U.S. patent application number 13/925912 was filed with the patent office on 2013-11-14 for imaging agents for detecting neurological disorders.
The applicant listed for this patent is Eli Lilly and Company. Invention is credited to Daniel Kurt Cashion, Gang Chen, Umesh B. Gangadharmath, Dhanalakshmi Kasi, Hartmuth C. Kolb, Changhui Liu, Vani P. Mocharla, Peter J.H. Scott, Anjana Sinha, Anna Katrin Szardenings, Joseph C. Walsh, Eric Wang, Chul Yu, Wei Zhang.
Application Number | 20130302248 13/925912 |
Document ID | / |
Family ID | 42288584 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130302248 |
Kind Code |
A1 |
Gangadharmath; Umesh B. ; et
al. |
November 14, 2013 |
Imaging Agents for Detecting Neurological Disorders
Abstract
Imaging agents of formula (I) and methods for detecting
neurological disorders comprising administering to a patient in
need compounds of formula (I) capable of binding to tau proteins
and .beta.-amyloid peptides are presented herein. The invention
also relates to methods of imaging A.beta. and tau aggregates
comprising introducing a detectable quantity of pharmaceutical
formulation comprising a radiolabeled compound of formula (I) and
detecting the labeled compound associated with amyloid deposits
and/or tau proteins in a patient. These methods and compositions
enable preclinical diagnosis and monitoring progression of AD and
other neurological disorders.
Inventors: |
Gangadharmath; Umesh B.;
(Los Angeles, CA) ; Kolb; Hartmuth C.; (Playa Del
Rey, CA) ; Scott; Peter J.H.; (Ypsilanti, MI)
; Walsh; Joseph C.; (Pacific Palisades, CA) ;
Zhang; Wei; (Las Cruces, NM) ; Szardenings; Anna
Katrin; (Torrance, CA) ; Sinha; Anjana; (San
Diego, CA) ; Chen; Gang; (Rancho Palos Verdes,
CA) ; Wang; Eric; (San Diego, CA) ; Mocharla;
Vani P.; (Sunnyvale, CA) ; Yu; Chul; (Los
Angeles, CA) ; Liu; Changhui; (Los Angeles, CA)
; Cashion; Daniel Kurt; (San Diego, CA) ; Kasi;
Dhanalakshmi; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eli Lilly and Company |
Indianapolis |
IN |
US |
|
|
Family ID: |
42288584 |
Appl. No.: |
13/925912 |
Filed: |
June 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12661777 |
Mar 23, 2010 |
8491869 |
|
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13925912 |
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61162421 |
Mar 23, 2009 |
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Current U.S.
Class: |
424/1.89 ;
544/128; 544/281; 544/353; 544/363; 546/114; 546/144; 546/167;
546/173; 548/178; 548/302.1; 548/304.4; 548/305.1 |
Current CPC
Class: |
A61K 51/0463 20130101;
A61K 51/0453 20130101; C07D 215/06 20130101; C07D 401/04 20130101;
A61K 51/0455 20130101; A61K 51/0459 20130101; C07D 413/14 20130101;
A61P 25/28 20180101; C07D 235/14 20130101; C07D 277/62 20130101;
C07D 487/04 20130101; C07D 403/06 20130101; C07B 59/002 20130101;
C07D 413/10 20130101 |
Class at
Publication: |
424/1.89 ;
548/178; 546/114; 546/167; 544/353; 546/173; 546/144; 544/363;
548/304.4; 548/305.1; 544/128; 544/281; 548/302.1 |
International
Class: |
A61K 51/04 20060101
A61K051/04 |
Claims
1. A compound of Formula (I) ##STR00362## wherein A is a bond,
(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl,
(C.sub.2-C.sub.4)alkene, or (C.sub.2-C.sub.4)alkyne; Z is aryl
selected from the group consisting of: ##STR00363## wherein X.sub.1
and X.sub.13 are each independently C, CH, N, O, or S;
X.sub.2-X.sub.12 and X.sub.14-X.sub.17 are each independently C, CH
or N; Y.sub.1 is N, O, or S; R.sub.1-R.sub.2 are each independently
H, halogen, hydroxy, nitro, cyano, amino, alkyl, alkoxy,
--(O--CH.sub.2--CH.sub.2).sub.n-- (PEG), monoalkylamino,
dialkylamino, monoarylamino, diarylamino, NR.sub.10COOalkyl,
NR.sub.10 COOaryl, NR.sub.10 COalkyl, NR.sub.10 CO aryl, COOalkyl,
COOaryl, COalkyl, COaryl, aryl, or saturated heterocyclyl, wherein
the last seventeen groups are unsubstituted or substituted by one
or more radicals selected from the group consisting of halogen,
alkyl, haloalkyl, cyano, hydroxyl, amino, monoalkylamino,
dialkylamino, alkoxy, R.sub.10, a radiolabel and alkyl substituted
with a radiolabel; or R.sub.1 and R.sub.2 together form a five- or
six-membered saturated or unsaturated ring which optionally
contains an additional heteratom in the ring which is selected from
N, O, and S, the ring being unsubstituted or substituted by one or
more radicals selected from the group consisting of halogen, alkyl,
haloalkyl, cyano, hydroxyl, amino, monoalkylamino, dialkylamino,
alkoxy, R.sub.10, a radiolabel and alkyl substituted with
radiolabel; R.sub.3-R.sub.9 are each independently H, halogen,
hydroxy, nitro, cyano, amino, alkyl, alkoxy,
--(O--CH.sub.2--CH.sub.2).sub.n--, monoalkylamino, dialkylamino,
monoarylamino, diarylamino, NR.sub.10COOalkyl, NR.sub.10 COOaryl,
NR.sub.10 COalkyl, NR.sub.10 CO aryl, COOalkyl, COOaryl, COalkyl,
COaryl, aryl, or heterocyclyl, wherein the last seventeen groups
are unsubstituted or substituted by one or more radicals selected
from the group consisting of halogen, alkyl, haloalkyl, cyano,
hydroxyl, amino, monoalkylamino, dialkylamino, alkoxy, R.sub.10, a
radiolabel and alkyl substituted with a radiolabel; R.sub.10 is H,
alkyl, alkene, aryl unsubstituted or substituted with halogen,
hydroxyl, cyano, nitro, amino, --OSO.sub.2alkyl, --OSO.sub.2aryl,
--OSi(alkyl).sub.3, --OTHP or a radiolabel; n is 1, 2, or 3; m is 0
or 1; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein at least one of R.sub.1-R.sub.9
comprises a radiolabel selected from the group consisting of
.sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.123I, .sup.124I,
.sup.125I, .sup.131I, .sup.76Br .sup.77Br.
3. The compound of claim 2, wherein the radiolabel is .sup.18F.
4. The compound of claim 1, wherein A is a bond.
5. The compound of claim 1, wherein A is C.sub.2 alkyne.
6. The compound of claim 5, wherein Z is ##STR00364## wherein at
least one of X.sub.9-X.sub.13 is nitrogen and m is 1.
7. (canceled)
8. The compound of claim 4, wherein Z is ##STR00365## and at least
one of X.sub.9-X.sub.16 is nitrogen and m is 1.
9. (canceled)
10. The compound of claim 8, wherein Z is quinoline.
11-12. (canceled)
13. The compound of claim 5, wherein Z is ##STR00366## and wherein
at least one X.sub.9-X.sub.16 is nitrogen and m is 1.
14-16. (canceled)
17. A pharmaceutical diagnostic formulation for detecting
neurological disorders comprising a radiolabeled compound of claim
2 or a pharmaceutically acceptable salt thereof in a suitable
vehicle or diluent.
18. The pharmaceutical diagnostic formulation of claim 17 for
detection of amyloid peptides.
19. The pharmaceutical diagnostic formulation of claim 17 for
detection of tau proteins of neurofibrillary tangles.
20. The pharmaceutical diagnostic formulation of claim 17 wherein
the neurological disorder is Alzheimer's disease.
21-30. (canceled)
31. The compound of claim 1, wherein A is C.sub.2 alkene.
32. The compound of claim 13, wherein Z is quinoline.
33. The compound of claim 10, wherein R.sub.1 is monoalkylamino,
wherein the monoalkylamino is unsubstituted or substituted by a
radical selected from the group consisting of halogen, hydroxyl,
alkoxy, R.sub.10, and a radiolabel.
34. The compound of claim 10, wherein R.sub.1 is dialkylamino,
wherein the dialkylamino is unsubstituted or substituted by a
radical selected from the group consisting of halogen, hydroxyl,
alkoxy, R.sub.10, and a radiolabel.
35. The compound of claim 1, which is selected from the group
consisting of: 2-(1-Methyl-1H-indol-5-yl)quinoline ##STR00367##
N-(3-Fluoropropyl)-4-(quinolin-2-ylethynyl)aniline ##STR00368##
N,N-dimethyl-4-[(E)-2-quinoxalin-2-ylvinyl]aniline ##STR00369##
N-Methyl-4-(quinolin-3-yl)aniline ##STR00370##
2-Fluoro-4-(6-methoxyquinolin-2-yl)-N-methylaniline ##STR00371##
N,N-Dimethyl-4-(quinolin-7-yl)aniline ##STR00372##
N-(2-fluoroethyl)-N-methyl-4-[(E)-2-quinazolin-2-ylvinyl]aniline
##STR00373## N,N-Dimethyl-4-(quinolin-6-ylethynyl)aniline
##STR00374##
2-(4-(4-(2-Fluoroethyl)piperizin-1-yl)-6-methoxyquinoline
##STR00375## and 4-(6-Methoxyquinolin-2-yl)-N-methylaniline
##STR00376## or a pharmaceutically acceptable salt thereof.
36. A compound which is
2-(4-(4-(2-(2-fluoroethoxy)ethyl)piperazin-1-yl)phenyl)-6-methoxyquinolin-
e ##STR00377## or a pharmaceutically acceptable salt thereof.
37. A method for detecting neurological disorders in a patient,
comprising administering an effective amount of the compound of
claim 2.
38. The method of claim 37, wherein the neurological disorder is
Alzheimer's disease (AD).
39. The method of claim 37, further comprising measuring the
affinity of the compound for senile plaques.
40. The method of claim 37, further comprising measuring the
affinity of the compound for tau aggregates.
41. The method of claim 39, wherein the detection is performed
using gamma imaging, magnetic resonance imaging, magnetic resonance
spectroscopy or fluorescence spectroscopy.
42. The method of claim 41, wherein the detection by gamma imaging
is PET or SPECT.
43. The method of claim 40, wherein the detection is performed
using gamma imaging, magnetic resonance imaging, magnetic resonance
spectroscopy or fluorescence spectroscopy.
44. The method of claim 43, wherein the detection by gamma imaging
is PET or SPECT
Description
CLAIM TO PRIORITY
[0001] The present application is based on and claims a priority
benefit of U.S. provisional application No. 61/162,421, filed Mar.
23, 2009, which is incorporated herein by reference in its
entirety.
[0002] The foregoing application, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention.
FIELD OF THE INVENTION
[0003] The present invention relates generally to imaging agents
for detecting neurological disorders. More specifically, the
present invention relates to the discovery of novel diagnostic
imaging agents targeting senile plaques (SPs) and/or
neurofibrillary tangles (NFTs) for detection, preclinical diagnosis
and for tracking progression of Alzheimer's disease (AD).
BACKGROUND OF THE INVENTION
[0004] Currently, Alzheimer's disease (AD), a leading cause of
dementia, develops in one percent of the population between the
ages 65 and 69, and increasing to 40-50% in those 95 years and
older. AD patients exhibit telltale clinical symptoms that include
cognitive impairment and deficits in memory function. In the
current working model, there are three `stages` that are associated
with AD. First, neuronal cells become sick as a result of
synaptic/metabolic malfunctioning that leads to neuronal
deficiencies. Secondly, in the histological stage, an accumulation
of neurofibrillary tangles and beta amyloid plaques begins, leading
to the untimely aggregation of insoluble proteins in the brain.
Finally, AD ultimately causes neuronal death and shrinkage in brain
volume. AD patients typically have a heavy senile plaque (SP)
burden found in the cerebral cortex which is verified by postmortem
histopathological examination. SPs are extracellular deposits
containing .beta.-amyloid peptide cleaved from a longer amyloid
precursor protein to produce a 40-43 amino acid peptide. Amyloid
aggregates in brain play a key role in a cascade of events leading
to AD. Interestingly, despite the development and presence of
senile plaques in elderly persons with normal cognitive function,
the severity of NFT and senile plaque deposition purportedly
correlates with a loss of cognitive function and neuronal circuitry
deterioration.
[0005] Major neuropathology observations of postmortem examination
of AD brains confirm the presence of AD through the detection of
extracellular .beta.-amyloid peptides and intracellular
neurofibrillary tangles (NFT). NFTs derive from filaments of
hyperphosphorylated tau proteins. The presense and severity of NTFs
correlate with severity of dementia and cognitive impairment
(Dickinson, D. W., Neurobiol. Aging 1997, 18 [4 suppl]:S21-S26).
The pathological process of AD must begin before the presentation
of the clinical symptoms of dementia.
[0006] Despite Alzheimer's disease being the fourth leading cause
of death in the United States, pharmaceutical intervention has yet
to commercialize a curative therapy. Recently, Marwan N. Sabbagh
published an overview of the current state of clinical development
of AD pharmacotherapy (The American Journal of Geriatric
Pharmacotherapy, 2009, 7(3), p. 167). Encouraging results from
completed Phase II trials of dimebon, huperzine A, intravenous
immunoglobulin, and methylthioninium chloride were reported at ICAD
2008. Nineteen compounds are currently in Phase II trials, and 3
compounds (AN1792, lecozotan SR, and SGS742) failed at this stage
of development.
[0007] In addition to pharmaceutical approaches for curbing the
effects of AD, researchers are attempting to detect AD by other
means, including establishing technologies for early detection.
Currently, there are many strategies that attempt to identify
AD-associated pathologies by targeting either the cell sickness or
histological stages of the disease. There is an array of AD imaging
agents that potentially confirm the well-established manifestation
of AD, however, this late stage diagnosis offers little defense
against further disease progression past 36 months. Interestingly,
the detection of senile plaques and tangles in the brain may not
necessarily prove that a patient has developed AD.
[0008] As summarized from a recent discussion group on Dec. 5, 2006
(Biochemical Pharmacology Discussion Group, cosponsored by the
American Chemical Society's New York section), researchers are now
focusing on methods that target AD precursors by blocking either
.beta.-amyloid protein (BAP) production or by controlling mutant
tau protein formation. Clearly, this focused research effort aims
to control the formation of AD precursors that potentially lead to
AD and this new strategy might delay full-onset AD more effectively
that current therapeutics. In parallel, neurological imaging must
mirror the therapeutic trend by identifying AD precursors in a duel
effort to compliment both AD therapeutic development and, in
addition, identify presymptomatic at-risk AD patients. Recent drug
development has been aimed at preventing the accumulation of SPs
and NFTs in presymptomatic AD patients. The ability to measure
levels of these lesions in the living human brain is thus desirable
for presymptomatic diagnosis of AD and also for monitoring the
progression of the disease.
[0009] Unfortunately, since AD cannot be confirmed in the patients
until a histological examination is performed, understanding the
link between the uptake of these tracers and the relevant
biochemical processes involved could remain unsolved for many
years.
[0010] Thus, in vivo imaging of NFTs in conjunction with imaging
SPs could prove useful for the early and accurate diagnosis of AD.
Quantitative evaluation of tau pathology could also be helpful for
tracking severity of dementia, because the formation of neuritic
pathology correlates well with clinical severity of dementia
(Dickson, 1997). NFT deposition in the entorhinal cortex is closely
associated with neuronal loss in very early AD patients (Gomez-Isla
et al., 1996). If novel treatments that prevent the pathological
formation of neurofibrillary pathology could be turned into
clinical applications, this imaging technique would be applicable
for the evaluation of treatment efficacy.
[0011] Currently, neurological imaging of AD has seen the emergence
of imaging tracers that appear to confirm the presence of AD based
on plaque and fibril mediated tracer uptake and, subsequently, are
currently undergoing extensive clinical examination. Many of these
tracers contain chemotypes that are derived from fluorescent
dyes.
[0012] Potential ligands for detecting A.beta. aggregates in the
living brain must cross the intact blood-brain barrier. Thus brain
uptake can be improved by using ligands with relatively smaller
molecular size and increased lipophilicity.
[0013] Previous neuropathological research suggests that the
deposition of NFTs occurs before the presentation of clinical
symptoms of AD. Even in the very early stages of AD, patients
display considerable numbers of NFTs in the entorhinal cortex and
hippocampus, sufficient for the neuropathological diagnosis of AD.
Thus, in vivo imaging of NFTs in conjunction with imaging SPs could
prove useful for the early and accurate diagnosis of AD, for
monitoring the progression of the disease and for evaluation of
treatment efficacy.
[0014] Optimization of current candidates and discovery of novel
compounds that specifically bind tau or A.beta. aggregates are of
high interest for development of in vivo tau- and A.beta. imaging
agents for detection of neurological disorders, and in particular
for imaging and detection of AD in patients.
SUMMARY OF THE INVENTION
[0015] This invention discloses a series of compounds of formula
(I) having enhanced binding properties to SPs and NFTs. The present
invention also provides diagnostic pharmaceutical compositions
comprising a radiolabeled compound of formula (I) and a
pharmaceutically acceptable carrier or diluent. The present
invention further relates to a method of imaging and detecting
amyloid deposits and/or tau aggregates, the method comprising
administering a detectable amount of a labeled compound of formula
(I) or its pharmaceutally acceptable salt to a subject in need
thereof and detecting the labeled compound associated with amyloid
deposits or tau aggregates.
[0016] One embodiment of the present invention is directed to a
biaryl or bis-aromatic compound of formula (I) or its
pharmaceutally acceptable salt.
[0017] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein at least
one of the aryl components is substituted with a side chain having
a radiolabel.
[0018] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein the
radiolabel is .sup.18F.
[0019] Another embodiment of the present invention is directed to
the compounds of formula (I) as described above, wherein at least
one of the aryl components is unsubstituted or substituted phenyl,
pyridine, pyrimidine or pyrazine.
[0020] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein at least
one of the aryl components is unsubstituted or substituted fused
heteroaryl.
[0021] Yet another embodiment of the present invention is directed
to the compound of formula (I) as described above, wherein at least
one fused heteroaryl is a bicycle.
[0022] Yet another embodiment of the present invention is directed
to the compound of formula (I) as described above, wherein at least
one fused heteroaryl is a tricycle.
[0023] Yet another embodiment of the present invention is directed
to diagnostic pharmaceutical compositions comprising a radiolabeled
compound of formula (I) or its pharmaceutally acceptable salt as
described above and, a pharmaceutically acceptable carrier or
diluent.
[0024] Yet another embodiment of the present invention is directed
to a method of imaging and detection of neurological disorders
associated with amyloid plaque and/or tau protein aggregation
comprising administering a detectable amount of a labeled compound
of formula (I) as described above or its pharmaceutally acceptable
salt to a subject in need thereof and detecting the labeled
compound associated with amyloid deposits and/or tau
aggregates.
[0025] Yet another embodiment of the present invention is directed
to a method of imaging and detection of neurological disorders
associated with amyloid plaque aggregation comprising administering
a detectable amount of a labeled compound of formula (I) as
described above or its pharmaceutally acceptable salt to a subject
in need thereof and detecting the labeled compound associated with
amyloid deposits.
[0026] Yet another embodiment of the present invention is directed
to a method of imaging and detection of neurological disorders
associated with tau protein aggregation comprising administering a
detectable amount of a labeled compound of formula (I) as described
above or its pharmaceutally acceptable salt to a subject in need
thereof and detecting the labeled compound associated tau
aggregates.
[0027] Yet another embodiment of the present invention is directed
to a method of imaging and detection of Alzheimer's disease
associated with amyloid plaque and/or tau protein aggregation
comprising administering a detectable amount of a labeled compound
of formula (I) as described above or its pharmaceutally acceptable
salt to a subject in need thereof and detecting the labeled
compound associated with amyloid deposits and/or tau
aggregates.
[0028] Yet another embodiment of the present invention is directed
to a method of imaging and detection of Alzheimer's disease
associated with amyloid plaque aggregation comprising administering
a detectable amount of a labeled compound of formula (I) as
described above or its pharmaceutally acceptable salt to a subject
in need thereof and detecting the labeled compound associated with
amyloid deposits.
[0029] Yet another embodiment of the present invention is directed
to a method of imaging and detection of Alzheimer's disease
associated with tau protein aggregation comprising administering a
detectable amount of a labeled compound of formula (I) as described
above or its pharmaceutally acceptable salt to a subject in need
thereof and detecting the labeled compound associated with tau
aggregates.
[0030] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0031] These and other embodiments will become apparent from and
encompassed by the following Detailed Description when taken in
conjunction with the accompanying drawings. The entire disclosures
of all patents and references cited throughout this application are
incorporated herein by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The application file contains at least one image executed in
color. Copies of this patent application publication with color
images will be provided by the Office upon request and payment of
the necessary fee.
[0033] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0034] FIG. 1a shows an example of a brain slice assay used to
determine the IC.sub.50 values of W366 and related compounds.
[0035] FIG. 1b shows an example of a brain slice assay used to
determine the IC.sub.50 values of W366 and related compounds.
[0036] FIG. 2 shows total tau binding of fluorescent compound T482
to AD brain sections confirmed by immunostaining with tau or
A.beta. antibodies (double labeling of T482 at 100 uM).
[0037] FIG. 3 shows ex vivo autoradiograph images of a preferred
compound, T482.
[0038] FIGS. 4a and 4b binding of fluorescent compound T540 to AD
brain sections, which contain A.beta. plaques and tau aggregates as
confirmed by immunostaining with A.beta. or tau antibodies (double
labeling of T540 at 100 uM).
[0039] FIG. 5 shows ex vivo autoradiograph images of a preferred
compound, T540, in three different types of brain sections:
A.beta.+/tau- brains contain A.beta. plaques, but no tau aggregates
(diagnosis by brain bank as non-AD donor); A.beta.+/tau+ brains
contain both A.beta. plaques and tau aggregates (diagnosed by brain
bank as AD patient), and normal (control) brains. The presence or
absence of A.beta. and/or tau was confirmed by immunostaining.
[0040] FIGS. 6a, 6b, and 6c show binding of fluorescent compound
T542 to AD brain sections confirmed by immunostaining with tau or
A.beta. antibodies.
[0041] FIG. 7 binding of fluorescent compound T544 to AD brain
sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0042] FIGS. 8a and 8b show binding of fluorescent compound T520 to
AD brain sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0043] FIGS. 9a and 9b show total binding of fluorescent compound
T522 to AD brain sections confirmed by immunostaining with tau or
A.beta. antibodies.
[0044] FIGS. 10a and 10b show binding of fluorescent compound T541
to AD brain sections confirmed by immunostaining with tau or
A.beta. antibodies.
[0045] FIG. 11 shows binding of fluorescent compound T527 to AD
brain sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0046] FIG. 12 shows binding of fluorescent compound T539 to AD
brain sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0047] FIGS. 13a and 13b show binding of fluorescent compound T499
to AD brain sections confirmed by immunostaining with tau or
A.beta. antibodies.
[0048] FIG. 14 shows binding of fluorescent compound T525 to AD
brain sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0049] FIG. 15 shows ex vivo autoradiograph images of a preferred
compound, T525.
[0050] FIG. 16 shows binding of fluorescent compound T543 to AD
brain sections confirmed by immunostaining with tau or A.beta.
antibodies.
[0051] FIG. 17 shows brain images (brain uptake) for tracer
T114.
[0052] FIG. 18 shows brain images (brain uptake) for tracer
T442.
[0053] FIG. 19 shows brain images (brain uptake) for tracer
T549.
[0054] FIG. 20 shows brain images (brain uptake) for tracer
T525.
[0055] FIG. 21 shows brain images (brain uptake) for tracer
T482.
[0056] FIG. 22 shows brain images (brain uptake) for tracer
T510.
DETAILED DESCRIPTION
[0057] The following description will describe the invention in
relation to advantageous embodiments thereof. The invention is in
no way limited to these advantageous embodiments as they are purely
included to exemplify the invention and the invention is intended
to include possible variations and modifications as would be
readily apparent to a person skilled in the art without departing
from the scope of the invention.
[0058] One of the embodiments of the present invention relates to a
compound of general formula (I)
##STR00001##
wherein A is a bond, (C.sub.1-C.sub.4)alkyl,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.2-C.sub.4)alkene, or
(C.sub.1-C.sub.4)alkyne; Z is aryl selected from the group
consisting of:
##STR00002##
wherein
[0059] X.sub.1 and X.sub.13 are each independently C, CH, N, O, or
S;
[0060] X.sub.2-X.sub.12 and X.sub.14-X.sub.18 are each
independently C, CH or N;
[0061] Y.sub.1 is N, O, or S;
[0062] R.sub.1-R.sub.2 are each independently H, halogen, hydroxy,
nitro, cyano, amino, alkyl, alkoxy,
--(O--CH.sub.2--CH.sub.2).sub.n--, monoalkylamino, dialkylamino,
monoarylamino, diarylamino, NR.sub.10COOalkyl, NR.sub.10 COOaryl,
NR.sub.10 COalkyl, NR.sub.10 CO aryl, COOalkyl, COOaryl, COalkyl,
COaryl, aryl, saturated heterocyclyl, wherein the last seventeen
groups are unsubstituted or substituted by one or more radicals
selected from the group consisting of halogen, alkyl, haloalkyl,
cyano, hydroxyl, amino, monoalkylamino, dialkylamino, alkoxy,
R.sub.10, a radiolabel or alkyl substituted with a radiolabel; or
R.sub.1 and R.sub.2 together form a five- or six-membered saturated
or unsaturated ring which optionally contains an additional
heteratom in the ring which is selected from N, O, and S, the ring
being unsubstituted or substituted by one or more radicals selected
from the group consisting of halogen, alkyl, haloalkyl, cyano,
hydroxyl, amino, monoalkylamino, dialkylamino, alkoxy, R.sub.10, a
radiolabel or alkyl substituted with radiolabel;
[0063] R.sub.3-R.sub.9 are each independently H, halogen, hydroxy,
nitro, cyano, amino, alkyl, alkoxy,
--(O--CH.sub.2--CH.sub.2).sub.n--, monoalkylamino, dialkylamino,
monoarylamino, diarylamino, NR.sub.10COOalkyl, NR.sub.10 COOaryl,
NR.sub.10 COalkyl, NR.sub.10 CO aryl, COOalkyl, COOaryl, COalkyl,
COaryl, aryl, heterocyclyl, wherein the last seventeen groups are
unsubstituted or substituted by one or more radicals selected from
the group consisting of halogen, alkyl, haloalkyl, cyano, hydroxyl,
amino, monoalkylamino, dialkylamino, alkoxy, R.sub.10, a radiolabel
or alkyl substituted with a radiolabel;
[0064] R.sub.10 is H, alkyl, alkene, aryl unsubstituted or
substituted with halogen, hydroxyl, cyano, nitro, amino,
--OSO.sub.2alkyl, --OSO.sub.2aryl, --OSi(alkyl).sub.3, --OTHP or a
radiolabel;
[0065] n is 1, 2, or 3;
[0066] m is 0 or 1, and
a pharmaceutically acceptable salt thereof.
[0067] Another embodiment of the present invention is directed to
the compound of formula (I) as described above wherein Z is
##STR00003##
and at least one of X.sub.9-X.sub.13 is nitrogen and m is 1.
[0068] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein at least
one of R.sub.1-R.sub.6 is --(O--CH.sub.2--CH.sub.2).sub.2--.
[0069] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein Z is
##STR00004##
and at least one of X.sub.9-X.sub.16 is nitrogen and m is 1.
[0070] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein Z is
##STR00005##
and at least one of X.sub.9-X.sub.18 is nitrogen and m is 1.
[0071] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein Z is
##STR00006##
and wherein at least one X.sub.9-X.sub.12 is nitrogen and m is
1.
[0072] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein Z is
##STR00007##
A is acetylene and at least one X.sub.9-X.sub.16 is nitrogen.
[0073] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein A is a bond
and Z is quinoline.
[0074] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein at least
one of R.sub.1-R.sub.9 is a saturated heterocycle.
[0075] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein m is 1 and
R.sub.1 is saturated heterocycle.
[0076] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein Z is
##STR00008##
wherein at least two of X.sub.9-X.sub.16 are nitrogens.
[0077] Yet another embodiment of the present invention is directed
to the compound of formula (I) as described above, wherein A is a
bond and Z is
##STR00009##
wherein at least three of X.sub.1-X.sub.16 are nitrogens.
[0078] Yet another embodiment of the present invention is directed
to the compound of formula (I) as described above, wherein A is a
bond and Z is
##STR00010##
wherein at least two of X.sub.9-X.sub.18 are nitrogens.
[0079] Another embodiment of the present invention is directed to
the compound of formula (I) as described above, wherein A is
C.sub.2 alkyne or acetylene and Z is
##STR00011##
wherein at least two of X.sub.9-X.sub.13 or Y.sub.1 are
nitrogens.
[0080] Another embodiment of the present invention relates to the
compound of formula (I) as described above, wherein at least one of
R.sub.1-R.sub.2 comprises a heterocycle.
[0081] Another embodiment of the present invention relates to the
compound of formula (I) as described above, wherein at least one of
R.sub.1-R.sub.2 comprises a saturated heterocycle.
[0082] Another embodiment of the present invention relates to the
compound of formula (I) as described above, wherein at least one of
R.sub.1-R.sub.9 comprises --(O--CH.sub.2--CH.sub.2).sub.2-- or
--(O--CH.sub.2--CH.sub.2).sub.3--.
[0083] Another embodiment of the present invention relates to the
compound of formula (I) as described above, wherein at least one of
R.sub.1-R.sub.9 comprises a radiolabel selected from the group
consisting of .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.76Br and .sup.77Br.
[0084] In another embodiment, the present invention provides the
compound of formula (I) as described above, wherein the compound
can be presented as formula (II). Formula (II) represents W366 and
related compounds containing labeling elements on the left hand
portion of the molecule:
##STR00012##
wherein
[0085] X is N or C;
[0086] Y is S or O;
[0087] Z is bond, S, O, alkyl, --(OCH.sub.2CH.sub.2).sub.n--, aryl
or heteroaryl;
[0088] L* is radioactive label;
[0089] Ar is aryl, heteroaryl, optionally substituted with O, S,
halogen, alkyl, or --(OCH.sub.2CH.sub.2).sub.n--;
[0090] G is H, S, O, halogen, alkyl, --(OCH.sub.2CH.sub.2).sub.n--
or aryl; and
[0091] n is 1, 2, or 3.
[0092] In another embodiment, the present invention provides the
compound of formula (I) as described above, wherein the compound
can be presented as formula (III), which represents W366 and
related compounds containing labeling elements on the right hand
portion of the molecule:
##STR00013##
wherein
[0093] X is N or C;
[0094] Y is S or O;
[0095] Z is S, O, alkyl, --(OCH.sub.2CH.sub.2).sub.n--, aryl or
heteroaryl optionally substituted with O, S, halogen, alkyl, aryl
or --(OCH.sub.2CH.sub.2).sub.n--;
[0096] L* is radioactive label;
[0097] J is bond, S, O, alkyl, --(OCH.sub.2CH.sub.2).sub.n--, aryl
or heteroaryl;
[0098] Ar is aryl, heteroaryl, optionally substituted with O, S,
halogen, alkyl, or --(OCH.sub.2CH.sub.2).sub.n--;
[0099] G is H, S, O, halogen, alkyl, --(OCH.sub.2CH.sub.2).sub.n--
or aryl; and
[0100] n is 1, 2, or 3.
