Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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AMIDINE DERIVATIVES FOR TREATING AMYLOH)OSIS
RELATED APPLICATION
This application claims the priority of U.S. Provisional Patent Application
No.
60/387,001, filed June 7, 2002, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Amyloidosis refers to a pathological condition characterized by the presence
of amyloid fibers. Amyloid is a generic term referring to a group of diverse
but
specific protein deposits (intracellular or extracellular) which are seen in a
number of
different diseases. Though diverse in their occurrence, all amyloid deposits
have
common morphologic properties, stain with specific dyes (e.g., Congo red), and
have
a characteristic red-green birefringent appearance in polarized light after
staining.
They also share common ultrastructural features and common X-ray diffraction
and
infrared spectra.
Amyloid-related diseases can either be restricted to one organ or spread to
several organs. The first instance is referred to as "localized amyloidosis"
while the
second is referred to as "systemic amyloidosis."
Some amyloidotic diseases can be idiopathic, but most of these diseases
appear as a complication of a previously existing disorder. For example,
primary
amyloidosis can appear without any other pathology or can follow plasma cell
dyscrasia or multiple myeloma.
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Secondary amyloidosis is usually seen associated with chronic infection (such
as
tuberculosis) or chronic inflammation (such as rheumatoid arthritis). A
familial form of
secondary amyloidosis is also seen in Familial Mediterranean Fever (FMF). This
familial
type of amyloidosis, as one of the other types of familial amyloidosis, is
genetically
S inherited and is found in specific population groups. In these two types of
amyloidosis,
deposits are found iri several organs and are thus considered systemic amyloid
diseases.
Another type of systemic amyloidosis is found in long-term hemodialysis
patients.
In each of these cases, a different amyloidogenic protein is involved in
amyloid deposition.
"Localized amyloidoses" are those that tend to involve a single organ system.
Different amyloids are also characterized by the type~of protein presentin the
deposit. For
example, neurodegenerative diseases such as scrapie, bovine spongiforna
encephalitis,
Creutzfeldt Jalcob disease, and the like are characterized by the appearance
and
. accumulation of a protease resistant form of a prion protein (referred to as
AScr or PrP 27)
in the central nervous system. Similarly, Alzheimer's disease, another
neurodegenerative
disorder, is characterized by neuritic plaques and neurofibrillary tangles. Tn
this case, the
plaque and blood vessel ainyloid is formed by the~deposition of fibrillary A(3
amyloid
protein. Other diseases such as adult-onset diabetes (Type T( diabetes) are
characterized by
the localized accumulation of amyloid in the pancreas.
Once these amyloids have formed, there is no known, widely accepted therapy or
ZO treatment which significantly dissolves amyloid deposits in situ.
Each amyloidogenic protein has the ability to organize into ~3-sheets and to
form
insoluble fibrils which may be deposited extracellularly or intracellulaxly. ~
Each
amyloidogenic protein, although different in amino acid sequence, has the same
property of
forming fibrils and binding to other elements such as proteoglycan, amyloid P
and
?5 complement component. Moreover, each aanyloidogenic protein has amino acid
sequences
which, although different, will show similarities such as regions with the
ability to bind to
the glycosaminoglycan (GAG) portion ofproteoglycan (referred to as the GAG
binding
site) as well as other regions which will promote ~i-sheet formation.
2
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In specific cases, amyloidotic fibrils, once deposited, can become. toxic to
the
surrounding cells. For example, the A~i fibrils organised as senile plaques
have been shown
to be associated with dead neuronal cells and microgliosis in patients with
Alzheimer's
disease. When tested in vitro, A(3. peptide was shown to be capable of
triggering an
activation pxocess of microglia (brain macrophages), which would explain the
presence of
microgliosis and brain inflammation found in the bxain of patients with
Alzheimer's disease.
In another type of amyloidosis seen in patients with Type B diabetes, the
amyloidogenic protein IAl'P has been shown to induce (3 islet cell toxicity in
vitro. Hence,
appearance of IAPP fibrils in the pancreas of Type II diabetic patients
contributes to the loss
of the ~3 islet cells (Langerhans) and organ dysfunction.
People suffering from Alzheimer's disease develop a progressive dementia in
adulthood,,accompanied by three main structural changes in the brain: diffuse
loss of
neurons in multiple parts of the brain; accumulation of intracellular pxotein
deposits termed
neurofibrillary tangles; and accumulation of extracellular protein deposits
termed amyloid
1 S or senile plaques,. surrounded by misshapen nerve terminals (dystrophic
neurites). A main
constituent of these amyloid plaques is the amyloid-(3 peptide (A(3), a 39-43
amino-acid
protein that is produced thxough cleavage of the ~i-amyloid precursor protein
(APP).
Although symptomatic treatments.exist for Alzheimer's disease, this disease
caimot be
prevented or cured at this time.
~0
SUMMARY OF THE INVENTION
The present invention relates to the use of amidine compounds in the treatment
of
amyloid-related diseases. Ixi particular, the invention relates to a method of
treating or
preventing an amyloid-related disease in a subject comprising administering to
the subject a
?5 therapeutic amount of an amidine compound. Among the compounds fox use in
the '
invention are those according to the following Formula, such that, when
administered;
amyloid fibril formation, neurodegeneration, or cellular toxicity is reduced
or inhibited:
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Rah N N-Ra2 ~ .
,,
Rb~ N N-Rba
Rc1 ~ Rc2
m q
(Formula ~
BRIEF DESCRIPTION OF DRAWINGS
FIG.. l -Effect ofpentamidine-type compounds on A(3(1-40) assembly determined
by ThT
assay.
FIG. 2 - Effect of pentamidiue-like compounds on Aji(1-40) assembly determined
by ThT
assay.
FIG. 3 - Effect of amidine-type compounds on A(3(1-40) assembly determined by
ThT
l0 assay.
FIG. 4 - Effect of pentamidine-type compounds on IAPP assembly determined by
ThT
assay.
DETAILED DESCRIPTION OF THE INVENTION
5 The present invention relates to the use of amidine compouuds in the
treatment of
amyloid-related diseases.
Amyloid-Related Diseases
AA (reactive) Amyloidosis
Generally, AA amyloidosis is a manifestation of a number of diseases that
provoke
0 a sustained acute phase response. Such diseases include chronic inflammatory
disorders,
' chronic local or systemic microbial infections, and malignant neoplasms.
4
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AA fibrils axe generally composed of 8,000 Dalton fragments (AA peptide or
protein) foamed by pxoteolytic cleavage of serum amyloid A protein (ApoSAA), a
circulating apolipoprotein which once secreted is complexed with HDL and which
is
synthesized in hepatocytes in response to such cytokines as TL-1, TL-6 and
TNF.
Deposition can be widespread in the body, with a preference for paxenchymal
organs. The
spleen is usually a deposition site, and the lcidneys may also be affected.
Deposition is also
common in the heart and gastrointestinal tract.
AA amyloid diseases include, but axe not limited to inflammatory diseases,
such as
rheumatoid arthritis, juvenile chronic arthritis, anlcylosing spondylitis;
psoriasis, psoriatic
axthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome, .and
Crobn's
disease. AA deposits axe also produced as a result of chronic microbial
infections, such as
leprosy, tuberculosis, bronchiectasis, decubitus ulcers,~chxonic
pyelonephritis, osteomyelitis,
and Wlaipple's disease. Certain malignant neoplasms can also result in AA
fibril amyloid
deposits. These include such conditions as Hodgkin's lymphoma, renal
carcinoma,
carcinomas. of gut, lung and urogenital tract, basal cell carcinoma, and hairy
cell leulcemia.
AL Atnylaidoses
AL arnyloid deposition is generally associated with almost any dyscxasia of
the B
lymphocyte lineage, ranging from malignancy of plasma cells (multiple myeloma)
to benign
monoclonal gammopathy. At times, the presence of amyloid deposits may be a
primary
?0 indicator ofthe underlying dyscrasia.
Fibrils of AL amyloid deposits axe composed of monoclonal immunoglobulin light
cliains or fragments thereof. More specifically, the fragments are derived
from the N-
terniinal region of the light chain (kappa or lambda) and contain. all or part
of the variable
(VL) domain thereof Deposits generally occur in the mesenchymal tissues,
causing
!5 peripheral and autonomic neuxopathy, carpal tunnel syndrome, macroglossia,
restrictive
cardiomyopathy, arthropathy of large joints, immune dyscrasias, myelomas, as
well as
occult dyscrasias: However, it should be noted that almost any tissue,
particularly visceral
organs such as the heart, may be involved..
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Hereditary ,Systemic At~zyloidoses
There are many forms of hereditary systemic amyloidoses. l~lthough they are
relatively rare conditions, adult onset of symptoms and their inheritance
patterns (usually
autosomal dominant) lead to persistence of such disorders in the general
population.
S Generally, the syndronries are attributable to point mutations in the
precursor protein leading
to production of variant amyloidogenic peptides or proteins. Table 1
summarizes the fibril
composition of exemplary forms of these disorders.
Table 1
Fibril Pe tidelProteir~Genetic variant Clix:ical .adronae
'
Transthyretin Met30, many othersFamilial amyloid
and fragments
(ATTR) ~ polyneuropathy
(FAP), (Mainly
eri heral nerves
Transthyxetin Thr4S, A1a60, Cardiac involvement
and fragments Ser84, Met111,
ATTR I1e122 ~ redominantwithcutneuro
ath
N-terminal fragmentArg26 'Familial amyloid
of
ApolipoproteinAl polyneuropathy
(apoAl) (FAP), (mainly
eri heral nerves
N-terminal fragmentArg26, ArgSO, Ostertag-type,
of Arg 60, othezs non-neuropathic
Apoliproprotein (predominantly
A1 (AapoAl) visceral
involvement
Lysozyme (Alys) ThrS6, His67 Ostertag-type,
non-neuropathic
(predominantly
visceral
' involvement
Fibrogen t1 chainLeuSS4, Val S26 Cranial neuropathy
fragment with lattic
corneal d stxo
by
Gelsolin fragmentAsn187, Tyr187 Cranial neuropathy
(Agel) with lattice
corneal d stro
h
Cystatin C fragmentG1u68 Hereditary cerebral
hemorrhage
(cerebral amyloid
angiopathy) -
IceIandic a
(3-amyloid proteinG1n693 Hereditary cerebral
(A/3) derived hemorrhage
from Amyloid Precursor (cerebral amyloid
Protein angiopathy) -
~
App Dutch type
'
(i-amyloid proteinI1e717, Phe717, Familial Alzheimer's
(A(3) derived GIy717 Disease
from Amyloid Precursor
Protein
APP
~i-amyloidproteinAsn670, Leu671 Familial Dementia-probably
(A(3) derived
from Amyloid Precursor. Alzheimer's Disease
Protein
APP
Pnion Protein Leu102, Va1167, Familial Creutzfeldt
(PrP) derived Asn178, Jakob
from
Prp precursor Lys200 disease; Gerstmann-Straussler-
protein
Sl-9I insert ~ Scheinker syndrome
(hereditary
spongiform encephalopathies;
rion diseases
AA derived from Familial Meditezranean
Serum fever,
amyloid A protein predominant renal
(ApoSAA) involvement
autosomal recessive
AA derived from Muckle-Well's
Serum , syndrome,
~
A oSAA ne hro ath , deafness,
am Ioid A rotein urticaria,
6
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Jimb ain
Unknown Cardiomyopathy
with persistent
atrial standstill
Unknown Cutaneous deposits
(bullous,
a ular, ustulodermal
Aata derived fromi Tan SY, Pepys MB. Amyloidosis. Histopathology, 25(S), 403-
414 (Nov 1994).
The data provided in Table 1 are exemplary and are not iutended to limit the
scope
of the invention. For example, more than 40 separate point mutations in the
transthyretin
gene have been described, all of which give rise to clinically similar forms
of familial
amyloid polyneuropathy.
Transthyretin (TTR) is a 14 kiloDalton protein that is also sometimes referred
to as
prealbumin. It is produced by the liver and choroid plexus, and it functions
in transporting
thyroid hormones and vitamin A. At least 50 variant forms of the protein, each
characterized by a, single amino acid change, are responsible for various
forms of familial
amyloid polyneuropathy. For example, substitution of proliue for leucine at
position 55
results in a particularly progressive form of neuropathy; substitution of
methionine fox
leucine at position 111 resulted in a severe cardiopathy in Danish patients.
Amyloid deposits isolated from heart tissue of patients with systemic
amyloidosis
LS . have revealed that the deposits are composed of a heterogeneous mixture
of TTR and .
fragments thereof, collectively referred to as ATTR, the full length sequences
of which have
been characterized. ATTR fibril components can be extracted from such plaques
and their
structure and sequence determined according to the methods lmown in the art
(e.g.,
Gustavsson, A., et al., Laboratory Invest. 73: 703-708, 1995; Kametani, F., et
al., Biochem.
t0 Biophys. Res. Commun. 125: 622-628,1984; Pras, M., et al., PNAS 80: S39-
42,1983).
Persons having point mutations in the molecule apolipoprotein Al (e.g.,
Gly~Arg26; Trp -~ Arg50; Leu -~ ~Arg60) exhibit a form of amyloidosis
("Ostextag type")
characterized by deposits of the protein apolipoprotein AI ox fragments
thereof (AApoAn.
These patients have low levels of high density lipoprotein (HDL) and present
with a
;5 peripheral neuropathy or renal failure.
7
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A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile-~Thr56 or
Asp-~His57) is the basis of another form~of C)stertag-type non-neuropathic
hereditary
amyloid reported in English families. Here, fibrils of the mutant lysozyme
protein (Alys)
are deposited, and patients generally exhibit impaired renal function. This
protein, unlike
S most of the fibril=forming proteins described hereiun, is usually present in
whole
(unfragmented) form (Benson, M.D., et al. CIBA Fdn. Symp. 199: 104-131, 1996).
[3-amyloid peptide (A(3) is a 3 9-4.3 amino acid peptide derived by
proteolysis from
a large protein known as Beta Amyloid Precursor protein (~iAPP). Mutations in
[3APP
result in familial forms of Alzheimer's disease, Down's syndrome'or senile
dementia,
characterized by cerebral deposition of plaques composed of Aj3 fibrils and
other
components, which are described in further detail below. Known mutations in
APP
associated with .Alzheimer's disease occur proximate fo the cleavage sites of
(3 or gamma-
secretase, or within A~. Fox example, position 717 is proximate to the site of
gamma-
secretase cleavage of APP in its processing to A[3, and positions 670/671 are
proximate to
l5 the site of ~3-secretase cleavage. Mutations at any of these residues may
result in
Alzheimer's disease, presumably by causing an increase in the amount of.the
42143 amino
acid for. of A(i generated from APP. . .
The structure and sequence of A~3 peptides of various lengths are well known
in the
art. Such peptides can be made according to methods known in the art (e.g.,
Glenner and
?0 Wong, Biochem Biophys. Res. Comm.129: 885-890, 1984; Glenuer and Wong,
Biochem
Biophys. Res. Comm. 122: l I3 1-1135, 1984). In addition, vari011S forms of
the peptides
are c6mmercially available.
As used herein, the term "~3 amyloid" or "amyloid-(3" refer to amyloid ~3
proteins or
peptides, amyloid .(i precursor proteins or peptides, intermediates, and
modifications and
:5 fragments thereof, unless otherwise specifically indicated. In particular,
"A~i" refers to any
peptide produced by proteolytic processing of the APP gene product, especially
peptides
which are associated with amyloid pathologies, including Aj31_39, A(3mo,
A(3m1, A~imz, and
A(1m3.
8
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Fox convenience of nomenclature, "Aj3l~z" may be referred to herein as "A~i(I-
42)
or simply as "A(342" or "A(342" (and likewise for any other amyloid peptides
discussed
herein). As used herein, the terms "(3 amyloid," "amyloid-Vii," and "A~i" axe
synonymous.
Unless otherwise specified, the term "amyloid" refers to amyloidogenic
proteins,
peptides, or fragments thereof which 'can be soluble (e.g.,.monomeric or
oligomeric) or
insoluble (e.g., having fibrillary structure or in amyloid plaque).
Gelsolin is a calcium biunding protein that binds to fragments and actin
filaments.
Mutations at position 187 (e.g., Asp-~Asn; Asp-~Tyr) of the protein result in
a form of
hereditary systemic amyloidosis, usually found in patients from Finland, as
well as persons
l0 of Dutch or Japanese origin. 1n afflicted individuals, fibrils formed from
gelsolin fragments
(Agel), usually consist of amino acids 173 243 (68 lcDa carboxyterminal
fragment) and are
deposited in blood vessels and basement membranes, resulting in corneal
dystrophy and
cranial neuropathy which progresses to peripheral neuropathy, dystrophic skin
changes and
deposition in other organs. (Kangas; H., et al. Human Mol. Genet. 5(9): 1237-
1243, 1996).
:5 . Other mutated proteins, such as mutant alpha chain of fibrinogen (AfibA)
and
mutant cystatin C (Acys) also form fibrils and pxoduce characteristic
hereditary disorders.
AfibA fibrils form deposits characteristic of a nonneuropathic hereditary
aznyloid with renal
disease; Acys deposits are characteristic of a hereditary cerebral asnyloid
angiopathy
reported in Iceland (Isselbacher, Harrison's Principles of Internal Medicine,
McGraw-HiII,
:0 San Francisco, 1995; Benson, et al.). In at least some cases, patients with
cerebral amyloid
angiopathy (CAA) have been shown to have amyloid fibrils containing a non-
mutant foam
of cystatin C in conjunction with amyloid beta protein (Nagai, A., et al.
Molec. Chem.
Neuropathol. 33: 63 78, 1998).
Certain forms of priors disease are now considered to be heritable, accounting
for up
S to '15% of cases, which were previously thought to be predominantly
infectious in nature.
