Transthyretin (TTR, thyroxine-binding prealbumin) is a 55 kDa homotetramer of four β-sheet-rich 127-residue polypeptide monomers that is synthesized primarily in the liver for secretion into the blood (Vieira, M. & Saraiva 2014). The TTR tetramer has two high-affinity binding sites for the thyroid hormone thyroxine (T4, 1) (Figure 1). Another serum protein, thyroxine-binding globulin (TBG), serves as the main transporter of T4 in the blood, whereas less than 1% of circulating TTR is bound to T4 (Vieira, M. & Saraiva 2014). TTR is not the main carrier of T4 in serum, but it serves as the main transport protein of the hormone in the central nervous system (CNS), where choroid plexus-derived TTR transports T4 from the cerebrospinal fluid (CSF) to the choroid plexus and brain (Kassem, NA et al. 2006). Accumulating evidence suggests that TTR may play an auxiliary role in sequestration of β-amyloid (Aβ) peptides in the CSF by promoting the clearance of Aβ peptides from the CNS to the periphery, and may provide a neuroprotective effect against Alzheimer's disease (AD) (Gimeno, A. et al. 2017; Giao, T. et al. 2020; Gales, L. et al. 2005). In blood, a significant portion of TTR (about 50%) forms a macromolecular complex with retinol-binding protein 4 (RBP4) associated with all-trans-retinol (vitamin A, 2) (Figure 1) (Kanai, M. et al. 1968; Hyung, SJ et al. 2010). This retinol-dependent RBP4-TTR interaction is essential for efficient systemic transport of all-trans-retinol, as it prevents glomerular filtration of the low molecular weight RBP4-all-trans-retinol complex (Kawaguchi, R. et al. 2015).

[0005] TTR molecules in blood are homotetramers formed from two dimers (Vieira, M. & Saraiva 2014). To form the homotetramer structure, two TTR monomers first assemble into a dimeric subunit, which then assembles with another dimeric subunit. The resulting dimeric structure of the dimers shows a tetramer with two identical C2-symmetric T4 binding sites located within the central channel of the tetramer and formed at the dimer-dimer interface (Vieira, M. & Saraiva 2014). The dimer-dimer interface of TTR is relatively weak, and its dissociation is the rate-limiting step in the entire TTR tetramer dissociation process (Sun, X. et al. 2018). The released dimeric subunits can then further dissociate into monomers that have the potential to misfold and oligomerize. Oligomerization can ultimately lead to the aggregation and formation of toxic amyloid fibrils, which is the basis of the pathophysiology of TTR amyloidosis (ATTR) (Sun, X. et al. 2018).

[0006] Autosomal dominant ATTR is a rare and progressive disease with severe organ damage due to extracellular deposition of the toxic TTR amyloid fibrils in tissues. The disease usually manifests clinically as TTR amyloid cardiomyopathy (ATTR-CM; which can lead to arrhythmias, arterial fibrillation, and biventricular heart failure) (Ruberg, FL et al. 2019; Yamamoto, H. & Yokochi, T. 2019) or peripheral polyneuropathy (ATTR-PN; which can cause loss of sensation, tingling, numbness, or pain, and can also damage the autonomic nervous system) (Waddington-Cruz, M. et al. 2019), which may result from inherited TTR mutations resulting in pre-pathogenic monomers. Non-inherited ATTR may result from misfolding of wild-type TTR (WT-TTR) monomers in elderly individuals (Park, GY et al. 2019). There are at least 77 TTR mutations associated with familial ATTR, which affect amyloidogenicity by either 1) decreasing the thermodynamic stability of the TTR tetramer (i.e., monomers are less likely to associate to form TTR tetramers and more likely to misfold into amyloidogenic intermediates), 2) lowering the kinetic barrier for tetramer dissociation (mutated TTR tetramers dissociate faster than WT-TTR, while also increasing the rate of monomer aggregation), or 3) destabilizing the TTR tetramer thermodynamically and kinetically (Leach, BI et al. 2018). The kinetically stable but thermodynamically unstable mutation V30M (Jesus, CS et al. 2016) is primarily associated with late-onset familial amyloidotic polyneuropathy (FAP) and is highly pathogenic. The most common amyloidogenic TTR mutation, V122I (Damrauer, SM et al. 2019), is present at a relatively high frequency (~3.4%) in African Americans and is primarily associated with familial amyloidotic cardiomyopathy (FAC). Its pathogenicity is thought to be due to its ability to kinetically destabilize TTR tetramers, making the dissociation rate approximately twice that of WT-TTR (Jiang, X. et al. 2001).The L55P mutation may thermodynamically and kinetically destabilize tetramer formation and actively promote the early onset of ATTR-CM and ATTR-PN (Sousa, MM et al. 2002). Conversely, compound heterozygotes carrying a pro-amyloidogenic TTR mutation (e.g., V30M) and disease-suppressing mutations that overstabilize the TTR tetramer, such as T119M or R104H (Kamata, M. et al. 2009), have been reported to either develop mild late-onset pathology or be completely protected from ATTR. The T119M mutation kinetically stabilizes the TTR tetramer, whereas the R104H mutation thermodynamically stabilizes the quaternary structure. This difference in stabilization mechanism is important, since the T119M mutation is resistant to tetramer dissociation and aggregation, providing a higher level of protection against TTR aggregation in vitro compared to R104H. Finally, age-related non-genetic misfolding and aggregation of WT-TTR is associated with senile systemic amyloidosis (SSA), a late-onset and widespread form of ATTR that is estimated to affect 10%–20% of people over the age of 80.

[0007] Currently available FDA-approved approaches for the treatment of ATTR-CM and ATTR-PN include two therapies that lower TTR in the blood (the antisense oligonucleotide inotersen (Mathew, V. & Wang, AK 2019) and the small interfering RNA (siRNA) patisiran (Hoy, SM 2018)) and the small molecule compound tafamidis (Vyndaqel & Vinmax, 3), which binds to and stabilizes the TTR tetramer (Figure 2) (Bulawa, CE et al. 2012;Coelho, T. et al. 2013;Coelho, T. et al. 2016;Cruz, MW 2019;Lamb, YN & Deeks, ED 2019;Park, J. et al. 2020). Ligand binding at the T4 site has been shown to kinetically stabilize the TTR tetramer by increasing the dissociation energy barrier of the native tetrameric state. Due to the additional presence of two T4 transport proteins (TGB and albumin), the majority of TTR in blood does not bind TTR, and the T4 binding sites are largely (>99%) unoccupied. Therefore, drug discovery approaches to identify T4-competitive small molecule compounds that can kinetically stabilize the TTR tetramer have attracted great interest as options for the treatment of ATTR. In addition to tafamidis, many structurally diverse molecular scaffolds have been reported to bind to the T4 site and stabilize the TTR tetramer, and representatives of this class are shown in Figure 2 (compounds 3-12). The two most advanced small molecule TTR tetramer stabilizers to date include the aforementioned FDA-approved compound 3 and AG10 (4), which is in clinical trials (Alhamadsheh, MM et al. 2011; Miller, M. et al. 2018; Penchala, SC et al. 2013). TTR stabilizer 3 has been approved for the treatment of FAP and ATTR-CM. A Phase III trial of 441 patients with ATTR-CM showed that treatment with compound 3 reduced the risk of death by 30% and cardiovascular-related hospitalizations by 32% compared to placebo (Maurer, MS et al. 2018).The TTR stabilizer 4 has been reported to be well tolerated and demonstrated near-complete stabilization of TTR in a 28-day Phase II proof-of-concept study in patients with ATTR-CM and symptomatic chronic heart failure (Judge, DP et al. 2019). Phase III clinical trials of compound 4 for the treatment of ATTR-CM and ATTR-PN are currently underway. In addition, the repurposed FDA-approved nonsteroidal anti-inflammatory drug (NSAID) diflunisal (5) (Berk, JL et al. 2013) and the catechol-O-methyltransferase (COMT) inhibitor tolcapone (7) (Sant'Anna, R. et al. 2016) have also been reported to exhibit TTR tetramer stabilizing activity and are being investigated for clinical efficacy against ATTR-PN.

[0008] In recent years, the blood RBP4-TTR-all-trans-retinol transport complex has become a target for pharmacological intervention in ocular diseases associated with enhanced retinal accumulation of cytotoxic lipofuscin bisretinoids such as A2E, isoA2E, A2-DHP-PE and atRAL di-PE (Figures 3, 4). Because apoRBP4 binds poorly to TTR, the formation of this transport complex requires the initial binding of compound 2 to RBP4 (holoRBP4) (Kawaguchi, R. et al. 2015). It has been reported that prevention of RBP4-TTR-all-trans-retinol ternary complex formation can be achieved by selective all-trans-retinol competitive RBP4 antagonists, which, due to their relatively small molecular weight (21 kDa), lead to a reduction in serum RBP4 promoted by rapid glomerular filtration (Kawaguchi, R. et al. 2015). Evidence suggests that lowering serum RBP4 with selective RBP4 antagonists may have therapeutic potential for a wide variety of indications. For example, it has been hypothesized that RBP4 antagonists may provide a mechanism to slow or halt the progression of focal atrophy in patients with dry age-related macular degeneration (AMD) and Stargardt's disease by preventing the entry of compound 2 into the eye and halting the accumulation of cytotoxic lipofuscin bisretinoids in the retina (Radu, RA et al. 2005). A potent and selective RBP4 antagonist blocks the formation of the RBP4-TTR-all-trans retinol ternary complex in vitro and significantly reduces serum RBP4 in rodents, dogs and non-human primates in vivo (Cioffi, CL et al. 2014;Cioffi, CL et al. 2015;Cioffi, CL et al. 2019;Racz, B. et al. 2020). Furthermore, Abca4, a model of excessive lipofuscinogenesis that recapitulates the Stargardt disease phenotype, has been shown to be a promising treatment for the condition. - / -Long-term oral administration of an RBP4 antagonist to knockout mice reduced the accumulation of cytotoxic bisretinoids in the retina, accompanied by stabilization of complement system protein expression in the retinal pigment epithelium (RPE) (Racz, B. et al. 2018;Dobri, N. et al. 2013). In addition, another administration experiment in wild-type BALB / cJ mice revealed that the reduction in blood RBP4 by an RBP4 antagonist correlated with a partial reduction in the concentration of bisretinoid precursors, without impairing the dynamics of the visual cycle (Racz, B. et al. 2018).

[0009] To date, only selective all-trans retinol competitive antagonists of RBP4 have been reported to prevent the formation of ternary complexes with TTR, lowering blood RBP4 in vivo and simultaneously inhibiting bisretinoid synthesis in the retina. Selective RBP4 antagonists may be a safe and effective bisretinoid lowering drug for the majority of patients with dry AMD and Stargardt's disease, but this class of compounds may be off-label for some patients with macular degeneration who are susceptible to diseases related to TTR aggregation. Selective RBP4 antagonists release unliganded TTR tetramers from the RBP4-TTR-all-trans retinol transport complex in the blood. Previous studies have suggested that RBP4-TTR-all-trans retinol interactions may stabilize TTR tetramers (Leach, BI et al. 2018; Jesus, CS et al. 2016) and that the massive release of unliganded TTR tetramers induced by selective RBP4 antagonists may promote TTR amyloid fibril formation in susceptible individuals who promote ATTR (Damrauer, SM et al. 2019; Jiang, X. et al. 2001; Sousa, MM et al. 2002).