[0101] In another embodiment, the present invention provides W366
and related compounds containing labeling elements and substituted
pyridyl moieties:
##STR00014##
[0102] In another embodiment, the present invention provides the
following compounds of formulas (II) and (III) containing labeling
element (either .sup.11C--NHMe or .sup.18F):
##STR00015## ##STR00016## ##STR00017## ##STR00018##
##STR00019##
[0103] The present invention also includes stereoisomers of
compounds of formula (I). Such stereoisomers include, but are not
limited to mixtures of enantiomers and diastereomers as well as
individual enantiomers and diastereomers.
[0104] When any variable occurs more than one time in any
constituent of formula (I), its definition in each instance is
independent of its definition at any other instance. Also,
combinations of substituents and/or variables are permissible only
if such combinations result in stable compounds.
[0105] The compounds of formula (I) may also be solvated,
especially hydrated. Hydration may occur during preparation of the
compounds or compositions comprising the compounds of formula (I),
or hydration may occur over time due to hydroscopic nature of the
compounds.
[0106] The compounds of formula (I) can be prepared by the
application or adaptation of known methods (i.e. methods heretofore
used (methods A-S, as presented in detail in EXPERIMENTAL EXAMPLES
section) or described in the chemical literature.
[0107] Another embodiment of the present invention relates to a
method of imaging amyloid deposits and/or tau aggregates. When the
compounds of formula (I) are used as imaging agents, they are
labeled with suitable radioactive isotopes, for example radioactive
halogens, such as .sup.18F, radioactive metals and other detectable
radioactive atoms such as .sup.11C.
[0108] In another embodiment, the present invention relates to
radiolabeled compounds of formulas (II) and (III) as imaging
agents. These imaging agents are unique as they contain new binding
moieties of tightly tethered through the alkyne linker. These
binding motifs may interact simultaneously with orthogonal binding
sites providing a more complete overview of the biochemical
phenomena associated with AD patients.
[0109] Another embodiment of the present invention is directed to
compounds containing both benzothiazole and acetylene binding
motifs, such as W366 and related compounds, which are designed to
interact with the orthogonal binding sites of senile plaques, and
potentially NFTs. In this regard, these imaging agents offer the
potential to provide a more complete dataset of biochemical
information. FIGS. 1a and 1b show examples of brain slice assay
used to determine the IC.sub.50 values of W366 and related
compounds.
[0110] Another embodiment of the present invention is directed to
compounds of formula (I) as imaging agents for tau aggregates.
[0111] It has been shown that solutions of the compounds of the
present invention when injected intravenously into normal mice,
exhibit excellent brain uptake. These compounds also display high
binding affinity to tau fibrils. Autoradiography using the present
compounds demonstrated labeling of NFTs in AD brain sections.
Fluorescence assay data shows the binding ability of these agents
to tau aggregates and A.beta. fibrils. In neuropathological
staining, compounds of the present invention stained amyloid
plaques and/or tau aggregates.
[0112] The results presented herein are based on the brain section
staining studies and autoradiography of tracers in brain sections
of three different types (Tau+/A.beta.+, Tau-/A.beta.+, and
Tau-/A.beta.-). These results are shown in FIGS. 2-16 and Tables
1-3.
[0113] Another embodiment of the present invention is directed to
quinoline compounds of formula (I), having extended side chains
containing radiolabel as illustrated in Scheme 2. As shown in
Scheme 2 and in Table 1, compounds of this class bind to tau
proteins. These compounds incorporate extended side chains,
especially containing piperidine or morpholine and/or polyethers
such as polyethylene glycols (PEG or --(OCH.sub.2CH.sub.2).sub.n--,
wherein n can be 1-10, preferably 1-3). These structural features
may play a role in binding affinity of these compounds to tau
aggregates. Fluorescence assay data show the binding ability of
these agents to tau aggregates.
[0114] FIGS. 12, 13a-13b, and 14 show binding of preferred
fluorescent compounds T539, T499, and T525 to AD brain sections
confirmed by immunostaining with tau or A.beta. antibodies. FIG. 15
show ex vivo autoradiograph images of T525, in three different
types of brain sections: A.beta.+/tau- brains contain A.beta.
plaques, but no tau aggregates (diagnosis by brain bank as non-AD
donor); A.beta.+/tau+ brains contain both .A.beta. plaques and tau
aggregates (diagnosed by brain bank as AD patient), and normal
(control) brains. The presence or absence of A.beta. and/or tau is
confirmed by immunostaining.
##STR00020## ##STR00021##
TABLE-US-00001 TABLE 1 Representative examples of quinoline
compounds of the present invention. Comp. Brain Section ID Stucture
FW MW Staining cLogP T046 ##STR00022## 270.3 T047 ##STR00023##
220.3 220.1 T048 ##STR00024## 291.4 291.2 binds to Ab (amyloid)
(100 uM) T049 ##STR00025## 256.7 256 T050 ##STR00026## 221.3 221.1
T051 ##STR00027## 251.3 251.1 T123 ##STR00028## 206.2 206.1 No
fluorescence is detected at 100 uM T124 ##STR00029## 231.3 231.1
T125 ##STR00030## 249.3 249.1 staining shows strong binding to tau
T126 ##STR00031## 224.2 224.1 T127 ##STR00032## 225.2 225.1 T128
##STR00033## 207.2 207.1 T138 ##STR00034## 254.3 254.1 T139
##STR00035## 278.4 278.1 binds to amyloid and Tau T407 ##STR00036##
458.4 T409 ##STR00037## 369.3 binds to amyloid T411 ##STR00038##
339.2 binds to tau, weakly binds to Ab. (100 uM) T412 ##STR00039##
377.4 binds to tau but not to amyloid T420 ##STR00040## 248.3 T427
##STR00041## 307.2 T428 ##STR00042## 290.3 T429 ##STR00043## 292.4
binds to Ab T433 ##STR00044## 472.3 T434 ##STR00045## 274.4 T442
##STR00046## binds to Tau and Ab 3.8 T443 ##STR00047## 429.4 T445
##STR00048## 238.3 binds to Tau T446 ##STR00049## 360.3 T447
##STR00050## 311.4 T453 ##STR00051## 258.3 binds to Ab T454
##STR00052## 304.4 binds to Ab T455 ##STR00053## 224.2 No
fluorescence T458 ##STR00054## 252.3 binds to amyloid and tau 4.1
T461 ##STR00055## 304.4 T463 ##STR00056## 279.3 T466 ##STR00057##
366.3 binds to tau and amyloid 4 T467 ##STR00058## 268.3 binds to
tau and amyloid 3.6 T475 ##STR00059## 275.4 binds to Ab mostly T476
##STR00060## 248.3 T477 ##STR00061## 348.3 binds to Ab montly 3.6
T480 ##STR00062## 267.3 T483 ##STR00063## 355.2 Mostly binds to Tau
T484 ##STR00064## 266.3 binds to Tau and Ab; 4.6 T485 ##STR00065##
391.3 weakly binds to Ab T490 ##STR00066## 221.3 2.5 T491
##STR00067## 279.3 T492 ##STR00068## 238.3 3.3 T498 ##STR00069##
423.5 5.2 T499 ##STR00070## 379.5 Binds to Tau mostly (+++). Weakly
binds to Ab (+) 4.9 T500 ##STR00071## 248.3 4.2 T501 ##STR00072##
270.8 No fluorescence 3.6 T502 ##STR00073## 369.4 3.5 T504
##STR00074## 248.3 binds to Ab strongly (++++) 4.1 T505
##STR00075## 248.3 binds to Ab weakly (+) 4.1 T507 ##STR00076##
307.4 Binds to Ab (+++) 4.1 T510 ##STR00077## 398.5 Binds both Tau
(+++) and Ab (++) 4.5 T513 ##STR00078## 267.3 Binds to Tau (+) and
Ab (++) 3.5 T514 ##STR00079## 234.3 3.4 T515 ##STR00080## 234.3
weakly to Ab (+) 3.4 T517 ##STR00081## 272.3 272.3 Binds to Ab
(+++) 4.8 T519 ##STR00082## 365.4 365.4 binds to Tau strongly
(+++); weakly to Ab (+). 4.7 T523 ##STR00083## 264.3 264.3 Binds to
Tau (++++). 4.1 T525 ##STR00084## 355.4 355.4 Binds to Tau (++);
Binds to Ab (+) 3.2 T530 ##STR00085## 453.6 Binds to Tau (++++);
Binds to Ab (+) 4.6 T531 ##STR00086## 264.4 264.4 Binds to Tau.
Binds to Ab (+) 4.4 T535 ##STR00087## 631.4 289.4 3.7 T536
##STR00088## 410.3 410.3 4 T537 ##STR00089## 349.4 349.4 3.4 T539
##STR00090## 441.5 441.5 2.9 T545 ##STR00091## 425.5 425.5 Binds to
Tau (+) and Ab (+) 3.7 T549 ##STR00092## 440.5 440.5 Binds to Tau
3.8 T550 ##STR00093## 453.6 453.6 Binds to Tau 4.4 T559
##STR00094## 321.4 321.4 No fluorescence 4.5 T565 ##STR00095##
440.5 440.5 2.9 T566 ##STR00096## 413.4 413.4 4.3 T569 ##STR00097##
310.4 310.4 5
[0115] Another embodiment of the present invention is directed to
acetylene compounds of formula (I) having bicyclic heteroaryl
moiety and extended side chains containing radiolabel as
illustrated in Scheme 3. As shown in Scheme 3 and in Table 2,
compounds of this class have a high binding affinity to tau
proteins. These compounds incorporate extended side chains,
especially containing polyethers such as PEG in a biaryl alkyne
core structure, wherein one of the aryl components is a substituted
benzimidazole. Such structural modification leads to an increased
selectivity of these compounds.
##STR00098##
TABLE-US-00002 TABLE 2 Representative examples of acetylene
compounds of the present invention Comp. Exact Brain Section ID
Stucture MW Mass Staining cLogP T110/ W294 ##STR00099## 265.33
T114/ W313 ##STR00100## 254.28 stains amyloid and tau T118/ W366
##STR00101## 298.33 binds to Ab T444 ##STR00102## 244.29 T448
##STR00103## 312.36 binds to Ab T454 ##STR00104## 304.36 binds to
Ab T464 ##STR00105## 233.27 T465 ##STR00106## 293.34 binds to tau
and Ab 4.2 T481 ##STR00107## 261.32 T482 ##STR00108## 307.36 Mostly
binds to Tau 4.6 T496 ##STR00109## 429.37 Weakly binds to Ab 4.3
T508 ##STR00110## 395.47 4.2 T516 ##STR00111## 240.28 Weakly binds
to Tau (+) and Ab (++) 3.6 T517 ##STR00112## 272.34 272.34 Binds to
Ab (+++) 4.8 T526 ##STR00113## 295.38 295.38 Binds to Tau (++);
Binds to Ab (+) 3.4 T527 ##STR00114## 506.45 Binds to Tau (+++);
Binds to Ab (+) 3.1 T528 ##STR00115## 375.34 261.32 Binds to Tau
(++++); Binds to Ab (+) 3.9 T534 ##STR00116## 407.36 293.34 4 T540
##STR00117## 307.36 307.36 Binds to Tau (++++). Binds to Ab (+) 4.2
T541 ##STR00118## 495.47 381.44 Binds to Tau (+++). Binds to Ab (+)
3.6 T546 ##STR00119## 385.48 385.48 4.1 T547 ##STR00120## 272.34
272.34 4.6 T551 ##STR00121## 379.31 265.28 3 T552 ##STR00122##
404.32 266.27 2 T553 ##STR00123## 380.3 266.27 2.8 T554
##STR00124## 412.3 298.27 Blue. Tau +++. Ab + 3.2 T564 ##STR00125##
506.45 392.43 2.9 T568 ##STR00126## 394.32 280.3 2.6
[0116] FIGS. 2 and 4a-4b show binding of fluorescent compounds T482
and T540 to AD brain sections confirmed by immunostaining with tau
or A.beta. antibodies. FIGS. 3 and 5 show ex vivo autoradiograph
images of the preferred compounds T482 and T540, in three different
types of brain sections: A.beta.+/tau- brains contain A.beta.
plaques, but no tau aggregates (diagnosis by brain bank as non-AD
donor); A.beta.+/tau+ brains contain both .A.beta. plaques and tau
aggregates (diagnosed by brain bank as AD patient), and normal
(control) brains. The presence or absence of A.beta. and/or tau is
confirmed by immunostaining.
[0117] Another embodiment of the present invention is directed to
compounds of formula (I) comprising a tricyclic aryl moiety. For
example, benzimidazole pyrimidines shown in Scheme 4, exhibit high
binding affinity to tau proteins. FIGS. 6a-6c, 7, 8a-8b, 9a-9b,
10a-10b, and 11 show binding of fluorescent compounds T542, T544,
T520, T522, T541, and T527 to AD brain sections confirmed by
immunostaining with tau or A.beta. antibodies.
##STR00127##
TABLE-US-00003 TABLE 3 Representative examples of benzimidazole and
tricyclic benzimidazole compounds of the present invention. Brain
Compound Section ID Structure MW Staining T506 ##STR00128## 401.4
T511 ##STR00129## 290.28 T512 ##STR00130## 290.28 T518 ##STR00131##
260.29 A.beta./1+ Tau/4+ T520 ##STR00132## 260.29 A.beta./1+ Tau/4+
T521 ##STR00133## 516.39 A.beta./1+ Tau/4+ T522 ##STR00134## 502.37
A.beta./1+ Tau/4+ T542 ##STR00135## 548.41 A.beta./3+ Tau/4+ T544
##STR00136## 548.41 A.beta./1+ Tau/3+ T557 ##STR00137## 622.49
A.beta./2+ Tau/4+ T452 ##STR00138## 213.21 T460 ##STR00139## 386.3
T469 ##STR00140## 350.41 T470 ##STR00141## 306.32 T473 ##STR00142##
420.36 T474 ##STR00143## 466.4 T487 ##STR00144## 398.35 T488
##STR00145## 234.3 T489 ##STR00146## 280.3 T493 ##STR00147## 364.3
T494 ##STR00148## 296.4 T495 ##STR00149## 366.3 T497 ##STR00150##
397.37 T524 ##STR00151## 273.28 Tau +++; Ab ++ T538 ##STR00152##
335.4 T543 ##STR00153## 473.5 Tau ++; Ab no binding T548
##STR00154## 359.2 T556 ##STR00155## 341.4
[0118] In another embodiment, the present invention relates to
compounds and compositions which comprise the formulae as disclosed
herein, wherein the compound is an amyloid and/or tau protein
binding compound. An amyloid and/or tau protein binding compound of
the invention may be administered to a patient in amounts suitable
for in vivo imaging of amyloid deposits and/or NTFs, and
distinguish between neurological tissue with amyloid deposits
and/or NTfs and normal neurological tissue.
[0119] A.beta. compounds are typically evaluated in a competitive
binding assay using synthetic A.beta.1-42 fibrils (IC.sub.50s). The
situation is more complicated for tau, because there are 6 isoforms
of tau potentially present in AD brains as products of alternate
splicing of a single tau gene. Most reports in the literature rely
therefore on only one recombinant isoform, Tau-441. To add more
complexity, the various tau isoforms are hyperphosphorylated in
vivo, something that is difficult to mimic in vitro. Furthermore,
structural information on these tau fibrils is lacking, making an
interpretation of binding of compounds difficult.
[0120] Native forms of tau (various isoforms, hyperphosphorylated)
and amyloid aggregates are present in brain sections and therefore
preferred for compound testing. Using the self-fluorescence of a
test compound can give an indication of whether the compound binds
to tau tangles/PHFs and/or amyloid plaques. This is further
confirmed by immunostaining with A.beta. and tau antibodies and
overlaying the images. The drawback is that the fluorescent signals
cannot be used for quantitation as some compounds might exhibit a
strong fluorescent signal than others and the coexistence of
A.beta. plaques and tau tangles in AD brains. However, it is
possible to "rate" the signal strength qualitatively and
distinguish compounds that show binding to these aggregates.
[0121] Furthermore, the selectivity can be evaluated in brains
containing only A.beta. plaques/no tau aggregates, A.beta.
plaques/and tau aggregates, and control brains. Unfortunately,
there are no AD brains with only tau and no A.beta. present. By
testing radiolabeled tracers in these brain sections, one can more
quantitative evaluate the relative binding strength (signal
strength) and selectivity of various test compounds as they all
contain the same radioactive tracer. For examples, if a test tracer
binds only to tau, and not amyloid, it should show no signal in the
A.beta. plaques only brain sections. If a compound binds only to
amyloid, it should show uptake in both types of brains. The
difficulty of identifying and further quantifying selective
compounds lies in the relative abundance of amyloid vs. tau, which
is difficult to measure.
[0122] In one of the embodiments of the present invention, the
self-fluorescence of the compound of formula (I) is used to
determine whether the compound binds to tau/amyloid in the brain
sections and to give it a qualitative rating. The next step is to
proceed to the autoradiography using different brain types for
further evaluation and quantitation.
[0123] Amyloid and/or tau protein probes of the invention may be
used to detect and quantitate amyloid deposits and/or NTFs in
diseases including, but not limited to Mediterranean fever,
MuckleWells syndrome, idiopathetic myeloma, amyloid polyneuropathy,
amyloid cardiomyopathy, systemic senile myloidosis, amyloid
polyneuropathy, hereditary cerebral hemorrhage with amyloidosis,
Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru,
Gerstamnn-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, chronic
traumatic encephalopathy (CTE), and Islets of Langerhans diabetes
Type II insulinoma.
[0124] The compounds and probes of the invention preferably exhibit
low toxicity at dosages effective to image (including diagnostic,
detection, quantification and evaluation) amyloid and/or related
afflictions.
[0125] In one of the embodiment, the present invention is directed
to a pharmaceutical diagnostic formulation comprising a
radiolabeled compound of formula (I) or a pharmaceutically
acceptable salt thereof in a suitable vehicle or diluent for
imaging and detection of neurological disorders.
[0126] In another embodiment, the present invention is directed to
a pharmaceutical diagnostic formulation for detection of amyloid
peptides.
[0127] In another embodiment, the present invention is directed to
a pharmaceutical diagnostic formulation for detection of tau
proteins of neurofibrillary tangles.
[0128] In another embodiment, the present invention is directed to
a pharmaceutical diagnostic formulation for detection of the
neurological disorder.
[0129] In another embodiment, the present invention is directed to
a pharmaceutical diagnostic formulation for detection of
Alzheimer's disease.
[0130] In another embodiment of the present invention, the
radioactive diagnostic agent composition may contain any additive
such as pH controlling agents (e.g., acids, bases, buffers),
stabilizers (e.g., ascorbic acid) or isotonizing agents (e.g.,
sodium chloride).
[0131] In yet another embodiment, the invention further relates to
a method for imaging and detection of senile plaques and/or
neurofibrillary tangles in a brain tissue, the method comprising
treating the tissue with a compound of formula (I).
[0132] In yet another embodiment, the invention further relates to
a method for ex vivo or in vitro detection of amyloid deposit in a
brain tissue, the method comprising treating the tissue with a
compound of formula (I) for detection of the amyloid deposit.
[0133] In yet another embodiment, the invention further relates to
a method for in vivo detection of amyloid deposits in a patient,
the method comprising administering an effective amount of the
compound of formula (I) to the patient, and detecting the binding
level of the compound to the amyloid deposit to the patient.
[0134] In yet another embodiment, the invention further relates to
a method for ex vivo or in vitro detection of tau proteins in a
brain tissue, the method comprising treating the tissue with a
compound of formula (I) for detecting of the neurofibrillary
tangles.
[0135] In yet another embodiment, the invention further relates to
a method for in vivo detection of neurofibrillary tangles in a
patient, the method comprising administering an effective amount of
the compound of formula (I) to the patient, and detecting the
binding level of the compound to tau proteins.
[0136] In yet another embodiment, the invention further relates to
a method of detecting a SPs and NTFs characteristic for a
neurological disorder.
[0137] In yet another embodiment, the invention further relates to
a method of detecting Alzheimer's disease (AD).
[0138] In yet another embodiment, the invention further relates to
a method of imaging and detection of neurological disorder
performed by using gamma imaging, magnetic resonance imaging,
magnetic resonance spectroscopy or fluorescence spectroscopy.
[0139] In yet another embodiment, the invention further relates to
a method of imaging and detection of SPs and NTFs, wherein the
detection is by PET or SPECT.
[0140] According to a particular embodiment of the present
invention, the compounds and methods of the present invention are
used for imaging, especially medical imaging.
[0141] Diagnostic techniques in nuclear medicine use radioactive
tracers which emit gamma rays from within the body. These tracers
are generally short-lived isotopes linked to chemical compounds
which permit specific physiological processes to be scrutinised.
They can be given by injection, inhalation or orally. The first
type is where single photons are detected by a gamma camera which
can view organs from many different angles. The camera builds up an
image from the points from which radiation is emitted; this image
is enhanced by a computer and viewed by a physician on a monitor
for indications of abnormal conditions.
[0142] Positron Emission Tomography (PET) is a precise and
sophisticated technique using isotopes produced in a cyclotron.
[0143] A positron-emitting radionuclide is introduced, usually by
injection, and accumulates in the target tissue. As it decays it
emits a positron, which promptly combines with a nearby electron
resulting in the simultaneous emission of two identifiable gamma
rays in opposite directions. These are detected by a PET camera and
give very precise indication of their origin. PET's most important
clinical role is in oncology, with fluorine-18 as the tracer, since
it has proven to be the most accurate non-invasive method of
detecting and evaluating most cancers. It is also well used in
cardiac and brain imaging.
[0144] A number of medical diagnostic procedures, including PET and
SPECT utilize radiolabeled compounds, are well known in the art.
PET and SPECT are very sensitive techniques and require small
quantities of radiolabeled compounds, called tracers. The labeled
compounds are transported, accumulated and converted in vivo in
exactly the same way as the corresponding non-radioactively
compound. Tracers, or probes, can be radiolabeled with a
radionuclide useful for PET imaging, such as .sup.11C, .sup.13N,
.sup.15O, .sup.18F, .sup.64Cu and .sup.124I, or with a radionuclide
useful for SPECT imaging, such as .sup.99Tc, .sup.77Br, .sup.61Cu,
.sup.153Gd, .sup.123I, .sup.125I, .sup.131I and .sup.32P.
[0145] PET creates images based on the distribution of molecular
imaging tracers carrying positron-emitting isotopes in the tissue
of the patient. The PET method has the potential to detect
malfunction on a cellular level in the investigated tissues or
organs. PET has been used in clinical oncology, such as for the
imaging of tumors and metastases, and has been used for diagnosis
of certain brain diseases, as well as mapping brain and heart
function. Similarly, SPECT can be used to complement any gamma
imaging study, where a true 3D representation can be helpful, for
example, imaging tumor, infection (leukocyte), thyroid or
bones.
[0146] According to another embodiment, the present invention is
also directed at a method of imaging amyloid deposits and NTFs.
When the compounds of this invention are used as imaging agents,
they are labeled with suitable radioactive isotopes or radiolabel
or radioactive label, for example, radioactive halogens, such as
.sup.18F or, radioactive metals and other detectable radioactive
atoms such as .sup.11C.
[0147] Regarding radiohalogens, .sup.125I isotopes are useful for
laboratory testing but they will generally not useful for
diagnostic purposes because of the relatively long half-life (60
days) and low gamma-emission (30-65 Key) of .sup.125I. The isotope
.sup.1231 has a half-life of thirteen hours and gamma energy of 159
Key, and it is therefore expected that labeling of ligands to be
used for diagnostic purposes would be with this isotope or with
.sup.18F (half-life of 2 hours). Other isotopes which may be used
include .sup.131I, .sup.77Br, and .sup.76Br.
[0148] In another embodiment, compounds of the present invention
also contain a radioactive isotope of carbon as the radiolabel.
This refers to a compound that comprises one or more radioactive
carbon atoms, preferably .sup.11C, with a specific activity above
that of the background level for that atom. It is well known that
naturally occurring elements are present in the form of varying
isotopes, some of which are radioactive. The radioactivity of the
naturally occurring elements is a result of the natural
distribution or abundance of these isotopes, and is commonly
referred to as a background level. The carbon labeled compounds of
the present invention have a specific activity that is higher than
the natural abundance, and therefore above the background level.
The carbon labeled compositions of the present invention can be
used for tracing, imaging, radiotherapy, and the like.
[0149] Those skilled in the art are familiar with the various ways
to detect labeled compounds for imaging purposes. For example,
positron emission tomography (PET) or single photon emission
computed tomography (SPECT) can be used to detect radiolabeled
compounds. The label that is introduced into the compound can
depend on the detection method desired. Those skilled in the art
are familiar with PET detection of a positron-emitting atom, such
as .sup.18F. The present invention is also directed to specific
compounds described herein where the .sup.18F atom is replaced with
a non-radiolabeled fluorine atom. Those skilled in the art are
familiar with SPECT detection of a photon-emitting atom, such as
.sup.123I or .sup.99mTc.
[0150] The radioactive diagnostic agent should have sufficient
radioactivity and radioactivity concentration which can assure
reliable diagnosis. The desired level of radioactivity can be
attained by the methods provided herein for preparing compounds of
Formula I. The imaging of amyloid deposits and NTFs can also be
carried out quantitatively so that the amount of amyloid deposits
and NTFs can be determined.
[0151] 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 a bolus i.v. injection. In the first step of the present
method of imaging, a labeled compound of Formula I is introduced
into a tissue or a patient in a detectable quantity. The compound
is typically 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.
[0152] In other embodiments of the invention, the labeled compound
is introduced into a patient in a detectable quantity and after
sufficient time has passed for the compound to become associated
with amyloid deposits and/or tau proteins, the labeled compound is
detected noninvasively. In another embodiment of the invention, a
labeled compound of Formula I is introduced into a patient,
sufficient time is allowed for the compound to become associated
with amyloid deposits, and then a sample of tissue from the patient
is removed and the labeled compound in the tissue is detected apart
from the patient. In another embodiment of the invention, a tissue
sample is removed from a patient and a labeled compound of Formula
I is introduced into the tissue sample. After a sufficient amount
of time for the compound to become bound to amyloid deposits and/or
tau proteins, the compound is detected.
[0153] A detectable quantity is a quantity of labeled compound
necessary to be detected by the detection method chosen. The amount
of a labeled compound 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 labeled compound
can be given to a patient until the compound is detected by the
detection method of choice. A label is introduced into the
compounds to provide for detection of the compounds.
[0154] The amount of time necessary can easily be determined by
introducing a detectable amount of a labeled compound of Formula I
into a patient and then detecting the labeled compound at various
times after administration.
[0155] The administration of the labeled compound to a patient can
be by a general or local administration route. For example, the
labeled compound may be administered to the patient such that it is
delivered throughout the body. Alternatively, the labeled compound
can be administered to a specific organ or tissue of interest. For
example, it is desirable to locate and quantitate amyloid deposits
in the brain in order to diagnose or track the progress of
Alzheimer's disease in a patient.
[0156] The term "pharmaceutically acceptable salt" as used herein
refers to those carboxylate salts or acid addition salts of the
compounds of the present invention which are, within the scope of
sound medical judgement, suitable for use in contact with the
tissues of patients without undue toxicity, irritation, allergic
response, and the like, commensurate with a reasonable benefit/risk
ratio, and effective for their intended use, as well as the
zwitterionic forms, where possible, of the compounds of the
invention. The term "salts" refers to the relatively nontoxic,
inorganic and organic acid addition salts of compounds of the
present invention. Also included are those salts derived from
non-toxic organic acids such as aliphatic mono and dicarboxylic
acids, for example acetic acid, 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, but are not limited to hydrobromide, hydrochloride,
sulfate, bisulfate, nitrate, acetate, trifluoroacetate, 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. (See, for example, Berge
S. M., et al., Pharmaceutical Salts, J. Pharm. Sci. 66:1-19 (1977),
which is incorporated herein by reference).
[0157] The term "alkyl" as employed herein by itself or as part of
another group refers to both straight and branched chain radicals
of up to 6 carbons, preferably up to 4 carbons.
[0158] The term "alkene" as employed herein by itself or as part of
another group refers to both straight and branched chain radicals
of up to 6 carbons, preferably up to 4 carbons, which have a double
bond between any two carbon atoms.
[0159] The term "alkyne" as employed herein by itself or as part of
another group refers to both straight and branched chain radicals
of up to 6 carbons, preferably up to 4 carbons, which have a triple
bond between any two carbon atoms. Alkynes are traditionally known
as acetylenes, although the name acetylene refers specifically to
C.sub.2H.sub.2.
[0160] The term "alkoxy" is used herein to mean a straight or
branched chain alkyl radical, as defined above, unless the chain
length is limited thereto, 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 4 carbon atoms in length,
more preferably 1 or 2 carbon atoms in length.
[0161] The term "halo" or "halogen" employed herein by itself or as
part of another group refers to chlorine, bromine, fluorine or
iodine, unless defined otherwise in specific uses in the text
and/or claims.
[0162] The term "radiohalogen" employed herein by itself or as part
of another group refers to .sup.18F, .sup.123I, .sup.125I,
.sup.131I, .sup.76Br and .sup.77Br.
[0163] The term "haloalkyl" as employed herein refers to any of the
above alkyl groups substituted by one or more chlorine, bromine,
fluorine or iodine with fluorine being preferred. Most preferably,
the alkyl is substituted with a single halo, such as fluorine, at
the distal end of the alkyl.
[0164] The term "radiohaloalkyl" refers to a haloalkyl group as
defined above that contains a halogen radioisotope. One example of
this type of group is .sup.18F--(C.sub.1-4)alkyl-.
[0165] The term "hydroxyalkyl" as employed herein by itself or as
part of another group refers to linear or branched alkyl groups
containing an --OH substituent.
[0166] The term "aryl" as employed herein by itself or as part of
another group refers to monocyclic, bicyclic, tricyclic or
polycyclic aromatic groups containing from 5 to 14 atoms in the
ring portion. Aryl compounds of the present invention include, but
are not limited to non-substituted or further substituted phenyl,
naphthyl, tetrahydronaphthyl and the like. The aryl group can also
contain at least one heteroatom, such as N, S or O, to form a
"heteroaryl." Examples of heteroaryls include, but are not limited
to non-substituted or further substituted thienyl, benzo[b]thienyl,
benzothiazolyl, furyl, pyranyl, isobenzofuranyl, benzofuranyl,
benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, benzopyrazolyl,
benzoimidazolyl, benzoimidazole pyrimidines, imidazoimidazole
pyridine, benzofuropyridine, benzofuropyrimidine, pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, indolyl, indazolyl, 4H-quinolizinyl,
isoquinolyl, quinolyl, quinazolinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, furazanyl, phenoxazinyl groups and the like.
[0167] The term "aryloxy" as employed herein refers to an "aryl"
group bonded to an oxygen atom, and include benzyloxy and phenoxy,
which can be further substituted and the like.
[0168] The term "tissue" means a part of a patient's body. It is an
ensemble of cells, not necessarily identical, but from the same
origin, that together carry out a specific function. Examples of
tissues include, but are not limited to the brain, heart, liver,
blood vessels, and arteries.
[0169] The term "patient" or "subject" means humans and other
animals.
[0170] Those skilled in the art are also familiar with determining
the amount of time sufficient for a compound to become associated
with amyloid deposits for imaging purposes.
[0171] The term "associated" means a chemical interaction between
the labeled compound and the amyloid deposit. Examples of
associations include covalent bonds, ionic bonds,
hydrophilic-hydrophilic interactions, hydrophobichydrophobic
interactions, and complexes.
[0172] The following examples are illustrative, but not limiting,
of the method and compositions of the present invention. Other
suitable modifications and adaptations of the variety of conditions
and parameters normally encountered and obvious to those skilled in
the art are within the spirit and scope of the invention.
EXPERIMENTAL EXAMPLES
[0173] Note: equivalents refer to molar equivalents. Actual volumes
are calculated by multiplying the molar equivalents by liters.