(Baldwin, et al., in Research Advances inAlzheinzer's Disease and Related
Disorders, John
Wiley and Sons, New Yorlc, 1995). In such priors disorders, patients develop
plaques
composed of abnormal isoforms of the normal priors protein (PrPs°).
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A predominant mutant isoform, PrPs~, also referred to as, AScr, differs from
the
normal cellular protein in its resistance to protease degradation,
insolubility after detergent
extraction, deposition in secondary lysosomes, post translational synthesis,
and high [3-
pleated sheet content. Genetic linkage has been established for at least five
mutations
resulting in Creutzfeldt Jacob disease (CJD), Gerstmann-Straussler-Scheinlcer
syndrome
(GSS), and fatal familial insomnia (FF~. (Baldwin, supra) Methods fox
extracting fibril
peptides from scrapie fibrils, determining sequences and making such peptides
are known in
the art (e.g., Beekes, M., et al. J. Gen. Virol. 76: 2567 76, 1995).
For~example, one form of GSS has been linked to a PrP m~itation at codon 102,
l0 while telencephalic GSS segregates with a mutation at codon 117. Mutations
at codons 198
and 217 result in a form of GSS in which neuritic plaques characteristic .of
Alzheimer's
disease contain PrP instead of A(3 peptide. Certain foams of familial CJD have
been
associated with mutations-at codons 200 and 210; mutations at codons 129
and'178 have
been found in both familial CJD and FFI. (Baldwin, supra):
5 Senile Systei~tic Amyloidosis
Amyloid deposition; either systemic or focal, increases with age. For example,
fibrils of wild type transthyretin (TTR) are commonly found in the heart
tissue of elderly
individuals. These may be asymptomatic, clinically silent, ox may result in
heart failure.
Asymptomatic fibrillar focal deposits may also occur in the brain (A~i),
corpora amylacea of
0 the prostate (Aj32 miexoglobulin), joints and seminal vesicles.
Cerebral Amyloidosis
Local deposition of amyloid is common in the brain, particularly in elderly
individuals. The most frequent type of amyloid in the brain is composed
primarily of AJ3
peptide fibrils, resulting in dementia or sporadic (non hereditary)
Alzheimer's disease. In
5 ' fact, the incidence of sporadic Alzheimer's disease greatly exceeds forms
shown to be
hereditary. Fibril peptides forming these plaques axe very similar to those
described above,
with reference to hereditary forms of Alzheimer's disease (AD).
' to
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Cerebral amyloid angiopathy (CAA) refers to the specific deposition of amyloid
fibrils in the walls of leptomingeal and cortical arteries, arterioles and in
capillaries and
veins. It is commonly associated with Alzheimer's disease, Down's syndrome and
normal
aging, as well as with. a.variety of familial conditions related to stroke or
dementia (see
Frangione et al., Amyloid: J. Protein Folding Disord. 8, Suppl: l, 36-42
(2001)). CAA can
occur sporadically or be hereditary. Multiple mutation sites in either A(3 or
the APP gene
have been identified and are clinically associated with either dementia or
cerebral
hemorrhage. Exemplary CAA disorders include, but are not limited to,
hereditary cerebral
hemorrhage with amyloidosis of Icelandic type (HCHWA-IJ; the Dutch variant of
HCHWA
t 0 (HCI3WA-D; a mutation in Aj3); the Flemish mutation of Aj3; the Arctic
mutation of A~;
the Italian mutation of A(3; the Iowa mutation of A(3; familial British
dementia; and familial
Danish dementia.
Dialysis-related Amyloidosis
Plaques composed of (32 microglobulin (AJ32M) fibrils commonly develop in
patients
5 receiving long term hemodialysis or peritoneal dialysis. X32 microglobulin
is a 11.8
kiloDalton polypeptide and is the light chain of Class I MHC antigens, which
are present on
all nucleated cells. Under normal circumstances,~it is continuously shed from
cell
membrmes and is normally filtered by the kidney. Failure of clearance, such as
in the case
of impaired renal function, Ieads to deposition in the kidney and other sites
(primarily in
.0 collagen-rich tissues of the joints). Unlike other fibril proteins, A~iaM
molecules are
generally present in unfragmented form in the fibrils. (Benson, supra).
Islet Amyloid Polypeptide arad Diabetes
Islet hyalinosis (amyloid deposition) was first described over a century ago
as the
presence of fibrous protein aggregates in the pancreas of patients with severe
hyperglycemia
5 (Opie, EL., JExp. Med. 5: 397-428,1990). Today, islet amyloid, composed
predominantly
of islet amyloid polypeptide (IAPP), or amylin, is a characteristic
histopathological marker
in over 90% of alI cases of Type II diabetes (also known as Non-Insulin
Dependent
Diabetes, or NIDDM). These fibrillar accumulations result from the aggregation
of the islet ,
amyloid polypeptide (IAI'P) or'amylin, which is a 37 amino acid peptide,
derived from a
0 larger precursor peptide, called pro-IA.pP. a
11
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IAPP co-localizes and is co-secreted with ilisulin in response to (3-cell
secretagogues. This pathological feature is not associated with insulin-
dependent (Type 1)
diabetes and is a unifying characteristic for the heterogeneous clinical
phenotypes diagnosed
as NIDDM (Type II diabetes).
Longitudinal studies in cats and ixnmunocytochemical investigations in
monlteys
have shown that a progressive increase in islet amyloid is associated with a
dramatic
decrease in the population of insulin-secreting (3-cells and increased
severity of the disease.
More recently, transgenic studies have strengthened the relationship between
IAPP plaque
formation and ~i-cell dysfunction, indicating that amyloid deposition is a
principal factor in
LO Type-II diabetes.
IAPP has also been shown to induce [3-islet cell toxicity ifa vitro,
indicating that
appearance of IAPP fibrils in the pancreas of Type II or Type I diabetic
patients (post-
transplantation) could contribute, to the loss of the (3 islet cells
(Langexhans) and organ
dysfunction. In patients with Type-II diabetes, the accumulation of pancreatic
LAPP leads
l5 to a buildup of IAPP-amyloid as insoluble fibrous deposits which eventually
replace the
insulin-producing (3 cells of the islet resulting in j3 cell depletion and
failure (Westermark,
P., Grimelius, L., Acta Path. Microbiol. Scahd., sect. A. 81: 291-300, 1973;
de Koning,
E3P., et al., Diabetologia 36: 378-384,1993; and Lorenzo, A., et al., Nature
368: 756-760,
1994).
la
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Diseases caused by the death of malfunctioning of a particular type or types
of cells
can be treated by transplanting into the patient healthy cells of the relevant
type of cell.
i
Tlus approach has been used for Type I diabetes patients. Often pancreatic
islet cells are
cultured in vitro prior to transplantation to increase their numbers, to allow
them to recover
after the isolation procedure or to reduce their immuuogenicity. However, in
many
instances islet cell transplantation is unsuccessful,.'due to death of the
transplanted cells.
One reason for this poor success rate is IAPP, which canform fibrils and
become toxic to
the cells in vitro. In addition, IAPP fibrils axe lilcely to continue to grow
after the cells are
transplanted and cause death or dysfunction of the cells. This may occur even
when the
cells are from a healthy donor and the patient receiving the transplant does
not have a
disease that is characterized by the pxesence of fibrils. For example,
compounds of the
present invention may also be used in. preparing tissues or cells for
transplantation according
to the methods described in Iuternational.Patent Application (PCT) number WO
01/03,680.
Hormone-derived Anayloidoses
l5 Endocrine organs may harbor amyloid deposits, particularly in aged
individuals.
Hormono--secreting tumors may also contain hormone-derived amyloid plaques,
the fibrils
of which are made up of polypeptide hormones such as calcitonin (medullary
carcinoma of
the thyroid), islet amyloid polypeptide (amylin; occurring in raaost patients
with Type II
diabetes), and atrial natriuretic peptide (isolated atrial amyloidosis).
Sequences and
;0 structures of these proteins are well known in the art.
Miscellaneous Arnyloidoses
There are a variety of other forms of amyloid disease that are normally
manifest as
localized. deposits of amyloid. In general, these diseases are pxobably the
result of the
localized production or lack of catabolism of specific fibril pxecursors or a
predisposition of
.5 a particular tissue (such as the joint) for fibril deposition. Examples of
such idiopathic
deposition include nodular AL amyloid, cutaneous amyloid, endocrine arnyloid,
and tumor-
related amyloid.
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The compounds of the invention may be administered therapeutically or
prophylactically to treat diseases associated with amyloid-j3 fibril
formation, aggregation or
deposition. The compounds of the invention xnay act to ameliorate the course
of an amyloid-
~i related disease using any of the following mechanisms (this list is meant
to be illustrative
S and not limiting): slowing the rate of amyloid-~i fibril formation or
deposition; lessening the
degree of amyloid-[3 deposition; inhibiting, reducing, ox preventing amyloid-
(3 fibril
formation; inhibiting neurodegeneration or cellular toxicity induced by
amyloid-(3;
inhibiting amyloid-(3 induced inflammation; or enhancing the clearance of
amyloid-(i from
the brain.
L O Compounds of the invention may be effective in controlling amyloid-(i
deposition
either following their entry into the brain (following penetration of the
blood brain barrier)
or from the periphery. When acting from the periphery, a compound may alter
the
equilibrium of A~i between the brain and the plasma so as to favor the exit of
A~i from the
brain. An increase in the exit of A(3 from the brain would result in a
decrease in Aø brain
L 5 concentration and therefore favor a decrease in A~i deposition.
Alternatively, compounds
that penetrate the brain could control deposition by acting directly on brain
A(3, e.g., by
maintaining it in.a non-fibrillar form or favoring its clearance from the
brain.
In a preferred embodiment, the method is used to treat Alzheimer's disease
(e.g.,
sporadic or familial AD). The method can also be used prophylactically or
therapeutically
'0 to treat other clinical occurrences of amyloid-(3 deposition, such as in.
Down's syndrome
individuals and in patients with cerebral atnyloid angiopathy ("CAA") or
hereditary cerebral
hemorrhage.
Additionally, abnormal accumulation of APP and of amyloid-(3 protein in muscle
fibers has been implicated in the pathology of sporadic inclusion body
myositis (1B1V~
(Askanas, V., et al. (1996? Proc. Natl. Acad. Sci. USA 93: 1314-1319; Askanas,
V. et al.
(1995) Curretzt C~piniota itz Rheumatology 7:,486-496. Accordingly, the
compounds of the
invention can be used prophylactically or therapeutically in the treatment of
disorders in
which amyloid-beta protein is abnormally deposited at non neurological
locations, such as
treatment of IBM by delivery of the compounds to muscle fibers.
14
CA 02488493 2004-12-07
WO 03/103598 PCT/US03/17992
The present invention therefore relates to the use of amidine compounds in the
prevention or treatment of amyloid-related diseases, including, inter alia,
Alzheimer's
disease, cerebral amyloid angiopathy, inclusion body myositis, Down's
syndrome, and type
II diabetes.
Preferred compounds of the invention have at least two amidine moieties
(preferably
arylanudines, more preferably benzamidines).
In one particular.embodiment, the present invention relates to the novel use
of
amidine compounds in the prevention or treatment of amyloid-related diseases,
such as
those disclosed in U.S. Patent Nos. 5,428,051, 4,963,589, 5,202,320,
5,935,982, 5,521,189,
5,686,456, 5,627,184, 5,.622,955, 5,606,058, 5,668,167, 5,667,975, 6,025,398,
6,214,883,
5,817,687, 5,792,782, 5,939,440, 6,017,941, 5,972,969, 6,046,226, 6,294,565
(B1),
6,156,779, 6,326,395, 6,008,247, 6,127,554, 6,172,104, 4,940,723, 5,594,138,
5,602,172,
5,206,236, 5,843,980, 4,933,347, 5,668,166, 5,817,686, 5,723,495, 4,619,942,
5,792,782,
5,639,755, 5,643,935, and 5,578,631, each of which are hereby incorporated
herein by
LS reference in their entirety.
Iu another embodiment, the invention relates to a method of treating or
preventing
an amyloid-related disease in a subject (preferably a human) comprising
administering to
the subject a therapeutic amount of a compound according to the following
Formula, such
that amyloid fibril-formation ox deposition, neurodegeneration, or cellular
toxicity is
;0 reduced or inhibited. In another embodiment, the invention relates to a
method of treating
or preventing an amyloid-related disease in a subject (preferably a human)
comprising
administering to the subject a therapeutic amount of a compound according to
the following
Formula, such that cognitive function is stabilized or further deterioration
in cognitive
function is prevented, slowed, or stopped in patients with brain amyloidosis,
e.g.,
.5 Alzheimer's disease or cerebral amyloid angiopathy:
Ra1 N N-Ra2
Rb~-N N-Rb2
~Ro1 m Rc2
q
(Formula X)
15~
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wherein each ofRal, Rbl, R~l, R~z, Rbz, and R°2 is independently a
hydrogen, a Z
group, or Ral and Rbl or Raz and Rbz are both taken together along with the
nitrogen atoms
to which they are bound to form a ring structure; '
each of YI and Yz is independently a dixect bond or a linking moiety;
m and q are each independently an integer selected from zero to five
inclusive, such
that l~rn+q<5, or in another embodiment, 2.<~m+q<5, or in another embodiment
15m+q<10,
or in another embodiment, 2~n+q<10; and
The A group is a Garner moiety selected from substituted or unsubstituted
aliphatic
and aromatic groups, and combinations thereof; preferably such that the Yl and
Yz moieties
~ are bonded to an aromatic group.
The A group preferably is a div.,alent group (i. e., m+q=2) such as an
allcylene group
(i. e., -(CHz)~- and substituted analogs thereof (including groups in which a -
CHz- moiety
is substituted by an oxygen atom), where k is 1 to 12 (preferably 6 to 9, more
preferably 7 to
9), au alkenylene group (preferably 2 to 12 carbon atoms; more preferably 6 to
9 carbon
l5 atoms, including groups with more than one double bond), an alkynylene
group (preferably
2 to 12 carbon atoms, more preferably 6 to 9 carbon atoms, including groups
with more than
one triple bond), an alkoxyalkylene group, an allcylaminoalkylene group, a
thioallcoxyalkylene group, an arylenediallcylene group, a
heteroarylenedialkylene group, an
arylene group, a heteroarylene group, an oligoethereal gioup such as an
;0 . oligo(alkyleneoxide) group, or an arylene-di(oligoallcyleneoxide) group,
each of which may,
be substituted (with a Z group as defined below, e.g., a hydroxyallcylene
group) or
uusubstituted.
The A group also includes the corresponding moieties of the Formulae I- N
herein.
16
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In preferred aspects of the invention, the invention relates to a method of
treating or
preventing an amyloid-related disease in a subject (preferably a human)
comprising
administering to the subj ect a therapeutic amount of a compound according to
one of the
following Formulae, such that amyloid fibril formation or deposition,
neurodegeneration, or
cellular toxicity is reduced or inhibited. In another embodiment, the
invention relates to a
method of ixeating or preventing an alnyloid-related disease in a subject
(preferably a
human) comprising administering to the subject a therapeutic amount of a
compound
according to one of the following Formulae, such that cognitive function is
stabilized or
further deterioration in cognitive function is prevented, slowed, or stopped
in patients with
brain amyloidosis, e.g., Alzheimer's disease or cerebral amyloid angiopathy:
Ra1 N (R ~n . /(R2)p N_Ra2
\~Y~ ~ ~ ~ ~ ~~1 Yz~/ .
Rb~ N \ X~~M~Xz \ N-Rbz
Rc1 m Rcz 9
(Formula n
Rah N
\~Y~
Rb~-N
(X~-R~)n
R m~
.5 (Formula I~
Rb~
q
(Formula ~
17
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~2
Rbz
q
(Formula T~
Rb' b2
9.
_ (Formula IVb)
Rc1 Rc2 * -
R~ N-A-N R2
..
N~Ra1 N~Ra2
(Formula V)
.0 wherein Rai, Rbl, R°i, Rte, lZb2, R°a, .Yi, and Yz are as
defined herein, and A is as
defined above;
18.
