A bispecific antagonist of retinol-binding protein 4 that stabilizes the transthyretin tetramer, its preparation, and its use in the treatment of common age-related complications.

Abstract

This invention provides a bispecific antagonist of retinol-binding protein 4, its preparation, and its use in the treatment of common age-related complications. [Solution] A compound having the following structure, or a pharmaceutically acceptable salt thereof, is provided. JPEG2026143489000061.jpg5929

Description

[Background Art]

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 054,218, filed on July 20, 2020, the content of which is incorporated herein by reference.

[0002] Throughout this application, certain publications are referenced in parentheses. Complete citations for these publications can be found immediately before the claims. The entire disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the present invention pertains.

[0003] Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Age-dependent accumulation of cytotoxic lipofuscin bisretinoids in the retina may significantly contribute to the atrophic pathogenesis of AMD. The essential vitamin all-trans-retinol (vitamin A, 1) (Figure 1) functions as a precursor for the biosynthesis of retinoic acid (2) (Steinmetz, AC et al. 2001; Clagett-Dame, M. & DeLuca, HF 2002; Wolf, G. 1984), 11-cis-retinal (3) (Kiser, PD et al. 2014; Tsin, A. et al. 2018), and many other important retinoids, which are involved in multiple cellular processes and numerous important biological functions throughout the body. Bisretinoid synthesis in the eye depends on the influx of all-trans-retinol (1) from serum into the retina. This influx requires the formation of a serum tertiary retinol-binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex. Decreasing the circulating levels of RBP4 and RBP1 with selective antagonists can regulate the visual cycle, reduce the rate of cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), and halt topographic atrophy in patients with atrophic (dry) age-related macular degeneration (AMD) and Stargardt disease (Radu, RA et al. 2005; Palczewski, K. 2010; Petrukhin, K. 2013; Petrukhin, K. 2007).

[0004] Competitive RBP4 antagonists that substitute for RBP1 inhibit the formation of the holo-RBP4-TTR complex, thereby inducing a decrease in circulating RBP4 and RBP1 levels via rapid renal clearance. A decrease in the inflow of 1 into the RPE reduces the accumulation of cytotoxic bisretinoids in the retina, which is thought to play a role in the pathogenesis of dry AMD and Stargardt disease (Radu, RA et al. 2005; Dobri, N. et al. 2013; Racz, B. et al. 2018; Young, RW 1987; Dorey, CK et al. 1989; Holz, FG et al. 2001; Holz, FG et al. 1999; Holz, FG et al. 2007; Schmitz-Valckenberg, S. et al. 2009; Finnemann, SC et al. 2002; Suter, M. et al. 2000; Sparrow, JR et al. 2003; Sparrow, JR et al. 2012; Delori, FC 1995; Weng, J. et al. al. 1999; Sparrow, J.R. & Cai, B. 2001; Bergmann, M. et al. 2004; Sparrow, JR et al. 1999; De, S. & Sakmar, TP 2002; Vives-Bauza, C. et al. 2008; Zhou, J. et al. 2006; Radu, RA et al. 2011; Ben-Shabat, S. et al. 2002; Rozanowska, M. et al. 1995; Sparrow, JR et al. 2002; Dontsov, AE et al. 2009).This approach is supported by proof-of-concept data obtained for fenretinide (5) (Figure 2) in a preclinical Abca4- / - transgenic mouse model of enhanced retinal lipofuscin development (Radu, RA et al. 2005) and in an expanded Phase II clinical trial in patients with dry AMD (Berni, R. & Formelli, F. 1992; Adams, WR et al. 1995; Mata, NL et al. 2013). The non-retinoid RBP4 antagonist A1120 (6) (Motani, A. et al. 2009) was found to reduce circulating RBP4 plasma levels in rodents by more than 70% and to decrease retinal bisretinoid accumulation in Abca4- / - mice (Dobri, N. et al. 2013). The selectively and orally bioavailable non-retinoid RBP4 antagonists 7 (Cioffi, CL et al. 2014) and BPN-14136(8) (Cioffi, CL et al. 2015) exhibited favorable pharmacokinetic (PK) profiles and induced dose-dependent reductions in plasma RBP4 levels in rodents in both acute and chronic administration studies. Compound 8 also reliably reduced serum RBP4 levels and showed excellent pharmacokinetic-pharmacodynamic (PK / PD) correlations in non-human primates via oral administration (Racz, B. et al. 2020). Compound 8 inhibited lipofuscin bisretinoid synthesis simultaneously with normalization of retinal complement system protein expression in the Abca4- / - mouse model at the dose that induced the maximum reduction in serum RBP4, without altering visual cycle dynamics (Racz, B. et al. 2018).

[0005] In addition to transporting protein 1 to target tissues, RBP4 has also been identified as an adipokine, and epidemiological evidence suggests that moderately elevated levels of the protein are positively correlated with type 2 diabetes (Graham, TE et al. 2006; Yang, Q. et al. 2005), obesity (Aeberli, I. et al. 2007), insulin resistance (Kowalska, I. et al. 2008), cardiovascular disease (Ingelsson, E. et al. 2009; Qi, Q. et al. 2007; Norseen, J. et al. 2012), and fatty liver (Lee, SA et al. 2016). Therefore, pharmacological reduction of circulating serum RBP4 levels may also be promising for the treatment of numerous metabolic diseases. Recently, it has been reported that the RBP4 antagonist 10 significantly reduced serum RBP4 levels in rodents (by over 80%), decreased circulating RBP4 concentration produced in adipose tissue, and showed efficacy in a transgenic adi-hRBP4 mouse model of hepatic adipose tissue, suggesting that it may have therapeutic efficacy in the treatment of non-alcoholic fatty liver disease (NAFLD) (Cioffi, CL et al. 2019).

[0006] Deposition of amyloid aggregates derived from mutant (TTRm) or wild-type (TTRwt) amyloids underlies TTR amyloidosis (ATTR) diseases such as senile systemic amyloidosis (SSA), peripheral polyneuropathy (ATTR-PN), and ATTR cardiomyopathy (ATTR-CM) (Johnson, SM et al. 2005; Foss, TR et al. 2005; Falk, RH et al. 1997; Brunjes, DL et al. 2016; Ton, VK et al. 2014). Breakage of the dimer-dimer interface of the TTR tetramer constitutes the first step in the TTR tetramer dissociation process, which leads to TTR misfolding. Approximately 50% of serum TTR is associated with holo-RBP4, and the formation of a tertiary holo-RBP4-TTR complex has been suggested to stabilize this fraction of serum TTR tetramers and protect them from dissociation and misfolding (White, JT & Kelly, JW 2001; Hyung, SJ et al. 2010). Based on in vitro observations that RBP4-TTR interactions can provide additional stabilization to tetrameric TTR (White, JT & Kelly, JW 2001; Hyung, SJ et al. 2010), it appears that the release of TTR tetramers from the RBP4-TTR-retinol complex induced by selective RBP4 antagonists may lead to tetramer destabilization and enhanced dissociation of the tetramer into dimeric subunits. Subsequently, the resulting dimers can further misfold, aggregate, oligomerize, and ultimately dissociate into monomers capable of forming insoluble TTR amyloid fibrils (White, JT & Kelly, JW 2001; Hyung, SJ et al. 2010). While selective RBP4 antagonists may be a safe and effective treatment for the majority of dry AMD patients, this class of compounds may potentially show the opposite effect in some AMD patients who are predisposed to developing ATTR.In addition to individuals with a rare genetic form of transthyretin amyloidosis caused by pro-amyloidogenic TTR mutations, the use of selective RBP4 antagonists may not be optimal in patients with senile systemic amyloidosis (SSA), a late-onset, non-hereditary disorder associated with misfolding and aggregation of wild-type TTR. SSA affects approximately 25% of patients over 80 years of age (Ruberg, FL & Berk, JL 2012; Connors, LH et al. 2011; Westermark, P. et al. 2003), and given the high population prevalence of this disease and dry AMD, significant comorbidities between the two conditions are anticipated. Furthermore, the use of selective RBP4 antagonists may not be optimal in older African American patients with dry AMD who are likely to carry a relatively high frequency of the pro-amyloidogenic V122I mutation in the TTR gene (Buxbaum, JN et al. 2017; Alexander, KM & Falk, RH 2016). It is undesirable for an effective chronic treatment for one of two conditions to show opposing indications for use in patients with the other condition, and developing an optimal treatment for dry AMD that can be safely used in patients with ATTR complications is a key objective.

[0007] The first rate-limiting step in the pathogenesis of ATTR is the sequential dissociation of the TTR tetramer (Johnson, SM et al. 2005; Foss, TR et al. 2005). Thyroxine (4) (Figure 1) binding has been reported to stabilize the TTR tetramer (Sekijima, Y. et al. 2003), but the majority of circulating TTR, including TTR in complex with holo-BP4 (up to 90%), is not bound to its native ligand (White, JT & Kelly, 2001). Current therapeutic approaches to treat ATTR-CM involve small-molecule kinetic stabilizers of the TTR tetramer that bind at the T4 binding site and increase the energy barrier of tetramer dissociation (Kerschen, P. & Plante-Bordeneuve, V. 2016; Almeida, MR et al. 2005; Almeida, MR et al. 2004; Johnson, SM et al. 2005; Nencetti, S. & Orlandini, E. 2012; Adams, D. et al. 2016). Two orally bioavailable kinetic stabilizers that have been clinically studied to date include the FDA-approved tafamidis (11) (Figure 3) (Coelho, T. et al. 2016; Nencetti, S. et al. 2013; Lamb, YN & Deeks, ED 2019; Bulawa, CE et al. 2012) and AG10 (12) (Penchala, SC et al. 2013; Miller, M. et al. 2018). The TTR stabilizer 11 is currently approved for the treatment of patients with familial amyloid polyneuropathy (FAP) and ATTR-CM, 12 has shown in a Phase III clinical trial that it nearly completely stabilizes TTR in patients with symptomatic chronic heart failure and ATTR-CM (Judge, DP et al. 2019).Furthermore, the FDA-approved nonsteroidal anti-inflammatory drug (NSAID) diflunisal (13) (Berk, JL et al. 2012) and the catechol-O-methyltransferase (COMT) inhibitor tolcapone (14) (Sant'Anna, R. et al. 2016), reused for other purposes, also exhibit TTR tetramer dynamic stabilization activity and are examples of additional small molecules being investigated for their clinical effects on ATTR-PN.

[0008] This invention describes a novel class of non-retinoid bispecific compounds capable of exhibiting dual retinol-binding protein 4 (RBP4) antagonist and transthyretin (TTR) tetramer dynamic stabilization activity for the treatment of dry age-related macular degeneration (AMD) and TTR amyloidosis (ATTR) complications. Here, we show that these compounds may offer therapeutic benefits associated with reduced circulating RBP4 levels while simultaneously stabilizing non-ligand TTR tetramers released from the holo-RBP4-TTR complex, thereby avoiding the potential risk of amyloid fibrillation, as illustrated in Figure 4. Furthermore, a polypharmacological approach consisting of a single bispecific molecule capable of exhibiting dual activity against both targets may offer advantages over the simultaneous administration of a single agent against each target. Such advantages include improved patient compliance, minimization of complex PKs, and avoidance of potential drug interactions that may arise from multiple drug administrations (Rodrigues, DA 2008). [Overview of the project] [Means for solving the problem]

[0009] The present invention relates to a compound having the following structure, [ka] [In the formula, X is either CR6 or N; R1, R2, R3, R4, and R6 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), -(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, -SO2R7, -NHSO2R7, -OC(O)R7, -SC(O)R7, -NHC(O)R8 or -NHC(S)R8, R7 is H, -(alkyl), -OH, -O(alkyl), -NH2, -NH(alkyl), or -N(alkyl)2. R8 is -(alkyl), -O-(alkyl), -NH2, -NH(alkyl), or -N(alkyl)2; Y is O, S, N, NH, or a bond; Z is O, S, N, NH, (CH2)O, or a bond; R5 is H, OH, halogen, alkyl, or (CH2)p and when Y is N, it bonds with Y to form a ring with Z; o and p are independently 0, 1, 2, or 3; m and n are independently 0, 1, 2, 3, or 4; A, C, and D are each independently N or CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); B and E are N, CR9, or CFG, where at least one of B or E is CFG; F is either absent or present, and if present, F is as follows: [ka] G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2]. or provide a pharmaceutically acceptable salt thereof. [Brief explanation of the drawing]

[0010] [Figure 1] All-trans-retinol (vitamin A) (1), retinoic acid (2) (an important retinoid involved in morphogenesis), 11-cis-retinal (3) (an important retinoid required for light transmission), and the thyroid hormone thyroxine (T4) (4). [Figure 2]Examples of previously reported RBP4 antagonists include fenretinide (5), A1120 (6), non-retinoid [3.3.0]-octahydrocyclopenta[c]pyrrolo RBP4 antagonist 7, BPN-14136 (8), [3.3.0]-octahydrocyclopenta[c]pyrrolo RBP4 antagonist 9, and [1,2,4]triazolo[4,3-a]pyridine 10. [Figure 3] Examples of reported low molecular weight TTR tetramer kinetic stabilizers include tafamidis (11), AG10 (12), diflunisal (13), and tolcapone (14). [Figure 4] Bispecific RBP4 antagonists and TTR tetramer kinetic stabilizers for treating RBP4 indications with potential ATTR complications. (A) Schematic diagram illustrating the use of a selective RBP4 antagonist to interfere with holo-RBP4-TTR protein interactions and induce serum depletion of RBP4 and RBP4. Co-release of non-ligand TTR may induce its aggregation and contribute to ATTR in predisposed patients. (B) Bispecific ligands with dual RBP4 antagonist and TTR tetramer kinetic stabilization activity may induce decreased circulating levels of RBP4 and RBP4 while preventing potential TTR aggregation and insoluble amyloid fibril formation. [Figure 5] Pharmacokinetic and pharmacodynamic properties of (±)-44 in mice. (A) Serum RBP4 levels after a single oral administration of 25 mg / kg of (±)-44. (B) Blood compound levels after a single oral administration of 5 mg / kg of (±)-44. Data are expressed as mean ± SD. Three mice were used in each treatment group for the study. [Figure 6]The analog (±)-44 reduces the formation of high molecular weight TTR morphologies in acid-induced agglutination assays. (A) TTR protein (5 μg) was agglutinated using acetate buffer (pH 4.0) and incubated at 37°C for 72 hours. After incubation in the presence of 50 μM tafamidis, 50 μM benzbromarone, and 50 μM (±)-44, the samples were crosslinked, subjected to SDS-PAGE, and then Western blotting with TTR antibody. Representative blots from at least three independent experiments are shown. (B, C) Bar graphs represent the pixel capacities of high molecular weight TTR aggregates (B) and monomers (C). The vertical axis represents the ratio of the mean ± SD of the pixel capacities of scanned bands of the immunoblot in any unit. Statistical significance was determined by one-way ANOVA using the Holm-Sidak post-hoc test;*, p≦0.05;**, p≦0.01;***, p≦0.001;****, p≦0.0001 compared to the TTR aggregate (pH 4.0) + DMSO group, #, p≦0.05;##, p≦0.01;###, p≦0.001;####, p≦0.001;####, p≦0.0001 compared to the TTR (pH 7.5) group without aggregates. [Figure 7] Effect of long-term oral (±)-44 administration on serum RBP4 levels in Abca4- / - mice. Serum RBP4 levels were measured at the indicated time points in Abca4- / - mice treated with vehicle (square) and Abca4- / - mice treated with (±)-44 (triangle). A statistically significant 79% decrease in RBP4 was observed in the (±)-44 treatment group at week 12 compared to baseline (two-way ANOVA with Sidak post-hoc test, p<0.0001). Error bars indicate SD. Each data point on the graph represents the serum RBP4 concentration from an individual animal. [Figure 8]Effect of long-term oral (±)-44 administration on lipofuscin fluorophore A2E levels in the eyes of Abca4- / - mice. Bisretinoids were extracted from the ocular cups of vehicle-treated wild-type mice (round), vehicle-treated Abca4- / - knockout mice (square), and (±)-44-treated Abca4- / - mice (triangular) after 12 weeks of administration and analyzed by HPLC. A significant 50% reduction in A2E concentration was detected in (±)-44-treated Abca4- / - mice compared to vehicle-treated knockout controls; one-way ANOVA by Holm-Sidak post-hoc test, p<0.0001 (****). [Figure 9] Effect of long-term oral (±)-44 administration on serum RBP4 levels in Abca4- / - / Rdh8- / - mice. Serum RBP4 levels were measured at the indicated time points in Abca4- / - / Rdh8- / - mice treated with vehicle (square) and Abca4- / - / Rdh8- / - mice treated with (±)-44 (triangle). A statistically significant 76% decrease in RBP4 was observed in the (±)-44 treatment group at week 10 compared to baseline (two-way ANOVA with Sidak post-hoc test, p<0.01). Error bars indicate SD. Each data point on the graph represents the serum RBP4 concentration from an individual animal. [Figure 10] Effect of long-term oral (±)-44 administration on lipofuscin fluorophore A2E levels in the eyes of Abca4- / - / Rdh8- / - mice. Bisretinoids were extracted from the ocular cups of vehicle-treated wild-type mice (round), vehicle-treated Abca4- / - / Rdh8- / - knockout mice (square), and (±)-44-treated Abca4- / - / Rdh8- / - mice (triangular) after 10 weeks of administration and analyzed by HPLC. A significant 77% reduction in A2E concentration was detected in (±)-44-treated Abca4- / - / Rdh8- / - mice compared to vehicle-treated double knockout controls; one-way ANOVA by Holm-Sidak post-hoc test, p<0.0001 (****). [Figure 11]Protection of photoreceptors in (±)-44-treated *Rdh8- / - *Abca4- / - mice. The reduction in ONL thickness in the retina of untreated *Rdh8- / - *Abca4- / - mice (squares), as compared with untreated wild-type C57BL / 6 mice (circles), is partially reversed by (±)-44 treatment (triangles). Data analyzed by two-way ANOVA with Sidak post-hoc test. [Figure 12] Structures of bisretinoids A2E and isoA2E, cytotoxic components of retinal lipofuscin. [Figure 13] Structures of bisretinoids atRAL di-PE (all-trans-retinal dimer-phosphatidylethanolamine) and A2-DHP-PE, cytotoxic components of retinal lipofuscin. R1 and R2 refer to various fatty acid moieties. MODE FOR CARRYING OUT THE INVENTION

[0011] (DETAILED DESCRIPTION OF THE INVENTION) The present invention provides a compound having the following structure:

Chemical Formula

[0012] In some embodiments, the compound is such that m is 1 or 2 and n is 0, 1, or 2.

