U.S. patent application number 14/818745 was filed with the patent office on 2015-11-26 for 8-hydroxy quinoline derivatives.
This patent application is currently assigned to PRANA BIOTECHNOLOGY LIMITED. The applicant listed for this patent is PRANA BIOTECHNOLOGY LIMITED. Invention is credited to Kevin Jeffrey BARNHAM, Elisabeth Colette Louise GAUTIER, Gaik Beng KOK, Guy KRIPPNER.
Application Number | 20150335635 14/818745 |
Document ID | / |
Family ID | 27809319 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150335635 |
Kind Code |
A1 |
BARNHAM; Kevin Jeffrey ; et
al. |
November 26, 2015 |
8-HYDROXY QUINOLINE DERIVATIVES
Abstract
The present invention describes a method for the treatment of a
neurological condition in a subject which comprises administering
to a subject in need thereof a therapeutically effect amount of a
compound of the formula ##STR00001## or pharmaceutically acceptable
salts, hydrates, or solvates thereof.
Inventors: |
BARNHAM; Kevin Jeffrey;
(Coburg, AU) ; GAUTIER; Elisabeth Colette Louise;
(Bentleigh, AU) ; KOK; Gaik Beng; (North Carlton,
AU) ; KRIPPNER; Guy; (Glen Waverley, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRANA BIOTECHNOLOGY LIMITED |
Parkville |
|
AU |
|
|
Assignee: |
PRANA BIOTECHNOLOGY LIMITED
Parkville
AU
|
Family ID: |
27809319 |
Appl. No.: |
14/818745 |
Filed: |
August 5, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14510652 |
Oct 9, 2014 |
|
|
|
14818745 |
|
|
|
|
11901941 |
Sep 19, 2007 |
8975278 |
|
|
14510652 |
|
|
|
|
10521902 |
Aug 10, 2005 |
7619091 |
|
|
PCT/AU2003/000914 |
Jul 16, 2003 |
|
|
|
11901941 |
|
|
|
|
Current U.S.
Class: |
514/313 ;
514/311; 514/314 |
Current CPC
Class: |
A61P 9/04 20180101; A61P
25/14 20180101; C07D 401/12 20130101; A61K 31/4709 20130101; A61K
31/4725 20130101; A61P 25/02 20180101; A61P 31/18 20180101; C07D
401/04 20130101; A61P 27/02 20180101; C07D 215/48 20130101; A61P
3/00 20180101; A61P 9/08 20180101; A61P 25/18 20180101; A61P 9/10
20180101; A61P 7/06 20180101; A61P 31/00 20180101; C07D 401/14
20130101; A61P 25/16 20180101; A61P 9/00 20180101; A61K 31/4709
20130101; A61P 31/04 20180101; A61K 45/06 20130101; A61P 25/28
20180101; C07D 215/38 20130101; A61P 7/04 20180101; A61P 27/12
20180101; A61P 5/30 20180101; A61P 35/00 20180101; A61P 37/04
20180101; C07D 417/14 20130101; C07D 215/24 20130101; C07D 417/12
20130101; A61P 1/04 20180101; A61P 25/32 20180101; A61P 25/20
20180101; A61P 27/06 20180101; A61P 25/08 20180101; A61P 21/04
20180101; A61P 21/00 20180101; C07D 215/26 20130101; Y02A 50/411
20180101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61P 43/00
20180101; A61K 2300/00 20130101; C07D 215/00 20130101; C07D 409/04
20130101; A61P 3/02 20180101; A61K 31/4725 20130101; A61P 39/02
20180101; C07D 215/28 20130101; A61P 25/36 20180101; A61P 25/00
20180101; A61P 29/00 20180101; A61P 13/12 20180101; A61P 31/12
20180101; A61P 17/02 20180101; A61K 31/47 20130101; A61K 31/47
20130101; A61P 31/06 20180101 |
International
Class: |
A61K 31/4709 20060101
A61K031/4709; A61K 45/06 20060101 A61K045/06; A61K 31/47 20060101
A61K031/47 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2002 |
AU |
2002950217 |
Claims
1.-14. (canceled)
15. A method for the treatment of Huntington's disease in a subject
which comprises administering to the subject a therapeutically
effective amount of a compound of the formula ##STR00284## or
pharmaceutically acceptable salts thereof, in which R.sup.1 is H;
R.sup.2 is optionally substituted phenyl; optionally substituted
naphthyl; optionally substituted tetrahydronaphthyl; optionally
substituted biphenyl; optionally substituted heterocyclyl;
COR.sup.6; CSR.sup.6; CN; (CH.sub.2)NR.sup.9R.sup.10; HCNOR.sup.9;
HCNNR.sup.9R.sup.10; OR.sup.11; SR.sup.11; NR.sup.11R.sup.12 or
SO.sub.2NR.sup.13R.sup.14; in which R.sup.13 and R.sup.14 are
either the same or different and selected from H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted aryl or optionally substituted heterocyclyl; and
R.sup.6 is H, C.sub.1-6 alkyl, optionally substituted C.sub.2-6
alkenyl, hydroxy, optionally substituted aryl, optionally
substituted heterocyclyl, SR.sup.7 or NR.sup.7R.sup.8; R.sup.7 and
R.sup.8 are either the same or different and selected from H,
optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6 alkenyl, optionally substituted aryl and optionally
substituted heterocyclyl; R.sup.9 is H, optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl,
optionally substituted aryl or optionally substituted heterocyclyl;
R.sup.10 is hydrogen, methyl, propyl, isopropyl, butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, optionally substituted
C.sub.2-6 alkenyl, optionally substituted aryl or optionally
substituted heterocyclyl; R.sup.11 is H, optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl,
optionally substituted aryl or optionally substituted heterocyclyl
or together with R.sup.12 form optionally substituted heterocyclyl;
R.sup.12 is optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl or
optionally substituted heterocyclyl or together with R.sup.11 form
optionally substituted heterocyclyl; R.sup.3, R and R' are either
the same or different and selected from H, optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl,
optionally substituted C.sub.1-6 alkoxy, optionally substituted
acyl, hydroxy, optionally substituted amino, optionally substituted
thio, optionally substituted sulphonyl, optionally substituted
sulphinyl, optionally substituted sulphonylamino, halo, SO.sub.3H,
amine, CN, CF.sub.3, optionally substituted aryl and optionally
substituted heterocyclyl; and R.sup.4 and R.sup.5 are chloro,
wherein the optional substituent is C.sub.1-6 alkyl, CF.sub.3,
fluorine, chlorine, iodine, cyano, C.sub.1-6 alkoxy, 5 or
6-membered aryl, heteroaryl, amino or C.sub.1-6 alkylamino.
16. The method according to claim 15, in which either R.sup.2 is
optionally substituted phenyl, optionally substituted naphthyl,
optionally substituted tetrahydronapthyl, optionally substituted
biphenyl, optionally substituted heterocyclyl,
CH.sub.2NR.sup.9R.sup.10 or COR.sup.6 or at least one of R, R.sup.3
and R' is optionally substituted C.sub.1-6 alkyl, optionally
substituted aryl, optionally substituted heterocyclyl,
CH.sub.2NR.sup.9R.sup.10 or COR.sup.6 in which R.sup.6 is
NR.sup.7R.sup.8 or NR.sup.11R.sup.12.
17. The method of claim 15, in which the compound of formula I is
##STR00285## ##STR00286## or pharmaceutically acceptable salts
thereof.
18. The method of claim 15, in which the compound is administered
in association with a pharmaceutically acceptable carrier.
19. The method of claim 15, in which the compound is administered
in association with a medicament, said medicament being an
inhibitor of the acetyl cholinesterase active site, an antioxidant,
an anti-inflammatory agent or an oestrogenic agent.
20. The method of claim 15, in which the compound is in hydrate
form.
Description
RELATED APPLICATION
[0001] The present application is a continuation in-part of
copending application U.S. Ser. No. 10/521,902, filed on Aug. 10,
2005, the contents of which are incorporated by reference, which is
a '371 of PCT Application PCT/AU03/00914, filed on Jul. 16,
2003.
FIELD OF THE INVENTION
[0002] The present invention relates to 8-hydroxy quinoline
derivatives, processes for their preparation and their use as
pharmaceutical or veterinary agents, in particular for the
treatment of neurological conditions, more specifically
neurodegenerative conditions such as Alzheimer's disease,
Huntington's disease and the like.
BACKGROUND OF THE INVENTION
[0003] All references, including any patents or patent
applications, cited in this specification are hereby incorporated
by reference. No admission is made that any reference constitutes
prior art. The discussion of the references states what their
authors assert, and the applicants reserve the right to challenge
the accuracy and pertinency of the cited documents. It will be
clearly understood that, although a number of prior art
publications are referred to herein, this reference does not
constitute an admission that any of these documents forms part of
the common general knowledge in the art, in Australia or in any
other country.
[0004] The life span is thought to be biologically fixed for each
species, and the length of the human life span is uncertain, but
may be up to 120 years. Since life expectancy has risen
significantly in this century, the elderly are an increasing
segment of our population, and their health care needs will
continue to grow for decades.
[0005] Although normal aging is characterized by modest reductions
in the mass and volume of the human brain, which may be due to the
atrophy and/or death of brain cells, these changes are far more
profound in the brains of patients who succumb to a
neurodegenerative condition. Most of these conditions are sporadic
and of unknown cause, but hundreds of different mutations in many
genes have been shown to cause familial (inherited) variants of
several neurodegenerative conditions. Many of the dozen or more
genes that harbour these mutations were discovered in the quest to
determine the genetic basis of neurodegenerative conditions just in
the last ten years. Neurodegenerative conditions evolve gradually
after a long period of normal brain function, due to progressive
degeneration (i.e., nerve cell dysfunction and death) of specific
brain regions. Since symptomatic expression of disease occurs when
nerve cell loss exceeds a "threshold" for the continuing function
(e.g., memory, movement) performed by the affected brain region,
the actual onset of brain degeneration may precede clinical
expression by many years.
[0006] Intellectual and higher integrative cognitive faculties
become progressively impaired and interfere with activities of
daily living in neurological conditions resulting in dementia. The
precise prevalence of dementia in the elderly population is
unknown, but may be 15% of people over 65 years old with 5%
severely and 10% mildly to moderately demented. The prevalence of
severe dementia increases from 1% at 65 years to 45% at 85 years.
There are many causes of dementia, but Alzheimer's Disease (AD)
accounts for 50% of demented patients over 65 years of age.
[0007] AD is a primary degenerative disease of the brain. It is
characterized by progressive decline of cognitive functions such as
memory, thinking, comprehension, calculation, language, learning
capacity and judgement. Dementia is diagnosed when the declines are
sufficient to impair personal activities of daily living. AD shows
an insidious onset with slow deterioration. This disease needs to
be clearly differentiated from age-related normal decline of
cognitive functions. The normal decline is much less, much more
gradual and leads to milder disabilities. The onset of AD is
usually after 65 years of age, although earlier onset is not
uncommon. As age advances, the incidence increases rapidly (it
roughly doubles every 5 years). This has obvious implications for
the total number of individuals living with this disorder as life
expectancy increases in the population.
[0008] The aetiology of AD is unclear. There is considerable
evidence of a heritable predisposition for some forms of AD
(reviewed in St George-Hyslop, 2000), and the expression of certain
isoforms of ApoE has also been linked to a higher risk of AD
(Corder et al, 1993; Czech et al 1994). The toxic accumulation of
aluminium has been suggested as a causative agent in AD, although
this hypothesis has now been superseded. The brains of AD patients
display abnormal deposits which include .beta.-amyloid protein
(A.beta.).
[0009] A.beta. is known to be present in the brains of individuals
with certain neurodegenerative diseases, but it is not known
whether it is symptomatic of an underlying disease process, or is
actually involved in the aetiology of the disease. For example,
some authors believe that the A.beta. deposits may be indicative of
a normal brain defense mechanism, wherein the brain attempts to
sequester the A.beta.; such deposits can be present in the brains
of normal individuals. There is a mutation of tau protein wherein
neurofibrillary tangles, but no amyloid plaques are present in the
brain; this condition is known as tauopathy.
[0010] One proposed approach to AD therapy is to inhibit production
of A.beta. in the brain. Proteolytic cleavage of APP by BACE1 and
.gamma.-secretase generates the full-length A.beta., which is then
released from cells (Nunan and Small, 2000). Alternatively, a
number of studies have shown that cholesterol can influence A.beta.
release (Simons et al., 1998; Hartmann, 2001; Fassbender et al.,
2001; Frears et al., 1999; Friedhoff et al., 2001). However, there
is some disagreement in the art as to the value of lowering
cholesterol levels, and some workers consider that cholesterol is
actually beneficial. For example, Ji et al, (2002) have suggested
that the binding of A.beta. to cholesterol might prevent A.beta.
toxicity by inhibiting its oligomerization.
[0011] In an alternative approach, it has been proposed that by
unravelling the proteolytic processing of the amyloid precursor
protein (APP), which generates the A.beta. amyloid monomer, a
number of possible therapeutic targets may be possible (Shearman et
al., 2000; Sinha et al., 1999);], and this approach is in an early
stage of clinical development. Attempts to promote the clearance of
A.beta. from the brain through immunization with A.beta., while
efficacious in a transgenic mouse model for AD (Schenk et al 1999),
have been found to have significant adverse effects (Brower,
2002).
[0012] It has also been suggested that deposition of amyloid-like
fibrils may also be important in other neurodegenerative diseases.
These include Parkinson's disease, dementia with Lewy body
formation, multiple system atrophy, Hallerboden-Spatz disease, and
diffuse Lewy body disease.
[0013] One of the competing theories of the aetiology of AD is that
the causative step(s) lies within the pathway of the intracerebral
biogenesis and accumulation of the A.beta. amyloid protein (see
recent reviews by Selkoe, 2001; Beyreuther et al., 2001; Bush,
2001). However, to date no drugs or agents which target this
pathway have been demonstrated to have a lasting effect on
modifying the clinical expression of the disease or in preventing
or ameliorating the decline in cognitive function associated with
neurodegenerative disorders, including Alzheimer's disease.
[0014] A further hypothesis is that AD is caused by the toxic
accumulation of A.beta. amyloid, due in part to excess binding of
copper and zinc, metal ions which are abundant in the regions most
affected. Moreover, it has been suggested that when Zn.sup.2+ and
Cu.sup.2+ ions interact with A.beta., aggregation of A.beta. into
fibrils and plaques occurs (Atwood et al., 1998; confirmed by
recent data from animals deficient in synaptic Zn.sup.2+ (Lee et
al., 2002). It has also been suggested that redox-active
Cu.sup.2+-A.beta. interactions can generate H.sub.2O.sub.2 from
O.sub.2 (Huang et al., 1999). Both Cu.sup.2+ and Zn.sup.2+ have
been shown to affect A.beta.-lipid membrane interactions (Curtain
et al., 2001). The brain is an organ that concentrates metal ions
and recent evidence suggests that a breakdown in metal homeostasis
plays a critical role in a variety of age-related neurodegenerative
diseases. Common features of these diseases include the deposition
of misfolded protein (each disease has its own specific amyloid
protein) and substantial cellular damage as a result of oxidative
stress. Indeed data is now rapidly accumulating that
metallochemical reactions could emerge as the common denominator
underlying amyloidogenic neurological disorders such as Alzheimer's
disease, amylotrophic lateral sclerosis (ALS), prion
diseases--including Creutzfeldt-Jakob Disease (CJD), transmissible
spongioform encephalopathies (TSE), cataracts, mitochondrial
disorders, Parkinson's disease and Huntington's disease. In these
instances, the pathological aggregation of a specific protein is
promoted by abnormal redox activity in a physiological environment
typefied by the presence of transition metals and available
reducing agents. [Bush, 2000 (Curr Opin Chem Biol. 2000 April;
4(2):184-91)].
[0015] Accordingly the present invention provides a means of
treating neurological conditions, including those characterised by
the abnormal interaction between proteins and metals.
[0016] A method of treatment of AD using iodochlorohydroxyquinoline
an antibiotic [also known as clioquinol (CQ)], is disclosed and
claimed in U.S. Pat. Nos. 5,994,323 and 6,001,852 by P. N.
Geromylatos S.A. and in U.S. patent application Ser. No. 09/972,913
by Bush et al. CQ was withdrawn as an antibiotic in 1970, because
of its association with an uncommon neurological syndrome, subacute
myelo-optic neuropathy (SMON), which was observed only in Japan in
the 1960s, in patients thought to have received the drug over long
periods and probably at doses higher than those recommended at the
time (Shiraki, 1975). However, recent evidence suggests that SMON
was caused by an overuse-related vitamin B12 deficiency in an
exceptionally vulnerable population, and therefore could be
rehabilitated for study in a clinical setting (Yassin et al., 2000;
Bush and Masters, 2001).
[0017] However, no in vivo results in animal models or in humans
are provided in the Geromylatos and Bush patents. U.S. Pat. No.
5,994,323 discloses a composition comprising CQ and Vitamin B12,
and its use for the treatment of "diseases or disorders responsive
to CQ administration while inhibiting detrimental side effects" of
CQ. These diseases include AD. U.S. Pat. No. 6,001,852 discloses a
method of treatment of AD using CQ, preferably together with
Vitamin B12. Both U.S. Pat. Nos. 5,994,323 and 6,001,852 suggest a
dosage of 10-750 mg per day; U.S. Pat. No. 5,994,323 recommends
that if treatment is over a long period, CQ should be given
intermittently, for up to 3 weeks at a time followed by a
"wash-out" period of 1-4 weeks.
[0018] In U.S. application Ser. No. 09/972,913, CQ is exclusively
referred to in terms of its ability to disaggregate A.beta.
deposits. No other mechanism of neurotoxicity is discussed.
PCT/US99/05291 assigned to General Hospital Corporation discloses
the use of CQ in combination with specific copper and zinc
chelators to promote dissolution of amyloid plaques and inhibition
of amyloid plaque formation and/or the production of ROS by
A.beta..
[0019] U.S. Pat. No. 6,001,852 also suggests that a composition
comprising CQ and Vitamin B12 could be used in the treatment of
Parkinson's disease; however, in this context it is suggested that
CQ acts primarily via clearing iron from the substantia nigra.
[0020] The efficacy of CQ in the treatment of AD rests upon its
ability to enter the CNS and then sequester the transition metals
Cu, Zn and Fe from various A.beta. entities, thereby reducing
A.beta. toxicity and liberating it for clearance. The effectiveness
of CQ is restricted by its poor aqueous solubility, which limits
its oral bioavailability. CQ is also known to undergo considerable
conjugative metabolism and has a history of toxicity, as discussed
above. The fact that CQ is a bidentate metal ligand makes necessary
the commitment of at least two molecules for every metal ion
captured.
[0021] We have now developed 8-hydroxyquinoline derivatives which
are more efficacious than CQ through the collective optimization of
one or more of the following properties: [0022] (a) metal chelation
(as herein defined); [0023] (b) aqueous solubility; [0024] (c)
reduced cell toxicity; [0025] (d) amyloid dispersion properties;
[0026] (e) membrane permeability appropriate for CNS penetration;
and [0027] (f) metabolic stability.
[0028] These derivatives include examples of therapeutics which are
concentrated in the CNS through active transport, contain
antioxidant activity in addition to their metal chelation
properties which in some cases leads to enhanced metal chelation
properties and demonstrate a prodrug strategy which masks the
8-hydroxy moiety to favour CNS penetration and make use of the
known esterase activity which resides on the inner surface of the
blood brain barrier (BBB).
SUMMARY OF THE INVENTION
[0029] According to the present invention there is provided a
method for the treatment, amelioration and/or prophylaxis of a
neurological condition which comprises the administration of an
effective amount of a compound of formula I:
##STR00002##
wherein
[0030] R.sup.1 is H, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted acyl, optionally
substituted aryl, optionally substituted heterocyclyl, an
antioxidant or a targeting moiety;
[0031] R.sup.2 is H; optionally substituted alkyl; optionally
substituted alkenyl; optionally substituted aryl; optionally
substituted heterocyclyl; optionally substituted alkoxy; an
antioxidant; a targeting moiety; COR.sup.E or CSR.sup.6 wherein
R.sup.6 is H, optionally substituted alkyl, optionally substituted
alkenyl, hydroxy, optionally substituted aryl, optionally
substituted heterocyclyl, an antioxidant, a targeting moiety,
OR.sup.7, SR.sup.7 or NR.sup.7R.sup.8 wherein R.sup.7 and R.sup.8
are either the same or different and selected from H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted aryl and optionally substituted heterocyclyl; CN;
(CH.sub.2).sub.nNR.sup.9R.sup.10, HCNOR.sup.9 or
HCNNR.sup.9R.sup.10, wherein R.sup.9 and R.sup.10 are either the
same or different and selected from H, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted aryl
and optionally substituted heterocyclyl and n is 1 to 4;
OR.sup.11SR.sup.11 or NR.sup.11R.sup.12; wherein R.sup.11 and
R.sup.12 are either the same or different and selected from H,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted aryl and optionally substituted heterocyclyl
or together form optionally substituted heterocyclyl; or
SO.sub.2NR.sup.13R.sup.14 wherein R.sup.13 and R.sup.14 are either
the same or different and selected from H, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted aryl
or optionally substituted heterocyclyl; and
[0032] R.sup.3, R.sup.4, R.sup.5, R and R are either the same or
different and selected from H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkoxy,
optionally substituted acyl, hydroxy, optionally substituted amino,
optionally substituted thio, optionally substituted sulphonyl,
optionally substituted sulphinyl, optionally substituted
sulphonylamino, halo, SO.sub.3H, amine, CN, CF.sub.3, optionally
substituted aryl, optionally substituted heterocyclyl, an
antioxidant and a targeting moiety,
[0033] salts, hydrates, solvates, derivatives, pro-drugs, tautomers
and/or isomers thereof
[0034] with the provisos that: [0035] (a) when R.sup.1 to R.sup.3,
R and R' are H, then R.sup.4 is not Cl or I and R.sup.5 is not I;
[0036] (b) when R.sup.1 to R.sup.3, R, R' and R.sup.5 are H, then
R.sup.4 is not CHO, CHOHCCl.sub.3,
[0036] ##STR00003## [0037] (c) when R.sup.1, R.sup.5, R' and R are
H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R.sup.4 is not
bromo, methyl, phenyl, hydroxymethyl or trifluoromethyl; [0038] (d)
when R.sup.1, R.sup.4, R.sup.5 and R are H, R.sup.2 is CO.sub.2H
and R.sup.3 is OH, then R' is not bromo, iodo, methyl, phenyl,
propyl, phenethyl, heptyl, benzylaminomethyl, 3-aminopropyl,
3-hydroxypropyl, 4-methoxyphenyl, 3-methylphenyl, 4-chlorophenyl,
3,4-dichlorophenyl, pyridin-3-yl, furo-2-yl, 4-chlorophenyl,
3,4-dichlorophenyl, 2-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl,
3-chlorophenyl, 2-methoxyphenyl or piperidin-2-yl; [0039] (e) when
R.sup.1, R.sup.4, R and R' are H, R.sup.2 is CO.sub.2H and R.sup.3
is OH, then R.sup.5 is not phenyl, 3-hydroxypropyl, phenethyl,
3-aminoprop-1-yl or hex-1-yl; [0040] (f) when R.sup.1, R.sup.4, R'
and R.sup.5 are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R
is not N-morpholinomethyl, bromo or phenyl; [0041] (g) when
R.sup.1, R and R' are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH,
then R.sup.4 and R.sup.5 are not chloro; [0042] (h) when R.sup.1,
R.sup.4 and R' are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then
R and R.sup.5 are not bromo; [0043] (i) when R', R, R' and R.sup.5
are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then R.sup.4 is not
hydroxymethyl, phenyl or bromo; [0044] (j) when R', R, R.sup.4 and
R.sup.5 are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then R' is
not 4-methoxyphenyl, 3-methylphenyl, pyridin-3-yl, benzyl, bromo,
4-chlorophenyl, 3,4-dichlorophenyl, 3-hydroxypropyl or
3-tert-butoxycarbonylaminopropyl; [0045] (k) when R.sup.1, R,
R.sup.4 and R' are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then
R.sup.5 is not phenyl or 3-tert-butoxycarbonylaminoprop-1-yl;
[0046] (l) when R.sup.1, R, R.sup.4, R' and R.sup.5 are H and
R.sup.2 is CO.sub.2Me, then R.sup.3 is not
toluene-4-sulphonylamino, piperazin-1-yl, morpholin-1-yl,
piperidin-1-yl, 4-methylpiperazin-1-yl, 3-benzoylaminoprop-1-yl,
phenethyl, 3-tert-butoxycarbonylaminopropyl, 3-hydroxypropyl, amino
or hex-1-yl; [0047] (m) when R.sup.1, R.sup.4, R' and R.sup.5 are
H, R.sup.2 is CO.sub.2Na and R.sup.3 is OH, then R is not phenyl;
[0048] (n) when R.sup.1, R, R.sup.4, R' and R.sup.5 are H and
R.sup.2 is CO.sub.2H, then R.sup.3 is not phenyl, 4-chlorophenyl,
phenethyl, 3-hydroxypropyl, amino, morpholin-1-yl, piperidin-1-yl,
4-methylpiperazin-1-yl, toluene-4-sulphonylamino,
3-benzoylaminoprop-1-yl, aminoprop-1-ynyl, hex-1-yl,
5-hydroxypent-1-yl, piperazin-1-yl or 2-(1-piperazinyl)pyrimidinyl;
[0049] (o) when R.sup.1, R' and R are H, R.sup.2 is CO.sub.2Me and
R.sup.3 is OH, then R.sup.4 and R.sup.5 are not chloro; [0050] (p)
when R.sup.1, R.sup.4, R' and R.sup.5 are H, R.sup.2 is CO.sub.2Me
and R.sup.3 is OH, then R is not bromo; [0051] (q) when R.sup.1, R'
and R.sup.4 are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then R
and R.sup.5 are not bromo; [0052] (r) when R.sup.1, R, R.sup.3, R'
and R.sup.5 are H and R.sup.2 is CO.sub.2H, then R.sup.4 is not
phenyl, 4-chlorophenyl or phenylethyl; [0053] (s) when R.sup.1,
R.sup.5, R', R.sup.4, R.sup.3 and R are H, then R.sup.2 is not
2H-tetrazol-1-yl; [0054] (t) when R', R.sup.5, R.sup.4 and R are H,
R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R' is not
3,5-dichlorophenyl or 4-fluorophenyl; and [0055] (u) at least one
of R.sup.1 to R.sup.5, R and R' is other than H, to a subject in
need thereof
[0056] Further according to the present invention there is provided
use of the compound of formula I in the manufacture of a medicament
for the treatment, amelioration and/or prophylaxis of a
neurological condition.
[0057] The invention also provides use of the compound of formula I
for the treatment, amelioration and/or prophylaxis of a
neurological condition.
[0058] The invention further provides the compound of formula I for
use in the treatment, amelioration and/or prophylaxis of a
neurological condition.
[0059] The invention still further provides use of the compound of
formula I as a pharmaceutical, preferably a neurotherapeutic or
neuroprotective agent, more preferably an antiamyloidogenic agent.
Preferably, the neurological condition is a neurodegenerative
condition, more preferably neurodegenerative amyloidosis such as
Alzheimer's disease, Huntington's disease, and the like.
[0060] Preferred compounds of formula I are as follows:
[0061] (i) Formula 1a
##STR00004##
wherein:
[0062] R, R.sup.1 and R.sup.3 are as defined in formula I above;
and
[0063] R.sup.2.sub.a is H; optionally substituted C.sub.1-6 alkyl;
optionally substituted C.sub.1-6 alkenyl; optionally substituted
aryl; optionally substituted heterocyclyl; an antioxidant; a
targeting moiety; COR.sup.6.sub.a or CSR.sup.6.sub.a, wherein
R.sup.6.sub.a is H, optionally substituted C.sub.1-6 alkyl,
optionally substituted C.sub.2-6 alkenyl, hydroxy, optionally
substituted aryl, optionally substituted heterocyclyl or
OR.sup.7.sub.a, SR.sup.7.sub.a or NR.sup.7.sub.aR.sup.8.sub.a
wherein R.sup.7.sub.a and R.sup.8.sub.a are either the same or
different and selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6 alkenyl, optionally
substituted aryl and optionally substituted heterocyclyl; CN;
CH.sub.2NR.sup.9.sub.aR.sup.10.sub.a, HCNOR.sup.9.sub.a or
HCNNR.sup.9.sub.aR.sup.10.sub.a, wherein R.sup.9.sub.a and
R.sup.10.sub.a are either the same or different and selected from
H, optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6 alkenyl, optionally substituted aryl or optionally
substituted heterocyclyl; OR.sup.11.sub.a, SR.sup.11.sub.a or
NR.sup.11.sub.aR.sup.12.sub.a wherein R.sup.11.sub.a and
R.sup.12.sub.a are either the same or different and selected from
H, optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6 alkenyl, optionally substituted aryl and optionally
substituted heterocyclyl or together form optionally substituted
heterocyclyl; or SO.sub.2NR.sup.13.sub.aR.sup.14.sub.a wherein
R.sup.13.sub.a and R.sup.14.sub.a are either the same or different
and selected from H, optionally substituted C.sub.1-6 alkyl,
optionally substituted C.sub.2-6 alkenyl, optionally substituted
aryl and optionally substituted heterocyclyl.
[0064] Another embodiment of the present invention is a compound of
the formula:
##STR00005##
or pharmaceutically acceptable salts, hydrates, or solvates and/or
prodrugs thereof in which
[0065] R.sup.1 is H;
[0066] R.sup.2 is optionally substituted C.sub.1-6 alkyl;
optionally substituted C.sub.2-6 alkenyl; optionally substituted
aryl; optionally substituted heterocyclyl; optionally substituted
C.sub.1-6 alkoxy; COR.sup.6 or CSR.sup.6, in which R.sup.6 is H,
optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6 alkenyl, hydroxy, optionally substituted aryl, optionally
substituted heterocyclyl, OR.sup.7, SR.sup.7 or NR.sup.7R.sup.8 in
which R.sup.7 and R.sup.8 are either the same or different and
selected from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl and
optionally substituted heterocyclyl; CN;
(CH.sub.2).sub.nNR.sup.9R.sup.10, HCNOR.sup.9 or
HCNNR.sup.9R.sup.10 in which R.sup.9 and R.sup.10 are either the
same or different and selected from H, optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl,
optionally substituted aryl and optionally substituted heterocyclyl
and n is 1 to 4; OR.sup.11, SR.sup.11 or NR.sup.11R.sup.12 in which
R.sup.11 and R.sup.12 are either the same or different and selected
from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl and
optionally substituted heterocyclyl or together form optionally
substituted heterocyclyl; or SO.sub.2NR.sup.13R.sup.14 in which
R.sup.13 and R.sup.14 are either the same or different and selected
from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl and
optionally substituted heterocyclyl;
[0067] R.sup.3, R and R' are either the same or different and
selected from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted C.sub.1-6
alkoxy, optionally substituted acyl, hydroxy, optionally
substituted amino, optionally substituted thio, optionally
substituted sulphonyl, optionally substituted sulphinyl, optionally
substituted sulphonylamino, halo, SO.sub.3H, amine, CN, CF.sub.3,
optionally substituted aryl and optionally substituted
heterocyclyl; and
[0068] R.sup.4 and R.sup.5 are chloro,
[0069] wherein the optional substituent is C.sub.1-6 alkyl,
CF.sub.3, fluorine, chlorine, iodine, cyano, C.sub.1-6 alkoxy, 5 or
6-membered aryl, saturated 5- or 6-membered heteromonocyclic group
containing 1 or 3 nitrogen atoms, an unsaturated condensed
heterocyclic group, saturated 3 to 6-membered heteromonocyclic
group containing 1 to 4 nitrogen atoms, indolyl, isoindolyl,
indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl,
benzotriazolyl, tetrazolopyridazinyl, benzoxazolyl,
benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, an unsaturated
5-membered heteromonocyclyl group containing 1 or 2 sulphur atoms;
an unsaturated 5- or 6-membered heteromonocyclyl group containing 1
or 2 sulphur atoms and 1 or 2 nitrogen atoms, amino or C.sub.1-6
alkylamino
[0070] with the proviso that when R and R' are H and R.sup.2 is
CO.sub.2H or CO.sub.2Me, then R.sup.3 is not OH.
