Note: Descriptions are shown in the official language in which they were submitted.
CA 02407777 2002-10-30
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NOVEL SPIRO[2.4]HEPTANE AMINO CARBOXY COMPOUNDS AND
DERIVATIVES THEREOF
FIELD OF THE INVENTION
This invention pertains to therapeutically active novel spiro[2.4]heptane
amino
carboxy compounds and derivatives thereof, a method for preparing the same,
pharmaceutical compositions comprising the compounds and a method of treating
diseases of the Central Nervous System (CNS) therewith.
to
BACKGROUND OF THE INVENTION
The acidic amino acid L-glutamate is recognized as the major excitatory
neurotransmitter in the CNS. The receptors that respond to L-glutamate are
called
excitatory amino acid receptors, The excitatory amino acid receptors are thus
of
great physiological importance, playing a role in a variety of physiological
processes, such as long-term potentiation (learning and memory), the
development
of synaptic plasticity, motor control, respiratory and cardiovascular
regulation, and
sensory perception.
Excitatory amino acid receptors are classified into two general types and both
are
activated by L-glutamate and its analogs. Receptors activated by L-glutamate
that
are directly coupled to the opening of cation channels in the cell membrane of
the
neurons are termed "ionotropic." This type of receptor has been subdivided
into at
least three subtypes, which are defined by the depolarizing actions of the
selective
agonists N-Methyl-D-aspartate (NMDA), a-Amino-3-hydroxy-5-methylisoxazole-4-
propionic acid (AMPA), and I~ainic acid (KA).
The second general type of receptor is the G-protein or second messenger-
linked
3o "metabotropic" excitatory amino acid receptor. This second type is coupled
to
multiple second messenger systems that lead to enhanced phosphoinositide
hydrolysis, activation of phospholipase D, increases or decreases in cAMP
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formation, and changes in ion channel function (Schoepp and Conn, Ti~e~cds in
Pharmacological Science, 14:13, 1993). Both types of receptors appear not only
to
mediate normal synaptic transmission along excitatory pathways but also to
participate in the modification of synaptic connections during development and
throughout life.
So far eight different clones of the G-protein-coupled mGluRs have been
identified
(Knopfel et al., 1995, J. Med. Chen2., 38, 1417-1426). These receptors
function to
modulate the presynaptic release of L-glutamate, and the postsynaptic
sensitivity of
to the neuronal cell to L-glutamate excitation. Based on pharmacology,
sequence
homology and the signal transduction pathway that they activate, the mGluRs
have
been subclassified into three groups. The mGluR1 and mGluRS receptors form
group I. They are coupled to hydrolysis of phosphatidylinositol (PI) and are
selectively activated by (RS)-3,5-dihydroxyphenylglycine (Brabet et al.,
Neu~opharmacology, 34, 895-903, 1995). Group II comprises mGluR2 and
mGluR3 receptors. They are negatively coupled to adenylate cyclase and are
selectively activated by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine
(DCG-IV; Hayashi et al., Nature, 366, 687-690, 1993). Finally, the mGluR4,
mGluR6, mGluR7 and mGluR8 receptors belong to group III. They are also
2o negatively coupled to adenylate cyclase and are selectively activated by
(S)-2-
amino-4-phosphonobutyric acid (L-AP4; Knopfel et al., 1995, .I. Med. Chem.,
38,
1417-1426).
Agonists and antagonists of these receptors are believed useful for the
treatment of
acute and chronic neurodegenerative conditions, and as antipsychotic,
anticonvulsant, analgesic, anxiolytic, antidepressant, and anti-emetic agents.
Antagonists and agonists of neural receptors are classified as selective for a
particular receptor or receptor subtype, or as non-selective. Antagonists may
also be
classified as competitive or non-competitive. While competitive and non-
3o competitive antagonists act on the receptors in a different manner to
produce similar
results, selectivity is based upon the observations that some antagonists
exhibit high
levels of activity at a single receptor type, and little or no activity at
other receptors.
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In the case of receptor-specific diseases and conditions, the selective
agonists and
antagonists are of the most value.
Compounds such as z-glutamate, quisqualate and ibotenate axe known to act as
non-
selective agonists on the mGluRs, while selective ionotropic glutamate
receptor
agonists such as NMDA, AMPA and kainate have little effect on these receptors.
Recently a few compounds without activity at the ionotropic glutamate
receptors but
with activity at the metabotropic receptors have been identified. These
include trans-
ACPD (tra~rs (1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid), the partial
agonist
l0 L-AP3 (L-2-amino-3-phosphonopropionic acid; Palmer, E., Monaghan, D. T. and
Cotman, C. W. Eur J. Pha~macol. 166, 585-587, 1989; Desai, M. A. and Conn, P.
J.
Neu~osciehce Lett. 109, 157-162, 1990; Schoepp, D. D. et al., J.
Neu~oche~raistry.
56, 1789-1796, 1991; Schoepp D. D. and Johnson B. G. .I. Neurochemistry 53,
1865-1613, 1989), L-AP4 (L-2-amino-4-phosphonobutyrate) which is an agonist at
the mGluR4 receptor (Thomsen C. et al., Eur. J. Pha~macol. 227, 361-362, 1992)
and some of the isomers of CCG (2-(carboxycyclopropyl)glycines) especially L-
CCG-I and z-CCG-II (Hayashi, Y. et al., Br: J. Pharmacol. 107, 539-543, 1992).
Very few selective antagonists at the mGluRs have been reported. However some
2o phenylglycine derivatives, S-4CPG (S-4-carboxyphenylglycine), S-4C3HPG (S-4
carboxy -3-hydroxyphenylglycine) and S-MCPG (S-a,-methyl-4
carboxyphenylglycine) have been reported to antagonize t~aias-ACPD- stimulated
phosphoinositide hydrolysis and thus possibly act as antagonists at mGluRl and
mGluRS subtypes (Thomsen, C. and Suzdak, P, Eur J. Pharmacol. 245, 299, 1993).
Research directed towards mGluRs is beginning to show that mGluRs may be
implicated in a number of normal as well as pathological mechanisms in the
brain
and spinal cord. For example, activation of these receptors on neurons can
influence
levels of alertness, attention and cognition; protect nerve cells from
excitotoxic
3o damage resulting from ischemia, hypoglycemia and anoxia; modulate the level
of
neuronal excitation; influence central mechanisms involved in controlling
movement; reduce sensitivity to pain; reduce levels of anxiety.
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The use of compounds active at the mGluRs for the treatment of epilepsy is
corroborated by investigations of the influence of traps-ACPD on the formation
of
convulsions (Sacaan and Schoepp, Neuroscience Lett. 139, 77, 1992) and that
phosphoinositide hydrolysis mediated via mGluR is increased after kindling
experiments in rats (Akiyama et al. Brain Res. 569, 71, 1992). Trafas-ACPD has
been shown to increase release of dopamine in the rat brain, which indicates
that
compounds acting on the mGluRs might be usable for the treatment of
Parkinson's
disease and Huntington's Chorea (Sacaan et al., J. Neurochemistry 59, 245,
1992).
l0 Traps-ACPD has also been shown to be a neuroprotective agent in a medial
cerebral
artery occlusion (MCAO) model in mice (Chiamulera et al. Eur: J. Pharmacol.
215,
353, 1992), and it has been shown to inhibit NMDA-induced neurotoxicity in
nerve
cell cultures (Koh et al., Proc. Natl. Acad. Sci. USA 88, 9431, 1991). The
mGluR-
active compounds are also implicated in the treatment of pain. This is proved
by the
fact that antagonists at the metabotropic glutamate receptors antagonizes
sensory
synaptic response to noxious stimuli of thalamic neurons (Eaton, S. A. et al.,
Eur J.
Neuroscience, 5, 186, 1993).
The use of compounds active at the mGluRs for treatment of neurological
diseases
2o such as senile dementia have also been indicated by the findings of Zheng
and
Gallagher (Neuron 9, 163, 1992) and Bashir et al. (Nature 363, 347, 1993) who
demonstrated that activation of mGluRs is necessary for the induction of long
term
potentiation (LTP) in nerve cells (septal nucleus, hippocampus) and the
finding that
long term depression is induced after activation of metabotropic glutamate
receptors
in cerebellar granule cells (Linden et al. Neuron 7, 81, 1991).
Thus, compounds that demonstrate either activating or inhibiting activity at
mGluRs
have therapeutic potential for the treatment of neurological disorders. These
compounds have application as new drugs to treat both acute and chronic
neurological disorders, such as stroke and head injuries; epilepsy; movement
disorders associated with Parkinson's disease and Huntington's chorea; pain;
anxiety; AIDS dementia; and Alzheimer's disease. Since the GluRs can influence
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levels of alertness, attention and cognition; protect nerve cells from
excitotoxic
damage resulting from ischemia, hypoglycemia and anoxia; modulate the level of
neuronal excitation; influence central mechanisms involved in controlling
movement; reduce sensitivity to pain; and reduce levels of anxiety, these
compounds
can also be used to influence these situations and also find use in learning
and
memory deficiencies such as senile dementia. mGluRs may also be involved in
addictive behavior, alcoholism, drug addiction, sensitization and drug
withdrawal
(Science, 280:2045, 1998), so compounds acting at mGluRs might also be used to
treat these disorders.
to
The current pharmaceutical options for treating neurological disorders tend to
be
very general and non-specific in their actions in that, although they may
reduce the
clinical symptoms associated with a specific neurological disorder, they may
also
negatively impact normal function of the central nervous system of patients.
Thus
new cellular targets and drugs that are more specific in their actions require
identification and development and a need remains for chemical compounds that
demonstrate specific binding characteristics towards mGluRs.
