U.S. patent application number 12/706787 was filed with the patent office on 2010-06-10 for compounds and amyloid probes thereof for therapeutic and imaging uses.
Invention is credited to David Alagille, Herve Da Costa, Gilles D. Tamagnan.
Application Number | 20100143251 12/706787 |
Document ID | / |
Family ID | 39789139 |
Filed Date | 2010-06-10 |
United States Patent
Application |
20100143251 |
Kind Code |
A1 |
Tamagnan; Gilles D. ; et
al. |
June 10, 2010 |
COMPOUNDS AND AMYLOID PROBES THEREOF FOR THERAPEUTIC AND IMAGING
USES
Abstract
The present invention provides compounds and amyloid probes
thereof that allow for an antemortem method of diagnosing AD and
quantitating the extent or progression of amyloid deposits
(plaques) by in vivo imaging of amyloid and/or amyloid deposits in
the regions of the brain. Preferably, an amyloid probe of the
invention can cross the blood-brain barrier and distinguish AD
brain from normal brain. An amyloid probe can be administered to a
patient in amounts suitable for in vivo imaging of amyloid
deposits. Amyloid probes of the invention can also be used to
detect and quantitate amyloid deposits in diseases including,
without limitation, Down's syndrome, familial AD and homozygotes
for the apolipoprotein E4 allele. In one aspect, the compounds may
be used in the treatment or prophylaxis of diseases that include,
without limitation, AD and type 2 diabetes mellitus. The compounds
and amyloid probes of the invention include analogs, salts,
pharmaceutical compositions, derivatives, prodrugs, racemic
mixtures or tautomeric forms thereof.
Inventors: |
Tamagnan; Gilles D.;
(Woodbridge, CT) ; Alagille; David; (New Haven,
CT) ; Da Costa; Herve; (New Haven, CT) |
Correspondence
Address: |
WEINGARTEN, SCHURGIN, GAGNEBIN & LEBOVICI LLP
TEN POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
39789139 |
Appl. No.: |
12/706787 |
Filed: |
February 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11800986 |
May 8, 2007 |
7700616 |
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12706787 |
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60906106 |
Mar 9, 2007 |
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60798603 |
May 8, 2006 |
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Current U.S.
Class: |
424/1.81 ;
424/9.1; 424/9.31; 424/9.6; 435/29; 514/259.1; 514/302; 544/281;
546/115 |
Current CPC
Class: |
A61K 31/424 20130101;
A61K 31/4188 20130101; A61K 31/4192 20130101; C07D 487/04 20130101;
C07D 498/04 20130101; C07D 513/04 20130101; C07D 249/20 20130101;
A61P 25/28 20180101; A61K 31/4184 20130101; A61K 51/0455 20130101;
A61K 51/0453 20130101; A61K 51/0459 20130101; A61K 31/421
20130101 |
Class at
Publication: |
424/1.81 ;
424/9.1; 424/9.31; 424/9.6; 435/29; 514/259.1; 514/302; 544/281;
546/115 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 49/00 20060101 A61K049/00; C12Q 1/02 20060101
C12Q001/02; A61K 31/519 20060101 A61K031/519; A61K 31/437 20060101
A61K031/437; C07D 487/04 20060101 C07D487/04; C07D 491/04 20060101
C07D491/04 |
Claims
1. An amyloid binding compound of the formula ##STR00093## wherein
R.sub.1 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl, alkenyl,
alkynyl, alkoxy, monoalkylamine, dialkylamine, hydroxyalkyl,
haloalkyl, alkylthio, alkylsulfonyl, aryl, heterocycles,
heteroaryl, aralkyl, carboxy, esterified carboxy, amidate carboxy,
OR.sub.6, NR.sub.5R.sub.6 or R.sub.6, R.sub.2 is H, F, Cl, Br, I,
NO.sub.2, CN, CF.sub.3, alkyl, alkenyl, alkynyl, alkoxy,
monoalkylamine, dialkylamine, hydroxyalkyl, haloalkyl, alkylthio,
alkylsulfonyl, aryl, heterocycles, heteroaryl, aralkyl, carboxy,
esterified carboxy, amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or
R.sub.6, R.sub.3 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl,
alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl, aralkyl, carboxy, esterified carboxy,
amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6, R.sub.4 is
H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl, alkenyl, alkynyl,
alkoxy, monoalkylamine, dialkylamine, hydroxyalkyl, haloalkyl,
alkylthio, alkylsulfonyl, aryl, heterocycles, heteroaryl, aralkyl,
carboxy, esterified carboxy, amidate carboxy, OR.sub.6,
NR.sub.5R.sub.6 or R.sub.6, R.sub.5 is C.sub.nH.sub.2n+1 or
--CH.sub.2--CH.dbd.CH--I and R.sub.6 is C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m are
independently 0, 1, 2, 3, 4, 5, 6 or 7, A is N or C, D is N or C, E
is CH or N, Y is N, Z is CH, B is O, N or CH and a, b, c, d, e and
f each independently represent an optional bond, provided that when
A and E are N, then B is CH, D is C and b and d are each a bond, or
provided that when B, D and E are N, then A is C, b and e are each
a bond, or provided that when E is N and B is O, then A and D are
C, a and c are each a bond, or further provided that when D is C,
then f is a bond or when D is N, then f is not a bond.
2. An amyloid binding compound of the formula ##STR00094## wherein
R.sub.1 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, OR.sub.6,
NR.sub.5R.sub.6 or R.sub.6, R.sub.2 is H, F, Cl, Br, I, NO.sub.2,
CN, CF.sub.3, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6, R.sub.3 is H,
F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, OR.sub.6, NR.sub.5R.sub.6 or
R.sub.6, R.sub.4 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3,
OR.sub.6, NR.sub.5R.sub.6 or R.sub.6, R.sub.5 is C.sub.nH.sub.2n+1
or --CH.sub.2--CH.dbd.CH--I and R.sub.6 is C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m are
independently 0, 1, 2, 3, 4, 5, 6 or 7, A is N or C, D is N or C, E
is CH or N, Y is N, Z is CH, B is O, N or CH and a, b, c, d, e and
f each independently represent an optional bond, provided that when
A and E are N, then B is CH, D is C and b and d are each a bond, or
provided that when B, D and E are N, then A is C, b and e are each
a bond, or provided that when E is N and B is O, then A and D are
C, a and c are each a bond, or further provided that when D is C,
then f is a bond or when D is N, then f is not a bond.
3. The amyloid binding compound of claim 1, wherein the compound
comprises the formula ##STR00095##
4. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable carrier.
5. An amyloid probe comprising the compound of claim 1, wherein the
compound comprises a detectable label.
6. The amyloid probe of claim 5, wherein at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 independently
comprises .sup.131I, .sup.124I, .sup.125I, .sup.3H, .sup.123I,
.sup.18F, .sup.19F, .sup.11C, .sup.75Br, .sup.13C, .sup.13N,
.sup.15O or .sup.76Br.
7. A pharmaceutical composition comprising the probe of claim 5 and
a pharmaceutically acceptable carrier.
8. A method of detecting the presence and/or location of an amyloid
deposit in a subject, the method comprising the steps of:
administering an effective amount of the probe of claim 5 to the
subject; and detecting the binding of the probe in the subject,
wherein the binding of the probe indicates the presence and/or
location of an amyloid deposit in the subject.
9. The method of claim 8, wherein the amyloid deposit is located in
the brain of the subject.
10. The method of claim 8, wherein detection is by gamma imaging,
magnetic resonance imaging, magnetic resonance spectroscopy or
fluorescence spectroscopy.
11. The method of claim 10, wherein detection by gamma imaging is
by PET or SPECT.
12. A method of detecting the presence and/or location of an
amyloid deposit in biopsy or post-mortem tissue from a subject, the
method comprising the steps of: incubating the tissue with a
solution comprising the probe of claim 5; and detecting the binding
of the probe in the tissue, wherein the binding of the probe
indicates the presence and/or location of an amyloid deposit in the
tissue.
13. The method of claim 12, wherein the tissue is from the brain of
the subject.
14. The method of claim 12, wherein detection is by gamma imaging,
magnetic resonance imaging, magnetic resonance spectroscopy or
fluorescence spectroscopy.
15. The method of claim 14, wherein detection by gamma imaging is
by PET or SPECT.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. application Ser.
No. 11/800,986 filed on May 8, 2007, and claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application No.
60/798,603 filed May 8, 2006 and entitled "COMPOUNDS AND AMYLOID
PROBES THEREOF FOR IN VIVO IMAGING," and U.S. Provisional Patent
Application No. 60/906,106 filed Mar. 9, 2007 and entitled
"COMPOUNDS AND AMYLOID PROBES THEREOF FOR IN VIVO IMAGING," the
whole of which are incorporated by reference herein in
entirety.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease (AD) is a neurodegenerative illness
characterized by memory loss and other cognitive deficits. McKhann
et al., Neurology, 34: 939 (1984). It is the most common cause of
dementia in the United States. AD can strike persons as young as
40-50 years of age, yet, because the presence of the disease is
difficult to determine without dangerous brain biopsies, the time
of onset is unknown. The prevalence of AD increases with age, with
estimates of the affected population reaching as high as 40-50% by
ages 85-90. Evans et al., JAMA, 262: 2551 (1989); Katzman,
Neurology, 43: 13 (1993).
[0003] In practice, AD is definitively diagnosed through
examination of brain tissue, usually at autopsy. Khachaturian,
Arch. Neurol., 42: 1097 (1985); McKhann et al., Neurology, 34: 939
(1984). Neuropathologically, this disease is characterized by the
presence of neuritic plaques (NP), neurofibrillary tangles (NFT)
and neuronal loss along with a variety of other findings. Mann,
Mech. Ageing Dev., 31: 213 (1985). Post-mortem slices of brain
tissue of victims of Alzheimer's disease exhibit the presence of
amyloid in the form of proteinaceous extracellular cores of the
neuritic plaques that are characteristic of AD.
[0004] The amyloid cores of these neuritic plaques are composed of
a protein called the .beta.-amyloid (A.beta.) that is arranged in a
predominately beta-pleated sheet configuration. Mori et al.,
Journal of Biological Chemistry, 267: 17082 (1992); Kirschner et
al., PNAS, 83: 503 (1986). Neuritic plaques are an early and
invariant aspect of the disease. Mann et al., J. Neurol. Sci., 89:
169; Mann, Mech. Ageing Dev., 31: 213 (1985); Terry et al., J.
Neuropathol. Exp. Neurol., 46: 262 (1987).
[0005] The initial deposition of A.beta. probably occurs long
before clinical symptoms are noticeable. The currently recommended
"minimum microscopic criteria" for the diagnosis of AD is based on
the number of neuritic plaques found in the brain. Khachaturian,
Arch. Neurol., 42: 1097 (1985). Unfortunately, assessment of
neuritic plaque counts must be delayed until after death.
[0006] Amyloid-containing neuritic plaques are a prominent feature
of selective areas of the brain in AD as well as Down's Syndrome
and in persons homozygous for the apolipoprotein E4 allele, who are
very likely to develop AD. Corder et al., Science, 261: 921 (1993);
Divry, P., J. Neurol. Psych., 27: 643 (1927); Wisniewski et al.,
Re-Examination of the Pathogenesis of the Senile Plaque: Progress
in Neuropathology, (Grune and Stratton, N.Y. 1973), pp. 1-26. Brain
amyloid is readily demonstrated by staining brain sections with
thioflavin S or Congo red. Puchtler et al., J. Histochem Cytochem,
10: 35 (1962). Congo red stained amyloid is characterized by a
dichroic appearance, exhibiting a yellow-green polarization color.
The dichroic binding is the result of the beta-pleated sheet
structure of the amyloid proteins. Glenner, G., N. Eng. J. Med.,
302: 1283 (1980). A detailed discussion of the biochemistry and
histochemistry of amyloid can be found in Glenner, G., N. Eng. J.
Med., 302: 1333 (1980).
[0007] Amyloidosis is a condition characterized by the accumulation
of various insoluble, fibrillar proteins, generically termed
"amyloid," in the tissues of a patient. Amyloidosis is a slowly
progressive condition, which can lead to significant morbidity and
death. A diverse group of diseases or disease processes fall under
the "amyloidosis" rubric, and can be termed amyloidosis-related
diseases. An amyloid deposit is formed by the aggregation of
amyloid proteins, followed by the further combination of aggregates
and/or amyloid proteins. Formation and accumulation of aggregates
of A.beta. peptides in the brain are critical factors in the
development and progression of AD. The fibrillar aggregates of
amyloid peptides, A.beta..sub.1-40 and A.beta..sub.1-42, are major
metabolic peptides derived from amyloid precursor proteins found in
senile plaques and cerebrovascular amyloid deposits in AD patients.
Xia et al., J. Proc. Natl. Acad. Sci. U.S.A., 97: 9299 (2000).
Prevention and reversal of A.beta. plaque formation are being
targeted as a treatment for this disease. Selkoe, D., JAMA, 283:
1615 (2000); Wolfe, M. S., et al., J. Med. Chem., 41: 6 (1998);
Skovronsky, D. M. et al., Trends Pharmacol. Sci., 21: 161
(2000).
[0008] In addition to the role of amyloid deposits in Alzheimer's
disease, the presence of amyloid deposits has been shown in
diseases such as glaucoma, Mediterranean fever, Muckle-Wells
syndrome, idiopathic myeloma, amyloid polyneuropathy, amyloid
cardiomyopathy, systemic senile amyloidosis, amyloid
polyneuropathy, hereditary cerebral hemorrhage with amyloidosis,
Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru,
Gerstmann-Straussler-Scheinker syndrome, medullary carcinoma of the
thyroid, Isolated atrial amyloid, .beta..sub.2-microglobulin
amyloid in dialysis patients, inclusion body myositis,
.beta..sub.2-amyloid deposits in muscle wasting disease, Islets of
Langerhans diabetes Type II insulinoma and other
amyloidosis-related diseases.
[0009] Thus far, diagnosis of AD has been achieved mostly through
clinical criteria evaluation, brain biopsies and post-mortem tissue
studies. Research efforts to develop methods for diagnosing AD in
vivo include (1) genetic testing, (2) immunoassay methods and (3)
imaging techniques.
[0010] Evidence that abnormalities in A.beta. metabolism are
necessary and sufficient for the development of AD is based on the
discovery of point mutations in the A.beta. precursor protein in
several rare families with an autosomal dominant form of AD. Hardy,
Nature Genetics, 1: 233 (1992); Hardy et al., Science, 256: 184
(1992). These mutations occur near the N and C-terminal cleavage
points necessary for the generation of A.beta. from its precursor
protein. St. George-Hyslop et al., Science, 235: 885 (1987); Kang
et al., Nature, 325: 733 (1987). Genetic analysis of a large number
of AD families has demonstrated, however, that AD is genetically
heterogeneous. St. George-Hyslop et al., Nature, 347: 194 (1990).
Linkage to chromosome 21 markers is shown in only some families
with early-onset AD and in no families with late-onset AD. More
recently, a gene on chromosome 14, whose product is predicted to
contain multiple transmembrane domains and resembles an integral
membrane protein, has been identified by Sherrington et al.,
Nature, 375: 754 (1995). This gene may account for up to 70% of
early-onset autosomal dominant AD. Preliminary data suggests that
this chromosome 14 mutation causes an increase in the production of
A.beta.. Scheuner et al., Soc. Neurosci. Abstr., 21: 1500 (1995). A
mutation on a very similar gene has been identified on chromosome 1
in Volga German kindreds with early-onset AD. Levy-Lahad et al.,
Science, 269: 973 (1995).
[0011] Screening for apolipoprotein E genotype has been suggested
as an aid in the diagnosis of AD. Scott, Nature, 366: 502 (1993);
Roses, Ann. Neurol., 38: 6 (1995). Difficulties arise with this
technology, however, because the apolipoprotein E4 allele is only a
risk factor for AD, not a disease marker. It is absent in many AD
patients and present in many non-demented elderly people. Bird,
Ann. Neurol., 38: 2 (1995).
[0012] Immunoassay methods have been developed for detecting the
presence of neurochemical markers in AD patients and to detect an
AD related amyloid protein in cerebral spinal fluid. Warner, Anal.
Chem., 59: 1203A (1987). These methods for diagnosing AD have not
been proven to detect AD in all patients, particularly, at early
stages of the disease, and are relatively invasive, requiring a
spinal tap. Also, attempts have been made to develop monoclonal
antibodies as probes for imaging of A.beta.. Majocha et al., J.
Nucl. Med., 33: 2184 (1992). The major disadvantage of antibody
probes is the difficulty in getting these large molecules across
the blood-brain barrier. Using antibodies for in vivo diagnosis of
AD would require marked abnormalities in the blood-brain barrier in
order to gain access into the brain. There is no convincing
functional evidence that abnormalities in the blood-brain barrier
reliably exist in AD. Kalaria, Cerebrovascular & Brain
Metabolism Reviews, 4: 226 (1992).
[0013] Radiolabeled A.beta. peptides have been used to label
diffuse, compact and neuritic type plaques in sections of AD brain.
However, these peptides share all of the disadvantages of
antibodies. Specifically, peptides do not normally cross the
blood-brain barrier in amounts necessary for imaging and because
these probes react with diffuse plaques, they may not be specific
for AD.
[0014] Data also suggest that amyloid binding compounds will have
therapeutic potential in AD and type 2 diabetes mellitus.
Morphological reactions including reactive astrocytosis, dystrophic
neurites, activated microglia cells, synapse loss and full
complement activation found around neuritic plaques all signify
that neurotoxic and cell degenerative processes are occurring in
the areas adjacent to these A.beta. deposits. Joachim et al., Am.
J. Pathol., 135: 309 (1989); Masliah et al., 137: 1293 (1990); Lue
et al., Dementia, 3: 308 (1992). A.beta.-induced neurotoxicity and
cell degeneration has been reported in a number of cell types in
vitro. Yankner et al., Science, 250: 279 (1990); Roher et al.,
BBRC, 174: 572 (1991); Frautschy et al., Proc. Natl. Acad. Sci.,
88: 83362 (1991); Shearman et al., 91: 1470 (1994). It has been
shown that aggregation of the A.beta. peptide is necessary for in
vitro neurotoxicity. Yankner, Neurobiol. Aging, 13: 615 (1992).
Thus far, several laboratories have reported results which suggest
that Congo red inhibits A.beta.-induced neurotoxicity and cell
degeneration in vitro. Burgevin et al., NeuroReport, 5: 2429
(1994); Lorenzo et al., Proc. Natl. Acad. Sci., 91: 12243 (1994);
Pollack et al., Neuroscience Letters, 184: 113 (1995); Pollack et
al., Neuroscience Letters, 197: 211 (1995). The mechanism appears
to involve both inhibition of fibril formation and prevention of
the neurotoxic properties of formed fibrils. Lorenzo et al., Proc.
Natl. Acad. Sci., 91: 12243 (1994). Congo red also has been shown
to protect pancreatic islet cells from the toxicity caused by
amylin. Lorenzo et al., Proc. Natl. Acad. Sci., 91: 12243 (1994).
Amylin is a fibrillar peptide similar to A.beta., which accumulates
in the pancreas in type 2 diabetes mellitus.
[0015] The inability to assess amyloid deposition in AD until after
death impedes the study of this devastating illness. Histological
analysis of biopsy or autopsy materials also has its drawbacks. As
such, a method of quantifying amyloid deposition before death is
needed both as a diagnostic tool in mild or clinically confusing
cases as well as in monitoring the effectiveness of therapies
targeted at preventing A.beta. deposition. Moreover, it remains of
utmost importance to develop a safe and specific method for
diagnosing AD before death by imaging amyloid in brain parenchyma
in vivo. Even though various attempts have been made to diagnose AD
in vivo, currently, there are few antemortem probes for brain
amyloid. To date, simple, noninvasive methods for detecting and
quantitating amyloid deposits in a patient have been eagerly
sought. Thus, a need exists for amyloid binding compounds which
enter the brain and bind selectively to amyloid. Similarly, amyloid
binding compounds are also needed for detecting and quantitating
amyloid deposits in other disease types including, for example,
those previously mentioned. Amyloid binding compounds may also be
needed for their therapeutic potential in the treatment and
prophylaxis of, for example, AD and type 2 diabetes mellitus.
SUMMARY OF THE INVENTION
[0016] The present invention relates to compounds and amyloid
probes thereof that allow for a safe and specific method of
diagnosing and quantitating AD before death by in vivo imaging of
amyloid and/or amyloid deposits in, for example, regions of the
brain and brain parenchyma. The invention also relates to methods
for identifying, detecting and/or quantitating AD amyloid deposits
and/or plaque in the brain before a patient's death, using
high-affinity amyloid probes and/or labeled compounds of the
invention, which have low toxicity, can cross the blood-brain
barrier and can distinguish an AD brain from a normal brain.
Preferably, the compounds or amyloid probes of the invention have a
low toxicity at an effective amount or dosage including dosages
effective for therapeutic or imaging (for example, identifying,
diagnosing, evaluating, detecting and/or quantitating amyloid
deposits or an amyloidosis-related disease state) uses. For
example, an amyloid probe of the invention can be administered to a
patient in an amount suitable for in vivo imaging of amyloid and/or
amyloid deposits (plaques) or aggregates as well as amyloid-like
aggregates and tau or synuclein aggregates. Moreover, the present
invention relates to compounds and amyloid probes thereof that bind
preferentially to or interact with amyloid proteins or precursors,
portions, fragments and peptides thereof and/or their deposits as
well as deposits that comprise one or more amyloid and/or
amyloidogenic proteins. Amyloid probes of the invention can also be
used to detect and quantitate amyloid deposits in such diseases as,
for example, AD, familial AD, homozygotes for the apolipoprotein E4
allele, glaucoma, Mediterranean fever, Muckle-Wells syndrome,
idiopathic myeloma, amyloid polyneuropathy, amyloid cardiomyopathy,
systemic senile amyloidosis, amyloid polyneuropathy, hereditary
cerebral hemorrhage with amyloidosis, Down's syndrome, Scrapie,
Creutzfeldt-Jacob disease, Kuru, Gerstmann-Straussler-Scheinker
syndrome, medullary carcinoma of the thyroid, Isolated atrial
amyloid, .beta..sub.2-microglobulin amyloid in dialysis patients,
inclusion body myositis, .beta..sub.2-amyloid deposits in muscle
wasting disease and Islets of Langerhans diabetes Type II
insulinoma.
[0017] In another aspect, the compounds of the invention may also
be used in the treatment or prophylaxis of diseases that include,
for example, AD and type 2 diabetes mellitus. The compounds of the
invention can also be used in the treatment or prophylaxis of a
disease state or malady characterized by or associated with amyloid
deposits or amyloidosis. Generally, prophylactic or prophylaxis
relates to a reduction in the likelihood of the patient developing
a disorder such as AD or proceeding to a diagnosis state for the
disorder. For example, the compounds of the invention can be used
prophylacticly as a measure designed to preserve health and prevent
the spread or maturation of disease in a patient. It is also
appreciated that the various modes of treatment or prevention of a
disease such as an amyloidosis-related disease or condition can
mean "substantial" treatment or prevention, which includes total
but also less than total treatment or prevention, and in which some
biologically or medically relevant result is achieved. Furthermore,
treatment or treating as well as alleviating can refer to
therapeutic treatment and prophylactic or preventative measures in
which the object is to prevent, slow down (lessen) a disease state,
condition or malady. For example, a subject can be successfully
treated for an amyloidosis-related disease if, after receiving
through administration an effective or therapeutic amount of one or
more compounds of the invention, the subject shows observable
and/or measurable reduction in or absence of one or more signs and
symptoms of the particular disease such as, but not limited to,
reduced morbidity and mortality, or improvement in quality of life
issues. The invention also provides for methods of administering
one or more compounds of the invention to a patient in an effective
amount for the treatment or prophylaxis of a disease such as, for
example, AD or type 2 diabetes mellitus.
[0018] The compounds of the invention can also be administered to a
patient along with other conventional therapeutic agents that may
be useful in the treatment or prophylaxis of amyloidosis-related
diseases. In one aspect, a method is provided for administering an
effective amount of one or more compounds of the invention to a
patient suffering from or believed to be a risk of suffering from a
disease characterized by amyloid deposition or amyloidosis.
Moreover, the invention relates to treating an amyloidosis-related
disease by administering an effective amount of one or more
compounds to a patient in need thereof. The methods of the
invention can also comprise administering, either sequentially or
in combination with one or more compounds of the invention, a
conventional therapeutic agent in an amount that can potentially or
synergistically be effective for the treatment or prophylaxis of an
amyloidosis-related disease. Exemplary therapeutic agents for use
in combination therapies with one or more compounds of the
invention include, but are not limited to, anti-inflammatory drugs,
therapeutic antibodies and cholesterol lowering drugs such as, for
example, statins. Fassbender et al., PNAS, 98: 5856 (2001);
DeMattos et al., PNAS, 98: 8850 (2001); Clark et al., An. Intern.
Med., 55: 15 (2004).
[0019] The compounds and amyloid probes of the invention also
include analogs, salts, pharmaceutical compositions, derivatives,
prodrugs or racemic mixtures thereof. Moreover, any methods, kits,
assays or uses (including, for example, those herein) for a
compound or amyloid probe of the invention can be performed with or
employ one or more such analogs, salts, pharmaceutical
compositions, derivatives, prodrugs or racemic mixtures.
Preferably, the compounds or amyloid probes of the invention are
amyloid binding compounds or a water soluble, non-toxic salt
thereof. In one aspect, the compounds of the invention are each
capable of being readily modified to be an amyloid probe that
comprises one or more detectable markers, tags or labels by
conventional techniques known to those of ordinary skill in the
art. Ellis et al., Aust. J. Chem., 26: 907 (1973); Wilson et al.,
J. Org. Chem., 51: 4833 (1986); Wilbur et al., J. Label. Compound.
Radiopharm., 19: 1171 (1982); Chumpradit et al., J. Med. Chem., 34:
877 (1991); Chumpradit et al., J. Med. Chem., 32: 1431 (1989);
Kabalka et al., J. Label. Compound. Radiopharm., 19: 795 (1982);
Koch et al., Chem. Ber., 124: 2091 (1991); H. Mach et al., J. Med.
Chem., 36: 3707 (1993); Arora et al., J. Med. Chem., 30: 918
(1987); March, J., Advanced Organic Chemistry: I Reactions,
Mechanisms, and Structure (3rd Ed., 1985); Morrison and Boyd,
Organic Chemistry (6th Ed., 1992).