Thus, 1 mmol times 5 vol equals 5 mmol.
1. General Experimental Procedures for the Preparation of Disclosed
Compounds
Method A: General Procedure for Suzuki Coupling Reactions
##STR00156##
[0175] A mixture of aryl/heterocyclic halide (1.0 equiv.), boronic
acid or boronate ester (1.1-1.5 equiv.), K.sub.2CO.sub.3 (3.0
equiv.) and Pd[PPh.sub.3].sub.4 (0.01-0.05 equiv) in DMF (30 mL)
was irradiated in a Biotage Emrys Initiator microwave reactor (250
W) at 100.degree. C. for 30 min. After cooling to room temperature,
the solvent was removed in vacuo. The residue was purified on flash
column chromatography over silica gel using EtOAc:Hexanes or
EtOAc:DCM or MeOH:DCM as the eluent to afford the desired biaryl
products.
Method B: General Procedure for Sonogashira Coupling Reaction
##STR00157##
[0177] A mixture of halide (1.0 equiv.), acetylene (1.1-1.5
equiv.), CuI (0.05 equiv.), Pd[PPh.sub.3].sub.4 or
PdCl.sub.2(PPh.sub.3).sub.2 (0.01-0.05 equiv.) and DIPEA (3.0
equiv.) in ACN (30 mL) was irradiated in a Biotage Emrys Initiator
microwave reactor (250 W) at 100.degree. C. for 30 min. After
cooling to room temperature, the solvent was removed in vacuo. The
residue was purified on flash column chromatography over silica gel
using EtOAc:Hexanes or EtOAc:DCM or MeOH:DCM as the eluent to
afford the disubstituted acetylene derivatives.
Method C: General Procedure for Phenolic Alkylation
##STR00158##
[0179] A mixture of phenol derivatives (1.0 equiv.), alkylating
agent (1.1 equiv.), and Cs.sub.2CO.sub.3 (3.0 equiv.) in DMF (10
mL) was heated at 60.degree. C. under Ar for 1-3 hrs. After the
reaction was completed, the solvents were removed in vacuo. The
residue was purified on flash column chromatography over silica gel
using EtOAc:Hexanes or EtOAc:DCM or MeOH:DCM as the eluent to
afford the alkylated products.
Method D: General Procedure for N-Alkylation Using NaH as the
Base
##STR00159##
[0181] To a solution of amine (1.0 equiv.) in DMF (10 mL) was added
NaH (1.5-6 equiv.), followed by alkylating agent (1.1-2 equiv.).
The reaction mixture was allowed to stir at room temperature for
1-15 hrs and monitored by LCMS. The reaction mixture was then
poured into water (50 mL) and extracted with EtOAc (4.times.20 mL).
The combined organic layers were washed with H.sub.2O (3.times.20
mL), brine (50 mL), dried (Na.sub.2SO.sub.4) and concentrated in
vacuo. The residue was purified on a flash column chromatography
over silica gel using EtOAc:Hexanes or EtOAc:DCM or MeOH:DCM as the
eluent to afford the N-alkylated products.
Method E: General Procedure for N-Alkylation Using Cs.sub.2CO.sub.3
as the Base
##STR00160##
[0183] To a solution of amine (1.0 equiv.) in DMF (10 mL) was added
alkylating agent (1.1-2 equiv.) and Cs.sub.2CO.sub.3 (2-3 equiv.).
The reaction mixture was allowed to stir at 60.degree. C. for 1-15
hrs. After the reaction was completed, the solvents were removed in
vacuo. Water (30 mL) was added was added to the residue. If the
desired products are precipitated as a solid, they are filtered,
washed with water and dried to give the pure products. If the
desired products are not precipitated out, the mixtures are
extracted with EtOAc (4.times.40 mL). The combined organic layers
were washed with H.sub.2O (3.times.40 mL), brine (50 mL), dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was
purified on a flash column chromatography over silica gel using
EtOAc:Hexanes or EtOAc:DCM or MeOH:DCM as the eluent to afford the
N-alkylated products.
Method F: General Procedure of Tosylation of Alcohol
[0184] ROH+Ts.sub.2O.fwdarw.ROTs
[0185] To a cooled solution of alcohol (1.0 equiv.) in DCM (20 mL)
was added Ts.sub.2O (1.5 equiv.) and Et.sub.3N (3.0 equiv.). The
reaction mixture was stirred at 0.degree. C. and then gradually
warmed to room temperature, and stirred at room temperature for 1-5
hrs. After the reaction was completed, DCM was removed in vacuo.
The residue was purified on flash column chromatography over silica
gel using EtOAc:Hexanes as the eluent to afford the final
tosylates.
Method G: General Procedure for Demethylation of Aryl Methyl
Ether
[0186] ArOMe.fwdarw.ArOH
[0187] To a cooled solution of aryl methyl ether (1.0 equiv.) in
DCM (10 mL) was added BBr.sub.3 (5.0 equiv.) slowly. The resulting
mixture was stirred at 0.degree. C. and then warmed gradually to
room temperature, and stirred at room temperature for 15 hrs. After
the reaction was completed, the reaction was quenched with
NaHCO.sub.3 solution (50 mL) and extracted with DCM (4.times.10
mL). The combined organic layers were washed with H.sub.2O
(3.times.10 mL), dried (Na.sub.2SO.sub.4) and concentrated in vacuo
to afford the expected phenols.
Method H: General Procedure for Click Reaction Between Azides and
Acetylenes Using CuI and DIPEA
##STR00161##
[0189] To a solution of azide derivatives (1.0 equiv.) in THF (29
mL) was added acetylene derivatives (1.0 equiv.), CuI (0.2 equiv.),
DIPEA (0.4 equiv.). The reaction mixture was allowed to stir at
room temperature under Ar until deemed completion of reaction by
LCMS. The reaction mixture was then concentrated in vacuo. The
residue was purified on a flash column chromatography over silica
gel using EtOAc:Hexanes as the eluent to afford the final
triazoles.
Method I: General Procedure for Silyl Deprotection Using
K.sub.2CO.sub.3
##STR00162##
[0191] To a solution of silyl protected compounds (1.0 equiv.) in
MeOH (20 mL) was added K.sub.2CO.sub.3 (1.2 equiv.). The reaction
mixture was allowed to stir at room temperature for 1 h. After the
reaction was completed, the solvent was removed in vacuo. The
residue was purified on a flash column chromatography over silica
gel using EtOAc:Hexanes as the eluent to give the deprotected
products.
Method J: General Procedure for Silyl Deprotection with TBAF
##STR00163##
[0193] To a solution of silyl protected compound (1.0 equiv.) in
THF (20 mL) was added a solution of TBAF in THF (1.0M, 1.0 equiv.).
The reaction mixture was allowed to stir at room temperature for 10
min. After the reaction was completed, the solvents were removed in
vacuo. The residue was purified on a flash column chromatography
over silica gel using EtOAc:Hexanes as the eluent to afford the
deprotected products.
Method K: General Procedure for the Deprotection of Boc, THP and
Ketal Derivatives
##STR00164##
[0195] To a solution of protected derivatives (1.0 equiv.) in
1,4-dioxane (20 mL) was added a solution of HCl in 1,4-dioxane
(4.0M, 3.8 mL). The reaction mixture was allowed to stir at room
temperature and monitored by LCMS. After the reaction was
completed, the solvents were removed in vacuo to afford the desired
deprotected products.
Method L: General Procedure for the Conversion of Nitropyridyl to
Fluoropyridyl Compounds
##STR00165##
[0197] To a solution of nitropyridyl derivatives (1.0 equiv.) in
DMSO (10 mL) was added KF (5 equiv.). The reaction mixture was
allowed to stir at 140.degree. C. for 1.5 hrs. After the reaction
was completed, the reaction was quenched with water (10 mL) and
extracted with DCM (4.times.10 mL). The combined organic layers
were washed with H.sub.2O (3.times.10 mL), dried (Na.sub.2SO.sub.4)
and concentrated in vacuo. The residue was purified on a flash
column chromatography over silica gel using EtOAc:Hexanes or
EtOAc:DCM or MeOH:DCM as the eluent to afford the F-pyridyl
compounds.
Method M: General Procedure for the Conversion of Fluoropyridyl to
Aminopyridyl Compounds
##STR00166##
[0199] A suspension of fluoropyridyl derivatives (1.0 equiv.) and
amine derivatives (excess) was irradiated in a Biotage Emrys
Initiator microwave reactor (250 W) at 120.degree. C. for 10 min.
After the reaction was completed, the reaction was quenched with
water (10 mL) and extracted with DCM (4.times.10 mL). The combined
organic layers were washed with H.sub.2O (3.times.10 mL), dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was
purified on a flash column chromatography over silica gel using
EtOAC:DCM as the eluent to afford the aminopyridyl compounds.
Method N: General Procedure for Click Reaction Between Azides and
Acetylenes Using CuSO.sub.4.H.sub.2O and Sodium Ascorbate
##STR00167##
[0201] To a solution of azide derivatives (1.0 equiv.) in a mixture
of tert-BuOH:H.sub.20 (1:1, 100 mL) was added acetylene (0.9-1.2
equiv.), CuSO.sub.4.5H.sub.2O (0.2 equiv.), and sodium L-ascorbate
(0.4 equiv.). The resulting reaction mixture was allowed to stir at
room temperature under Ar until deemed complete by LCMS. After the
reaction was completed, the solvents were removed in vacuo. The
residue was mixed with water (100 mL), cooled to 0.degree. C., and
filtered. The solid collected was washed with ether (5.times.10 mL)
and dried in vacuo to afford the final triazoles.
Method O: General Procedure for Fluorination
##STR00168##
[0203] To a solution of precursor (alkyl tosylates/bromides, 1.0
equiv.) in acetonitrile (1.0 mL) was added a solution Bu.sub.4NF in
THF (4M, 1.0 equiv.). The reaction mixture was allowed to stir at
90.degree. C. for 30 min and cooled. The reaction mixture was
diluted with water/acetonitrile (1 mL), filtered through 0.45 .mu.m
filter prior to purification by HPLC using ACN:Water both
containing 0.05% TFA to afford the fluorinated product.
Method P: General Procedure for Boc and Ketal Deprotections Using
TFA
##STR00169##
[0205] To a solution of protected compound (1.0 equiv.) in DCM (100
mL) was added TFA (10 mL). The resulting reaction mixture was
stirred at room temperature for 15 hrs, and then poured into water
(200 mL). The mixture was extracted with DCM (2.times.100 mL). The
combined organic layers were washed with sat. NaHCO.sub.3 solution
(50 mL), brine (50 mL), dried (Na.sub.2SO.sub.4) and concentrated
in vacuo. The residue was purified on a flash column chromatography
over silica gel using EtOAc:Hexanes as the eluent to afford the
deprotected products.
Method Q: General Procedure for One-Pot Reductive Amination of
Anilines from Alcohols
##STR00170##
[0207] To a solution of alcohols (0.2 mmol) in DCE (1 mL) was added
Dess-martin reagent (0.2 mmol). The mixture was stirred at room
temperature for 15 minutes and filtered off solid with syringe
filter. The filtrate was added to a solution of substituted
anilines (0.1 mmol) and NaBH(OAc).sub.3 (0.3 mmol) in DCE (1 mL).
The mixture was stirred 5 to 10 min at room temperature and quickly
quenched with 1 N NaOH solution (1 mL). The DCE layer was separated
and concentrated in vacuo. The residue was purified by flash
chromatography (silica gel, 0-20% EtOAc/DCM) to give the desired
mono alkylated anilines.
Method R: General Procedure for Reductive Dimethylation of Anilines
with Paraformaldehyde
##STR00171##
[0209] To a suspension of paraformaldehyde (1.0 mmol) in THF (5 mL)
was added concentrated sulfuric acid (98%, 0.1 mL, 1.9 mmol). The
mixture was stirred at room temperature while a suspension of
anilines (0.1 mmol) and NaBH.sub.4 (1.0 mmol) in THF (5 mL) was
added to above paraformaldehyde suspension. The mixture was stirred
at room temperature for 30 minutes and quenched by adding 1 N NaOH
solution (1 mL). The mixture was concentrated and the residue was
partitioned between DCM and water. The DCM layer was separated and
concentrated in vacuo. The residue was purified by flash
chromatography (silica gel, 0-10% EtOAc/DCM) to give the desired
N,N-dimethyl anilines.
Method S: General Procedure for the Preparation of Benzimidazole
Derivatives
##STR00172##
[0211] A solution of 2-aminoaniline (1.0 equiv.),
benzoaldehyde/aldehyde derivatives (1.0 equiv.) and
1,4-benzoquinone (1.0 equiv.) in EtOH (10 mL) was heated at
95.degree. C. for 4-6 hrs, and then cooled and concentrated in
vacuo. The residue was purified on flash column chromatography
(silica gel, 0-5% EtOAc/DCM) to give the desired products.
2. The Preparation of Claimed Compounds According to the General
Procedures Described Above
Preparation of W366
##STR00173## ##STR00174##
[0212] Preparation of 2-iodo-6-methoxybenzo[d]thiazole (2)
[0213] To a 100 mL round bottomed flask equipped with a magnetic
stir bar, ACN (33.0 mL) and PTSA (6.3 g, 33.33 mmol), 1 (2.0 g,
11.11 mmol) was added at 0.degree. C. and stirred for 15 min. To
this, a solution of NaNO.sub.2 (1.5 g, 22.22 mmol) and KI (4.6 g,
27.78 mmol) in H20 (7 mL) was added and stirred for 4 hr at RT. To
the reaction mixture was added H.sub.20 (175 mL) and was then made
basic with sat. NaHCO.sub.3 (pH=9) and Na.sub.2S.sub.2O.sub.3 (2M,
23 mL). The resulting reaction mixture was extracted with EtOAc
(3.times.50 mL). The combined organic extracts were washed with
water (50 mL), brine (50 mL), dried over MgSO.sub.4 and
concentrated in vacuo. The residue was purified over silica gel
using EtOAc:Hexanes (0.5:9.5) as the eluent to afford 2 (1.62 g,
50%) as a white crystalline solid. .sup.1H NMR (400 MHz,
CDCl.sub.3), .delta.: 3.85 (s, 3H), 7.00 7.04 (m, 1H), 7.24-7.27
(m, 1H), 7.89 (d, J=9.19 Hz, 1H); MS (ESI) 291.9 (M+H.sup.+).
Preparation of 4-bromo-2-fluoro-N-methylaniline (4)
[0214] To a 100 mL round bottomed flask equipped with a magnetic
stir bar was added at RT MeOH (26.0 mL), 3 (0.5 g, 2.63 mmol),
NaOMe (0.71 g, 13.16 mmol) and paraformaldehyde (0.394 g, 13.16
mmol). The reaction was refluxed for 2 hr. The reaction mixture was
then cooled to 0.degree. C. and NaHB.sub.4 (0.5 g, 13.16 mmol) was
added in portions. After the addition, the reaction mixture was
again refluxed for 1 hr. After the reaction is complete, MeOH was
removed, water (50 mL) added and extracted with EtOAc (3.times.30
mL). The combined organic extracts were washed with water (30 mL),
brine (30 mL), dried over MgSO.sub.4 and concentrated in vacuo. The
residue was purified over silica gel using EtOAc:Hexanes (0.5:9.5)
as the eluent to afford 4 (0.5 g, 93%) as a brown oil. .sup.1H NMR
(400 MHz, CDCl.sub.3), .delta.: 2.86 (s, 3H), 6.64 6.68 (m, 1H),
7.11-7.15 (m, 2H); MS (ESI): 203.9 (M+H.sup.+).
Preparation of 2-fluoro-N-methyl-4-((trimethylsilyl)ethynyl)aniline
(6)
[0215] A 5 mL microwave tube was charged with 4 (0.5 g, 2.45 mmol),
5 (0.7 mL, 4.9 mmol), [Pd(PPh.sub.3).sub.4](0.3 g, 0.245 mmol), CuI
(0.07 g, 0.37 mmol) and NH(C.sub.2H.sub.5).sub.2 (0.8 mL, 7.35
mmol) in DMF (2.5 mL). The suspension was irradiated in a Biotage
Emrys Optimizer microwave reactor (250 W) at 100.degree. C. for 15
min. After cooling to room temperature water (50 mL) was added and
then extracted with EtOAc (3.times.30 mL). The combined organic
extracts were washed with water (30 mL), brine (30 mL), dried over
MgSO.sub.4 and the crude mixture was used for the next step. MS
(ESI): 222.1 (M+H.sup.+).
Preparation of 4-ethynyl-2-fluoro-N-methylaniline (7)
[0216] To a 25 mL round bottom flask equipped with a magnetic stir
bar, 6 (crude 0.4 g), MeOH (9 mL) and K.sub.2CO.sub.3 (0.5 g, 3.62
mmol) were added. The reaction was stirred at RT for 30 min. To the
reaction mixture, silica gel added (approximately 10 g) and
concentrated in vacuo. The residue was purified over silica gel
using EtOAc:Hexanes (1:3) as the eluent to afford 7 (0.26 g, 96%)
as brown oil. MS (ESI): 150.1 (M+H.sup.+).
Preparation of
2-fluoro-4-{(6-methoxybenzo[d]thiazol-2-yl)ethynyl)-N-methylaniline
(8)
[0217] A 5 mL microwave tube was charged with 2 (0.2 g, 0.68 mmol),
7 (0.1 g, 0.68 mmol) [Pd(PPh.sub.3).sub.4](0.08 g, 0.034 mmol), CuI
(0.02 g, 0.05 mmol) and TEA (0.28 mL, 2.04 mmol) in ACN (2.0 mL).
The suspension was irradiated in a Biotage Emrys Optimizer
microwave reactor (250 W) at 100.degree. C. for 5 min. After
cooling to room temperature the solvent was evaporated in vacuo.
The residue was purified over silica gel using
Hexanes:Dichloromethane (DCM) (0-100%) as the eluent to afford the
coupling product 8 (0.084 g, 39%) as yellow solid. .sup.1H NMR (400
MHz, CDCl.sub.3), .delta.: 2.92 (s, 3H), 3.87 (s, 3H), 6.64 (t,
J=8.4 Hz, 1H), 7.11 (dd, J=12.0, 4.0 Hz, 1H), 7.22 (dd, J=10.4, 2
Hz, 1H), 7.27 (d, J=2.4, 1H), 7.33-7.35 (m, 1H), 7.96 (d, J=12 Hz,
1H); MS (ESI): 313.0 (M+H.sup.+).
Preparation of
2-((3-fluoro-4-(methylamino)phenyl)ethynyl)benzo[d]thiazol-6-ol
(W366)
[0218] To a 25 mL round bottomed flask equipped with a magnetic
stir bar containing DCM (5.2 mL) was placed 8 (0.08 g, 0.26 mmol).
The reaction mixture was cooled to 0.degree. C. and BBr.sub.3 (0.75
mL of 1M in DCM) was added drop wise. The reaction was stirred at
RT for 8 hr. The reaction was then neutralized with sat.
NaHCO.sub.3 and extracted into DCM (2.times.10 mL). The combined
organic extracts were washed with water (10 mL), brine (10 mL),
dried over MgSO.sub.4 and concentrated in vacuo. The residue was
purified over silica gel using EtOAc:Hexanes (1:3) as the eluent to
afford W366 (0.02 g, 26%) as a brown solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6), .delta.: 2.5 (d, J=10.4 Hz, 3H), 6.06-6.08 (m, 1H),
6.42 (t, J=8.8 Hz, 1H), 6.74 (dd, J=8.8, 2.4 Hz, 1H), 7.05-7.14 (m,
2H), 7.54 (d, J=8.8, 1H), 9.76 (br s, 1H); MS (ESI): 299.0
(M+H.sup.+).
Preparation of W378 Standard
##STR00175##
[0219] Preparation of 2-iodo-5-methoxythiazolo[5,4-b]pyridine
(10)
[0220] To a 25 mL round bottomed flask equipped with a magnetic
stir bar, ACN (4.0 mL), PTSA (0.79 g, 4.14 mmol) and 9 (0.25 g,
1.38 mmol) were added at 0.degree. C. and stirred for 15 min. To
this, a solution of NaNO.sub.2 (0.19 g, 2.76 mmol) and KI (0.57 g,
3.45 mmol) in H.sub.2O (0.9 mL) was added and stirred for 4 hr at
RT. The reaction mixture was then added to H.sub.2O (15 mL), made
basic with sat. NaHCO.sub.3 (pH=9) and Na.sub.2S.sub.2O.sub.3 (2M,
3 mL) was added. The resulting reaction mixture was extracted with
EtOAc (3.times.20 mL). The combined organic extracts were washed
with water (20 mL), brine (20 mL), dried over MgSO.sub.4 and
concentrated in vacuo. The residue was purified over silica gel
using EtOAc:Hexanes (1:4) as the eluent to afford 10 (0.12 g, 30%)
as a white crystalline solid. .sup.1H NMR (400 MHz, CDCl.sub.3),
.delta.: 3.97 (s, 3H), 6.77 (d, J=8.8 Hz, 1H), 8.08 (d, J=8.8 Hz,
1H); MS (ESI): 292.9 (M+H.sup.+).
Preparation of
2-fluoro-4-((5-methoxythiazolo[5,4-b]pyridin-2-yl)ethynyl)-N-methylanilin-
e (W378)
[0221] A 5 mL microwave tube was charged with 10 (0.12 g, 0041
mmol), 7 (0.06 g, 0041 mmol) [Pd(PPh.sub.3).sub.4](0.05 g, 0.004
mmol), CuI (0.012 g, 0.06 mmol) and TEA (0.2 mL, 1.23 mmol) in ACN
(2.0 mL). The suspension was irradiated in a Biotage Emrys
Optimizer microwave reactor (250 W) at 100.degree. C. for 5 min.
After cooling to room temperature the solvent was evaporated in
vacuo. The residue was purified over silica gel using Hexanes:DCM
(0-100%) as the eluent to afford the coupling product W378 (0.04 g,
31%) as yellow solid. MS (ESI): 314.0 (M+H.sup.+)
Preparation of W366 Labeling Precursor
##STR00176##
[0222] Preparation of
4-((6-methoxybenzo[d]thiazol-2-yl)ethynyl)-N-methyl-2-nitroaniline
[0223] A 5 mL microwave tube is charged with 2 (1 equiv),
4-ethynyl-N-methyl-2-nitroaniline (1 equiv) [Pd(PPh.sub.3).sub.4]
(0.05 equiv), CuI (0.05 equiv) and TEA (5 equiv) in ACN (5 vol).
The suspension is irradiated in a Biotage Emrys Optimizer microwave
reactor (250 W) at 100.degree. C. for 5 min. After cooling to room
temperature, the solvent is evaporated in vacuo. The residue is
purified over silica gel using Hexanes:DCM (0-100%) as the eluent
to afford the coupling product.
Preparation of
2-((4-(methylamino)-3-nitrophenyl)ethynyl)benzo[d]thiazol-6-ol
[0224] To a round bottomed flask equipped with a magnetic stir bar
containing DCM (5 vol) is placed
4-((6-methoxybenzo[d]thiazol-2-yl)ethynyl)-N-methyl-2-nitroaniline
(1 equiv). The reaction mixture is cooled to 0.degree. C.,
BBr.sub.3 (5 equiv of 1M in DCM) is added dropwise and the reaction
is stirred at RT for 8 hours. The reaction is neutralized with sat.
NaHCO.sub.3, extracted into DCM (2.times.5 vol). The combined
organic extracts are washed with water (5 vol), brine (5 vol),
dried over MgSO.sub.4 and concentrated in vacuo. The residue is
purified over silica gel using EtOAc:Hexanes (1:3) as an eluent to
afford the desired product.
Preparation of
4-((6-(ethoxymethoxy)benzo[d]thiazol-2-yl)ethynyl)-N-methyl-2-nitroanilin-
e
[0225] To a round bottom flask under Ar containing
2-((4-(methylamino)-3-nitrophenyl)ethynyl)benzo[d]thiazol-6-ol (1
equiv) is added THF (5 vol). To this solution is added NaH (1.3
equiv). The solution is stirred at RT for 30 min. To this solution
is added MOM-Cl (1.5 equiv). The reaction is stirred overnight at
RT. The reaction is poured onto water (10 vol) and extracted into
DCM (10 vol). The organic layer is washed with water (10 vol),
dried (MgSO.sub.4), filtered and concentrated to dryness. The
material is purified using EtOAc:Hexanes as the eluent over silica
gel to afford the final product.
Preparation of
N-(4-((6-(ethoxymethoxy)benzo[d]thiazol-2-yl)ethynyl)-2-nitrophenyl)-N-me-
thylformamide (W366 labeling precursor)
[0226] To a round bottom flask containing
4-((6-(ethoxymethoxy)benzo[d]thiazol-2-yl)ethynyl)N-methyl-2-nitroaniline
(1 equiv) is added formic acid (5 equiv), acetic anhydride (5
equiv) and DCM (5 vol). The reaction is warmed to 60.degree. C. for
7 days. The r.times.n is then concentrated in vacuo and the
material is purified using EtOAc:Hexanes as the eluent over silica
gel to afford the final product.
Preparation of 2-(Pyridin-4-yl)quinoline (T123)
##STR00177##
[0228] 2-(Pyridin-4-yl)quinoline T123 was prepared using general
procedure A from 2-chloroquinoline (82 mg, 0.5 mmol) and
pyridin-4-ylboronic acid (61.5 mg, 0.5 mmol). The product was
obtained as an off white solid (100 mg, 97%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.75 (dd, J=5.0, 1.6 Hz, 2H), 8.26 (d, J=8.8
Hz, 1H), 8.16 (d. J=8.4 Hz, 1H), 8.04 (m, 2H), 7.87 (d, J=8.4 Hz,
1H), 7.83 (d, J=8.0 Hz, 1H), 7.74 (m, 1H), 7.55 (m, J=1H); MS
(ESI): 207 (M+H.sup.+).
Preparation of 5-(Quinolin-2-yl)picolinonitrile (T124)
##STR00178##
[0230] 5-(Quinolin-2-yl)picolinonitrile T124 was prepared using
general procedure A from 2-chloroquinoline (82 mg, 0.5 mmol) and
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinonitrile (115
mg, 0.5 mmol). The product was obtained as yellow solid (3 mg, 3%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.48 (dd, J=2.4, 0.8 Hz,
1H), 8.69 (dd, J=8.0, 2.0 Hz, 1H), 8.33 (d, J=8.8 Hz, 1H), 8.19 (d,
J=8.4 Hz, 1H), 7.93 (d, J=8.4 Hz, 1H), 7.90 (dd, J=8.4, 1.2 Hz,
1H), 7.87 (dd, J=8.4, 0.8 Hz, 1H), 7.81 (m, 1H), 7.63 (m, 1H); MS
(ESI):232 (M+H.sup.+).
Preparation of N,N-Dimethyl-5-(quinolin-2-yl)pyridin-2-amine
(T125)
##STR00179##
[0232] N,N-Dimethyl-5-(quinolin-2-yl)pyridin-2-amine T125 was
prepared using general procedure A from 2-chloroquinoline (82 mg,
0.5 mmol) and (6-(dimethylamino)-pyridin-3-yl)boronic acid (83 mg,
0.5 mmol). The product was obtained as yellow solid (90 mg, 72%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.93 (dd, J=2.4, 1H),
8.40 (dd, J=8.8, 2.4 Hz, 1H), 8.15 (d, J=8.4 Hz, 1H), 8.09 (d,
J=8.6 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 7.70 (m, 1H), 7.47 (m, 1H),
6.65 (dd, J=8.8, 0.8 Hz, 1H), 3.18 (s, 6H); MS (ESI): 250
(M+H.sup.+).
Preparation of 2-(4-Fluoropyridin-3-yl)quinoline (T126)
##STR00180##
[0234] 2-(4-Fluoropyridin-3-yl)quinoline T126 was prepared using
general procedure A from 2-chloroquinoline (82 mg, 0.5 mmol) and
(5-fluoropyridin-3-yl)boronic acid (70 mg, 0.5 mmol). The product
was obtained as white solid (80 mg, 71%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.93 (m, 1H), 8.63 (m, 1H), 8.24 (d, J=8.8 Hz,
1H), 8.13 (dd, J=8.8, 0.8 Hz, 1H), 7.82 (m, 2H), 7.74 (m, 1H), 7.06
(m, 1H); MS (ESI): 225 (M+H.sup.+).
Preparation of 2-(6-Fluoropyridin-3-yl)quinoxaline (T127)
##STR00181##
[0236] 2-(6-Fluoropyridin-3-yl)quinoxaline T127 was prepared using
general procedure A from 2-(6-fluoropyridin-3-yl)quinoxaline (82
mg, 0.5 mmol) and
2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(111 mg, 0.5 mmol). The product was obtained as white solid (93 mg,
82%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.32 (s, 1H), 9.00
(m, 1H), 8.66 (m, 1H), 8.15 (m, 2H), 7.82 (m, 2H), 7.15 (m, 1H); MS
(ESI): 226 (M+H.sup.+).
Preparation of 2-(Pyridin-3-yl)quinoxaline (T128)
##STR00182##
[0238] 2-(Pyridin-3-yl)quinoxaline T128 was prepared using general
procedure A from 2-chloroquinoline (82 mg, 0.5 mmol) and
(5-fluoropyridin-3-yl)boronic acid (70 mg, 0.5 mmol). The product
was obtained as white solid (80 mg, 71%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 9.40 (d, J=2.0 Hz, 1H), 9.33 (s, 1H), 8.75
(dd, J=4.8 Hz, 1 Hz, 8.51 (m, 1H), 8.15 (m, 2H), 7.79 (m, 2H), 7.49
(m, 1H); MS (ESI): 208 (M+H.sup.+).
Preparation of
##STR00183##
[0240] 2-(6-Fluoropyridin-3-yl)-6-methoxyquinoline T138 was
prepared using general procedure A from 2-chloro-6-methoxyquinoline
(65 mg, 0.33 mmol) and
2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(75 mg, 0.33 mmol). The product was obtained as yellow solid (30
mg, 35%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.91 (m, 1H),
8.63 (m, 1H), 8.16 (d, J=8.4 Hz, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.80
(d, J=8.8 Hz, 1H), 7.41 (dd, J=9.2, 2.8 Hz, 1H), 7.11 (d, J=2.8 Hz,
1H), 7.08 (m, 1H); 3.96 (s, 3H); MS (ESI): 255 (M+H.sup.+).
Preparation of
##STR00184##
[0242] 4-(6-Methoxyquinolin-2-yl)-N,N-dimethylaniline T139 was
prepared using general procedure A from 2-chloro-6-methoxyquinoline
(65 mg, 0.33 mmol) and (4-(dimethylamino)phenyl)boronic acid (55
mg, 0.33 mmol). The product was obtained as yellow solid (30 mg,
33%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.06 (d, J=8.8 Hz,
2H), 8.02 (dd, J=8.4, 6.4 Hz, 2H), 7.78 (d, J=8.8 Hz, 1H), 7.33
(dd, J=9.2, 2.8 Hz, 1H), 7.06 (d, J=2.8 Hz, 1H), 6.83 (m, 2H), 3.93
(s, 3H), 3.03 (s, 6H); MS (ESI): 279 (M+H.sup.+).
Preparation of
##STR00185##
[0244] tert-Butyl
5-(quinolin-2-yl)-1H-benzo[d]imidazole-1-carboxylate T432 was
prepared using general procedure A from 2-chloroquinoxaline (24 mg,
0.145 mmol) and tert-butyl
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate
(50 mg, 0.145 mmol). The product was obtained as white wax (30 mg,
60%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.55 (dd, J=1.6,
0.8 Hz, 1H), 8.41 (dd, J=8.8, 2.0 Hz, 1H), 8.32 (d, J=8.8 Hz, 1H),
8.29 (s, 1H), 8.26 (d, J=8.4 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.95
(d, J=8.8 Hz, 1H), 7.75 (m, 1H), 7.55 (m, 1H), 1.76 (s, 9H); MS
(ESI): 346 (M+H.sup.+).