SUMMARY OF THE INVENTION
It is an object of this invention to provide novel spiro[2.4]heptane amino
carboxy
compounds and derivatives thereof, that demonstrate activity at the various
metabotropic glutamate receptors. In one aspect of the present invention there
is
provided a compound of Formula (I) and stereoisomers thereof or
pharmaceutically
acceptable salts or hydrates thereof,
R1 R4
R2
R3 ~R5
5
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wherein:
Rl is (CH~)n(CH)r"XY, where: n is 0-3, m is 0 or 1, X is C02H and Y is NHS,
with
the proviso that, when m = 0, then n = 0 and the groups X and Y are directly
attached to the ring,
RZ and R3 can be same or different and selected from the group comprising H,
halo,
alkyl, cycloalkyl, aryl or heterocycle, or when R2 and R3 are present on
adjacent
carbon atoms and taken together then R2 and R3 can form a cycloalkyl (3-6
carbon
to atoms), heterocycle, or an aromatic ring or heteroaromatic ring,
R4 is selected from the group comprising H, halo, alkyl, cycloalkyl, aryl, or
heterocyclic,
R5 is selected from the group comprising of carboxyl, phosphono, phosphino,
sulfono, sulfino, borono, tetrazol, isoxazol, -CHZ-carboxyl, -CHI-phosphono,
-CHZ-phosphino, -CH2-sulfono, -CHI-sulfino, -CHI-borono, -CHZ-tetrazol, -CHZ-
isoxazol, and higher homologues thereof.
In accordance with further aspect of the present invention, there is provided
a
process for the preparation of a compound of Formula I,
R1 R4
R2
R3 v \R5
stereoisomers thereof, or pharmaceutically acceptable salts or hydrates
thereof,
wherein:
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Rl is (CHZ)n(CH)mXY, where: n is 0-3, m is 0 or 1, X is C02H and Y is NH2,
with
the proviso that, when m = 0, then n = 0 and the groups X and Y are directly
attached to the ring,
R2 and R3 can be same or different and selected from the group comprising H,
halo,
alkyl, cycloalkyl, aryl or heterocycle, or when R2 and R3 are present on
adjacent
carbon atoms and taken together then R2 and R3 can form a cycloalkyl (3-6
carbon
atoms), heterocycle, an aromatic ring or heteroaromatic ring,
to R4 is selected from the group comprising H, halo, alkyl, cycloalkyl, aryl
or
heterocycle,
R5 is selected from the group comprising carboxyl, phosphono, phosphino,
sulfono,
sulfino, borono, tetrazol, isoxazol, -CHZ-carboxyl, -CH2-phosphono, -CH~
phosphino, -CHZ-sulfono, -CHI-sulfino, -CH2-borono, -CHz-tetrazol, -CHz-
isoxazol,
and higher homologues thereof, which comprises:
(a) hydrolyzing a compound of formula II:
R6 R4
R2 ' (II)
R3 R5
wherein: R2, R3, R4, R5 are as defined above, and R6 is:
CN
-(CH2)~ (CH)~
NHR
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wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0 and the
groups CN and NHR7 are directly attached to the ring, R7 represents a
hydrogen atom or an acyl group. Preferred values for R7 are hydrogen and
(C1-CZ) alkanoyl groups, such as acetyl,
(b) hydrolyzing a compound of formula III:
R8 R4
R2
(III)
R3 ~R5
wherein: R2, R3, R4, RS are as defined above, and R8 is:
R10
O
N
- (CH2)~ (CH N
~R9
O
wherein: n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0
and the cyclic group containing R9 and R10 is directly attached to the 5-
membered ring, R9 and R10 each independently represent a hydrogen atom,
a (CZ-C6) alkanoyl group, a (Cl-C4) alkyl group, a (C~-C4) alkenyl group or a
phenyl (C1-C~) alkyl group in which the phenyl is unsubstituted or
2o substituted by halogen, (C1-Cd) alkyl or (Cl-C4) alkoxy, or a salt thereof;
or
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(c) deprotecting a compound of formula IV:
R11 R4
R2 ~ (IV)
R3 ~R5
wherein: R2, R3, R4, R5 are as defined above, and Rll is:
02R 13
(CH2)~ (CH NHR12
m
1 o wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n = 0 and the
groups R13 and NHR12 are directly attached to the ring, R13 represents a
hydrogen atom or a carboxyl protecting group, or a salt thereof, and R12
represents a hydrogen atom or a nitrogen protecting group; whereafter, if
necessary and/or desired the following steps are carried out:
(i) resolving the compound of Formula I;
(ii) converting the compound of Formula I into a non-toxic
metabolically-labile ester or amide thereof; and/or;
(iii) converting the compound of Formula I or a non-toxic metabolically-
labile ester or amide thereof into a pharmaceutically acceptable salt
thereof.
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According to further aspect of the invention there is provided a compound of
formula:
R6 R4
R2
R3 R5
wherein: R2, R3, R4, R5 are as defined above, and R6 is:
CN
-(CHz)~ (CFi)~
NHR7
wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0 and the
groups CN
and NHR7 are directly attached to the ring, R7 represents a hydrogen atom or
an
acyl group. Preferred values for R7 are hydrogen and (C1-CZ) alkanoyl groups,
such
as acetyl,
According to further aspect of the invention there is provided a compound of
formula:
R8 R4
R2
(III)
R3 R5
wherein: R2, R3, R4, R5 are as defined above, and R8 is:
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R10
O
N
- (CH2)~ (CH 1N
~R9
O
wherein: n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0
and the
cyclic group containing R9 and R10 is directly attached to the five membered
ring,
R9 and R10 each independently represent a hydrogen atom, a (C2-C6) alkanoyl
group, a (C1-Cø) alkyl group, a (C2-C~) alkenyl group or a phenyl (Cl-C4)
alkyl
group in which the phenyl is unsubstituted or substituted by halogen, (Cl-Cd)
alkyl
or (C1-C4) alkoxy, or a salt thereof.
According to further aspect of the invention there is provided a compound of
formula:
R11 R4
R2 ' (IV)
R3 R5
wherein: R2, R3, R4, R5 are as defined above, and Rll is:
02R 13
(CH2)" (C m NHR12
wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then ~n = 0 and the
groups
R13 and NHR12 are directly attached to the ring, R13 represents a hydrogen
atom or
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a carboxyl protecting group, or a salt thereof, and R12 represents a hydrogen
atom
or a nitrogen protecting group.
DETAILED DESCRIPTION OF THE INVENTION
The terms and abbreviations used in the instant examples have their normal
meanings unless otherwise designated. For example "°C" refers to
degrees Celsius;
"N" refers to normal or normality; "mmol" refers to millimole or millimoles;
"g"
refers to gram or grams; "mL" means milliliter or milliliters; "M" refers to
molar or
to molarity; "MS" refers to mass spectrometry; "IR" refers to infrared
spectroscopy;
and "NMR" refers to nuclear magnetic resonance spectroscopy.
Alkyl: refers to a saturated straight chain, or branched hydrocarbon group
containing
1-10 carbons. Typical alkyl groups include, but not limited to methyl, ethyl,
propyl,
isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like.
Cycloalkyl: refers to a mono- or polycyclic hydrocarbon group that contains 3
to 15
carbons and may optionally contain one or two double bonds. Typical cycloalkyl
groups include, but not limited to cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl
2o and the like.
Heteroeyclic or heterocycle: refers to a stable mono- or polycyclic compound
which
may optionally contain one or two double bonds or may optionally contain one
or
more aromatic rings. Each heterocycle consists of carbon atoms and from one to
four heteroatoms independently selected from a group including nitrogen,
oxygen
and sulfur and include any oxidized form of nitrogen and sulfur and
quaternized
form of any basic nitrogen. Typical heterocyclic groups include, but not
limited to
pyrimidinyl, tetrahydroquinolyl, tetrahydroisoquinonlinyl, purinyl, pyrimidyl,
indolinyl, benzimidazolyl, imidazolyl, imidazolinoyl, imidazolidinyl,
quinolyl,
3o isoquinolyl, indolyl, pyridyl, pyrrolyl, pyrrolinyl, prrazolyl, pryazinyl,
quinoxolyl,
piperidinyl, piperazinyl, morpholinyl, thiamorpholinyl, furyl, thienyl,
triazolyl,
thiazolyl, and the like. Further heterocycles are described in A. R. Katrizky
and C.
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W. Rees, eds., Comprehe>zsive Heterocyclic Che~aistry: The Structure,
Reactions,
Synthesis grad Use of Heterocyclic Cornpounds, Vol. 1-~, Pergamon Press, N.Y.
(1984).
Ate: refers to a mono- or polycyclic group which contains 6, 10, 12, or 14
carbons
In which at least one ring is aromatic. Typical aryl groups include, but not
limited
to, phenyl, naphthyl, anthracyl, azulenyl, and the like.
Heteroaromatic; refers to a mono- or polycyclic group which contains 1 to 15
carbon
l0 atoms and from 1 to 4 heteroatoms, each of which is selected independently
from a
group including sulfur, nitrogen and oxygen, and which additionally contains 1
to 3,
five or six membered rings, at least one of which is aromatic.
As would be understood by the skilled artisan throughout the synthesis of the
compounds of Formula I, it may be necessary to employ an amino-protecting
group
or a carboxy-protecting group in order to reversibly preserve a reactively
susceptible
amino or carboxy functionality while reacting other functional groups on the
compound.
2o Examples of such amino-protecting groups include formyl, trityl,
phthalimido,
trichloroacetyl, chloroacetyl, bromoacetyl, iodoacetyl, and urethane-type
blocking
groups such as benzyloxycarbonyl, 4-phenylbenzyloxycarbonyl, 2-
methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-
fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,
2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-
bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,
4-cyanobenzyloxycarbonyl, t-butoxycarbonyl, 2-(4-xenyl)-isopropoxycarbonyl,
1,1-
diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-
yloxycarbonyl, 2-(p-toluyl)-prop-2-yloxycarbonyl, cyclopentanyloxy-carbonyl,
1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-
methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 2-(4-
toluylsulfono)-ethoxycarbonyl, 2-(methylsulfono)ethoxycarbonyl, 2-
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(triphenylphosphino)-ethoxycarbonyl, fluorenylmethoxycarbonyl ("FMOC"), 2-
(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-
1-
enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,
2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,
cyclopropylmethoxycarbonyl, 4-(decycloxy)benzyloxycarbonyl,
isobornyloxycarbonyl, 1-piperidyloxycarbonlyl and the like;
benzoylmethylsulfono
group, 2-nitrophenylsulfenyl, diphenylphosphine oxide and like amino-
protecting
groups. The species of amino-protecting group employed is not critical so long
as
the derivatized amino group is stable to the condition of subsequent
reactions) on
other positions of the intermediate molecule and can be selectively removed at
the
appropriate point without disrupting the remainder of the molecule including
any
other amino-protecting group(s). Preferred amino-protecting groups are t-
butoxycarbonyl (t-Boc), allyloxycarbonyl and benzyloxycarbonyl (CbZ). Further
examples of these groups are found in E. Haslam in Protective Groups i~z
Organic
Synthesis; McOmie, J. G. W., Ed. 1973, at Chapter 2; and Greene, T.W. and
Wuts,
P. G. M., Protective Groups in Organic Synthesis, Second edition; Wiley-
Interscience: 1991; Chapter 7.