[0071] In an embodiment, it is preferred that
[0072] R.sup.2 is optionally substituted C.sub.1-6 alkyl;
optionally substituted C.sub.2-6 alkenyl; optionally substituted
aryl; optionally substituted heterocyclyl; optionally substituted
C.sub.1-6 alkoxy; COR.sup.6 or CSR.sup.6 in which R.sup.6 is H,
optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6 alkenyl, hydroxy, optionally substituted aryl, optionally
substituted heterocyclyl, SR.sup.7 or NR.sup.7R.sup.8 in which
R.sup.7 and R.sup.8 are either the same or different and selected
from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl and
optionally substituted heterocyclyl; CN;
(CH.sub.2)--NR.sup.9R.sup.10, HCNOR.sup.9 or HCNNR.sup.9R.sup.10,
in which R.sup.9 and R.sup.10 are either the same or different and
selected from H, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.2-6 alkenyl, optionally substituted aryl and
optionally substituted heterocyclyl and n is 1 to 4; OR.sup.11,
SR.sup.11 or NR.sup.11R.sup.12 in which R.sup.11 and R.sup.12 are
either the same or different and selected from H, optionally
substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6
alkenyl, optionally substituted aryl and optionally substituted
heterocyclyl or together form optionally substituted heterocyclyl;
or SO.sub.2NR.sup.13R.sup.14 in which R.sup.13 and R.sup.14 are
either the same or different and selected from H, optionally
substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6
alkenyl, optionally substituted aryl and optionally substituted
heterocyclyl.
[0073] Another embodiment includes compounds wherein at least one
of R.sup.2, R, R.sup.3 and R' is optionally substituted C.sub.1-6
alkyl, optionally substituted aryl, optionally substituted
heterocyclyl, CH.sub.2NR.sup.9R.sup.10, COR.sup.6 or
NR.sup.11R.sup.12 in which R.sup.6 is NR.sup.7R.sup.8.
[0074] Preferred compounds of formula Ia are as follows:
[0075] (a) Formula IIa
##STR00006##
wherein: [0076] R.sup.1 is as defined in formula I above; and
[0077] R.sup.2'.sub.a is optionally substituted C.sub.1-6 alkyl,
optionally substituted C.sub.2-6 alkenyl, optionally substituted
aryl or optionally substituted heterocyclyl.
[0078] Formula IIa may represent compounds wherein an antioxidant
moiety is attached to the C2 position of the 8-hydroxyquinoline in
such a way that exposure to a prooxidative environment, that is,
hydroxy radicals, will result in a molecule with enhanced metal
chelation properties.
##STR00007## [0079] Representative examples are shown below:
[0080] (b) Formula IIIa
##STR00008##
wherein: [0081] R.sup.1 and R.sup.3 are as defined in formula I
above; and [0082] R.sup.6'.sub.a is optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, hydroxy,
OR.sup.7 '.sub.a, SR.sup.7 '.sub.a,
N.sub.2R.sup.7'.sub.aR.sup.8'.sub.a, or
NR.sup.7'.sub.aR.sup.8'.sub.a wherein R.sup.7'.sub.a and
R.sup.8'.sub.a are either the same or different and selected from
H, optionally substituted C.sub.1-6 alkyl, optionally substituted
aryl and optionally substituted heterocyclyl.
[0083] Formula IIIa represents compounds wherein a hydrophilic
amide moiety is attached to the C2 position of the
8-hydroxyquinoline so as to generally enhance solubility, while
maintaining membrane permeability. Compounds of formula IIIa also
show enhanced metal chelation properties.
[0084] Representative examples are shown below:
##STR00009##
[0085] (c) Formula IVa
##STR00010##
wherein: [0086] R.sup.1 is as defined in formula I above; and
[0087] R.sup.2''.sub.a is CN;
CH.sub.2NR.sup.9'.sub.aR.sup.10.sub.a, HCNOR.sup.9'.sub.a or
HCNNR.sup.9'.sub.aR.sup.10'.sub.a wherein R.sup.9'.sub.a and
R.sup.10' are either the same or different and selected from H,
optionally substituted C.sub.1-6 alkyl, optionally substituted
alkenyl, optionally substituted aryl and optionally substituted
heterocyclyl.
[0088] Formula IVa represents compounds which have improved metal
chelation and optimised activity in the panel of assays described
hereinafter.
[0089] Representative examples are shown below:
##STR00011##
[0090] (d) Formula Va
##STR00012##
wherein:
[0091] R.sup.1 is as defined in formula I above; and
[0092] R.sup.11'.sub.a and R.sup.12'.sub.a are either the same or
different and selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6 alkenyl, optionally
substituted aryl and optionally substituted heterocyclyl or
together form optionally substituted heterocyclyl.
[0093] (e) Formula VIa
##STR00013##
wherein:
[0094] R.sup.1 is as defined in formula I above; and
[0095] R.sup.13'.sub.a and R.sup.14'.sub.a are either the same or
different and selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6 alkenyl, optionally
substituted aryl or optionally substituted heterocyclyl.
[0096] Preferred compounds of the present invention also include
compounds of
[0097] (ii) Formula Ib
##STR00014##
wherein:
[0098] R.sup.1, R', R, R.sup.2 and R.sup.3 are as defined in
formula I above;
[0099] R.sup.4.sub.b and R.sup.5.sub.b are either the same or
different and selected from H; optionally substituted C.sub.1-6
alkyl; optionally substituted C.sub.2-6 alkenyl; halo; CN;
CF.sub.3; optionally substituted aryl; optionally substituted
heterocyclyl; an antioxidant; a targeting moiety; SO.sub.3H;
SO.sub.2NR.sup.13.sub.aR.sup.14.sub.a wherein R.sup.13.sub.a and
R.sup.14.sub.a are as defined in formula Ia above; OR.sup.15.sub.b,
SR.sup.15.sub.b, SO.sub.2R.sup.15.sub.b,
CONR.sup.15.sub.bR.sup.16.sub.b and NR.sup.15.sub.bR.sup.16.sub.b
wherein R.sup.15.sub.b and R.sup.16.sub.b are either the same or
different and selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6 alkenyl, optionally
substituted C.sub.1-6 acyl, optionally substituted aryl and
optionally substituted heterocyclyl, including provisos (a) to (c),
(e), (g), (h), (I), (k), (o), (q), (r), and (u) as defined
above.
[0100] Preferred compounds of formula Ib are as follows:
[0101] (a) Formula IIb
##STR00015##
wherein:
[0102] R.sup.1, R', R, R.sup.2 and R.sup.3 are as defined in
formula I above; and
[0103] R.sup.4 '.sub.b and R.sup.5 '.sub.a are as defined in
formula Ib above provided that at least one is halo,
[0104] including provisos (a), (c), (g), (h), (i), (o), (q) and (u)
defined above.
[0105] (b) Formula IIIb
##STR00016##
wherein:
[0106] R.sup.1 is as defined in formula I above;
[0107] R.sup.4'.sub.b is H or halo; and
[0108] R.sup.5''.sub.b is optionally substituted aryl or optionally
substituted heterocyclyl.
[0109] A representative example is shown below:
##STR00017##
[0110] (c) Formula IVb
##STR00018##
wherein:
[0111] R.sup.1 is as defined in formula I above;
[0112] R'' is C.sub.1-6 alkoxy, halo, C.sub.1-6 alkyl, C.sub.2-6
alkenyl or C.sub.1-6 haloalkyl; and R.sup.5 '.sub.b is H or
halo.
[0113] A representative example is shown below:
##STR00019##
[0114] (d) Formula Vb
##STR00020##
wherein
[0115] R.sup.1 is as defined in formula I above; and
R'' is as defined in formula IVb above.
[0116] (e) Formula VIb
##STR00021##
wherein:
[0117] R.sup.2 to R.sup.5, R and R' are as defined in formula I
above; and
[0118] R.sup.1 ''.sub.b is optionally substituted C.sub.1-6 alkyl,
optionally substituted aryl, optionally substituted aryl acyl,
C.sub.1-6 alkyl acyl or optionally substituted heterocyclyl.
[0119] Formula VIb represents compounds wherein the 8-hydroxyl
group on the quinoline is blocked to form a prodrug, in particular
an ester prodrug. The 8-hydroxy represents a principal site of
metabolism for the compound of Formula I: conjugation with
glucuronic acid or sulphate gives a hydrophilic species ready to be
excreted. Such conjugates probably do not pass the blood brain
barrier. The ester prodrug may protect the compound of Formula I
from conjugation. Esterases integral to the blood brain barrier may
then release the C8-hydroxy on passage through that barrier
activating the compound for its role in the CNS.
[0120] Another preferred compound of the present invention is a
compound of (iii) Formula Ic
##STR00022##
wherein
[0121] R.sup.1, R.sup.2, R.sup.3, R and R' are as defined in
formula I; and
[0122] at least one of R.sup.4.sub.c and R.sup.5.sub.c is halo and
the other is selected from H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkoxy,
optionally substituted acyl, hydroxy, optionally substituted amino,
optionally substituted thio, optionally substituted sulphonyl,
optionally substituted sulphinyl, optionally substituted
sulphonylamino, SO.sub.3H, amine, CN, CF.sub.3, optionally
substituted aryl, optionally substituted heterocyclyl, an
antioxidant and a targeting moiety, salts, hydrates, solvates,
derivatives, pro-drugs, tautomers and/or isomers thereof
[0123] with the provisos that:
[0124] (a) when R.sup.1 to R.sup.3, R and R' are H, then R.sup.4 is
not chloro or iodo and R.sup.5.sub.c is not iodo;
[0125] (b) when R.sup.1, R.sup.5.sub.c, R' and R are H, R.sup.2 is
CO.sub.2H and R.sup.3 is OH, then R.sup.4.sub.c is not bromo;
[0126] (c) when R.sup.1, R and R' are H, R.sup.2 is CO.sub.2H and
R.sup.3 is OH, then R.sup.4.sub.c and R.sup.5.sub.c are not
chloro;
[0127] (d) when R', R.sup.4.sub.c and R' are H, R.sup.2 is
CO.sub.2H or CO.sub.2Me and R.sup.3 is OH, then R and R.sup.5 are
not bromo;
[0128] (e) when R.sup.1, R, R' and R.sup.5.sub.c are H, R.sup.2 is
CO.sub.2Me and R.sup.3 is OH, then R.sup.4.sub.c is not bromo; and
[0129] (f) when R.sup.1, R and R' are H, R.sup.2 is CO.sub.2Me and
R.sup.3 is OH, then R.sup.4.sub.c and R.sup.5.sub.c are not
chloro.
[0130] Preferred compounds of Formula Ic are as follows:
[0131] (a) Formula IIc
##STR00023##
wherein
[0132] R.sup.2, R, R', R.sup.4.sub.c and R.sup.5.sub.c are as
defined in formula Ic; and R.sup.3' is H, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkoxy, optionally substituted acyl, optionally substituted amino,
optionally substituted thio, optionally substituted sulphonyl,
optionally substituted sulphinyl, optionally substituted
sulphonylamino, halo, SO.sub.3H, amine, CN, CF.sub.3, optionally
substituted aryl, optionally substituted heterocyclyl, an
antioxidant or a targeting moiety, with the proviso that at least
one of R, R.sup.2 and R.sup.3' is other than H.
[0133] Representative examples are shown below:
##STR00024##
wherein
[0134] R.sup.1 is as defined in formula I and R.sup.4.sub.c is as
defined in formula Ic; and
[0135] R.sup.5.sub.c'' is optionally substituted aryl or optionally
substituted heterocyclyl;
[0136] (b) Formula IVc
##STR00025##
wherein
[0137] R.sup.1 is as defined in formula I, R.sup.5.sub.c is as
defined in formula Ic and R'' is as defined in formula IVb; and
[0138] (c) Formula Vc
##STR00026##
wherein
[0139] R.sup.2, R.sup.3, R and R' are as defined in formula I,
R.sup.4.sub.c and R.sup.5.sub.c are as defined in formula Ic and
R.sup.1.sub.b is as defined in formula VIb.
[0140] In a particularly preferred embodiment, the compound of
formula I is a compound of formula Ib, III) or Ic wherein
R.sup.4.sub.b and R.sup.5.sub.b, R.sup.4.sub.b' and R.sup.5.sub.b'
or R.sup.4.sub.c and R.sup.5.sub.c, respectively are both halo,
more preferably chloro substituents. Preferably, at least one of
R.sup.2, R, R.sup.3 and R' is optionally substituted alkyl,
optionally substituted aryl, optionally substituted heterocyclyl,
(CH.sub.2).sub.nNR.sup.9R.sup.10 wherein R.sup.9 and R.sup.10 are
as defined above and n is 1 to 4, COR.sup.E wherein R.sup.6 is
NR.sup.7R.sup.8, OR.sup.7 or SR' wherein R.sup.7 and R.sup.8 are as
defined above or NR.sup.11R.sup.12, OR.sup.11, SR.sup.11 wherein
R.sup.11 and R.sup.12 are as defined above.
[0141] While not wishing to be bound by theory, it is believed that
substituents R, R.sup.3 and R' have a limited effect,
electronically or sterically, in the chelating properties of the
compounds of the present invention. Substitution at those positions
can therefore be used to modulate other parameters such as
cytotoxicity and physicochemical properties including the number of
hydrogen bond donors and acceptors, lipophilicity (C log P, E log P
and Log D), solubility and polar surface area. Modulation of these
parameters contribute to the optimisation of the pharmacokinetic
profile of the compounds. It is also postulated that substituent
R.sup.2 in addition to modulating cytotoxicity and physicochemical
properties could also affect activity if the substituent provides
chelating properties. Examples of particularly preferred compounds
having R.sup.2 substituents with chelating properties are shown
below.
##STR00027##
[0142] Other preferred compounds have the formula:
##STR00028##
[0143] In a further aspect, the invention provides a pharmaceutical
or veterinary composition comprising the compound of formula I as
defined above, together with a pharmaceutically or veterinarily
acceptable carrier.
[0144] Accordingly, the invention provides a compound of formula II
which is a compound of formula I with the provisos that: [0145] (a)
when R' and R.sup.3 to R.sup.5, R and R' are H, then R.sup.2 is not
H, methyl,
##STR00029##
[0145] CO.sub.2H, CN, CONCH.sub.2CO.sub.2H, COCH.sub.3,
CH.sub.2NH.sub.2, CNOH, (pyrid-2-yl), 2-hydroxyphenyl, CHNNH.sub.2,
NH-(pyrid-2-yl),
##STR00030##
or SO.sub.3H;
[0146] (b) when R.sup.1 and R.sup.4 to R.sup.7 are H, then R.sup.3
is not OH and R.sup.2 is not CO.sub.2H; [0147] (c) when R.sup.1 to
R.sup.3, R.sup.6 and R.sup.7 are H, then (i) when R.sup.5 is I,
R.sup.4 is not Cl, SO.sub.3H or I; (ii) when R.sup.5 is H, R.sup.4
is not SO.sub.3H, NH.sub.2 or Cl; (iii) R.sup.4 and R.sup.5 are
both not Cl, Br or CH.sub.3; and (iv) when R.sup.2 to R.sup.7 are
H, then R.sup.1 is not
[0147] ##STR00031## [0148] (d) when R1 to R.sup.3, R and R' are H,
then R.sup.4 is not Cl or I and R.sup.5 is not I; [0149] (e) when
R1 to R.sup.3, R, R' and R.sup.5 are H, then R.sup.4 is not CHO,
CHOHCCl.sub.3,
[0149] ##STR00032## [0150] (f) when R.sup.1, R.sup.5, R' and R are
H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R.sup.4 is not
bromo, methyl, phenyl, hydroxymethyl or trifluoromethyl; [0151] (g)
when R', R.sup.4, R.sup.5 and R are H, R.sup.2 is CO.sub.2H and
R.sup.3 is OH, then R' is not bromo, iodo, methyl, phenyl, propyl,
phenethyl, heptyl, benzylaminomethyl, 3-aminopropyl,
3-hydroxypropyl, 4-methoxyphenyl, 3-methylphenyl, 4-chiorophenyl,
3,4-dichlorophenyl, pyridin-3-yl, furo-2-yl, 4-chlorophenyl,
3,4-dichlorophenyl, 2-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl,
3-chlorophenyl, 2-methoxyphenyl or piperidin-2-yl; [0152] (h) when
R.sup.1, R.sup.4, R and R' are H, R.sup.2 is CO.sub.2H and R.sup.3
is OH, then R.sup.5 is not phenyl, 3-hydroxypropyl, phenethyl,
3-aminoprop-1-yl or hex-1-yl; [0153] (i) when R.sup.1, R.sup.4, R'
and R.sup.5 are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R
is not N-morpholinomethyl, bromo or phenyl; [0154] (j) when
R.sup.1, R and R' are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH,
then R.sup.4 and R.sup.5 are not chloro; [0155] (k) when R.sup.1,
R.sup.4 and R' are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then
R and R.sup.5 are not bromo; [0156] (l) when R', R, R' and R.sup.5
are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then R.sup.4 is not
hydroxymethyl, phenyl or bromo; [0157] (m) when R', R, R.sup.4 and
R.sup.5 are H, R2 is CO2Me and R3 is OH, then R' is not
4-methoxyphenyl, 3-methylphenyl, pyridin-3-yl, benzyl, bromo,
4-chiorophenyl, 3,4-dichlorophenyl, 3-hydroxypropyl or
3-tert-butoxycarbonylaminopropyl; [0158] (n) when R.sup.1, R,
R.sup.4 and R' are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then
R.sup.5 is not phenyl or 3-tert-butoxycarbonylaminoprop-1-yl;
[0159] (o) when R.sup.1, R, R.sup.4, R' and R.sup.5 are H and
R.sup.2 is CO.sub.2Me, then R.sup.3 is not
toluene-4-sulphonylamino, piperazin-1-yl, morpholin-1-yl,
piperidin-1-yl, 4-methylpiperazin-1-yl, 3-benzoylaminoprop-1-yl,
phenethyl, 3-tert-butoxycarbonylaminopropyl, 3-hydroxypropyl, amino
or hex-1-yl; [0160] (p) when R.sup.1, R.sup.4, R' and R.sup.5 are
H, R.sup.2 is CO.sub.2Na and R.sup.3 is OH, then R is not phenyl;
[0161] (q) when R.sup.1, R, R.sup.4, R' and R.sup.5 are H and
R.sup.2 is CO.sub.2H, then R.sup.3 is not phenyl, 4-chlorophenyl,
phenethyl, 3-hydroxypropyl, amino, morpholin-1-yl, piperidin-1-yl,
4-methylpiperazin-1-yl, toluene-4-sulphonylamino,
3-benzoylaminoprop-1-yl, aminoprop-1-ynyl, hex-1-yl,
5-hydroxypent-1-yl, piperazin-1-yl or 2-(1-piperazinyl)pyrimidinyl;
[0162] (r) when R.sup.1, R' and R are H, R.sup.2 is CO.sub.2Me and
R.sup.3 is OH, then R.sup.4 and R.sup.5 are not chloro; [0163] (s)
when R.sup.1, R.sup.4, R' and R.sup.5 are H, R.sup.2 is CO.sub.2Me
and R.sup.3 is OH, then R is not bromo; [0164] (t) when R.sup.1, R'
and R.sup.4 are H, R.sup.2 is CO.sub.2Me and R.sup.3 is OH, then R
and R.sup.5 are not bromo; [0165] (u) when R.sup.1, R, R.sup.3, R'
and R.sup.5 are H and R.sup.2 is CO.sub.2H, then R.sup.4 is not
phenyl, 4-chlorophenyl or phenylethyl; [0166] (v) when R.sup.1,
R.sup.5, R', R.sup.4, R.sup.3 and R are H, then R.sup.2 is not
2H-tetrazol-1-yl; [0167] (w) when R.sup.1, R.sup.5, R.sup.4 and R
are H, R.sup.2 is CO.sub.2H and R.sup.3 is OH, then R' is not
3,5-dichlorophenyl or 4-fluorophenyl; and [0168] (x) at least one
of R.sup.1 to R.sup.5, R and R' is other than H; [0169] (y) when
R.sup.1 to R.sup.3, R.sup.5, R' and R are H, then R.sup.4 is not
chloro, NH.sub.2 or SO.sub.3H; and [0170] (z) when R.sup.1, R.sup.3
to R.sup.5, R and R' are H, then R.sup.2 is not CH.sub.3.
[0171] Preferably, the invention provides a compound of formula Ic,
with the additional provisos that: [0172] (d) when R.sup.1 to
R.sup.3, R and R' are H, then R.sup.4, and R.sup.5, are both not
chloro or bromo; and [0173] (e) when R.sup.1 to R.sup.3,
R.sup.5.sub.c, R and R' are H, then R.sup.4.sub.c is not chloro,
more preferably a compound of formula IIc.
[0174] The compound of formula II defined above may be prepared
using the processes described in detail hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0175] FIG. 1 is a scatterplot showing the levels of soluble and
insolule A.beta. fractions obtained from transgenic mice brains
following treatment with PBT 1 and PBT 1038 [methodology as per
assay 11];
[0176] FIG. 2 is a scatterplot showing the levels of soluble and
insolule A.beta. fractions obtained from transgenic mice brains
following treatment with PBT 1033 and PBT 1051 [methodology as per
assay 11];
[0177] FIG. 3 is a scatterplot showing the levels of soluble and
insolule A.beta. fractions obtained from transgenic mice brains
following treatment with PBT 1052 [methodology as per assay
11];
[0178] FIG. 4(a) is a graph showing the dose normalised plasma
concentrations of PBT 1033 following IV (2 mg/Kg) and oral (30
mg/Kg) administration to rats;
[0179] FIG. 4(b) is a graph showing the dose normalised plasma
concentrations of PBT 1038 following IV (2 mg/Kg) and oral (30
mg/Kg) administration to rats;
[0180] FIG. 4(c) is a graph showing the dose normalised plasma
concentrations of PBT 1050 following IV (2 mg/Kg) and oral (30
mg/Kg) administration to rats;
[0181] FIG. 4(d) is a graph showing the dose normalised plasma
concentrations of PBT 1051 following IV (2 mg/Kg) and oral (30
mg/Kg) administration to rats;
[0182] FIG. 5 is a graph summarising the effect of CQ (PBT 1), PBT
1033, PBT 1038, PBT 1051 and PBT 1052 on soluble and insoluble
A.beta. in transgenic mouse brains [methodology as per assay
11];
[0183] FIG. 6 is a graph showing the immunohistochemistry of PBT
1033, PBT 1038, PBT 1051 and PBT 1052 on amyloid plaque abundance
in transgenic mice brains [methodology as per assay 15];
[0184] FIG. 7 is a flow chart of subjects studied;
[0185] FIG. 8 are graphs showing mean change (.+-.SE) over time
from baseline in cognitive abilities (as assessed with ADAS-cog) in
(A) two arms of CQ vs placebo and (B) stratification by severity
within treatment arms [less-severely affected (ADAS-cog<25),
more-severely affected (ADAS-cog.gtoreq.25) (*p.ltoreq.0.05;
**p.ltoreq.0.01);
[0186] FIG. 9 are graphs showing mean change (.+-.SE) over time
from baseline in plasma A.beta..sub.42 levels in (A) the arms of CQ
vs placebo and (B) stratification by severity as in FIG. 8.
(***p.ltoreq.0.001);
[0187] FIG. 10 are graphs showing mean change (.+-.SE) over time
from baseline in (A) plasma Zn (B) plasma Cu in the two arms of CQ
vs placebo; and
[0188] FIG. 11 is a graph showing relative changes in behavioral
(ADAS-cog) and biochemical (plasma/CSF A.beta.) levels over the
course of AD.
[0189] FIGS. 12 and 13 are graphs showing the rotarod time for mice
in Groups A and D and Groups A and B, respectively;
[0190] FIG. 14 is a graph showing the time to clasping for mice in
Groups A and B;
[0191] FIG. 15 is a graph showing the clasping duration for mice in
Groups A and B;
[0192] FIG. 16 is a graph showing the body weight of mice in Groups
A and B;
[0193] FIG. 17 is a graph showing the cumulative survival of mice
in Groups A and B;
[0194] FIG. 18 is a graph showing the lateral ventricular area for
mice in Groups A to D;
[0195] FIG. 19 is a graph showing the brain weight of mice in
Groups A to D;
[0196] FIG. 20 is a photo of a Western Blot Analysis of mutant
Hungtington protein from mice in Groups A and B, WT and male R6/2
mice; and
[0197] FIG. 21 is a photo showing slices taken from the mice brains
for WT, R6/2, and R6/2 mice treated with PBT 1033;
[0198] FIG. 22 is a graph showing a cytotoxicity screen of B-1033
and D-105 on C6 cells;
[0199] FIG. 23 is a graph showing a cytotoxicity screen of B-1033
and D-105 on U87MG cells;
[0200] FIG. 24 is a graph showing a cytotoxicity screen of B-1033
and D-105 on SMA 560 cells;
[0201] FIG. 25 is a graph showing a cytotoxicity screen of B-1033
and D-105 on 3T3 cells; and
[0202] FIG. 26a to d are graphs showing the effects of compound
B-1033 in the C6 glioma model (a,c) and the SMA 560 glioma model
(b, d).
DETAILED DESCRIPTION OF THE INVENTION
[0203] For the purposes of this specification it will be clearly
understood that the word "comprising" means "including but not
limited to", and that the word "comprises" has a corresponding
meaning.
[0204] The term "alkyl" used either alone or in combination with
terms such as "optionally substituted alkyl" "haloalkyl" or "alkyl
acyl" refers to straight chain, branched chain or cyclic
hydrocarbon groups having from 1 to 10 carbon atoms, preferably 1
to 6 carbon atoms, more preferably 1 to 4 carbon atoms.
Illustrative of such alkyl groups are methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,
neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl or
cyclohexyl.
[0205] The term "alkenyl" used either alone or in combination with
other terms such as "optionally substituted alkenyl", denotes
linear, branched or mono- or poly-cyclic radicals having at least
one carbon-carbon double bond of 2 to 20 carbon atoms, preferably 2
to 14 carbon atoms, and more preferably 2 to 6 carbon atoms.
Examples of alkenyl radicals include allyl, ethenyl, propenyl,
butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl,
cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl,
cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl,
1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl,
1,3-butadienyl, 1,4-pentadienyl, 1,3-cyclopentadienyl,
1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl,
1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl,
1,3,5,7-cycloocta-tetraenyl and the like.
[0206] The term "acyl" used either alone or in combination with
other terms such as "optionally substituted acyl", "aryl acyl" or
"alkyl acyl", denotes as part of a carboxylic acid or derivative
thereof, e.g., amides, esters, anhydrides, and the like. It
includes carbamoyl, aliphatic acyl group, acyl group containing an
aromatic ring which is referred to as aromatic acyl or an acyl
group containing a heterocyclic ring which is referred to as
heterocyclic acyl. The acyl group preferably contains 1 to 20
carbon atoms, and more preferably 1 to 14 carbon atoms. Examples of
acyl include carbamoyl; straight chain or branched alkanoyl, such
as, formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl,
pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl,
nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl,
tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl,
octadecanoyl, nonadecanoyl or icosanoyl; alkoxycarbonyl, such as,
methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl,
t-pentyloxycarbonyl or heptyloxycarbonyl; cycloalkylcarbonyl, such
as, cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentyl, carbonyl
or cyclohexylcarbonyl; alkylsulfonyl, such as, methylsulfonyl or
ethylsulfonyl; alkoxysulfonyl, such as, methoxysulfonyl or
ethoxysulfonyl; aroyl, such as, benzoyl, toluoyl or naphthoyl;
aralkanoyl, such as, phenylalkanoyl, for example, phenylacetyl,
phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl or
phenylhexanoyl or naphthylalkanoyl, for example, naphthylacetyl,
naphthylpropanoyl or naphthylbutanoyl; aralkenoyl, such as,
phenylalkenoyl, for example, phenylpropenoyl, phenylbutenoyl,
phenylmethacrylyl, phenylpentenoyl or phenylhexenoyl or
naphthylalkenoyl, for example, naphthylpropenoyl, naphthylbutenoyl
or naphthylpentenoyl; aralkoxycarbonyl, such as,
phenylalkoxycarbonyl, for example, benzyloxycarbonyl;
aryloxycarbonyl, such as, phenoxycarbonyl or naphthyloxycarbonyl,
aryloxyalkanoyl, such as, phenoxyacetyl or phenoxypropionyl,
arylcarbamoyl, such as, phenylcarbamoyl; arylthiocarbamoyl, such
as, phenylthiocarbamoyl, arylglyoxyloyl, such as, phenylglyoxyloyl
or naphthylglyoxyloyl; arylsulfonyl, such as, phenylsulfonyl or
naphthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl, such
as, thienylacetyl, thienylpropanoyl, thienylbutanoyl,
thienylpentanoyl, thienylhexanoyl, thiazolylacetyl,
thiadiazolylacetyl or tetrazolylacetyl, heterocyclicalkenoyl, such
as, heterocyclicpropenoyl, heterocyclicbutenoyl,
heterocyclicpentenoyl or heterocyclichexenoyl; or
heterocyclicglyoxyloyl, such as, thiazolylglyoxyloyl or
thienylglyoxyloyl.
[0207] The term "heterocyclyl group" used either alone or in
combination with other terms such as "optionally substituted
heterocyclyl" refers to monocyclic or polycyclic heterocyclic
groups containing at least one heteroatom ring atom selected from
nitrogen, sulphur and oxygen and containing up to 18 ring atoms and
up to 17 carbon ring atoms. It is preferred that the heterocyclyl
group is mono or bicyclic or tricyclic heterocyclic group
containing up to 20 ring atoms and up to 17 ring carbon atoms, and
at least 1 ring heteroatom selected from nitrogen, oxygen and
sulphur The preferred heterocyclic group contains two or more ring
heteroatoms; it is preferred that at least one of the ring
heteroatoms is nitrogen.
[0208] The term heterocyclic, as used herein, also includes
heteroaromatics that is an aryl, as defined herein, with at least
one of the carbon ring atoms being replaced with at least one
oxygen, sulfur or nitrogen ring atom.
[0209] The terms "saturated heterocyclic", or "saturated
heteromonocyclic", and the like, whether in combination or alone as
used herein, refers to a heterocyclic group in which there are no
double or triple bonds present in or bonded to the heterocyclic
ring.
[0210] But, the terms "unsaturated heterocyclic", "unsaturated
heteromonocyclic" and the like, as used herein, whether in
combination or alone, refers to a heterocyclic group which contains
at least one double or triple bond and preferably at least one
double bond, present in or bonded to the heterocyclic ring. Unless
indicated to the contrary, these terms include heteroaromatics.
[0211] Suitable heterocyclic groups include N-containing
heterocyclic groups, such as, unsaturated 3 to 6-membered and more
preferably 5 or 6 membered heteromonocyclic groups containing 1 to
4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl,
pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl
or tetrazolyl;
[0212] saturated 3 to 6-membered and more preferably 5 or 6
membered heteromonocyclic groups containing 1 to 4 nitrogen atoms,
such as, pyrrolidinyl, imidazolidinyl, piperidino or
piperazinyl;
[0213] unsaturated condensed heterocyclic groups containing 1 to 5
nitrogen atoms, such as indolyl, isoindolyl, indolizinyl,
benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or
tetrazolopyridazinyl; [0214] unsaturated 3 to 6-membered and more
preferably 5 or 6 membered heteromonocyclic group containing an
oxygen atom, such as, pyranyl or furyl;
[0215] unsaturated 3 to 6-membered and more preferably 5 to 6
membered heteromonocyclic group containing 1 or 2 sulphur atoms,
such as, thienyl;
[0216] unsaturated 3 to 6-membered heteromonocyclic group and more
preferably 5 or 6 membered containing 1 or 2 oxygen atoms and/or 1
to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or
oxadiazolyl;
[0217] saturated 3 to 6-membered and more preferably 5-6 membered
heteromonocyclic group containing 1 or 2 oxygen atoms and 1 or 3
nitrogen atoms, such as, morpholinyl;
[0218] unsaturated condensed heterocyclic group containing 1 to 2
oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or
benzoxadiazolyl;
[0219] unsaturated 3 to 6-membered and more preferably 5-6 membered
heteromonocyclic group containing 1 or 2 sulphur atoms and 1 or 3
nitrogen atoms, such as, thiazolyl or thiadiazolyl;
[0220] saturated 3 to 6-membered and more preferably 5-6 membered
heteromonocyclic group containing 1 or 2 sulphur atoms and 1 or 3
nitrogen atoms, such as, thiazolidinyl; and
[0221] unsaturated condensed heterocyclic group containing 1 or 2
sulphur atoms and 1 or 3 nitrogen atoms, such as, benzothiazolyl or
benzothiadiazolyl.