Preparation of
##STR00186##
[0246] 2-(1H-Indol-5-yl)quinoline T433 was prepared using general
procedure P. The reaction was performed on a 10 mg scale of T432.
T433 was isolated as a yellow solid (5 mg, 35%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 8.83 (t, 1H), 8.70-8.51 (m, 4H),
8.24-8.22 (m, 1H), 8.14-8.08 (m, 3H), 8.00 (d, J=8.8 Hz, 0.5H),
7.88 (m, 1H), 7.68 (d, J=8.4 Hz, 0.5H); MS (ESI): 246
(M+H.sup.+).
Preparation of
##STR00187##
[0248] 2-(1-Methyl-1H-indol-5-yl)quinoline T453 was prepared using
general procedure A from 2-chloroquinoline (32 mg, 0.2 mmol) and
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole
(51 mg, 0.2 mmol). The product was obtained as off-white solid (22
mg, 42%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.43 (dd,
J=2.0, 0.8 Hz, 1H), 8.17 (d, J=8.8 Hz, 2H), 8.12 (dd, J=8.8, 2.0
Hz, 1H), 7.95 (d, J=8.8 Hz, 1H), 7.80 (dd, J=8.4, 1.6 Hz, 1H), 7.70
(m, 1H), 7.48 (m, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.09 (d, J=3.2 Hz,
1H), 6.60 (dd, J=3.2, 0.8 Hz, 1H), 3.82 (s, 3H); MS (ESI): 259
(M+H.sup.+).
Preparation of 2-(1-(3-fluoropropyl)-1H-indol-5-yl)quinoline
(T461)
##STR00188##
[0250] 2-(1H-indol-5-yl)quinoline was prepared using general
procedure A from 2-chloroquinoline (32 mg, 0.2 mmol) and
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole
(51 mg, 0.2 mmol). The product was obtained as off-white solid (22
mg, 42%). MS (ESI): 245 (M+H.sup.+).
[0251] 2-(1-(3-fluoropropyl)-1H-indol-5-yl)quinoline T461 was
prepared using general procedure E. The reaction was performed on a
24 mg scale of 2-(1H-indol-5-yl)quinoline. T461 was isolated as a
yellow wax (0.8 mg, 2.6%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.70 (d, J=8.4 Hz, 1H), 8.67 (d, J=8.8 Hz, 1H), 8.44 (d,
J=1.2 Hz, 1H), 8.10 (d, J=9.2 Hz, 1H), 8.03-7.98 (m, 3H), 7.78 (t,
J=7.2 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.24 (d, J=3.2 Hz, 1H), 6.71
(d, J=3.2 Hz, 1H), 4.47 (t, J=5.2 Hz, 1H), 4.36 (m, 3H), 2.29-2.16
(m, 2H); MS (ESI): 305 (M+H.sup.+).
Preparation of 4-(4-fluoroquinolin-2-yl)-N-methylaniline (T466)
##STR00189##
[0252] tert-Butyl
methyl(4-(4-nitroquinolin-2-yl)phenyl)carbamate
[0253] 4-Nitroquinoline 1-oxide (940 mg, 4.9 mmol) in chloroform
(12 mL) was cooled to 0.degree. C. To this solution, POBr.sub.3
(1.77 g, 6.2 mmol) was added in small portions. The mixture was
stirred at 0.degree. C. for 2 h and diluted with DCM (50 mL), and
poured on ice (50 g). To this suspension, 1 M NaOH was added to
adjust pH to about 9. Layers were separated and the organic layer
was washed with water (2.times.50 mL) and dried over MgSO.sub.4 and
concentrated. The crude product was purified by silica
chromatography to afford 2-bromo-4-nitroquinoline as a yellow solid
(620 mg, 50%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.63-7.94
(m, 2H), 8.06 (s, 1H), 8.17 (t, J=7.16, 6.84 Hz, 1H), 8.38 (d,
J=8.76 Hz, 1H).
[0254] 2-Bromo-4-nitroquinoline (50 mg, 0.2 mmol) and tert-butyl
methyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
(66 mg, 0.2 mmol) were reacted using General Procedure (A) to
afford tert-butyl methyl(4-(4-nitroquinolin-2-yl)phenyl)carbamate
as a yellow oil (52 mg, 68%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.43-8.40 (m, 2H), 8.28 (m, 1H), 8.19 (m, 2H), 7.87 (m,
1H), 7.73 (m, 1H), 7.46 (m, 2H), 3.35 (s, 3H), 1.59 (s, 3H), 1.50
(s, 9H); MS (ESI): 380 (M+H.sup.+).
4-(4-fluoroquinolin-2-yl)-N-methylaniline T466
[0255] tert-Butyl methyl(4-(4-fluoroquinolin-2-yl)phenyl)carbamate
was prepared using general procedure M from tert-butyl
methyl(4-(4-nitroquinolin-2-yl)phenyl)carbamate (10 mg, 0.026 mmol)
and KF (60 mg, 1 mmol). tert-butyl
(4-(4-fluoroquinolin-2-yl)phenyl)(methyl)carbamate was isolated as
a clear oil (4.5 mg, 49%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.16 (m, 1H), 8.12-8.07 (m, 3H), 7.78 (m, 1H), 7.59-7.54
(m, 2H), 7.41 (m, 1H), 3.32 (s, 3H), 1.48 (s, 9H); MS (ESI): 353
(M+H.sup.+).
[0256] tert-Butyl(4-(4-fluoroquinolin-2-yl)phenyl)(methyl)carbamate
(4.5 mg, 0.013 mmol) was treated with TFA by using general
procedure P. The crude product was purified by HPLC to afford T466
as an orange-colored solid (1.8 mg, 55%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.57 (d, J=8.4 Hz, 1H), 8.02-7.97 (m, 3H),
7.75 (t, J=7.6 Hz, 1H), 7.56 (d, J=10.0 Hz, 1H), 6.72 (d, J=8.4 Hz,
2H), 2.95 (s, 3H); MS (ESI): 253 (M+H.sup.+).
Preparation of N-Methyl-4-(quinolin-3-yl)aniline (T477)
##STR00190##
[0258] 3-Bromoquinoline (42 mg, 0.2 mmol) and tert-butyl
methyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
(66 mg, 0.2 mmol) were reacted using General Procedure (A) to
afford tert-butyl methyl(4-(quinolin-3-yl)phenyl)carbamate a clear
wax (44 mg, 66%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.18
(d, J=2.4 Hz, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.15 (m, 1H), 7.89 (m,
1H), 7.73 (m, 1H), 7.68 (m, 2H), 7.59 (m, 1H), 7.42 (m, 2H), 3.3
(s, 3H), 1.50 (s, 9H); MS (ESI): 335 (M+H.sup.+).
[0259] tert-Butyl methyl(4-(quinolin-3-yl)phenyl)carbamate (44 mg,
0.13 mmol) was then treated with TFA by using the general procedure
P. The crude product was purified by HPLC to afford T477 as an
orange-colored wax (13 mg, 29%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 9.46 (d, J=2.0 Hz, 1H), 8.79 (d, J=1.6 Hz, 1H), 8.47 (d,
J=8.4 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.95 (m, 1H), 7.83 (m, 1H),
7.62 (m, 2H), 6.82 (m, 2H), 2.94 (s, 3H); MS (ESI): 235
(M+H.sup.+).
Preparation of
##STR00191##
[0261] 5-(4-Fluoroquinolin-2-yl)-N,N-dimethylpyridin-2-amine T480
was prepared using general procedure M from
N,N-dimethyl-5-(4-nitroquinolin-2-yl)pyridin-2-amine (4 mg, 0.014
mmol) and KF (16 mg, 0.28 mmol). The crude product was purified by
silica chromatography to afford
5-(4-fluoroquinolin-2-yl)-N,N-dimethylpyridin-2-amine as a
light-brown solid (1.4 mg, 37%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.89 (d, J=2.8 Hz, 1H), 8.36 (dd, J=11.6 Hz, 1H), 8.09 (m,
1H), 8.04 (m, 1H), 7.73 (m, 1H), 7.51 (m, 1H), 7.47 (d, J=11.6 Hz,
1H), 6.64 (d, J=9.2 Hz, 1H), 3.19 (s, 6H); MS (ESI): 268
(M+H.sup.+).
Preparation of
##STR00192##
[0263] 4-(4-Fluoroquinolin-2-yl)aniline T492 was prepared by using
general procedure M from 4-(4-Nitroquinolin-2-yl)aniline (6 mg,
0.02 mmol) and KF (26 mg, 0.45 mmol), The crude product was
purified by silica chromatography to afford
4-(4-fluoroquinolin-2-yl)aniline as a yellow solid (2 mg, 42%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.11 (m, 1H), 8.03 (m,
1H), 8.00 (m, 2H), 7.72 (m, 1H), 7.51 (m, 1H), 7.48 (d, J=11.6 Hz,
1H), 6.80 (m, 2H), 3.93 (br s, 2H); MS (ESI): 239 (M+H.sup.+).
Preparation of
##STR00193##
[0265] 4-(Isoquinolin-3-yl)-N,N-dimethylaniline T500 was by
prepared using general procedure A from 3-chloroisoquinoline (41
mg, 0.25 mmol) and (4-(dimethylamino)phenyl)boronic acid (41 mg,
0.25 mmol). The product T500 was obtained as a white solid (11 mg,
17%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.27 (s, 1H), 8.05
(m, 2H), 7.94 (s, 1H), 7.93 (d, J=6.8 Hz, 1H), 7.81 (d, J=8.4 Hz,
1H), 7.63 (m, 1H), 7.49 (m, 1H), 6.84 (m, 2H), 3.03 (s, 6H); MS
(ESI): 249 (M+H.sup.+).
Preparation of
##STR00194##
[0267] 3-Chloroisoquinoline (33 mg, 0.2 mmol) and tert-butyl
methyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
(66 mg, 0.2 mmol) were reacted using general procedure A to afford
tert-butyl (4-(isoquinolin-3-yl)phenyl)(methyl)carbamate as a clear
oil (12 mg, 18%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.33
(s, 1H), 8.09 (m, 2H), 8.05 (s, 1H), 7.99 (m, 1H), 7.87 (m, 1H),
7.69 (m, 1H), 7.58 (m, 1H), 7.38 (m, 2H), 3.32 (s, 3H), 1.48 (s,
9H); MS (ESI): 335 (M+H.sup.+).
[0268] 4-(Isoquinolin-3-yl)-N-methylaniline T501 was prepared using
general procedure K. The reaction was performed on a 12 mg scale of
tert-Butyl (4-(isoquinolin-3-yl)phenyl)(methyl). T501 was isolated
as a off-white solid (7 mg, 72%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 9.71 (s, 1H), 8.68 (s, 1H), 8.47 (d, J=8.4 Hz,
1H), 8.29 9d, J=8.0 Hz, 1H), 8.19 (m, 1H), 8.01-7.95 (m, 3H), 7.34
9d, J=8.8 Hz, 2H), 3.02 (s, 3H); MS (ESI): 235 (M+H.sup.+).
Preparation of
##STR00195##
[0270] N,N-Dimethyl-4-(quinolin-7-yl)aniline T504 was prepared
using general procedure A from 7-bromoquinoline (52 mg, 0.25 mmol)
and (4-(dimethylamino)phenyl)boronic acid (41 mg, 0.25 mmol). The
product T504 was obtained as a yellow solid (52 mg, 83%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.89 (dd, J=4.4, 1.6 Hz, 1H),
8.28 (m, 1H), 8.12 (dq, J=8.4, 0.8 Hz, 1H), 7.81 (d, J=1.2 Hz, 2H),
7.68 (m, 2H), 7.33 (dd, J=8.4, 4.4 Hz, 1H), 6.84 (m, 2H), 3.00 (s,
3H); MS (ESI): 249 (M+H.sup.+).
Preparation of
##STR00196##
[0272] N,N-Dimethyl-4-(quinolin-6-yl)aniline T505 was prepared
using general procedure A from 6-bromoquinoline (52 mg, 0.25 mmol)
and (4-(dimethylamino)phenyl)boronic acid (41 mg, 0.25 mmol). The
product T505 was obtained as a yellow solid (42 mg, 67%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.85 (dd, J=4.4, 1.6 Hz, 1H),
8.15 (m. 2H), 7.97 (dd, J=8.4, 1.6 Hz, 1H), 7.92 (d, J=2.0 Hz, 1H),
7.63 (m, 2H), 7.38 (dd, J=8.4, 4.4 Hz, 1H), 6.84 (m, 2H), 3.01 (s,
6H); MS (ESI): 249 (M+H.sup.+).
Preparation of
##STR00197##
[0274] N-Methyl-4-(quinolin-6-yl)aniline T514 was prepared using
general procedure A from 6-bromoquinoline (41 mg, 0.2 mmol) and
N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (46
mg, 0.2 mmol). The product T514 was obtained as a black solid (20
mg, 43%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.86 (dd,
J=4.4, 1.6 Hz, 1H), 8.18 9m, 1H), 8.13 (d, J=9.2 Hz, 1H), 7.96 (dd,
J=8.8, 2.4 Hz, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.59 (m, 2H), 7.40 (dd,
J=8.4, 4.4 Hz, 1H), 6.74 (m, 2H), 2.91 (s, 3H); MS (ESI): 235
(M+H.sup.+).
Preparation of
##STR00198##
[0276] N-Methyl-4-(quinolin-7-yl)aniline T515 was prepared using
general procedure A from 7-bromoquinoline (41 mg, 0.2 mmol) and
N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (46
mg, 0.2 mmol). T515 was obtained as a yellow wax (18 mg, 38%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.90 (dd, J=4.0, 1.2 Hz,
1H), 8.26 (m, 1H), 8.13 (m, 1H), 7.82-7.81 (m, 2H), 7.64 (m, 2H),
7.34 (dd, J=8.4, 4.4 Hz, 1H), 6.73 (m, 2H), 2.90 (s, 3H); MS (ESI):
235 (M+H.sup.+).
Preparation of
##STR00199##
[0278] 4-(6-Methoxyquinolin-2-yl)-N-methylaniline T523 was prepared
using general procedure A from 2-chloro-6-methoxyquinoline (39 mg,
0.2 mmol) and
N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (46
mg, 0.2 mmol). T523 was obtained as a brown solid (27 mg, 51%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.03-7.99 (m, 4H), 7.75
(d, J=8.4 Hz, 1H), 7.33 (dd, J=9.2, 2.8 Hz, 1H), 7.04 (d, J=2.8 Hz,
1H), 6.71 (m, 2H), 3.92 (s, 3H), 2.89 (s, 3H); MS (ESI): 235
(M+H.sup.+).
Preparation of
##STR00200##
[0280] 1-(4-(6-Fluoropyridin-3-yl)phenyl)ethanone T405 was prepared
using general procedure A from 1-(4-bromophenyl)ethanone (117 mg,
0.5 mmol) and
2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(100 mg, 0.5 mmol). T405 was obtained as a yellow solid (64 mg,
59%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.47 (m, 1H), 8.06
9m, 2H), 8.04-7.99 (m, 1H), 7.65 (m, 2H), 7.05 (ddd, J=8.4, 3.2,
0.8 Hz, 1H), 2.65 (s, 3H); MS (ESI): 216 (M+H.sup.+).
Preparation of
5-((1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)pyridin-2-amine
(T568)
##STR00201##
[0282] tert-Butyl(5-ethynylpyridin-2-yl)carbamate was prepared from
tert-butyl (5-((trimethylsilyl)ethynyl)pyridin-2-yl)carbamate (200
mg, 0.69 mmol) using general procedure J. The product was obtained
as a white solid (98 mg, 65%). .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 8.44 (dd, J=2.0, 0.8 Hz, 1H), 7.69 (dd, J=8.4, 2.4 Hz, 1H),
7.33 (dd, J=8.4, 0.8 Hz, 1H), 6.09 (s, 1H), 3.17 (s, 1H), 1.26 (s,
9H).
[0283] tert-Butyl(5-ethynylpyridin-2-yl)carbamate (22 mg, 0.1 mmol)
and 2-bromo-1-(2-fluoroethyl)-1H-benzo[d]imidazole (24.3 mg, 0.1
mmol) was reacted using general procedure B to afford tert-butyl
(5-((1-(2-fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)pyridin-2-yl)carb-
amate (12 mg, 31%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.51 (d,
J=1.6 Hz, 1H), 8.47 (m, 1H), 7.88 (dd, J=8.8, 2.4 Hz, 7.40-7.34 (m,
3H), 7.31 (d, J=12.8 Hz, 6.22 (s, 1H), 4.80 (t, J=4.8 Hz, 1H), 4.68
(t, J=4.8 Hz, 1H), 4.53 (m, 1H), 4.47 (m, 1H), 1.55 (s, 9H).
[0284]
5-((1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)pyridin-2-am-
ine was prepared using general procedure P from tert-butyl
(5-((1-(2-fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)pyridin-2-yl)carb-
amate (10 mg, 0.026 mmol). The product T568 was obtained as a
yellow solid (0.5 mg, 5%). .sup.1H NMR (300 MHz, MeOH-d4): .delta.
8.32 (br s, 1H), 8.01 (dd, J=9.2, 1.6 Hz, 1H), 7.71-7.66 (m, 2H),
7.45 (t, J=7.2 Hz, 1H), 7.412 (m, 1H), 6.98 (d, J=9.2 Hz, 1H),
4.89-4.76 (m, 4H); MS (ESI): 281 (M+H.sup.+).
Preparation of
##STR00202##
[0286]
tert-Butyl-4-(6-methoxyquinolin-2-yl)phenyl)(N-methyl)carbamate
T406 was prepared using general procedure A. Reaction was performed
on a 0.1 g scale. T406 was isolated as off white solid (0.118 g,
62%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.10-8.07 (m, 4H),
7.80 (d, J=8.4 Hz, 1H), 7.36 (dt, J=8.4, 2.0 Hz, 3H), 7.07 (d,
J=2.8 Hz, 1H), 3.93 (s, 3H), 3.30 (s, 3H), 1.46 (s, 9H), 2.56 (br
s, 2H); MS (ESI): 365.2 (M+H.sup.+).
Preparation of
##STR00203##
[0288] 4-(6-Methoxyquinolin-2-yl)-N-methylaniline bis TFA salt T407
was prepared using general procedure P. Reaction was performed on a
0.045 g scale. The crude mixture was purified by HPLC (Acetnitrile:
H.sub.2O: 0.05% TFA) system. T407 was isolated as an orange solid
(0.018 g, 33%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.72 (d,
J=9.2 Hz, 1H), 8.17 (d, J=9.2 Hz, 1H), 8.10 (d, J=9.2 Hz, 1H), 7.95
(dt, J=9.2, 2.8 Hz, 2H), 7.64 (dd, J=9.2, 2.8 Hz, 1H), 7.56 (d,
J=2.8 Hz, 1H), 6.81 (dt, J=9.2, 2.8 Hz, 2H), 3.98 (s, 3H), 2.89 (s,
3H); MS (ESI): 265.1 (M+H.sup.+).
Preparation of
##STR00204##
[0290] tert-Butyl 4-(6-fluoroethoxy)quinolin-2-yl)-N-methylaniline
carbamate T408 was prepared using general procedure A and general
procedure C sequentially. Reaction was performed on a 0.042 g
scale. T408 was isolated as an off white solid (0.070 g, 76%, in
two steps). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.10-8.08
(m, 4H), 7.80 (d, J=8.8 Hz, 1H), 7.40 (dd, J=9.2, 2.8 Hz, 1H), 7.37
(br d, J=8.8 Hz, 2H), 7.08 (d, J=2.4 Hz, 1H), 4.88 (t, J=4.0 Hz,
1H), 4.76 (t, J=4.0 Hz, 1H), 4.37 (t, J=4.4 Hz, 1H), 4.30 (t, J=4.4
Hz, 1H), 3.30 (s, 3H), 1.46 (s, 9H); MS (ESI): 397.2
(M+H.sup.+).
Preparation of
##STR00205##
[0292] 4-(6-Fluoroethoxy)quinolin-2-yl)-N-methylaniline
dihydrochloride T409 was prepared using general procedure K.
Reaction was performed on a 0.045 g scale. T409 was isolated as an
orange solid (0.035 g, 83%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 8.74 (d, J=8.4 Hz, 1H), 8.18 (dd, J=15.2, 9.2 Hz, 2H), 7.98
(d, J=7.2 Hz, 2H), 7.71 (br d, J=9.2 Hz, 1H), 7.61 (br s, 1H), 6.88
(dt, J=9.2, 2.8 Hz, 2H), 4.84 (t, J=4.0 Hz, 1H), 4.74 (t, J=4.0 Hz,
1H), 4.46 and 4.39 (t, J=4.0 Hz, 1H), 2.91 (s, 3H); MS (ESI): 297.1
(M+H.sup.+).
Preparation of
##STR00206##
[0294] tert-Butyl(4-(quinolin-2-yl)phenyl)carbamate T410 was
prepared using general procedure A. Reaction was performed on a
0.164 g scale. T410 was isolated as an off white solid (0.203 g,
64%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.16 (t, J=8.0 Hz,
2H), 8.12 (dt, J=8.8, 2.4 Hz, 2H), 7.83 (d, J=8.4 Hz, 1H), 7.79
(dd, J=8.0, 1.2 Hz, 1H), 7.69 (ddd, J=8.4, 7.2, 1.6 Hz, 1H),
7.55-7.47 (m, 3H), 6.63 (br s, 1H), 1.53 (s, 9H); MS (ESI): 321.1
(M+H.sup.+).
Preparation of
##STR00207##
[0296] N-(2-Fluoroethyl)-4-(quinolin-2-yl)aniline dihydrochloride
T411 was prepared using general procedure A and general procedure D
sequentially. Reaction was performed on a 0.036 g scale. T411 was
isolated as an orange solid (0.018 g, 48%, in two steps). .sup.1H
NMR (400 MHz, CD.sub.3OD): .delta. 9.11 (br s, 1H), 8.72 (d, J=8.8
Hz, 1H), 8.38 (dt, J=9.2, 2.8 Hz, 1H), 8.10 (d, J=8.8 Hz, 2H),
8.007 (td, J=8.8, 1.6 Hz, 1H), 7.78 (td, J=8.8, 1.6 Hz, 1H), 6.83
(dt, J=9.2, 2.8 Hz, 2H), 4.68 (t, J=4.8 Hz, 1H), 4.56 (t, J=4.8 Hz,
1H), 3.57 (q, J=4.8 Hz, 1H), 3.51 (q, J=4.8 Hz, 1H); MS (ESI):
267.1 (M+H.sup.+).
Preparation of
##STR00208##
[0298]
tert-butyl-(2-(2-(2-azidoethoxy)ethoxy)ethyl)(4-(quinolin-2-yl)phen-
yl-carbamate (AS-5306-190 Boc) was prepared using general procedure
D. Reaction was performed on 0.020 g scale. AS-5332-190 Boc was
isolated as an off semi solid (0.020, 70%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.21 (br d, J=8.8 Hz, 2H), 8.11 (dt, J=8.8,
2.0 Hz, 2H), 7.85 (d, J=8.4 Hz, 1H), 7.81 (dd, J=9.2, 1.6 Hz, 1H),
7.71 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.51 (ddd, J=8.4, 7.2, 1.2 Hz,
1H), 7.40 (br d, J=8.4 Hz, 2H), 3.86 (t, J=6.0 Hz, 2H), 3.65 (t,
J=6.0 Hz, 2H), 3.62-3.59 (m, 6H), 3.34 (t, J=5.2 Hz, 2H), 1.44 (s,
9H); MS (ESI): 478.1 (M+H.sup.+).
Preparation of
##STR00209##
[0300]
N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)(4-(quinolin-2-yl)aniline T412
was prepared using general procedure K. Reaction was performed on a
0.020 g scale. The crude product was neutralized with NaHCO.sub.3
and purified. T412 was isolated as yellow oil (0.010 g, 63%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.26-8.14 (m, 4H),
7.88-7.72 (m, 3H), 7.57-7.48 (m, 1H), 7.45 (d, J=8.0 Hz, 1H), 6.75
(d, J=8.8 Hz, 2H), 3.74 (t, J=4.8 Hz, 2H), 3.70-3.60 (m, 6H), 3.39
(t, J=5.2 Hz, 4H); MS (ESI): 378.1 (M+H.sup.+).
Preparation of
##STR00210##
[0302] 4-(Isoquinolin-1-yl)-N,N-dimethylaniline T420 was prepared
using general procedure A. Reaction was performed on a 0.105 g
scale. T420 was isolated as off white solid (0.115 g, 72%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.55 (d, J=5.6 Hz, 1H), 8.25 (d,
J=8.8 Hz, 1H), 7.85 (d, J=7.6 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H), 7.66
(dt, J=8.8, 2.0 Hz, 2H), 7.53 (d, J=6.0 Hz, 1H), 7.54 (t, J=7.6 Hz,
1H), 6.86 (dt, J=8.8, 2.0 Hz, 2H), 3.05 (s, 6H); MS (ESI): 249.1
(M+H.sup.+).
Preparation of
##STR00211##
[0304] tert-Butyl(4-(isoquinolin-1-yl)phenyl)-N-methyl)carbamate
T426 was prepared using general procedure A. Reaction was performed
on a 0.082 g scale. T426 was isolated as a colorless oil (0.0.78 g,
46%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.56 (dd, J=6.8,
0.8 Hz, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.84 (d, J=8.4 Hz, 1H),
7.67-7.64 (m, 3H), 7.60 (d, J=5.6 Hz, 1H), 7.50 (td, J=8.4, 1.6 Hz,
1H), 7.39 (br d, J=8.8 Hz, 2H), 3.31 (s, 3H), 1.47 (s, 9H); MS
(ESI): 335.1 (M+H.sup.+).
Preparation of
##STR00212##
[0306] 4-(Isoquinolin-1-yl)phenyl)-N-methylaniline dihydrochloride
T427 was prepared using general procedure K. Reaction was performed
on a 0.048 g scale. T427 was isolated as yellow solid (0.038 g,
72%). .sup.1H NMR (400 MHz, CD.sub.3CN): .delta. 8.42 (d, J=7.6 Hz,
1H), 8.35 (d, J=6.4 Hz, 1H), 8.24 (d, J=8.0 Hz, 1H), 8.18 (d, J=7.2
Hz, 1H), 8.14 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.94 (ddd, J=8.4, 7.2,
1.2 Hz, 1H), 7.72 (dt, J=8.8, 2.0 Hz, 2H), 7.08 (d, J=8.8 Hz, 2H),
2.93 (s, 3H); MS (ESI): 235.1 (M+H.sup.+).
Preparation of
##STR00213##
[0308] 3(4-(Quinolin-2-yl)phenyl)oxazolidin-2-one T428 was isolated
as a by-product during N-alkylation of T-410 using NaH as the base
(0.010 g, 33%); .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.20
(dt, J=9.2, 2.4 Hz, 2H), 8.15 (d, J=8.4 Hz, 2H), 7.86 (d, J=8.4 Hz,
1H), 7.81 (dd, J=9.2, 1.2 Hz, 1H), 7.71 (ddd, J=8.4, 7.2, 1.2 Hz,
1H), 7.70 (dt, J=9.2, 2.4 Hz, 2H), 7.51 (ddd, J=8.4, 7.2, 1.2 Hz,
1H), 4.52 (t, J=8.0 Hz, 2H), 4.14 (t, J=8.0 Hz, 2H); MS (ESI):
291.1 (M+H.sup.+).
Preparation of
##STR00214##
[0310]
N-(2-(2-(2-Fluoroethoxy)ethoxy)ethyl-4-(quinolin-2-yl)aniline T442
was prepared using general procedure D. Reaction performed on a
0.031 g scale. T442 was isolated as a yellow oil (0.015 g, 42%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.12 (d, J=8.0 Hz, 2H),
8.05 (dd, J=8.0 Hz, 2H), 7.79 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.0 Hz,
1H), 7.66 (dt, J=8.4, 1.6 Hz, 1H), 7.45 (dt, J=8.4, 1.6 Hz, 1H),
6.75-6.72 (m, 3H), 4.64 (m, 1H), 4.52 (m, 1H), 4.38 (br s, 1H),
3.80-3.67 (m, 8H), 3.38 (m, 2H), MS (ESI): 355.2 (M+H.sup.+).
Preparation of
##STR00215##
[0312] 2-Fluoro-4-(quinolin-2yl)aniline T445 was prepared using
general procedure A. Reaction was performed on a 0.090 g scale.
T445 was isolated as white solid (0.120 g, 91%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 8.17 (d, J=8.8 Hz, 2H), 7.92 (dd, J=12.8,
2.0 Hz, 1H), 7.82-7.62 (m, 3H), 7.70 (t, J=8.0 Hz, 1H), 7.49 (t,
J=8.0 Hz, 1H), 6.88 (t. J=8.0 Hz, 1H), 3.95 (br s, 2H); MS (ESI):
239.1 (M+H.sup.+).
Preparation of
##STR00216##
[0314] A mixture of T445 (0.024 g, 0.95 mmol) and paraformaldehyde
(0.06 g, 2.0 eq) in DCE-AcOH (10:1, 5 ml) was stirred at room
temperature for 2 hrs, and then Sodium triacetoxyborohydride (0.061
g, 3.0 eq) was added. The resulting mixture was stirred overnight.
After the reaction was complete, the solvents were removed in vacuo
and product was purified on a Combiflash purification system
(silica gel, 0-10% EtOAc:DCM). T458 was isolated as off white semi
solid (0.005 g, 20%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.14 (dt, J=8.4, 5.6 Hz, 2H), 7.93 (dd, J=13.6, 2.0 Hz, 1H), 7.87
(d, J=8.0 Hz, 1H), 7.78 (d, J=8.4 Hz, 1H), 7.77 (ddd, J=8.4, 6.8,
1.2 Hz, 1H), 7.68 (ddd, J=8.4, 6.8, 1.2 Hz, 1H), 6.77 (t, J=8.8 Hz,
1H), 2.95 (s, 3H); MS (ESI): 253.1 [M+H.sup.+].
Preparation of
##STR00217##
[0316] N-Methyl-2-nitro-4-(quinolin-2-yl)aniline T463 was prepared
using general procedure A. Reaction was performed on a 0.045 g
scale. T463 was isolated as yellow solid (0.068 g, 88%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.97 (d, J=2.4 Hz, 1H), 8.49
(dd, J=9.2, 2.0 Hz, 1H), 8.25 (br s, 1H), 8.19 (d, J=8.4 Hz, 1H),
8.12 (d, J=8.0 Hz, 1H), 7.86 (d, J=8.8 Hz, 2H), 7.80 (dd, J=9.6,
1.2 Hz, 1H), 7.71 (ddd, J=8.8, 6.8, 1.6 Hz, 1H), 7.50 (ddd, J=8.8,
6.8, 1.6 Hz, 1H), 7.00 (d, J=9.2 Hz, 1H), 3.10 (d, J=5.2 Hz, 3H);
MS (ESI): 280.1 (M+H.sup.+).
Preparation of
##STR00218##
[0318] 2-Fluoro-4-(6-methoxyquinolin-2-yl)aniline T467 was prepared
using general procedure A. Reaction was performed on a 0.1 g scale.
T467 was isolated as off white solid (0.112 g, 81%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.08 (br d, J=8.8 Hz, 2H), 7.87 (dd,
J=12.4, 2.0 Hz, 1H), 7.77 (br d, J=7.6 Hz, 1H), 7.73 (d, J=8.8 Hz,
1H), 7.36 (dd, J=9.2, 2.8 Hz, 1H), 7.06 (d, J=2.4 Hz, 1H), 6.87 (t,
J=8.4 Hz, 1H), 3.32 (s, 3H); MS (ESI): 269.0 (M+H.sup.+).