Examples of carboxyl-protecting groups include methyl,p-nitrobenzyl,p-
2o methylbenzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl,
2,4,6.-
trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-
methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2',4,4'-
tetramethoxybenzhydryl, t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'-
dimethoxytrityl,
4,4',4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl, t-
butyldimethylsilyl,
phenacyl, 2,2,2-trichloroethyl, (3-(di(n-butyl)methylsilyl)ethyl,p-
toluenesulfonoethyl, 4-nitrobenzylsulfonoethyl, allyl, cinnamyl, 1-
(trimethylsilylmethyl)prop-1-en-3-yl and like moieties. Preferred carboxyl-
protecting groups are allyl, benzyl and t-butyl. Further examples of these
groups are
found in E. Haslam, supra, at Chapter 5; and T. W. Greene and P. G. M. Wuts,
3o supra, at Chapter 5.
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The present invention provides a compound of the formula (I), stereoisomers
thereof, or pharmaceutically acceptable salts or hydrates thereof:
R~ R4
R2 (I)
R3 v 'R5
wherein:
Rl is (CH2)n(CH)mXY, where: n is 0-3, m is 0 or 1, X is C02H and Y is NH2,
with
the proviso that, when m = 0, then n = 0 and the groups X and Y are directly
attached to the ring,
R2 and R3 can be same or different and selected from the group comprising H,
halo,
alkyl, cycloalkyl, aryl or heterocycle, or when R2 and R3 are present on
adjacent
carbon atoms and taken together then R2 and R3 can form a cycloalkyl (3-6
carbon
atoms), heterocycle, or an aromatic ring or heteroaromatic ring,
R4 is selected from the group comprising H, halo, alkyl, cycloalkyl, aryl or
heterocycle,
R5 is selected from the group comprising of carboxyl, phosphono, phosphino,
2o sulfono, sulfino, borono, tetrazol, isoxazol, -CHI-carboxyl, -CH2-
phosphono,
-CHZ-phosphino, -CHz-sulfono, -CHI-sulfino, -CHZ-borono, -CHZ-tetrazol, -CH~-
isoxazol, and higher homologues thereof;
Compounds of the present invention include, but are not limited to the
following
examples:
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HZN COzH HZN~COZH
H02C NHz H02C NHZ
COzH COZH
C02H COzH
HOzC NH2
HOZC ,~ NHZ COzH HOzC ~.NHz \
C02H
Ph Ph
HOZC
The present invention includes the pharmaceutically acceptable salts of the
compounds defined by Formula I. A compound of this invention can possess a
sufficiently acidic, a sufficiently basic, or both functional groups, and
accordingly
react with any of a number of organic and inorganic bases, and inorganic and
organic acids, to form a pharmaceutically acceptable salt.
The term "pharmaceutically acceptable salt" as used herein, refers to salts of
the
compounds of the above formula which are substantially non-toxic to living
organisms. Typical pharmaceutically acceptable salts include those salts
prepared
by reaction of the compounds of the present invention with a pharmaceutically
acceptable mineral or organic acid or an organic or inorganic base. Such salts
are
known as acid addition and base addition salts.
Acids commonly employed to form acid addition salts are inorganic acids such
as
hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric
acid,
and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic
acid,
oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric
acid,
benzoic acid, acetic acid, and the like. Examples of such pharmaceutically
acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
phosphate,
monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate,
bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,
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hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate,
oxalate malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-
dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,
hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate,
phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate,
glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-
sulfonate,
napththalene-2-sulfonate, mandelate and the like. Preferred pharmaceutically
acceptable acid addition salts are those formed with mineral acids such as
hydrochloric acid and hydrobromic acid, and those formed with organic acids
such
to as malefic acid and methanesulfonic acid.
Salts of amine groups may also comprise quarternary ammonium salts in which
the
amino nitrogen carries a suitable organic group such as an alkyl, alkenyl,
alkynyl, or
aralkyl moiety.
Base addition salts include those derived from inorganic bases, such as
ammonium
or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and
the like.
Such bases useful in preparing the salts of this invention thus include sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate,
2o sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium
hydroxide,
calcium carbonate, and the like. The potassium and sodium salt forms are
particularly preferred.
It should be recognized that the particular counterion forming a part of any
salt of
this invention is usually not of a critical nature, so long as the salt as a
whole is
pharmacologically acceptable and as long as the counterion does not contribute
undesired qualities to the salt as a whole. This invention further encompasses
the
pharmaceutically acceptable solvates of the compounds of Formula I. Many of
the
Formula I compounds can combine with solvents such as water, methanol, ethanol
and acetonitrile to form pharmaceutically acceptable solvates such as the
corresponding hydrate, methanolate, ethanolate and acetonitrilate.
17
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The compounds of the present invention have multiple asymmetric (chiral)
centers.
As a consequence of these chiral centers, the compounds of the present
invention
occur as racemates, mixtures of enantiomers and as individual enantiomers, as
well
as diastereomers and mixtures of diastereomers. All asymmetric forms,
individual
isomers and combinations thereof, are within the scope of the present
invention.
The prefixes "R" and "S" are used herein as commonly used in organic chemistry
to
denote the absolute configuration of a chiral center, according to the Cahn-
Ingold-
Prelog system. The stereochemical descriptor R (rectus) refers to that
configuration
to of a chiral center with a clockwise relationship of groups tracing the path
from
highest to second-lowest priorities when viewed from the side opposite to that
of the
lowest priority group. The stereochemical desciptor S (sinister) refers to
that
configuration of a chiral center with a counterclockwise relationship of
groups
tracing the path from highest to second-lowest priority when viewed from the
side
opposite to the lowest priority group. The priority of groups is decided using
sequence rules as described by Cahn et al., Ahgew. Chem., 78, 413-447, 1966
and
Prelog, V. and Helmchen, G.; Angew. Chem. Iht. Ed. Ercg., 21, 567-583, 1982).
In addition to the R,S system used to designate the absolute configuration of
a chiral
center, the older D-1. system is also used in this document to denote relative
configuration, especially with reference to amino acids and amino acid
derivatives.
In this system a Fischer projection of the compound is oriented so that carbon-
1 of
the parent chain is at the top. The prefix "D" is used to represent the
relative
configuration of the isomer in which the functional (determining) group is on
the
right side of the carbon atom at the chiral center and "L", that of the isomer
in which
it is on the left.
As would be expected, the stereochemistry of the Formula I compounds is
critical to
their potency as agonists or antagonists. The relative stereochemistry is
established
early during synthesis, which avoids subsequent stereoisomer separation
problems
later in the process. Further manipulation of the molecules then employs
18
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WO 01/85669 PCT/CA01/00650
stereospecific procedures so as to maintain the preferred chirality. The
preferred
methods of this invention are the methods employing those preferred compounds.
Non-toxic metabolically-labile ester and amide of compounds of Formula I are
ester
or amide derivatives of compound of Formula I that are hydrolyzed an ~iuo to
afford
said compounds of Formula I and a pharmaceutically acceptable alcohol or
amine.
Examples of metabolically-labile esters include esters formed with (Cl-C6)
alkanols
in which the alkanol moiety may be optionally substituted by a (Ci-C$) alkoxy
group, for example methanol, ethanol, propanol and methoxyethanol. Examples of
1o metabolically-labile amides include amides formed with amines such as
methylamine.
Preparation of Compounds of Formula (I)
According to another aspect, the present invention provides a process for the
preparation of a compound of Formula I, or a pharmaceutically acceptable
metabolically-labile ester or amide thereof, or a pharmaceutically acceptable
salt
thereof, which comprises:
2o (a) hydrolyzing a compound of formula: II
R6 R'l
R2
R3 R5
wherein: R2, R3, R4, RS are as defined above, and R6 is:
CN
(CH2)n (CH)~ .
NHR7
19
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wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0 and the
groups CN
and NHR7 are directly attached to the ring, R7 represents a hydrogen atom or
an
acyl group. Preferred values for R7 are hydrogen and (Cl-CZ) alkanoyl groups,
such
as acetyl,
(b) hydrolyzing a compound of formula III
R5 /R4
R2 -~~ ~I (III)
R3 ~R5
wherein: R2, R3, R4, RS are as defined above, and R~ is:
R10
O
N
- (CH2)~ (CH ~N
'~' ~R9
O
wherein: n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n= 0
and the
cyclic group containing R9 and R10 is directly attached to five membered ring,
R9
and R10 each independently represent a hydrogen atom, a (CZ-C6) alkanoyl
group, a
(C1-C4) alkyl group, a (CZ-C4) alkenyl group or a phenyl (C1-C~) alkyl group
in
2o which the phenyl is unsubstituted or substituted by halogen, (C1-C4) alkyl
or (C1-C4)
alkoxy, or a salt thereof; or
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
(c) deprotecting a compound of formula IV
R11 R4
R2 ~ (IV)
R3 ~R5
wherein: R2, R3, R4, R5 are as defined above, and R11 is:
02R 13
(CH2)" (CH
NHR12
wherein:
n is 0-3, m is 0 or 1, with the proviso that, when m = 0, then n = 0 and the
groups -
CO~R13 and NHR12 are directly attached to the ring, R13 represents a hydrogen
atom or a carboxyl protecting group, or a salt thereof, and R12 represents a
hydrogen atom or a nitrogen protecting group;
whereafter, if necessary and/or desired:
(i) resolving the compound of Formula I;
(ii) converting the compound of Formula I into a non-toxic metabolically-
labile
2o ester or amide thereof; and/or;
(iii) converting the compound of Formula I or a non-toxic metabolically-labile
ester or amide thereof into a pharmaceutically acceptable salt thereof.