[0222] Preferably the heterocyclyl is an unsaturated 5- or
6-membered heteromonocyclic group containing 1, 2 or 3 nitrogen
ring atoms such as imidazolyl, triazolyl, pyrazolyl or pyridinyl;
an unsaturated condensed heterocyclic group such as quinolyl or
benzothiadiazolyl; an unsaturated 5-membered heteromonocyclyl group
containing 1 or 2 sulphur ring atoms such as thiophenyl; or an
unsaturated 5- or 6-membered heteromonocyclyl group containing 1 or
2 sulphur atoms and 1 or 2 nitrogen atoms such as thiazolyl.
[0223] The term "aryl" used either alone or in combination with
other terms such as "optionally substituted aryl" or "aryl acyl"
denotes a carbocyclic aromatic system containing one, two or three
rings wherein such rings may be attached together in a pendent
manner or may be fused and containing up to 18 ring carbon atoms.
This term excludes heteroaromatics. In addition, as defined, this
term includes cyclic compounds in which at least one of the rings
is aromatic, while any additional ring structures may be
unsaturated or saturated. The term "aryl embraces aromatic radicals
such as phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl.
Preferably, the aryl is a 5- or 6-membered aryl such as phenyl.
[0224] The term "halo", used alone or in combination with other
groups refers to fluorine, chlorine, bromine or iodine.
[0225] The term "optionally substituted thio," when used alone or
in combination with other terms, refers to optional substituents,
such as radicals containing a linear or branched alkyl of 1 to 10
carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to
4 carbon atoms, attached to a divalent sulphur atom. Examples of
alkylthio radicals include methylthio, ethylthio, propylthio,
butylthio and hexylthio.
[0226] The term optionally substituted "sulfinyl" alone or in
combination with other groups, refers to optional substituents such
radicals containing a linear or branched alkyl radical, of 1 to 10
carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to
4 carbon atoms, attached to a divalent --S(.dbd.O)-- radical.
Examples include methylsulfinyl, ethylsulfinyl, butylsulfinyl and
hexylsulfinyl.
[0227] The term "optionally substituted sulfonyl" refers to
optional substituents such as radicals containing a linear or
branched alkyl radical of 1 to 10 carbon atoms, preferably 1 to 6
carbon atoms, more preferably 1 to 4 carbon atoms, attached to a
divalent --SO.sub.2-- radical. Examples include methylsulfonyl,
ethylsulfonyl and propylsulfonyl.
[0228] The term "alkoxy" when used alone or in combination with
other groups, refers to straight chain or branched oxy-containing
radicals preferably each having alkyl portions of 1 to about 6
carbon atoms. Examples of alkoxy include methoxy, ethoxy, propoxy,
butoxy and tert-butoxy.
[0229] The term "optionally substituted" when used to modify a
substituent refers to a group which may or may not be further
substituted with one or more groups selected from alkyl, alkenyl,
alkynyl, aryl, aldehyde, halo, haloalkyl, haloalkenyl, haloalkynyl,
haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy,
haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl,
nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino,
alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino,
diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl,
alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy,
alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy,
heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl,
carboalkoxy, carboaryloxy, mercapto, alkylthio, benzylthio,
acylthio, cyano, phosphorus-containing groups and the like.
Preferably, the optional substituent is C.sub.1-6 alkyl, more
preferably C.sub.14 alkyl; CF.sub.3; fluorine; chlorine; iodine;
cyano; C.sub.1-6 alkoxy, more preferably C.sub.14 alkoxy; aryl;
heteroaryl; amino; or alkylamino. The term "antioxidant" is used
herein in its broadest sense and refers to a group which has the
capacity to react with a reactive oxygen species such as a hydroxyl
radical in such a way as to generate a non toxic product. Examples
include phenols such as 3,4,5-trimethoxyphenyl and
3,5-di-t-butyl-4-hydroxyphenyl, indole amines such as melatonin and
flavonoids. Other examples may be found the literature (Wright,
2001; Karbownik, 2001; Gilgun-Sherki, 2001).
[0230] The term "targeting moiety" is used herein in its broadest
sense and refers to a group which will facilitate the brain
delivery of the drug by way of an active transport mechanism. The
targeting moiety is recognised by specific transporter enzymes
integral to the blood brain barrier and these transporter enzymes
then provide a mechanism for the drug to be imported into the
brain. Typically such transporters are sodium dependant and their
substrates contain carboxylic acids such as ascorbic acid and
L-glutamate. Conjugation of the targeting moiety to the drug is
enacted so as to retain the acid moiety. Examples can be found in
the literature (Manfredini, 2002, Tamia, 1999).
[0231] The term "metal chelator" is used herein in its broadest
sense and refers to compounds having two or more donor atoms
capable of binding to a metal atom, preferably Cu, Zn or Fe wherein
at least two of the donor atoms are capable of simultaneous binding
to the metal atom and the resultant metal complex has a
thermodynamic stability greater than or equal to that of the metal
ion; biological ligand complex. The said use of metal chelators as
treatments for neurological disorders in accordance with the
present invention is distinguished from the previously known
concept of "chelation therapy". "Chelation therapy" is a term
associated clinically with the removal of bulk metals such as in
Wilson's disease, .beta.-thallesemia and haemochromatosis. The
break down in metal homeostasis in these diseases can be described
as a catastrophic event, much like a dam bursting, leading to
overwhelming flooding of the problem metal. The mechanism of action
of such compounds is that bulk metal is sequestered by the
chelators and cleared by excretion. By way of comparison the
breakdown in metal homeostasis associated with neurological
conditions of the present invention is more akin to the constant
drip of a leaky tap, which if left long enough will eventually
cause local damage over a long period of time. The intention of the
"metal chelator" of the present invention is to disrupt an abnormal
metal-protein interaction to achieve a subtle repartitioning of
metals and a subsequent normalization of metal distribution with
the aim that once the toxic cycle is short-circuited, endogenous
clearance processes can cope more effectively with the accumulating
amyloidogenic protein.
[0232] The salts of the compound of Formula I or II are preferably
pharmaceutically acceptable, but it will be appreciated that
non-pharmaceutically acceptable salts also fall within the scope of
the present invention, since these are useful as intermediates in
the preparation of pharmaceutically acceptable salts. Examples of
pharmaceutically acceptable salts include salts of pharmaceutically
acceptable cations such as sodium, potassium, lithium, calcium,
magnesium, ammonium and alkylammonium; acid addition salts of
pharmaceutically acceptable inorganic acids such as hydrochloric,
orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,
sulfamic and hydrobromic acids; or salts of pharmaceutically
acceptable organic acids such as acetic, propionic, butyric,
tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic,
gluconic, benzoic, succinic, oxalic, phenylacetic,
methanesulphonic, trihalomethanesulphonic, toluenesulphonic,
benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic,
edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic,
ascorbic and valeric acids.
[0233] In addition, some of the compounds of the present invention
may form solvates with water or common organic solvents. Such
solvates are encompassed within the scope of the invention.
[0234] By "pharmaceutically acceptable derivative" is meant any
pharmaceutically acceptable salt, hydrate, ester, amide, active
metabolite, analogue, residue or any other compound which is not
biologically or otherwise undesirable and induces the desired
pharmacological and/or physiological effect.
[0235] The term "pro-drug" is used herein in its broadest sense to
include those compounds which are converted in vivo to compounds of
Formula I or II. Use of the pro-drug strategy optimises the
delivery of the drug to its site of action, for example, the brain.
In one aspect, the term refers to the presence of a C.sub.1-6 alkyl
or arylester moiety which is designed to resist hydrolysis until
the pro-drug has crossed the blood-brain barrier ("BBB"), where
esterases on the inner surface of the BBB act to hydrolyse the
ester and liberate the C8 hydroxyl of the compounds of formula I or
II. In a second aspect, the term refers to the attachment at C2 of
the 8-hydroxyquinoline core of an antioxidant group, in particular
the 3,4,-5-trimethoxyphenyl moiety or derivatives thereof. Exposure
to the prooxidative environment of the brain will then lead to
hydroxylation of the 3,4,5-trimethoxyphenyl group to give a
2-hydroxy-3,4,5-trimethoxyphenyl substituent, the hydroxyl group of
which acts to enhance the chelation properties of the compounds of
formula I or II.
[0236] The term "tautomer" is used herein in its broadest sense to
include compounds of Formula I or II which are capable of existing
in a state of equilibrium between two isomeric forms. Such
compounds may differ in the bond connecting two atoms or groups and
the position of these atoms or groups in the compound.
[0237] The term "isomer" is used herein in its broadest sense and
includes structural, geometric and stereoisomers. As the compound
of Formula I or II may have one or more chiral centres, it is
capable of existing in enantiomeric forms.
[0238] The compositions of the present invention comprise at least
one compound of Formula I or II together with one or more
pharmaceutically acceptable carriers and optionally other
therapeutic agents. Each carrier, diluent, adjuvant and/or
excipient must be pharmaceutically "acceptable" in the sense of
being compatible with the other ingredients of the composition and
not injurious to the subject. Compositions include those suitable
for oral, rectal, nasal, topical (including buccal and sublingual),
vaginal or parenteral (including subcutaneous, intramuscular,
intravenous and intradermal) administration. The compositions may
conveniently be presented in unit dosage form and may be prepared
by methods well known in the art of pharmacy. Such methods include
the step of bringing into association the active ingredient with
the carrier which constitutes one or more accessory ingredients. In
general, the compositions are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers, diluents, adjuvants and/or excipients or finely divided
solid carriers or both, and then if necessary shaping the
product.
[0239] The term "neurological condition" is used herein in its
broadest sense and refers to conditions wherein various cell types
of the nervous system are degenerated and/or have been damaged as a
result of neurodegenerative disorders or injuries or exposures. In
particular, compounds of formula I or II can be used for the
treatment of resulting conditions, wherein damage to cells of the
nervous system has occurred due to surgical interventions,
infections, exposure to toxic agents, tumours, nutritional deficits
or metabolic disorders. In addition, compounds of the formula I or
II can be used for the treatment of the sequelae of
neurodegenerative disorders, such as Alzheimer's disease,
Parkinson's disease, multiple sclerosis, amylotrophic lateral
sclerosis, epilepsy, drug abuse or drug addiction (alcohol,
cocaine, heroin, amphetamine or the like), spinal cord disorders
and/or injuries, dystrophy or degeneration of the neural retina
(retinopathies) and peripheral neuropathies, such as diabetic
neuropathy and/or the peripheral neuropathies induced by toxins
[0240] The term "neurodegenerative disorder" as used herein refers
to an abnormality wherein neuronal integrity is threatened.
Neuronal integrity can be threatened when neuronal cells display
decreased survival or when the neurons can no longer propagate a
signal. Neurological disorders that can be treated with the
compounds of the present invention include but are not limited to
acute intermittent porphyria; adriamycin-induced cardiomyopathy;
AIDS dementia and HIV-1 induced neurotoxicity; Alzheimer's disease;
amylotrophic lateral sclerosis; atherosclerosis; cateract; cerebral
ischaemia; cerebral palsy; cerebral tumour; chemotherapy-induced
organ damage; cisplatin-induced nephrotoxicity; coronary artery
bypass surgery; Creutzfeldt-Jacob disease and its new variant
associated with "mad cow" disease; diabetic neuropathy; Down's
syndrome; drowning; epilepsy and post-traumatic epilepsy;
Friedrich's ataxia; frontotemporal dementia; glaucoma;
glomerulopathy; haemochromatosis; haemodialysis; haemolysis;
haemolytic uraemic syndrome (Weil's disease); haemorrhagic stroke;
Hallerboden-Spatz disease; heart attack and reperfusion injury;
Huntington's disease; Lewy body disease; intermittent claudication;
ischaemic stroke; inflammatory bowel disease; macular degeneration;
malaria; methanol-induced toxicity; meningitis (aseptic and
tuberculous); motor neuron disease; multiple sclerosis; multiple
system atrophy; myocardial ischaemia; neoplasia; Parkinson's
disease; peri-natal asphyxia; Pick's disease; progressive
supra-nuclear palsy; radiotherapy-induced organ damage; restenosis
after angioplasty; retinopathy; senile dementia; schizophrenia;
sepsis; septic shock; spongiform encephalopathies; subharrachnoid
haemorrhage/cerebral vasospasm; subdural haematoma; surgical
trauma, including neurosurgery; thalassemia; transient ischaemic
attack (TIA); traumatic brain injury (TBI); traumatic spinal
injury; transplantation; vascular dementia; viral meningitis; and
viral encephalitis.
[0241] Additionally, compounds of the present invention may also be
used to potentiate the effects of other treatments, for example to
potentiate the neuroprotective effects of brain derived nerve
growth factor.
[0242] The invention is particularly directed to conditions which
induce oxidative damage of the central nervous system, including
acute and chronic neurological disorders such as traumatic brain
injury, spinal cord injury, cerebral ischaemia, stroke (ischaemic
and haemorragic), subharrachnoid haemorrage/cerebral vasospasm,
cerebral tumour, Alzheimer's disease, Creutzfeldt-Jacob disease and
its new variant associated with "mad cow" disease, Huntington's
disease, Parkinson's disease, Friedrich's ataxia, cataract,
dementia with Lewy body formation, multiple system atrophy,
Hallerboden-Spatz disease, diffuse Lewy body disease, amylotrophic
lateral sclerosis, motor neuron disease, multiple sclerosis, fatal
familial insomnia, Gertsmann Straussler Sheinker disease and
hereditary cerebral haemorrhage with amyoidoisis-Dutch type.
[0243] More particularly, the invention is directed to the
treatment of neurodegenerative amyloidosis. The neurodegenerative
amyloidosis may be any condition wherein neurological damage
results from an abnormal interaction between a biological ligand
such as a protein and redox active metal ions promoting reactive
oxygen species formation, radicalization and/or the deposition of
amyloid. The amyloid may be formed from a variety of protein or
polypeptide precursors, including but not limited to A.beta.,
synuclein, huntingtin, SOD, amyloid precursor protein (APP) or
prion protein.
[0244] Thus the condition is preferably selected from the group
consisting of sporadic or familial Alzheimer's disease, amyotrophic
lateral sclerosis, motor neuron disease, cataract, Parkinson's
disease, Creutzfeldt-Jacob disease and its new variant associated
with "mad cow" disease, Huntington's disease, dementia with Lewy
body formation, multiple system atrophy, Hallerboden-Spatz disease,
and diffuse Lewy body disease.
[0245] More preferably the neurodegenerative amyloidosis is an
A.beta.-related condition, such as Alzheimer's disease or dementia
associated with Down syndrome or one of several forms of autosomal
dominant forms of familial Alzheimer's disease (reviewed in St
George-Hyslop, 2000). Most preferably the A.beta.-related condition
is Alzheimer's disease.
[0246] In a particularly preferred embodiment of all aspects of the
invention, prior to treatment the subject has moderately or
severely impaired cognitive function, as assessed by the
Alzheimer's Disease Assessment Scale (ADAS)-cog test, for example
an ADAS-cog value of 25 or greater.
[0247] In addition to slowing or arresting the cognitive decline of
a subject, the methods and compounds of the invention may also be
suitable for use in the treatment or prevention of
neurodegenerative conditions, or may be suitable for use in
alleviating the symptoms of neurodegenerative conditions. The
compounds may be able to provide at least a partial reversal of the
cognitive decline experienced by patients. If administered to a
subject who has been identified as having an increased risk of a
predisposition to neurodegenerative conditions, or to a subject
exhibiting pre-clinical manifestations of cognitive decline, such
as Mild Cognitive Impairment or minimal progressive cognitive
impairment, these methods and compounds may be able to prevent or
delay the onset of clinical symptoms, in addition to the effect of
slowing or reducing the rate of cognitive decline.
[0248] Currently Alzheimer's disease and other dementias are
usually not diagnosed until one or more warning symptoms have
appeared. These symptoms constitute a syndrome known as Mild
Cognitive Impairment (MCI), which was recently defined by the
American Academy of Neurology, and refers to the clinical state of
individuals who have memory impairment, but who are otherwise
functioning well, and who do not meet clinical criteria for
dementia (Petersen et al., 2001). Symptoms of MCI include:
[0249] (1) Memory loss which affects job skills
[0250] (2) Difficulty performing familiar tasks
[0251] (3) Problems with language
[0252] (4) Disorientation as to time and place (getting lost)
[0253] (5) Poor or decreased judgement
[0254] (6) Problems with abstract thinking
[0255] (7) Misplacing things
[0256] (8) Changes in mood or behaviour
[0257] (9) Changes in personality
[0258] (10) Loss of initiative
[0259] MCI can be detected using conventional cognitive screening
tests, such as the Mini Mental Status Exam, and the Memory
Impairment Screen, and neuropsychological screening batteries.
[0260] The term "subject" as used herein refers to any animal
having a disease or condition which requires treatment with a
pharmaceutically-active agent. The subject may be a mammal,
preferably a human, or may be a domestic or companion animal. While
it is particularly contemplated that the compounds of the invention
are suitable for use in medical treatment of humans, it is also
applicable to veterinary treatment, including treatment of
companion animals such as dogs and cats, and domestic animals such
as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and
sheep, or zoo animals such as primates, felids, canids, bovids, and
ungulates.
[0261] Suitable mammals include members of the Orders Primates,
Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and
Artiodactyla. Members of the Orders Perissodactyla and Artiodactyla
are particularly preferred because of their similar biology and
economic importance.
[0262] For example, Artiodactyla comprises approximately 150 living
species distributed through nine families: pigs (Suidae), peccaries
(Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae),
chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer
(Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats
and antelope (Bovidae). Many of these animals are used as feed
animals in various countries. More importantly, many of the
economically important animals such as goats, sheep, cattle and
pigs have very similar biology and share high degrees of genomic
homology.
[0263] The Order Perissodactyla comprises horses and donkeys, which
are both economically important and closely related. Indeed, it is
well known that horses and donkeys interbreed.
[0264] As used herein, the term "therapeutically effective amount"
is meant an amount of a compound of the present invention effective
to yield a desired therapeutic response, for example, to prevent or
treat a neurological condition.
[0265] The specific "therapeutically effective amount" will,
obviously, vary with such factors as the particular condition being
treated, the physical condition of the subject, the type of subject
being treated, the duration of the treatment, the nature of
concurrent therapy (if any), and the specific formulations employed
and the structure of the compound or its derivatives.
[0266] The compounds of the present invention may additionally be
combined with other medicaments to provide an operative
combination. It is intended to include any chemically compatible
combination of pharmaceutically-active agents, as long as the
combination does not eliminate the activity of the compound of
formula I or II. It will be appreciated that the compound of the
invention and the other medicament may be administered separately,
sequentially or simultaneously.
[0267] Other medicaments may include, for example, where the
condition is a .beta.-amyloid related condition, particularly
Alzheimer's disease, an inhibitor of the acetylcholinesterase
active site, for example phenserine, galantamine, or tacrine; an
antioxidant, such as Vitamin E or Vitamin C; an anti-inflammatory
agent such as flurbiprofen or ibuprofen optionally modified to
release nitric oxide (for example NCX-2216, produced by NicOx) or
an oestrogenic agent such as 17-.beta.-oestradiol.
[0268] Methods and pharmaceutical carriers for preparation of
pharmaceutical compositions are well known in the art, as set out
in textbooks such as Remington's Pharmaceutical Sciences, 20th
Edition, Williams & Wilkins, Pennsylvania, USA.
[0269] As used herein, a "pharmaceutical carrier" is a
pharmaceutically acceptable solvent, suspending agent or vehicle
for delivering the compound of formula I or II to the subject. The
carrier may be liquid or solid and is selected with the planned
manner of administration in mind. Each carrier must be
pharmaceutically "acceptable" in the sense of being compatible with
other ingredients of the composition and non injurious to the
subject.
[0270] The compound of formula I or II may be administered orally,
topically, or parenterally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants, and vehicles. The term parenteral as used herein
includes subcutaneous injections, aerosol for administration to
lungs or nasal cavity, intravenous, intramuscular, intrathecal,
intracranial, injection or infusion techniques.
[0271] The present invention also provides suitable topical, oral,
and parenteral pharmaceutical formulations for use in the novel
methods of treatment of the present invention. The compounds of the
present invention may be administered orally as tablets, aqueous or
oily suspensions, lozenges, troches, powders, granules, emulsions,
capsules, syrups or elixirs. The composition for oral use may
contain one or more agents selected from the group of sweetening
agents, flavouring agents, colouring agents and preserving agents
in order to produce pharmaceutically elegant and palatable
preparations. Suitable sweeteners include sucrose, lactose,
glucose, aspartame or saccharin. Suitable disintegrating agents
include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan
gum, bentonite, alginic acid or agar. Suitable flavouring agents
include peppermint oil, oil of wintergreen, cherry, orange or
raspberry flavouring. Suitable preservatives include sodium
benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl
paraben, propyl paraben or sodium bisulphite. Suitable lubricants
include magnesium stearate, stearic acid, sodium oleate, sodium
chloride or talc. Suitable time delay agents include glyceryl
monostearate or glyceryl distearate. The tablets contain the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipients which are suitable for the manufacture of tablets.
[0272] These excipients may be, for example, (1) inert diluents,
such as calcium carbonate, lactose, calcium phosphate or sodium
phosphate; (2) granulating and disintegrating agents, such as corn
starch or alginic acid; (3) binding agents, such as starch, gelatin
or acacia; and (4) lubricating agents, such as magnesium stearate,
stearic acid or talc. These tablets may be uncoated or coated by
known techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed.
Coating may also be performed using techniques described in the
U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic
therapeutic tablets for control release.
[0273] The compound of formula I or II as well as the
pharmaceutically-active agent useful in the method of the invention
can be administered, for in vivo application, parenterally by
injection or by gradual perfusion over time independently or
together. Administration may be intravenously, intraarterial,
intraperitoneally, intramuscularly, subcutaneously, intracavity,
transdermally or infusion by, for example, osmotic pump. For in
vitro studies the agents may be added or dissolved in an
appropriate biologically acceptable buffer and added to a cell or
tissue.
[0274] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, anti-microbials,
anti-oxidants, chelating agents, growth factors and inert gases and
the like.
[0275] Generally, the terms "treating", "treatment" and the like
are used herein to mean affecting a subject, tissue or cell to
obtain a desired pharmacologic and/or physiologic effect. The
effect is therapeutic in terms of a partial or complete cure of a
disease and/or alleviation of symptoms associated with the disease.
"Treating" as used herein covers any treatment of, amelioration of,
or prevention of disease in a vertebrate, a mammal, particularly a
human, and includes: (a) preventing the disease from worsening in a
subject that may, have the disease, or (b) inhibiting the disease,
i.e., arresting its development; or (c) relieving or ameliorating
the effects of the disease, i.e., cause regression of the effects
of the disease. Prophylaxis or synonym thereto refers to reducing
the risks of a subject from having the disease, especially those
predisposed to the disease.
[0276] The invention includes various pharmaceutical compositions
useful for ameliorating disease. The pharmaceutical compositions
according to one embodiment of the invention are prepared by
bringing a compound of formula I or II, analogues, derivatives or
salts thereof, or combinations of compound of formula I or II and
one or more pharmaceutically-active agents into a form suitable for
administration to a subject using carriers, excipients and
additives or auxiliaries. Frequently used carriers or auxiliaries
include magnesium carbonate, titanium dioxide, lactose, mannitol
and other sugars, talc, milk protein, gelatin, starch, vitamins,
cellulose and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water, alcohols,
glycerol and polyhydric alcohols. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial, anti-oxidants, chelating agents and inert gases.
Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 20th ed. Williams and Wilkins (2000) and
The British National Formulary 43rd ed. (British Medical
Association and Royal Pharmaceutical Society of Great Britain,
2002; http://bnf.rhn.net), the contents of which are hereby
incorporated by reference. The pH and exact concentration of the
various components of the pharmaceutical composition are adjusted
according to routine skills in the art. See Goodman and Gilman's
The Pharmacological Basis for Therapeutics (7th ed., 1985).
[0277] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units may be tablets,
capsules and suppositories. For treatment of a subject, depending
on activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the subject,
different daily doses can be used. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administration of
subdivided doses at specific intervals.
[0278] The pharmaceutical compositions according to the invention
may be administered locally or systemically in a therapeutically
effective dose. Amounts effective for this use will, of course,
depend on the severity of the disease and the weight and general
state of the subject. Typically, dosages used in vitro may provide
useful guidance in the amounts useful for in situ administration of
the pharmaceutical composition, and animal models may be used to
determine effective dosages for treatment of the cytotoxic side
effects. Various considerations are described, e.g., in Langer,
Science, 249: 1527, (1990). Formulations for oral use may be in the
form of hard gelatin capsules wherein the active ingredient is
mixed with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They may also be in the form of soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, such as peanut oil, liquid paraffin or olive
oil.
[0279] Aqueous suspensions normally contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspension. Such excipients may be (1) suspending agent such as
sodium carboxymethyl cellulose, methyl cellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) dispersing
or wetting agents which may be (a) naturally occurring phosphatide
such as lecithin; (b) a condensation product of an alkylene oxide
with a fatty acid, for example, polyoxyethylene stearate; (c) a
condensation product of ethylene oxide with a long chain aliphatic
alcohol, for example, heptadecaethylenoxycetanol; (d) a
condensation product of ethylene oxide with a partial ester derived
from a fatty acid and hexitol such as polyoxyethylene sorbitol
monooleate, or (e) a condensation product of ethylene oxide with a
partial ester derived from fatty acids and hexitol anhydrides, for
example polyoxyethylene sorbitan monooleate.
[0280] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension. This
suspension may be formulated according to known methods using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables.
[0281] Compounds of formula I or II may also be administered in the
form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles, and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine, or phosphatidylcholines.
[0282] The compounds of formula I or II may also be presented for
use in the form of veterinary compositions, which may be prepared,
for example, by methods that are conventional in the art. Examples
of such veterinary compositions include those adapted for:
[0283] (a) oral administration, external application, for example
drenches (e.g. aqueous or non-aqueous solutions or suspensions);
tablets or boluses; powders, granules or pellets for admixture with
feed stuffs; pastes for application to the tongue;
[0284] (b) parenteral administration for example by subcutaneous,
intramuscular or intravenous injection, e.g. as a sterile solution
or suspension; or (when appropriate) by intramammary injection
where a suspension or solution is introduced in the udder via the
teat;
[0285] (c) topical applications, e.g. as a cream, ointment or spray
applied to the skin; or
[0286] (d) intravaginally, e.g. as a pessary, cream or foam.
[0287] Dosage levels of the compound of formula I or II of the
present invention are of the order of about 0.5 mg to about 20 mg
per kilogram body weight, with a preferred dosage range between
about 0.5 mg to about 10 mg per kilogram body weight per day (from
about 0.5 gms to about 3 gms per patient per day). The amount of
active ingredient that may be combined with the carrier materials
to produce a single dosage will vary depending upon the host
treated and the particular mode of administration. For example, a
formulation intended for oral administration to humans may contain
about 5 mg to 1 g of an active compound with an appropriate and
convenient amount of carrier material which may vary from about 5
to 95 percent of the total composition. Dosage unit forms will
generally contain between from about 5 mg to 500 mg of active
ingredient.
[0288] Optionally the compounds of the invention are administered
in a divided dose schedule, such that there are at least two
administrations in total in the schedule. Administrations are given
preferably at least every two hours for up to four hours or longer;
for example the compound may be administered every hour or every
half hour. In one preferred embodiment, the divided-dose regimen
comprises a second administration of the compound of the invention
after an interval from the first administration sufficiently long
that the level of active compound in the blood has decreased to
approximately from 5-30% of the maximum plasma level reached after
the first administration, so as to maintain an effective content of
active agent in the blood. Optionally one or more subsequent
administrations may be given at a corresponding interval from each
preceding administration, preferably when the plasma level has
decreased to approximately from 10-50% of the immediately-preceding
maximum.
[0289] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and
the severity of the particular disease undergoing therapy.
EXAMPLES
[0290] The invention will now be described in detail by way of
reference only to the following non-limiting examples.
[0291] General
[0292] 8-Hydroxyquinoline-2-carboxylic acid 1 (Shrader et al,
1988), 8-hydroxyquinoline-2-carbonitrile 2 (Shrader et al, 1988),
2-chloro-8-hydroxyquinoline 3 (Wang et al, 1996; Fleming et al,
1971), 2-aminomethylthiazole 4 (Dondoni et al, 1987, 1996),
2,5,7-trichloro-8-hydroxyquinoline 10 (Ostrovskaya et al, 1986),
5,7-dichloro-8-benzyloxy-quinoline-2-carboxylic acid 18 (Carissimi,
M., 1972), 7-chloro-5-iodo-8-hydroxyquinoline 20 (Gershon et al,
1971), 4-chloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester
25 (Richard et al, 1997) and 1-methyl-1H-histamine hydrochloride
(Durant et al, 1976) were prepared according to the literature. The
following compounds/reagents were sourced commercially: quinolines:
2-methyl-quinolin-8-ol, 8-hydroxy-quinoline (8-HQ) and
5,7-dibromo-8-hydroxy-quinoline were purchased from Fluka;
4,8-dihydroxy-quinoline-2-carboxylic acid,
5-chloro-7-iodo-8-hydroxy-quinoline,
5,7-dichloro-2-methyl-quinolin-8-ol and
5,7-diiodo-8-hydroxyquinoline were purchased from Aldrich; amines:
histamine, 2-aminoethylyridine, 2-aminothiazole,
2-(2-aminoethyl)pyridine, 2-(aminomethyl)pyridine,
5-methyl-2-aminothiazole, 2-aminophenol, 1,2-diaminoethane,
glycine, 1,2-phenylenediamine, di-(2-picolyl)amine and
2-(2-methylaminoethyl)pyridine were all purchased from Aldrich;
aldehydes: 4-imidazolecarboxaldehyde, 2-thiazolecarboxaldehyde and
2-pyridinecarboxaldehyde were all purchased from Aldrich; azoles:
pyrazole, imidazole, methylimidazole and 1H-1,2,3-triazole were
purchased from Aldrich; boronic acids:
2-(trifluoromethyl)phenylboronic acid, 2-methoxyphenylboronic acid,
o-tolylboronic acid, 2-fluorophenylboronic acid,
3-methoxyphenylboronic acid, 4-methoxyphenylboronic acid,
m-tolylboronic acid, 4-(dimethylamino)phenylboronic acid,
2-formylphenylboronic acid, thianaphthene-2-boronic acid,
3,5-difluorophenylboronic acid, 2,4-difluorophenylboronic acid,
3-thiopheneboronic acid, 3-fluorophenylboronic acid,
4-fluorophenylboronic acid and 3-nitrophenylboronic acid were all
purchased from Aldrich; and organozinc reagents: 2-pyridylzinc
bromide, 2-(methylthio)phenylzinc iodide,
2-(ethoxycarbonyl)phenylzinc iodide and 6-methylpyridylzinc bromide
(0.5 M solution in THF) were commercially available (Aldrich).
3-Pyridylboronic acid was purchased from Frontier Scientific.
Solvents were analytical grade and used as supplied. THF was
distilled from sodium and benzophenone under argon. .sup.1H NMR
spectra (.delta., relative to TMS) were recorded on a Varian Unity
300 spectrometer unless otherwise indicated; J-Values are given in
hertz. Mass spectral data were recorded on a Micromass Quattro II
mass spectrometer.
Example 1
Preparation of 8-hydroxy-quinoline-2-carboxylic acid amides (Scheme
1)
##STR00033##
[0293] Procedure A:
[0294] 1,3-Dicyclohexylcarbodiimide (182 mg, 0.87 mmol) was added
to a stirred solution of 1-hydroxybenzotriazole hydrate (119 mg,
0.87 mmol) and 8-hydroxy-quinoline-2-carboxylic acid 1 (150 mg,
0.87 mmol) in DMF and dichloromethane (1:1, 10 mL). After 30 min,
histamine (182 mg, 0.87 mmol) was added and the mixture stirred at
RT for a further 16 h. The volatiles were then removed in vacuo and
the remaining residue gave, after purification by column
chromatography on silica (ethyl acetate/i-PrOH/2 N NH.sub.4OH,
6:2:1), 8-hydroxy-quinoline-2-carboxylic
acid[2-(1H-imidazol-4-yl)-ethyl]-amide A1 as a cream-colored
solid.