[0020] The invention relates to compounds and amyloid probes that
can target amyloid deposits (plaques) in vivo or in vitro. The
compounds and/or probes of the invention can be administered to a
subject in effective amounts for therapeutic (for example, treating
or preventing an amyloidosis-related disease state) or imaging (for
example, identifying, diagnosing, evaluating, detecting and/or
quantitating amyloid deposits or an amyloidosis-related disease
state) applications. The compounds or amyloid probes of the
invention can comprise the structure or formula
##STR00001##
in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or
different and can independently be H, F, Cl, Br, I, NO.sub.2, CN,
CF.sub.3, alkyl, alkenyl, alkynyl, alkoxy, monoalkylamine,
dialkylamine, hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl,
aryl, heterocycles, heteroaryl, aralkyl, carboxy, esterified
carboxy, amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
R.sub.5 can be C.sub.nH.sub.2n+1 or --CH.sub.2--CH.dbd.CH--I ((E)
or (Z) configuration) and R.sub.6 can be C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m can each
independently be 0, 1, 2, 3, 4, 5, 6 or 7 and a dashed bond (---)
represents an optional bond, Y and Z can each independently be CH
or N and A.sub.linker can be any suitable linker including, for
example, one or more heteroaryl, aralkyl, aryl, alkyl, alkenyl,
alkynyl and/or heterocycle groups, each of which can independently
comprise one or more single, double or triple bonds (for example,
alkenyl or alkynyl groups) and substituents that include, for
example, NO.sub.2, CN, CF.sub.3, alkyl, alkyl, alkenyl, alkoxy,
monoalkylamine, dialkylamine, hydroxylalkyl, halo, haloalkyl,
alkylthio, alkylsulfonyl, heteroatom, heteroaryl, aralkyl, aryl,
heterocyclic, carboxy, esterified carboxy and/or amidate carboxy
groups. Preferably, an amyloid probe of the invention can comprise
one or more substituents as a label (marker or tag). Exemplary
labels include radionuclides, radioisotopes or isotopes. For
example, an amyloid probe of the invention comprises one or more
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6, each of
which can independently comprise (for example, R.sub.1 can be
.sup.131I or CH.sub.2--CH.sub.2--.sup.131I) 131I, .sup.124I,
.sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F, .sup.11C,
.sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
CH.sub.2--CH.sub.2-label, O--CH.sub.2--CH.sub.2-label,
CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group. A detectable label for an amyloid probe can be
included as an additional substituent (for example, group) to a
compound of the invention or as an alternative substituent for any
substituents that are present. For example, a label (tag or marker)
included as an additional substituent to the group
--CH.sub.2--CH.dbd.CH.sub.2 of a compound of the invention can be
CH.sub.2--CH.sub.2--CH.sub.2--.sup.131I. Moreover, a detectable
label provided as an alternative substituent for one or more
substituents present for a compound of the invention can, by way of
example, include CH.sub.2--CH.sub.2--CH.sub.3 to
CH.sub.2--CH.sub.2--CH.sub.2--.sup.131I, or
--CH.sub.2--CH.dbd.CH--I to --CH.sub.2--CH.dbd.CH--.sup.123I.
[0021] In one aspect, the invention relates to a compound or
amyloid probe thereof comprising the structure or formula
##STR00002##
in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or
different and can independently be H, F, Cl, Br, I, NO.sub.2, CN,
CF.sub.3, alkyl, alkenyl, alkynyl, alkoxy, monoalkylamine,
dialkylamine, hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl,
aryl, heterocycles, heteroaryl, aralkyl, carboxy, esterified
carboxy, amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
R.sub.5 can be C.sub.nH.sub.2n+1 or --CH.sub.2--CH.dbd.CH--I ((E)
or (Z) configuration) and R.sub.6 can be C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m can each
independently be 0, 1, 2, 3, 4, 5, 6 or 7, A and D can each
independently be N or C, E, Y and Z can each independently be CH or
N, B can be S, O, N or CH and a, b, c, d, e and f each
independently represent an optional bond, provided that when A and
E are N, then B can be CH, D can be C and b and d can each be a
bond (to provide double bonds), or provided that when B, D and E
are N, then A can be C, b and e can each be a bond (to provide
double bonds), or provided that when E is N and B is O or S, then A
and D can be C, a and c can each be a bond (to provide double
bonds), or further provided that when D is C, then f can be a bond
(to provide a triple bond of C.ident.C) or when D is N, then f is
not a bond (to provide a double bond of N.dbd.N). For example, an
amyloid probe of the invention can comprise one or more
substituents as a radiolabel (marker or tag). Preferably, an
amyloid probe of the invention comprises one or more of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6, each of which can
independently comprise (for example, R.sub.1 can be .sup.131I or
CH.sub.2--CH.sub.2--.sup.131I) .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.19F, .sup.11C, .sup.75Br, .sup.13N,
.sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2-label, CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group.
[0022] In one aspect, the invention relates to an in vivo or in
vitro method for detecting amyloid deposits. The invention also
relates to an in vivo or in vitro method for detecting in a subject
one or more amyloid deposits comprising one or more amyloid or
amyloidogenic proteins. For example, a method of the invention can
comprise administering to a subject thought to be of risk for or
suffering from a disease associated with amyloid deposits or
amyloidosis, a detectable quantity or effective amount of a
compound or amyloid probe thereof (or analogs, salts,
pharmaceutical compositions, derivatives, prodrugs or racemic
mixtures thereof) comprising
##STR00003##
in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or
different and can independently be H, F, Cl, Br, I, NO.sub.2, CN,
CF.sub.3, alkyl, alkenyl, alkynyl, alkoxy, monoalkylamine,
dialkylamine, hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl,
aryl, heterocycles, heteroaryl, aralkyl, carboxy, esterified
carboxy, amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
R.sub.5 can be C.sub.nH.sub.2n+1 or --CH.sub.2CH.dbd.CH--I ((E) or
(Z) configuration) and R.sub.6 can be C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m can each
independently be 0, 1, 2, 3, 4, 5, 6 or 7, A and D can each
independently be N or C, E, Y and Z can each independently be CH or
N, B can be S, O, N or CH and a, b, c, d, e and f each
independently represent an optional bond, provided that when A and
E are N, then B can be CH, D can be C and b and d can each be a
bond (to provide double bonds), or provided that when B, D and E
are N, then A can be C, b and e can each be a bond (to provide
double bonds), or provided that when E is N and B is O or S, then A
and D can be C, a and c can each be a bond (to provide double
bonds), or further provided that when D is C, then f can be a bond
(to provide a triple bond of C.ident.C) or when D is N, then f is
not a bond (to provide a double bond of N.dbd.N), and detecting the
binding of the compound or amyloid probe thereof to an amyloid
deposit comprising one or more amyloid or amyloidogenic proteins.
For example, an amyloid probe used in conjunction with a method of
the invention can comprise one or more substituents as a radiolabel
(marker or tag). Preferably, an amyloid probe of the invention
comprises one or more of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5 and R.sub.6, each of which can independently comprise (for
example, R.sub.1 can be .sup.131I or CH.sub.2--CH.sub.2--.sup.131I)
.sup.131I, .sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F,
.sup.19F, .sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O,
.sup.76Br, CH.sub.2--CH.sub.2-label, O--CH.sub.2--CH.sub.2-label,
CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group.
[0023] The invention also relates to a compound or amyloid probe
thereof comprising the structure or formula
##STR00004##
wherein
[0024] R.sub.1 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl,
alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl, aralkyl, carboxy, esterified carboxy,
amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
[0025] R.sub.2 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl,
alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl, aralkyl, carboxy, esterified carboxy,
amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
[0026] R.sub.3 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl,
alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl, aralkyl, carboxy, esterified carboxy,
amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
[0027] R.sub.4 is H, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl,
alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl, aralkyl, carboxy, esterified carboxy,
amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
[0028] R.sub.5 is or --CH.sub.2--CH.dbd.CHA ((E) or (Z)
configuration) and R.sub.6 is C.sub.nH.sub.2n+1,
--[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5, where n and m are
independently 0, 1, 2, 3, 4, 5, 6 or 7,
[0029] A is N or C,
[0030] D is N or C,
[0031] E is CH or N,
[0032] Y is CH or N,
[0033] Z is CH or N,
[0034] B is S, O, N or CH and
[0035] a, b, c, d, e and f each independently represent an optional
bond, provided that when A and E are N, then B is CH, D is C and b
and d are each a bond (to provide double bonds), or provided that
when B, D and E are N, then A is C, b and e are each a bond (to
provide double bonds), or provided that when E is N and B is O or
S, then A and D are C, a and c are each a bond (to provide double
bonds), or further provided that when D is C, then f is a bond (to
provide a triple bond of C.ident.C) or when D is N, then f is not a
bond (to provide a double bond of N.dbd.N). For example, an amyloid
probe of the invention can comprise one or more substituents as a
radiolabel (marker or tag). Preferably, an amyloid probe of the
invention comprises one or more of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6, each of which can independently
comprise (for example, R.sub.1 can be .sup.131I or
CH.sub.2--CH.sub.2--.sup.131I) .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br,
.sup.13C, .sup.13N, .sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2-label, CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be
I.sup.131I, .sup.124I, .sup.125I, .sup.18F, .sup.19F, .sup.11C,
.sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br, .sup.11C or
.sup.13C, or .sup.11C or .sup.13C can be a label (mark or tag) as a
substituent of a lower alkyl group, (CH.sub.2).sub.nOR, CL.sub.3,
CH.sub.2--CH.sub.2-L, O--CH.sub.2--CH.sub.2-L,
CH.sub.2--CH.sub.2--CH.sub.2-L, O--CH.sub.2--CH.sub.2--CH.sub.2-L,
CN, (C.dbd.O)--R, (C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl,
CR.dbd.CR-aryl or CR.sub.2--CR.sub.2-aryl in which L can be a
halogen (for example, .sup.13CH.sub.2--CH.sub.2--F) and R can be H,
F, Cl, Br, I or a lower alkyl group.
[0036] In one aspect, a compound of the invention can comprise one
or more of the exemplary structures or formulas of Table 1. The
compound or amyloid probe of the structure or formula
##STR00005##
can also comprise a compound or amyloid probe of Table 1.
TABLE-US-00001 TABLE 1 ##STR00006## R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is
CnH.sub.2n + 1, R.sub.3 is CnH.sub.2n + 1, F, Cl, Br, I or OR.sub.1
and R.sub.6 is CnH.sub.2n + 1, F, Cl, Br, I or OR.sub.1, where
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4, or 5 ##STR00007##
R.sub.1 is CnH.sub.2n + 1, where n is 0, 1, 2, 3, 4 or 5
##STR00008## R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is CnH.sub.2n + 1, R.sub.3 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1, R.sub.6 is
CnH.sub.2n + 1, F, Cl, Br, I or OR.sub.1 and X is O or S, where
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4 or 5 ##STR00009##
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and R.sub.2 is CnH.sub.2n + 1, where n is
0, 1, 2, 3, 4 or 5 ##STR00010## R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I and R.sub.2 is
CnH.sub.2n + 1, where n is 0, 1, 2, 3, 4 or 5 ##STR00011## R.sub.4
is F, Cl, Br, I or OR.sub.1, R.sub.5 is H, OR.sub.1 or
NR.sub.1R.sub.2, R.sub.6 is H, OR.sub.1 or NR.sub.1R.sub.2 and X is
O or S, where R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I
or (Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is CnH.sub.2n + 1 and n is 0,
1, 2, 3, 4 or 5 ##STR00012## R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is
CnH.sub.2n + 1, R.sub.3 is CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1
or OR.sub.1 and R.sub.6 is CnH.sub.2n + 1, F, Cl, Br, I or
OR.sub.1, where R.sub.1 is CnH.sub.2n + 1, (E)-
CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1, 2,
3, 4 or 5 ##STR00013## R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is
CnH.sub.2n + 1, R.sub.3 is CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1
or OR.sub.1, R.sub.6 is CH.sub.2n + 1, F, Cl, Br, I or OR.sub.1 and
R.sub.7 is CH.sub.2n + 1, F, Cl, Br, I or OR.sub.1, where R.sub.1
is CH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4, or 5 ##STR00014##
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is CnH.sub.2n + 1, R.sub.3 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1, R.sub.6 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1 and X is O or S,
where R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or (Z)-
CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4 or 5 ##STR00015##
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is CnH.sub.2n + 1, R.sub.3 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1, R.sub.6 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1 and X is O or S,
where R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or (Z)-
CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4 or 5 ##STR00016##
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is CnH.sub.2n + 1, R.sub.3 is
CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1 or OR.sub.1, R.sub.4 is F,
Cl, Br, I or OR.sub.1 and R.sub.6 is CnH.sub.2n + 1, F, Cl, Br, I,
NR.sub.1 or OR.sub.1, where R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1,
2, 3, 4, or 5 ##STR00017## ##STR00018## R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I, R.sub.2 is
CnH.sub.2n + 1, R.sub.3 is CnH.sub.2n + 1, F, Cl, Br, I, NR.sub.1
or OR.sub.1 and R.sub.4 is F, Cl, Br, I or OR.sub.1, where R.sub.1
is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1, 2, 3, 4, or 5 ##STR00019##
R.sub.1 is CnH.sub.2n + 1, (E)-CH.sub.2CH.dbd.CH--I or
(Z)-CH.sub.2CH.dbd.CH--I and R.sub.3 is CnH.sub.2n + 1, F, Cl, Br,
I, NR.sub.1 or OR.sub.1, where R.sub.1 is CnH.sub.2n + 1,
(E)-CH.sub.2CH.dbd.CH--I or (Z)-CH.sub.2CH.dbd.CH--I and n is 0, 1,
2, 3, 4, or 5
Preferably, an amyloid probe of one or more of the compounds of
Table 1 can comprise one or more substituents as a radiolabel
(marker or tag). For example, the label can replace any substituent
of a compound of the invention or be provided as an additional
substituent. An amyloid probe of one or more of the compounds of
Table 1 can comprise one or more of .sup.131I, .sup.124I, .sup.3H,
.sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br, .sup.13C,
.sup.13N, .sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2-label, CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group.
[0037] In another aspect, the compound or amyloid probe of the
structure or formula
##STR00020##
as well as a compound or amyloid probe of Table 1 can comprise any
one of the compounds of Table 2 or an amyloid probe thereof
comprising a label (for example, .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br,
.sup.13C, .sup.13N, .sup.15O, or .sup.76Br). Similarly, a compound
or probe of the invention can comprise one or more of the exemplary
structures or formulas of Table 2.
TABLE-US-00002 TABLE 2 ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053##
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059##
[0038] In one aspect, an amyloid probe of the invention can
comprise one or more of the exemplary structures or formulas of
Table 3. A probe comprising the structures or formulas of Table 3
can also be provided from a compound of Table 1 or any one of the
compounds of Table 2. For example, an amyloid probe of the
structure or formula
##STR00060##
can comprise one or more substituents as a label (marker or tag).
Exemplary labels include radionuclides, radioisotopes or isotopes.
For example, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and
R.sub.6, each of which can independently comprise (for example,
R.sub.1 can be .sup.131I or CH.sub.2--CH.sub.2--.sup.131I)
.sup.131I, .sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F,
.sup.19F, .sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O,
.sup.76Br, CH.sub.2--CH.sub.2-label, O--CH.sub.2--CH.sub.2-label,
CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group.
TABLE-US-00003 TABLE 3 ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074##
[0039] In one aspect, an amyloid probe of the invention can
comprise one or more of the exemplary structures or formulas of
Table 4. A probe of the structure or formula
##STR00075##
comprising one or more substituents as a label, marker or tag (for
example, a radionuclide, radioisotope or isotope) can also comprise
a probe of Table 4.
TABLE-US-00004 TABLE 4 ##STR00076## X = N or O n = 0 or 1 m = 0 or
1 Tag = .sup.123I or .sup.18F ##STR00077## X = N or O n = 0 or 1 m
= 0 or 1 Tag = .sup.123I or .sup.18F
[0040] The invention also relates to an in vivo method for
detecting amyloid deposits in a subject. For example, the method
can comprise administering a detectable quantity (effective amount)
of a labeled compound of the invention and detecting the binding of
the compound to an amyloid deposit in the subject. In one aspect,
the amyloid deposit is located in the brain of a subject. The
subject can be suffering from or suspected of suffering from a
disease associated with amyloid deposits or amyloidosis such as,
for example, AD, familial AD, homozygotes for the apolipoprotein E4
allele or Down's syndrome.
[0041] In one aspect, detection can be performed via a
scintigraphic approach. For example, detection accomplished by
gamma imaging, magnetic resonance imaging, magnetic resonance
spectroscopy and/or fluorescence spectroscopy. Preferably, the
scintigraphic approach for detecting an amyloid probe of the
invention comprises either PET or SPECT imaging and standard
protocols used in conjunction therewith. The compound or amyloid
probe of the invention can also be administered as a pharmaceutical
composition. Exemplary pharmaceutical compositions comprise a
compound or amyloid probe of the invention and a pharmaceutically
acceptable carrier. Preferably, administering a compound or probe
of the invention to a subject in need thereof can be by intravenous
injection or bolus intravenous injection. Other exemplary routes of
administration can include oral, rectal, parenteral (intramuscular
or subcutaneous), intracisternal, intravaginal, intraperitoneal,
local (powders, ointments or drops) or as a buccal or nasal spray
as well as ocular drops.
[0042] A method of the invention can also comprise determining a
ratio (for example, an amyloid deposit uptake ratio). In one
aspect, the ratio can be that of the radioactive uptake of a
compound or probe of the invention to a brain area other than the
cerebellum as compared to the radioactive uptake of the compound or
probe to the cerebellum. The method can comprise comparing the
ratio from a subject suffering from or thought to be at risk for a
disease associated with associated with amyloid deposits or
amyloidosis to that of a healthy (non-diseased) subject. In another
aspect, the invention relates to a method of inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady. For example, the method
comprises administering to a subject having, suspected of having
and/or at risk for a disease or malady associated with amyloid
deposits or amyloidosis, a compound or amyloid probe of the
invention in an effective amount.
[0043] A method of the invention relates to inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady. Preferably, the method
comprises administering to a subject in need thereof a compound or
amyloid probe of the invention (or analogs, salts, pharmaceutical
compositions, derivatives, prodrugs or racemic mixtures thereof) in
an effective amount, for example, an amount capable inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady. Examples of amyloidosis
associated diseases or maladies include, but are not limited to,
AD, familial AD, homozygotes for the apolipoprotein E4 allele,
glaucoma, Mediterranean fever, Muckle-Wells syndrome, idiopathic
myeloma, amyloid polyneuropathy, amyloid cardiomyopathy, systemic
senile amyloidosis, amyloid polyneuropathy, hereditary cerebral
hemorrhage with amyloidosis, Down's syndrome, Scrapie,
Creutzfeldt-Jacob disease, Kuru, Gerstmann-Straussler-Scheinker
syndrome, medullary carcinoma of the thyroid, Isolated atrial
amyloid, .beta..sub.2-microglobulin amyloid in dialysis patients,
inclusion body myositis, .beta..sub.2-amyloid deposits in muscle
wasting disease and Islets of Langerhans diabetes Type II
insulinoma.
[0044] In one aspect, the invention relates to a method for
detecting amyloid deposits in biopsy or post-mortem subject tissue
(in vitro). The method comprises incubating formalin-fixed tissue
with a solution of a compound or probe of the invention to allow
for binding with the deposit or formation of a labeled deposit and
detecting the compound, probe or labeled deposit. The solution can
be composed of 25 to 100% ethanol (with the remainder being water)
saturated with the compound or amyloid probe of the invention.
Preferably, in vitro detection can be accomplished by microscopic
techniques. Examples of microscopic techniques include bright
field, fluorescence, laser confocal or cross-polarization
microscopy.
[0045] The invention also relates to a method of distinguishing an
AD brain or a brain having amyloid deposits (plaques) from a normal
brain comprising incubating (separately) homogenates of weighed
tissue from the cerebellum and another area of the same brain other
than the cerebellum, from a subject suspected of having AD or
amyloid deposits, with a compound or probe of the invention so that
binding with amyloid in the tissues occurs. The method also
comprises quantifying the amount of amyloid bound to the compound
or probe by separating the tissue-bound from the tissue-unbound,
quantifying the tissue-bound and converting the units of
tissue-bound (labeled deposit) to units of micrograms of amyloid
per 100 mg of tissue by comparison with a standard. The method can
also comprise calculating a ratio of the amount of amyloid in the
area of the brain other than the cerebellum to the amount of
amyloid in the cerebellum and comparing the ratio of the amount of
amyloid in tissue from the subject suspected of having AD or
amyloid deposits with ratios for the amount of amyloid in the
tissue from normal subjects. In one aspect, the method comprises
determining the presence of AD or amyloid deposits if the ratio
from the brain of a subject suspected of having AD or amyloid
deposits is above about 40%, 50%, 60%, 70%, 80% or 90% (preferably,
for example, above 50% and, more preferably, for example, above
90%) of the ratios obtained from the brains of normal subjects.
[0046] The invention also relates to methods for preparing
compounds of the invention. In one aspect, one or more of the
compounds can be modified to be an amyloid probe of the invention.
The amyloid probes of the invention are particularly useful for the
in vivo diagnosis and/or study of the progression or regression of
disease states or maladies in a patient. Exemplary disease states
or maladies include, for example, AD, familial AD, homozygotes for
the apolipoprotein E4 allele, glaucoma, Mediterranean fever,
Muckle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy,
amyloid cardiomyopathy, systemic senile amyloidosis, amyloid
polyneuropathy, hereditary cerebral hemorrhage with amyloidosis,
Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru,
Gerstmann-Straussler-Scheinker syndrome, medullary carcinoma of the
thyroid, Isolated atrial amyloid, .beta..sub.2-microglobulin
amyloid in dialysis patients, inclusion body myositis,
.beta..sub.2-amyloid deposits in muscle wasting disease and Islets
of Langerhans diabetes Type II insulinoma. An amyloid probe may
also comprise one or more compounds of the invention and at least
one detectable marker, tag or label such as, for example, a
radionuclide, radioisotope or isotope. The selection of detectable
markers, tags or labels for an amyloid probe of the invention can
vary depending on the particular modality chosen for in vivo
imaging, the disease state or malady being diagnoses or studied or
the route of administration of the probe.
[0047] The invention relates to an in vivo or in vitro method for
detecting in a subject one or more amyloid deposits. In one aspect,
the amyloid deposit can comprise one or more amyloid or
amyloidogenic protein. The method comprises administering to a
subject suffering from a disease associated with amyloidosis, a
detectable quantity (effective amount) of a compound or amyloid
probe of the invention (or analogs, salts, pharmaceutical
compositions, derivatives, prodrugs or racemic mixtures thereof).
For example, an amyloid probe of the invention can comprise one or
more substituents as a label (radiolabel, marker or tag).
Preferably, an amyloid probe of the invention comprises one or more
radionuclides, radioisotopes or isotopes (labels). Examples of
radiolabels for an amyloid probe of the invention include, but are
not limited to, .sup.131I, .sup.124I, .sup.125I, .sup.3H,
.sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br, .sup.13C,
.sup.13N, .sup.15O, .sup.76Br. The method also comprises detecting
the binding of the compound or probe to an amyloid deposit
(plaque). An amyloid deposit can comprise amyloid or amyloidogenic
proteins (or precursors, portions, fragments and peptides thereof).
Examples of precursor and amyloidogenic proteins as well as
amyloidosis-related diseases are generally described in
International Publication No. WO 2007/035405, which is incorporated
by reference herein.
[0048] Moreover, the invention relates to an in vivo method for
detecting at least one amyloid deposit. For example, the method can
comprise administering to a subject suffering from or thought to be
at risk of suffering from a disease associated with amyloidosis, a
detectable quantity (effective amount) of a compound or probe of
the invention (or analogs, salts, pharmaceutical compositions,
derivatives, prodrugs or racemic mixtures thereof). In one aspect,
the compound or amyloid probe binds to the amyloid deposit. The
method also comprises irradiating the subject and collecting
imaging data emitted by the compound or amyloid probe. Optionally,
the method comprises processing the imaging data in order to
diagnose and/or study of the progression or regression (when
accompanied by a therapy protocol) of disease states or maladies in
a subject.
[0049] The invention also relates to the use of a compound or probe
of the invention for detecting amyloid deposits in a subject
suffering from a disease associated with amyloidosis. The invention
further relates to the use of a compound or amyloid probe of the
invention in the preparation of a medicament for use in the
detection of amyloid deposits in a subject. In one aspect, one or
more amyloid deposits are located in the brain. For example, a
subject can be suffering from amyloidosis characterized by amyloid
deposits (plaques) in the regions of the brain. Other organs or
tissues that can comprise amyloid deposits and are able to be
studied, detected or imaged using the compounds or probes of the
invention as well as methods, kits, assays or uses thereof include,
for example, mesodermal tissue, tenosynovium, joints, aortic,
thyroid, islets of Langerhans, aging pituitary, latrogenic, cardiac
atria, cornea, lens, vitreous humor, retina, sclera, pancreas and
parenchymatous organ. Preferably, a compound or amyloid probe of
the invention can be detected via approaches that include gamma
imaging, magnetic resonance imaging, magnetic resonance
spectroscopy or fluorescence spectroscopy.
[0050] In one aspect, the invention relates to a method of
diagnosing an amyloidosis-related disease or a neurodegenerative
disease such as, for example, AD. Preferably, the method comprises
contacting an ocular tissue with a labeled compound of the
invention (probe), which binds to an amyloid deposit and/or amyloid
protein or precursor, portion, fragment or peptide thereof and/or
one or more A.beta. and/or amyloidogenic proteins as well as any
receptors of the same in the ocular tissue. The method also
comprises optionally allowing the compound to distribute into the
lens and then imaging the ocular tissue. For example, the labeled
compound can comprises an amyloid probe of the invention and an
increase in binding of the probe to the ocular tissue compared to a
normal control level of binding indicates that the mammal is
suffering from or is at risk of developing an amyloidosis-related
disease or a neurodegenerative disease (for example, AD). A
compound or probe of the invention can be administered in an
effective amount to a subject, for example, as ocular drops.
[0051] In another aspect, the invention provides a method for
prognosis of an amyloidosis-related disease or a neurodegenerative
disease such as, for example, AD. For example, the method can
comprise contacting ocular tissue of a mammal with a compound or
probe, which binds to an amyloid deposit and/or amyloid protein or
precursor, portion, fragment or peptide thereof and/or one or more
A.beta. and/or amyloidogenic proteins as well as any receptors of
the same. The method can also optionally comprise allowing the
compound or probe to distribute into the lens and imaging the
ocular tissue. Preferably, the method comprises quantitating the
level of association of the compound or probe with the ocular
tissue and comparing the level of association with a normal control
level of association, where increasing levels of association over
time indicates an adverse prognosis. The methods of the invention
also contemplate administering a compound or probe of the invention
to a subject as ocular drops.
[0052] The invention also relates to a method for diagnosing an
amyloidosis-related disease or a predisposition thereto in a
mammal. The method comprises detection of an amyloid deposit and/or
amyloid proteins or precursors, portions, fragments or peptides
thereof (including A.beta. precursor proteins, A.beta.,
A.beta..sub.1-42, prion proteins and .alpha.-synuclein) and/or one
or more A.beta. and/or amyloidogenic proteins with a labeled
compound or probe in a supranuclear or deep cortical region of an
ocular lens. For example, the method comprises comparing an amount
of the "amyloid" compared to a normal control value and an increase
indicates that the subject is suffering from or is at risk of
developing an amyloidosis-related disease. In one aspect, detection
can be by quasi-elastic light scattering or spectroscopic
techniques (for example, Raman), although radioscintigraphy,
magnetic resonance imaging (MRI), assays, chemilumensence, near
infrared luminescence, fluorescence, gamma imaging, magnetic
resonance imaging, magnetic resonance spectroscopy, fluorescence
spectroscopy, SPECT, computed tomography (CT scan) and/or positron
emission tomography (PET) can also be used.