[0013] In some embodiments, the compound is such that m is 1 and n is 1.

[0014] In some embodiments, Y and Z are each independently CH2, O, S, or NH in the compound.

[0015] In some embodiments, the compound is O and Z is CH2.

[0016] In some embodiments, A and B are compounds N, C and D are compounds CR9, and E is compounds CFG.

[0017] In some embodiments, A, B, C, and D are compounds CR9 and E is compound CFG.

[0018] In some embodiments, A is N, B is CFG, and C, D, and E are CR9 compounds, respectively.

[0019] In some embodiments, A is N, B, C, and D are CR9, respectively, and E is CFG, a compound.

[0020] In some embodiments, A, C, and D are CR9, B is N, and E is CFG, respectively, of the compound.

[0021] In some embodiments, X is either CR6 or N; A compound in which R1, R2, R3, R4, and R6 are each independently H, t-butyl, cyclopentyl, cyclohexyl, CF3, F, Cl, CN, or -OCH3.

[0022] In some embodiments, R1 or R4 is a compound that is CF3.

[0023] In some embodiments, X is CR6, A compound in which R1 is CF3, and R2, R3, R4, and R6 are each independently H, t-butyl, cyclopentyl, cyclohexyl, CF3, F, Cl, CN, or -OCH3.

[0024] In some embodiments, the compound has the following structure: [ka] [In the formula, C is CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); F is either absent or present, and if present, F is as follows: [ka] G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2. or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, C is CR9, R9 is a compound consisting of H, -alkyl, -O(alkyl), or -NH(alkyl).

[0026] In some embodiments, R9 is a compound that is alkyl.

[0027] In some embodiments, the compound has the following structure: [ka] [ka] [In the formula, F is a substituted or unsubstituted heteroaryl group.

[0028] In some embodiments, F is a compound having the following structure: [ka] R12 is H, -alkyl, -(alkenyl), or -(alkynyl).

[0029] In some embodiments, the compound has the following structure: [ka] [In the formula, C is CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); F is either absent or present, and if present, F is as follows: [ka] G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2. or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, C is CR9, R9 is a compound consisting of H, alkyl, -O(alkyl), or -NH(alkyl).

[0031] In some embodiments, R9 is a compound that is alkyl.

[0032] In some embodiments, the compound has the following structure: [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt of the compound.

[0033] In some embodiments, the compound has the following structure: [ka] or a pharmaceutically acceptable salt of the compound.

[0034] In some embodiments, the compound has the following structure: [ka] or a pharmaceutically acceptable salt of the compound.

[0035] In some embodiments, the compound has the following structure: [ka] or a pharmaceutically acceptable salt of the compound.

[0036] In some embodiments, the compound has the following structure: [ka] or a pharmaceutically acceptable salt of the compound.

[0037] In some embodiments, the compound has the following structure: [ka] or a pharmaceutically acceptable salt of the compound.

[0038] The present invention provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0039] The present invention provides a method for stabilizing TTR tetramers in mammals, comprising administering an effective amount of the compound or composition of the present invention to a mammal in order to effectively stabilize TTR tetramers in mammals.

[0040] The present invention provides a method for treating a disease characterized by excessive lipofuscin accumulation in the retina, or TTR amyloidosis (ATTR) disease, or a disease characterized by excessive lipofuscin and TTR amyloidosis (ATTR) disease, comprising administering an effective amount of the compound or composition of the present invention to a mammal suffering from the disease.

[0041] In some embodiments of the method, the disease is further characterized by bisretinoid-mediated macular degeneration.

[0042] In some embodiments of the method, the amount of the compound is effective in reducing the serum concentration of RBP4 in mammals, or the amount of the compound is effective in reducing the retinal concentration of the bisretinoid in lipofuscin in mammals.

[0043] In some embodiments of the method, the amount of compound is effective in stabilizing the TTR tetramer in mammals.

[0044] In some embodiments of the method, the bisretinoid is A2E.

[0045] In some embodiments of the method, the bisretinoid is isoA2E.

[0046] In some embodiments of the method, the bisretinoid is A2-DHP-PE.

[0047] In some embodiments of the method, the bisretinoid is atRAL di-PE.

[0048] In some embodiments of the method, the disease characterized by the accumulation of excessive lipofuscin in the retina is age-related macular degeneration.

[0049] In some embodiments of the method, the disease characterized by an excessive accumulation of lipofuscin in the retina is dry (atrophic) age-related macular degeneration.

[0050] In some embodiments of the method, a disease characterized by an excessive accumulation of lipofuscin in the retina is Stargardt disease.

[0051] In some embodiments of the method, a disease characterized by the accumulation of excessive lipofuscin in the retina is Best's disease.

[0052] In some embodiments of the method, the disease characterized by the accumulation of excessive lipofuscin in the retina is adult vitiligo maculopathy.

[0053] In some embodiments of the method, the disease characterized by the accumulation of excessive lipofuscin in the retina is Stargardt-like macular dystrophy.

[0054] In some embodiments of the method, the administration is effective in reducing photoreceptor degeneration.

[0055] In some embodiments of the method, the method is even more effective in stabilizing mammalian TTR tetramers.

[0056] In some embodiments of the method, the mammal is further suffering from TTR amyloidosis (ATTR) disease, and the method is effective in treating TTR amyloidosis (ATTR) disease in mammals.

[0057] In some embodiments of the method, TTR amyloidosis (ATTR) disease is senile systemic amyloidosis (SSA).

[0058] In some embodiments of the method, TTR amyloidosis (ATTR) disease is peripheral polyneuropathy (ATTR-PN).

[0059] In some embodiments of the method, TTR amyloidosis (ATTR) disease is cardiomyopathy (ATTR-CM).

[0060] In some embodiments of the method, TTR amyloidosis (ATTR) disease is characterized by the deposition of amyloid aggregates.

[0061] In some embodiments, bisretinoid-mediated macular degeneration is age-related macular degeneration or Stargardt disease.

[0062] In some embodiments, bisretinoid-mediated macular degeneration is age-related macular degeneration.

[0063] In some embodiments, bisretinoid-mediated macular degeneration is dry (atrophic) age-related macular degeneration.

[0064] In some embodiments, bisretinoid-mediated macular degeneration is Stargardt disease.

[0065] In some embodiments, bisretinoid-mediated macular degeneration is the best disease.

[0066] In some embodiments, bisretinoid-mediated macular degeneration is adult vitiligo maculopathy.

[0067] In some embodiments, bisretinoid-mediated macular degeneration is Stargardt-like macular dystrophy.

[0068] Bisretinoid-mediated macular degeneration may involve the accumulation of lipofuscin deposits in the retinal pigment epithelium.

[0069] As used herein, “bisretinoid lipofuscin” refers to lipofuscin containing cytotoxic bisretinoids. Cytotoxic bisretinoids include, but are not limited to, A2E, isoA2E, atRAL di-PE (all-trans-retinal dimer-phosphatidylethanolamine), and A2-DHP-PE (A2-dihydropyridine-phosphatidylethanolamine) (Figures 7-8).

[0070] 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).

[0071] In some embodiments, TTR amyloidosis (ATTR) disease is characterized by the deposition of amyloid aggregates.

[0072] In some embodiments, TTR amyloidosis (ATTR) disease is characterized by the deposition of amyloid aggregates derived from a variant (TTRm) or wild-type (TTRwt).

[0073] In some embodiments, TTR amyloidosis (ATTR) disease is senile systemic amyloidosis (SSA).

[0074] In some embodiments, TTR amyloidosis (ATTR) disease is peripheral polyneuropathy (ATTR-PN).

[0075] In some embodiments, TTR amyloidosis (ATTR) disease is cardiomyopathy (ATTR-CM).

[0076] In some embodiments, the compounds of the present invention exhibit dual retinol-binding protein 4 (RBP4) antagonist and transthyretin (TTR) tetramer dynamic stabilization activity.

[0077] In some embodiments, the compounds of the present invention exhibit retinol-binding protein 4 (RBP4) antagonist activity.

[0078] In some embodiments, the compounds of the present invention exhibit transthyretin (TTR) tetramer dynamic stabilization activity.

[0079] In some embodiments, the compounds of the present invention reduce circulating RBP4 levels while simultaneously stabilizing the non-ligand TTR tetramer released from the holo-RBP4-TTR complex.

[0080] In some embodiments, the compounds of the present invention reduce circulating RBP4 levels.

[0081] In some embodiments, the compounds of the present invention stabilize the non-ligand TTR tetramer released from the holo-RBP4-TTR complex.

[0082] In some embodiments, the compounds or compositions of the present invention may be used to treat dry age-related macular degeneration (AMD) and TTR amyloidosis (ATTR) complications.

[0083] In some embodiments, the compounds or compositions of the present invention may be used to treat dry age-related macular degeneration (AMD) and senile systemic amyloidosis (SSA).

[0084] In some embodiments, the compounds or compositions of the present invention may be used to treat dry age-related macular degeneration (AMD) and peripheral polyneuropathy (ATTR-PN).

[0085] In some embodiments, the compounds or compositions of the present invention may be used to treat dry age-related macular degeneration (AMD) and cardiomyopathy (ATTR-CM).

[0086] In some embodiments, the compounds or compositions of the present invention may be used for the treatment of type 2 diabetes.

[0087] In some embodiments, the compounds or compositions of the present invention may be used to treat obesity.

[0088] In some embodiments, the compounds or compositions of the present invention may be used in the treatment of insulin resistance.

[0089] In some embodiments, the compounds or compositions of the present invention may be used in the treatment of cardiovascular diseases.

[0090] In some embodiments, the compounds or compositions of the present invention may be used to treat fatty liver disease.

[0091] In some embodiments, the compounds or compositions of the present invention may be used to treat non-alcoholic fatty liver disease (NAFLD).

[0092] In some embodiments, the mammal is a human.

[0093] Those skilled in the art can prepare the deuterium analog using the techniques disclosed herein.

[0094] Unless otherwise specified, the structures of the compounds of the present invention include an asymmetric carbon atom, and the compounds are understood to exist as racemates, racemic mixtures, scalemic mixtures, and isolated single enantiomers. All such isomeric forms of these compounds are expressly included in the present invention. Unless otherwise specified, each stereogenic carbon may have an R or S configuration. Therefore, unless otherwise indicated, it should be understood that isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are within the scope of the present invention. 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, resolution can be performed by preparative chromatography on a chiral column.

[0095] Unless otherwise specified, the present invention is intended to include all isotopes of atoms present in the compounds disclosed herein. Isotopes include these atoms that have the same atomic number but different mass numbers. As a general example, but not limited to, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0096] It should be noted that, when used without further notation, any notation of carbon in the overall structure of this application is intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compound containing 13C or 14C may specifically have the structure of any compound disclosed herein.

[0097] It should also be noted that, unless otherwise specified, any notation for hydrogen (H) in the overall structure of this application, when used without further designation, is intended to represent all isotopes of hydrogen, such as 1H, 2H(D), or 3H(T). Furthermore, any compound containing 2H or 3H may specifically have the structure of any compound disclosed herein, unless otherwise specified.

[0098] Isotope-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, using appropriate isotope-labeling reagents instead of the unlabeled reagents used.

[0099] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen with an atomic weight of 2.0144. Hydrogen atoms in compounds occur spontaneously as a mixture of isotopes 1H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.0156%. Therefore, in a composition containing molecules of a naturally occurring compound, the level of deuterium at a particular hydrogen atom site in that compound is expected to be 0.0156%. Thus, a composition containing a compound enriched such that the level of deuterium at any site of hydrogen atoms in the compound is greater than its natural abundance of 0.0156% is novelr than its naturally occurring counterpart.

[0100] The terms “substituted,” “substituted,” and “substituted” refer to the functional groups described above in which one or more bonds to hydrogen atoms are replaced by bonds to non-hydrogen or non-carbon atoms, provided that the normal valence is maintained and the substitution results in a stable compound. A substituted group also includes groups in which one or more bonds to carbon or hydrogen atoms are replaced by one or more bonds, including double or triple bonds, to heteroatoms. Examples of substituents include the functional groups and 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. If multiple substituent parts are disclosed or claimed, the substituted compound may be substituted individually or independently by one or more of the disclosed or claimed substituent parts. To be independently substituted means that the (two or more) substituents may be the same or different.

[0101] In the compounds used in the method of the present invention, substituents may be substituted or unsubstituted unless otherwise defined.

[0102] In the compounds used in the methods of the present invention, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclic, heterocycloalkyl, alkylheteroalkyl, alkylaryl, monocyclic, dicyclic, heteromonocyclic, and heterodicyclic groups may be further substituted by substituting one or more hydrogen atoms with other nonhydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, and carbamoyl groups.

[0103] The substituents and substitution patterns of the compounds used in the methods of the present invention are selected by those skilled in the art and are understood to provide compounds that are chemically stable and readily synthesizeable from readily available starting materials by techniques known in the art. If the substituent itself is substituted with two or more groups, these groups may be on the same carbon or different carbons, as long as a stable structure is obtained.

[0104] When selecting compounds to be used in the method of the present invention, those skilled in the art will recognize that various substituents, namely R1, R2, etc., should be selected in accordance with the well-known principles of chemical structure bonding.

[0105] As used herein, “alkyl” includes both branched and linear saturated aliphatic hydrocarbon groups having a specific number of carbon atoms, and may be unsubstituted or substituted. Thus, C1-Cn, as in “C1-Cn alkyl,” is defined as a group containing 1, 2, ..., n-1, or n carbon atoms arranged linearly or branchedly. For example, C1-C6, as in “C1-C6 alkyl,” is defined as a group containing 1, 2, 3, 4, 5, or 6 carbon atoms arranged linearly or branchedly, specifically including methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, and hexyl. Unless otherwise specified, the number of carbon atoms is 1 to 10. Alkyl groups may be unsubstituted or substituted with one or more substituents, including but not limited to halogens, alkoxys, alkylthios, trifluoromethyl, difluoromethyl, methoxy, and hydroxyl. “Haloalkyl” includes any alkyl group containing at least one halogen atom.

[0106] The term "alkenyl" refers to a linear or branched non-aromatic hydrocarbon radical that contains at least one carbon-carbon double bond and may have up to the maximum possible number of non-aromatic carbon-carbon double bonds. Thus, C2-Cn alkenyls are defined as groups having 1, 2..., n-1, or n carbon atoms. For example, "C2-C6 alkenyls" each mean an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms and at least one carbon-carbon double bond, and in the case of a C6 alkenyl, up to, for example, three carbon-carbon double bonds. Alkenyl groups include ethenyl, propenyl, butenyl, and cyclohexenyl. As described above with respect to alkyls, the linear, branched, or cyclic portion of an alkenyl group may contain double bonds and may be substituted, if substituted alkenyl groups are shown. Embodiments may be C2-C12 alkenyls or C2-C8 alkenyls.

[0107] The term "alkynyl" refers to a linear or branched hydrocarbon radical that contains at least one carbon-carbon triple bond and may have up to the maximum possible number of non-aromatic carbon-carbon triple bonds. Thus, C2-Cn alkynyls are defined as groups having 1, 2..., n-1, or n carbon atoms. For example, "C2-C6 alkynyl" refers to an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl, and butynyl. As described above with respect to alkyl, the linear or branched portion of the alkynyl group may contain triple bonds and may be substituted, if substituted alkynyl groups are shown. Embodiments may be C2-Cn alkynyls. Embodiments may be C2-C12 alkynyls or C3-C8 alkynyls.

[0108] As used herein, “aryl” is intended to mean any stable monocyclic, bicyclic, or polycyclic carbocyclic ring having up to 10 atoms in each ring, where at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include, but are not limited to, phenyl, p-truenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl, anthryl, or acenaphthyl. When the aryl substituent is bicyclic and one of the rings is non-aromatic, the bond is understood to be mediated through an aromatic ring.

[0109] As used herein, the term “heteroaryl” refers to a stable monocyclic, bicyclic, or polycyclic ring having up to 10 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from the group consisting of O, N, and S. A bicyclic aromatic heteroaryl group includes a phenyl, pyridine, pyrimidine, or pyridazine ring fused to (a) a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) a 5-membered or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with one oxygen atom or one sulfur atom; or (d) a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N, or S.Heteroaryl groups within this definition include benzimidazolyl, benzofuranil, benzofuranil, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzooxazolyl, carbazolyl, carbonil, cinnolinil, furanil, indolinyl, indolyl, indazinyl, indazolyl, isobenzofuranil, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, oxazolyl, oxazoline, and isoxazoline. Oxetanil, pyranil, pyrazinil, pyrazolyl, pyridadinil, pyridopyridinil, pyridadinil, pyridyl, pyrimidyl, pyrrolyl, quinazolinil, quinolyl, quinoxalinil, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinil, aziridinil, 1,4-dioxanil, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranil, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydro Lofuranil, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazolyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrof This includes, but is not limited to, lanyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. When a heteroaryl substituent is bicyclic and one of the rings is non-aromatic or does not contain a heteroatom, it is understood that the bond is via an aromatic ring or a heteroatom-containing ring, respectively.If a heteroaryl contains a nitrogen atom, it is understood that its corresponding N-oxide is also included in this definition.

[0110] As used herein, “cycloalkyl” includes a cyclic ring of an alkane having a total number of carbon atoms of 3 to 8, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl). “Cycloalkylalkyl” includes any alkyl group having at least one cycloalkyl ring.

[0111] As used herein, “heteroalkyl” includes both branched-chain and linear saturated aliphatic hydrocarbon groups having at least one heteroatom in the chain or branch. “Alkyl heteroalkyl” includes any alkyl group containing at least one heteroalkyl group.

