US20050261365A1

US20050261365A1 - Transthyretin stabilization

Info

Publication number: US20050261365A1
Application number: US11/134,527
Authority: US
Inventors: Jeffery Kelly; H. Petrassi
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2004-05-20
Filing date: 2005-05-20
Publication date: 2005-11-24
Also published as: EP1768968A1; JP2007538103A; CA2566688A1; ZA200610417B; US20050272704A1; US20080319175A1; EP2314584A1; WO2005118511A2; WO2005112913A1; CA2566923A1; KR20070032700A; US20110092545A1; BRPI0510142A; US8338459B2; BRPI0510033A; US20050282780A1; JP2007538095A; RU2371440C2; CN1976693A; WO2005118511A3

Abstract

Dibenzofuran-4,6-dicarboxylic acid core structures having an aromatic substituent appended onto the at the C1 position using three different types of linkages are disclosed herein and shown to afford exceptional amyloidogenesis inhibitors that display increased affinity and greatly increased binding selectivity to TTR over all the other plasma proteins, relative to lead compound 1. It is further disclosed herein that these compounds function by imposing kinetic stabilization on the TTR tetramer.

Description

TECHNICAL FIELD

The present invention relates to inhibitors of transthyretin amyloid fibril formation. More particularly, the invention relates to derivatized dibenzofurans as inhibitors of transthyretin amyloid fibril formation.

BACKGROUND

Several structurally distinct classes of small molecule transthyretin (TTR) stabilizers have been discovered, of which dibenzofuran-4,6-dicarboxylic acid (1) is particularly interesting, FIG. 1 (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Razavi, H.; et al. Angew Chem 2003, 42, 2758-2761; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. USA 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. USA 1998, 95, 12956-12960; Baures, P. W.; et al. Bioorg. Med. Chem. 1998, 6, 1389-1401; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; McCammon, M. G.; et al. Structure 2002, 10, 851-863; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Sacchettini, J. C.; et al. Nature Rev. Drug Disc. 2002, 1, 267-275; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Miller Sean, R.; et al. Lab. Inv. 2004, 84, 545-552). This inhibitor (1; 7.2 μM) decreases the extent of WT-TTR (3.6 μM) amyloid formation (pH 4.4) by 90% over a time course of 72 h. The X-ray co-crystal structure of TTR•1₂reveals that it exerts its impressive activity by binding exclusively to the outer portion of each thyroxine binding site (Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321). What is needed is to modify 1 with substructures that project into the inner cavity of the thyroxine binding pocket so as to increase their affinity and selectivity for binding to TTR in human blood.

SUMMARY

Dibenzofuran-4,6-dicarboxylic acid core structures having an aromatic substituent appended onto the dibenzofuran ring at the C1 position using three different types of linkages are disclosed herein and shown to afford exceptional amyloidogenesis inhibitors that display increased affinity and greatly increased binding selectivity to TTR over all the other plasma proteins, relative to lead compound 1 (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571). It is further disclosed herein that these compounds function by imposing kinetic stabilization on the TTR tetramer.
Transthyretin (TTR) amyloidogenesis requires rate limiting tetramer dissociation and partial monomer denaturation to produce a misassembly competent species. This process has been followed by turbidity to identify transthyretin amyloidogenesis inhibitors including dibenzofuran-4,6-dicarboxylic acid (1). An X-ray cocrystal structure of TTR•1₂reveals that it only utilizes the outer portion of the two thyroxine binding pockets to bind to and inhibit TTR amyloidogenesis. Herein, structure-based design was employed to append aryl substituents using three different chemical linkages at C1 of the dibenzofuran ring to complement the unused inner portion of the thyroxine binding pockets. Twenty-eight amyloidogenesis inhibitors of increased potency and dramatically increased plasma TTR binding selectivity resulted that function by imposing kinetic stabilization on the native tetrameric structure of TTR, creating a barrier that is insurmountable under physiological conditions. Since kinetic stabilization of the TTR native state by interallelic trans-suppression is known to ameliorate disease, there is reason to be optimistic that the dibenzofuran-based inhibitors will do the same. Preventing the onset of amyloidogenesis is the most conservative strategy to intervene clinically, as it remains unclear which of the TTR misassembly intermediates result in toxicity. The exceptional binding selectivity enables these inhibitors to occupy the thyroxine binding site(s) in a complex biological fluid like blood plasma, required for inhibition of amyloidogenesis in humans. It is now established that the dibenzofuran-based amyloidogenesis inhibitors have high selectivity, affinity, and efficacy.
One aspect of the invention is directed to a compound represented by Formula I:

In Formula I, X is absent or is a diradical selected from the group consisting of —O—, —S—, and —NH—; and R², R³, R⁴, and R⁵are radicals independently selected from the group consisting of —H, —OH, —F, —Cl, —Br, —CF₃, and —CO₂H. In a first subgenus of this first aspect of the invention, the compound is represented by Formula II:

Within the subgenus of Formula II, preferred embodiments may include species wherein R²is a radical selected from the group consisting of —H, —F, —Cl, and —CF₃; additional preferred embodiments may include species wherein R⁴is a radical selected from the group consisting of —H, —Cl, and —CO₂H; additional preferred embodiments may include species wherein R⁵is a radical selected from the group consisting of —H, —F, and —Cl. Preferred species of the subgenus of Formula II include compounds selected from the group represented by the following structures:
In a second subgenus of this first aspect of the invention, the compound is represented by Formula III:

Within the subgenus of Formula III, preferred embodiments may include species wherein R³is a radical selected from the group consisting of —H, —F, —Cl, —Br, and —CF₃; additional preferred embodiments may include species wherein R⁵is a radical selected from the group consisting of —H, —F, —Cl, and —Br. Preferred species of the subgenus of Formula III include compounds selected from the group represented by the following structures:
In a third subgenus of this first aspect of the invention, the compound is represented by Formula IV:

Within the subgenus of Formula IV, preferred embodiments may include species wherein R²is a radical selected from the group consisting of —H, —F, and —Cl; additional preferred embodiments may include species wherein R³is a radical selected from the group consisting of —H, —F, —Cl, —CF₃, and —CO₂H; additional preferred embodiments may include species wherein R⁴is a radical selected from the group consisting of —H, and —CO₂H; additional preferred embodiments may include species wherein R⁵is a radical selected from the group consisting of —H, —F, —Cl, and —CF₃.
Preferred species of the subgenus of Formula IV include compounds selected from the group represented by the following structures:
A further aspect of the invention is directed to a process comprising the step of contacting transthyretin with a concentration of a compound selected from Formulas I-IV, described above, sufficient for inhibiting amyloid fibril formation.

