U.S. patent application number 11/134527 was filed with the patent office on 2005-11-24 for transthyretin stabilization.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Kelly, Jeffery W., Petrassi, H. Michael.
Application Number | 20050261365 11/134527 |
Document ID | / |
Family ID | 34970266 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050261365 |
Kind Code |
A1 |
Kelly, Jeffery W. ; et
al. |
November 24, 2005 |
Transthyretin stabilization
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.
Inventors: |
Kelly, Jeffery W.; (La
Jolla, CA) ; Petrassi, H. Michael; (Cardiff,
CA) |
Correspondence
Address: |
THE SCRIPPS RESEARCH INSTITUTE
OFFICE OF PATENT COUNSEL, TPC-8
10550 NORTH TORREY PINES ROAD
LA JOLLA
CA
92037
US
|
Assignee: |
The Scripps Research
Institute
La Jolla
CA
|
Family ID: |
34970266 |
Appl. No.: |
11/134527 |
Filed: |
May 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60573720 |
May 20, 2004 |
|
|
|
Current U.S.
Class: |
514/468 ;
549/461 |
Current CPC
Class: |
C07C 229/60 20130101;
A61P 43/00 20180101; C07C 229/42 20130101; A61P 25/28 20180101;
C07C 65/24 20130101; C07C 39/367 20130101; A61P 9/00 20180101; A61P
35/00 20180101; C07D 307/91 20130101; A61P 7/00 20180101; A61P
25/00 20180101; A61P 25/02 20180101; C07C 229/58 20130101; C07C
251/48 20130101; A61P 3/04 20180101; A61P 3/10 20180101 |
Class at
Publication: |
514/468 ;
549/461 |
International
Class: |
C07D 307/91; A61K
031/343 |
Claims
What is claimed is:
1. A compound represented by Formula I: 11wherein: X is absent or
is a diradical selected from the group consisting of --O--, --S--,
and --NH--; and R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are radicals
independently selected from the group consisting of --H, OH, --F,
--Cl, --Br, --CF.sub.3, and --CO.sub.2H.
2. A compound according to claim 1 represented by Formula II:
12
3. A compound according to claim 2 wherein: R.sup.2 is a radical
selected from the group consisting of --H, --F, --Cl, and
--CF.sub.3.
4. A compound according to claim 2 wherein: R.sup.4 is a radical
selected from the group consisting of --H, --Cl, and
--CO.sub.2H.
5. A compound according to claim 2 wherein: R.sup.5 is 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: 131415
7. A compound according to claim 1 represented by the following
structure: 16
8. A compound according to claim 7 wherein: R.sup.3 is a radical
selected from the group consisting of --H, --F, --Cl, --Br, and
--CF.sub.3.
9. A compound according to claim 7 wherein: R.sup.5 is 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: 17
11. A compound according to claim 1 represented by the following
structure: 18
12. A compound according to claim 11 wherein: R.sup.2 is a radical
selected from the group consisting of --H, --F, and --Cl.
13. A compound according to claim 11 wherein: R.sup.3 is a radical
selected from the group consisting of --H, --F, --Cl, --CF.sub.3,
and --CO.sub.2H.
14. A compound according to claim 11 wherein: R.sup.4 is a radical
selected from the group consisting of --H, and --CO.sub.2H.
15. A compound according to claim 11 wherein: R.sup.5 is a radical
selected from the group consisting of --H, --F, --Cl, and
--CF.sub.3.
16. A compound according to claim 11 selected from the group
represented by the following structures: 1920
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.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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 .mu.M) decreases the extent of WT-TTR (3.6 .mu.M)
amyloid formation (pH 4.4) by 90% over a time course of 72 h. The
X-ray co-crystal structure of TTR.cndot.1.sub.2 reveals 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
[0003] 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.
[0004] 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.cndot.1.sub.2 reveals 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.