[0053] In one aspect, a method of diagnosing an amyloidosis-related
disease or a predisposition thereto in a mammal can comprise
illuminating the subject's lens tissue with an excitation light
beam and detecting scattered light emitted from the tissue (for
example, to detect a compound or probe of the invention. Exemplary
protocols, means, devices, apparatuses or systems for studying or
diagnosing amyloidosis-related diseases, particularly, those
associated with detecting amyloid deposits and/or amyloid proteins
or precursors, portions, fragments or peptides thereof (including
A.beta. precursor proteins, A.beta., A.beta..sub.1-42, prion
proteins and .alpha.-synuclein) and/or one or more A.beta. and/or
amyloidogenic proteins with a labeled compound or probe in a
supranuclear, deep cortical region of an ocular lens and/or ocular
tissues are generally described in U.S. Pat. Nos. 7,107,092 and
6,849,249, both of which are incorporated by reference herein.
DESCRIPTION OF THE DRAWINGS
[0054] Other features and advantages of the invention may also be
apparent from the following detailed description thereof, taken in
conjunction with the accompanying drawings, which may depict
preferred aspects by way of example, not by way of limitations.
[0055] FIG. 1 includes exemplary structures for compounds of the
invention, which can be modified to comprise one or more amyloid
probes that can be useful for in vivo imaging of amyloid and/or
amyloid deposits.
[0056] FIG. 2 includes exemplary structures for compounds of the
invention, which can be modified to comprise one or more amyloid
probes that can be useful for in vivo imaging of amyloid and/or
amyloid deposits.
[0057] FIG. 3 includes exemplary structures for compounds of the
invention, which can be modified to comprise one or more amyloid
probes that can be useful for in vivo imaging of amyloid and/or
amyloid deposits.
[0058] FIG. 4 includes experimental IC50 binding data for several
exemplary compounds of the invention, which can be modified to
comprise one or more amyloid probes that can be useful for in vivo
imaging of amyloid and/or amyloid deposits.
[0059] FIG. 5 includes experimental IC50 binding data for several
exemplary compounds of the invention, which can be modified to
comprise one or more amyloid probes that can be useful for in vivo
imaging of amyloid and/or amyloid deposits.
[0060] FIG. 6 includes SPECT images of a normal (healthy) and AD
diagnosed brain as obtained from an amyloid probe of the
invention.
[0061] FIG. 7 includes autoradiographic images of brain tissue
demonstrating in vitro binding of 125-I MNI-187 to amyloid deposits
and lack of binding for brain tissue pretreated and saturated with
conventional amyloid ligands (IMPY and PIB, Newberg et al., J. Nuc.
Med., 47: 748 (2006), Kung et al., Brain Res., 956: 202 (2002),
Kung et al., European J. Nuc. Med. Mol. Imaging, 31: 1136 (2004),
Blennow et al., European J. Nuc. Med. Mol. Imaging, 12: 753 (2006)
and Engler et al., The living Brain and Alzheimer's, (Eds.,
Springer-Verlag Berlin Heidelberg 2004), pp. 123-137.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention provides compounds and amyloid probes
thereof comprising detectable markers for antemortem in vivo
imaging of amyloid deposits such as, for example, amyloid plaques.
In one aspect, the compounds and amyloid probes of the invention
are amyloid binding compounds. The amyloid probes of the invention
can be used in vivo to diagnosis and study the progression or
regression of disease states or maladies that include, for example,
AD, Down's syndrome, familial AD, glaucoma, Mediterranean fever,
Muckle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy,
amyloid cardiomyopathy, systemic senile amyloidosis, amyloid
polyneuropathy, hereditary cerebral hemorrhage with amyloidosis,
Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru,
Gerstmann-Straussler-Scheinker syndrome, medullary carcinoma of the
thyroid, Isolated atrial amyloid, .beta..sub.2-microglobulin
amyloid in dialysis patients, inclusion body myositis,
.beta..sub.2-amyloid deposits in muscle wasting disease and Islets
of Langerhans diabetes Type II insulinoma and homozygotes for the
apolipoprotein E4 allele.
[0063] In one aspect, the invention relates to a compound or
amyloid probe thereof comprising the structure or formula
##STR00078##
in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or
different and can independently be H, F, Cl, Br, I, NO.sub.2, CN,
CF.sub.3, alkyl, alkenyl, alkynyl, alkoxy, monoalkylamine,
dialkylamine, hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl,
aryl, heterocycles, heteroaryl, aralkyl, carboxy, esterified
carboxy, amidate carboxy, OR.sub.6, NR.sub.5R.sub.6 or R.sub.6,
R.sub.5 can be C.sub.nH.sub.2n+1 or --CH.sub.2--CH.dbd.CH-halo ((E)
or (Z) configuration in which halo can be any halogen) and R.sub.6
can be C.sub.nH.sub.2n+1, --[CH.sub.2--CH.sub.2--O].sub.m--R.sub.5,
where n and m can each independently be 0, 1, 2, 3, 4, 5, 6 or 7, A
and D can each independently be N or C, E, Y and Z can each
independently be CH or N, B can be S, O, N or CH and a, b, c, d, e
and f each independently represent an optional bond, provided that
when A and E are N, then B can be CH, D can be C and b and d can
each be a bond (to provide double bonds), or provided that when B,
D and E are N, then A can be C, b and e can each be a bond (to
provide double bonds), or provided that when E is N and B is O or
S, then A and D can be C, a and c can each be a bond (to provide
double bonds), or further provided that when D is C, then f can be
a bond (to provide a triple bond of C.ident.C) or when D is N, then
f is not a bond (to provide a double bond of N.dbd.N), and
detecting the binding of the compound or amyloid probe thereof to
an amyloid deposit comprising one or more amyloid or amyloidogenic
proteins. For example, an amyloid probe used in conjunction with a
method of the invention can comprise one or more substituents as a
radiolabel (marker or tag). Preferably, an amyloid probe of the
invention comprises one or more of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6, each of which can independently
comprise (for example, R.sub.1 can be .sup.131I or
CH.sub.2--CH.sub.2--.sup.131I) .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br,
.sup.13C, .sup.13N, .sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2-label, CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group.
[0064] The in vivo methods of the invention can be performed on a
subject having, suspected of having or at risk for an
amyloidosis-related disease or a disease or malady associated with
amyloid deposits and/or amyloidosis. An amyloid probe of the
invention can comprise a label (marker or tag) that includes, for
example, radionuclides, radioisotopes or isotopes. For example, a
label can replace any substituent of a compound of the invention or
be provided as an additional substituent for an amyloid probe. In
one aspect, an amyloid probe of a compound of the invention can
also comprise one or more of .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br,
.sup.13C, .sup.13N, .sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2-label, CH.sub.2--CH.sub.2--CH.sub.2-label,
O--CH.sub.2--CH.sub.2--CH.sub.2-label,
--[OCH.sub.2--CH.sub.2].sub.n-label, O--CH.sub.2--CH.dbd.CH-label
((E) or (Z) configuration), N--CH.sub.2--CH.dbd.CH-label ((E) or
(Z) configuration) in which "label" can independently be .sup.131I,
.sup.124I, .sup.125I, .sup.3H, .sup.123I, .sup.18F, .sup.19F,
.sup.11C, .sup.75Br, .sup.13C, .sup.13N, .sup.15O, .sup.76Br,
.sup.11C or .sup.13C, or .sup.11C or .sup.13C can be a label (mark
or tag) as a substituent of a lower alkyl group,
(CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2F) and R can be H, F, Cl, Br, I or a lower
alkyl group.
[0065] The invention also relates to compounds or amyloid probes
that can be characterized as amyloid binding compounds (including
analogs, salts, pharmaceutical compositions, derivatives, prodrugs
or racemic mixtures thereof). In one aspect, a compound or amyloid
probe of the invention can be a water-soluble, non-toxic salt
thereof. Preferably, a compound or probe of the invention binds to
amyloid deposits (plaques). For example, a compound or probe of the
invention can bind to amyloid (including A.beta.) and/or
amyloidogenic proteins or precursors, portions, fragments and
peptides thereof, which can comprise one or more amyloid deposits
or plaques. A compound or amyloid probe of the invention can
preferentially bind to amyloid deposits that are present in disease
states or maladies characterized by or associated with
amyloidosis.
[0066] A compound or amyloid probe of the invention can bind to
amyloid deposits of amyloid (including A.beta.) and/or
amyloidogenic proteins with a dissociation constant (for example,
an equilibrium dissociation constant, K.sub.d) from, for example,
about 0.0001 to 10 .mu.M as measured by binding to a synthetic
amyloid peptide or AD brain tissue. The invention contemplates
measurement of a dissociation constant (for example, K.sub.d and
K.sub.i) or performing competition, saturation and kinetics
experiments by conventional techniques routine to one of ordinary
skill in the art. Moreover, a compound or probe of the invention
can compete with a reference compound for binding to amyloid
deposits with a dissociation constant of inhibition (for example,
K.sub.i) from, for example, about 0.01 nM to >10,000 nM. For
example, a compound of the invention (MM-187) demonstrated a
high-affinity for A.beta. based on its IC50 binding value of 0.17
nM as evaluated using human AD brain tissue.
[0067] In one aspect, a method of the invention can be used to
determine the presence and location of amyloid deposits in an organ
or body area, preferably, the brain, of a patient. An exemplary
method of the invention comprises administration of a detectable
quantity of an amyloid probe to a patient. For example, an amyloid
probe may be derived from a compound of the invention such as those
having the exemplary structures included in FIGS. 1, 2 and 3 or
Tables 1, 2, 3 and 4. An amyloid probe may be administered to a
patient as a pharmaceutical composition or a pharmaceutically
acceptable salt, preferably, water-soluble, thereof.
[0068] "Pharmaceutically acceptable salt" can refer to an acid or
base salt of a compound or amyloid probe of the invention, which
possesses the desired pharmacological activity and is neither
biologically nor otherwise undesirable. Pharmaceutically acceptable
salt can also refer to those carboxylate salts or acid addition
salts of the compounds or amyloid probes of the invention, which
are suitable for use in contact with the tissues of patients
without undue toxicity, irritation, allergic response and the like.
The salt can refer to the relatively nontoxic, inorganic and
organic acid addition salts of compounds or probes of the present
invention and may be formed with acids that include, without
limitation, acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride
hydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate,
maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
oxalate, thiocyanate, tosylate and undecanoate. Examples of a base
salt include, without limitation, ammonium salts, alkali metal
salts such as sodium and potassium salts, alkaline earth metal
salts such as calcium and magnesium salts, salts with organic bases
such as dicyclohexylamine salts, N-methyl-D-glucamine and salts
with amino acids such as arginine and lysine. In various aspects,
the basic nitrogen-containing groups can be quarternized with
agents including lower alkyl halides such as methyl, ethyl, propyl
and butyl chlorides, bromides and iodides, dialkyl sulfates such as
dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides
such as decyl, lauryl, myristyl and stearyl chlorides, bromides and
iodides and aralkyl halides such as phenethyl bromides. Also
included are those salts derived from non-toxic organic acids such
as aliphatic mono and dicarboxylic acids, phenyl-substituted
alkanoic acids, hydroxy alkanoic and alkanedioic acids, aromatic
acids and aliphatic and aromatic sulfonic acids. These salts can be
prepared in situ during the final isolation and purification of the
compounds or by separately reacting the purified compound in its
free base form with a suitable organic or inorganic acid and
isolating the salt thus formed. Further representative salts
include the hydrobromide, hydrochloride, sulfate, bisulfate,
nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate,
laurate, borate, benzoate, lactate, phosphate, tosylate, citrate,
maleate, fumarate, succinate, tartrate, naphthylate mesylate,
glucoheptonate, lactiobionate and laurylsulphonate salts,
propionate, pivalate, cyclamate, isethionate and the like. These
may include cations based on the alkali and alkaline earth metals
such as sodium, lithium, potassium, calcium, magnesium and the like
as well as nontoxic ammonium, quaternary ammonium and amine cations
including, but not limited to, ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine and the like. Berge S. M., et al.,
Pharmaceutical Salts, J. Pharm. Sci., 66: 1 (1977).
[0069] In one aspect, a compound or amyloid probe of the invention
is administered to a patient in an amount or dosage suitable for
therapeutic use or in vivo imaging. Generally, a unit dosage
comprising a compound or amyloid probe of the invention will vary
depending on patient considerations. Such considerations include,
for example, age, protocol, condition, sex, extent of disease,
contraindications, concomitant therapies and the like. An exemplary
unit dosage based on these considerations can also be adjusted or
modified by a physician skilled in the art. For example, a unit
dosage for a patient comprising an amyloid probe can vary from
1.times.10.sup.-15 g/kg to 10 g/kg, preferably, 1.times.10.sup.-15
g/kg to 1.0 g/kg. Moreover, a unit dosage comprising an amyloid
probe can also be from 1 .mu.Ci/kg to 10 mCi/kg and, preferably,
0.1 mCi/kg. Dosage of a compound or probe of the invention can also
vary from 0.001 .mu.g/kg to 10 .mu.g/kg or, preferably, from 0.01
.mu.g/kg to 1.0 .mu.g/kg. An effective amount for detection of a
compound or probe of the invention administered to a subject as
ocular drops can also be adjusted or modified by one skilled in the
art. Similarly, an effective amount for therapeutic use of a
compound or probe of the invention administered to a subject as
ocular drops can also be adjusted or modified by one skilled in the
art.
[0070] For administration as ocular drops, if a probe of the
invention emits light in the range of a normal human lens
autofluorescence (blue-green range), the level of autofluorescence
is factored into a spectroscopic reading. By way of example, a 10%
increase in fluorescence (after probe administration) compared to
the level in the absence of the probe (autofluorescence) indicates
a pathological state or predisposition to developing an
amyloidosis-related disease (for example, AD). Preferably, baseline
autofluorescence is established (prior to probe administration) for
each subject. A diagnostic level of fluorescence can be at least
25% (preferably, at least 50% and, more preferably, at least 100%)
greater than a normal control value. For example, detection of an
amyloid probe of the invention via fluorescence spectroscopy, which
is 2-fold or more greater than a normal control value, indicates a
pathological state. Given that normal human lens tissue
autofluorescences in the blue-green range (495 nm-520 nm), the
probe can preferably emit a wavelength of light outside the
blue-green spectra. In one aspect, the probe can emits a wavelength
of light greater than 520 nm (for example, fluorescence in the red,
orange-red or infrared range). Alternatively, an amyloid probe of
the invention can emit a wavelength less than 450 nm (for example,
in the violet or ultra-violet (UV) range). Other protocols, means,
devices, apparatuses or systems for studying or diagnosing
amyloidosis-related diseases, particularly, those associated with
detecting amyloid deposits and/or amyloid proteins or precursors,
portions, fragments or peptides thereof (including A.beta.
precursor proteins, A.beta., A.beta..sub.1-42, prion proteins and
.alpha.-synuclein) and/or one or more A.beta. and/or amyloidogenic
proteins with a labeled compound or probe administered to a subject
as ocular drops are generally described in U.S. Pat. Nos. 7,107,092
and 6,849,249, both of which are incorporated by reference
herein.
[0071] Administration of a compound or amyloid probe of the
invention to a subject may be local or systemic and accomplished
intravenously, intraarterially, intrathecally (via the spinal
fluid) or the like. Administration may also be intradermal or
intracavitary, depending upon the body site under examination. In
one aspect, after a sufficient time has elapsed for an amyloid
probe of the invention to bind with the amyloid, for example, 5
minutes to 48 hours, the area of the subject under investigation is
examined by routine imaging techniques or modalities such as
magnetic resonance spectroscopy (MRS), magnetic resonance
spectroscopy imaging (MRI), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), planar
scintillation imaging or combinations thereof as well as any
emerging imaging modalities. The exact protocol will necessarily
vary depending upon factors specific to the patient and depending
upon the body site under examination, method of administration and
type of amyloid probe or detectable marker used, although the
determination of specific procedures would be routine to the
skilled artisan.
[0072] For brain imaging, preferably, the amount (total or specific
uptake) of a bound amyloid probe of the invention (such as a probe
that is radioactively labeled with a detectable marker) is measured
and compared (as a ratio) with the uptake of a labeled compound of
the invention, which may be an amyloid probe, bound to the
cerebellum of the patient. This ratio is then compared to the same
ratio in one or more age-matched normal brains. Preferably, an
amyloid probe of the invention is administered intravenously to a
patient in an amount or dosage appropriate for in vivo imaging of
amyloid and/or amyloid deposits. The compounds and amyloid probes
of the invention can also be administered via a pharmaceutically
acceptable carrier. In one aspect, a compound of the invention can
be administered for the treatment or prophylaxis of a disease such
as AD. For example, a compound of the invention can be included in
a composition comprising a pharmaceutically acceptable carrier. An
exemplary composition contains human serum albumin and a compound
of the invention.
[0073] The amyloid probes of the invention can also be administered
in the form of injectable compositions, but may also be formulated
into well known drug delivery systems such as, for example, oral,
rectal, parenteral (intravenous, intramuscular, or subcutaneous),
intracisternal, intravaginal, intraperitoneal, local (powders,
ointments or drops) or as a buccal or nasal spray as well as ocular
drops. As described, administration of a compound or amyloid probe
of the invention may also be local or systemic and accomplished
intravenously, intraarterially, intrathecally (via the spinal
fluid) or the like. A typical composition for administration can
comprise a pharmaceutically acceptable carrier for the compound or
amyloid probe of the invention. A pharmaceutically acceptable
carrier includes such carriers as, for example, aqueous solutions,
non-toxic excipients including salts, preservatives, buffers and
the like, which are described in Remington's Pharmaceutical
Sciences, 15th Ed. Easton: Mack Publishing Co., pp. 1405-1412 and
1461-1487 (1975) and The National Formulary XIV., 14th Ed.
Washington: American Pharmaceutical Association (1975).
[0074] Exemplary pharmaceutically acceptable carriers for a
compound or amyloid probe of the invention can also include
non-aqueous solvents such as propylene glycol, polyethylene glycol
and vegetable oil or injectable organic esters such as ethyl
oleate. An aqueous carrier can also include, without limitation,
water, alcoholic/aqueous solutions, saline solutions and parenteral
vehicles such as sodium chloride or Ringer's dextrose. Intravenous
carriers for administration of a compound or amyloid probe of the
invention include, for example, fluid and nutrient replenishers.
Preservatives for a compound or amyloid probe of the invention also
may include antimicrobial solutions, anti-oxidants, chelating
agents and inert gases. The pH and exact concentration of the
various components for a pharmaceutical composition can also be
adjusted according to routine skills in the art. Goodman and
Gilman's The Pharmacological Basis for Therapeutics (7th
Edition).
[0075] In one aspect, amyloid probes of the invention are those
that, in addition to binding (for example, preferentially or
specifically) amyloid in vivo and capable of crossing the blood
brain barrier, are non-toxic at appropriate dosage levels and have
a satisfactory duration of effect. Moreover, a pharmaceutical
composition comprising an amyloid probe can be administered to a
subject in whom amyloid or amyloid fibril formation is anticipated,
for example, patients clinically diagnosed with AD or another
disease associated with amyloid deposition. An amyloid probe of a
pharmaceutical composition can be derived from a compound of the
invention such as those having the exemplary structures included in
FIGS. 1, 2 and 3 or Tables 1, 2, 3 and 4.
[0076] The invention employs amyloid probes which, in conjunction
with noninvasive neuroimaging techniques or modalities such as MRS,
MRI, PET or SPECT, are used to quantify amyloid deposition in vivo.
The methods of the invention also involve imaging a patient to
establish a baseline of amyloid deposition. The term "baseline" can
refer to the amount and distribution of a patient's amyloid
deposition prior to initiation of an anti-amyloid therapy. An
exemplary method of the invention comprises at least one imaging
session of a patient following administration of an anti-amyloid
therapy. In one aspect, a method of the invention may involve
imaging a patient before and after treatment with at least one
anti-amyloid or therapeutic agent such as, for example,
anti-inflammatory or cholesterol lowering drugs including statins.
In vivo imaging may also be performed at any time during the
treatment.
[0077] Amyloid probes can comprise labeled (marked or tagged)
amyloid binding compounds for imaging or detection (for example,
identifying, diagnosing, evaluating and/or quantitating in vivo or
in vitro) amyloid deposits (plaques) and/or an amyloidosis-related
disease state. Amyloid probes can bind (associated or interact) to
amyloid deposits including deposits that comprise amyloid proteins
or precursors, portions, fragments and peptides thereof and/or one
or more A.beta. and/or amyloidogenic proteins as well as any
receptors of such. Amyloid probes can also bind to amyloid proteins
or precursors, portions, fragments and peptides thereof and/or one
or more A.beta. and/or amyloidogenic proteins as well as any
receptors of such. The binding of amyloid probes to amyloid
deposits or amyloid proteins or precursors, portions, fragments and
peptides thereof and/or one or more A.beta. and/or amyloidogenic
proteins (as well as any receptors of such) can be of high-affinity
and a specific or preferential nature as would be understood by one
of ordinary skill in the art and evaluated by conventional
techniques related to binding (for example, dissociation
constants). The amyloid probes of the invention can include
analogs, salts, pharmaceutical compositions, derivatives, prodrugs
or racemic mixtures thereof.
[0078] A compound of the invention comprises amyloid binding
compounds. An amyloid binding compound of the invention can be
labeled with any suitable marker (radiolabel or tag) to provide or
comprise an amyloid probe. Amyloid binding compounds of the
invention can bind (associated or interact) to amyloid deposits
including deposits that comprise amyloid proteins or precursors,
portions, fragments and peptides thereof and/or one or more A.beta.
and/or amyloidogenic proteins as well as any receptors of such. The
compounds of the invention can also bind to amyloid proteins or
precursors, portions, fragments and peptides thereof and/or one or
more A.beta. and/or amyloidogenic proteins as well as any receptors
of such. The binding of amyloid binding compounds to amyloid
deposits or amyloid proteins or precursors, portions, fragments and
peptides thereof and/or one or more A.beta. and/or amyloidogenic
proteins (as well as any receptors of such) can be of high-affinity
and a specific or preferential nature as would be understood by one
of ordinary skill in the art and evaluated by conventional
techniques related to binding (for example, dissociation
constants). The amyloid binding compounds of the invention can
include analogs, salts, pharmaceutical compositions, derivatives,
prodrugs or racemic mixtures thereof. Moreover, amyloid binding
compounds of the invention can be useful as therapeutic agents for
the treatment or prophylaxis of amyloidosis or an
amyloidosis-related disease state. In one aspect, the amyloid
binding compounds of the invention are capable of preventing cell
degeneration and toxicity associated with amyloid fibril formation.
For example, an amyloid binding compound of the invention can
inhibit cell degeneration and toxicity associated with fibril
formation in an amyloidosis associated disease or malady.
Preferably, an amyloid binding compound can be administered
therapeutically to a subject (for example, a patient in need of
treatment for an amyloidosis-related disease state) in an effective
amount (for example, an amount capable of inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady) to treat a patient
suffering from or thought to be at risk for an amyloidosis-related
disease state.
[0079] The term "in vivo" or "in vitro" in the context of detection
or imaging can refer to any method that permits the detection of an
amyloid probe of the invention or labeled compound such as, for
example, a compound having an exemplary structure included in FIG.
1, 2 or 3 or Tables 1, 2, 3 and 4. Similarly, an "in vivo method
for detecting" or "in vitro method for detecting" as well as "use
in detection" can comprise any type of detection for a compound or
amyloid probe of the invention. Exemplary techniques for detection
for a compound or probe of the invention include scintigraphy,
radioscintigraphy, magnetic resonance imaging (MRI),
chemilumensence, near infrared luminescence, fluorescence, SPECT,
computed tomography (CT scan), positron emission tomography (PET)
or combinations thereof and detection and related techniques are
understood by those of ordinary skill in the art. Moreover,
detection can include any future developed techniques related to
the field of imaging. For gamma-based imaging, the radiation
emitted from the organ or area being examined is measured and
expressed either as total uptake or as a ratio in which total
uptake in one tissue is normalized to (for example, divided by) the
total uptake in another tissue of the same subject during the same
in vivo imaging procedure. Total uptake in vivo is defined as the
entire signal detected in a tissue by an in vivo imaging technique
without the need for correction by a second administration of an
identical quantity of a probe or labeled compound along with a
large excess of unlabeled, but otherwise chemically identical,
compound. Similarly, in vitro methods can involve obtaining a fresh
or frozen tissue specimen and incubating a section of the tissue or
a homogenate of the tissue with a labeled compound of the invention
and then separating bound and free radiolabel by washing the tissue
section or filtering and washing the tissue homogenate. The bound
radioactivity can be measured by standard autoradiographic
techniques or by liquid scintillation or gamma counting and
compared to controls from the same tissue to which an excess of
unlabeled compounds has been added.
[0080] A "subject" or "patient" is a mammal, preferably, a human,
and, most preferably, a human suspected of having a disease
associated with amyloid deposition such as AD and/or dementia. The
term "subject" and "patient" can be used interchangeably. Moreover,
any substituents for the compounds and amyloid probes of the
invention are generally understood to be described herein in the
alternative or, as appropriate, in a conjunctive manner. For
example, a compound or amyloid probe of the invention can comprise
substituents such as F, Cl, Br, I in the alternative or, as
appropriate, in a conjunctive manner.
[0081] For purposes of in vivo or in vitro imaging, the type of
detection instrument available is a major factor in selecting a
given detectable marker. For example, radioactive isotopes and
.sup.18F or .sup.123I are particularly suitable for in vivo imaging
in the methods of the invention. The type of instrument used will
also guide the selection of a radionuclide or stable isotope. In
one aspect, the radionuclide chosen must have a type of decay
detectable by a given type of instrument. Moreover, other
considerations such as the half-life of the radionuclide are taken
into account when selecting a detectable marker for in vivo
imaging.
[0082] The half-life of a detectable marker should be long enough
so that the marker is still detectable at the time of maximum
uptake by the target, but short enough so that the subject does not
sustain deleterious radiation. The amyloid probes of the invention
can be detected using gamma imaging in which emitted gamma
irradiation of the appropriate wavelength is detected. Conventional
methods of gamma imaging include, but are not limited to, SPECT and
PET. Preferably, for SPECT detection, the chosen detectable marker
will lack a particulate emission, but will produce a large number
of photons in a 140-300 keV range. For PET detection, the
detectable marker will be a positron-emitting radionuclide such as
.sup.18F, which will annihilate to form two 511 keV gamma rays that
can then be detected by a PET camera.
[0083] In one aspect, compounds or amyloid probes of the invention,
which are useful for in vivo imaging and quantification of amyloid
deposition, are administered to a patient. These compounds or
probes are to be used in conjunction with non-invasive neuroimaging
techniques such as MRS, MRI, PET, SPECT and combinations thereof.
Preferably, a compound of the invention may be labeled with
.sup.19F or .sup.13C to yield an amyloid probe for MRS/MRI using
general organic chemistry techniques known to the art. March, J.,
Advanced Organic Chemistry: I Reactions, Mechanisms, and Structure
(3rd Ed., 1985); Morrison and Boyd, Organic Chemistry (6th Ed.,
1992). The compounds of the invention also may be radiolabeled with
.sup.18F, .sup.11C, .sup.78Br or .sup.76Br for PET by techniques
well known in the art and described by Fowler, J. and Wolf, A. in
Positron Emission Tomography and Autoradiography (Phelps, M.,
Mazziota, J., and Schelbert, H., eds.) pp. 391-450 (Raven Press, NY
1986). The compounds of the invention also may be radiolabeled with
.sup.123I for SPECT by any of several techniques known to the art.