BRIEF DESCRPTION OF DRAWINGS

FIG. 1A illustrates an X-ray crystallographic structure of TTR•1₂(Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321).
FIG. 1B illustrates a line drawing representation of the design of the 1-substituted-dibenzofuran-4,6-dicarboxylic acids placed in the thyroxine binding pocket where X represents either an NH, O or direct C_aryl-C_aryllinkage. R represents the substituents of the aryl ring designed to complement TTR's inner binding cavity.
FIG. 2 illustrates a table highlighting the concentration dependent acid-substituted dibenzofuran activity against WT-TTR (3.6 μM) amyloid fibril formation (f.f.) at pH 4.4 (72 h).
FIG. 3 illustrates a chart showing a summary of dibenzofuran-based amyloid inhibition activity (3.6 μM) against WT-TTR (3.6 μM) fibril formation (pH 4.4, 72 h) and binding stoichiometry to TTR in human blood plasma.
FIG. 4 illustrates a scheme for the synthesis of 1-hydroxy-dibenzofuran-4,6-dicarboxylate dimethyl ester and the corresponding triflate.
FIG. 5 illustrates a scheme for the synthesis of 1-phenyl-, phenoxy-, and phenylamine-dibenzofuran-4,6-dicarboxylate dimethyl esters and the corresponding dicarboxylates.
FIG. 6 illustrates a chart showing dibenzofuran-based inhibitor activity (7.2 μM) against WT-TTR (3.6 μM) amyloid fibril formation (f.f.) at pH 4.4 (72 h).
FIG. 7 illustrates a table illustrating dibenzofuran plasma TTR binding stoichiometry plotted vs. fibril formation inhibition efficacy.
FIG. 8 illustrates a plot of the absorbance at 280 nm versus distance from the center in the sedimentation velocity study on TTR (3.6 μM) after being preincubated with 27 (7.2 μM) and after another incubation period where the pH was dropped to 4.4 for 72 h, a time frame that results in maximal amyloid formation in the absence of inhibitor.
FIG. 9 illustrates a plot of the absorbance at 280 nm versus distance from the center in the equilibrium ultracentrifugation studies on TTR (3.6 μM) after being preincubated with 27 (7.2 μM) and after another incubation period where the pH was dropped to 4.4 for 72 h, a time frame that results in maximal amyloid formation in the absence of inhibitor.
FIG. 10 illustrates a plot of the timecourse analysis of WT-TTR (3.6 μM) fibril formation mediated by partial acid denaturation in the absence (▴) and presence of 7.2 μM (⋄) and 3.6 μM (◯) inhibitors 25, 47, and 64, as measured by turbidity at 500 nm (see shading scheme within Figure to differentiate inhibitors).
FIG. 11 illustrates a plot of the timecourse analysis of WT-TTR (3.6 μM) tetramer dissociation (6.0 M urea) in the absence (▴) and presence of 7.2 μM (⋄) and 3.6 μM (◯) concentrations of inhibitors 25, 47, and 64 (see color scheme within Figure to differentiate inhibitors).