CA 02488493 2004-12-07
WO 03/103598 PCT/US03/17992
each of Rl and RZ is independently a hydrogen or a Z group, or two adjacent or
proximate R1 and R2 groups, along with the corresponding Xl and X2 groups, if
present
(e.g., in Formula I>], talcen together with the ring (e.g., phenyl ring) to
which they are bound
forma fused ring structure, e.g., an aromatic or heteroaromatic (e.g.,
benzofuxan) structure,
or a cycloallcyl or heterocylic structure;
each of R3 and R~ is independently selected from the group consisting of
hydrogen,
substituted or unsubstituted straight or branched alkyl (preferably Cl-CS),
cycloalkyl
(prefexably C3-C$), carbocyclic, aryl (e.g., phenyl), heterocyclic, and
heteroaryl;
each of Rl'~ and R2~ is independently selected from the group consisting of
substituted or unsubstituted straight or branched all~yl, cycloalkyl,
heterocyclic, aryl
{including phenyl), and heteroaryl;
each of Xl and Xa is independently a direct bond, or an oxygen, a NR' group
(where
R' is hydrogen (i.e., NH), a Cl-CS allcyl, Ca-CS alkenyl, C2-CS allcynyl, or
aryl group), a
sulfonamide group (i.e., NHS02 or SO~NH), a carbonyl, amide (i.e., NHCO ox
CONH), a
Ci-CS allcylene group (e.g., -CH2 ), Ca-C$ allcenylene group (e.g., E or 2'-CH-
-CH ),
C2-CS alkyi~ylene group, or a sulfur atom, or combinations thereof (e.g., -
OCH~-,
-CH20-, E or ~'-OCH=CH- or-CH=CHO-);
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M is a,divalent group such as an allcylene group, i. e., -(Cl;T2)~- and
substituted
. analogs thereof (including groups in which a-CHz-moiety is substituted by an
oxygen
atom), whexe lc is 1 to 12 (preferably 5 to 10, more preferably 6 to 9, most
preferably 7 to
8), an allcenylene group (preferably 2 to 12 carbon atoms, more preferably 6
to 9 carbon
atoms, including groups with more than one double bond), an alkynylene group
(preferably
2 to 12 carbon atoms, more preferably 6 to 9 carbon atoms, including groups
with more than
one triple bond), an allcoxyalkylene group, an alltylaminoalkylene group; a
thioallcoxyalkylene group, an arylenedialkylene group, axi allcylenediarylene
group, a
heteroarylenediallcylene group, an arylene group, a heteroarylene group, an
oligoethereal
group such as an.oligo(alkyleneoxide} group, or an arylene-
di(oligoalkyleneoxide) group,
each of which rnay be substituted (with, for example, a Z group as defined
herein, e.g., a
hydroxyalkylene group such as -(CH2)0-6(CHOH)(CH2)0~6-; or other such
substituted
moieties, e.g.,
-(CH2)0-6(CHZ)(CH2)0-6-~ ~cluding-(CH2)0-6(CHC02alkyl)(CH2)0-5 ) or
unsubstituted;
Z is a substituted ox uusubstituted moiety selected from straight or branched
alkyl
(preferably Cl-CS}, cycloalkyl (pxeferably C3-C$), alkoxy (preferably Cl-C6),
thioallryl
(preferably Cl-C6), alkenyl (preferably CZ-C6), alkynyl (preferably C~-C6),
heterocyclic,
carbocyclic, aryl (e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g.,
benzyl), 'aryloxyallcyl
'0 (e.g., phenyloxyaltcyl), arylacetamidoyl, altcylaryl, heteroaralkyl,
allcylcarbonyl and
arylcarbonyl or other such acyl group, heteroarylcarbonyl, or heteroaryl
group,
' (CR'R")~3NR'R".(e.g., NHZ), (CR'R")~3CN (e.g., -CN), NOa, halogen (e.g., F,
Cl, Br, or
1], (CR'R")~3C(halogen)3 (e.g.,-CF3), (CR'R")~3CH(halogen)2,
(CR'R")~3CHa(halogen),
(CR'R")a-sCONR'R", (CR'R")o-s(~~'R»~ (CR~R»)o-3s(0)i-aNR'R",
;5 (CR'R")o-sCHO, (CR'R")o 30(CR'R")o-sH~ (CR'R")o--sS(0)o=3R' (e.g.,-SOaH), .
(CR.'R.")a-s0(CR'R")o-sH (e.g,,-CH20CH3 and-OCH3), (CR'R")o--sS(CR'R")o-sH
(e.g., -SH and-SCH3), (CR'R")~30H (e.g., -OH),.(CR'R")~3COR',
(CR'R")~3(substituted or unsubstituted phenyl), (CR'R")o...3(C3-C$
cycloalkyl),
(CR'R")~3COZR' (e.g., -COaI~, or (CR'R")~30R' group, or the side chain of any
naturally
0 occurring amino acid;
CA 02488493 2004-12-07
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in another embodiment, Z is a substituted or unsubstituted moiety selected
from
shraight or branched allcyl (preferably Cl-CS), cycloallcyl (preferably C3-
CS), allcoxy .
(preferably Cl-C6), thioallcyl (preferably Cl-C6), alltenyl (preferably Ca-
C6), alkynyl
(preferably Ca-C6), heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy
(e.g., phenoxy),
5, arallcyl (e.g., benzyl), aryloxyallcyl (e.g.,.phenyloxyallcyl),
arylacetamidoyl, allcylaryl,
heteroarallcyl, alkylcarbonyl and arylcarbonyl or other such acyl group,
hetexoarylcarbonyl,
or heteroaryl group, (CR'R")a.loNR'R" (e.g., NHa), (CR'R")~IOCN (e.g., -CN),
NOa,
halogen (e.g., F, Cl, Br, or n, (CR'R")o-loC(halogen)3 (e.g., -CF3), (CR'R")o-
ioCH(halogen)a, (CR'R")o_ioCHa(halogen), (CR'R")~IOCONR.'R", (CR'R")~
~ ~o(CN~NR'R",
(CR'R")o-ios(0)i-aNR'R"~ (CR'R")o-ioCHO, (CR'R")o-io0(CR'R")o-ioH>
(CR'R")o-ios(0)o-sR' (e.g., -503~~ (CR'R")o-to0(CR'R")o-ioH (e~g ~ -CHa0CH3
and-OCH3), (CR'R")o-ioS(CR.'R")o-3H (e.g.,-SH and-SCH3), (CR'R")o-ioOH (e.g.,
_
OH), (CR'R")~IOCOR', (CR'R")~lo(substituted or unsubstituted phenyl),
(CR'R")o...to(C3-C$ cycloalkyl), (CR'R")o_ioCOaR' (e.g., -COaH), ox
(CR'R")o_ioOR' group,
or the side chain. of any naturally occurring amino acid;
wherein R' and R" are each independently hydrogen, a Cl-CS alkyl,
Ca-C$ allcenyl, Ca-CS allcynyl, or aryl group, or R' and R" taken together are
a benzylidene
group or a -(CHa)a0(CHa)a- group;
~0 m and q are each independently an integer selected from zero to five
inclusive;
ill Formula I, m and q are each independently an integer selected from zero to
four
inclusive, and n and p are each independently au integer selected from zero to
four
inclusive, such that m+n<5 and p+q<5, wherein either m or q is at least one;
and preferably
m and q are one; ~ ,
!S in Formula II, lxi is an integer selected from one to six inclusive, and n
is an integer
selected from zero to five inclusive, such that m+~6;
in Formula III, m, n, p, and q are each independently an integer selected from
zero to .
three inclusive, m+~ul., p+q~l., and m+q>_l, (preferably m=q=1 );
21
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WO 03/103598 PCT/US03/17992
in Formula IV and IVb, m and n are each independently an integer selected from
zero to three inclusive, p and q are each independently an integer selected
from zero to four
inclusive, m+n~4, p+q<5, and m+q>_1 (preferably m=q=1);
and pharmaceutically acceptable salts thereof.'
The chemical stxuctures herein axe drawn according to the conventional
standards
known in the art. Thus, where an atom, such as a carbon atom, as drawn appears
to have an
unsatisfied valency, then that valency is assumed to be satisfied by a
hydrogen atom even
though that hydrogen atom is not necessarily explicitly drawn.
In an alternate embodiment, the invention relates to novel compounds, and
novel
methods of their use as described herein, which are within the scope of the
Formulae
disclosed herein, and which are not disclosed in the above-referenced U.S.
Patents.
The groups Rat', Rbl, R°i, Raa, Rbz, and Rya in the above Formulae are
pzeferably a
hydrogen, or a substituted or unsubstituted Cl-C8 alkyl or Cl-C$ alkoxy group
or a hydroxy
group. Preferred Ral and R~ groups are hydxogen, hydroxyl, alkyloxy groups
(especially
LS lower~alkyloxy groups, e.g, methoxy), aryloxy, acyloxy, and aroyloxy (i.e.,
R-(C=O)-O-,
wherein R is aliphatic ox aromatic).
The phrase "R.a and Rb both taken together along with the nitrogen atoms to
which
they are bound to form a ring structure" means that the two Ra and Rb groups
are a moiety
which joins the two nitrogen atoms in a heterocycle, such as the following
ring structures:
/\~Zr .
~,--N
;p '~'z ~R~, whereinr is an integer from zero to 4 inclusive,
~/.~/y r .
°z'~.~N/ Ro
wherein r is an integer from zero to 2 inclusive,
,~Zr ,
N\
N
~~ \Rc wherein r is an ~inte er from zezo to 6 '
g inclusive,
22
CA 02488493 2004-12-07
WO 03/103598 PCT/US03/17992
/ /Z~
or '~ ~~ ,wherein r is an integer from Zero to 4 inclusive.
In another embodiment of the invention, fox example, in compounds of Formula
II,
Rsl and Rb~ or R~ and Rb2 are both taken together along with the nitrogen
atoms to which
they are bound to form a ring structure which is a nonaromatic ring, or an
alicyclic ring, or a
monocyclic ring, or a non-fused ring.
In some embodiments of Formula II, e.g., Ral, Rbl, R~i, Rte, Rb2, anal
R°a axe
preferably a hydxogen, or a substituted or unsubsfiituted Cl-C8 alleyl group,
wherein the
allcyl substituent is any member of the group Z defined above, but not an aryl
(e.g., phenyl)
or alkyl group. Likewise, in certain embodiments of Formula II, Rl is a moiety
selected
l0 , from the Z group defined above other than an substituted aryl (e.g.,
phenyl) or heteroaryl
group.
The groups Rt and RZ are preferably a Hydrogen, a substituted ox unsubstituted
C1-
Cs alkyl group, a substituted or unsubstituted CZ-C$ alkenyl group, a halogen
(particularly
bromine), a substituted or unsubstituted aryl or heteroaryl group, a
substituted or
.5 unsubstituted amino group, a nitro group, or a substituted or unsubstituted
Cl-C$ alkoxy
group (particularly methoxy).
Each Y group may be a dixect bond, or a "linking moiety" (or "Iinlang group")
which is a group that is covalently bound to at least two other moieties and
may be, for
example, a single divalent atom or an oligomethylene group. A linking moiety
which is a
;0 linear chain of carbon atoms may be optionally substituted or unsaturated.
23
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WO 03/103598 PCT/US03/17992
Preferably a linking moiety is relatively small compared to the rest of the
molecule,
and more preferably less than about 2S0 molecular weight, and even more
preferably less
than about 7S molecular weight. Especially preferred linking moieties axe -
(CHZ)ri
(wherein n is 1, 2, or 3), NR'- (where R' is hydrogen, a Cl-CS alkyl, CZ-CS
all{enyl, Cz-CS
S all~ynyl, or aryl group), -S-, -O-; NH-CHz-, and -CH=CH- (both E and Z
configuxations), or combinations thereof. The linking moiety may also be
(CR°R"~n,
CR°OR"'(CR"R~n, CR°SH(CR"RY)n, CR°NR""R"(CRyRZ)n,
(CR°R''~n0(CR"Ry)n, wherein
each n is independently either 0,1, 2, or 3, and R°, RW, R", Ry, and Rz
are each
independently hydrogen, a substituted or unsubstituted Cl-CS branched or
straight chain
alkyl or allcoxy, C2-CS branched or straight chain alkenyl, aryloxycarbonyl,
arylaminocarbonyl, arylalkyl, acyl, aryl, or C3-C$ ring group.
"I~ihibition" of amyloid deposition includes preventing or stopping of amyloid
formation, e.g., fibrillogenesis, inhibiting or slowing down of further
amyloid deposition in
a subject with amyloidosis, e.g., already having amyloid deposits, and
reducing or reversing
amyloid fibrillogenesis or deposits in a subject with ongoing amyloidosis.
Inhibition of
amyloid deposition is determined relative to an untreated subject, or relative
to the treated
subject prior to treatment, or, e.g., determined by clinically measurable
improvement in
pancreatic function in a diabetic patient, or in the case of a patient with
brain amyloidosis,
e.g., an Alzheimer's or cerebral amyloid angiopathy patient, stabilization of
cognitive
function or prevention of a fin -ther decrease in cognitive function (i.e.,
preventing, slowing,
or stopping disease progression).
The term "alkyl" includes saturated aliphatic groups, including straight-chain
alkyl
groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,
nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, teat butyl, isobutyl, etc.),
cycloallcyl (alicyclic)
p5 groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
ete.), alkyl '
substituted cycloalltyl groups, and cycloalkyl substituted allcyl groups.
Unless otherwise
specified, the term alkyl further includes allryl groups, which can further
include oxygen,
nitrogen; sulfux or phosphorous atoms replacing one or more carbons of the
hydrocarbon
backbone.
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In certain embodiments,~a straight chain or branched chain alkyl has 6 or
fewer
caxbou atoms in its backbone (e.g., Cl-Cs for straight chain, C3-Cs for
branched chain), and
more preferably A~ or fewer. Lilcewise, preferred cycloallcyls have from 3-8
caxbon atoms in
their ring structure, and more preferably have 5 or 6 carbons in the ring
structure. The term ,
Cl-C6 includes alkyl groups containing ~1 to 6 carbon atoms. An "alkylene"
group is a
divalent moiety derived from the corresponding alkyl group.
Moreover, unless otherwise specified the term alkyl includes both
"unsubstituted
alkyls" and "substituted allcyls," the latter, of which refexs to alkyl
moieties having
substituents replacing one or more hydrogens on one or more carbons of the
hydrocarbon
LO backbone. .Such substituents cau include, for.example, alkenyl, allcynyl,
halogen, hydroxyl,
allcylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
caxboxylate,
alkylcarbonyl, atylcarbonyl, allcoxycarbonyl, aminocaxbonyl,
alkylamin.ocarbonyl,
dialkylaminocarbonyl, allcylthiocaxbonyl, allcoxyl, phosphate, phosphonato,
phosphinato,
cyano, amino (including allcyl amino, diallcylamino, axylamino, diarylamino,
and
:5 alkylarylamino), .acylamino (including allcylcarbonylamiuo,
arylcaxbonylaxnino, carbamoyl
and uxeido), amidino, imino, sulthydryl, allcylthio, arylthio,
tbiocarboxylate, sulfates,
alkylsulfiuyl, sulfonato, sulfamoyl, sulfonamido, vitro, trifluoromethyl,
cyano, azido,
heterocyclyl, allcylaryl, or an aromatic or heteroaxomatic moiety.
Cycloalkyls~may be
further substituted, e.g., with the substituents described above.
;0 An "arylalkyl" moiety is an alkyl, group substituted with an aryl (e.g.,
phenyhnethyl
(i.e., benzyl)). An "alkylaryl" moiety is ari aryl group substituted with au
all{yl group (e.g.,
p:-methylphen~yl (i.e., p-tolyl)). The term "rz-allcyl" meaus a straight chain
(i.e., unbranched)
unsubstituted alkyl group.
The term "alkenyl" includes unsaturated aliphatic groups analogous in length
and
possible substitution to the alkyls described above, but that contain at least
one double bond..
For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g ,
ethylenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl,
etc.), branched-
chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclobutenyl,
cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl, etc.), allzyl or alkenyl
substituted cycloallcenyl
p ~ groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. The
term allcenyl may
further include alkenyl groups which include oxygen, nitrogen, sulfur or
phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone.
CA 02488493 2004-12-07
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Zn certain embodiments, a straight chain or branched chain alkenyl group has 6
or
fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-Cs for
branched
chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their
ring
structure, and more preferably have 5 ox 6 carbons in the zing structure. The
term CZ-C6
includes allcenyl groups containing 2 to 6 carbon atoms. An "alkenylene" group
is a
divalent moiety derived from the corresponding alkenyl group.
Moreover, unless otherwise specified the term allcenyl includes both
"unsubstituted
allcenyls" and "substituted alkenyls," the latter of which refers to alkenyl
moieties having
substituents replacing one or more hydrogens on one or more carbons of the
hydrocarbon
LO backbone. Such substituents can include, for example, alkyl groups, alkynyl
groups,
halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, allcoxycarbonyloxy,
aryloxycaxbonyloxy,. carboxylate (and lower allcyl esters thereof),
alkylcarbonyl,
arylcaxbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
diallcylaminocarbonyl,
alkylthiocarbonyl, allcoxyl, phosphate, phosphonato, phosphinato, cyano, amino
(including
.5 alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino
(including alkylcarbonylamino, arylcaxbonylamino, carbarnoyl and ureido),
amidino, imino,
sulfhydryl, allcylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl,
sulfonato, sulfamoyl,
sulfonamido, vitro, triffuoromethyl, cyano, azido, heterocyclyl; alkylaxyl, ox
au aromatic or
heteroaromatic moiety.
0 The term "alkynyl" includes unsaturated aliphatic groups analogous in length
and
possible substitution to the alkyls described above, but which contain at
least one triple
bond. For example, the term "allcynyl" includes straight-chain alkynyl groups
(e.g.,
ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl,
decynyl, etc.),
branched-chain alkynyl groups, and cyeloalltyl or cycloalkenyl substituted
allcynyl groups.
5 Unless specified otherwise, the term alkynyl further includes alkynyl groups
which include
oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of
the
. ' hydrocarbon backbone. In certain embodiments, a straight chain or branched
chain alkynyl
group has 6 or fewer carbon atoms in~its backbone (e.g., Cz-Cs for straight
chain, C3-Cs for
branched chain). The term CZ-Cs includes allcynyl groups containing 2 to 6
carbon atoms.
7 Ai'i "allcynylene" group is a divalent moiety derived from the corresponding
allcynyl group.
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Moreover, unless otherwise specified the term alkynyl includes both
"unsubstituted
allcynyls" and "substituted allcynyls," the latter of which refers to allcynyl
moieties having
substituents replacing one or more hydrogens on one or more carbons of the
hydrocarbon
backbone.
Such substituents can include? for example, allcyl groups, allcynyl groups,
halogens,
.hydroxyl, allcylcarbonyloxy, arylcarbonyloxy, allcoxycarbonyloxy,
aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbanyl, dialkylaminocarbonyl, all~ylthiocarbonyl, allcoxyl,
phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino, diallcylamino,
arylamina,
l0 diarylamino, and alkylarylamino), acylamino (including allcylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino, suli~hydryl,
alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfmyl, sulfonato, sulfamoyl, sulfonamido,
vitro,
trifluoromethyl, cyano, azido, heterocyclyl, all~ylaryl, ax an aromatic or
heteroaromatic
moiety.