[0010] This invention describes novel TTR tetramer kinetic stabilizers that selectively bind to TTR tetramers. Therefore, these compounds can be applied to treat ATTR-CM, ATTR-PN, FAP, FAC or SSA, and other ATTR. In addition, this specification shows that these compounds can reduce the level of RBP4, and therefore may be used as a treatment for AMD, dry AMD, Stargardt's disease, Best's disease, adult vitelliform maculopathy, and other conditions characterized by increased lipofuscin accumulation in the retina. Summary of the Invention

[0011] The present invention relates to a method for producing a composition comprising the steps of:

[0012] [ka]

[0013] (In the formula, X1 is N or CR5; R5 is H, OH, halogen or alkyl; X2, X3 and X4 are each independently NH, N, S, O or CR6; each R6 is independently H, OH, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2, or -CO2H; R1, R2, R3, and R4 each independently represent -H, -F, -Cl, -Br, -I, -NO2, -CN, -CF3, -CF2H, -OCF3, -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl). , -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -SH, -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -NH2, -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl), -NH-(heteroaryl), -C(O)R7, -S(O)R7, -SOR7, -NHSO2R7, -OC(O)R7, -SC(O)R7, -NHC(O)R7 or -NHC(S)R7; R7 is H, -(alkyl), -OH, -O(alkyl), -NH2, -NH(alkyl) or -N(alkyl); B is,

[0014] [ka]

[0015] or not present, R8 is H, OH, halogen, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2 or -CO2H; C is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR9, OH, OR9, NH2, NHR9, or NR9R. 10 , SO2R 11 , CH2NHR9, CH2NR9R 10 or CH2COOR9, R9 and R 10 are each independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl), -CHNH(alkyl), -CHCOOH, -SOCH, -OH, -O(alkyl), -NH, -NH(alkyl) or -N(alkyl); R 11 is alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, NH, NH(alkyl), NH(cycloalkyl), NH(heterocycle), NH(aryl), NH(heteroaryl) or NHCOR 12 and R 12 is alkyl, haloalkyl, cycloalkyl, heterocycle, aryl or heteroaryl. or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0016] [Figure 1] The thyroid hormones thyroxine (T4) (1) and all-trans-retinol (vitamin A) (2). [Diagram 2] Representative examples of different classes of TTR tetramer stabilizers that bind at the T4 binding site. This sample set of TTR tetramer stabilizers includes tafamidis (3), AG10 (4), diflunisal (5), diflunisal iodide (6), tolcapone (7), benzbromarone (8), diclofenac (9), N-phenylphenoxazine 10, dibenzofuran 11, and bisaryloxime ether 12. [Diagram 3] Structures of the bisretinoids A2E and isoA2E, the cytotoxic components of retinal lipofuscin. [Figure 4]Structures of the cytotoxic components of retinal lipofuscin, the bisretinoids atRAL di-PE (all-trans retinal dimer-phosphatidylethanolamine) and A2-DHP-PE. R1 and R2 refer to various fatty acid moieties. [Figure 5A] Analog 18a reduces the formation of higher molecular weight forms of TTR in an acid-induced aggregation assay. TTR protein (5 μg) was aggregated in acetate buffer (pH 4.0) and incubated at 37° C. for 72 h. The concentration of TTR tetramer during incubation was 9 μM. After incubation in the presence of DMSO, 50 μM 3 (tafamidis) or 50 μM compound 18a and cross-linking with glutaraldehyde, samples were subjected to SDS-PAGE followed by Western blot analysis of TTR. Blots representative of at least three independent experiments are shown (A). [Figure 5B-5D] Graphs represent the average pixel volume in arbitrary units ± SD of bands scanned from immunoblots for high molecular weight aggregates (B), dimers (C) and monomers (D) of TTR. Statistical significance was determined by one-way ANOVA with Holm-Sidak post-hoc test; compared to TTR aggregate + DMSO group (pH 4.0) *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; compared to TTR group without aggregation (pH 7.5) #, p ≤ 0.05; ##, p ≤ 0.01; ###, p ≤ 0.001; ####, p ≤ 0.0001 [Figure 6A] Pharmacokinetic and pharmacodynamic properties of compound 18a in mice. (A) Serum RBP4 levels following a single 25 mg / kg oral dose of compound 18a. Data are presented as mean ± SD. Three mice per treatment group were used in the PK-PD study. [Figure 6B] Pharmacokinetic and pharmacodynamic properties of compound 18a in mice. (B) Compound levels in blood following a single 5 mg / kg oral dose of compound 18a. Data are presented as mean ± SD. Three mice per treatment group were used in the PK-PD study. [Figure 7] RBP4 levels in Abca4− / − mice orally treated with compound 18a·dihydrochloride for 8 weeks. [Figure 8] Bisretinoid-reducing effect of compound 18a·dihydrochloride in Abca4− / − mice treated with the compound for 8 weeks. [Figure 9A-9C] Lipofuscin autofluorescence in mouse retinal sections. A–C, Autofluorescence images of mouse retinal sections prepared from eyes of 129S1 / SvLmJ untreated mice (A), vehicle-treated 129S-Abca4tm1Ght / J mice (B), and compound 18a·2HCl-treated 129S-Abca4tm1Ght / J mice (C) administered 27 mg / kg compound 18a·2HCl in the diet for 6 weeks. Images were taken using a confocal microscope equipped with a 40 oil objective at excitation wavelengths of 405 nm (blue, DAPI), 488 nm (green) and emission wavelengths of 420–470 nm (blue, DAPI), 500–600 nm (green). Merged images showing the localization of lipofuscin autofluorescence peaks (green) within the retinal layers (blue, DAPI) and retinal tissue (DIC) imaged in differential interference contrast mode. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS / OS, inner and outer segments of the photoreceptor layer; RPE, retinal pigment epithelium. Scale bar, 50 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The present invention relates to a method for producing a composition comprising the steps of:

[0018] [ka]

[0019] (In the formula, X1 is N or CR5; R5 is H, OH, halogen or alkyl; X2, X3 and X4 are each independently NH, N, S, O or CR6; each R6 is independently H, OH, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2, or -CO2H; R1, R2, R3, and R4 each independently represent -H, -F, -Cl, -Br, -I, -NO2, -CN, -CF3, -CF2H, -OCF3, -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl). , -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -SH, -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -NH2, -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl), -NH-(heteroaryl), -C(O)R7, -S(O)R7, -SOR7, -NHSO2R7, -OC(O)R7, -SC(O)R7, -NHC(O)R7 or -NHC(S)R7; R7 is H, -(alkyl), -OH, -O(alkyl), -NH2, -NH(alkyl) or -N(alkyl); B is, [ka] or not present, R8 is H, OH, halogen, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2 or -CO2H; C is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR9, OH, OR9, NH2, NHR9, or NR9R. 10 , SO2R11 , CH2NHR9, CH2NR9R 10 or CH2COOR9, R9 and R 10 are each independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl), -CHNH(alkyl), -CHCOOH, -SOCH, -OH, -O(alkyl), -NH, -NH(alkyl) or -N(alkyl); R 11 is alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, NH, NH(alkyl), NH(cycloalkyl), NH(heterocycle), NH(aryl), NH(heteroaryl) or NHCOR 12 and R 12 is alkyl, haloalkyl, cycloalkyl, heterocycle, aryl or heteroaryl. or a pharma- ceutically acceptable salt thereof.

[0020] In some embodiments, the compound or a pharma- ceutically acceptable salt thereof is X1 is N or CR5; R5 is H, OH, halogen or alkyl; X2, X3 and X4 are each independently NH, N, S, O or CR6; each R6 is independently H, OH, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2, or -CO2H; R1, R2, R3, and R4 are each independently -H, -F, -Cl, -Br, -I, -NO2, -CN, -CF3, -CF2H, -OCF3, -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkyl aryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl), -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -SH, -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -NH, -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl) or -NH-(heteroaryl); B is, [ka] or not present, R8 is H, OH, halogen, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2 or -CO2H; C is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR9, OH, OR9, NH2, NHR9, or NR9R. 10 , SO2R 11 , CH2NHR9, CH2NR9R 10 or CH2COOR9, R9 and R 10are each independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl), -CHNH(alkyl), -CHCOOH, -SOCH, -OH, -O(alkyl), -NH, -NH(alkyl) or -N(alkyl); R 11 is alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, NH, NH(alkyl), NH(cycloalkyl), NH(heterocycle), NH(aryl), NH(heteroaryl) or NHCOR 12 and R 12 is alkyl, haloalkyl, cycloalkyl, heterocycle, aryl or heteroaryl.

[0021] In some embodiments, the compound or a pharma- ceutically acceptable salt thereof is X1 is N, X2, X3 and X4 are each independently NH, N, S, O or CR6; each R6 is independently H, halogen, alkyl, alkenyl, alkynyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2, or -CO2H; R1, R2, R3 and R4 are each independently -H, -F, -Cl, -Br, -I, -CN, -CF3, -CF2H, -OCF3, -(alkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl), -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -NH2, -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl) or -NH-(heteroaryl); BC is -CO2H, -CONH2 or [ka] It is.

[0022] In some embodiments, the compound, or a pharma- ceutically acceptable salt thereof, has the structure: [ka] has.

[0023] In some embodiments, the compound, or a pharma- ceutically acceptable salt thereof, has the structure: [ka] has.

[0024] In some embodiments, X3 of the compound is NH and X2 and X4 are CR6.

[0025] In some embodiments, X3 of the compound is O and X2 and X4 are CR6.

[0026] In some embodiments, X3 of the compound is S and X2 and X4 are CR6.

[0027] In some embodiments, R6 of the compound is H, OH, alkyl, alkenyl, alkynyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH-(alkyl), -N(alkyl)2, or -CO2H.

[0028] In some embodiments, R6 of the compound is alkyl.

[0029] In some embodiments, R6 of the compound is methyl.

[0030] In some embodiments, R6 of the compound is -CF3.

[0031] In some embodiments, BC of the compound is -CO2H, -CONH2, or [ka] It is.

[0032] In some embodiments, BC of the compound is -CO2H.

[0033] In some embodiments, R1, R2, R3, and R4 of the compound are each independently -H, -F, -Cl, -Br, -I, -NO2, -CN, -CF3, -CF2H, -OCF3, -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -OH, -OAc, -O-(alkyl), -S-(alkyl).

[0034] In some embodiments, R1, R2, R3, and R4 of the compound are each independently H, F, Cl, CH3, CF3, or OCH3.

[0035] In some embodiments, R1 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0036] In some embodiments, R2 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0037] In some embodiments, R3 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0038] In some embodiments, R4 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0039] In some embodiments, R1 of the compound is H, F, Cl, CH3, CF3, or OCH3, and R2, R3, and R4 are each H.

[0040] In some embodiments, R1 of the compound is F, Cl, CH3, CF3, or OCH3, R3 is CH3, and R2 and R4 are each H.

[0041] In some embodiments, R1 of the compound is F, and R2, R3, and R4 are each independently H, F, Cl, CH3, CF3, or OCH3.

[0042] In some embodiments, R1 of the compound is F, and R2, R3, and R4 are each H.

[0043] In some embodiments, R1 of the compound is Cl, and R2, R3, and R4 are each independently H, F, Cl, CH3, CF3, or OCH3.

[0044] In some embodiments, R1 of the compound is Cl, and R2, R3, and R4 are each H.

[0045] In some embodiments, BC of the compound is -CO2H, -CONH2, or [ka] It is.

[0046] In some embodiments, R1 of the compound is F or Cl, R2, R3, and R4 are each H, and BC is -CO2H.

[0047] In some embodiments, R1 of the compound is F or Cl, R2, R3, and R4 are each H, and BC is -CONH2.

[0048] In some embodiments, R1 of the compound is F or Cl, R2, R3, and R4 are each H, and BC is [ka] It is.

[0049] In some embodiments, the compound, or a pharma- ceutically acceptable salt thereof, has the structure: [ka] has.

[0050] In some embodiments, X1 of the compound is N or CR5.

[0051] In some embodiments, BC of the compound is -CO2H, -CONH2, or [ka] It is.

[0052] In some embodiments, the compound, or a pharma- ceutically acceptable salt thereof, has the structure: [ka] has.

[0053] In some embodiments, R1, R2, R3, and R4 of the compound are each independently H, F, Cl, CH3, CF3, or OCH3.

[0054] In some embodiments, R1 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0055] In some embodiments, R2 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0056] In some embodiments, R3 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0057] In some embodiments, R4 of the compound is H, F, Cl, CH3, CF3, or OCH3.

[0058] In some embodiments, R1 of the compound is H, F, Cl, CH3, CF3, or OCH3, and R2, R3, and R4 are each H.

[0059] In some embodiments, R1 of the compound is F, Cl, CH3, CF3, or OCH3, R3 is CH3, and R2 and R4 are each H.

[0060] In some embodiments, R1 of the compound is F, and R2, R3, and R4 are each H.

[0061] In some embodiments, R1 of the compound is Cl, and R2, R3, and R4 are each H.

[0062] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0063] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0064] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0065] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0066] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0067] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0068] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0069] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0070] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0071] The present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharma- ceutically acceptable carrier.

[0072] The invention provides a method for stabilizing the TTR tetramer in a mammal, comprising administering to the mammal an amount of a compound of the invention or a composition of the invention effective to stabilize the TTR tetramer.

[0073] The invention provides a method for preventing the formation of TTR aggregates or preventing the formation of high molecular weight aggregates in a mammal, comprising administering to the mammal an amount of a compound of the invention or a composition of the invention effective to prevent the formation of TTR aggregates or prevent the formation of high molecular weight aggregates.

[0074] The present invention provides a method for treating TTR amyloidosis (ATTR) in a mammal suffering from said disease, comprising administering to said mammal an effective amount of a compound of the present invention or a composition of the present invention.

[0075] In some embodiments of this method, the method is further effective to stabilize the mammalian TTR tetramer.

[0076] In some embodiments of this method, the TTR amyloidosis (ATTR) is peripheral polyneuropathy (ATTR-PN).

[0077] In some aspects of this method, the TTR amyloidosis (ATTR) is TTR amyloid cardiomyopathy (ATTR-CM).

[0078] In some embodiments of this method, the TTR amyloidosis (ATTR) is late-onset familial amyloidotic polyneuropathy (FAP).