[0112] The terms “heterocyclic,” “heterocyclyl,” or “heterocyclic” refer to monocyclic or polycyclic ring systems that can be saturated or contain one or more degrees of unsaturation and contain one or more heteroatoms. Preferred heteroatoms include N, O, and / or S, including N-oxides, sulfur oxides, and dioxides. Preferably, the rings are 3- to 10-membered and are saturated or have one or more degrees of unsaturation. Heterocyclic rings may be unsubstituted or substituted, and multiple degrees of substitution are possible. Such rings may be optionally condensed with one or more other “heterocyclic” rings, heteroaryl rings, aryl rings, or cycloalkyl rings. Examples of heterocyclic rings include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, and 1,3-oxathiolane.

[0113] As used herein, “heterocycloalkyl” is intended to mean a 5-10 membered non-aromatic ring containing 1-4 heteroatoms selected from the group consisting of O, N, and S, and including a bicyclic group. Thus, “heterocyclyl” includes, but is not limited to, imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothiophenyl, etc. If the heterocyclyl contains nitrogen, it is understood that its corresponding N-oxide is also included in this definition.

[0114] The term "alkylaryl" refers to the alkyl group described above, in which one or more bonds to hydrogen contained therein are replaced by bonds to the aryl group described above. The "alkylaryl" group is understood to be bonded to the core molecule via a bond from the alkyl group, with the aryl group acting as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl(phenylmethyl), p-trifluoromethylbenzyl(4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, and 2-phenylpropyl.

[0115] As used herein, “monocyclic” includes any stable polycyclic carbocyclic ring containing up to 10 atoms, which may be unsubstituted or substituted. Examples of such non-aromatic monocyclic elements include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocyclic elements include, but are not limited to, phenyl. As used herein, “heteromonocyclic” includes any monocyclic ring containing at least one heteroatom.

[0116] As used herein, “bicyclic” includes any stable polycyclic carbon ring of up to 10 atoms, condensed into a polycyclic carbon ring of up to 10 atoms, where each ring is independently unsubstituted or substituted. Examples of such non-aromatic bicyclic elements include, but are not limited to, decahydronaphthalene. Examples of such aromatic bicyclic elements include, but are not limited to, naphthalene. As used herein, “heterobicancyclic” includes any bicyclic ring containing at least one heteroatom.

[0117] The compounds used in the method of the present invention are well known in organic synthesis and can be prepared by techniques familiar to those skilled in the art. However, these may not be the only means of synthesizing or obtaining the desired compounds.

[0118] The compounds used in the methods of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A.I. Vogel, AR. Tatchell, BS. Furnis, A.J. Hannaford, P.W. 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 the references therein, which are incorporated herein by reference. However, these may not be the only means of synthesizing or obtaining the desired compounds.

[0119] The various R groups attached to the aromatic rings of the compounds disclosed herein can 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), the details of which are incorporated herein by reference.

[0120] Another aspect of the present invention includes the compound or composition of the present invention as a pharmaceutical composition.

[0121] As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject that provides biological activity or other direct effect in the treatment, cure, alleviation, diagnosis, or prevention of a disease, or that affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds listed in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (US Department of Health and Human Services, 30th edition, 2010), which are incorporated herein by reference. Pharmaceutically active agents having a pendant carboxylic acid group can be modified according to the present invention using standard esterification reactions and methods readily available to those skilled in the art of chemical synthesis. If a pharmaceutically active agent does not have a carboxylic acid group, those skilled in the art can design a carboxylic acid group and incorporate it into the pharmaceutically active agent, and then esterification can be performed, provided that the modification does not interfere with the biological activity or effect of the pharmaceutically active agent.

[0122] The compounds used in the methods of the present invention may be in the form of salts. As used herein, “salt” refers to a salt of the present compound modified by making an acid or base salt of the compound. In the case of a compound used to treat a disease or medical disorder, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic acid salts of acidic residues such as phenols; and alkali or organic acid salts of acidic residues such as 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, and ascorbicates. Phenolate salts include sodium, potassium, or lithium salts. Carboxylate salts include sodium, potassium, or lithium salts. In this regard, the term “pharmaceutically acceptable salt” 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 when finally isolating and purifying the compounds of the present invention, or they can be prepared by separately reacting the purified compounds of the present invention, in the form of their free base or free acid, with a suitable organic or inorganic acid or base, and then isolating the salts thus produced. Typical 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, and laurylsulfonate. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0123] Salts or pharmaceutically acceptable salts are intended for all compounds disclosed herein.

[0124] As used herein, “to treat” means to prevent, slow, stop, or reverse the progression of a disease. Treatment may also mean improving one or more symptoms of a disease.

[0125] The compounds used in the methods of the present invention may be administered in various forms, including those detailed herein. Treatment with the compounds may be a component of combination therapy or adjunct therapy, i.e., a subject or patient requiring a drug is treated or administered with another drug for a disease in combination with one or more of the compounds of the present invention. This combination therapy may be a sequential therapy in which the patient is first treated with one drug, and then another drug or two drugs are administered simultaneously. Depending on the dosage form used, these may be administered independently via the same route, or via two or more different routes of administration.

[0126] As used herein, “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable solvent, suspension, or vehicle for delivering the compound to animals or humans. The carrier may be liquid or solid and is selected with the planned method of administration in mind. Liposomes, as well as capsules, coatings, and various syringes, are also pharmaceutically acceptable carriers.

[0127] The dosage of compounds administered in treatment varies depending on factors such as the pharmacodynamic properties of the specific chemotherapeutic agent, as well as its mode and route of administration; the recipient's age, sex, metabolic rate, absorption efficiency, health status, and weight; the nature and severity of symptoms; the types of treatments being administered concurrently; the frequency of treatment with that agent; and the desired therapeutic effect.

[0128] The drug delivery units of the compounds used in the method of the present invention may comprise a single compound or a mixture thereof with additional agents. The compounds may be administered in oral dosage forms such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, or introduced into or directly into the disease site by, for example, injection, topical application, or other methods, all of which are dosage forms well known to those skilled in the art in the field of pharmacy.

[0129] The compounds used in the methods of the present invention may be administered in mixture with appropriate pharmaceutically acceptable diluents, swellers, excipients, or carriers (collectively referred to herein as pharmaceutically acceptable carriers) that are appropriately selected with respect to the intended dosage form and in accordance with conventional pharmaceutical practice. The units are in a form suitable for oral, rectal, topical, intravenous, direct injection, or parenteral administration. The compounds may be administered alone or in mixture with a pharmaceutically acceptable carrier. This carrier may be solid or liquid, and the type of carrier is generally selected based on the type of dosage used. The activator may be administered co-administered in the form of tablets or capsules, liposomes, as aggregated powder, or in liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin, and agar. Capsules or tablets may be easily formulated and made easy to swallow or chew; other solid forms include granules and active pharmaceutical ingredient powders. Tablets may contain appropriate binders, lubricants, diluents, disintegrants, colorants, flavorings, flow inducers, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable oils and fats, 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 dosage forms may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms may optionally contain flavorings and colorings. Parenteral and intravenous preparations may also contain minerals and other materials to make them suitable for the type of injection or delivery system of choice.

[0130] The techniques and compositions for producing useful dosage forms in this invention are described 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 the aforementioned publications are incorporated herein by reference.

[0131] Tablets may contain appropriate binders, lubricants, disintegrants, colorants, flavorings, flow inducers, and melting agents. For example, when administered orally in the form of a tablet or capsule dosing unit, the active drug component may be combined with an oral, non-toxic, and pharmaceutically acceptable inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, or sorbitol. Appropriate binders include natural sugars such as starch, gelatin, glucose, or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, and waxes. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, and xanthan gum.

[0132] The compounds used in the methods of the present invention may also be administered in the form of liposome delivery systems such as small monolayer vesicles, large monolayer lamellar vesicles, and multilayer vesicles. Liposomes may be formed from various phospholipids such as cholesterol, stearylamine, or phosphatidylcholine. The compounds may be administered as components of tissue-targeted emulsions.

[0133] The compounds used in the methods of the present invention may also be bound to soluble polymers as targetable drug carriers or prodrugs. Such polymers include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide-phenol, polyhydroxyethyl aspartamidophenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be bound to a class of biodegradable polymers useful for achieving controlled drug release, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0134] Gelatin capsules may contain an active ingredient compound and a powdered carrier such as lactose, starch, cellulose derivatives, magnesium stearate, or stearic acid. Compressed tablets can be manufactured using similar diluents. Both tablets and capsules may be manufactured as immediate-release products or as sustained-release products to provide continuous release of the drug over several hours. Compressed tablets may be coated with sugar or film to mask any unpleasant taste and protect the tablet from the air, or with enteric coating to selectively disintegrate in the gastrointestinal tract.

[0135] In the case of oral administration by liquid formulation, the oral drug component is combined with any orally accepted, non-toxic, and pharmaceutically acceptable inert carrier, such as ethanol, glycerol, or water. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable oils and fats, 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 dosage forms may, for example, include suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0136] Liquid dosage forms for oral administration may contain colorants and flavorings to enhance patient tolerance. Generally, water, suitable oils, physiological saline, aqueous glucose solutions, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are suitable carriers for parenteral solutions. Parenteral solutions preferably contain a water-soluble salt of the active ingredient, a suitable stabilizer, and, if necessary, a buffer. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid are suitable stabilizers, either alone or in combination. Citric acid and its salts, as well as sodium EDTA, are also used. Furthermore, parenteral solutions may 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.

[0137] The compounds used in the method of the present invention may also be administered in an intranasal form via the use of a suitable intranasal vehicle, or via a transdermal route using those forms of transdermal skin patches well known to those skilled in the art. Because they are administered in the form of a transdermal delivery system, the dosage is generally continuous rather than intermittent throughout the administration regimen.

[0138] Parenteral and intravenous medications may also contain minerals and other materials to ensure they are compatible with the chosen type of injection or delivery system.

[0139] Each embodiment disclosed herein is intended to be applicable to each other disclosed embodiment. Therefore, all combinations of the various elements described herein are within the scope of the invention. Any of the disclosed general or specific compounds are applicable to any of the disclosed compositions, processes, or methods.

[0140] The present invention will be better understood by referring to the details of the experiments that follow, but those skilled in the art will readily understand that the specific detailed experiments are merely illustrative of the present invention, which will be more fully described in the subsequent claims.

[0141] Experiment details General Chemistry Unless otherwise specified, all reactions were carried out under a dry nitrogen atmosphere. The indicated reaction temperatures are the reaction vessel temperatures, with room temperature (rt) indicated as 25°C. Commercial reagents and anhydrous solvents were used as received from the supplier, and no attempts were made to further purify or dry these components. Solvent removal under reduced pressure was performed using a Buchi rotary evaporator and a Teflon-linked KNF vacuum pump at a pressure of approximately 28 mmHg. Thin-layer chromatography was performed using 1''×3'' AnalTech No.02521 silica gel plates with fluorescent indicators. Visualization of the TLC plates was performed by observation using either short-wavelength ultraviolet light (254 nm lamp), 10% phosphomolybdic acid in ethanol, or iodine vapor. Preparative thin-layer chromatography was performed using Analtech, 20×20 cm, 1000 micron preparative TLC plates. Flash column chromatography was performed using a Teledyne Isco CombiFlash Companion Unit and a Biotage® Selekt System equipped with Teledyne Isco RediSep Rf and Biotage Sfar silica gel columns. Where necessary, the product was purified by reverse-phase chromatography using a Teledyne Isco CombiFlash Companion Unit and a Biotage® Selekt System equipped with a RediSep Gold C18 reverse-phase column. Proton NMR spectra were acquired using a 400 MHz Varian nuclear magnetic resonance spectrometer. Chemical shifts (μ) are reported in parts per million (ppm), and binding constant (J) values ​​are reported in Hz. The following spectral pattern notations are used: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet; br, broad. Tetramethylsilane was used as an internal reference. The provided melting points were uncorrected and were obtained using a MEL-TEMP electrothermal melting point device.Mass spectrometry was performed using ESI ionization with a Waters ACQUITY UPLC MS triple quadrupole mass spectrometer. High-pressure liquid chromatography (HPLC) purity analysis was performed using a Waters Breeze2 HPLC system with two-component solvent systems A and B, with gradient elution [A, H2O containing 0.1% formic acid; B, CH3CN containing 0.1% formic acid] and flow rate = 0.5 mL / min, with UV detection at 254 nm (system equipped with a photodiode array (PDA) detector). An ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 50 mm was used. All final compounds tested in in vitro and in vivo biological studies were purified to a purity of 95% or higher, and these purity levels were measured by both 1H NMR and HPLC.

[0142] Scheme 1. [ka] Reagents and conditions: (a) NaBH4, CH3OH, 0°C to room temperature, 8 hours; (b) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (c) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (d) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (e) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (f) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0143] Example 1: 6-methyl-2-(4-(2-(trifluoromethyl)phenoxy)piperidine-1-yl)pyrimidine-4-carboxylic acid 21. Step A: NaBH4 (1.14 g, 30.1 mmol) was added to a solution of tert-butyl 4-oxopiperidine-1-carboxylate 15 (5.0 g, 25.1 mmol) in CH3OH (50 mL) cooled to 0°C. The mixture was stirred for 8 hours while gradually warming to room temperature. The mixture was concentrated under reduced pressure, and the resulting residue was diluted with H2O (100 mL) and extracted with CH2Cl2 (2 × 100 mL). The combined organic extract was dried over Na2SO4, filtered, and evaporated under reduced pressure to obtain tert-butyl 4-hydroxypiperidine-1-carboxylate 16 as a white solid (4.5 g, 89%): ¹H NMR (400 MHz, CDCl3): δ 3.91-3.75 (m, 3H), 3.05-2.96 (m, 2H), 1.90-1.79 (m, 2H), 1.49-1.40 (m, 11H); ES MS: m / z 224 [M + Na]+. Step B: To a 50 mL solution of tert-butyl 4-hydroxypiperidine-1-carboxylate 16 (4.5 g, 22.3 mmol) cooled to 0°C in CH2Cl2, Et3N (4.7 mL, 33.5 mmol) and DMAP (0.127 g, 1.10 mmol) were added, followed by TsCl (5.10 g, 26.8 mmol). The resulting solution was stirred for 16 hours while gradually warming to room temperature under an N2 atmosphere. The mixture was diluted with saturated NaOH aqueous solution (50 mL) and extracted with ELISA (3 × 100 mL). The combined organic extract was washed with H2O (100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0% to 50% RINKAN in hexane) to obtain tert-butyl 4-(tosyloxy)piperidine-1-carboxylate 17 as a colorless liquid (6.6 g, 84%): ¹H NMR (400 MHz, CDCl3) δ 7.82 (d, 2H), 7.49 (d, 2H), 4.69 (b, s, 1H), 3.49 (b, m, 2H), 3.15 (b, m, 2H), 2.43 (bs, 3H), 1.70 (b, m, 2H), 1.51 (b, m, 2H), 1.38 (s, 9H); ESI MS m / z 356 [M + H]+. Step C: To a solution of tert-butyl 4-(tosyloxy)piperidine-1-carboxylate 17 (0.250 g, 0.703 mmol) in anhydrous DMF (4 mL), Cs2CO3 (0.450 g, 1.38 mmol) and 2-(trifluoromethyl)phenol (95.0 mg, 0.586 mmol) were added, and the resulting solution was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature and then diluted with H2O (20 mL). The water-soluble mixture was extracted with RINKAN (3 × 25 mL), the combined organic extract was washed with H2O (3 × 25 mL) and brine (25 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%-30% RINKAN in hexane) to obtain tert-butyl 4-(2-(trifluoromethyl)phenoxy)piperidine-1-carboxylate 18 as a white solid (0.118 g, 56%): ¹H NMR (400 MHz, acetone-d6) δ 7.65-7.55 (m, 2H), 7.30 (d, 1H), 7.08 (t, 1H), 4.92-4.82 (m, 1H), 3.67-3.57 (m, 2H), 3.50-3.40 (m, 2H), 2.0-1.90 (m, 2H), 1.80-1.70 (m, 2H), 1.45 (s, 9H); ESI MS m / z 346 [M + H]+. Step D: Add TFA (0.33 mL, 4.31 mmol) to a 10 mL solution of tert-butyl 4-(2-(trifluoromethyl)phenoxy)piperidine-1-carboxylate 18 (0.118 g, 0.341 mmol) cooled to 0°C in CH2Cl2 (10 mL), and stir the resulting solution for 16 hours while gradually warming it to room temperature. Carefully pour the mixture into a 10 mL solution of saturated water-soluble NaHCO3 and neutralize it. Separate the two phases of the mixture, and further extract the aqueous layer with CH2Cl2 (3 × 20 mL). The combined organic extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 4-(2-(trifluoromethyl)phenoxy)piperidine 19 as a white solid (80.0 mg, 95%): 1H NMR (400 MHz, acetone-d6) δ 7.70-7.60 (m, 2H), 7.37 (d, 1H), 7.14 (t, 1H), 5.10-5.03 (m, 1H), 3.50-3.40 (m, 4H), 2.47-2.37 (m, 2H), 2.21-2.11 (m, 2H); ESI MS m / z 246 [M + H]+. Step E: A mixture of 4-(2-(trifluoromethyl)phenoxy)piperidine 19 (0.100 g, 0.408 mmol), methyl 2-chloro-6-methylpyrimidine-4-carboxylate (76.1 mg, 0.408 mmol), and i-Pr2NEt (0.21 mL, 1.22 mmol) in THF (10 mL) was heat-treated under reflux for 16 hours in an N2 atmosphere. The reaction mixture was concentrated under reduced pressure, and the resulting residue was chromatographed on silica gel (0% to 100% SiO in hexane) to obtain methyl 6-methyl-2-(4-(2-(trifluoromethyl)phenoxy)piperidine-1-yl)pyrimidine-4-carboxylate 20 as an off-white solid (0.140 g, 87%): MS(ESI+) m / z 396 [M + H]+. Step F: A solution of methyl 6-methyl-2-(4-(2-(trifluoromethyl)phenoxy)piperidine-1-yl)pyrimidine-4-carboxylate 20 (0.100 g, 0.253 mmol) and LiOH (18.1 mg, 0.758 mmol) in CH3OH (5 mL), THF (5 mL), and H2O (5 mL) was stirred at room temperature for 16 hours. The mixture was acidified to pH=5 with 2N HCl aqueous solution and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 6-methyl-2-(4-(2-(trifluoromethyl)phenoxy)piperidine-1-yl)pyrimidine-4-carboxylic acid 21 as an off-white solid (87.8 mg, 91%): ¹H NMR (400 MHz, CDCl3) δ 7.56-7.53 (m, 2H), 7.22 (d, J = 6 Hz, 2H), 7.02-7.51 (m, 1H), 4.86 (brs, 1H), 3.99-3.85 (m, 4H), 2.37 (s, 3H), 2.05-1.95 (m, 2H), 1.81-1.80 (m, 2H); ESI MS m / z 382 [M + H]+; HPLC >99%(AUC), tR = 16.8 min.