.5 Unless the number of carbons is otherwise specified, "lower alkyl" as used
herein
means an all~yl group, as defined above, but having from one to five carbon
atoms in its
backbone structure. "Lower alkenyl" and "lower alkynyl" have chain lengths af,
for
example, 2-5 carbon atoms.
The term "acyl" xefers to a carbonyl group that is attached through its carbon
atom
;0 to ahydrogen (i.e., a formyl), an aliphatic group (e.g., acetyl), an
aromatic group (e,g,,
benzoyl), and the like. .The term "substituted acyl" includes acyl groups
where one or more
of the hydrogen atoms on one or more carbon atoms are replaced by, for
example, an alkyl
group, allcynyl group, halogen, hydroxyl, allcylcarbonyloxy, axylcarbonyloxy,
alkoxybarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl,
5 ~ a~koxycarbonyl, aminocaxbonyl, alkylaminocarbonyl, diall~ylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino
(including
allcyl amino, diallcylamino, arylamino,~diarylamin.o, and alkylarylarnino),
acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imin.o,
sulfliydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl,
0 sulfonamido, vitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl,
or an aromatic or
heteroaromatic moiety.
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The term "acylamino" includes moieties wherein an amino moiety is bonded to an
acyl group. For example, the acylamino group includes allcylcarbonylamino,
axylcarbonylamino, carbamoyl and ureido groups.
The terms "alltoxyallcyl", "allcylaminoalltyl" and "thioalkoxyalkyl" include
allcyl
S groups, as described above, which further include oxygen, nitrogen or sulfur
atoms
replacing one or more carbons of the hydrocarbon backbone.
The terms "alkoxy" or "alkyloxy" include substituted and unsubstituted alkyl,
alkenyl, and alkynyl groups covalently linl~ed to an oxygen atom. Examples of
allcoxy
groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy
groups.
l0 Examples of substituted allcoxy groups include halogenated alkoxy groups.
The allcoxy groups can be substituted with groups such as alkenyl, allcynyl,
halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, allcoxycarbonyloxy,
aryloxycarbonyloxy,
.carboxylate, allcylcarbonyl, arylcarbonyl, allcoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, allcylthiocarbonyl, allcoxyl,
phosphate,
S phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino,
diarylamiuo, and alkylarylamino), acylamino (including allcylcarbonylamino,
arylcarbonylamino, carbamoyl and uxeido), amidino, imino~ sulfhydryl,
alkylthio, arylthio,
thiocarboxylafe, sulfates, allcylsulfinyl, sulfonato, sulfamoyl, suLfonamido,
vitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or
heteroaromatic
0 moieties. Examples of halogen substituted alkoxy groups include, but are not
limited to,
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,
dichloromethoxy,
trichloromethoxy, etc., as well as perhalogenated alkyloxy groups.
The term "amine" or "amino" includes compounds or moieties in which a nitrogen
atom is covalently bonded to at least one carbon or heteroatom:
The term "alkylamino" includes groups wherein the nitrogen is bound to at
least one
allcyl group. The term "diallcylairiino" includes groups. wherein the nitrogen
atom is bound
to at least two alkyl groups.
The term "arylamino" and "diairylamino" include groups wherein the nitrogen is
bound to at least one or two aryl groups, respectively.
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The term "allcylarylamino" refers to an amino group which is bound to at least
one
alkyl group and at least one aryl group.
The term "alkaminoallcyl" refers to an allcyl, allcenyl, or allcynyl group
substituted
with an alkylamino group.
S The term "amide" or "aminocarbonyl" includes compounds or moieties which
contain a nitrogen atom which is bound to the carbon of a carbonyl or a
tbiocarbonyl group.
The term "carbonyl" or "carboxy" includes compounds and moieties wluch contain
a carbon connected with a double bond to an oxygen atom. Examples of moieties
which
contain a carbonyl include aldehydes, ketones, carboxylic acids, amides,
estexs, anhydrides,
etc.
The term "ether" ox "ethereal" ilicludes compounds or moieties wluch contain
an
oxygen bonded to two carbon atoms. For example, au ether or ethereal group
includes
"allcoxyallcyl" which refers to all allcyl, alkenyl, or alkynyl group
substituted with an alkoxy
.group. _ .
LS The term "hydroxy" or "hydroxyl" includes the groups -OH or -O- (with an
appropriate counter ion).
The term "halogen" includes fluorine, bromine, chlorine, iodine, etc. The term
"perhalogenated" generally refers to a moiety wherein all hydrogens are
replaced by
halogen atoms.
!0 Arylenediallcylene or arylenedialkyl groups include those groups which have
an
arylene group to which are bound two other alkylene groups, which may be the
same or
different, and which two alkylene groups are in turn bound to other moieties.
Examples of .
arylenedialkylene or arylenedialkyl groups include the .following:
Rh
CR ~I~ ~ In
h , ( 2)f (~RZ)f
~ R2)f / \ (CR2)9 . ~ (CRZ)9 _ C
R
~ , Or ~ 2)g ~ ~d
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~(CR2)F
\ \
/ / (~R2)f \ \(CR2)g
'(CR2\ g I , j / v
,
\(CR2)~
\ \
CR
\ \ ( 2~g ~ / /
/ / (CR2)g
(CR2)f . , or
wherein each R group is independently a hydrogen (preferred) or is selected
from
the group Z defined above, and 1<~<8, 1<g~8, 0<~~9..
Allcylenediarylene groups include groups which have an alkylene (or
cycloalkylene)
group to which are bound two other arylene groups, which may be the same or
different,
and which two allzylene groups are in turn bound to other moieties. Examples
of
allcylenediarylene groups include the following:
_~1
(CRz)f-'~~(CRz)y ~ / (CR2)g
and
. Ih ~ i
0 WR2)f / \ (C3~C Y ) ofo \ / (CR
afk ( ~
wherein each R group is,
independently a hydrogen (preferred) or is selected.from the. group Z defined
above,
1<_y_10 (preferably 1<y<4),15f_<8, 1Sg<_8, 0_<l~<4, and 0_<i~4..
Heteroarylenediallcylene or heteroarylenedialkyl groups include those groups
which
have a heteroarylene group to which are bound two other allcylene groups,
which may be
the same or,different, and which two allcylene groups are in turn bound to
other moieties.
Examples of heteroarylenediallcylene or heteroarylenedialkyl groups include
the following:
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UR2~f ~ ~ ~ (C\R2~g ,
wherein 0_<h<_3, and 0_<,i<-3, and X = NR'
(iwhexein R' is hydrogen, a Cl-CS allcyl, C2-C5 allcenyl, Cz-CS alkynyl, or
aryl group), 0, ox'
S, 15f<_8,1<g<_8, .
Rh~ CR2)f Rn '
>CR (CR2) ~ ~ CR
2)g f___C~( 2)g
or X . \ , whexein 0~, and X = NR'
(wherein R' is hydxogen, a Cl-Cs alkyl, CZ-CS alkenyl, C2-CS allcynyl, or aryl
group), O, or
S,1<f~8,1_g<_8,
Rh\~~R2)f R~\ \
1 (CR2) ~ ~ ~ i
(CR2)f N (CR2)g
or \ , wherein 0~3, 18,1<g_<8,or
Rh\N~CRZ)f Rh\N~
cR
\ 2)g (CR2)f N (CR2)g
ox \ , wherein 0_~,
wherein each R group is independently a, hydrogen (preferred) ox is selected
from the group
l0 Z defined above, 1<_~8, 1<_g<-8, and h and i are as indicated.
An arylene group is an aromatic group which is capable, of being connected
covalently to othex substituents through at least two positions, W eluding the
following
examples:
\ \
> > > >
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J
\ \ ( \ \ . / /
/ . /, ~ / / . ~ or
wherein each R group is independently a hydrogen (preferred) or is selected
from the group
Z defined above, and 0_<1~.~; for example: ,
OH
S A heteroarylene group is a heteroaromatic group which is capable of being
connected covaleritly to other substituents through at least two positions,
including the
following examples:
. .
O ,
wherein 0~3, and 0~<_3, and X = NR.' (wherein R' is
hydrogen, a Cl-CS alkyl, CZ-CS allcenyl, CZ-Cs alleynyl, or aryl group), O, or
S,
R~, , Rn .
/\X\ or X , wherein 0~<.2, and X = NR.' (wherein R' is
hydrogen, a Ci-Cs alltyl, Cz-C5 alkenyl, C2-CS alltynyl, or aryl group), O, or
S;
Rh\ \ ..
J ~~
or N , wherein 0_~3, or
R~'\Nw
J
or N , wherein 0<_h<2,
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wherein each R group is independently a hydrogen (preferred) or is selected
from the group
Z defined above, and h and i are as indicated; for example, the following
groups;
0 ~ N
.N~ ,0 w O
N
> > > >
H3C CH3 CH3
N I ~ ~ ~ N~~N
CH3 ~ O ~ O , or ~~.
Likewise, the invention relates to the following beteroarylene groups
Rh
~i~ ~ ~ , ~ _~.,
~2~f ,~ X ~ ~ ~C\~2~g -
wherein X - NR' (wherein R' is
hydrogen, a Cl-CS alkyl, Cz-CS alkenyl, Ca-CS alkynyl, or axyl group), 0, or
S; 0-<f<8,
0<g<_8; and each R group is independently a hydrogen (preferred) or is
selected from the
group Z defined above.
0 In general, the term "aryl" includes groups, including 5- and 6-membered
single-
ring aromatic groups that may include from zero to four heteroatoms, for
example, groups
derived from benzene, pyrrole, furan, tliiophene, thiazole, isothiaozole,
imidazole, triazole,
tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine, and
the like.
Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., groups
derived
from tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole,
benzothiazole,
benzoimidazole,~benzothi.ophene, methylenedioxyphenyl, quinoline,
isoquinoline,
napthyridine, indole, benzofuran, purine, benzofuxan, deazapurine, or
indolizine.
Those aryl groups having heteroatoms in the ring structure may also be
referred to as
0 "aryl heterocycles," '~eterocycles," "heteroaryls" or "heteroaromatics".
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An aromatic ring can be substituted at one or more ring positions with such .
substituents as described above, as for example, halogen, hydroxyl, allcyl
(e.g. tolyl),
alkoxy, allcylcarbonyloxy, arylcarbonyloxy, allcoxycarbonyloxy,
aryloxycarbonyloxy,
carboxylate, allcylcarbonyl, allcylaminoacarbonyl, arylalkyl aminocarbonyl,
S alkenylaminocarbonyl, allcylcarbonyl, arylcarbonyl, arylallcylcarbonyl,
allcenylcarbonyl,
allcoxycarbonyl, aminocaxbonyl, allrylthiocarbonyl, phosphate, phosphonato,
phosphinato,
cyano, amino (including allcyl amino, diallcylainino, arylamino, diarylamino,
and .
alkylaxylamino), acylamino (including allrylcarbonylamino, arylcarbonylamino,
caxbamoyl
and ureido), amidino, imino, sulfhydryl, allcylthio, arylthio,
thiocarboxylate, sulfates,
allcylsulfinyl, sulfonato, sulfamoyl, sulfonamido, vitro, trifluoromethyl,
cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
,Aryl groups can also be fused or bridged with alicyclic ox heterocyclic rings
which
are not aromatic so as to form a polycycle (e.g., tetralin).
The term "heterocyclic" or "heterocycle" includes heteroaryls as well as any
ring
LS formed which incorporate a heteroatom or au atom which is not carbon.
The~ring may be
saturated or unsaturated and may contain one or more double bonds. Examples of
preferred
heterocyclic groups include pyridyl, furanyl, thiophenyl, morpholinyl, and
indolyl groups.
The term "heteroatom" includes atoms of any element other than carbon or
hydrogen.
Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
!0 ' An "axylene" group is a divalent moiety derived from an aryl group.
An oligoethereal group, such as an oligo(allcyleneoxide) group, includes
polyethyleneglycol (PEG) and short chain analogs thereof including
-[(CRZ)SO]t(CRZ)~--, wherein I_<t<_6 and 1<_s<_6, and each R group is
independently a
hydrogen (preferred) or is selected from the group Z defined above.
:5 An arylene-di(oligoalkyleneoxide) group is an aryl group which has two
oligoallcyleneoxide groups bound to it which in turn are bound to other
moieties, and
include the following examples:
[(CR2)sG]t(CR2)S AnY~-L(CR2)SGIt(CR2)s
a
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wherein "Aryl" is au arylene moiety, 1_<t_<5z 0<s<_6, and each R group is
independently a
hydrogen (preferred) or is selected from the group Z defined above. Pxeferred
arylene-
di(oligoalltyleneoxide) groups include:
Rh .
L(CRZ)solt(CR2)S ~ ~ I(CR2)S~lt(C~'2)s
,
f~R2)Solt(CR2)S ~ ~
~~CRz)sOlttCR2)s
' ' , or
Rn
L(CR2)sOlt(CR2)S
f(~ R~)S~lt(cRz)s
wherein 1<t_<6, Oss_<6, 0<l~<4, and each R group is independently a hydrogen
(preferred) or
is selected from the group Z defined above.
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The term "substituted" means that the moiety has substituents placed on the
moiety
other than hydrogen which allow the molecule to perFonn its intended function.
Examples
of substituents include moieties selected from straight or branched allcyl
(preferably Cl-CS},
cycloallcyl (preferably C3-C$), allcoxy (preferably Cl-C6), thioallcyl
(preferably Ct-C6),
alkenyl (preferably Cz-C6), allcynyl (preferably C2-C6), heterocyclic,
caxbocyclic, aryl
(e.g., phenyl), aryloxy (e.g., phenoxy), axalkyl (e.g., benzyl), aryloxyalkyl
(e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl,
allcylcarbonyl.and
arylcarbonyl or other such acyl group, hetexoarylcarbonyl, or heteroaryl
group,
(CR'R")o...3NR'R" (e.g., NH2), (CR'R")~3CN (e.g., --CN), NOa, halogen (e.g.,
F, Cl, Br, or
n, (CR'R")~3C(halogen)3 (e.g., -CF3), (CR'R")o 3CH(halogen)z,
(CR'R")o-.3CH2(halogen), (CR'R")~3CONR'R", (CR'R")o 3(CNH)NR'R",
(CR'R")o 3s(O)1-z~'R"~ (CR'R")o-3CH0, (CR'R")o-s0(CR-'R")o-3H,
(CR'R")o-3S(O)o-sR.' (e.g., -S03H), (CR'R")o-30(CR'R"}o-3H (e.g., -CH20CH3 and
-
OCH3), (CR'R")o-sS(CR'R")o-3H (e.g., ~H and-SCH3), (CR'R")a-sOH (e.g., -OH),
(CR'R")~3COR', (CR'R")~3(substituted or unsubstituted phenyl),
(CR'R")o..s(C3-Cs cycloallcyl), (CR'R")~3C02R' (e.g., -COZH), or (CR'R")~30R'
group, or
the side chain of any naturally occurring amino acid; wherein R' and R" are
each
independently hydrogen, a Cl-CS alkyl, G2-CS alkenyl, Ca-Cs allcynyl, or aryl
group, or R'
and R" taken together are a benzylidene group ox a -(CHa)20(CHZ)2- group.
Preferably,
substitutions enhance the ability of the compounds of the invention to perform
its intended
function, e.g., inhibit formation of amyloid deposits.
In compounds of the invention, it is preferred that m=1 and that n--0, 1, or
2. In
compounds ~of Formula I, preferably p=0, l, or 2, and q--1. It is especially
preferred that
molecules according to Formula I are symmetric, thus Ral Rte, Rbl=Rb~,
R°i R°2, m=q,
n~, and Yl=Ya. Likewise, it is preferred that Rl Ra, and Xl=X2 in molecules of
Formula
I.
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One group of preferred compounds of the invention are those of Formula Ia:
a1 a2
R ~N N'R
b1 ~ ~ Rb2
R ~N / / N.
- ~ ~ ~z
M ~ R (Formula Ia)
wherein M is
Rt,1 Rnz
X
wherein, in a preferred aspect, Ral and Rbl together, ox Raz and Rbz together,
represent a Cz
to C3 alkylene; R°1 and R°z ara H; Rhl is H; and Rhz is OCH3 ox
0(C6H~)R, wherein R is H
ox lower-allcyl, and X is 0, NR' (wherein R' is hydrogen, a Cl-C5 alkyl, Cz-CS
alkenyl, Cz-
CS alkynyl, or aryl group), or S.
In another group of preferred compounds of Formula Ia, Ral and Rbl together,
or Raz
and Rbz together, represent a Cz linear, saturated alkylene; R°' and
R°z are -(lower alkyl)-
OH; and Rhl and Rha are each H. The "lower alltyl" group of R~l and R°z
are preferably
ethylene.
In yet another group of preferred cbmpounds of Formula Ia, Ral and Rbl
together, or
R~z and Rbz together, represent a C4 alkylene; R~l and R°z are H
(preferred), lower alkyl,
. cycloaJkyl, aryl, hydroxyalkyl, a~ninoalkyl or alkylaminoalkyl; Rhl and Rhz
are
independently selected from the group consisting of H (preferred),
lower.alkyl, halogen,
alkoxy, aryloxy, or arylalkoxy.
In still yet another group of preferred compounds of Formula Ia, Ral, R~z, Rbl
and
Rbz are H; R°1 and R~z are isopropyl or -(CHz)3N(CH3)2; and Rhl and R~z
are H.
In a further group of preferred compounds of Formula Ia, Ral and Rbl together,
or
Raz and Rbz together, represent a phenylene group which is optionally
substituted with up to
three -C0~°NReRf groups where Rd is lower allcyl and R' and Rf are each
irodependently
selected from the group consisting of H or lower allcyl; and R°l, R~z,
Rhl, and R~ are H.