Preparation of
##STR00219##
[0320] N--N-Dimethyl-4-(quinolin-2-yl)aniline T476 was prepared
using general procedure A. Reaction was performed on a 0.092 g
scale. T-467 was isolated as yellow solid (0.120 g, 86%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.11 (br t, J=7.6 Hz, 4H), 7.81
(d, J=8.8 Hz, 1H), 7.75 (dd, J=9.6, 1.6 Hz, 1H), 7.66 (ddd, J=9.2,
6.8, 1.6 Hz, 1H), 7.44 (ddd, J=9.2, 6.8, 1.6 Hz, 1H), 6.82 (dt,
J=9.2, 2.8 Hz, 1H), 3.94 (s, 6H); MS (ESI): 249.1 (M+H.sup.+).
Preparation of
##STR00220##
[0322] 2-Fluoro-4-(6-methoxyquinolin-2-yl)-N-methylaniline
dihydrochloride T483 was prepared using general procedure D and
general procedure K sequentially. Reaction was performed on a 0.030
g scale. T483 was isolated as an orange color solid (0.025 g, 86%
in two steps). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.07 (d,
J=9.2 Hz, 1H), 8.65 (d, J=8.8 Hz, 1H), 8.21 (dd, J=13.6, 2.4 Hz,
1H), 8.09 (dd, J=8.4, 2.4 Hz, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.64
(dd, J=9.6, 2.8 Hz, 1H), 7.49 (d, J=2.8 Hz, 1H), 6.85 (t, J=8.8 Hz,
1H), 3.98 (s, 3H), 2.91 (s, 3H); MS (ESI): 283.1 (M+H.sup.+).
Preparation of
##STR00221##
[0324] 4-(4-Fluoroquinolin-2-yl)-N--N-dimethylaniline T484 was
prepared using general procedure L. Reaction was performed on a
0.030 g scale. T484 was isolated as a light yellow color solid
(0.012 g, 44%), .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.11 (d,
J=8.4 Hz, 1H), 8.04 (dt, J=9.2, 2.8 Hz, 2H), 8.01 (dd, J=8.8, 0.8
Hz, 1H), 7.70 (td, J=8.4, 1.2 Hz, 1H), 7.50-7.46 (m, 2H), 6.80 (dt,
J=9.2, 2.8 Hz, 2H), 3.04 (s, 6H); MS (ESI): 267.1 (M+H.sup.+).
Preparation of
##STR00222##
[0326] 3-Fluoropropyl
4-(4-(6-methoxyquinolin-2-yl)phenyl)piperazine-1-carboxylate T498
was prepared using general procedure E. Reaction performed on a
0.032 g scale. T498 was isolated as off white solid (0.030 g, 70%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.07 (br t, J=8.0 Hz,
4H), 7.78 (d, J=8.8 Hz, 1H), 7.35 (dd, J=9.2, 2.8 Hz, 1H), 7.06 (d,
J=2.8 Hz, 1H), 7.02 (dt, J=9.2, 2.8 Hz, 2H), 4.61 (t, J=6.0 Hz,
1H), 4.49 (t, J=6.0 Hz, 1H), 4.26 (t, J=6.0 Hz, 2H), 3.93 (s, 3H),
3.65 (t, J=4.8 Hz, 4H), 2.57 (t, J=4.8 Hz, 4H), 2.09 (quintet,
J=6.0 Hz, 1H), 2.02 (quintet, J=6.0 Hz, 1H); MS (ESI): 224.1
(M+H.sup.+).
Preparation of
##STR00223##
[0328]
2-(4-(4-(3-Fluoropropyl)piperazin-1-yl)phenyl)-6-methoxyquinoline
T499 was prepared using general procedure E. Reaction performed on
a 0.032 g scale. T499 was isolated as off white solid (0.005 g,
13%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.05 (dt, J=9.2,
2.8 Hz, 2H), 8.03 and 8.00 (d, J=7.6 and 9.2 Hz, 2H), 7.77 (d,
J=8.8 Hz, 1H), 7.54 (dd, J=9.2, 3.2 Hz, 1H), 7.05 (d, J=2.8 Hz,
1H), 7.02 (dt, J=9.2, 2.8 Hz, 2H), 4.60 (t, J=6.0 Hz, 1H), 4.48 (t,
J=6.0 Hz, 1H), 3.92 (s, 3H), 3.34 (br s, 4H), 2.68 (br s, 4H), 2.56
(br s, 2H), 1.97 (br d, J=24.8 Hz, 2H); MS (ESI): 380.2
(M+H.sup.+).
Preparation of
##STR00224##
[0330]
tert-Butyl-(4-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)quinoline-2-yl-
)-phenyl)-carbamate T509 was prepared using general procedure C and
general procedure A sequentially. Reaction was performed on a 0.045
g scale T509 was isolated as off white solid (0.010 g, 8.4% in two
steps). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.05 (dt, J=8.0,
2.0 Hz, 2H), 8.04 (t, J=10.4 Hz, 2H), 7.78 (d, J=8.8 Hz, 1H), 7.49
(d, J=8.4 Hz, 1H), 7.38 (dd, J=9.2, 2.8 Hz, 1H), 7.07 (d, J=2.8 Hz,
1H), 6.59 (s, 1H), 4.61 (td, J=4.4, 0.4 Hz, 1H), 4.49 (td, J=4.4,
0.4 Hz, 1H), 4.26 (t, J=4.8 Hz, 2H), 3.93 (t, J=4.8 Hz, 2H),
3.80-3.76 (m, 3H), 3.74-3.70 (m, 3H), 1.53 (s, 9H); MS (ESI): 471.2
(M+H.sup.+).
Preparation of
##STR00225##
[0332]
4-(6-(2-(2-(Fluoroethoxy)ethoxy)ethoxy)quinoline-2-yl)-N--N-dimethy-
laniline T510 was prepared using general procedure C and general
procedure A sequentially. Reaction was performed on a 0.037 g
scale. T510 was isolated as a light yellow color solid (0.006 g,
7.2% in two steps). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.05
(d, J=8.0 Hz, 2H), 8.0 (d, J=8.0 Hz, 2H), 7.76 (d, J=8.8 Hz, 1H),
7.34 (dd, J=9.2, 2.8 Hz, 1H), 7.05 (d, J=2.4 Hz, 1H), 6.81 (m, 1H),
4.61 (m, 1H), 4.49 (m, 1H), 4.25 (t, J=4.4 Hz, 2H), 3.93 (t, J=4.4
Hz, 2H), 3.80-3.70 (m, 5H), 3.02 (s, 6H); MS (ESI): 399.2
(M+H.sup.+).
Preparation of
##STR00226##
[0334] 2-(6-Fluoropyridin-3-yl)-N--N-dimethylquinolin-6-amine T513
was prepared using general procedure A. Reaction was performed on a
0.0.036 g scale. T513 was isolated as a yellow color solid (0.015
g, 54%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.86 (m, 1H),
8.57 (td, J=10.0, 2.4 Hz, 1H), 8.02 (d, J=8.8 Hz, 1H), 7.97 (d,
J=9.6 Hz, 1H), 7.69 (d, J=8.8 Hz, 1H), 7.38 (dd, J=9.2, 2.8 Hz,
1H), 7.02 (dd, J=8.4, 0.8 Hz, 1H), 6.80 (d, J=2.8 Hz, 1H), 3.09 (s,
6H); MS (ESI): 268.1 [M+H.sup.+].
Preparation of
##STR00227##
[0336] 2-(4-(4-(2-Fluoroethyl)piperizin-1-yl)-6-methoxyquinoline
T519 was prepared using general procedure E. Reaction performed on
a 0.050 g scale. T519 was isolated as a off white solid (0.010 g,
17.5%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.01 (dt, J=6.8,
2.0 Hz, 2H), 7.95 (d, J=10.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 1H), 7.27
(dd, J=9.2, 2.4 Hz, 1H), 6.99 (d, J=2.4 Hz, 1H), 6.96 (dt, J=6.8,
2.0 Hz, 2H), 4.62 (t, J=4.0 Hz, 1H), 4.50 (t, J=4.0 Hz, 1H), 3.86
(s, 3H), 3.27 (t, J=5.2 Hz, 4H), 2.75 (t, J=5.2 Hz, 1H), 2.69-2.66
(m, 5H); MS (ESI): 366.1 (M+H.sup.+).
Preparation of
##STR00228##
[0338]
2-(4-(4-(2-(2-Fluoroethoxy)ethyl)piperazin-1-yl)phenyl)-6-methoxyqu-
inoline T530 was prepared using general procedure D. Reaction
performed on a 0.032 g scale. T530 was isolated as off white semi
solid (0.004 g, 9%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.04 (dt, J=8.0, 1.2 Hz, 2H), 8.01 (dd, J=10.4, 7.6 Hz, 2H), 7.77
(d, J=8.0 Hz, 1H), 7.32 (dd, J=9.2, 2.8 Hz, 1H), 7.05 (d, J=2.8 Hz,
1H), 7.01 (dt, J=8.0, 1.2 Hz, 2H), 4.62 (t, J=4.4 Hz, 1H), 4.50 (t,
J=4.4 Hz, 1H), 3.92 (s, 3H), 3.78 (t, J=4.4 Hz, 1H), 3.72-3.64 (m,
4H), 3.19 (t, J=4.8 Hz, 2H), 2.70 (m, 3H); MS (ESI): 454.1
(M+H.sup.+).
Preparation of
##STR00229##
[0340] 2-(4-Dimethylamino)phenyl)quinoline-6-ol T531 was prepared
using general procedure A. Reaction was performed on a 0.0.236 g
scale. T531 was isolated as a yellow solid (0.218 g, 53%). .sup.1H
NMR (400 MHz, CD.sub.3CN): .delta. 8.08 (td, J=8.4, 2.0 Hz, 3H),
7.86 (dd, J=8.8, 5.2 Hz, 1H), 7.28 (ddd, J=8.8, 5.6, 2.8 Hz, 1H),
7.14 (d, J=2.8 Hz, 1H), 6.86 (dt, J=8.8, 2.0 Hz, 2H), 3.03 (s, 6H);
MS (ESI): 365.1 (M+H.sup.+).
Preparation of
##STR00230##
[0342] 4-Fluoro-2-(4-(4-methylpiperazin-1yl)phenylquinoline T559
was prepared using general procedure L. Reaction was performed on a
0.005 g scale. T559 was isolated as light yellow solid (0.004 g,
89%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.06-7.96 (m, 4H),
7.66 (td, J=8.4, 1.6 Hz, 1H), 7.44 (td, J=8.4, 1.6 Hz, 1H), 7.43
(d, J=11.2 Hz, 1H), 6.96 (dt, J=9.2, 2.4 Hz, 2H), 3.28 (t, J=4.8
Hz, 4H), 2.54 (t, J=4.8 Hz, 4H), 2.31 (s, 3H); MS (ESI): 322.1
(M+H.sup.+).
Preparation of
##STR00231##
[0344] 6-Methoxy-2-(4-(piperazine-1-yl)phenyl)quinoline AS-5332-52
was prepared using general procedure A. Reaction was performed on a
0.194 g scale. AS-5332-52 was isolated as a off white solid (0.269
g, 84%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.01 (dt,
J=8.0, 4.0 Hz, 2H), 7.98 (d, J=9.2 Hz, 1H), 7.95 (d, J=9.2 Hz, 1H),
7.72 (d, J=8.8 Hz, 1H), 7.27 (dd, J=8.0, 4.0 Hz, 1H), 7.01-6.96 (m,
3H), 3.87 (s, 2H), 3.24 (td, J=5.2, 2.8 Hz, 4H), 3.06 (td, J=5.2,
2.8 Hz, 4H); MS (ESI): 320.1 (M+H.sup.+).
Preparation of
##STR00232##
[0346] 2-(4-(4-Methylpiperazin-1-yl)phenyl)-4-nitroquinoline
AS-5332-80 was prepared using general procedure A. Reaction was
performed on a 0.06 g scale. AS-5332-80 was isolated as red color
solid (0.062 g, 75%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.35 (dd, J=9.2, 0.4 Hz, 2H), 8.25 (dd, J=9.2, 0.4 Hz, 1H), 8.13
(dt, J=9.2, 2.0 Hz, 2H). 7.80 (td, J=8.0, 1.2 Hz, 1H), 7.64 (td,
J=8.0, 1.2 Hz, 1H), 7.03 (dt, J=8.8, 2.0 Hz, 2H), 3.36 (t, J=6.4
Hz, 4H), 2.59 (t, J=6.4 Hz, 4H), 2.36 (s, 3H); MS (ESI): 349
(M+H.sup.+).
Preparation of
##STR00233##
[0348] N-methyl-N-(2-nitro-4-(quinolin-2-yl)phenyl)formamide
AS-5332-30. A mixture of acetic anhydride (0.600 g, 22 equiv.) and
HCO.sub.2H (0.252 g, 22 equiv.) was heated at 60.degree. C. for 15
min. To this mixture was added a solution of T463 (0.078 g) in DCM
(5 mL). The resulting mixture was heated at 80.degree. C. for 2
days. The volatiles were removed in vacuo. The crude product was
purified on a Combiflash purification system (silica gel, 0-20%
EtOAc:DCM). AS-5332-30 was isolated as yellow solid (0.054 g, 70%).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.84 and 8.82 (d, J=2.0
Hz, 1H each), 8.52-8.49 (m, 1H), 8.29 and 8.27 (d, J=8.8 Hz, 1H
each), 8.26 (s, 1H), 8.18 and 8.15 (d, J=8.8 Hz, 1H each), 7.89 (d,
J=8.4 Hz, 1H), 7.88-7.84 (m, 2H), 7.79-7.75 (m, 1H), 7.61-7.57 (m,
1H), 7.47 (d, J=8.4 Hz, 1H), 3.7 and 3.28 (s, 3H each); MS (ESI):
308.1 (M+H.sup.+).
Preparation of
##STR00234##
[0350] tert-Butyl
2-fluoro-4-(6-methoxyquinolin-2-yl)phenyl)carbamate AS-5332-32): To
a solution of T467 (0.050 g, 0.186 mmol) in THF (3.0 mL) was added
Boc anhydride (0.82 g, 0.373 mmol). The resulting reaction mixture
was heated at 100.degree. C. overnight. The volatiles were removed
in vacuo and residue was purified on a Combiflash purification
system silica gel, 0-20% EtOAc:DCM). AS-5332-32 was isolated as off
white solid (0.040 g, 58%). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta. 8.22 (br, 1H), 8.09 (d, J=8.4 Hz, 1H), 8.12 (d, J=9.2 Hz
1H), 7.98 (dd, J=8.8, 2.0 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.77 (d,
J=8.8 Hz, 1H), 7.35 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.06 (d, J=2.8 Hz
1H), 6.83 (br, 1H), 3.93 (s, 3H), 1.54 (s, 9H); MS (ESI): 369.2
(M+H.sup.+).
Preparation of
##STR00235##
[0352] N--N-Dimethyl-4-(4-nitroquinolin-2-yl)aniline AS-5332-36 was
prepared using general procedure A. Reaction was performed on a
0.126 g scale. AS-5332-36 was isolated as yellow solid (0.103 g,
70%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.34-8.32 (m, 2H),
8.20 (d, J=8.8 Hz, 1H), 8.13 (dt, J=9.2, 2.8 Hz, 2H), 7.78 (ddd,
J=8.4, 7.2, 1.2 Hz, 1H), 7.62 (ddd, J=8.4, 7.6, 1.2 Hz, 1H), 6.82
(br d, J=9.2 Hz, 2H), 3.07 (s, 6H); MS (ESI): 294.1
(M+H.sup.+).
Preparation of
##STR00236##
[0354] 4-(6-Methoxyquinolin-2-yl)-N-methyl-2-nitroaniline
AS-5332-42 was prepared using general procedure A. Reaction was
performed on a 0.050 g scale. AS-5332-42 was isolated as yellow
solid (0.080 g, 100%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.92 (d, J=2.0 Hz 1H), 8.50 (br, 1H), 8.23 (br, 1H), 8.12 (d, J=8.4
Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.37 (dd, J=9.2 Hz, 2.8 Hz, 1H),
7.08 (d, J=2.8 Hz, 1H), 7.00 (d, J=8.8 Hz 1H), 3.94 (s, 3H), 3.11
(d, J=4.8 Hz, 3H); MS (ESI): 310.1 (M+H.sup.+).
Preparation of
##STR00237##
[0356]
N-(4-(6-Methoxyquinolin-2-yl)-2-nitrophenyl)-N-methylformamide
AS-5332-43: A mixture of acetic anhydride (0.305 g, 22 eq) and
HCO.sub.2H (0.137 g, 22 eq) was heated at 60.degree. C. for 15 min.
To this mixture was added a solution of AS-5332-42 (0.042 g) in DCM
(5 mL). The resulting mixture was heated at 80.degree. C. for 3
days. The volatiles were removed in vacuo. The residue was purified
on a Combiflash purification system (silica gel, 0-20% EtOAc:DCM)
to give AS-5332-43 as a yellow solid (0.034 g, 74%). .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta.: 8.80 and 8.76 (d, J=2.0 Hz, 1H),
8.47-8.46 (m, 1H), 8.25 and 8.24 (s, 1H each), 8.17 (t, J=8.4 Hz,
1H), 8.05 (d, J=9.2 Hz, 1H), 7.85 and 7.83 (d, J=5.2 Hz, 1H each),
7.45 (d, J=8.4 Hz, 1H), 7.44-7.40 (m, 1H), 7.11 and 7.10 (d, J=0.8
Hz, 1H each), 3.96 and 3.95 (s, 3H each), 3.46 and 3.27 (s, 3H
each); MS (ESI): 338.1 (M+H.sup.+).
Preparation of
##STR00238##
[0358] tert-Butyl
4-(6-methoxyquinolin-2-yl)-2-nitrophenyl(methyl)carbamate
AS-5332-46: To a solution AS-5332-42 (0.030 g, 0.186 mmol) in THF
(3.0 mL) was added Boc anhydride (0.063 g, 0.0.291 mmol) and DMAP
(0.012 g, 0.097 mmol). The resulting reaction mixture was heated at
100.degree. C. for 30 min. The volatiles were removed in vacuo and
residue was purified on a Combiflash purification system (silica
gel, 0-7% EtOAc:DCM) to afford AS-5332-43 as a off white solid
(0.040 g, 100%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.69
(d, J=2.0 Hz, 1H), 8.40 (d, J=8.0 Hz, 1H), 8.16 (d, J=8.8 Hz, 1H),
8.07 (d, J=9.2 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.44 (d, J=8.0 Hz,
1H), 7.41 (dd, J=9.2, 2.8 1H), 7.10 (d, J=2.8 Hz, 1H), 3.95 (s,
3H), 3.34 (s, 3H), 1.32 (s, 9H); MS (ESI): 410.1 (M+H.sup.+).
Preparation of
##STR00239##
[0360] N,N-dimethyl-5-(6-nitroquinolin-2-yl)pyridin-2-amine
AS-5332-49 was prepared using general procedure A. for Suzuki
coupling (method A) was followed. Reaction was performed on a 0.104
g scale. AS-5332-49 was isolated as a orange red color solid (0.1
g, 68%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.99 (d, J=2.4
Hz, 1H), 8.72 (d, J=2.4 Hz, 1H), 8.44-8.41 (m, 2H), 8.27 (d, J=8.8
Hz, 1H), 8.14 (d, J=8.8 Hz, 1H), 7.93 (d, J=8.8 Hz, 1H), 6.66 (d,
J=8.8 Hz, 1H), 3.21 (s, 6H); MS (ESI): 295.1 (M+H.sup.+).
Preparation of
##STR00240##
[0362] 2-(4-Azidophenyl)quinoline*TFA: T446 To a solution of
4-(quinolin-2-yl)aniline dihydrochloride (29.0 mg, 0.1 mmol) in 1 N
HCl (1 mL) was added NaNO.sub.2 solution (7.0 mg in 0.3 mL water,
0.1 mmol) at 0.degree. C. The mixture was stirred at 0.degree. C.
for 2 hrs before NaN.sub.3 (7.8 mg in 1.0 mL water, 0.12 mmol) was
added at 0.degree. C. The mixture was stirred at 0.degree. C. for 1
hour and concentrated. The residue was purified by HPLC
(acetonitrile/water) to give T446 as a light yellow solid (23.0 mg,
64%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.31 (d, J=8.8 Hz,
1H), 8.10 (m, 2H), 8.00 (m, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.85 (dd,
J=8.2, 1.4 Hz, 1H), 7.69 (m, 1H), 7.50 (m, 1H), 7.16 (m, 2H); MS
(ESI): 247 (M+H.sup.+).
Preparation of
##STR00241##
[0364]
2-(4-(4-(3-Fluoropropyl)-1H-1,2,3-triazol-1-yl)phenyl)quinoline*TFA-
: T443 was prepared using general procedure N. Reaction was
performed on a 4.0 mg scale of T446. T443 was isolated as a brown
solid (2.7 mg, 39%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
8.72 (d, J=8.8 Hz, 1H), 8.50 (s, 1H), 8.34-8.38 (m, 2H), 8.19-8.24
(m, 2H), 8.09-8.14 (m, 3H), 7.94 (m, 1H), 7.74 (m, 1H), 4.59 (t,
J=6.0 Hz, 1H), 4.47 (t, J=6.0 Hz, 1H), 2.94 (t, J=7.6 Hz, 1H), 2.14
(m, 2H); MS (ESI): 333 (M+H.sup.+).
Preparation of
##STR00242##
[0366] 4-(Quinolin-2-ylethynyl)aniline: T444 was prepared using
general procedure B. Reaction was performed on a 16.0 mg scale of
2-chloroquinoline. T444 was isolated as a light yellow solid (6.0
mg, 25%). .sup.1H NMR (400 HMz, CDCl.sub.3): .delta. 8.11 (d, J=8.4
Hz, 2H), 7.79 (dd, J=8.0, 1.4 Hz, 1H), 7.72 (m, 1H), 7.57 (d, J=8.4
Hz, 1H), 7.53 (m, 1H), 7.48 (m, 2H), 6.66 (m, 2H), 3.91 (br s, 2H);
MS (ESI): 245 (M+H.sup.+).
Preparation of
##STR00243##
[0368] N-(4-(Quinolin-2-yl)phenyl)benzene-1,4-diamine*3TFA: T447 To
a solution of 4-(quinolin-2-yl)aniline dihydrochloride (7.6 mg,
0.026 mmol) in DCM (1.0 mL) was added
4-(tert-butoxycarbonylamino)phenylboronic acid (12.4 mg, 0.052
mmol), Cu(OAc).sub.2 (4.8 mg, 0.026 mmol) and triethylamine (0.036
mL, 0.26 mmol). The mixture was stirred at room temperature for 3
hrs. LCMS showed that the desired product was formed. To the
mixture was added 4 N HCl in dioxane (1.0 mL) and stirred for
another hour. The mixture was concentrated in vacuo and purified by
HPLC (acetonitrile/water) to give T447 as a brown solid (4.3 mg,
25%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.77 (d, J=8.8 Hz,
1H), 8.16 (m, 2H), 8.10 (d, J=8.4 Hz, 1H), 8.01 (m, 2H), 7.96 (m,
1H), 7.73 (m, 1H), 7.21-729 (m, 6H); MS (ESI): 312 (M+H.sup.+).
Preparation of
##STR00244##
[0370] N-(3-Fluoropropyl)-4-(quinolin-2-ylethynyl)aniline: T454 was
prepared using general procedure Q. Reaction was performed on a 4.0
mg scale of T444. T454 was isolated as a light yellow solid (2.3
mg, 46%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.11 (d, J=8.4
Hz, 2H), 7.79 (dd, J=8.0, 1.4 Hz, 1H), 7.26-7.58 (m, 4H), 6.59 (m,
2H), 4.66 (t, J=5.6 Hz, 1), 4.54 (t, J=5.6 Hz, 1H), 4.05 (m, 1H),
3.36 (m, 2H), 2.05 (m, 2H); MS (ESI): 305 (M+H.sup.+).
Preparation of
##STR00245##
[0372] 4-((1H-Benzo[d]imidazol-2-yl)ethynyl)aniline: T464 was
prepared using general procedure B. Reaction was performed on a
60.0 mg scale of 2-bromo-1H-benzo[d]imidazole. T464 was isolated as
a light yellow solid (35.9 mg, 51%). .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 7.50 (br s, 2H), 7.31 (m, 2H), 7.24 (m, 2H),
6.64 (m, 2H); MS (ESI): 234 (M+H.sup.+).
Preparation of
##STR00246##
[0374]
4-((1H-Benzo[d]imidazol-2-yl)ethynyl)-N-(3-fluoropropyl)aniline:
T465 was prepared using general procedure Q. Reaction was performed
on a 33.3 mg scale of T464. T465 w isolated as a white solid (8.9
mg, 21%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 7.50 (m, 2H),
7.35 (m, 2H), 7.26 (m, 2H), 6.61 (m, 2H), 4.58 (t, J=5.6 Hz, 1H),
4.46 (t, J=5.6 Hz, 1H), 3.27 (m, 2H), 1.95 (m, 2H); MS (ESI): 294
(M+H.sup.+).
Preparation of
##STR00247##
[0376] 2-(6-(Benzyloxy)naphthalen-2-yl)-1H-benzo[d]imidazole: T469
was prepared using general procedure A. Reaction was performed on a
100 mg scale of 2-bromo-1H-benzo[d]imidazole. T469 was isolated as
a white solid (75.0 mg, 42%). .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 12.95 (s, 1H), 8.63 (d, J=1.2 Hz, 1H), 8.22 (dd, J=8.4, 2.0
Hz, 1H), 7.93 (t, J=9.2 Hz, 2H), 7.64 (m, 1H), 7.50 (m, 4H), 7.40
(m, 2H), 7.34 (m, 1H), 7.29 (m, 1H), 7.17 (m, 2H), 5.24 (s, 2H); MS
(ESI): 351 (M+H.sup.+).
Preparation of
##STR00248##
[0378] 6-(1H-Benzo[d]imidazol-2-yl)naphthalen-2-ol*formate: T470 To
a solution of 2-(6-(benzyloxy)naphthalen-2-yl)-1H-benzo[d]imidazole
(73 mg, 0.21 mmol) in THF (2 mL) was added MeOH (2 mL), Pd--C (10%,
30 mg), and formic acid (0.30 mL). The mixture was flushed with
argon and sealed in a microwave vial. The mixture was heated at
100.degree. C. for 5 minutes in a microwave synthesizer. The
mixture was filtered off Pd--C and the filtrate was concentrated to
give T470 as a white solid (60 mg, 94%). .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 8.46 (t, J=1.0 Hz, 1H), 8.12 (s, 1H), 8.05
(dd, J=8.8, 2.0 Hz, 1H), 7.85 (d, J=9.6 Hz, 1H), 7.79 (d, J=8.8 Hz,
1H), 7.62 (m, 2H), 7.29 (m, 2H), 7.16 (m, 2H); MS (ESI): 261
(M+H.sup.+).
Preparation of
##STR00249##
[0380]
2-(6-(2-Fluoroethoxy)naphthalen-2-yl)-1H-benzo[d]imidazole*TFA:
T473 was prepared using general procedure. Reaction was performed
on a 16 mg scale of T470. T473 was isolated as a light yellow solid
(1.9 mg, 8.6%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.63 (d,
J=1.6 Hz, 1H), 8.01-8.11 (m, 3H), 7.82 (m, 2H), 7.62 (m, 2H), 7.45
(m, 1H), 7.38 (dd, J=9.0, 2.6 Hz, 1H), 4.87 (t, J=4.0 Hz, 1H), 4.75
(t, J=4.0 Hz, 1H), 4.44 (t, J=4.0 Hz, 1H), 4.37 (t, J=4.0 Hz, 1H);
MS (ESI): 307 (M+H.sup.+).
Preparation of
##STR00250##
[0382]
2-(6-(2-Fluoroethoxy)naphthalen-2-yl)-1-(2-fluoroethyl)-1H-benzo[d]-
imidazole*TFA: T474 was prepared using general procedure C.
Reaction was performed on a 16 mg scale T470. T474 was isolated as
a light yellow solid (9.4 mg, 39%). .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 8.41 (d, J=1.6 Hz, 1H), 8.12 (d, J=8.4 Hz,
1H), 8.02 (m, 2H), 7.82-7.89 (m, 2H), 7.71 (m, 2H), 7.47 (d, J=2.4
Hz, 1H), 7.38 (dd, J=9.0, 2.6 Hz, 1H), 4.95-5.00 (m, 2H), 4.86-4.92
(m, 3H), 4.75 (m, 1H), 4.44 (m, 1H), 4.37 (m, 1H); MS (ESI): 353
(M+H.sup.+).
Preparation of
##STR00251##
[0384] 4-((1H-Benzo[d]imidazol-2-yl)ethynyl)-N,N-dimethylaniline:
T481 was prepared using general procedure R. Reaction was performed
on a 26.0 mg scale of T464. T481 was isolated as a light yellow
solid (11.2 mg, 39%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
7.51 (m, 2H), 7.44 (m, 2H), 7.25 (m, 2H), 6.73 (m, 2H), 3.00 (s,
6H); MS (ESI): 262 (M+H.sup.+).
Preparation of
##STR00252##
[0386]
4-((1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)-N,N-dimethy-
laniline: T482 was prepared using general procedure E. Reaction was
performed on a 10.1 mg scale of T481. T482 was isolated as a light
yellow solid (9.6 mg, 81%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 7.76 (m, 1H), 7.48 (m, 2H), 7.37 (m, 1H), 7.28 (m, 2H),
4.87 (t, J=5.2 Hz, 1H), 4.76 (t, J=5.2 Hz, 1H), 4.66 (t, J=5.2 Hz,
1H), 4.60 (t, J=5.2 Hz, 1H), 3.02 (s, 6H); MS (ESI): 308
(M+H.sup.+).
Preparation of
##STR00253##
[0388] 5-(Quinolin-2-yl)pyridin-2-amine: T490 was prepared using
general procedure A. Reaction was performed on a 106 mg scale of
2-chloroquinoline. T490 was isolated as a white solid (135 mg,
94%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.85 (d, J=2.4 Hz,
1H), 8.37 (dd, J=8.4, 2.4 Hz, 1H), 8.18 (d, J=8.8 Hz, 1H), 8.11 (d,
J=8.8 Hz, 1H), 7.79 (m, 2H), 7.71 (m, 1H), 7.50 (m, 1H), 6.65 (dd,
J=8.4, 0.8 Hz, 1H), 4.66 (br s, 2H); MS (ESI): 222 (M+H.sup.+).
Preparation of
##STR00254##
[0390]
N-(2-(2-(2-Fluoroethoxy)ethoxy)ethyl)-N-methyl-5-(quinolin-2-yl)pyr-
idin-2-amine: T502 was prepared using general procedure S. Reaction
was performed on a 7.4 mg scale of T502-precursor. T502 as light
yellow oil (3.8 mg, 73%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.92 (dd, J=2.4, 0.8 Hz, 1H), 8.38 (dd, J=8.8, 2.4 Hz, 1H),
8.15 (d, J=8.4 Hz, 1H), 8.09 (dd, J=8.4, 1.2 Hz, 1H), 7.79 (m, 2H),
7.69 (m, 1H), 7.47 (m, 1H), 6.67 (dd, J=8.8, 0.8 Hz, 1H), 4.61 (t,
J=4.2 Hz, 1H), 4.49 (t, J=4.2 Hz, 1H), 3.86 (t, J=5.8 Hz, 2H),
3.65-3.78 (m, 8H), 3.19 (s, 3H); MS (ESI): 370 (M+H.sup.+).
