Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVEL COMPOUNDS AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to novel compounds for use as inhibitors of
insulin-regulated
aminopeptidase (IRAP) and methods for inhibiting same, as well as compositions
comprising said compounds. In particular, the compounds of the present
invention may be
useful in therapeutic applications including enhancing memory and learning
functions.
DESCRIPTION OF THE PRIOR ART
The reference in this specification to any prior publication (or information
derived from it),
or to any matter which is known, is not, and should not be taken as an
acknowledgment or
admission or any form of suggestion that that prior publication (or
information derived
from it) or known matter forms part of the common general knowledge in the
field of
endeavour to which this specification relates.
Insulin-regulated aminopeptidase (IRAP) is a 165 kDa glycoprotein that is
widely
distributed in many tissues including fat, muscles, kidney, adrenal, lung and
heart (Keller
et al., 1995; Rogi et al., 1996; Zhang et al., 1999). In the brain, it occurs
as a smaller 140
kDa protein, probably due to differential glycosylation (Keller et al., 1995;
Zhang et al.,
1999). It is a type II integral membrane protein belonging to the MI family of
zinc-
dependent metallopeptidases and possesses a large C-terminal extracellular
tail which
contains the catalytic site, a single transmembrane domain and a smaller N-
terminal
intracellular domain (Keller et al., 1995; Rogi et al., 1996). Initially
cloned from a rat
epididymal fat pad cDNA library as a marker protein (vp165) for a specialised
vesicle
containing the insulin-responsive glucose transporter GLUT4 (Keller et al.,
1995), the
same protein was cloned concurrently from human placental cDNA library as
oxytocinase
(Rogi et al., 1996), an enzyme which was thought to have an important role in
degrading
oxytocin. The AT4 receptor has also recently been identified as the
transmembrane
enzyme insulin regulated aminopeptidase (IRAP) via mass spectral analysis of
tryptic
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peptides generated from AT4 receptor protein purified from bovine adrenal
membranes
(Albiston et al., 2001). Analysis of the biochemical and pharmacological
properties of
IRAP confirm that it is, in fact, the AT4 receptor (Albiston et al., 2001).
Although isolated
by three independent groups from different tissue sources and thought to
subserve distinct
physiological roles, properties and characteristics of this protein remain
consistent.
The AT4 ligands, angiotensin IV (Ang IV), its analogues Nle-Ang IV and
Norleucinal Ang
IV, and the structurally distinct peptide LVV-hemorphin 7 (LVV-H7), all bind
with high
affinity and relative specificity to a pharmacologically distinct binding
site, termed the AT4
receptor. All the AT4 ligands, Ang IV, Nle-Ang IV, and LVV-H7, were
demonstrated in
vitro to be inhibitors of the aminopeptidase activity of IRAP as assessed by
cleavage of the
synthetic substrate Leu-l3-naphthylamide (Albiston et al., 2001; Lew et al.,
2003). Both
Ang IV and LVV-H7 display competitive kinetics indicating that AT4 ligands
mediate
their effects by binding to the catalytic site of IRAP. Using the same system
it has also
been demonstrated that although the peptides Ang IV and LVV-H7 bind to the
catalytic
site they are not cleaved by IRAP (Lew et al., 2003).
Central administration of the peptide AT4 ligands, Ang IV, its more stable
analogues, or
LVV-H7, in normal animals has been shown to lead to improved performance of
memory
tasks in both passive avoidance and spatial learning paradigms. The initial
effects were
observed in the passive avoidance paradigm in rats where an intracerebro-
ventricular dose
(1 nmol) of Ang IV increased the latency in re-entering the dark chamber after
an aversive
stimulus (Braszko et al., 1988; Wright et al., 1993; Wright et al., 1996).
Chronic infusion
(6 days) of an Ang IV analogue into the lateral ventricle of rats at a dose of
between 0.1
and 0.5 nmol/h enhanced performance in the swim maze, a spatial memory
paradigm. In
the Barnes maze, another spatial learning task, treatment of rats with 100
pmoles or 1 nmol
of the peptide AT4 ligands, Nlel-Ang IV or LVV-H7, decreased the time taken to
achieve
learner criteria in this paradigm (Lee et al., 2004). Control animals treated
with artificial
cerebrospinal fluid took 7 days to achieve learner criteria, whereas animals
treated with
Nlel-Ang IV or LVV-H7, at a concentration of either 100 pmoles or 1 nmole,
achieved
learner criteria in 3-4 days (Lee et al., 2004). This observation strongly
indicates that the
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two peptides tested not only improved memory, but also enhanced spatial
learning.
Not only did peptide AT4 ligands enhance memory and learning in normal
animals, the
peptides reversed memory deficits induced (1) chemically by a muscarinic
antagonist or
(2) mechanically by knife-cut lesion of the perforant pathway. A more stable
analogue of
Ang IV, Nle-Ang IV, given acutely into the lateral ventricles, reversed the
memory deficits
induced by the muscarinic receptor antagonist, scopolamine, in a spatial
learning paradigm
(Pederson et al., 1998; Pederson et al., 2001). In the swim maze paradigm,
memory
deficits induced by bilateral perforant pathway lesion can be reversed by an
acute dose (1
nmol) of another Ang IV analogue, Norleucinal Ang IV (Wright et al., 1999).
The other
AT4 ligand, LVV-H7, given acutely prior to the conditioning trial in the
passive avoidance
paradigm, has also been found to reverse the memory deficit induced by
scopolamine
(Albiston et al., 2004).
The mechanisms for IRAP inhibitors facilitating memory are not fully
understood, but
recent studies implicate neuroendocrine mechanisms of action. Inhibition of
IRAP may
extend the half-life of neuropeptides that modify learning and memory
processes
(Albiston, 2003). A number of IRAP peptide substrates including arginine-
vasopressin,
oxytocin, met-enkephalin, somatostatin, dynorphin and lys-bradykinin have
previously
been associated with memory (Herbst, 1997; Lew, 2003). Moreover, studies have
shown
that peptidergic neurotransmission is altered in neurodegenerative diseases
leading to
memory loss. IRAP is found in high concentrations in brain regions involved in
processing cognitive function including the cerebral cortex, hippocampus,
basal forebrain
and amygdale (Fernando, 2005) where it is co-expressed in neurons with the
glucose
transporter, GLUT4. It has recently been demonstrated that IRAP inhibitors
increase
activity-evoked glucose uptake into the pyramidal neurons of the hippocampus
(J
neurochem submitted). Glucose is a potent modulator of learning and memory in
both
humans and rodents with increases in glucose demand in the hippocampus
occurring
during memory processing (McNay, 2000; Dash, 2006). Therefore, one potential
mechanism by which compounds may facilitate memory is through the potentiation
of
glucose uptake into neurons.
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IRAP therefore provides a target for the development of agents which may
enhance or
improve memory and learning. Accordingly, inhibitors of IRAP, which may
disrupt or
interfere with IRAP functional activity may have useful therapeutic and/or
prophylactic
applications in the treatment of cognitive and memory disorders or in
enhancing memory
and learning.
WO 2006/026832 discloses a class of benzo-fused compounds for use as IRAP
inhibitors.
Nevertheless, there remains the need for identification of compounds which may
be useful
in the treatment or prevention of memory disorders or improve memory or
learning.
SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context
requires
otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will
be understood to imply the inclusion of a stated integer or step or group of
integers or steps
but not the exclusion of any other integer or step or group of integers or
steps.
The singular forms "a", "an" and "the" include plural aspects unless the
context clearly
dictates otherwise.
It has now been found that certain benzo-fused compounds within the scope of
the
disclosure of WO 2006/026832 have an improved IRAP inhibitory activity when
compared
to the most active compound exemplified therein.
Accordingly, in a first aspect, the present invention provides a compound of
Formula (I)
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R3 A
Y
R4 R2
I 1 (1)
X R'
R5
R6
wherein A is aryl, heteroaryl carbocyclyl or heterocyclyl, each of which
may be optionally substituted, when R' is NHCOR8; or
quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,
quinoxalinyl, 1,8-naphthyridyl, phthalazinyl or pteridinyl,
each of which may be optionally substituted, when R' is
NR7Rg, NHCOR8, N(COR8)2, N(COR7)(COR8), N=CHOR8
or N=CHR8;
X is 0, NR or S, wherein R is hydrogen, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted aryl, optionally substituted
acyl, optionally substituted heteroaryl, optionally substituted
carbocyclyl or optionally substituted heterocyclyl;
R' and Rg are independently selected from hydrogen, optionally
substituted alkyl, optionally substituted aryl, or R7 and R8,
together with the nitrogen atom to which they are attached
form a 3-8-membered ring which may be optionally
substituted;
R2 is CN, COZR9, C(O)O(O)R9, C(O)R9 or C(O)NR9R10 wherein
R9 and R10 are independently selected from alkyl, alkenyl,
alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, each of
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which may be optionally substituted, and hydrogen; or R9 and
R10, together with the nitrogen atom to which they are
attached, form a 3-8-membered ring which may be optionally
substituted;
R3-R6 are independently selected from hydrogen, halo, nitro, cyano
alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,
carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy,
alkynyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, amino,
acyl, acyloxy, carboxy, carboxyester, methylenedioxy,
amido, thio, alkylthio, alkenylthio, alkynylthio, arylthio,
heteroarylthio, heterocyclylthio, carbocyclylthio, acylthio and
azido, each of which may be optionally substituted where
appropriate, or any two adjacent R3-Rb, together with the
atoms to which they are attached, form a 3-8-membered ring
which may be optionally substituted; and
Y is hydrogen or Ci_loalkyl,
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
In another aspect, the present invention provides a method for inhibiting IRAP
activity
which comprises contacting IRAP with an inhibitory amount of a compound of
formula
(I), or a pharmaceutically acceptable salt, solvate or prodrug thereof.
Inhibition of IRAP
activity can be performed in vitro or in vivo, such as in vivo in a subject.
The invention
therefore also provides a method for inhibiting IRAP activity in a subject in
need thereof,
which comprises administering to said subject an inhibitory effective amount
of a
compound of formula (I), or a pharmaceutically acceptable salt, solvate or
prodrug thereof.
Compounds described herein may be useful in treating a disease or condition in
which
excessive or undesirable IRAP activity plays a role. Thus, the invention
further provides a
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method for treating a disease or condition in which IRAP activity is
implicated, in a
subject in need thereof, comprising the step of administering to said subject
an effective
amount of a compound of formula (I), or a pharmaceutically acceptable salt,
solvate or
prodrug thereof. The invention also provides for the use of a compound of
formula (I), or
a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a
medicament
for treating a disease or condition in which IRAP activity is implicated, as
well as for the
use of a compound of formula (I), or a pharmaceutically acceptable salt,
solvate or prodrug
thereof, for treating a disease or condition in which IRAP activity is
implicated. In one
embodiment, the disease is Alzheimer's disease.
As described above, IRAP inhibitors have been shown to improve memory and
enhance
spatial learning as well as reversing memory deficits.
Accordingly, in another aspect, the present invention provides a method for
enhancing
memory and/or learning in a subject, comprising the step of administering to
said subject a
compound of formula (I) or a pharmaceutically acceptable salt, solvate or
prodrug thereof.
The invention thus also provides for the use of a compound of formula (I), or
a
pharmaceutically acceptable salt, solvate or prodrug thereof in the
manufacture of a
medicament for enhancing memory and/or learning in a subject, as well as for
the use of a
compound of formula (I), or a pharmaceutically acceptable salt, solvate or
prodrug thereof,
for enhancing memory and/or learning in a subject.
The invention also provides agents and compositions comprising a compound
according to
Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof
together with
a pharmaceutically acceptable carrier or excipient.
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DESCRIPTION OF THE INVENTION
As used herein, the term "alkyl" or "alk", used either alone or in compound
words denotes
straight chain, or branched alkyl, preferably Ci.20 alkyl, e.g. CI.10 or CI_6.
Examples of
straight chain and branched alkyl include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-
butyl, t-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1-dimethyl-propyl, hexyl, 4-
methylpentyl, 1-
methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-
dimethylbutyl, 3,3-
dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2,-trimethylpropyl,
1,1,2-
trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-
dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl,
1,4-
dimethyl-pentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-
trimethylbutyl, octyl, 6-
methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-,
5-, 6- or 7-
methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl,
1-, 2-, 3-, 4-, 5-,
6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-
propylheptyl,
undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-,
6- or 7-ethylnonyl,
1-, 2-, 3-, 4- or 5-propylocytl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl,
dodecyl, 1-, 2-, 3-, 4-,
5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-
ethyldecyl, 1-, 2-, 3-, 4-,
5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2-pentylheptyl and the
like. Where an
alkyl group is referred to generally as "propyl", butyl" etc, it will be
understood that this
can refer to any of straight or branched isomers where appropriate. An alkyl
group may be
optionally substituted by one or more optional substituents as herein defined.