[0144] Scheme 2. [ka] Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (b) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (c) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (d) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0145] Example 2: (±)-6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-27. Step A: To a solution of (±)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (±)-22 (1.00 g, 5.34 mmol) in CH2Cl2 (20 mL) cooled to 0°C, Et3N (1.1 ml, 8.02 mmol) and DMAP (32.0 mg, 0.262 mmol) were added, followed by TsCl (1.10 g, 5.88 mmol). The resulting solution was stirred for 16 hours while gradually warming to room temperature under an N2 atmosphere. The mixture was diluted with saturated NaOH aqueous solution (20 mL) and extracted with ELISA (3 × 50 mL). The combined organic extracts were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0% to 50% RINKAN in hexane) to obtain (±)-tert-butyl 3-(tosyloxy)pyrrolidine-1-carboxylate (±)-23 as a colorless liquid (1.50 g, 82%): ¹H NMR (400 MHz, CDCl3) δ 7.81 (d, 8.9 Hz, 2H), 7.34 (d, 7.8 Hz, 2H), 5.05 (bs, 1H), 3.43 (m, 4H), 2.43 (bs, 3H), 2.06 (m, 2H), 1.45 (s, 9H); ESI MS m / z 342 [M + H]+. Step B: To a solution of (±)-tert-butyl 3-(tosyloxy)pyrrolidine-1-carboxylate (±)-23 (0.100 g, 0.293 mmol) in anhydrous DMF (4 mL), Cs2CO3 (0.290 g, 0.902 mmol) and 2-(trifluoromethyl)phenol (73.0 mg, 0.450 mmol) were added, and the resulting mixture was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature and then diluted with H2O (20 mL). The water-soluble mixture was extracted with RINKAN (3 × 25 mL), the combined organic extract was washed with H2O (3 × 25 mL) and brine (25 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0% to 30% RINKAN in hexane) to obtain (±)-tert-butyl 3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-carboxylate (±)-24 as a white solid (80.0 mg, 83%): ¹H NMR (400 MHz, acetone-d6) δ 7.62 (m, 2H), 7.34 (d, 1H), 7.15 (t, 1H), 5.20 (s, 1H), 3.65-3.40 (m, 4H), 2.19 (m, 2H), 1.44 (s, 9H); ESI MS m / z 332 [M + H]+. Step C: Add TFA (0.18 mL, 2.41 mmol) to a solution of (±)-tert-butyl 3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-carboxylate (±)-24 (80.0 mg, 0.241 mmol) cooled to 0°C in CH2Cl2 (5 mL), and stir the resulting solution for 8 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a solution of saturated water-soluble NaHCO3 (10 mL). The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extract was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-3-(2-(trifluoromethyl)phenoxy)pyrrolidine (±)-25 as a white solid (50.0 mg, 90%): ESI MS m / z 232 [M + H]+. Step D: A mixture of (±)-3-(2-(trifluoromethyl)phenoxy)pyrrolidine(±)-25 (0.100 g, 0.432 mmol), methyl 2-chloro-6-methylpyrimidine-4-carboxylate (80.6 mg, 0.432 mmol), and i-Pr2NEt (0.23 mL, 1.29 mmol) in THF (10 mL) was heat-treated under reflux for 16 hours in an N2 atmosphere. The reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~100% RINKAN in hexane) to obtain (±)-methyl 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-26 as an off-white solid (0.145 g, 88%): 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.98-6.95 (m, 2H), 5.10 (s, 1H), 4.00-3.86 (m, 3H), 3.89 (s, 3H), 3.76-3.74 (m, 2H), 2.38 (s, 3H), 2.22-2.19(m, 1H); ESI MS m / z 382 [M + H]+. Step E: A solution of (±)-methyl 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-26 (48.8 g, 0.128 mmol) and LiOH (9.21 mg, 0.384 mmol) in CH3OH (5 mL), THF (5 mL), and H2O (5 mL) was stirred at room temperature for 16 hours. The mixture was acidified to pH 5 with 2N hydrochloric acid aqueous solution and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-27 as an off-white solid (0.043 g, 91%): 1H NMR (400 MHz, DMSO-d6) δ 7.61-7.54 (m, 2H), 7.34-7.33 (m, 1H), 7.00-6.93 (m, 2H), 5.31 (s, 1H), 3.77-3.86 (m, 3H), 3.50-3.48 (m, 1H), 2.30 (s, 3H), 2.29-2.20 (m, 2H); ESI MS m / z 368 [M + H]+; HPLC >99%(AUC), tR = 14.5 min.

[0146] Scheme 3. [ka] Reagents and conditions: (a) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (c) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (d) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0147] Example 3: 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)azetidine-1-yl)pyrimidine-4-carboxylic acid 32. Step A: To a solution of tert-butyl 3-(tosyloxy)azetidine-1-carboxylate 28 (0.500 g, 1.52 mmol) in DMF (20 mL), Cs2CO3 (990 mg, 3.05 mmol) and 2-(trifluoromethyl)phenol (0.272 g, 1.68 mmol) were added, and the resulting solution was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature, then diluted with H2O (50 mL), and extracted with RINKAN (3 × 50 mL). The combined organic extract was washed with H2O (3 × 50 mL) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%-30% RINKAN in hexane) to obtain tert-butyl 3-(2-(trifluoromethyl)phenoxy)azetidine-1-carboxylate 29 as a colorless liquid (0.400 g, 82%): 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 7.3 Hz, 1H), 7.61-7.45 (m, 1H), 7.06-7.03 (m, 1H), 6.65 (d, J = 7.9 Hz, 1H), 4.99-4.91 (m, 1H), 4.35-4.30 (m, 2H), 4.10-4.05 (m, 2H), 1.44 (s, 9H); ESI MS m / z 318 [M + H]+. Step B: Add TFA (0.96 mL, 12.0 mmol) to a 20 mL solution of tert-butyl 3-(2-(trifluoromethyl)phenoxy)azetidine-1-carboxylate 29 (0.400 g, 1.20 mmol) cooled to 0°C in CH2Cl2, and stir the resulting solution for 16 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a 10 mL solution of saturated water-soluble NaHCO3. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extract was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 3-(2-(trifluoromethyl)phenoxy)azetidine 30 as a white solid (0.240 g, 87%): ESI MS m / z 218 [M + H]+. Step C: A mixture of 3-(2-(trifluoromethyl)phenoxy)azetidine 30 (0.100 g, 0.460 mmol), methyl 2-chloro-6-methylpyrimidine-4-carboxylate (85.9 mg, 0.460 mmol), and i-Pr2NEt (0.24 mL, 1.38 mmol) in THF (10 mL) was heat-treated under reflux for 16 hours in an N2 atmosphere. The reaction mixture was concentrated under reduced pressure, and the resulting residue was chromatographed on silica gel (0% to 100% SiO in hexane) to obtain methyl 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)azetidine-1-yl)pyrimidine-4-carboxylate 31 as an off-white solid (0.152 g, 90%): MS(ESI+) m / z [M + H]+. Step D: A solution of methyl 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)azetidine-1-yl)pyrimidine-4-carboxylate 31 (0.100 g, 0.272 mmol) and LiOH (19.5 mg, 0.816 mmol) in CH3OH (5 mL), THF (5 mL), and H2O (5 mL) was stirred at room temperature for 16 hours. The mixture was acidified to pH 5 with 2N hydrochloric acid aqueous solution and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 6-methyl-2-(3-(2-(trifluoromethyl)phenoxy)azetidine-1-yl)pyrimidine-4-carboxylic acid 32 as an off-white solid (83.6 mg, 87%): 1H NMR (400 MHz, CDCl3) δ 7.59-7.54 (m, 2H), 7.14 (s, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 5.28 (m, 1H), 4.61-4.60 (m, 2H), 4.15-4.12 (m, 2H), 2.40 (s, 3H); ESI MS m / z 354 [M + H]+; ESI MS m / z 354 [M + H]+; HPLC >99%(AUC), tR = 14.1 min.

[0148] Scheme 4. [ka] Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (b) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (c) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (d) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0149] Example 4: 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-yl)pyrimidine-4-carboxylic acid 38. Step A: To a 50 mL solution of tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate 33 (3.0 g, 16.0 mmol) cooled to 0°C in CH2Cl2 (50 mL), Et3N (4.5 ml, 32.0 mmol) and DMAP (97.0 mg, 0.736 mmol) were added, followed by TsCl (3.35 g, 17.6 mmol). The resulting solution was stirred for 16 hours while gradually warming to room temperature under an N2 atmosphere. The mixture was diluted with saturated NaOH aqueous solution (50 mL) and extracted with ELISA (3 × 100 mL). The combined organic extracts were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain tert-butyl 3-((tosyloxy)methyl)azetidine-1-carboxylate 34 as a colorless liquid (5.0 g, 92%): ESI MS m / z 342 [M + H]+. Step B: To a solution of tert-butyl 3-((tosyloxy)methyl)azetidine-1-carboxylate 34 (5.0 g, 14.6 mmol) in DMF (50 mL), Cs2CO3 (9.5 g, 29.32 mmol) and 2-(trifluoromethyl)phenol (2.3 g, 14.6 mmol) were added, and the resulting mixture was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature, then diluted with H2O (100 mL), and extracted with ELISA (3 × 50 mL). The combined organic extracts were washed with H2O (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-carboxylate 35 as a brown liquid (4.5 g, 93%): ESI MS m / z 332 [M + H]+. Step C: Add TFA (10.3 mL, 135 mmol) to a 50 mL solution of tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-carboxylate 35 (4.50 g, 13.59 mmol) cooled to 0°C in CH2Cl2, and stir the resulting solution for 8 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a 10 mL solution of saturated water-soluble NaHCO3. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extract was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 3-((2-(trifluoromethyl)phenoxy)methyl)piperidine 36 as a white solid (2.8 g, 90% crude): ESI MS m / z 232 [M + H]+. Step D: A mixture of 3-((2-(trifluoromethyl)phenoxy)methyl)azetidine 36 (1.0 g, 4.32 mmol), methyl 2-chloro-6-methylpyrimidine-4-carboxylate (0.807 g, 4.32 mmol), and i-Pr2NEt (2.25 mL, 12.9 mmol) in THF (20 mL) was heat-treated under reflux for 16 hours in an N2 atmosphere. The reaction mixture was concentrated under reduced pressure, and the resulting residue was subjected to chromatography on silica gel (0% to 100% RINKAN in hexane) to obtain methyl 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-yl)pyrimidine-4-carboxylate 37 as an off-white solid (1.64 g, 86%): ¹H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.05 (s, 1H), 7.00-6.95 (m, 2H), 4.33 (t, J = 8.8 Hz, 2H), 4.22-4.21 (m, 2H), 4.08-4.04 (m, 2H), 3.91 (s, 3H), 3.18-3.16(m, 1H), 2.40(s, 3H); ESI MS m / z 382 [M + H]+. Step E: A solution of methyl 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-yl)pyrimidine-4-carboxylate 37 (1.0 g, 2.62 mmol) and LiOH (0.188 g, 7.86 mmol) in CH3OH (10 mL), THF (10 mL), and H2O (10 mL) was stirred at room temperature for 16 hours. The mixture was acidified to pH 5 with 2N hydrochloric acid aqueous solution and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)azetidine-1-yl)pyrimidine-4-carboxylic acid 38 as an off-white solid (0.914 g, 95%): 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 7.00-6.94 (m, 2H), 4.33 (t, J = 8.4 Hz, 2H), 4.24-4.22 (m, 2H), 4.07-4.03 (m, 2H), 3.17-3.15(m, 1H), 2.42(s, 3H); ESI MS m / z 368 [M + H]+; HPLC 98.2%(AUC), tR = 13.5 min.

[0150] Scheme 5. [ka] Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (b) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (c) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (d) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0151] Example 5: (±)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-44. Step A: (±)-tert-butyl 3-(hydroxymethyl)pyrrolidine-1-carboxylate (±)-39 (2.0 g, 9.93 mmol), Et3N (2.8 ml, 83.7 mmol), and DMAP (60.4 mg, 0.494 mmol) were dissolved in CH2Cl2 (50 mL) at 0°C, to which TsCl (2.27 g, 11.9 mmol) was added. The resulting mixture was stirred for 16 hours while gradually warming to room temperature under an N2 atmosphere. The mixture was diluted with saturated water-soluble NaOH solution (50 mL) and then extracted with ELISA (3 × 100 mL). The combined organic extracts were washed with H2O (50 mL) and brine (50 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~30% RINKAN in hexane) to obtain (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40 as a white solid (3.1g, 88%): 1H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 3.97 (d, J = 6.8 Hz, 2H), 3.28-3.25 (m, 1H), 3.24-3.19 (m, 2H), 3.13-3.10 (m, 1H), 2.85-2.81 (m, 1H), 2.39 (s, 3H), 1.82-1.81 (m, 1H), 1.49-1.47(m, 1H), 1.33(s, 9H); ESI MS m / z 356 [M + H]+. Step B: To a solution of (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40 (1.0 g, 2.81 mmol) in DMF (10 mL), Cs2CO3 (2.74 g, 8.41 mmol) and 2-(trifluoromethyl)phenol (0.410 g, 2.53 mmol) were added, and the resulting mixture was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature and then diluted with H2O (30 mL). The water-soluble mixture was extracted with ELISA (3 × 50 mL), the combined organic extract was washed with H2O (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~30% RINKAN in hexane) to obtain (±)-tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-carboxylate (±)-41 as a white solid (0.816 g, 84%): 1H NMR (400 MHz, DMSO-d6) δ 7.61-7.56 (m, 2H), 7.22 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 4.09-4.02 (m, 2H), 3.44-3.35 (m, 1H), 3.36-3.33 (m, 1H), 3.25-3.19 (m, 1H), 3.11-3.01 (m, 1H), 2.62-2.55(m, 1H), 1.97(brs, 1H), 1.96-1.64(m, 1H), 1.35(s, 9H); ESI MS m / z 346 [M + H]+. Step C: To a 0°C solution of (±)-tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-carboxylate (±)-41 (0.800 g, 2.32 mmol) in CH2Cl2 (10 mL), TFA (3.5 mL, 46.3 mmol) was added, and the resulting mixture was stirred under an N2 atmosphere for 16 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a saturated water-soluble NaHCO3 (50 mL) solution, and the resulting two-phase mixture was separated. The aqueous layer was further extracted with CH2Cl2 (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine(±)-42 as a white solid (0.520 g, 90%): 1H NMR (400 MHz, DMSO-d6) δ 9.25 (brs, 1H), 7.62-7.57 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 4.16-4.05 (m, 2H), 3.38-3.33 (m, 1H), 3.28-3.16 (m, 2H), 2.98(t, J = 8.0 Hz, 1H), 2.77-2.69(m, 1H), 2.11-2.03(m, 1H), 1.78-1.69(m, 1H); ESI MS m / z 346 [M + H]+; ESI MS m / z 246 [M + H]+. Step D: To a solution of (±)-tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-carboxylate (±)-42 (0.265 g, 1.08 mmol) in THF (5 mL), i-Pr2NEt (0.6 mL, 3.24 mmol) and methyl 2-chloro-6-methylpyrimidine-4-carboxylate (0.242 g, 1.29 mmol) were added, and the resulting mixture was stirred under reflux for 16 hours. The mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~50% ELISA in hexane) to obtain (±)-methyl6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-43 as a white solid (0.362 g, 85%): 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 6.99 (s, 1H), 6.97-6.92 (m, 2H), 4.10-4.06 (m, 1H), 4.00-3.96 (m, 1H), 3.91 (s, 3H), 3.89-3.86 (m, 1H), 3.79-3.76(m, 1H), 3.66-3.61(m, 1H), 3.51-3.47(m, 1H), 2.86-2.82(m, 1H), 2.39(s, 3H), 2.24-2.20(m, 1H), 1.99-1.94(m, 1H); ESI MS m / z 396 [M + H]+. Step E: (±)-methyl6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-43 (0.250 g, 0.632 mmol) was dissolved in CH3OH (4 mL), THF (4 mL), and H2O (2 mL). LiOH (0.151 g, 6.32 mmol) was added to the solution and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous mixture was diluted with an additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The acidified mixture was extracted with HCl (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-1-(4-(2-(trifluoromethyl)phenyl)piperidine-1-carbonyl)pyrrolidine-2-carboxylic acid (±)-44 as a white solid (0.160 g, 66%): 1H NMR (400 MHz, DMSO-d6) δ 7.59 (m, 2H), 7.24 (m, 1H), 7.06-7.02 (m, 1H), 6.93 (s, 1H), 4.16-4.11 (m, 2H), 3.95-3.61 (m, 2H), 3.50-3.44 (m, 1H), 3.35-3.09 (m, ESI MS m / z 382 [M + H]+; HPLC 98.7%(AUC), tR = 14.5 min.

[0152] Scheme 6. [ka] Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (b) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (c) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (d) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0153] Example 6: (R)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (R)-50. Compound (R)-50 was prepared from tert-butyl (R)-3-(hydroxymethyl)pyrrolidine-1-carboxylate (R)-45 using a procedure similar to that described for the synthesis of (±)-44: ¹H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.6 Hz, ¹H), 7.45 (t, J = 7.6 Hz, ¹H), 7.12 (s, ¹H), 7.01-6.94 (m, ²H), 4.07-4.06 (m, ²H), 3.86-3.77 (m, ²H), 3.62-3.51 (m, ²H), 2.89-2.86 (m, ¹H), 2.43 (s, ³H), 2.27-2.23 (m, ¹H), 2.14-2.01(m, 1H); ESI MS m / z 382 [M + H]+; HPLC >99%(AUC), tR = 14.5 min.