DETAILED DESCRIPTION

All but one of the C1-aryl substituted dibenzofurans (7.2 μM) are exceptional inhibitors of WT-TTR (3.6 μM) acid-mediated fibril formation in vitro (pH 4.4, 37° C.), even those bearing unsubstituted aryl rings (FIG. 6). The only dibenzofuran-based inhibitor that was not as effective against TTR amyloidogenesis was 34, bearing potentially four negative charges. Because all the compounds completely inhibited TTR fibril formation (within the ±5% experimental error), no structure-activity relationships (SAR) are deducible from the 7.2 μM inhibitor data. However, FIGS. 3 and 7 reveal a range of inhibitor efficacy at an inhibitor concentration equal to that of TTR (3.6 μM), allowing some SAR conclusions to be drawn, limited by the 31 analogs prepared and the experimental error. Notably, all the inhibitors (except 34) display increased potency relative to the parent compound (1) at 3.6 μM (FIG. 3). Most importantly, of the thirty inhibitors exhibiting increased potency, all but two (31 and 35) exhibit dramatically increased binding selectivity to TTR in human blood plasma. The exceptional binding selectivity exhibited by the C1-substituted inhibitors is ideal for inhibiting TTR amyloidogenesis in complex biological systems.
Comparing the four C1-aryl substitution patterns (H, 3-CF₃, 3,5-F₂, and 3,5-Cl₂) found in all three inhibitor series reveals that the inhibitors having their aryl rings directly linked to C1 of the dibenzofuran skeleton, hereafter referred to as the biaryls, display slightly increased inhibitor potency relative to their biarylamine and biarylether counterparts (FIG. 3). This may be due to structural differences that enable the rings to be oriented differently within the inner binding cavity; however, we caution that this preference may not hold in a very large analog series. It could be argued with the same qualifiers that the other two series produce the most selective TTR binders in plasma; however, the SAR here is confounded by the fact that as many as one hundred proteins are competing for these inhibitors. While it is not surprising that the most potent inhibitors have 2-F or 3,5-Cl₂substituents (36, 63, and 67), likely picking up the halogen binding pockets in the thyroxine binding site, it is somewhat surprising that the 3-CO₂H substituted aryl inhibitors 33 and 69 are amongst the most potent (although their plasma binding selectivity to TTR is modest). Previous crystallographic results on simple biphenyl and biphenylamine inhibitors demonstrate that it can be preferable to have the carboxyl-bearing aryl ring in the inner binding cavity (enabling H-bonding to S117 and T119) (Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374).
Appending aryl groups to the C1 position not only increases inhibitor potency, but more importantly dramatically increases plasma binding selectivity to TTR, presumably by increasing binding affinity for TTR over the other plasma proteins. The superior binding selectivity of the C1-aryl substituted dibenzofuran-based inhibitors to TTR in plasma is clearly demonstrated by the fact that ˜⅔ of the compounds prepared display a TTR binding stoichiometry greater than one. This is exceedingly interesting as the screening hit 1, utilizing only the outer cavity of TTR for binding, displays no measurable binding selectivity to TTR in plasma. Previous experience with amyloidogenesis inhibitors of diverse chemical structure reveals that very few members display binding stoichiometries exceeding 1 (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Razavi, H.; et al. Angew. Chem. 2003, 42, 2758-2761; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. USA 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. USA 1998, 95, 12956-12960; Baures, P. W.; Peterson, S. A.; Kelly, J. W. Bioorg. Med. Chem. 1998, 6, 1389-1401; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; McCammon, M. G.; et al. Structure 2002, 10, 851-863; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Sacchettini, J. C.; Kelly, J. W. Nature Rev. Drug Disc. 2002, 1, 267-275; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Miller S. R.; et al. Lab. Inv. 2004, 84, 545-552). The area of FIG. 7 shaded in gray contains dibenzofuran-based compounds that meet the criteria for high in vitro activity (<40% fibril formation) and high binding selectivity (>1 equiv bound to TTR in plasma). The most important point is that the activity and binding selectivity of almost all of the dibenzofuran-based inhibitors, especially those in the gray box (FIG. 7), are sufficient to kinetically stabilize TTR in plasma should they display desirable oral bioavailability, pharmacokinetic, and toxicity profiles.
It is not surprising that inhibitor efficacy in vitro (3.6 μM) and inhibitor binding selectivity (10.8 μM) to TTR in plasma do not correlate (FIG. 7). Compounds that exhibit superior binding selectivity to TTR over all of the other plasma proteins should be excellent inhibitors of fibril formation. However, the converse is not necessarily true: excellent inhibitors need not display high TTR plasma binding selectivity. Excellent inhibitors that display high TTR plasma binding selectivity are the most useful compounds in humans because these can selectively stabilize the TTR native state over the dissociative transition state and impart kinetic stabilization on TTR in a protein-rich biological fluid. Their binding constants to TTR are important because the extent of kinetic stabilization is proportional to the binding constants. However, focusing on binding constants and potency in vitro can be misleading because compounds can be excellent TTR amyloidogenesis inhibitors in vitro, but bind to other plasma proteins and therefore be rendered useless in humans (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571). Potent in vitro inhibitors not displaying good binding selectivity to TTR likely interact with other plasma proteins, such as albumin (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571). Because the dibenzofuran inhibitors display unprecedented binding selectivity and inhibitor potency as a group relative to inhibitors characterized heretofore (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Razavi, H.; et al. Angew. Chem. 2003, 42, 2758-2761; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. USA 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. USA 1998, 95, 12956-12960; Baures, P. W.; Peterson, S. A.; Kelly, J. W. Bioorg. Med. Chem. 1998, 6, 1389-1401; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; McCammon, M. G.; et al. Structure 2002, 10, 851-863; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Sacchettini, J. C.; Kelly, J. W. Nature Rev. Drug Disc. 2002, 1, 267-275; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Miller S. R.; et al. Lab. Inv. 2004, 84, 545-552), these are ideal for further pharmacological evaluation. Because TTR is the tertiary carrier of T₄, more than 99% of its binding sites are unoccupied; therefore, inhibitor binding to TTR should not perturb T₄homeostasis.
The C1-substituted dibenzofuran-based TTR amyloidogenesis inhibitors are promising because of their amyloid inhibition potency in vitro, their superb binding selectivity to TTR in plasma, their slow TTR dissociation rates (which must be the case to see high plasma selectivity by the method utilized herein), their ability to impose kinetic stabilization upon the TTR tetramer, their chemical stability in plasma, and their chemical stability at low pH (making them excellent candidates for oral administration). These inhibitors are useful for the treatment of TTR amyloid diseases, including SSA, FAP, and FAC, because kinetic stabilization of TTR is known to ameliorate FAP (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Coelho, T.; et al. J. Rheumatol. 1993, 20, 179-179; Coelho, T.; et al. Neuromuscular Disord. 1996, 6, 27-27).
Design and Synthesis:
FIG. 1A depicts the two symmetry equivalent binding modes of 1 (green and yellow) within one of the TTR thyroxine binding sites, the surface of which is outlined in gray (Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321). The carboxylates at the 4 and 6 positions make electrostatic interactions with the ε-NH₃ ⁺ groups of Lys 15 and 15′ at the entrance to the thyroxine binding site. Removal of one of the carboxylates renders dibenzofuran much less active, as does varying the carboxylate spacing from the aromatic ring in most cases (FIG. 2). In addition, the dibenzofuran ring nicely complements the shape and hydrophobicity of the outer portion of the thyroxine binding cavity. Inspection of the TTR•1₂crystal structure in FIG. 1A reveals that there is a large amount of unoccupied volume in the inner portion of the thyroxine binding site with 1 bound. Based on this structure, it is clear that a substituent, such as an aryl ring, could be projected into the inner portion of the binding site by attaching it to the C1 position of the dibenzofuran ring of 1. As shown in FIG. 1B, such a substituent could be linked to a dibenzofuran scaffold through a heteroatom (N or O) or directly via a C_aryl-C_arylbond (not shown). Aromatic substituents (FIG. 3) were chosen to interact with either the halogen binding pockets or hydrogen bonding substructures within the inner cavity based on the envisioned orientation of the phenyl ring in the inner binding cavity and previous SAR data from other chemical series thought to position their aryl rings similarly (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Razavi, H.; et al. Angew Chem 2003, 42, 2758-2761; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. USA 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. USA 1998, 95, 12956-12960; Baures, P. W.; et al. Bioorg. Med. Chem. 1998, 6, 1389-1401; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; McCammon, M. G.; et al. Structure 2002, 10, 851-863; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Sacchettini, J. C.; et al. Nature Rev. Drug Disc. 2002, 1, 267-275; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Miller Sean, R.; et al. Lab. Inv. 2004, 84, 545-552).