[0295] The above reaction was repeated using amines with 1 or
4,8-dihydroxy-quinoline-2-carboxylic acid: histamine gave B1;
2-(2-aminoethyl)pyridine gave A2, 2-(aminomethyl)pyridine gave
A5/B2, 2-aminothiazole gave A3,5-methyl-2-aminothiazole gave A4,
2-aminophenol gave A6, 1,2-diaminoethane gave A7, glycine gave
A8/B3, 1,2-phenylenediamine gave B4 and di-(2-picolyl)amine gave
A10. Using A8 as the starting acid, coupling with amines
2-(aminomethyl)pyridine gave B5 and histamine gave B6. Yields and
data are given in Table 1.
Procedure B:
[0296] 8-Hydroxy-quinoline-2-carboxylic acid 1 (100 mg, 0.59 mmol)
or 4,8-dihydroxyquinoline-2-carboxylic acid (121 mg, 0.59 mmol) and
phosphorus oxychloride (5 mL) were heated under reflux for 1 h,
cooled, and concentrated. THF (20 mL) was added to the residue and
the mixture cooled (0.degree. C.) before the addition of Et.sub.3N
(0.5 mL) and the amine (1.18 mmol). The mixture was allowed to warm
to RT. After 16 h, the volatiles were removed in vacuo and the
resulting residue afforded, after column chromatography on silica,
the 8-hydroxy-quinoline-2-carboxylic acid amide. Yields and data
are given in Table 1.
Example 2
Preparation of 2-Acetyl-8-hydroxy-quinoline C1 (Scheme 2)
##STR00034##
[0298] Methylmagnesium bromide (1.2 mL of a 3 M solution in diethyl
ether, 3.5 mmol) was added dropwise into a stirred solution of
8-hydroxyquinoline-2-carbonitrile 2 (100 mg, 0.588 mmol) in diethyl
ether (10 mL) at -15.degree. C. The resulting solution was allowed
to warm to RT over 2 h and stirred at RT for a further 4 h. The
reaction mixture was then quenched with saturated NH.sub.4Cl and
extracted with ethyl acetate (10 mL.times.3). The extracts were
combined, dried (Na.sub.2SO.sub.4) and concentrated to afford the
title compound as a pale orange solid (108 mg, 98%) C1. Spectral
data of this compound are given in Table 1.
Example 3
Preparation of 8-Hydroxy-quinoline-2-carboxaldehyde Oxime D1
(Scheme 3)
##STR00035##
[0300] A solution of 2-methyl-quinolin-8-ol (536 mg, 3.37 mmol) in
dioxane (8 mL) was added dropwise over 3 h into a stirred mixture
of SeO.sub.2 (665 mg, 5.99 mmol) in dioxane (25 mL) at
50-55.degree. C. The resulting mixture was then heated at
80.degree. C. for 16 h, cooled, and the solids filtered off. The
filtrate was concentrated and the residue purified by column
chromatography on silica (dichloromethane/MeOH, 1:0-40:1). This
afforded 8-hydroxy-quinoline-2-carboxaldehyde 4 as a straw-coloured
solid (358 mg, 61%). 4: .sup.1H NMR (CDCl.sub.3): .delta. 10.24 (s,
1H), 8.34 (d, J=8.6, 1H), 8.22 (br, 1H), 8.07 (d, J=8.6, 1H), 7.64
(dd, J=7.5 and 8.0, 1H), 7.44 (d, J=8.0, 1H), 7.30 (d, J=7.5, 1H).
The mixture of 4 (100 mg, 0.578 mmol), NaOAc (63 mg, mmol),
hydroxylamine hydrochloride (60 mg, 0.863 mmol) and water (5 mL)
was heated at 100.degree. C. for 15 min. The precipitate was
isolated by filtration. This provided the title oxime (ID 969) D1
as an off-white solid (87 mg, 80%); spectral data of this compound
are shown in Table 2.
Example 4
2-Aminomethyl-quinolin-8-ol E1 (Scheme 4)
##STR00036##
[0302] 8-Hydroxy-quinoline-2-carboxaldehyde oxime D1 (167 mg, 0.888
mmol) and MeOH (50 mL) was treated under hydrogenolysis conditions
(atmospheric H.sub.2, catalytic 10% Pd/carbon) at RT. After 4 h,
the catalyst was filtered off and the volatiles removed which
afforded 2-aminomethyl-quinolin-8-ol E1 as a light brown solid (126
mg, 82%); spectral data of this compound are given in Table 2.
N-(8-Hydroxy-quinolin-2-ylmethyl)-guanidine E3 (Scheme 4)
[0303] N,N'-Bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine
(54 mg, 0.174 mmol) was added to a stirred mixture of
2-aminomethyl-quinolin-8-ol E1 (25 mg, 0.144 mmol) in THF (5 mL).
After 16 h at RT, the volatiles were removed in vacuo and the
residue provided, after column chromatography on silica (ethyl
acetate/hexane, 1:2), the (Boc).sub.2-derivative of
N-(8-Hydroxy-quinolin-2-ylmethyl)-guanidine as a colorless solid
(52 mg, 87%). A solution of this solid (47 mg, 0.113 mmol) and
concentrated hydrochloric acid (0.5 mL) in dioxane (1 mL) was then
stirred at RT for 16 h, and concentrated. H.sub.2O (2 mL) was
added, the pH adjusted to 8 (conc. NH.sub.4OH) and the mixture
concentrated. The solid was dissolved in MeOH and the solution
triturated with ethyl acetate. The resulting solid was filtered off
and the filtrate was concentrated to a solid. The latter, after
column chromatography on silica (ethyl acetate/i-PrOH/H.sub.2O,
12:4:1), afforded the title compound E3 as an off-white solid (23
mg, 94%); spectral data are given in Table 2.
2-Acetamidomethyl-quinolin-8-ol E2 (Scheme 4)
[0304] A solution of 2-aminomethyl-quinolin-8-ol E1 (30 mg, 0.172
mmol) and Ac.sub.2O (1 mL) in pyridine (2 mL) was stirred at RT
overnight and concentrated. Subsequent column chromatography on
silica (ethyl acetate) gave 2-acetamido-8-acetoxy-quinoline as a
colorless solid (35 mg, 79%). A solution of
2-acetamido-8-acetoxy-quinoline (33 mg, 0.128 mmol) and
K.sub.2CO.sub.3 (50 mg, 0.362 mmol) in MeOH (1 mL) and H.sub.2O
(0.5 mL) was stirred at RT for 16 h. Volatiles were removed in
vacuo and H.sub.2O (2 mL) added. The pH of the mixture was adjusted
to 7 (2 N HCl) and the solid was isolated by filtration, washed
with H.sub.2O (1 mL.times.2) and dried. The title compound E2 was
isolated as a cream solid (21 mg, 76%); spectral data are given in
Table 2.
Example 5
Reductive Amination of 8-hydroxyquinoline-2-carboxaldehyde (Scheme
5)
##STR00037##
[0306] Sodium triacetoxyborohydride (225 mg, 1.061 mmol) was added
to a stirred solution of 8-hydroxy-quinoline-2-carboxaldehyde 4
(200 mg, 1.156 mmol) and histamine (128 mg, 1.152 mmol) in
dichloroethane (10 mL). The mixture was left to stir at RT for 16
h, neutralized (aqueous NaHCO.sub.3), and concentrated. The
resulting residue, after column chromatography on silica (ethyl
acetate/i-PrOH/2 N NH.sub.4OH, 6:2:1), afforded
2-{[2-(1H-imidazol-4-yl)-ethylamino]-methyl}-quinolin-8-ol F1 as a
straw-colored solid (190 mg, 61%). The above method was repeated
using other amines: 2-(aminomethyl)pyridine gave F2 and
2-(2-methylaminoethyl)pyridine gave F3, data given in Table 2.
Example 6
Reductive Amination with Amines from Example 5 (Scheme 6)
##STR00038##
[0308] Following the procedure of Example 5, aldehydes:
2-imidazolecarboxaldehyde gave G1/H1, 2-pyridinecarboxaldehyde gave
G2/H2 and 2-thiazolecarboxaldehyde gave H3 when treated with F1 (G
series) or F2 (H series). Results and spectral data are given in
Table 2.
Example 7
2-(Azole)-8-hydroxyquinolines I1-I4 (Scheme 7)
##STR00039##
[0310] A mixture of 2-chloro-quinolin-8-ol 3 (80 mg, 0.447 mmol)
and pyrazole (152 mg, 2.233 mmol) was heated at 175.degree. C. in a
steel autoclave for 48 h. The crude product was then purified by
column chromatography on silica (ethyl acetate/hexane, 1:1) to give
2-pyrazol-1-yl-quinolin-8-ol (compound ID 964) I1 as a white solid
(68 mg, 72%).
[0311] The above procedure was repeated using imidazole,
2-methylimidazole and 1H-1,2,3-triazole to give 12, 13 and 14. The
crude product for 14 was washed with MeOH (10 mL.times.3) to give
2-[1,2,3]triazol-1-yl-quinolin-8-ol (compound ID 994) 14 as an
off-white solid (67 mg, 71%). Spectral data of these products are
given in Table 3.
Example 8
Preparation of 5-chloro-7-aryl-8-hydroxyquinolines K1-K17 (Scheme
8)
##STR00040##
[0313] 2-Bromopropane (0.46 mL, 4.90 mmol) was added into a stirred
mixture of the 5-chloro-7-iodo-quinolin-8-ol (1.00 g, 3.27 mmol),
K.sub.2CO.sub.3 (1.86 g, 13.5 mmol) and DMSO (10 mL). After 16 h at
RT, saturated NH.sub.4Cl (10 mL) was added and the mixture
extracted with dichloromethane (10 mL.times.3). The extracts were
combined and concentrated. Diethyl ether (40 mL) was added to the
residue and the resulting mixture washed successively with 2 N
NaOH, H.sub.2O and brine, and dried (Na.sub.2SO.sub.4). Subsequent
column chromatography on silica (ethyl acetate/hexane, 1:1)
afforded 5-chloro-7-iodo-8-isopropoxy-quinoline 5 as a solid (1.06
g, 93%). 5: .sup.1H NMR (CDCl.sub.3): .delta. 8.93 (dd, J=1.5 and
4.2, 1H), 8.52 (dd, J=1.5 and 8.4, 1H), 7.98 (s, 1H), 7.53 (dd,
J=4.2 and 8.4, 1H), 5.38 (m, 1H), 1.43 (d, J=6.0, 6 H). To a
stirred mixture of 5-chloro-7-iodo-8-isopropoxy-quinoline 5 (200
mg, 0.58 mmol), phenylboronic acid (77 mg, 0.62 mmol), 2 N
Na.sub.2CO.sub.3 (7.2 mL), EtOH (1.2 mL) and benzene (6 mL) was
added, under a blanket of argon, Pd(PPh.sub.3).sub.4 (20 mg). The
mixture was stirred under reflux for 16 h, cooled and concentrated.
This provided, after column chromatography on silica (ethyl
acetate/hexane, 1:9), 5-chloro-7-phenyl-8-isopropoxy-quinoline as a
yellow solid. To a stirred solution of the 8-isopropoxy-quinoline
(0.339 mmol) in dichloromethane (2 mL) at -78.degree. C. was added
BCl.sub.3 (1.36 mL of a 1 M solution in dichloromethane, 1.36
mmol). After 2 h, the reaction mixture was allowed to warm to RT
and stirred for a further 2 h. MeOH (5 mL) was added and the
mixture was concentrated to dryness. This process was repeated four
times. Further washing of the remaining residue with diethyl ether
(2 mL.times.3) provided K1 in 91% yield. Data in Table 4.
[0314] In a similar fashion, reaction of 5 with boronic acids:
2-(trifluoromethyl)phenylboronic acid, 2-methoxyphenylboronic acid
(Note cleavage to the 2-hydroxyphenyl derivative), o-tolylboronic
acid, 2-fluorophenylboronic acid, 3-methoxyphenylboronic acid,
4-methoxyphenylboronic acid, m-tolylboronic acid,
4-(dimethylamino)phenylboronic acid, 2-formylphenylboronic acid,
thianaphthene-2-boronic acid, 3,5-difluorophenylboronic acid,
2,4-difluorophenylboronic acid, 3-thiopheneboronic acid,
3-fluorophenylboronic acid, 4-fluorophenylboronic acid and
3-nitrophenylboronic acid; and isopropoxy cleavage with BCl.sub.3
gave 5-chloro-7-aryl-8-hydroxyquinolines K2-K17. Data in Table
4.
Example 9
Preparation of 5-aryl-7-bromo-8-hydroxyquinolines L1-L2 (Scheme
9)
##STR00041##
[0316] Reaction of 5,7-dibromo-quinolin-8-ol with 2-bromopropane
following the method described in Example 8 gave
5,7-dibromo-8-isopropoxy-quinoline 6 (97%): .sup.1H NMR
(CDCl.sub.3): .delta. 8.94 (dd, J=1.5 and 4.2, 1H), 8.48 (dd, J=1.5
and 8.4, 1 H), 8.00 (s, 1H), 7.52 (dd, J=4.2 and 8.4, 1H), 5.22 (m,
1H), 1.43 (d, J=6.1, 6 H); mass spectrum: m/z 344, 346, 348
(M.sup.++1, 50, 100 and 50%, respectively). Reaction of 6 with aryl
boronic acids, and cleavage of the isopropoxy group following the
method outlined in Example 8 gave compounds L1 and L2 (data in
Table 4).
Example 10
Preparation of 5,7-diaryl-8-hydroxyquinolines M1-M5 and
5-aryl-7-iodo-8-hydroxyquinolines N2-N5 (Scheme 10)
##STR00042##
[0317] Preparation of 5,7-diaryl-8-hydroxyquinolines M1-M5 and
5-aryl-7-iodo-8-hydroxyquinolines N2-N5 (Scheme 10)
[0318] Reaction of 5,7-diiodo-quinolin-8-ol with 2-bromopropane
following the method described in Example 8 gave
5,7-dibromo-8-isopropoxy-quinoline 7 (93%): .sup.1H NMR
(CDCl.sub.3): .delta. 8.86 (dd, J=1.5 and 4.4, 1H), 8.46 (s, 1H),
8.33 (dd, J=1.5 and 8.5, 1H), 7.49 (dd, J=4.4 and 8.5, 1H), 5.40
(m, 1H), 1.43 (d, J=6.1, 6 H). To a stirred mixture of 7 (200 mg,
0.51 mmol), phenylboronic acid (143 mg, 1.17 mmol), 2 N
Na.sub.2CO.sub.3 (7.2 mL), EtOH (1.2 mL) and benzene (6 mL) was
added, under a blanket of argon, Pd(PPh.sub.3).sub.4 (21 mg). The
mixture was stirred under reflux for 16 h, cooled and concentrated.
This provided, after column chromatography on silica (ethyl
acetate/hexane, 1:9), 5,7-diphenyl-8-isopropoxy-quinoline as a
yellow solid (157 mg, 91%). Cleavage of the isopropoxy group
following the method outlined in Example 8 gave
5,7-diphenyl-8-hydroxy-quinoline M1 in 91% yield. (See table 4 for
data).
[0319] Reaction of 7 with aryl boronic acids, and cleavage of the
isopropoxy group following the method outlined in Example 8 gave
compounds M2-M5 (data in Table 4). In those cases where the boronic
acid contained an ortho substituent, the Suzuki reaction yielded a
mixture of 5-aryl-7-iodo-8-isopropoxyquinolines and
5,7-diaryl-8-isopropoxyquinoline, which could be separated prior to
isopropoxy cleavage to provide both
5-aryl-7-iodo-8-hydroxyquinolines N2-N5 and
5,7-diaryl-8-hydroxyquinolines M2-M5.
Example 11
Preparation of 5,5'-Dichloro-8,8'-dihydroxy-7,7'-biquinoline 01
(Scheme 11)
##STR00043##
[0321] A solution of 5 (0.576 mmol), bis(pinacolato)diboron (1.1
equiv.), 2 N Na.sub.2CO.sub.3 (2 mL) and KOAc (3 equiv) was stirred
in the presence of a catalytic amount of PdCl.sub.2(dppf) in DMF
(10 mL) at 80.degree. C. for 3 h. The reaction mixture was then
quenched with saturated NH.sub.4C1 and extracted with diethyl ether
(10 mL.times.3), dried (Na.sub.2SO.sub.4), and concentrated. Column
chromatography of the resulting residue (silica; ethyl
acetate/hexane, 1:1) afforded
5,5'-dichloro-8,8'-diisopropoxy-7,7'-biquinoline (compound ID 971)
as a solid (56 mg, 22%). Cleavage of the isopropoxy groups with
BCl.sub.3 following the procedure outlined in Example 8 gave 01 in
22% yield.
Example 12
Preparation of 2-aryl-8-hydroxyquinolines P1-P4 (Scheme 12)
##STR00044##
[0322] 2-Iodo-quinolin-8-ol 8
[0323] Acetyl chloride (0.422 mL, 5.95 mmol) was added dropwise
over 20 min into a stirred slurry of 2-chloro-quinolin-8-ol 3 (500
mg, 2.79 mmol), NaI (649 mg, 4.33 mmol) and AcCN (3 mL) at
RT..sup.5 The mixture was then stirred at 35-40.degree. C. for 3 h,
then overnight at 70.degree. C., and concentrated. H.sub.2O (10 mL)
was added, and mixture was extracted with dichloromethane (10
mL.times.3). The extracts were combined and washed successively
with a 1:1 solution of saturated NaHCO.sub.3 and sodium thiosulfate
(5 mL.times.2), and H.sub.2O (10 mL.times.2), and dried
(Na.sub.2SO.sub.4). The residue obtained after solvent removal
gave, after column chromatography on silica (ethyl acetate/hexane,
1:8-1:3), 2-Iodo-quinolin-8-ol 8 as a white solid (268 mg, 35%) and
a 1:2 inseparable mixture of 8-acetoxy-2-iodo-quinoline and
8-acetoxy-2-chloro-quinoline (360 mg) 8: .sup.1H NMR (CDCl.sub.3):
.delta. 7.80-7.77 (m, 2H), 7.49 (dd, J=8.1 and 8.1, 1H), 7.73 (d,
J=8.1, 1H), 7.21 (d, J=8.1, 1H), 1.77 (br, 1H); mass spectrum: m/z
272 (M.sup.++1, 100%).
2-(Pyrid-2-yl)-8-hydroxyquinoline M1
[0324] Reaction of 2-iodo-quinolin-8-ol 8 with 2-bromopropane
following the method described in Example 8 gave
2-iodo-8-isopropoxyquinoline 9 in 84% yield. 9: .sup.1H NMR
(CDCl.sub.3): .delta. 7.75-7.67 (m, 2H), 7.45 (dd, J=7.0 and 8.0,
1H), 7.33 (dd, J=1.2 and 8.0, 1H), 7.12 (dd, J=1.2 and 7.0, 1H),
4.80 (m, 1H), 1.49 (d, J=5.9, 6 H). To a stirred solution of 9 (29
mg, 0.093 mmol) and PdCl.sub.2(PPh.sub.3).sub.2 (5 mg) in THF (2.5
mL) under an argon atmosphere at RT was added dropwise over 5 min
2-pyridylzinc bromide (0.370 mL of a 0.5 M solution in THF, 0.185
mmol). After 2 h, saturated NH.sub.4Cl (5 mL) was added and the
mixture extracted with dichloromethane (10 mL.times.3). The
combined extracts were washed with H.sub.2O (10 mL) and brine (10
mL), dried (Na.sub.2SO.sub.4), and concentrated. Subsequent column
chromatography on silica (dichloromethane/MeOH, 19:1) gave
2-(pyrid-2-yl)-8-isopropyloxyquinoline as a yellow solid. The
isopropyl ether was cleaved according to the procedure of Example
8, to give 2-(Pyrid-2-yl)-8-hydroxyquinoline P1 (22 mg, 89%) (data
in Table 5). This reaction was repeated using:
2-(methylthio)phenylzinc iodide, 2-(ethoxycarbonyl)phenylzinc
iodide and 6-methylpyridylzinc bromide to give P2, P3 and P4.
Spectral data tabulated (Table 5).
Example 13
Preparation of 5,7-dichloro-2-methylamino-8-hydroxyquinoline (PBT
1047) and 5,7-dichloro-2-(methyl-pyridin-2-yl-amino)-quinolin-8-ol
(PBT 1056) (Scheme 13)
##STR00045##
[0325] 5,7-Dichloro-2-methylamino-8-hydroxyquinoline (PBT 1047)
[0326] 2,5,7-Trichloro-8-hydroxyquinoline 10 (200 mg, 0.805 mmol)
and a solution of methylamine in ethanol (12 mL of a 33% solution)
were heated in a sealed vessel at 90.degree. C. for 26 h, and
cooled. The precipitate was then isolated via filtration and washed
with diethyl ether. This provided pure
5,7-dichloro-2-methylamino-8-hydroxyquinoline (PBT 1047) as a pale
yellow solid (186 mg, 95%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz):
.delta. 8.80 (br, 1H), 7.98 (d, J=9.1, 1H), 7.48 (br, 1H), 7.25 (s,
1H), 6.90 (d, J=9.1, 1H), 2.98 (d, J=4.8, 3 H); mass spectrum: m/z
243, 245 (M.sup.++1, 100 and 66%, respectively).
5,7-Dichloro-8-hydroxy-2-(methyl-pyridin-2-yl-amino)-quinoline (PBT
1056)
[0327] A solution of 5,7-dichloro-2-methylamino-8-hydroxyquinoline
(1.02 g, 4.21 mmol), anhydrous potassium carbonate (2.4 g) and
2-bromopropane (0.6 mL) in dimethyl sulphoxide (10 mL) was stirred
at RT for 2 days. Saturated ammonium chloride solution was added
and the mixture was extracted with dichloromethane (30 mL.times.3).
The extracts were combined, dried, and concentrated. The residue,
after column chromatography (silica gel, dichloromethane), gave
5,7-dichloro-2-methylamino-8-isopropoxy-quinoline 11 as an
off-white solid (938 mg, 78%). .sup.1H NMR (CDCl.sub.3): .delta.
8.13 (d, J=9.0, 1H), 7.28 (s, 1H), 6.69 (d, J=9.0, 1H), 5.10 (m,
1H), 4.90 (br, 1H), 3.11 (d, J=5.0, 3 H), 1.43 (s, 3H), 1.41 (s,
3H).
[0328] To a solution of
5,7-dichloro-2-methylamino-8-isopropoxy-quinoline 11 (200 mg, 0.701
mmol), racemic-BINAP (17.5 mg, 4 mol %), Pd.sub.2(dba).sub.3 (12.8
mg, 2 mol %) and sodium tert-butoxide (78.6 mg, 0.818 mmol) in dry
toluene (10 mL) under an argon atmosphere was added 2-bromopyridine
(0.056 mL, 0.584 mmol). The orange-brown solution was then heated
at 80.degree. C. for 3 h. More 2-bromopyridine (0.010 mL, 0.104
mmol) was added and heating resumed for a further 2 h. The reaction
mixture was quenched with saturated ammonium chloride, extracted
with dichloromethane (20 mL.times.3), the extracts combined, dried,
and concentrated. The residue gave, after column chromatography
(silica gel, dichloromethane/methanol (1:0-100:1),
5,7-dichloro-8-isopropoxy-2-(methyl-pyridin-2-yl-amino)-quinoline
12 as an off-white solid (175 mg, 69%). .sup.1H NMR (CDCl.sub.3,
400 MHz): .delta. 8.47 (dd, J=1.7 and 5.0, 1H), 8.21 (d, J=9.3,
1H), 7.72 (m, 1H), 7.40 (s, 1H), 7.32 (m, 2H), 7.09 (dd, J=5.0 and
7.0, 1H), 5.14 (m, 1H), 3.80 (s, 3H), 1.41 (s, 3H), 1.40 (s,
3H).
[0329] The isopropyl ether 12 (171 mg, 0.472 mmol) was cleaved with
boron trichloride according to the procedure of Example 8 to give,
after methanol treatment,
5,7-dichloro-8-hydroxy-2-(methyl-pyridin-2-yl-amino)-quinoline as
the hydrochloride (170 mg). Water (10 mL) was added and the pH of
the mixture was adjusted to 8 with saturated NaHCO.sub.3. The solid
was then isolated via filtration. Subsequent column chromatography
(silica gel, dichloromethane/methanol (9:1)) yielded the title
compound (PBT 1056) as an off-white solid (140 mg, 93%). .sup.1H
NMR (CD.sub.3OD, 400 MHz): .delta. 8.43 (dd, J=2.0 and 5.0, 1H),
8.18 (d, J=9.4, 1H), 7.89 (ddd, J=2.0, 8.0 and 8.0, 1H), 7.41 (d,
J=8.0, 1H), 7.35 (s, 1H), 7.27 (d, J=9.4, 1H), 7.25 (dd, J=5.0 and
8.0, 1H), 3.75 (s, 3H).
Example 14
Preparation of 5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline
(Scheme 14)
##STR00046##
[0331] A mixture of 2,5,7-trichloro-8-hydroxyquinoline 10 (1.14 g,
4.61 mmol), 2-bromopropane (1.10 mL, 11.5 mmol) and anhydrous
potassium carbonate (1.56 g, 11.5 mmol) in DMF (15 mL) was heated
at 60.degree. C. overnight. The mixture was then poured into water,
extracted with ethyl acetate (20 mL.times.3), the extracts
combined, and dried. Solvent removal gave a brown oil (3.15 g).
Subsequent column chromatography (silica gel, ethyl acetate/hexane
(1:9)) afforded 2,5,7-trichloro-8-isopropoxy-quinoline 13 as a
white solid (1.15 g, 87%), m.p. 83-85.degree. C. .sup.1H NMR
(CDCl.sub.3, 200 MHz): .delta. 8.43 (d, J=10, 1H), 7.64 (s, 1H),
7.45 (d, J=10, 1H), 5.15 (m, 1H), 1.46 (s, 3H), 1.42 (s, 3H).
[0332] A mixture of 2,5,7-trichloro-8-isopropoxy-quinoline 13 (5.93
g, 20.5 mmol), sodium iodide (12.3 g, 82 mmol) and acetyl chloride
(1.4 mL, 20 mmol) in acetonitrile (30 mL) was heated under reflux
overnight. The mixture was then poured into water and extracted
with ethyl acetate (30 mL.times.3). The combined extracts was
washed with 10% sodium thiosulphate solution, water, brine, dried
with magnesium sulphate and concentrated to give an orange solid
(6.9 g). Purification via column chromatography (silica gel, ethyl
acetate/hexanes (1:19)) gave the iodide,
5,7-dichloro-2-iodo-8-isopropoxy-quinoline 14, as a white solid
(4.57 g, 58%), m.p. 97-99.degree. C. .sup.1H NMR (CDCl.sub.3, 200
MHz): .delta. 8.05 (d, J=8.6, 1H), 7.80 (d, J=8.6, 1H), 7.62 (s,
1H), 5.02 (m, 1H), 1.45 (s, 3H), 1.42 (s, 3H).
[0333] Palladium chloride bis(triphenylphosphine) (362 mg, 0.51
mmol) was added to a stirred solution of
5,7-dichloro-2-iodo-8-isopropoxy-quinoline 14 (2.80 g, 7.35 mmol)
in anhydrous THF (150 mL) at room temperature under an atmosphere
of nitrogen. 2-Pyridylzinc bromide (29.4 mL of a 0.5 M solution in
THF, 14.7 mmol) was then added dropwise over 15 minutes and the
mixture was stirred at RT for 2 h. Saturated ammonium chloride was
added and the mixture extracted with ethyl acetate (30 mL.times.3),
the combined extracts dried, and concentrated. The residue
afforded, after column chromatography (silica gel, ethyl
acetate/hexanes (1:9)),
5,7-dichloro-8-isopropoxy-2-(2-pyridyl)quinoline 15 as a white
solid (1.83 g, 75%), m.p. 112-114.degree. C. .sup.1H NMR
(DMSO-d.sub.6, 200 MHz): .delta. 8.78-8.58 (m, 4H), 7.85 (m, 1H),
7.64 (s, 1H), 7.39 (m, 1H), 5.25 (m, 1H), 1.52 (s, 3H), 1.50 (s,
3H).
[0334] Boron trichloride (27 mL of a 1 M solution in
dichloromethane, 27.6 mmol) was added dropwise to a solution of
5,7-dichloro-8-isopropoxy-2-(2-pyridyl)quinoline 15 (1.83 g, 5.51
mmol) in dichloromethane (30 mL) at 0.degree. C. under an
atmosphere of nitrogen. The mixture was stirred at 0.degree. C. for
1 h and then allowed to warm to RT. After 24 h, some starting
material was still present (by TLC analysis). More boron
trichloride (14 mL) was added and stirring resumed for a further 4
h. The reaction was quenched with methanol (10 mL) and the
volatiles removed in vacuo. The process was repeated until the
residue reached constant weight. This gave
5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline as the hydrochloride
salt. The hydrochloride salt (1.68 g) and water (20 mL) was then
treated with saturated sodium bicarbonate until the pH of the
solution was 8. The mixture was the extracted with ethyl acetate
(30 mL.times.3) and dried. The residue obtained after solvent
removal was washed with methanol. This provided
5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline (PBT 1052) as an
off-white solid (1.25 g, 78%), m.p. >230.degree. C. .sup.1H NMR
(DMSO-d.sub.6, 200 MHz): .delta. 10.8 (br, 1H), 9.21 (d, J=9, 1H),
8.30-8.62 (m, 3H), 8.12 (m, 1H), 7.88 (s, 1H), 7.62 (m, 1H).
Example 15
Preparation of 5,7-dichloro-2-dimethylaminomethyl-quinolin-8-ol
hydrochloride (PBT 1033) (Scheme 15)
##STR00047##
[0335] 5,7-Dichloro-8-hydroxyquinoline-2-carboxaldehyde 17
[0336] A solution of 5,7-dichloro-2-methyl-quinolin-8-ol 16 (1.5 g,
6.58 mmol) in 1,4-dioxane (20 mL) was added dropwise over 3 h to a
stirred suspension of selenium dioxide (1.3 g, 11.72 mmol) in
1,4-dioxane (60 mL) at 50-55.degree. C. The resulting mixture was
then heated at 80.degree. C. overnight, cooled, and the solids
filtered off (celite). The filtrate was concentrated and the
residue, after washing with diethyl ether (10 mL.times.3), gave 17
as a yellow solid (quantitative yield). This material was used in
the subsequent step without further purification. .sup.1H NMR
(CDCl.sub.3, 400 MHz): .delta. 10.26 (s, 1H), 8.69 (d, J=8.8, 1H),
8.37 (br, 1H), 8.17 (d, J=8.8, 1H), 7.76 (s, 1H).
5,7-Dichloro-2-dimethylaminomethyl-quinolin-8-ol hydrochloride (PBT
1033)
[0337] Triethylamine (0.55 mL) was added dropwise to a stirred
solution of 5,7-dichloro-8-hydroxyquinoline-2-carboxaldehyde 17
(1.0 g, 4.13 mmol) and dimethylamine hydrochloride (365 mg, 4.48
mmol) in 1,2-dichloroethane (50 mL). After 5 minutes, sodium
triacetoxyborohydride (1.2 g, 5.66 mmol) was added portionwise over
5 minutes. The mixture was then allowed to stir at RT overnight.
Dichloromethane (100 mL) was added, the mixture washed with
saturated sodium bicarbonate (50 mL.times.3), dried
(Na.sub.2SO.sub.4), and concentrated. The resulting residue was
extracted with diethyl ether (50 mL.times.4), the ethereal extracts
combined and concentrated. Concentrated hydrochloric acid (5 mL)
was then added and the mixture concentrated in vacuo. The process
was repeated twice. The residue, after washing with
dichloromethane, gave
5,7-dichloro-2-dimethylaminomethyl-quinolin-8-ol hydrochloride (PBT
1033) as a pale straw-coloured solid (0.96 g, 73%). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz): .delta. 10.80 (s, 1H), 10.40 (br, 1H),
8.60 (d, J=8.6, 1H), 7.92 (s, 1H), 7.78 (d, J=8.6, 1H), 4.83 (d,
J=5.3, 2 H), 2.94 (s, 3H), 2.92 (s, 3H).
Preparation of 5,7-dichloro-2-ethylaminomethyl-quinolin-8-ol
hydrochloride (PBT 1051) (Scheme 15)
[0338] The procedure described in Example 15 was repeated on
5,7-dichloro-8-hydroxyquinoline-2-carboxaldehyde 17 (1.00 g, 4.13
mmol) substituting dimethylamine hydrochloride with ethylamine
hydrochloride. This provided
5,7-dichloro-2-ethylaminomethyl-quinolin-8-ol hydrochloride (PBT
1051) as a pale straw-coloured solid (0.60 g, 47%). .sup.1H NMR
(DMSO-d.sub.6): .delta. 9.40 (br, 2H), 8.59 (d, J=8.8, 1 H), 7.90
(s, 1H), 7.76 (d, J=8.8, 1H), 4.64 (s, 2H), 3.14 (q, J=7.2, 2 H),
1.32 (t, J=7.2, 3 H).