Kulkarni, Int. J. Rad. Appl. & Inst., (Part B) 18: 647
(1991).
[0084] A label, detectable label, radiolabel, tag, marker,
detectable marker, tracer, radiotracer or equivalent term as
generally understood by those of ordinary skill in the art can
represent any substituent (group, moiety, position) suitable for
imaging and/or assaying (for example, identifying, diagnosing,
evaluating, detecting and/or quantitating) in vivo or in vitro. For
example, an amyloid probe of the invention can comprise labels,
radiolabels, tags, markers, detectable markers, tracers,
radiotracers or equivalent terms suitable for in vivo or in vitro
detection via radioscintigraphy, magnetic resonance imaging (MRI),
assays, chemilumensence, near infrared luminescence, fluorescence,
spectroscopy, gamma imaging, magnetic resonance imaging, magnetic
resonance spectroscopy, fluorescence spectroscopy, SPECT, computed
tomography (CT scan), positron emission tomography (PET). Suitable
labels, radiolabels, tags, markers, detectable markers, tracers,
radiotracers or equivalent terms are known by those skilled in the
art and can include, for example, radioisotopes, radionuclides,
isotopes, fluorescent groups, biotin (in conjunction with
streptavidin complexation) or photoaffinity groups. Preferably, a
label, detectable label, radiolabel, tag, marker, detectable
marker, tracer, radiotracer of an amyloid probe of the invention
can comprise .sup.131I, .sup.124I, .sup.125I, .sup.3H, .sup.123I,
.sup.18F, .sup.19F, .sup.11C, .sup.11C, .sup.75Br, .sup.13C,
.sup.13N, .sup.15O, .sup.76Br, CH.sub.2--CH.sub.2-Q,
O--CH.sub.2--CH.sub.2-Q, CH.sub.2--CH.sub.2--CH.sub.2-Q or
O--CH.sub.2--CH.sub.2--CH.sub.2-Q, --[OCH.sub.2--CH.sub.2].sub.n-Q,
O--CH.sub.2--CH.dbd.CH-Q ((E) or (Z) configuration),
N--CH.sub.2--CH.dbd.CH-Q ((E) or (Z) configuration) in which "Q"
can independently be .sup.131I, .sup.124I, .sup.125I, .sup.3H,
.sup.123I, .sup.18F, .sup.19F, .sup.11C, .sup.75Br, .sup.13C,
.sup.13N, .sup.15O, .sup.76Br, .sup.11C or .sup.13C, or .sup.11C or
.sup.13C can be a label, detectable label, radiolabel, tag, marker,
detectable marker, tracer, radiotracer as a substituent of a lower
alkyl group, (CH.sub.2).sub.nOR, CL.sub.3, CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2-L, CH.sub.2--CH.sub.2--CH.sub.2-L,
O--CH.sub.2--CH.sub.2--CH.sub.2-L, CN, (C.dbd.O)--R,
(C.dbd.O)N(R).sub.2, O(CO)R, OR, COOR, aryl, CR.dbd.CR-aryl or
CR.sub.2--CR.sub.2-aryl in which L can be a halogen (for example,
.sup.13CH.sub.2--CH.sub.2--F) and R can be H, F, Cl, Br, I or a
lower alkyl group. "Photoaffinity group" or "photoaffinity labeled"
can refer to a substituent on a compound or probe of the invention,
which can be activated by photolysis at an appropriate wavelength
to undergo a cross-linking photochemical reaction with a
macromolecule associated therewith. An example of a photoaffinity
group is a benzophenone substituent.
[0085] Suitable radioisotopes are known to those skilled in the art
and include, for example, isotopes of halogens (such as chlorine,
fluorine, bromine and iodine) and metals including technetium and
indium. Exemplary labels, radiolabels, tags, markers, detectable
markers, tracers, radiotracers can also include .sup.3H, .sup.11C,
.sup.14C, .sup.18F, .sup.32P, .sup.35S, .sup.123I, .sup.125I,
.sup.131I, .sup.124I, .sup.19F, .sup.75Br, .sup.13C, .sup.13N,
.sup.15O, .sup.76Br. The amyloid probes of the invention may be
labeled (radiolabeled, tagged, marked, detectablely marked, traced
or radiotraced) either directly (that is, by incorporating the
label directly into a compound of the invention) or indirectly
(that is, by incorporating the label into a compound of the
invention through a chelating agent, where the chelating agent has
been incorporated into the compound). Furthermore, a label for an
amyloid probe can be included as an additional substituent (group,
moiety, position) to a compound of the invention or as an
alternative substituent for any substituents that are present. For
example, a label included as an additional substituent to the group
--CH.sub.2--CH.dbd.CH.sub.2 of a compound of the invention can be
CH.sub.2--CH.sub.2--CH.sub.2--.sup.131I. Moreover, a label provided
as an alternative substituent for one or more substituents present
for a compound of the invention can, by way of example, include
CH.sub.2--CH.sub.2--CH.sub.3 to
CH.sub.2--CH.sub.2--CH.sub.2--.sup.131I, or CH.sub.2--CH.dbd.CH--I
to --CH.sub.2--CH.dbd.CH--.sup.123I. A label, detectable label,
radiolabel, tag, marker, detectable marker, tracer or radiotracer
may appear at any substituent (group, moiety, position) on a
compound or probe of the invention.
[0086] In one aspect, labeling can be isotopic or nonisotopic. With
isotopic labeling, one substituent (group, moiety, position)
already present in a compound of the invention can be substituted
with (exchanged for) a radioisotope or isotope. With nonisotopic
labeling, a radioisotope or isotope can be added to a compound of
the invention without substituting with (exchanging for) an already
existing group. Direct and indirect labeled compounds as well as
isotopic and nonisotopic labeled compounds are contemplated by an
amyloid probe of the invention comprising one or more labels,
radiolabels, tags, markers, detectable markers, tracers or
radiotracers and equivalents thereof. Preferably, a label,
detectable label, radiolabel, tag, marker, detectable marker,
tracer or radiotracer can be reasonably stable, both chemically and
metabolically, applying recognized standards in the art. Moreover,
although the compounds or probes of the invention may be labeled in
any fashion (for example, via conventional techniques) with a
variety of different substituents, as those skilled in the art can
appreciate, such labeling may be performed in a manner so as to
retain the high-affinity (binding affinity) and a specific or
preferential nature of binding to amyloid deposits or amyloid
proteins or precursors, portions, fragments and peptides thereof
and/or one or more A.beta. and/or amyloidogenic proteins (as well
as any receptors of thereof). In one aspect, the affinity and
specificity of a compound of the invention is not significantly
affected by labeling to comprise an amyloid probe. By not
significantly affected, affinity and specificity may not be
affected by more than, for example, about 3 log units (preferably,
not more than, for example, about 2 log units or, more preferably,
not more than, for example, about 1 log unit). Furthermore, by not
significantly affected, affinity and specificity may not be
affected by more than, for example, about 500% (preferably, not
more than, for example, about 250% or, more preferably, affinity
and specificity may not be affected at all).
[0087] In addition, the compounds of the invention may be labeled
with any suitable radioactive iodine isotope such as, but not
limited to, .sup.131I, .sup.125I or .sup.123I by iodination of a
diazotized amino derivative directly via a diazonium iodide
(Greenbaum, F., Am. J. Pharm., 108: 17 (1936)), by conversion of
the unstable diazotized amine to the stable triazene or by
conversion of a non-radioactive halogenated precursor to a stable
tri-alkyl tin derivative, which then can be converted to an iodo
compound by several methods well known to the art. Satyamurthy and
Barrio, J. Org. Chem., 48: 4394 (1983), Goodman et al., J. Org.
Chem., 49: 2322 (1984), Mathis et al., J. Labell. Comp. and
Radiopharm., 1994: 905; Chumpradit et al., J. Med. Chem., 34: 877
(1991); Zhuang et al., J. Med. Chem., 37: 1406 (1994); Chumpradit
et al., J. Med. Chem., 37: 4245 (1994). For example, a stable form
or derivative of a compound of the invention can be reacted with a
halogenating agent containing .sup.131I, .sup.125I, .sup.123I,
.sup.75Br, .sup.76Br or .sup.18F. Thus, the stable form or
derivative of a compound of the invention and analogs, salts,
pharmaceutical compositions, derivatives, prodrugs, racemic
mixtures or tautomeric forms thereof are precursors useful for the
synthesis of many of the amyloid probes of the invention.
[0088] The compounds of the invention also may be radiolabeled with
known metal detectable markers such as Technetium-99m (.sup.99mTc).
Modification of the substituents to a compound of the invention in
order to introduce ligands that bind such metal ions can be
effected without undue experimentation by one of ordinary skill in
the art. The metal radiolabeled compound of the invention can then
be used as an amyloid probe to detect amyloid deposits. Preparing
amyloid probes comprising a detectable marker such as .sup.99mTc is
well known in the art. Zhuang et al., Nuclear Medicine &
Biology, 26(2): 217 (1999); Oya et al., Nuclear Medicine &
Biology, 25(2): 135 (1998); Horn et al., Nuclear Medicine &
Biology, 24(6): 485 (1997).
[0089] In one aspect, a method of the invention may use isotopes
detectable by nuclear magnetic resonance (NMR) spectroscopy for
purposes of in vivo imaging and spectroscopy. Elements particularly
useful in magnetic resonance spectroscopy include .sup.1H, .sup.19F
and .sup.13C. Suitable detectable markers for preparing an amyloid
probe of the invention also include beta-emitters, gamma-emitters,
positron-emitters and x-ray emitters. Moreover, exemplary
detectable markers include .sup.131I, .sup.124I, .sup.125I,
.sup.3H, .sup.123I, .sup.18F, .sup.11C, .sup.75Br, .sup.13C,
.sup.13N, .sup.15O and .sup.76Br. Suitable stable isotopes for use
in MRI or MRS, according to the invention, include .sup.19F and
.sup.13C. In another aspect, suitable radioisotopes for in vitro
quantification of amyloid in homogenates of biopsy or post-mortem
tissue include .sup.125I, .sup.14C and .sup.3H. Preferably, an
amyloid probe of the invention comprises .sup.11C, .sup.124I or
.sup.18F for use in PET in vivo imaging, .sup.123I for use in SPECT
imaging, .sup.19F for MRS/MRI and .sup.3H or .sup.14C for in vitro
studies. Nonetheless, any conventional method or detectable markers
for visualizing amyloid probes can be used in accordance with the
invention and may be appreciated by those of ordinary skill in the
art.
[0090] In one aspect of the invention relating to detecting amyloid
deposits in biopsy tissue, a method is provided that involves
incubating formalin-fixed tissue with a solution of a compound or
amyloid probe of the invention. Preferably, the solution is 5-20%
ethanol (with the remainder being 0.9% saline) saturated with a
compound or amyloid probe of the invention. Alternatively, such a
solution may be used for detection or quantitation of amyloid
deposits in non-biopsied tissues. Given that the detection of
amyloid deposits can be performed in biopsied tissue, the solution
used for incubation can also be from 5-100% ethanol (with the
remainder being water). Upon incubation, the compound or probe
stains or labels the amyloid deposit in the tissue and the stained
or labeled deposit can be detected or visualized by any standard
method. Such detection means include microscopic techniques such as
bright-field, fluorescence, laser-confocal and cross-polarization
microscopy. A method of quantifying the amount of amyloid in biopsy
tissue involves incubating an amyloid probe or labeled compound of
the invention or a water-soluble, non-toxic salt thereof with
homogenate of biopsy or post-mortem tissue. The tissue is obtained
and homogenized by techniques well known in the art.
[0091] Preferably, a detectable marker for an amyloid probe or
labeled compound of the invention is a radiolabel, although other
labels such as enzymes, chemiluminescent and immunofluorescent
labels are well known to skilled artisans. In one aspect, a
detectable marker such as .sup.125I, .sup.14C or .sup.3H can be
used to label a compound of the invention such as a compound having
an exemplary structure included in FIG. 1, 2 or 3 or Tables 1, 2, 3
and 4. Tissue containing amyloid deposits will bind to the
compounds or amyloid probes of the invention. For biopsied tissues,
the bound tissue can then be separated from the unbound tissue by
any mechanism known to the skilled artisan such as filtering. The
bound tissue may also be quantified through any means known to the
skilled artisan. The units of tissue-bound probes or labeled
compounds of the invention are then converted to units of
micrograms of amyloid per 100 mg of tissue by comparison to a
standard curve generated by incubating known amounts of amyloid
with a probe or labeled compound of the invention.
[0092] In one aspect, a method of the invention can determine the
presence and location of amyloid deposits in an organ, tissue or
body area of a subject. For example, a method of the invention can
be used to detect the presence and location of amyloid deposits in
the brain of a subject suffering from an amyloidosis-related
disease or malady including, but not limited to, AD, familial AD,
homozygotes for the apolipoprotein E4 allele, glaucoma,
Mediterranean fever, Muckle-Wells syndrome, idiopathic myeloma,
amyloid polyneuropathy, amyloid cardiomyopathy, systemic senile
amyloidosis, amyloid polyneuropathy, hereditary cerebral hemorrhage
with amyloidosis, Down's syndrome, Scrapie, Creutzfeldt-Jacob
disease, Kuru, Gerstmann-Straussler-Scheinker syndrome, medullary
carcinoma of the thyroid, Isolated atrial amyloid,
.beta..sub.2-microglobulin amyloid in dialysis patients, inclusion
body myositis, .beta..sub.2-amyloid deposits in muscle wasting
disease and Islets of Langerhans diabetes Type II insulinoma.
[0093] The ability of a compound or probe of the invention to
preferentially (or specifically) bind to amyloid plaques may vary
depending on concentration, although the determination of specific
concentrations to achieve binding that can be effective for therapy
and/or imaging (for example, identifying, diagnosing, evaluating,
detecting and/or quantitating amyloid deposits or an
amyloidosis-related disease state) would be routine to the skilled
artisan. For example, the probes or labeled compounds may be
specific for A.beta. deposits at concentrations less than 50 nM.
These low concentrations are also detectable with imaging studies
including PET. The use of the probes or labeled compounds of the
invention also permits detection in amyloid deposits such as those
found in plaques and cerebrovascular amyloid. Give that it has been
reported that A.beta. levels in the frontal cortex are increased
prior to neurofibrillary tangle formation, the invention
contemplates that probes or labeled compounds of the invention,
used as detectable labels, would be specific for the earliest
changes in AD cortex. Naslund et al. JAMA, 283: 1571 (2000).
[0094] When the compounds of the invention are modified to be used
as amyloid probes, they may be labeled with suitable radioactive
halogen isotopes. Although .sup.125I isotopes are useful for
laboratory testing, they will generally not be useful as a
detectable marker for actual diagnostic purposes given the
relatively long half-life (60 days) and low gamma-emission (30-65
Kev) of .sup.125I. The isotope .sup.123I has a half-life of
thirteen hours and a gamma energy of 159 KeV such that amyloid
probes comprising this detectable marker can be readily used for
diagnostic purposes. Other isotopes which may be used for in vivo
imaging include .sup.131I (half-life of 8.3 days). Suitable bromine
isotopes for an amyloid probe of the invention also include
.sup.77Br, .sup.75Br and .sup.76Br.
[0095] The compounds and probes of the invention lend themselves
easily to formation from materials that could be provided to users
in kits. For example, kits for forming the amyloid probes can
contain, without limitation, a vial containing a physiologically
suitable solution of an intermediate of a compound of the invention
in a concentration and at a pH suitable for optimal complexing
conditions. The user would add to the vial an appropriate quantity
of a detectable marker, for example, Na.sup.123I and an oxidant
such as hydrogen peroxide. The resulting probe may then be
administered intravenously to a patient such that amyloid plaque in
the brain can be imaged antemortem by a means for measuring the
gamma ray or photo emissions from the probe.
[0096] In one aspect, a method of the invention may be used to
diagnose AD in mild or clinically confusing cases. For example, the
method provides for longitudinal studies of amyloid deposition in
high risk populations including, without limitation, patients
suffering from or believed to be at risk of suffering from Down's
syndrome, familial AD or homozygotes for the apolipoprotein E4
allele. Corder et al., Science, 261: 921 (1993). The method also
provides for the temporal sequence of amyloid deposition to be
followed to determine if deposition occurs long before dementia
begins or if deposition is unrelated to dementia. The method of the
invention can also be used to monitor the effectiveness of
therapies targeted at preventing amyloid deposition.
[0097] As indicated, the specific method of detection of a compound
or probe of the invention can vary, depending upon the chemical and
physical nature of the species utilized and detected. For
gamma-emitting species, standard, commercially available single
photon and positron detection methods can be utilized. For magnetic
nuclear spin detection, standard, commercially available magnetic
resonance imaging and spectroscopy techniques can be utilized.
[0098] In the methods of the invention, data collection using
conventional and developing technologies can be conducted according
to standard clinical imaging protocols involving whole body imaging
techniques such as repeatedly moving the subject through the
scanner over the course of the scanning period. In one aspect, data
collection may be achieved by imaging selectively over one or more
regions of interest in the body, for example, by emphasizing the
brain, lungs, liver, heart or kidneys using a limited range of
patient body coverage in an imaging scanner. Following the
administration of a compound or probe of the invention, imaging
data collection can begin immediately and proceed for several hours
post administration using a dynamic imaging protocol. Late-time
snapshots of about 30 minutes could also be taken following the in
vivo distribution of the compound or amyloid probe using standard
static late time imaging protocols. Imaging data can then be
collected and stored electronically in an automated and routine
fashion, for later processing and analysis. Data processing and
analysis can make use of commercially available software packages,
which are typically installed by the manufacturer on the single
photon, positron emission or magnetic resonance scanners' operating
system computers.
[0099] Examples of these processes and methods for detecting,
collecting and processing imaging data are established in the art
for positron emission methodologies. Price et al., J. Cereb. Blood
Flow Metab., 25: 1528 (2005) and Lopresti et al., Nuclear Medicine,
46: 1959 (2005). Analogous data collection and processing of single
photon, positron and magnetic resonance species are similarly
conducted for systemic amyloid deposits using standard,
commercially available scanners, data collection methodologies and
data processing techniques in body regions including the brain.
[0100] The invention also provides a method for the treatment or
prophylaxis of a disease characterized by amyloid deposition and/or
amyloidosis comprising administering to a patient in need thereof
an effective amount of a compound of the invention. In one aspect,
the method can include providing a patient suffering from or
believed to be at risk of suffering from a disease characterized
by, for example, amyloid deposition and/or amyloidosis. The method
may also comprise administering to the patient an effective amount
of a compound of the invention. The compound of the invention can
also be administered as part of a composition comprising a
pharmaceutically acceptable carrier.
[0101] In another aspect, a method for detecting or quantitating a
disease characterized by amyloid deposition and/or amyloidosis
comprising administering to a patient in need thereof an effective
amount of an amyloid probe of the invention. For example, the
method can comprise a patient suffering from or believed to be at
risk of suffering from a disease characterized by, without
limitation, amyloid deposition and/or amyloidosis. The method may
also comprise administering to the patient an effective amount of
an amyloid probe of the invention and, optionally, imaging the
probe in vivo. Exemplary means for imaging of an amyloid probe of
the invention in vivo include, without limitation, MRS, MRI, PET,
SPECT or combinations thereof.
[0102] "Effective amount" can refer to the amount required to
produce a desired effect. One example of an effective amount
includes amounts or dosages that enable detecting, quantitation and
imaging of amyloid deposits in vivo or in vitro. In one aspect, the
amyloid deposits can comprise one or more amyloid or amyloidogenic
proteins. Another example of an effective amount includes amounts
or dosages that yield acceptable toxicity and bioavailability
levels for imaging or therapeutic (pharmaceutical) use including,
but not limited to, the treatment or prophylaxis of amyloidosis or
an amyloidosis-related disease state. Another example of an
effective amount includes amounts or dosages that are capable of
preventing cell degeneration and toxicity associated with amyloid
fibril formation.
[0103] A method of the invention relates to inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady. Preferably, the method
comprises administering to a subject in need thereof a compound or
amyloid probe of the invention (or analogs, salts, pharmaceutical
compositions, derivatives, prodrugs or racemic mixtures thereof) in
an effective amount, for example, an amount capable inhibiting cell
degeneration and toxicity associated with fibril formation in an
amyloidosis associated disease or malady. Examples of amyloidosis
associated diseases or maladies include, but are not limited to,
AD, familial AD, homozygotes for the apolipoprotein E4 allele,
glaucoma, Mediterranean fever, Muckle-Wells syndrome, idiopathic
myeloma, amyloid polyneuropathy, amyloid cardiomyopathy, systemic
senile amyloidosis, amyloid polyneuropathy, hereditary cerebral
hemorrhage with amyloidosis, Down's syndrome, Scrapie,
Creutzfeldt-Jacob disease, Kuru, Gerstmann-Straussler-Scheinker
syndrome, medullary carcinoma of the thyroid, Isolated atrial
amyloid, .beta..sub.2-microglobulin amyloid in dialysis patients,
inclusion body myositis, .beta..sub.2-amyloid deposits in muscle
wasting disease and Islets of Langerhans diabetes Type II
insulinoma.
[0104] The invention also provides a method of distinguishing a
normal brain from one comprising amyloid deposits indicative of a
disease state or malady. In one aspect, the method comprises
obtaining tissue samples from the cerebellum and another area of
the brain of a normal subject. Furthermore, the method includes
obtaining comparable tissue samples from subjects suffering from or
suspected of suffering from a disease such as, for example, AD.
These tissue samples are made into separate homogenates using
methods well known to the skilled artisan and are then incubated
with an amyloid probe of the invention. The amount of tissue that
binds to the probe is calculated for each tissue sample type, for
example, cerebellum, non-cerebellum, normal or abnormal and a ratio
for the binding of non-cerebellum to cerebellum tissue is
calculated. These ratios are may also be compared to each other. In
one aspect, if the ratio from the brain suspected of having a
disease such as AD is above about 40%, 50%, 60%, 70%, 80% or 90%
(preferably, for example, above 50% and, more preferably, for
example, above 90%) of the ratios obtained from normal brains, the
diagnosis of a disease state is made. The normal ratios can be
obtained from previously obtained data or, alternatively, they may
be recalculated at the same time the suspected brain tissue is
studied via a method of the invention.
[0105] In one aspect, a pharmaceutical composition comprising an
amyloid probe can also be prepared easily and simply by a user with
a kit. For example, the invention provides a kit comprising as
materials therefor a non-radiolabeled compound of the invention.
Optionally, the compound can be in a dry condition and, also
optionally, one or more inert, pharmaceutically acceptable carriers
and/or auxiliary substances may be added thereto. A kit of the
invention can also include materials such as a reducing agent and,
optionally, a chelator. These materials may also be combined.
Moreover, the kit can comprise instructions for carrying out a
method that involves reacting the materials with a detectable
marker including, without limitation, .sup.123I, .sup.125I,
.sup.124I, .sup.131I, .sup.18F, .sup.75Br, .sup.76Br or .sup.99mTc.
An exemplary .sup.99mTc detectable marker can be in the form of a
pertechnetate solution that is, optionally, included with a kit of
the invention. Similarly, the detectable marker can also be
included with the kit. The kit can also include instructions for
performing an in vivo imaging protocol with an amyloid probe
prepared therefrom.
[0106] In one aspect, a pertechnetate solution for a kit of the
invention can be obtained from a molybdenum-technetium-generator.
Such generators are available in a number of institutions that
perform radiodiagnostic procedures. As indicated, the materials for
a kit of the invention may be combined, provided they are
compatible. Such a monocomponent kit, in which the combined
materials are preferably lyophilized, is suitable to be reacted by
the user with the pertechnetate solution in a simple manner that
will be appreciated by those of ordinary skill in the art.
[0107] The invention also provides a method for preparing an
amyloid probe comprising a .sup.99mTc detectable marker by reacting
.sup.99mTc as a pertechnetate in the presence of a reducing agent
and, optionally, a suitable chelator. For example, the reducing
agent serves to reduce the .sup.99mTc perteclnetate, which is
eluted from a molybdenum-technetium-generator in a physiological
solution such as saline. Suitable reducing agents are, for example,
dithionite, formamidine sulphinic acid, diaminoethane disulphinate
or metallic agents such as Sn(II), Fe(II), Cu(I), Ti(III) or
Sb(III). In one aspect, .sup.99mTc is reacted with a compound of
the invention as a salt or in the form of Tc bound to comparatively
weak chelators. For the latter, a .sup.99mTc complex is formed by
ligand exchange. Examples of suitable chelators for a method of the
invention include, without limitation, dicarboxylic acids such as
oxalic acid, malonic acid, succinic acid, maleic acid, orthophtalic
acid, malic acid, lactic acid, tartaric acid, citric acid, ascorbic
acid, salicylic acid or derivatives thereof, phosphorus compounds
such as pyrophosphates and enolates. Preferably, citric acid,
tartaric acid, ascorbic acid, glucoheptonic acid or derivatives
thereof can be used as a chelate of .sup.99mTc given that each
undergoes a ligand exchange particularly easily.
[0108] In one aspect, [Tc.sup.vO].sup.+3N.sub.2S.sub.2 complexes
are prepared based on stannous (II) chloride reduction of
[.sup.99mTc]-pertechnetate. The method of labeling can rely on a
.sup.99mTc ligand exchange reaction between .sup.99mTc
(Sn)-glucoheptonate and the N.sub.2S.sub.2 ligand. Preparation of
stannous (II) chloride and preserving it in a consistent stannous
(II) form is necessary for the success of the labeling reaction. To
stabilize the air-sensitive stannous ion, it may be preferably to
use a lyophilized kit in which the stannous ion is in a lyophilized
powder form mixed with an excess amount of glucoheptonate under an
inert gas such as nitrogen or argon. The preparation of a
lyophilized stannous chloride/sodium glucoheptonate kit may ensure
that the method of labeling is reproducible and predictable. The
N.sub.2S.sub.2 ligands can be air-sensitive (thiols are easily
oxidized by air) such that they may need be preserved by using
lyophilized kits containing 100-500 .mu.n of the ligands under
argon or nitrogen.
[0109] When desired, an amyloid probe of the invention or
pharmaceutical composition thereof may contain any additive such as
pH controlling agents (for example, acids, bases, buffers),
stabilizers (for example, ascorbic acid) or isotonizing agents (for
example, sodium chloride). It will also be appreciated that the
methods of the invention can be performed in conjunction with other
in vivo techniques such as, for example, PET or SPECT imaging for
evaluating one or more additional characteristics of the subject
including, but not limited to, neuronal cell loss, glucose
metabolic activity or behavioral characteristics. Exemplary
behavioral characteristics can often be assessed by MMSE and
Buschke scores. In one aspect, one or more in vivo techniques can
be used to detect or quantitate amyloid and/or amyloid deposits and
monitor regional decreases in glucose metabolism in parietal and
temporal lobes of a patient.