The protection of carboxylic acid and amine groups is generally described in
McOmie, Protecting Groups in Organic Chemistry, Plenum Press, NY, 1973, and
Greene and Wuts, Protecting Groups in Organic, Synthesis, 2nd. Ed., John Wiley
&
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WO 01/85669 PCT/CA01/00650
Sons, NY, 1991. Examples of carboxyl protecting groups include alkyl groups
such
as methyl, ethyl, t-butyl and t-amyl; aralkyl groups such as benzyl, 4-
nitrobenzyl,
4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl,
2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, benzhydryl and trityl; silyl
groups
such as trimethylsilyl and t-butyldimethylsilyl; and allyl groups such as
allyl and
1-(trimethylsilylmethyl)prop-1-en-3-yl. Examples of amine protecting groups
include acyl groups, such as groups of formula Rlm CO in which Rla represents
(C1-C6) alkyl, (C3-Clo) cycloalkyl, phenyl (C1-C6) alkyl, phenyl, (C1-C6)
alkoxy,
phenyl (C1-C6) alkoxy, or a (C3-Clo) cycloalkoxy, Wherein a phenyl group may
optionally be substituted by one or two substituents independently selected
from
amino, hydroxy, nitro, halogeno, (Cl-C6) alkyl, (C1-C6) alkoxy, carboxyl, (C1-
C6)
alkoxycarbonyl, carbamoyl, (C1-C6) alkanoylamino, (C1-C6) alkylsulphonylamino,
phenylsulphonylamino, toluenesulphonylamino, and (C1-C6) fluoroalkyl.
The compounds of Formula II are conveniently hydrolyzed in the presence of an
acid, such as hydrochloric acid or sulfuric acid, or a base, such as an alkali
metal
hydroxide, for example sodium hydroxide. The hydrolysis is conveniently
performed in an aqueous solvent such as water and at a temperature in the
range of
50 to 200 °C.
he compounds of Formula III are conveniently hydrolyzed in the presence of a
base,
for example an alkali metal hydroxide such as lithium, sodium or potassium
hydroxide, or an alkaline earth metal hydroxide such as barium hydroxide.
Suitable
reaction media include water. The temperature is conveniently in the range of
from
50 to 150 °C.
The compounds of Formula IV may be deprotected by a conventional method.
Thus, an alkyl carboxyl protecting group may be removed by hydrolysis. The
hydrolysis may conveniently be performed by heating the compound of Formula V
3o in the presence of either a base, for example an alkali metal hydroxide
such as
lithium, sodium or potassium hydroxide, or an alkaline metal hydroxide, such
as
barium hydroxide, or an acid such as hydrochloric acid. The hydrolysis is
22
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
conveniently performed at a temperature in the range from 10 to 300 °C.
An
arylalkyl carboxyl protecting group may conveniently be removed by
hydrogenolysis. The hydrogenolysis may conveniently be effected by reacting
the
compound of Formula V with hydrogen in the presence of a Group VIII metal
catalyst, for example a palladium catalyst such as palladium on charcoal.
Suitable
solvents for the reaction include alcohols such as ethanol. The reaction is
conveniently performed at a temperature in the range from 0 to 100 °C.
An acyl,
amine protecting group is also conveniently removed by hydrolysis, for example
as
described for the removal of an alkyl carboxyl protecting group.
The compounds of Formula II may be prepared by reacting a compound of formula
V with an alkali metal cyanide, such as lithium, sodium or potassium cyanide,
and
either ammonium carbonate in an aqueous alcohol, such as aqueous ethanol, or
with
an ammonium halide, such as ammonium chloride, conveniently in the presence of
ultrasound. If the reaction is conducted with ammonium carbonate, the reaction
is
conveniently performed at a temperature in the range from 35 °C to 150
°C. If
desired, the compounds of Formula TI may then be alkylated, for example using
a
compound of formula RCI, wherein: R is (Cl-C6) straight or branched chain
alkyl, or
(C1-C6) alkanoyl group. The alkylated compounds may be separated into their
2o diastereomers. If the reaction is conducted with an ammonium halide in the
presence of ultrasound, the ammonium halide is mixed with chromatography grade
alumina in the presence of a suitable diluent such as acetonitrile. The
mixture is then
irradiated with ultrasound, whereafter the compound of Formula V is added, and
the
mixture is again irradiated. The alkali metal cyanide is then added, followed
by
further irradiation with ultrasound.
R14 R4
R2 '
(V)
R3 R5
23
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
wherein:
RZ, R3, R4, R5 are as defined above, and RI4 is =O, or -(CH~,)n-CHO, wherein:
n
is 0-3,
Individual isomers of compounds of Formula II may be made by reacting a
compound of the Formula V with the stereoisomers of the chiral agent (S) and
(R)-
phenylglycinol and a reactive cyanide such as trimethylsilyl cyanide.
to The compounds of Formula III may be prepared by reacting a compound of
Formula
V with an alkali metal cyanide, such as lithium, sodium or potassium cyanide,
and
ammonium carbonate or ammonium carbamate. Convenient solvents include water,
dilute ammonium hydroxide, alcohols such as methanol, aqueous methanol and
aqueous ethanol. Conveniently the reaction is performed at a temperature in
the
range of from IO to I50 °C. If desired, the compounds of Formula III
may then be
N-alkylated, for example using an appropriate compound of formula R9 Cl and/or
R10 Cl.
Compounds of the formula V, wherein RI4 is =O or -(CHZ)~-CHO, can be
2o prepared:
(i) by cycloaddition of compound of the formula VI with ethyl acrylate,
followed by hydrolysis of the ester group;
R14
R2 R15 (VI)
R3
wherein: R2, R3, and R14. are as defined above, and R15 = CI, Br, or I, or
24
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
(ii) by cyclopropanation or 2 + 2 cycloaddition of the compound of
formula (VII)
R14
R2 ~ R16 (VII)
R3
wherein: R~, R3, and R14 are as defined above, and R16 is =CHR17, wherein: R17
is H, alkyl, aryl, halo, heterocyclic, alkyl ester (C1-C3) or aryl ester or
alkyl aryl
ester, or
(iii) by ring closure of the diester VIII or IX under Dieckmann
condensation conditions followed by further manipulations of the
resulting compounds within the knowledge of a worker skilled in the
art;
R2 R3 R18 R2
R3
Et02C R4
~~ R4
Et02C R5 Et02C
Et02C R5
R18
(VIII) (I~)
is
wherein:
R4, and R5 are as defined above, R2, R3 and R18 is H, halo, alkyl, cycloalkyl,
aryl
or heterocycle, or when taken together, R2 and R3 can form a cycloalkyl (3-6 .
carbon atoms), heterocycle, an aromatic ring or heteroaromatic ring, or when
taken
together, R18 and R2 can form a cycloalkyl (3-6 carbon atoms), heterocycle.
With
the proviso, that (i) at least one of RZ, R3 or R18 must be H or (ii) when, R2
and
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
R3, taken together to form a cycloalkyl (3-6 carbon atoms), heterocycle, an
aromatic
ring or heteroaromatic ring, then R18 is H or (iii) when, R18 and R2 are taken
together to form a cycloalkyl (3-6 carbon atoms), heterocycle, an aromatic
ring or
heteroaromatic then R3 is H.
Compounds of the formula V, when Rl4 is -(CHZ)n CHO, may also be prepared via
Wittig reaction of compound V where R14 is =O, with appropriate Wittig salt,
followed by further manipulations of the resulting compounds within the
knowledge
of a worker skilled in the art.
to
Compounds of the formula VII, wherein: R14 is =O can be prepared:
(a) by condensation of the compound of formula X with aldehyde of the t
ype HCOR17,
R2
O (X)
R3
wherein: R2, R3 and R17 are as defined above, or
2o (b) by Wittig reaction of the compound of formula XI with appropriate
Wittig reagent,
R19
R2 ~ O (XI)
R3
wherein: R19 is =O, R2 and R3 are as defined above, or
26
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
Compounds of the formula VII, wherein: R14 is -(CH2)"-CHO, wherein: n is 0-3
may be prepared by Wittig reaction of compound of formula XII,
R20
R2 0 (XII)
R3
wherein:
R20 is -(CHz)n-CHO, R2 and R3 are as defined above, in this case the aldehyde
group is protected with an appropriate protecting group before treating the
compound XII with appropriate Wittig reagent. The protecting group can be
removed after the Wittig reaction to obtain free aldehyde. The protecting
groups for
to carbonyl group are well known to the worker skilled in the art, and
examples of
these groups can be found in Greene, T.W. and Wuts, P. G. M., Proteetive
Groups i~c
Organic SytZtheszs, Second edition; Wiley-Interscience: 1991; Chapter 4.
Compounds of formulae XI and XII are commercially available or may be
synthesized by standard reactions by a person expert in the art.
Many of the intermediates described herein, for example the compounds of
Formula
II, III and IV are believed to be novel, and are provided as further aspects
of the
invention.
Biological and Therapeutic Activity Of Compounds Of Formula (I)
The compounds of formula I of the present invention are agonists or
antagonists at
certain metabotropic excitatory amino acid receptors (mGluRs). Therefore,
another
aspect of the present invention is a method of affecting mGluRs in mammals,
which
comprises administering to a mammal requiring modulated excitatory amino acid
neurotransmission a pharmacologically-effective amount of a compound of
Formula
I. The term "pharmacologically-effective amount" is used to represent an
amount of
27
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
the compound of the invention that is capable of affecting the mGluRs. By
affecting, a compound of the invention is acting as an agonist or antagonist.
When a
compound of the invention acts as an agonist, the interaction of the compound
with
the excitatory amino acid receptor mimics the response of the interaction of
this
receptor with its natural ligand (i.e. z-glutamate). When a compound of the
invention acts as an antagonist, the interaction of the compound with the
excitatory
amino acid receptor blocks the response of the interaction of this receptor
with its
natural ligand (i.e. L-glutamate).
l0 The particular dose of compound administered according to this invention
will, of
course, be determined by the particular circumstances surrounding the case,
including the compound administered, the route of administration, the
particular
condition being treated, and similar considerations. The compounds can be
administered by a variety of routes including oral, rectal, transdermal,
subcutaneous,
intravenous, intramuscular, or intranasal routes. Alternatively, the compound
may be
administered by continuous infusion. A typical daily dose will contain from
about
0.001 mg/kg to about 100 mg/kg of the active compound of this invention.
Preferably, daily doses will be about 0.05 mg/kg to about 50 mg/kg, more
preferably
from about 0.1 mg/kg to about 20 mg/kg.