[0079] In some aspects of this method, the TTR amyloidosis (ATTR) is familial amyloidotic cardiomyopathy (FAC).

[0080] In some embodiments of this method, the TTR amyloidosis (ATTR) is senile systemic amyloidosis (SSA).

[0081] In some aspects of this method, the TTR amyloidosis (ATTR) is characterized by the deposition of amyloid aggregates.

[0082] The invention provides a method for treating a disease characterized by excess lipofuscin accumulation in the retina, comprising administering to said mammal an effective amount of a compound of the invention or a composition of the invention.

[0083] In some aspects of this method, the disease is further characterized by bisretinoid induced macular degeneration.

[0084] In some aspects of this method, the amount of compound is effective to reduce a serum concentration of RBP4 in the mammal or is effective to reduce a retinal concentration of a bisretinoid in lipofuscin in the mammal.

[0085] In some aspects of this method, the bisretinoid is A2E.

[0086] In some aspects of this method, the bisretinoid is isoA2E.

[0087] In some embodiments of this method, the bisretinoid is A2-DHP-PE.

[0088] In some embodiments of this method, the bisretinoid is atRAL di-PE.

[0089] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is age-related macular degeneration.

[0090] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is dry (dry) age-related macular degeneration.

[0091] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Stargardt's disease.

[0092] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Best's disease.

[0093] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is adult vitellogenic maculopathy.

[0094] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Stargardt-like macular dystrophy.

[0095] The invention provides a method for treating TTR amyloidosis (ATTR), a disease characterized by the accumulation of excess lipofuscin in the retina, or both TTR amyloidosis (ATTR) and a disease characterized by the accumulation of excess lipofuscin, comprising administering to the mammal an effective amount of a compound of the invention or a composition of the invention.

[0096] In some aspects of this method, the amount of compound is effective to stabilize a mammalian TTR tetramer.

[0097] In some aspects of this method, the amount of compound is effective to prevent formation of TTR aggregates or to prevent formation of high molecular weight aggregates.

[0098] In some aspects of this method, the amount of compound is effective to reduce a serum concentration of RBP4 in the mammal or is effective to reduce a retinal concentration of a bisretinoid in lipofuscin in the mammal.

[0099] In some aspects of this method, the amount of compound is effective to stabilize TTR tetramers in said mammal and reduce the serum concentration of RBP4 in said mammal.

[0100] In some aspects of this method, the amount of compound is effective to prevent formation of TTR aggregates or formation of high molecular weight aggregates in said mammal and to reduce the serum concentration of RBP4 in said mammal.

[0101] In some aspects of this method, the amount of compound is effective to stabilize TTR tetramers in said mammal and reduce retinal concentrations of bisretinoids in lipofuscin in said mammal.

[0102] In some aspects of this method, the amount of compound is effective to prevent formation of TTR aggregates or prevent formation of high molecular weight aggregates in the mammal and reduce retinal concentrations of bisretinoid in lipofuscin in the mammal.

[0103] In some embodiments of this method, the TTR amyloidosis (ATTR) is peripheral polyneuropathy (ATTR-PN).

[0104] In some aspects of this method, the TTR amyloidosis (ATTR) is TTR amyloid cardiomyopathy (ATTR-CM).

[0105] In some embodiments of this method, the TTR amyloidosis (ATTR) is late-onset familial amyloidotic polyneuropathy (FAP).

[0106] In some aspects of this method, the TTR amyloidosis (ATTR) is familial amyloidotic cardiomyopathy (FAC).

[0107] In some embodiments of this method, the TTR amyloidosis (ATTR) is senile systemic amyloidosis (SSA).

[0108] In some aspects of this method, the TTR amyloidosis (ATTR) is characterized by the deposition of amyloid aggregates.

[0109] In some aspects of this method, the disease is further characterized by bisretinoid induced macular degeneration.

[0110] In some aspects of this method, the amount of compound is effective to reduce a serum concentration of RBP4 in the mammal or is effective to reduce a retinal concentration of a bisretinoid in lipofuscin in the mammal.

[0111] In some aspects of this method, the bisretinoid is A2E.

[0112] In some aspects of this method, the bisretinoid is isoA2E.

[0113] In some embodiments of this method, the bisretinoid is A2-DHP-PE.

[0114] In some embodiments of this method, the bisretinoid is atRAL di-PE.

[0115] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is age-related macular degeneration.

[0116] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is dry (dry) age-related macular degeneration.

[0117] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Stargardt's disease.

[0118] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Best's disease.

[0119] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is adult vitellogenic maculopathy.

[0120] In some aspects of this method, the disease characterized by excess lipofuscin accumulation in the retina is Stargardt-like macular dystrophy.

[0121] Bisretinoid-induced macular degeneration may involve the accumulation of lipofuscin deposits in the retinal pigment epithelium.

[0122] As used herein, "bisretinoid lipofuscin" refers to lipofuscin that contains cytotoxic bisretinoids, including, but not limited to, A2E, isoA2E, atRAL di-PE (all-trans retinal dimer-phosphatidylethanolamine), and A2-DHP-PE (A2-dihydropyridine-phosphatidylethanolamine) (Figures 3-4).

[0123] As used herein, "high molecular weight aggregates" refers to all forms of TTR aggregates having a molecular weight greater than 198 kDa.

[0124] Transthyretin (TTR) amyloidosis (ATTR) is a neurodegenerative disease that includes, but is not limited to, senile systemic amyloidosis (SSA), peripheral polyneuropathy (ATTR-PN) or cardiomyopathy (ATTR-CM).

[0125] In some aspects, TTR amyloidosis (ATTR) is characterized by the deposition of amyloid aggregates.

[0126] In some aspects, TTR amyloidosis (ATTR) is characterized by the deposition of amyloid aggregates derived from either mutant (TTRm) or wild-type (TTRwt).

[0127] In some embodiments, the TTR amyloidosis (ATTR) is senile systemic amyloidosis (SSA).

[0128] In some embodiments, the TTR amyloidosis (ATTR) is peripheral polyneuropathy (ATTR-PN).

[0129] In some embodiments, the TTR amyloidosis (ATTR) is cardiomyopathy (ATTR-CM).

[0130] In some embodiments, compounds of the present invention exhibit transthyretin (TTR) tetramer kinetic stabilizing activity.

[0131] In some embodiments, the compounds of the invention reduce circulating levels of RBP4 while stabilizing unliganded TTR tetramers released from the holoRBP4-TTR complex.

[0132] In some embodiments, the compounds of the present invention reduce RBP4 in the blood.

[0133] In some embodiments, the compounds of the invention stabilize the unliganded TTR tetramer released from the holo RBP4-TTR complex.

[0134] In some embodiments, the compounds of the invention or compositions of the invention may be used to treat dry age-related macular degeneration (AMD) with comorbid TTR amyloidosis (ATTR).

[0135] In some embodiments, the compounds of the invention or compositions of the invention may be used to treat dry age-related macular degeneration (AMD) and senile systemic amyloidosis (SSA).

[0136] In some embodiments, the compounds of the invention or compositions of the invention may be used to treat peripheral polyneuropathy (ATTR-PN) in dry age-related macular degeneration (AMD).

[0137] In some embodiments, the compounds of the present invention or compositions of the present invention may be used to treat dry age-related macular degeneration (AMD) and cardiomyopathy (ATTR-CM).

[0138] In some aspects, the compounds of the invention or the compositions of the invention may be used to treat type 2 diabetes.

[0139] In some aspects, the compounds of the invention or compositions of the invention may be used to treat obesity.

[0140] In some aspects, the compounds of the invention or compositions of the invention may be used to treat insulin resistance.

[0141] In some aspects, the compounds of the invention or compositions of the invention may be used to treat cardiovascular disease.

[0142] In some aspects, the compounds of the invention or compositions of the invention may be used to treat fatty liver.

[0143] In some embodiments, the compounds of the invention or compositions of the invention may be used to treat non-alcoholic fatty liver disease (NAFLD).

[0144] In some embodiments, the mammal is a human.

[0145] Those skilled in the art may prepare deuterium analogs thereof using the techniques disclosed herein.

[0146] Unless otherwise specified, when the compounds of the present invention contain asymmetric carbon atoms, it is understood that the compounds exist as racemates, racemic mixtures, mixtures of enantiomers in various ratios, and isolated single enantiomers. All such isomers of these compounds are expressly included in this invention. Unless otherwise specified, each asymmetric carbon is in the R or S configuration. It is therefore understood that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless otherwise specified. Such isomers can be obtained in substantially pure form by classical separation techniques and stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution can be carried out by preparative chromatography on a chiral column.

[0147] Unless otherwise specified, the present invention is intended to include all isotopes of atoms present in the compounds disclosed herein. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0148] Throughout this application, carbons in structures are designated as follows, unless further noted: 12 C. 13 C or 14 It should be noted that all isotopes of carbon, such as C, are intended to represent. 13 C or 14 The compound containing C may specifically be of the structure of any of the compounds disclosed herein.

[0149] Throughout this application, hydrogen (H) in structures is, unless further noted, 1 H, 2 H(D) or3 Note that all isotopes of hydrogen, such as H(T), are intended to represent. 2 H or 3 A compound containing H may specifically be any of the structures of the compounds disclosed herein, unless otherwise specified.

[0150] Isotopically labeled compounds can generally be prepared by conventional techniques known to those of skill in the art, substituting an appropriate isotopically labeled reagent for the non-labeled reagent employed.

[0151] deuterium( 2 H or D) is a stable, non-radioactive isotope of hydrogen with an atomic mass of 2.0144. 1 H (hydrogen or protium), D ( 2 H or deuterium) and T( 3 Deuterium occurs naturally as a mixture of hydrogen atoms (H, H, or tritium). The abundance of deuterium in nature is 0.0156%. Thus, in a composition that includes a naturally occurring compound, the percentage of deuterium at any particular hydrogen atom site in that compound is expected to be 0.0156%. Thus, a composition that includes a compound having deuterium at any hydrogen atom site in the compound enriched to be greater than the natural abundance of 0.0156% is novel relative to its naturally occurring counterpart.

[0152] The terms "substitution," "substituted," and "substituent" refer to a functional group in which one or more bonds to a hydrogen atom are replaced by an atom other than hydrogen or a bond to an atom other than carbon, provided that the normal valences are maintained and the substitution results in a stable compound. Substituent also includes groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituents include the functional groups previously described, as well as halogens (i.e., F, Cl, Br, and I); alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups such as phenoxy; arylalkyloxy groups such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups such as methylsulfanyl, ethylsulfanyl, and propylsulfanyl; cyano; amino groups such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituents are described or claimed, the substituted compound can be independently substituted one or more times with one or more of the described or claimed substituents. By independently substituted, it is meant that the (two or more) substituents can be the same or different.

[0153] In the compounds used in the methods of the invention, unless otherwise specified, the substituents may be substituted or unsubstituted.

[0154] In the compounds used in the methods of the invention, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycle, heterocycloalkyl, alkylheteroalkyl, alkylaryl, monocyclic, bicyclic, heteromonocyclic, and heterobicyclic can be further substituted by replacing one or more hydrogen atoms with another non-hydrogen group, including, but not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, and carbamoyl.

[0155] It is understood that the substituents and substitution patterns of the compounds used in the methods of the present invention can be selected by one of ordinary skill in the art to provide chemically stable compounds, and can be readily synthesized from readily available starting materials by techniques known in the art. When a substituent is itself substituted with multiple groups, it is understood that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0156] In selecting compounds for use in the methods of the present invention, one of skill in the art will recognize that the various substituents (i.e., R1, R2, etc.) should be selected in accordance with well-known principles of bonding in chemical structures.

[0157] As used herein, "alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, which may be unsubstituted or substituted. Thus, "C1-C n C1 to C in "Alkyl" nis defined to include groups having 1, 2,..., n-1, or n carbons, linear or branched. For example, C1-C6 in "C1-C6 alkyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons, linear or branched, specifically including methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, and hexyl. Unless otherwise specified, contains 1 to 10 carbons. Alkyl groups may be unsubstituted or substituted with one or more substituents, including but not limited to halogen, alkoxy, alkylthio, trifluoromethyl, difluoromethyl, methoxy, and hydroxyl. "Haloalkyl" includes alkyl groups having at least one halogen atom.

[0158] The term "alkenyl" refers to a straight or branched chain non-aromatic hydrocarbon group having at least one carbon-carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present. Thus, C2-C n Alkenyl is defined to include groups having 1, 2,...n-1 or n carbons. For example, "C2-C6 alkenyl" refers to an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms and at least one carbon-carbon double bond, e.g., in the case of C6 alkenyl, up to three carbon-carbon double bonds. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As explained for alkyl, the straight, branched or cyclic portions of the alkenyl group may contain double bonds and may be substituted, as referred to as a substituted alkenyl group. In some embodiments, C2-C6 alkenyl is an alkyl ... 12 alkenyl or C2-C8 alkenyl.