The synthesis of C1-substituted dibenzofuran-based inhibitors commenced with the radical phenolic homo-coupling of commercially available 2,4-ditertbutyl-6-bromophenol (2) to afford the dibenzofuran derivative 3 using potassium hexacyanoferrate (III) as previously reported (FIG. 4) (Tashiro, M. Y., et al. Synthesis 1980, 6, 495-496). This tetra-t-butyl dibenzofuran derivative was subjected to transalkylation in toluene to form 1-hydroxydibenzofuran (4) in 33% overall yield from 2 (alternative strategies for the synthesis of this intermediate have appeared) (Labiad, B.; Villemin, D. Synthesis 1989, 143-144; Lee, Y. R.; et al. Org. Lett. 2000, 2, 1387-1389). After protection of the phenol, silyl ether 5 was selectively ortho-metalated at the 4- and 6-positions with sec-butyllithium (the triisopropylsilyl group on the 1-oxygen precludes it from acting as a metalation director) (Snieckus, V. Chem. Rev. 1990, 90, 879-933). The dianion was quenched with gaseous CO₂and esterified to afford 6, which was then deprotected with TBAF and converted to triflate 8 in high overall yield.
Selected anilines were coupled to triflate 8 using a palladium mediated N-arylation reaction developed by Buchwald and Hartwig to afford dibenzofuran-based biarylamine analogues 9-23 (FIG. 5) (Louie, J. D., et al. J. Org. Chem. 1997, 62, 1268-1273; Wolfe, J. P. B., Stephen L. Tetrahedron Lett. 1997, 38, 6359-6362). To append an aryl ether to the C1-position of dibenzofuran, phenol 7 and several phenylboronic acids were cross-coupled using the copper-mediated biaryl ether coupling methodology of Chan and Evans, affording compounds 39-43 (Chan, D. M. T.; et al. Tetrahedron Lett. 1998, 39, 2933-2936; Evans, D. A. et al. Tetrahedron Lett. 1998, 39, 2933). Compound 8 was also subjected to Suzuki coupling conditions in the presence of several phenylboronic acids to afford the dibenzofuran-based biaryl analogues 49-59 (Suzuki, A. Modern Arene Chemistry 2002, 53-106). Saponification of the methyl esters in these precursors afforded the desired dibenzofuran-4,6-dicarboxylic acid amines (24-38), ethers (44-48), and biaryls (60-70) to be evaluated as potential TTR amyloidogenesis inhibitors.
Results:
Two of the most important characteristics of an effective small molecule amyloidogenesis inhibitor are that they must be able to bind with high affinity and selectively to TTR in blood and stabilize its native tetrameric quaternary structure (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Razavi, H.; et al. Angew Chem 2003, 42, 2758-2761; Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571). Inhibition efficacy (compounds 24-38, 44-48, and 60-70) was first evaluated using recombinant TTR in a partially denaturing buffer that promotes amyloidogenesis (pH 4.4, 37° C.). As a follow up, the ability of effective inhibitors to bind to TTR selectively over all the other proteins in human plasma was assessed.
Evaluating the Dibenzofuran-Based Compounds as Amyloidogenesis Inhibitors.
TTR amyloid inhibition efficacy was probed using a stagnant fibril formation assay described previously, wherein partial denaturation was triggered by acidification (pH 4.4, 37° C.) (Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654-8660). Briefly, a test compound (7.2 or 3.6 μM) is incubated with TTR (3.6 μM) for 30 min in pH 7 buffer. Amyloidogenesis is then initiated by lowering to pH 4.4, where maximal fibril formation is observed with WT-TTR after 72 h (37° C.). The turbidity in the presence of a potential inhibitor (T_test) is compared to that of a solution lacking a test compound (T_control) Exceptional inhibitors exhibit 0% fibril formation, whereas compounds not functioning as an inhibitor would exhibit 100% fibril formation. From experience we know that excellent inhibitors allow <10% fibril formation at a small molecule concentration of 7.2 μM and <40% fibril formation at a concentration equal to that of WT-TTR (3.6 μM) (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321; Razavi, H.; et al. Angew Chem 2003, 42, 2758-2761; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. USA 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. USA 1998, 95, 12956-12960; Baures, P. W.; et al. Bioorg. Med. Chem. 1998, 6, 1389-1401; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; McCammon, M. G.; et al. Structure 2002, 10, 851-863; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Saccheftini, J. C.; et al. Nature Rev. Drug Disc. 2002, 1, 267-275; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Miller Sean, R.; et al. Lab. Inv. 2004, 84, 545-552). Of the 31 compounds evaluated, all but one (34) completely inhibit fibril formation at a concentration twice that of TTR (7.2 μM inhibitor), FIG. 6 (at 7.2 μM there is enough test compound added to occupy both of the binding sites of TTR (TTR•I₂), provided their dissociation constants are both in the low nM range at pH 4.4). Small molecules typically bind to TTR with negative cooperativity, hence K_d1and K_d2are often are separated by one or two orders of magnitude. Therefore, when both the ligand and TTR are at equal concentrations, a population of TTR•I, TTR•I₂and unliganded TTR is observed, dictated by the dissociation constants. It is now established by other studies that inhibitor occupancy of only one of the two TTR binding sites is sufficient to stabilize the entire tetramer against amyloidogenesis (Wiseman, R. L.; et al. J. Am. Chem. Soc. 2005, in press). This is further supported by the observation herein that twenty-six of these dibenzofurans are excellent TTR amyloidogenesis inhibitors (<40% fibril formation) at a concentration equal to that of TTR (3.6 μM each, FIG. 3). Representative small molecules from all three series (25, 26, 27, 30, 45, 47, 62; 3.6 μM) were subjected to the acid-mediated amyloidogenic conditions (pH 4.4, 37° C., 72 h) in the absence of TTR, revealing no measurable precipitation.
Evaluating the Plasma TTR Binding Selectivity of the Dibenzofuran-Based Inhibitors.
Inhibitor binding selectivity to TTR in human blood plasma was assessed using a previously established antibody capture method (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571). In this evaluation, inhibitors (10.8 μM, ˜2-3× the natural concentration of TTR) are incubated in human blood plasma for 24 h at 37° C. Quenched sepharose resin is then added to the plasma to remove any small molecules that would bind to the resin as opposed to TTR. TTR and any TTR-bound small molecule is then immunocaptured using a polyclonal TTR antibody covalently attached to sepharose resin. After washing the resin (5×10 min washes), the antibody-TTR complex is dissociated at high pH and analyzed by RP-HPLC. The relative stoichiometry between TTR and inhibitor is then calculated from their HPLC peak areas using standard curves. Wash-associated losses are typically observed for inhibitors that have high dissociation rates; therefore, this analysis can underestimate their true binding stoichiometry, but gives faithful results for compounds exhibiting low dissociation constants and off-rates. Twenty-one inhibitors exhibit a binding stoichiometry exceeding one (two being the maximal binding stoichiometry), nineteen of which exhibit <40% fibril formation at a concentration of 3.6 μM (FIG. 7, shaded box). The high plasma TTR binding selectivities observed are remarkable considering the parent dibenzofuran-4,6-dicarboxylic acid (1) displays no binding selectivity to TTR, demonstrating the importance of the C1 aryl substituent in terms of endowing binding selectivity to TTR over all the other plasma proteins.
Stabilization of the Tetrameric Quaternary Structure Under Amyloidogenic Conditions.
To ensure that these C1-arylated dibenzofurans inhibit TTR fibril formation by native state stabilization (i.e. tetramer stabilization), we studied the TTR quaternary structure by analytical ultracentrifugation after a preincubation period of 72 h under amyloidogenic conditions (pH 4.4, 37° C.). In the presence of 27 (7.2 μM), TTR (3.6 μmM) was found to have hydrodynamic molecular weights of 57.1±0.3 and 55.1±0.4 kDa by sedimentation velocity (FIG. 8) and equilibrium analytical ultracentrifugation (FIG. 9), respectively, comparable to the expected molecular weight of tetrameric TTR (55.0 kDa). In the absence of 27, TTR aggregated into very high molecular weight oligomers that sedimented rapidly in the ultracentrifugation experiment (data not shown).
Do the Dibenzofuran-Based Inhibitors Impose Kinetic Stabilization on TTR?
The ability of these inhibitors to impose kinetic stability on tetrameric TTR is best evaluated by assessing the rate of TTR tetramer dissociation (Hammarstrom, P.; et al. Science 2003, 299, 713-716; Hammarstrom, P.; et al. Proc. Natl. Acad. Sci. USA 2002, 99, 16427-16432). Under acidic conditions tetramer dissociation leads to amyloidogenesis, whereas in the presence of chaotropes (6M urea), tetramer dissociation leads to monomer unfolding. The influence of inhibitors 25, 47 and 64, representing the three structural classes of dibenzofuran-based inhibitors, on the rates of tetramer dissociation under acid- and urea-mediated denaturing conditions was probed. TTR amyloidogenesis mediated by partial acidification is dramatically slowed in a dose-dependent fashion relative to control (no inhibitor) by 25, 47 and 64 (FIG. 4A). The rate of TTR tetramer dissociation in 6M urea is easily monitored by linking the slow quaternary structural changes to rapid tertiary structural changes that are easily monitored by spectroscopic methods (Hammarstrom, P.; et al. Proc. Natl. Acad. Sci. USA 2002, 99, 16427-16432). The rate of TTR tetramer (3.6 μM) dissociation monitored by far-UV CD is markedly slowed in a dose-dependent fashion in 6M urea by 25, 47 and 64. These results are consistent with differential stabilization of the ground state vs. the dissociative transition state by the binding of 25, 47, and 64.
Experimental:
The procedures used for bacterial expression of TTR (Lai, Z.; et al. Biochemistry 1996, 35, 6470-6482), the stagnant fibril formation assay (Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654-8660; Lai, Z.; et al. Biochemistry 1996, 35, 6470-6482), the blood plasma binding selectivity assay (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. USA 2001, 98, 5566-5571), and analytical ultracentrifugation (Lashuel, H. A.; et al. Biochemistry 1998, 37, 17851-17864) have all been described in detail previously.