Example 16
Preparation of 5,7-dichloro-8-hydroxy-quinoline-2-carboxylic acid
[2-(1H-imidazol-4-yl)-ethyl)-amide (PBT 1038) (Scheme 16)
##STR00048##
[0339] 5,7-Dichloro-8-hydroxyquinoline-2-carboxylic acid 19
[0340] A mixture of 5,7-dichloro-8-benzyloxy-quinoline-2-carboxylic
acid 18 (2.56 g, 7.35 mmol) and concentrated hydrochloric acid (25
mL) was stirred at RT for 48 h, and then concentrated to dryness.
The resulting residue was washed with diethyl ether (20
mL.times.2). This provided
5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 as a yellow
solid (1.78 g, 94%). NMR (CDCl.sub.3/DMSO-d.sub.6 (19:1), 400 MHz):
.delta. 10.60 (br, 1H), 8.53 (d, J=8.8, 1H), 8.22 (d, J=8.8, 1H),
7.60 (s, 1H).
5,7-Dichloro-8-hydroxyquinoline-2-carboxylic acid
[2-(1H-imidazol-4-yl)-ethyl]-amide (PBT 1038)
[0341] According to the procedure described in Example
1,5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 (597 mg,
2.31 mmol), dicyclohexylcarbodiimide (483 mg, 2.31 mmol),
1-hydroxybenzotriazole hydrate (316 mg, 2.31 mmol), histamine
dihydrochloride (425 mg, 2.31 mmol) and triethylamine (0.5 mL)
gave, after column purification (silica gel, ethyl
acetate/isopropanol/water (12:4:1)),
5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid
[2-(1H-imidazol-4-yl)-ethyl]-amide (PBT 1038) as a pale
straw-coloured solid (276 mg, 34%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz): .delta. 11.40 (br, 2H), 9.74 (m, 1H), 8.64 (d, J=8.6, 1H),
8.28 (d, J=8.6, 1H), 7.92 (s, 1H), 7.53 (s, 1H), 6.83 (s, 1H), 3.59
(m, 2H), 2.81 (m, 2H).
Preparation of 5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid
[2-(1-methyl-1H-imidazol-4-yl)-ethyl]-amide (PBT 1050) (Scheme
16)
[0342] Following the procedure of Example
1,5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 (1.00 g,
3.88 mmol) was treated with dicyclohexylcarbodiimide (0.96 g, 4.60
mmol), 1-hydroxybenzotriazole hydrate (0.53 g, 5.20 mmol),
1-methyl-1H-histamine hydrochloride (1.24 g, 7.67 mmol) and
triethylamine (0.65 mL) for 24 h. The solid was isolated via
filtration and dissolved in hot methanol. Upon cooling, this
provided 5,7-dichloro-8-hydroxy-quinoline-2-carboxylic acid
[2-(1-methyl-1H-imidazol-4-yl)-ethyl]-amide (PBT 1050) as
colourless needles (0.99 g, 70%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz): .delta. 10.25 (s, 1H), 9.10 (m, 1H), 8.14 (s, 1H), 7.83 (d,
J=8.6, 1H), 7.44 (d, J=8.6, 1H), 7.12 (s, 1H), 6.68 (s, 1H), 2.95
(s, 3H), 2.85 (m, 2H), 2.15 (m, 2H).
Example 17
Preparation of 7-chloro-5-(pyridin-3-yl)-quinolin-8-ol (PBT 1057)
(Scheme 17)
##STR00049##
[0344] Following the procedure described in Example
8,7-chloro-5-iodo-8-hydroxy-quinoline 20 and 2-bromopropane gave
7-chloro-5-iodo-8-isopropoxy-quinoline 21 (80%). .sup.1H NMR
(CDCl.sub.3/DMSO-d.sub.6 (19:1), 400 MHz): .delta. 9.09 (m, 1H),
8.55 (m, 1H), 8.10 (s, 1H), 7.63 (m, 1H), 5.15 (m, 1H), 1.49 (s,
3H), 1.47 (s, 3H).
[0345] A mixture of 21 (180 mg, 0.518 mmol), 3-pyridylboronic acid
(76 mg, 0.622 mmol), KF (60 mg, 1.04 mmol), Pd(Ph.sub.3P).sub.4 (10
mg) and toluene-water (1:1, 10 mL) was heated under reflux under an
argon atmosphere for 16 h. The mixture was cooled, quenched with
saturated ammonium chloride, extracted with dichloromethane (10
mL.times.3), the extracts combined, dried, and concentrated. The
residue gave, after column chromatography (silica gel,
dichloromethane/methanol (40:1)),
7-chloro-5-(pyridin-3-yl)-8-isopropoxyquinoline 22 as a pale cream
solid (20 mg, 14%); 132 mg of starting material was also recovered.
22: .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 9.00 (m, 1H), 8.08
(m, 1H), 7.93 (d, J=7.7, 1H), 7.70-7.56 (m, 2H), 7.55 (s, 1H),
7.50-7.42 (m, 2H), 5.23 (m, 1H), 1.49 (s, 3H), 1.48 (s, 3H).
[0346] Cleavage of the isopropyl ether 22 (20 mg, 0.07 mmol) with
boron trichloride following the method outlined in Example 8, gave
7-chloro-5-(pyridin-3-yl)-quinolin-8-ol (PBT 1057) as a pale
straw-coloured solid (17 mg, 94%). .sup.1H NMR (CD.sub.3OD, 400
MHz): .delta. 9.16 (m, 2H), 9.04 (d, J=5.8, 1H), 8.93 (dd, J=1.2
and 8.6, 1 H), 8.84 (m, 1H), 8.30 (dd, J=5.8 and 8.1, 1H), 8.09 (s,
1H), 8.08 (dd, J=5.1 and 8.6, 1H).
Example 18
Preparation of 3,5,7-trichloro-8-hydroxyquinoline (PBT 1058)
(Scheme 18)
##STR00050##
[0348] m-Chloroperbenzoic acid (3.10 g of a 70% reagent, 26 mmol)
was added portionwise to a stirred solution of
5,7-dichloro-8-hydroxyquinoline 23 (5.00 g, 23 mmol) in chloroform
(150 mL) at 0.degree. C. After 1 h, the mixture was warm to RT and
allowed to stir for a further 48 h. The mixture was concentrated
and the residue partitioned between ethyl acetate and 1 N
NaHCO.sub.3 (200 mL, 1:1); some of the 1-N-oxide 24 remained as a
precipitate and was isolated via filtration. The filtrate was then
extracted with ethyl acetate (40 mL.times.3), the extracts
combined, dried, and concentrated to give more 1-N-oxide 24. A
total of 4.76 g (90%) of 1-N-oxide 24 was obtained. .sup.1H NMR
(DMSO-d.sub.6, 400 MHz): .delta. 8.74 (d, J=5.9, 1H), 8.24 (d,
J=8.8, 1H), 8.02 (s, 1H), 7.70 (dd, J=5.9 and 8.8, 1H).
[0349] A solution of 24 (2.01 g, 8.8 mmol) and phosphorus
oxychoride (40 mL) was heated under reflux for 18 h. Excess
phosphorus oxychoride was removed in vacuo, concentrated
hydrochloric acid (80 mL) was added, the solution heated under
reflux for 2 h, and cooled. The mixture was then poured into ice
and aqueous ammonia, adjusting the pH to 8. The precipitate was
isolated via filtration and washed with water. This material was
then dissolved in dichloromethane and successively filtered through
short pads of silica gel and celite. The solvent was removed
providing 3,5,7-trichloro-8-hydroxy-quinoline (PBT 1058) as an
off-white solid (0.92 g, 42%), m.p. 144-147.degree. C. (lit.
(Gershon et al, 1999) 159-160.degree. C.). .sup.1H NMR
(CD.sub.3OD): .delta. 8.85 (d, J=2.2, 1H), 8.53 (d, J=2.2, 1H),
7.71 (s, 1H); mass spectrum: m/z 248, 250, 252 (M.sup.++1, 100, 100
and 33%, respectively).
Example 19
Preparation of 3-amino-5,7-dichloro-8-hydroxyquinoline (PBT 1060)
(Scheme 19)
##STR00051##
[0350] 4,5,7-Trichloro-8-methoxy-quinoline-3-carboxylic acid ethyl
ester 26
[0351] To a stirred solution of
4-chloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester (1.00 g,
3.76 mmol) in chloroform (50 mL) was added, dropwise over 1 h, a
solution of sulfuryl chloride (15 mL) in chloroform (15 mL) whilst
maintaining the temperature at 25-30.degree. C. The solution was
then heated at 60-70.degree. C. for 48 h. During this time, further
sulfuryl chloride (2 mL) was added at regular intervals (2, 24 and
30 h). The solution was allowed to cool to RT and added to
ice-aqueous ammonia, adjusting the pH to 8. The mixture was then
extracted with dichloromethane (20 mL.times.3), the extracts
combined and concentrated. Column chromatography (silica gel,
dichloromethane/methanol (100:1)) gave the title compound 26 as a
cream solid (0.36 g, 29%). .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta. 9.04 (s, 1H), 7.78 (s, 1H), 4.51 (q, J=7.1, 2 H), 4.14 (s,
3H), 1.46 (t, J=7.1, 3 H).
5,7-Dichloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester
27
[0352] A suspension of zinc powder (0.70 g) was stirred at
20.degree. C. in a solution of
4,5,7-trichloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester
26 (364 mg, 1.09 mmol) and acetic acid (2.2 ml) in 1,4-dioxane (15
ml). After 20 minutes, ethyl acetate (20 ml) was added and the
resultant mixture filtered through a pad of celite. The filtrate
was washed with saturated aqueous sodium chloride solution (10 ml),
dried (MgSO.sub.4), filtered, and the solvent removed under vacuum.
The residue was chromatographed on flash silica (ethyl
acetate/hexane, 1:9), yielding 130 mg (39%) of the title compound
27 as a white solid. .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.
9.51 (d, J=2.0, 1H), 9.15 (d, J=2.0, 1H), 7.72 (s, 1H), 4.51 (q,
J=7.2, 2 H), 4.18 (s, 3H), 1.47 (t, J=7.2, 3 H).
5,7-Dichloro-8-methoxy-quinoline-3-carboxylic acid hydrazide 28
[0353] A solution of 5,7-dichloro-8-methoxy-quinoline-3-carboxylic
acid ethyl ester 27 (404 mg, 1.35 mmol) and hydrazine monohydrate
(1.0 g) in ethanol (10 ml) was heated under reflux for 5 hours.
Upon cooling, a white crystalline solid was deposited. This was
isolated via filtration, washed with ethanol, and dried, yielding
the hydrazide 28 (307 mg, 80%) as a white solid. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz): .delta. 10.32 (br, 1H), 9.33 (d, J=2.4,
1H), 8.89 (d, J=2.4, 1H), 8.02 (s, 1H), 4.66 (br, 2H), 4.06 (s,
3H).
3-Amino-5,7-dichloro-8-methoxy-quinoline 29
[0354] Sodium nitrite (180 mg, 2.61 mmol) was added at 0.degree. C.
to a stirred suspension of
5,7-dichloro-8-methoxy-quinoline-3-carboxylic acid hydrazide 28
(248 mg, 0.87 mmol) in 1 M hydrochloric acid (2 ml), acetic acid (5
ml) and water (20 ml). Stirring was continued at 0.degree. for 1 h,
the ice bath removed and upon warming to RT, the heterogeneous
mixture was heated under reflux. The mixture became homogeneous
after about 30 minutes and heating was continued for a total of 6
h. Upon cooling, the volatiles were removed under vacuum and the
residue partitioned between ethyl acetate (20 ml) and 10% aqueous
ammonia solution (10 ml). The layers were separated and the aqueous
layer washed with more ethyl acetate (5 ml.times.2). The combined
ethyl acetate layers were dried (Na.sub.2SO.sub.4), filtered and
the ethyl acetate removed under vacuum. The residue gave, after
flash chromatography (ethyl acetate/hexane, 1:1), the title
compound 29 (106 mg, 50%) as a white solid. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz): .delta. 8.48 (d, J=2.4, 1H), 7.63 (s, 1H),
7.28 (d, J=2.4, 1H), 6.16 (br, 2H), 3.97 (s, 3H).
3-Amino-5,7-dichloro-quinolin-8-ol
[0355] Boron tribromide (2.0 ml of a 1 M solution in
dichloromethane, 2.0 mmol) was added to a stirred suspension of
3-amino-5,7-dichloro-8-methoxy-quinoline (104 mg, 0.43 mmol) in
dichloromethane (10 ml) at -30.degree. C. Stirring was continued
for 14 h with the cold bath being allowed to reach RT. The mixture
was then cooled to 0.degree. C. and water (1 ml) added. The
dichloromethane was then removed and ethyl acetate (10 ml) and more
water (5 ml) were added. The yellow precipitate that formed was
collected by filtration and dried to give 74 mg of a mixture of
starting material and product (NMR analysis). The ethyl acetate
layer from the filtrate was dried (Na.sub.2SO.sub.4), filtered, and
the solvent removed under vacuum to yield 42 mg of solid which was
also a mixture of starting material and product (NMR analysis). The
two solid samples were combined and the components separated by
flash chromatography (ethyl acetate/2-propanol, 1:0-3:1). This
provided title compound as the hydrobromide (30 mg), and recovered
starting material (57 mg). To a mixture of
3-amino-5,7-dichloro-quinolin-8-ol hydrobromide and water (10 mL)
was added saturated NaHCO.sub.3 until the pH was 8. The solid was
isolated yielding the title compound (PBT 1060) as a cream solid
(25 mg, 26%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz): .delta. 8.15
(br, 1H), 7.24 (br, 1H), 7.14 (br, 1H), 5.70 (br, 2H); mass
spectrum: m/z 229, 231 (M.sup.++1, 100 and 66%, respectively).
3-Amino-5,7-dichloro-quinolin-8-ol hydrobromide: .sup.1H-NMR
(CD.sub.3OD, 400 MHz): .delta. 8.43 (d, J=2.6, 1H), 7.46 (d, J=2.6,
1H), 7.42 (s, 1H).
TABLE-US-00001 TABLE 1 Data for Examples 1 and 2. Method Mass
Product of Yield spectral ID Preparation.sup.a Product (%) .sup.1H
NMR data data A1 A ##STR00052## 52 (CDCl.sub.3/DMSO-d.sub.6, 19:1):
.delta. 9.81 (m, 1 H), 8.30-8.23 (m, 2 H), 7.91 (d, J = 9.0, 1 H),
7.86 (s, 1 H), 7.68 (d, J = 9.0, 1 H), 7.51 (m, 1 H), 7.36 (m, 1
H), 7.19 (d, J = 4.0, 1 H), 6.94 (s, 1 H), 3.80 (m, 2 H), 3.03 (m,
2 H) A2 A ##STR00053## 85 (CDCl.sub.3): .delta. 9.61 (m, 1 H), 8.60
(d, J = 5.4, 1 H), 8.27-8.18 (m, 2 H), 8.03 (m, 1 H), 7.93 (d, J =
8.0, 1 H), 7.77 (d, J = 8.3, 1 H), 7.63 (d, J = 8.0, 1 H), 7.51 (d,
J = 8.0, 1 H), 7.35 (d, J = 8.0, 1 H), 7.24 (m, 1 H), 3.90 (m, 2
H), 3.42 (m, 2 H) 294 (M.sup.+ + 1) A3 A ##STR00054## 65
(CDCl.sub.3): .delta. 9.53 (m, 1 H), 8.42- 8.25 (m, 2 H), 7.65-7.25
(m, 6 H) 272 (M.sup.+ + 1) A4 A ##STR00055## 81
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 10.35 (br, 1 H), 8.38-8.29
(m, 2 H), 7.58 (m, 1 H), 7.48 (s, 1 H), 7.40 (d, J = 8.3, 1 H),
7.27-7.20 (m, 2 H), 2.48 (s, 3 H) 286 (M.sup.+ + 1) A5 A
##STR00056## 81 (CDCl.sub.3): .delta. 10.54 (t, J = 4.0, 1 H), 8.72
(br, 1 H), 8.63 (d, J = 5.6, 1 H), 8.30-8.18 (m, 2 H), 7.9 (d, J =
7.8, 1 H), 7.64-7.30 (m, 5 H), 5.10 (m, 2 H) 280 (M.sup.+ + 1) A6 A
##STR00057## 65 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 11.34 (br,
1 H), 8.36 (s, 1 H), 7.59 (m, 1 H), 7.56 (d, J = 9.0, 1 H), 7.41
(d, J = 9.0, 1 H), 7.23 (d, J = 4.0, 1 H), 7.19 (d, J = 4.0, 1 H),
7.09 (m, 1 H), 6.96 (m, 1 H), 5.00 (br, 2 H) A7 A ##STR00058## 71
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 10.16 (m, 1 H), 8.60 (br,
1 H), 8.25 (m, 1 H), 7.92 (d, J = 7.8, 1 H), 7.67 (d, J = 7.8, 1
H), 7.57-7.35 (m, 2 H), 5.20 (br, 2 H), 3.89 (m, 2 H), 2.60 (m, 2
H) A8 A ##STR00059## 62 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta.
10.35 (m, 1 H), 8.37 (s, 1 H), 7.81 (m, 1 H), 7.59-7.39 (m, 2 H),
4.23 (m, 2 H), 3.60 (br, 2 H) A9 B ##STR00060## 60 (CDCl.sub.3):
.delta. 9.01 (m, 1 H), 8.31 (m, 1 H), 8.25 (br, 1 H), 7.77 (d, J =
3.4, 1 H), 7.55 (dd, J = 8.0 and 8.0, 1 H), 7.40 (d, J = 8.0, 1 H),
7.33 (d, J = 3.4, 1 H), 7.27 (m, 1 H), 7.24 (d, J = 7.3, 1 H), 5.05
(m, 2 H) 286 (M.sup.+ + 1) A10 B ##STR00061## 63 (CDCl.sub.3):
.delta. 9.60 (br, 1 H), 8.75 (d, J = 4.2, 1 H), 8.58 (d, J = 4.5, 1
H), 8.28 (d, J = 8.6, 1 H), 8.08 (d, J = 8.6, 1 H), 7.77 (m, 1 H),
7.68 (m, 1 H), 7.58-7.15 (m, 7 H) 371 (M.sup.+ + 1) B1 A
##STR00062## 77 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.60 (m, 1
H), 8.29 (s, 1 H), 7.91 (d, J = 8.3, 1 H), 7.68-7.65 (d, J = 9.0, 1
H), 7.50-7.29 (m, 2 H), 7.15- 7.07 (m, 2 H), 3.40 (m, 2 H), 3.30
(br, 2 H), 3.10 (m, 2 H). B2 A ##STR00063## 31
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 10.11 (m, 1 H), 9.55 (br,
1 H), 8.63 (d, J = 4.4, 1 H), 7.95 (m, 1 H), 7.70- 7.65 (m, 2 H),
7.58 (s, 1 H), 7.45 (m, 1 H), 7.28-7.34 (m, 2 H), 7.14 (m, 1 H),
4.96 (m, 2 H) B3 A ##STR00064## 78 (CDCl.sub.3/DMSO-d.sub.6, 19:1):
.delta. 10.35 (m, 1 H), 8.37 (s, 1 H), 7.81 (m, 1 H), 7.59-7.39 (m,
2 H), 4.23 (m, 2 H), 3.60 (br, 2 H) B4 A ##STR00065## 97
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 10.99 (br, 1 H), 9.63 (br,
1 H), 7.77 (s, 1 H), 7.69 (d, J = 8.5, 1 H), 7.50- 7.32 (m, 3 H),
7.18-7.05 (m, 2 H), 6.85-6.78 (m, 2 H), 4.20 (br, 2 H) 296 (M.sup.+
+ 1) B5 A ##STR00066## 51 (CDCl.sub.3/DMSO-d.sub.6, 9:1): .delta.
10.02 (m, 1 H), 8.49 (s, 1 H), 7.95 (m, 1 H), 7.72-7.60 (m, 3 H),
7.22- 7.10 (m, 4 H), 4.57 (m, 2 H), 4.22 (m, 2 H), 3.20 (br, 2 H)
352 (M.sup.+ + 1) B6 A ##STR00067## 47 (CDCl.sub.3/DMSO-d.sub.6,
19:1): .delta. 9.92 (m, 1 H), 7.67 (d, J = 8.8, 1 H), 7.53 (m, 1
H), 7.50-7.30 (m, 4 H), 7.12 (d, J = 8.0 Hz, 1 H), 6.76 (s, 1 H),
4.09 (m, 2 H), 3.48 (m, 2 H), 2.60 (m, 2 H) 390 (M.sup.+ + 1) C1
##STR00068## 98 (CDCl.sub.3): .delta. 8.31 (d, J = 9.0, 1 H), 8.18
(d, J = 9.0, 1 H), 8.15 (br, 1 H), 7.60 (dd, J = 9.0 and 9.0, 1 H),
7.42 (d, J = 9.0, 1 H), 7.28 (d, J = 9.0, 1 H), 2.88 (s, 3 H)
.sup.aSee Experimental Section: A = General Procedure A; B =
General Procedure B.
TABLE-US-00002 TABLE 2 Data for Examples 3, 4, 5 and 6 Mass Product
Yield spectral ID Product (%) .sup.1H NMR data data D1 ##STR00069##
80 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 11.00 (br, 1 H), 8.41
(s, 1 H), 8.16 (d, J = 8.6, 1 H), 8.03 (d, J = 8.6, 1 H), 7.47 (m,
1 H), 7.34 (d, J = 8.3, 1 H), 7.23 (d, J = 7.5, 1 H), 2.40 (br, 1
H) E1 ##STR00070## 82 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.12
(d, J = 8.0, 1 H), 7.46-7.30 (m, 4 H), 7.17 (d, J = 7.3, 1 H), 4.20
(br s, 2 H), 3.20 (br, 2 H) E2 ##STR00071## 60 (CDCl.sub.3):
.delta. 8.25 (d, J = 8.3, 1 H), 7.53- 7.48 (m, 2 H), 7.38 (d, J =
8.3, 1 H), 7.30 (d, J = 7.6, 1 H), 6.70 (br, 1 H), 4.82 (m, 2 H),
3.53 (s, 1 H), 2.14 (s, 3 H) E3 ##STR00072## 82
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.89 (br, 1 H), 8.20 (d, J
= 8.5, 1 H), 7.56 (m, 1 H), 7.50-7.40 (m, 2 H), 7.33 (d, J = 8.0, 1
H), 7.21 (d, J = 7.5, 1 H), 5.30 (br, 1 H), 4.83 (m, 2 H), 2.88 (br
s, 3 H) F1 ##STR00073## 61 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta.
8.02 (d, J = 8.6, 1 H), 7.50 (s, 1 H), 7.35-7.20 (m, 4 H), 7.06 (d,
J = 7.3, 1 H), 6.74 (s, 1 H), 6.30 (br, 2 H), 4.10 (s, 2 H), 3.03
(m, 2 H), 2.84 (m, 2 H) F2 ##STR00074## 86 (CDCl.sub.3): .delta.
8.63 (d, J = 4.7, 1 H), 8.11 (d, J = 8.3, 1 H), 7.66 (m, 1 H), 7.46
(d, J = 8.3, 1 H), 7.41 (d, J = 7.8, 1 H), 7.34 (d, J = 8.3, 1 H),
7.28 (d, J = 8.3, 1 H), 7.22-7.16 (m, 2 H), 4.18 (m, 2 H), 4.02 (m,
2 H), 2.60 (br, 2 H) F3 ##STR00075## 68 (CDCl.sub.3): .delta. 8.53
(d, J = 4.9, 1 H), 8.06 (d, J = 8.3, 1 H), 7.59 (m, 1 H), 7.45 (d,
J = 8.3, 1 H), 7.40 (d, J = 7.8, 1 H), 7.29 (d, J = 8.3, 1 H), 7.16
(d, J = 8.3, 1 H), 7.14 (m, 1 H), 3.91 (s, 2 H), 3.08 (m, 2 H),
2.91 (m, 2 H), 2.39 (s, 3 H) G1 ##STR00076## 77
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.02 (d, J = 8.6, 1 H),
7.50 (s, 1 H), 7.35-7.20 (m, 4 H), 7.06 (d, J = 7.3, 1 H), 6.74 (s,
1 H), 6.30 (br, 2 H), 4.10 (s, 2 H), 3.03 (m, 2 H), 2.84 (m, 2 H)
G2 ##STR00077## 79 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.61
(d, J = 4.9, 1 H), 8.00 (d, J = 8.3, 1 H), 7.68 (s, 1 H), 7.59 (dd,
J = 7.5 and 7.5, 1 H), 7.42-7.13 (m, 7 H), 6.71 (s, 1 H), 4.04 (s,
2 H), 3.93 (s, 4 H), 3.90 (br, 1 H), 2.89 (br s, 4 H) H1
##STR00078## 66 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.59 (d, J
= 4.8, 1 H), 8.10 (d, J = 8.5, 1 H), 7.71- 7.64 (m, 2 H), 7.57 (d,
J = 8.5, 1 H), 7.48-7.37 (m, 2 H), 7.32-7.14 (m, 3 H), 6.96 (s, 1
H), 3.98 (s, 2 H), 3.84 (s, 2 H), 3.80 (br, 1 H), 3.72 (s, 2 H) 346
(M.sup.+ + 1) H3 ##STR00079## 51 (CDCl.sub.3/DMSO-d.sub.6, 19:1):
.delta. 8.11 (d, J = 8.3, 1 H), 7.80-7.65 (m, 2 H), 7.53 (d, J =
8.0, 1 H), 7.45-7.10 (m, 5 H), 6.76 (s, 1 H), 5.30 (br, 1 H), 4.08
(m, 4 H), 2.94 (m, 4 H) 363 (M.sup.+ + 1) H2 ##STR00080## 73
(CDCl.sub.3): .delta. 8.57 (m, 2 H), 8.10 (d, J = 8.5, 1 H),
7.70-7.63 (m, 2 H), 7.62-7.54 (m, 3 H), 7.41 (dd, J = 8.0 and 8.0,
1 H), 7.31-7.14 (m, 4 H), 4.03 (s, 2 H), 3.93 (s, 4 H), 3.40 (br, 1
H) 357 (M.sup.+ + 1)
TABLE-US-00003 TABLE 3 Data for Example 7. Product Yield Mass
spectral ID Product (%) .sup.1H NMR data data I1 ##STR00081## 72
(CDCl.sub.3): .delta. 8.73 (d, J = 8.7, 1 H), 8.28 (d, J = 9.0, 1
H), 8.25 (d, J = 9.0, 1 H), 7.81 (s, 1 H), 7.66 (s, 1 H), 7.46-
7.36 (m, 2 H), 7.23 (m, 1 H), 6.56 (m, 1 H) I2 ##STR00082## 75
(CDCl.sub.3): .delta. 8.47 (s, 1 H), 8.34 (d, J = 8.9, 1 H), 7.82
(s, 1 H), 7.70 (br, 1 H), 7.57 (d, J = 8.9, 1 H), 7.48 (dd, J = 7.5
and 7.5, 1 H), 7.39 (m, 1 H), 7.28-7.25 (m, 2 H) I3 ##STR00083## 71
(DMSO-d.sub.6) (400 MHz): .delta. 8.96 (br, 1 H), 7.58 (d, J = 9.4,
1 H), 7.54-7.49 (m, 2 H), 7.37 (d, J = 7.8, 1 H), 7.31 (dd, J = 7.8
and 7.8, 1 H), 7.18 (dd, J = 1.4 and 7.8, 1 H) I4 ##STR00084## 68
(CDCl.sub.3): .delta. 8.34 (dd, J = 1.5 and 8.8, 1 H), 7.86 (br, 1
H), 7.58-7.48 (m, 2 H), 7.42-7.40 (m, 2 H), 7.28 (d, J = 7.8, 1 H),
7.10 (br, 1 H), 2.71 (s, 3 H)
TABLE-US-00004 TABLE 4 Data for Examples 8, 9, 10 and 11. Product
Yield Mass spectral ID Product (%) .sup.1H NMR data data K1
##STR00085## 89 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.16 (m, 1
H), 9.03 (d, J = 8.6, 1 H), 7.95 (dd, J = 5.3 and 8.6, 1 H), 7.82
(m, 1 H), 7.58-7.42 (m, 3 H), 5.65 (br, 1 H) 256 (M.sup.+ + 1,
100%), 258 (M.sup.+ + 1, 33%) K2 ##STR00086## 55
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.23 (d, J = 5.1, 1 H),
9.13 (m, 1 H), 8.02 (m, 1 H), 7.83 (d, J = 8.0, 1 H), 7.69 (s, 1
H), 7.64 (m, 1 H), 7.41 (d, J = 7.3, 1 H), 5.60 (br, 1 H) 324
(M.sup.+ + 1, 100%), 326 (M.sup.+ + 1, 33%) K3 ##STR00087## 96
(DMSO-d.sub.6) (400 MHz): .delta. 8.99 (d, J = 4.0, 1 H), 8.57 (d,
J = 8.4, 1 H), 7.78 (dd, J = 4.0 and 8.4, 1 H), 7.62 (s, 1 H), 7.35
(m, 1 H), 7.18 (m, 1 H), 7.16-6.86 (m, 2 H) 270 [(M - H).sup.-,
100%], 272 [(M - H).sup.-, 33%], K4 ##STR00088## 90
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.19 (d, J = 5.0, 1 H),
9.13 (d, J = 8.3, 1 H), 7.96 (dd, J = 5.0 and 8.3, 1 H), 7.73 (s, 1
H), 7.40-7.22 (m, 4 H), 4.10 (br, 1 H), 2.23 (s, 3 H) 270 (M.sup.+
+ 1, 100%), 272 (M.sup.+ + 1, 33%) K5 ##STR00089## 95
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.19 (m, 1 H), 9.12 (d, J
= 8.5, 1 H), 8.00 (dd, J = 5.1 and 8.5, 1 H), 7.83 (s, 1 H), 7.54
(m, 1 H), 7.47 (m, 1 H), 7.30 (dd, J = 8.3 and 8.5, 1 H), 7.22 (dd,
J = 8.5 and 8.5, 1 H), 7.00 (br, 1 H) 274 (M.sup.+ + 1, 100%), 276
(M.sup.+ + 1, 33%) K6 ##STR00090## 98 (CDCl.sub.3/DMSO-d.sub.6,
19:1): .delta. 9.17 (d, J = 4.7, 1 H), 9.10 (d, J = 8.3, 1 H), 7.96
(dd, J = 4.9 and 8.3, 1 H), 7.91 (s, 1 H), 7.43 (dd, J = 8.1 and
8.1, 1 H), 7.28-7.24 (m, 2 H), 6.95 (m, 1 H), 5.00 (br, 1 H), 3.88
(s, 3 H) 286 (M.sup.+ + 1, 100%), 288 (M.sup.+ + 1, 33%) K7
##STR00091## 95 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.16- 9.00
(m, 2 H), 7.92 (dd, J = 4.9 and 8.6, 1 H), 7.88 (d, J = 6.3, 1 H),
7.22- 7.66 (m, 2 H), 7.07-7.05 (m, 2 H), 5.70 (br, 1 H), 3.85 (s, 3
H) 286 (M.sup.+ + 1, 100%), 288 (M.sup.+ + 1, 33%) K8 ##STR00092##
95 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.19- 9.13 (m, 2 H),
8.05-7.93 (m, 2 H), 7.54-7.48 (m, 2 H), 7.40 (dd, J = 7.3 and 7.3,
1 H), 7.27 (d, J = 7.3, 1 H), 6.65 (br, 1 H), 2.45 (s, 3 H) K9
##STR00093## 97 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.04 (m, 1
H), 8.81 (d, J = 8.5, 1 H), 7.97- 7.88 (m, 4 H), 7.82 (m, 1 H),
7.75 (s, 1 H), 4.20 (br, 1 H), 3.26 (s, 6 H) K10 ##STR00094## 68
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.24 (s, 1 H), 8.74 (d, J
= 8.3, 1 H), 8.52 (d, J = 8.0, 1 H), 8.36 (s, 1 H), 8.24 (d, J =
8.0, 1 H), 7.97 (dd, J = 7.3 and 7.3, 1 H), 7.82-7.70 (m, 3 H),
4.20 (br 1 H) K11 ##STR00095## 23 (DMSO-d.sub.6) (400 MHz): .delta.