The term "alkenyl" as used herein denotes groups formed from straight chain or
branched
hydrocarbon residues containing at least one carbon to carbon double bond
including
ethylenically mono-, di- or poly-unsaturated alkyl groups as previously
defined, preferably
CZ_20 alkenyl (e.g. C2_10 or C2_6). Examples of alkenyl include vinyl, allyl,
1-methylvinyl,
butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 1-hexenyl, 3-hexenyl, 1-
heptenyl, 3-
heptenyl, 1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl,
1,3-
butadienyl, 1-4,pentadienyl, 1,3-hexadienyl and 1,4-hexadienyl. An alkenyl
group may be
optionally substituted by one or more optional substituents as herein defined.
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As used herein the term "alkynyl" denotes groups formed from straight chain or
branched
hydrocarbon residues containing at least one carbon-carbon triple bond
including
ethynically mono-, di- or poly- unsaturated alkyl groups as previously
defined. Unless the
number of carbon atoms is specified the term preferably refers to C2_20
alkynyl (e.g. Cz4o
or Cz_b). Examples include ethynyl, 1-propynyl, 2-propynyl, and butynyl
isomers, and
pentynyl isomers. An alkynyl group may be optionally substituted by one or
more optional
substituents as herein defined.
Terms written as "[group]oxy" refer to a particular group when linked by
oxygen, for
example, the terms "alkoxy", "alkenoxy",,"alkynoxy" and "aryloxy" and
"acyloxy"
respectively denote alkyl, alkenyl, alkynyl, aryl and acyl groups as
hereinbefore defined
when linked by an oxygen atom. Terms written as "[group]thio" refer to a
particular group
when linked by sulfur, for example, the terms "alkylthio", "alkenylthio",
alkynylthio" and
"arylthio" respectively denote alkyl, alkenyl, alkynyl, aryl groups as
hereinbefore defined
when linked by a sulfur atom. Similarly, a term written as "[groupA]groupB" is
intended
to refer to a groupA when linked by a divalent form of groupB, for example,
"hydroxyalkyl" is a hydroxy group when linked by an alkylene group.
The term "halogen" ("halo") denotes fluorine, chlorine, bromine or iodine
(fluoro, chloro,
bromo or iodo).
The term "aryl" (or "carboaryl)", or the abbreviated form "ar" used in
compound words
such as "aralkyl", denotes any of mono-, bi- or polcyclic, (including
conjugated and fused)
hydrocarbon ring systems containing an aromatic residue. Examples of aryl
include
phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl
(tetralinyl),
anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl,
phenanthrenyl,
fluorenyl, pyrenyl, idenyl, isoindenyl, indanyl, azulenyl and chrysenyl.
Particular
examples of aryl include phenyl and naphthyl. An aryl group may be optionally
substituted by one or more optional substituents as herein defined.
The term "carbocyclyl" includes any of non-aromatic monocyclic, bicyclic and
polycyclic,
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(including fused, bridged or conjugated) hydrocarbon residues, e.g. C3.20
(such as C3_1o or
C3_8). The rings may be saturated, for example cycloalkyl, or may possess one
or more
double bonds (cycloalkenyl) and/or one or more triple bonds (cycloalkynyl).
Examples of
particular carbocyclyl are monocyclic 5-6-membered or bicyclic 9-10 membered
ring
systems. Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl,
cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl and
decalinyl. A
carbocyclyl group may be optionally substituted by one or more optional
substituents as
herein defined. In particular, a monocarbocyclyl group may be substituted by a
bridging
group to form a bicyclic bridged group.
The term "heterocyclyl" when used alone or in compound words includes any of
monocyclic, bicyclic or polycyclic, (including fuse, bridged or conjugated)
hydrocarbon
residues, such as C3.20 (e.g. C3_10 or C3.8) wherein one or more carbon atoms
are
independently replaced by a heteroatom so as to provide a group containing a
non-aromatic
heteroatom containing ring. Suitable heteroatoms include, 0, N, S, P and Se,
particularly
0, N and S. Where two or more carbon atoms are replaced, this may be by two or
more of
the same heteroatom or by different heteroatoms. The heterocyclyl group may be
saturated
or partially unsaturated, e.g. possess one or more double bonds. Particularly
preferred
heterocyclyl are monocyclic 5-6- and bicyclic 9-10- membered heterocyclyl.
Suitable
examples of heterocyclyl groups may include azridinyl, oxiranyl, thiiranyl,
azetidinyl,
oxetanyl, thietanyl, 2H-pyrrolyl, pyrrolidinyl, 1-, 2- and 3-pyrrolinyl,
piperidyl,
piperazinyl, morpholinyl, indolinyl, imidazolidinyl, imidazolinyl,
pyrazolidinyl,
thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydropyrrolyi,
tetrahydrothiophenyl (tetramethylene sulfide), pyrazolinyl, dioxalanyl,
thiazolidinyl,
isoxazolidinyl, dihydropyranyl, oxazinyl, thiazinyl, thiomorpholinyl,
oxathianyl, dithianyl,
trioxanyl, thiadiazinyl, dithiazinyl, trithianyl, azepinyl, oxepinyl,
thiepinyl, indenyl,
indanyl, 3H-indolyl, isoindolinyl, 4H-quinolazinyl, chromenyl, chromanyl,
isochromanyl,
benzoxazinyl (2H-1,3, 2H-1,4-, 1H-2,3-, 4H-3,1- 4H-1,4) pyranyl and
dihydropyranyl. A
heterocyclyl group may be optionally substituted by one or more optional
substituents as
defined herein.
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The term "heteroaryl" includes any of monocyclic, bicyclic, polycyclic, (fused
or
conjugated) hydrocarbon residues, wherein one or more carbon atoms are
replaced by a
heteroatom so as to provide a residue having at least one aromatic heteroatom-
containing
ring. Exemplary heteroaryl have 3-20 ring atoms, e.g. 3-10. Particularly
preferred
heteroaryl are 5-6 monocyclic and 9-10 membered bicyclic ring systems.
Suitable
heteroatoms include, 0, N, S, P and Se, particularly 0, N and S. Where two or
more
carbon atoms are replaced, this may be by two or more of the same heteroatom
or by
different heteroatoms. Suitable examples of heteroaryl groups may include
pyridyl,
pyrrolyl, thienyl, imidazolyl, furanyl, benzothienyl, isobenzothienyl,
benzofuranyl,
isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl,
pyridazinyl,
indolizinyl, quinolyl, isoquinolyl, phthalazinyl, 1,5-naphthyridinyl,
quinozalinyl,
quinazolinyl, quinolinyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl,
triazolyl,
oxadialzolyl, oxatriazolyl, triazinyl, tetrazolyl and furazanyl. A heteroaryl
group may be
optionally substituted by one or more optional substituents as defined herein.
The term "acyl" either alone or in compound words denotes a group containing
the moiety
C=0 (and not being a carboxylic acid, ester or amide) Preferred acyl includes
C(O)-R,
wherein R is hydrogen or an alkyl, alkenyl, alkynyl, aryl, heteroaryl,
carbocyclyl, or
heterocyclyl residue. Examples of acyl include formyl, straight chain or
branched
alkanoyl (e.g. CI_ZO) such as, acetyl, propanoyl, butanoyl, 2-methylpropanoyl,
pentanoyl,
2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,
undecanoyl,
dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl,
heptadecanoyl,
octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as
cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and
cyclohexylcarbonyl;
aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as
phenylalkanoyl (e.g.
phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl,
phenylpentanoyl and
phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl
and
naphthylbutanoyl]; aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl,
phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl and phenyihexenoyl and
naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and
naphthylpentenoyl);
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aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylthiocarbamoyl
such as
phenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and
naphthylglyoxyloyl;
arylsulfonyl such as phenylsulfonyl and napthylsulfonyl; heterocycliccarbonyl;
heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl,
thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and
tetrazolylacetyl;
heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl,
heterocyclicpentenoyl and heterocyclichexenoyl; and heterocyclicglyoxyloyl
such as
thiazolyglyoxyloyl and thienylglyoxyloyl. The R residue may be optionally
substituted as
described herein.
In this specification "optionally substituted" is taken to mean that a group
may be
unsubstituted or further substituted or fused (so as to form a condensed bi-
or polycyclic
group) with one, two, three or more of organic and inorganic groups, including
those
selected from: alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl,
heteroaryl, acyl,
aralkyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, alkylcarbocyclyl,
halo, haloalkyl,
haloalkenyl, haloalkynyl, haloaryl, halocarbocyclyl, haloheterocyclyl,
haloheteroaryl,
haloacyl, haloaryalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl,
hydroxycarbocyclyl, hydroxyaryl, hydroxyheterocyclyl, hydroxyheteroaryl,
hydroxyacyl,
hydroxyaralkyl, alkoxyalkyl, alkoxyalkenyl, alkoxyalkynyl, alkoxycarbocyclyl,
alkoxyaryl, alkoxyheterocyclyl, alkoxyheteroaryl, alkoxyacyl, alkoxyaralkyl,
alkoxy,
alkenyloxy, alkynyloxy, aryloxy, carbocyclyloxy, aralkyloxy, heteroaryloxy,
heterocyclyloxy, acyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy,
haloaryloxy,
halocarbocyclyloxy, haloaralkyloxy, haloheteroaryloxy, haloheterocyclyloxy,
haloacyloxy,
nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl,
nitroheteroaryl,
nitrocarbocyclyl, nitroacyl, nitroaralkyl, amino (NH2), alkylamino,
dialkylamino,
alkenylamino, alkynylamino, arylamino, diarylamino, aralkylamino,
diaralkylamino,
acylamino, diacylamino, heterocyclamino, heteroarylamino, carboxy,
carboxyester, amido,
alkylsulphonyloxy, arylsulphenyloxy, alkylsulphenyl, arylsulphenyl, thio,
alkylthio,
alkenylthio, alkynylthio, arylthio, aralkylthio, carbocyclylthio,
heterocyclylthio,
heteroarylthio, acylthio, sulfoxide, sulfonyl, sulfonamido, aminoalkyl,
aminoalkenyl,
aminoalkynyl, aminocarbocyclyl, aminoaryl, aminoheterocyclyl, aminoheteroaryl,
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aminoacyl, aminoaralkyl, thioalkyl, thioalkenyl, thioalkynyl, thiocarbocyclyl,
thioaryl,
thioheterocyclyl, thioheteroaryl, thioacyl, thioaralkyl, carboxyalkyl,
carboxyalkenyl,
carboxyalkynyl, carboxycarbocyclyl, carboxyaryl, carboxyheterocyclyl,
carboxyheteroaryl,
carboxyacyl, carboxyaralkyl, carboxyesteralkyl, carboxyesteralkenyl,
carboxyesteralkynyl,
carboxyestercarbocyclyl, carboxyesteraryl, carboxyesterheterocyclyl,
carboxyesterheteroaryl, carboxyesteracyl, carboxyesteraralkyl, amidoalkyl,
amidoalkenyl,
amidoalkynyl, amidocarbocyclyl, amidoaryl, amidoheterocyclyl, amidoheteroaryl,
amidoacyl, amidoaralkyl, formylalkyl, formylalkenyl, formylalkynyl,
formylcarbocyclyl,
formylaryl, formylheterocyclyl, formylheteroaryl, formylacyl, formylaralkyl,
acylalkyl,
acylalkenyl, acylalkynyl, acylcarbocyclyl, acylaryl, acylheterocyclyl,
acylheteroaryl,
acylacyl, acylaralkyl, sulfoxidealkyl, sulfoxidealkenyl, sulfoxidealkynyl,
sulfoxidecarbocyclyl, sulfoxidearyl, sulfoxideheterocyclyl,
sulfoxideheteroaryl,
sulfoxideacyl, sulfoxidearalkyl, sulfonylalkyl, sulfonylalkenyl,
sulfonylalkynyl,
sulfonylcarbocyclyl, sulfonylaryl, sulfonylheterocyclyl, sulfonylheteroaryl,
sulfonylacyl,
sulfonylaralkyl, sulfonamidoalkyl, sulfonamidoalkenyl, sulfonamidoalkynyl,
sulfonamidocarbocyclyl, sulfonamidoaryl, sulfonamidoheterocyclyl,
sulfonamidoheteroaryl, sulfonamidoacyl, sulfonamidoaralkyl, nitroalkyl,
nitroalkenyl,
nitroalkynyl, nitrocarbocyclyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl,
nitroacyl,
nitroaralkyl, cyano, sulfate, sulfonate, phosphonate and phosphate groups.