Time course analysis of WT-TTR fibril formation inhibition by compounds 25, 47, and 64. Compounds 25, 47, and 64 were dissolved in DMSO to provide 7.2 mM primary stock solutions (10× stocks), from which 5- and 10-fold DMSO dilutions yielded 1.44 mM (2×) and 0.72 mM (1×) secondary stock solutions, respectively. 495 μL of 0.4 mg/mL (7.2 μM) WT-TTR solution (10 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.2), and 5 μL of either the 1.44 or 0.72 mM inhibitor secondary stock solutions were added to disposable UV cuvettes, vortexed briefly, then incubated for 30 min at 25° C. The pH was then adjusted to 4.4 with addition of 500 μL of acidic buffer (100 mM acetate, 100 mM KCl, 1 mM EDTA, pH 4.2), and the final 1 mL solutions were vortexed again and incubated in the dark at 37° C. without agitation. At 0, 4, 8, 12, 25, 49, 74, 100, 122, 145, and 169 h time points after acidification, the solutions were vortexed and the turbidity at 500 nm was measured. Control samples containing 5 μL of pure DMSO were prepared and analyzed as above for comparison. Small molecule and TTR control samples were prepared in groups of 10 to prevent disturbance of the cuvettes during incubation. Samples were discarded after their turbidities were measured.
Time course analysis of WT-TTR tetramer dissociation inhibition by compounds 25, 47, and 64 evaluated by linked-monomer unfolding in urea. Compounds 25, 47, and 64 were dissolved in DMSO to provide 10 mM primary stock solutions, from which 10-fold EtOH dilutions yielded 1 mM secondary stock solutions. 200 μL of 1.0 mg/mL (18 μM) WT-TTR solution (50 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.2), and either 7.2 or 3.6 μL (2× and 1×, respectively) of 1 mM inhibitor secondary stock solutions were added to 2 mL eppendorf tubes, vortexed briefly, and incubated for 15 min at 25° C. 100 μL of the TTR•inhibitor solutions were added to 900 μL of urea buffer (6.67 M urea, 50 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH 7.2), and the final 1 mL solutions were vortexed again and incubated in the dark at 25° C. without agitation. At 0, 4, 11, 24, 49, 73, 97, 122, 146, and 170 h time points after mixing with urea, the circular dichroism spectra were measured between 218 and 215 nm, with scanning every 0.5 nm and averaging for 5 s. After measurements were taken, samples were returned to their respective eppendorf tubes and incubation was continued. Control samples containing 7.2 μL of 10% DMSO in EtOH were prepared and analyzed as above for comparison. The CD amplitude values were averaged between 215 and 218 nm to determine the extent of β-sheet loss throughout the experiment. TTR tetramer dissociation is linked to the rapid (˜500,000× faster) monomer denaturation as measured through this β-sheet loss (Hammarstrom, P.; et al. Proc. Natl. Acad. Sci. USA 2002, 99, 16427-16432).
Inhibitor Synthesis: Reagents for chemical synthesis were purchased from commercial suppliers and used without further purification unless otherwise stated. Thin-layer chromatography on silica gel 60 F₂₅₄coated aluminum plates (EM Sciences) or analytical reverse phase high performance liquid chromatography (HPLC) were used to monitor reaction progress. HPLC was performed using a Waters 600E multisolvent delivery system employing a Waters 486 tunable absorbance detector and a Waters 717 plus auto sampler. A C18 Western Analytical column was used (model 033-715, 150 Å pore size, 3 μm particles) for all reverse phase HPLC analyses. An acetonitrile/water/trifluoroacetic acid solvent system was used; solvent A in the proportions of 4.8%, 95%, and 0.2%, respectively, while solvent B was of 95%, 4.8%, and 0.2%, respectively. Following 2 min of isocratic flow at 100% A, a linear gradient of 0 to 100% B over 8 min was run at 1.5 mL/min. All flash chromatography was accomplished using 230-400 mesh silica gel 60 (EM Sciences). ¹H- and ¹³C-NMR spectra were recorded at 300, 400, 500 or 600 MHz on Bruker spectrometers. Chemical shifts are reported in parts per million downfield from the internal standard (Me₄Si, 0.0 ppm).
(Dibenzofuran-1-yloxy)-triisopropyl-silane (5). To a dry 250 mL round bottom flask was added phenol 4 (Tashiro, M. Y., et al. Synthesis 1980, 6, 495-496) (492 mg, 2.67 mmol) and a stir bar and the flask was capped with a septum. CH₂Cl₂(5 mL) was added followed by DMAP (391 mg, 3.2 mmol) and triisopropylsilyl chloride (800 μL, 3.73 mmol). The resulting colorless solution became a white suspension overnight. The reaction was transferred to a 250 mL separatory funnel and washed with H₂O (3×10 mL). The aqueous layers were combined and extracted with CH₂Cl₂(3×30 mL). The organic layers were combined, dried with MgSO₄, and concentrated under reduced pressure to afford a pale yellow oil. The oil was purified by flash chromatography over silica (100% hexanes) to afford 0.70 g (77%) of 5 as a colorless oil. MALDI-FTMS 341.1932 m/z (M+H)⁺, C₂₁H₂₉O₂Si requires 341.1931.
1-Triisopropylsilanyloxy-dibenzofuran-4,6-dicarboxylic acid dimethyl ester (6). Silyl ether 5 (654 mg, 1.92 mmol) was added to a dry 50 mL round bottom flask followed by Et₂O (7.4 mL) and TMEDA (0.87 mL, 5.77 mmol). The flask was cooled to −78° C. in an acetone/CO₂(s) bath for 10 min before adding sec-butyl lithium (4.44 mL of a 1.3 M solution in cyclohexane, 5.77 mmol) over 10 minutes. The resulting orange suspension was allowed to warm to room temperature and stirred for 24 h. The flask was cooled again to −78° C. as described above and a 15 psi stream of CO₂(g) was bubbled through the reaction suspension (the CO₂was dried by passing it through a drying tube containing activated silica). Following initial addition of CO₂(g), the cooling bath was removed and the reaction was stirred for 30 min. The reaction mixture was poured into a 1 L beaker containing ice water (50 mL). The solution was brought to pH 9 by the slow addition of 0.05 M KOH, and then cooled to 0° C. with an ice/H₂O bath. The solution was acidified to pH 2 with 0.5 M HCl causing a white solid to precipitate. The aqueous suspension (pH 2) was transferred into a 1 L separatory funnel and extracted with EtOAc (5×50 mL). The combined extracts were dried with MgSO₄and concentrated under reduced pressure to afford the crude diacid as an oil. The 100 mL flask containing the crude diacid was equipped with a stir bar, capped with a septum and evacuated. The flask was then back-filled with argon. Anhydrous MeOH (2 mL) and ACS reagent grade benzene (8 mL) were added via syringe. Trimethylsilyidiazomethane (TMSCHN₂; 2.5 mL of a 2 M solution in hexanes, 5 mmol) was added slowly via syringe through the septum. Upon completion of the TMSCHN₂addition the reaction was stirred for 10 min and the solvent removed under reduced pressure to afford a red oil. The residue was purified by flash chromatography over silica (15% EtOAc in hexanes) to afford 0.36 g (43%) of 6 as a white solid. MALDI-FTMS 479.1874 m/z (M+Na)⁺, C₂₅H₃₂O₆SiNa requires 479.1860.
1-Hydroxy-dibenzofuran-4,6-dicarboxylic acid dimethyl ester (7). A dry 100 mL round bottom flask was equipped with a stir bar, charged with 6 (363 mg, 0.95 mmol), capped with a septum, evacuated, and back-filled with argon. Anhydrous THF (6.3 mL) and tetra-butylammonium fluoride (1 M in THF, 1.2 mL, 1.19 mmol) were added to the reaction by syringe. The reaction was stirred for 1 h at room temperature and then poured into 30 mL of H₂O in a 250 mL separatory funnel. The aqueous layer was extracted with CHCl₃(4×20 mL). The organic layers were combined, dried with MgSO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography over silica (30% EtOAc in hexanes) to afford 0.23 g (97%) of 7 as a white solid. LC-MS m/z 301, C₁₆H₁₂O₆requires 301.
1-Trifluoromethanesulfonyloxy-dibenzofuran-4,6-dicarboxylic acid dimethyl ester (8). The triflation procedure previously described by Stille was used to synthesize 8 (Echavarren, A. M.; et al. J. Am. Chem. Soc. 1987, 109, 5478-5486). Phenol 7 (120 mg, 0.4 mmol) was added to a dry 10 mL round bottom flask, which was then fitted with a septum. The solvent, anhydrous pyridine (2 mL), was added by syringe through the septum. The reaction mixture was cooled to 0° C. with an ice/H₂O bath. To initiate the reaction, trifluoromethanesulfonic anhydride (81 μL, 12 mmol) was added by syringe through the septum. The ice bath was removed and the reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture was poured into a 250 mL beaker containing 30 mL of an ice/H₂O slurry and transferred into a 125 mL separatory funnel. The aqueous layer was extracted with Et₂O (4×40 mL). The organic layers were combined, washed with saturated CuSO₄(4×20 mL) and brine (2×20 mL), dried over MgSO₄, and then the Et₂O was removed under reduced pressure to afford a slightly yellow solid. The solid was purified by flash chromatography over silica (30% EtOAc in hexanes) to afford 159 mg (92%) of 8 as a white solid. FAB-MS (NBA/NaI) m/z 433.0215 (M+H)⁺, C₁₇H₁₂F₃O₈S requires 433.0205.
Representative Procedure for the Palladium Catalyzed Cross Coupling of 8 with Substituted Anilines.
The aryl coupling procedure reported by Buchwald and Hartwig was used to prepare compounds 9-23. A flame dried 10 mm by 13 cm borosilicate test tube, equipped with a stir bar and capped with a septum, was charged with 8 (140 mg, 0.324 mmol), palladium dibenzylidene acetone, Pd₂(dba)₃(15 mg, 0.016 mmol), (±)-binap (15 mg, 0.024 mmol), Cs₂CO₃(147 mg, 0.456 mmol), and aniline (32 μL, 0.356 mmol). Upon addition of all reagents the tube was purged with argon for 10 min. Anhydrous toluene (2.4 mL) was then added through the septum and the reaction mixture was heated to 100° C. for 36 h in an oil bath. The reaction mixture was filtered through Celite, and the solvent was removed from the filtrate under reduced pressure. The resulting dark oil was purified by flash chromatography over silica (30% EtOAc in hexanes) to afford biaryl amine 17 as a white solid (0.12 g, 68%). Refer to the supporting information for specific synthetic details and characterization data for compounds 10-23 analogous to that reported for 9 below.