[0154] Example 7: (S)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (S)-56. Compound (S)-56 was prepared from tert-butyl(S)-3-(hydroxymethyl)pyrrolidine-1-carboxylate(S)-51 following a procedure similar to that described for the synthesis of (±)-44: ¹H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz, ¹H), 7.46 (t, J = 8.4 Hz, ¹H), 7.12 (s, ¹H), 7.01-6.94 (m, ²H), 4.07-4.06 (m, ²H), 3.86-3.77 (m, ²H), 3.62-3.51 (m, ²H), 2.90-2.85 (m, ¹H), 2.44 (s, ³H), 2.26-2.24 (m, ¹H), 2.14-2.01(m, 1H); ESI MS m / z 382 [M + H]+; HPLC >99%(AUC), tR = 14.5 min.

[0155] Scheme 7. [ka] Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 0°C to room temperature, 16 hours; (b) 2-(trifluoromethyl)phenol, Cs2CO3, DMF, 80°C, 16 hours; (c) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (d) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (e) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0156] Example 8: (±)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)piperidine-1-yl)pyrimidine-4-carboxylic acid (±)-62. Step A: To a solution of (±)-tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (±)-57 (1.0 g, 4.65 mmol) cooled to 0°C in CH2Cl2 (20 mL), Et3N (0.81 ml, 5.80 mmol) and DMAP (52.0 mg, 0.426 mmol) were added, followed by TsCl (0.883 g, 4.65 mmol). The resulting solution was stirred for 16 hours while gradually warming it to room temperature. The reaction mixture was diluted with saturated NaOH aqueous solution (50 mL) and then extracted with ELISA (3 × 100 mL). The combined organic extracts were washed with H2O (100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-tert-butyl 3-((tosyloxy)methyl)piperidine-1-carboxylate (±)-58 as a colorless liquid (1.6 g, 94%): ESI MS m / z 370 [M + H]+. Step B: To a solution of (±)-tert-butyl 3-((tosyloxy)methyl)piperidine-1-carboxylate (±)-58 (0.500 g, 1.35 mmol) in DMF (20 mL), Cs2CO3 (0.650 g, 2.00 mmol) and 2-(trifluoromethyl)phenol (0.219 g, 1.35 mmol) were added, and the resulting mixture was stirred at 80°C for 16 hours under an N2 atmosphere. The reaction mixture was cooled to room temperature, then diluted with H2O (50 mL), and extracted with Depositphotos (3 × 50 mL). The combined organic extracts were washed with H2O (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-tert-butyl 3-((2-(trifluoromethyl)phenoxy)methyl)piperidine-1-carboxylate (±)-59 as a brown oil (0.400 g, 82%): ESI MS m / z 360 [M + H]+. Step C: (±)-tert-butyl 3-((2-(trifluoromethyl)phenoxy)piperidine-1-carboxylate (±)-59 (0.400 g, 1.11 mmol) was added to a 0°C CH2Cl2 (20 mL) solution with TFA (0.85 mL, 11.1 mmol), and the resulting solution was stirred for 8 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a saturated water-soluble NaHCO3 (10 mL) solution. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extract was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-3-((2-(trifluoromethyl)phenoxy)methyl)piperidine (±)-60 as a white solid (0.250 g, 86%): ESI MS m / z 260 [M + H]+. Step D: To a solution of (±)-3-((2-(trifluoromethyl)phenoxy)methyl)piperidine(±)-60 (0.100 g, 0.385 mmol) in THF (5 mL), i-Pr2NEt (0.20 mL, 1.16 mmol) and methyl 2-chloro-6-methylpyrimidine-4-carboxylate (71.8 mg, 0.385 mmol) were added, and the resulting mixture was stirred under reflux under an N2 atmosphere for 16 hours. The mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~50% RINKAN in hexane) to obtain (±)-methyl6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)piperidine-1-yl)pyrimidine-4-carboxylate (±)-61 as a white solid (0.137 g, 87%): 1H NMR (400 MHz, DMSO-d6) δ 7.59-7.55 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.05 (t, J = 7.6 Hz, 1H), 6.94 (s, 1H), 4.79 (d, J = 12.8 Hz, 1H), 4.55 (d, J = 12.8 Hz, 1H), 4.04-3.96 (m, 2H), 3.80(s, 3H), 2.93-2.83(m, 3H), 2.29(s, 3H), 1.85-1.71(m, 4H); ESI MS m / z 410 [M + H]+. Step E: (±)-methyl6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)piperidine-1-yl)pyrimidine-4-carboxylate (±)-61 (0.100 g, 0.244 mmol) was dissolved in CH3OH (4 mL), THF (4 mL), and H2O (2 mL). LiOH (58.4 mg, 2.44 mmol) was added to the solution and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous mixture was diluted with an additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The acidified mixture was extracted with HCl (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)piperidine-1-yl)pyrimidine-4-carboxylic acid (±)-62 as a white solid (96.4 mg, 66%): ¹H NMR (400 MHz, DMSO-d6) δ 7.60-7.56 (m, 2H), 7.23 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 6.91 (s, 1H), 4.77 (d, J = 10.4 Hz, 1H), 4.57 (d, J = 13.2 Hz, ESI MS m / z 396 [M + H]+; HPLC >99%(AUC), tR = 16.2 minutes.

[0157] Scheme 8. [ka] Reagents and conditions: (a) 1-bromo-2-(trifluoromethyl)benzene, X-Phos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 110°C, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (c) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (d) (i) LiOH, CH3OH, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0158] Example 9: (±)-6-methyl-2-(7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonane-2-yl)pyrimidine-4-carboxylic acid (±)-67. Step A: A mixture of (±)-tert-butyl2,7-diazaspiro[4.4]nonane-2-carboxylate (±)-63 (0.250 g, 1.11 mmol) and 1-bromo-2-(trifluoromethyl)benzene (0.273 g, 1.22 mmol) in 1,4-dioxane was degassed with N2 for 5 minutes, followed by the addition of Cs2CO3 (1.08 g, 3.31 mmol), X-Phos (0.105 mg, 0.223 mmol), and Pd2(dba)3 (0.101 g, 0.112 mmol). The reaction mixture was stirred in a sealed container at 110°C for 16 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (0% to 30% siRNA in hexane) to obtain (±)-methyl tert-butyl 7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (±)-64 as an off-white amorphous solid (0.230 g, 56%): ESI MS m / z 371 [M + H]+. Step B: Add TFA (0.20 mL, 2.61 mmol) to a 5 mL solution of (±)-methyl tert-butyl 7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (±)-64 (0.100 g, 2.69 mmol) cooled to 0°C in CH2Cl2 (5 mL), and stir the resulting solution for 8 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a 10 mL solution of saturated water-soluble NaHCO3. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-2-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonane(±)-65 as a white solid (65.0 mg, 90%): ESI MS m / z 271 [M + H]+. Step C: To a solution of (±)-2-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonane(±)-65 (0.150 g, 0.554 mmol) in THF (5 mL), i-Pr2NEt (0.29 mL, 1.66 mmol) and methyl 2-chloro-6-methylpyrimidine-4-carboxylate (0.103 g, 0.554 mmol) were added, and the resulting mixture was stirred under reflux under an N2 atmosphere for 16 hours. The mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~50% HCl in hexane) to obtain (±)-methyl6-methyl-2-(7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonan-2-yl)pyrimidine-4-carboxylate (±)-66 as a white solid (0.205 g, 88%): ¹H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 6.4 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.87 (t, J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.74-3.61 (m, 4H), 3.47-3.44(m, 2H), 3.30(s, 2H), 2.39(s, 3H), 2.08-1.93(m, 4H); ESI MS m / z 421 [M + H]+. Step D: (±)-methyl 6-methyl-2-(7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonan-2-yl)pyrimidine-4-carboxylate (±)-66 (0.100 g, 0.237 mmol) was dissolved in CH3OH (4 mL), THF (4 mL), and H2O (2 mL). LiOH (56.9 mg, 2.37 mmol) was added to the solution and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous mixture was diluted with an additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The acidified mixture was extracted with HCl (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-6-methyl-2-(7-(2-(trifluoromethyl)phenyl)-2,7-diazaspiro[4.4]nonan-2-yl)pyrimidine-4-carboxylic acid (±)-67 as a white solid (65.5 mg, 68%): ¹H NMR (400 MHz, CDCl₃) δ 7.56 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.01-6.99 (m, 1H), 6.93-6.88 (m, 1H), 3.67-3.55 (m, 4H), 3.46(t, J = 6.8 Hz, 2H), 3.31-3.26(m, 2H), 2.44(s, 3H), 2.10-1.97(m, 4H); ESI MS m / z 407 [M + H]+; HPLC 98.4%(AUC), tR = 15.5 min.

[0159] Scheme 9. [ka] Reagents and conditions: (a) 2-(trifluoromethyl)benzenethiol, Cs2CO3, DMF, 80°C, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (c) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (d) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0160] Example 10: (±)-6-methyl-2-(3-(((2-(trifluoromethyl)phenyl)thio)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-71. Compound (±)-71 was prepared from (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40 and 2-(trifluoromethyl)benzenethiol following a similar procedure to that described for the synthesis of (±)-44: 1H NMR (400 MHz, acetone-d6) δ 7.72 (t, J = 7.2 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.05 (s, 1H), 3.89-3.70 (m, 2H), 3.59-3.55 (m, 1H), 3.42-3.74 (m, 1H), 3.25 (d, J = 7.2 Hz, 2H), 2.64-2.62(m, 1H), 2.40(s, 3H), 2.27-2.24(m, 1H), 1.92-1.87(m, 1H); ESI MS m / z 398 [M + H]+; HPLC 97.1%(AUC), tR = 14.9 min.

[0161] Scheme 10. [ka] Reagents and conditions: (a) 2-(trifluoromethyl)aniline, NaBH(OAc)3, HOAc, CH2Cl2, room temperature, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 12 hours; (c) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, DMF, 80°C, 16 hours; (d) (i) LiOH, CH3OH, THF, H2O, room temperature, 12 hours; (ii) 2N HCl aqueous solution.

[0162] Example 11: (±)-6-methyl-2-(3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-76. Step A: NaBH(OAc)3 (0.960 g, 4.53 mmol) was added to a solution of (±)-tert-butyl3-formylpyrrolidine-1-carboxylate (±)-72 (0.300 g, 1.51 mmol) and 2-(trifluoromethyl)aniline (0.242 g, 1.51 mmol) in CH2Cl2 (10 mL), and the mixture was stirred at room temperature for 16 hours. The mixture was washed with saturated water NaHCO3 solution (5 mL) and brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (±)-tert-butyl 3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-carboxylate (±)-73 as oil, which was used as is in the next step (0.400 g, crude yield 77%): ESI MS m / z 345 [M + H]+. Step B: (±)-tert-butyl 3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-carboxylate (±)-73 (0.400 g, 1.16 mmol) was added to a 5 mL solution of CH2Cl2 cooled to 0°C, and TFA (0.89 mL, 11.6 mmol) was added. The resulting solution was stirred for 12 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a 10 mL solution of saturated water-soluble NaHCO3. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL). The combined organic extract was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-N-(pyrrolidine-3-ylmethyl)-2-(trifluoromethyl)aniline (±)-74 as a yellow oil, which was used directly in the next step (0.360 g). ESI MS m / z 245 [M + H]+. Step C: To a solution of ((±)-N-(pyrrolidine-3-ylmethyl)-2-(trifluoromethyl)aniline(±)-74 (0.360 g, 1.16 mmol)) in THF (5 mL), i-Pr2NEt (0.61 mL, 3.48 mmol) and methyl 2-chloro-6-methylpyrimidine-4-carboxylate (0.216 g, 1.16 mmol) were added, and the resulting mixture was stirred under reflux under an N2 atmosphere for 16 hours. The mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%-50% RINKAN in hexane) to obtain (±)-methyl6-methyl-2-(3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-75 as a white solid (0.290 g, 63%): 1H NMR (400 MHz, CDCl3) δ 7.43-7.40 (m, 1H), 7.35-7.31 (m, 1H), 7.00 (s, 1H), 6.71-6.68 (m, 2H), 4.40 (brs, 1H), 3.91 (s, 3H), 3.90-3.86 (m, 1H), 3.79-3.58 (m, 1H), 3.41-3.19 (m, 4H), 2.68-2.62(m, 1H), 2.40(s, 3H), 2.22-2.16(m, 1H), 1.82-1.77(m, 1H); ESI MS m / z 395 [M + H]+. Step D: LiOH (56.9 mg, 2.37 mmol) was added to a solution of (±)-methyl 6-methyl-2-(3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylate ((±)-75, 0.110 g, 0.278 mmol) in CH3OH (4 mL), THF (4 mL), and H2O (2 mL), and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous mixture was diluted with an additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The acidified mixture was extracted with RINKAN (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-6-methyl-2-(3-(((2-(trifluoromethyl)phenyl)amino)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-76 as a white solid (90.0 mg, 68%): 1H NMR (400 MHz, DMSO-d6) δ 7.36-7.32 (m, 2H), 6.84-6.79 (m, 2H), 6.20 (t, J = 7.6 Hz, 1H), 5.53 (brs, 1H), 3.63-3.58 (m, 2H), 3.41-3.39 (m, 1H), 3.25-3.17(m, 3H), 2.58-2.57(m, 1H), 2.23(s, 3H), 1.98-1.95(m, 1H), 1.70-1.67(m, 1H); ESI MS m / z 381 [M + H]+; HPLC >99%(AUC), tR = 14.5 minutes.

[0163] Scheme 11. [ka] Reagents and conditions: (a) 1-(bromomethyl)-2-(trifluoromethyl)benzene, NaH, DMF, 0°C to room temperature, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (c) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (d) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0164] Example 12: (±)-6-methyl-2-(3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-80. Step A: NaH (0.267 g, 6.68 mmol) was added to a solution of tert-butyl 3-hydroxypyrrolidine-1-carboxylate (±)-22 (0.500 g, 2.67 mmol) in DMF (5 mL) cooled to 0°C. The mixture was stirred at 0°C under an N2 atmosphere for 30 minutes, then 1-(bromomethyl)-2-(trifluoromethyl)benzene (0.766 g, 3.20 mmol) was added, and the resulting mixture was stirred for 16 hours while gradually warming to room temperature. The mixture was cooled back to 0°C, diluted with H2O (20 mL), and carefully quenched. The water-soluble mixture was extracted with Â(3 × 30 mL), the combined organic extract was washed with H₂O(3 × 30 mL) and brine(30 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-tert-butyl 3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-carboxylate(±)-77 as an off-white solid (0.900 g, crude yield 97%), which was used directly in the next step: ESI MS m / z 346 [M + H]+. Step B: Add TFA (2.0 mL, 26.0 mmol) to a 5 mL solution of (±)-tert-butyl 3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-carboxylate (±)-77 (0.900 g, 2.61 mmol) cooled to 0°C in CH2Cl2, and stir the resulting solution for 8 hours while gradually warming it to room temperature. The mixture was neutralized by carefully pouring it into a 30 mL solution of saturated NaHCO3. The two-phase mixture was separated, and the aqueous layer was further extracted with CH2Cl2 (3 × 30 mL). The combined organic extracts were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine(±)-78 as a white solid (0.450 g, crude yield 70%): ESI MS m / z 246[M + H]+. Step C: To a solution of (±)-3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine(±)-78 (0.100 g, 0.407 mmol) in THF (5 mL), i-Pr2NEt (0.25 mL, 1.23 mmol) and methyl 2-chloro-6-methylpyrimidine-4-carboxylate (76.2 mg, 0.408 mmol) were added, and the resulting solution was stirred at 80°C for 16 hours under an N2 atmosphere. The mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~50% HCl in hexane) to obtain (±)-methyl 6-methyl-2-(3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-yl)pyrimidine-4-carboxylate (±)-79 as a white solid (0.110 g, 68%): 1H NMR (400 MHz, acetone-d6) δ 7.75-7.68 (m, 2H), 7.62 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 6.97 (s, 1H), 4.78-4.76 (m, 2H), 4.41-4.39 (m, 1H), 3.85 (s, 3H), 3.74-3.66 (m, 4H), 2.36(s, 3H), 2.31-2.04(m, 2H); ESI MS m / z 396 [M + H]+. Step D: (±)-methyl 6-methyl-2-(3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-yl)pyrimidine-4-carboxylate ((±)-79, 90.0 mg, 0.228 mmol) was dissolved in CH3OH (4 mL), THF (4 mL), and H2O (2 mL). LiOH (54.5 mg, 2.28 mmol) was added to the solution and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous layer was diluted with additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The mixture was extracted with HCl (3 × 30 mL), the combined organic extract was washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain (±)-6-methyl-2-(3-((2-(trifluoromethyl)benzyl)oxy)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-80 as a white solid (60.0 mg, 85%): 1H NMR (400 MHz, DMSO-d6) δ 7.67-7.59 (m, 3H), 7.47-7.45 (m, 1H), 6.90 (s, 1H), 4.64-4.58 (m, 2H), 4.28-4.23 (m, 1H), 3.66-3.46 (m, 4H), 2.25 (s, 3H), 2.05-1.98(m, 2H); ESI MS m / z 382 [M + H]+; HPLC 98.1%(AUC), tR = 14.4 min.

[0165] Scheme 12. [ka] Reagents and conditions: (a) Substituted phenol, Cs2CO3, DMF, 80°C, 16 hours; (b) TFA, CH2Cl2, 0°C to room temperature, 8 hours; (c) Methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (d) (i) LiOH, CH3OH, THF, H2O, room temperature, 16 hours; (ii) 2N HCl aqueous solution.

[0166] Example 13: (±)-2-(3-((2-(Tert-butyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-83. Compound (±)-83 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 2-(tert-butyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: ¹H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.0 Hz, ¹H), 7.17-7.12 (m, ²H), 6.91-6.82 (m, ²H), 4.06-4.00 (m, ³H), 3.93-3.82 (s, ¹H), 3.63-3.51 (m, ²H), 2.93-2.90 (m, ¹H), 2.44 (s, ³H), 2.31-2.28 (m, ¹H), 2.00-1.98(m, 1H), 1.38(s, 9H); ESI MS m / z 370 [M + H]+; HPLC 96.4%(AUC), tR = 16.2 min.