[0110] In one aspect, the invention also contemplates the use of
stable .eta..sup.5-substituted cyclopentadienyltricarbonyl rhenium
and technetium organometallic complexes for radiolabeling one or
more compounds of the invention. These complexes may be abbreviated
collectively as CpMet(CO).sub.3 complexes in which Met is metal or
referred to individually as cyclopentadienyltricarbonylrhenium
(CpRe(CO).sub.3) for the rhenium and CpTc(CO).sub.3 for the
technetium analogs. As compared to the more widely used high
oxidation state metal-oxo complexes, CpMet(CO).sub.3 complexes
exhibit high chemical and metabolic stability, are lipophilic and
relatively small and, unlike many inorganic chelates, do not
possess additional stereocenters. Thus, these complexes can be
useful for the development of amyloid probes or metal-labeled
compounds of the invention. The preparation and use of low valent
(for example, Met(CO).sub.3.sup.+) technetium and rhenium are also
known to those of skill in the art. For example, the suitability of
CpMet(CO).sub.3 conjugates as amyloid ligands has been shown by a
series of CpRe(CO).sub.3 and manganese conjugates with nanomolar
affinity.
[0111] A practical radiochemical preparation of substituted
CpMet(CO).sub.3 complexes was the double ligand transfer (DLT)
reaction, originally reported in 1992. Improved versions of the DLT
reaction that minimize the formation of unwanted byproducts are
also known to those in the art. While not being bound by theory,
this transformation involves the in situ reduction/carbonylation of
the permetalate species, followed by selective ring transfer from
an appropriately substituted ferrocene precursor. The reaction can
occur in a single pot and, in most cases, is limited to ferrocenes
substituted with electronwithdrawing groups. However, use of a DLT
reaction for labeling compounds of the invention may require
additional steps in order to conjugate the substituted
CpMet(CO).sub.3 to the compound. The invention also contemplates
alternative routes to CpRe(CO).sub.3 complexes without a
requirement for substitution with an electron withdrawing group.
For example, one technique known in the art involves a
"three-component condensation" reaction and the stannane
approach.
[0112] To extend the DLT methodology further and to expand
structure-activity relationships for organometallic amyloid probes,
a direct version of the DLT reaction can also be used according to
the invention. In general, for one or more compounds of the
invention, a direct version of the DLT reaction can be applied to a
series of ferrocenyl phenyl benzoxazole conjugates and the binding
affinity of the compounds to amyloid deposits can be measured.
Although such rhenium compounds could be made more efficiently
without going through a ferrocene intermediate, the purpose may be
to develop methods that could be applied to short-lived
.gamma.-emitting radiotracers. Additional ligands can also be made
by the three-component condensation. Besides the innate interest in
the rhenium compounds as organometallic amyloid probes, they serve
as analogs of radioactive rhenium and technetium agents that may be
useful for SPECT imaging. The conditions for metal incorporation
such as high temperature, pressure in organic solvent and
chromatographic purification are also known to those of ordinary
skill in the art.
[0113] In one aspect of the invention, an amyloid probe is
introduced into a tissue or a patient in a detectable quantity. The
probe may be part of a pharmaceutical composition and is
administered to the tissue or the patient by methods well known to
those skilled in the art. For example, the compound can be
administered either orally, rectally, parenterally (intravenous, by
intramuscularly or subcutaneously), intracisternally,
intravaginally, intraperitoneally, intravesically, locally
(powders, ointments or drops) or as a buccal or nasal spray as well
as ocular drops.
[0114] In another aspect, an amyloid probe of the invention is
introduced into a patient in a detectable quantity and after
sufficient time has passed for the compound to become associated
with amyloid deposits, the probe is detected. The protocol is
noninvasive (without incision) as the probe inside the patient is
detected by, for example, an imaging device, apparatus, means or
system outside the patient. Alternatively, an amyloid probe of the
invention is introduced into a patient, sufficient time is allowed
for the probe to become associated with amyloid deposits and then a
sample of tissue from the patient is removed and the probe in the
tissue is detected apart from the patient. A tissue sample can also
be removed from a patient and an amyloid probe introduced into the
tissue sample. After a sufficient amount of time has passed for the
amyloid probe to become bound to amyloid deposits, the probe is
detected by a suitable imaging modality.
[0115] The administration of an amyloid probe to a patient can be
by a general or local administration route. For example, the
amyloid probe may be administered to the patient such that it is
delivered throughout the body. Alternatively, the amyloid probe can
be administered to a specific organ or tissue of interest. In one
aspect, it may be desirable to locate and quantitate amyloid
deposits in the brain in order to diagnose or track the progress of
AD in a patient.
[0116] A compound or amyloid probe of the invention can also be
modified, for example, by the covalent attachment of an organic
moiety or conjugate to improve pharmacokinetic properties, toxicity
or bioavailability (e.g., increased in vivo half-life). The
conjugate can be a linear or branched hydrophilic polymeric group,
fatty acid group or fatty acid ester group. A polymeric group can
comprise a molecular weight that can be adjusted by one of ordinary
skill in the art to improve, for example, pharmacokinetic
properties, toxicity or bioavailability. Exemplary conjugates can
include a polyalkane glycol (e.g., polyethylene glycol (PEG),
polypropylene glycol (PPG)), carbohydrate polymer, amino acid
polymer or polyvinyl pyrolidone and a fatty acid or fatty acid
ester group, each of which can independently comprise from about
eight to about seventy carbon atoms. Conjugates for use with a
compound or amyloid probe of the invention can also serve as
linkers to, for example, any suitable substituents or groups,
radiolabels (marker or tags), halogens, proteins, proteins,
enzymes, polypeptides, other therapeutic agents (for example, a
pharmaceutical or drug), nucleosides, dyes, oligonucleotides,
lipids, phospholipids and/or liposomes. In one aspect, conjugates
can include polyethylene amine (PEI), polyglycine, hybrids of PEI
and polyglycine, polyethylene glycol (PEG) or methoxypolyethylene
glycol (mPEG). A conjugate can also link a compound of the
invention to, for example, a label or marker (radionuclide,
radioisotope and/or isotope) to comprise a probe of the invention.
Conjugates for use with a compound or probe of the invention can,
in one aspect, improve in vivo half-life. Other exemplary
conjugates for use with a compound or probe of the invention as
well as applications thereof and related techniques include those
generally described by U.S. Pat. No. 5,672,662, which is hereby
incorporated by reference herein.
[0117] Lipids can include synthetic or naturally-occurring
compounds, which are generally amphipathic and biocompatible. The
lipids typically comprise a hydrophilic component and a hydrophobic
component. Exemplary lipids include fatty acids, neutral fats,
phosphatides, glycolipids, aliphatic alcohols, waxes, terpenes,
steroids and surfactants. "Lipid composition" can refer to a
composition which comprises a lipid compound, typically in an
aqueous medium. Exemplary lipid compositions include suspensions,
emulsions and vesicle compositions. Similarly, liposome can refer
to a generally spherical cluster or aggregate of amphipathic
compounds (including lipid compounds) typically in the form of one
or more concentric layers, for example, bilayers. They may also be
referred to herein as lipid vesicles. The liposomes may be
formulated, for example, from ionic lipids and/or non-ionic
lipids.
[0118] The terms "tissue" or "organ" can mean a part of a patient's
body. Examples of tissues or organs include the brain, heart,
liver, blood vessels, arteries, mesodermal tissue, tenosynovium,
joints, aortic, thyroid, islets of Langerhans, aging pituitary,
latrogenic, cardiac atria, cornea, lens, vitreous humor, retina,
sclera, pancreas or parenchymatous organ. A detectable or imaging
effective quantity is a quantity of an amyloid probe or labeled
compound of the invention necessary to be detected by the detection
method chosen. For example, a detectable quantity can be an
administered amount sufficient to enable detection of binding of
the probe to amyloid and/or amyloid deposits. The amount of an
amyloid probe to be introduced into a patient in order to provide
for detection can readily be determined by those skilled in the
art. For example, increasing amounts of the amyloid probe can be
given to a patient until the probe is detected by the detection
method of choice. A detectable marker is introduced to the
compounds of the invention to provide for an amyloid probe that can
be detected by suitable imaging modalities. In one aspect, a method
of the invention determines the presence and location of amyloid
deposits in an organ or body area, preferably, the brain of a
patient. The method comprises administration of a detectable
quantity of an amyloid probe or pharmaceutical composition
thereof.
[0119] Those skilled in the art are also familiar with determining
the amount of time sufficient for a compound or amyloid probe to
become associated with amyloid deposits. The amount of time
necessary can easily be determined by introducing a detectable
amount of an amyloid probe of the invention into a patient and then
detecting the probe at various times after administration.
[0120] The terms "associated" and/or "binding" can mean a chemical
or physical interaction between a compound or amyloid probe of the
invention and an amyloid deposit. In one aspect, an amyloid deposit
can comprise amyloid proteins or precursors, portions, fragments
and peptides thereof and/or one or more A.beta. and/or
amyloidogenic proteins. Preferably, the compounds of the invention
and probes thereof are amyloid binding compounds. Examples of
associations or interactions include covalent bonds, ionic bonds,
hydrophilic-hydrophilic interactions, hydrophobic-hydrophobic
interactions and complexes. Associated can also refer generally to
"binding" or "affinity" as each can be used to describe various
chemical or physical interactions. Measuring binding or affinity is
also routine to those skilled in the art. For example, compounds or
probes of the invention can bind to or interact with amyloid
proteins or precursors, portions, fragments and peptides thereof
and/or their deposits as well as deposits that can comprise one or
more amyloid and/or amyloidogenic proteins. Those skilled in the
art are familiar with the various ways to detect labeled compounds.
For example, MRI, PET or SPECT can be used to detect amyloid probes
of the invention. The label that is introduced to a compound of the
invention to yield an amyloid probe can depend on the detection
method desired. As indicated, if PET is selected as a detection
method, the amyloid probe must possess a positron-emitting atom
such as .sup.11C or .sup.18F.
[0121] In one aspect, the amyloid probe should also have sufficient
radioactivity and radioactivity concentration to assure reliable
diagnosis. Without limitation, for .sup.99mTc, the probe may be
included usually in an amount from 0.1 to 100 mCi in about 0.5 to
5.0 ml at the time of administration. The amount of a compound of
the invention may be such as is sufficient to form a stable chelate
compound or amyloid probe with the radioactive metal.
[0122] The imaging of amyloid deposits can also be carried out
quantitatively so that the amount of amyloid deposits can be
determined. In one aspect, amyloid probes for imaging include a
radioisotope such as .sup.123I, .sup.125I, .sup.124I, .sup.131I,
.sup.18F, .sup.75Br, or .sup.76Br. The invention also provides a
method of imaging amyloid deposits. One of the key prerequisites
for an in vivo imaging agent of the brain is the ability to cross
the intact blood-brain barrier after, for example, a bolus
intravenous injection.
[0123] In another aspect, a method of inhibiting amyloid plaque
aggregation is provided. For example, the invention provides a
method of inhibiting the aggregation of amyloid proteins to form
amyloid deposits by administering to a patient an amyloid
inhibiting amount of a compound of the invention.
[0124] Those skilled in the art are readily able to determine an
amyloid inhibiting amount by simply administering a compound of the
invention to a patient in increasing amounts until the growth of
amyloid deposits is decreased or stopped. The rate of growth can be
assessed using in vivo imaging, as described, or by taking a tissue
sample from a patient and observing the amyloid deposits therein.
The compounds of the invention can be administered to a patient at
dosage levels in the range of about 0.1 to about 1,000 mg per day.
For a normal human adult having a body weight of about 70 kg, a
dosage in the range of about 0.01 to about 100 mg per kilogram of
body weight per day is sufficient. The specific dosage used,
however, can vary or may be adjusted as considered appropriate by
those of ordinary skill in the art. For example, the dosage can
depend on a number of factors including the requirements of the
patient, the severity of the condition being treated and the
pharmacological activity of the compound being used. The
determination of optimum dosages for a particular patient is well
known to those skilled in the art.
[0125] The term "alkyl" by itself or as part of another group can
refer to both straight and branched chain radicals of up to 8
carbons, preferably, 5 carbons, more preferably, 4 carbons such as
methyl, ethyl, propyl, isopropyl, butyl, t-butyl and isobutyl. A
compound or amyloid probe of the invention can also comprise one or
more alkyl substituents (for example, as A.sub.linker) included via
general organic chemistry techniques known to the art. March, J.,
Advanced Organic Chemistry: I Reactions, Mechanisms, and Structure
(3rd Ed., 1985); Morrison and Boyd, Organic Chemistry (6th Ed.,
1992).
[0126] The term "alkenyl" can refer to an unsaturated straight or
branched chain hydrocarbon radical comprising at least one carbon
to carbon double bond. Examples include without limitation ethenyl,
propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl,
n-pentenyl and n-hexenyl. Moreover, "alkynyl" can refer to an
unsaturated straight or branched chain hydrocarbon radical
comprising at least one carbon to carbon triple bond. Examples
include without limitation ethynyl, propynyl, iso-propynyl,
butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl. A
compound or amyloid probe of the invention can also comprise one or
more alkenyl or alkynyl substituents (for example, as A.sub.linker)
included via general organic chemistry techniques known to the art.
March, J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0127] The term "alkoxy" can mean a straight or branched chain
alkyl radical, as indicated, bonded to an oxygen atom including,
but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy and the
like. Preferably, the alkoxy chain is 1 to 6 carbon atoms in length
and, more preferably, 1-4 carbon atoms in length. A compound or
amyloid probe of the invention can also comprise one or more alkoxy
substituents (for example, as A.sub.linker) included via general
organic chemistry techniques known to the art. March, J., Advanced
Organic Chemistry: I Reactions, Mechanisms, and Structure (3rd Ed.,
1985); Morrison and Boyd, Organic Chemistry (6th Ed., 1992).
[0128] The term "monoalkylamine" by itself or as part of another
group can refer to an amino group that is substituted with one
alkyl group as indicated. In one aspect, the term "methylamino" can
refer to a neutral group or ring substituent in which N is
connected to a compound of the invention via the ring or a chain of
the compound and N is further bound to a methyl and a hydrogen.
Moreover, the N may be charged and may form a salt. A compound or
amyloid probe of the invention can also comprise one or more
monoalkylamine substituents (for example, as A.sub.linker) included
via general organic chemistry techniques known to the art. March,
J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0129] The term "dialkylamine" by itself or as part of another
group can refer to an amino group that is substituted with two
alkyl groups as indicated. In one aspect, the term "dimethylamino"
can refer to a neutral group or ring substituent in which N is
connected to a compound of the invention via the ring or a chain of
the compound and N is further bound to two methyl groups. In
addition, the N may be charged and may form a salt. A compound or
amyloid probe of the invention can also comprise one or more
dialkylamine substituents (for example, as A.sub.linker) included
via general organic chemistry techniques known to the art. March,
J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0130] The term "hydroxy(C.sub.1-5)alkyl" can refer to an alkyl
chain connected to a compound of the invention via the ring or a
chain of the compound in which the distal portion of the alkyl
chain of the group contains a hydroxy moiety. The alkyl chain can
contain any number of carbons, but, preferably, the number of
carbons in the alkyl chain is from 1 to 5. A compound or amyloid
probe of the invention can also comprise one or more
hydroxy(C.sub.1-5)alkyl substituents (for example, as A.sub.linker)
included via general organic chemistry techniques known to the art.
March, J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0131] The term "halo" or "halogen" by itself or as part of another
group can refer to chlorine, bromine, fluorine or iodine. A
compound or amyloid probe of the invention can also comprise one or
more halo substituents (for example, as A.sub.linker) included via
general organic chemistry techniques known to the art. March, J.,
Advanced Organic Chemistry: I Reactions, Mechanisms, and Structure
(3rd Ed., 1985); Morrison and Boyd, Organic Chemistry (6th Ed.,
1992).
[0132] The term "haloalkyl" can refer to any of the mentioned alkyl
groups substituted by one or more chlorine, bromine, fluorine or
iodine with fluorine and chlorine such as chloromethyl, iodomethyl,
trifluoromethyl, 2,2,2-trifluoroethyl and 2-chloroethyl. A compound
or amyloid probe of the invention can also comprise one or more
haloalkyl substituents (for example, as A.sub.linker) included via
general organic chemistry techniques known to the art. March, J.,
Advanced Organic Chemistry: I Reactions, Mechanisms, and Structure
(3rd Ed., 1985); Morrison and Boyd, Organic Chemistry (6th Ed.,
1992).
[0133] The term "alkylthio" by itself or as part of another group
can refer to a thioether of the structure: R.sup.x--S in which
R.sup.x is a C.sub.1-4 alkyl as indicated. A compound or amyloid
probe of the invention can also comprise one or more alkylthio
substituents (for example, as A.sub.linker) included via general
organic chemistry techniques known to the art. March, J., Advanced
Organic Chemistry: I Reactions, Mechanisms, and Structure (3rd Ed.,
1985); Morrison and Boyd, Organic Chemistry (6th Ed., 1992).
[0134] The term "alkylsulfonyl" by itself or as part of another
group can refer to a sulfone of the structure: R.sup.y--SO.sub.2 in
which R.sup.y is a C.sub.1-4 alkyl as indicated. A compound or
amyloid probe of the invention can also comprise one or more
alkylsulfonyl substituents (for example, as A.sub.linker) included
via general organic chemistry techniques known to the art. March,
J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0135] The term "aryl" by itself or as part of another group can
refer to monocyclic or bicyclic aromatic groups containing from 6
to 12 carbons in the ring portion, preferably, 6-10 carbons in the
ring portion such as phenyl, naphthyl or tetrahydronaphthyl. A
compound or amyloid probe of the invention can also comprise one or
more aryl substituents (for example, as A.sub.linker) included via
general organic chemistry techniques known to the art. March, J.,
Advanced Organic Chemistry: I Reactions, Mechanisms, and Structure
(3rd Ed., 1985); Morrison and Boyd, Organic Chemistry (6th Ed.,
1992).
[0136] The term "carboxy" can refer to the group --COOR.sub.A in
which R.sub.A may be hydrogen or any suitable substituent
including, for example, F, Cl, Br, I, NO.sub.2, CN, CF.sub.3,
alkyl, alkenyl, alkynyl, alkoxy, monoalkylamine, dialkylamine,
hydroxyalkyl, haloalkyl, alkylthio, alkylsulfonyl, aryl,
heterocycles, heteroaryl or aralkyl groups. Carboxy can also
generally refer to esterified carboxy (--COOR.sub.A in which
R.sub.A can be alkyl) or amidate carboxy (--CONHR.sub.A in which
may be hydrogen or any suitable substituent including, for example,
F, Cl, Br, I, NO.sub.2, CN, CF.sub.3, alkyl, alkenyl, alkynyl,
alkoxy, monoalkylamine, dialkylamine, hydroxyalkyl, haloalkyl,
alkylthio, alkylsulfonyl, aryl, heterocycles, heteroaryl or aralkyl
groups) groups. A compound or amyloid probe of the invention can
also comprise one or more carboxyl substituents (for example, as
A.sub.linker) included via general organic chemistry techniques
known to the art. March, J., Advanced Organic Chemistry: I
Reactions, Mechanisms, and Structure (3rd Ed., 1985); Morrison and
Boyd, Organic Chemistry (6th Ed., 1992).
[0137] The term "heterocycle" or "heterocyclic ring" can represent
a stable 4 to 7-membered mono-heterocyclic ring system that may be
saturated or unsaturated, and consist of carbon atoms and from one
to three heteroatoms selected from the group consisting of N, O and
S. Moreover, the nitrogen and sulfur heteroatom may optionally be
oxidized. Especially useful are rings containing one nitrogen
combined with one oxygen or sulfur or two nitrogen heteroatoms.
Examples of such heterocyclic groups include piperidinyl, pyrrolyl,
pyrrolidinyl, imidazolyl, imidazinyl, imidazolidinyl, pyridyl,
pyrazinyl, pyrimidinyl, oxazolyl, oxazolidinyl, isoxazolyl,
isoxazolidinyl, thiazolyl, thiazolidinyl, isothiazolyl,
homopiperidinyl, homopiperazinyl, pyridazinyl, pyrazolyl, and
pyrazolidinyl, most preferably thiamorpholinyl, piperazinyl and
morpholinyl. A compound or amyloid probe of the invention can also
comprise one or more heterocycle or heterocyclic ring substituents
(for example, as A.sub.linker) included via general organic
chemistry techniques known to the art. March, J., Advanced Organic
Chemistry: I Reactions, Mechanisms, and Structure (3rd Ed., 1985);
Morrison and Boyd, Organic Chemistry (6th Ed., 1992).
[0138] The term "heteroatom" can mean an oxygen atom ("O"), a
sulfur atom ("S") or a nitrogen atom ("N"). It will also be
recognized that when the heteroatom is nitrogen, it may form an
NR.sup.aR.sup.b moiety in which R.sup.a and R.sup.b are,
independently from one another, hydrogen, C.sub.1-4 alkyl, C.sub.2
aminoalkyl, C.sub.1-4 halo alkyl or halo benzyl. Moreover, R.sup.a
and R.sup.b can be taken together to form a 5 to 7-member
heterocyclic ring that optionally comprises O, S or NR.sup.c in
which R.sup.c is hydrogen or C.sub.1-4 alkyl. A compound or amyloid
probe of the invention can also comprise one or more heteroatom
substituents (for example, as A.sub.linker) included via general
organic chemistry techniques known to the art. March, J., Advanced
Organic Chemistry: I Reactions, Mechanisms, and Structure (3rd Ed.,
1985); Morrison and Boyd, Organic Chemistry (6th Ed., 1992).
[0139] The term "heteroaryl" can refer to groups having 5 to 14
ring atoms, 6, 10 or 14 n electrons shared in a cyclic array and
containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur
heteroatoms in which examples of heteroaryl groups are thienyl,
benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl,
pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl,
phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,
4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl,
4aH-carbazolyl, carbazolyl, .beta.-carbolinyl, phenanthridinyl,
acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl,
phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups. A
compound or amyloid probe of the invention can also comprise one or
more heteroaryl substituents (for example, as A.sub.linker)
included via general organic chemistry techniques known to the art.
March, J., Advanced Organic Chemistry: I Reactions, Mechanisms, and
Structure (3rd Ed., 1985); Morrison and Boyd, Organic Chemistry
(6th Ed., 1992).
[0140] The term "aralkyl" or "arylalkyl" by itself or as part of
another group can refer to alkyl groups as indicated having an aryl
substituent such as benzyl, phenylethyl or 2-naphthylmethyl. A
compound or amyloid probe of the invention can also comprise one or
more aralkyl or arylalkyl substituents (for example, as
A.sub.linker) included via general organic chemistry techniques
known to the art. March, J., Advanced Organic Chemistry: I
Reactions, Mechanisms, and Structure (3rd Ed., 1985); Morrison and
Boyd, Organic Chemistry (6th Ed., 1992).
[0141] Exemplary radiotracers can be used to, for example, study
amyloid distributions via radioscintigraphy, magnetic resonance
imaging (MRI), chemilumensence, near infrared luminescence,
fluorescence, spectroscopy, gamma imaging, magnetic resonance
imaging, magnetic resonance spectroscopy, fluorescence
spectroscopy, SPECT, computed tomography (CT scan), positron
emission tomography (PET) or combinations thereof. The invention
also contemplates the use of conventional imaging protocols, means,
devices, apparatuses or systems for performing radioscintigraphy,
magnetic resonance imaging (MRI), chemilumensence, near infrared
luminescence, fluorescence, SPECT, computed tomography (CT scan),
positron emission tomography (PET) or combinations thereof.
Exemplary imaging protocols, means, devices, apparatuses or systems
include those generally described in U.S. Pat. Nos. 6,072,177,
6,803,580, 5,900,636, 6,271,524, 5,532,489, 5,272,343, 5,241,181,
5,512,755, 5,345,082, 5,023,895, 4,864,140, 5,323,006, 4,675,526
and 4,395,635, each of which are incorporated by reference
herein.
[0142] The examples herein are provided to illustrate advantages of
the present invention and to further assist a person of ordinary
skill in the art with preparing or using the compounds or amyloid
probes of the invention or salts, pharmaceutical compositions,
derivatives, prodrugs, racemic mixtures or tautomeric forms
thereof. The examples herein are also presented in order to more
fully illustrate the preferred aspects of the invention. The
examples should in no way be construed as limiting the scope of the
invention, as defined by the appended claims. The examples can
include or incorporate any of the variations, aspects or aspects of
the invention described above. The variations, aspects or aspects
described above may also further each include or incorporate the
variations of any or all other variations, aspects or aspects of
the invention. For example, a compound of the invention can
comprise any suitable detectable marker, tag or label to yield an
amyloid probe that can be used to diagnose and study the
progression or regression of disease states or maladies that
include, for example, AD, familial AD, homozygotes for the
apolipoprotein E4 allele, glaucoma, Mediterranean fever,
Muckle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy,
amyloid cardiomyopathy, systemic senile amyloidosis, amyloid
polyneuropathy, hereditary cerebral hemorrhage with amyloidosis,
Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru,
Gerstmann-Straussler-Scheinker syndrome, medullary carcinoma of the
thyroid, Isolated atrial amyloid, .beta..sub.2-microglobulin
amyloid in dialysis patients, inclusion body myositis,
.beta..sub.2-amyloid deposits in muscle wasting disease and Islets
of Langerhans diabetes Type II insulinoma.
Example I
Series I Compounds of the Invention
##STR00079##
[0143] 4'-dimethylaminoacetophenone (1)
[0144] In a mixture EtOH/HCl 37% 80 ml/5 ml was dissolved
4'-aminoacetophenone (13.5 g, 0.1 mole), then formaldehyde 37% (15
ml) was added followed by PtO.sub.2 (150 mg). The resulting
solution was hydrogenated at 50 PSI for 1 h, filtered through
celite, evaporated and purified by flash chromatography (SiO.sub.2,
hexane/AcOEt, 8/2) giving 1 as a white solid in 62% yield.
[0145] NMR .sup.1H (CDCl.sub.3), .delta.=2.30 (s, 3H, CH.sub.3);
2.85 (s, 6H, 2CH.sub.3); 6.44 (d, 2H, J=9.0 Hz, 2CHAr); 7.67 (d,
2H, J=9.0 Hz, 2CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=26.3 (1C,
CH.sub.3); 40.4 (2C, 2CH.sub.3); 110.9 (2C, CHAr); 125.6 (1C, Cq),
130.9 (2C, CHAr); 153.7 (1C, Cq); 196.7 (1C, Cq).
2,2-dibromo-4'-dimethylaminoacetophenone (2)
[0146] In 20 ml of concentrated H.sub.2SO.sub.4 was dissolved 1
(3.8 g, 1 eq), then at 0.degree. C. bromine (1.19 ml, 1 eq) was
added dropwise and the resulting mixture was stirred at RT for 6 h
before being poured into 200 ml of ice/H.sub.2O. The resulting
precipitate was collected by filtration, washed with H.sub.2O,
dissolved in CH.sub.2Cl.sub.2, dried over Na.sub.2SO.sub.4 and
concentrated in vacuum to give 2 as a green solid in 68% yield,
which was directly used in the next step without any
purification.
2-bromo-4'-dimethylacetophenone (3)
[0147] In 30 ml of THF was dissolved 2 (5.1 g, 1 eq), then at
0.degree. C. was added dropwise a mixture of diethylphosphite (2.04
ml, 1 eq) and Et.sub.3N (2.4 ml, 1.1 eq) in 12 ml of THF. The
resulting mixture was stirred 6 h at RT, then evaporated and poured
into 200 ml of ice/H.sub.2O and the resulting precipitated was
filtered, washed with H.sub.2O and dried in vacuum. 3 was obtained
as a green solid in 89% yield.