[0005] One aspect of the invention is directed to a compound
represented by Formula I: 1
[0006] In Formula I, X is absent or is a diradical selected from
the group consisting of --O--, --S--, and --NH--; and R.sup.2,
R.sup.3, R.sup.4, and R.sup.5 are radicals independently selected
from the group consisting of --H, --OH, --F, --Cl, --Br,
--CF.sub.3, and --CO.sub.2H. In a first subgenus of this first
aspect of the invention, the compound is represented by Formula II:
2
[0007] Within the subgenus of Formula II, preferred embodiments may
include species wherein R.sup.2 is a radical selected from the
group consisting of --H, --F, --Cl, and --CF.sub.3; additional
preferred embodiments may include species wherein R.sup.4 is a
radical selected from the group consisting of --H, --Cl, and
--CO.sub.2H; additional preferred embodiments may include species
wherein R.sup.5 is 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: 345
[0008] In a second subgenus of this first aspect of the invention,
the compound is represented by Formula III: 6
[0009] Within the subgenus of Formula III, preferred embodiments
may include species wherein R.sup.3 is a radical selected from the
group consisting of --H, --F, --Cl, --Br, and --CF.sub.3;
additional preferred embodiments may include species wherein
R.sup.5 is 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: 7
[0010] In a third subgenus of this first aspect of the invention,
the compound is represented by Formula IV: 8
[0011] Within the subgenus of Formula IV, preferred embodiments may
include species wherein R.sup.2 is a radical selected from the
group consisting of --H, --F, and --Cl; additional preferred
embodiments may include species wherein R.sup.3 is a radical
selected from the group consisting of --H, --F, --Cl, --CF.sub.3,
and --CO.sub.2H; additional preferred embodiments may include
species wherein R.sup.4 is a radical selected from the group
consisting of --H, and --CO.sub.2H; additional preferred
embodiments may include species wherein R.sup.5 is a radical
selected from the group consisting of --H, --F, --Cl, and
--CF.sub.3.
[0012] Preferred species of the subgenus of Formula IV include
compounds selected from the group represented by the following
structures: 910
[0013] 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
[0014] FIG. 1A illustrates an X-ray crystallographic structure of
TTR.cndot.1.sub.2 (Klabunde, T.; et al. Nature Struct. Biol. 2000,
7, 312-321).
[0015] 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.sub.aryl-C.sub.aryl linkage. R represents the
substituents of the aryl ring designed to complement TTR's inner
binding cavity.
[0016] FIG. 2 illustrates a table highlighting the concentration
dependent acid-substituted dibenzofuran activity against WT-TTR
(3.6 .mu.M) amyloid fibril formation (f.f.) at pH 4.4 (72 h).
[0017] FIG. 3 illustrates a chart showing a summary of
dibenzofuran-based amyloid inhibition activity (3.6 .mu.M) against
WT-TTR (3.6 .mu.M) fibril formation (pH 4.4, 72 h) and binding
stoichiometry to TTR in human blood plasma.
[0018] FIG. 4 illustrates a scheme for the synthesis of
1-hydroxy-dibenzofuran-4,6-dicarboxylate dimethyl ester and the
corresponding triflate.
[0019] FIG. 5 illustrates a scheme for the synthesis of 1-phenyl-,
phenoxy-, and phenylamine-dibenzofuran-4,6-dicarboxylate dimethyl
esters and the corresponding dicarboxylates.
[0020] FIG. 6 illustrates a chart showing dibenzofuran-based
inhibitor activity (7.2 .mu.M) against WT-TTR (3.6 .mu.M) amyloid
fibril formation (f.f.) at pH 4.4 (72 h).
[0021] FIG. 7 illustrates a table illustrating dibenzofuran plasma
TTR binding stoichiometry plotted vs. fibril formation inhibition
efficacy.
[0022] 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 .mu.M) after being preincubated with 27 (7.2 .mu.M) 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.
[0023] 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 .mu.M) after being preincubated with 27 (7.2
.mu.M) 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.
[0024] FIG. 10 illustrates a plot of the timecourse analysis of
WT-TTR (3.6 .mu.M) fibril formation mediated by partial acid
denaturation in the absence (.tangle-solidup.) and presence of 7.2
.mu.M (.diamond.) and 3.6 .mu.M (.largecircle.) inhibitors 25, 47,
and 64, as measured by turbidity at 500 nm (see shading scheme
within Figure to differentiate inhibitors).
[0025] FIG. 11 illustrates a plot of the timecourse analysis of
WT-TTR (3.6 .mu.M) tetramer dissociation (6.0 M urea) in the
absence (.tangle-solidup.) and presence of 7.2 .mu.M (.diamond.)
and 3.6 .mu.M (.largecircle.) concentrations of inhibitors 25, 47,
and 64 (see color scheme within Figure to differentiate
inhibitors).