A variety of physiological functions have been shown to be subject to
influence by
excessive or inappropriate stimulation of excitatory amino acid transmission.
The
Formula I compounds of the present invention are believed (through their
interactions at the mgluRs) to have the ability to treat a variety of
neurological
disorders in mammals associated with this condition, including acute
neurological
disorders such as cerebral deficits subsequent to cardiac bypass surgery and
grafting,
cerebral ischemia (e.g. stroke and cardiac arrest), spinal cord trauma, head
trauma,
perinatal hypoxia, and hypoglycemic neuronal damage. The Formula I compounds
are believed to have the ability to treat a variety of chronic neurological
disorders,
such as Alzheimer's disease, Huntington's Chorea, amyotrophic lateral
sclerosis,
AIDS-induced dementia, ocular damage and retinopathy, cognitive disorders, and
idiopathic and drug-induced Parkinson's. The present invention also provides
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CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
methods for treating these disorders which comprises administering to a
patient in
need thereof an effective amount of a compound of Formula I.
The Formula I compounds of the present invention (through their interactions
at the
mgluRs) are also believed to have the ability to treat a variety of other
neurological
disorders in mammals that are associated with glutamate dysfunction, including
muscular spasms, convulsions, migraine headaches, urinary incontinence,
psychosis,
drug tolerance, withdrawal, and cessation (i.e. opiates, benzodiazepines,
nicotine,
cocaine, or ethanol), smoking cessation, anxiety and related disorders (e.g.
panic
attack), emesis, brain edema, chronic pain, sleep disorders, Tourette's
syndrome,
attention deficit disorder, and tardive dyskinesia. Therefore, the present
invention
also provides methods for treating these disorders which comprise
administering to a
patient in need thereof an effective amount of the compound of Formula I.
The Formula I compounds of the present invention (through their interactions
at the
mgluRs) are also believed to have the ability to treat a variety of
psychiatric
disorders, such as schizophrenia, anxiety and related disorders (e.g. panic
attack),
depression, bipolar disorders, psychosis, and obsessive compulsive disorders.
The
present invention also provides methods for treating these disorders which
comprises administering to a patient in need thereof an effective amount of a
compound of Formula I.
Functional Assays Employing Cloned Subtypes of Metabotropic Receptors
The pharmacological properties of the compounds of the present invention can
be
determined via appropriate functional assays using recombinant metabotropic
glutamate receptors. For example adenylate cyclase assays or
phosphatidylinositol
hydrolysis assays, performed using standard procedures, can be used to
determine
agonist or antagonist activity towards mGluRs.
In general assay methods include monitoring of adenylate cyclase activity and
phosphatidyl inositol hydrolysis in a cell line that expresses the appropriate
mGluR,
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including but not limited to CHO cell lines.
(a~ Adenylate Cyclase Activity
Adenylate cyclase activity is determined in initial experiments in transfected
mammalian cells, using standard techniques. See, e.g., N. Adham, et al.,
Supra; R.
L. Weinshank, et al. Proc. Natl. Acad. Sci. (USA), 89:3630-3634 (1992), and
the
references cited therein.
Mammalian cells (the cell line AV12-664 is especially preferred) are stably
1o transfected with a plasmid comprising the cloned metabotropic glutmate
receptor.
The cells are maintained in as appropriate medium, for example one consisting
of
Dulbecco's Modified Eagle's Medium (DMEM) containing 5% dialyzed fetal calf
serum, 10 mM HEPES buffer (pH 7.3), 1 mM sodium pyruvate, 1 mM glutamine,
and 200 p,.g/mL hygromycin.
For the assay the cells are disassociated from stock culture flasks with
trypsin, and
plated in 24-well plastic tissue culture dishes (15 mm wells) at a density of
500,000-
700,000 cells per well using the same culture medium. After a twenty four hour
incubation in a humidified CO~, incubator, the cell monolayers are washed with
buffer (for example Dulbecco's phosphate-buffered saline containing 0.5 mM
IBMX
and 3 mM glucose) and then incubated in the same buffer at 37 °C for 30
minutes.
The monolayers are then washed with six exchanges of buffer.
Test compounds) and forskolin, or forskolin alone, dissolved in buffer, are
added
after the final wash. After incubating for 20 minutes at 37 °C, 0.5 mL
of 8 mM
EDTA is added to each well. The plates are then placed in a boiling water bath
for
about four minutes. The supernatant fluids are recovered from the wells and
lyophilized. Cyclic AMP (CAMP) determinations are carried out on the
lyophilized
samples using commercially available radioimmunoassay kits, following the
3o manufacturer's instructions. The CAMP levels in wells containing test
compounds)
are then compared to the forskolin controls.
CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
(b) Phosphatidylihositol Assay
Phosphatidylinositol hydrolysis is measured in clonal cell lines (for example
AV12)
harbouring a plasmid expressing the cloned metabotropic glutamate receptor in
response to addition of glutamate agonists, as a functional assay for
metabotropic
glutamate receptor activity according to D. Schoepp, Trends in Pharmaceutical
Sciences, 11:508, 1990.
Twenty four well tissue culture vessels are seeded with approximately 250,000
cells
to per well in an appropriate medium for example Dulbecco's Minimal Essential
Media
(D-MEM) (in the absence of glutamic acid) containing 2 mM glutamine and 10%
dialyzed fetal calf serum. After 24 hours growth at 37 °C, the media is
removed and
replaced with fresh media containing four microcuries of [3 H]myoinositol per
well
and the cultures are incubated a further 16 to 20 hours. The media is then
removed
and the cells in each well are washed with serum free medium containing 10 mM
lithium chloride, 10 mM myoinositol, and 10 rnM HEPES (2 x 1 mL washes). After
the final wash, 0.5 mL of washing solution is added containing the appropriate
concentrations) of test compound(s).
If the particular assay is also testing antagonists, a ten minutes incubation
is
performed prior to antagonist induction. Cells are incubated for about one
hour at
37 °C. in 95%:5% OZ :C02 or as appropriate for time course. The
reactions are
terminated by removing media and adding 1 mL of cooled 1:1 acetone:methanol
followed by incubation on ice for a minimum of twenty minutes.
These extracts are then collected and placed in 1.5 mL centrifuge tubes. Each
well
is washed with 0.5 mL water and this wash is added to the appropriate extract.
After
mixing and centrifugation, each aqueous supernatant is processed by
chromatography on a QMA SEP-PAK~ column, which is prewetted and
equilibrated by passing 10 mL of water, followed by 8 mL of 1M
triethylammonium
hydrogen carbonate (TEAB), followed by 10 rnL of water through the column.
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CA 02407777 2002-10-30
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The assay supernatants containing the water soluble [3H]inositol phosphate are
passed over the columns. This is followed by a 10 mL water wash and a 4 mL
wash
with 0.02 M TEAB to remove [3H]inositol precursors. [3H]inositol phosphate is
eluted with 4 mL of 0.1 M TEAB into scintillation vials and counted in the
presence
of scintillation cocktail. Total protein in each sample is measured using
standard
technidues. Measurements are taken as the amount of [3H]inositol phosphate
released per milligram of protein.
The assays are carried out in the absence and in the presence of the compound
being
1o tested. The measurements of [3H]inositol phosphate per milligram of protein
are
compared in order to confirm agonist and antagonist activity of the compound
being
tested.
These types of assays, employing cell lines expressing different subtype of
cloned
metabotropic receptors, may be used to determine which compounds have
selective
affinity in that they modulate one subtype of receptor with a greater activity
than
another subtype.
(c~ Testing in Chinese hamster cell lines
The Chinese hamster ovary cell lines expressing mGluRla, mGlu RZ and mGluRøa
receptors have been described previously (Amarori and Nakanishi, Neuron 8, 757-
765, 1992; Tanabe et al., Neuron 8, 169-179, 1992, and J. Neurochem. 63, 2038-
2047, 1993). They are maintained at 37 °C in a humidified 5% COZ
incubator in
Dubecco's Modified Eagle Medium (DMEM) containing a reduced concentration of
(S)-glutamine (2mM) and are supplemented with 1% proline, penicillin (100
U/mL),
streptomycin (100 mg/mL) and 10% dialyzed fetal calf serum (all GIBCO,
Paisley).
Two days before assay 1.8 x 106 cells are evenly distributed into the wells of
24 well
plates.
Phosphatidylinositol (PI) hydrolysis can be measured as described previously
(Hayashi et al., Nature 366, 687-690,1992, and J. Neuroscience 14, 3370-3377,
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CA 02407777 2002-10-30
WO 01/85669 PCT/CA01/00650
1994). Briefly, the cells are labeled with [3H]inositol (2p Ci/mL) 24 h prior
to the
assay. For agonist assays, the cells are incubated with test compound
dissolved in
phosphate-buffered saline (PBS)-LiCI for 20 min, and agonist activity is
determined
from the level of 3H-labeled mono-, bis- and tris-inositol phosphates
generated, as
measured following ion-exchange chromatography, compared with the level
generated in the absence of the test compound. For antagonist assays, the
cells are
preincubated with ligand dissolved in PBS-LiCI for 20 min prior to incubation
with
test compound and 10 ~, M (S)-Glu for 20 min. The antagonist activity is then
determined as the inhibitory effect of the (S)-Glu mediated response.
The assay of cyclic AMP formation can be performed as described previously
(Hayashi et al., 1992, 1994). Briefly, the cells are incubated for 10 min in
PBS
containing test coumpound and 10 p. M forskolin and 1 mM 3-isobutyl-1-
methylxanthine (IBMX) (both Sigma, St. Louis, MO, USA). The agonist activity
is
then determined as the inhibitory effect on the forskolin-induced cyclic AMP
formation. For antagonist assay, the cells are preincubated with ligand
dissolved in
PBS containing 1 mM IBMX for 20 min prior to a IO min incubation in PBS
containing test compound, 20 p, M(mGlu2) or 50 p. M (mGlu4a) (S)-Glu, 10 p, M
forskolin and 1 mM IBMX. The antagonist activity is then determined as the
potentiating effect on the forskolin-induced cyclic AMP formation.
Testing for demonstration of the pharmacological activity of certain compounds
on
representative mGlu receptor subtypes can be performed using Sprague Dawley
rat
tissues.