9.47 (d, J = 6.0, 1 H), 9.09 (d, J = 8.0, 1 H), 8.51 (s, 1 H), 8.40
(s, 1 H), 8.25 (m, 1 H), 8.10 (m, 1 H), 8.00 (m, 1 H), 7.48-7.44
(m, 2 H) K12 ##STR00096## 93 (CDCl.sub.3/DMSO-d.sub.6, 19:1):
.delta. 9.04 (m, 1 H), 7.82 (m, 1 H), 7.81 (m, 1 H), 7.74 (s, 1 H),
7.38-7.30 (m, 2 H), 6.88 (m, 1 H), 4.60 (br, 1 H) K13 ##STR00097##
43 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.05 (m, 1 H), 8.81 (d,
J = 8.6, 1 H), 7.81 (m, 1 H), 7.67 (s, 1 H), 7.57 (m, 1 H),
7.07-6.95 (m, 2 H), 3.25 (br, 1 H) K14 ##STR00098## 91
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.00 (m, 1 H), 8.53 (m, 1
H), 8.16 (m, 1 H), 8.06 (s, 1 H), 7.82 (m, 1 H), 7.75 (dd, J = 4.2
and 8.5, 1 H), 7.66 (dd, J = 2.9 and 5.9, 1 H) K15 ##STR00099## 97
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.13 (m, 1 H), 9.03 (m, 1
H), 7.93 (m, 1 H), 7.87 (m, 1 H), 7.52-7.43 (m, 3 H), 7.14 (m, 1
H), 5.35 br, 1 H) K16 ##STR00100## 69 (CDCl.sub.3/DMSO-d.sub.6,
19:1): .delta. 9.14 (d, J = 4.9, 1 H), 9.05 (d, J = 8.6, 1 H), 7.94
(dd, J = 5.2 and 8.6, 1 H), 7.86 (s, 1 H), 7.75-7.69 (m, 2 H),
7.24- 6.75 (m, 2 H), 5.20 (br, 1 H) 509 (M.sup.+ + 1, 100%), 511
(M.sup.+ + 1, 33%) K17 ##STR00101## 41 (CDCl.sub.3/DMSO-d.sub.6,
19:1): .delta. 9.06 (m, 1 H), 8.78 (m, 1 H), 8.64 (m, 1 H), 8.26
(m, 1 H), 8.14 (d, J = 8.0, 1 H), 7.83 (dd, J = 4.6 and 8.8, 1 H),
7.80 (s, 1 H), 7.72 (dd, J = 8.0 and 8.0, 1 H), 4.60 (br, 1 H) L1
##STR00102## 88 (CDCl.sub.3): .delta. 9.00-8.88 (m, 2 H), 8.02 (s,
1 H), 7.83 (m, 1 H), 7.60- 7.38 (m, 5 H), 3.80 (br, 1 H) 300
(M.sup.+ + 1, 100%), 302 (M.sup.+ + 1, 100%) L2 ##STR00103## 68
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.18 (m, 1 H), 8.74 (m, 1
H), 7.92-7.84 (m, 2 H), 7.04-6.95 (m, 3 H), 5.00 (br, 1 H) 336
(M.sup.+ + 1, 100%), 338 (M.sup.+ + 1, 33%) M1 ##STR00104## 91
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.15 (m, 1 H), 8.92 (d, J
= 8.3, 1 H), 7.89 (m, 1 H), 7.81 (m, 1 H), 7.78-7.72 (m, 2 H),
7.60-7.41 (m, 8 H), 4.60 (br, 1 H) 298 (M.sup.+ + 1) M2
##STR00105## 70 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.10 (m, 1
H), 9.52 (d, J = 8.3, 1 H), 7.81 (m, 1 H), 7.59 (s, 1 H), 7.45-7.23
(m, 8 H), 3.50 (br, 1 H), 2.25 (s, 3 H), 2.03 (s, 3 H) M3
##STR00106## 29 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta..delta.
9.25 (m, 1 H), 8.59 (d, J = 8.3, 1 H), 7.88- 7.81 (m, 2 H),
7.56-7.32 (m, 5 H), 7.18-7.03 (m, 3 H), 3.90 (br, 1 H), 3.73 (s, 6
H) M4 ##STR00107## 4 432 (M - H).sup.- M5 ##STR00108## 39
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.13 (m, 1 H), 8.60 (m, 1
H), 7.82 (m, 1 H), 7.76 (s, 1 H), 7.64-7.18 (m, 8 H), 3.30 (br, 1
H) N2 ##STR00109## 16 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.07
(d, J = 4.4, 1 H), 8.42 (d, J = 8.3, 1 H), 8.08 (s, 1 H), 7.81 (dd,
J = 3.2 and 8.3, 1 H), 7.47-7.30 (m, 3 H), 7.20 (d, J = 7.4, 1 H),
5.80 (br, 1 H), 2.01 (s, 3 H) N3 ##STR00110## 46
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.06 (d, J = 3.9, 1 H),
8.53 (d, J = 8.5, 1 H), 8.08 (s, 1 H), 7.83 (dd, J = 5.2 and 8.6, 1
H), 7.51 (m, 1 H), 7.27 (m, 1 H), 7.15 (d, J = 8.3, 1 H), 7.07 (d,
J = 8.3, 1 H), 4.50 (br, 1 H), 3.70 (s, 3 H) 378 (M.sup.+ + 1) N4
##STR00111## 79 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 9.08 (m, 1
H), 8.26 (m, 1 H), 8.07 (s, 1 H), 7.89 (m, 1 H), 7.80 (dd, J = 5.1
and 8.5, 1 H), 7.75-7.65 (m, 2 H), 7.36 (m, 1 H), 5.75 (br, 1 H)
416 (M.sup.+ + 1) N5 ##STR00112## 59 (CDCl.sub.3/DMSO-d.sub.6,
19:1): .delta. 9.11 (m, 1 H), 8.54 (m, 1 H), 8.10 (s, 1 H), 7.88
(dd, J = 5.1 and 8.7, 1 H), 7.54 (m, 1 H), 7.42-7.22 (m, 3 H), 5.30
(br, 1 H) 366 (M.sup.+ + 1) O1 ##STR00113## 22 355 [(M - H).sup.-,
100%], 357 [(M - H).sup.-, 66%]
TABLE-US-00005 TABLE 5 Data for the 2-aromatic group-substituted
8-HQ Derivatives (prepared via the Negishi Coupling Reaction).sup.a
Product Yield Mass spectral ID Product (%) .sup.1H NMR data data P1
##STR00114## 89 (CDCl.sub.3): .delta. 8.98 (d, J = 3.9, 1 H), 8.60
(d, J = 8.8, 1 H), 8.40-8.15 (m, 3 H), 7.75 (m, 1 H), 7.60 (m, 1
H), 7.50- 7.35 (m, 3 H) 223 (M.sup.+ + 1) P2 ##STR00115## 80
(CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.68 (d, J = 8.3, 1 H),
7.93 (d, J = 8.3, 1 H), 7.76-7.54 (m, 5 H), 7.50 (d, J = 7.5, 1 H),
7.41 (dd, J = 7.3 and 7.3, 1 H), 2.50 (br, 1 H), 2.49 (s, 3 H) 268
(M.sup.+ + 1) P3 ##STR00116## 33 (CDCl.sub.3): .delta. 8.66 (d, J =
8.8, 1 H), 8.22 (d, J = 7.1, 1 H), 7.80-7.38 (m, 7 H), 4.20 (q, J =
7.0, 2 H), 1.70 (br, 1 H), 1.18 (t, J = 7.0, 3 H) 294 (M.sup.+ + 1)
P4 ##STR00117## 95 (CDCl.sub.3/DMSO-d.sub.6, 19:1): .delta. 8.64
(d, J = 8.5, 1 H), 8.42 (d, J = 7.6, 1 H), 8.28 (d, J = 8.5, 1 H),
8.10 (m, 1 H), 7.78 (m, 1 H), 7.48-7.16 (m, 3 H), 2.68 (s, 3 H),
2.59 (br, 1 H) 279 (M.sup.+ + 1)
Example 20
Assessment of Compounds of Formula I or II
[0356] The following Assays were used in the assessment of the
compounds of formula I or II for suitability for use in the methods
of the invention.
Assay 1. Fluorometric H.sub.2O Assay
[0357] A fluorometric assay was used to test the ability of a test
compound to inhibit hydrogen peroxide generation by A.beta. in the
presence of copper based on dichlorofluoroscein diacetate (DCF;
Molecular Probes, Eugene Oreg.). The DCF solution (5 mM) in 100%
dimethyl sulphoxide (previously purged with argon for 2 hr at
20.degree. C.) was deacetylated in the presence of 0.25M NaOH for
30 min and neutralised at pH 7.4 to a final concentration of 1 mM.
Horseradish peroxidase (HRP) stock solution was prepared to 1 .mu.M
at pH 7.4. The reactions were carried out in PBS, pH 7.4 in a 96
well plate (total volume=250 .mu.l/well). The reaction solutions
contained A.beta. 1-42 at concentrations in the range of 50 nM to 1
.mu.M, copper-glycine chelate (Cu-Gly), was prepared by adding
CuCl.sub.2 to glycine in the ratio of 1:6 and added to the A.beta.
in the proportion 2Cu-Gly: 1A.beta.), reducing agents including
dopamine (5 .mu.M) or ascorbic acid, deacetylated DCF 100 .mu.M,
and HRP, 0.1 .mu.M. 1-10 .mu.M EDTA or another chelator may also be
present as a control for free copper, but was not required for the
assay to function. The reaction mixture was incubated at 37 C for
60 min. Catalase (4000 units/ml) and H.sub.2O.sub.2 (1-2.5 .mu.M)
standards in PBS pH 7.4 may be included as positive controls.
Fluorescence was recorded using a plate reader with excitation and
emission filters at 485 nM and 530 nM respectively. H.sub.2O.sub.2
concentration may be established by comparing fluorescence with the
H.sub.2O.sub.2 standards. Inhibition of A.beta. H.sub.2O.sub.2
production was assayed by including a given concentration of test
compound(s) in the test wells.
Assay 2. Neurotoxicity Assays
Primary Cortical Neuronal Cultures
[0358] Cortical cultures were prepared as previously described
(White et al., 1998). Embryonic day 14 BL6Jx129sv mouse cortices
were removed, dissected free of meninges and dissociated in 0.025%
(wt/vol) trypsin. Dissociated cells were plated in 48 well culture
plates at a density of 2.times.10.sup.6 cells/mL in MEM with 25%
(vol/vol) FCS and 5% (vol/vol) HS and incubated at 37.degree. C., 2
hrs. Media was then replaced with Neurobasal media (Invitrogen Life
Technologies) and B27 supplements (Invitrogen Life Technologies).
Cultures were maintained at 37.degree. C. in 5% CO.sub.2. Prior to
experimentation, the culture medium was replaced with Neurobasal
media and B27 minus antioxidants (Invitrogen Life
Technologies).
Primary Cerebellar Granule Neuronal Cultures
[0359] Cerebella from post-natal day 5-6 (P5-6) mice were removed
and dissected free of meninges and dissociated in 0.025% trypsin.
Cerebellar granule neurons (CGN) were plated in 24 well culture
plates at 350 000 cells/cm.sup.2 in BME (Invitrogen Life
Technologies) supplemented with 10% Fetal Calf Serum (FCS), 2 mM
glutamine and 25 mM KCl. Gentamycin sulphate (100 .mu.g/mL) was
added to all plating media and cultures were maintained at
37.degree. C. in 5% CO.sub.2.
Assay 3. Assays for Cell Viability
(a) MTS Assay for Cell Viability
[0360] Cell viability is determined using the MTS assay. Culture
medium is replaced with fresh neurobasal medium plus B27
supplements minus antioxidants. 1/10th volume MTS solution (Cell
Titre 96 Aqueous One, Promega Corporation) and incubated at at
37.degree. C., 2 hrs. 200 microlitre aliquots are measured with a
spectrophotometer at 560 nm.
(b) LDH Assay for Cell Viability
[0361] Cell death is determined from culture supernatants free of
serum and cell debris using the lactate dehydrogenase (LDH)
Cytotoxicity Detection Kit (Boehringer Ingelheim) according to the
manufacturer's instructions.
(c) Assay for A.beta. Neurotoxicity and A.beta. Neuroprotection
[0362] Neuronal cortical cells were cultured for five days as per
Assay 2. On day six the neurobasal (NB) media (Invitrogen Life
Technologies) and B27 supplement (Invitrogen Life Technologies)
were replaced with NB media and B27 supplement (no antioxidants).
On day six, test compounds were individually added to the neuronal
cell cultures:
[0363] The test compounds were dissolved in 100% DMSO to a
concentration of 2.5 mM (10 mM if excess compound was weighed out
per vial--then diluted to 2.5 mM). 2.5 mM stock solution was
serially diluted 1 in 10 to give working solutions of 250 uM, 25
uM, 2.5 uM.
A.beta. Preparation:
[0364] A.beta. was initially dissolved in 20 mM NaOH to a
concentration of 1 mM and sonicated for 5 minutes. The peptide was
then diluted in H.sub.2O and 10.times.PBS to a final concentration
of 200 uM A.beta. is in 1.times.PBS. The peptide was again
sonicated for 5 minutes and then spun at 14000 rpm for 5 min and
transferred to a fresh tube.
[0365] The test compounds were dissolved in 100% DMSO to a
concentration of 2.5 mM (10 mM if excess compound was weighed out
per vial--then diluted to 2.5 mM). 2.5 mM stock solution was
serially diluted 1 in 10 [in NB media and B27 (no antioxidants)] to
give working solutions of 250 uM, 25 uM, 2.5 uM. Test compounds
were not added directly to cells, instead they were added to a 48
well `Drug Plate` as comprised below:
[0366] Preparation of "Drug Plate":
To a 48 well plate add: [0367] Well 1: 515 ul NB+B27(no
antioxidant)*+24 ul 25 uM test compound+60 ul A.beta. diluent**
[0368] Well 2: 515 ul NB+B27(no antioxidant)+24 ul 250 uM test
compound+60 ul A.beta. diluent [0369] Well 3: 515 ul NB+B27(no
antioxidant)+24 ul test compound diluent***+60 ul A.beta.1-42
[0370] Well 4: 515 ul NB+B27(no antioxidant)+24 ul 2.5 uM test
compound+60 ul A.beta.1-42 [0371] Well 5: 515 ul NB+B27(no
antioxidant)+24 ul 25 uM test compound+60 ul A.beta.1-42 [0372]
Well 6: 515 ul NB+B27(no antioxidant)+24 ul 250 uM test compound+60
ul A.beta.1-42 diluent [0373] Well 7: 515 ul NB+B27(no
antioxidant)+24 ul test compound diluent+60 ul A.beta.1-42 diluent
[0374] Well 8: 600 ul NB+B27(no antioxidant) [0375] N.B. 60 ul
A.beta.1-42 equals 20 ul A.beta.1-42 per well equals 20 uM
A.beta.1-42
[0376] The Drug Plate was incubated at 37.degree. C. for 15 mins.
200 ul of each well was added in triplicate to the corresponding
cell plate. The cell plate was incubated at 37 C, for 4 days. * NB
media+B27 (no antioxidants),** A.beta. diluent 2 mM NaOH,
1.times.PBS*** PBT diluent 10% DMSO in NB+B27(no antioxidant)
Completion of the Assay:
[0377] On the 4.sup.th day after treating the cells the assay is
completed by adding MTS to the cells.
(d) Assay for Test Compound Cytoxicity
[0378] Neuronal cortical cells were cultured for five days as per
Assay 2 in NB media and B27 supplement.
[0379] On day six the test compounds were added to the neuronal
cell cultures in NB media and B27 supplement minus
antioxidants.
[0380] Test compounds were dissolved in 100% DMSO to a
concentration of 2.5 mM (10 mM if excess compound was weighed out
per vial--then diluted to 2.5 mM). 2.5 mM stock solution was
serially diluted 1 in 10 to give working solutions of 250 uM, 25
uM, 2.5 uM. Test compounds were not added directly to cells,
instead they were added to a 48 well `Drug Plate` as comprised
below:
[0381] Preparation of "Drug Plate":
[0382] To a 48 well plate add: [0383] Well 1: 576 ul NB+B27(no
antioxidant)*+24 ul 2.5 uM test compound [0384] Well 2: 576 ul
NB+B27(no antioxidant)+24 ul 25 uM test compound [0385] Well 3: 576
ul NB+B27(no antioxidant)+24 ul 250 uM test compound [0386] Well 4:
576 ul NB+B27(no antioxidant)+24 ul 2.5 uM test compound [0387]
Well 5: 576 ul NB+B27(no antioxidant)+24 ul 25 uM test compound
[0388] Well 6: 576 ul NB+B27(no antioxidant)+24 ul 250 uM test
compound [0389] Well 7: 576 ul NB+B27(no antioxidant)+24 ul test
compound diluent** [0390] Well 8: 600 ul NB+B27(no antioxidant)
[0391] The Drug Plate was incubated at 37.degree. C. for 15 mins.
200 ul of each well was added in triplicate to the corresponding
cell plate. The cell plate was incubated at 37 C, for 4 days. * NB
media and B27 (no antioxidants),** PBT diluent 10% DMSO in NB+B27
(no antioxidants)
[0392] On completion of the assay, 1/10 volume MTS was added per
well of plate (ie 25 ul/250 ul). The plates were incubated at 37 C
for 2 hrs, and then absorbance was read at 560 nm.
Assay 4. Caspase Assay
[0393] To measure caspase activity in neuronal cultures, growth
medium is removed, cells are washed twice with control salt
solution (pH 7.4) and ice-cold cell extraction buffer is added
directly to the cultures. The extraction buffer consists of 20 mM
Tris (pH 7.4), 1 mM sucrose, 0.25 mM EDTA, 1 mM dithiothreitol
(DTT), 0.5 mM PMSF, 1% Triton X-100 (Tx-100) and 1 .mu.g/mL of
pepstatin and aprotinin. After incubation for 15 min on ice, the
extraction buffer is removed, centrifuged for 5 min at 4.degree. C.
in a microcentrifuge and 100 .mu.L of supernatant is added to each
well of a 96 well plate. 100 .mu.L of 200 .mu.M substrate (either
DEVD-pNA, VEID-pNA or IETD-pNA for caspases 3, 6 and 8
respectively) is added to each well to give a final concentration
of 100 .mu.M substrate. Plates are incubated at 37.degree. C. for
2, 4, 6 or 24 hr and the absorbance is determined at a wavelength
of 415 nm (Abs415). The absorbance reading is compared to a known
standard of pNA alone.
Assay 5. Annexin V Assay
[0394] To determine the level of annexin V binding to cells,
cultures are washed twice with control salt solution (pH 7.4)
followed by the addition of annexin V-FITC at a concentration of
approximately 0.5 .mu.g/mL in control salt solution (pH 7.4).
Propidium iodide (10 .mu.g/mL) is also added to the cultures at the
same time. Cells are incubated in the dark for 30 min at ambient
temperature and subsequently washed three times with fresh control
salt solution. Analysis of FITC fluorescence (ex. 488 nm, em. 510
nm) is determined using a Leica DMIRB microscope. Photographs are
taken with a Leica MPS 60 camera attachment using ASA400 colour
film, and negatives are scanned into Adobe Photoshop v2.0.1.
Assay 6. Lipoprotein Oxidation Assay
[0395] Two different assays of metal-mediated lipid peroxidation
can be utilized. The first assay involves measuring the oxidative
activity of metallated proteins. This is determined by mixing
dialyzed metallated or native protein (at designated
concentrations) with 0.5 mg/mL LDL for 24 hr (37.degree. C.). Lipid
peroxidation (LPO) is measured using a lipid peroxidation assay kit
(LPO 486, Oxis International Inc. Portland, Oreg.) as per kit
instructions. The level of LPO is determined by comparing
absorbance (486 nm) with LDL alone (100% LPO). The second assay is
used to measure the LPO activity of native proteins in the presence
of free, non-protein-bound Cu. This involves adding non-metallated
peptides (140 .mu.M) to 0.5 mg/mL LDL together with 20 .mu.M Cu-gly
and assaying for LPO as for the metallated proteins. The level of
LPO is determined by comparing the absorbance (486 nm) with
LDL+Cu-gly (100% LPO). As a negative control, LDL is also exposed
to dialysed Cu-gly solutions comparable to those used to
Cu-metallate the proteins.
Assay 7. Cytotoxicity Induced by Cu-Metallated Proteins
[0396] Proteins or synthetic peptides are mixed with metal-glycine
solutions at equimolar or two-fold metal to protein concentration.
Metal-protein mixtures are incubated overnight at 37.degree. C. and
then extensively dialysed (24 hr against two changes of dH.sub.2O
(3 L/change) at room temperature) using mini-dialysis cups with a
3,500 kilodalton cut-off (Pierce, Rockford, Ill.). Dialysis of
proteins against PBS pH 7.4 resulted in metallated proteins with
identical activity to dH.sub.2O dialysis.
[0397] To determine their neurotoxic effects, metallated proteins,
native proteins or peptides are added to two day-old primary
cortical neuronal cultures. The cultures are also exposed to Cu-gly
(5 or 10 .mu.M) or LDL. Positive control cultures are treated with
Cu-gly+LDL or the LPO product, 4-hydroxy-nonenol (HNE, Sigma
Chemicals). Cultures are assayed for cell death using the lactate
dehydrogenase (LDH) assay kit (Roche Molecular Biochemicals,
Nunawading, Australia) according to the manufacturer's
instructions.
Assay 8. Acridine Orange Assay for A.beta.-Mediated Loss of
Lysosomal Acidification
[0398] Cultured mouse cortical neurons are treated with A.beta.1-42
(20 .mu.M) for 16 h and then stained with 5 mg/ml acridine orange
(AO) for 5 min at 37.degree. C. 15 min at 37.degree. C. The
AO-induced fluorescence is measured with a red filter on a
fluorescence microscope. AO is a lysosomotropic weak base which
accumulates in the endosomal/lysosomal compartments and displays
orange fluorescence during incubation. AO is sequestered inside the
lysosomes as long as there is a substantial proton gradient over
the lysosomal membranes. Treatment of cells with A.beta.1-42
disrupts the lysosomal membrane proton gradient and relocalises AO
into the cytosol, as indicated by the loss of orange fluorescence
within 16-24 hr.
Assay 9. Human Brain Amyloid Solubilisation Assay
[0399] This assay was performed in order to assess the ability of a
test compound to mobilise A.beta. from the insoluble to the soluble
phase of an extract of tissue from post mortem human AD brain.
[0400] Up to 0.5 g of plaque-bearing cortex without meninges was
homogenized using a DIAX 900 homogenizer (Heudolph and Co, Kelheim,
Germany) or other suitable device for three 30-second periods at
full speed in 2 ml of ice-cold phosphate-buffered saline, pH 7.4.
To obtain the phosphate-buffered saline-extractable fraction, the
homogenate was centrifuged at 100,000.times.g for 30 min and the
supernatant removed. Alternatively, the tissue was freeze dried
then pulverised to form a powder which was then weighed out into
aliquots for extraction as above. Supernatant, either freeze-dried
and resuspended or in unconcentrated form, was dissolved in 200
.mu.l of Tris-Tricine sodium dodecyl sulfate (SDS) sample buffer pH
8.3 containing 8% SDS, 10% 2-mercaptoethanol. Aliquots (10 .mu.l)
were then boiled for 10 minutes before SDS-polyacrylamide gel
electrophoresis. The insoluble fraction of the cortical samples was
obtained by resuspending the initial pelleted sample in 1 ml of
phosphate-buffered saline. A 50-.mu.l aliquot of this suspension
was then boiled in 200 ml of sample buffer as above.
[0401] Tris-Tricine polyacrylamide gel electrophoresis was
performed by loading appropriately diluted samples on to 10% to 20%
gradient gels (Novex, San Diego, Calif.) followed by transfer on to
0.2-.mu.m nitrocellulose membrane (Bio-Rad, Hercules, Calif.).
A.beta. was detected by using monoclonal antibody W02, which
detects residues 5 through 8, 17 (or another suitable antibody) in
conjunction with horseradish peroxidase-conjugated rabbit
anti-mouse IgG (Dako, Denmark), and visualized by using enhanced
chemiluminescence (eg ECL; Amersham Life Science, Buckinghamshire,
UK). Each gel included three lanes containing 0.5, 1, and 2 ng of
synthetic A.beta..sub.40 (Keck Laboratory, Yale University, New
Haven, Conn.) as reference standards.
[0402] Blot films were scanned by using a suitable imaging system
such as the UVP gel documentation system, and densitometry
performed using suitable software, eg UVP Labworks. The dynamic
range of the film/scanner was determined by using a step tablet
(No. 911ST600, Kodak, Rochester N.Y.), a calibrated film exposed by
the manufacturer to provided steps of known increasing intensity.
The quantifiable range of signal intensity for densitometric
analysis of the mono- and dimeric A.beta. bands was based on the
comparison with a curve obtained by scanning and densitometry of
the step tablet. Samples wherein the signal intensity is low after
preliminary assay may be re-assayed by using synthetic standards of
lower or higher concentration.
[0403] All samples were analysed at least twice, and gel loadings
and dilutions were adjusted to fit within the quantifiable region
of the standard curve. The proportion of `soluble` to `insoluble`
A.beta. may be used to determine the efficiency of extraction of a
test compound compared with the efficiency of a known compound,
such as clioquinol (PBT 1). The insoluble A.beta. being comprised
of the pelletable fraction derived from the insoluble amyloid
plaque from the above cortical samples and the soluble fraction
comprising monomeric and/or oligomeric soluble A.
Assay 10. Metal Partitioning
[0404] To assay effects upon the partitioning of various metals,
including zinc and copper, following extraction of brain tissue in
the presence of a test compound, soluble and insoluble fractions
from an extract of human brain tissue are prepared as for the
amyloid solubilisation assay. Metals in the two fractions are
analysed by inductively-coupled plasma mass spectrometry, following
appropriate pretreatment with nitric acid and/or hydrogen peroxide
where necessary.
Assay 11. Effect of Administration of Test Compounds on A.beta.
Deposits in Transgenic Animals
[0405] Transgenic mouse models are available for a number of
neurological disorders, including Alzheimer's disease (Games et
al., 1995; Hsiao et al., 1996); Parkinson's disease (Masliah et
al., 2000); familial amyotrophic lateral sclerosis (ALS) (Gurney et
al., 1994); Huntington's disease (Reddy et al., 1998); and
Creutzfeld-Jakob disease (CJD) (Telling et al., 1994). We have
found that one of the transgenic models for Alzheimer's disease,
the APP2576 transgenic mouse (Hsiao et al., 1996) also has a high
incidence of cataract. These animal models were suitable for
testing the methods of the invention.
[0406] Transgenic mice of the strain APP2576 (Hsiao et al 1996)
were used. Eight to nine month old female mice were selected and
divided into groups for treatment.
[0407] Mice were sacrificed at intervals, and their brains examined
to determine whether the treatment with test compounds decreased
brain amyloid formation, and the identification of the most
effective administration protocol. The levels of soluble and
insoluble A.beta. in the brain and serum were determined using
calibrated Western blots as per the methodology described for Assay
9. Brain Amyloid Solubilisation Assay.
[0408] Other mice in each group were tested over a period of up to
eight months for cognitive performance, using a Morris water maze
according to standard methods. The general health and well-being of
the animals was also measured every day by a blinded operator,
using a five point integer scale which subjectively rates a
combination of features, including motor activity, alertness and
general health signs.
Assay 12. Solubility Assay
[0409] Stock solutions of compounds of formula I or II (1 mM) were
prepared in dimethyl sulfoxide. Compounds which did not dissolve
were classed as not soluble (N). The DMSO stock solutions were
diluted 1 in 100 into PBS pH 7.4. Compounds which gave a clear
solution were classed as soluble (Y), while those compounds which
gave a translucent suspension after dissolution in DMSO were
classed as "crashed out" (C).
Assay 13. Physiochemical Properties
Polar Surface Area Calculations (PSA)
[0410] Polar surface area values were calculated using the
web-based program available through "Molinspiration", a package for
calculation of molecular properties.
Turbidimetric Solubility Measurements
[0411] The solubility estimate was measured at both pH 2.0 and pH
6.5. This is within the pH range that can be anticipated along the
proximal gastrointestinal tract in humans.
[0412] The compounds were dissolved in DMSO to appropriate
concentrations and then spiked into either 0.01M HCl (approx.
pH=2.0) or pH 6.5 isotonic phosphate buffer, the final DMSO
concentration being 1%. Samples were then analysed via Nephelometry
to determine a solubility range. [as per D. Bevan and R. S. Lloyd,
Anal. Chem. 2000, 72, 1781-1787].
c Log P Values
[0413] Theoretical Log P values were determined using the ACD Log P
software. The values quoted have been calculated from an untrained
database and refer to the unionised species.
E Log D
[0414] Effective Log D values were measured using a chromatographic
method employing a SUPELCOSIL LC-ABZ column using an octanol
saturated mobile phase at pH 7.4. [0415] See F. Lombardo et al, J.
Med. Chem. 2000, 43, 2922-2928.
Assay 14. Blood Brain Barrier Penetration
[0416] The test compounds were dissolved in DMSO and phosphate
buffered saline (PBS) was added to obtain solutions at a
concentration of 50 .mu.M in PBS containing 1.25-2.5% DMSO. A trace
amount of .sup.14C-sucrose was added to each stock infusion
solution (approx 0.01 .mu.Ci/mL) to act as Blood-Brain Barrier
(BBB)-impermeable marker in order to assess the integrity of the
BBB during each perfusion and to estimate the volume of the
residual vascular space (RVS) in samples of brain tissue (ie: the
volume of fluid remaining inside the lumen of blood vessels at the
end of each perfusion).
[0417] Adult male Spague Dawley rats (180-190 g) were anaesthetized
with intraperitoneal injections of Urethane (25% w/v) at a dose of
1.0 mL/100 g body weight. The right common carotid artery was
surgically exposed and cannulated for perfusion of the cerebral
circulation. The right external carotid artery (which supplies
tissues outside the skull) was then ligated distal to its
bifurcation from the right common carotid artery so that all of the
infusion solution would pass into the brain via the remaining right
internal carotid artery. The heart was then exposed and transected
immediately prior to the commencement of the infusion. The rate of
the infusion was controlled by a pump set to deliver at 3.2 mL/min
(approx. 85% of the normal blood supply to the brain for this size
of rat). The infusion cannula initially contained a 0.5 mL pre-wash
of heparinised PBS (10 IU/ml) that acts to flush blood vessels and
to prevent blood from clotting and blocking small vessels.
[0418] After 1.5 minutes, the infusion pump automatically stopped,
the cannula was withdrawn from the carotid artery and a sample of
the infusion solution (1-1.5 mL) was then collected from the tip of
the infusion cannula. The brain was then dissected free and divided
into 3 parts; the right hemisphere together with the right
midbrain, the left hemisphere together with the left midbrain and
the hindbrain (cerebellum, pons and brainstem). Only the right part
of the brain was used for subsequent measurements because perfusion
via the right internal carotid artery preferentially supplies the
right hemisphere and right midbrain (the left hemisphere and
hindbrain receive a variable collateral perfusion). The brain
tissue samples from each animal were frozen at -30.degree. C.,
homogenized and weighed aliquots analysed by LC-MS to give total
brain concentration. The analysis was carried out using the
Micromass Triple Quad instrument. The mobile phase consisted of an
acetonitrile/water gradient (containing 0.05% Formic acid) and the
column was a Phenomenex Luna CN.
[0419] Small aliquots from each brain tissue sample and the
corresponding infusion solution were analysed by liquid
scintillation counting to determine the level of .sup.14C-sucrose.
The residual vascular space (RVS) in each brain tissue sample was
calculated by dividing the measured concentration of sucrose in
brain tissue (dpm/mg) by its concentration in the corresponding
infusion solution (dpm/.mu.L). This is the volume of fluid that
remains inside blood vessels at the end of each perfusion.
Multiplying this RVS by the concentration of the test compound in
the infusion solution gives the total residual amount of the test
compound that is present inside blood vessels in each brain tissue
sample (ie: that which has not crossed the BBB). Subtracting this
from the total brain concentration gives the amount of drug in each
brain tissue sample that is outside the blood vessels (ie: which
has crossed the BBB). Dividing this RVS-corrected brain
concentration gives the brain uptake ratio (Equation. 1).