Optional
substitution may also be taken to refer to where a CH2 group in a chain or
ring is replaced
by a carbonyl group (C=0) or a thiocarbonyl group (C=S), where 2 adjacent or
non-
adjacent carbon atoms (e.g. 1,2- or 1,3) are substituted by one end each of a-
O-(CH2)s-O-
or -NR"-(CH2)s-NR"- group, wherein s is 1 or 2 and each R" is independently H
or Cl-
6alkyl, and where 2 adjacent or non-adjacent atoms, independently selected
from C and N,
are substituted by one end each of a C1_5alkylene or CZ_Salkenylene group (so
as to form a
bridged group).
Exemplary optional substituents include those selected from: alkyl, (e.g.
C1.6alkyl such as
methyl, ethyl, propyl, butyl), cycloalkyl (e.g. C3_6cycloalkyl, such as
cyclopropyl,
cyclobutyl, cyclopentyl or cyclohexyl), hydroxyalkyl (e.g. hydroxyCl-6alkyl,
such as
hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g.
CI.6alkoxyC1_6alkyl, such
CA 02611072 2007-11-19
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as methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl,
ethoxypropyl), alkoxy (e.g. C1.6alkoxy, such as methoxy, ethoxy, propoxy,
butoxy),
alkoxyalkoxy (e.g. C1_6alkocyC1_6 alkoxy, such as methoxymethoxy,
methoxyethoxy,
methoxypropoxy, ethoxymethoxy, ethoxyethoxy, ethoxypropoxy, propoxymethoxy,
propoxyethoxy, propoxypropoxy) cycloalkoxy (e.g. cyclopropoxy, cyclobutoxy,
cyclopentoxyl, cyclohexyloxy), halo, haloalkyl( e.g. haloC1_6alkyl, such as
chloromethyl,
difluoromethyl, trifluoromethyl, trichloromethyl, tribromomethyl), haloalkoxy
(e.g.
haloCI_6alkoxy), hydroxy, thio (-SH), sulfonyl, sulfonamido, phenyl (which
itself may be
further substituted e.g., by one or more C1_6alkyl, halo, hydroxy,
hydroxyCl_6alkyl, Ci.
6alkoxy, C,_6alkoxyC,.6alkyl, CI.6alkoxyC1.6alkoxy, haloC,.6alkyl,
haloC1.6alkoxy, cyano,
nitro, OC(O)CI-6alkyl, NH2, NHC1.6alkyl, NHC(O)CI_6alkyl and
NC1.6alkylCI_6alkyl),
benzyl (wherein benzyl itself may be further substituted e.g., by one or more
of CI-6alkyl,
halo, hydroxy, hydroxyC1_6alkyl, C1_6alkoxy, CI.6alkoxyC1_6alkyl, CI-
6alkoxyQ_6alkoxy,
haloC].6alkyl, haloCi.6alkoxy, cyano, nitro, OC(O)CI-6alkyl, NH2, NHC1_6alkyl,
NHC(O)C1_6alkyl and NCI.6a1ky1CI_6alkyl), phenoxy (wherein phenyl itself may
be further
substituted e.g., by one or more of C1_6alkyl, halo, hydroxy, hydroxyQ.6alkyl,
C1_6alkoxy,
CI.6alkoxyC1_6alkyl, C1_6alkoxyC1_6alkoxy, haloCl_6alkyl, haloQ-6alkoxy,
cyano, nitro,
OC(O)CI_6alkyl, NHZ, NHCi-6alkyl, NHC(O)CI.6alkyl and NCI.6alkylCI.6alkyl),
benzyloxy
(wherein benzyl itself may be further substituted e.g., by one or more of
C1.6alkyl, halo,
hydroxy, hydroxyQ-6alkyl, Ct-6alkoxy, Cl.6alkoxyCl.6alkyl, C1_6alkoxyQ-
6alkoxy, haloCj.
6alkyl, haloCl.6alkoxy, cyano, nitro, OC(O)CI-6alkyl, NH2, NHC1.6alkyl,
NHC(O)CI_6alkyl
and NC,.6alkylCt.6alkyl), NH2, alkylamino (e.g. -NHC1_6alkyl, such as
methylamino,
ethylamino, propylamino etc), dialkylamino (e.g. -NH(CI_6alkyl)2, such as
dimethylamino,
diethylamino, dipropylamino), acylamino (e.g. -NHC(O)C1_6alkyl, such as -
NHC(O)CH3),
phenylamino (i.e. NHphenyl, wherein phenyl itself may be further substituted
e.g., by one
or more of CI_6alkyl, halo, hydroxy, hydroxyC1.6alkyl, hydroxyQ-6alkoxy Cl-
6alkoxy, Cl_
6alkoxyQ_6alkyl, C1.6alkoxyCI.6alkoxy, haloCI.6alkyl, haloQ.6alkoxy, cyano,
nitro,
OC(O)CI-6alkyl, NH2, NHCI.6a1ky1, NHC(O)Cl-6alkyl and NC,-6alkylCI.6alkyl),
nitro,
cyano, formyl, -C(O)-alkyl (e.g. -C(O)CI..6alkyl, such as acetyl), O-C(O)-
alkyl (e.g. -
OC(O)CI-6alkyl, such as acetyloxy), benzoyl (wherein benzyl itself may be
further
substituted e.g., by one or more of C1.6alkyl, halo, hydroxy, hydroxyCl-
6alkyl, CI.6alkoxy,
CA 02611072 2007-11-19
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CI.6alkoxyC,.6alkyl, C1_6alkoxyCI.6alkoxy, haloC1.6alkyl, haloCI_balkoxy,
cyano, nitro,
OC(O)CI.6alkyl, NHZ, NHCI.6alkyl, NHC(O)CI-6alkyl and NCi.6a1ky1CI.6alkyl),
benzoyloxy (wherein benzyl itself may be further substituted e.g., by one or
more of Cl_
6alkyl, halo, hydroxy, hydroxyCl.6alkyl, Ci.6alkoxy, C1.6a1koxyC1_6alkyl,
C1_6alkoxyC,_
6alkoxy, haloC1_6alkyl, haloCI-6alkoxy, cyano, nitro, OC(O)CI_6alky1, NH2,
NHCI-6alkyl,
NHC(O)CI-6alkyl and NC1.6a1ky1C1_6alkyl), CO2H, CO2alky1 (e.g. CO2C1.6alkyl
such as
methyl ester, ethyl ester, propyl ester, butyl ester), CO2phenyl (wherein
phenyl itself may
be further substituted e.g., by one or more of C1.6alkyl, halo, hydroxy,
hydroxyC1_6alkyl,
C1.6alkoxy, C1.6alkoxyCI.6alkyl, CI.6alkoxyCl-6alkoxy, haloCI.6alkyl,
haloC1_6alkoxy,
cyano, nitro, OC(O)C1.6alkyl, NH2, NHC1_6alkyl, NHC(O)CI-6alkyl and
NCI.6alkylCl_
6alkyl), CO2benzyl (wherein benzyl itself may be further substituted e.g., by
one or more
of CI_6alkyl, halo, hydroxy, hydroxyC].6alkyl, C1_6alkoxy, Ci.6alkoxyCi-
6alkyl, C1_
6alkoxyC1.6alkoxy, haloQ.6alkyl, haloQ.6alkoxy, cyano, nitro, OC(O)Ci.6alkyl,
NH2,
NHC1_6alkyl, NHC(O)CI-6alkyl and NC1_6alkylCl.6alkyl), CONH2, C(O)NHphenyl
(wherein phenyl itself may be further substituted e.g., by one or more of
C1_6alkyl, halo,
hydroxy, hydroxyC1_6alkyl, C1_6alkoxy, CI.6alkoxyC1_6alky1,
C1_6alkoxyC1_6alkoxy, haloCi.
6alkyl, haloCi.6alkoxy, cyano, nitro, OC(O)CI_6alky1, NH2, NHC1_6alky1,
NHC(O)CI-6alkyl
and NC1_6a1ky1C1:6alkyl), C(O)NHbenzyl (wherein benzyl itself may be further
substituted
e.g., by one or more of Cl-6alkyl, halo, hydroxy, hydroxyCi.6alkyl,
CI.6alkoxy, CI_
6alkoxyCl.6alkyl, CI_6alkoxyCI_6alkoxy, haloC,.6alkyl, haloC1.6alkoxy, cyano,
nitro,
OC(O)CI_6alkyl, NH2, NHC1_6alky1, NHC(O)CI-6alkyl and NCI_6a1ky1CI_6alkyl),
C(O)NHalkyl (e.g. C(O)NHC1.6 alkyl such as methyl amide, ethyl amide, propyl
amide,
butyl amide) C(O)Ndialkyl (e.g. C(O)N(Cl.6alkyl)2) aminoalkyl (e.g.,
HNCI_6alkyl-, Cl.
6a1ky1HN-C1_6alkyl- and (CI.6alkyl)ZN-C1_6alkyl-), thioalkyl (e.g.,
HSCt.6alkyl-),
carboxyalkyl (e.g., HOzCCi.6alkyl-), carboxyesteralkyl (e.g.,
C1_6a1ky1O2CCI.6alkyl-),
amidoalkyl (e.g., H2N(O)CC1_6alkyl-, H(C1.6alkyl)N(O)CC1_6alky1-), formylalkyl
(e.g.,
OHCCI.6alkyl-), acylalkyl (e.g., C1_6alkyl(O)CCI.6alkyl-), nitroalkyl (e.g.,
O2NCI-6alkyl-),
replacement of CH2 with C=O, replacement of CH2 with C=S, substitution of 2
adjacent
or non-adjacent carbon atoms (e.g. 1,2 or 1,3) by one end each of a-O-(CH2)s-O-
or -NR'-
(CH2)s-NR'- group, wherein s is 1 or 2 and each R' is independently H or Q-
_6alkyl, and
CA 02611072 2007-11-19
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substitution of 2 adjacent or non-adjacent atoms, independently selected from
C and N, by
a C2_5alkylene or C2.5alkenylene group.
The term "sulfoxide", either alone or in a compound word, refers to a group -
S(O)R
wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, heterocyclyl,
carbocyclyl, and aralkyl. Examples of R include hydrogen, C1_2palkyl, phenyl
and benzyl.
The term "sulfonyl", either alone or in a compound word, refers to a group
S(O)2-R,
wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, heterocyclyl,
carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C1.20alkyl,
phenyl and
benzyl.
The term "sulfonamide", or "sulfonamyl" of "sulfonamido", either alone or in a
compound
word, refers to a group S(O)2NRR wherein each R is independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,
carbocyclyl, acyl, and
aralkyl. Examples of R include hydrogen, C1.z.oalkyl, phenyl and benzyl. In an
embodiment at least one R is hydrogen. In another form, both R are hydrogen.
A "sulfate" group refers to a group -OS(O)20R wherein each R is independently
selected
from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,
carbocyclyl, acyl,
and aralkyl. Examples of R include hydrogen, C1_20alkyl, phenyl and benzyl.
The term "sulfonate" refers to a group SO3R wherein each R is independently
selected
from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,
carbocyclyl, acyl,
and aralkyl. Examples of R include hydrogen, C1_ZOalkyl, phenyl and benzyl.
The term "thio" is intended to include groups of the formula "-SR" wherein R
can be
hydrogen (thiol), alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl,
heterocyclyl,
aralkyl, and acyl. Examples of R include hydrogen, C1_20alkyl, phenyl and
benzyl.
CA 02611072 2007-11-19
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The term, "amino" is used here in its broadest sense as understood in the art
and includes
groups of the formula -NRARB wherein RA and RB may be any independently
selected
from hydrogen, hydroxy alkyl, alkoxyalkyl, alkenyl, alkynyl, aryl,
carbocyclyl, heteroaryl,
heterocyclyl, aralkyl, and acyl, each of which may be optionally substituted.
RA and RB,
together with the nitrogen to which they are attached, may also form a
monocyclic, or
polycyclic ring system e.g. a 3-10 membered ring, particularly, 5-6 and 9-10
membered
systems. Examples of "amino" include -NH2, -NHalkyl (e.g. -NHC1_20alkyl), -
NHalkoxyalkyl, -NHaryl (e.g. -NHphenyl), -NHaralkyl (e.g. -NHbenzyl), -NHacyl
(e.g. -
NHC(O)C1_20alkyl, -NHC(O)phenyl), -Ndialkyl (wherein each alkyl, for example
C1_20i
may be the same or different) and 5 or 6 membered rings, optionally containing
one or
more same or different heteroatoms (e.g. 0, N and S). Reference to groups
written as
"[group]amino" is intended to reflect the nature of the RA and RB groups. For
example,
"alkylamino" refers to -NRARB where one of RA or RB is alkyl. "Dialkylamino"
refers to -
NRARB where Rp' and RB are each (independently) an alkyl group.
The term "amido" is used here in its broadest sense as understood in the art
and includes
groups having the formula C(O)NRARB, wherein RA and RB are as defined as
above.
Examples of amido include C(O)NH2, C(O)NHalkyl (e.g. C1_20alkyl), C(O)NHaryl
(e.g.