Preparation of
##STR00255##
[0392] tert-Butyl 5-(quinolin-2-yl)pyridin-2-yl-carbamate T503: To
a solution of 5-(quinolin-2-yl)pyridin-2-amine (130 mg, 0.59 mmol)
in DCM (5 mL) was added Boc.sub.2O (154 mg, 0.71 mmol), DIEA (76
mg, 0.59 mmol) an DMAP (14 mg, 0.11 mmol). The mixture was stirred
at room temperature for 24 hrs. LCMS showed that mono-Boc, di-Boc
product, and starting material were present. The solvent was
removed and the residue was dissolved in a mixture of ethyl acetate
and DCM. As DCM evaporated, and needle crystals were formed. The
crystals were collected by filtration, washed with ethyl acetate,
and dried to give T503 white needles (67 mg, 35%). .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 10.03 (s, 1H), 9.11 (dd, J=2.4, 0.8 Hz,
1H), 8.60 (dd, J=8.8, 2.4 Hz, 1H), 8.43 (d, J=8.8 Hz, 1H), 8.15 (d,
J=8.8 Hz, 1H), 8.04 (dd, J=8.2, 1.0 Hz, 1H), 7.96 (m, 2H), 7.76 (m,
1H), 7.57 (m, 1H), 1.47 (s, 9H); MS (ESI): 322 (M+H.sup.+).
Preparation of
##STR00256##
[0394] 4-((6-Fluoropyridin-3-yl)ethynyl)-N,N-dimethylaniline: T516
was prepared using general procedure B. Reaction was performed on a
90 mg scale of 5-bromo-2-fluoropyridine. T516 was isolated as a
light yellow solid (50 mg, 41%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.34 (m, 1H), 7.85 (m, 1H), 7.39 (m, 2H), 6.90 (m, 1H),
6.66 (m, 2H), 3.00 (s, 6H); MS (ESI): 241 (M+H.sup.+).
Preparation of
##STR00257##
[0396]
N-(2-(2-(2-Fluoroethoxy)ethoxy)ethyl)-5-(quinolin-2-yl)pyridin-2-am-
ine: T525 was prepared using general procedure D. Reaction was
performed on a 22.0 mg scale of T490. T525 was isolated as light
yellow oil (3.3 mg, 9.3%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.86 (d, J=2.4 Hz, 1H), 8.35 (dd, J=8.8, 2.4 Hz, 1H), 8.15
(d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.79 (d, J=8.4 Hz, 2H),
7.69 (m, 1H), 7.48 (m, 1H), 6.57 (dd, J=8.8, 0.8 Hz, 1H), 5.15 (m,
1H), 4.65 (t, J=4.0 Hz, 1H), 4.53 (t, J=4.0 Hz, 1H), 3.81 (t, J=4.0
Hz, 1H), 3.68-3.77 (m, 7H), 6.63 (dd, J=10.6, 5.0 Hz, 2H); MS
(ESI): 356 (M+H.sup.+).
Preparation of
##STR00258##
[0398]
2-((5-((4-(Dimethylamino)phenyl)ethynyl)pyridin-2-yl)(methyl)amino)-
ethanol: T526 was prepared using general procedure M from
2-fluoropyridine derivatives and 2-(methylamino)ethanol. Reaction
was performed on a 45 mg scale of T516. T526 was isolated as a
light yellow solid (40 mg, 72%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.22 (dd, J=2.4, 0.8 Hz, 1H), 7.57 (dd, J=8.8, 2.4 Hz, 1H),
7.37 (m, 2H), 6.65 (m, 2H), 6.90 (d, J=8.8, 0.8 Hz, 1H), 4.58 (br
s, 1H), 3.85 (t, J=4.8 Hz, 2H), 3.74 (t, J=4.8 Hz, 2H), 3.09 (s,
3H), 2.98 (s, 6H); MS (ESI): 296 (M+H.sup.+).
Preparation of
##STR00259##
[0400] 2-(4-(piperazin-1-yl)phenyl)quinoline*3TFA: T535 was
prepared using general procedure A. Reaction was performed on a
17.0 mg scale of 2-chloroquinoline. T535 was isolated as a light
yellow solid (10.0 mg, 42%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 8.74 (d, J=8.8 Hz, 1H), 8.11-8.21 (m, 5H), 7.96 (t, J=7.2
Hz, 1H), 7.75 (t, J=7.2 Hz, 1H), 7.26 (m, 2H), 3.66 (br t, J=5.4
Hz, 4H), 3.40 (br t, J=5.4 Hz, 4H); MS (ESI): 290 (M+H.sup.+).
Preparation of
##STR00260##
[0402]
2-Bromo-1-(4-(4-(quinolin-2-yl)phenyl)piperazin-1-yl)ethanone T536:
To a solution of 2-(4-(piperazin-1-yl)phenyl)quinoline*3TFA (8.6
mg, 0.014 mmol) in DCM (2 mL) was added TEA (9.0 mg, 0.089 mmol),
followed by 2-bromoacetyl bromide (12.0 mg, 0.059 mmol). The
mixture was stirred at room temperature for 1 hour and quenched by
adding NaHCO.sub.3 solution. The DCM layer was separated and
concentrated. The residue was purified by flash chromatography
(silica gel, 0-30% ethyl acetate/DCM) to give T536 as a white solid
(3.7 mg, 66%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.11-8.18
(m, 4H), 7.84 (d, J=8.8 Hz, 1H), 7.80 (dd, J=8.0, 1.6 Hz, 1H), 7.72
(m, 1H), 7.49 (m, 1H), 7.05 (m, 2H), 3.91 (s, 2H), 3.83 (br t,
J=5.2 Hz, 2H), 3.72 (br t, J=5.2 Hz, 2H), 3.39 (br t, J=5.2 Hz,
2H), 3.32 (br t, J=5.2 Hz, 2H); MS (ESI): 410 (M+H.sup.+).
Preparation of
##STR00261##
[0404]
2-Fluoro-1-(4-(4-(quinolin-2-yl)phenyl)piperazin-1-yl)ethanone:
T537 was prepared using general procedure 0. Reaction was performed
on a 2.8 mg scale of T536. T537 was isolated as a white solid (1.9
mg, 80%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.11-8.18 (m,
4H), 7.84 (d, J=8.8 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 7.70 (m, 1H),
7.49 (m, 1H), 7.05 (m, 2H), 5.11 (s, 1H), 4.99 (s, 1H), 3.84 (br s,
2H), 3.67 (br s, 2H), 3.33 (br t, J=5.0 Hz, 2H); MS (ESI): 350
(M+H.sup.+).
Preparation of
##STR00262##
[0406]
N-(3-Fluoropropyl)-4-((1-methyl-1H-benzo[d]imidazol-2-yl)ethynyl)an-
iline: T540 was prepared using general procedure E. Reaction was
performed on a 6.7 mg scale of T465. T540 was isolated as a white
solid (5.9 mg, 84%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.74 (m, 1H), 7.46 (m, 2H), 7.27-7.32 (m, 3H), 6.59 (m, 2H), 4.66
(t, J=5.6 Hz, 1H), 4.54 (t, J=5.6 Hz, 1H), 4.15 (br s, 1H), 3.91
(s, 3H), 3.36 (m, 2H), 2.02 (m, 2H); MS (ESI): 308 (M+H.sup.+).
Preparation of
##STR00263##
[0408]
5-((4-(Dimethylamino)phenyl)ethynyl)-N-(2-(2-(2-fluoroethoxy)ethoxy-
)ethyl)-N-methylpyridin-2-amine: T546 was prepared using general
procedure 0. Reaction was performed on a 30.2 mg scale of
T546-precursor. T546 was isolated as a light yellow gum (11.6 mg,
53%). .sup.1H NMR (400 Hz, CDCl.sub.3): .delta. 8.27 (d, J=2.4 Hz,
1H), 7.52 (dd, J=8.8, 2.4 Hz, 1H), 7.36 (m, 2H), 6.64 (br d, J=8.8
Hz, 2H), 6.47 (d, J=8.8 Hz, 1H), 4.59 (t, J=4.2 Hz, 1H), 4.47 (t,
J=4.2 Hz, 1H), 3.60-3.79 (m, 10H), 3.11 (s, 3H), 2.97 (br s, 6H);
MS (ESI): 386 (M+H.sup.+).
Preparation of
##STR00264##
[0410]
6-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-2-(4-(4-methylpiperazin-1-yl-
)phenyl)quinoline: T550 was prepared using general procedure A.
Reaction was performed on a 38.6 mg scale of
2-chloro-6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)quinoline. T550 was
isolated as a white crystal (7.2 mg, 13%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.99-8.08 (m, 4H), 7.77 (d, J=9.2 Hz, 1H),
7.37 (dd, J=9.2, 2.8 Hz, 1H), 7.07 (d, J=2.8 Hz, 1H), 7.02 (m, 2H),
4.62 (t, J=4.4 Hz, 1H), 4.50 (t, J=4.4 Hz, 1H), 4.26 (t, J=5.0 Hz,
2H), 3.94 (t, J=5.0 Hz, 2H), 3.70-3.81 (m, 6H), 3.43 (br s, 4H),
2.78 (br s, 4H), 2.50 (br s, 3H); MS (ESI): 454 (M+H.sup.+).
Preparation of
##STR00265##
[0412] 2-(4-(6-Fluoropyridin-3-yl)phenyl)-1H-benzo[d]imidazole:
T468 was prepared using general procedure S. Reaction was performed
on a 0.029 g scale of 2-aminoaniline. The desired product T468 was
isolated as a yellow solid (0.043 g, 55%). .sup.1H NMR (400 MHz,
MeOH-d.sub.4): .delta. 8.61 (d, J=2.8 Hz, 1H), 8.33 (dd, J=8.4, 2.8
Hz, 1H), 8.24 (d, J=8.8 Hz, 2H), 8.03 (d J=8.8 Hz, 2H), 7.82 (dd,
J=8.8, 2.4 Hz, 2H), 7.61 (dd, J=8.8, 2.4 Hz, 2H), 7.22 (dd, J=8.8,
2.8 Hz, 1H); MS (ESI): 290 (M+H.sup.+).
Preparation of
##STR00266##
[0414] 2-(4-(2-Fluoropyridin-4-yl)phenyl)-1H-benzo[d]imidazole:
T460 was prepared using general procedure S. Reaction was performed
on a 0.027 g scale of 2-aminoaniline. The desired product T460 as a
yellow solid (0.010 g, 14%). .sup.1H NMR (400 MHz, MeOH-d.sub.4):
.delta. 8.25 (d, J=5.2 Hz, 1H), 8.19 (d, J=8.8 Hz, 2H), 8.04 (d,
J=8.8 Hz, 2H), 8.03 (d, J=8.8 Hz, 2H), 7.72 (q, J=3.2 Hz, 2H), 7.64
(dt, J=5.2, 1.6 Hz, 1H), 7.49 (q, J=3.2 Hz, 2H), 7.42 (s, 1H); MS
(ESI): 290 (M+H.sup.+).
Preparation of
##STR00267##
[0416]
4'-(1H-Benzo[d]imidazol-2-yl)-N,N-dimethyl-[1,1'-biphenyl]-4-amine.
EW5338-028 was prepared using general procedure S. Reaction was
performed on a 0.052 g scale of 2-aminoaniline. EW5338-028 was
isolated as a yellow solid (0.076 g, 50%). .sup.1H NMR (400 MHz,
MeOH-d.sub.4): .delta. 8.08 (d, J=8.8 Hz, 2H), 7.72 (d, J=8.8 Hz,
2H), 7.51-7.62 (m, 4H), 7.23 (dd, J=8.8, 2.8 Hz, 2H), 6.84 (d,
J=8.8 Hz, 2H), 2.97 (s, 6H); MS (ESI): 314 (M+H.sup.+).
Preparation of
##STR00268##
[0418]
4'-(1H-Benzo[d]imidazol-2-yl)-[1,1'-biphenyl]-4-carbonitrile:
EW5338-043 was prepared using general procedure S. Reaction was
performed on a 0.052 g scale of 2-aminoaniline. EW5338-043 was
isolated as a yellow solid (0.076 g, 50%). .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.66 (s, 2H), 8.28 (d, J=8.4 Hz, 2H),
7.92-8.02 (m, 6H), 7.16-7.22 (m, 2H); MS (ESI): 296
(M+H.sup.+).
Preparation of
##STR00269##
[0420]
4'-(1H-Benzo[d]imidazol-2-yl)-N,N-dimethyl-[1,1'-biphenyl]-3-amine:
EW5338-036 was prepared using general procedure S. Reaction was
performed on a 0.052 g scale of 2-aminoaniline. EW5338-036 was
isolated as a yellow solid (0.076 g, 50%). .sup.1H NMR (400 MHz,
MeOH-d.sub.4): .delta. 8.08 (d, J=8.8 Hz, 2H), 7.56-7.62 (m, 3H),
7.20-7.51 (m, 4H), 6.99 (s, 1H), 6.84-6.91 (m, 2H), 3.02 (s, 6H);
MS (ESI): 314 (M+H.sup.+).
Preparation of
##STR00270##
[0422] 2-(Benzofuran-2-yl)-1H-benzo[d]imidazole: T488 was prepared
using general procedure S. Reaction was performed on a 0.34 g scale
of 2-aminoaniline. T488 was isolated as a yellow solid (0.1 g,
14%). .sup.1H NMR (400 MHz, MeOH-d.sub.4): .delta. 7.54-7.74 (m,
5H), 7.36-7.44 (m, 1H), 7.26-7.34 (m, 3H); MS (ESI): 235
(M+H.sup.+).
Preparation of
##STR00271##
[0424] 2-(Benzo[b]thiophen-2-yl)-1H-benzo[d]imidazole: T493 was
prepared using general procedure S. Reaction was performed on a 0.4
g scale of 2-aminoaniline. T493 was isolated as a yellow solid (0.7
g, 76%). .sup.1H NMR (400 MHz, MeOH-d.sub.4): .delta. 8.25 (d,
J=0.8 Hz, 1H), 7.98-8.06 (m, 2H), 7.73 (dd, J=8.8, 2.4 Hz, 2H),
7.48-7.56 (m, 4H); MS (ESI): 251 (M+H.sup.+).
Preparation of
##STR00272##
[0426] 2-(Benzofuran-2-yl)-4-fluoro-1H-benzo[d]imidazole: T495 was
prepared using general procedure S. Reaction was performed on a
0.34 g scale of 2-aminoaniline. T495 was isolated 0 as a solid (0.3
g, 50%). .sup.1H NMR (400 MHz, MeOH-d.sub.4): .delta. 7.72-7.78 (m,
1H), 7.71 (d, J=1.2 Hz, 1H), 7.62-7.68 (m, 1H), 7.43-7.49 (m, 2H),
7.30-7.38 (m, 2H), 7.09 (dd, J=8.0, 0.8 Hz, 1H); MS (ESI): 253
(M+H.sup.+).
Preparation of
##STR00273##
[0428]
1-(2-Fluoroethyl)-2-(4-(6-fluoropyridin-3-yl)phenyl)-1H-benzo[d]imi-
dazole: T538 was prepared using general procedure E. Reaction was
performed on a 0.01 g scale of T468. T538 was isolated as a white
solid (0.012 g, 100%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.49 (dt, J=2.4, 0.8 Hz, 1H), 8.30 (dd, J=8.4, 2.4 Hz, 1H),
7.93-7.97 (m, 1H), 7.90 (d, J=8.8 Hz, 2H), 7.72 (d, J=8.8 Hz, 2H),
7.38-7.51 (m, 3H), 7.05 (dd, J=8.8, 0.4 Hz, 1H), 4.84 (dt, J=46.4,
5.2 Hz, 2H), 4.61 (dt, J=24, 4.8 Hz, 2H); MS (ESI): 336
(M+H.sup.+).
Preparation of
##STR00274##
[0430]
4'-(1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-3/1)-N,N-dimethyl-[1,1'-
-biphenyl]-4-amine. T543 was prepared using general procedure E.
Reaction was performed on a 0.030 g scale of EW5338-028. T543 was
isolated as a yellow solid (0.007 g, 20%). .sup.1H NMR (400 MHz,
MeOH-d.sub.4): .delta. 7.83-8.20 (m, 6H), 7.67-7.73 (m, 4H), 6.97
(d, J=8.8 Hz, 2H), 4.96 (dt, J=46.4, 5.2 Hz, 2H), 4.61 (dt, J=24,
4.8 Hz, 2H), 3.05 (s, 6H); MS (ESI): 360 (M+H.sup.+).
Preparation of
##STR00275##
[0432]
4'-(1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)-[1,1'-biphenyl]-4-c-
arbonitrile: T556 was prepared using general procedure E. Reaction
was performed on a 0.036 g scale of EW5338-043. T556 was isolated
as a yellow solid (0.009 g, 22%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.89 (d, J=8.0 Hz, 3H), 7.72-7.79 (m, 6H),
7.42-7.48 (m, 1H), 7.34-7.41 (m, 2H), 4.83 (dt, J=46, 4.8 Hz, 2H),
4.59 (dt, J=24, 5.2 Hz, 2H); MS (ESI): 342 (M+H.sup.+).
Preparation of
##STR00276##
[0434]
4'-(1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)-N,N-dimethyl-[1,1'--
biphenyl]-3-amine. T548 was prepared using general procedure E.
Reaction was performed on a 0.036 g scale of EW5338-036. T548 was
isolated as a yellow solid (0.014 g, 33%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.85-7.89 (m, 1H), 7.79 (d, J=8.4 Hz, 2H),
7.75 (d, J=8.4 Hz, 2H), 7.42-7.47 (m, 1H), 7.31-7.37 (m, 3H),
6.95-7.02 (m, 2H), 6.78 (dd, J=8.4, 0.8 Hz, 1H), 3.02 (s, 6H); MS
(ESI): 360 (M+H.sup.+).
Preparation of
##STR00277##
[0436] 2-(Benzofuran-2-yl)-1-(2-fluoroethyl)-1H-benzo[d]imidazole:
T489 was prepared using general procedure E. Reaction was performed
on a 0.052 g scale of T488. T489 was isolated as a yellow solid
(0.076 g, 50%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.81-7.88 (m, 1H), 7.69-7.74 (m, 1H), 7.55-7.63 (m, 2H), 7.45-7.51
(m, 1H), 7.30-7.44 (m, 4H), 4.92-5.03 (m, 2H), 4.85-4.95 (m, 2H);
MS (ESI): 281 (M+H.sup.+).
Preparation of
##STR00278##
[0438]
2-(Benzo[b]thiophen-2-yl)-1-(2-fluoroethyl)-1H-benzo[d]imidazole:
T494 was prepared using general procedure E. Reaction was performed
on a 0.052 g scale of T493. T494 was isolated as a yellow solid
(0.076 g, 50%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.85-7.92 (m, 3H), 7.77 (s, 1H), 7.81-7.95 (m, 3H), 7.60-7.75 (m,
2H), 4.91 (dt, J=46.4, 4.8 Hz, 2H), 4.75 (dt, J=24, 4.8 Hz, 2H); MS
(ESI): 297 (M+H.sup.+).
Preparation of
##STR00279##
[0440] 6-(6-Fluoropyridin-3-yl)-1H-benzo[d]imidazole: T532 was
prepared using general procedure A. Reaction was performed on a
0.08 g scale of 6-bromobenzoimidazole. T532 was isolated as a
yellow solid (0.025 g, 29%). .sup.1H NMR (400 MHz, MeOH-d.sub.4):
.delta. 9.16 (s, 1H), 8.15 (d, J=2.8 Hz, 1H), 8.26 (dd, J=10, 2.4
Hz, 1H), 8.0-8.04 (m, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.83 (dd, J=8.4,
1.6 Hz, 1H), 7.19 (dd, J=8.4, 1.6 Hz, 1H); MS (ESI): 214
(M+H.sup.+).
Preparation of
##STR00280##
[0442]
4-(1-(2-Fluoroethyl)-1H-benzo[d]imidazol-5-yl)-N,N-dimethylaniline:
T533 was prepared using general procedure A. Reaction was performed
on a 0.08 g scale of 6-bromo-N-2-fluoroethylbenzoimidazole. T533
was isolated as a yellow solid (0.025 g, 29%). .sup.1H NMR (400
MHz, D.sub.2O): .delta. 9.23 (s, 1H), 8.05 (d, J=1.2 Hz, 1H),
7.81-7.89 (m, 4H), 7.64 (d, J=8.8 Hz, 2H), 4.90 (dt, J=27.2, 5.2
Hz, 2H), 4.78-4.83 (m, 2H), 3.25 (s, 6H); MS (ESI): 284
(M+H.sup.+).
Preparation of
##STR00281##
[0444] 2-(6-Fluoropyridin-3-yl)quinoline: T455 was prepared using
general procedure A. Reaction was performed on a 0.1 g scale. T455
was isolated as a white solid (0.14 g, 100%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.94 (d, J=2.4 Hz, 1H), 8.66 (ddd, J=10.4,
7.6, 2.4 Hz, 1H), 8.27 (d, J=8.0 Hz, 1H), 8.14 (dd, J=8.4, 1.2 Hz,
1H), 7.75 (ddd, J=8.4, 6.8, 1.6 Hz, 1H), 7.85 (d, J=8.8 Hz, 2H),
7.56 (ddd, J=8.0, 6.3, 1.2 Hz, 1H), 7.09 (ddd, J=8.8, 3.2, 0.2 Hz,
1H); MS (ESI): 225.0 (M+H.sup.+).
Preparation of
##STR00282##
[0446] 4-Fluoro-2-(1-methyl-1H-benzo[d]imidazol-6-yl)quinoline
2,2,2-trifluoroacetate: T485 was prepared using general procedure
L. Reaction was performed on a 0.017 g scale. Product was purified
by HPLC using ACN(0.05% TFA)/H2O (0.05% TFA). T485 was isolated as
a white solid (0.09 g, 58%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 9.24 (s, 1H), 8.65-8.64 (m, 1H), 8.44 (dd, J=8.8, 2.4 Hz,
1H), 8.13-8.07 (m, 2H), 7.95 (d, J=11.6 Hz, 1H), 7.90-7.88 (m, 1H),
7.81 (ddd, J=8.4, 6.8, 1.6 Hz, 1H), 7.62 (ddd, J=8.0, 6.8, 0.8 Hz,
1H), 4.14 (s, 3H); MS (ESI): 278.1 (M+H.sup.+).
Preparation of
##STR00283##
[0448]
5-(1-(2-Fluoroethyl)-1H-benzo[d]imidazol-2-yl)-N,N-dimethylpyridin--
2-amine 2,2,2-trifluoroacetate: T487 was prepared using general
procedure A. Reaction was performed on a 0.02 g scale. Product was
purified by HPLC using ACN (0.05% TFA)/H.sub.2O (0.05% TFA). T487
was isolated as a white solid (0.018 g, 77%). .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 8.55 (d, J=2.4 Hz, 1H), 7.98 (ddd, J=9.2, 2.4,
0.4 Hz, 1H), 7.93-7.91 (m, 1H), 7.81-7.78 (m, 1H), 7.63 (ddd,
J=5.6, 2.4, 1.2 Hz, 1H), 6.93 (dd, J=9.2, 0.8 Hz, 1H), 5.00 (t,
J=4.4 Hz, 1H), 4.89 (m, 1H), 4.83 (s, 6H), 4.84 (m, 1H), 4.79 (t,
J=4.4 Hz, 1H); MS (ESI): 285.1 (M+H.sup.+).
Preparation of
##STR00284##
[0450] N,N-Dimethyl-4-(quinolin-6-ylethynyl)aniline: T517 was
prepared using general procedure B. Reaction was performed on a 0.1
g scale. T517 was isolated as a yellow solid (0.1 g, 76%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 8.87 (dd, J=4.4, 2.0 Hz, 1H),
8.09 (d, J=8.0 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.95 (d, J=1.6 Hz,
1H), 7.78 (dd, J=8.8, 2.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 2H), 7.39
(dd, J=8.4, 4.4 Hz, 1H), 6.67 (d, J=9.2 Hz, 2H), 3.00 (s, 6H); MS
(ESI): 273.1 (M+H.sup.+).
Preparation of
##STR00285##
[0452]
2-Fluoro-4-(1-(2-fluoroethyl)-1H-benzo[d]imidazol-2-yl)aniline:
T524 was prepared using general procedure A. Reaction was performed
on a 0.1 g scale. T524 was isolated as a white solid (0.09 g, 76%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.81-7.78 (m, 1H),
7.43-7.37 (m, 2H), 7.33-7.29 (m, 3H), 6.87 (dd, J=8.8, 8.4 Hz, 1H),
4.84 (t, J=4.8 Hz, 1H), 4.72 (t, J=5.2 Hz, 1H), 4.54 (t, J=4.8 Hz,
1H), 4.48 (t, J=5.2 Hz, 1H); MS (ESI): 274.1 (M+H.sup.+).
Preparation of
##STR00286##
[0454]
4-(5-(6-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)quinolin-2-yl)pyridin-2-
-yl)morpholine: T539 was prepared using general procedure A.
Reaction was performed on a 0.037 g scale. T539 was isolated as
white solid (0.04 g, 77%). .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 8.99 (d, J=2.0 Hz, 1H), 8.41 (dd, J=8.8, 2.8 Hz, 1H), 8.25
(d, J=8.8 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.91 (d, J=8.8 Hz, 1H),
7.41-7.37 (m, 2H), 6.98 (d, J=9.6 Hz, 1H), 4.58 (t, J=4.0 Hz, 1H),
4.46 (t, J=4.0 Hz, 1H), 4.25 (t, J=4.4 Hz, 2H), 3.84 (t, J=4.8 Hz,
2H), 3.74-3.69 (m, 6H), 3.66-3.56 (m, 4H), 3.58-3.56 (m, 4H); MS
(ESI): 442.1 (M+H.sup.+).
Preparation of
##STR00287##
[0456]
6-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-2-(6-(pyrrolidin-1-yl)pyridi-
n-3-yl)quinoline: T545 was prepared using general procedure A.
Reaction was performed on a 0.039 g scale. T545 was isolated as
white solid (0.035 g, 66%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 8.86 (d, J=2.4 Hz, 1H), 8.34 (dd, J=8.8, 2.4 Hz, 1H), 8.01
(d, J=8.4 Hz, 1H), 7.97 (d, J=9.2 Hz, 1H), 7.73 (d, J=8.8 Hz, 1H),
7.36 (dd, J=9.2, 2.8 Hz, 1H), 7.06 (d, J=2.04 Hz, 1H), 6.49 (d,
J=8.8 Hz, 1H), 4.62 (t, J=4.0 Hz, 1H), 4.50 (t, J=4.0 Hz, 1H), 4.26
(t, J=4.8 Hz, 2H), 3.94 (t, J=4.0 Hz, 2H), 3.80-3.71 (m, 6H),
3.55-3.52 (m, 4H), 2.05-2.02 (m, 4H); MS (ESI): 426.1
(M+H.sup.+).
Preparation of
##STR00288##
[0458] 4-(Isoquinolin-1-ylethynyl)-N,N-dimethylaniline: T547 was
prepared using general procedure B. Reaction was performed on a
0.064 g scale. T547 was isolated as yellow solid (0.1 g, 76%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.52 (d, J=7.2 Hz, 1H),
8.49 (d, J=6.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.73-7.65 (m, 2H),
7.622-7.56 (m, 3H), 6.70 (d, J=9.2 Hz, 2H), 3.03 (s, 6H); MS (ESI):
273.1 (M+H.sup.+).
Preparation of
##STR00289##
[0460]
4-(4-(6-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)quinolin-2-yl)phenyl)mo-
rpholine: T549 was prepared using general procedure A. Reaction was
performed on a 0.033 g scale. T549 was isolated as white solid
(0.035 g, 76%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.02 (d,
J=8.8 Hz, 2H), 7.96 (dd, J=9.6, 8.8 Hz, 2H), 7.72 (d, J=8.4 Hz,
1H), 7.31 (dd, J=9.2, 3.2 Hz, 1H), 7.01 (d, J=2.4 Hz, 1H), 6.96 (d,
J=9.2 Hz, 2H), 4.56 (t, J=4.0 Hz, 1H), 4.44 (t, J=4.4 Hz, 1H), 4.20
(t, J=4.8 Hz, 2H), 3.88 (t, J=4.8 Hz, 2H), 3.83 (t, J=4.8 Hz, 4H),
3.75-3.65 (m, 6H), 3.19 (t, J=4.8 Hz, 4H), MS (ESI): 441.1
(M+H.sup.+).
Preparation of
##STR00290##
[0462] T450: A mixture of 1,2-phenylenediamine (80 mg, 0.740 mmol)
and 4-dimethylamino-benzoyl chloride (80 mg, 0.436 mmol) in DMF
(1.0 mL) was heated at 200.degree. C. for 15 mins in a microwave.
The crude product was purified by prepHPLC and neutralized with
NaHCO3 to afford T450 (20 mg, 19.35%). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.03-8.01 (d, 2H), 7.72-7.69 (m, 2H),
7.47-7.45 (m, 2H), 6.95-6.93 (m, 2H), 3.06 (s, 6H); MS (ESI): 238.1
(M+H.sup.+).
Preparation of
##STR00291##
[0464] T452 A mixture of 2-bromobenzimidazole (0.05 g, 0.254 mmol),
2-fluoropyridine-5-boronic acid (0.036 g, 0.254 mmol), Potassium
carbonate (0.190 ml, 0.381 mmol), and PdCl.sub.2(dppf).sub.2DCM
(10.36 mg, 0.013 mmol) in DMF (1.0 mL) was heated at 150.degree. C.
for 15 min. The crude product was purified by prepHPLC to afford
T452 (6 mg, 11.09%). .sup.1H NMR (400 MHz, CD.sub.3CN) .quadrature.
9.01 (s, 1H), 8.70-8.65 (m, 1H), 7.81-7.79 (m, 2H), 7.45-7.43 (m,
2H), 7.29-7.26 (m, 1H); MS (ESI): 214.0 (M+H.sup.+).
Preparation of
##STR00292##
[0466] T497 was prepared using general procedure D. The reaction
was performed on a 20 mg scale of T450. T497 TFA salt was isolated
(6 mg, 25.1%). .sup.1H NMR (400 MHz, CD.sub.3CN) .delta. 7.96-7.93
(m, 1H), 7.77-7.75 (m, 1H), 7.70-7.67 (m, 2H), 7.56-7.53 (m, 2H),
6.94-6.90 (m, 2H), 4.94-4.92 (m, 1H), 4.83-4.80 (m, 2H), 4.76-4.74
(m, 1H), 3.04 (s, 6H); MS (ESI): 284.10 (M+H.sup.+).
Preparation of
##STR00293##
[0468] T555: To a solution of 5-bromo-7-azaindole (0.1 g, 0.508
mmol), 4-Dimethylaminophenyl boronic acid (0.084 g, 0.508 mmol),
Copper(I) iodide (9.67 mg, 0.051 mmol), and Potassium carbonate
(0.508 ml, 1.015 mmol) in DMF (2.0 mL) was added a solution of
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.021
g, 0.025 mmol) in DCM (2.0 mL). The resulting mixture was heated at
120.degree. C. in a microwave for 30 min. and then cooled to room
temperature. The crude product was purified by prep HPLC to afford
T555 TFA salt (0.010 g, 5.61%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 11.80 (s, 1H), 8.48-8.47 (m, 1H), 8.22-8.21 (m, 1H),
7.61-7.58 (m, 2H), 7.51-7.50 (m, 1H), 6.98-6.96 (m, 2H), 6.51-6.50
(m, 1H), 2.97 (s, 6H); MS (ESI): 238.7 (M+H.sup.+).