An especially preferred compound of Formula Ia has Rht, R~'z, Rbt, R°i,
Rbz, and R~z
being H, and Ral and R~z groups being hydxoxy or methoxy.
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.Another group of preferred compounds are those of Formula Zb:
Ra1-N . N_Ra2
y~ f ~1~
Rbl--N ~ ~ I N-Rb2
v c1 M
R R1 R2 Rc2 ~ ~o~ula Ib)
wherein M is
Rn~ Rn2
X
wherein X is Oa NR' (whexein R' is hydrogen, a Cl-CS alkyl, C2-CS all~enyl,
CZ'-CS allcynyl,
or aryl group), or S; Rhl and R~ are each independently selected from the
group consisting
ofH, Iowerallcyl, aryl, allcylaryl,~aminoalltyl, aminoaryl, halogen, allcoxy,
aryloxy, or
oxyarylalkyl; Rl and RZ are each independently selected from the group
consisting of H,
Ioweralkyl, alkoxy, allcylaryl, aryl, ~aryloxy, aminoallcyl, aminoaryl, or
halogen; and each
Rai, R°~, Rbl, and Rb2 group is independently selected from the group
consisting of H,
loweralltyl, allcoxyalkyl, hydroxyallcyl, aminoallcyl, allcylaminoalkyl,
cycloallcyl, aryl,
hydroxy, or allcylaryl; or Ral and Rbl together, or R~ and Rba together,
represent CZ-Clo
allcyl, hydroxyalkyl, or alkylene; and each R°1 and R°~ group is
independently H, hydro~y,
loweralkyl, alkoxyallcyl, hydroxyalkyl, aminoall~yl, alkylamirio,
alkylaminoallcyl,
cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or allcylaryl.
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Another group of preferred compounds are those of Formula Ic:
(Formula Ic)
wherein M is
Rh~ Rnz
X
wherein X is. S, O, or NR' (wherein R' is hydrogen, a Ci-Cs allcyl, CZ-Cs
allcenyl, Cz-Cs
allcynyl, or aryl group); Rbt, Rb2, R~i, and Rya are each independently
selected from the
group consisting of H, loweraltcyl, alkoxy, alkoxyallcyl, cycloallcyl, aryl,
hydroxyallcyl,
aminoalkyl or altcylaminoalkyl; Rl and R2 are H, lower allcyl, alkoxy,
allcoxyalkyl,
liydroxyallcyl, cycloalkyl, aryl, aminoalkyl, allcylaminoalkyl or halogen; Ral
and R~ are
OY, or Ral and Rbl together, or R°a and Rb2 together represent '
Rs I
i
wherein Rs is
YON
R~RbN
Y is H or lower alltyl; each of Xl and Xa are -(CHa)n , where n is an integer
from 0 to 2; and
Rhl and Rl'~ are each independently selected from the group consisting of H,
lower alkyl,
halogen, alkoxy, aryloxy, or oxyarylalkyl.
Yet another group of preferred compounds are those of Formula Ic, wherein M is
-(CHZ)n- where n is an integer from 2 to 16 (or 2 to 12, or 2 to 10); each of
Xl and Xa is O,
NH, or S; Ral, Rte, Rbl,'and Rb2 are H; or Ra1 and Rbl together, or R~ and Rb2
together
represent -(CHZ)m-, 'wherein m is 2, 3, or 4; each of Rl and RZ are H, OCH3,
NOZ or NHZ;
R°1 and R°2 are~H, CH3 or CH2CH3.In another embodiment, when Xl
is O or S, both Rl and
R°i cannot be H; and when Xa is 0 or S, both R2 and R°2
cannot be H.
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Another group of preferred compounds are those of Formula Id:
~b2
(.Formula Id)
wherein each Ral, Raz, Rbi' and Rb2 are independently selected from the group
consisting of
H, loweralkyl, alkoxyallcyl, hydroxyallcyl, aminoalkyl, allcylaminoallcyl,
cycloalkyl, aryl, or
allcylaryl; or two R&1 and Rbt together, or R~ and Rb2 together represent Cz-
Cto allcylene; R°z
and R°Z are independently H, hydroxy, lowerallcyl, alkoxyalkyl,
hydroxyalkyl, aminoalkyl,
alkylaminoallcyl, cycloallcyl, aryl, or aJkylaryl; and R' is H, loweralkyl,
allcoxyalkyl,
hydroxyallcyl, arninoalkyl, allrylaminoalkyl, cycloallcyl, aryl, or
allcylaryl.
Another group of preferred compounds are those of Formula Ie:
~ (Formula Ie)
wherein M is an alkylene group (e.g., CZ to C16), and Xl and XZ are oxygen.
In another group of preferred compounds of Formula Ie, Ral and Rbl together,
or R~
and Rbz together, represent a C2 linear, saturated alkylene; R°1 and
R°2 are H.
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Another group of preferred compounds of the invention are those of Formula
IIa:
1)
n
(Formula ITa)
wherein E is
Ra1-N N N
b1
R -N N N
Rc1 or Rc1 or Rc1
wherein Yi, Yz, Z, and Ri are as defined above; n is 0 - 4; Y2 is preferably
0, NH, S, a
substituted or unsubstituted methylene group, or a direct bond; Z may be a
hydrogen atom,
or Z is preferably alkyl, aryl, alkoxy, aryloxy, hydroxy, a substituted or
unsubstituted
amino, nitro, sulfo, or halogen group; Ral, Rbl, and R°1 are
independently hydrogen, lower
alkyl, aromatic, hydroxyl, or allcoxy; and B is a direct bond or a substituted
or unsubstituted
allcylene group containing from 1 to 16 carbon atoms, or abiphenylene group,
or a
combination biphenylene-alkylene group, the group -[(CHZ)"O]m(CHZ)"- where m
is 1 to 6
and n is 2 to 6, or a heterocyclic group.
Compounds of Formula IIb axe also within the invention:
NH
R
H2N ~ / ~,UH2)n
4 (Formula IIb)
wherein n = 2, 3, 4, 5, 6, 7, S, 9, or I0; and R = hydrogen, hydroxy, halogen,
phenyl,
biphenyl, naphthyl, allcoxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, or
aryloxy.
41
CA 02488493 2004-12-07
WO 03/103598 PCT/US03/17992
Another group of preferred compounds are of Formula ITfa:
Ra1-N _ N_Raz
b1 \ ~ ~ N ~ / b2
R -N ~ -R
'Rc1 N M N Rcz..
H H (Formula 111'a)
wherein M is
Rh1 Rh2
X
wherein X is S, O, or NR' (wherein R' is hydrogen, a Ci-CS alkyl, Cz-CS
alkenyl, Ca-CS
allcynyl, or aryl group); Ral, Rte, Rbt, and Rb2 are each independently
selected from the
group consisting of H, lower alkyl, allcoxyallcyl, cycloallcyl, aryl,
alltylaryl, hydxoxyalltyl,
aminoallcyl, or alkylaminoallcyl; or Ral and Rbl together, or R~ and Rb2
together represent a
CZ to Ci° allcyl, hydroxyalkyl, or alkylene; or Ral and Rbi together,
or R~ and Rb2 together
are:
~R10) i
n j
wherein n is a number from I to 3, and R1° is H or -CONHR11NR.1SR16,
wherein R11 is
lower 'alkyl and Rls and R16 are each independently selected from the group
consisting of H
and lower alkyl; and R~l and R°2 are H, hydroxy, lower allcyl,
cycloalkyl, aryl, allcylaryl,
alkoxyalkyl, hydroxycycloalkyl, alkoxycycloalkoxy, hydzoxyallcyl, aminoalkyl
or ,
allcylaminoalkyl; and Rhi and R~ are.each independently selected from the
group consisting
ofH, lower allcyl, halogen, aryl, arylalkyl, aminoalkyl, aminoaryl, alkoxy,
aryloxy, or
oxyarylalkyl.
42
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Yet another group of preferred compounds are of Formula IZIb:
Ra1
Rbv
N
R
(Formula TIIb)
wherein each pair of Ral with Rbl and R~ with Rb2 together represent -(CH2)m-
wherein m is
from two to four; R°1 and R°a are independently H or
lowerallcyl; and M, which may be
substituted with a lower allcyl group, is selected from the group consisting
of -CH=CH-CHZ-
CH2-, -CHZ-CH=CH-CHz-, and -CH--CH-CH--CH-.
(Formula aIc)
wherein Ri and RZ are independently H or -CONHRSNR.6R7, wherein RS is lower
allcyl, R6
and R7 axe each independently selected from the group consisting of H and
lower allcyl; Ral,
Ra2~ Rbl, and Rb2 are independently selected from the group consisting of H,
lower allcyl,
alkoxyalkyl, hydroxyallcyl, aminoallcyl, allcylaminoallcyl, cycloallcyl, aryl,
ox alkylaryl, or
Ral and Rbl together, or R~ and Rba together repxesent CZ-Clo alkylene;
R°1 and R°z are
independently H, hydroxy, lower alkyl, alkoxyalkyl, hydroxyalkyl, aminoallcyl,
,
alkylaminoallcyl, cycloallcyl, aryl, or allcylaryl; R°3 and R'~ axe
independently H, hydroxy,
loweralkyl, alkoxyalkyl, hydroxyallcyl, aminoalkyl, allcylaminoallcyl,
cycloallcyl, aryl, or
allcylaryl; and R' is H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoallcyl,
alkylaminoalkyl,
cycloallcyl, aryl, allcylaryl, or halogen.
In another embodiment, the present invention relates to pharmaceutical
compositions comprising compounds according to any of the Formulae herein. for
the
treatment of an amyloid-related disease, as well as methods of manufacturing
such
pharmaceutical compositions.
43
Another group of preferred compounds are those of Formula llIc:
CA 02488493 2004-12-07
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The compounds of the invention can be formulated to ensure proper distribution
i~a
vivo. For example, the blood brain barrier (BBB) excludes many highly
hydrophilic
compounds. To ensure that the more hydrophilic therapeutic compounds of the
invention
cross the BBB, they can be formulated, for example, in liposomes. For methods
of
manufacturing liposomes, see,,e.g., U.S. Patent Nos. 4;522,811; 5,374,548; and
5,399,331.
The liposomes may comprise one or more moieties which are selectively
transported into
specific cells or oxgans ("targetingmoieties"), fihus providing targeted drug
delivery (see,
e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent
No.
5,416,016 to Low et al.); mannosides (CJmezawa et al. (1988) Biochem. Biophys.
Res.
Common. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;
M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein
A receptox
(Briscoe et al. (1995) Am. J. Physiol. 1233:134); gp120 (Schreier et al.
(1994) J. Biol.
Chem. 269:9090); see, also K.~Keinanen; M. L. Laukkanen (1994) FEES Lett.
346:123; J. J.
l5 Killion; I. J. Fidler (I994) Immunomethods 4:273. In a preferred
embodiment, the
therapeutic compounds of the invention are formulated in. liposomes; in a more
preferred
embodiment, the liposomes include a targeting moiety.
To ensure that compounds of the invention cross the.BBB, they may be coupled
to a
C
BBB transport vector (for review of BBB txansport vectors and mechanisms, see
Bickel, et
.0 al., Ado Drug1?eliveYy Reviews, vol. 46, pp. 247-279, 2001). Exemplary
transport vectors
include canonized albumin or the 0X26 monoclonal aunbody to the transferrin
receptor;
these proteins,undergo absorptive-mediated and receptor-mediated transcytosis
through the
BBB, respectively. '
44
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Examples of other BBB transport vectors that taxget receptor-mediated
transport
systems into the brain include factoxs such as insulin, ixisulin-like growth
factors (IGF-T,
IGF-Il], angiotensin II, atrial and brain natriuretic peptide (ANP, BNP),
interleulcin I (TL-1)
and transferrin. Monoclonal antibodies to the receptors which bind these
factors may also
be used as BBB transport vectoxs. BBB transport vectors targeting mechanisms
for
absorptive-mediated trauscytosis include catioiuc moieties such as cationized
LDL; albumin
or horseradish peroxidase coupled with polylysine, cationized albumin or
cationized
immunoglobulins. Small basic oligopeptides such as the dynorphin. analogue E-
2078 and
the ACTH analogue ebiratide can also cross the brain via absorptive-mediated
transcytosis
and are potential transport vectors.
Other BBB transport vectors target systems for transporting nutrients into the
brain.
Examples of such BBB transport vectors include hexose moieties, e.g. glucose,
monocaxboxylic acids, e.g. lactic acid, neutral amino acids, e.g.
phenylalanine, amines, e.g.
choline, basic amino acids, e.g. arginine, nucleosides, e.g. adenosine, purine
bases, e.g.
15~ adenine, and thyroid hormone, e.g. triiodothyridine. Antibodies to the
extracellular domain
of nutrient transporters can also be used as transport vectors. Other possible
vectoxs include
angiotensin II and ANP, which may be involved in regulating BBB permeability.
In some cases; the bond linking the therapeutic compound to the transport
vector
may be cleaved following transport into the brawn. in order to liberate the
biologically active
compound. Exemplary linlcers include disulfide bonds, ester-based linkages,
thioether
linkages, amide bonds, acid-labile linkages, and Schiffbase linkages.
Avidin/6iotin linkers,
in which avidin is covalently coupled to the BBB drug transport vector; may
also be used.
Avidin itself may be a drug transport vector.
To adrrlinister the therapeutic compound by other than parenteral
administration; it
may be necessary to coat the compound with, or co-administer the compound
with, a
material to prevent its inactivation. For example, the therapeutic compound
may be
administered to a subject in an appropriate carrier, for example, liposomes,
or a diluent.
Pharmaceutically acceptable diluents include saline and aqueous buffer
solutions.
Liposomes include water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejau et al., (1984) J. Neuroimmunol. 7:27).
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The therapeutic compound may also be administered parenterally,
intraperitoneally,
intraspinally, or intracerebxally. Dispersions can be prepared in glycerol,
liquid
polyethylene glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage
and use, these preparations may contain a preservative to prevent the growth
of
microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases, the
composition must
be sterile and must be fluid to the extent that easy syringability exists. It
must be stable
. under the conditions of manufacture and storage and must b a preserved
against the
contaminating action of microorganisms such as bacteria and fungi.
The vehicle can be a solvent or dispersion medium containing, for example,
water,
ethanol,.polyol (fox example, glycerol, propylene glycol, and liquid
polyethylene glycol,
and the Iike), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be
.5 maintained, for example, by the use of a coating such as lecithin, by the
maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of
the action of microorganisms can be achieved by various antibacterial and
antifungal agents,
for example, paxabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and
the like. In
many cases, it will be preferable to include isotonic agents, for example,
sugars, sodium
0 chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.
Prolonged
absorption of the injectable compositions can be brought about by including in
the
composition an agent which delays absorption, for example, aluminum
monostearate or
gelatin.
Sterile injectable solutions can be prepared by incorporating the therapeutic
compound in the required amount in an appropriate solvent with one or a
combination of
ingredients enumerated above, as required, followed by filtered sterilization.
Generally,
dispersions are prepared by incorporating the therapeutic compound into a
sterile vehicle
which contains a basic dispersion medium and the required other ingredients
from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-
drying which
yields a powder of the active ingredient (i. e., the therapeutic compound)
plus any additional
desired ingredient from a previously sterile-filtered solution thereof
46
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The therapeutic compound can be orally administered, for example, with an,
inert
diluent or an assimilable edible carrier. The therapeutic compound and other
ingredients
may also be enclosed in a hard or soft shell gelatin capsule, compressed into
tablets, or
incorporated directly into the subject's diet. Fox oral therapeutic
administration, the
therapeutic compound may be incorporated with excipients and used in the form
of
ingestible tablets, buccal tablets, troches, capsules, elixiurs, suspensions,
syrups, wafers, and
the like. The percentage of the therapeutic compound in the compositions and
preparations .
may, of course, be varied. The amount of the therapeutic compound in such
therapeutically
useful compositions is such that a suitable dosage will be obtained.
0 It is especially advantageous to formulate parenteral compositions in dosage
unit
form for ease of administration and uniformity ~of dosage. Dosage unit form as
used herein
refers to physically discrete units suited as unitary dosages for the subjects
to be treated;
each unit containing a predetermined quantity of therapeutic compound
calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical
vehicle. The specification for the dosage unit forms of the invention are
dictated by and
directly dependent on (a) the unique characteristics of the therapeutic
compound and the
particular therapeutic effect to be achieved, and (b) the limitations inherent
in the art of
compounding such a therapeutic compound for the treatment of amyloid
deposition in .
subjects.
~0 The present invention therefore includes pharmaceutical formulations
comprising
the compounds of the Formulae described herein, including pharmaceutically
acceptable
salts thereof, in pharmaceutically acceptable carriers, for aerosol, oral and
paxenteral
administration. Also, the present invention includes such compounds, or salts
thereof, ,
which have been lyophilized and which may be reconstituted to form
pharmaceutically
25 . ~ acceptable formulations for administration, as by intravenous,
intramuscular, or
subcutaneous injection. Adxniiiistration may also be intradermal or
transdermal.
In accordance with the present invention, a compound of the Formulae described
herein, and pharmaceutically acceptable salts thereo f may be administered
orally.or through
inhalation as a solid, or may be administered intramuscularly or intravenously
as a solution,
30 suspension or emulsion. Alternatively, the compounds or salts may also be
administered by
inhalation, intravexlously or intramuscularly as a liposomal suspension.