C(O)NHphenyl), C(O)NHaralkyl (e.g. C(O)NHbenzyl), C(O)NHacyl (e.g.
C(O)NHC(O)C1_20alkyl, C(O)NHC(O)phenyl), C(O)Nalkylalkyl (wherein each alkyl,
for
example C1_20, may be the same or different) and 5 or 6 membered rings,
optionally
containing one or more same or different heteroatoms (e.g.. 0, N and S).
The term "carboxy ester" is used here in its broadest sense as understood in
the art and
includes groups having the formula COZR, wherein R may be selected from groups
including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl,
heterocyclyl, aralkyl, and
acyl. Particular examples of carboxy ester include CO2C1_20alkyl, COZaryl
(e.g.
COZphenyl), COZaralkyl (e.g. CO2 benzyl).
In some embodiments, when R' is NHCOR8, A is selected from optionally
substituted 5-6-
membered aryl, heteroaryl, carbocyclyl and heterocyclyl, e.g. phenyl, pyridyl
(attached at
CA 02611072 2007-11-19
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C2, 0 or C4), pyrazinyl, pyrimidinyl (attached at C2, C4 or C5), pyridazinyl
(attached at
0 or C4), s-triazinyl (attached at C2, C4 or C6), as-triazinyl (attached at
C3, C5 or C6), v-
triazinyl (attached at C4, C5 or C6), furanyl (attached at C2 or C3), pyrrolyl
(attached at
C2 or C3), thienyl (attached at C2 or C3), cyclopentyl, cyclohexyl,
cyclopentadienyl,
cyclohexadienyl, pyranyl, piperidinyl, piperazinyl, morpholinyl, pyrolidinyl
and pyrrolinyl.
In certain embodiments A is optionally substituted 5-6-membered aryl or
optionally
substituted heteroaryl, particularly optionally substituted phenyl or
optionally substituted
pyridyl.
In other embodiments, when R' is NHCOR8, A is selected from optionally
substituted
naphthyl, an optionally substituted 9-10 membered bicyclic heteroaryl group,
including N-
containing 10-membered groups.
Some particular examples of NHCORB include NHCOC1.6alkyl(e.g. NHCOMe, NHCOEt,
NHCOPr), NHCOphenyl and NHCObenzyl. In further examples, where R' is NHCOCI.
6alkyl, it is NHCOMe. In other examples it is NHCOC2_6alkyl(e.g. NHCOEt,
NHCOPr or
NHCOBu).
In other embodiments, when R' is NR7R8, NHCORs, N(COR8)2, N(COR7)(COR8),
N=CHOR8 or N=CHR8, A is a bicyclic N-containing heteroaryl group such as
quinolinyl,
isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridyl,
phthalazinyl or
pteridinyl, each of which may be substituted or unsubstituted. The group may
be attached
via any carbon atom of the ring system. In certain embodiments, the group may
be
attached via the 2-, 3-, 6- or 7- position as appropriate. In other
embodiments, the group
may be attached via the 1-, 4-, 5- or 8- position as appropriate. Some
particular examples
contemplated herein are 3- and 4-quinolinyl, particularly 3-quinolinyl. In
further
embodiments, A is unsubstituted.
In some embodiments, when R' is NR7R8, R' is NHR8, wherein R8 is optionally
substituted
alkyl or optionally substituted aryl. In other embodiments when R' is NR7 RB,
R7 and R8
together with the N atom to which they are attached, form a 3-8-membered ring,
e.g.
CA 02611072 2007-11-19
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together are an alkylene group such as -(CHz)q- where q is 2, 3, 4 or 5.
In yet further embodiments of the invention, A is a bicyclic N-containing
heteroaryl group
as described above and R' is NHCORs, such as NHCOC1_6alkyl. In a particular
embodiment, A is 3-quinolinyl and R' is NHCOMe.
In certain embodiments, when A is quinolinyl, isoquinolinyl, cinnolinyl,
quinazolinyl,
quinoxalinyl, 1,8-naphthyridyl, phthalazinyl or pteridinyl, particularly
quinolinyl, then R'
is NHR8, N<(CH2)q, NHCOR8, N(COR8)2, N(COR7)(COR8), N=CHORg or N=CHR8.
In yet other embodiments of the invention, when A is aryl, carbocyclyl, or
heterocyclyl
then R' is NR7Rx, N(COR8)2, N(COR7)(COR8), N=CHOR8 or N=CHRB.
Exemplary optional substituents for A include alkyl, haloalkyl (e.g. mono-, di-
or
trifluoromethyl, mono-, di-or trichloromethyl and mono-, di-, tri-, tetra,
penta- or
hexafluoroethyl and mono-, di-, tri-, tetra, penta- or hexachloroethyl),
hydroxyalkyl,
aminoalkyl, thioalklyl, arylalkyl (e.g. benzyl and phenylethyl), alkoxy,
haloalkoxy,
hydroxyalkoxyl, aminoalkoxy, alkythio, haloalkythio, hydroxyalkylthio,
aminoalkylthio,
thioalkythio, amino, C(O)alkyl, OC(O)alkyl, aryl (eg. phenyl), carboxy,
carboxy ester (e.g.
CO2alkyl, CO2ary1), C(O)aryl, OC(O)aryl, nitro, cyano, heteroaryl (e.g.
pyridyl),
heteroarylalkyl, heterocyclyl, heterocyclylalkyl, hydroxy, thio,
methylenedioxy, halo (e.g.
Cl, Br) and amido. Where such a substituent is or contains an "alkyl" moiety,
it may be Cl_
loalkyl, particularly CI-6alkyl, such as methyl, ethyl, i-propyl, n-propyl, n-
butyl, sec-butyl
or t-butyl.
In one embodiment X is O. In another embodiment X is NR'. In yet another
embodiment
X is S. In particular examples X is O. Where X is NR', some examples of R'
include
hydrogen, Cl.loalkyl, benzyl, phenylethyl, OC(O)Ci_loalkyl and OC(O)phenyl.
In one embodiment, Y is hydrogen.
CA 02611072 2007-11-19
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Particular examples of R7 and R8 include hydrogen, Cl.lo alkyl, and phenyl
wherein Cl.lo
alkyl and phenyl may be optionally substituted.
Some exemplary substituents for R7 and R8 (including exemplified R7 and R8 as
above)
include alkyl, haloalkyl (e.g. mono-, di- or trifluoromethyl, mono-, di-or
trichloromethyl
and mono-, di-, tri-, tetra, penta- or hexafluoroethyl and mono-, di-, tri-,
tetra, penta- or
hexachloroethyl), hydroxyalkyl, aminoalkyl, thioalkyl, arylalkyl (e.g. benzyl
and
phenylethyl) alkoxy, haloalkoxy, hydroxyalkoxyl, aminoalkoxy alkythio,
haloalkythio,
hydroxyalkylthio, aminoalkylthio, thioalkythio, amino, C(O)alkyl OC(O)alkyl,
aryl (e.g.
phenyl) carboxy, carboxy ester C(O)aryl OC(O)aryl, nitro, cyano, heteroaryl
(e.g. pyridyl)
heteroarylalkyl, heterocyclyl, heterocyclylalkyl, hydroxy, thio,
methylenedioxy, halo (e.g.
Cl, Br) and amido or together form a 5-6-membered ring e.g. (piperidyl,
morpholinyl).
Where such a substituent contains an "alkyl" moiety, preferred alkyl are
Cl_loalkyl,
particularly C1.6alkyl. Particularly preferred Rl include NH2, NHC1.6alkyl,
N(Q_6alkyl)
(C1.6alkyl) and NHC(O)Cl-6alkyl, most preferably NH2.
For RZ, examples of R9 and R10 include hydrogen, Cl.10 alkyl, aryl (e.g.
phenyl) heteroaryl
(e.g. pyridyl). One particularly preferred RZ is cyano (CN). Particular
examples of RZ are
CO2CI.1o alkyl, (e.g. CO2Me, CO2Et, CO2Pr, COZBu etc) and amido, e.g. CONH2.
Examples of suitable R3-R6 include hydrogen, chloro, bromo, Cl.lo alkyl
(including
cycloalkyl), CZ_to alkenyl (including cycloalkenyl), C2.10 alkynyl (including
cycloalkynyl),
Ci.lo alkoxy (e.g. methoxy, ethoxy, n- and i-propoxy and n-, sec- and t-
butoxy), phenyl,
halophenyl, hydroxyphenyl, aminophenyl, alkylphenyl, hydroxy, NH2,
NHCI.Ioalkyl, N
C1.1oalkylCl.loalkyl (wherein each alkyl may be the same or different), nitro,
haloalkyl,
including trifluromethyl, trichloromethyl, acyl (e.g. C(O)C1_loalkyl), acyloxy
(e.g.
OC(O)C1_ioalkyl or OC(O)aryl such as OC(O)phenyl), carboxy ester (e.g.
CO2Ci.loalkyl
and CO2 phenyl), CO2H, amido (e.g. CONHCI.,oalkyl), nitro, cyano, thio,
alkylthio (e.g.
SC1_10alkyl) and 2 of adjacent R3-R6 form methylenedioxy. None, one, two,
three or four
of R3-R6 may be hydrogen. In one preferred form, 2 or 3 or 4 of R3-R6 are
hydrogen. In
one particular embodiment, R3, R4 and R6 are all H. In a further embodiment
thereof, R3,
CA 02611072 2007-11-19
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R4 and R6 are all H and R5 is hydroxy.
Where any two adjacent R3-R6 form a 3-8-membered ring together with the atoms
to which
they are attached, the ring may be carbocylic (saturated or partially
unsaturated),
heterocyclic, aryl or heteroaryl. In some preferred embodiments, the ring
formed has 5-6-
members. Particularly preferred forms of this embodiment are where R5 and R6
form a
ring. Examples of any two adjacent R3-R6 taken together include -(CH2)õ- where
n is 1-7,
preferably 1-4, particularly 3 or 4, -O-CH2-O-, O-(CH2)2-O -CH=CH2-CH2=CH-,
-CH2-NHCH2-, -(CH2)2-NH-CH2-, -CH2-NH-(CH2)2-, -(CH2)2-NH-, -NH-(CH2)2-, -NH-
CH=CH-, -CH=CH-NH-, -O-CH=CH-, -CH=CH-O-, -S-CH=CH-, -CH=CH-S-, -N=CH-
CH=N- O-CH=CH-CH2-, and -CH2-CH=CH-O-. Where appropriate, an N atom within
such a ring may be further substituted with alkyl (eg. C1_10), aryl (eg.
phenyl), arylalkyl
(eg. benzyl or phenylethyl), acyl (eg. C(O)C1_loalkyl) hydroxyalkyl (eg.
C1_lo), haloalkyl
(eg. Cl_lo), carbocyclylalkyl, heteroarylalkyl, heterocyclylalkyl,
carbocyclyl, heteroaryl or
heterocyclyl.
In yet other embodiments of the invention, X is oxygen, R2 is C02R9 (such as
CO2C1_
6alkyl, e.g. CO2Me or CO2Et), R3, R4, R6 are all hydrogen, R5 is OH and Y is
hydrogen.
Some exemplary compounds contemplated by the invention are:
A
C OZ Et
HO O NHR
(i) A = 3-pyridyl, R = C(=O)CH3
(ii) A = 3-quinolinyl, R = H
(iii) A = 3-quinolinyl, R = C(=0)CH3
, _ .~...... ,
CA 02611072 2007-11-19
-22-
Compounds for use in the invention may be prepared via methods known in the
art of
synthetic organic chemistry, see for example WO 02/092594.
Thus, for the preparation of compounds where X is 0, an appropriately
substituted phenol
compound may be reacted with an appropriate aldehyde and malononitrile in the
presence
of a base such as piperidine or N,N-diisopropylethylamine in accordance with
the
generalised Scheme 1 below:
R3
R3 A
R4
R4 / CN
+ A-CHO + CN base > (
5
R R6 OH CN R5 ` O NH2
6
Scheme I
Alternatively, the aldehyde may be reacted with malononitrile in the presence
of a base,
and the resulting arylidene intermediate reacted with the appropriate phenol.
In an alternative methodology, the appropriately substituted ortho-aroyl
phenol, aniline or
benzenethiol can be reacted with malononitrile (or appropriate cyanoacetate,
cyanoacetamide or acylacetonitrile) to access the corresponding 4H-chromene,
1,4-
dihydroquinoline or 4H-thiochromene as in Scheme 2 below (where P is H or a
protecting
group as appropriate).
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R3 A Rs A
R4
~ 0 NC RZ R4 / R2
~ -~ -~ I I
\
R XP R5 \ X NHZ
Rg Rs
Scheme 2
Suitable dihydroquinoline compounds can also be prepared by reduction of an
appropriately substituted or protected quinoline.