Phosphatidylinositol (PI) hydrolysis can be measured as described below:
Briefly,
cross-chopped slices are prepared from neonatal Sprague Dawley rat tissue
(age: p7-
p14). The slices are pre-labelled with [3H] myo-inositol. Following pre-
labelling,
the slices are incubated with the test drugs and standard (known Group I
agonists i.e.
ACPD) for a period of 45 minutes. The incubation is terminated by the addition
of
chloroform/methanol/HCl (100:200:2). The resulting mixture is separated into
two
phases by the addition of chloroform and distilled water. The aqueous fraction
is
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applied to ion exchange columns, and inositol phosphates are eluted using 800
mM
Ammonium Formate/100 mM Formic Acid. The eluent is then analyzed using
liquid scintillation counting. The amount of inositol phosphate accumulation
is
expressed as a percentage of that induced by ACPD.
The assay of cyclic AMP formation can be performed as described previously
(Tovey et al., Clihica Chimica Acta, 56, 221-234, 1974). The assay can be
modelled
on the cyclic AMP assay kit available from Amersham, which in turn, is based
on
the assay created by Tovey et al. Briefly, samples are prepared from Sprague
to Dawley rat (225-250g) cortical slices. Slices are incubated with the drug,
and then
challenged with forskolin to induce cyclic AMP release. Following termination
of
the reaction by boiling, the slices are homogenized and centrifuged. Samples
of
supernatant are then incubated for 2-3 hours with a known quantity of [3H]CAMP
and a binding protein. When the incubation is complete, the bound cyclic AMP
is
separated from the free cyclic AMP by centrifugation with charcoal. The
resulting
supernatant (containing free cyclic AMP) is then analyzed by liquid
scintillation
counting. The amount of unbound cyclic AMP can be calculated from a standard
curve previously determined with various samples of free cyclic AMP.
2o In performing such experiments with some of the compounds of the present
invention, it has been demonstrated that some compounds of the present
invention
act as modulators of the cAMP-linked metabotropic glutamate receptors, while
showing less activity with phosphatidylinositol-linked metabotropic glutamate
receptors and vice versa.
Administration of Compounds of Formula (I)
According to another aspect, the present invention provides a method of
modulating
one or more metabotropic glutamate receptor functions in a warm-blooded mammal
3o which comprises administering an effective amount of a compound of Formula
I, or
a non-toxic metabolically-labile ester or amide thereof, or a pharmaceutically
acceptable salt thereof.
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The compounds of the present invention are preferably formulated prior to
administration. Therefore, another aspect of the present invention is a
pharmaceutical formulation comprising a compound of Formula I and a
pharmaceutically-acceptable carrier, diluent, or excipient. The present
pharmaceutical formulations are prepared by known procedures using well-known
and readily available ingredients. In making the compositions of the present
invention, the active ingredient will usually be mixed with a carrier, or
diluted by a
carrier, or enclosed within a carrier, and may be in the form of a capsule,
sachet,
1o paper, or other container. When the carrier serves as a diluent, it may be
a solid,
semi-solid, or liquid material that acts as a vehicle, excipient, or medium
for the
active ingredient.
The compounds of Formula I are usually administered in the form of
pharmaceutical
compositions. These compounds can be administered by a variety of routes
including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular,
and
intranasal. These compounds are effective as both injectable and oral
compositions.
Such compositions are prepared in a manner well known in the pharmaceutical
art
and comprise at least one active compound.
The present invention also provides pharmaceutical compositions containing
compounds as disclosed in the claims in combination with one or more
pharmaceutically acceptable, inert or physiologically active, diluent or
adjuvant.
The compounds of the invention can be freeze-dried and, if desired, combined
with
other pharmaceutically acceptable excipients to prepare formulations for
administration. These compositions may be presented in any form appropriate
for
the administration route envisaged. The parenteral and the intravenous route
are the
preferential routes for administration.
3o Compounds of the general Formula I may be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit formulations
containing conventional non-toxic pharmaceutically acceptable carriers,
adjuvants
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and vehicles. The term parenteral as used herein includes subcutaneous
injections,
intravenous, intramuscular, intrasternal injection or infusion techniques. In
addition,
there is provided a pharmaceutical formulation comprising a compound of
general
Formula I and a pharmaceutically acceptable carrier. One or more compounds of
general Formula I may be present in association with one or more non-toxic
pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if
desired
other active ingredients. The pharmaceutical compositions containing compounds
of general Formula I may be in a form suitable for oral use, for example, as
tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
granules,
emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any known to
the
art for the manufacture of pharmaceutical compositions and such compositions
may
contain one or more agents selected from the group consisting of sweetening
agents,
flavouring agents, colouring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets contain the
active
ingredient in admixture with non-toxic pharmaceutically acceptable excipients
that
are suitable for the manufacture of tablets. These excipients may be for
example,
inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium
phosphate or sodium phosphate: granulating and disintegrating agents for
example,
corn starch, or alginic acid: binding agents, for example starch, gelatin or
acacia, and
lubricating agents, for example magnesium stearate, stearic acid or talc. The
tablets
may be uncoated or they may be coated by known techniques to delay
disintegration
and absorption in the gastrointestinal tract and thereby provide a sustained
action
over a longer period. For example, a time delay material such as glyceryl
monostearate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules
wherein the
active ingredient is mixed with an inert solid diluent, for example, calcium
carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein
the
active ingredient is mixed with water or an oil medium, for example peanut
oil,
liquid paraffin or olive oil.
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Aqueous suspensions contain active materials in admixture with excipients
suitable
for the manufacture of aqueous suspensions. Such excipients are suspending
agents,
for example sodium carboxylmethylcellulose, methyl cellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth
and gum acacia: dispersing or wetting agents may be a naturally-occurring
phosphatide, for example, lecithin, or condensation products of an alkylene
oxide
with fatty acids, for example polyoxyethylene stearate, or condensation
products of
ethylene oxide with long chain aliphatic alcohols, for example hepta-
to decaethyleneoxycetanol, or condensation products of ethylene oxide with
partial
esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial esters
derived
from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives,
for example ethyl, or h-propyl- p-hydroxy benzoate, one or more coloring
agents,
one or more flavoring agents or one or more sweetening agents, such as sucrose
or
saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a
2o vegetable oil, for example peanut oil, olive oil, sesame oil or coconut
oil, or in a
mineral oil such as liquid paraffin. The oily suspensions may contain a
thickening
agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such
as those set forth above, and flavouring agents may be added to provide
palatable
oral preparations. These compositions may be preserved by the addition of an
anti
oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous
suspension
by the addition of water provide the active ingredient in admixture with a
dispersing
or wetting agent, suspending agent and one or more preservatives. Suitable
3o dispersing or wetting agents and suspending agents are exemplified by those
already
mentioned above. Additional excipients, for example sweetening, flavouring and
colouring agents, may also be present.
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Pharmaceutical compositions of the invention may also be in the form of oil-in-
water emulsions. The oil phase may be a vegetable oil, for example olive oil
or
peanut oil, or a mineral oil, for example liquid paraffin or mixtures of
these.
Suitable emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for example soy
bean,
lecithin, and esters or partial esters derived from fatty acids and hexitol,
anhydrides,
for example sorbitan monooleate, and condensation products of the said partial
esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
The
to emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example
glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a
demulcent, a preservative and flavoring and coloring agents. The
pharmaceutical
compositions may be in the form of a sterile mjectable aqueous or oleaginous
suspension. This suspension may be formulated according to known art using
those
suitable dispersing or wetting agents and suspending agents that have been
mentioned above. The sterile injectable preparation may also be a sterile
injectable
solution or a suspension in a non-toxic parentally acceptable diluent or
solvent, for
2o example as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are conventionally
employed as a
solvent or suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In addition, fatty acids
such as
oleic acid find use in the preparation of injectables.
The compounds) of the general Formula I may be administered, together or
separately, in the form of suppositories for rectal administration of the
drug. These
compositions can be prepared by mixing the drug with a suitable non-irritating
excipient which is solid at ordinary temperatures but liquid at the rectal
temperature
and will therefore melt in the rectum to release the drug. Such materials are
cocoa
butter and polyethylene glycols.
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Compounds) of general Formula I may be administered, together or separately,
parenterally in sterile medium. The drug, depending on the vehicle and
concentration used, can either be suspended or dissolved in the vehicle.
Advantageously, adjuvants such as local anaesthetics, preservatives and
buffering
agents can be dissolved in the vehicle.
The dosage to be administered is not subject to defined limits, but it will
usually be
an effective amount. It will usually be the equivalent, on a molar basis of
the
pharmacologically active free form produced from a dosage formulation upon the
to metabolic release of the active free drug to achieve its desired
pharmacological and
physiological effects. The compositions are preferably formulated in a unit
dosage
form, each dosage containing from about. 0.05 to about 100 mg, more usually
about
1.0 to about 30 mg, of the active ingredient. The term "unit dosage form"
refers to
physically discrete units suitable as unitary dosages for human subjects and
other
mammals, each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in association with a
suitable
pharmaceutical excipient.
The active compound is effective over a wide dosage range. For examples,
dosages
per day normally fall within the range of about 0.01 to about 30 mg/kg of body
weight. A typical daily dose will contain from about 0.01 mg/kg to about 100
mg/kg of the active compound of this invention. Preferably, daily doses will
be
about 0.05 mg/kg to about 50 mg/kg, more preferably from about 0.1 mg/kg to
about
mg/kg. In the treatment of adult humans, the range of about 0.1 to about 15
25 mg/kg/day, in single or divided dose, is especially preferred. However, it
will be
understood that the amount of the compound actually administered will be
determined by a physician, in the light of the relevant circumstances,
including the
condition to be treated, the chosen route of administration, the actual
compound
administered, the age, weight, and response of the individual patient, and the
3o severity of the patient's symptoms, and therefore the above dosage ranges
are not
intended to limit the scope of the invention in any way. In some instances
dosage
levels below the lower limit of the aforesaid range may be more than adequate,
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while in other cases still larger doses may be employed without causing any
harmful
side effect, provided that such larger doses are first divided into several
smaller
doses for administration throughout the day.
The compositions are preferably formulated in a unit dosage form, each dosage
containing from about 5 mg to about 500 mg, more preferably about 25 mg to
about
300 mg of the active ingredient. The term "unit dosage form" refers to a
physically
discrete unit suitable as unitary dosages for human subjects and other
mammals,
each unit containing a predetermined quantity of active material calculated to
to produce the desired therapeutic effect, in association with a suitable
pharmaceutical
carrier, diluent, or excipient. The following formulation examples are
illustrative
only and are not intended to limit the scope of the invention in any way.