DETAILED DESCRIPTION
[0026] All but one of the C1-aryl substituted dibenzofurans (7.2
.mu.M) are exceptional inhibitors of WT-TTR (3.6 .mu.M)
acid-mediated fibril formation in vitro (pH 4.4, 37.degree. 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 .mu.M inhibitor
data. However, FIGS. 3 and 7 reveal a range of inhibitor efficacy
at an inhibitor concentration equal to that of TTR (3.6 .mu.M),
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 .mu.M (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.
[0027] Comparing the four C1-aryl substitution patterns (H,
3-CF.sub.3, 3,5-F.sub.2, and 3,5-Cl.sub.2) 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-Cl.sub.2 substituents (36, 63, and 67), likely picking up
the halogen binding pockets in the thyroxine binding site, it is
somewhat surprising that the 3-CO.sub.2H 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).
[0028] 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 .about.2/3 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.
[0029] It is not surprising that inhibitor efficacy in vitro (3.6
.mu.M) and inhibitor binding selectivity (10.8 .mu.M) 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 T.sub.4, more than 99% of
its binding sites are unoccupied; therefore, inhibitor binding to
TTR should not perturb T.sub.4 homeostasis.
[0030] 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).
[0031] Design and Synthesis:
[0032] 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
.epsilon.-NH.sub.3.sup.+ 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.cndot.1.sub.2 crystal 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.sub.aryl-C.sub.aryl bond (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).
[0033] 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 CO.sub.2 and esterified to afford 6, which
was then deprotected with TBAF and converted to triflate 8 in high
overall yield.
[0034] 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.
[0035] Results:
[0036] 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.degree. 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.
[0037] Evaluating the Dibenzofuran-Based Compounds as
Amyloidogenesis Inhibitors.
[0038] 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.degree. C.)
(Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654-8660).
Briefly, a test compound (7.2 or 3.6 .mu.M) is incubated with TTR
(3.6 .mu.M) 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.degree. C.). The turbidity in
the presence of a potential inhibitor (T.sub.test) is compared to
that of a solution lacking a test compound (T.sub.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
.mu.M and <40% fibril formation at a concentration equal to that
of WT-TTR (3.6 .mu.M) (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 .mu.M inhibitor), FIG. 6 (at 7.2 .mu.M there
is enough test compound added to occupy both of the binding sites
of TTR (TTR.cndot.I.sub.2), 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.sub.d1 and
K.sub.d2 are 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.cndot.I, TTR.cndot.I.sub.2 and
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 .mu.M each, FIG. 3).
Representative small molecules from all three series (25, 26, 27,
30, 45, 47, 62; 3.6 .mu.M) were subjected to the acid-mediated
amyloidogenic conditions (pH 4.4, 37.degree. C., 72 h) in the
absence of TTR, revealing no measurable precipitation.
[0039] Evaluating the Plasma TTR Binding Selectivity of the
Dibenzofuran-Based Inhibitors.
[0040] 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 .mu.M,
.about.2-3.times. the natural concentration of TTR) are incubated
in human blood plasma for 24 h at 37.degree. 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.times.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 .mu.M (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.
[0041] Stabilization of the Tetrameric Quaternary Structure Under
Amyloidogenic Conditions.
[0042] 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.degree. C.). In the
presence of 27 (7.2 .mu.M), TTR (3.6 .mu.mM) 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).
[0043] Do the Dibenzofuran-Based Inhibitors Impose Kinetic
Stabilization on TTR?
[0044] 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 .mu.M) 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.
[0045] Experimental:
[0046] 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.
[0047] 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.times. stocks), from which 5- and 10-fold DMSO dilutions
yielded 1.44 mM (2.times.) and 0.72 mM (1.times.) secondary stock
solutions, respectively. 495 .mu.L of 0.4 mg/mL (7.2 .mu.M) WT-TTR
solution (10 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH
7.2), and 5 .mu.L 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.degree. C. The pH was then
adjusted to 4.4 with addition of 500 .mu.L 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.degree. 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 .mu.L 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.