1-Phenylamino-dibenzofuran-4,6-dicarboxylic acid dimethyl ester (9).

MALDI-FTMS 375.1094 m/z (M⁻)⁺, C₂₂H₁₇NO₅requires 375.1106.
Representative Procedure for the Copper-Mediated Cross-Coupling of Phenol 7 with Substituted Phenylboronic Acids to Afford 1-Phenoxydibenzofurans 39-43.
The biaryl ether coupling was directly adapted from the procedures reported by Chan and Evans. A 20 mL scintillation vial equipped with a magnetic stir bar was charged with phenol 7 (150 mg, 0.50 mmol), copper (II) acetate (91 mg, 0.5 mmol), freshly activated 4 Å molecular sieves (˜250 mg), and phenylboronic acid (180 mg, 1.5 mmol). Dichloromethane (5 mL) was added followed by pyridine (201 μL, 2.5 mmol), resulting in an aqua colored suspension. The cap was very loosely applied such that the reaction suspension was partly open to the atmosphere. The reaction was monitored by TLC. After completion, the reaction mixture was adsorbed onto ˜6 g of silica gel, adding silica gel to the reaction mixture, then removing the solvent under reduced pressure. Chromatography (30% EtOAc in hexanes) of the reaction mixture over silica afforded biaryl ether 39 as a white solid (29 mg, 15%). Refer to the supporting information for specific synthetic details and characterization data for compounds 40-43 analogous to that reported for 39 below.