47
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Pharmaceutical formulations are also provided which are suitable for
administration
as an aerosol, by inhalation. These formulations comprise a solution or
suspension of the
desired compound of any Formula herein, or a salt thereof, or a plurality of
solid particles of
the compound or salt. The desired formulation may be placed in a small chamber
and
nebulized. Nebulization may be accomplished by compressed air or by ultrasonic
energy to
form a plurality of liquid droplets or solid particles comprising the
compounds or salts. The .
liquid droplets or solid particles should have a particle size in the range of
about 0.5 to
about 5 microns. The solid particles can be obtained by processing the solid
compound of
any Formula described herein, or a salt thereof, in any appropriate manner
laiown in the art,
such as by micronization. Most preferably, the size of the solid particles or
droplets will be
from about 1 to about 2 microns. rn this respect, commercial nebulizers are
available to
achieve this purpose.
Preferably, when the pharmaceutical formulation suitable for administration as
an
aerosol is in the form of a liquid, the formulation will comprise a water-
soluble compound
l5 of any Formula described herein, or a salt thereof, in a carrier which
comprises water. A
surfactant may be present Which lowers the surface tension of the formulation
sufficiently to
result in the formation of droplets within the desired size range when subj
ected to
nebulization.
Active compounds are administered at a therapeutically effective dosage
sufficient
r0 to inhibit amyloid deposition in a subject. A "therapeutically effective"
dosage preferably
inhibits amyloid deposition by at least about 20%, more preferably by at least
about 40%,
even more preferably by at least about 60%, and still more preferably by at
least about 80%
~~ relative to untreated subjects. In the case of an Alzheimer's patient, a
",therapeutically
effective" dosage stabilizes cognitive function or prevents a further decrease
in cognitive
5 function (i.e., preventing, slowing,. or stopping disease progression).
48
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The ability of a compound to inhibit amyloid deposition can b~e evaluated in
an
animal model system that may be predictive of efficacy in inhibiting amyloid
deposition in
human diseases, such as a transgenic mouse expressing human APP or other
relevant animal
models where A(3 deposition is seen. Likewise, the ability of a compound to
prevent or
reduce cognitive impairment in a model system may be indicative of efficacy in
humans.
Alternatively, the ability of a compound can be evaluated by examining the
ability of the
compound to inhibit amyloid fibril formation ire vit'ro, e.g., using a
~.brillogenesis assay
such as that described herein, including a ThT, CD, or EM assay. Also the
binding of a
compound to amyloid fibrils may be measured using a MS assay as described
herein.
.0 The present invention is also related to prodrugs of the compounds of the
Formulae
disclosed herein. Prodrugs are compounds wl~.ch are converted in vivo to
active forms (see,
e.g , R.B. Silverman, 1992, "The Organic Chemistry of Drug Design and Drug
Action,"
Academic Pxess, Chp. 8). Prodrugs can be used to alter the biodistribution
(e.g., to allow
compounds which would not typically enter the reactive site of the protease)
or the
5 pharmacokinetics for a particular compound. Fox example, a carboxylic acid
group, can be
esterified, e.g., with a methyl group or an ethyl group to yield an ester.
When the ester is
administered to .a subject, the ester is cleaved, enzymatically or non-
enzymatically,
reductively, oxidatively, or hydrolytically, to reveal the anionic group. An
anionic group
can. be esterified with moieties (e.g., acyloxymethyl esters) which are
cleaved to reveal an
intermediate compound which subsequently decomposes to yield the active
compound. The
prodrug moieties may be metabolized in vivo by esterases or by other
mechanisms to
carboxylic acids.
Examples ofprodrugs and their uses are well known in the art (See, e.g., Berge
et al.
(1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). .The prodrags can be
prepared ire
situ during the final isolation and purification of the compounds, or by
separately reacting
the purified compound in its free acid form with a suitable derivatizing
agent. Carboxylic
acids can be converted into esters via treatment with an alcohol in the
presence of a catalyst.
49
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Examples of cleavable carboxylic acid prodrug moieties include substituted and
unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., ethyl
esters, propyl
estexs, butyl estexs, pentyl esters, cyclopentyl esters, hexyl esters,
cyclohexyl esters), lower
alkenyl esters, dilower alkyl-amino lower-alltyl esters (e.g.,
dirnethylaminoethyl ester),
acylamino lower allcyl esters, acyloxy lower alkyl esters (e.g.,.
pivaloyloxymethyl ester),
aryl estexs (phenyl ester), axyl-louver alkyl estexs (e.g., benzyl ester),
substituted (e.g., with
methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters,
amides, lower-alkyl
amides, dilower alkyl amides, and hydroxy amides.
It will be noted that the stntctures of some of the compounds of this
invention
include stereogenic carbon atoms. It is to be understood accordingly that the
isomexs
arising from such asymmetry (e.g., all enantiomers and diastereomers) are
included within
the scope of this invention unless indicated otherwise. That is, unless
otherwise stipulated,
any chiral carbon center may be of either (R)- or (~-stereochemistry. Such
isomers can. be
obtained in substantially pure form by classical separation techniques and by
l5 stereochemically controlled synthesis. Furthermore, allcenes caminclude
either the E- or Z
geometry, where appropriate.
Certain embodiments of the present compounds can contain a basic functional
group, such as amino or alkylsmixio, and are, thus, capable of forming
pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically
;0 acceptable salts" in this respect, refers to the relatively non-toxic,
inorganic and organic
acid addition salts of compounds of the present invention. These salts can be
prepared ih
situ during the final isolation and purification of the compounds of the
invention, ox by
separately reacting a purified compound of the invention in its free base form
with a suitable
organic or inorganic acid, and isolating the salt thus formed.
5 Representative salts include the hydrohalide (including liydrobxomide and .
hydrochloride), sulfate, bisulfate, phosphate, nitrate, acetate, valerate,
oleate, palinitate,
stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, 2-
hydroxyethylsulfonate, and
laurylsulphonate salts and the lilte. (See, e.g., Berge et al. (1977)
'pharmaceutical Salts", J.
0 Pharm. Sci. 66:1-19).
CA 02488493 2004-12-07
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In other cases, the compounds of the present invention may contain one or more
acidic functional gxoups and, thus, are capable of forming pharmaceutically
acceptable salts
with pharmaceutically acceptable bases. The term 'pharmaceutically acceptable
salts" in
these instances refers to the relatively non-toxic, inorganic and organic base
addition salts
S of compounds of the present invention.
These salts can lilcewise be prepared in situ during the final isolation and
purification
of the compounds, or by separately reacting the purified compound in its free
acid form.
with a suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically
acceptable metal canon, with ammonia, or with a pharmaceutically acceptable
organic
primary, secondary or tertiary amine. Repxesentanve alkali or alkaline earth
salts include
the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the
like.
Representative organic amines useful for the foxxn.anon of base addition salts
include
ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,
piperazine and
the like.
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments and
methods
described herein. Such equivalents are intended to be encompassed by the scope
of the.
following claims. All patents, patent applications, and literature references
cited herein are
hereby expressly incorporated by reference in their entirety. This invention
is fiuther
0 illustrated by the following examples which should not be construed as
limiting.
EXAMPLES
51~
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The synthesis of anzidine compounds of the invention is described in U.S.
Patent
Nos. 5,428,051, 4,963,589, 5,202,320, 5,935,982, 5,521,189, 5,686,456,
5,627,184,
5,622,955, 5,606,058, 5,668,167, 5,667,975, 6,025,398, 6,214,883, 5,817,687,
5,792,782,
5,939,440, 6,017,941, 5,972,969, 6,046,226, 6,294,565 (B1), 6,156,779,
6,326,395,
6,008,247, 6,127,554, 6,172,104, 4,940,723, 5,206,236, 5,843,980, 4,933,347,
5,668,166,
5,817,686, 5,723,495, 4,619,942, 5,792,782, 5,639,755, 5,643,935, 5,602,172,
5,594,138,
and S,S78,63I. Many of the compounds may also be purchased from Sigma-Aldrich
Co.
(Milwaukee, USA). The compounds may also be synthesized according to art
recognized
techniques. ,
Test compounds were purchased from commercial sources or synthesized and
screened by thioflavin T fluorescent assay ("ThT assay"). Alternatively, one
could screen
test compounds by circular dichroism ("CD"), electron microscopy ("EM"), or
mass
spectroscopy ("MS") assays. The MS assay gives data on the ability of
compounds to bind
to an amyloid protein , whereas the ThT, EM, and CD assays give data on
inhibition of
fibrillogenesis.
The thioflavin T fluorescent assay for fibrillogenesis is based on the
principle that
the fluorescent dye, thioflavin T, binds specifatcally to fibrillar, but not
to unaggregate Af3
peptide (LeVine III, H., 1993, Protein Science 2:404-410). Upon binding,
thioflavin T
develops a characteristic fluorescence (Naiki, H., et al.,1996, Lab. Invest.
74: 374-383)
which can be easily detected. The dye is believed to interact with the stacked
cross-ji
pleated sheets, the common structural motif of all amyloid (LeVine III, H.,
1995, Amyloid:
Int. J. Exp. Clin Invest..2:1.6). Tluoflavin T is widely used to assay the
effect of compounds
on fibxillogenesis of A(3 peptide and other amyloid proteins (Bronfinan, P.C.,
et al.,1995,
Neuroscience Lett. 218:201 203). In this assay, test compounds are incubated
with a
2S solution of A(3 (1-40) (20 iZM) or IAPP (10 wM) containing 5 ~.iM
Thioflavin T, in 0.02M
Tris/0.02M.acetate/O.15M NaC110.005% azidelpH 7.40 at 37°C in sealed
384 well
microplates. Readings (ex 430 nm/em~485nm) are taken at various time intervals
with a
micxoplate fluorescence wader. An increase in fluorescence signifies the
appearance of
alnyloid or intermediates in the production of amyloid, as illustrated in the
Figures (in
general, a compound which inhibits fibrillogenesis produces lower fluorescence
in the assay
because the fluorescence of ThT is greater when bound to fibrils).
52
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PYOtocol: Aji peptide: A(3 (1-40) 95% purity (American Peptide Company, Inc,
Sunnyvale, Cal. USA) is disaggregated in trifluoroacetic acid and filtered
through a 0.02
p.M filter, (Whatman Anotop 25 plus, 0.02 wm, Catalogue no: 6809 4102) in
hexafluoroisopropanol (I~'IP). Solutions of Aji (1-40) or IAPP at 600 p,m in
HFIP are
~ stored at-SO°C. Assay mixture: The mixture is pxepaxed as two
solutions which are
combined upon addition to the 384 well microplate (Corning Costar cat. 3705).
i) Solution A consists of test compounds in 0.02M Tris/0.02M acetate/0.15M
NaCI/0.01 % azide at pH 7.40 or buffer alone (control),
ii) Solution B consists of A~i (1-40) 40 ~,M or IAPP 20 pM, Thioflaviu T 10 mM
in
0.02M Tris/0.02M acetatel0.15M NaCI at pH 7.40. This solution is prepared by
drying the
A~i peptide under nitrogen and then xesuspending this in. 0.04M Tris base with
15 minutes
sonication. An equal volume of 0.04M acetic acid containing 0.3 M NaCI is
added and the
solution is adjusted to 7.400.02. A small volume of 20 mM Thioflavin T is
added to the
solution to give a final 5 p,M concentration of Thioflavin T.
iii) The micxoplate is loaded with 40 p,L of solution A followed by 40 uL of
solution
B which gives a final 20 ~M A[3 (1- 40) or 10 ~,M IAPP, 5 p,M Thioflavin T,
and a given
concentration of test compound in 0.02M Tris/0.02M acetate/O.ISM NaCI/0.005%
azide,
pH 7.40.
The plate is sealed and loaded into the microplate fluorescence reader.
Fluorescence
measurement data analysis: The HTS-7000 Bio Assay Reader, Perkin Elmer, is
used to
perform kinetic nuLS of about I day. Readings were taken at 15 minute
intervals; with one
minute shaking before each read. Bandpass filters used were: excitation 430
nm, emission
485 mm.
Similarly, in the electron microscopy ("EM") assay, each sample was sonicated
for 1
?5 min to disrupt large clumps before testing. The sample (5-p,L aliquot) was
placed on freshly
cleaved mica and allowed to air dry. The mica was placed .in a Balzers High-
vacuum,
Freeze-Etch Unit (model 301), shadowed with platinum {30° angle), and
coated with a
carbon film. The replica was removed from the mica by flotation and
transferred onto a
300-mesh copper grid. Samples were examined using a Joel 2000 FX transmission
electron
microscope.
53
CA 02488493 2004-12-07
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Tn the circular dichroism ("CD") assay, samples were transferred to 0.1-cm
path-
length quartz cuvettes and CD scans were taken using a Jasco J-715
spectropolarimeter.
Readings were taken at 37 °C, between 190 and 240 nm, with a resolution
of O.I nrn and a
bandwidth of 1 nm.
And in the mass spectroscopy ("MS") assay, samples were prepared as aqueous
solutions containing 20% ethanol, 200 g,M of a test compound and 20 wM of
solubilized
A~i40. The pH value of each sample was adjusted to 7.4 ( X0.2) by addition of
0.1%
aqueous sodium hydroxide. The solutions were then analyzed by electrospray
ionization
mass spectroscopy using a Waters ZQ 4000 mass spectrorrieter. Samples were
introduced
by direct infusion at a flow-rate of 25 ~L/min within 2 hr. after sample
preparation. The
source temperature was kept at 70 °C and the cone voltage was 20 V for
all the analysis.
Data were processed using Masslynx 3.5 software. The MS assay gives data on
the abilify
of compounds to bind to A~i, whereas the ThT, EM and CD assays give data on
inlubition of
fibrillogenesis.
Some selected compounds of the present invention are presented in Table 2
below.
Although particular salts are depicted (such as the hydrochloride), the free
base and other
pharmaceutically acceptable salts are within the present invention.
Table 2 Structures and Activities of Some Compounds of the Invention in
Soluble A(3
Assays
Structure . No a A s y CD EM A~ay
H~O~O \ / ~2 ~ 1 + ~ +
H~O ~O \ / ~ ~2 2HC1 1 + + + +
5
O 0 --
H~~ \ / ~z 1 + ~ .+ +
. [HOCH2CHZS03H]2.
54
CA 02488493 2004-12-07
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Structure No A h CD EM
a y
NH .
C-NHz
Z ,
(HCI)2
NHz
I
I , .
C-NHZ
'
j HCl 3
~
C-NHa
,
NH .
HN ~
~~~S~F 4.
~O HCI
HzN
~ ~2
HzN
NH
5 ..~ +
i ~ ~a
0
2 HC1 , + + _ +
O O \ 6
/
~z 1
H N ."
z
. H~
, ~ ~.. + + +
z2 HCl ~
~ . /
O \
/
~
~
H~,
2
Olvfe
Me0 8 + + - +
2HQ
O
\ / ~
H \ /
~
2 5
~~ \ 9 + + _ +
O 0~~~2HC1
HZN "' .
~o ~Z
~ .2
HCl ~'~ + _ _
~z
H
~
z
O \ !
_ NH
~~ ~ + + + +
2HC1 1
\ /
H
~
NHZ
CA 02488493 2004-12-07
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Structure , No a .A s y CD EM Allay
HILT ~ ,
Ha \ ~ o ~o ~ ' ~2 2HC1 12 + + +
~ ..
\ / p ~° ~ \ / ~z ~Cl 13 + . + . + +
O--p ~-° ~ , N~ ~~ 14 + + _ - _
~H .
H
\ / N~ 2HC1 15 + +
l /5
H
H
C~° 'E~'° ~N~ ~a 16 + ~ + .. +
6. , .
-\H H
O \ / N~ . ZHC1 17 + + - +
L
\H . . H
\ / ° ° \ / ~ N~ 2HQ -
is + + +
~H H
~2HCl
L \ J9 ~ % '~ 19 + + +
~H H
\ / N~ ~a 20 nd . - +
m ,
\H H
Br Br
NH
H~~~o~o ~ / ~Z 2HC1 21 pr nd
~(a
Bz
Br , Br ,
~N~p ~-o / N~ 2HC1 ~ 22 pr nd
H Br 5 Br ~ J .
HN ~ .
H2~ ~~ \ / ~2 23 + - +
H~ \ /, ° \ ~ O \ / ~~ ~Ci 24 + + +
25 ~ + + nd
H H
56
CA 02488493 2004-12-07
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Structure ~ No a A h y CD EM AMaY
Hr1
Ha \ / . '~ ~ - 2HC1 26 ~ + ,
\ / ~a
2HC1 2'7 r nd
\ / N~ P
g .
-( \) ~i 2HC1 '
HN ~ I ~y~ ~a 28 + + ~ + -
~a
NH
II
C-NH2
29
OCH3
CN / \ pip \ / NJ ~Cl)2 30
H ~ ..
H
OIi
~__~~~ 3I: + ~ - +
a~o~ ~a
Hz HEIZ .
HN / \ p ° \ / 32 +
CIHgN~
HN
~- ~/ \--o cH3 33 +
CIH3N~
c~x~~°~o~°~r~xc~ 34 ~. + - +
N
CN~-O~p~o--~-l(N7 zHCi 35 , + - ' +
g
o \ / 36 + +
~ . .
o~ 37 + +
-O~ , 38 -+~ r - +
~~p~C00H 39 + . + s
p~3N \
xK .
xc~~2 / \ ~o / \ cxzcx~cnox q.0 -
S7
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Structure CN.°o a A h ~ CD ~ EM AMaY
'~ °~..~rrHz 41 + ~ +
2 NH4C1
HCl ~O~~COOH 42 ~ +
Hz
HN ~ \ ~
o Br 43 +
HZN~ ,
HN
HBr . 0 44 +
HzN Br
~Hbt
HCl ~-~--0 COOH 45 + ~
HZN
HN\~~NHZ .
4s - - - ~a
-~ \~ ~ ~~ .