[0167] Example 14: (±)-2-(3-((2-cyclopentylphenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-84. Compound (±)-84 was prepared from 2-cyclopentylphenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40 following a similar procedure to that described for the synthesis of (±)-44: ¹H NMR (400 MHz, DMSO-d6) δ 7.16–7.07 (m, J = 8.0 Hz, 2H), 6.94 (s, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.83 (t, J = 7.2 Hz, 1H), 3.98 (d, J = 6.4 Hz, 2H), 3.76–3.63 (m, 2H), 3.53–3.39 (m, 2H), 3.22–3.15 (m, 1H), ESI MS m / z 382 [M + H]+; HPLC 95.2%(AUC), tR = 16.4 min.

[0168] Example 15: (±)-2-(3-((2-cyclohexylphenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-85. Compound (±)-85 was prepared from 2-cyclohexylphenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40 following a similar procedure to that described for the synthesis of (±)-44: ¹H NMR (400 MHz, DMSO-d6) ¹H NMR (400 MHz, CDCl3) δ 7.17-7.15 (m, 1H), 7.13-7.11 (m, 1H), 7.12 (s, 1H), 6.92-6.88 (m, 1H), 6.82 (d, J = 8.4 Hz, 1H), 4.01-3.93 (m, 2H), 3.87-3.80 (m, 2H), 3.69-3.48 (m, 2H), ESI MS m / z 396 [M + H]+; HPLC 98.3%(AUC), tR = 17.1 min.

[0169] Example 16: (±)-2-(3-((3-chloro-2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-86. Compound (±)-86 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 3-chloro-2-(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, DMSO-d6) δ 7.56 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.94 (s, 1H), 4.19-4.10 (m, 2H), 3.76-3.63 (m, 2H), 3.51-3.44 (m, 1H), 3.33-3.32 (m, 1H), 2.77-2.70 (m, 1H), 2.32(s, 3H), 2.15-2.07(m, 1H), 1.87-1.79(m, 1H); ESI MS m / z 416 [M + H]+; HPLC 99.0%(AUC), tR = 15.1 min.

[0170] Example 17: (±)-2-(3-((4-fluoro-2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-87. Compound (±)-87 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 4-fluoro-2-(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, DMSO-d6) δ 7.48-7.44 (m, 2H), 7.28-7.25 (m, 1H), 6.92 (s, 1H), 4.12-4.08 (m, 2H), 3.72-3.68 (m, 1H), 3.64-3.60 (m, 1H), 3.47-3.42 (m, 1H), 3.33-3.29 (m, 1H), 2.71-2.68 (m, 1H), 2.32 (s, 3H), 2.10-2.06(m, 1H), 1.83-1.78(m, 1H); ESI MS m / z 400 [M + H]+; HPLC 98.7%(AUC), tR = 14.8 min.

[0171] Example 18: (±)-2-(3-((5-fluoro-2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-88. Compound (±)-88 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 5-fluoro-2-(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, DMSO-d6) δ 7.66-7.62 (m, 1H), 7.20 (d, J = 11.2 Hz, 1H), 6.93 (s, 1H), 6.91-6.86 (m, 1H), 4.18-4.10 (m, 2H), 3.73-3.63 (m, 2H), 3.51-3.44 (m, 1H), 3.35-3.32 (m, 1H), 2.76-2.69 (m, 1H), 2.31(s, 3H), 2.14-2.06(m, 1H), 1.87-1.79(m, 1H); ESI MS m / z 400 [M + H]+; HPLC 98.0%(AUC), tR = 14.7 min.

[0172] Example 19: (±)-2-(3-((2-fluoro-6-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-89. Compound (±)-89 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 2-fluoro-6-(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, DMSO-d6) δ 7.64-7.60 (m, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.29-7.24 (m, 1H), 6.94 (s, 1H), 4.19-4.13 (m, 2H), 3.77-3.73 (m, 1H), 3.67-3.61 (m, 1H), 3.51-3.45 (m, 1H), 3.34-3.34 (m, 1H), 2.77-2.72(m, 1H), 2.32(s, 3H), 2.13-2.09(m, 1H), 1.89-1.82(m, 1H); ESI MS m / z 400 [M + H]+; HPLC 96.7%(AUC), tR = 14.8 min.

[0173] Example 20: (±)-2-(3-((5-methoxy-2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-90. Compound (±)-90 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 5-methoxy-2-(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.8 Hz, 1H), 6.94 (s, 1H), 6.75 (s, 1H), 6.58 (d, J = 8.4 Hz, 1H), 4.13-4.07 (m, 2H), 3.79 (s, 3H), 3.74-3.64 (m, 2H), 3.51-3.45 (m, 1H), 3.36-3.31 (m, 1H), 2.73-2.70 (m, 1H), 2.31(s, 3H), 2.12-2.08(m, 1H), 1.87-1.80(m, 1H); ESI MS m / z 412 [M + H]+; HPLC >99%(AUC), tR = 14.6 min.

[0174] Example 21: (±)-2-(3-((3,5-bis(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)-6-methylpyrimidine-4-carboxylic acid (±)-91. Compound (±)-91 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 3,5-bis(trifluoromethyl)phenol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, DMSO-d6) δ 7.61-7.59 (m, 3H), 6.94 (s, 1H), 4.23-4.14 (m, 2H), 3.76-3.71 (m, 1H), 3.69-3.63 (m, 1H), 3.53-3.47 (m, 1H), 3.42-3.38 (m, 1H), 2.78-2.71 (m, 1H), 2.32 (s, 3H), 2.16-2.08 (m, 1H), 1.89-1.81(m, 1H); ESI MS m / z 450 [M + H]+; HPLC 97.0%(AUC), tR = 16.0 min.

[0175] Example 22: (±)-6-methyl-2-(3-(((4-(trifluoromethyl)pyridine-3-yl)oxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-92. Compound (±)-92 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 4-(trifluoromethyl)pyridine-3-ol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.37 (d, J = 4.8 Hz, 1H), 7.43 (d, J = 4.8 Hz, 1H), 7.13 (s, 1H), 4.21-4.20 (m, 2H), 3.87-3.78 (m, 2H), 3.65-3.64 (m, 1H), 3.54-3.49 (m, 1H), 2.93-2.86 (m, 1H), 2.44(s, 3H), 2.29-2.24(m, 1H), 2.02-1.97(m, 1H); ESI MS m / z 383 [M + H]+; HPLC >99%(AUC), tR = 12.6 min.

[0176] Example 23: (±)-6-methyl-2-(3-(((2-(trifluoromethyl)pyridine-3-yl)oxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-93. Compound (±)-93 was prepared from (±)-40 by following a similar procedure to that described for the synthesis of (±)-44, using 2-(trifluoromethyl)pyridine-3-ol and (±)-tert-butyl 3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (±)-40: 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.4 Hz, 1H), 7.45-7.42 (m, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.13 (s, 1H), 4.11-4.07 (m, 2H), 3.86-3.85 (m, 2H), 3.63-3.61 (m, 1H), 3.51-3.47 (m, 1H), 2.91-2.84 (m, 1H), 2.44(s, 3H), 2.28-2.25(m, 1H), 2.00-1.97(m, 1H); ESI MS m / z 383 [M + H]+; HPLC 99.0%(AUC), tR = 12.7 min.

[0177] Scheme 13. [ka] Reagents and conditions: (a) (i) Methyl 2-chloropyrimidine-4-carboxylate, i-Pr2NEt, THF, reflux, 16 hours; (ii) LiOH, CH3OH, H2O, room temperature, 16 hours; (ii) 2N aqueous HCl solution; (b) (i) Methyl 6-chloro-4-methyl picolinate, XantPhos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 80°C, 16 hours; (ii) LiOH, CH3OH, H2O, room temperature, 16 hours; (iii) 2N aqueous HCl solution; (c) (i) Methyl 6-chloropicolinate, XantPhos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 80°C, 16 hours; (ii) LiOH, H2O, CH3OH, THF, room temperature, 16 hours; (iii) 2N HCl aqueous solution; (d) (i) Methyl 2-chloronicotinate, XantPhos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 80°C, 16 hours; (ii) LiOH, H2O, CH3OH, THF, room temperature, 16 hours; (iii) 2N HCl aqueous solution; (e) (i) Methyl 3-bromo-4-fluorobenzoate, XPhos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 110°C, 16 hours; (ii) LiOH, H2O, CH3OH, THF, room temperature, 16 hours; (iii) 2N HCl aqueous solution.

[0178] Example 24: (±)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-94. Compound (±)-94 was prepared from methyl 2-chloropyrimidine-4-carboxylate and (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (±)-42 following a similar procedure to that described for the synthesis of (±)-44: ¹H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 4.8 Hz, ¹H), 7.61-7.57 (m, ²H), 7.26 (d, J = 8.4 Hz, ¹H), 7.06 (t, J = 7.6 Hz, ¹H), 7.00 (d, J = 5.2 Hz, ¹H), 4.15 (brs, ²H), 3.77-3.63 (m, ²H), 3.53-3.47 (m, ¹H), 3.37-3.33(m, 1H), 2.79-2.72(m, 1H), 2.17-2.09(m, 1H), 1.90-1.83(m, 1H); ESI MS m / z 368 [M + H]+; HPLC >99%(AUC), tR = 14.3 min.

[0179] Example 25: (±)-4-methyl-6-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)picolinic acid (±)-95. Step A: XantPhos (0.153 g, 0.265 mmol) and Pd2(dba)3 (74.6 mg, 0.082 mmol) were added to a mixture of (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (±)-42 (0.200 g, 0.815 mmol), methyl 6-chloro-4-methyl picolinate (0.151 g, 0.815 mmol), and Cs2CO3 (0.796 g, 2.44 mmol) in N2-degassed anhydrous 1,4-dioxane (10 mL). The mixture was heated in a sealed container at 80°C for 16 hours. The reaction mixture was cooled to room temperature, then diluted with H2O (30 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic extract was washed with H2O (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~50% RINKAN in hexane) to obtain (±)-methyl 4-methyl-6-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)picolinate as an off-white solid (0.180 g, 56%): 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.23-7.20 (m, 1H), 7.00-6.92 (m, 2H), 6.34 (s, 1H), 4.08-3.99 (m, 2H), 3.89 (s, 3H), 3.76-3.72 (m, 1H), 3.69-3.63 (m, ESI MS m / z 395 [M+H]+. Step B: LiOH (0.115 g, 4.81 mmol) was added to a solution of (±)-methyl 4-methyl-6-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)picolinate (0.190 g, 0.481 mmol) in CH3OH (6 mL), THF (6 mL), and H2O (3 mL), and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to remove the volatile solvent, and the resulting aqueous mixture was diluted with an additional H2O (10 mL) and acidified to pH=3 with 2N HCl aqueous solution. The acidified mixture was extracted with HCl (3 × 20 mL), the combined organic extract was washed with brine (20 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (±)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-95 as a white solid (0.136 g, 77%): 1H NMR (400 MHz, DMSO-d6) δ 7.57-7.54 (m, 2H), 7.15 (d, J = 8.4 Hz, 1H), 7.05-7.01 (m, 2H), 6.18 (s, 1H), 4.02-3.92 (m, 2H), 3.68-3.46 (m, 3H), 3.34-3.32 (m, 1H), 2.64-2.61(m, 1H), 2.13(s, 3H), 2.10-2.01(m, 1H), 1.81-1.68(m, 1H); ESI MS m / z 381 [M + H]+; HPLC >99%(AUC), tR = 12.8 min.

[0180] Example 26: (±)-6-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)picolinic acid (±)-96. Compound (±)-96 was prepared from 6-chloropicolinate and (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (±)-42 following a similar procedure to that described for the synthesis of (±)-95: 1H NMR (400 MHz, CDCl3) δ 7.63-7.54 (m, 2H), 7.46-7.42 (m, 2H), 7.00-6.95 (m, 2H), 6.62-6.60 (m, 1H), 4.10-4.04 (m, 2H), 3.77-3.60 (m, 2H), 3.51-3.39 (m, 2H), 2.92-2.80 (m, 1H), 2.25-2.22 (m, 1H), 2.08-1.96 (m, 1H); ESI MS m / z 367 [M + H]+; HPLC 95.8%(AUC), tR = 12.7 min.

[0181] Example 27: (±)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)nicotinic acid (±)-97. Compound (±)-97 was prepared from 2-chloronicotinate and (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (±)-42 following a similar procedure to that described for the synthesis of (±)-95: ¹H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 4.4 Hz, 1H), 8.23 ​​(d, J = 7.6 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.47-7.43 (m, 1H), 7.00-6.91 (m, 3H), 4.10-4.02 (m, 2H), 3.61-3.37 (m, 4H), 2.93-2.86 (m, 1H), 2.29-2.21 (m, 1H), 1.98-1.89(m, 1 H); ESI MS m / z 367 [M + H]+; HPLC 98.3%(AUC), tR = 12.0 min.

[0182] Example 28: (±)-4-fluoro-3-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)benzoic acid (±)-98. Step A: Cs2CO3 (1.2g, 3.66mmol), XPhos (58.1mg, 0.12mmol), and Pd2(dba)3 (37.9mg, 0.037mmol) were added to a mixture of (±)-3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (±)-42 (0.300g, 1.22mmol) and methyl 3-bromo-4-fluorobenzoate (0.342g, 1.47mmol) in N2-degassed 1,4-dioxane. The mixture was stirred in a sealed container at 110°C for 16 hours and then cooled to room temperature. The mixture was concentrated under reduced pressure, and the resulting residue was subjected to chromatography on silica gel (0%-40% RINKAN in hexane) to obtain methyl 4-fluoro-3-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)benzoate as an off-white solid (0.198 mg, 41%): ¹H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.6 Hz, ¹H), 7.45 (t, J = 7.6 Hz, ¹H), 7.37-7.32 (m, ²H), 7.00-6.95 (m, ³H), 4.04 (d, J = 7.2 Hz, ²H), 3.85 (s, ³H), 3.64-3.59 (m, ¹H), 3.52-3.46 (m, ²H), 3.40-3.35(m, 1H), 2.85-2.81(m, 1H), 2.22-2.17(m, 1H), 1.93-1.88(m, 1H); ESI MS m / z 398 [M + H]+. Step B: LiOH (0.144 g, 6.04 mmol) was added to a mixed solution of methyl 4-fluoro-3-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)benzoate (0.120 g, 0.63 mmol) in CH3OH (4 mL), THF (4 mL), and H2O (2 mL). The mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure to remove the volatile solvent. The resulting aqueous layer was diluted with H2O (50 mL) and acidified to pH=3 with 2N HCl aqueous solution. The water-soluble mixture was extracted with RINKAN (3 × 50 mL), the combined organic extract was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 4-fluoro-3-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)benzoic acid (±)-98 as a white solid (78.0 mg, 67%): δ 7.59-7.55 (d, J = 7.6 Hz, 2H), 7.24-7.20 (m, 3H), 7.13-7.02 (m, 2H), 4.13-4.07 (m, 2H), 3.52-3.48 (m, 1H), 3.39 (brs, 2H), 3.29-3.25 (m, 1H), 2.73-2.70 (m, 1H), 2.11-2.08(m, 1H), 1.81-1.76(m, 1H); ESI MS m / z 384 [M + H]+; HPLC 98.5%(AUC), tR = 16.1 min.

[0183] Scheme 14. [ka] Reagents and conditions: (a) Methanesulfonamide, HBTU, i-Pr2NEt, DMF, room temperature, 18 hours; (b) NH4Cl, HBTU, i-Pr2NEt, DMF, room temperature, 18 hours; (c) NHCH3·HCl, T3P, i-Pr2NEt, DMF, room temperature, 18 hours; (d) Cyclopropylamine, HBTU, i-Pr2NEt, DMF, room temperature, 18 hours.

[0184] Example 29: (±)-6-methyl-N-(methylsulfonyl)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-99. Step A: Methanesulfonamide (19.1 mg, 0.197 mmol) was added to a mixture of 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid(±)-44 (50.0 mg, 0.131 mmol), HBTU (74.5 mg, 0.197 mmol), and i-Pr2NEt (0.08 mL, 0.393 mmol) in DMF (4 mL). The resulting solution was stirred at room temperature under an N2 atmosphere for 18 hours. The mixture was diluted with H2O (10 mL) and extracted with ELISA (3 × 20 mL). The combined organic extracts were washed with H2O (3 × 20 mL) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude residue was subjected to chromatography (0% to 80% RINKAN in hexane) on silica gel to obtain 6-methyl-N-(methylsulfonyl)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-99 as a white solid (30.0 mg, 50%): 1H NMR (400 MHz, acetone-d6) δ 10.36 (brs, 1H), 7.61-7.57 (m, 2H), 7.27-7.25 (m, 1H), 7.10-7.06 (m, 2H), 4.23 (brs, 2H), 3.92-3.82 (m, 2H), 3.65-3.49 (m, 2H), 3.34 (s, 3H), 2.92-2.82(m, 2H), 2.42(s, 3H), 2.25-2.22(m, 1H); ESI MS m / z 459 [M + H]+; HPLC 98.4%(AUC), tR = 15.9 min.

[0185] Example 30: (±)-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-100. Compound (±)-100 was prepared from NH4Cl and 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid (±)-44 following a similar procedure to that described for the synthesis of (±)-99: ¹H NMR (400 MHz, CDCl3) δ 7.71 (brs, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.14 (s, 1H), 7.00-6.94 (m, 2H), 5.57 (brs, 1H), 4.06-4.02 (m, 2H), 3.88-3.83 (m, 1H), 3.79-3.73 (m, 1H), ESI MS m / z 381 [M + H]+; HPLC >99%(AUC), tR = 14.3 min.