[0148] NMR .sup.1H (CDCl.sub.3), .delta.=2.97 (s, 6H, 2CH.sub.3);
4.45 (s, 2H, CH.sub.2); 6.57 (d, 2H, J=9.0 Hz, 2CHAr); 7.72 (d, 2H,
CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=31.3 (1C, CH2); 40.4 (2C,
CH3); 111.1 (2C, CHAr); 121.8 (1C, Cq); 131.6 (2C, CHAr); 154.1
(1C, Cq); 189.7 (1C, Cq).
An Exemplary Method for the Synthesis of 4 Through 7
[0149] In the minimum volume of EtOH was dissolved the commercially
available 2-amino-6-substituted-benzothiazole (2 mmol), then 3 (2
mmol) was added and the resulting mixture was refluxed for 2 h
before addition of NaHCO.sub.3 (3 mmol). After 6 h more of reflux,
the mixture was hydrolyzed with H.sub.2O (5 ml), extracted using
AcOEt (4.times.25 ml), dried over Na.sub.2SO.sub.4, concentrated in
vacuum and purified by flash chromatography (SiO.sub.2,
hexane/AcOEt, 1/1).
7-bromo-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(4)
[0150] NMR .sup.1H (DMSO-d.sub.6), .delta.=2.93 (s, 6H, 2CH.sub.3);
6.77 (d, 2H, J=8.5 Hz, 2CHAr); 7.67 (d, 2H, J=8.5 Hz, 2CHAr); 7.71
(d, 1H, J=8.5 Hz, CHAr); 7.88 (d, 1H, J=8.5 Hz, CHAr); 8.29 (s, 1H,
CHAr); 8.50 (s, 1H, CHAr). NMR .sup.13C (DMSO-d.sub.6),
.delta.=40.4 (2C, CH.sub.3); 107.2 (1C, CHAr); 112.6 (2C, CHAr);
114.9 (1C, CHAr); 116.6 (1C, Cq); 122.1 (1C, Cq); 126.0 (2C, CHAr);
127.6 (1C, CHAr); 129.7 (1C, CHAr); 131.5 (1C, Cq); 146.8 (1C, Cq);
147.7 (1C, Cq); 150.1 (1C, Cq). HRMS Calcd for
C.sub.17H.sub.15N.sub.3BrS: 372.0170, found: 372.0171. Anal. Calcd
for C.sub.17H.sub.14N.sub.3BrS: C, 54.85%; H, 3.79%; N, 11.29%,
found: C, 54.49%; H, 3.73%; N, 11.06%.
7-fluoro-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(5)
[0151] NMR .sup.1H (CDCl.sub.3), .delta.=2.97 (s, 6H, 2CH.sub.3);
6.71 (d, 2H, J=8.5 Hz, CHAr); 7.08 (td, 1H, J=8.8, 2.8 Hz, CHAr);
7.33 (dd, 1H, J=8.8, 2.4 Hz, CHAr); 7.43 (dd, 1H, J=8.0, 4.4 Hz,
CHAr), 7.70 (s, 1H, CHAr). HRMS Calcd for
C.sub.17H.sub.15N.sub.3FS: 312.0971, found: 312.0959. Anal. Calcd
for C.sub.17H.sub.14N.sub.3FS, 0.2H.sub.2O: C, 64.82%; H, 4.61%; N,
13.34%, found: C, 64.89%; H, 4.41%; N, 13.25%.
7-methoxy-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(6)
[0152] NMR .sup.1H (CDCl.sub.3), .delta.=2.88 (s, 6H, 2CH.sub.3);
3.66 (s, 3H, CH.sub.3); 6.62 (d, 2H, J=9.0 Hz, CHAr); 6.74 (dd, 1H,
J=8.8, 2.4 Hz, CHAr); 6.96 (d, 1H, J=2.4 Hz, CHAr); 7.17 (d, 1H,
J=8.8 Hz, CHAr); 7.51 (s, 1H, CHAr); 7.58 (d, 2H, J=9.0 Hz, CHAr).
NMR .sup.13C (CDCl.sub.3), .delta.=40.9 (2C, CH3); 56.2 (IC,
CH.sub.3); 105.4 (IC, CHAr); 108.9 (1C, CHAr); 112.9 (2C, CHAr);
113.2 (1C, CHAr); 113.4 (1C, CHAr); 122.8 (1C, Cq); 126.3 (2C,
CHAr); 126.8 (IC, Cq); 131.5 (1C, Cq); 147.2 (1C, Cq); 148.1 (1C,
Cq); 150.2 (1C, Cq); 157.2 (IC, Cq). MS m/z: 324.2 (M+1). Anal.
Calcd for C.sub.18H.sub.17N.sub.3OS, 2 HCl, 2H.sub.2O: C, 50.00%;
H, 5.36%; N, 9.72%, found: C, 49.66%; H, 5.44%; N, 9.39%.
7-methyl-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(7)
[0153] NMR .sup.1H (CDCl.sub.3), .delta.=2.39 (s, 3H, CH.sub.3);
3.00 (s, 6H, 2CH.sub.3); 6.80 (d, 2H, J=8.8 Hz, CHAr); 7.20 (d, 1H,
J=8.0 Hz, CHAr); 7.45 (d, 1H, J=8.0 Hz, CHAr); 7.47 (s, 1H, CHAr);
7.76 (d, 2H, J=8.8 Hz, CHAr); 7.79 (s, 1H, CHAr). NMR .sup.13C
(CDCl.sub.3), .delta.=21.7 (1C, CH.sub.3); 41.0 (2C, CH.sub.3);
105.4 (1C, CHAr); 112.5 (1C, CHAr); 113.0 (2C, CHAr); 122.8 (1C,
Cq); 124.7 (1C, CHAr); 126.5 (2C, CHAr); 127.4 (1C, CHAr); 130.5
(1C, Cq); 130.6 (1C, Cq); 134.9 (1C, Cq); 147.1 (1C, Cq); 148.3
(1C, Cq); 150.3 (1C, Cq). FIRMS Calcd for C.sub.18H.sub.18N.sub.3S:
308.1221, found: 308.1207. Anal. Calcd for
C.sub.18H.sub.17N.sub.3S, CH.sub.2Cl.sub.2: C, 58.16%; H, 4.88%, N,
10.71%, found: C, 57.85%; H, 4.85%; N, 10.87%.
Exemplary Compounds of the Invention in Series I
##STR00080## ##STR00081##
[0154] Example II
Series II Compounds of the Invention
##STR00082## ##STR00083##
[0155] 2-(2-(4-dimethylaminophenyl)diazenyl)benzeneamine (8a)
[0156] o-Phenylenediamine (2 g, 1 eq),
4-dimethylaminonitrozoaniline (2.77 g, 1 eq) and NaOH (740 mg, 1
eq) were mixed neat and heated at 70.degree. C. for 20 min with
constant triturating. The resulting paste was extracted with
toluene, concentrated in vacuum and purified by flash
chromatography (SiO.sub.2, hexane/AcOEt, 8/2) giving 8a as a red
solid in 43% yield.
[0157] NMR .sup.1H (CDCl.sub.3), .delta.=2.89 (s, 6H, 2CH.sub.3);
5.44 (bs, 2H, NH.sub.2); 6.59-6.62 (m, 3H, 3CHA3); 6.67 (td, 1H,
J=8.0, 1.2 Hz, CHAr); 7.02 (td, 1H, J=8.0, 1.2 Hz, CHAr); 7.64 (dd,
1H, J=8.0, 1.2 Hz, CHAr); 7.70 (d, 2H, J=9.2 Hz, CHAr). NMR
.sup.13C (CDCl.sub.3), .delta.=40.4 (2C, CH.sub.3); 111.7 (2C,
CHAr); 116.8 (1C, CHAr); 117.5 (1C, CHAr); 125.6 (2C, CHAr); 126.1
(1C, CHAr); 130.7 (1C, CHAr); 137.8 (1C, Cq); 143.1 (1C, Cq); 143.9
(1C, Cq); 151.9 (1C, Cq).
1-(2-amino-4 and -5-bromophenyl)-2-(4-dimethylaminophenyl)diazene
(8b)
[0158] 4-bromo-1,2-phenylenediamine (1.5 g, 1 eq),
4-dimethylaminonitrozoaniline (1.2 g, 1 eq) and NaOH (450 mg, 1 eq)
were mixed neat and heated at 70.degree. C. for 20 min with
constant triturating. The resulting paste was extracted with
toluene, concentrated in vacuum and purified by flash
chromatography (SiO.sub.2, hexane/AcOEt, 8/2) giving a mixture of
two isomers (3 and 4 bromo) as a red solid in 39% yield. The less
polar isomer was isolated and characterized.
[0159] NMR .sup.1H (CDCl.sub.3), .delta.=2.99 (s, 6H, 2CH3); 5.59
(s, 2H, NH2); 6.67 (d, 2H, J=9.2 Hz, CHAr); 6.79-6.82 (m, 2H,
CHAr); 7.50 (d, 1H, J=8.4 Hz, CHAr); 7.71 (d, 2H, J=9.2 Hz,
CHAr).
1-(2-amino-4 and -5-iodophenyl)-2-(4-dimethylaminophenyl)diazene
(8c)
[0160] 4-iodo-1,2-phenylenediamine (2 g, 1 eq),
4-dimethylaminonitrozoaniline (1.26 g, 1 eq) and NaOH (340 mg, 1
eq) were mixed neat and heated at 70.degree. C. for 20 min with
constant triturating. The resulting paste was extracted with
toluene, concentrated in vacuum and purified by flash
chromatography (SiO.sub.2, hexane/AcOEt, 8/2) giving a mixture of
two isomers (3 and 4 iodo) as a red solid in 32% yield.
1-(2-amino-4 and -5-chlorophenyl)-2-(4-dimethylaminophenyl)diazene
(8d)
[0161] Using the same methodology as for 8c, compound 8d was
obtained as a mixture of two isomers (3 and 4 chloro) as a red
solid in 38% yield.
1-(2-amino-4 and -5-fluorophenyl)-2-(4-dimethylaminophenyl)diazene
(8e)
[0162] Using the same methodology as for 8c, compound 8e was
obtained as a mixture of two isomers (3 and 4 fluoro) as a red
solid in 37% yield.
1-(2-amino-4 and -5-methoxyphenyl)-2-(4-dimethylaminophenyl)diazene
(8f)
[0163] Using the same methodology as for 8c, compound 8f was
obtained as a mixture of two isomers (3 and 4 methoxy) as a red
solid in 53% yield.
1-(2-amino-4 and -5-nitrophenyl)-2-(4-dimethylaminophenyl)diazene
(8g)
[0164] Using the same methodology as for 8c, compound 8g was
obtained as a mixture of two isomers (3 and 4 nitro) as a red solid
in 64% yield.
4-(2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine (9a)
[0165] In CH.sub.2Cl.sub.2 was dissolved 8a (1 g, 1 eq) and a
solution of Pb(OAc).sub.4 (2.03 g, 1.1 eq) in which
CH.sub.2Cl.sub.2 was added dropwise. The resulting solution was
stirred 30 min at RT, then hydrolyzed with 15 ml of
Na.sub.2CO.sub.3 salt, extracted with CH.sub.2Cl.sub.2, dried by
Na.sub.2SO.sub.4, evaporated and purified by flash chromatography
(SiO.sub.2, hexane/AcOEt, 8/2) giving 9a as an orange solid in 37%
yield.
[0166] NMR .sup.1H (CDCl.sub.3), .delta.=2.84 (s, 6H, 2CH.sub.3);
6.62 (d, 2H, J=9.2 Hz, CHAr); 7.23 (dd, 2H, J=6.5, 2.8 Hz, CHAr);
7.78 (dd, 2H, J=6.5, 2.8 Hz, CHAr); 8.06 (d, 2H, J=9.2 Hz, CHAr).
NMR .sup.13C (CDCl.sub.3), .delta.=40.4 (2C, CH.sub.3); 111.9 (2C,
CHAr); 117.9 (2C, CHAr); 121.7 (2C, CHAr); 126.3 (2C, CHAr); 130.2
(1C, Cq); 144.7 (2C, Cq); 150.7 (1C, Cq).
4-(5-bromo-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9b)
[0167] Using the same methodology as for 9a (starting from the
mixture 8b), 9b was obtained as an orange solid in 33% yield.
[0168] NMR .sup.1H (CDCl.sub.3), .delta.=2.97 (s, 3H, CH.sub.3);
6.70 (d, 2H, J=9.2 Hz, CHAr); 7.36 (dd, 1H, J=9.0, 2.0 Hz, CHAr);
7.67 (dd, 1H, J=8.0, 0.4 Hz, CHAr); 7.99 (d, 1H, J=0.4 Hz, CHAr);
8.08 (d, 2H, J=9.2 Hz, CHAr). NMR .sup.13C (CDCl.sub.3),
.delta.=40.3 (2C, CH.sub.3); 111.1 (1C, CHAr); 119.0 (2C, CHAr);
120.2 (1C, CHAr); 123.4 (1C, Cq); 124.2 (2C, CHAr); 126.2 (1C,
CHAr); 143.8 (1C, Cq); 146.3 (1C, Cq); 151.0 (1C, Cq).
4-(5-iodo-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9c)
[0169] Using the same methodology as for 9a (starting from the
mixture 8c), 9c was obtained as an orange solid in 10% yield.
[0170] NMR .sup.1H (CDCl.sub.3), .delta.=2.95 (s, 6H, 2CH.sub.3);
6.68 (dd, 2H, J=8.8, 2.0 Hz, CHAr); 7.51 (dd, 1H, J=8.8, 2.0 Hz,
CHAr); 7.56 (dd, J=8.8, 0.4 Hz, CHAr); 8.06 (dd, 2H, J=8.8, 2.0 Hz,
CHAr); 8.22 (s, 1H, CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=40.4
(2C, CH.sub.3); 91.0 (1C, Cq); 111.9 (2C, CHAr); 119.4 (1C, CHAr);
121.7 (2C, CHAr); 126.9 (1C, CHAr); 135.0 (1C, CHAr); 143.6 (1C,
Cq); 146.2 (1C, Cq); 150.9 (1C, Cq).
4-(5-chloro-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9d)
[0171] Using the same methodology as for 9a (starting from the
mixture 8d), 9d was obtained as an yellow solid in 34% yield.
[0172] NMR .sup.1H (CDCl.sub.3), .delta.=2.93 (6H, s); 6.66 (2H, d,
J=7.4 Hz); 7.21 (1H, dd, J=1.4 Hz, J=7.2 Hz); 7.72 (1H, d, J=7.2
Hz); 7.77 (1H, d, J=1.4 Hz); 8.04 (2H, d, J=7.4 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=40.5 (2C); 112.3 (2C); 117.3, 119.4, 122.1
(2C); 128.1, 130.3, 132.4, 143.5, 145.4, 151.3.
4-(5-fluoro-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9e)
[0173] Using the same methodology as for 9a (starting from the
mixture 8e), 9e was obtained as an yellow solid in 23% yield.
[0174] NMR .sup.1H (CDCl.sub.3), .delta.=2.96 (6H, s); 6.70 (2H, d,
J=9.1 Hz); 7.09 (1H, td, J=2.1 Hz, J=8.7 Hz); 7.41 (1H, dd, J=2.0
Hz, J=8.7 Hz); 7.79 (1H, m); 6.70 (2H, d, J=9.1 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=40.4 (2C); 101.4 (d); 111.9 (2C); 117.6 (d);
119.4, 119.5, 121.1 (2C); 131.2, 141.8, 144.6 (d); 150.8.
4-(5-methoxy-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9f)
[0175] Using the same methodology as for 9a (starting from the
mixture 8f), 9f was obtained as an yellow solid in 28% yield.
[0176] NMR .sup.1H (CDCl.sub.3), .delta.=2.95 (6H, s); 3.81 (3H,
s); 6.71 (2H, d, J=9.2 Hz); 6.97 (1H, dd, J=2.3 Hz, J=9.2 Hz); 7.04
(1H, d, J=2.3 Hz); 7.68 (1H, d, J=9.2 Hz); 8.04 (2H, d, J=9.2 Hz).
NMR .sup.13C (CDCl.sub.3), .delta.=40.5 (2C); 55.5, 94.8, 112.1
(2C); 118.7, 121.2, 121.3 (2C); 126.1, 140.8, 145.6, 150.5,
158.8.
4-(5-nitro-2H-benzo[d][1,2,3]thiazol-2-yl)-N,N-dimethylbenzenamine
(9g)
[0177] Using the same methodology as for 9a (starting from the
mixture 8g), 9g was obtained as an yellow solid in 23% yield.
[0178] NMR .sup.1H (CDCl.sub.3), .delta.=3.03 (6H, s); 6.70 (2H, d,
J=7.2 Hz); 7.92 (1H, d, J=9.2 Hz); 8.14-8.18 (3H, m); 8.82 (1H, d,
J=2.0 Hz). NMR .sup.13C (CDCl.sub.3), .delta.=40.4 (2C); 113.4
(2C); 118.1, 119.7, 122.6 (2C); 128.1, 131.4, 135.9, 148.2,
151.3.
4-(5-hydroxy-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(9h)
[0179] Under N.sub.2, 130 mg (5 mmol) of 9f was dissolved in (5 ml)
CH.sub.2Cl.sub.2, at 0.degree. C. was slowly added 0.458 ml (50
mmol) of BBr.sub.3. The solution was stirred overnight at room
temperature. The reaction was poured into ice/water and alkalinized
with Na.sub.2CO.sub.3 (aqueous) and the organic layer was extracted
and dried over Na.sub.2SO.sub.4. After removing the solvent by
evaporation, the residue was purified by chromatography
(dichloromethane/ethyl acetate, 95/5). The solvent was evaporated
to obtain 91 mg (74%) of 9h as yellow solid.
[0180] NMR .sup.1H (CD.sub.3OD), .delta.=4.52 (6H, s); 6.37 (1H,
sl); 8.35 (2H, d, J=8.9 Hz); 8.53-8.55 (2H, m); 9.21 (1H, d, J=9.7
Hz); 9.53 (2H, d, J=8.9 Hz). NMR .sup.13C (CD.sub.3OD),
.delta.=42.1 (2C); 99.8, 114.8 (2C); 121.0, 123.4, 123.7 (2C);
133.2, 143.1, 148.6, 153.7, 159.4.
4-(5-amino-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(91)
[0181] Under N.sub.2, 350 mg (1.2 mmol) of 9g with 1.1 g (5 mmol)
of tin(II) chloride was stirred at reflux 2 hours in (70 ml)
ethanol. The reaction was extracted with Na.sub.2CO.sub.3
solution/ethyl acetate, and after removing the solvent by
evaporation, the residue was purified by chromatography
(hexane/ethyl acetate, 6/4). The solvent was evaporated to obtain
120 mg (38%) of 91 as yellow solid.
[0182] NMR .sup.1H (CDCl.sub.3), .delta.=2.96 (6H, s); 3.81 (2H,
sl); 6.72 (2H, d, J=9.2 Hz); 6.79 (1H, dd, J=2.0 Hz, J=9.0 Hz);
6.88 (1H, d, J=2.0 Hz); 7.64 (1H, d, J=8.9 Hz); 8.03 (2H, d, J=9.0
Hz). NMR .sup.13C (CDCl.sub.3), .delta.=40.5 (2C); 96.7, 112.1
(2C); 118.6, 120.6, 121.2 (2C); 130.5, 140.4, 145.2, 146.1,
150.3.
N-((E)-3-(tributylstannyl)allyl)-2-(4-(dimethylamino)phenyl)-2H-benzo[d][1-
,2,3]triazol-5-amine (9j)
[0183] Under N.sub.2, to 90 mg (0.35 mmol) of 91 in (5 ml) THF was
added 0.186 ml (1 mmol) of N,N-diisopropylethylamine and 535 mg (1
mmol) of a tin compound. The reaction was heated at 60.degree. C.
overnight, then water was added and extracted with ethyl acetate.
After removing the solvent by evaporation, the residue was purified
by chromatography (hexane/ethyl acetate/triethylamine, 22/2.5/0.5).
The solvent was evaporated to obtain 48 mg (23%) of 9j as yellow
oil.
[0184] NMR .sup.1H (CDCl.sub.3), .delta.=0.79 (9H, m); 1.01-1.41
(18H, m); 2.95 (6H, s); 3.82 (2H, m); 3.96 (1H, m); 6.06 (1H, d,
J=15.2 Hz); 6.14 (1H, d, J=15.2 Hz); 6.72-6.80 (3H, m); 7.58-7.72
(2H, m); 8.03 (2H, d, J=9.2 Hz). NMR .sup.13C (CDCl.sub.3),
.delta.=9.5 (3C); 13.7 (3C); 27.3 (3C); 29.1 (3C); 40.7 (2C); 48.7,
112.4 (2C); 118.3, 120.6, 121.2 (2C); 126.0, 127.4, 128.8, 129.3,
140.3, 143.6, 146.4, 150.0.
2-(4-(dimethylamino)phenyl)-N-((E)-3-iodoallyl)-2H-benzo[d][1,2,3]triazol--
5-amine (9k)
[0185] Under N.sub.2, to 40 mg (0.7 mmol) of 9j in (3 ml)
CH.sub.2Cl.sub.2 was added at 0.degree. C., 2 mg iodine in (0.5 ml)
CH.sub.2Cl.sub.2. The reaction was stirred 15 min at room
temperature and water was added, after extraction the solvent was
evaporated. The residue was purified by chromatography
(hexane/ethyl acetate, 8/2). The solvent was evaporated to obtain
11 mg (38%) of 9k as yellow solid.
[0186] NMR .sup.1H (CDCl.sub.3), .delta.=2.96 (6H, s); 3.75 (2H,
m); 4.03 (1H, m); 6.33 (1H, d, J=14.5 Hz); 6.55 (1H, d, J=14.5 Hz);
6.66-6.73 (4H, m); 7.61 (1H, J=9.0 Hz); 8.02 (2H, d, J=9.2 Hz). NMR
.sup.13C (CDCl.sub.3), .delta.=40.5 (2C); 48.4, 78.3, 92.7, 112.1
(2C); 118.6, 120.6, 121.1 (2C); 120.5, 140.1, 142.1, 146.0, 146.3,
150.3.
1-(2-amino-4 and -5-chlorophenyl)-2-(4-nitrophenyl)diazene
(10a)
[0187] 4-chloro-1,2-phenylenediamine (1.5 g, 1 eq),
4-nitronitrozoaniline (1.2 g, 1 eq) and NaOH (450 mg, 1 eq) were
mixed neat and heated at 70.degree. C. for 20 min with constant
triturating. The resulting paste was extracted with toluene,
concentrated in vacuum and purified by flash chromatography
(SiO.sub.2, hexane/AcOEt, 8/2) giving a mixture of two isomers (3
and 4 chloro) as a red solid in 21% yield.
1-(2-amino-4 and -5-iodophenyl)-2-(4-nitrophenyl)diazene (10b)
[0188] Using the same methodology as for 10a compound, 10b was
obtained as a mixture of two isomers (3 and 4 nitro) as a red solid
in 43% yield.
1-(2-amino-4 and -5-methoxyphenyl)-2-(4-nitrophenyl)diazene
(10c)
[0189] Using the same methodology as for 10a, compound 10c was
obtained as a mixture of two isomers (3 and 4 nitro) as a red solid
in 6% yield.
1-(2-amino-4 and -5-iodophenyl)-2-(4-nitrophenyl)diazene (10d)
[0190] Using the same methodology as for 10a, compound 10d was
obtained as a mixture of two isomers (3 and 4 nitro) as a red solid
in 77% yield.
5-chloro-2-(4-nitrophenyl)-2H-benzo[d][1,2,3]triazole (11a)
[0191] Under N.sub.2, to 400 mg (1.4 mmol) of 10a in (10 ml)
CH.sub.2Cl.sub.2 at 0.degree. C. was slowly added 642 mg (1.4 mmol)
of Pb(OAc).sub.4 solubilized in (2 ml) of CH.sub.2Cl.sub.2. The
solution was stirred 20 min at room temperature and
Na.sub.2CO.sub.3 solution was added. Organic layer was extracted
and dried over Na.sub.2SO.sub.4. After removing the solvent by
evaporation, the residue was purified by chromatography
(hexane/ethyl acetate, 7/3). The solvent was evaporated to obtain
470 mg (38%) of 11a as pink solid.
[0192] NMR .sup.1H (CDCl.sub.3), .delta.=7.34 (1H, dd, J=1.4 Hz,
J=7.3 Hz); 7.82 (1H, d, J=7.3 Hz); 7.87 (1H, d, J=1.4 Hz); 8.36
(2H, d, J=7.3 Hz); 8.49 (2H, d, J=7.3 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=92.7, 116.3 (2C); 117.4, 119.4, 122.3 (2C);
127.3, 132.7, 141.3, 143.5, 146.7.
5-iodo-2-(4-nitrophenyl)-2H-benzo[d][1,2,3]triazole (11b)
[0193] Using the same methodology as for 11a, compound 11b was
obtained as a red solid in 20% yield.
[0194] NMR .sup.1H (CDCl.sub.3), .delta.=7.26 (1H, dd, J=1.5 Hz,
J=7.2 Hz); 7.59 (1H, d, J=7.2 Hz); 7.76 (1H, d, J=1.5 Hz); 8.54
(2H, d, J=7.3 Hz); 8.63 (2H, d, J=7.3 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=92.4, 93.7, 118.2 (2C); 119.7, 122.4 (2C);
126.8, 134.3, 140.1, 142.5, 144.8.
5-methoxy-2-(4-nitrophenyl)-2H-benzo[d][1,2,3]triazole (11c)
[0195] Using the same methodology as for 11a, compound 11c was
obtained as a yellow solid in 61% yield.
[0196] NMR .sup.1H (CDCl.sub.3), .delta.=3.86 (3H, s); 7.02 (1H, d,
J=2.0 Hz); 7.07 (1H, dd, J=2.0 Hz, J=9.3 Hz); 7.71 (1H, d, J=9.3
Hz) 8.33 (2H, d, J=7.6 Hz); 8.42 (2H, d, J=7.6 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=53.4, 92.1, 118.1 (2C); 121.6, 122.2, 122.9
(2C); 124.1, 140.1, 142.1, 144.6, 157.9.
5-nitro-2-(4-nitrophenyl)-2H-benzo[d][1,2,3]triazole (11d)
[0197] Using the same methodology as for 11a, compound 11c was
obtained as a yellow solid in 75% yield.
[0198] NMR .sup.1H (CDCl.sub.3), .delta.=7.76 (1H, dd, J=1.3 Hz,
J=7.4 Hz); 7.89 (1H, d, J=7.4 Hz); 7.91 (1H, d, J=1.3 Hz); 8.41
(2H, d, J=7.4 Hz); 8.52 (2H, d, J=7.4 Hz). NMR .sup.13C
(CDCl.sub.3), .delta.=117.6, 118.3 (2C); 120.3, 122.6 (2C); 128.4,
131.6, 136.3, 147.6, 152.4, 155.3.