[0159] The term "alkynyl" refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-C nAlkynyl is defined to include groups having 1, 2,..., n-1 or n carbons. For example, "C2-C6 alkynyl" refers to an alkynyl group having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described for alkyl, the straight or branched chain of the alkynyl group may contain triple bonds and may be substituted, in the case of a substituted alkynyl group. In one embodiment, C2-C6 alkynyl is .... n In one embodiment, C2-C 12 It is alkynyl or C3-C8 alkynyl.

[0160] In this specification, "aryl" is intended to mean a stable monocyclic, bicyclic or polycyclic carbocyclic ring of up to 10 atoms in each ring, where at least one ring is aromatic and may be unsubstituted or substituted. Examples of such aryl groups include, but are not limited to, phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydronaphthyl, indanyl, phenanthryl, anthryl or acenaphthyl. When an aryl group is bicyclic and one ring is non-aromatic, it is understood that it is attached through the aromatic ring.

[0161] As used herein, the term "heteroaryl" refers to a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, in which at least one ring is aromatic and has from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include a phenyl, pyridine, pyrimidine or pyridazine ring (a) fused to a 6-membered aromatic (unsaturated) heterocycle having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocycle having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocycle having one oxygen and one nitrogen atom or one sulfur and one nitrogen atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocycle having one heteroatom selected from O, N or S.Heteroaryl groups of this definition include benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetyl, oxazol ... Tanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl Heteroaryl groups include, but are not limited to, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. When a heteroaryl group is bicyclic and one ring is non-aromatic or has no heteroatoms, it is understood that the bond is at the aromatic ring or at the ring having a heteroatom, respectively.When the heteroaryl group contains a nitrogen atom, it is understood that the N-oxide thereof is also included.

[0162] As used herein, "cycloalkyl" includes alkane rings having a total of three to eight carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl). "Cycloalkylalkyl" includes alkyl groups having at least one cycloalkyl ring.

[0163] As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having at least one heteroatom in the chain or branch. "Alkylheteroalkyl" includes alkyl groups having at least one heteroalkyl group.

[0164] The term "heterocycle", "heterocyclyl" or "heterocyclic" refers to a monocyclic or polycyclic ring system that is saturated or has one or more degrees of unsaturation and has one or more heteroatoms. Preferred heteroatoms include N, O and / or S (including N-oxides, S-oxides and dioxides). Preferably, the ring is 3-10 membered and is saturated or has one or more degrees of unsaturation. Heterocycles may be unsubstituted or substituted, with multiple substitutions permitted. Such rings may be fused, as appropriate, with one or more other "heterocycles", heteroaryl rings, aryl rings or cycloalkyl rings. Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

[0165] As used herein, "heterocycloalkyl" is intended to mean a 5-10 membered non-aromatic ring having 1-4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. Thus, "heterocyclyl" includes, but is not limited to, the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothiophenyl, etc. If the heterocycle contains a nitrogen, it is understood that the N-oxide thereof is also included.

[0166] The term "alkylaryl" refers to an alkyl group as previously described in which one or more bonds to a hydrogen in the alkyl group are replaced by a bond to an aryl group as described above. It is understood that the "alkylaryl" group is connected to a core molecule via an alkyl group, and the aryl group is a substituent of the alkyl group. Examples of arylalkyl include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl, and the like.

[0167] As used herein, a "monocyclic ring" includes a stable polycyclic carbocyclic ring of up to 10 atoms, which may be unsubstituted or substituted. Examples of such non-aromatic monocyclic rings include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocyclic rings include, but are not limited to, phenyl. As used herein, a "heteromonocyclic ring" includes a monocyclic ring having at least one heteroatom.

[0168] As used herein, a "bicycle" includes a stable polycyclic carbocycle of up to 10 atoms fused with a polycyclic carbocycle of up to 10 atoms, each ring being independently unsubstituted or substituted. An example of such a non-aromatic bicycle includes, but is not limited to, decahydronaphthalene. An example of such an aromatic bicyclic element includes, but is not limited to, naphthalene. As used herein, a "heterobicycle" includes a bicycle having at least one heteroatom.

[0169] The compounds used in the methods of the present invention can be prepared by techniques that are well known in organic synthesis and will be familiar to those skilled in the art, however, these may not be the only means to synthesize or obtain the desired compounds.

[0170] The compounds used in the methods of the invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, AI Vogel, AR Tatchell, BS Furnis, AJ Hannaford, PWG Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated herein by reference, however, these may not be the only means of synthesizing or obtaining the desired compounds.

[0171] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the ring by standard procedures, such as those described in Advanced Organic Chemistry: Part B: Reactions and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), which is incorporated herein by reference.

[0172] Another aspect of the present invention includes the compounds or compositions of the present invention as pharmaceutical compositions.

[0173] As used herein, the term "pharmaceutically active agent" refers to a substance or compound suitable for administration to a subject, which provides biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or which affects the structure or function of a subject. Pharmaceutically active agents include those described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009), which are incorporated herein by reference, and in "Approved Drug Products with Therapeutic Equivalence Evaluations" (US Department of Health and Human Services, 30 th Pharmaceutically active agents having pendant carboxylic acid groups can be modified in accordance with the present invention by standard esterification reactions and methods that are readily available and known to those skilled in the art of chemical synthesis. If the pharma- ceutical active agent does not have a carboxylic acid group, one skilled in the art can design and incorporate a carboxylic acid group into the pharma- ceutical active agent, which may then be subsequently esterified, so long as the modification does not interfere with the biological activity or effect of the pharma- ceutical active agent.

[0174] The compounds used in the methods of the invention may be in the form of a salt. As used herein, a "salt" is a salt of a compound of the invention modified by making an acid salt or base salt of the compound. For compounds used to treat a disease or medical disorder, the salt is pharma- ceutically acceptable. Examples of pharma-ceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues (e.g., amines); alkali or organic salts of acidic residues (e.g., phenols); alkali or organic salts of acidic residues (e.g., carboxylic acids). Salts can be made using organic or inorganic acids. Such acid salts include chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts include sodium, potassium, or lithium salts. Carboxylate salts include sodium, potassium, or lithium salts. In this respect, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid or base addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or by separately reacting the free base or free acid form of the purified compounds of the present invention with a suitable organic or inorganic acid or base and isolating the salt formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0175] Salts or pharma- ceutically acceptable salts are contemplated for all compounds disclosed herein.

[0176] As used herein, "treatment" means preventing, slowing, halting or reversing the progression of a disease. Treatment may also mean the amelioration of one or more symptoms of a disease.

[0177] The compound used in the method of the present invention can be administered in various forms, including those described in detail in this specification. Treatment with the compound can be part of a combination therapy or adjunctive therapy, i.e., the subject or patient in need of the drug is treated or given one or more of the compounds in combination with another drug against the disease. This combination therapy can be a sequential treatment, where the patient is first treated with one drug, and then the other drug is administered, or two drugs are administered at the same time. They can be administered independently, by the same route, or by two or more different routes of administration, depending on the dosage form applied.

[0178] In this specification, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspension or vehicle for delivering the compound of the present invention to animals or humans. The carrier may be liquid or solid and is selected with the planned method of administration in mind. Liposomes are also pharmaceutically acceptable carriers, as are capsules, coatings and various syringes.

[0179] The dosage of the compound administered in the treatment will vary depending on factors such as the pharmacodynamic characteristics of the particular chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, efficiency of absorption, health status and weight of the recipient; the nature and extent of the condition; the type of treatment being performed; the frequency of treatment; and the desired therapeutic effect.

[0180] The dosage units of the compounds used in the methods of the present invention may contain a single compound or a mixture with another drug. The compounds may be administered in oral formulations such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, or may be introduced directly into or onto the disease site, for example, by injection, topical application, or other methods, all using dosage forms well known to those skilled in the pharmaceutical arts.

[0181] The compounds used in the methods of the present invention are appropriately selected according to the intended administration form and can be administered in admixture with suitable pharmaceutical diluents, fillers, excipients or carriers (collectively referred to herein as pharma- ceutically acceptable carriers) consistent with conventional pharmaceutical practice. The dosage units are in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or in admixture with a pharma-ceutically acceptable carrier. The carrier can be solid or liquid and is generally selected based on the administration method used. The active agents can be administered simultaneously in the form of tablets or capsules, liposomes, as agglomerated powders or in liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsules or tablets can be easily formulated to be easy to swallow or chew; other solid forms include granules and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow-inducing agents and melting agents. Examples of suitable liquid formulations include solutions or suspensions in water, pharma- ceutically acceptable fats and oils, alcohols or other organic solvents (including esters, emulsions, syrups or elixirs), suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Such liquid formulations may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners and melting agents. Oral formulations may contain flavorings and colorants as necessary. Parenteral and intravenous dosage forms may also contain minerals and other materials to make them compatible with the selected injection or delivery system.

[0182] Techniques and compositions for preparing dosage forms useful in the present invention may be found in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al. 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993);Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology;JG Hardy, SS Davis, Clive G. Wilson, Eds.);Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.), all of which are incorporated herein by reference.

[0183] Tablets may contain suitable binders, lubricants, disintegrants, colorants, flavoring agents, flow-inducing agents, and melting agents. For example, for oral administration in a tablet or capsule, the active pharmaceutical ingredient may be combined with an oral, non-toxic, pharma- ceutically acceptable inert carrier, such as lactose, gelatin, agar, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, and the like. Suitable binders include starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, natural and synthetic gums (e.g., acacia, tragacanth, or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. The disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0184] The compounds used in the methods of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The compounds may also be administered as a component of a tissue-targeted emulsion.

[0185] The compounds used in the methods of the invention can also be coupled to soluble polymers as targetable drug carriers or as prodrugs. Such polymers include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. In addition, the compounds can also be coupled to biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, poly-ε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0186] Gelatin capsules may contain the active ingredient compound and powdered carriers (e.g., lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc.). Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be prepared as immediate release formulations or as sustained release formulations that continuously release the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask unpleasant tastes and protect the tablet from the atmosphere, or enteric-coated to selectively disintegrate in the gastrointestinal tract.

[0187] For oral administration in liquid formulations, the oral drug components are combined with an oral non-toxic pharma- ceutically acceptable inert carrier (e.g., ethanol, glycerin, water, etc.). Examples of suitable liquid formulations include solutions or suspensions in water, pharma- ceutically acceptable fats and oils, alcohols or other organic solvents (including esters, emulsions, syrups or elixirs), suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Such liquid formulations may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners and melting agents.

[0188] Liquid preparations for oral administration can be formulated with coloring and flavoring agents to enhance patient acceptance. In general, water, suitable oil, saline, aqueous dextrose (glucose) and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and buffer substances, if necessary. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA can also be used. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 17th ed., 1989, a standard reference text in this field.

[0189] The compounds used in the methods of the present invention can also be administered in the form of intranasal administration using suitable intranasal vehicles, or via transdermal routes in the form of transdermal patches well known to those skilled in the art. When administered in the form of a transdermal delivery system, administration of the formulation is generally continuous rather than intermittent.

[0190] Parenteral and intravenous forms may contain minerals and other materials to make them compatible with injection or the delivery system of choice.

[0191] Each embodiment disclosed in this specification is considered applicable to any other disclosed embodiment. Accordingly, all combinations of the various elements described herein are within the scope of the invention. Any of the generic or specific compounds disclosed are applicable to any of the disclosed compositions, processes, or methods.