FoYmulatioh 1
Hard gelatin capsules are prepared using the following ingredients:
Quantity (mg/capsule)
Active Ingredient 250
Starch, dried 200
Magnesium stearate 10
Total 460
The above ingredients are mixed and filled into hard gelatin capsules in 460
mg
quantities.
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Formulation 2
A tablet is prepared using the ingredients below:
Quantity (mgltablet)
Active Ingredient 250
Cellulose, microcrystalline 400
Silicon dioxide, fumed 10
Stearic acid 5
Total 665
The components are blended and compressed to form tablets each weighing 665
mg.
For»zulation 3
to An aerosol solution is prepared containing the following components:
Weight %
Active Ingredient 0.25
Ethanol 29.75
Propellant 22 (Chlorodifluoromethane) 70.00
Total 100
The active compound is mixed with ethanol and the mixture added to a portion
of
the Propellant 22, cooled to -30 °C and transferred to a filling
device. The required
amount is then fed to a stainless steel container and diluted with the
remainder of the
propellant. The valve units are then fitted to the container.
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Formulation 4
Tablets each containing 60 mg of active ingredient are made as follows:
Quantity (mgltablet)
Active Ingredient 60
Starch 45
Microcrystalline cellulose35
Polyvinylpyrrolidone 4
Sodium carboxymethyl 4.5
starch
Magnesium stearate 0.5
Talc 1.0
Total I50
The active ingredient, starch, and cellulose are passed through a No. 45 mesh
U.S.
sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with
the
resultant powders that are then passed through a No. 14 mesh U.S. sieve. The
granules so produced are dried at 50~C and passed through a No. 18 mesh U.S.
sieve. The sodium carboxymethyl starch, magnesium stearate, and talc,
previously
1o passed through a No. 60 mesh U.S. sieve, are then added to the granules
which, after
mixing, are compressed on a tablet machine to yield tablets each weighing 150
mg.
Formulation S
Capsules each containing 80 mg medicament are made as follows:
Quantity (mg/capsule)
Active Ingredient 80
Starch 59
Microcrystalline cellulose59
Magnesium stearate 2
Total 200
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The active ingredient, cellulose, starch, and magnesium stearate are blended,
passed
through a No. 45 sieve, and filled into hard gelatin capsules in 200 mg
quantities.
Formulatioi2 6
Suppositories each containing 225 mg of active ingredient may be made as
follows:
Quantity (mg/suppository)
Active Ingredient 225
Saturated fatty acid glycerides 2000
Total 2225
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended
in
to the saturated fatty acid glycerides previously melted using the minimum
heat
necessary. The mixture is then poured into a suppository mold of nominal 2 g
capacity and allowed to cool.
Formulation 7
Suspensions each containing 50 mg of medicament per 5 mL dose are made as
follows:
Active Ingredient 50 mg
Sodium carboxylmethyl cellulose 50 mg
Syrup ~ 1.25 mL
Benzoic acid solution 0.10 mL
Flavour q.v.
Color q.v.
Purified water to total 5 mL
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The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the
sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic
acid solution, flavor and color are diluted with some of the water and added,
with
stirring. Sufficient water is then added to produce the required volume.
Fonnulatior~ 8
An intravenous formulation may be prepared as follows:
Quantity
Active Ingredient 100 mg
Mannitol 100 mg
5 N Sodium hydroxide 200 mL
Purified water to total 5 mL
ZO
Fo~nulat~oh 9
A topical formulation may be prepared as follows:
Quantity
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White soft paraffin100 g
The white soft paraffin is heated until molten. The liquid paraffin and
emulsifying
wax are incorporated and stirred until dissolved. The active ingredient is
added and
stirring is continued until dispersed. The mixture is then cooled until solid.
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Formulation 10
Sublingual or buccal tablets, each containing 10 mg of active ingredient, may
be
prepared as follows:
Quantity (mg/tablet)
Active Ingredient 10.0
Glycerol 210.5
Water 143.0
Sodium Citrate 4.5
Polyvinyl Alcohol 26.5
Polyvinylpyrrolidone 15.5
Total 410.0
The glycerol, water, sodium citrate, polyvinyl alcohol, and
polyvinylpyrrolidone are
admixed together by continuous stirring and maintaining the temperature at
about 90
°C. When the polymers have gone into solution, the solution is cooled
to about
50°-55 °C. and the medicament is slowly admixed. The homogenous
mixture is
to poured into forms made of an inert material to produce a drug-containing
diffusion
matrix having a thickness of about 2-4 mm. This diffusion matrix is then cut
to
form individual tablets having the appropriate size.
Another preferred formulation employed in the methods of the present invention
employs transdermal delivery devices ("patches"). Such transdermal patches may
be
used to provide continuous or discontinuous infusion of the compounds of the
present invention in controlled amounts.
The construction and use of transdermal patches for the delivery of
pharmaceutical
2o agents is well known in the art (see, for example, U.S. Pat. No. 5,023,252
issued
Jun. 11, 1991) herein incorporated by reference. Such patches may be
constructed
for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
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Frequently, it will be desirable or necessary to introduce the pharmaceutical
composition to the brain, either directly or indirectly. Direct techniques
usually
involve placement of a drug delivery catheter into the host's ventricular
system to
bypass the blood-brain barrier. One such implantable delivery system, used for
the
transport of biological factors to specific anatomical regions of the body, is
described in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991, which is herein
incorporated by reference.
Indirect techniques, which are generally preferred, usually involve
formulating the
1o compositions to provide for drug latentiation by the conversion of
hydrophilic drugs
into lipid-soluble drugs or prodrugs. Latentiation is generally achieved
through'
blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present
on the
drug to render the drug more lipid soluble and amenable to transportation
across the
blood-brain barrier. Alternatively, the delivery of hydrophilic drugs may be
enhanced by intra-arterial infusion of hypertonic solutions that can
transiently open
the blood-brain barrier.
EXAMPLES
The following Examples illustrate the invention. The following abbreviations
are
used in the Examples: EtOAc, ethyl acetate; THF, tetrahydrofuran; EtOH,
ethanol;
TLC, thin layer chromatography; GC, gas chromatography; HPLC, high pressure
liquid chromatography; m-CPBA, m-chloroperbenzoic acid; Et20, diethyl ether;
DMSO, dimethyl sulfoxide; DBU, 1,8-diazabicyclo-[5.4.0]undec-7-ene, MTBE,
methyl t-butyl ether; and FDMS, field desorption mass spectrometry.
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Example 1: Spiro[2.4] heptane-4-amino-1,4-dicarboxylic acid Isomers
(7) and (8)
O O O
CI + ~C02Et DBU CO Et K
C02H
Benzene
1 2 3 4
O O
KCN _ HN~ HN
(NH4)2C03 O ,,NH 0~,,,,, ~NH
~~~~C02H + C02H
6
NaOH ~ NaOH
H02C NH2 H02C, NH2
CO~H C02H
7 8
5
Preparation of Ethyl spiro[2.4]heptane-4-oxo-1-carboxylate (3)
To a solution of 2-chlorocyclopentanone (1) (10 g, 0.084 mol) and ethyl
acrylate (2) (16.8 g, 0.168 mol) in benzene (50 mL), DBU (15 mL, 0.10 mol) was
to added at room temperature (RT). After stirring for 2 h and 30 minutes, the
mixture
was poured into water. The mixture was extracted with ~;tUAc, washed with HZU,
dried with MgS04, and evaporated to dryness. The residue was purified by
chromatography on a silica gel column with 5-75% EtOAc/hexanes as the eluent.
Product (3) was obtained as a colourless oil (5.99 g, 40%).
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Preparation of [2.4] heptane-4-oxo-1-carboxylic acid (4)
Compound (3) (5.0 g) was hydrolyzed in KOH (1N, 32.5 mL) and EtOH
(200 mL) at RT overnight. The acid was obtained as a solid (100%).
Preparation of Hydantoins (5) and (6)
Compound (4) 3.92 g was placed in a pressure bottle with KCN, (NHø)~C03
and 1:1 HZO:EtOH. The reaction was stirred at 80 °C overnight. The
hydantoins
were isolated by addition of 6M HCl and the two isomers were separated by
chromatography with MeOH/CHC13/HZO/NH3 (26:70:4:1). Hydantoin 5 comes out
to of the column first (1.8 g, solid), followed by hydantoin 6 (1.8 g, solid).
Preparation of Amino Acids (7) and (8)
Hydantoin (5) (800 mg) was dissolved in 2 N NaOH in a sealed pressure
tube and is heated to 100 °C for 46 h. The resulting solution was
cooled and
acidified (6N HCl). The solution was evaporated to dryness and extracted with
EtOH. The ethanolic extract was treated with 4 mL of propylene oxide to
precipitate
the crude amino acid. The amino acids were purified by cation exchange
chromatography. 430 mg of compound (7) was derived from hydantoin (5).
Following the same procedure as for hydantoin 5, 380 mg of compound 8 was
2o derived from 800 mg of hydantoin (6).
Analysis calculated for amino acid (7) C9H13N04.H20: % C, 49.16; %H,
6.96; %N, 6.45. Found: %C, 49.35; %H, 6.58; %H, 7.19.
Analysis calculated for amino acid (8) C9H13N04.H~0: % C, 53.07; %H,
6.68; %N, 6.88. Found: %C, 53.16; %H, 6.51; %H, 6.95.
1H NMR (200 MHz, D20): ~ 5.2-4.9 (m 1H), 4.6-4.2 (m 6H), 3.7, 3.6 (AB
system, 2H) ppm.
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Example 2: Spiro [2.4] heptane4-amino-2-phenyl-1,4-dicarboxylic acid Isomers
(16 ) and (17)
O CHO O
O CO2Et
NaOH i Ph EDSA
Ph
9 10 11 12
~KOH
O C02H
2H H KCN
+ E
(NH4)zC03
Ph
15 14 13
NaOH NaOH
H02C,, NH2 C02H H02C ; NH2 C02H
~Ph ~Ph
17 16
Preparation of 2-Phenylmethylenecyclopentanone (11)
A mixture of cyclopentanone (9) (6.0g, 0.071 mol), benzaldehyde (10), (7.35
to g, 0.069 mol) and NaOH (1N, 66.6 mL) in ether (70 mL) was stirred at room
temperature for 64 h. The organic layer was separated from the aqueous layer
and
washed with water. The residue was purified after evaporation of the solvent
by
chromatography and the product was obtained in solid form. (7.29 g, 60%).