HN \ I NH ~~z 47 _
~2
48 +
HZ NH HCI ~ HCI H NH2
HN N ~ I ~ ~ I HN "NH 4g +
Hz~ HCI HCI -~NHz
o ~ ~
\ \
~ 50
HN NH HN NH
HZ CF3C03H CF3COzH ~2
\ I O~/~/~/0 \ I. ,
51
NH HCI HCI HN
HZN~NH HNi 'NHZ
NH
.
NHz ~ .52
NCI
CH30
58
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Structure NO a ~S y CD EM Say
NH HCI
NH: ..
53
H2 \ ~ , ' .
HCI H ,
I ~ ~ 54
HzN. Hz
HCI H NH HCI
p
N
HZ ~ I H , H ~ I NHZ 55
NH CF3COZH CF3COZH NH
ors o os,b
i
HTN \ I ~ ~ ~ 1 ~= 56
H4 HQ
NH
57 +
N~ I
N \ 58 +
HN I ~ I ~ ' NH
NHZ NHS
NH2 59 +
NH
60 + +
59
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Structure . Code ThT CD EM ' MS
No. Assay Assay
61 + + +
HN o ~ / Q NH 62 + + +
N / \ .\ I N
H H
63 +
NHZ NHz
HN \ I ~ ~ I ~ ~NH 64 +
~O O'r
y
65 - + . +
N
H
/ \
HN - Hz 66 +
\ / ° ° \ /
H2 . NH
H2N
HN \ ~ p N~~ 67
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. Structure , Code ThT CD EM MS
No. Assay Assay
68
H2N
NHz , .
HN~ [ ~ ~ 69. ,
/ O
HzN . . .
HN// \ . / O 7~ + + +
y
H H. . .
N N
s~ o . ~ s/ N~ 71 +
.
H '
N
\ I ~ H~ N . 72 ,
~o \ /
NHz , NH2
HN ~ I ~ \ ~ ~ ~ i NH 73 + ,
N
M H
\ / NH 7
z
HZN HN
NH
0
C ~ ~ NH 75 ' +
2
HzN HN
NH .
8r
r . Hz -~- s -E
Hs - o ~ ~ 76 -
~ ~ ~ NH
HN
I \
z
HZN \~ ~ / H , NH 77
H
\ /
61
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Structure No.e. A s y CD EM AMaY
HHN ~ / O \ / ~3 . ," + +
NHZ
HN
OH
O~O \
\ / ~9 , +
NH2
H2N . HN
NH .
NH2 101+ .
80 +
HN I \ ~~~~ ~ \ \O- ,
H .H ~
NHz
HN l ~ , N
v \ / . sl +
N
1
H
82 -
Hz
HN
% O 1Hz . 83 . - + +
\ ~ /
\ r/ NH
H
NHZ NH2
HN ( ~ \ ~ \ / ~ \ NH 8~ +
N
C ~ s~ ~~~ s~ 'N~ $5
H O O
/ ~ .
Hz Hz
O 0
HN \ / \ / NH ~86 +
O H . H O
62
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Structure N.. NO a ~T~ y CD ' EM AMS~y
N ~ N ~ ~ 'H H7 +
H ~1 ~ / \ IN
H H
w ~I \ / N ' ,
- , \ , ~ s8 . +
H H
/ ~ H ~ HI
~H ~ / N ~ / I w ~~ 89 +
H H
~N r I r I N
H \ ° \ \ ° ~ H 90 +.
~ r r '
H
N \ ~ 0 \ \ ° \ ~ N 91 +
NH I r r H
N~ ~ I O . 0 , ~ N 92 ~ + , + +
v TJ
NCH H,N
NH
HN \ p
~N,H 93
H/N
NHZ
H ~ ~ \ \ / °~° \ / HZ 94 ~ ~ +
NH
H
HN ~ /~ ~~NH
O ~ 0
Ho ~ ~ ~ ~~NOH 95 ' +
63
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Structure . Na a A h y CD EM
HCI HCI HCI
96 +
NHz NHz
HN. ,
\ \ ~ I ~ . NH 97 +
HN H H NH
H ~ ~ N N ~ / NH 98
z , z
\ \
HN I / 'o I / NH ~ 99 +
NHz NHz
100 ' +
N
HN ~ / \ ~ ~ OH
N l0I +
1
N H2 H
w \ .- / ' w
HN ~ / N . . ~ I N / ~ NH 102 +
NHz H H NHz
~ ~ /
HN I / N N N ~ / NH 103 - +
NH2
NHz
64
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Structure ~ No a Ass y CD EM AMY
~N w..
I \ ~ \ I N ~ /, OMe
HN / N 1 104 +
H
NHz M .
N / N -
I \ ~ \ I .N ~ l p~ '
HN / N ~ 105 +
H
NH2
106 +
\ ~O \
iO~N ~ I. / I / N~O~ 107 -
NHZ NHS
\ ~N~ \
HN ~ / I ( / NH 108 +
NH2 _ H2
I
w ~ \
HN I / O I / NH 109 +
NHa v ' H2 ,
110 +
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Structure' ~ No a A s y CD EM AMS~y
11I ~ +
HN ~ I \ I ~ . I ~ / \ _
NH ~ ~ ' ~ NH 112 + +
2
Hz
HN ~ ~ \ ~ ~ ~ ~ / ~ / NH . 113 ~ +
NH2 ~ H N '
H ~ I \ ~ ~ ~ ~ / I \
V \_/
~ NH 114 - + +
NHz
H2N
HN ~ I ~ I ~ I ~ ~ I \ -
H 115 + +
NHz
. Hz
HN ~ I \ ' S\ I ~ N I ~
NH ~ ~ ~ NH I16 , + +
2
HZN
I0~ ~- NH -
I17 + +
. . NH2 . .
HN ~ ~ l0' ~ NH 118 -
HEN NHS
119 +
66
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Structure ~ ~ Code ThT , CD ~M MS
No. Assay Assay
H .
/ \ ./ . \ l NH2 120 - + +
H2N ~ .
O
I21 - + +
~N ,
HN I / . HN ~ ~ 122 .+ + +
NH2 .
123 - + +
i / . HN~NHZ
N
NH 124. , - + +
NHZ
_ N w
NH' 125 - + +
NHz ,
_ N
\ / ~ ~ ~s NH 126 +
H
NH2
127 +
67
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. Structu~~e No a A h ~ CD EM . A say
HN~ ~NH ~ 128 + + +
~NHz H2N~NH , ~ '
129 +
130 +
131 +
CF3COZH NH H CF3COZH
NH HN ~ ~ 132
s~ ~ ~ . ~ Br
H HCl HCl H
NH HN I ~ 133
CH30 ~ ~ OCH3
HN ~ / o~o \ / ~ NH
_ 134 + - +
/ \ \ /
,, / \ / \ . ~ +
~O~ ~~ 135
HN~ . ~NH
68
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Structure No a A h y CD EM essay
OH / ' OH . , ,
H I \ . O I \ NH
\ ~ / ~~ \ 136. +
/N
HC NHz I ~ O NH Ct 0 ~ ~ NHzHCI , 137
,H~N~H I ~ ~ I ~ H'~NH 138
NHz CF3COZH CFaCO2H NHz
\ O~~O ~ \ .
HZN / / NHa 139
HO'N CF3COZH CF3COZH N~pH
\ O O ~ \
HZN / \~~ r NH2 140
I~
HO'N CF3COZH CF3C02H N'OH
141
H2N~~ ~/~~NH2
142
NW ~ ~ NH
HZ I I \ NH' 143
N
CF3COZH / ~ H H ~ CF3COZH
HN NH3CI
\ 144 ,
N02
Tin each indicated assay, "+" = active; "-" = inactive; 'fir" = promoting;
"nd" or blank entry
= not determined.
69
HO'N CF3C02H CF3CO2H N.OH
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The following compotands i1i Table 3 may also be employed according to the
methods
described herein.
Table 3 Additional Exemplary Compounds for Use in The Methods of The Invention
r
H N \ ~ O~O~o~O \ / NH ~ 2HCI
2 2
. CN . \ / O~O~O~o \ / N~ . 2HCI .
N ~N
H .
HN / I . NH
HEN \ ~ O~0 ~ O~O \ , NHz ~ ~HCI
CN \ / O~O~ ~ I O~./O \ ~ ~~ ' 2HCI
HN
O~O \ I: O~ NH
H2N ~ , ~ \ , 2HCI
NHz
N
O~O ~ I O~ N
C \ , O \ ~ ~ . ~ 2HCI
N
C ~ . Q ' 2~~ ~ / / ~ ~ 2HCI
N N
\~H \ I C~H~C \ I H~/ ~ ~ 2HCI
2 , 2
, C~ z~C ~ \ ~ ~~% .~ ' 2HC1
N N
C C , S \ ~ ~ 2HCI
0
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N , \ / 0-~CHz~ ~ O \ / . N~N~ . 2HCI
H H
~Cz \ / O~CHz~ ~ O \ / C~N~ ~ 2HCI
N ~ ~ N
H H
N
.~ _ N,
/ O , CHz)" O . \ / S~N~ ' 2HCI
H H
N
N Cz \ / . 0'~CHz~ ~ 0 \ / C? N--~rN~ . 2HCl
H H
C=C \ / O-~CHz) ~ O . \ / . C=C-~°N~ ~ 2HC1
H H
H NH
H N~N \ /. O'~CHz) ° 0 ~ / N NH , . 2HC1
z z
HN _
z Hz NH
H2 ~-C \ / 0-~CHz)" O \ / C~NH ~ 2HCI
2
HN
NH
H ~S \ / ~~cH2) ° ~ \ / s- ~NH ~ zHCI
2 2
H N' N Cz \ / O~CHz) n O \ / CZ N-~NH ~ 2HCl
2 Hz
HN _
Hz ~-C C \ / O-~CHz) n O \ / C=C--~NHz . 2HCI
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NH O ~ 0 NH
S-NH ' NH-S ~ ~ ~ 2 HCI
NH2 ~ p NHz .
NH O O NH
S-NH~O~NH-S ~ ~ 2 HCI
NHZ O O NH2
N O O N
S-NH ~ ~NH-S ~ ~ ~ 2 HCI
NH O O NH
O O
N~ ~ ~ S-NHS ~NH-S ~ ~ N 2 HCI
C ~I O
NH p p NH
NH ~ ~ I o-NH ~ NH-o ~ ~ ~ H 2 HCI
NH2 U . ~NH2
NH / ~ ~ O ~ ~ NH
C-NHS ~NH-C 2 HCI
NH2 O NH2
N O . O N
C-NH NH-C ~ ~ ~ 2 HCI
NH NH
O O
N ~ ~ ~ C-NH NH-C ~ ~ N 2 HCI
C
NH NH
72
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NH ~ \ o-'NH , ~ ~ \ NH
NH2 II NH-S 2 HC(
O O NH2
NH ~ \ ~ o-NH -~ / \ N
C II NH S 2 HCI
N' O II
O NH .
NH / \ O ~ ~ \ NH
S-NH NH-S 2 HCI
NH2 ~ p NH2
NH. O
\ S-NHS ~O~ 101 ~ \ l H
NH2 11 O NH-S 2 HCI
O , 101 NH2 .
O
N / \ S-NH NH-O / \ N 2 HCI
NH , ~ , II
O NH
O '
~N ~ ~ \ _ O
S NH~O~O.~NH~S ~ \ N 2 HCI
NH
O NH
NH ~ \ o'_NH .-~. / \ NH
NHS NH C 2 HCl
NH2
NH / \ ~-NH ~ O j .\ N
C NH-C 2 HCI
N
NH
73
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NH / \ ~ ~ . .O / \ NH
C-NH NH-C 2 HCI
NH2 NHZ
NH ~ / \ O
C NH~O~O~NH-o ~ ~ NH 2 HCI
NH2 . , \NH2
N ~ ~ O NH_.o ~ ~ /N
C-NH C 2 HCI
NH
NH
O. . . O
N , ~ ~ .C-NH~O~O~NH~C ~' ~ N - 2 HCI
C
NH NH
The following charts are results from. the ThT assay.
~ . 2 HCl
. H~ . \ / ~ ~~ \ / NHa
LO
Ikbefia fi6rillogenesis
100
0 80
o~ 6D y200 uM
»_:
'° 40 0100 uM
c
'- 20
0
3 4 5 6 7 8. 8 10
Carbon chain length (n)
\ / ~ ~~ \ / N~ ~Cl
H
H
74
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IAPP fibrillogenesis
100
0 80
0 60
w
c
0
1 2 3 4 5 6 7
Carbon chain length (n)
The present invention also xelates to novel compounds and the synthesis
thereof.
Accordingly, the following examples are presented to illustrate how some of
those
S , compounds may be prepared. . .
Ge~aer~al Aspeets . '
Chen>icals were purchased from Aldrich. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 Fzs4 plastic-backed plates. Solvents were
reagent
10 grade unless otherwise specified., The iH (S00 MHz) and 13C (125 MHz) were
recorded on a
Varian Inova SpO. The chemical shifts are reported on the 8 scales in parts
per million
(ppm). The infra-red (llt) spectra were carried out on a Perlcin-Elmer Spectra
One
spectrometer (neat compound on NaCI plate).
15 1,4-bis(4-an:idifzoanilino)butane
NH
HzN 4 ~ l NHz
Step 1: 1,4-bis(4-cyanoanilino)butatae
H N NH Et3N, DMSO
C--~-- N
N~E ~ z ~ z zeflux ~
A mixture of 4-fluorobenzonitrile (3 g, 0.025 mol), 1, 4-diamin.obutane(0.6 g,
0.006
20 ~ mol), triethylamine (S mL) and DMSO (I6 mL) was heated at 150 °C
with stirring for 3h.
The mixture was then poured into iced water (250 mL) and the precipitate was
collected by
filtration. Recrystallization of the crude product (O.S8 g) from DMSO/Hz0
(6:1) gave the
product as a light yellowsolid, 0.48 g, yield 27.6%.
75~
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Step 2: 1, 4-bis(4-anziditzoanilino)butane
1. HCl (gas) ' NH
~ NH ~3 ~ EtOH/dioxane
~a \ ~ 2. NH- IEtOH~ HaN~~~ ~NH2
3
A mixture of 1,4-bis(4-cyanoanilino)butane (0.44 g, 1.52 mmol) in ethanol (30
mL)
and dioxane (10 mL) was cooled to 0 °C and saturated with HCl gas. The
resulting mixture
was stirred at room temperature until the IR indicated the disappearance of
the nitrite
absorbance peak at 2200 crri 1. Diethyl ether (100 mL) was added, and the
fornned
precipitate was collected and washed with diethyl ether. The solid thus
obtained was then
placed into a 50-mL round bottom flask. Ethanolic ammonia (2 M, 30 mL) was
slowly
added via syringe. The resulting mixture was refluxed for 3h, and then was
cooled to room
temperature. Diethyl ether (100 mL) was added to induce precipitation. The
precipitate thus
formed was collected, washed with ether, and recrystallized from H20 to give
0.50 g of
product, yield 99%.
1S Linear dibetzzanziditze and diimidazolino compounds
H~~p~ \ ~ ~z2HC1 . ~~-O~O \ / N~ 2HCl
2 n N
_ 1H H
n 310 n=4~I0
Step 1: a, w-bis(4-cyanophenoxy)alkanes
Sodium (1.2 g, 0.05 mot) was cut into small pieces and slowly added to a
stinted
solution of dry ethanol (40 mL). After complete dissolution of the sodium, 4-
cyanophenol
(6 g, 0.05 mot) was added and followed by the dropwise addition of 1,4-
dibromobutane (5.4
g, 0.025 mot). The resulting miXture was stirred at refluX for 1~2 days and
then cooled to
room temperature. The white solid formed in the reaction was collected by
vacuum
filtration, washed with water and dried under vacuum. The obtained product,
1,4-bis(4-
cyanophenoxy)butane (7.18 g~ 98% yield), was used directly for the next step
without
purification. Analogous compounds with n = 3, 5, 6, 7, 8, 9, and 10 were
prepared and
yields ranged from 70 - 95%. The 1H and 13C NMR of the compounds were
consistent with
the structures.
Step 2: Dibenzamidines atzd diitnidazolino compounds
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A mixture of a, cu-bis(4-cyanophenoxy)alkane (3.42 mmol), dioxane (15 mL) and
ethanol (40 mL) was cooled to 0 °C. Dry HCl gas was bubbled through the
mixture until
saturation. The mixture was stined at room temperature until the IR. nitrite
absorbance at
2200 crri 1 subsided.
Diethyl ether (100 mL) was then added and a white precipitate was formed. The
precipitate was collecfied by vacuum filtration, washed with diethyl ether,
and placed into a
SO-mL round bottom flask. Ethanolic ammonia solution (2 M, 30 mL; in the
preparation of
dibenzamidines) or ethylenediamine in MeOH (1.S M, 30 mL; in the preparation
of
diimidazolino compounds) was added slowly via syringe. The resulting mixture
was stirred
at reffux for 3h. After the mixture was cooled to room temperature, diethyl
ether (100 mL)
was added. The white precipitate that formed was collected and washed with
diethyl ether.
The solid was then recrystallized with HCl (2 N) giving the desired product.
Dibenzamidine
compounds with n = 3 -10 were prepared azld yields ranged from 60 - 85%.
Diimidazolino
compounds with n = 4 -10 were prepared and yields ranged from 50 92%.
1-(4-amidino)phenoxy-8-bromooctahe, hydnobYOnaide
HN
HBr. / \ ~ Br
HZN
Step 1: 1-(4-cyano)phenoxy-8-bromooctane
In a 100-mL round-bottom flask were placed 4-cyanophenol (2.38 g, 20 mmol),
I~ZC03 (anhydrous, 25 mmol) and DMF (SO mL). The mixture was stirred at room
temperature for 30 min. When the mixture became cloudy, 8-bromooctainol (20
mmol) was
added dropwise via syringe. The mixture was then refluxed for 5h, cooled to
room
temperature, and poured into ~ iced water (200 mL). White precipitate was
formed and
collected by vacuum filtration. The pure product (4.I g, 88.7% yield) was
obtained as a
white solid after silica geI flash column chromatography (eluent: 20 - 40%
ethyl acetate in
hexane).