[0192] The present invention will be better understood by reference to the following experimental details, however, those skilled in the art will readily appreciate that these detailed experiments are merely illustrative of the invention as more fully described in the appended claims. EXAMPLES

[0193] Experimental details General Chemistry All reactions were carried out under dry nitrogen unless otherwise stated. Reaction temperatures indicated are reaction bath temperatures, room temperature is 25°C. Commercial grade reagents and anhydrous solvents were used as received without further purification or drying. Removal of solvents by reduced pressure was performed using a Buchi rotary evaporator and a Teflon-linked KNF vacuum pump was used to achieve a pressure of approximately 28 mmHg. Thin layer chromatography was performed using 1 inch x 3 inch AnalTech No. 02521 silica gel plates and used fluorescent indicators. Visualization of TLC plates was performed with either short wave UV light (254 nm lamp), 10% phosphomolybdic acid in ethanol or iodine vapor. Preparative thin layer chromatography was performed using 20 x 20 cm, 1000 micron preparative TLC plates from Analtech. Flash column chromatography was performed on a Biotage® Select System equipped with a Teledyne Isco CombiFlash Companion Unit and a Teledyne Isco RediSep Rf and a Biotage Sfaer silica gel column. If necessary, products were purified by reversed phase chromatography using a Biotage® Select System equipped with a Teledyne Isco CombiFlash Companion Unit and a RediSep Gold C18. Proton NMR spectra were obtained on a 400 MHz Varian nuclear magnetic resonance spectrometer. Chemical shifts (δ) are given in parts per million (ppm), coupling constant (J) values ​​are given in Hz, and the following spectral patterns are indicated by signals: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; dd, double doublet; dt, double triplet; dq, double quartet; br, broad. Tetramethylsilane was used as an internal standard. Peak enumeration, multiplicity assignments, and coupling constant calculations were performed using Mnova v.14 software (Mestrelab Research). Carbon NMR spectra were obtained on a 500 MHz Bruker AV III nuclear magnetic resonance spectrometer, and tetramethylsilane was used as the internal standard.Fluorine NMR spectra were obtained on a 400 MHz Bruker AV III nuclear magnetic resonance spectrometer. Melting points were obtained using a Stanford Research Systems OptiMelt melting point apparatus (MPA100) equipped with an automated melting point system and are uncorrected. Mass spectrometry was performed on a Waters AQUITY UPLC MS triple quadrupole mass spectrometer with ESI ionization. High pressure liquid chromatography (HPLC) purification analysis was performed using a Waters Breeze2 HPLC system equipped with a photodiode array (PDA) detector, gradient elution with binary solvent systems A and B [A, HO with 0.1% formic acid; B, CHCN with 0.1% formic acid] at a flow rate of 0.5 mL / min with UV detection at 254 nm. An ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 50 mm, was used. High resolution mass spectrometry (HRMS) was performed using an Agilent 6530 Accurate-Mass Q-TOF. All final compounds undergoing in vitro and in vivo biological testing were purified to greater than 95% purity. 1 1 H NMR and HPLC were used.

[0194] [ka]

[0195] Reagents and conditions: (a) substituted methyl bromobenzoate, XPhos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, reflux, 16 h; (b) TFA, CH2Cl2, 0 °C to room temperature, 16 h; (c) 3-chloropentane-2,4-dione, i-Pr2NEt, DMF, 0 °C to room temperature, 16 h; (d) N2H2·H2O (64–65% in H2O), CH3OH, room temperature, 1 h; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 h; (ii) neutralized to pH 7 with 2N HCl.

[0196] Example 1 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzoic acid 18a Step A: A mixture of tert-butyl piperazine-1-carboxylate 13 (2.00 g, 10.7 mmol) and methyl 3-bromo-4-fluorobenzoate (2.25 g, 9.67 mmol) in anhydrous 1,4-dioxane (50 mL) was degassed with N2 for 5 min. Then, Cs2CO3 (10.0 g, 32.2 mmol), X-Phos (0.600 g, 1.29 mmol) and Pd2(dba)3 (0.491 g, 0.53 mmol) were added and the mixture was stirred at reflux under N2 for 16 h. The mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0-30% EtOAc in hexanes) to give tert-butyl 4-(2-fluoro-5-(methoxycarbonyl)phenyl)piperazine-1-carboxylate 14a as a brown oil (3.0 g, 83%), which was used directly in the next step. ESI MS m / z 339 [M + H] +

[0197] Step B: To a solution of tert-butyl 4-(2-fluoro-5-(methoxycarbonyl)phenyl)piperazine-1-carboxylate 14a (3.00 g, 8.87 mmol) in CHCl (30 mL) cooled to 0° C., TFA (6.7 mL, 88.7 mmol) was added, and the resulting solution was gradually warmed to room temperature and stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure, diluted with H0 (30 mL), basified with saturated aqueous NaHCO (50 mL), and extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was triturated with Et0 and filtered to give pure methyl 4-fluoro-3-(piperazin-1-yl)benzoate 15a as a white solid (1.20 g, 60%). 1 ESI MS m / z 239 [M+H]+

[0198] Step C: To a solution of methyl 4-fluoro-3-(piperazin-1-yl)benzoate 15a (1.20 g, 5.02 mmol) in anhydrous DMF (10 mL) cooled to 0° C., i-Pr2NEt (0.9 mL, 5.02 mmol) and 3-chloropentane-2,4-dione (0.672 g, 5.02 mmol) were added simultaneously, and the resulting solution was gradually warmed to room temperature and stirred at room temperature for 16 h under N2 atmosphere. The mixture was diluted with H2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0-50% EtOAc in hexanes) to give methyl 3-(4-(2,4-dioxopentan-3-yl)piperazin-1-yl)-4-fluorobenzoate 16a as a brown oil (0.600 g, 35%). 1 H NMR (400 MHz, CDCl3) δ 7.64-7.62 (m, 2H), 7.06-7.01 (m, 1H), 3.86 (s, 1H), 3.31-3.09 (m, 4H), 3.09-3.05 (m, 4H), 2.27 (s, 1H), 2.24 (s, 6H);ESI MS m / z 337 [M + H] +

[0199] Step D To a solution of methyl 3-(4-(2,4-dioxopentan-3-yl)piperazin-1-yl)-4-fluorobenzoate 16a (0.500 g, 1.48 mmol) in CH3OH (10 mL) was added N2H2·H2O (0.2 mL, 2.67 mmol, 64-65% solution in H2O) and the resulting mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue was chromatographed on silica gel (0-50% EtOAc in hexanes) to give methyl 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzoate 17a as a brown solid (0.420 g, 85%). 1H NMR (400 MHz, CDCl3) δ 7.67-7.63 (m, 2H), 7.04-7.01 (m, 1H), 3.86 (s, 1H), 3.16-3.15 (m, 4H), 3.15-3.11 (m, 4H), 2.24 (s, 6H);ESI MS m / z 333 [M+H] +

[0200] Step E To a solution of methyl 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzoate 17a (0.420 g, 1.26 mmol) in CH3OH (4 mL), THF (4 mL) and H2O (2 mL) was added LiOH (91 mg, 3.79 mmol). The reaction mixture was stirred at room temperature for 16 h and concentrated under reduced pressure. The aqueous phase was diluted with H2O (30 mL) and neutralized to pH 7 with 2N hydrochloric acid. The aqueous mixture was extracted with EtOAc (3 x 50 mL) and the combined organic solution was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was chromatographed on silica gel (0-10% CHOH in CHCl) to give 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzoic acid 18a as a white solid (0.390 g, 97%). Melting point = 220-222°C; 1 H NMR (400 MHz, DMSO-d6) δ 7.53-7.55 (m, 2H, H1and H2), 7.23-7.18 (dt, J = 12 Hz, 3.2 Hz, 1H, H3), 3.04 (m, 4H, H4), 3.01 (m, 4H, H5), 2.10 (s, 6H, H6); 13 C NMR (500 MHz, DMSO-d6) δ 128.73, 121.71, 124.10, 124.03, 120.25, 120.21, 116.39, 116.22; 19 F NMR (400 MHz, DMSO-d6) δ -116.00 (s, F);ESI MS m / z 319 [M + H] + ;HRMS (ESI + ) C16 H 19 FN4O2[M + H] + Calculated value = 319.157, [M + H] + Actual value = 319.1562; Combustion analysis (%CHN): C 16 H 19 Calculated for FN4O2 0.5 H2O 0.5 HCl: %C = 55.61; %H = 5.98; %N = 16.21; Found: %C = 55.88; %H = 5.74; %N = 15.97; HPLC >99% (AUC), t R = 11.5 minutes

[0201] Example 2 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)benzoic acid 18b Compound 18b was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromobenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.48 (s, 1H), 7.36 (m, 2H), 7.23 (m, 1H), 3.23 (m, 4H), 3.04 (m, 4H), 2.14 (s, 6H);ESI MS m / z 301[M + H] + ;HPLC >99% (AUC), t R = 10.7 minutes

[0202] Example 3 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-methoxybenzoic acid 18c Compound 18c was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-4-methoxybenzoate in a similar manner to the synthesis of compound 18a. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 3.83 (s, 3H), 2.99 (s, 8H), 2.08 (s, 6H);ESI MS m / z 331 [M + H] + ;HPLC 94.6% (AUC), t R = 10.2 minutes

[0203] Example 4 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-methylbenzoic acid 18d Compound 18d was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-4-methylbenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 1.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 3.01 (m, 4H), 2.90 (m, 4H), 2.46 (s, 3H), 2.12 (s, 6H);ESI MS m / z 315 [M + H] + ;HPLC >99% (AUC), t R = 11.2 minutes

[0204] Example 5 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-(trifluoromethyl)benzoic acid 18e Compound 18e was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-4-trifluoromethylbenzoate in a similar manner to the synthesis of compound 18a. 1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 2.97 (m, 4H), 2.94 (m, 4H), 2.11 (s, 6H);ESI MS m / z 369 [M+H] + ;HPLC 98.7% (AUC), t R = 11.9 minutes

[0205] Example 6 4-Chloro-3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)benzoic acid 18f Compound 18f was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-4-chlorobenzoate in a similar manner to the synthesis of compound 18a. 1 ESI MS m / z 335 [M + H] + ;HPLC 98.0% (AUC), t R = 11.5 minutes

[0206] Example 7 18 g of 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-2-fluorobenzoic acid Compound 18g was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-2-fluorobenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.29 (m, 1H), 7.19 (m, 1H), 7.12 (t, J = 9.6 Hz, 1H), 3.03 (br, 8H), 2.12 (s, 6H);ESI MS m / z 319 [M + H] + ;HPLC 98.3% (AUC), tR = 10.3 minutes

[0207] Example 8 5-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-2-fluorobenzoic acid 18h Compound 18h ​​was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 5-bromo-2-fluorobenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.29 (m, 1H), 7.19 (m, 1H), 7.12 (t, J = 9.6 Hz, 1H), 3.13 (br, 4H), 2.99 (br, 4H), 2.10 (s, 6H);ESI MS m / z 319 [M+H] + ;HPLC >99% (AUC), t R = 10.6 minutes

[0208] Example 9 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-5-fluorobenzoic acid 18i Compound 18i was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 3-bromo-5-fluorobenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.28 (s, 1H), 6.99 (m, 2H), 3.24 (m, 4H), 2,98 (m, 4H), 2.10 (s, 6H);ESI MS m / z 319 [M + H] + ;HPLC 97.6% (AUC), t R = 11.3 minutes

[0209] Example 10 5-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluoro-2-methylbenzoic acid 18j Compound 18j was prepared from tert-butyl piperazine-1-carboxylate 13 and methyl 4-fluoro-5-bromo-2-methylbenzoate in a similar manner to the synthesis of compound 18a. 1 H NMR (400 MHz, DMSO-d6) δ 7.52 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 13.2 Hz, 1H), 3.04 (br, 8H), 2.45 (s, 1H), 2.13 (s, 6H);ESI MS m / z 333 [M + H] + ;HPLC 98.7% (AUC), t R = 11.6 minutes

[0210] [ka]

[0211] Reagents and conditions: (a) NH2OH·HCl, CH3OH, room temperature, 16 h; (b) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 h; (ii) neutralized to pH 7 with 2N HCl.

[0212] Example 11 3-(4-(3,5-dimethylisoxazol-4-yl)piperazin-1-yl)-4-fluorobenzoic acid 20 Step A: To a solution of methyl 3-(4-(2,4-dioxopentan-3-yl)piperazin-1-yl)-4-fluorobenzoate 16a (80.0 mg, 0.23 mmol) in CH3OH (2 mL) was added NH2OH·HCl (32.0 mg, 0.47 mmol) and the resulting solution was stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure and the resulting residue was chromatographed on silica gel (0-50% EtOAc in hexanes) to give methyl 3-(4-(3,5-dimethylisoxazol-4-yl)piperazin-1-yl)-4-fluorobenzoate 19 as a brown solid (20.1 mg, 25%). ESI MS m / z 334 [M + H] +

[0213] Step B: To a solution of methyl 3-(4-(3,5-dimethylisoxazol-4-yl)piperazin-1-yl)-4-fluorobenzoate 19 (8.1 mg, 0.023 mmol) in CHOH (1 mL), THF (1 mL) and H2O (0.5 mL) was added LiOH (2.7 mg, 0.11 mmol). The reaction mixture was stirred at room temperature for 16 h and concentrated under reduced pressure. The aqueous phase was diluted with H2O (15 mL) and neutralized to pH 7 with 2N hydrochloric acid. The aqueous mixture was extracted with EtOAc (3 x 10 mL) and the combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0-10% CHOH in CHCl) to give 3-(4-(3,5-dimethylisoxazol-4-yl)piperazin-1-yl)-4-fluorobenzoic acid 20 as a white solid (2.5 mg, 34%). 1 H NMR (400 MHz, DMSO-d6) δ 7.71-7.59 (m, 1H), 7.19-7.10 (m, 2H), 3.18-3.3.17 (m, 4H), 3.12-3.11 (m, 4H), 2.38 (s, 3H), 2.25 (s, 3H);ESI MS m / z 320 [M + H] + ;HPLC 96.8% (AUC), t R = 13.7 minutes

[0214] [ka]

[0215] Reagents and conditions: (a) NH4Cl, HBTU, i-Pr2NEt, DMF, room temperature, 18 hours; (b) NaN3, tetrachlorosilane, CH3CN, 80°C, 18 hours.