4-(5-chloro-2H-benzo[d][1,2,3]triazol-2-yl)benzenamine (12a)
[0199] Under N.sub.2, 100 mg (0.4 mmol) of 11a with 330 mg (1.4
mmol) of tin(II) chloride was stirred at reflux 5 hours in (20 ml)
ethanol. The reaction was extracted with Na.sub.2CO.sub.3
solution/ethyl acetate and after removing the solvent by
evaporation, the residue was purified by chromatography
(hexane/ethyl acetate, 7/3). The solvent was evaporated to obtain
56 mg (63%) 12a as white solid.
[0200] NMR .sup.1H (CDCl.sub.3), .delta.=3.87 (2H, sl); 6.70 (2H,
d, J=8.7 Hz); 7.52-7.59 (2H, m); 8.01 (2H, d, J=8.7 Hz); 8.24 (1H,
s). NMR .sup.13C (CDCl.sub.3), .delta.=115.1 (2C); 117.0, 119.2,
122.1 (2C); 128.0, 131.9, 132.2, 143.2 145.0, 147.6.
4-(5-iodo-2H-benzo[d][1,2,3]triazol-2-yl)benzenamine (12b)
[0201] Using the same methodology as for 12a, compound 12b was
obtained as a yellow solid in 52% yield.
[0202] NMR .sup.1H (CDCl.sub.3), .delta.=3.87 (2H, sl); 6.70 (2H,
d, J=8.7 Hz); 7.52-7.59 (2H, m); 8.01 (2H, d, J=8.7 Hz); 8.24 (1H,
s). NMR .sup.13C (CDCl.sub.3), .delta.=91.3, 115.1 (2C); 119.5,
122.1 (2C); 127.1, 131.8, 135.3, 143.6, 146.2, 147.7.
4-(5-methoxy-2H-benzo[d][1,2,3]triazol-2-yl)benzenamine (12c)
[0203] Using the same methodology as for 12a, compound 12c was
obtained as a yellow solid in 76% yield.
[0204] NMR .sup.1H (CDCl.sub.3), .delta.=3.81 (2H, sl); 3.83 (3H,
s); 6.73 (2H, d, J=8.9 Hz); 7.00 (1H, dd, J=2.3 Hz, J=9.2 Hz); 7.05
(1H, d, J=2.0z); 7.69 (1H, d, J=9.2 Hz); 7.99 (2H, d, J=8.9 Hz).
NMR .sup.13C (CDCl.sub.3), .delta.=55.5, 94.7, 113.4, 115.1 (2C);
118.8, 121.5, 121.6 (2C); 126.3, 145.6, 146.9, 158.9.
N,N-dimethyl-4-(5-(trimethylstannyl)-2H-benzo[d][1,2,3]triazol-2-yl)benzen-
amine (14)
[0205] To a solution of 12 (100 mg, 1 eq) was added dropwise at
-78.degree. C. nBuLi (138 ml, 1.1 eq, 2.5M hexane) and the
resulting mixture was stirred for 15 min at -78.degree. C.
Me.sub.3SnCl (378 ml, 1.2 eq, 1M THF) was then added dropwise and
the resulting solution was stirred for about 30 min at -78.degree.
C. and 1 h at RT before hydrolysis with 2 ml of H.sub.2O.
Extraction with 3.times.15 ml AcOEt and purification by flash
chromatography (SiO.sub.2, hexane/AcOEt, 9/1) giving 14 as a yellow
solid in 13% yield.
[0206] NMR .sup.1H (CDCl.sub.3); .delta.=0.28 (s, 9H, CH.sub.3);
2.96 (s, 6H, CH.sub.3); 6.72 (d, 2H, J=8.8 Hz, CHAr); 7.39 (d, 1H,
J=8.4 Hz, CHAr); 7.80 (dd, 1H, J=8.4, 0.8 Hz, CHAr); 7.98 (s, 1H,
CHAr); 8.12 (d, 2H, J=8.8 Hz, CHAr). NMR .sup.13C (CDCl.sub.3);
.delta.=-9.3 (3C, CH.sub.3); 40.4 (2C, CH.sub.3); 112.0 (2C, CHAr);
117.0 (1C, CHAr); 121.7 (2C, CHAr); 125.5 (1C, CHAr); 130.3 (1C,
Cq); 132.5 (1C, CHAr); 140.8 (1C, Cq); 144.8 (1C, Cq); 144.9 (1C,
Cq); 150.7 (1C, Cq).
Compound (15)
[0207] Using the same methodology as for 8 (starting from
4-iodoaniline), 15 was obtained as a red solid in 41% yield.
Exemplary Compounds of the Invention in Series II
##STR00084##
[0208] Example III
Series III Compounds of the Invention
##STR00085##
[0209] 2-(3-methoxyphenyl)oxazol-4(5H)-one (16)
[0210] 2 g (1 eq) of 3-methoxybenzamide was mixed with 3.1 ml (2.7
eq) of chloroacethylchloride and the resulting mixture was heated
at 110.degree. C. for 1 h. After cooling, the resulting solid was
recrystallized in CHCl.sub.3. The resulting solid was added to a
suspension of NaH (489 mg, 1.1 eq) in DME at 0.degree. C. After 30
min, the resulting mixture was refluxed for 5 h, cooled to RT and
hydrolyzed, extracted with AcOEt, washed with NaCl salt and dried.
The resulting solid was recrystallized in a mixture hexane/AcOEt
giving 16 as a white solid in 26% yield.
[0211] NMR .sup.1H (CDCl.sub.3), .delta.=3.85 (s, 3H, CH.sub.3);
4.74 (s, 2H, CH.sub.2); 7.22 (dd, 1H, J=8.0, 2.4 Hz, CHAr); 7.42
(t, 1H, J=8.0 Hz, CHAr); 7.69 (s, 1H, CHAr); 7.77 (d, 1H, J=8.0 Hz,
CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=55.9 (1C, CH.sub.3); 70.2
(1C, CH.sub.2); 114.4 (1C, CHAr); 122.6 (1C, CHAr); 122.7 (1C,
CHAr); 127.9 (1C, Cq); 130.4 (1C, CHAr); 160.1 (1C, Cq); 188.1 (1C,
Cq); 189.9 (1C, Cq).
2-(3-methoxyphenyl)oxazol-4-yl trifluoromethanesulfonate (17)
[0212] Compound 16 (516 mg, 1 eq) was dissolved in
CH.sub.2Cl.sub.2. To the resulting solution, at 0.degree. C., was
added 501 .mu.L (1.6 eq) of 2,6-lutidine, followed by the dropwise
addition of Tf.sub.2O (681 ml, 1.5 eq). The resulting mixture was
stirred for 4 h at RT, then an additional 501 .mu.L (1.6 eq) of
2,6-lutidine and 681 ml (1.5 eq) of Tf.sub.2O were added. After 1
h, the mixture was evaporated and the residue purified by flash
chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/hexane, 9/1) giving 17
as a red oil in 75% yield.
[0213] NMR .sup.1H (CDCl.sub.3), .delta.=3.69 (s, 3H, CH.sub.3);
6.87 (dd, 1H, J=8.0, 2.5 Hz, CHAr); 7.19 (t, 1H, J=8.0 Hz, CHAr);
7.34 (t, 1H, J=2.5 Hz, CHAr); 7.42 (d, 1H, J=8.0 Hz, CHAr); 7.56
(s, 1H, CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=55.7 (1C,
CH.sub.3); 111.6 (1C, CHAr); 118.2 (1C, CHAr); 119.3 (1C, CHAr);
127.6 (1C, Cq); 130.4 (1C, CHAr); 146.3 (1C, Cq); 159.9 (1C, Cq);
160.3 (1C, Cq).
tert-butyl 4-(2-(3-methoxyphenyl)oxazol-4-yl)phenylcarbamate
(18)
[0214] In a sealable tube, 17 (436 mg, 1 eq), tert-butyl
4-(trimethylstannyl)phenylcarbamate (600 mg, 1.25 eq), LiCl (280
mg, 4 eq), Pd(PPh.sub.3).sub.4 (77 mg, 5%) were mixed in 10 ml of
dioxane. The tube was sealed and heated for 1 night at 100.degree.
C., cooled to RT, evaporated and purified by flash chromatography
(SiO.sub.2, hexane/AcOEt, 85/15) giving 18 as a white solid in 73%
yield.
[0215] NMR .sup.1H (CDCl.sub.3), .delta.=1.45 (s, 9H, 3CH.sub.3);
3.81 (s, 3H, CH.sub.3); 6.54 (bs, 1H, NH); 6.93 (dd, 1H, J=8.0, 2.5
Hz, CHAr); 7.29 (t, 1H, J=8.0 Hz, CHAr); 7.35 (d, 2H, J=8.5 Hz,
CHAr); 7.56 (s, 1H, CHAr); 7.61 (d, 1H, J=8.0 Hz, CHAr); 7.66 (d,
2H, J=8.5 Hz, CHAr); 7.81 (s, 1H, CHAr). NMR .sup.13C (CDCl.sub.3),
.delta.=28.7 (3C, CH.sub.3); 55.8 (1C, CH.sub.3); 111.5 (1C, CHAr);
117.3 (1C, CHAr); 118.9 (1C, CHAr); 119.3 (2C, CHAr); 126.2 (1C,
Cq); 126.7 (2C, CHAr); 129.0 (1C, Cq); 130.2 (1C, CHAr); 133.2 (1C,
CHAr); 138.6 (1C, Cq); 142.0 (1C, Cq); 153.0 (1C, Cq); 160.2 (1C,
Cq); 162.1 (1C, Cq).
4-(2-(3-methoxyphenyl)oxazol-4-yl)benzenamine (19)
[0216] In 10 ml of CH.sub.2Cl.sub.2 was dissolved 300 mg of
compound 19. To the resulting solution, at 0.degree. C., was added
dropwise 1 ml of TFA. The resulting solution was stirred at RT for
3 h before the addition of an additional 1 ml of TFA. After 1
night, the mixture was hydrolyzed with NaHCO.sub.3 salt, extracted
with CH.sub.2Cl.sub.2 and purified by flash chromatography
(SiO.sub.2, hexane/AcOEt, 7/3) giving 19 as a white solid in 75%
yield.
[0217] NMR .sup.1H (CDCl.sub.3), .delta.=3.79 (s, 3H, CH.sub.3);
6.64 (d, 2H, J=8.4 Hz, CHAr); 6.91 (ddd, 1H, J=8.0, 2.8, 0.8 Hz,
CHAr); 7.28 (t, 1H, J=8.0 Hz, CHAr); 7.53 (d, 2H, J=8.4 Hz, CHAr);
7.54-7.55 (m, 1H, CHAr); 7.60 (dt, 1H, J=7.6, 1.2 Hz, CHAr); 7.74
(s, 1H, CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=55.8 (1C,
CH.sub.3); 111.4 (1C, CHAr); 115.5 (2C, CHAr); 117.0 (1C, CHAr);
119.2 (1C, CHAr); 121.7 (1C, Cq); 127.1 (2C, CHAr); 129.2 (1C, Cq);
130.2 (1C, CHAr); 132.2 (1C, CHAr); 142.5 (1C, Cq); 146.9 (1C, Cq);
160.1 (1C, Cq); 161.7 (1C, Cq).
4-(2-(3-methoxyphenyl)oxazol-4-yl)N,N-dimethylbenzenamine (20)
[0218] In DMF was dissolved 19 (217 mg, 1 eq). To the resulting
solution, K.sub.2CO.sub.3 (450 mg, 4 eq) and CH.sub.3 I (203 .mu.L,
4 eq) were added successively. The resulting mixture was stirred
overnight, then hydrolyzed with H.sub.2O, extracted with AcOEt and
purified by flash chromatography (SiO.sub.2, hexane/AcOEt, 8/2)
giving 20 as a white solid in 79% yield.
[0219] NMR .sup.1H (CDCl.sub.3), .delta.=2.78 (s, 6H, 2CH.sub.3);
3.69 (s, 3H, CH.sub.3); 6.58 (d, 2H, J=8.5 Hz, CHAr); 6.80 (ddd,
1H, J=8.5, 2.5, 1.0 Hz, CHAr); 7.17 (t, 1H, J=8.0 Hz, CHAr);
7.45-7.46 (m, 1H, CHAr); 7.48-7.61 (m, 3H, CHAr); 7.62 (s, 1H,
CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=41.0 (2C, CH.sub.3); 55.9
(1C, CH.sub.3); 111.5 (1C, CHAr); 112.9 (2C, CHAr); 117.1 (1C,
CHAr); 119.4 (1C, CHAr); 119.8 (1C, Cq); 127.1 (2C, CHAr); 129.4
(1C, Cq); 130.2 (1C, CHAr); 132.2 (1C, CHAr); 142.8 (1C, Cq); 150.8
(1C, Cq); 160.3 (1C, Cq); 161.9 (1C, Cq).
Example IV
Series IV Compounds of the Invention
##STR00086##
[0220] 2-bromo-6-methoxybenzo[d]thiazole (22)
[0221] Under N.sub.2, to a solution of copper bromide (34.7 mmol),
isoamylnitrile (52.1 mmol) and PEG (20 ml) were added a warm
solution of 2-amino-6-methoxybenzothiazole (21, 0.35 mmol) in
acetonitrile (150 ml) during about 30 min. The resulting mixture
was stirred at room temperature for 2 h, then the reaction was
hydrolyzed and extracted with ethyl acetate. The organic layer was
dried over Na.sub.2SO.sub.4 and the solvent was removed via a
rotary evaporator. The residue was purified by chromatography on
silica gel using 90:10 hexane/ethyl acetate as the eluent to yield
4.83 g (57%) of 22 as a white solid.
[0222] NMR .sup.1H (CDCl.sub.3), .delta.=3.59 (3H, s); 6.78 (1H,
dd, J=2.8 Hz, J=9.6 Hz); 6.91 (1H, d, J=2.8 Hz); 7.57 (1H, d, J=9.6
Hz). NMR .sup.13C (CDCl.sub.3), .delta.55.2; 103.2; 115.4; 122.8;
134.9; 137.2; 146.4; 157.6.
6-methoxy-2-(4-nitrophenyl)benzo[d]thiazole (23)
[0223] Under N.sub.2, in a sealed tube, to 22 (19.7 mmol) in DMF 50
ml, 1-iodo-4-nitrobenzene (21.6 mmol), cesium carbonate (19.6
mmol), palladium acetate (0.98 mmol), copper bromide (0.2 mmol) and
tributylphosphine (1.9 mmol) were added. The reaction was stirred
at 150.degree. C. overnight and, after cooling to room temperature,
the mixture was extracted with ethyl acetate. The organic layer was
then washed (3 times) with water, dried over Na.sub.2SO.sub.4 and
the solvent removed via a rotary evaporator. The residue was
purified by chromatography on silica gel using 9:1 hexane/ethyl
acetate as the eluent to yield 4.62 g (81%) of 23 as a yellow
solid.
[0224] NMR .sup.1H (DMSO), .delta.=3.83 (3H, s); 7.21 (1H, J=8.6
Hz); 7.73 (s, 1H); 8.04 (2H, d, J=8.7 Hz); 8.25 (1H, d, J=8.6 Hz);
8.35 (2H, d, J=8.7 Hz). NMR .sup.13C (DMSO), .delta.=55.3; 104.7;
115.7; 116.7; 123.4; 124.1 (2C); 124.5; 127.8; 128.6 (2C); 144.0;
147.5; 157.5.
2-(4-nitrophenyl)benzo[d]thiazol 6-ol (24)
[0225] Under N.sub.2, 23 (10.4 mmol) was dissolved in
CH.sub.2Cl.sub.2 and, at -40.degree. C., BBr.sub.3 (31.4 mmol) was
slowly added. The mixture was then stirred 24 hours at room
temperature. The reaction mixture was quenched with water and
extracted with ethyl acetate (.times.3 100 ml). The organic layers
were combined, dried over Na.sub.2SO.sub.4 and the solvent was
removed via a rotary evaporator. The residue was purified by
chromatography on silica gel using 7:3 hexane/ethyl acetate as the
eluent to yield 1.53 g (53%) of 24 as a yellow solid.
[0226] NMR .sup.1H (DMSO), .delta.=7.06 (1H, dd, J=2.4 Hz, J=8.8
Hz); 7.47 (1H, d, J=2.4 Hz); 7.93 (1H, d, J=8.8 Hz); 8.23 (2H, d,
J=8.8 Hz); 8.34 (2H, d, J=8.8 Hz); 10.07 (1H, sl). NMR .sup.13C
(DMSO), .delta.=106.9; 115.9; 117.0; 124.6 (2C); 127.8 (2C); 136.9;
138.8; 147.3; 148.3; 156.7; 160.9.
2-(4-nitrophenyl)benzo[d]thiazol-6-yl acetate (25)
[0227] Under N2, 24 (4.0 mmol) was dissolved in THF (100 ml) and,
at 0.degree. C., NaH (8.2 mmol) was slowly added. The mixture was
stirred for 30 min at 0.degree. C. and acetyl chloride (6.0 mmol)
was added dropwise. The resulting mixture was then stirred at room
temperature overnight. The reaction was hydrolyzed and extracted
with ethyl acetate. The organic layer was dried over
Na.sub.2SO.sub.4 and the solvent was removed via a rotary
evaporator. The residue was purified by chromatography on silica
gel using 7:3 hexane/ethyl acetate as the eluent to yield 1.24 g
(74%) of 25 as a yellow solid.
[0228] NMR .sup.1H (CDCl.sub.3), .delta.=2.29 (3H, s), 7.20 (1H,
dd, J=2.2 Hz, J=8.7 Hz), 7.65 (1H, d, J=2.2 Hz,), 8.02 (1H, d,
J=8.7 Hz), 8.16 (2H, d, J=8.8 Hz), 8.27 (2H, d, J=8.8 Hz). NMR
.sup.13C (CDCl.sub.3), .delta.=21.5, 115.2, 121.8, 124.7 (2C),
124.8, 128.6 (2C), 136.5, 139.3, 149.2, 149.5, 152.3, 159.9,
169.7.
2-(4-aminophenyl)benzo[d]thiazol-6-yl acetate (26)
[0229] Under H.sub.2, 25 (2.7 mmol) was dissolved in a mixture of
THF/MeOH (5/20 ml) and was then hydrogenated at room temperature
for 1 h under 30 PSI. Pd (c) was filtered and the solvent removed
via a rotary evaporator. The residue was purified by chromatography
on silica gel using 7:3 hexane/ethyl acetate as the eluent to yield
560 mg (71%) of 26 as a white solid.
[0230] NMR .sup.1H (CDCl.sub.3), .delta.=2.23 (3H, s); 4.01 (2H,
sl); 6.72 (2H, d, J=8.7 Hz,); 7.16 (1H, dd, J=2.2 Hz, J=8.8 Hz);
7.59 (1H, d, J=2.1 Hz); 7.86 (2H, d, J=8.7 Hz); 7.95 (1H, d, J=8.8
Hz). NMR .sup.13C (CDCl.sub.3), .delta.=21.3, 114.4, 114.9 (2C);
120.5; 123.0; 123.9; 127.4; 129.3 (2C); 135.4; 147.6; 149.5; 152.3;
164.8.
2-(4-((E)-3-(tributylstannyl)allylamino)phenyl)benzo[d]thiazol-6-yl
acetate (27)
[0231] Under N.sub.2, to 26 (0.35 mmol) in acetone (5 ml),
K.sub.2CO.sub.3 (1 mmol) was slowly added at 0.degree. C. The
reaction was stirred for 30 min with 31 (0.5 mmol) in 2 ml of
acetone added. The resulting mixture was stirred at reflux
overnight. The reaction was cooled at room temperature and acetone
was removed in vacuo. Water was then added and the resulting
reaction was extracted with ethyl acetate. The organic layer was
dried over Na.sub.2SO.sub.4 and the solvent was removed via a
rotary evaporator. The residue was purified by chromatography on
silica gel using 65:20:15 hexane/diethyl ether/triethylamine as the
eluent to yield 105 mg (49%) of 27 as a white solid.
[0232] NMR .sup.1H (CDCl.sub.3), .delta.=0.75-0.83 (15H, m);
1.17-1.27 (6H, m); 1.32-1.44 (6H, m); 2.26 (3H, s); 3.81 (2H, m);
4.22 (1H, sl); 5.98 (1H, dt, J=4.8 Hz, J=9.5 Hz); 6.16 (1H, d, J=19
Hz); 6.57 (2H, d, J=8.8 Hz); 7.07 (1H, dd, J=2.4 Hz, J=8.6 Hz);
7.51 (1H, d, J=1.5 Hz); 7.79 (2H, d, J=8.8 Hz); 7.86 (1H, d, J=8.8
Hz). NMR .sup.13C (CDCl.sub.3), .delta.=9.6 (3c); 13.9 (3C); 21.4;
27.4 (3C); 29.2 (3C); 49.1; 112.8 (2C); 114.4; 120.4; 122.5; 122.8;
129.2 (2C); 130.6; 135.3; 143.9; 147.5; 150.8; 152.5; 169.3;
169.9.
2-(4-((E)-3-iodoallylamino)phenyl)benzo[d]thiazol-6-yl acetate
(28)
[0233] Under N.sub.2, to 27 (0.11 mmol) in CHCl.sub.3 (3 ml), at
0.degree. C., I.sub.2 (1.25 mmol) in 1 ml of CHCl.sub.3 was slowly
added. The reaction was stirred for 45 min at room temperature and
the mixture was extracted with a solution of 10% of
Na.sub.2S.sub.2O.sub.3 in water. The organic layer was dried over
Na.sub.2SO.sub.4 and the solvent was removed via a rotary
evaporator. The residue was purified by chromatography on silica
gel using 60:40 hexane/ethyl acetate as the eluent to yield 51 mg
(96%) of 28 as a white solid.
[0234] NMR .sup.1H (CDCl.sub.3), .delta.=2.34 (3H, s); 3.81-3.83
(2H, m); 4.26 (1H, sl); 6.37 (1H, d, J=14.5 Hz); 6.62-6.65 (2H, d,
J=8.8 Hz); 6.98 (1H, s); 7.15 (1H, dd, J=2 Hz, J=8.8 Hz); 7.59 (1H,
d, J=2 Hz); 7.88 (2H, d, J=8.8 Hz); 7.64 (1H, d, J=8.5 Hz). NMR
.sup.13C (CDCl.sub.3), .delta.=21.4; 48.0; 78.5; 112.9 (2C); 114.5;
120.5; 122.7; 123.0; 125.7; 129.4 (2C); 136.0; 142.2; 149.9; 151.8;
152.8; 169.8.
2-(4-((E)-3-iodoallylamino)phenyl)benzo[d]thiazol-6-ol (29)
[0235] 28 was stirred at 80.degree. C. for 1.5 h with NaOH 2M.
After cooling at room temperature, the reaction was acidified with
HCl 1N (Ph=7-8) and the aqueous layer extracted with ethyl acetate
(.times.2). The organic layer was dried over Na.sub.2SO.sub.4 and
the solvent was removed via a rotary evaporator. The residue was
purified by chromatography on silica gel using 70:30 hexane/ethyl
acetate as the eluent to yield 29 mg (81%) of 29 as a white
solid.
[0236] NMR .sup.1H (MeOH), .delta.=3.80 (2H, dd, J=5.2 Hz, J=1.5
Hz); 6.42 (1H, dt, J=5 Hz, J=15 Hz); 6.61-6.67 (1H, m); 6.69 (2H,
d, J=8.5 Hz); 6.94 (1H, dd, J=2.5 Hz, J=9 Hz); 7.26 (1H, d, J=2.5
Hz); 7.69 (1H, d, J=8.5 Hz); 7.77 (2H, d, J=8.5 Hz). NMR .sup.13C
(MeOH), .delta.=54.6, 77.4, 107.4, 113.4 (2C); 116.6, 122.9, 123.0,
129.5 (2C); 136.6, 144.2, 148.6, 152.0, 156.4, 167.9.
(E)-3-(tributylstannyl)prop-2-en-1-ol (30)
[0237] Under N.sub.2, to propargylic alcohol (51.5 mmol) at room
temperature, tributyltin hydride (67.2 mmol) then AIBN (2.6 mmol)
were added. The mixture was heated for 2 h at 80.degree. C. After
cooling at room temperature, the residue was purified by
chromatography on silica gel using 95:5 hexane/ethyl acetate as the
eluent to yield 4.65 g (26%) of 30 as a colorless oil.
[0238] NMR .sup.1H (CDCl.sub.3), .delta.=0.75-0.98 (15H, m);
1.20-1.34 (6H, m); 1.40-1.60 (6H, m); 2.03-2.15 (1H, m); 4.10-4.14
(2H, m); 6.13-6.15 (2H, m). NMR .sup.13C (CDCl.sub.3), .delta.=9.4
(3C); 13.7 (3C); 27.3 (3C); 29.1 (3C) 66.3; 128.2; 147.1.
(E)-3-(tributylstannyl)allyl 4-methylbenzenesulfonate (31)
[0239] Under N.sub.2, to 30 (2.9 mmol) in ether (50 ml), at
.+-.25.degree. C., potassium trimethylsilanolate was added. After
stirring for 30 min, p-toluenesulfonyl chloride in ether (10 ml)
was added dropwise. The resulting mixture was stirred for 2 h at
-25.degree. C. After hydrolyzing, the reaction was extracted with
ethyl acetate and the organic layer was dried over
Na.sub.2SO.sub.4. The solvent was also removed via a rotary
evaporator. The residue was purified by chromatography on silica
gel using 95:2.5:2.5 hexane/ethyl acetate/triethylamine as the
eluent to yield 650 mg (45%) of 31 as a colorless oil.
[0240] NMR .sup.1H (CDCl.sub.3), .delta.=0.73-0.78 (15H, m);
1.15-1.21 (6H, m); 1.31-1.36 (6H, m); 2.33 (3H, s); 4.42 (2H, m);
5.80 (1H, dt, J=5 Hz, J=19 Hz); 6.18 (1H, d, J=19 Hz); 7.22 (2H, d,
J=8 Hz); 7.68 (2H, d, J=8 Hz). NMR .sup.13C (CDCl.sub.3),
.delta.=9.8 (3C); 14.0 (3C); 22.6; 27.6 (3C); 29.4 (3C); 73.7;
128.3 (2C); 130.2 (2C); 136.8; 138.1; 139.4; 145.0.