[0048] 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 .mu.L of 1.0
mg/mL (18 .mu.M) WT-TTR solution (50 mM sodium phosphate, 100 mM
KCl, and 1 mM EDTA, pH 7.2), and either 7.2 or 3.6 .mu.L (2.times.
and 1.times., respectively) of 1 mM inhibitor secondary stock
solutions were added to 2 mL eppendorf tubes, vortexed briefly, and
incubated for 15 min at 25.degree. C. 100 .mu.L of the
TTR.cndot.inhibitor solutions were added to 900 .mu.L 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.degree. 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 .mu.L 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 .beta.-sheet loss
throughout the experiment. TTR tetramer dissociation is linked to
the rapid (.about.500,000.times. faster) monomer denaturation as
measured through this .beta.-sheet loss (Hammarstrom, P.; et al.
Proc. Natl. Acad. Sci. USA 2002, 99, 16427-16432).
[0049] 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 F.sub.254 coated 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 .ANG.
pore size, 3 .mu.m 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).
.sup.1H- and .sup.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 (Me.sub.4Si,
0.0 ppm).
[0050] (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. CH.sub.2Cl.sub.2 (5 mL) was
added followed by DMAP (391 mg, 3.2 mmol) and triisopropylsilyl
chloride (800 .mu.L, 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 H.sub.2O (3.times.10
mL). The aqueous layers were combined and extracted with
CH.sub.2Cl.sub.2 (3.times.30 mL). The organic layers were combined,
dried with MgSO.sub.4, 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).sup.+,
C.sub.21H.sub.29O.sub.2Si requires 341.1931.
[0051] 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 Et.sub.2O (7.4 mL) and
TMEDA (0.87 mL, 5.77 mmol). The flask was cooled to -78.degree. C.
in an acetone/CO.sub.2(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.degree. C. as described above and a 15 psi stream of
CO.sub.2(g) was bubbled through the reaction suspension (the
CO.sub.2 was dried by passing it through a drying tube containing
activated silica). Following initial addition of CO.sub.2(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.degree. C. with an
ice/H.sub.2O 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.times.50 mL). The combined extracts were dried with
MgSO.sub.4 and 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 (TMSCHN.sub.2; 2.5 mL of a 2 M
solution in hexanes, 5 mmol) was added slowly via syringe through
the septum. Upon completion of the TMSCHN.sub.2 addition 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).sup.+, C.sub.25H.sub.32O.sub.6SiNa requires 479.1860.
[0052] 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 H.sub.2O in a 250
mL separatory funnel. The aqueous layer was extracted with
CHCl.sub.3 (4.times.20 mL). The organic layers were combined, dried
with MgSO.sub.4, 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, C.sub.16H.sub.12O.sub.6 requires 301.
[0053] 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.degree. C. with an ice/H.sub.2O bath. To initiate the
reaction, trifluoromethanesulfonic anhydride (81 .mu.L, 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/H.sub.2O slurry and transferred
into a 125 mL separatory funnel. The aqueous layer was extracted
with Et.sub.2O (4.times.40 mL). The organic layers were combined,
washed with saturated CuSO.sub.4 (4.times.20 mL) and brine
(2.times.20 mL), dried over MgSO.sub.4, and then the Et.sub.2O 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).sup.+,
C.sub.17H.sub.12F.sub.3O.sub.8S requires 433.0205.
[0054] Representative Procedure for the Palladium Catalyzed Cross
Coupling of 8 with Substituted Anilines.
[0055] 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, Pd.sub.2(dba).sub.3 (15 mg, 0.016 mmol),
(.+-.)-binap (15 mg, 0.024 mmol), Cs.sub.2CO.sub.3 (147 mg, 0.456
mmol), and aniline (32 .mu.L, 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.degree. 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).
[0056] MALDI-FTMS 375.1094 m/z (M.sup.-).sup.+,
C.sub.22H.sub.17NO.sub.5 requires 375.1106.
[0057] Representative Procedure for the Copper-Mediated
Cross-Coupling of Phenol 7 with Substituted Phenylboronic Acids to
Afford 1-Phenoxydibenzofurans 39-43.
[0058] 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 .ANG. molecular sieves (.about.250 mg), and
phenylboronic acid (180 mg, 1.5 mmol). Dichloromethane (5 mL) was
added followed by pyridine (201 .mu.L, 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 .about.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)
[0059] MALDI-FTMS 399.0825 m/z (M+Na).sup.+,
C.sub.22H.sub.16O.sub.6Na requires 399.0839.
[0060] Representative Procedure for the Palladium Catalyzed
Cross-Coupling of Triflate 8 with Substituted Phenylboronic
Acids.