[0216] Example 12 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzamide 21 Step A To a mixture of 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzoic acid 18a (0.100 g, 0.314 mmol), HBTU (0.178 g, 0.471 mmol) and i-Pr2NEt (0.218 mL, 1.26 mmol) in DMF (4 mL) was added NH4Cl (16.7 mg, 0.314 mmol). The resulting solution was stirred at room temperature under N2 atmosphere for 18 h. The mixture was diluted with H2O (10 mL) and extracted with EtOAc (3 x 20 mL). The combined organic phase was washed with H2O (3 x 20 mL) and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting crude residue was chromatographed on silica gel (0% to 80% EtOAc in hexanes) to afford 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzamide 21 as a white solid (71.7 mg, 72%). 1 H NMR (400 MHz, DMSO-d6) δ 11.84 (br, 1H), 7.94 (bs, 1H), 7.54-7,48 (m, 2H), 7.31 (s, 1H), 7.14 (m, 1H), 3.06-3.01 (m, 8H), 2.10 (s, 6H);ESI MS m / z 318 [M + H] + ;HPLC >99% (AUC), t R = 10.5 minutes

[0217] Example 13 1-(3,5-dimethyl-1H-pyrazol-4-yl)-4-(2-fluoro-5-(2H-tetrazol-5-yl)phenyl)piperazine 22 Step A A mixture of 3-(4-(3,5-dimethyl-1H-pyrazol-4-yl)piperazin-1-yl)-4-fluorobenzamide 21 (0.180 g, 0.526 mmol), NaN3 (0.142 g, 0.375 mmol) and tetrachlorosilane (98.5 mg, 0.579 mmol) in CH3CN (4 mL) was stirred at 80 °C for 18 h in a sealed vessel. The reaction mixture was cooled to room temperature and diluted with saturated NaHCO3 (5 mL). The aqueous mixture was extracted with CHCl3 (3 x 50 mL) and the combined organic phase was washed with brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0% to 10% CHOH in CHCl) to give 1-(3,5-dimethyl-1H-pyrazol-4-yl)-4-(2-fluoro-5-(2H-tetrazol-5-yl)phenyl)piperazine 22 as a white solid (60.3 mg, 30%). 1 H NMR (400 MHz, DMSO-d6) δ 7.69 (m, 1H), 7.67 (m, 1H), 7.36-7. (dd, J = 4Hz, J = 8.4 Hz, 1H);3.12 (m, 4H), 3.09 (m, 4H), 2.13 (s, 6H);ESI MS m / z 343 [M+H] + ; HPLC >99% (AUC), t R = 11.1 minutes

[0218] Example 14 Fluorescence Polarization TTR Tetramer Binding Assay Compound binding to TTR was assessed by a fluorescence polarization assay. The assay measured the competitive displacement of the fluorescent probe FITC-diclofenac on TTR (Clabiochem-Millipore, cat. No. 52957) isolated from human plasma. FITC-diclofenac was synthesized at LeadGen Labs, LLC. To each well, FP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% CHAPS, 0.01% Prionex) containing 200 nM TTR and 100 nM FITC-diclofenac was added along with the test compound. Nonspecific binding was measured in the presence of 500 μM unlabeled diclofenac (Sigma-Aldrich). Reactions with test compounds were incubated overnight at 4° C., and FP was measured on a Spectramax M5e plate reader (Molecular Devices).

[0219] Example 15 TTR Aggregation Assay The ability of test compounds to prevent TTR aggregation was evaluated in acidic conditions that favor TTR aggregation and fibril formation. 2 μl of a solution of 167 μM human TTR (ACRO Biosystems #H5223) was incubated with 7 μl of 50 mM sodium acetate (pH=4.0) (Sigma #S7545), 100 mM KCl (Sigma #S5405) in the presence or absence of 1 μl of TTR inhibitor at 37° C. for 72 hours. At the end of the incubation, 3.5 μl of 500 mM sodium phosphate (Sigma #S5136) buffer (pH=8.0) was added to each sample for neutralization, and 0.6 μl of 5% CHAPS (Sigma #C5070) was added as a detergent to prevent reassociation of the proteins. Cross-linking was performed by adding 1.5 μl of 5% glutaraldehyde solution (Sigma #G6257). After 4 min, 2.5 μl of freshly prepared 5% NaBH4 was added to stop the reaction. Samples were subjected to TTR Western blotting using a prealbumin antibody (1:500; Dako #A0002). Band intensities of TTR monomer and TTR aggregates were quantified from scanned images of the blots.

[0220] Example 16 Binding of compounds to RBP4 in vitro Compounds that bind to RBP4 were evaluated in a radiometric scintillation proximity assay (SPA) as previously described (Cioffi, CL et al. 2014; Cioffi, CL et al. 2015; Cioffi, CL et al. 2020). The assay involves the detection of radiolabeled [ 3 Competitive displacement of [H]-all-trans-retinol was measured. Proteins were biotinylated using ThermoFisher's EZ-link Sulfo-NHS-LC-Biotinylation kit (Cat #21335) as recommended by the manufacturer. Binding assays were performed in a final volume of 100 μL SPA buffer (1× PBR, pH 7.4, 1 mM EDTA, 0.1% BRA, 0.5% CHAPS). The assay reaction consisted of 10 nM [H]-all-trans-retinol. 3 [H]-all-trans-retinol (48.7 Ci / mmol; PerkinElmer, Waltham, MA) was included along with 0.3 mg / well of streptavidin-PVT beads (PerkinElmer, RPNQ0006) and 50 nM biotinylated human RBP4. Nonspecific binding was assessed by adding 20 μM unlabeled retinol (Sigma, cat #95144) to control wells. After 16 h of incubation at room temperature with gentle shaking, radioactivity counts were measured using a CHAMELEON plate reader (Hidex Oy, Turku, Finland).

[0221] [Table 1] a IC of the fluorescence polarization (FP) assay obtained in the presence of a fixed concentration of 25 μM fluorescein isothiocyanate (FITC)-conjugated TTR FP probe 50 value. b Fixed concentration 10 nM 3 IC of the SPA assay obtained in the presence of H-retinol 50 value. c For compounds that were tested multiple times (three or more), the IC 50 Data are expressed as mean ± standard deviation. For compounds tested in duplicate, IC 50 Data are presented as the average of two independent experiments, not as the mean ± standard deviation.

[0222] Example 17 Kinetic Solubility Assay Kinetic water solubility of compound 18a in PBS (pH 7.4) was determined on Eurofins using UV detection (230 nm). Water solubility (μM) was determined by comparing the area of ​​the main peak in calibration standards (200 μM) containing organic solvent (methanol / water, 60 / 40, v / v) with the area of ​​the corresponding peak in the buffer sample. Furthermore, chromatographic purity (%) was defined as the peak area of ​​the main peak relative to the total integrated peak area in the HPLC chromatogram of the calibration standard. Chromatograms of calibration standards of each test compound were generated along with UV / VIS spectra with labeled absorption maxima.

[0223] Standards for kinetic solubility experiments Metoprolol - 192.6 μM Rifampicin - 200μM Ketoconazole - 152.8 μM Phenytoin - 101.8μM Simvastatin - 14.2μM Diethylstilbestrol - 7.0 μM Tamoxifen - 1.9 μM

[0224] Example 18: CYP450 Inhibition Assay Inhibitory potency (IC) of compound 18a against human cytochrome P450 (CYP) isoforms 2C9, 2C19, 2D6, and 3A4 50 Each recombinant human CYP isoform was tested with standard positive and negative controls using fluorescent detection to measure CYP activity. IC values ​​for each standard inhibitor were 50All observed values ​​were within the expected range for each isoform (see below).

[0225] IC of standard CYP inhibitors 50 concentration: CYP Inhibitor IC 50 (μM): 2C9 Sulfaphenazole IC 50 = 3.4 μM 2C19 Tranylcypromine IC 50 = 2.8 μM 2D6 Quinidine IC 50 =0.058μM 3A4 Ketoconazole IC 50 =0.0084μM

[0226] Preformulated NADPH regenerating solution, recombinant CYP isoforms 2C19 and 3A4 (Lot #3007790 and 2276593, respectively), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-cyano-7-ethoxycoumarin (CEC), and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were obtained from Corning Life Sciences (Bedford, MA). Recombinant CYP isoform 2D6 (Lot #49242) was obtained from Invitrogen (Carlsbad, CA). CYP isoform 2C9 (Lot #0446966-1) was obtained from Cayman Chemical (Ann Arbor, MI). 7-Methoxy-4-trifluoromethylcoumarin (MFC), trans-2-phenylcyclopropylamine hydrochloride (TCP), sulfaphenazole (SFZ), ketoconazole (KTZ), and quinidine (QDN) were obtained from Sigma (St. Louis, MO). All solvents and buffers were used as commercial products without further purification.

[0227] method Test compounds were prepared as 10 mM stock solutions in acetonitrile. Inhibition of four human P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) expressed in insect cells as cRNAs was tested by a fluorescence-based assay. Nine serial dilutions (0-100 μM) of each test compound stock solution were prepared in black microtiter plates. The dilution series was incubated at 37°C with each CYP isoform and a standard fluorogenic probe substrate for each isoform. The probe substrate concentrations added were at or near the Km value for each CYP isoform. The reaction mixture contained potassium phosphate buffer (pH 7.4) and an NADPH regenerating system. The final reaction volume was 0.20 mL, and after the appropriate incubation time (15-45 min), the reaction was terminated with 75 μL of stop solution (0.5 M Tris base in acetonitrile). Fluorescence measurements were performed at appropriate excitation and emission wavelengths. Control wells without test compound, blank wells with stop solution prior to addition of the isoforms, and dilution series with standard inhibitors of each isoform were also performed. All experiments were performed in duplicate. IC 50 Values ​​were calculated using nonlinear regression of the data using a four-parameter logistic model (dose-response equation) fit with XLFit 5.2 in IDBS software (Emeryville, CA), supported by linear interpolation of the data at the concentration where the noninhibitory rate was approximately 50%.

[0228] Example 19 Plasma Protein Binding Assay Plasma protein binding (PPB) of compound 18a in PBS (pH 7.4) was determined at Eurofins using equilibrium dialysis of plasma with HPLC-UV / Vis detection.

[0229] Mean Plasma Protein Binding of Control Propranolol in Human, Rat (Sprague Dawley), Mouse (CD-1), and Dog (Beagle) Plasma Using the peak areas of the test compound in the buffer and the test sample, the following formula:

number

[0230] Example 20 Metabolic stability Metabolic stability in microsomes Metabolic stability measurements of the novel compounds and testosterone (positive control) were performed in the presence of human, rat, mouse and monkey liver microsomes. Values ​​shown are the percentage of parent remaining after 30 min incubation. All measurements were performed in duplicate. Testosterone assay results were within acceptable limits.

[0231] Metabolic clearance in microsomes Mixed-gender human liver microsomes (Lot #1710084), male Sprague-Dawley rat liver microsomes (Lot #1610290), male CD-1 mouse liver microsomes (Lot #1710069) and male cynomolgus monkey liver microsomes (Lot #1510193) were purchased from XenoTech. Reaction mixtures, excluding NADPH, were prepared as follows: Test compounds were added to the reaction mixture at a final concentration of 1 μM. A control compound, testosterone, was run simultaneously with the test compounds in a separate reaction. An aliquot of the reaction mixture (without cofactor) was equilibrated in a shaking water bath at 37° C. for 3 minutes. The reaction was initiated by the addition of cofactor and the mixture was incubated in a shaking water bath at 37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30 and 60 minutes. Test compounds and testosterone samples were immediately mixed with 400 μL of ice-cold acetonitrile (ACN) / H2O (50 / 50) with 0.1% formic acid and internal standard to terminate the reaction. Samples were then mixed and centrifuged to precipitate proteins. All samples were assayed by LC-MS / MS with electrospray ionization. Peak area response ratios (PARR) to the internal standard were compared to the PARR at time 0 to determine the percentage remaining at each time point. Half-lives were calculated using GraphPad software by fitting to a single-phase exponential decay equation.

[0232] [Table 2]

[0233] a Kinetic solubility measured in PBS (pH=7.4). b Microsomal specific clearance (CL int );H=human;R=rat;M=mouse;cyno=cynomolgus monkey. c Hepatic microsomal metabolic stability, percentage of parent drug remaining after 30 min incubation in the presence of microsomes; HLM = human liver microsomes; RLM = rat liver microsomes; MLM = mouse liver microsomes; cynoLM = cynomolgus monkey liver microsomes.d CiPA hERG QPatch assay; compounds were tested in a five point concentration-response experiment (n=2). e %PPB=plasma protein bound; H=human, R=rat, M=mouse.

[0234] Example 21: In vivo PK assay Mouse PK experiment information and data Drug-naive adult male CD-1 mice were administered a single dose of test compound intravenously (IV) or orally (PO).