Preparation of
##STR00294##
[0470] T558: To a solution of 6-bromoimidazo[1,2-a]pyrimidine (0.08
g, 0.404 mmol), 4-dimethylaminophenylboronic acid (0.087 g, 0.525
mmol), Copper(I) iodide (7.69 mg, 0.040 mmol) and Potassium
carbonate (0.404 ml, 0.808 mmol) in DMF (2.0 mL) was added a
solution of
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.016
g, 0.020 mmol) in DCM (2.0 mL). The resulting mixture was
microwaved at 120.degree. C. for 30 min., cooled and filtered. The
filtrate was concentrated in vacuo. The residue was purified on
prep HPLC to afford T558 TFA salt (0.008 g, 0.023 mmol, 5.62%
yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.44 (m, 1H),
9.26 (m, 1H), 8.14-8.11 (m, 2H), 7.69-7.66 (m, 2H), 6.90-6.88 (m,
2H), 2.99 (s, 3H); MS (ESI): 239.1 (M+H.sup.+).
Preparation of
##STR00295##
[0472] T496 was prepared using general procedure B. Reaction was
performed on a 50 mg scale. Filtered and purified on prep HPLC to
afford T496 TFA salt (0.02 g, 30.8%). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.52-8.50 (m, 1H), 8.07-8.04 (m, 2H),
7.90-7.82 (m, 2H), 7.74-7.69 (m, 3H), 7.40-7.37 (m, 1H), 7.34-7.32
(m, 1H), 4.92-4.90 (m, 1H), 4.87-4.86 (m, 1H), 4.80 (m, 2H); MS
(ESI): 316.1 (M+H.sup.+).
Preparation of
##STR00296##
[0474] T508 was prepared using general procedure D from T481 and
2-(2-(2-fluoroethoxy)-ethoxy)ethyl 4-methylbenzenesulfonate. The
reaction was performed on a 60 mg scale of T481. The crude product
was purified by prep HPLC to afford T508 (5 mg, 5.51%). .sup.1H NMR
(400 MHz, CD.sub.3CN) .delta. 7.83-7.77 (m, 2H), 7.59-7.52 (m, 4H),
6.79-6.77 (m, 2H), 4.66-4.63 (m, 2H), 4.43-4.41 (m, 1H), 4.31-4.29
(m, 1H), 3.96-3.94 (m, 2H), 3.57-3.52 (m, 3H), 3.48-3.44 (m, 3H),
3.05 (s, 6H); MS (ESI): 396.20 (M+H.sup.+).
Preparation of
##STR00297##
[0476] T527 was prepared using general procedure B from
2-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-benzo[.alpha.]imidazole
and 5-ethynyl-7-azaindole. The reaction was performed on a 105 mgs
scale of
2-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-benzo[d]imidazole.
The crude product was purified by Prep HPLC to afford T527 TFA salt
(0.01 g, 6.24%). .sup.1H NMR (400 MHz, CD.sub.3CN) .delta. 10.14
(s, 1H), 8.59-8.58 (m, 1H), 8.32 (m, 1H), 7.82-7.80 (m, 1H),
7.73-7.71 (m, 1H), 7.52-7.45 (m, 3H), 6.61-6.59 (m, 1H), 4.69-4.67
(m, 2H), 4.42-4.40 (m, 1H), 4.30-4.28 (m, 1H), 3.98-3.95 (m, 2H),
3.58-3.52 (m, 3H), 3.49-3.44 (m, 3H); MS (ESI): 393.10
(M+H.sup.+).
Preparation of
##STR00298##
[0478] T528 was prepared using general procedure B. Reaction was
performed on a 63 mgs scale. The crude product was purified by prep
HPLC to afford T528 TFA salt (0.005 g, 4.21%). .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 8.28 (m, 1H), 7.98 (m, 1H), 7.33-7.28 (m,
3H), 6.66-6.64 (m, 2H), 6.42-6.41 (m, 1H), 2.88 (s, 6H); MS (ESI):
261.1 (M+H.sup.+).
Preparation of
##STR00299##
[0480] T534 was prepared using general procedure B from
2-bromo-1-(2-fluoroethyl)-1H-benzo[d]imidazole and tert-butyl
methyl-4-(ethynyl)phenylcarbamate. Reaction was performed on a 53
mgs scale of 2-bromo-1-(2-fluoroethyl)-1H-benzo[d]imidazole. The
crude product was purified by ISCO column to afford tert-butyl
4-((1-(2-fluoroethyl)-1H-benzo[d]imidazol-2-yl)ethynyl)phenyl(methyl)carb-
amate (0.03 g, 35.3%). It was dissolved in acetonitrile (0.5 mL).
To this solution was added a solution of 20% sulfuric acid (1.5 mL,
5.63 mmol). The resulting mixture was stirred at room temperature
for 20 minutes, diluted with water (2.0 mL) and purified by
preparative HPLC to afford T534 as TFA salt (0.004 g, 12.88%).
.sup.1H NMR (400 MHz, CD.sub.3CN) .delta. 7.81-7.79 (m, 1H),
7.67-7.65 (m, 1H), 7.52-7.48 (m, 4H), 6.66-6.64 (m, 2H), 4.95-4.93
(m, 1H), 4.83-4.478 (m, 2H), 4.74-4.73 (m, 1H), 2.82 (s, 3H); MS
(ESI): 294.1 (M+H.sup.+).
Preparation of
##STR00300##
[0482] T541 was prepared using general procedure B from
2-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-benzo[d]imidazole
and tert-butyl methyl-4-(ethynyl)phenylcarbamate. Reaction was
performed on a 72 mgs scale of
2-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-benzo[d]imidazole.
The crude product was purified by ISCO column to afford tert-butyl
4-((1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethyny-
l)phenyl(methyl)carbamate (0.02 g, 19.21%). It was then dissolved
in acetonitrile (1.0 mL). To this solution was added 20% Sulfuric
acid (1.0 mL, 3.75 mmol). The reaction mixture was stirred at room
temperature for 30 mins. The crude product was purified by prep
HPLC to afford T541 TFA salt (0.004 g, 19.44%). .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 7.78-7.76 (m, 1H), 7.70-7.68 (m, 1H),
7.51-7.45 (m, 4H), 6.66-6.64 (m, 2H), 4.63-4.60 (m, 2H), 4.44-4.42
(m, 1H), 4.32-4.30 (m, 1H), 3.95-3.92 (m, 2H), 3.57-3.53 (m, 3H),
3.48-3.45 (m, 3H), 2.82 (m, 3H). MS (ESI): 382.1 (M+H.sup.+).
Preparation of
##STR00301##
[0484] T551 was prepared using general procedure B from
2-ethynyl-1-(2-fluoroethyl)-1H-benzo[d]imidazole and
3-bromopyridine. Reaction was performed on a 40 mgs scale of
2-ethynyl-1-(2-fluoroethyl)-1H-benzo[d]imidazole. The crude product
was purified by prep HPLC to afford T551 TFA salt (0.006 g, 7.44%).
.sup.1H NMR (400 MHz, CD.sub.3CN): .delta. 8.92-8.91 (m, 1H),
8.70-8.69 (m, 1H), 8.14-8.11 (m, 1H), 7.79-7.77 (m, 1H), 7.64-7.62
(m, 1H), 7.55-7.40 (m, 3H), 4.94-4.92 (m, 1H), 4.82-4.80 (m, 2H),
4.76-4.73 (m, 1H); MS (ESI): 266.1 (M+H.sup.+).
Preparation of
##STR00302##
[0486]
2-(2-amino-1H-benzo[d]imidazol-1-yl)-1-(4-(diethylamino)phenyl)etha-
none: A solution of 2-aminobenzimidazole (197 mg, 1.5 mmol) and
2-Bromo-4'-(diethylamino)acetophenone (402 mg, 1.5 mmol) in MeOH (7
mL) was stirred at r.t. for 18 hours. The volatiles were removed in
vacuo and NaHCO.sub.3 (sat. aq., 30 mL) was added. The aqueous
mixture was extracted with EtOAc (3.times.30 mL). The combined
EtOAc extracts were dried with MgSO.sub.4 and concentrated in
vacuo. The residue was purified on silica gel eluting with a
gradient up to 5:95 (MeOH:DCM) to isolate
2-(2-amino-1H-benzo[d]imidazol-1-yl)-1-(4-(diethylamino)phenyl)ethanone
(176 mg, 36%) as a beige solid.
Preparation of
##STR00303##
[0488]
4-(1H-benzo[d]imidazo[1,2-a]imidazol-2-yl)-N,N-diethylaniline
trifluoroacetate T506: A solution of
2-(2-amino-1H-benzo[d]imidazol-1-yl)-1-(4-(diethylamino)phenyl)ethanone
(50 mg, 0.155 mmol) was heated to reflux in AcOH (2 mL) for several
hours. The volatiles were removed in vacuo. The residue was
dissolved in ACN and purified by semi-prep HPLC to isolate T506 (15
mg, 24%) as a beige solid. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 1.19 (t, 6H), 3.48 (q, 4H), 6.92 (m, 2H), 7.42-7.51 (m,
2H), 7.62 (m, 3H), 7.91 (m, 1H), 8.06 (s, 1H); MS (ESI): 305.1
(M+H.sup.+).
Preparation of
##STR00304##
[0490] T552 was prepared using general procedure B. Reaction was
performed on a 40 mgs scale. The crude product was purified by prep
HPLC to afford T552 TFA salt (0.006 g, 6.98%). .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 9.08-9.07 (m, 1H), 8.42-8.40 (m, 1H),
8.19-8.16 (m, 1H), 7.71-7.67 (m, 2H), 7.40-7.29 (m, 2H), 4.86 (m,
2H), 4.79-4.74 (m, 2H); MS (ESI): 291.0 (M+H.sup.+).
Preparation of
##STR00305##
[0492] T553 was prepared using general procedure B. Reaction was
performed on a 40 mgs scale. The crude product was purified by prep
HPLC to afford T553 TFA salt (0.006 g, 7.42%). .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 9.23 (s, 1H), 9.05 (s, 2H), 7.82-7.79 (m,
1H), 7.67-7.65 (m, 1H), 7.52-7.43 (m, 2H), 4.94-4.92 (m, 1H),
4.84-4.80 (m, 2H), 4.78-4.75 (m, 1H); MS (ESI): 267.1
(M+H.sup.+).
Preparation of
##STR00306##
[0494] T554 was prepared using general procedure B. Reaction was
performed on a 40 mgs scale. The crude product was purified by prep
HPLC to afford T554 TFA salt (0.006 g, 6.85%). .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 11.63-11.61 (m, 1H), 11.56 (s, 1H),
8.17-8.16 (m, 1H), 7.64-7.59 (m, 2H), 7.33-7.23 (m, 2H), 4.84-4.83
(m, 1H), 4.76-4.68 (m, 3H); MS (ESI): 299.6 (M+H.sup.+).
Preparation of
##STR00307##
[0496] T564 was prepared using general procedure B from
5-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-pyrrolo[2,3-b]pyridine
and tert-butyl 5-ethynyl-1H-pyrrolo[2,3-b]pyridine-1-carboxylate,
followed by a hydrolysis with NaOH. The reaction was performed on a
85 mg scale of
5-bromo-1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-1H-pyrrolo[2,3-b]p-
yridine. T564 TFA salt was isolated (0.007 g, 5.40%). .sup.1H NMR
(400 MHz, CD.sub.3CN) 811.53 (s, 1H), 8.50-8.49 (m, 2H), 8.41-8.4
(m, 2H), 8.17 (m, 2H), 7.59-7.58 (m, 1H), 7.53-7.52 (m, 1H),
6.69-6.68 (m, 1H), 6.54-6.53 (m, 1H), 4.53-4.51 (m, 1H), 4.48-4.46
(m, 2H), 4.41-4.39 (m, 1H), 3.86-3.84 (m, 2H), 3.64-3.62 (m, 1H),
3.59-3.53 (m, 5H); MS (ESI): 393.5 (M+H.sup.+).
Preparation of
##STR00308##
[0498] 4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-methylaniline
bistrifluoroacetate T522: To a suspension of
4-(benzo[4,5]imidazo[1,2-a]pyrimin-2-yl)-aniline (25 mg, 0.10 mmol)
in MeOH (3 mL) at r.t. was added paraformaldehyde (110 mg, 3.7
mmol) followed by NaCNBH.sub.3 (40 mg, 0.63 mmol). The mixture was
heated in a microwave reactor at 100.degree. C. for 20 minutes. The
volatiles were removed in vacuo. The residue was dissolved in EtOAc
(15 mL), washed with NaHCO.sub.3 (2.times.15 mL), and brine (15
mL). The EtOAc layer was dried with MgSO.sub.4, filtered and
evaporated to obtain an oil that was purified by semi-prep HPLC.
4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N,N-dimethylaniline
bistrifluoroacetate (2.0 mg, 4%) was obtained as an orange solid.
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.17 (s, 6H), 6.91 (m,
2H), 7.64 (m, 1H), 7.73-7.80 (m, 2H), 8.07 (d, J=7.6 Hz, 1H), 8.26
(m, 1H), 8.34 (m, 2H), 9.33 (d, J=7.6 Hz, 1H). MS (ESI): 275.1
(M+H.sup.+).
Preparation of
##STR00309##
[0500]
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N,N-dimethylaniline
bistrifluoroacetate T521 was also obtained from the preceding
reaction (1 mg, 2%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.92
(s, 3H), 6.75 (m, 2H), 7.63 (m, 1H), 7.71-7.79 (m, 2H), 8.02 (d,
J=7.6 Hz, 1H), 8.24-8.30 (m, 3H), 9.30 (d, J=7.6 Hz, 1H). MS (ESI):
289.1 (M+H.sup.+)
Preparation of
##STR00310##
[0502] 4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)aniline T520: To a
solution of 4-(4-nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (35
mg, 0.12 mmol) in MeOH:THF:H2O (1:1:3, 2 mL) was added a large
excess of Na.sub.2S.sub.2O.sub.4. The reaction was quenched with
NaHCO.sub.3 (sat. aq.) and extracted with EtOAc. The EtOAc layer
was washed with H.sub.2O and then brine. The EtOAc layer was dried
with MgSO4. The residue was purified by semi-prep HPLC to give T520
as TFA salt (3 mg, 7%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.81 (m, 2H), 7.27 (m, 1H), 7.36 (m, 2H), 7.45 (m, 2H), 7.67 (m,
1H), 7.88 (m, 1H), 9.01 (d, J=4.8 Hz, 1H). MS (ESI): 261.1
(M+H.sup.+).
Preparation of
##STR00311##
[0504] 4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)aniline T518: To a
suspension of 2-(4-nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine
(58 mg, 0.20 mmol) in ethanol (3 mL) was added SnCl.sub.2.2H.sub.2O
(361 mg, 1.6 mmol). The solution was refluxed for 1.5 hours and
then the volatiles were removed under vacuum. The residue was
dissolved in DCM, washed with 1 N NaOH, and then H.sub.2O. The DCM
layer was dried with MgSO.sub.4. The crude product was purified on
flash chromatography (silica gel, 5% MeOH/DCM) to provide T518 as a
yellow solid (35 mg, 67%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 5.94 (s, 2H), 6.70 (m, 2H), 7.34 (m, 1H), 7.47 (m, 1H),
7.61 (d, J=7.6 Hz, 1H), 7.75 (m, 1H), 8.08 (m, 2H), 8.21 (m, 1H),
9.34 (d, J=7.6 Hz, 1H). MS (ESI): 261.1 (M+H.sup.+).
Preparation of
##STR00312##
[0506] 2-(4-Nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine T511: A
solution of (E)-3-(dimethylamino)-1-(4-nitrophenyl)prop-2-en-1-one
(410 mg, 1.9 mmol) and 1H-benzo[d]imidazol-2-amine (248 mg, 1.9
mmol) in AcOH (10 ml) was heated to reflux overnight. The volatiles
were removed by rotary evaporation and the residue was partitioned
between DCM and aqueous NaHCO.sub.3. The mixture was filtered to
obtain pure 2-(4-nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (85
mg, 15%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.47 (m, 1H), 7.59 (m, 1H), 7.90 (d, J=7.2 Hz, 1H), 7.96
(m, 1H), 8.38 (m, 1H), 8.44 (m, 2H), 8.61 (m, 2H), 9.72 (d, J=7.2
Hz, 1H). MS (ESI): 291.0 (M+H.sup.+)
Preparation of
##STR00313##
[0508] 4-(4-Nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine T512:
The DCM layer from the preceding reaction was washed with H.sub.2O
and dried (MgSO.sub.4). The residue was purified by flash
chromatography (silica gel, 100% EtOAc) to give
4-(4-nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (120 mg, 22%)
as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.65
(m, 1H), 7.09-7.14 (m, 2H), 7.47-7.52 (m, 1H), 7.90 (m, 1H), 8.08
(m, 2H), 8.54 (m, 2H), 8.91 (d, J=4.0 Hz, 1H). MS (ESI): 291.1
(M+H.sup.+)
Preparation of
##STR00314##
[0510]
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(3-fluoropropyl)anilin-
e T542: To 3-fluoropropan-1-ol (4 mg, 0.05 mmol) in 0.5 mL DCM was
added Dess-Martin reagent (42 mg, 0.1 mmol). The mixture was
stirred at rt for 1 h and filtered directly into a mixture of
4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)aniline (4 mg, 0.015
mmol) and NaBH(OAc).sub.3 (43 mg, 0.2 mmol) with stirring. After
vigorously stirred for 5 min, reaction was quenched by adding 0.5 M
NaOH (2 mL). The mixture was extracted with EtOAc (3.times.10 mL)
and the organic phase was dried over MgSO4 and concentrated. The
crude product was purified by HPLC to afford
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(3-fluoropropyl)anili-
ne as a yellow solid (2.7 mg, 33%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.76 (d, J=7.2 Hz, 1H), 7.88 (m, 4H), 7.55 (d,
J=7.2 Hz, 1H), 7.53 (m, 1H), 7.40 (m, 1H), 6.47 (d, J=9.2 Hz, 1H),
4.68 (t, J=5.2 Hz, 1H), 4.56 (t, J=5.2 Hz, 1H), 3.34 (t, J=6.8 Hz,
2H), 2.09 (m, 1H), 2.02 (m, 1H); MS (ESI): 321 (M+H.sup.+).
Preparation of
##STR00315##
[0512]
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)-N-(3-fluoropropyl)anilin-
e T544 was prepared using the procedure for
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(3-fluoropropyl)aniline
from 4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)aniline (10 mg,
0.038 mmol) and 3-fluoropropan-1-ol (8 mg, 0.1 mmol). The product
T544 was obtained as a yellow solid (7 mg, 33%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 8.93 (d, J=4.4 Hz, 1H), 8.19 (d, J=8.4
Hz, 1H), 7.65 (m, 1H), 7.45 (m, 2H), 7.35 (d, J=8.0 Hz, 1H), 7.29
(m, 1H), 7.17 (d, J=4.4 Hz, 1H), 6.83 (m, 2H), 4.73 (t, J=5.2 Hz,
1H), 4.61 (t, J=5.2 Hz, 1H), 3.47 (t, J=6.8 Hz, 2H), 2.16 (m, 1H),
2.08 (m, 1H); MS (ESI): 321 (M+H.sup.+).
Preparation of
##STR00316##
[0514]
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(2-(2-(2-fluoroethoxy)-
ethoxy)ethyl)-aniline T557:
4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(2-(2-(2-fluoroethoxy)ethoxy-
)ethyl)aniline was prepared using the procedure for
4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-N-(3-fluoropropyl)aniline
from 4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)aniline (10 mg,
0.038 mmol) and 2-(2-(2-hydroxyethoxy)ethoxy)ethyl
4-methylbenzenesulfonate (23 mg, 0.075 mmol). The product T557 was
obtained as a yellow solid (1.2 mg, 5.1%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 9.31 (d, J=7.6 Hz, 1H), 8.25 (m, 2H), 8.02 (d,
J=7.2 Hz, 1H), 7.78-7.75 (m, 1H), 7.72 (m, 1H), 7.61 (m, 1H), 6.80
(d, J=9.2 Hz, 2H), 4.56 (m, 1H), 4.45 (m, 1H), 3.75 (m, 1H), 3.71
(t, J=5.2 Hz, 2H), 3.69-3.65 (m, H), 3.47-3.43 (m, H); MS (ESI):
395 (M+H.sup.+).
Preparation of
##STR00317##
[0516] Synthesis of GC-5333-63: 4-Bromoaniline (10 g, 58 mmol) was
dissolved in MeOH (20 ml). To the reaction mixture was added
paraformaldehyde (5.18 ml, 174 mmol) and 25% sodium methoxide
solution (48.3 ml, 291 mmol). The mixture was heated at 65.degree.
C. for 1 h and allowed to cool to room temperature. Sodium
borohydride (6.17 ml, 174 mmol) was added into the reaction mixture
portionwise. The reaction mixture was heated for another 2 h. The
mixture was concentrated, diluted with water (50 mL), extracted
with EtOAc (3.times.50 mL). The organic layers were combined, dried
and concentrated in vacuo. The residue was purified on flash column
chromatography (silica gel, 10% EtOAc/DCM) to afford GC-5333-63
(7.5 g, 69%). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.27-7.25
(m, 2H), 6.50-6.48 (m, 2H), 3.80. (br, 1H), 2.81 (s, 3H); MS (ESI):
186.1 (M+H.sup.+).
[0517] GC-5333-65 was prepared using general procedure D. Reaction
was performed on a 4 g scale. GC-5333-65 was eluted out in 20%
EtOAc: Hexanes mixture in a gradient elution on a Biotage
purification system (500 mg, 10%) as a colorless oil. .sup.1H-NMR
(400 MHz, CDCl.sub.3): .delta. 7.31-7.29 (m, 2H), 6.59-5.58 (m,
2H), 4.59 (dt, J=47.2, 5.2 Hz, 2H), 3.62 (dt, J=24.8, 5.2 Hz, 2H),
2.99 (s, 3H); MS (ESI): 232.1 (M+H.sup.+).
[0518] T478 was prepared using general procedure A. Reaction was
performed on a 30 mg scale. T78 was isolated as a solid (8 mg,
23%). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.75-7.72 (m, 2H),
7.57-7.52 (m, 2H), 7.24-7.19 (m, 2H), 6.81-6.76 (m, 3H), 4.63 (dt,
J=47.2, 5.2 Hz, 2H), 3.70 (dt, J=24.8, 5.2 Hz, 2H), 3.01 (s, 3H);
MS (ESI): 270.1 (M+H.sup.+).
3. Preparation of Radiolabeling Precursors
Preparation of
##STR00318##
[0520]
2-((tert-Butoxycarbonyl)(4-(quinolin-2-yl)phenyl)amino)ethyl-4-meth-
yl-benzene-sulfonate T411P was prepared using general procedure D.
Reaction was performed on 0.187 g scale. T411P was isolated as an
oil (0.014 g, 6%); .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.28-8.19 (m, 2H), 8.09 (dt, J=8.8, 2.4 Hz, 1H), 7.88-7.82 (m, 2H),
7.76-7.70 (m, 3H), 7.54 (ddd, J=8.0, 6.8, 0.8 Hz, 1H), 7.30-7.24
(m, 4H), 4.21 (t, J=5.6 Hz, 2H), 3.90 (t, J=5.6 Hz, 2H), 2.34 (s,
3H), 1.40 (s, 9H); MS (ESI): 519.1 [M+H.sup.+], 541.1
(M+Na.sup.+).
Preparation of
##STR00319##
[0522]
2,2-Dimethyl-4-oxo-5-(4-(quinolin-2-yl)phenyl)3,8,11-trioxa-azatrid-
ecan-13yl-4-methylbenzenesulfonate T442P was prepared using general
procedure D. [00303] Reaction performed on a 0.032 g scale. T442P
was isolated as a light yellow oil (0.028 g, 46%); .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 8.21 (d, J=8.4 Hz, 2H), 8.11 (d, J=8.8
Hz, 2H), 7.86 (d, J=8.8 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.75 (dt,
J=8.0, 2.0 Hz, 2H), 7.70 (d, J=6.8 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H),
7.38 (d, J=8.4 Hz, 1H), 7.27 (d, J=8.0 Hz, 1H), 4.12 (t, J=4.8 Hz,
2H), 3.83 (t, J=4.8 Hz, 2H), 3.64-3.59 (m, 4H), 3.52 (s, 4H), 2.38
(s, 3H), 1.43 (s, 9H); MS (ESI): 607.2 M+H.sup.+).
Preparation of
##STR00320##
[0524]
3-(Tosyloxy)propyl-4-(4-(6-methoxyquinolin-2-yl)phenyl)piperazine-1-
-carboxylate T498P was prepared using General experimental
procedure E for N-alkylation using Cs.sub.2CO.sub.3 as the base
(method E) was used. Reaction performed on a 0.032 g scale. Product
eluted out in 20% EtOAc:DCM mixture in a gradient elution on a
Combiflash purification system. T498P was isolated as a light
yellow color solid (0.010 g, 18%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.06 (br t, J=8.8 Hz, 4H), 7.79 (dt, J=8.4 and
1.6 Hz, 2H), 7.77 (d, J=8.4 Hz, 1H), 7.36-7.32 (m, 3H), 7.06 (d,
J=2.4 Hz, 1H), 7.00 (dt, J=8.8 and 1.6 Hz, 2H), 4.14 (q, J=7.2 Hz,
4H), 3.93 (s, 3H), 3.60 (br s, 4H), 3.22 (br s, 4H), 2.43 (s, 3H),
2.01 (q, J=8.0 Hz, 2H); LC-MS (ESI): (M+H.sup.+).
Preparation of
##STR00321##
[0526]
2-(2-(2-((2-(4-(Dimethylamino)phenyl)quinolin-6-yl)oxy)ethoxy)ethox-
y)ethyl-4-methylbenzenesulfonate T510P was prepared using general
procedure E. Reaction performed on a 0.050 g scale. T510P was
isolated as yellow solid (0.030 g, 29%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.05 (dt, J=8.0, 2.0 Hz, 2H), 7.98 (d, J=8.8
Hz, 2H), 7.77 (dt, J=8.8, 2.0 Hz, 2H), 7.76 (d, J=8.8 Hz, 1H), 7.33
(dd, J=9.2, 2.8 Hz, 1H), 7.28 (dd, J=8.4, 0.4 Hz, 2H), 7.04 (d,
J=2.8 Hz, 1H), 6.81 (dt, J=8.8, 2.0 Hz), 4.21 (t, J=4.8 Hz, 2H),
4.15 (t, J=4.8 Hz, 2H), 3.88 (t, J=4.8 Hz, 2H), 3.70-3.66 (m, 3H),
3.63-3.60 (m, 3H), 3.01 (s, 3H), 3.39 (s, 6H); MS (ESI): 551.2
(M+H.sup.+), 324 (M+Na.sup.+).
Preparation of
##STR00322##
[0528]
2-(4-(4-(2-(6-Methoxyquinolin-2-yl)phenyl)piperazin-1-yl)ethoxy)eth-
oxy)ethyl-4-methylbenzenesulfonate T530P was prepared using general
procedure E. Reaction was performed on a 0.1 g scale. T530P was
isolated as off white oil (0.046 g, 24%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.99 (dt, J=8.8, 2.0 Hz, 2H), 8.01 (d, J=8.4
Hz, 1H), 7.97 (d, J=9.2 Hz, 1H), 7.73 (dt, J=8.4, 2.0 Hz, 1H), 7.71
(d, J=9.2 Hz, 1H), 7.29-7.25 (m, 2H), 6.99 (d, J=2.8 Hz, 1H), 6.95
(dt, J=8.8, 2.0 Hz, 2H), 4.09 (t, J=4.8 Hz, 2H), 3.86 (s, 3H), 3.64
(t, J=4.8 Hz, 2H), 3.61-3.52 (m, 6H), 3.25 (t, J=4.8 Hz, 4H),
2.64-2.60 (m, 6H); MS (ESI): 606.1 (M+H.sup.+).
Preparation of
##STR00323##
[0530]
2-(2-((4-(Mimethylamino)phenyl)ethynyl)-1H-benzo[d]imidazol-1-yl)et-
hyl-4-methylbenzenesulfonate: T482P was prepared using general
procedure E. Reaction was performed on a 140 mg scale of T481.
T482P was isolated as a white solid (135 mg, 55%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 7.67 (m, 1H), 7.44-7.48 (m, 4H),
7.25-7.27 (m, 3H), 7.05 (d, J=8.4 Hz, 2H), 6.68 (m, 2H), 4.57 (t,
J=5.6 Hz, 2H), 4.43 (t, J=5.6 Hz, 2H), 3.04 (s, 6H), 2.33 (s, 3H);
MS (ESI): 460 (M+H.sup.+).
Preparation of
##STR00324##
[0532] 2-(Methyl(5-(quinolin-2-yl)pyridin-2-yl)amino)ethanol: T491
was prepared using general procedure M. Reaction was performed on a
110 mg scale of T455. T491 was isolated as a light yellow solid
(120 mg, 88%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.86 (dd,
J=2.4, 0.8 Hz, 1H), 8.41 (dd, J=8.8, 2.4 Hz, 1H), 8.15 (d, J=8.4
Hz, 1H), 8.09 (dd, J=8.4, 0.8 Hz, 1H), 7.78 (m, 2H), 7.69 (m, 1H),
7.48 (m, 1H), 6.69 (dd, J=8.8, 0.8 Hz, 1H), 4.92 (br s, 1H), 3.90
(t, J=4.6 Hz, 2H), 3.81 (t, J=4.6 Hz, 2H), 3.15 (s, 3H); MS (ESI):
280 (M+H.sup.+).
Preparation of
##STR00325##
[0534]
2-(2-(2-(Methyl(5-(quinolin-2-yl)pyridin-2-yl)amino)ethoxy)ethoxy)e-
thyl 4-methylbenzenesulfonate: T502P was prepared using general
procedure D. Reaction was performed on a 94 mg scale of T491. T502P
was isolated as light yellow oil (86 mg, 49%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 8.92 (dd, J=2.4, 0.8 Hz, 1H), 8.37 (dd,
J=8.8, 2.4 Hz, 1H), 8.15 (d, J=8.8 Hz, 1H), 8.09 (dd, J=8.4, 1.0
Hz, 1H), 7.80 (m, 2H), 7.69 (m, 1H), 7.48 (m, 1H), 6.65 (dd, J=8.4,
0.8 Hz, 1H), 4.15 (m, 2H), 3.84 (t, J=6.2 Hz, 2H), 3.66-3.72 (m,
4H), 3.57 (t, J=1.4 Hz, 2H), 3.17 (s, 3H), 2.42 (s, 3H); MS (ESI):
522 (M+H.sup.+).
Preparation of
##STR00326##
[0536]
2,2-Dimethyl-4-oxo-5-(5-(quinolin-2-yl)pyridin-2-yl)-3,8,11-trioxa--
5-azatridecan-13-yl 4-methylbenzenesulfonate: T525P was prepared
using general procedure D. Reaction was performed on a 67.0 mg
scale of T503. T525P was isolated as colorless oil (80.5 mg, 65%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.10 (dd, J=2.8, 0.8 Hz,
1H), 8.44 (dd, J=4.6, 2.6 Hz, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.14 (d,
J=8.4 Hz, 1H), 7.71-7.86 (m, 6H), 7.54 (m, 1H), 7.29 (m, 2H), 4.22
(t, J=6.4 Hz, 2H), 4.10 (m, 2H), 3.70 (t, J=6.4 Hz, 2H), 3.61 (m,
2H), 3.53 (m, 2H), 3.49 (m, 2H), 2.40 (s, 3H); MS (ESI): 608
(M+H.sup.+).
Preparation of
##STR00327##
[0538] 4-((1-Methyl-1H-benzo[d]imidazol-2-yl)ethynyl)aniline:
CL-5311-144 Intermediate for T540P was prepared using general
procedure E. Reaction was performed on a 277 mg scale of T464.
CL-5311-144 was isolated as a light yellow solid (140 mg, 48%).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 7.58 (m, 1H), 7.47 (m,
1H), 7.09-7.38 (m, 4H), 6.66 (m, 2H), 3.91 (s, 3H); MS (ESI): 248
(M+H.sup.+).