Brain Uptake Ratio = [ brain ng mg - 1 ] - [ RVS ng l - 1 ] [
infusion solution ng L - 1 ] . Equation 1 ##EQU00001##
[0420] A total of 5-6 brain perfusion experiments were performed
for each of the test compounds and mean brain uptake ratios were
calculated.
[0421] Ratios of greater than 50% indicate compounds that enter the
brain extremely rapidly; ratios between 10 and 50% indicate
compounds that enter the brain well; ratios less than 10% (not
observed) would indicate compounds that enter the brain very slowly
and would not be suitable for therapeutic administration; ratios
less than 1% (not observed) would indicate compounds that are
effectively excluded from the brain.
Assay 15. Transgenic Mouse Brain Immunohistochemistry
[0422] The APP2576 transgenic mouse (Hsiao et al., 1996) as
referred to in Assay 11 were utilized in this assay. The
contralateral formalin-fixed mouse brain tissue was coronally cut.
Sections (10 .mu.m) were taken from the corresponding sites and
treated with 80% formic acid for antigen retrieval. The primary
antibody used was monoclonal antibody 1E8, which recognizes
epitopes between residues 18 and 22 of A.beta. (SmithKline Beecham,
UK). Immunoreactivity was developed with secondary antibody linked
to horseradish peroxidase (using a 3,39-diaminobenzidinechromagen)
(Dako) and alkaline phosphatase (using 5-bromo-4-chloro 3-indoxyl
phosphate and nitroblue tetrazolium chloride chromagen) (Dako).
Plaque abundance per section was assessed by two operators blinded
to treatment according to the following scale:
[0423] 0=no plaques apparent
[0424] 1=plaques present but very sparse
[0425] 2=several plaques present
[0426] 3=numerous plaques visible in restricted areas
[0427] 4=plaques abundant and not restricted to any particular
area.
Intermediate values eg 2.5 were assigned where applicable.
Students' t `test was used for comparisons between groups.
Assay 16. Pharmacokinetic Profile
(a) PBT-1033
[0428] Intravenous infusion of PBT-1033; 2 mg/Kg (1 mL of a 0.5
mg/mL solution in 7.5% DMSO with 0.1 m Captisol in Citrate Buffer
adjusted to pH 3.0) was administered over 5 minutes to 2 rats and
arterial blood was sampled up to 24 hours. [0429] Oral
administration of PBT-1033; 30 mg/Kg (as a suspension in CMC-SSV*)
via administered via oral gavage to 2 rats and arterial blood was
sampled up to 26 hours. [0430] Plasma concentrations of PBT-1033
were determined by LCMS (LOQ 3.7 nM). For rat 020710-D, an
overlapping peak was present for PBT-1033. * Standard Suspending
Vehicle--0.5% w/v Na-Carboxymethyl Cellulose (CMC), 5% v/v benzyl
alcohol, 4% v/v Tween 80 in 0.9% NaCl.
[0430] CL total = Dose IV AUC IV ##EQU00002## V d .beta. = CL total
.beta. ##EQU00002.2## BA ( % ) = AUC oral * Dose IV AUC IV * Dose
oral ##EQU00002.3##
[0431] Calculations: [0432] CL.sub.total=total plasma clearance
after IV administration [0433] V.sub.d.beta.=volume of distribution
during the elimination phase after IV administration [0434] BA=oral
bioavailability [0435] AUC.sub.IV=area under the plasma
concentration versus time profile from time zero to infinity after
IV administration [0436] AUC.sub.oral=area under the plasma
concentration versus time profile from time zero to infinity after
oral administration [0437] .beta.=terminal elimination rate
constant after IV administration [0438] The results are shown in
FIG. 4(a).
(b) PBT-1038
[0438] [0439] Intravenous infusion of PBT-1038; (0.5 mg/Kg in 7.5%
DMSO in Citrate Buffer pH 3.0) was administered over 5 minutes to 2
rats and arterial blood was sampled up to 24 hours. [0440] Oral
administration of PBT-1038; (30 mg/Kg as a 0.05% CMC suspension)
via administered via oral gavage to 2 rats and arterial blood was
sampled up to 24 hours. [0441] Plasma concentrations of PBT-1038
were determined by MS (LOQ 3 nM)
[0442] Calculations:
[0443] As described above for PBT-1033. [0444] The results are
shown in FIG. 4(b).
(c) PBT-1050
[0444] [0445] Intravenous infusion of PBT-1050; (2 mg/Kg in 7.5%
DMSO in Citrate Buffer pH 3.0) was administered over 5 minutes to 2
rats and arterial blood was sampled up to 24 hours. [0446] Oral
administration of PBT-1050; (30 mg/Kg as a 0.05% CMC suspension)
was administered via oral gavage to 2 rats and arterial blood was
sampled up to 24 hours. [0447] Plasma concentrations of PBT-1050
were determined by MS (LOQ 3 nM)
[0448] Calculations:
[0449] As described above for PBT-1033 [0450] The results are shown
in FIG. 4(c).
(d) PBT-1051
[0450] [0451] Intravenous infusion of PBT-1051; 2 mg/Kg (1 mL of a
0.6 mg/mL solution in 7.5% DMSO in Citrate Buffer pH 3.0) was
administered over 5 minutes to 2 rats and arterial blood was
sampled up to 24 hours. [0452] Oral administration of PBT-1051; 30
mg/Kg (as a suspension in CMC-SSV*) was administered via oral
gavage to 2 rats and arterial blood was sampled up to 24 hours.
[0453] Plasma concentrations of PBT-1051 were determined by LCMS
(LOQ 3.7 nM) * Standard Suspending Vehicle--0.5% w/v
Na-Carboxymethyl Cellulose (CMC), 5% v/v benzyl alcohol, 4% v/v
Tween 80 in 0.9% NaCl.
[0454] Calculations:
[0455] As described above for PBT-1033. [0456] The results are
shown in FIG. 4(d).
TABLE-US-00006 [0456] TABLE 6 Screening Tests of Compound of
formula I or II for the treatment of Alzheimer's disease. Table 6
Parameter Assay Sol. (Y, C, N) Assay 1 clogP Assay 13 Peroxide IC50
Assay 1 Viable 10 uM Assay 8 BAS score Assay 9 N/A = not assayed. =
not effective at solubilising plaques relative to PBS. + =
effective at solubilising plaques at more than 1 concentration
relative to PBS. ++ = extremely effective at solubilising plaques
relative to PBS. This would mean better than twice the amount of
PBS at most concentrations tested on each of 2 or more
experiments
[0457] The results of screening tests referred to in Table 6 with
respect to compounds of Formula IIA-VIb are tabulated
hereinbelow.
TABLE-US-00007 Formula IIa Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 49 ##STR00118## 826-81-3 Y 2.58
100 N/A 89 ##STR00119## 189506-06-7 Y 3.7 3, 2.5 + 89 74.28 91
##STR00120## Y 3.86 50, >10 N/A 1004 ##STR00121## >10, 6.6
N/A 1005 ##STR00122## 2.4 N/A 1006 ##STR00123## 0.53 ++
##STR00124## ##STR00125## ##STR00126## ##STR00127## 1007
##STR00128## 0.58 N/A 1019 ##STR00129## >10, >10 N/A 1020
##STR00130## 1.3 N/A 1021 ##STR00131## 0.27 N/A 1029 ##STR00132##
N/A N/A 1035 ##STR00133## >10 N/A
TABLE-US-00008 Formula IIIa Peroxide Viable ID Structure CAS Sol
(Y, C, N) clogp IC50 10 uM BAS Score 52 ##STR00134## 59-00-7 Y 3
>100 N/A 57 ##STR00135## 1571-30-8 Y 2.67 40 N/A 58 ##STR00136##
6759-78-0 Y 1.95 100 N/A 95 ##STR00137## Y 6.66 10 N/A 948
##STR00138## Y 1.61 0.19, 0.15 + 948 106.66 949 ##STR00139## Y 2.38
0.43, 0.9 + 949 84.82 950 ##STR00140## Y 2.51 0.25, 0.15 + 950 92.8
951 ##STR00141## Y 3.26 1.43 - 951 91.86 952 ##STR00142## C 2.47
<0.81, 0.27 + 952 99.52 953 ##STR00143## C 2.97 <4.24, 0.62 +
953 67.8 954 ##STR00144## Y 1.93 0.18, 0.12 - 954 104.9 955
##STR00145## Y 2.71 0.26, 0.18 - 955 100 956 ##STR00146##
125686-78-4 Y 1.7 >10 N/A 956 89 957 ##STR00147## Y 1.42 >10
+ 957 95.86 976 ##STR00148## 149003-27-2 Y 2.35 3.7 - 986
##STR00149## Y 2.8 3.6 + 986 81.73 987 ##STR00150## Y 1.08 1.8 +
987 89.03 988 ##STR00151## Y 1.76 >10 - 988 93.27 992
##STR00152## Y 2.03 >10 N/A ##STR00153## ##STR00154##
##STR00155##
TABLE-US-00009 Formula IVa Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 966 ##STR00156## Y 0.2 4.3 + 966
88.61 967 ##STR00157## Y 0.89 7.8 N/A 967 90.69 968 ##STR00158##
17018-81-4 Y 1.03 0.26 ++ 968 97.12 969 ##STR00159## 5603-22-5 Y
2.83 0.54 + 969 94.55 989 ##STR00160## Y 1.14 0.42 - 989 43.24 990
##STR00161## Y 2.51 0.4 + 990 57.45 991 ##STR00162## Y 1.11 0.47 +
1002 ##STR00163## 1.95 0.39 N/A 1003 ##STR00164## 2.19 0.55 N/A
1008 ##STR00165## 1.2 0.26 N/A 1009 ##STR00166## 1.88 0.32 N/A 1010
##STR00167## 2.35 0.33 N/A 1011 ##STR00168## 1.68 0.32 N/A
##STR00169## ##STR00170## ##STR00171##
TABLE-US-00010 Formula Va Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 53 ##STR00172## 82361-90-8 Y 6.27
0.3 + 53 95.8 54 ##STR00173## 70125-16-5 Y 1.75 1 + 54 99.57 56
##STR00174## 65165-14-2 Y 4.69 0.7, 0.25 + 56 24.61 56 100.6 964
##STR00175## Y 2.97 7.1 N/A 965 ##STR00176## Y 1.94 >10 N/A 993
##STR00177## Y 2.21 >10 N/A 994 ##STR00178## Y 1.75 >10 N/A
##STR00179## ##STR00180## ##STR00181## ##STR00182##
TABLE-US-00011 Formula VIa Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 50 ##STR00183## 20946-17-2 0.71 90
N/A ##STR00184##
TABLE-US-00012 Formula IIb Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score ##STR00185## ##STR00186##
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## ##STR00193## ##STR00194## 1 ##STR00195## 130-26-7 Y
3.73 0.4-0.5 ++ 41 ##STR00196## 84-88-8 Y -0.71 0.5 + 41 81.33 42
##STR00197## 148-24-3 Y 2.08 0.7 + 42 97.66 43 ##STR00198##
547-91-1 Y 0.19 0.6 - 43 91.02 44 ##STR00199## 21302-43-2 Y 1.53
>10 71.05 + 45 ##STR00200## 773-76-2 Y 3.34 0.7, 0.4 ++ 45 75.19
45 66.51 46 ##STR00201## 83-73-8 Y 4.14 1 - 46 91.97 47
##STR00202## 521-74-4 Y 3.69 0.9, 0.5 + 47 93.59 48 ##STR00203##
130-16-5 Y 2.91 0.8, 0.8 - 48 85 59 ##STR00204## 37873-29-3 Y 3.02
0.7 + 59 84.95 59 42.59 814 ##STR00205## <1.1, >10 + 1026
##STR00206## 0.23 N/A 1028 ##STR00207## 0.32 N/A 1031 ##STR00208##
0.76 N/A 1032 ##STR00209## 1 N/A 1033 ##STR00210## 0.38, 0.35 +
1034 ##STR00211## 0.44 N/A 1036 ##STR00212## >5, 0.24 N/A 1037
##STR00213## >10 N/A 1038 ##STR00214## 0.26 + 1039 ##STR00215##
>10 N/A 1043 ##STR00216## 0.64 N/A 1047 ##STR00217## >10 N/A
1050 ##STR00218## 0.28 N/A 1051 ##STR00219## 0.38 + 1052
##STR00220## 0.64 + 1056 ##STR00221## 0.69 68.25 1057 ##STR00222##
0.43 95.02 1058 ##STR00223## 0.68 54.60 1060 ##STR00224## 0.50
TABLE-US-00013 Formula IIIb Peroxide Viable ID Structure CAS Sol
(Y, C, N) clogp IC50 10 uM BAS Score 808 ##STR00225## C 4.3 >10
N/A 808 71.89 810 ##STR00226## C 4.23 >10, <0.7 + 810 70.7
810 90.15 811 ##STR00227## C 4.06 >10 N/A 811 78.46 812
##STR00228## C 4.45 >10 N/A 812 75.36 813 ##STR00229## C 4.6
>10 N/A 813 8 813 66 814 ##STR00230## C 4.23 <1.1, >10 +
814 31.13 815 ##STR00231## C 4.45 >10 N/A 815 53.68 849
##STR00232## Y 3.67 4.5 N/A 849 98.83 850 ##STR00233## C 4.45
>10 N/A 850 71.28 851 ##STR00234## C 4.47 <0.7 - 851 84.92
851 86.08 854 ##STR00235## C 4.5 <0.78 + 854 100 854 71.39 854
34.95 859 ##STR00236## C 4.8 <0.67 + 859 73.14 859 36.01 859
34.07 864 ##STR00237## Y 5.2 0.77 + 864 93.12 947 ##STR00238## Y
3.14 1.14 + 947 70.4 970 ##STR00239## C 5.54 6.7 N/A 970 32.33 971
##STR00240## C 4.57 >10 N/A 971 84.29 972 ##STR00241## C 3.95
>10 N/A 972 30.59 973 ##STR00242## C 4.6 >10 N/A 973
42.38
TABLE-US-00014 Formula IVb Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 806 ##STR00243## C 4.67 <1.2,
<0.9 ++ 806 97 806 100 853 ##STR00244## Y 4.97 0.77 + 853 94.79
860 ##STR00245## Y 5.76 0.79 + 860 89.58 860 64.83 861 ##STR00246##
C 5.06 0.91 + 861 37.83 863 ##STR00247## C 4.23 <0.73 + 863
34.97 865 ##STR00248## C 5.01 >10 N/A 865 34.07
TABLE-US-00015 Formula Vb Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 809 ##STR00249## C 5.35 <4, 1.8
+ 809 26.31 852 ##STR00250## Y 5.75 2.1 + 852 33.52 862
##STR00251## C 4.09 <0.77 + 862 51.52 862 52.69 974 ##STR00252##
Y 7.17 0.6 + 975 ##STR00253## Y 5.67 3.2 +
TABLE-US-00016 Formula VIb Peroxide Viable ID Structure CAS Sol (Y,
C, N) clogp IC50 10 uM BAS Score 39 ##STR00254## 14683-61-5 Y 90
N/A 62 ##STR00255## 29266-96-4 Y >10 N/A 800 ##STR00256## C
>10 N/A 801 ##STR00257## C >10 N/A 802 ##STR00258## C >10
N/A 803 ##STR00259## C >10 N/A 804 ##STR00260## C >10 N/A 805
##STR00261## C >10 N/A 807 ##STR00262## C >10 N/A 816
##STR00263## C >10 N/A 817 ##STR00264## C >10 N/A 818
##STR00265## C >10 N/A 819 ##STR00266## C >10 N/A 820
##STR00267## C >10 N/A 821 ##STR00268## C >10 N/A 822
##STR00269## C >10 N/A 823 ##STR00270## C >10 N/A 824
##STR00271## C >10 N/A 825 ##STR00272## C >10 N/A 826
##STR00273## C >10 N/A 827 ##STR00274## C >10 N/A 855
##STR00275## Y >10 N/A 856 ##STR00276## C >10 N/A 857
##STR00277## C >10 N/A 858 ##STR00278## Y >10 N/A 866
##STR00279## Y >10 N/A 867 ##STR00280## Y >10 N/A 868
##STR00281## C >10 N/A 1022 ##STR00282## >10 N/A
[0458] Additional results with respect to various assays utilizing
the methodology described are tabulated below on representative
compounds identified as their PBT numbers:
TABLE-US-00017 TABLE 8 Test Compound Cytoxicity [Methodology as per
Assay 2(d)] PBT 0.1 um 1 uM 10 uM 1 78 90 77 41 95 100 100 42 90 87
79 45 100 92 67 47 nd 88 86 53 96 95 100 54 89 97 61 56 108 74 31
59 89 97 55 89 87 92 101 806 99 78 38 810 100 85 57 853 93 79 39
854 94 81 36 864 100 88 37 947 100 94 51 948 100 79 85 950 95 88 89
952 98 91 53 953 101 85 53 968 103 87 82 969 91 92 90 986 96 85 60
987 92 90 87 990 95 88 57 1002 nd 82 34 1003 nd 97 38 1005 nd 100
95 1006 nd 92 52 1007 nd 90 43 1008 nd 86 28 1009 nd 94 32 1010 nd
88 27 1011 nd 89 31 1020 nd 85 83 1021 nd 93 81 1031 nd 85 81 1032
nd 83 42 1033 nd 80 70 1037 nd 88 87 1038 nd 93 83 1039 nd 94 87
1044 nd 92 89 1045 nd 90 86 1049 nd 93 89 1050 nd 93 88 1051 nd 87
56 1052 nd 63 32 1053 nd 100 105 1055 nd 112 57 1056 96.44 68.25
1057 101.84 95.02 1058 82.47 54.60
TABLE-US-00018 TABLE 7 A.beta. Neuroprotection [Methodology as per
Assay 2(c)] % inhibition Abeta PBT toxicity 1 16 41 12 42 14 45 28
47 13 53 -75 54 100 56 17 59 22 89 -31 806 36 810 11 853 31 854 22
864 13 947 3 948 9 950 5 952 13 953 25 968 6 969 2 986 7 987 5 990
18 1002 17 1003 17 1005 1 1006 7 1007 9 1008 9 1009 16 1010 12 1011
21 1020 4 1021 1 1031 8 1032 -2 1033 38 1037 4 1038 19 1039 9 1044
3 1045 10 1049 6 1050 6 1051 23 1052 19 1053 -2 1055 37 1056 39
1057 4 1058 30
TABLE-US-00019 TABLE 9 Levels of Soluble A.beta. and Insoluble
A.beta. in Transgenic Mouse Brains. [Methodology as per Assay 11.]
Soluble fraction. Insoluble fraction. Test % change compared %
change compared Compound with control. with control. PBT 1 +50 -49
PBT 1033 -37 -29 PBT 1038 negligible -37% PBT 1051 negligible -21
PBT 1052 negligible -22
TABLE-US-00020 TABLE 10 Blood Brain Barrier Penetration
[Methodology as per Assay 14.] Test Compound Uptake Ratio PBT-1
Between 10 and 50% PBT-1033 >50% PBT-1038 >50% PBT-1050
>50% PBT-1051 >50% PBT-1052 Between 10 and 50%
TABLE-US-00021 TABLE 11 Physiochemical Properties [Methodology as
per Assay 13] PSA Solubility.sub.pH 6.5 Solubility (.mu.g/mL) E Log
Compound (.ANG..sup.2) (.mu.g/mL) 0.01M HCl cLog P D.sub.7.4 PBT1
33.1 <3.1 3.1-6.2 4.32 1.85 PBT1033 35.8 3.1-6.2 <3.1 3.51
1.32 PBT1038 90.9 12.5-25 3.1-6.2 2.69 2.92 PBT1050 80.0 12.5-25
25-50 2.56 2.98 PBT1051 44.6 <3.1 <3.1 3.58 2.64 PBT1052 46.0
<3.1 6.3-12.5 4.22 2.85
TABLE-US-00022 TABLE 12 Transgenic Mouse Brain Immunohistochemistry
[Methodology as per Assay 15] % difference from control Mean plaque
(sham treated score animal) P value Sham 3.5 1033 2.06 -41 0.018
1038 3.0 -17 NSD 1051 2.13 -39 0.0037 1052 3.2 -8 NSD
TABLE-US-00023 TABLE 13(a) Pharmacokinetic Parameters following
Intravenous and Oral Administration of PBT 1033 to rats.
[Methodology as per Assay 16] 030701-A 030701-B 030701-C 030710-D
Parameter IV IV Mean .+-. SD PO PO Mean .+-. SD Measured 1.84 1.62
1.73 .+-. 0.16 34.11 31.21 32.66 .+-. 2.05 Dose (mg/Kg)
C.sub.max(.mu.M) 0.70 2.48 1.59 .+-. 1.26 2.02 1.36 1.69 .+-. 0.47
T.sub.max(min) 20 5 12.50 .+-. 10.61 45 60 52.50 .+-. 10.61
t.sub.1/2(min) 52.25 53.29 52.77 .+-. 0.73 -- -- --
Cl.sub.total.sup.a 90.98 145.69 118.34 .+-. 38.69 -- -- --
(mL/min/Kg) V.sub.dB(L/Kg) 6.86 11.20 9.03 .+-. 3.07 -- -- --
BA(%).sup.b -- -- -- 25.45 25.04 25.25 .+-. 0.29 .sup.aTotal plasma
clearance .sup.bOral BA calculated using the truncated
AUC.sub.0-1560.
TABLE-US-00024 TABLE 13(b) Pharmacokinetic Parameters following
Intravenous and Oral Administration of PBT 1038 to rats.
[Methodology as per Assay 16] 030410-B 030410-E 030410-C 030415-E
Parameter IV IV Mean .+-. SD PO PO Mean .+-. SD Measured 0.34 0.35
0.34 .+-. 0.00 38.74 34.47 36.60 .+-. 3.02 Dose (mg/Kg)
C.sub.max(.mu.M) 8.06 2.84 5.45 .+-. 3.70 57.77 68.48 63.12 .+-.
7.57 T.sub.max(min) -- -- -- 45 45 45.00 .+-. 0.00
TABLE-US-00025 TABLE 13(c) Pharmacokinetic Parameters following
Intravenous and Oral Administration of PBT 1050 to rats.
[Methodology as per Assay 16] 030415-A 030415-B 030415-C 030415-D
Parameter IV IV Mean .+-. SD PO PO Mean .+-. SD Measured 2.37 2.05
2.21 .+-. 0.23 35.14 26.25 30.69 .+-. 6.29 Dose (mg/Kg)
C.sub.max(.mu.M) 33.93 16.88 25.41 .+-. 12.06 61.00 7.03 34.02 .+-.
38.17 T.sub.max(min) -- -- -- 45 120 82.5 .+-. 53.03
TABLE-US-00026 TABLE 13(d) Pharmacokinetic Parameters following
Intravenous and Oral Administration of PBT 1051 to rats.
[Methodology as per Assay 16] 030506-A 030506-B 030506-C 030506-D
Parameter IV IV Mean .+-. SD PO PO Mean .+-. SD Measured 3.16 2.77
2.96 .+-. 0.28 34.24 26.45 30.35 .+-. 5.51 Dose (mg/Kg)
C.sub.max(.mu.M) 2.96 3.03 2.99 .+-. 0.05 3.18 1.50 2.34 .+-. 1.18
T.sub.max(min) -- -- -- 60 30 45 .+-. 21.21 t.sub.1/2(min) 46.07
46.52 46.30 .+-. 0.32 200.09 365.72 282.9 .+-. 117.1
Cl.sub.total.sup.a 153.24 135.58 144.4 .+-. 12.5 -- -- --
(mL/min/Kg) V.sub.dB(L/Kg) 10.19 9.10 9.64 .+-. 0.77 -- -- --
BA(%).sup.b -- -- -- 37.96 17.55 27.75 .+-. 14.43 .sup.aTotal
plasma clearance .sup.bOral BA calculated using the truncated
AUC.sub.0-1440. This value may be an overestimation of the true
bioavailability.
Example 21
Clinical Trial of Compound of Formula I or II for the Treatment of
Alzheimer's Disease
[0459] A Phase II clinical trial of the compound of formula I or II
for the treatment of AD was undertaken to study the effects of oral
PBT-1 treatment in a randomised, double-blind, placebo-controlled
pilot phase 2 clinical trial of moderately severe AD patients.
Thirty-six subjects were randomized [18 placebo and 18 PBT-1, with
32 completions], and stratified into more- and less-severely
affected groups. The effect of treatment was statistically
significant in preventing cognitive deterioration over 36 weeks in
the more-severely affected patients (baseline ADAS-cog.gtoreq.25).
The performance of the less-severely affected group
(ADAS-cog<25) deteriorated negligibly over this interval, so
cognitive changes could not be discriminated in this stratum.
Plasma A.beta..sub.42 declined in the PBT-1 group but increased in
the placebo group (p<0.001). Plasma Zn levels rose significantly
(.apprxeq.30%) in the PBT-1 group.
Dosage
[0460] Several considerations drove the choice of dose. In previous
studies on transgenic mice, doses of 20-30 mg/kg of PBT-1 orally
daily for five days per week were markedly effective at inhibiting
A.beta. accumulation after 2-3 months of treatment. The human
equivalent dose of 1500-2250 mg/day is close to the prescribed
antibiotic dose of PBT-1 (600 mg po qid). However, this magnitude
of dose, administered for months, would raise concerns about SMON
toxicity.
[0461] The starting dose of 3.3 mg/kg/day, assuming 75 kg average
weight, is within the same order of magnitude of the effective dose
in the transgenic mouse model, but only about one tenth of the
antibiotic dose.
[0462] Since there is no data from the transgenic mouse study of
the effectiveness of doses less than 20 mg/kg/day, we reasoned that
a beneficial effect might require a longer period of treatment than
the 9-12 week duration of the mouse study (Cherny et al., 2001).
Therefore a trial length of 36 weeks at an average dose which is
approximately one-third of what is effective in the transgenic mice
is chosen. The final dose of 10 mg/kg/day is half of an effective
dose in mice.
[0463] The starting dose of 3.3 mg/kg/day was within the same order
of magnitude of the effective dose in the transgenic mouse model,
but only about one tenth of the anti-infective dose. The study was
powered to detect biochemical effects on metal and AO levels that
would be in the same magnitude as those seen in the transgenic
study.
EXPERIMENTAL PROCEDURES
[0464] Ethical Issues:
[0465] In compliance with Australian laws concerning consent from
individuals whose cognitive function may be impaired to the extent
of being unable to make informed judgements or decisions, "Consent
to Special Procedures" administered by the Victorian Civil and
Administrative Tribunal was obtained for each participant not able
to consent on their own behalf. In addition, third party consent
was obtained from all carers. All subjects were stabilized on
donepezil prior to commencement of the study. The study was
approved by the Royal Melbourne Hospital Research Foundation's
Clinical Research and Ethics Committee.
[0466] Study Population:
[0467] The study took place at the AD clinical trials unit, Mental
Health Research Institute of Victoria and at the Royal Melbourne
Hospital. Criteria for inclusion in the study were: informed
consent; a diagnosis of probable AD by NINCDS-ADRDA criteria
(McKhann et al., 1984); AD Assessment Scale-cognitive (ADAS-cog)
(Rosen et al., 1984) score of 18-45; Mini Mental State Examination
(MMSE) (Foistein et al., 1975) score of 10-24; on donepezil 5 mg or
10 mg for at least 6 months; relative or carer willing and able to
support the trial; able to complete trial examinations; primary
sensorial functions intact.
[0468] Patients were excluded if they had a history or clinical
evidence of peripheral or optic neuropathy or had co-existing
illnesses or past history that may have affected cognitive
function, nerve conduction or illnesses that may have confounded
the adverse event profile.
[0469] The following factors were obtained at baseline to determine
if they correlated with outcome measures: age, sex, premorbid IQ
[estimated from the National Adult Reading Test (NART)], years of
education, and apolipoprotein E (ApoE) allotype.
[0470] Study Design:
[0471] The study was a double blind, placebo-controlled, parallel
group randomized design. Thirty-six patients and their carers were
recruited to participate, with patients randomized at a 1:1 ratio
to receive either PBT-1 or placebo. The duration of the study was
36 weeks. PBT-1 oral dosage was 125 mg bid from weeks 0-12,
increased to 250 mg bid from weeks 13-24, and finally, 375 mg bid
from weeks 25-36.
[0472] Study Procedures:
[0473] Screening procedures consisted of a complete medical
history, physical, neurological and ophthalmic examination, blood
and urine tests and psychometric tests (ADAS-cog, MMSE). Nerve
conduction tests and visual evoked responses were conducted between
the screening and baseline visits to provide a baseline
measurement. Blood was collected for ApoE allotyping, baseline
plasma levels of metals and AO prior to randomization. All patients
continued their study entry dose of donepezil and all patients
received 100 mg vitamin B.sub.12 intramuscularly every four
weeks.
[0474] Blood samples were collected by antecubital venepuncture
except on weeks 12, 24 and 36 when they were collected by an
indwelling catheter. The procedural change did not affect
biochemical readouts except for Zn levels which were found to be
consistently .about.10% depressed (probably as a result of
differences in platelet activation). Zn data from these intervals
were therefore omitted from analysis.
[0475] Outcome Measures:
[0476] The primary clinical efficacy variable was a change from
baseline score on the ADAS-cog conducted at baseline and at weeks
4, 12, 24 and 36. This measure was chosen to allow comparability of
treatment effects with current therapeutics such as donepezil,
where efficacy trials also used ADAS-cog as their primary outcome
measure (Rogers et al., 1998). Although numerous neuropsychological
tests could be considered as secondary measures, it was necessary
to avoid fatiguing the subjects at review. Therefore the only other
cognitive test was the Mini-Mental State Exam (MMSE). The CIBIC+
(clinician interview based impression of change incorporating
caregiver information), a subjective observational index was also
conducted. Plasma A.beta., and plasma zinc and copper were all
taken every four weeks.
[0477] Double Antibody Capture Enzyme-Linked Immunosorbent Assay
(ELISA) for A.beta. Detection:
[0478] Polystyrene plates were coated with mAb G210 (for A.beta.40)
or mAb G211 (for A1342). Plates were washed and biotinylated mAb
WO2 was added. Bound antibody was detected with
streptavidin-labelled Europium (Perkin Elmer, Vic Australia). The
values obtained from triplicated wells were calculated based on
standard curves generated on each plate. Plasma samples
supplemented with synthetic A.beta.1-40 and A.beta.1-42 were also
assayed to confirm measurement reliability across the concentration
range of interest.
[0479] Metal Levels:
[0480] Metals were measured by inductively coupled plasma mass
spectrometry as previously described (Cherny et al., 2001).
[0481] Therapeutic Drug Monitoring:
[0482] At weeks 12, 24 and 36, PBT-1 blood levels were assayed by
HPLC with appropriate validation studies (Centre for Pharmaceutical
Research, University of South Australia).
[0483] Safety Measures:
[0484] Standard adverse event reporting was conducted and
biochemical tests, renal and liver function, complete blood
examination, serum vitamin B.sub.12 and folate levels were
documented at each visit. To assess for peripheral and optic
neuropathy a neurological examination was conducted at each visit,
and visual evoked responses, nerve conduction studies and
ophthalmic examination were conducted at screening, week 16 and
prior to the final trial visit. An ECG was done at screening and
weeks 12, 24 and 36.
[0485] Data Preparation and Statistical Analysis:
[0486] Data monitoring and management were undertaken by
independent contractors (Kendle International and Health Research
Solutions, Melbourne). Evidence for efficacy was indicated by a
significant difference in change from baseline between treatment
arms. Analysis of variance was the principal method of evaluating
statistical significance with the treatment arm illness severity at
baseline being the primary design factor. Potentially significant
covariates were introduced as necessary. Differences between groups
on categorical measures were analysed using exact statistical
methods in order to maximise power. Based on the assumption of a
correlation of 0.60 between measurement occasions, power to detect
an effect of one standard deviation difference in change between
groups from baseline to week 36 would have been approximately 80%
if 15 subjects were recruited per group. Since an attrition rate of
15% has been observed in similar populations, 18 patients were
recruited into each arm.
Results
[0487] Subject Recruitment and Demographics:
[0488] Thirty-six subjects were recruited over a 12 month period
commencing April 2000 (FIG. 7). Of these, 32 had sufficient data
for per protocol analysis. Two subjects were lost from each
arm.
[0489] The baseline illness severity factor was created, as
planned, by division of the sample into two groups at the median
ADAS-cog score at baseline (values <25, .gtoreq.25), yielding
less-severely and more-severely affected groups (n=8 and 8 in the
treatment arm and n=7 and 9 in the placebo arm, respectively).