[0186] Example 31: (±)-N,6-dimethyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-101. Step A: Methylamine hydrochloride (44.0 mg, 0.393 mmol) was added to a solution of 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid(±)-44 (50.0 mg, 0.131 mmol), T3P (50% w / w in CH2Cl2) (83.4 mg, 0.262 mmol), and i-Pr2NEt (0.2 mL, 1.05 mmol) in CH2Cl2 (3 mL). The mixture was stirred at ambient temperature for 18 hours and then concentrated under reduced pressure. The obtained residue was subjected to chromatography (0% to 60% RINKAN in hexane) on silica gel to obtain N,6-dimethyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-101 as a white solid (40.0 mg, 77%): 1H NMR (400 MHz, acetone-d6) δ 8.25 (brs, 1H), 7.62-7.59 (m, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.08-7.05 (m, 2H), 4.23-4.19 (m, 2H), 3.88-3.83 (m, 1H), 3.79-3.74 (m, 1H), 3.60-3.58 (m, 1H), ESI MS m / z 395 [M + H]+; HPLC >99%(AUC), tR = 14.7 minutes.

[0187] Example 32: (±)-N-cyclopropyl-6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-102. Compound (±)-102 was prepared from cyclopropylamine and 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxylic acid ((±)-44) following a similar procedure to that described for the synthesis of (±)-100: 1H NMR (400 MHz, acetone-d6) δ 8.17(brs, 1H), 7.62-7.57(m, 2H), 7.26(d, J = 8.4 Hz, 1H), 7.10-7.04(m, 2H), 4.21(brs, 2H), 3.87-3.82(m, 1H), 3.76-3.72(m, 1H), 3.58-3.55(m, 1H), 3.49-3.44(m, 1H), 2.88-2.78(m, 2H), 2.36(s, 3H), 2.23-2.13(m, 1H), 2.02-1.95(m, 1H), 0.75-0.73(m, 2H), 0.58(brs, 2H); ESI MS m / z 421 [M + H]+; HPLC >99%(AUC), tR = 15.3 min.

[0188] Scheme 15. [ka] Reagents and conditions: (a) NaN3, tetrachlorosilane, CH3CN, 80°C, 18 hours.

[0189] Example 33: (±)-4-methyl-6-(2H-tetrazole-5-yl)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine(±)-103. Step A: A mixture of 6-methyl-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine-4-carboxamide(±)-100 (0.200 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 in a sealed container at 80°C for 18 hours. The reaction mixture was cooled to room temperature and diluted with saturated NaHCO3 (5 mL). The water-soluble mixture was extracted with CHCl3 (3 × 50 mL), the combined organic extract was washed with brine (50 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was subjected to chromatography on silica gel (0%~10% CH3OH in CH2Cl2) to obtain 4-methyl-6-(2H-tetrazole-5-yl)-2-(3-((2-(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-yl)pyrimidine(±)-103 as a white solid (66.0 mg, 30%): 1H NMR (400 MHz, acetone-d6) δ 7.62-7.58 (m, 2H), 7.28-7.25 (m, 2H), 7.08 (t, J = 7.6 Hz, 1H), 4.28-4.21 (m, 3H), 3.91-3.86 (m, 1H), 3.80-3.74 (m, 1H), 3.653.58 (m, 1H), 3.52-3.47(m, 1H), 2.92-2.85(m, 1H), 2.42(s, 3H), 2.27-2.21(m, 1H); ESI MS m / z 406 [M + H]+: HPLC 97.4%(AUC), tR = 14.6 min.

[0190] Example 34: In vitro binding of compounds to RBP4. Binding of compounds to RBP4 was evaluated using the previously described radiometric scintillation proximity (SPA) assay (Cioffi, CL et al. 2014; Cioffi, CL et al. 2015; Cioffi, CL et al. 2019). This assay measured competitive substitution of radiolabeled retinol from naturally occurring RBP4 purified from human urine (Fitzgerald, 30R-AR022L). The protein was biotinylated using ThermoFisher's EZ-link Sulfo-NHS-LC-Biotinylation kit (Cat #21335) according to the manufacturer's recommendations. Binding assays were performed in a final volume of 100 μL in SPA buffer (1×PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The assay reaction involved radioligand, 10 nM 3H-retinol (48.7 Ci / mmol; PerkinElmer, Waltham, MA), 0.3 mg / well of Streptavidin-PVT beads (PerkinElmer, RPNQ0006), and 50 nM biotinylated human RBP4. To assess nonspecific binding, 20 μM unlabeled retinol (Sigma, cat # 95144) was added to the control well. After incubation at room temperature (rt) for 16 hours with gentle shaking, radioactivity counts were measured using a CHAMELEON plate reader (Hidex Oy, Turku, Finland).

[0191] Example 35: Evaluation of antagonist activity in HTRF RBP4-TTR interaction assay. The ability of analogs to act as antagonists in all-trans-retinol-dependent RBP4-TTR interactions was measured by HTRF (Homogenous Time-Resolved Fluorescence) assay as previously described (Cioffi, CL et al. 2014; Cioffi, CL et al. 2015; Cioffi, CL et al. 2019). Untagged TTR (Calbiochem, cat #529577) and maltose-binding protein-tagged RBP4 expressed in E. coli were used in this assay. TTR was labeled with Eu3+ Cryptate using an HTRF Cryptate labeling kit from CisBio (Cisbio, cat #62EUSPEA, Bedfored, MA). The assay was performed in a final assay volume of 16 μl in buffer containing 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Other components of the reaction mixture included 26.7 nM anti-MBP antibody (Cisbio, cat #61MBPDAA) conjugated with 60 nM MBP-RBP4, 5 nM TTR-Eu, d2, and 1 μM all-trans-retinol (Sigma, cat #95144). All reactions were performed in the dark under dim red light. After incubation overnight at 4°C, plates were read using a SpectraMax M5e Multimode Plate Reader (Molecular Devices, Sunnyvale, CA). Fluorescence was excited at 337 nm; emission was measured at 668 and 620 nm with a count delay of 75 μs. The HTRF signal was expressed as the ratio of fluorescence intensities: Flu668 / Flu620 × 10,000.

[0192] Example 36: Fluorescence-polarized TTR tetramer binding assay. Binding of compounds to TTR was evaluated by a fluorescence-polarized assay. This assay measured the competitive substitution of the fluorescent probe, FITC-diclofenac, from TTR isolated from human plasma (Clabiochem-Millipore, cat. No. 52957). FITC-diclofenac was synthesized at LeadGen Labs, LLC according to a published procedure (Alhamadsheh, MM et al. 2011). Each well contained 200 nM TTR and 100 nM FITC-diclofenac in FP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% CHAPS, 0.01% Prionex) along with the test compound. Nonspecific binding was determined in the presence of 500 μM unlabeled diclofenac (Sigma-Aldrich). The reaction mixture with the test compound was incubated overnight at 4°C, and the fission products (FP) were measured using a Spectramax M5e plate reader (Molecular Devices). [Table 1-1] [Table 1-2] [Table 1-3]

[0193] Example 37: TTR Aggregation Assay. The ability of test compounds to prevent TTR aggregation was evaluated under acidic conditions favorable to TTR aggregation and fiber formation. A 2 μl solution of 167 μM human TTR (ACROBiosystems #H5223) was incubated at 37°C for 72 hours with 7 μl of 50 mM sodium acetate pH 4.0 (Sigma # S7545) and 100 mM KCl (Sigma # S5405) in or without 1 μl of a TTR inhibitor. At the end of incubation, each sample was neutralized by adding 3.5 μl of 500 mM sodium phosphate (Sigma # S5136) buffer pH=8.0, and 0.6 μl of 5% CHAPS (Sigma # C5070) was added as a surfactant to prevent protein recombination. Crosslinking was performed by adding 1.5 μl of 5% glutaraldehyde solution (Sigma # G6257). After 4 minutes, the reaction was stopped by adding 2.5 μl of freshly prepared 5% NaBH4. The sample was subjected to TTR Western blotting using prealbumin antibody (1:500; Dako #A0002). The band intensities of TTR monomers and TTR aggregates were quantified from scanned images of the blot.

[0194] In VitroADME assay information Example 38: Dynamic Solubility Assay The kinetic water solubility of compound (±)-44 in PBS (pH 7.4) was performed using Eurofins with UV detection (230 nm). Water solubility (μM) was determined by comparing the peak area of ​​the principal peak in a calibration standard (200 μM) containing an organic solvent (methanol / water, 60 / 40, v / v) with the peak area of ​​the corresponding peak in the buffer sample. Chromatographic purity (%) was defined as the peak area of ​​the principal peak relative to the total integrated peak area of ​​the HPLC chromatogram of the calibration standard. Chromatograms of the calibration standard and UV / VIS spectra labeled with maximum absorbance values ​​were prepared for each test substance. Standard substances for kinetic solubility testing: Metoprolol-192.6μM Rifampicin-200μM Ketoconazole - 152.8 μM Phenytoin - 101.81 μM Simvastatin - 14.2 μM Diethylstilbestrol - 7.0 μM Tamoxifen - 1.9 μM

[0195] Example 39: CYP450 Inhibition Assay Results of the inhibitory activity (IC50 value) of compound (±)-44 against human cytochrome P450 (CYP) isoforms 2C9, 2C19, 2D6, and 3A4. Each recombinant human CYP isoform was tested with standard positive and negative controls using fluorescence detection for measuring CYP activity. The measured IC50 values of the respective standard inhibitors were all within the expected range for each isoform (see below). IC50 concentrations of standard CYP inhibitors: CYP inhibitor IC50 (μM): 2C9 Sulfaphenazole IC50 = 3.4 μM 2C19 Tranylcypromine IC50 = 2.8 μM 2D6 Quinidine IC50 = 0.058 μM 3A4 Ketoconazole IC50 = 0.0084 μM

[0196] Pre-formulated NADPH regeneration solutions, 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 HCl (TCP), sulfafenazole (SFZ), ketoconazole (KTZ), and quinidine (QDN) were obtained from Sigma (St. Louis, MO). All solvents and buffers were obtained from commercial sources and used without further purification.

[0197] method: The test compounds were prepared as 10 mM stock solutions in acetonitrile. Four human P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) expressed via cDNA in insect cell microsomes were tested for inhibition by the test compounds using a fluorescence-based assay. Nine serial dilutions (concentrations from 0 to 100 μM) using each test compound stock solution were prepared in two sets on black microtiter plates. This dilution series was incubated at 37°C with the individual CYP isoforms and standard fluorescence-generating probe substrates for each isoform. The concentration of the added probe substrates was at or near the Km value for each CYP isoform. The reaction mixture contained potassium phosphate buffer, pH 7.4, and an NADPH-regeneration system. The final reaction volume was 0.20 mL, and after an appropriate incubation time (15–45 minutes), 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. Replication of control wells without the test compound, replication of blank wells containing stop solution before isoform addition, and replication of dilution series containing standard inhibitors for each isoform were also performed. IC50 values ​​were calculated using a four-parameter logistic model (dose-response equation) fitted with XLFit 5.2 from IDBS Software (Emeryville, CA), supported by linear interpolation of data points at concentrations indicating inhibition levels of approximately 50% of the non-inhibition rate, and supported by nonlinear regression of the data.

[0198] Example 40: Plasma protein binding assay Plasma protein binding (PPB) for compound analysis of compound (±)-44 in PBS (pH 7.4) was performed using Eurofins with equilibrium dialysis of plasma equipped with HPLC-UV / Vis detection.

[0199] Mean plasma protein binding of control propranolol in human, rat (Sprague Dawley), mouse (CD-1), and dog (Beagle) plasma.

[0200] The binding and recovery rates were calculated using the peak areas of the test compound in the buffer and test samples, according to the following formula.

number

[0201] Example 41: Metabolic Stability Microsomal metabolic stability Metabolic stability determinations for novel compounds and testosterone (positive control) were performed in the presence of human, rat, mouse, and monkey liver microsomes. The values ​​shown represent the percentage of parental cells remaining after 30 minutes of incubation. All measurements were repeated. Testosterone assay results were within acceptable limits.

[0202] Microsomal metabolic clearance Mixed male and female 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. The reaction mixture, excluding NADPH, was prepared as shown below. The test samples were added to the reaction mixture at a final concentration of 1 μM. The control compound, testosterone, was carried out concurrently with the test samples in a separate reaction. Aliquots (without coenzyme) of the reaction mixture were equilibrated in a 37°C shaking bath for 3 minutes. The reaction was initiated by the addition of coenzyme, and the mixture was incubated in a 37°C shaking bath. Aliquots (100 μL) were taken at time points 0, 10, 20, 30, and 60. The test substance and testosterone samples were immediately combined with 400 μL of ice-cold 50 / 50 acetonitrile (ACN) / H2O containing 0.1% formic acid and an internal standard to terminate the reaction. The samples were then mixed and centrifuged to precipitate the proteins. All samples were assayed by LC-MS / MS using electrospray ionization. The remaining percentage at each time point was determined by comparing the peak area response ratio (PARR) relative to the internal standard with the PARR at time 0. Half-lives were calculated using GraphPad software and fitted to a monophase exponential decay equation. [Table 2]

[0203] Example 42: In VivoPK assay Information and data from mouse PK research The test substance was administered as a single dose to drug-free adult male CD-1 mice via intravenous (IV) or forced oral (PO) administration.

[0204] Testing facilities and laboratories: Absorption Systems, LLC, 436 Creamery Way, Suite 600, Exton, PA 19341-2556

[0205] Test Article and Vehicle Information: Vehicle for IV administration: 3% DMA / 45% PEG300 / 12% ethanol / 40% sterile water Vehicle for PO administration: 2% Tween 80 in 0.9% physiological saline Dosing Formulation: Dosing formulations were prepared by stepwise addition of each vehicle component (in the order described) to a weighed quantity of test compound that would provide the desired final concentration. Each formulation was prepared by mixing the weighed amount of test compound with an appropriate volume of vehicle. Dose Solution Analysis: Dose solutions were analyzed by LC-MS / MS. Dose solutions were diluted with mouse blood and analyzed in triplicate. All concentrations are expressed as mg / mL of free base. The nominal dose level was used in all calculations for Group 1.

[0206] Test System: Species and Strain: Mouse; Male CD-1 Average Weight: 0.034 kg for the IV arm; 0.027 kg for the PO arm Number: 6 animals in total (3 animals per dose group (Group 1 (IV) and Group 2 (PO)), per study)

[0207] Compliance: This nonclinical study was conducted in accordance with Absorption Systems' established practices and standard operating procedures, and the study protocol. This study was exploratory in nature and was not conducted in accordance with the principles set forth in the Good Laboratory Practice (GLP) Regulations, 21 Code of Federal Regulations (CFR) Part 58 of the United States Food and Drug Administration (FDA). The report is stored in a valid scientific data management system. Electronic signatures comply with 21 CFR Part 11.

[0208] Experimental Design: Blood samples were collected from mice before administration and at 5, 15, and 30 minutes, and 1, 2, 4, 8, 24, and 48 hours after administration. Hemolyzed blood samples were extracted by protein precipitation with acetonitrile. After protein extraction with acetonitrile, compound levels were measured by LC-MS / MS. Pharmacokinetic parameters were calculated from the time course of blood concentration. Pharmacokinetic parameters were determined using a non-compartment model with Phoenix WinNonlin (v8.0) software. The maximum blood concentration (C0) after IV administration was estimated by extrapolating the first two time points back to t=0. The maximum blood concentration (Cmax) and time to reach the maximum blood concentration (tmax) after PO administration were observed from the data. The area under the time-concentration curve (AUC) was calculated using the linear trapezoidal rule, which involved calculating up to the last quantifiable data point and extrapolating to infinity where applicable. The blood half-life (t1 / 2) was calculated from the slope of the terminal elimination phase (0.693). The mean residence time (MRT) was calculated by dividing the area under the moment curve (AUMC) by the AUC. Clearance (CL) was calculated from dose / AUC. The steady-state volume of distribution (Vss) was calculated from CL*MRT. Bioavailability was determined by dividing the individual dose-normalized PO AUC∞ value by the average dose-normalized IV AUC∞ value. Samples below the limit of quantification (1.00 ng / mL) were treated as zero in the pharmacokinetic data analysis. [Table 3]

[0209] Example 43: Serum RBP4 Measurement Assay Blood samples were collected from the tail vein. Whole blood was placed in a centrifuge tube, allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 2000g for 15 minutes at 48°C to collect serum. Aliquots of plasma samples collected in the mouse pharmacokinetic study were analyzed for RBP4 concentration using the RBP4 (mouse / rat) dual ELISA kit (AdipoGen, San Diego, CA) according to the manufacturer's instructions. In the adi-hRBP4 transgenic mouse experiment, blood samples were collected from the tail vein. Whole blood was placed in a centrifuge tube, allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 2000g for 15 minutes at +4°C to collect serum. Mouse serum RBP4 (mainly produced in the liver) was measured using the RBP4 (mouse / rat) dual ELISA kit (AdipoGen, San Diego, CA; catalog number AG-45A-0012YTP-KI01).

[0210] Statement on Animal Care and Use: All procedures in this protocol comply with the U.S. Department of Agriculture's (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3); the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, DC, 1996; and the National Institutes of Health, Office of Laboratory Animal Welfare. Wherever possible, the procedures in this study are designed to avoid or minimize discomfort, distress, and pain to the animals.

[0211] Example 44. Effect of (±)-44 on the accumulation of N-retinylidene-N-retinylethanolamine (A2E) in the eyes of Abca4- / - mice. (±)-44 was incorporated into the Picolab 5053 diet to allow for a daily dose of 25 mg / kg. Abca4- / - mice were administered the compound in the diet for 12 weeks. A control group of age-matched wild-type 129S1 / SvLmJ mice was maintained on standard Picolab 5053 samples. Age-matched reference group mice were used to define baseline A2E levels in mice without Abca4 excision. Blood samples for assessing RBP4 serum levels were collected from (±)-44-treated and control-diet-treated Abca4- / - mice before administration and after 12 weeks of treatment. After 12 weeks of treatment, optic cups from treated and untreated Abca4- / - mice, as well as from reference wild-type mice, were collected for quantitative A2E analysis.