Preparation of
##STR00328##
[0540]
3-(4-((1-Methyl-1H-benzo[d]imidazol-2-yl)ethynyl)phenylamino)propyl
4-methylbenzenesulfonate: T540P was prepared using general
procedure Q. Reaction was performed on an 80.0 mg scale of
CL-5311-144. T540P was isolated as a light yellow solid (83.0 mg,
56%). .sup.1H NMR (400 MHz, CD.sub.2Cl.sub.2): .delta. 7.77 (m,
2H), 7.68 (m, 1H), 7.44 (m, 2H), 7.24-7.38 (m, 5H), 6.53 (m, 2H),
4.14 (t, J=6.0 Hz, 2H), 4.09 (br t, J=6.0 Hz, 2H), 3.89 (s, 3H),
3.24 (m, 2H), 2.44 (s, 3H), 1.95 (m, 2H); MS (ESI): 460
(M+H.sup.+).
Preparation of
##STR00329##
[0542]
2-(2-(2-((5-((4-(Dimethylamino)phenyl)ethynyl)pyridin-2-yl)(methyl)-
amino)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate: T546P was
prepared using general D. Reaction was performed on a 35 mg scale
of T526. T546P was isolated as a colorless gum (30.2 mg, 47%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.26 (dd, J=2.4, 0.8 Hz,
1H), 7.78 (m, 2H), 7.50 (dd, J=8.8, 2.4 Hz, 1H), 7.35 (m, 2H), 7.31
(m, 2H), 6.64 (m, 2H), 6.44 (d, J=8.8 Hz, 1H), 4.12 (t, J=4.8 Hz,
2H), 3.74 (t, J=5.6 Hz, 2H), 3.61-3.64 (m, 4H), 3.52-3.53 (m, 4H),
3.07 (s, 3H), 2.96 (s, 6H), 2.42 (s, 3H); MS (ESI): 538
(M+H.sup.+).
Preparation of
##STR00330##
[0544] 2-(4-(4-Methylpiperazin-1-yl)phenyl)quinolin-6-ol:
CL-5311-146 Intermediate for T550P was prepared using general
procedure A. Reaction was performed on a 208 mg scale of
2-chloroquinolin-6-ol. CL-5311-146 was isolated as a grey solid
(214 mg, 58%). .sup.1H NMR (400 Hz, DMSO-d.sub.6): .delta. 9.88 (s,
1H), 8.10 (d, J=8.8 Hz, 1H), 8.06 (m, 2H), 7.89 (d, J=8.8 Hz, 1H),
7.81 (d, J=9.2 Hz, 1H), 7.25 (dd, J=9.2, 2.8 Hz, 1H), 7.09 (d,
J=2.8 Hz, 1H), 7.02 (m, 2H), 3.22 (br, 4H), 2.45 (br s, 4H), 2.22
(s, 3H); MS (ESI): 320 (M+H.sup.+).
Preparation of
##STR00331##
[0546]
2-(2-(2-(2-(4-(4-Methylpiperazin-1-yl)phenyl)quinolin-6-yloxy)ethox-
y)-ethoxy)ethyl 4-methylbenzenesulfonate: T550P was prepared using
general procedure C. Reaction was performed on a 101 mg scale of
CL-5311-146. T550P was isolated as a white solid (90.0 mg, 47%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.97-8.08 (m, 4H),
7.75-7.79 (m, 3H), 7.35 (dd, J=9.2, 2.8 Hz, 1H), 7.29 (m, 2H), 7.06
(d, J=2.8 Hz, 1H), 7.02 (m, 2H), 4.24 (t, J=4.6 Hz, 2H), 4.15 (t,
J=4.8 Hz, 2H), 3.89 (t, J=4.8 Hz, 2H), 3.67-3.70 (m, 4H), 3.61-3.64
(m, 2H), 3.35 (br s, 4H), 2.66 (br s, 4H), 2.40 (s, 3H), 2.39 (s,
3H); MS (ESI): 606 (M+H.sup.+).
Preparation of
##STR00332##
[0548]
2-(2-(4'-(Dimethylamino)-[1,1'-biphenyl]-4-yl)-1H-benzo[d]imidazol--
1-yl)ethyl-4-methylbenzenesulfonate T543P was prepared using
general procedure E. Reaction was performed on a 0.082 g scale.
T543P was isolated s a yellow solid (0.050 g, 38%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 7.78 (d, J=7.6 Hz, 1H), 7.62-7.71
(m, 4H), 7.56 (d, J=8.4 Hz, 2H), 7.39 (d J=8.4 Hz, 2H), 7.22-7.34
(m, 3H), 7.05 (d, J=8.8, Hz, 2H), 6.83 (d, J=8.8, Hz, 2H), 3.02 (s,
6H), 2.32 (s, 3H); MS (ESI): 512 (M+H.sup.+).
Preparation of
##STR00333##
[0550] 2-chloroquinolin-6-ol: DHK-6-71 was prepared using general
procedure G. Reaction was performed on a 2 g scale. DHK-6-71 was
isolated as yellow solid (1.72 g, 93%). MS (ESI): 180.0
(M+H.sup.+).
[0551] 2-(4-morpholinophenyl)quinolin-6-ol: DHK-6-77 was prepared
using general procedure A. Reaction was performed on a 0.2 g scale.
DHK-6-77 was isolated as yellow solid (0.31 g, 91%). MS (ESI):
307.1 (M+H.sup.+).
[0552]
2-(2-(2-((2-(4-morpholinophenyl)quinolin-6-yl)oxy)ethoxy)ethoxy)eth-
yl 4-methylbenzenesulfonate: T549P was prepared using general
procedure C. Reaction was performed on a 0.19 g scale. T549P was
isolated as white solid (0.1 g, 27%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.07 (d, J=8.8 Hz, 2H), 8.03 (d, J=8.8 Hz,
1H), 8.00 (d, J=9.2 Hz, 1H), 7.78 (d, J=8.0 Hz, 3H), 7.35 (dd,
J=9.2, 2.8 Hz, 1H), 7.30 (d, J=8.0 Hz, 2H), 7.07 (d, J=3.2 Hz, 1H),
7.01 (d, J=8.8 Hz, 2H), 4.23 (t, J=4.8 Hz, 2H), 4.15 (t, J=4.4 Hz,
H), 3.89 (t, J=4.8 Hz, 3H), 3.88 (t, J=4.8 Hz, 3H), 3.71-3.62 (m,
4H), 3.64-3.62 (m, 2H), 3.25 (t, J=4.8 Hz, 4H), 2.40 (s, 3H); MS
(ESI): 593.1 (M+H.sup.+).
Preparation of
##STR00334##
[0554] 5-((6-Nitropyridin-3-yl)ethynyl)benzo[d]thiazole: T114P was
prepared using general procedure A. Reaction was performed on a
0.04 g scale. T114P was isolated as yellow solid (0.070 g, 99%).
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 9.53 (s, 1H), 8.91 (d,
J=1.6 Hz, 1H), 8.46-8.39 (m, 3H), 8.32 (d, J=8.0 Hz, 1H), 7.68 (dd,
J=8.4, 1.2 Hz, 1H); MS (ESI): 282.0 (M+H.sup.+).
Preparation of
##STR00335##
[0556] T508P was prepared using general procedure D. The reaction
was performed on a 0.2 g scale. T508P was isolated as a solid (0.18
g, 42.9%). .sup.1H NMR (400 MHz, CD.sub.3CN): .delta. 7.74-7.72 (m,
2H), 7.63-7.61 (m, 1H), 7.51-7.48 (m, 3H), 7.41-7.38 (m, 2H),
7.29-7.24 (m, 2H), 6.77-6.75 (m, 2H), 4.52-4.49 (m, 2H), 3.94-3.92
(m, 2H), 3.86-3.83 (m, 2H), 3.46-3.36 (m, 6H), 3.01 (s, 6H), 2.42
(s, 3H); MS (ESI): 548.1 (M+H.sup.+).
Preparation of
##STR00336##
[0558] T527P was prepared using general procedure D from butyl
5-((1H-benzo[d]imidazol-2-yl)ethynyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxy-
late and
2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(4-methylben-
zenesulfonate). The reaction was performed on a 0.21 g scale of
butyl
5-((1H-benzo[d]imidazol-2-yl)ethynyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxy-
late. T527P was isolated as a colorless oil (0.07 g, 18.53%).
.sup.1H NMR (400 MHz, CD.sub.3CN) .delta. 8.69 (m, 1H), 8.27-8.26
(m, 1H), 7.88-7.87 (m, 1H), 7.72-7.66 (m, 3H), 7.55-7.53 (m, 1H),
7.38-7.28 (m, 4H), 6.71-6.70 (m, 1H), 4.59-4.56 (m, 2H), 4.48-4.44
(m, 2H), 3.93-3.86 (m, 4H), 3.47-3.45 (m, 2H), 3.41-3.36 (m, 4H),
2.40 (s, 3H), 1.83-1.79 (m, 2H), 1.57-1.51 (m, 2H), 1.02-1.00 (m,
3H); MS (ESI): 645.0 (M+H.sup.+).
Preparation of
##STR00337##
[0560] (E)-3-(Dimethylamino)-1-(4-nitrophenyl)prop-2-en-1-one: A
solution of 1-(4-nitrophenyl)ethanone (2.2 g, 13 mmol) and
N,N-dimethylformamide dimethyl acetal (25 ml) was heated to
120.degree. C. in a sealed tube overnight. The volatiles were
removed. The residue was dissolved in DCM and washed twice with
H.sub.2O. The DCM layer was dried with MgSO.sub.4. The crude
product was purified by flash chromatography (silica gel, 100%
EtOAc) to isolate
(E)-3-(dimethylamino)-1-(4-nitrophenyl)prop-2-en-1-one (2.2 g), as
a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.99 (s,
3H), 3.23 (s, 3H), 5.70 (d, J=12.4 Hz, 1H), 7.94 (d, J=12.4 Hz,
1H), 8.03 (m, 2H), 8.26 (m, 2H). MS (ESI): 221 (M+H.sup.+)
Preparation of
##STR00338##
[0562]
tert-Butyl(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)phenyl)carbama-
te: A solution of 4-(benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)aniline
(350 mg, 1.3 mmol) in di-tert-butyl dicarbonate (4 mL) was heated
in a sealed tube at 120.degree. C. for 15 minutes. The reaction
mixture was diluted with DCM and purified directly by flash
chromatography (silica gel, 100% EtOAc) to give tert-butyl
(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)phenyl)carbamate (249
mg, 53%) as an orange solid. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 1.59 (s, 9H), 6.89 (m, 2H), 6.97 (m, 1H), 7.16 (m, 1H),
7.54-7.58 (m, 3H), 8.11 (m, 1H), 8.88 (d, J=4.4 Hz, 1H). MS (ESI):
361 (M+H.sup.+).
Preparation of
##STR00339##
[0564]
3-((4-(Benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)phenyl)(tert-butoxyca-
rbonyl)amino)propyl 4-methylbenzenesulfonate T544P The title
compound was prepared using general procedure D from
4-(benzo[4,5]imidazo[1,2-a]pyrimidin-4-yl)aniline and
propane-1,3-diyl bis(4-methylbenzenesulfonate). T554P was isolated
as a solid (130 mg, 21%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 1.51 (s, 9H), 2.09 (m, 2H), 2.46 (s, 3H), 3.87-3.90 (m,
2H), 4.12-4.15 (m, 2H), 6.94 (m, 1H), 7.04 (bs, 1H), 7.19 (m, 1H),
7.35 (m, 1H), 7.52-7.65 (m, 5H), 7.77-7.79 (m, 2H), 8.18 (m, 1H),
8.97 (d, J=4.4 Hz, 1H). MS (ESI): 573.1 (M+H.sup.+).
Preparation of
##STR00340##
[0566] N,N-Dimethyl-5-(4-nitroquinolin-2-yl)pyridin-2-amine T480P
was prepared using general procedure A from
2-Bromo-4-nitroquinoline (50 mg, 0.2 mmol) and
(6-(dimethylamino)pyridin-3-yl)boronic acid (34 mg, 0.2 mmol). The
product was obtained as a yellow solid (40 mg, 68%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.99 (d, J=2.4 Hz, 1H), 8.36-8.34
(m, 2H), 8.30 (s, 1H), 8.19 (m, 1H), 7.81 (m, 1H), 7.65 (m, 1H),
6.66 (d, J=9.0 Hz, 1H), 3.21 (s, 6H); MS (ESI): 295
(M+H.sup.+).
Preparation of
##STR00341##
[0568] 4-(4-Nitroquinolin-2-yl)aniline T492P was prepared by using
general procedure A from 2-Bromo-4-nitroquinoline (50 mg, 0.2 mmol)
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (44 mg,
0.2 mmol). The product was obtained as a dark brown solid (31 mg,
58%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.36 (m, 1H), 8.32
(s, 1H), 8.20 (m, 1H), 8.06 (m, 2H), 7.80 (m, 1H), 7.65 (m, 1H),
6.81 (m, 2H), 3.99 (br s, 2H); MS (ESI): 266 (M+H.sup.+).
Preparation of
##STR00342##
[0570] N-Methyl-4-(4-nitroquinolin-2-yl)aniline T466P was prepared
using general procedure A from 2-bromo-4-nitroquinoline (50 mg, 0.2
mmol) and
N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (46
mg, 0.2 mmol). The product T466P was obtained as a brown solid (37
mg, 66%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.34 (m, 1H),
8.32 (s, 1H), 8.19 (m, 1H), 8.09 (m, 2H), 7.79 (m, 1H), 7.63 (m,
1H), 6.72 (m, 2H), 2.93 (s, 3H); MS (ESI): 280 (M+H.sup.+).
Preparation of
##STR00343##
[0572]
6-Methoxy-2-(4-(4-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)piperazi-
n-1-yl)-phenyl)quinoline AS-5332-79 was prepared using general
procedure E. Reaction performed on a 0.032 g. AS-5332-79 was
isolated as a off white solid (0.025 g, 54%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.05 (dt, J=8.8, 2.8 Hz, 2H), 8.00 (d, J=10.4
Hz, 2H), 7.76 (d, J=8.8 Hz, 1H), 7.32 (dd, J=9.2, 2.8 Hz, 1H), 7.04
(d, J=2.8 Hz, 1H), 7.01 (dt, J=9.2, 2.8 Hz, 2H), 4.58 (t, J=4.4 Hz,
1H), 3.92 (s, 3H), 3.86-3.77 (m, 1H), 3.51-3.45 (m, 2H), 3.30 (t,
J=4.8 Hz, 4H), 2.63 (t, J=4.8 Hz, 4H), 2.53-2.49 (m, 2H), 1.88-1.80
(m, 4H), 1.73-1.68 (m, 1H), 1.59-1.49 (m, 4H); MS (ESI): 462.4
(M+H.sup.+).
Preparation of
##STR00344##
[0574]
2-(4-(4-(3-Chloropropyl)piperazin-1-yl)phenyl)-6-methoxyquinoline
AS-5332-94, T499P (Cl) was prepared using general procedure E.
Reaction performed on a 0.025 g. T-99P (Cl) was isolated as a off
white solid (0.010 g, 32%). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta. 8.6-7.99 (m, 4H), 7.77 (d, J=8.8 Hz, 1H), 7.33 (dd, J=9.2,
2.8 Hz, 1H), 7.06-7.00 (m, 3H), 3.92 (s, 3H), 3.66 (t, J=6.4 Hz,
2H), 3.29 (t, J=5.2 Hz, 4H), 2.62 (t, J=4.8 Hz, 4H), 2.55 (t, J=7.6
Hz, 2H), 1.99 (m, 2H); MS (ESI): 396.1 (M+H.sup.+).
4. General Procedures for Radiochemistry
Description of Radiolabeling Manufacturing Process and Process
Controls
General Process for the Production of [F-18] Fluoride Ion
18F-Radiolabeling:
[0575] Aqueous [F-18] Fluoride ion produced in the cyclotron
target, is passed through an anion exchange resin cartridge. The
[O-18]H20 readily passes through the anion exchange resin while
[F-18] fluoride is retained. The [F-18] fluoride is eluted from the
column using a solution of potassium carbonate (3 mg) in water (0.4
mL) and is collected in a reaction vessel. Kryptofix.RTM. 222 (20
mg) dissolved in acetonitrile (1 mL) is added to the aqueous [F-18]
fluoride mixture in the reaction vessel. The Kryptofix sequesters
the potassium ions preventing the formation of strong K+/F
on-pairs. This increases the chemical reactivity of the [F-18]
fluoride ions. The mixture is dried by heating between
70-115.degree. C. under a stream of inert gas and/or reduced
pressure (250 mbar) and additional aliquots of acetonitrile may
added to insure the fluoride mixture is completely dry. This
evaporation step removes the water and converts the [F-18] to an
anhydrous form, which is much more reactive than aqueous [F-18]
fluoride.
[0576] Fluorine-18 [F-18] is produced by proton bombardment of the
stable isotope, oxygen-18 (O-18) in water. For bombardment, the
chemical form of the enriched O-18 is [O-18]H.sub.2O. The [F-18]
Fluorine produced is aqueous [F-18] fluoride ion. The target water
is loaded into an approximately 1-2 mL target and pressurized to
approximately 350 psi. The tantalum target body is outfitted with a
high strength, durable metal foil. The foil is an alloy referred to
as, Havar.RTM.. The major components of Havar.RTM. are cobalt,
nickel, chromium, and iron. This thin Havar.RTM. foil window
permits entry of the protons, yet is sufficiently durable to
withstand the pressurized water and proton irradiation. The
facility utilizes two Siemens RDS-111 Eclipse cyclotron that
produces 11 MeV protons with a 40-60 microamp beam current. Both
targets are made of tantalum metal and are used exclusively for the
production of F-18. After proton bombardment, the [O-18]H.sub.2O
containing the [F-18] fluoride ion is transferred to a shielded
enclosure ("hot cell"). The aqueous [F-18] Fluoride is then
separated from the [O-18]H.sub.2O.
Extraction of [F-18] Fluoride and Conversion to Anhydrous Form
[0577] Aqueous [F-18] Fluoride ion produced in the cyclotron
target, as described in the preceding Section, is passed through an
anion exchange resin cartridge. The [O-18]H.sub.2O readily passes
through the anion exchange resin while [F-18] fluoride is retained.
The [F-18] fluoride is eluted from the column using a solution of
potassium carbonate (3 mg) in water (0.4 mL) and is collected in a
reaction vessel. Kryptofix.RTM. 222 (20 mg) dissolved in
acetonitrile (1 mL) is added to the aqueous [F-18] fluoride mixture
in the reaction vessel. The Kryptofix sequesters the potassium ions
preventing the formation of strong K.sup.+/F ion-pairs. This
increases the chemical reactivity of the [F-18] fluoride ions.
[0578] The mixture is dried by heating between 70-115.degree. C.
under a stream of inert gas and/or reduced pressure (250 mbar) and
additional aliquots of acetonitrile may added to insure the
fluoride mixture is completely dry. This evaporation step removes
the water and converts the [F-18] to an anhydrous form, which is
much more reactive than aqueous [F-18] fluoride.
Reaction of Anhydrous [F-18] Fluoride with W366 Precursor
[0579] A solution of the nitro precursor, (1 to 20 mg), dissolved
in anhydrous DMSO (0.5-2.5 mL) is added to the reaction vessel
containing the anhydrous [F-18] Fluoride. The vessel is heated to
approximately 150.+-.10.degree. C. for 15.+-.5 minutes to induce
displacement of the aromatic nitro leaving group by [F-18] fluoride
as illustrated in the scheme below. The reaction mixture is then
treated with 2N HCl (1 mL) and refluxed at 105.degree. C. for 10
min.
##STR00345##
HPLC Purification of [F-18]W366
[0580] The reaction mixture containing crude [F-18]W366 is cooled
and first passes through an Al.sub.2O.sub.3 cartridge followed by a
mixture of MeCN (1.+-.0.5 mL) and H.sub.2O (2.+-.1.0 mL). The final
solution is then transferred to the HPLC sample loop and is
purified via chromatographic separation using a semi-preparative
HPLC column (Either ACE C18 Pyramid, 7.mu., 250.times.10 mm,
Phenomenex Luna, C18, 5.mu., 10.times.250 mm or Phenomenex Synergi
Hydro-RP C18, 250.times.10 mm, using a gradient system, up to 5.5
mL/min, however lower flow rates may be used if there is a high
backpressure, or the system may start at a lower flow rate and then
increase to the maximum flow rate). The first column uses a linear
gradient starting at 5% MeCN (0.1% formic acid):95% H20 (0.1%
formic acid) containing 100 mg/L of ascorbic acid and to a 95:5 mix
of the solvents at 30 minutes. The column effluent is monitored
using UV (220, 254 or 280 nm) and radiometric detectors connected
in series. The purified [F-18]W366 is collected from the column at
the retention time window determined for the W366 reference
standard which coincides with the time that the radiometric
detectors begin showing the main peak. After the product elutes, it
is collected, loaded onto the HPLC load loop and purified again.
(Either ACE C18 Pyramid, 7.mu., 250.times.10 mm, Phenomenex Luna,
C18, 5.mu., 10.times.250 mm or Phenomenex Synergi Hydro-RP C18,
250.times.10 mm, using a isocratic solvent system suitable to
purify W366, such as 40% MeCN:water with 0.1% formic acid and 100
mg/L of ascorbic acid, up to 5.5 mL/min, however lower flow rates
may be used if there is a high backpressure, or the system may
start at a lower flow rate and then increase to the maximum flow
rate).
Formulation, Sterile Filtration and Aseptic Filling of Purified
[F-18]W366
[0581] The purified [F-18]W366 fraction elutes from the second HPLC
purification column, is diluted with water (40 100 mL) containing
5.+-.5 mg/mL of ascorbic acid, and is captured onto a C18 SepPak
cartridge. The C18 SepPak cartridge is washed with water (10 mL)
containing 5.+-.5 mg/mL ascorbic acid followed by eluting the
product with 0.5-0.9 mL of EtOH. The sample is then diluted with
sterile water (4.5-9.0 mL of water) containing 25.+-.25 mg/mL of
ascorbic acid affording a final formulation of [F-18]W366 in a
maximum of 10% EtOH:water. The solution is then processed through a
0.45 .mu.m sterile filter into the preloaded collection vial.
Biological Data
[0582] The disclosed compounds compete favorably for binding
against 18F-PiB, as shown below. Briefly, 5 micron thick human
brain slices from regions of the brain bearing high amyloid plaque
and fibril burden were incubated with approximately 20 uCi of a
radiolabeled tracer in 2.5%:2.5%:95% DMSO:EtOH:PBS in the presence
of blocker (2.5 and 0.25 uM total concentration) or absence of
blocker (control). The slices were incubated at rt for 90 min. The
slices were then quickly washed in PBS, followed by 70% EtOH:PBS
for 2 min, then 30% EtOH:PBS for 2 min and then quickly washed with
PBS. The slices were dried for 30 min and then exposed on
autoradiographic film for 20 min. The brain slices were then
removed from the slide and the radioactivity counted in a gamma
counter. The counts are normalized and the percent blocking is
determined in order to determine IC.sub.50 values. The lower the
number, the more effective the compounds displaced the tracer.
Reaction of Anhydrous [F-18] Fluoride with T114 Precursor
##STR00346##
[0583] [F-18] Fluoride was prepared using K.sub.2CO.sub.3 and
Kryptofix-2.2.2 according to the general procedure described above.
A solution of T114P (10 mg) dissolved in anhydrous DMSO (1.0 mL) is
added to the reaction vessel containing the anhydrous [F-18]
Fluoride. The vessel is heated to approximately to 150.degree. C.
for 20 minutes. Reaction was loaded to a vial containing water (4
mL) and the resultant solution is then transferred to the HPLC
sample loop (5 mL) and purified via chromatographic separation
using a semi-preparative HPLC column (Phenomenex Gemini C18,
250.times.10 mm). This column uses a flow rate of 5 mL/min and an
isocratic solvent system of 40% MeCN: 60% H.sub.2O containing 0.85
mL of 12N HCl per 1000 mL of water. The column effluent is
monitored using UV (254 nM) and radiometric detectors connected in
series. The purified [F-18] T114 is collected from the column at
the retention time window determined for the T114 reference
standard which coincides with the time that the radiometric
detectors begin showing the main peak. The retention time of the
[F-18] T114 in this system is approximately 39 minutes.
Formulation of Purified [F-18] T114
[0584] The purified [F-18] T114 fraction eluted from the HPLC
purification column is diluted with water (50 mL) and filtered thru
a C18 SepPak cartridge. The C18 SepPak cartridge is washed with
water (10 mL) followed by elution of the product with 0.5 mL of
ethyl alcohol. The sample is then diluted with 4.5 mL of water to
afford a final formulation of [F-18] T114 in a maximum of 10% ethyl
alcohol in water.
General Procedure for [F-18] Labeling of Aliphatic Tosylates
[0585] [F-18] Fluoride was prepared using K.sub.2CO.sub.3 and
Kryptofix-2.2.2 according to the general procedure described above.
After cooling, a solution of the tosylate precursor (5 mgs to 20
mgs) in anhydrous DMSO or MeCN (1 mL) was added to the residue of
"dry" reactive [F-18] fluoride ion in the reaction vessel of the
Explora RN synthesis module and the reaction was heated (80.degree.
C. to 110.degree. C.) for 10 to 15 mins. The reaction was cooled to
70.degree. C.
[0586] If the precursor contains an acid labile protecting group,
1N HCl (1 mL) was added to the reaction mixture and heated to
100.degree. C. After 5 minutes, the reaction was cooled to room
temperature and 2M NaOAc (0.5 mL) was added. The resulting mixture
was added to a separate vial containing water (1.5 mL) and loaded
to the HPLC sample loop to initiate purification.
[0587] If the precursor contains a basic labile protecting group,
1:1 MeOH: 1N NaOH (1 mL) was added to the reaction mixture and
heated to 100.degree. C. After 5 minutes, the reaction was added to
a separate vial containing water (2 mL) and loaded to the HPLC
sample loop to initiate purification.
[0588] If the precursor contains no protecting groups, the
resulting reaction mixture is added to a separate vial containing
water (3 mL) and loaded to the HPLC sample loop to initiate
purification.
[0589] Purification was performed by semi-preparative HPLC
(Phenomenex Gemini C18, 250.times.10 mm, flow rate 5 mL/min). The
elution of the final product is initiated at 5% MeCN (0.05% TFA) in
H.sub.2O (0.05% TFA) until the final concentration of MeCN (0.05%
TFA) is reached within 15 to 20 minutes. Once the final
concentration of the MeCN (0.05% TFA) is reached then the elution
is allowed to run isocratic until the [F-18] product is collected.
Once collected, the final formulation described above is
followed.
TABLE-US-00004 TABLE 4 Radiolabeling results of aliphatic tosylates
Radio- Cmpd Yield chem. HPLC # Precursor [F-18] Product (mCi)
Purity conditions T114 ##STR00347## ##STR00348## 48 >98% as
described above T442 ##STR00349## ##STR00350## 40 >98% Final
conc: 25% MeCN (0.05% TFA) in water (0.05% TFA) T482 ##STR00351##
##STR00352## 28 >98% Final conc: 30% MeCN (0.05% TFA) in water
(0.05% TFA) T510 ##STR00353## ##STR00354## 30 >98% Final conc:
30% MeCN (0.05% TFA) in water (0.05% TFA) T525 ##STR00355##
##STR00356## 258 >98% Final conc: 20% MeCN (0.05% TFA) in water
(0.05% TFA) T527 ##STR00357## ##STR00358## 15 >98% Final conc:
25% MeCN in water (0.08% HCl) T549 ##STR00359## ##STR00360## 78
>98% Final conc: 25% MeCN (0.05% TFA) in water (0.05% TFA)
##STR00361##
Human AD Brain Section Autoradiography
[0590] 5 micron thick human AD brain slices were first examined
using antibodies for A.beta. and Tau to determine whether the
tested human brain contains A.beta. and Tau. Thus, three types of
human brain slices were selected for autoradiography:
A.beta.+/Tau+; A.beta.+/Tau-; and A.beta.-/Tau-(control).
[0591] The experimental protocol is as follows:
Pick one brain section for each type and air-dry in hood. A
solution of diluted F-18 labeled tracer (40 .mu.Ci/mL, 500 .mu.L)
which was obtained from the dilution of F-18 tracer with
1.times.PBS containing 2.5% EtOH and 2.5% DMSO was applied onto
each slides to cover the whole tissue section. The resulting slides
were incubated at room temperature for 90 minutes, drained, and
placed onto a slide holder. The slides were then washed
sequentially with 1.times.PBS for 1 min; 70% EtOH in 1.times.PBS
for 2 min; 30% EtOH in 1.times.PBS for 2 min; and 1.times.PBS for 1
min. The slides were dried in the hood for 30 min, and then placed
on Fuji imaging plates and exposed overnight. The imaging plates
were then scanned and the signal was measured using Fuji software
to produce an autoradiography image of the brain section.
(PBS--Phosphate Buffer Saline)
Protocol for Synthetic Beta-Amyloid and Tau Kds
[0592] Various concentrations solution of F-18 labeled and its
parent cold compound in 1.times.PBS containing 5% Ethanol and 5%
DMSO (pH 7.4) were incubated with synthetic beta-Amyloid or
synthetic tau at room temperature in glass tubes for 90 min. The
reaction mixture in each tube was filtered under vacuum through a
microfiber filter. Each tube was washed with a solution of 20% EtOH
in PBS. The PBS wash solution was filtered under vacuum through the
filters. Each filter was then washed with a solution of 20% EtOH in
PBS and then placed into a gamma counter vial for CPM counting. The
data obtained was plotted for Kd determination.
Tau Fluorescent Compounds Staining on Human Brain Sections (Double
or Triple Labeled with Tau and .beta.-Amyloid Immunohistochemistry
(IHC))
[0593] Serial sections (6 .mu.m thick) from OCT-embedded frozen
blocks of front lobe were used for staining (OCT--optimal cutting
temperature). After fixation and quenching of autofluorescence,
tissue sections were incubated with in 100 .mu.M of tau compound in
50% ethanol PBS for 60 min. Then sections were dipped briefly into
water, rinsed in PBS, blocked with 5% normal horse serum in PBS for
1 hour at room temperature. After blocking, the tissue was
incubated with tau or .beta.-amyloid primary antibody at 4.degree.
C. overnight in a humid chamber. Next day, the sections were washed
with PBS and then incubated with secondary antibody for 1 hour. The
sections were washed and covered, and they were observed with a
Nikon (Tokyo, Japan) Eclipse microscope equipped with violet, blue,
and green filters.
Mouse and Rat Brain microPET Imaging
[0594] Wild type mice and rats were injected intravenously with the
candidate tracers. Mice (weight range 25-45 g) were injected with
doses between 180 and 300 .mu.Ci in 200 uL of saline solution. Rats
(weight range 300-400 g) were injected with doses between 300 and
500 .mu.Ci of tracer in 400 .mu.L of saline solution. Anesthesia
was induced and maintained with Isoflurane. CT and PET scans were
performed with a MM INVEON scanner (SIEMENS.TM.). Acquisition for
CT images preceded PET scanning and lasted 5 minutes. Only several
minutes after the beginning of the PET acquisition, the radioactive
dose was injected into the animal through the tail vein. Images
were generated as dynamic scans that typically lasted 30 minutes.
The initial image analysis consisted of determining whether there
was uptake of the tracer in the brain which would establish its
ability to cross the blood-brain barrier. All measurements were
performed at the time point of 5 minutes following the injection of
the tracer. The degree of uptake in the brain was estimated
relative to the uptake of the traces in the region of neck muscles.
The ratio between the percentage of injected dose per gram in the
brain and that of the muscular neck region was provided as an
estimate of brain uptake. Brain images of the representative
tracers of formula (I) are shown in FIGS. 17-22.
[0595] Having thus descried in detail advantageous embodiments of
the present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
* * * * *