1-Phenoxy-dibenzofuran-4,6-dicarboxylic acid dimethyl ester (39)

MALDI-FTMS 399.0825 m/z (M+Na)⁺, C₂₂H₁₆O₆Na requires 399.0839.
Representative Procedure for the Palladium Catalyzed Cross-Coupling of Triflate 8 with Substituted Phenylboronic Acids.
A flame dried 10 mm by 13 cm test tube, equipped with a stir bar and capped with a septum, was charged with 8 (100 mg, 0.23 mmol), Pd(PPh₃)₄(14 mg, 0.01 mmol), LiCl (29 mg, 0.69 mmol), Na₂CO₃(300 μL of a 2 M aqueous solution) and toluene (3 mL). Phenylboronic acid (43 mg, 0.35 mmol) was dissolved in EtOH (0.5 mL) and added to the reaction mixture. MeOH replaced EtOH in this procedure for all other compounds because transesterification was observed; therefore compound 49 was isolated as the diethyl ester and all other compounds as dimethyl esters. After the reagents were added, the tube was purged with argon and the reaction mixture heated to 100° C. for 12 h in an oil bath. The reaction mixture was then filtered through Celite. The solvent was removed under reduced pressure from the filtrate and the resulting dark residue was purified by flash chromatography over silica to afford biaryl 49 as a white solid (52 mg, 63%). Refer to the supporting information for specific synthetic details and characterization data for compounds 50-59 analogous to that reported for 49 below.
1-Phenyl-dibenzofuran-4,6-dicarboxylic acid diethyl ester (49). MALDI-FTMS 411.1197 m/z (M+Na)⁺, C₂₄H₂₀O₅Na requires 411.1203.
Representative Procedure for Ester Hydrolysis to Afford Final Inhibitors 24-38, 44-48, and 60-70.
Methyl ester 9 (25 mg, 0.067 mmol) was saponified in THF: MeOH: H₂O (3:1:1, 1 mL) in a 20 mL scintillation vial equipped with a stir bar. LiOH.H₂O (22 mg, 0.53 mmol) was added to the suspension and the reaction was allowed to stir until completion (typically 4 h) as determined by TLC or analytical reverse phase HPLC monitoring. The reaction mixture was diluted with brine (2 mL) and acidified to pH 2 with 1 M HCl (pH paper) resulting in a biphasic solution. The upper layer (THF) was removed and the aqueous layer was extracted with THF (3×3 mL). The combined organic layers were dried with MgSO₄and then concentrated under reduced pressure to afford diacid 24 as a white solid (21 mg, 92%). Refer to the supporting information for specific synthetic details and characterization data for compounds 25-38, 44-48, and 60-70 analogous to that reported for 24 below.
1-Phenylamino-dibenzofuran-4,6-dicarboxylic acid (24). MALDI-FTMS 347.0794 m/z (M⁻)⁺, C₂₀H₁₃NO₅requires 347.0788.
FIG. 1A shows an X-ray crystallographic structure of TTR•1₂(Klabunde, T.; et al. Nature Struct. Biol. 2000, 7, 312-321). The residues lining the binding site are displayed as stick models (oxygen in red, nitrogen in blue, and carbon in gray), with the protein's Connolly surface depicted in gray. Compound 1 is shown in both of its C₂symmetry equivalent binding modes (yellow and green). The binding channel has 3 sets of depressions referred to as the halogen binding pockets (HBPs) because they interact with the iodines of thyroxine. Compound 1 occupies only the outer portion of the binding pocket and fills both HBP1 and 1′. The carboxylic acids of 1 are in proximity to the e-NH₃ ⁺ of K15 and K15′.
FIG. 1B shows a line drawing representation of the design of the 1-substituted-dibenzofuran-4,6-dicarboxylic acids placed in the thyroxine binding pocket where X represents either an NH, O or direct C_aryl-C_aryllinkage. R represents the substituents of the aryl ring designed to complement TTR's inner binding cavity.
FIG. 2 is a table highlighting the concentration dependent acid-substituted dibenzofuran activity against WT-TTR (3.6 μM) amyloid fibril formation (f.f.) at pH 4.4 (72 h). Values represent the extent of f.f. and thus inhibitor efficacy relative to WT-TTR fibril formation in the absence of inhibitor (assigned to be 100%): complete inhibition is equivalent to 0% f.f.
FIG. 3 is a chart showing a summary of dibenzofuran-based amyloid inhibition activity (3.6 mM) against WT-TTR (3.6 mM) fibril formation (pH 4.4, 72 h) and binding stoichiometry to TTR in human blood plasma. % Fibril formation (f.f.) values in the middle column represent the extent of f.f., and thus inhibitor efficacy, relative to WT-TTR f.f. in the absence of inhibitor (assigned to be 100%). Complete inhibition is equivalent to 0% f.f. The right column depicts the observed stoichiometry of inhibitor (dosed at 10.8 mM, ˜2-3× the concentration of plasma TTR) bound to TTR in blood plasma as determined using the antibody capture method.
FIG. 4 is a scheme for the synthesis of 1-hydroxy-dibenzofuran-4,6-dicarboxylate dimethyl ester and the corresponding triflate: a) K₃[Fe(CN)₆], KOH, H₂O, benzene; b) AlCl₃, toluene, 33% for both steps; c) TIPSCI, DMAP, CH₂Cl₂, 77%; d) sec-BuLi, Et₂O, −78° C., gaseous CO₂, TMSCHN₂, 43%; e) TBAF, THF, 97%; f) Tf₂O, pyridine, 92%.
FIG. 5 is a scheme for the synthesis of 1-phenyl-, phenoxy-, and phenylamine-dibenzofuran-4,6-dicarboxylate dimethyl esters and the corresponding dicarboxylates: a) Pd₂(DBA)₃, (±)binap, Cs₂CO₃, toluene 100° C.; b) LiOH—H₂O, THF/MeOH/H₂O (3:1:1); c) Cu^II(OAc)₂, pyridine, 4 Å MS, CH₂Cl₂; d) Pd(PPh₃)₄, LiCl, aq. Na₂CO₃, toluene, MeOH, 80° C.
FIG. 6 is a chart showing dibenzofuran-based inhibitor activity (7.2 μM) against WT-TTR (3.6 μM) amyloid fibril formation (f.f.) at pH 4.4 (72 h). Values represent the extent of f.f. and thus inhibitor efficacy relative to WT-TTR fibril formation in the absence of inhibitor (assigned to be 100%): complete inhibition is equivalent to 0% f.f.
FIG. 7 is a table illustrating dibenzofuran plasma TTR binding stoichiometry plotted vs. fibril formation inhibition efficacy. The lightly shaded area corresponds to the definitions of high activity and high selectivity (<40% fibril formation and a binding stoichiometry >1), while the darkly shaded area corresponds to exceptional compounds (<30% fibril formation and a binding stoichiometry >1.25). Data points identify the three different linkers: NH (▴), ◯ ( ), and direct C_aryl-C_aryllinkage (●). Dibenzofuran-4,6-dicarboxylic acid (1) data point (●) shown for comparison.
FIG. 8 is a plot of the absorbance at 280 nm versus distance from the center in the sedimentation velocity study on TTR (3.6 mM) after being preincubated with 27 (7.2 mM) and after another incubation period where the pH was dropped to 4.4 for 72 h, a time frame that results in maximal amyloid formation in the absence of inhibitor. Velocity analysis—overlay of data sets taken 15 min apart at 50,000 rpm. The data (symbols) fit to a single ideal species model (solid line) with MW 57.1±0.2 kDa.
FIG. 9 is a plot of the absorbance at 280 nm versus distance from the center in the equilibrium ultracentrifugation studies on TTR (3.6 mM) after being preincubated with 27 (7.2 mM) and after another incubation period where the pH was dropped to 4.4 for 72 h, a time frame that results in maximal amyloid formation in the absence of inhibitor. Equilibrium analysis—equilibrium concentration gradient observed after a 24 h application of centrifugal force to the sample employing at a speed of 17,000 rpm. The data (◯) fit to a single ideal species model (solid line) with MW 55.0±0.2 kDa. The residuals, the difference between experimental and fitted data, are shown in the inset.
FIG. 10 is a plot of the timecourse analysis of WT-TTR (3.6 μM) fibril formation mediated by partial acid denaturation in the absence (▴) and presence of 7.2 μM (⋄) and 3.6 μM (◯) inhibitors 25, 47, and 64, as measured by turbidity at 500 nm (see color scheme within Figure to differentiate inhibitors). It is hard to discern which compound is most efficacious in the black and white plot. At the end of the plot or after 169 hours have passed, compound 47 shows the most fibrils formed followed by compound 64 followed by compound 25.
FIG. 11 is a plot of the timecourse analysis of WT-TTR (3.6 μM) tetramer dissociation (6.0 M urea) in the absence (▴) and presence of 7.2 μM (⋄) and 3.6 μM (◯) concentrations of inhibitors 25, 47, and 64 (see color scheme within Figure to differentiate inhibitors). Slow tetramer dissociation is not detectable by far-UV CD spectroscopy, but this process is linked to rapid (˜500,000× faster) monomer denaturation as monitored by loss of β-sheet content easily followed by circular dichroism spectroscopy. It is hard to discern which compound is most efficacious in the black and white plot. At the end of the plot or after 169 hours have passed, compound 25 is most effective in dissociating tetramers followed by compound 64 followed by compound 47.