[0235] Testing Facility and Site: Absorption Systems, LLC, 436 Creamery Way, Suite 600, Exton, PA 19341-2556 Test compounds and vehicles: Vehicle for IV administration: 3% DMA / 45% PEG300 / 12% ethanol / 40% sterile water. Vehicle for PO administration: 2% Tween® 80 in 0.9% saline. Formulations: Formulations were prepared by stepwise addition of the individual components of the vehicle (in the order listed) to a volume of test compound that resulted in the desired final concentration. Each formulation was prepared by mixing a weighed amount of test compound with the appropriate volume of vehicle. Dose solution analysis: Dose solutions were analyzed by LC-MS / MS. Dose solutions were diluted and analyzed in mouse blood (performed in triplicate). All concentrations are given as mg / mL of free base. Nominal doses were used in all calculations for Group 1.

[0236] Test system: Species and strain: Mouse; male CD-1. Mean weight: 0.034 kg in the IV group; 0.027 kg in the PO group. Number: 3 animals total, with the same 3 animals in each dosing group (Group 1 (IV) and Group 2 (PO)). Compliance: This non-clinical study followed established practices and standard operating procedures and study protocols for absorption systems. This study is exploratory in nature and was not conducted in accordance with the principles set forth in the U.S. Food and Drug Administration's (FDA) Good Laboratory Practices (GLP) regulations, 21 Code of Federal Regulations (CFR) Part 58. Reports will be stored in a validated scientific data management system. Electronic signatures will comply with 21 CFR Part 11.

[0237] Experimental design Blood was collected from mice before dosing and at 5, 15, 30 min, 1, 2, 4, 8, 12, 24, and 48 h after dosing. Hemolyzed blood samples were extracted by protein precipitation with acetonitrile. After protein removal with acetonitrile, the amount of compound was measured by LC-MS / MS. Pharmacokinetic parameters were calculated from the time course of blood concentration. Pharmacokinetic parameters were determined with a non-compartmental model in Phoenix WinNonlin (v8.0) software. Maximum blood concentration (C0) after IV dosing was estimated by extrapolating the data of the first two time points to t=0. Maximum blood concentration (Cmax) and time to maximum blood concentration (tmax) after PO dosing were observed from the data. Area under the blood concentration-time curve (AUC) was calculated using the linear trapezoidal rule, calculated to the last quantifiable data and extrapolated to infinity if possible. The blood half-life (t 1 / 2) was calculated from 0.693 / slope of terminal elimination. The mean residence time (MRT) was calculated by dividing the area under the moment curve (AUMC) by AUC. The clearance (CL) was calculated as dose / AUC. The volume of distribution at steady state (Vss) was calculated as CL × MRT. Bioavailability was calculated as PO AUC normalized to individual doses. ∞ IV AUC values ​​normalized to mean dose ∞ Samples below the limit of quantitation (1.00 ng / mL) were treated as zero in the pharmacokinetic data analysis.

[0238] [Table 3]

[0239] Data are presented as the mean (mean (SD)) with standard deviation in brackets. Dosing groups consisted of drug-naive adult male CD-1 mice. IV administration: test compound administered at a dose of 2 mg / kg; vehicle for test compound = 3% DMA / 45% PEG300 / 12% ethanol / 40% sterile water; PO administration: test compound administered at a dose of 5 mg / kg; vehicle = 2% Tween® 80 in 0.9% saline. a Initial blood concentration of the compound at time zero. b Full body clearance. c The apparent half-life for the terminal elimination of a compound from the blood. d Steady-state volume of distribution. e The area under the blood concentration-time curve from 0 to the last time point at which a compound was quantifiable in blood. f The area under the blood concentration-time curve from 0 to infinity. g Peak blood concentration of the compound. h Time to peak plasma concentration of compound. i Bioavailability; F = (AUC INFpo × Dose iv ) ÷ AUC INFiv × Dose po )

[0240] Example 22 Serum RBP4 Measurement Assay Blood samples were collected from the tail vein. Whole blood was placed in a centrifuge tube and allowed to clot at room temperature for 30 minutes, then centrifuged at 2000g at 48°C for 15 minutes to collect serum. Aliquots of plasma samples collected in mouse pharmacokinetic studies were analyzed for RBP4 concentration using the RBP4 (mouse / rat) Dual ELISA Kit (AdipoGen, San Diego, CA) according to the manufacturer's instructions. Whole blood was collected in a centrifuge tube and allowed to clot at room temperature for 30 minutes, then centrifuged at 2000g at 4°C for 15 minutes to collect serum. Mouse serum RBP4 (mainly produced in the liver) was measured using the RBP4 Dual ELISA Kit (AdipoGen, San Diego, CA; cat #AG-45A-0012YTP-KI01).

[0241] Example 23 Abca4 - / - Validity in the model Compound 18a·dihydrochloride was administered to Abca4 rats for 12 weeks after feeding. - / - This was done in mice. Abca4 - / - An age-matched control group of mice was fed standard Picolab® 5053 chow without any compound. A reference group of age-matched wild-type mice was fed mice without Abca4 deletion (these Abca4 + / + Mice were used to determine basal levels of A2E in mice (fed on standard Picolab® 5053 diet). Blood samples to assess serum levels of RBP4 were collected from compound-treated Abca4 mice. - / - Abca4 mice treated with diet and control - / - Mice were sampled prior to dosing, at 4 weeks, and at 8 weeks after the end of compound treatment. A significant decrease in serum RBP4 was recorded in compound-treated mice (Figure 7). After 8 weeks of treatment, treated and untreated Abca4 - / - Mouse eyecup, as well as reference Abca4 + / + Eyecups of mice were collected for quantitative A2E analysis by HPLC. - / - Compared to compound-treated Abca4 mice - / - In mice, A2E was significantly reduced (Figure 8). A 98.5% reduction in A2E abundance induced by compound 18a dihydrochloride resulted in A2E levels lower than those in untreated wild-type animals (Figure 8).

[0242] In addition to biochemical characterization of A2E levels by HPLC, the inventors also + / + Mice (Figure 9A), untreated Abca4 - / - Mice (Figure 9B) and compound 18a·dihydrochloride-treated Abca4 - / - We assessed A2E fluorescence in retinal sections from mice (Figure 9C). Abca4 gene deletion significantly reduced Abca4 expression compared to wild-type controls (Figure 9A). - / -Compound 18a dihydrochloride induced a significant increase in A2E autofluorescence in mice (Fig. ​(Fig.9B).9B). Treatment with compound 18a dihydrochloride markedly reduced the intensity of A2E autofluorescence (Fig.9C), consistent with a strong inhibition of bisretinoid synthesis.

[0243] Animal Care and Use Statement: All procedures conformed to the United States Department of Agriculture (USDA) Animal Welfare Act (9 CFR parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals, Office of Laboratory Animal Resources, National Academy Press, Washington, DC, 1996, and the National Institutes of Health Office of Laboratory Animal Welfare. Procedures in this study were designed to avoid or minimize, whenever possible, animal discomfort, distress, and pain.

[0244] Consideration To identify a novel class of TTR tetramer kinetic stabilizers that can lower RBP4 in blood, we performed structure-based drug design using the clinically investigated compound 4 (Figure 2) as a lead compound. 1) Compound 4 has been reported to effectively bind to both WT-TTR and the pro-amyloidogenic V122I-TTR variant, and 2) Compound 4 has a TTR binding affinity similar to that of compound 3 (the K d = 4.8 ± 1.9 nM; K for compound 3 d = 4.4 ± 1.3 nM) (Miller, M. et al. 2018; Penchala, S. C. et al. 2013), it was also reported to be more potent and selective than compound 3 in stabilizing the TTR tetramer in buffer and human serum, and therefore compound 4 was selected as a benchmark molecular scaffold for developing a novel series of ligands.

[0245] Compounds were initially evaluated in two assays designed to measure (1) the binding affinity of compounds to the unliganded TTR tetramer (fluorescence polarization assay, FP) and (2) the binding affinity of compounds to RBP4 not associated with TTR (scintillation proximity assay, SPA). The results are shown in Table 1. Compound 18a (FP IC50 =220nM) is benchmark 3 (FP IC 50 = 410 nM) and was approximately twice as potent in TTR as in Benchmark 4 (FP IC 50 = 160 nM) (Table 1). Compound 18a is inactive in the in vitro RBP4 scintillation proximity assay (SPA), which measures the binding affinity of all-trans retinol competitive ligands at RBP4 (RBP4 SPA IC 50 Compound 18a showed good selectivity for RBP4 (Table 1).

[0246] The ability of TTR ligands to act as kinetic stabilizers of the TTR tetramer was evaluated in vitro by a low pH-induced TTR aggregation SDS-PAGE assay (Coelho, T. et al. 2016;Cruz, MW 2019). Incubation of TTR tetramers at pH=4.0 for 72 hours initiates dissociation into monomeric intermediates that misassemble and oligomerize into amyloid fibrils and other high molecular weight forms (Lamb, YN & Deeks, ED 2019). The ability of compound 18a to act as a kinetic stabilizer of the TTR tetramer was evaluated by its ability to inhibit low pH-mediated TTR aggregate formation using previously published protocols (Park, J. et al. 2020;Cioffi, CL et al. 2020). FDA-approved compound 3 was used as a positive control in the aggregation experiments. As shown in Figure 5A, the amount of high molecular weight forms of TTR after incubation of TTR tetramers with DMSO at pH 4 and 37 °C for 72 h is significantly increased compared to the DMSO control incubated at pH 7.5 for 72 h. Consistent with its ability to act as a kinetic TTR stabilizer, treatment with compound 18a (50 μM) significantly inhibited the formation of high molecular weight forms of TTR, as did compound 3 (50 μM) (Figure 5A). The stronger intensity of the TTR monomer and dimer bands in samples treated with compound 18a or compound 3 compared to DMSO correlated with the corresponding reduction in TTR aggregates induced by compound 18a or compound 3. Quantification of band intensities on Western blots confirmed that a 3.1-fold reduction in the amount of aggregates was induced in the presence of compound 18a, whereas compound 3 induced a 3.0-fold reduction (Figure 5B). The significant increase in the dimer band intensity and the considerable increase in the TTR monomer band intensity caused by compound 18a or compound 3 were associated with the inhibition of low-pH-induced TTR aggregate formation (FIGS. 5C and 5D). These results indicated that compound 18a could act as a TTR tetramer kinetic stabilizer.

[0247] Compound 18a exhibited excellent kinetic solubility in phosphate buffered saline (PBS) (pH 7.4), and the observed microsomal stability and CLint Values ​​suggest very low predicted hepatic clearance in multiple species (Table 2). Percent plasma protein binding (%PPB) data indicate low unbound fraction in human, rat, and mouse (Table 2). Furthermore, compound 18a lacked limiting inhibitory activity at the standard CYP panel, the hERG channel, or the nuclear peroxisome proliferator-activated receptor-γ (PPARγ) receptor (Table 2).

[0248] Compound 18a showed good plasma clearance (0.354 L / hr / kg) with a half-life of 5.08 hours after a single dose (2 mg / kg IV) in CD-1 male mice (Table 3). The compound was well absorbed and slowly eliminated from plasma, with a C of 0.001 after oral administration of a single dose (5 mg / kg). max was 1563ng / ml, and the corresponding T max The average exposure time (AUC INF The oral bioavailability was 16073 hr·ng / mL and excellent (%F=103%).

[0249] Two hours after a single 25 mg / kg oral dose of compound 18a, serum RBP4 was reduced by up to 66% (Figure 6A). There was no effect of compound administration on serum TTR levels (data not shown). The in vivo kinetics of serum RBP4 reduction demonstrated a good correlation between the presence of compound 18a in the blood after oral administration (Figure 6B) and the reduction in serum RBP4 (Figure 6A). The maximum reduction in RBP4 observed at 1 and 2 hours correlated very well with the rapid oral absorption resulting in high concentrations of compound 18a in the blood at these time points (Figures 6A, 6B). Similarly, the low levels of compound 18a in the blood after 24 hours correspond well with serum RBP4 levels (Figures 6A, 6B). This data indicates a very good PK / PD relationship between compound 18a exposure and serum RBP4 reducing activity in mice.

[0250] Decreased serum RBP4 and Abca4 in Stargardt disease induced by different classes of selective RBP4 antagonists- / - A direct correlation between the bisretinoid lowering activity in mouse models has been previously established (Radu, RA et al. 2005;Racz, B. et al. 2018;Dobri, N. et al. 2013). Based on the very good in vivo RBP4 lowering activity of compound 18a, it is expected that this compound may be effective in suppressing the formation of cytotoxic lipofuscin bisretinoids in the retina, justifying the evaluation of selective TTR ligands as a class of potential therapeutic agents for the treatment of Stargardt's disease, dry AMD, and other conditions characterized by enhanced lipofuscin accumulation in the retina.