Step 2: 8-(4-amidinophenoxy)octanol
The corresponding amidine compounds were obtained by serial treatments with
saturated ethanolic hydrochloride solution and ethanolic ammonia analogously
as described
above.
,Step 3: 1-(4-amidiraophenoxy)-8-bromooctane, hydrobromide .
77
CA 02488493 2004-12-07
WO 03/103598 PCT/US03/17992
In a 50-mL round-bottom flask were placed 8-(4-amidinophenoxy)octanol (2.I4 g,
6.8mmo1) and dichloromethane (30 mL). The mixture was cooled to 0 °C,
and PBr3 (3.4
mrnol, 0.5 eq.) was added dropwise via syringe. Then the mixture was stirred
overnight at
room temperature. The white solid starting material gradually dissolved anal
turned into a
yellow oil phase immiscible with the dichloromethane. Upon completion of the
reaction,
water was added to quench the reaction, and the dichloromethane vVas
evaporated under
reduced pressure to give a white solid as crude product. Pure product (white
solid, 780 mg,
31% yield) was obtained after silica gel ' flash column chromatography (eluent
CHC13/MeOH/AcOH 94/5/1) and subsequent recrystallization from HBr/CH3CN (2 I~.
9-(4-antidinophenoxy)nortanoic acid, hydrochloride
HCl
O COOH
Step 1: 9-(4-cyanophenoxy)nonaraol
Tn. a 100-rnL round bottom flask, 4-cyanophenol (2.38 g,, 20 mmol) and K2C03
(anhydrous, 25 mmol) were mixed in DMF (50 mL). The mixture was stirred at
room
temperature for 30 min. When the mixture became cloudy, 9-bromononanol (20
mmol) was
added dropwise via syringe. The mixture was then refluxed for Sh, cooled to
room
temperature, and poured into iced water (200 mL). The white precipitate that
formed was
collected by vacuum filtration. The pure product (4.8 g, 98 % yield) was
obtained as a white
solid after silica gel flash column chromatography (eluent: 20 - 40% ethyl
acetate in
hexane).
Step 2: 9-(4-eyanopheraoxy)nonanoic acid
To a solution of 9-(4-cyanophenoxy)nonanol (2.5 g, 10.2 mmol) in DMF (50 mL),
PDC (19 g, 61 mmol, 6 eq.) was added. The mixture was stirred at SO °C
overnight, then
cooled to room temperature, and poured into iced water (150 mL). The mixture
was
extracted with ethyl acetate {4 x 50 mL). The combined organic layers were
washed with
brine and dried over sodium sulfate. Purification via silica gel flash column
chromatography
(eluent 25 - 50 % ethyl acetate in hexane) gave product as a white solid, 1.65
g, 62% yield.
Step 3: 9-(4-cyanopltenoxy)rtortanoic acid, ethyl ester
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In a 100-rnL round-bottom flask, thionyl chloride (0.88 mL, 12 mmol) was added
to .
anhydrous ethanol (SO mL). The nurture was stirred fox 10 min, then 9-(4-
cyanophenoxy)nonanoic acid (1.65 g, 6.02 mmol) was added in one portion. The
reaction
was monitored by TLC. Upon completion of the reaction,, ethanol was removed
under
reduced pressure. Ether (100 mL) and saturated sodium bicarbonate solution
(100 mL) was
added. The organic phase was separated and dried over sodium sulfate. The
product (1.6 g,
87.7 % yield) was obtained as a white solid after evaporation of the solvent.
Step 4: 9-(4-amidinophenoxy)nonanoic acid hydrochloride
9-(4-cyanophenoxy)nonanoic acid ethyl ester, (1.6 g, 5.28 mmol) was dissolved
in a
l0 mixture of ethanol and dioxane (SOI10 mL) in a sealed 100-mL round-bottom
flaslc. The
mixture was saturated with HCl (g) at 0 °C and stirredat room
temperature until IR. showed
the disappearance of the nitrile absorbance at 2200 cni t. Ethanol/dioxane was
then removed
under reduced pressure, and ether (100 mL) was added to induce precipitation.
The
precipitate was collected and immediately placed into a dry 100-mL flask.
Ethanolic
L5 ammonia (2 M, 40 mL) was added via syringe. The mixture was refluxed for 3
h, followed
by removal of the solvent and addition of ether to induce precipitation. The
solid that
formed was collected and recrystalllized from HCl (2 I~. Final product was
obtained as a
colorless needle crystal, 0.56 g, 32.3 %. 1H NMR (500 MHz, DMSO-d6): 11.96
(s,1H),
9.16 (s, 2H), 8.85 (s, 2H), 7.80 (d, 2H, J= 8.5 Hz), 7.13 (d,,2H, J= 8.S~Hz),
4.06 (t, ZH, J=
20 6.5 Hz), 2.18 (t, 2H, J= 7.5 Hz),1.73-I.70 (m, 2H), 1.49-1.46 (m, 2H), 1.40-
1.38 (m, 2H),
1.30-1.27 (m, 6H);13C NMR (125 MHz, DMSO-d6): 174.46, 164.71,163.08, 130:15,
119.23, 114.74, 68.08, 33.66, 28.66, 28.57, 28.47, 28.40, 25.34, 24.46.
Some Substituted Pentamidines
R.1 RZ
\ / O O \ / 2 HCl
H N ~ _,~-J~~NH2
Rz Rt
25 . 2
Step 1: 1,5-Bis(4-eyatio-2-metlaoxyphenoxy)penta~ae
Rl R1 Ri
N OH + ~ . Br Na, EtOH N O O ~CN
reftu~x ' ~ ~ \
R2 . RZ RZ
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Sodium (0.3 g, 0.014 mol) was cut into small pieces and slowly added to a
stixred
solution of dry ethanol (30 mL). After complete dissolution of the sodium, 4-
hydroxy-3-
methoxybenzonitrile (2 g, 0.013 mol) was added and followed by the dropwise
addition of
1, 5-dibromopentane (0.9 mL, 0.007 mol). The resulting mixture was stirred at
reflux for 2
days, and then cooled to room temperature. The light brown precipitate in the
mixture was
collected, washed with water and dried under vacuum. The pxoduct obtained
(1.45 g, 73%)
was used directly for the next , step without purification. The 1H and 13C NMR
of the
compounds were consistent with the structures.
Step 2: CorrespondingPentamidaraes
l0 A mixture of substituted 1,5-bis(4-cyanophenoxy)pentane (in this example,
Ri =
methoxy and RZ = hydxogen) (1.8 g, 4.91 mrnol), dioxane (15 mL) and ethanol
(50 mL) was
cooled to 0 °C. Dry HCl gas was bubbled through the mixture until
saturation. The mixtuxe
was stirred at room temperature until IR showed the disappearance of the
nitrile alisorbance
at 2200 ciri 1. Then diethyl ether (100 mL) was added and the white
precipitate that formed
L 5 was collected by vacuum filtration and washed with diethyl ether.
The white solid obtained was placed into a 50-mL round-bottom flask and
ammonia
ethanol solution (2 M, 30 mL) was added slowly via syringe. The resulting
mixture was
stirred at reflex for 3h. After the mixture was cooled to room temperature,
diethyl ether (100
mL) was added and a white precipitate formed. The precipitate was collected
and washed
Z0 with diethyl ether. The solid was then recrystallized from 2 N HCl giving
the desired -
product (0.92 g, 40% yield). In lilce manner, the corresponding compound with
Rl =
bromine and RZ = bromine was synthesized in 53% yield.
Compound # 139
.\ I \
NC CN 1) NazC03, NHzOH.HCI
. HzO, EtOH, d
2) Preparative HPLC
Hz \ I \ I N Hz
i
HO~ ~ CFaCOZH CF3C02H ~OH
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A mixture of 1,S-bis(4-cyanophenoxy)pentane (153 mg, 0.5 mmol), sodium
carbonate (180 mg, 1.7 mmol) and hydroxylamine hydrochloride (278 mg, 4 mmol)
in 80
ethanol (10 mL) -was heated at reflex for 2h. The mixture was cooled to room
temperature.
Some solid precipitated and was removed by filtration. The filtrate was
concentrated to
dryness under reduced pressure. The crude product was purified by preparative
RP-HPLC
(Vydac C18, 215 nm, 50 mL/min, 0 % to 90 % MeCN in H20 containing 0.1 % TFA)
and
lyophilized to give a white solid, 127.2 rng, 42%. The heptane and nonane
anolags were
prepared iri the same way. ,
0 Compouxcd # SS
o
0
ci
I HzN~NH2 --a s ~y~ /
NC ~ Et3N ~ I H H I
DMF ~ NC CN
O O
N~
1) HCI,1,4-dloxane, EtOH H2 ~ I H H ~ I NHz
2) (NH4)C03, EfOH
3) Preparative HPLC NH CF3COZH CF3COZH NH
Step 1: To a cold solution (0 °C) ~of 1,5-diaminopentane (0.35 mL, 3
mmol) and
triethylamine (0.98 mL, 7 mmol) in DMF (10 mL) was added 4-cyanobenzoyl
chloride (1 g,
6 mmol). The mixture was stirred overnight at room temperature, and then
diluted with
l5 water. The beige solid that precipitated was collected by filtration and
dried in vacuo, giving
the corresponding amide, x g, 92 %.
Step 2: A suspension of the 1,5-bis-(4-cyanobenzamido)pentane (465 mg, 1.3
mmol), in a mixture of absolute ethanol (25 mL) anid 1,4-dioxane (20 mL), was
cooled to 0
°C, saturated with dry HCI, and the resulting mixture was stirred for
60 houxs at room
ZO temperature. The solvent was evaporated under reduced pxessure. A brownish
solid was
obtained: A mixture of the solid and ammonium carbonate (2.5 g, 25 mmol) in
ethanol (25
mL) was stirred overnight at room temperature. A small amount of activated
charcoal was
added, then the mixture was filtered over Celite. The solvent was evaporated
under reduced
pressure. The crude product was purified by preparative RP-HPLC (Vydac C18,
215 nm, SO
25 mL/min, 0 % to 90 % MeCN in. Ha0 containing 0.1 % TFA) and lyophilized to
give the title
compound as a white solid, 410 mg, S 1 %. The heptane and nonane analogs were
prepared
in the same way.
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Compound # 54
OH
~. Br~~Br ~.. '
\ , K2C03 O
O ~ poC \ I \ I H
O O
Step 1: A mixture of4-hydroxybenzaldehyde (2.7 g, 22 mmol), 1,5-dibromopentane
(1.35 mL, 10 mmol) and potassium carbonate (5.2 g) in dry DMF (25 mL) was
heated at
100 °C with an oil bath for S hours. The mixture was cooled to room
temperature, then
water (100 mL) was added. The solid that formed was collected by filtration,
rinsed with
water and dried in vacuo. The desiured bis-aldehyde was obtained as a brownish
solid, 2.8 g,
89 %.
H ~ I ~ I H ,~ U-Pr0)z-~~CN -~ / ~ O
I(pC03
O O O THF
NC CN
t0 Step 2: Diisopropyl (cyanomethyl)phosphonate (0.86 mL, 4.2 mmol) was added
to
suspension of sodium hydride (4.4 mmol) in THF at (0 °C).The mixture
was stirred at room
temperature fox 1 hour. A solution of the bis-aldehyde (2 mmol) in._THF was
added. The
mixture was stirred at room temperature for 2h, then diluted with ethyl
acetate, washed
subsequently with water, saturated sodium bicarbonate, brine and dried ovex
magnesium
LS sulfate. The solvent was evaporated wider reduced pressuxe. The crude solid
was washed
with a mixture of ethyl acetate and hexane (1 to 10, 10 mL) the dried in vacuo
to affoxd the
bis-nitrile, 0.51 g, 71 % yield.
o
' ~ J ~ I ,
I I 1) HCI, EtOH H2 I I N H2
NC CN 2) tJH3, EtOH HCI
NH HCI H
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Step 3: A suspension of the bis-nitrite (0.48 g,1.34 mmol) in ethanol (20 mL)
was
saturated with HCl at 0 °C. The mixture was stirred at room temperature
for 3 days. The
solvent was evaporated under reduced pressure. The solid was them dissolved in
2 N NH3 in
ethanol (20 rnL) and the mixture was heated at reflux for 2h. The mixture was
cooled to
room~temperature and the, solvent was evaporated under reduced pressure. The
resulting
solid was dried in vacuo, then recrystallized from 2 N HCl with the addition
of a few drops
of ethanol. The solid was collected by filtration, rinsed with water and dried
overnight in
vacuo, giving the title compound as a light yellow solid, 0.44 g, 71 %.
Compouhd # 137 ,
OH
NC I ~ I ~ CN ,
Br B K2C03 /
r
DMF
CN 100°C
Step 1: A mixture of 4-hydroxybenzylcyanide (2.56 g, 19.2 xnmol), 1,7-
dibromoheptane (1.49 mL, 8.7 mmol), potassium carbonate (11 g) in DMF (30 mI,)
was
heated with an oil bath at 100 °C for 3 hours. The mixture was cooled
to room temperature
and diluted with water (150 xnL). A solid precipitated. The solid was
collected by filtration.
t5 and rinsed with water. It was then dissolved in ethyl acetate, washed
subsequently with 10%
NaOH (3 x 20 mL), brine (3 0 nnh) and dried over magnesium sulfate. The
solvent was
evaporated under xeduced pressure. The resulting solid was dried ita vacuo to
give the 1,7-
bis(4-cyanomethylphenoxy)heptane as a tan solid, 2.58 g, 82 %.
NC ~ ~ CN
O~~O I / 1) NCI, EtOH,
1,4-d(oxane
2) NH3, EtOH, D
H~ ~ ~ ~~NH
HCI NH I ~~~ ~~i \~ HCI
0 NH4C) 0 ~ NHZ
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Step 2: A solution of 1,5 bis(4-cyauomethylphenoxy)heptane (750 mg, 5.07
mnnol}
in a mixture of 1,4-dioxane (10 mL) and absolute ethanol (10 mL) was saturated
with HCl
at 0 °C. The mixture was then stirred at room temperature for the 3
days: The solvent was
evaporated under reduced pressure and the residue was dried in vacuo. The
residue was
' dissolved in 2 N ammonia in ethanol (20 mL) and the mixtwre was heated at
reflex for 3h.
The solvent was evaporated under reduced pressure. The crude solid was
recrystallized from
2 N HCl 1 acetone. The crystals were collected and dried irc vacuo. The title
compound was
obtained as an off white solid, 655.3 mg, 60 %.
Compound # SI .
o~° s ~ oho ,
~ \I \I
CN CN 1) BH3:THF, D NH HCI
. 2) HCi, MeOH z HCI N2
Step 1: A solution of boraneaetrahydrofuraiz complex (10 mL, 10 mmol) was
added
to a solution of the bis-nitrile (510 mg, 1.53 mmol) at 0 °C. The
mixture was then heated at
reflex for 18 hours, then cooled with an ice bath. The excess of reagent was
quenched by
the slow addition of methanol (10 mL). The resulting mixture was heated at
reflex for 15
minutes, then the solvent was removed under reduced pressure. The residue was
coevapotated 3 times with methanol, then suspended in tnix_ture of methanol
(20 inI,) and
concentrated HCl (6 rizL). The mixture was heated at reflex for 1.5 hour. The
mixture was
then reduce to about 5 mL under reduced pressure. A fme white solid had
formed. The
mixture was diluted with ethauol and cooled to -10 °C. The solid was
collected by filtration,
rinsed with, cold ethanol and dried overnight irt vacuo. The 1,5-bis (4-(2-
aminoethyl)phenoxy)pentane dihydrochloride was obtained as a fine white
powder, 564.6
mg, 89%.
o~~o
I ~ I
a
NBoc
NHz HCI HCI H2
N ~ NHBoc
NBoc BocN '
Hunlg's base HN~ ~--NH
THF, dlchioromethane NHBOc BocHN
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Step 2: N,N'-bis(tent-butoxycarbonyl)-1H-pyrazole-1-caxboxamidine (0.78 g, 2.5
mmol) was added to a suspension of tb.e 1,5-bis(4-(2-
aminoethyl)phenoxy)pentane
dihydrochloride (470 mg, 1.13 rizmol) and Hunig's base (0.435 mL) in a mixture
of THF (5
mL) and dichloromethane (20 mL). The mixture was stirred at~room temperahu~e
for 2 days.
Excess xeagent was quenched with 1,2-ethylenediamine. The mixture was diluted
with .
bhloroform, washed subsequently with 1 N HCI, saturated sodium
carbonate,brine, and
dried over magnesium sulfate. The solvent was removed under reduced pressure.
The crude
product was purified by flash chromatography on silica gel (0.5 % to 1 % MeOH
in CHCI3)
giving a white foamy solid 246.5 mg, 26 %.
i ~ o~.~o i ~ s ~ i
w w ~ ~I
NBoc ' . BocN ~ HCI ~ NH HN
HN--~ ~--NH 1,4-dloxane HN--~ HCI HCI ~-NH
(Q NHBOc BocHN NH2 H2N
Step 3: A solution of 4 M HCl in 1,4-dioxane (5 mL) was added to a solution of
the
protected bis guanidino compound (246 mg, 0.297 mmol) in 1,4-dioxane (10 mL).
The
mixture was stirred at room temperature for one day. The solvent was
evaporated under
xeduced pressure. The product was dissolved in water, then the aqueous
soultion was
lyophilized, giving the title compound as a white solid, 146.4 mg, 99 %.