Claims

1. A compound represented by Formula I:

wherein:

X is absent or is a diradical selected from the group consisting of —O—, —S—, and —NH—; and

R², R³, R⁴, and R⁵are radicals independently selected from the group consisting of —H, OH, —F, —Cl, —Br, —CF₃, and —CO₂H.

2. A compound according to claim 1 represented by Formula II:

3. A compound according to claim 2 wherein:

R²is a radical selected from the group consisting of —H, —F, —Cl, and —CF₃.

4. A compound according to claim 2 wherein:

R⁴is a radical selected from the group consisting of —H, —Cl, and —CO₂H.

5. A compound according to claim 2 wherein:

R⁵is a radical selected from the group consisting of —H, —F, and —Cl.

6. A compound according to claim 2 selected from the group represented by the following structures:

7. A compound according to claim 1 represented by the following structure:

8. A compound according to claim 7 wherein:

R³is a radical selected from the group consisting of —H, —F, —Cl, —Br, and —CF₃.

9. A compound according to claim 7 wherein:

R⁵is a radical selected from the group consisting of —H, —F, —Cl, and —Br.

10. A compound according to claim 7 selected from the group represented by the following structures:

11. A compound according to claim 1 represented by the following structure:

12. A compound according to claim 11 wherein:

R²is a radical selected from the group consisting of —H, —F, and —Cl.

13. A compound according to claim 11 wherein:

R³is a radical selected from the group consisting of —H, —F, —Cl, —CF₃, and —CO₂H.

14. A compound according to claim 11 wherein:

R⁴is a radical selected from the group consisting of —H, and —CO₂H.

15. A compound according to claim 11 wherein:

R⁵is a radical selected from the group consisting of —H, —F, —Cl, and —CF₃.

16. A compound according to claim 11 selected from the group represented by the following structures:

17. A process comprising the step of contacting transthyretin with a concentration of a compound selected from claims 1-16 sufficient for inhibiting amyloid fibril formation.