[0061] 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(PPh.sub.3).sub.4 (14 mg, 0.01 mmol), LiCl (29 mg, 0.69
mmol), Na.sub.2CO.sub.3 (300 .mu.L 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.degree. 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.
[0062] 1-Phenyl-dibenzofuran-4,6-dicarboxylic acid diethyl ester
(49). MALDI-FTMS 411.1197 m/z (M+Na).sup.+,
C.sub.24H.sub.20O.sub.5Na requires 411.1203.
[0063] Representative Procedure for Ester Hydrolysis to Afford
Final Inhibitors 24-38, 44-48, and 60-70.
[0064] Methyl ester 9 (25 mg, 0.067 mmol) was saponified in THF:
MeOH: H.sub.2O (3:1:1, 1 mL) in a 20 mL scintillation vial equipped
with a stir bar. LiOH.H.sub.2O (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.times.3 mL). The combined
organic layers were dried with MgSO.sub.4 and 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.
[0065] 1-Phenylamino-dibenzofuran-4,6-dicarboxylic acid (24).
MALDI-FTMS 347.0794 m/z (M.sup.-).sup.+, C.sub.20H.sub.13NO.sub.5
requires 347.0788.
[0066] FIG. 1A shows an X-ray crystallographic structure of
TTR.cndot.1.sub.2 (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 C.sub.2 symmetry 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-NH.sub.3.sup.+
of K15 and K15'.
[0067] 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.sub.aryl-C.sub.aryl linkage. R represents the substituents
of the aryl ring designed to complement TTR's inner binding
cavity.
[0068] FIG. 2 is a table highlighting the concentration dependent
acid-substituted dibenzofuran activity against WT-TTR (3.6 .mu.M)
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.
[0069] 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,
.about.2-3.times. the concentration of plasma TTR) bound to TTR in
blood plasma as determined using the antibody capture method.
[0070] FIG. 4 is a scheme for the synthesis of
1-hydroxy-dibenzofuran-4,6-- dicarboxylate dimethyl ester and the
corresponding triflate: a) K.sub.3[Fe(CN).sub.6], KOH, H.sub.2O,
benzene; b) AlCl.sub.3, toluene, 33% for both steps; c) TIPSCI,
DMAP, CH.sub.2Cl.sub.2, 77%; d) sec-BuLi, Et.sub.2O, -78.degree.
C., gaseous CO.sub.2, TMSCHN.sub.2, 43%; e) TBAF, THF, 97%; f)
Tf.sub.2O, pyridine, 92%.
[0071] 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) Pd.sub.2(DBA).sub.3,
(.+-.)binap, Cs.sub.2CO.sub.3, toluene 100.degree. C.; b)
LiOH--H.sub.2O, THF/MeOH/H.sub.2O (3:1:1); c) Cu.sup.II(OAc).sub.2,
pyridine, 4 .ANG. MS, CH.sub.2Cl.sub.2; d) Pd(PPh.sub.3).sub.4,
LiCl, aq. Na.sub.2CO.sub.3, toluene, MeOH, 80.degree. C.
[0072] FIG. 6 is a chart showing dibenzofuran-based inhibitor
activity (7.2 .mu.M) against WT-TTR (3.6 .mu.M) 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.
[0073] 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 (.tangle-solidup.), .largecircle. ( ), and
direct C.sub.aryl-C.sub.aryl linkage (.circle-solid.).
Dibenzofuran-4,6-dicarboxylic acid (1) data point (.circle-solid.)
shown for comparison.
[0074] 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.
[0075] 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 (.largecircle.) 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.
[0076] FIG. 10 is a plot of the timecourse analysis of WT-TTR (3.6
.mu.M) fibril formation mediated by partial acid denaturation in
the absence (.tangle-solidup.) and presence of 7.2 .mu.M
(.diamond.) and 3.6 .mu.M (.largecircle.) 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.
[0077] FIG. 11 is a plot of the timecourse analysis of WT-TTR (3.6
.mu.M) tetramer dissociation (6.0 M urea) in the absence
(.tangle-solidup.) and presence of 7.2 .mu.M (.diamond.) and 3.6
.mu.M (.largecircle.) 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 (.about.500,000.times. faster)
monomer denaturation as monitored by loss of .beta.-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.
* * * * *