5
Compounds of formula (I), where R' is other than NH2 may be prepared by
converting the
amine to the desired group using chemical transformations known in the art,
for example
as described in Comprehensive Organic Transformation, A Guide to Functional
Group
Preparations, R. C. Larock, VCH, 1989 and Advanced Organic Chemistry,
Reactions,
Mechanisms and Structure, J. March, 3`d Edition, 1985 or 4Ih Edition, 1992
(the entire
contents of which are incorporated herein by reference). Thus, treatment of
the amine
(NH2) with a suitable acylating agent such as a carboxylic acid, anhydride or
chloride in
the presence of an appropriate base or catalyst provides access to compounds
of formula
(I) where R' is NHCOR8. Alkyl and aryl amines can be prepared by treatment of -
NH2
with an appropriate alkyl or aryl halide. Imines may be formed by treating the
amine
group (NH2) with a suitable carbonyl containing compound such as an aldehyde
or ketone
in accordance with art known methods.
Similarly, compounds where R2 is other than CN, i.e. carboxylic acid, esters,
amides,
anhydrides and ketones, may be prepared by transformations known in the art,
see in
particular Larock, supra, Chapter 9, pp 963-995. Alternatively, reaction of
the appropriate
phenol, aldehyde and the appropriate cyanoacetate, cyanoacetamide or
acylacetonitrile
affords access to compounds where R2 is COZR9, C(O)NR9R10, C(O)R9 and
C(O)O(O)R9.
Compounds where Y is alkyl can be prepared by treating the benzopyran with DDQ
and
CA 02611072 2007-11-19
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the subsequent intermediate with CuBr-DMS and an alkyllithium compound.
It will be recognised that during the processes for the preparation of
compounds
contemplated by the present invention, it may be necessary or desirable to
protect certain
functional groups which may be reactive or sensitive to the reaction or
transformation
conditions undertaken (e.g. OH (including diols), NHZ, CO2H, SH, C=O).
Suitable
protecting groups for such functional groups are known in the art and may be
used in
accordance with standard practice. As used herein, the term "protecting
group", refers to
an introduced functionality which temporarily renders a particular functional
group
inactive under certain conditions. Such protecting groups and methods for
their
installation and subsequent removal at an appropriate stage are described in
Protective
Groups in Organic Chemistry, 3rd Edition, T.W.Greene and P. G. Wutz, John
Wiley and
Sons, 1999, the entire contents of which are incorporated herein by reference.
Exemplary
forms of protected groups include:
for amino (NHZ) - carbamates (such as Cbz, Boc, Fmoc), benzylamines,
acetamides (e.g.
acetamide, trifluoroacetamide);
for carbonyl - acetals, ketals, dioxanes, dithianes, and hydrazones;
for hydroxy - ethers (e.g. alkyl ethers, alkoxylalkyl ethers, allyl ethers,
silyl ethers, benzyl
ethers, tetrahydropyranyl ethers), carboxylic acid esters, acetals (e.g.
acetonide and
benzylidene acetal);
for thio (SH) -ethers (e.g. alkyl ethers, benzyl ethers), esters
for CO2H - esters (e.g. alkyl esters, benzyl esters).
The level of IRAP inhibitory activity of the compounds disclosed herein can be
initially
determined in an in vitro assay, which measures the ability of the test
compound to inhibit
the aminopeptidase activity of IRAP, by assessing the rate or extent of
cleavage or
degradation of an IRAP aminopeptidase substrate such as Leu-(3-naphthylamide
or Leu-4-
methylcoumaryl-7-amide. Comparison can then be made to a control assay,
whereby the
rate or extent of cleavage is determined in the absence of the compound. A
comparative
reduction in the rate or extent of cleavage of the substrate in the presence
of the compound
can be taken to be a measure of the inhibitory effect of the compound.
CA 02611072 2007-11-19
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Thus, there is also provided a method of determining the IRAP inhibitory
activity of a
compound, comprising:
(a) incubating IRAP, an IRAP substrate and a compound as described herein; and
(b) assessing the rate or extent of cleavage of the substrate;
wherein a reduction or inhibition in the rate or extent of cleavage of the
substrate, when
compared to a control, is indicative of IRAP inhibitory activity of the
compound.
Advantageously, in one or more embodiments of the invention, a compound of
Formula
(I), or a pharmaceutically acceptable salt, prodrug or solvate, may exhibit
selectivity or
specificity for IRAP over other enzymes.
In one or more embodiments, compounds of the invention may potentiate, enhance
or
otherwise increase glucose uptake into neurons.
Disorders and conditions where undesirable or excessive IRAP aminopeptidase
activity is
implicated or involved may include memory or learning disorders associated
with
Alzheimer's disease and other forms of dementia and memory loss (be they age-
related,
induced through head trauma, hypoxic damage, surgery, cerebral infarcts or
chemical
means such as neurotoxins). It should also be appreciated that the compounds
of the
present invention may also be useful in enhancing or improving memory or
learning in
normal individuals, ie. those not suffering from cognitive pathologies such as
those
described above.
Memory or learning (e.g. spatial learning) enhancement refers to an
improvement in the
ability of a subject to memorize or learn information and can be determined by
well
established tests. A positive improvement in the "score" or result obtained in
such a test
compared to a score/result obtained prior to administration of the compounds
is taken to be
an enhancement in memory or learning as appropriate. In certain embodiments of
the
invention, the improvement can be expressed as a % (score after administration
of
compound / score prior to administration of compound) and may represent an
CA 02611072 2007-11-19
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improvement of at least about 10%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%,
100%,
150% or 200%.
A number of well known and established tests exist for laboratory animals and
rats and
mice can be tested using these, which include the Barnes Maze paradigm
(Greferath et al.,
2000), Barnes Circular Maze test (Lee et al., 2004) or modifications thereof,
the Y maze
test, or passive avoidance test. Suitable examples of these are briefly
outlined below.
1. Barnes maze
Normal male Sprague-Dawley rats are implanted with cannulas in the lateral
ventricles and
allowed to recover for at least three days. For the Barnes Circular Maze test,
the maze
comprises a raised rotatable white circular platform of diameter 1.2m, with 18
evenly
spaced holes in the periphery (diameter 0.09m). An escape tunnel comprising a
black box
of internal diameter of 0.16m width, 0.29m length and 0.09m depth is
positioned
immediately beneath a peripheral hole. Visual cues are placed at various
positions around
the maze.
For each trial, the animal is placed on the maze platform under the starting
chamber, which
is a cylindrical chamber located in the centre of the platform, and left for
20s. Following
the twenty seconds disorientation period the chamber is raised. The animals
are then
allowed 240s in which to find and utilise the escape tunnel. Each rat receives
three
consecutive trials per day over ten days. On the first day of the testing
period, each rat is
placed directly into the escape tunnel for a 2min familiarisation period. The
rat is then
replaced into its cage for approximately one minute, after which the animal is
injected with
either the test compound or artificial cerebrospinal fluid and then replaced
into its cage for
a further 5min. Three consecutive trials are then carried out, with a recovery
period of
2min between each trial in its home cage. On subsequent days (days 2 to 8),
the
familiarisation period prior to the three trials is eliminated from the
protocol.
Animals can be administered the compounds of the invention
intracerebroventricularly
(icv) via a chronic indwelling cannula. These compounds are given 5 mins
before the first
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trial on the first day of testing.
2. Y maze
The Y-maze test is another behavioural paradigm which measures spatial memory
performance, and exploits the natural instinct of rodents to explore novel
environments.
The Y maze consists of three identical alleys (30cm in length, 10cm in width
and 17cm in
height) with the three arms separated by 120 angles. To minimize stress, the
maze is
located in a sound-attenuated room under dim illumination, with the floor of
the maze
covered with sawdust. After each trial, the sawdust is mixed to eliminate
olfactory cues.
For spatial orientation, visual cues are placed on the walls of the testing
room.
The test consists of two trials, separated by a time interval known as the
intertrial interval.
During the acquisition trial, the animals are placed at the distal end of one
arm, their heads
pointing away from the centre of the maze. The animals are allowed to visit
only two
accessible arms of the maze for 3 minutes. At the end of the acquisition
trial, animals are
replaced in their cages for the intertrial interval. During the retention
trial, the animals
have access to all three arms for 5 minutes. The first arm entered (novel vs
familiar), the
number of entries and duration of time spent in the novel arm is documented.
A shorter intertrial interval (2 hours) separating the acquisition phase from
the retention
phase enables amnesiant effects to be detected. Control animals still remember
the
location of the novel arm and will preferentially spend more time (45-50% of
time) in that
arm. A longer intertrial interval (e.g. 6 hours) results in the animals not
remembering the
location of the novel arm and hence spending equal amount of time in all three
arms.
Animals are surgically implanted with an infusion cannula in the dorsal third
ventricle.
Each animal is tested twice, and the intertrial interval of greater than 6h
can be adopted to
test for memory-enhancing properties of the test compounds. Animals are
administered
the compounds intracerebroventricularly via a chronic indwelling cannula.
These
compounds are given 5 mins before the first acquisition trial. The animals are
then
CA 02611072 2007-11-19
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returned to their home cages for at least 6 hours and then tested again. The
time spent in
the novel arm will be a measure of the memory enhancing properties of the test
compound.
3. Passive avoidance
Passive avoidance trials can be used to test the effect of IRAP inhibitors on
aversive
conditioning behaviour in amnesic animals. The passive avoidance task involves
aversive
conditioning behaviour to measure facilitation of memory retention and
retrieval. The
testing can be carried out in an apparatus that consists of a light and a dark
compartment
separated by a guillotine door. The floor of the dark compartment contains an
electrified
grid. The passive avoidance task is divided into two trials separated by a 24-
48h inter-trial
interval. During the first trial, known as the acquisition trial, the animal
is placed in the lit
compartment and the guillotine door is closed once the animal enters the dark
compartment. Inside the dark chamber, the animal receives a low-level electric
shock
(0.5mA for 2s) via the grid floor. The animal is then returned to its home
cage for 24h or
48h before being tested. The latency periods to re-enter the dark compartment
are taken as
a measure of the ability of the animals to remember the aversive stimuli.
Animals are surgically implanted with an infusion cannula in the dorsal third
ventricle.
Each animal is tested twice, and the intertrial interval of 24h and 48h can be
adopted to test
for memory-enhancing properties of the test compounds. Animals are
administered the
compounds intracerebroventricularly via a chronic indwelling cannula. These
compounds
are given 5 mins before the acquisition trial. The animals are then returned
to their home
cages for 24h or 48h and then tested again. The latency in entering the dark
chamber will
be a measure of the memory-enhancing property of the test compound.
4. Age-induced memory deficit model
Spatial learning impairment in aged rats is well documented and this deficit
can be
detected in the Barnes maze paradigm (Greferath et al., 2000). The effect of
IRAP
inhibitors on aged-induced learning deficits can be tested in the Barnes maze
paradigm.
For drug treatment, the animals are implanted with Alzet minipumps (secured
CA 02611072 2007-11-19
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subcutaneously in the neck region) which delivers the test compounds
chronically into the
cerebral ventricles.
5. Animal model of Alzheamer's dementia
The effects of the IRAP inhibitors can be tested in a mouse model of
Alzheimer's disease,
the Tg2576 transgenic mouse (with the Swedish mutation Lys-670-Asn; Met-671-
Leu).
This mouse model of Alzheimer's disease is commercially available from Taconic
Biotechnology, and the mice have demonstrated deficits in spatial learning
(water maze)
and working memory (spontaneous alternation Y maze).
Memory and learning can be tested in humans by any one of a number of well
established
neuropsychological tests such as California Verbal Learning Test, Wechsler
Memory
Scale-III, Hopkins Verbal Learning Test - RevisedTM, Rey Auditory Verbal
Learning Test,
and Rey-Osterrieth Complex Figure Design Test.
Subjects to be treated by the compounds and methods of the invention include
mammalian
subjects: humans, non-primates, livestock animals (including cows, horses,
sheep, pigs and
goats), companion animals (including dogs, cats, rabbits, guinea pigs), and
captive wild
animals. Laboratory animals such as rabbits, mice, rats, guinea pigs and
hamsters are also
contemplated as they may provide a convenient test system. Non-mammalian
species such
as birds, amphibians and fish may also be contemplated in certain embodiments
of the
invention. In particular embodiments, the subject is a human subject.
The compounds of the invention are administered to the subject in an IRAP
inhibiting, or
otherwise treatment effective amount. An IRAP inhibiting amount is an amount
which
will at least partially interact with IRAP or disrupt IRAP activity. IRAP
activity as used
herein includes IRAP functional interaction with endogenous ligands,
particularly where
the functional interaction directly or indirectly promotes memory and/or
learning loss. A
treatment effective amount is intended to include an amount which, when
administered
according to the desired dosing regimen, results in a measurable improvement
or
.---- . ,
CA 02611072 2007-11-19
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enhancement in memory or learning, or at least partially attains the desired
therapeutic or
prophylactic effect of one or more of: alleviating, eliminating or reducing
the frequency of
one or more of the symptoms of, preventing or delaying the onset of,
inhibiting the
progression of, or halting or reversing, partially or altogether, the onset or
progression of
the particular disorder or condition being treated.