[0212] Example 45. Effect of (±)-44 on the accumulation of N-retinylidene-N-retinylethanolamine (A2E) in the eyes of double knockout Abca4- / - / Rdh8- / - mice. (±)-44 was incorporated into the Picolab 5053 diet to allow for a daily dose of 25 mg / kg. Abca4- / - / Rdh8- / - mice were given a 10-week long-term administration of the compound incorporated into the diet. A control group of age-matched wild-type C57BL / 6J mice was maintained with standard Picolab 5053 samples. Age-matched reference group mice were used to define baseline levels of A2E in mice without Abca4 and Rdh8 excision. Blood samples to assess serum RBP4 levels were collected from (±)-44-treated and control-diet-treated Abca4- / - mice before administration and after 10 weeks of administration. After 10 weeks of administration, optic cups from treated and untreated Abca4- / - / Rdh8- / - mice, as well as from reference wild-type mice, were collected for quantitative A2E analysis.

[0213] Examples 46(±)-44 conferred partial conservation of photoreceptor cells in a double knockout Abca4- / - / Rdh8- / - mouse model. (±)-44 was incorporated into the Picolab 5053 diet to allow for a daily dose of 25 mg / kg. Abca4- / - / Rdh8- / - mice were given a 10-week long-term administration of the compound incorporated into the diet. A control group of age-matched wild-type C57BL / 6J mice was maintained on standard Picolab 5053 samples. Ten weeks after administration, the entire eye was harvested and fixed with 2% glutaraldehyde / 4% paraformaldehyde. The eye was embedded in paraffin and incised to a thickness of 8 μm. Sections were counterstained with hematoxylin and eosin (H&E). Morphological observation and light microscopy were performed. The thickness of the outer nucleus layer (ONL) was measured vertically along the vertical meridian to the end of the optic nerve head at 200 μm intervals using a digital image processing system. The ONL area was calculated by multiplying the sum of the ONL thicknesses of the upper and lower retina by the measurement interval. Protection of photoreceptors is evident at multiple points in the upper retina (Figure 11).

[0214] Discussion Here, we describe a novel class of non-retinoid bispecific compounds capable of exhibiting dual RBP4 antagonist and kinetic stabilization activity of the TTR tetramer. The compounds were evaluated in three assays designed to measure the compound's functional antagonist efficacy for disruption of the holo-RBP4-TTR complex: (1) compound binding affinity to non-TTR-associated RBP4 (scintillation proximity assay, SPA), (2) compound binding affinity to the non-ligand TTR tetramer (fluorescence polarization assay, FP), and (3) holo-RBP4-TTR complex (homogeneous time-resolved fluorescence assay, HTRF). The results are shown in Table 1. Compound (±)-44 exhibited lower potency in RBP4 SPA binding affinity and RBP4-TTR HTRF functional antagonist activity compared to benchmark 8 ((±)-44 RBP4 SPA IC50 = 80.0 nM; RBP4-TTR HTRF IC50 = 0.25 μM), but (±)-44 showed an attractive balance of dual activity for both targets ((±)-44 TTR FP IC50 = 2.85 μM). The R-enantiomer of (±)-44 showed approximately twofold enantioprecision in RBP4 SPA binding affinity ((R)-50 RBP4 SPA IC50 = 65.0 nM; (S)-56 RBP4 SPA IC50 = 150.0 nM), but there was no clear distinction between the enantiomers in terms of RBP4-TR HTRF or TTR FP activity.

[0215] Compound (±)-44 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 (Table 2). Plasma protein binding (PPB) data showed a low proportion of unbound compounds (Table 2). Furthermore, (±)-44 lacked restriction inhibitory activity in a standard CYP panel (Table 2). Importantly, unlike previously reported analog 8, which showed incidental PPARγ agonist activity, (±)-44 was found to lack PPARγ agonist activity (Table 2).

[0216] Compound (±)-44 showed very low plasma clearance (0.0499 L / hour / kg) and a half-life of 9.9 hours after single administration (2 mg / kg IV and 5 mg / kg PO) to CD-1 male mice (Table 3). The compound was well absorbed and slowly excreted from plasma after oral administration, with an observed Cmax of 3033 ng / ml and a corresponding Tmax of 0.83 hours (Table 3). Very high exposure (AUCINF was 52439 hours·ng / mL) was observed, with an estimated %F of 52%.

[0217] Following a single oral administration of (±)-44 at 25 mg / kg, a maximum decrease of 81% in serum RBP4 was observed 6 hours after dose administration (Figure 5, A). There was no effect of compound administration on serum TTR levels (data not shown). The in vivo kinetics of serum RBP4 reduction demonstrated a general correlation between the presence of (±)-44 in circulation after oral administration (Figure 5, B) and the decrease in serum RBP4 (Figure 5, A). The magnitude of the maximum RBP4 reduction (81%) and the duration of the RBP4 reduction effect (64% reduction at 24 hours) correlated well with the compound's pharmacokinetic characteristics, such as high Cmax, long exposure time, and slow clearance (Table 3).

[0218] A very good correlation has previously been established between the ability of different classes of RBP4 antagonists to induce serum RBP4 reduction and their preclinical effects in an Abca4- / - mouse model of enhanced retinal lipofuscin development (Radu, RA et al. 2005; Dobri, N. et al. 2013; Racz, B. et al. 2018). Based on its excellent RBP4-reducing activity, it seems reasonable to expect that (±)-44 is effective in reducing the formation of cytotoxic lipofuscin retinoids in the retina.

[0219] Inhibition of acid-induced TTR aggregation in vitro is a well-established approach to characterizing TTR kinetic stabilizers (Petrassi, HM et al. 2005; Green, NS et al. 2005). Incubation of TTR at 37°C under acidic conditions for a prolonged period of 72 hours leads to tetramer destabilization and dissociation, followed by partial monomer denaturation and misassociation with amyloid fibrils and other high molecular weight aggregates (Hurshman, AR et al. 2004). Compound (±)-44 was evaluated as a kinetic TTR stabilizer by its ability to prevent acid-mediated TTR aggregate formation, using modifications of previously published protocols and with tafamidis and benzbromarone as positive controls (Klabunde, T. et al. 2000; Niemietz, C. et al. 2018). Tafamidis is a potent TTR kinetic stabilizer approved for the treatment of familial amyloid polyneuropathy, while benzbromarone, a uremic drug, was found in our previous experiments to be a potent TTR ligand with an IC50 of 293 nM in the FP TTR binding assay, equivalent to the reported potency of tafamidis in this assay (Penchala, SC et al. 2013). Incubation with DMSO at pH 4.0 for 72 hours significantly increased the high molecular weight morphology of TTR, but no such morphology was observed after incubation for a similar period at neutral pH (Figure 6, A). Similar to the activity of the two potent TTR ligands, tafamidis and benzbromarone, (±)-44 significantly reduced the formation of high molecular weight TTR species (Figure 6, A), indicating its potential to function as a TTR kinetic stabilizer. The higher intensity of the TTR monomer band in samples treated with tafamidis, benzbromarone, and (±)-44 compared to DMSO reflects a corresponding reduction in TTR aggregation induced by tafamidis, benzbromarone, and (±)-44.Quantitative analysis of band intensity revealed that the formation of high molecular weight aggregates induced by tafamidis, benzbromarone, and (±)-44 was reduced by 3.6, 5.6, and 4.7 times, respectively (Figure 6, B). A significant increase in TTR monomer band intensity, associated with the reduction in aggregate formation, was observed in samples treated with tafamidis, benzbromarone, and (±)-44 (Figure 6, C). Overall, the aggregation experiments established that the bispecific analog (±)-44 can act as a TTR kinetic stabilizer.

[0220] The Abca4- / - mouse model serves as an established model for evaluating the preclinical efficacy of compounds that inhibit bisretinoid formation (Petrukhin, K. 2013). Long-term (12-week) daily administration of 25 mg / kg of (±)-44 to Abca4- / - mice resulted in a 79% reduction in serum RBP4 at 12 weeks compared to baseline wild-type mice, and an 82% reduction compared to untreated Abca4- / - mice (Figure 7). After 12 weeks of administration, optic cups from treated and untreated Abca4- / - mice, as well as from reference wild-type mice, were collected for quantitative A2E analysis. This analysis revealed a statistically significant reduction in A2E of approximately 50% in (±)-44-treated Abca4- / - mice compared to control-feed-treated Abca4- / - mice (p<0.0001) (Figure 8).

[0221] Mice lacking both the Abca4 transporter and retinol dehydrogenase 8 (Rdh8) combine damage caused by enhanced bisretinoid accumulation with increased exposure to retinal dehydride (Maeda, A. et al. 2008). Long-term (10 weeks) daily administration of (±)-44 at 25 mg / kg to Abca4- / - / Rdh8- / - mice resulted in a 76% reduction in serum RBP4 compared to baseline at 10 weeks (Figure 9). Furthermore, compared to untreated Abca4- / - / Rdh8- / - mice, the reduction in serum RBP4 at 10 weeks was 84% ​​in (±)-44-treated Abca4- / - / Rdh8- / - mice. After 10 weeks of administration, analysis of the optic cups of treated Abca4- / - / Rdh8- / - mice and untreated Abca4- / - / Rdh8- / - mice, as well as reference wild-type mice, revealed a statistically significant 77% reduction in A2E (p<0.0001) in treated Abca4- / - / Rdh8- / - mice compared to control-feed-treated Abca4- / - / Rdh8- / - animals (Figure 10).

[0222] The Abca4- / - mouse model does not mimic certain key aspects of Stargardt disease and dry AMD, such as photoreceptor degeneration. In contrast, mice lacking both the Abca4 transporter and the enzyme retinol dehydrogenase 8 (Rdh8) develop severe photoreceptor degeneration, in addition to enhanced lipofuscin bisretinoid accumulation. (±)-44 was characterized for its ability to reduce photoreceptor degeneration in the Abca4- / - / Rdh8- / - mouse model. Long-term daily administration (10 weeks) of 25 mg / kg of (±)-44 to Abca4- / - / Rdh8- / - mice resulted in photoreceptor protection at multiple points in the upper retina (Figure 11).

[0223] The compounds described herein are shown to have properties useful for treating the above-mentioned indications.

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Claims

1. Compounds having the following structure, 【Chemistry 1】 [In the formula, X is either CR6 or N; R1, R2, R3, R4, and R6 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), -(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, -SO2R7, -NHSO2R7, -OC(O)R7, -SC(O)R7, -NHC(O)R8 or -NHC(S)R8, R7 is H, -(alkyl), -OH, -O(alkyl), -NH2, -NH(alkyl), or -N(alkyl)2. R8 is -(alkyl), -O-(alkyl), -NH2, -NH(alkyl), or -N(alkyl)2; Y is O, S, N, NH, or a bond; Z is O, S, N, NH, (CH2)O, or a bond; R5 is H, OH, halogen, alkyl, or R5 is (CH2)p and when Y is N, it bonds with Y to form a ring with Z; o and p are independently 0, 1, 2, or 3; m and n are independently 0, 1, 2, 3, or 4; A, C, and D are each independently N or CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); B and E are N, CR9, or CFG, where at least one of B or E is CFG; F is either absent or present, and if present, F is as follows: 【Chemistry 2】 G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Here, each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2] or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein m is 1 or 2, and n is 0, 1, or 2.

3. The compound according to claim 1 or 2, wherein m is 1 and n is 1.

4. The compound according to claim 1, 2, or 3, wherein Y and Z are each independently CH2, O, S, or NH.

5. The compound according to any one of claims 1 to 4, wherein Y is O and Z is CH2.

6. A and B are N, C and D are CR9, and E is CFG; or A, B, C, and D are CR9, and E is CFG; or, A is N, B is CFG, and C, D, and E are each CR9; or A is N, B, C, and D are CR9 respectively, and E is CFG; or, The compound according to any one of claims 1 to 5, wherein A, C, and D are each CR9, B is N, and E is CFG.

7. X is either CR6 or N; The compound according to any one of claims 1 to 6, wherein R1, R2, R3, R4, and R6 are each independently H, t-butyl, cyclopentyl, cyclohexyl, CF3, F, Cl, CN, or -OCH3.

8. The compound according to any one of claims 1 to 7, wherein R1 or R4 is CF3.

9. X is CR6, The compound according to any one of claims 1 to 8, wherein R1 is CF3, and R2, R3, R4, and R6 are each independently H, t-butyl, cyclopentyl, cyclohexyl, CF3, F, Cl, CN, or -OCH3.

10. The compound according to claim 1 or 2 having the following structure: 【Transformation 3】 【Chemistry 4】 [In the formula, C is CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); F is either absent or present, and if present, F is as follows: 【Transformation 5】 G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2] or a pharmaceutically acceptable salt thereof.

11. C is CR9, The compound according to claim 10, wherein R9 is H, -alkyl, -O(alkyl), or -NH(alkyl).

12. The compound according to any one of claims 10 or 11, wherein R9 is an alkyl group.

13. The compound according to any one of claims 10 to 12, having the following structure. 【Transformation 6】 [In the formula, F is a substituted or unsubstituted heteroaryl group.

14. F has the following structure: 【Transformation 7】 The compound according to claim 13, wherein R12 is H, -(alkyl), -(alkenyl), or -(alkynyl).

15. The compound according to claim 1 having the following structure: 【Transformation 8】 [In the formula, C is CR9; R9 is H, halogen, -OH, alkyl, cycloalkyl, cycloalkylalkyl, -O-(alkyl), -S-(alkyl), -NH2, -NH(alkyl), -NH(alkyl)2, -CO2H, -CO(O-alkyl); F is either absent or present, and if present, F is as follows: 【Chemistry 9】 G is H, substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterodicyclic, aryl, heteroaryl, alkyl, cycloalkyl, cycloalkylalkyl, CO2H, COOR10, OH, OR10, NH2, NHR10, NR10R11, SO2(alkyl), SO2(cycloalkyl), SO2(cycloalkylalkyl), CH2NHR10, CH2NR10R11, or CH2COOR10. Each R10 and R11 is independently H, alkyl, cycloalkyl, -C(O)-alkyl, -C(O)-cycloalkyl, -C(O)OH, -C(O)-O-alkyl, -C(O)-O-cycloalkyl, -C(O)NH2, -C(O)NH(alkyl), -C(O)NH(cycloalkyl), -C(O)N(alkyl)2, -CH2NH(alkyl), -CH2COOH, -SO2CH3, -OH, -O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2] or a pharmaceutically acceptable salt thereof.

16. C is CR9, and R9 is H, -alkyl, -O(alkyl), or -NH(alkyl); or, The compound according to claim 15, wherein R9 is an alkyl group.

17. The compound according to claim 1 having the following structure: 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 or a pharmaceutically acceptable salt of the compound.

18. The compound according to claim 1 having the following structure: 【Chemistry 15】 or a pharmaceutically acceptable salt of the compound.

19. The compound according to claim 1 having the following structure: 【Chemistry 16】 or a pharmaceutically acceptable salt of the compound.

20. The compound according to claim 1 having the following structure: 【Chemistry 17】 or a pharmaceutically acceptable salt of the compound.

21. The compound according to claim 1 having the following structure: [Chemistry 18] or a pharmaceutically acceptable salt of the compound.

22. The compound according to claim 1 having the following structure: 【Chemistry 19】 or a pharmaceutically acceptable salt of the compound.

23. A pharmaceutical composition comprising a compound according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

24. A method for stabilizing a TTR tetramer in a mammal, comprising administering to the mammal an amount effective in stabilizing the TTR tetramer of any one of claims 1 to 22 or the composition according to claim 23.

25. A method for treating a disease characterized by excessive lipofuscin accumulation in the retina, or TTR amyloidosis (ATTR) disease, or both a disease characterized by excessive lipofuscin and TTR amyloidosis (ATTR) disease, comprising administering an effective amount of the compound according to any one of claims 1 to 22 or the composition according to claim 23 to a mammal suffering from the disease.

26. The method according to claim 25, wherein the disease is further characterized by bisretinoid-mediated macular degeneration.

27. The method according to any one of claims 24 to 26, wherein the amount of the compound is effective in reducing the serum concentration of RBP4 in the mammal, or the amount of the compound is effective in reducing the retinal concentration of bisretinoids in lipofuscin in the mammal.

28. The method according to any one of claims 24 to 27, wherein the amount of the compound is effective in stabilizing the TTR tetramer in the mammal.

29. The method according to any one of claims 26 to 28, wherein the bisretinoid is A2E.

30. The method according to any one of claims 26 to 28, wherein the bisretinoid is isoA2E.

31. The method according to any one of claims 26 to 28, wherein the bisretinoid is A2-DHP-PE.

32. The method according to any one of claims 26 to 28, wherein the bisretinoid is atRAL di-PE.

33. The method according to any one of claims 25 to 32, wherein the disease characterized by the accumulation of excessive lipofuscin in the retina is age-related macular degeneration.

34. The method according to any one of claims 25 to 32, wherein the disease characterized by the accumulation of excessive lipofuscin in the retina is dry (atrophic) age-related macular degeneration.

35. The method according to any one of claims 25 to 32, wherein the disease characterized by the accumulation of excessive lipofuscin in the retina is Stargardt disease.

36. The method according to any one of claims 25 to 32, wherein the disease characterized by the excessive accumulation of lipofuscin in the retina is Best's disease.

37. The method according to any one of claims 25 to 32, wherein the disease characterized by the excessive accumulation of lipofuscin in the retina is adult vitiligo maculopathy.

38. The method according to any one of claims 25 to 32, wherein the disease characterized by the accumulation of excessive lipofuscin in the retina is Stargardt-like macular dystrophy.

39. The method according to any one of claims 24 to 38, wherein the administration is effective in reducing photoreceptor degeneration.

40. The method according to any one of claims 24 to 39, wherein the method is further effective in stabilizing the TTR tetramer in the mammal.

41. The method according to any one of claims 24 to 40, wherein the mammal is further suffering from TTR amyloidosis (ATTR) disease, and the method is effective in treating the TTR amyloidosis (ATTR) disease in the mammal.

42. The method according to claim 41, wherein the TTR amyloidosis (ATTR) disease is senile systemic amyloidosis (SSA).

43. The method according to claim 41, wherein the TTR amyloidosis (ATTR) disease is peripheral polyneuropathy (ATTR-PN).

44. The method according to claim 41, wherein the TTR amyloidosis (ATTR) disease is cardiomyopathy (ATTR-CM).

45. The method according to claim 41, wherein the TTR amyloidosis (ATTR) disease is characterized by the deposition of amyloid aggregates.

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