Example V
Radioiodination as Synthesis of
4-(5-[.sup.123I]-2H-benzo[d][1,2,3]triazol-2-yl)N,N-dimethylbenzenamine
(13)
[0241] A sterile, pyrogen-free solution of no-carrier-added
.sup.123I-13 in physiological saline is prepared by reacting a
trimethylstannyl precursor such as 14 with sodium [.sup.123I]iodide
in the presence of an oxidizing agent, for example, peracetic acid
followed by HPLC isolation. The resulting radiolabeled product is
purified by means of HPLC and formulated. To a shipping vial with
dry Na[.sup.123I]I/NaOH are added, in the following order, 50%
aqueous MeOH, 0.8M H.sub.3PO.sub.4 in the amount just enough to
neutralize the NaOH plus extra 10111, a solution of 14 (100 .mu.g,
0.19 .mu.mol) in 50 .mu.l of MeOH and 50 .mu.L of 6.4% aqueous
peracetic acid freshly prepared by a 5-fold dilution of 32%
CH.sub.3C(O)OOH. The total volume of added reagents is 3204. After
standing for 14-16 min at room temperature, the reaction mixture in
the vial is quenched by the addition of 100 .mu.l of a 100 mg/ml
solution of Na.sub.2S.sub.2O.sub.5 in saturated aqueous NaHCO.sub.3
and the vial headspace is flushed with 60 ml of air into a charcoal
filter. The vial is emptied and rinsed with 0.3-0.4 ml of 85%
aqueous MeCN. The rinse is combined with the quenched reaction
mixture and the resultant liquid is injected onto a reverse-phase
HPLC column. The column (C18, 10.mu., 4.6.times.250 mm) is eluted
with a mixture of acetonitrile, water and triethylamine (60:40:0.2
v/v) at a flow rate of 1.0 ml/min. The fraction eluting at the
retention time of the authentic 13 is collected into a 50 ml flask
containing 50 .mu.l of 34 mM L-ascorbic acid (stabilizer). The
solvent is removed via a rotary evaporator at 45-50.degree. C.
under reduced pressure/argon gas flow. The dry residue in the flask
is dissolved in 800 .mu.l of 50% ethanol and the resulting solution
is filtered through a 0.2 .mu.m sterilizing filter into an empty
sterile vial. The formulation is finalized by the addition of 6-8
ml of sterile 0.9% NaCl for injection through the same filter.
Quality control testing includes visual inspection, determination
of specific concentration, identity and radiochemical purity (by
HPLC), pH, pyrogenicity and sterility (by compendia) tests, USP
XXII, 1990). All tests, including inoculation in two media for
sterility, are performed before release for administration to
subjects. Sterility is confirmed after 1 and 2 weeks of incubation
at 37.degree. C. The invention also contemplates other conventional
methods known to those of ordinary skill in the art for preparing
an amyloid probe from a compound of the invention. Ellis et al.,
Aust. J. Chem., 26: 907 (1973); Wilson et al., J. Org. Chem., 51:
4833 (1986); Wilbur et al., J. Label. Compound. Radiopharm., 19:
1171 (1982); Chumpradit et al., J. Med. Chem., 34: 877 (1991);
Chumpradit et al., J. Med. Chem., 32: 1431 (1989); Kabalka et al.,
J. Label. Compound. Radiopharm., 19: 795 (1982); Koch et al., Chem.
Ber., 124: 2091 (1991); H. Mach et al., J. Med. Chem., 36: 3707
(1993); Arora et al., J. Med. Chem., 30: 918 (1987).
Example VI
Binding Assays Using Human AD Brain Tissues by Quantitative
Autoradiography
[0242] Postmortem human cerebral cortical tissue from the frontal
lobe was obtained from the Center for Neurodegenerative Disease at
Emory University (Atlanta, Ga.). Fresh-frozen tissue sections were
cut at a thickness of 20-25 .mu.m and thaw-mounted onto
gelatin-coated glass slides. The sections were then air-dried and
stored at -80.degree. C. until used. Prepared sections were thawed
and incubated at room temperature in 0.05M Tris-HCl buffer, pH 7.7
with 10% ethanol containing 0.02 nM [.sup.125I]-IMPY (a
conventional amyloid ligand) having the structure
##STR00087##
The radioligand was displaced with increasing concentrations (0.1
nm-5 .mu.M) of cold inhibitor (in 200 .mu.l of 100% ethanol).
Nonspecific binding was determined in the presence of 5 .mu.M
thioflavin-T (THFT). Borosilicate glass tubes were used for the
incubation containers to minimize hydrophobic adsorption to the
walls. After 5 h, the sections were washed with 100% ethanol for 30
min at room temperature and allowed to air dry. The radiolabeled
sections and .sup.14C-plastic standards (calibrated for .sup.125I,
American Radiolabeled Chemicals, Inc., St Louis, Mo.) were apposed
to autoradiograhphic film (Biomax MS, Eastman Kodak, Rochester,
N.Y.) for 24 h. The resulting autoradiograms were digitized using
an Epson 1680 Scanner with transparency unit and analyzed
densitometrically with AIS software (Imaging Research, St.
Catherines, Ontario) to determine binding density. Binding curves
and corresponding K.sub.i or K.sub.d values can also generated
using non-linear regression with GraphPad Prism software. IC50
binding data for compounds of the invention based on this binding
assay are shown in FIGS. 4 and 5.
Example VII
Developing SPECT Imaging Agents to Quantify Amyloid and/or Amyloid
Deposits (Plaque) Burden in Alzheimer Disease Patients
[0243] In one aspect, recent interest in AD therapy with drugs
targeting reduction of .beta.-amyloid burden such as, for example,
the compounds of the invention, has underscored the need for
non-invasive scintigraphic methods for interrogating amyloid
deposition for both drug development and elucidating
pathophysiological changes in AD patients. The present invention
synthesized and radiolabeled a series of ligands with iodine-123
and iodine-125, although any labels, markers or tags as described
herein can be used.
[0244] Compounds of the invention represented by MNI-187 and
MNI-195 (Chart 1), were synthesized, their affinities for the
.beta.-amyloid protein were evaluated using human AD brain tissue
and compared to that of IMPY, a conventional amyloid ligand. The
compounds of the invention demonstrated binding affinities 2 to
10-fold better than that of IMPY.
##STR00088##
[0245] The labeling of the compounds of the invention was carried
out under standard conditions (Na.sup.123/125I, oxidizer, acidic
medium) at ambient temperature. Radiochemical yields averaged
35-90%. The labeled compounds were readily purified by
reverse-phase HPLC, and their radiochemical purity exceeded 95%.
Lipophilicity and protein binding of the obtained amyloid probes of
the invention (for example, for SPECT imaging) are comparable to
those conventionally used in humans (for example, for MNI-187, LogD
at pH 7.4 was 2.7, and the free ligand fraction in plasma was
4%).
[0246] The compounds or amyloid probes of the invention including
those in Chart 1 and probes thereof can be used therapeutically or
in subject scintigraphic imaging.
Example VIII
Series I Compounds of the Invention
##STR00089## ##STR00090##
[0247] 4'-dimethylaminoacetophenone (1)
[0248] In a mixture EtOH/HCl 37% 80 ml/5 ml was dissolved
4'-aminoacetophenone (13.5 g, 0.1 mol), then formaldehyde 37% (15
ml) was added followed by PtO.sub.2 (150 mg). The resulting
solution was hydrogenated at 50 PSI for 1 h, filtered through
celite, evaporated and purified by flash chromatography (SiO.sub.2,
hexane/AcOEt, 8/2) giving 1 as a white solid in 62% yield.
[0249] NMR .sup.1H (CDCl.sub.3), .delta.=2.30 (s, 3H, CH.sub.3);
2.85 (s, 6H, 2CH.sub.3); 6.44 (d, 2H, J=9.0 Hz, 2CHAr); 7.67 (d,
2H, J=9.0 Hz, 2CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=26.3 (1C,
CH.sub.3); 40.4 (2C, 2CH.sub.3); 110.9 (2C, CHAr); 125.6 (1C, Cq);
130.9 (2C, CHAr); 153.7 (1C, Cq); 196.7 (1C, Cq).
2,2-dibromo-4'-dimethylaminoacetophenone (2)
[0250] In 20 ml of concentrated H.sub.2SO.sub.4 was dissolved 1
(3.8 g, 1 eq), then at 0.degree. C. bromine (1.19 ml, 1 eq) was
added dropwise and the resulting mixture was stirred at RT for 6 h
before being poured into 200 ml of ice/H.sub.2O. The resulting
precipitate was collected by filtration, washed with H.sub.2O,
dissolved in CH.sub.2Cl.sub.2, dried over Na.sub.2SO.sub.4 and
concentrated in vacuum to give 2 as a green solid in 68% yield,
which can be directly used in the next step without any
purification.
2-bromo-4'-dimethylacetophenone (3)
[0251] In 30 ml of THF was dissolved 2 (5.1 g, 1 eq), then at
0.degree. C. was added dropwise a mixture of diethylphosphite (2.04
ml, 1 eq) and Et.sub.3N (2.4 ml, 1.1 eq) in 12 ml of THF. The
resulting mixture was stirred 6 h at RT, then evaporated and poured
into 200 ml of ice/H.sub.2O and the resulting precipitated was
filtered, washed with H.sub.2O and dried in vacuum. 3 was obtained
as a green solid in 89% yield.
[0252] NMR .sup.1H (CDCl.sub.3), .delta.=2.97 (s, 6H, 2CH.sub.3);
4.45 (s, 2H, CH.sub.2); 6.57 (d, 2H, J=9.0 Hz, 2CHAr); 7.72 (d, 2H,
CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=31.3 (1C, CH.sub.2); 40.4
(2C, CH.sub.3); 111.1 (2C, CHAr); 121.8 (1C, Cq); 131.6 (2C, CHAr);
154.1 (1C, Cq); 189.7 (1C, Cq).
7-bromo-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(4)
[0253] In the minimum volume of EtOH was dissolved commercially
available 2-amino-6-bromobenzothiazole (2 mmol), then 3 (2 mmol)
was added and the resulting mixture was refluxed for 2 h before
addition of NaHCO.sub.3 (3 mmol). After 6 h more of reflux, the
mixture was hydrolyzed with H.sub.2O (5 ml), extracted using AcOEt
(4.times.25 ml), dried over Na.sub.2SO.sub.4, concentrated in
vacuum and purified by flash chromatography (SiO.sub.2,
hexane/AcOEt, 1/1).
[0254] NMR .sup.1H (DMSO-d.sub.6), .delta.=2.93 (s, 6H, 2CH.sub.3);
6.77 (d, 2H, J=8.5 Hz, 2CHAr); 7.67 (d, 2H, J=8.5 Hz, 2CHAr); 7.71
(d, 1H, J=8.5 Hz, CHAr); 7.88 (d, 1H, J=8.5 Hz, CHAr); 8.29 (s, 1H,
CHAr); 8.50 (s, 1H, CHAr). NMR .sup.13C (DMSO-d.sub.6),
.delta.=40.4 (2C, CH.sub.3); 107.2 (1C, CHAr); 112.6 (2C, CHAr);
114.9 (1C, CHAr); 116.6 (1C, Cq); 122.1 (1C, Cq); 126.0 (2C, CHAr);
127.6 (1C, CHAr); 129.7 (1C, CHAr); 131.5 (1C, Cq); 146.8 (1C, Cq);
147.7 (1C, Cq); 150.1 (1C, Cq). HRMS Calcd for
C.sub.17H.sub.15N.sub.3BrS: 372.0170, found: 372.0171. Anal. Calcd
for C.sub.17H.sub.14N.sub.3BrS: C, 54.85%; H, 3.79%; N, 11.29%;
found: C, 54.49%; H, 3.73%; N, 11.06%.
7-(trimethylstannyl)-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(5)
[0255] To a solution of 4 (200 mg, 1 eq) in DME (5 ml) was added
hexamethylditin (527 mg, 3 eq) and Pd(PPh.sub.3).sub.4 (61 mg, 10%)
and the resulting mixture was refluxed for 6 h. After cooling to
RT, 20 ml of AcOEt were added and the mixture washed twice with 5
ml of H.sub.2O, purified by flash chromatography (SiO.sub.2,
hexane/AcOEt/Et.sub.3N, 9/1/0.1) to give 5 as a yellow oil in 35%
yield.
[0256] NMR .sup.1H (CDCl.sub.3), .delta.=0.16 (s, 9H, CH.sub.3);
2.79 (s, 6H, CH.sub.3); 6.60 (d, 2H, J=8.8 Hz, CHAr); 7.30-7.36 (m,
2H, CHAr); 7.55-7.58 (m, 2H, CHAr); 7.64 (s, 1H, CHAr). NMR
.sup.13C (CDCl.sub.3), .delta.=-9.1 (3C, CH.sub.3); 40.6 (2C,
CH.sub.3); 105.0 (1C, Cq); 112.2 (1C, Cq); 112.7 (2C, CHAr); 122.4
(1C, Cq); 126.1 (2C, CHAr); 130.3 (1C, CHAr); 131.1 (1C, Cq); 132.3
(1C, Cq); 133.0 (1C, Cq); 138.4 (1C, CHAr); 147.5 (1C, Cq); 148.0
(IC, CHAr); 149.9 (1C, CHAr).
7-iodo-2-(4-dimethylaminophenyl)-imidazo[2,1-b]benzothiazole
(6)
[0257] 5 (64 mg, 1 eq) was dissolved in CH.sub.2Cl.sub.2 (5 ml) and
to that was added dropwise I.sub.2 (39 mg, 1.1 eq) in 2 ml of
CH.sub.2Cl.sub.2, the resulting mixture was stirred at RT for 1 h
before hydrolysis with 5 ml of Na.sub.2S.sub.2O.sub.3 10%.
Extraction was performed using CH.sub.2Cl.sub.2 (2.times.5 ml) with
drying over Na.sub.2SO.sub.4 and concentrating was carried out in
vacuum. Purification by flash chromatography (SiO.sub.2,
hexane/AcOEt, 9/1) was used to give 6 as a white solid in 15%
yield.
[0258] NMR .sup.1H (CDCl.sub.3); .delta.=3.02 (s, 6H, 2CH.sub.3);
6.35 (d, 1H, J=8.4 Hz, CHAr); 7.20 (s, 1H, 2CHAr); 7.36 (dd, 1H,
J=8.8, 2.0 Hz, CHAr); 7.50 (d, 2H, J=8.0 Hz, CHAr); 7.71 (d, 1H,
J=8.0 Hz, CHAr); 7.99 (s, 1H, CHAr).
Example IX
Series II Compounds of the Invention
##STR00091##
[0259] 1-(2-amino-4 and
-5-iodophenyl)-2-(4-dimethylaminophenyl)diazene (7)
[0260] 4-iodo-1,2-phenylenediamine (2 g, 1 eq),
4-dimethylaminonitrozoaniline (1.26 g, 1 eq) and NaOH (340 mg, 1
eq) were mixed neat and heated at 70.degree. C. for 20 min with
constant triturating. The resulting paste was extracted with
toluene, concentrated in vacuum and purified by flash
chromatography (SiO.sub.2, hexane/AcOEt, 8/2) giving a mixture of
two isomers (3 and 4 iodo) as a red solid in 32% yield.
4-(5-iodo-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-dimethylbenzenamine
(8)
[0261] In CH.sub.2Cl.sub.2 was dissolved 7 (1 g, 1 eq) and a
solution of Pb(OAc).sub.4 (2.03 g, 1.1 eq) in which
CH.sub.2Cl.sub.2 was added dropwise. The resulting solution was
stirred 30 min at RT, then hydrolyzed with 15 ml of
Na.sub.2CO.sub.3 salt, extracted with CH.sub.2Cl.sub.2, dried by
Na.sub.2SO.sub.4, evaporated, purified by flash chromatography
(SiO.sub.2, hexane/AcOEt, 8/2) giving 11 as an orange solid in 10%
yield.
[0262] NMR .sup.1H (CDCl.sub.3), .delta.=2.95 (s, 6H, 2CH.sub.3);
6.68 (dd, 2H, J=8.8, 2.0 Hz, CHAr); 7.51 (dd, 1H, J=8.8, 2.0 Hz,
CHAr); 7.56 (dd, J=8.8, 0.4 Hz, CHAr); 8.06 (dd, 2H, J=8.8, 2.0 Hz,
CHAr); 8.22 (s, 1H, CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=40.4
(2C, CH.sub.3); 91.0 (1C, Cq); 111.9 (2C, CHAr); 119.4 (1C, CHAr);
121.7 (2C, CHAr); 126.9 (1C, CHAr); 135.0 (1C, CHAr); 143.6 (1C,
Cq); 146.2 (1C, Cq); 150.9 (1C, Cq).
Example X
##STR00092##
[0263] 7-methoxy-2-(4-bromophenyl)-imidazo[2,1-b]benzothiazole
(1)
[0264] In the minimum volume of EtOH was dissolved the commercially
available 2-amino-6-methoxybenzothiazole (2 mmol), then
2,4'-dibromoacetophenone (2 mmol) was added and the resulting
mixture was refluxed for 5 h before addition of NaHCO.sub.3 (3
mmol). After 2 h more of reflux, the precipitate was filtered off,
washed with a mixture 1/1 AcOEt/hexane and dried in vacuum and then
used in the next step, optionally, without further purification.
The result was a white solid as a 54% yield.
[0265] NMR .sup.1H (CDCl.sub.3), .delta.=3.82 (s, 3H, OCH.sub.3);
6.78-7.85 (m, 8H, 8CHAr). NMR .sup.13C (CDCl.sub.3), .delta.=55.4
(1C, OCH.sub.3); 106.7 (1C, CHAr); 107.9 (1C, CHAr); 112.8 (1C,
CHAr); 113.1 (1C, CHAr); 124.1 (2C, CHAr); 123.2 (1C, Cq); 126.6
(1C, Cq); 131 (1C, Cq); 132.1 (2C, CHAr); 133.8 (1C, Cq); 147.3
(1C, Cq); 156.1 (1C, Cq).
7-methoxy-2-(4-(trimethylstannyl)phenyl)-imidazo[2,1-b]benzothiazole
(2)
[0266] To a solution of 1 (300 mg, 1 eq) in DME (5 ml) was added
hexamethylditin (1.4 mg, 5 eq) and Pd(PPh.sub.3).sub.4 (99 mg, 10%)
and the resulting mixture was refluxed for 1 night. After cooling
to RT, 20 ml of AcOEt were added and the mixture washed twice with
5 ml of H.sub.2O, purification was performed by flash
chromatography (SiO.sub.2, hexane/AcOEt/Et.sub.3N, 9/1/0.1) giving
2 as a white oil in 19% yield.
[0267] NMR .sup.1H (CDCl.sub.3), .delta.=0.16 (s, 9H, CH.sub.3);
3.70 (s, 2H, OCH.sub.3); 6.83 (dd, 1H, J=8.8, 2.4 Hz, CHAr); 7.03
(d, 1H, J=2.4 Hz, CHAr); 7.32 (d, 1H, J=8.8 Hz, CHAr); 7.39 (d, 2H,
J=8.0 Hz, CHAr); 7.67 (d, 2H, J=8.0 Hz, CHAr); 7.74 (s, 1H, CHAr).
NMR .sup.13C (CDCl.sub.3), .delta.=-9.4 (3C, CH.sub.3); 55.9 (C,
OCH.sub.3); 106.9 (1C, CHAr); 108.7 (1C, CHAr); 113.1 (1C, CHAr);
113.4 (1C, CHAr); 124.6 (2C, CHAr); 126.4 (1C, Cq); 131.5 (1C, Cq);
133.8 (1C, Cq); 136.2 (2C, CHAr); 141.4 (1C, Cq); 147.3 (1C, Cq);
157.2 (1C, Cq).
Example XI
Data Acquisition and Image Reconstruction
[0268] Subjects can receive Lugol's solution (10 drops in solution)
approximately 30 min prior to a bolus 123-I MNI-187 (Chart 1)
injection to minimize radioactive uptake by the thyroid.
Alternatively, another compound or amyloid probe of the invention
can be administered as a bolus injection. Five fiducial markers
filled with 1 .mu.Ci of 123-I can be attached to both sides of the
subject's heads at the level of the canthomeatal line prior to
imaging to facilitate post hoc computer reorientation of transaxial
images, aiding in the standardization of brain orientation.
Subjects can be dosed by intravenous injection 5 mCi of bolus 123-I
MNI-187 (Chart 1) or another compound or amyloid probe of the
invention. Serial dynamic SPECT projection data can be acquired
using a three-headed detector SPECT system (PICKER PRISM 3000XP,
Philips, Cleveland, Ohio) fitted with low-energy, high-resolution
fanbeam collimators. Scans can be acquired for 10 min acquisition
time.times.6 scans, then 20 min.times.6 scans, for a total of 15
SPECT scans acquired over 8 h. Projection data can be acquired into
a 20% symmetric photopeak window centered on 159 keV for a total of
120 raw projection images sampled every 3 degrees. Uniformity
corrected projection data can be reconstructed using filtered
back-projection and a ramp filter. A standardized three dimensional
Butterworth filter can be applied to the reconstructed images.
Images can also be reoriented to obtain an axial image set aligned
parallel to the canthomeatal line. Attenuation correction can be
performed using a Chang zero order (homogeneous) correction applied
to the reconstructed data using an empiric .mu. determined for a
distributed 123-I source in an anthropomorphic brain phantom.
Venous sampling can also be performed at the end of each SPECT
acquisition for measurement of 123-I MNI-187 (Chart 1) or another
amyloid probe of the invention in plasma (both protein bound and
free). Images of a normal (healthy) and AD diagnosed brain as
obtained from such an exemplary protocol using 123-I MNI-187 (Chart
1) as an amyloid probe are provide in FIG. 6.
Example XII
Human Brain Tissue
[0269] Postmortem human cerebral cortical tissue from the frontal
lobe are used. Neurological diagnoses using CERAD criteria (Mirra
et al., Neurology, 41: 479 (1991)) will have been made by a
neuropathologist using immunostained and/or silver stained
paraffin-embedded sections from adjacent tissue blocks. Fresh
blocks will have been cut from frontal cortex, quick-frozen, and
stored at -80.degree. C. until used.
In vitro Probe Binding, Homogenate (Filtration) Assay
[0270] Frozen human brain tissue is thawed. The gray matter can be
dissected free, weighed and homogenized in 10 volumes (1:10
weight:volume) of phosphate buffer, pH 7.4 (PB) using a polytron
set to 20,00 rpm for 30 minutes. The following mixture of reagents
can be added to borosilicate glass tubes in triplicate, 50 .mu.l of
[.sup.125I]labeled ligand (IMPY) (final concentration 0.02 nM),
(final concentration 2 nM), and as needed for competition
experiments, 50 .mu.l of compounds of the invention (10.sup.-5 to
10.sup.-10M) in a final volume of 1 ml of PB with 10% ethanol.
Nonspecific binding can be defined in the presence of 3.2 .mu.M
IMPY (blank). Assays can be initiated by the addition of 50 .mu.l
of tissue homogenates. The mixture can be incubated at 50.degree.
C. for 48 hours (except for kinetics assays) and the membranes may
be trapped by vacuum filtration through Whatman GF/B filters using
a cell harvester and rinsed with 3.times.3 ml of 50% ethanol.
Filters containing the bound ligand can be counted in a liquid
scintillation counter. Typically in this assay, non-specific
binding is less than 25% of the total bound ligand and free ligand
observes "Zone A" behavior. Goldstein et al., Principles of drug
action: The basis of pharmacology, (1973). The results of
competition, saturation and kinetics experiments can be analyzed by
nonlinear regression using Prism (GraphPad Software, Inc.) to
calculate K.sub.i, K.sub.d and rate constants, respectively.
In vitro Ligand Binding, Autoradiography Assay
[0271] Twenty micron cryostat sections are cut from frozen blocks
of brain tissue and thaw-mounted onto gelatin-coated glass slides
and stored at .+-.20.degree. C. Stored sections can be thawed and
incubated at 50.degree. C. in 0.05M Tris-HCl buffer, pH 7.7 with
10% ethanol containing 0.02 nM [.sup.125I] IMPY or 2 nM
[.sup.18F]IMPY analog. The higher concentration for the latter
ligand may be needed to ensure a signal detectable by the
autoradiographic film. Nonspecific binding can be determined in the
presence of 3.2 .mu.M cold IMPY. After a 48 h incubation, the
sections can be washed with 100% ethanol for 30 min at room
temperature and allowed to air dry. The labeled sections and
.sup.14C-plastic standards (calibrated either for .sup.125I) can be
apposed to autoradiographic film for 1-3 days. Miller et al.,
Neurosci. Lett., 81: 345 (1987) and Baskin et al., Neurosci. Lett.,
104: 171 (1989). The resulting autoradiograms can be digitized and
analyzed densitometrically to determine binding levels.
Non-specific binding is typically less than 5% of the total bound
ligand.
Immunohistochemistry
[0272] After in vitro binding autoradiography, some sections are
subsequently immunostained for amyloid (A.beta.). The sections can
be pre-treated sequentially in 3% hydrogen peroxide (5 min, RT) and
70% formic acid (2 min, RT), each followed with rinses in TBS
buffer (0.05M Tris, 0.9% NaCl, pH 7.2), then pre-blocked with 8%
normal goat serum (NGS), 0.1% Triton-X and 10 .mu.g/ml avidin
(Vector Laboratories) in TBS for 30 minutes at 4.degree. C.
Sections can be incubated in a solution containing 2% NGS, 50
.mu.g/ml biotin and primary antibody over 2 nights at 4.degree. C.
Mouse monoclonal antibodies BA27 (specific for A.sup.1-40, Takeda
Pharmaceuticals) or BC05 (specific for A.sup.1-42, Takeda
Pharmaceuticals) can be used at dilutions of 1:150,000. Kung, J.
Mol. Neurosci. 19: 7 (2002). Sections can be rinsed in TBS and
incubated with 1:200 biotinylated goat anti-mouse secondary
antibody (Vector Laboratories), 2% NGS and 0.2% Triton-X in TBS for
1 h at 4.degree. C. Sections will again be rinsed in TBS and
treated with ABC Elite (Vector Laboratories) for 1 h at room
temperature. After rinses in TBS, immunoreactivity can be
visualized by incubation in 0.5 mg/ml 3,3'-diaminobenzadine
tetrahydrochloride (Sigma-Aldrich) and 0.01% hydrogen peroxide in
0.05M Tris buffer, pH 7.6 for 10 min followed by TBS rinse. Stained
sections can be dehydrated through ascending concentrations of
ethanol, cleared in Histo-Clear (National Diagnostics) and
coverslipped with Permaslip (Alban Scientific). Coverslipped
sections will then be analyzed under a Leica brightfield microscope
and photographed with a digital camera. Scanned autoradiograms can
be compared to the micrographs of A.sup.1-40 and A.sup.1-42
immunostaining using Canvas 8.0 software (ACD Systems, Inc.). The
number of deposits or plaques labeled with labeled ligands and one
or both of the A antibodies can be tabulated in randomly selected
microscopic fields.
[0273] The disclosures of each and every patent, patent application
and publication (for example, journals, articles and/or textbooks)
cited herein are hereby incorporated herein by reference in their
entirety. Also, as used herein and in the appended claims, singular
articles such as "a", "an" and "one" are intended to refer to
singular or plural. While the present invention has been described
herein in conjunction with a preferred aspect, a person with
ordinary skill in the art, after reading the foregoing
specification, can effect changes, substitutions of equivalents and
other types of alterations to the compounds and amyloid probes of
the invention or salts, pharmaceutical compositions, derivatives,
prodrugs or racemic mixtures thereof as set forth herein. Each
aspect described above can also have included or incorporated
therewith such variations or aspects as disclosed in regard to any
or all of the other aspects. The present invention is also not to
be limited in terms of the particular aspects described herein,
which are intended as single illustrations of individual aspects of
the invention. Many modifications and variations of this invention
can be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods within the scope of the invention, in addition to those
enumerated herein, will be apparent to those skilled in the art
from the foregoing descriptions. It is to be understood that this
invention is not limited to particular methods, reagents,
compounds, compositions, probes or biological systems, which can,
of course, vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular aspects
only, and is not intended to be limiting. Thus, it is intended that
the specification be considered as exemplary only with the breadth,
scope and spirit of the invention indicated only by the appended
claims, definitions therein and any equivalents thereof.
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