Suitable dosage amounts and dosing regimens can be determined by the attending
physician and may depend on the particular condition being treated, the
severity of the
condition as well as the general age, health and weight of the subject.
The active ingredient may be administered in a single dose or a series of
doses. While it is
possible for the active ingredient to be administered alone, it is preferable
to present it as a
composition, preferably as a pharmaceutical composition, with one or more
pharmaceutically acceptable adjuvants. Thus, the present invention also
relates to the use
of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug
thereof in
the manufacture of a medicament for inhibiting IRAP activity in a subject.
The formulation of such compositions is well known to those skilled in the
art, see for
example, Remington's Pharmaceutical Sciences, 18a' Edition, Mack Publishing,
1990. The
composition may contain any suitable carriers, diluents or excipients. These
include all
conventional solvents, dispersion media, fillers, solid carriers, coatings,
antifungal and
antibacterial agents, dermal penetration agents, surfactants, isotonic and
absorption agents
and the like. It will be understood that the compositions of the invention may
also include
other supplementary physiologically active agents.
The carrier 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 dermal,
buccal and
sublingual), vaginal or parental (including subcutaneous, intramuscular,
intravenous,
intracerebroventricular and intradermal) administration. The compositions may
conveniently be presented in unit dosage form and may be prepared by any
methods well
CA 02611072 2007-11-19
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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 or finely divided solid
carriers or both,
and then if necessary shaping the product.
Compositions of the present invention suitable for oral administration may be
presented as
discrete units such as capsules, sachets or tablets each containing a
predetermined amount
of the active ingredient; as a powder or granules; as a solution or a
suspension in an
aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water-in-oil
liquid emulsion.
A tablet may be made by compression or moulding, optionally with one or more
accessory
ingredients. Compressed tablets may be prepared by compressing in a suitable
machine
the active ingredient in a free-flowing form such as a powder or granules,
optionally mixed
with a binder (e.g. inert diluent), preservative disintegrant (e.g. sodium
starch glycolate,
cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl
cellulose) surface-
active or dispersing agent. Moulded tablets may be made by moulding in a
suitable
machine a mixture of the powdered compound moistened with an inert liquid
diluent. The
tablets may optionally be coated or scored and may be formulated so as to
provide slow or
controlled release of the active ingredient therein using, for example,
hydroxypropylmethyl
cellulose in varying proportions to provide the desired release profile.
Tablets may
optionally be provided with an enteric coating, to provide release in parts of
the gut other
than the stomach.
Compositions suitable for topical administration in the mouth include lozenges
comprising
the active ingredient in a flavoured base, usually sucrose and acacia or
tragacanth gum;
pastilles comprising the active ingredient in an inert basis such as gelatin
and glycerin, or
sucrose and acacia gum; and mouthwashes comprising the active ingredient in a
suitable
liquid carrier.
CA 02611072 2007-11-19
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Compositions suitable for topical administration to the skin may comprise the
compounds
dissolved or suspended in any suitable carrier or base and may be in the form
of lotions,
gel, creams, pastes, ointments and the like. Suitable carriers include mineral
oil, propylene
glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan
monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and
water. Transdermal patches may also be used to administer the compounds of the
invention.
Compositions for rectal administration may be presented as a suppository with
a suitable
base comprising, for example, cocoa butter, glycerin, gelatin or polyethylene
glycol.
Compositions suitable for vaginal administration may be presented as
pessaries, tampons,
creams, gels, pastes, foams or spray formulations containing in addition to
the active
ingredient such carriers as are known in the art to be appropriate.
Compositions suitable for parenteral administration (e.g. subcutaneous,
intramuscular,
intravenous or intracerebroventricular) may include aqueous and non-aqueous
isotonic
sterile injection solutions which may contain anti-oxidants, buffers,
bactericides and
solutes which render the composition isotonic with the blood of the intended
recipient; and
aqueous and non-aqueous sterile suspensions which may include suspending
agents and
thickening agents. The compositions may be presented in unit-dose or multi-
dose sealed
containers, for example, ampoules and vials, and may be stored in a freeze-
dried
(lyophilised) condition requiring only the addition of the sterile liquid
carrier, for example
water for injections, immediately prior to use. Extemporaneous injection
solutions and
suspensions may be prepared from sterile powders, granules and tablets of the
kind
previously described.
Preferred unit dosage compositions are those containing a daily dose or unit,
daily sub-
dose, as herein above described, or an appropriate fraction thereof, of the
active ingredient.
It should be understood that in addition to the active ingredients
particularly mentioned
above, the compositions of this invention may include other agents
conventional in the art
CA 02611072 2007-11-19
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having regard to the type of composition in question, for example, those
suitable for oral
administration may include such further agents as binders, sweeteners,
thickeners,
flavouring agents disintegrating agents, coating agents, preservatives,
lubricants and/or
time delay agents. Suitable sweeteners include sucrose, lactose, glucose,
aspartame or
saccharine. 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 coating agents include polymers or copolymers of acrylic acid and/or
methacrylic
acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
Suitable
preservatives include sodium benzoate, vitamin E, alpha-tocopherol, 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 present invention also relates to prodrugs of formula (I). Any compound
that is a
prodrug of a compound of formula (I) is within the scope and spirit of the
invention. The
term "prodrug" is used in its broadest sense and encompasses those derivatives
that are
converted in vivo, either enzymatically or hydrolytically, to the compounds of
the
invention. Such derivatives would readily occur to those skilled in the art,
and include, for
example, compounds where a free thiol or hydroxy group is converted into an
ester, such
as an acetate, or thioester or where a free amino group is converted into an
amide.
Procedures for acylating the compounds of the invention, for example to
prepare ester and
amide prodrugs, are well known in the art and may include treatment of the
compound
with an appropriate carboxylic acid, anhydride or chloride in the presence of
a suitable
catalyst or base. Esters of carboxylic acid (carboxy) groups are also
contemplated.
Suitable esters C1_6alkyl esters; CI_6alkoxymethyl esters, for example
methoxymethyl or
ethoxymethyl; CI_6alkanoyloxymethyl esters, for example, pivaloyloxymethyl;
phthalidyl
esters; C3_8cyc1oalkoxycarbonylC1_6alkyl esters, for example, 1-
cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters, for example, 5-
methyl-1,3-
dioxolen-2-onylmethyl; and C1_6alkoxycarbonyloxyethyl esters, for example, 1-
methoxycarbonyloxyethyl. Prodrugs of amino functional groups include amides
(see, for
CA 02611072 2007-11-19
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example, Adv. BioSci., 1979, 20, 369, Kyncl, J. et ao, enamines (see, for
example, J.
Pharm. Sci., 1971, 60, 1810, Caldwell, H. et al), Schiff bases (see, for
example, US Patent
No 2,923,661 and Antimicrob. Agents Chemother., 1981, 19, 1004, Smyth, R. et
ao,
oxazolidines (see, for example, J. Pharm. Sci, 1983, 72, 1294, Johansen, M. et
ao,
Mannich bases (see, for example, J. Pharm. Sci. 1980, 69, 44, Bundgaard, H. et
al and J.
Am. Chem. Soc., 1959, 81, 1198, Gottstein, W. et al), hydroxymethyl
derivatives (see, for
example, J. Pharm. Sci, 1981, 70, 855, Bansal, P. et al) and N-(acyloxy)alkyl
derivatives
and carbamates (see, for example, J. Med. Chem., 1980, 23, 469, Bodor, N. et
al, J. Med.
Chem., 1984, 27, 1037, Firestone, R. et al, J. Med. Chem., 1967, 10, 960,
Kreiger, M. et al,
US Patent No 5,684,018 and J Med. Chem., 1988, 31, 318-322, Alexander, J. et
al). Other
conventional procedures for the selection and preparation of suitable prodrugs
are known
in the art and are described, for example, in WO 00/23419; Design of Prodrugs,
H.
Bundgaard, Ed., Elsevier Science Publishers, 1985; Methods in Enzymology, 42:
309-396,
K. Widder, Ed, Academic Press, 1985; A Textbook of Drug Design and
Development,
Krogsgaard-Larsen and H. Bundgaard, Eds, Chapter 5, p113-191 (1991); Advanced
Drug
Delivery Reviews, 8; 1-38 (1992); Journal of Pharmaceutical Sciences, 77;285
(1988), H.
Bundgaard, et al; Chem Pharm Bull, 32692 (1984), N. Kakeya et al and The
Organic
Chemistry of Drug Desig and Drug Action, Chapter 8, pp352-401, Academic press,
Inc.,
1992.
Suitable pharmaceutically acceptable salts of compounds of formula (I)
include, but are not
limited to salts of pharmaceutically acceptable inorganic acids such as
hydrochloric,
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, maleic, citric, lactic, mucic, gluconic,
benzoic, succinic,
oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic,
salicyclic
sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric,
pantothenic, tannic,
ascorbic, fendizoic, 4-4'-methylenebis-3-hydroxy-2-naphthoic acid, o-(p-
hydroxybenzoyl)benzoic, 4'-4"-dihydroxytriphenylmethane-2-carboxylic acid and
valeric
acids. Base salts include, but are not limited to, those formed with
pharmaceutically
acceptable cations, such as sodium, potassium, lithium, calcium, magnesium,
ammonium
CA 02611072 2007-11-19
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and alkylammonium. Basic nitrogen-containing groups may be quaternised with
such
agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl
chlorides, bromides
and iodides or dialkyl sulfates such as dimethyl and diethyl sulfate.
The compounds of the invention may be in crystalline form either as the free
compounds
or as solvates and it is intended that both forms are within the scope of the
present
invention. The term "solvate" refers to a complex or aggregate formed by one
or more
molecules of a solute, i.e. compounds contemplated by the invention, and one
or more
molecules of a solvent. Suitable solvents are well understood in the art and
include for
example, of water, i.e. to form hydrates, and common organic solvents such as
alcohols
(methanol, ethanol, isopropanol) and acetic acid. Methods of solvation are
generally
known within the art, for example, recrystallization from an appropriate
solvent.
Tautomeric forms of compounds described herein, such as keto-enol tautomers,
are also
contemplated to be part of the invention where appropriate.
It will also be recognised that certain compounds of formula (I) may possess
asymmetric
centres and are therefore capable of existing in more than one stereoisomeric
form, such as
enantiomers and diastereomers. The invention thus also relates to optically
active
compounds and compounds in substantially pure isomeric form at one or more
asymmetric centres, e.g., diastereoisomers and enantiomers having greater than
about 90%
ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures,
including
racemic mixtures, thereof. Such isomers may be prepared by asymmetric
synthesis, for
example using chiral intermediates, enzymes, or mixtures may be resolved by
conventional
methods, e.g., chromatography, recrystallization, or use of a resolving agent.
The compounds of the invention may also be used to treat non-human subjects
and may
therefore be presented as veterinary compositions. These may be prepared by
any suitable
means known in the art. Examples of such compositions include those adapted
for:
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(a) oral administration, external application (e.g. drenches including aqueous
and non-
aqueous solutions or suspensions), tablets, boluses, powders, granules,
pellets for
admixture with feedstuffs, pastes for application to the tongue;
(b) parenteral administration, e.g. subcutaneous, intramuscular, intravenous
or
intracerebroventricular injection as a sterile solution or suspension;
(c) topical application e.g. creams, ointments, gels, lotions etc.
Those skilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all such variations and modifications which fall
within the spirit
and scope. The invention also includes all of the steps, features,
compositions and
compounds referred to or indicated in this specification, individually or
collectively, and
any and all combinations of any two or more of said steps or features.
The following examples are provided for the purpose of illustrating certain
embodiments
of the invention and are not intended to limit the generalities hereinbefore
described.
CA 02611072 2007-11-19
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EXAMPLES
EXAMPLE 1
Four compounds (1-4) were prepared and tested for IRAP inhibitory activity and
compared
against the most potent exemplified compound of WO 2006/026832.
Preparation of Comparative Compound 1 from WO 2006/026832
(i) 3-Pyridine carboxaldehyde (2.2g) and ethylcyanoacetate (2.3g) were heated
at
reflux in toluene together with acetic acid (100 l) and piperidine (40 1).
Nitrogen
was blown over the surface to help remove water. A precipitate formed on
cooling
and standing, with the addition of pet. spirit. A cream-coloured crystalline
solid
(3.0g) was collected.