[0490] The groups did not differ across demographic, biological and
clinical parameters at baseline (Table 14), other than the
treatment arm having a higher mean premorbid IQ than the placebo
group as estimated using the NART (111.4 compared to 104.9;
t(30)=2.27, p=0.031) and a lower level of thyroid stimulating
hormone (TSH) (1.14 compared to 2.00 mU/L; t(30)=4.400,
p<0.001). The NART and TSH were subsequently provisionally
entered into analyses as co-variates but were found to be not
significant in any analysis.
[0491] Clinical Effects:
[0492] Changes in the ADAS-cog score at weeks 4, 12, 24 and 36 from
baseline were subject to two-way analysis of variance with factors
of treatment arm and baseline illness severity. The means of the
changes in ADAS-cog score showed greater deterioration in the
placebo treated group at each examination interval, compared to the
PBT-1-treated group (FIG. 8A). This trend came close to statistical
significance at week 4 [F(1,28)=3.55, p=0.070] and week 24
[F(1,28)=3.31, p=0.080] (FIG. 8A). As planned in the protocol, the
effect of severity of illness was examined by stratification of the
sample into subjects less- or more-severely affected (baseline
ADAS-cog values <25, .gtoreq.25). Simple effects tests within
level of severity showed the trend in the pooled groups to be
separable into non-significant results for the less-severe stratum
on all weeks and significant differences in the more-severe stratum
at weeks 4 [F(1,28)=7.73, p=0.010] and week 24 [F(1,28)=6.63,
p=0.016] (FIG. 8B). This trend was maintained at week 36 but
narrowly escaped statistical significance [F(1,28)=3.62, p=0.068].
In the more-severely affected groups, the difference in mean change
from baseline ADAS-cog score of PBT-1 over placebo at weeks 24 and
36 was a difference of 7.37 (95% CI: 1.51-13.24) and 6.36 (95% CI:
-0.50-13.23) respectively (FIG. 8B).
[0493] Effects on Plasma A.beta., Zn and Cu:
[0494] At baseline, there were no significant differences in plasma
A.beta..sub.42 levels between treatment arms or severity strata.
The variance in individual levels at baseline in plasma
A.beta..sub.40/42 was large and led to reduced power of the study
to detect any significant differences in mean changes between
groups. However, reference of individual AR levels to baseline
reference levels markedly decreased variance, and revealed
significant treatment effects. Plasma A.beta..sub.42 showed a
significant decline from baseline in the PBT-1-treated group from
week 20 onwards; over the same time, plasma A.beta..sub.42 in the
placebo group increased (FIG. 9A). Stratification by illness
severity as above demonstrated that changes were evident only in
the less-severely affected (FIG. 9B).
[0495] Administration of PBT-1 was associated with a significant
elevation (.apprxeq.30%) of total plasma Zn (FIG. 10A) but with no
effect on plasma Cu (FIG. 10B). Mean baseline levels of Zn (9.4
.mu.M) in the pooled AD groups were below age-related normative
values (Wood and Zheng, 1997). The increase in plasma Zn induced by
PBT-1 treatment therefore represented a normalization of levels. In
contrast, mean baseline levels of Cu (13.1 .mu.M) were within the
age-related normative range (Rahil-Khazen et al., 2000).
Correlation of plasma A.beta..sub.42/40 levels with Zn/Cu levels
assayed on the same or subsequent occasions showed no significant
associations.
[0496] An important result of treatment of AD subjects with PBT-1
is the paradoxical elevation in plasma Zn (FIG. 10A), which is
consistent with a restoration in the ZnT3-mediated communication of
synaptic zinc with the blood. This also indicates that, in contrast
to a typical metal chelator such as desferrioxamine, the mechanism
of action of PBT-1 at this dose is not that of a gross tissue
chelator. The relatively weak affinity of PBT-1 for the metals
appears to be insufficient to cause marked systemic metal depletion
in the presence of a re-established equilibrium of metal
homeostasis.
[0497] Blood levels of PBT-1: Steady state pre-dose levels of PBT-1
at total daily dosages of 250, 500 and 750 mg were 4.03.+-.2.10,
6.74.+-.3.70, 7.60.+-.2.15 .mu.g/ml, respectively, and did not show
significant correlations with ADAS-cog, metal or A.beta. levels
assayed on the same or subsequent occasions.
TABLE-US-00027 TABLE 14 Baseline demographics and key clinical
variables Group Total Sample Clioquinol Placebo Variable (n = 32)
(n = 16) (n = 16) P Value Age mean 72.50 73.19 71.81 P =
0.65.dagger. (SD; min-max) (8.37; 56-87) (8.61; 58-87) (8.35;
56-87) Sex 17 8 9 P = 1.00.dagger-dbl. (n; % male) (53.1%) (47.1%)
(52.9%) ApoE status ApoE4 heterozygote n (%) 15 7 8 P =
1.00.dagger-dbl. (46.9%) (43.8%) (50.0%) ApoE4 homozygote n (%) 3 2
1 (9.4%) (12.5%) (6.3%) Estimated premorbid IQ 108.1 111.4 104.9 P
= 0.03.dagger. NART (8.86; 91-124) (8.04; 94-121) (8.26; 91-124)
mean, (SD; min-max) ADAS-Cog 26.31 25.56 27.06 P = 0.57.dagger.
(7.27; 15-46) (7.67; 15-46) (7.01; 19-41) Age of first diagnosis
70.09 70.88 69.31 P = 0.59.dagger. mean, (SD; min-max) (7.98;
54-83) (8.50; 57-83) (7.61; 54-83) Duration of illness (years) 2.41
2.31 2.56 P = 0.66.dagger. mean (SD; min-max) (1.19; 1-5) (1.08;
1-4) (1.32; 1-5) .dagger.Independent sample t-test (all tests 30
df) .dagger-dbl.Exact, two-tailed test.
Example 22
PBT1033 Effects in Transgenic Huntington's Mice (R6/2)
[0498] This example describes an in vivo study of PBT1033 in
transgenic Huntington's mice (R6/2) using the procedure of Nyuyen,
Harby and Massa, 2005. During this study, 44 samples were collected
for behavioral tests, including Rotarod performance, clasping
tests, body weight and lifespan observations, and 24 samples were
utilized for pathology assessments, including brain sectioning and
staining for measuring lateral ventricle size, brain weight, whole
brain western blotting for mutant Htt aggregate accumulation, and
immunocytochemistry for Htt aggregates.
Methods
[0499] 1. SAMPLE GROUPS
GROUP A: 10+6(pathology) R6/2 MICE, treated daily with PBT1033
GROUP B: 10+6(pathology) R6/2 MICE, treated daily with SSV solution
GROUP C: 12+6(pathology) wild type (WT) mice, treated daily with
PBT1033 GROUP D: 12+6(pathology) WT mice, treated daily with SSV
solution
[0500] 2. MICE SOURCE
[0501] The first generation of the colony of transgenic mice R6/2
was ordered. WT males were bred with females that were transplanted
with HDexon1/+ ovaries.
[0502] From 2.sup.nd generation, R6/2 positive males were used to
breed with WT females. Samples taken were the 3 to 6th
generation.
[0503] 3. PBT1033 in SSV solution was orally administered to mice
at 0.6 mg/0.1 ml (100 ml)/20 g. The PBT1033 solution was taken up
into a 1-cc syringe attached to a feeding needle and the drug was
delivered by oral gavage.
Components of SSV (Standard Suspending Vehicle)
TABLE-US-00028 [0504] Composition Conc. 500 ml NaCl 0.9%(w/v) 4.5 g
Na-CMC 0.5%(w/v) 2.5 g Benzyl alcohol 0.5% (v/v) 2.5 ml TWeen 80
0.4% (v/v) 2.0 ml
[0505] 4. Rotarod was assessed as described by Hockly et al., the
contents of which are incorporated by reference, with the
modification that the SDI Rota-Rod (San Diego Instruments, San
Diego), was set to linearly increase in speed to 40 rpm in 4-min
ramp time. Performance is at 4.sup.th, 8.sup.th, 10.sup.th, and
12.sup.th week, with three days (one for training) per period and
three times per day.
[0506] 5. Body Weight: once per week, start at age 3 weeks.
[0507] 6. Clasping testing and Behavioral observation: once per
week from 5.sup.th week and daily during the drug treating.
[0508] 7. Lifespan observation: Group A and B were treated until
they died.
[0509] 8. Brain lateral ventricle size: mouse brain perfusion,
sectioning and staining at age 10 weeks. Mice brains were perfused
and fixed with 4% PFA and stained with H&E.
[0510] 9. Whole Brain Western Blotting: whole mouse brain was
homogenized in buffer (10 mM Tris HCl (pH 7.4) 11 mM EDTA Na2/1%
Triton X100/complete protease inhibitor mixture and then
centrifuged at 7000.times.g for 90 s. The supernatant was boiled in
SDS sample buffer for 5 mm at 100.degree. C.
[0511] 40 micrograms of total protein was run on a 10% SDS gel with
a 4% stacking gel. Antibody: anti-GAPDH (1:5000), EM48 anti-Htt
antibody (1:1000)
[0512] 10. Brain weight: brain weighed before freezing at
-80.degree. C.
[0513] 11. Immunohistochemistry was performed in accordance with
the procedure in Assay 15, hereinabove.
SUMMARY
[0514] 1. There was significant difference for rotarod performance
between A and B groups (FIGS. 12 and 13). The rotarod performance
time in Group B was less in the 10th week (18.+-.1 seconds,
compared with 28.+-.4 seconds in Group A, P<0.05) and 12 week
(13.+-.1 seconds in Group B, compared with 26.+-.4 seconds in Group
A, P<0.05).
[0515] 2. As shown in FIGS. 14 and 15, the clasping latency and
duration in Group B were significantly greater than Group A.
[0516] 3. As shown in FIG. 16, the body weight of mice in Group B
was significantly decreased as the week increased (P<0.05, vs A,
C, and D groups) of birth.
[0517] 4. FIG. 17 shows the life spans of the A and B groups.
Animals in Group A survived significantly longer than mice in Group
B.
[0518] 5. Lateral ventricle area in Group B was significantly
larger (0.62.+-.0.037 mm2, P<0.05, compared with A, C, and D
groups) (FIG. 18). The lateral ventricle area of Group A was
0.36.+-.0.086 mm.sup.2.
[0519] 6. Brain Weight data also showed the difference between
treated and untreated groups (FIG. 19). The brain weight of group B
(0.321.+-.0.0298) was less than group A (0.373.+-.0.0228,
P<0.05).
[0520] 7. Western analysis shows that mutant Huntingtin (Htt)
protein aggregates are detected in untreated HDexon1 mice, but not
in treated animals (FIG. 20).
[0521] 8. FIG. 21 shows the mice brain photos for WT, R6/2 and R6/2
mice treated with PBT1033.
Example 23
Clinical Development of PBT 1033
[0522] 1033 was administered to healthy volunteers in a 2-part
study comprising single dose administration (Stage A) and multidose
administration (Stage B). Overall, 1033 was generally well
tolerated as a single dose in young male volunteers and up to 7
days of treatment in elderly healthy volunteers.
[0523] The objectives of the following trials were to determine the
safety, tolerability and pharmacokinetics of single and multiple
oral doses of 1033 in healthy volunteers. Double-blind studies were
conducted. The protocol included safety measures designed to
capture the potential human adverse effects. A total of 65 healthy
subjects have been exposed to doses of 1033 (41 single dose, 24
multiple dose).
TABLE-US-00029 Disposition of Subjects - Stages A & B Number of
Subjects (N) 1033 (mg) Stage Sex Placebo 25 50 100 200 300 400 500
600 800 Total A M 14 5 6 6 6 6 -- 6 -- 6 55 B M 4 -- -- -- 3 -- 3
-- 3 3 16 F 4 -- -- -- 3 -- 3 -- 3 3 16 Total 8 -- -- -- 6 -- 6 --
6 6 32
Safety and Tolerability Stage A--Single Dose
[0524] In Stage A, fifty five (55) healthy male subjects aged 18-50
years were randomised (3:1) to receive a single oral dose of either
placebo or 1033 at one of 7 dose levels (25, 50, 100, 200, 300, 500
or 800 mg).
[0525] Analysis from Stage A demonstrated that 1033, when
administered as a single oral dose, was well tolerated in healthy
male volunteers aged 18 to 50 years. There was no difference in the
incidence of adverse events between the 1033 (43.9%) and placebo
arms (42.9%).
[0526] No clinically significant or relevant changes were observed
for clinical laboratory evaluations and physical assessments.
[0527] In this study a relationship between the dose range and the
number and intensity of the adverse effects could not be
established.
Safety and Tolerability Stage B--Multiple Dose
[0528] In Stage B, thirty two (32) healthy male and female subjects
aged 45-75 years (eight subjects per dose level) were randomised
(3:1) to receive a daily oral dose over 7 consecutive days of
either placebo or 1033 at one of 4 dose levels (200, 400, 600 or
800 mg). Each dose level comprised of 4 males and 4 females; the
randomisation schedule ensuring that 1 subject/sex received placebo
and 3 subjects/sex received 1033.
[0529] Analysis of Stage B indicates that 1033 was generally well
tolerated in healthy elderly subjects.
Example 24
Effect of PBT 1033 Against Glioma Tumours
[0530] The following Table provides the property and structure of
1033.
TABLE-US-00030 In vivo Efficacy and Safety Profile Cytotoxicity
ClogP (% viable ElogD Toxicity.sup.b at 1 Parent (E) or at Mice and
MW/ ClogD 30 mg/ plasma B:P 10 Um).sup.a PSA (C) kg
concentration.sup.c Ratio.sup.d ##STR00283## Neuronal cells: 84, 72
M17 cells: 94, 54.3 271.1 36.36 3.51 (C) 1.07 None Up to 500 ng/ml
12.85 at 5 min, 9.45 at 60 min
1033 was screened for in vitro efficacy and in vivo efficacy.
[0531] Emulsion carrier was used as a control for the in vitro and
in vivo test systems. All the agents were tested initially via in
vitro testing to determine an efficacy profile with three glioma
cell lines and a control cell line. The results are shown in FIGS.
22 to 26.
Experimental Design
In Vitro Efficacy Protocol
[0532] The in vitro efficacy of the test articles were analyzed via
the MTT cell viability assay.
The following cell lines were used to determine cell viability on
exposure to the test articles:
[0533] C6--rat glioma cell line (FIG. 22), VMDK--mouse glioma cell
line (FIG. 24),
[0534] U87MG--human glioma cell line (FIG. 23), 3T3-- Control cell
line (FIG. 25).
[0535] Cells were plated in 96 well plates with 100 .mu.l of cell
culture medium and be allowed to adhere over 24 hours allowing for
approximately 50% confluence. At 24 hours, the cell medium was
replaced with fresh cell culture medium containing agents or the
carrier emulsions.
[0536] The cells will then be incubated and grown for a designated
period (72 hours) after which the MTT solution were added to the
wells and incubated at 37.degree. C. for 1-2 hours. The absorbance
of each well will then be measured with a plate reader at 570 nm.
The efficacy profiles were calculated relevant to the cells
incubated in the absence of the agents over the course of the
experiment.
In Vivo Efficacy Protocol
[0537] 2 doses were used: at the maximum tolerated dose and one
level below the maximum tolerated dose.
[0538] 3 mouse models are employed: [0539] C6--xenograft mouse
model (glioma model) [0540] SMA560--VMDK mouse model (glioma model)
[0541] U87MG--nude mouse model
C6--CBA Xenograft Model (ATCC Number: CCL-107)
[0542] This model is used to screen 1033.
SMA560--VMDK Mouse Model (ATCC Number: CCL-163)
[0543] This model is used to screen 1033 screened previously with
the C6 xenograft model.
U87MG Nude Mouse Model (ATCC Number: CRL-9589)
[0544] This model is used to screen 1033.
[0545] Initially, CBA mice are used to receive an intracranial
inoculation of the C6 glioma cells. Briefly, 1.times.10.sup.6 cells
are inoculated into the left hemisphere via at day 5 post C6 cell
inoculation. The mice receive daily intraperitoneal (ip)
administration of test articles in a carrier emulsion or carrier
emulsion alone as a control for 8 days until day 12. At day 14, the
mice are euthanized via CO.sub.2 inhalation and the brain removed
for histological processing.
[0546] The VMDK mouse strain is then used to screen the identical
test articles and carrier emulsions as per the C6 xenograft model
in the CBA mice. The VMDK mice received an inoculation of
1.times.10.sup.5 SMA560 cells into the left hemisphere via standard
methods. At day 5 post SMA560 cell inoculation, the mice receive
daily ip administration of agents in a carrier emulsion or carrier
emulsion alone as a control for 12 days until day 16. Identical
doses of test articles and carrier emulsions as used in the C6
xenograft model are used with the SMA560 model. At day 18, the mice
are euthanized via CO.sub.2 inhalation and the brain removed for
histological processing.
[0547] A nude mouse model utilizing the U87MG human glioma cell
line is used to screen agents. The nude mouse Nu/nu strain receives
an inoculation of 1.times.10.sup.6 U87MG cells into the left
hemisphere. At day 5 post U87MG cell inoculation, the mice receive
daily ip administration of the agent or carrier emulsion alone as a
control for 12 days until day 16. At day 18, the mice are
euthanized via CO.sub.2 inhalation and the brain removed for
histological processing.
[0548] Haematoxylin and eosin stained sections are used to measure
tumor dimensions in order to determine the efficacy of the test
agents on tumor growth relative to the control mice.
[0549] The results of the effect of compound 1033 is shown in FIGS.
26(a) to (d).
[0550] The graphs can be summarized as follows: Y-axis refers to
the tumor area in pixels.
[0551] The numbers in the bars refer to the total mice in each
group (mice that were found dead very early or brains could not be
sampled due to the head being chewed by other mice were not
included).
[0552] The numbers with the asterix refer to the groups were mice
may have been culled 1 or 2 days early due to being ill or found
dead. These have been included in the final calculations as their
tumors were fairly large and were they were culled close to the
final cull point.
[0553] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
[0554] References cited herein are listed on the following pages,
and are incorporated herein by this reference.
REFERENCES
[0555] Alvarez, A., Alarcon, R., Opaza, C., Campos, E. O., Munoz,
F. J., Calderon, F. H., Dajas, F., Gentry, M. K., Doctor, B. P., De
Mello, F., Inestrosa, N. C. (1998) Stable complexes involving
acetylcholinesterase and amyloid-.beta. peptide change the
biochemical properties of the enzyme and increase the neurotoxicity
of alzheimer's fibrils. The Journal of Neuroscience,
18(9):3213-3223 [0556] Ariga, T., Kobayashi, K., Hasegawa, A.,
Kiso, M., Ishida, H., and Miyatake, T. (2001) Characterization of
high-affinity binding between gangliosides and amyloid 0-protein.
Arch. Biochem. Biophys. 388, 225-230 [0557] Avdulov, N. A.,
Chochina, S. V., Igbavboa, U., O'Hare, E. O., Schroeder, F.,
Cleary, J. P., and Wood, W. G. (1997) Lipid binding to amyloid
b-peptide aggregates: preferential binding of cholesterol a. J.
Neurochem. 68, 2086-2091 [0558] Beyreuther K, Christen Y, Masters C
L (eds) Neurodegenerative Disorders: Loss of Function Through Gain
of Function. Springer. Berlin. 2001. 189 [0559] Brower V.
Harnessing the immune system to battle Alzheimer's: Some of the
most promising approaches to fight Alzheimer's diseases aim to
develop vaccines. EMBO Rep 2002; 3:207-9 [0560] Bush A I, Masters C
L. Clioquinol's return. Science 2001; 292:2251-2252 [0561] Bush A
I. Therapeutic targets in the biology of Alzheimer's disease.
Current Opinion in Psychiatry 2001; 14:341-348 [0562] Carissimi, M.
(1972) U.S. Pat. No. 3,682,927 [0563] Cherny R A, Atwood C S,
Xilinas M E et al. Treatment with a copper-zinc chelator markedly
and rapidly inhibits .beta.-amyloid accumulation in Alzheimer's
disease transgenic mice. Neuron 2001; 30:665-676 [0564] R. C.
Corcoran and S. H. Bang, Tetrahedron Lett., 1990, 31, 6757-6758.
[0565] Corder, E. H., Saunders, A. M., Strittmatter, W. J.,
Schmechel, D. E., Gaskell, P. C., Small, G. W., Haines, J. L., and
Pericak-Vance, M. A. (1993) Gene dose of apolipoprotein E type 4
allele and the risk of Alzheimer's disease in the late onset
familial disease. Science 261, 921-923 [0566] Cronin-Golomb A,
Sugiura R, Corkin S, Growdon J H. Incomplete achromatopsia in
Alzheimer's disease. Neurobiol Aging 1993; 14: 471-477 [0567]
Curtain, C. C., Ali, F., Volitakis, I., Cherny, R. A., Norton, R.
S., Beyreuther, K., Barrow, C. J., Masters, C. L., Bush, A. I., and
Barnham, K. J. (2001) Alzheimer's disease amyloid .beta. binds
copper and zinc to generate an allosterically ordered
membrane-penetrating structure containing superoxide dismutase-like
subunits. J. Biol. Chem. 276, 20466-20473 [0568] Czech, C., Forstl,
H., Hentschel, F., Monning, U., Besthorn, C., Geigerkabisch, C.,
Sattel, H., Masters, C., and Beyruether, K. (1994) Apolipoprotein
E-4 gene dose in clinically disgnosed Alzhiemer's disease:
prevalence, plasma cholesterol levels and cerebrovascular change.
Eur. Arch. Psychiatry Clin. Neurosci. 243, 291-292 [0569] De
Ferrari, G. V., Canales, M. A., Shin, I., Weiner, L. M., Silman,
I., Inestrosa, N. C. (2001) A structural motif or
acetylcholinesterase that promotes amyloid beta-peptide fibril
formation. Biochemistry 40(35):10447-57 [0570] Dodart J-C, Bales K
R, Gannon K S et al. Immunization reverses memory deficits without
reducing brain A.beta. burden in Alzheimer's disease model. Nat
Neurosci 2002; 5: 452-457 [0571] A. Dondoni, G. Fantin, M.
Fogagnolo, A. Medici and P. Pedrini, Synthesis, 1987, 998 1001.
[0572] A. Dondoni, F. L. Merchan, P. Merino, I. Rojo and T. Tejero,
Synthesis, 1996, 641-646 Durant, G. J., Emmett, J. C., Ganellin, C.
R., Roe, A. M. and Slater, R. A. J. Med. Chem., 1976, 19, 923.
[0573] Eckert, G. P., Cairns, N. J., Maras, A., Gattaz, W. F., and
Muller, W. E. (2000) Cholesterol modulates the membrane-disordering
effects of .beta.-amyloid peptides in the hippocampus specific
changes in Alzheimer's disease. Dement. Geriatr. [0574] Cogn.
Disord. 11, 181-186 [0575] Fassbender, K., Simons, M., Bergmann,
C., Stroick, M., Lutjohann, D., Keller. P., Runz, H., Kuhl, S.,
Bertsch, T., von Bergmann. K., Hennerici, M., Beyreuther, K., and
Hartmann, T. (2001) Simvastatin strongly reduces levels of
Alzheimer's disease .beta.-amyloid peptides A.beta.42 and A.beta.40
in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 98, 5856-5861
[0576] Fleming, W. C. and Pettit, G. R. J. Org. Chem., 1971, 36,
3490-3493. [0577] Folstein M F, Folstein S E, McHugh P R.
Mini-mental state: a practical method for grading the cognitive
state of patients for the clinician. J. Psychiatr. Res. 1975;
12:189-198 [0578] Frears, E. R., Stephens, D. J., Walters, C. E.,
Davies, H., and Austen, B. M. (1999) The role of cholesterol in the
biosynthesis of b-amyloid. NeuroReport 10, 1699-1705 [0579]
Friedhoff, L. T., Cullen, E. I., Geoghagen, N. S., and Buxbaum, J.
D. (2001) Treatment with controlled-release lovastatin decreases
serum concentrations of human .beta.-amyloid (A.beta.) peptide.
Int. J. Neuropsychopharmacol. 4, 127-130 [0580] Gershon, H.,
Clarke, D. D. and Gershon, M. Monashafte fur Chemie, 1999, 130,
653. [0581] Gershon, H. and McNeil, W. M. J. Heterocycl. Chem.,
1971, 8, 821. [0582] Gordon, L. M., Curtain, C. C (1988). In: Aloia
R. C, Curtain C. C, Gordon L. M. (eds) Advances in Membrane
Fluidity 1: Methods for Studying Membrane Fluidity. Alan R. Liss
New York, pp 25-89 [0583] Hartmann, T. (2001) Cholesterol, A.beta.
and Alzheimer's disease. Trends Neurosci. 24, S45-S48 [0584]
Hertel, C., Terzi, E., Hauser, N., Jakob-Rotne, R., Seelig, J., and
Kemp, J. A. (1997) Inhibition of the electrostatic interaction
between .beta.-amyloid peptide and membranes prevents
.beta.-amyloid-induced toxicity. Proc. Natl. Acad. Sci. USA. 94,
9412-9416 [0585] Hobara N, Taketa, K. Electrophoretic studies of
clioquinol binding to human serum proteins. Biochem Pharmacol 1976;
25: 1601-1606 [0586] Hockly, E. Woodman, B., Mahal, A., Lewis, C.
M. & Bates, G. (2003), Brain Res. Bull. 61, 469-479. [0587]
Hope, M. J., Bally, M. B., Webb, G., Cullis, P. R. (1985) Biochim.
Biophys. Acta. 812, 55-56 [0588] Hsiao, K., Chapman, P., Nilsen,
S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., Cole, G.
(1996) Correlative memory deficits, A.beta. elevation, and amyloid
plaques in transgenic mice Science; 274(5284):99-102. [0589] Huang
X, Atwood C S, Hartshorn M A et al. The A.beta. peptide of
Alzheimer's disease directly produces hydrogen peroxide through
metal ion reduction. Biochemistry 1999; 38:7609-7616 [0590]
Hubbell, W. L. and McConnell, H. M. (1971) J. Amer. Chem. Soc. 93,
314-326 [0591] Inestrosa, N. C. Alvarez, A., Perez, C. A., Moreno,
R. D., Vicente, M., Linker, C., Casaneuva, O. I., Soto, C.,
Garrido, J. (1996) Acetylcholinesterase accelerates assembly of
amyloid-beta-peptides into Alzheimer's fibrils: possible role of
the peripheral site of the enzyme. Neuron 16(4):881-91 [0592]
Jensen M, Schroder J, Blomberg M et al. Cerebrospinal fluid A1342
is increased early in sporadic Alzheimer's disease and declines
with disease progression. Ann Neurol 1999; 45: 504-511 [0593] Ji,
S. R., Wu, Y., and Sui, S. F. (2002) Cholesterol is an important
factor affecting the membrane insertion of .beta.-amyloid peptide
(A.beta. 1-40), which may potentially inhibit the fibril formation.
J. Biol. Chem. 277, 6273-6279 [0594] Lee J-Y, Cole T B, Palmiter R
D, Suh S W, Koh J-Y. Contribution by synaptic zinc to the
gender-disparate plaque formation in human Swedish mutant APP
transgenic mice. Proc Natl Acad Sci USA 2002: Early edition. [0595]
Mahfoud, R., Garmy, N., Maresca, M., Yahi, N., Puigserver, A, and
Fantini, J. (2002) Identification of a common sphingolipid-binding
domain in Alzheimer, Prion, and HIV-1 proteins. J. Biol. Chem. 277,
11292-11296 [0596] McKhann G, Drachman D, Folstein M F, Katzman R,
Price D, Stadlen E. Clinical diagnosis of Alzheimer's disease:
Report of the NINCDS-ADRDA work group under the auspices of the
Department of Health and Human Services Task Force on Alzheimer's
Disease. Neurology 1984; 34:939-944 [0597] McLean C A, Cherny R A,
Fraser F W et al. Soluble pool of A.beta. amyloid as a determinant
of severity of neurodegeneration in Alzheimer's disease. Ann Neurol
1999; 46:860-866 [0598] Nguyen, T, Hamby, A and Maisa, S. T (2005),
"Clinoquinol down-regulates mutant hungtingtin expression in vitro
and mitigates pathology in a Huntington's disease mouse model",
PNAS, Vol 102; No. 33; 11840-11845. [0599] Nunan, J., and Small, D.
H. (2000) Regulation of APP cleavage by .alpha.-, .beta.- and
.delta.-secretases. FEBS Lett. 483, 6-10 [0600] Ostrovskaya, V. M.,
Krasavin, I. A., Inshakova, V. A., Mamaev, V. P. and Krivopalov, V.
P. (1986) SU Patent 1216184 A1. [0601] Petersen, R. C, Stevenas, J.
C., Ganguli, M., Tangalos, E. G., Cummings, J. L., and DeKosky, S.
T. Practice parameter: Early detection of dementia: Mild cognitive
impairment Neurology 2001 56 1133-1142 [0602] Rahil-Khazen R,
Bolann B J, Ulvik Rj. Trace element reference values in serum
determined by inductively coupled plasma atomic emission
spectrometry. Clin Chem Lab Med 2000; 38 (8); 765-72. [0603]
Regland B, Lehmann W, Abedini I et al. Treatment of Alzheimer's
disease with clioquinol. Dement Geriatr Cogn Disord 2001; 12:408-14
[0604] Richard, J. A., Breen, G. F., Crawford, L. P., Grinter, T.
J., Harris, M. A., Haynes, J. F., Moores, C. J., Saunders, R. N.,
Share, A. C., Walsgrove, T. C. and Wicks, C. Organic Process
Research & Development, 1997, 1, 185. [0605] Rogers S L, Farlow
M R, Doody R S, Mohs R, Friedhoff L T. A 24-week, double-blind,
placebo-controlled trial of donepezil in patients with Alzheimer's
disease. Donepezil Study Group. Neurology 1998; 50:136-45 [0606]
Rosen W G, Mohs R C, Davis K L. A new rating scale for Alzheimer's
disease. Am J Psychiatry 1984; 141:1356-64 [0607] Schenk, D.,
Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K.,
Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M.,
Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F.,
Shopp, G., Vasquez, N., Vandervert, C., Walker, S., Wogulis, M.,
Yednock, T., Games, D., and Seubert, P. (1999) Immunization with
amyloid-.beta. attenuates Alzheimer's disease like pathology in the
PDAPP mouse. Nature 400, 173-177 [0608] Selkoe, D. J. Alzheimer's
disease: genes, proteins and therapy. Physiol Rev 81 (2): 741-766
[0609] Shearman M S, Beher D, Clarke E E et al. L-685,458, an
aspartyl protease transition state mimic, is a potent inhibitor of
amyloid .beta.-protein precursor .beta.-secretase activity.
Biochemistry 2000; 29:8698-704 [0610] Shrader, W. D. Celebuski, J.
Kline S. J. and Johnson, D. Tetrahedron Lett., 1988, 29, 1351-1354.
[0611] Shin, I., Silman, I., Weiner, L. M. (1996) Interaction of
partially unfolded forms of Torpedo acetylcholinesterase with
liposomes. Protein Sci 5(1):42-51 [0612] Shiraki, H. The
neuropathology of subacute myelo-optico-neuropathy (SMON) in the
humans: With special reference to the quinoform intoxication. Jpn J
Med Sci Biol 1975; 28 (suppl): 101-164 [0613] Simons M, Schwarzler
F, Lutjohann D et al. Treatment with simvastatin in
normocholesterolemic patients with Alzheimer's disease: a 26-week
randomised, placebo-controlled, double-blind trial. Ann of Neurol
In Press. [0614] Sinha S, Anderson J P, Barbour R et al.
Purification and cloning of amyloid precursor protein
.beta.-secretase from human brain. Nature 1999; 402:537-40 [0615]
St George-Hyslop, P. H. (2000) Molecular genetics of Alzheimer's
disease. Biol. Psychiatry 47, 183-199 [0616] T. C. Wang, Y. L.
Chen, K. H. Lee and C. C. Tzeng, Tetrahedron Lett., 1996, 37,
6369-6370. [0617] White et al., J Neuroscience, (1998) 18,
6207-6217 [0618] Valdez-Gonzalez, T., Inagawa, J., and Ido, T.
(2001) Neuropeptides interact with glycolipid receptors: a surface
plasmon resonance study. Peptides 22; 1099-1106. [0619] Wright, J.
S. Johnson, E. R. and DiLabio, G. A. J. Am. Chem. Soc 2001 123
1173-1183. Yassin M S, Ekblom J, Xilinas M, Gottfries C G, Oreland
L. Changes in uptake of vitamin B(12) and trace metals in brains of
mice treated with clioquinol. J Neurol Sci 2000; 173:40-44
* * * * *
References