[0251] These results show a novel class of TTR tetramer kinetic stabilizers that selectively bind to TTR tetramers. Thus, these compounds can be applied to treat ATTR-CM, ATTR-PN, FAP, FAC or SSA and other ATTR. These ligands may also reduce the blood levels of RBP4 in vivo. Thus, in addition to diseases characterized by ATTR, these compounds may be effective in suppressing the formation of cytotoxic lipofuscin bisretinoids in the retina while simultaneously preventing possible TTR amyloid fibril formation. Thus, these selective TTR tetramer ligands may also have applications as therapeutic agents for Stargardt's disease, dry AMD and other conditions characterized by enhanced lipofuscin accumulation in the retina, especially for patients prone to ATTR comorbidities such as sporadic SSA or hereditary TTR amyloidosis.

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Claims

1. structure: 【Chemistry 1】 (In the formula, X １ is N or CR ５ and R ５ is H, OH, halogen or alkyl, X ２ , X ３ and X ４ are each independently NH, N, S, O, or CR ６ and Each R ６ are independently H, OH, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ H and R １ , R ２ , R ３ and R ４ are each independently -H, -F, -Cl, -Br, -I, or -NO ２ , -CN, -CF ３ , -CF ２ H, -OCF ３ , -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl), -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -SH, -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -NH ２ , -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl), -NH-(heteroaryl), -C(O)R ７ , -S(O)R ７ , -SO ２ R ７ , -NHSO ２ R ７ , -OC(O)R ７ , -SC(O)R ７ , -NHC(O)R ７ or -NHC(S)R ７ and R ７ is H, -(alkyl), -OH, -O(alkyl), -NH ２ , -NH(alkyl) or -N(alkyl) ２ and B is, 【Chemistry 2】 or not present, R ８ is H, OH, halogen, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ H, C is H, a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO ２ H, COOR ９ , O.H., O.R. ９ , N.H. ２ , N.H.R. ９ , N.R. ９ R １０ , S.O. ２ R １１ , C.H. ２ NHR ９ , C.H. ２ N.R. ９ R １０ or CH ２ COOR ９ and R ９ and R １０ are each independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH ２ , -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl) ２ , -CH ２ NH(alkyl), -CH ２ COOH, -SO ２ CH ３ , —OH, —O(alkyl), —NH ２ , -NH(alkyl) or -N(alkyl) ２ and R １１ is an alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, NH ２ , NH(alkyl), NH(cycloalkyl), NH(heterocycle), NH(aryl), NH(heteroaryl) or NHCOR １２ and R １２ is alkyl, haloalkyl, cycloalkyl, heterocycle, aryl or heteroaryl. or a pharma- ceutically acceptable salt thereof.

2. X １ is N or CR ５ and R ５ is H, OH, halogen or alkyl, X ２ , X ３ and X ４ are each independently NH, N, S, O, or CR ６ and Each R ６ are independently H, OH, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ H and R １ , R ２ , R ３ and R ４ are each independently -H, -F, -Cl, -Br, -I, or -NO ２ , -CN, -CF ３ , -CF ２ H, -OCF ３ , -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl), -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -SH, -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -NH ２ , -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl) or -NH-(heteroaryl); B is, 【Chemistry 3】 or not present, R ８ is H, OH, halogen, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ H, C is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO ２ H, COOR ９ , O.H., O.R. ９ , N.H. ２ , N.H.R. ９ , N.R. ９ R １０ , S.O. ２ R １１ , C.H. ２ NHR ９ , C.H. ２ N.R. ９ R １０ or CH ２ COOR ９ and R ９ and R １０ are each independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH ２ , -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl) ２ , -CH ２ NH(alkyl), -CH ２ COOH, -SO ２ CH ３ , —OH, —O(alkyl), —NH ２ , -NH(alkyl) or -N(alkyl) ２ and R １１ is an alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, NH ２ , NH(alkyl), NH(cycloalkyl), NH(heterocycle), NH(aryl), NH(heteroaryl) or NHCOR １２ and R １２ 2. The compound of claim 1, wherein is alkyl, haloalkyl, cycloalkyl, heterocycle, aryl or heteroaryl, or a pharma- ceutically acceptable salt thereof.

3. X １ is N, X ２ , X ３ and X ４ are each independently NH, N, S, O, or CR ６ and Each R ６ are independently H, halogen, alkyl, alkenyl, alkynyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ H, R １ , R ２ , R ３ and R ４ are each independently -H, -F, -Cl, -Br, -I, -CN, or -CF ３ , -CF ２ H, -OCF ３ , -(alkyl), -(alkenyl), -(alkynyl), -(aryl), -(heteroaryl), -(cycloalkyl), -(cycloalkylalkyl), -(heteroalkyl), heterocycle, heterocycloalkyl, -(alkylheteroalkyl), -(alkylaryl), -OH, -OAc, -O-(alkyl), -O-(alkenyl), -O-(alkynyl), -O-(aryl), -O-(heteroaryl), -NH ２ , -NH-(alkyl), -NH-(alkenyl), -NH-(alkynyl), -NH-(aryl) or -NH-(heteroaryl); B-C is -CO ２ H, -CONH ２ or 【Chemistry 4】 2. The compound of claim 1, wherein:

4. Structure: a) 【Chemistry 5】 or b) 【Chemistry 6】 4. The compound of claim 3, having the formula:

5. X ３ is NH, X ２ and X ４ is CR ６ or X ３ is O and X ２ and X ４ is CR ６ or X ３ is S and X ２ and X ４ is CR ６ That is, The compound according to claim 4.

6. R ６ is H, OH, alkyl, alkenyl, alkynyl, haloalkyl, -O-(alkyl), -S-(alkyl), -NH ２ , -NH-(alkyl), -N(alkyl) ２ Or -CO ２ The compound of claim 5, wherein R 6 is H, preferably R 6 is alkyl, more preferably R 6 is methyl or -CF 3 .

7. B-C is -CO ２ H, -CONH ２ or 【Chemistry 7】 Preferably, BC is -CO2H.

8. R １ , R ２ , R ３ and R ４ are each independently -H, -F, -Cl, -Br, -I, or -NO ２ , -CN, -CF ３ , -CF ２ H, -OCF ３ , -(alkyl), -(haloalkyl), -(alkenyl), -(alkynyl), -OH, -OAc, -O-(alkyl), -S-(alkyl), preferably R 1 , R 2 , R 3 and R 4 are each independently H, F, Cl, CH 3 , CF 3 or OCH 3 .

9. R １ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ２ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ３ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ４ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ That is, Preferably, a) R 1 is H, F, Cl, CH 3 , CF 3 or OCH 3 , and R 2 , R 3 and R 4 are each H; b) R 1 is F, Cl, CH 3 , CF 3 or OCH 3 , R 3 is CH 3 , and R 2 and R 4 are each H; c) R 1 is F and R 2 , R 3 and R 4 are each independently H, F, Cl, CH 3 , CF 3 or OCH 3 ; d) R 1 is F and R 2 , R 3 and R 4 are each H; or e) R 1 is Cl and R 2 , R 3 and R 4 are each independently H, F, Cl, CH 3 , CF 3 or OCH 3 ; or f) R 1 is Cl and R 2 , R 3 and R 4 are each H; The compound according to claim 8.

10. B-C is -CO ２ H, -CONH ２ or 【Chemistry 8】 Preferably, a) R 1 is F or Cl, R 2 , R 3 and R 4 are each H, and BC is —CO 2 H; or b) R 1 is F or Cl, R 2 , R 3 and R 4 are each H, and BC is —CONH 2 , or c) R 1 is F or Cl, R 2 , R 3 and R 4 are each H, and B-C is 【Chemistry 9】 That is, The compound of claim 1.

11. structure: 【Chemistry 10】 having Preferably X 1 is N or CR 5 .

2. The compound of claim 1 or a pharma- ceutically acceptable salt thereof.

12. B-C is -CO ２ H, -CONH ２ or 【Chemistry 11】 12. The compound of claim 11, wherein

13. structure: 【Chemistry 12】 (In the formula, R 1 , R 2 , R 3 and R 4 are each independently H, F, Cl, CH 3 , CF 3 or OCH 3 .) 13. The compound of claim 12 having the formula:

14. R １ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ２ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ３ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ or R ４ is H, F, Cl, CH ３ , C.F. ３ Or OCH ３ That is, Preferably, a) R 1 is H, F, Cl, CH 3 , CF 3 or OCH 3 , and R 2 , R 3 and R 4 are each H; b) R 1 is F, Cl, CH 3 , CF 3 or OCH 3 , R 3 is CH 3 , and R 2 and R 4 are each H; c) R 1 is F and R 2 , R 3 and R 4 are each H; or d) R 1 is Cl and R 2 , R 3 and R 4 are each H; The compound of claim 13.

15. The compound has the structure: 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 2. The compound of claim 1, wherein:

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 and a pharma- ceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, for stabilizing TTR tetramers in a mammal, comprising administering to the mammal a quantity of a composition effective to stabilize the TTR tetramer, or for preventing the formation of TTR aggregates or high molecular weight aggregates in a mammal, comprising administering to the mammal a quantity of a composition effective to prevent the formation of TTR aggregates or high molecular weight aggregates.

18. 1. A pharmaceutical composition for treating TTR amyloidosis (ATTR) in a mammal, comprising administering to said mammal an effective amount of the composition, Preferably, (a) the composition is further effective to stabilize a FTR tetramer in the mammal, and / or (b) the TTR amyloidosis (ATTR) is peripheral polyneuropathy (ATTR-PN), TTR amyloid cardiomyopathy (ATTR-CM), late-onset familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) or senile systemic amyloidosis (SSA), or the TTR amyloidosis (ATTR) is characterized by deposition of amyloid aggregates; 17. The pharmaceutical composition of claim 16.

19. 1. A pharmaceutical composition for the treatment of a disease in a mammal characterized by the accumulation of excess lipofuscin in the retina, comprising administering to said mammal an effective amount of a composition comprising: (a) the disease is further characterized by bisretinoid induced macular degeneration; (b) the amount of the composition is effective to reduce a serum concentration of RBP4 in the mammal, or the amount of the composition is effective to reduce a retinal concentration of a bisretinoid in lipofuscin in the mammal. (c) the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE; or (d) the disease characterized by excess accumulation of lipofuscin in the retina is age-related macular degeneration, dry (atrophic) age-related macular degeneration, Stargardt's disease, Best's disease, adult vitelliform maculopathy, or Stargardt-like macular dystrophy; 17. The pharmaceutical composition of claim 16.

20. A pharmaceutical composition for treating TTR amyloidosis (ATTR), a disease characterized by the accumulation of excess lipofuscin in the retina, or both TTR amyloidosis (ATTR) and a disease characterized by the accumulation of excess lipofuscin in the retina, comprising administering to a mammal an effective amount of the composition, Preferably a) the amount of said composition is effective to stabilize a TTR tetramer in said mammal; b) the amount of the composition is effective to prevent the formation of FAP aggregates or to prevent the formation of high molecular weight aggregates; or c) the amount of the composition is effective to reduce a serum concentration of RBP4 in the mammal, or the amount of the composition is effective to reduce a retinal concentration of a bisretinoid in lipofuscin in the mammal.

17. The pharmaceutical composition of claim 16.

21. (a) an amount of the composition effective to stabilize a TTR tetramer in the mammal and reduce a serum concentration of RBP4 in the mammal; (b) the amount of the composition is effective to prevent the formation of FAP aggregates or high molecular weight aggregates in the mammal and to reduce the serum concentration of RBP4 in the mammal; (c) the amount of the composition is effective to stabilize FTR tetramers in the mammal and reduce retinal concentrations of bisretinoids in lipofuscin in the mammal; or (d) the amount of said composition is effective to prevent formation of FAP aggregates or prevent formation of high molecular weight aggregates in said mammal and to reduce retinal concentrations of bisretinoid in lipofuscin of said mammal.

22. 21. The pharmaceutical composition of claim 20, wherein the TTR amyloidosis (ATTR) is peripheral polyneuropathy (ATTR-PN), TTR amyloid cardiomyopathy (ATTR-CM), late-onset familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) or senile systemic amyloidosis (SSA), or the TTR amyloidosis (ATTR) is characterized by deposition of amyloid aggregates, preferably the disease is further characterized by bisretinoid-induced macular degeneration.

23. 21. The pharmaceutical composition of claim 20, wherein the amount of the composition is effective to reduce the serum concentration of RBP4 in the mammal, or the amount of the composition is effective to reduce the retinal concentration of a bisretinoid in lipofuscin in the mammal, preferably wherein the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE.

24. The pharmaceutical composition according to claim 20, wherein the disease characterized by accumulation of excess lipofuscin in the retina is age-related macular degeneration, dry (atrophic) age-related macular degeneration, Stargardt's disease, Best's disease, adult vitellogenic maculopathy or Stargardt-like macular dystrophy.

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