(ii) (Reference: Org. Prep and Proc., 1999, 31, 305)
The ethylacrylate ester product of (i) above (1.0g) was dissolved, together
with
resorcinol (0.55g) in 15m1 ethanol at 80 C. Piperidine (5 drops) was added to
the
solution and the mixture heated at reflux for 4 hours. A white solid was
collected
and the title compound separated from the dimeric product to afford 40mg of
the
desired compound.
The desired product was obtained in 48% yield, free of dimeric product when
1.5
equivalents of resorcinol was used on a 4.9mmol scale.
Compound 1
Ethy12-amino-7-hydroxy-4-quinolin-4-yl-4H-chromene-3-carboxylate
(i) Ethyl 2-cyano-3-quinolinyl-4-ylacrylate was obtained in a similar manner
to that
described above by reaction of 4-quinolinecarboxaldehyde and ethyl
cyanoacetate.
The product precipitated from the reaction mixture and was isolated by
filtration
CA 02611072 2007-11-19
- 38
(1.432g, 61%).
(ii)
N~s N
f I
0 0
~
I OEt
HO 0H OEt II
`
CN HO 0 NH2
A mixture of resorcinol (126mg, 1.14mmo1) and ethyl 2-cyano-3-quinolin-4-
ylacrylate (254mg, 1.Olmmol) was suspended in absolute ethanol (3.OmL).
Piperidine (201AL) was added and the mixture heated at reflux for 3h. A
precipitate
formed on cooling. The mixture was filtered. The residue was dissolved in a
mixture of ethyl acetate (20mL) and ethanol (5mL). Silica gel 60 (0.9g) was
added
and the mixture evaporated to dryness. Purified by flash chromatography over
silica gel 60, 40-63 m (eluent: 75% ethyl acetate / petroleum spirits (8 x
20mL
fractions), packing height: 18 cm, column diameter: 1 cm). The fractions
containing the major band (Rf 0.43, eluent: 75% ethyl acetate / petroleum
spirits,
fractions 3-5) were combined and evaporated to dryness. The residue was dried
in
a vacuum oven (80 C, 200mbar) for 6h to give the title compound (115mg, 32%)
as a yellow powder.
HPLC (214 nm) tR = 6.02 min (97%).
'H NMR (400 MHz, DMSO-d6) 6 0.61 (t, J= 7.2Hz, 3H), 3.64-3.79 (m, 2H), 5.78
(s, 1 H), 6.3 7(dd, J= 8.4, 2.4Hz, 1 H), 6.48 (d, J= 2.4Hz, 1 H), 6.82 (d, J=
8.4Hz, 1 H), 7.19 (d, J= 4.4Hz, 1 H), 7.64=7.80 (m, 4H), 8.00 (dd, J= 8.4,
1.2Hz,
1 H), 8.64 (br d, J= 8.4Hz, 1 H), 8.74 (d, J= 4.8Hz, 1 H), 9.67 (s, 1 H).
CA 02611072 2007-11-19
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Compound 2
Ethyl 2-(acetylam ino)-7-hydroxy-4-pyrid in-3-yl-4H-chrom en e-3-carboxylate
N ~N
I
O ~ 0
~
OEt O Et
~
I I
AcO 0 NH(Ac)2 HO / O NHAc
Ethyl 7-(acetyloxy)-2-(diacetylamino)-4-pyridin-3-yl-4H-chromene-3-carboxylate
(6.989g, 15.9mmo1) was dissolved in absolute ethanol (5OmL). Hydrazine hydrate
(9501iL, 19.5mmol) was added and the mixture stirred at room temperature for
6h.
The reaction mixture was evaporated to dryness to give an orange solid. The
residue was dissolved in a mixture of ethyl acetate (200mL) and ethanol
(50mL).
Silica gel 60 (35g) was added and the mixture evaporated to dryness. Purified
by
flash chromatography over silica gel 60, 40-63 m (eluent: 75% ethyl acetate /
petroleum spirits (54 x 100mL fractions), packing height: 15 cm, column
diameter:
5 cm). The fractions containing the major band (Rf 0.26, eluent: 75% ethyl
acetate
/ petroleum spirits, fractions 15-53) were combined and evaporated to dryness
to
give the title compound (3.307g, 59% yield) as a pale yellow powder.
HPLC (214 nm) tR = 5.05 min (99%).
'H NMR (400 MHz, DMSO-d6) 8 1.09 (t, J= 7.0Hz, 3H), 2.08 (s, 3H), 3.99 (q, J=
6.9Hz, 2H), 4.99 (s, 1 H), 6.47 (d, J= 2.4Hz, 1 H), 6.55 (dd, J= 8.4, 2.4Hz, 1
H),
7.10 (d, J= 8.4Hz, 1H), 7.27 (dd, J= 7.8, 4.6Hz, IH), 7.57-7.62 (m, 1H), 8.36
(dd, J= 4.8, 1.6Hz, 1 H), 8.54 (d, J= 2.0Hz, IH), 9.75 (br s, 1 H), 10. 3 5(br
s,
1H).
LC/MS tR = 5.43 min (355.5 [M+H]+).
MP 216-218 C
CA 02611072 2007-11-19
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Compound 3
Ethyl 2-amino-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
(i) Ethyl 2-cyano-3-quinolin-3-ylacrylate was obtained in a similar manner to
that
described above by reaction of 3-quinolinecarboxaldehyde and ethyl
cyanoacetate.
The product precipitated from the reaction mixture and was isolated by
filtration
(1.023g, 92%).
(ii)
N
N
O
~
~ ~-
OEt
HO / OH O Et I I
CN HO O NH2
A mixture of resorcinol (512mg, 4.65mmol), ethyl 2-cyano-3-quinolin-3-
ylacrylate
(1.003g, 3.98mmol), piperidine (80 L, 0.81mmol) and absolute ethanol (12mL)
was heated at reflux for 3h. A precipitate formed on cooling. Water (36mL) was
added and the mixture filtered. The residue was washed with water (2 x 30mL),
dried in a vacuum desiccator over silica gel, washed with ethyl acetate (5mL)
and
dried under high vacuum (180 C, 0.5mbar) for 90min to give an orange powder
(794mg, 55% yield). A portion of this product (596mg) was suspended in ethanol
(20mL). Silica gel 60 (3.1 g) was added and the mixture evaporated to dryness.
Purified by flash chromatography over silica gel 60, 40-63 m (eluent: 75%
ethyl
acetate / petroleum spirits (19 x 40mL fractions), ethyl acetate (16 x 40mL
fractions), ethanol (7 x 40mL fractions), packing height: 15 cm, column
diameter:
2.5 cm). The pale yellow fractions containing the major band (Rf0.33, eluent:
75%
ethyl acetate / petroleum spirits, fractions 4-37) were combined and
evaporated to
CA 02611072 2007-11-19
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dryness. The residue was dried under high vacuum (150 C, 0.75mbar) for 15min
to give the title compound (282mg, 47% recovery) as a yellow powder.
HPLC (214 nm) tR = 5.86 min (92%).
'H NMR (400 MHz, DMSO-d6) S 1.00 (t, J= 7.0Hz, 3H), 3.92 (q, J= 7.1Hz, 2H),
5.05 (s, 1 H), 6.45-6.52 (m, 2H), 7.02 (d, J= 8.8Hz, 1 H), 7.54 (t, J= 7.4Hz,
1 H),
7.63-7.78 (m, 3H), 7.93 (t, J= 8.4Hz, 2H), 7.98 (d, J= 2.0Hz, 1 H), 8.75 (d,
J=
2.0Hz, 1 H), 9.70 (s, 1 H).
LC/MS tR = 6.25 (363.4 [M+H]+) min.
MP 229-231 C
Compound 4
Ethyl 2-(acetylamino)-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
I I
N N
I I
0 O
-~-
I I I
OEt OEt
AcO 0 NH(Ac)2 HO 0 NHAc
Ethyl 7-(acetyloxy)-2-(diacetylamino)-4-quinolin-3 -yl-4H-chromene-3-
carboxylate
(268mg, 0.55mmol) was dissolved in absolute ethanol (1.7mL). Hydrazine hydrate
(35 L, 0.72mmol) was added and the mixture stirred at room temperature for lh.
The reaction mixture was evaporated to dryness. 'H NMR analysis showed
incomplete conversion of the starting material. The product was suspended in
absolute ethanol (1.7mL) and hydrazine hydrate (35 L, 0.72mmol) added. The
mixture was stirred at room temperature for 2h. The mixture was filtered. The
residue was washed with ethanol (1.OmL) and ethyl acetate (2.OmL). The residue
CA 02611072 2007-11-19
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was dissolved in boiling ethanol (16mL) and the volume reduced to half by
heating
under a stream of nitrogen. The mixture was cooled to 4 C and filtered. The
residue was dried at the pump to give the title compound (65mg, 29% yield) as
pale
yellow needles.
HPLC (214 nm) tR = 5.84 min (98.5%).
'H NMR (400 MHz, DMSO-d6) S 1.07 (t, J= 7.2Hz, 3H), 2.11 (s, 3H), 3.98 (q, J=
7.1 Hz, 2H), 5.20 (s, 1 H), 6.51 (d, J= 2.0Hz, 1 H), 6.55 (dd, J= 8.4, 2.4Hz,
1 H),
7.16 (d, J= 8.4Hz, 1H), 7.57 (t, J= 7.4Hz, 1H), 7.69 (t, J= 7.6Hz, 1H), 7.89
(d,
J= 8.0Hz, IH), 7.96 (d, J= 8.0Hz, 1 H), 8.13 (d, J= 2.0Hz, 1 H), 8.89 (d, J=
2.0Hz, 1 H), 9.78 (br s, 1 H), 10.41 (br s, 1 H).
LC/MS tR = 6.27min (405.4 [M+H]+).
MP 248-251 C
EXAMPLE 2
An in vitro enzymatic assay was used to initially assess IRAP inhibitory
activity.
IRAP Enzymatic assay
Crude membranes were prepared from HEK 293T cells transfected with IRAP or
empty
vector, then solubilized in buffer consisting of 50 mM Tris-HCI, 1% Triton X-
100, pH 7.4
at 4 C under agitation over 5 h. After solubilization, the membranes were
pelleted by
centrifugation at 23,100 g for 15 min at 4 C, and the supernatant was reserved
as the
source of IRAP activity. The enzymatic activities of IRAP were determined by
the
hydrolysis of the synthetic substrate Leu-MCA (Sigma-Aldrich, Missouri, USA)
monitored
by the release of a fluorogenic product, MCA, at excitation and emission
wavelengths of
380 and 440 nm, respectively. Assays were performed in 96-well plates; each
well
contains between 0.2 - 10 g solubilized membrane protein, a range of
concentration of
substrate in a final volume of 100 L 50 mM Tris-HCl buffer (pH 7.4). Non-
specific
hydrolysis of the substrate was corrected by subtracting the emission from
incubations
with membranes transfected with empty vector. Reactions proceeded at 37 C for
30min
CA 02611072 2007-11-19
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within a thermostatted FLEX station fluorescence microplate reader (Molecular
Devices,
Sunnyvale, CA). The kinetic parameters (Km and V) were determined by non-
linear fitting
of the Michaelis-Menten equation (GraphPad Prism, GraphPad Software Inc., CA,
USA);
final concentrations of Leu-MCA of 15.6 M - 1 mM. Inhibitor constants (K;)
for the
competitive inhibitors were calculated from the relationship IC50 = K;
(1+[S]/K,n), where
IC50 values were determined over a range of inhibitor concentrations (10"9 to
10"4 M). Kn,
values of IRAP for Leu-MCA were determined from the kinetic studies. Binding
affinities
of the compounds to IRAP were examined by monitoring the inhibition of the
hydrolysis
of Leu-MCA in the presence of increasing concentrations of the compounds (10"8
to 10'3
M). All data obtained were from at least three separate experiments performed
in duplicate.
The results are depicted in Table 2-1.
Compounds Kl(uM) IC50 Structures
"
1 0.9 1.5 M
~ Y I OEt
HO 0 NH2
N
0
2 0.48 0.79 M oEt
HO -0- NHAc
CA 02611072 2007-11-19
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Compounds Ki(uM) ICSO Structures
IN-N
3 0.36 0.6 M oEt
z
HO o H
I~
D
4 0.02 40 nM i~ I oEt
HO J p NHAC
Compound 1
from
WO 2006/026832 2.0 3 M ! ,,, cH
3
(Comparative) HQ i NH
a
In order to see whether the compounds are selective or specific for IRAP, the
inhibitory
activities of Compounds 2-4 for other zinc-dependent metallopeptidases were
determined
in 96-well microtiter plates with absorbance monitored on a Wallac Victor 3
spectrophotometer.
Glucose-6-phosphate dehydrogenase and hexokinase activity. Glucose-6-phosphate
dehydrogenase and hexokinase activity in the absence and presence of the
compounds was
determined in a fluorimetric assay. Ten mM Tris MgC12 buffer (total volume of
200 ml)
containing glucose-6-phosphate dehydrogenase, 0.3M ATP, 30mM NADP+ was added
to
96 well microtitre plate together with 20p1 of compound, a reading at
excitation 350 nm
CA 02611072 2007-11-19
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and emission 510 nm was taken followed by the addition of hexokinase and
another
reading.
Leukotriene A4 hydrolase assay. Recombinant human LTA4H (Cayman Chemicals
Michigan USA) (1-20 g) was incubated at room temperature with alanine p-
nitroanilide
as substrate in 50 mM Tris-HC1 buffer, pH 8.0, containing 100 mM KCl with or
without
increasing concentrations of an inhibitor. The absorbance at 405 nm was
measured at 10
min intervals.
Aminopeptidase N assay. 5 mU of aminopeptidase M (Sigma Aldrich) was incubated
with 100 M of substrate alanine-(3-naphthylamide (Sigma Aldrich) in Tris
buffered saline
(50 mM Tris-HCI, 150 mM NaCl pH 7.5) at 25 C. IRAP inhibitors (1-10 M) were
added
after 1 minute and fluorescence at 405 nm was followed.
Angiotensin converting enzyme assay. The enzyme (final concentration 0.02
pmol/100
L) was incubated with 200 L of assay solution that included 5 mmol/L HHL in
100
mmol/L potassium buffer (pH 8.3) that contained 300 mmol/L NaCI and 10'4 mol/L
ZnSOa
for 3 hours at 37 C. The enzymatic reaction was stopped by the addition of 1.5
mL of
0.28N NaOH, 100 L of o-phthaldialdehyde (20 mg/mL) in methanol was added and
the
fluorescent reaction stopped by the addition of 200 L of 3N HCI. The product,
L-His-Leu,
was measured fluorometrically (360 nm excitation and 500 nm emission).
Limited inhibition of control enzymes was observed. The results are depicted
in Table 2-2
and are expressed as % inhibition of the catalytic activity of the enzyme at
the given
concentration.
CA 02611072 2007-11-19
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Table 2-2
Compound 2 Compound 3 Compound 4
100 M 10 M 100 M 10 M 100 M 10 M
Glucose-6-phosphate
dehydrogenase/ glucose 0 ND* 0 ND 0 ND
hexokinase
Leukotriene A4 hydrolase 0 ND 4 ND 25 0
Aminopeptidase N ND 13 ND 12 14 3
Angiotensin converting 3 9 3 0 0 0
enzyme I
*ND = not determined
EXAMPLE 3
The effects of Compound 2 on performance in different memory tasks in vivo
were
investigated.
Surgical preparation of rats
All experiments were performed according to the National Health and Medical
Research
Council of Australia "Code of practise for the care and use of animals for
scientific
purposes". Male Spraque Dawley rats, (250 - 270g) are housed individually and
given
water and standard rat chow ad libitum. On the day of surgery, the rats were
anaesthetized
with 5% isofluorane, placed in a stereotaxic frame and maintained on 2%
isofluorane for
the duration of the cannula implantation procedure. The rats were
stereotaxically
implanted with chronic indwelling cannula (Plastics One) into the cerebral
lateral
ventricles using the following flat skull coordinates 0.8 mm posterior to
Bregma, 1.55 mm
lateral to midline and 3.5 mm ventral to the dura. The cannula was then
secured to the
skull with stainless steel screws and dental cement. Seven days following
surgery, proper
cannula placement was verified by a bolus injection of angiotensin 11 (1 nmol/
l). Lack of
CA 02611072 2007-11-19
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a robust dipsogenic response within 5 min of angiotensin II administration
would suggest
misplacement of the cannula and the animal was then excluded from further
studies.
Novel Object Recognition Task (Bevins et al, 2006)
The rats are allowed at least 5 days post-operative recovery prior to use in
any behavioural
paradigms. On the day of the acquisition trial, the rats were habituated for 5
min in the
testing box (made from grey perspex of dimensions 60 cm width x 60 cm length x
50 cm
height) in diffuse dim light and then returned to their home cage. The animals
were then
rested for at least 2 h and then injected with 1 nmol or 0.1 nmol Compound 2
in 2 l of
10% dimethyl sulfoxide (DMSO). Control animals received 2 l of 10% DMSO.
Following drug administration, the rats were returned to their home cage for 5
min and
then placed in the testing box facing the opposite direction to 2 identical
objects that have
been secured to the floor in adjacent corners of the box. The rats were
allowed 5 min to
explore the objects and the definition of explore is that the animal's nose is
less than 2 cm
from object when it is facing the object. Animals that displayed a lack of
interest in the
object, exploration time of less than 15 secs were excluded from the study at
this stage.
The animals were then returned to their home cage during the intertrial
interval of 20h. On
day 2 of testing, one of the objects were replaced by a novel object made from
the same
material but of a different shape - the rats were given 2 mins in the box. The
recognition
index was determined as the time spent exploring the novel object minus the
time spent on
the familiar object divided by the time spent on both object.
Spontaneous Alternation Plus Maze (McNay et al, 2000)
The plus maze was composed of four arms with each arm measuring 75 x 10 x 20
cm. The
floor and walls of the central platform and the floors of the arms were made
of black
plastic. Spontaneous alternation testing was conducted by placing the rat on
the center
platform of the maze and allowing 20 min of unimpeded exploration. The number
and
sequence of arm entries were recorded for calculation of a percent alternation
score. An
alternation consisted of 4 different arm choices of 5 consecutive arm entries.
A 4/5
alternation score was computed by dividing the number of observed alternations
in
CA 02611072 2007-11-19
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overlapping quintuplets by the number of possible alternations and multiplying
the quotient
by 100.
Elevated Plus Maze
The elevated plus maze was used to investigate the potential effect of IRAP
inhibitors on
stress or anxiety. The elevated plus maze consists of two open arms (70xlOcm)
with a
5cm high surrounding wall and two enclosed arms (70x10cm) with a 27cm high
surrounding wall. The floor of the open and closed arms are white laminate,
the open arm
walls are clear perspex, and the closed arms walls dark grey perspex. The maze
is elevated
85cm above the ground in the centre of a room that is lit by overhead lights
generating
124lux. Naive rats, treated either with 1 nmol Compound 2 dissolved in 10%
DMSO or
vehicle, were placed 5 min after the icv injection, on the central platform
facing one of the
closed arms and behaviour monitored for 10min. The time spent in the closed
arms
compared to the open arms was a measure of the anxiety status of the animals.
Locomotor Cell Activity
Locomotor activity of rats treated with I nmol Compound 2
intracerebrocentricularly was
monitored in special cages measuring 40 x 40 x 40 cm (Coulbourn Instruments,
Philadelphia, USA) equipped with harmless infrared photobeams. Activity was
measured
when pairs of photobeams spaced 2.54 cm apart providing a 1.27 cm spatial
resolution
were crossed. Data was collected and analysed using TruScan Photo Beam
Activity
svstem (Coulbourn Instruments, Philadelphia, USA). Each rat was placed in the
arena for
30min.
The results are depicted in Tables 3-Al - 3-A2 and 3-B1 - 3-B3.
Rats treated with Compound 2, at 1 and 0.1 nmole intracerebroventricularly,
exhibited
better recognition of a novel object after 24h compared to vehicle treated
control rats, n=
10/group and * p<0.05 (Table 3-A1). Rats treated with 0.1 nmole exhibited
significantly
enhanced spontaneous alternation scores compared to vehicle treated control
rats,
n=8/group and **p<0.01 (Table 3-A2). Rats treated with Compound 2 at 0.1 and I
nmol
CA 02611072 2007-11-19
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intracerebroventricularly, were not significantly more or less stressed than
vehicle treated
control rats as determined by time spent in the open arm of the elevated plus
maze. n=8 per
group (Table 3-B 1). Rats treated with Compound 2 at 0.1 and 1 nmol
intracerebroventricularly, did not exhibit increased or decreased locomotor
activity
compared to vehicle treated rats as determined by crossing of infrared beams
and total
distance travelled in a locomotor cell over a period of 30 mins. n=8 per group
(Table 3-
B2).
Table 3-Al
Treatment Recognition Index
Vehicle 5.04 + 5.43
0.1 nmol 35.82 + 7.30*
1 nmol 45.09 + 9.97*
Table 3-A2
Treatment Spontaneous Alternation Score (%)
Vehicle 62.07 + 1.55
0.1 nmol 73.70 + 3.14 **
1 nmol 64.34 + 2.49
Table 3-Bl
Treatment Time spent in the open arm (% control)
Vehicle 100 + 31.26
0.1 nmol 121.62 + 28.26
1 nmol 127.88 + 19.18
CA 02611072 2007-11-19
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Table 3-B2
Treatment Distance travelled (% control)
Vehicle 100 + 3
0.1 nmol 85.12 + 12.99
1 nmol 90.16 + 6.73
EXAMPLE 4
Hippocampal glucose uptake assay. Eight week old male Spraque-Dawley rats were
deeply anaesthetised with Isofluorothane and killed by decapitation. The
brains were
rapidly removed and placed into ice-cold, 95% 02, 5% C02-bubbled artificial
cerebrospinal fluid (aCSF: 124 mM NaCI, 2.5 mM KCI, 2 mM CaC12, 2 mM MgSOa, 26
mM NaHCO3, 1.25 mM NaH2PO4 pH 7.4) supplemented with 10 mM D-glucose (Sigma).
Under aCSF, the hippocampal hemispheres were rapidly dissected out and 200 m
slices
prepared on McIllwain tissue chopper with an ice-cold blade and stage. Slices
from each
hemisphere were kept together and transferred to freshly 95% 02, 5% C02-
bubbled aCSF
supplemented with ] 0 mM D-glucose at 37 C for 1 hour. All subsequent steps
were carried
out at 37 C in a gently shaking chamber with continuous infusion of 95% 02, 5%
CO2.
Slices from one hemisphere were used as a basal control while slices from the
other
hemisphere were stimulated, providing an internal control for each animal.
Hemisphere
pairs were transferred to 95% 02, 5% CO2 bubbled aCSF supplemented with 0.1 mM
D-
glucose and 2 mM 2-deoxyglucose and ImM dbCAMP for 15 min following which
slices
from one hemisphere were stimulated by the addition of 100 nM HF1419 while the
other
hemisphere provided an internal control for each animal and 0.1 Ci 2-Deoxy-d-
[2,6-
3H]glucose (3H-2DG) (specific activity 49Ci/mmol; Amersham Biosciences, NSW,
Australia) was added to a final concentration of 0.1 M in both basal and
stimulated
hemispheres for five minutes. Following uptake, hemispheres were rapidly
rinsed in four
changes of ice cold PBS and slices transferred to pre-weighed filter paper and
allowed to
dry. Dried slices from each hemisphere were weighed and solubilized overnight
with
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Soluene-350 (Perkin Elmer, MO, USA). Tritium content was measured in a Liquid
Scintillation Analyser 1900TR (Perkin Elmer, MA, USA).
An increase in dibutyryl cAMP-evoked glucose uptake in response to Compound 2
was
observed.
The results are depicted in Table 4-1.
The effect of Compound 2 was tested in vitro on the metabolic demands of
neurones.
Table 4-1
Fold change in 3H-2DOG uptake
dbcAMP 1
dbcAMP & Compound 2 1.7
CA 02611072 2007-11-19
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BIBLIOGRAPHY
Albiston et al., JBiol Chem 276: 48263-48266, 2001.
Albiston et al., Behav Brain Res 154: 239-243, 2004.
Bevins et al., Nat Protoc 1(3): 1306-11, 2006.
Braszko, et al., Neuroscience 27: 777-783, 1988.
Dash et al., J. Neurosci. 26: 8048-56, 2006.
Fernando et al., Journal of Comparative Neurology 487: 372-390, 2005.
Greferath et al., Neuroscience 100: 363-373, 2000.
Herbst et al., Am. J. Physiol. 272: E600-6, 1997.
Keller el al., JBiol Chem 270: 23612-23618, 1995.
Lee et al., Neuroscience 124: 341-349, 2004.
Lew et al., Journal of Neurochemistry 86: 344-350, 2003.
McNay et al., Proc Natl Acad Sci USA, 97(6): 2881-5, 2000.
Pederson et al., Regul Pept 74: 97-103, 1998.
Pederson et al., Regul Pept 102: 147-156, 2001.
Rogi et al., J Biol Chem 271: 56-61, 1996.
Waters et al., JBiol Chem 272: 23323-23327, 1997.
Wright et al., Brain Res Bull 32: 497-502, 1993.
Wright et al., Brain Res 717: 1-11, 1996.
Wright et al., JNeurosci 19: 3952-3961, 1999.
Zhang et al., JPharmacol Exp Ther 289: 1075-1083, 1999.