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Preparation of Ethyl spiro[2.4]heptane-4-oxo-2-phenyl-1-carboxylate (12)
A mixture of (11) (6.82g, 0.04 mol) and EDSA in benzene (50 mL) was
heated to reflux overnight. ~EDSA was made as follows: A solution of
(Ethoxycar°bonylmetlzyl~dimethyl-sulfoniurn bromide (13.798, 0.06 rnol)
irz CHCl3
(50 mL~ was treated with KZC03 (saturated, 40 mL~ for 1 hour at room
temperature.
The chloroform layer was dried with MgSO~ The solvent was evapor°ated
to give ara
oil (5.928, 0.04 mol~J. After cooling the reaction mixture to room
temperature, the
organic layer was washed with brine, dried over anhydrous MgSO~ and
concentrated
in vacuo. Purification by column chromatography (CHZCl2/Hexanes 1:1) afforded
l0 9.968 (95%) of the title compound (12) as a solid.
Preparation of hydantoins (14 and 15) from 12
The ketone ester (12) (9.928) was treated in EtOH (250 mL) with KOH (1N,
45.0 mL) at room temperature overnight. The mixture was neutralized with KHSOd
and the resulting precipitates were collected by filtration to give a solid
(13, 8.958,
100%). This solid (13, 2.308, 10.0 mmol) was treated with KCN (1.688, 25.0
mmol)
and (NH~)2C03 (6.698, 70 mmol) in H2O-EtOH (1:1, 20 mL) at 85°C
overnight. The
resulting mixture was acidified cautiously (6N HCl) and filtered to give a
solid
containing two isomers. The isomers (14 and 15) were separated by
chromatography
(CHC13/MeOH/H20/NH3/H20, 70:26:4:1). Hydantoin (14) (0.88 g) comes off first
and then hydantoin (15) (0.638).
Preparation of Spiro[2.4]heptan-4-amino-2-phenyl-1,4-dicarboxylic acids (16
and 17)
The hydantoin (14) (0.88 g) was hydrolyzed at 136°C in a pressure
tube
overnight. The resulting solution was cooled and acidified (6N HCl). The
solution
was evaporated to dryness and extracted with EtOH. The ethan~lic extracts were
treated with 4 mL of propylene oxide to precipitate the crude amino acid. The
crude
product was purified by cation ion-exchange chromatography. 210 mg (26%) of
(16) was obtained. In a similar manner, 50 mg of (17) was obtained (9%) from
606
mg of hydantoin (15).
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Analysis calculated for compound (17) (C15H1~N04.H~0Ø2NHdCl: % C,
63.99; %H, 5.37; %N, 9.33. Found: %C. 58.84; %H, 6.20; %H, 5.21.
1H NMR (200 MHz, DSO) ~ 7.15 (s 5H), 2.9 (d 1H), 2.65 (d 1H), 2.5-1.6 (m
6H) ppm.
Example 3: 1-Amino-1-carboxyindan-3-spiro-cyclopropanecarboxylic acid
0 0 0
EtOOCCH=PPh3
Dimethylsulfoxide methylide
/ / /
0
EtOOC
Et
2
18 19 20
KCN,
(NH4)2C03
0
H02C NHZ HN
NaOH O~ INH
/
H02C
C02Et
22 21
Preparation of Compound (19)
5g of 1,3 Indandione (18) was suspended in toluene (180) mL and
(carbethoxymethylene) triphenylphosphorane (13.1 g, 37.63 mmol) was added. The
resulting solution was refluxed for 3.5 h. The solution was cooled and the
solvent
evaporated. The crude mixture was purified by column chromatography (hexane
EtOAc (3:1) yielding 4.38 g of pure compound (19).
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Preparation of Compound (20)
Compound (19) (1.12 g, 5.18 mmol) was dissolved in dry THF (20 mL).
Dimethylsulfoxide methylide (0.5 M, 12.44 mL, 6.2 mmol) was added dropwise
under nitrogen gas. The resulting solution as stirred at room temperature for
2 h.
H20 (10 mL) was added and the mixture extracted with Et20 (2 x 30 mL). The
combined extracts were washed with brine and dried. Column chromatography of
the crude mixture using hexanes: EtOAc (3:1) to give compound (20). (0.13 g,
0.55
mmol)
20 Preparation of Compound (21)
Compound (20) (0.13 g, 0.55 mmol), ethanol (4.5 mL) and 1 N NaOH (0.65
mL) were stirred at 70 °C for 4 hrs. and the solvent was evaporated in
vacuo. The
residue was dissolved in 4 mL (1:1, EtOH: H20) and treated with KCN (90 mg)
and
(NH4)2C03 (238 mg).The resulting mixture was stirred at 85 °C for 60 h,
under
pressure. The solution was then cooled to room temperature and acidified with
6N
HCI. The ethanol was evaporated and the aqueous solution extracted with EtOAc.
The product was used for the next step without any purification.
Preparation of Compound (22)
2o Compound (21) (crude) was dissolved in 2 M NaOH (5 mL) and the
resulting solution was stirred at 150 °C for 24 h under pressure. The
solution was
then cooled and acidified with 6 N HCI. A precipitate formed which was
filtered and
washed with water. The filtrate was then concentrated i~c vacuo and put on an
anion
exchange column using 0.5 M HOAc as an eluent to give 56 mg of compound (22)
as a white powder. Yield was 31.8% from compound (20).
Analysis calculated for compound (22): C13H13NO4: % C, 57.90; %H, 5.69;
%N, 5.18. Found: %C, 57.44; %H, 5.39; %H, 4.82.
. 1H NMR (200 MHz, DZO) 8 7.4-7.2 (m, 4H), 2.45 (m, 1H), 1.9-1.7 (m, 2H),
2.05, 0.95 (AB system
2H) ppm.
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Example 4 Spiro[4,2]heptane-1-carboxy-4-glycine Isomers (27) and (028)
0 OMe 0
H
CH30CH2PPh3Cl ~ PTSA
"-C02Et
C02Et
3 ~ C02Et
23
24
O
KCN H2N CO~H H2N~COZH
NaOH
(NH~2C03
C02H ~.CO
25 26 27
Preparation of intermediate 23
Sodium bis(trirnethylsilyl)amide (26.7 mL) was added to a stirred suspension
of (rnethoxy methyl)triphenylphosphonium chloride (9.5024g) in dry THF (80 mL)
at 0 °C under N2. The resulting red mixture was stirred at 0 °C
for 35 minutes,
to followed by addition of a solution of compound (3) (4.31g) in dry THF (40
mL)
over 10 minutes. The reaction mixture was stirred at 0 °C for 2 h and
at room.
temperature for 1 hour. Water (30 mL) was added to the reaction mixture and
then
the mixture was partitioned between brine (200 mL) and EtOAc (200 mL). The
organic phase was washed with brine (2 x 150 mL) and the combined aqueous
phase
was extracted again with EtOAc (3 x 150 mL). The combined' EtOAc extracts were
dried and concentrated under uacuo. The crude product was purified by column
chromatography (hexanes: EtOAc, 9: 1) to obtain 3.11g
of compound 23.
2o Preparation of Compound 24
Compound 2 (4.4 g) in dioxane-HZO (90 mL 2:I) was heated to reflux for 2
h, and stirred overnight at room temperature. The reaction mixture was diluted
with
53
SUBSTITUTE SHEET (RULE 26)
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water and extracted with Et20. The organic extracts were washed with NaHC03
(50%) followed by water, dried over anhydrous MgS04 and concentrated in vacuo
to
give 3.0 g of the compound 24 as an oil.
Preparation of Compounds 25
A mixture of 24 (3.0 g, 0.015 mol), KCN (2.08 g, 0.032 mol) and (NH4)2C03
(10.7 g, 0.11 mol) in EtOH-H20 (30 mL 1:1) was heated at 60 °C
overnight. The pH
was adjusted to 5 with 6N HCI. The reaction mixture was extracted with EtOAc
and
the organic solvent was evaporated to give compound 25 as 4ark oil.
Preparation of Compound 26 and 27
Compound 25, obtained from the above reaction, was treated with 3N NaOH
under reflux conditions overnight. The reaction mixture was acidified with 6 N
HCl
and then HCl and HZO were evaporated. The residue was treated with EtOH.
Filtration and evaporation of EtOH gave a foam. The foam was submitted to
cation-
exchange resin chromatography and eluted with 10% pyridine to obtain 250 mg of
the amino acid 26 and 14 mg of amino acid 27.
1H NMR (200 MHz, D20) ~ 3.3 (d, 1H), 2.4-2.1 (m, 1H), 1.9-1.3 (m, 7H), 1.05,
0.9
(AB system 2H) ppm.
Example 5: Testing of Exemplary Compounds:
Cyclic AMP assay:
Rationale:
Group II/III metabotropic glutamate receptors (mGluRs) are negatively coupled
to
adenylate cyclase, and agonists of these receptors lead to a decrease in
intracellular
cyclic AMP accumulation.
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Method:
The assay has been modeled on the cyclic AMP assay kit available from
Amersham.
This kit, in turn, is based on the assay created by Tovey et al. (1974).
Briefly, the
samples were prepared from Sprague Dawley rat (225-250g) cortical slices.
Slices
were incubated with the test compound (drug), and then challenged with
forskolin to
induce cyclic AMP release. Following termination of the reaction by boiling,
the
slices were homogenized and centrifuged. Samples of supernatant were then
incubated for 2-3 hours with a known quantity of [3H]CAMP and a binding
protein.
When the incubation was complete, the bound cyclic AMP was separated from the
to free cyclic AMP by centrifugation with charcoal. The resulting supernatant
(containing free cyclic AMP) was then analyzed by liquid scintillation
counting.
The amount of unbound cyclic AMP was calculated from a standard curve
previously determined with various samples of free cyclic AMP.
Results I~terpretatior~:
If the drugs tested inhibit forskolin-induced cyclic AMP accumulation, they
are
considered to be Group II/III agonists. Conversely, if they inhibit the
decrease in
forskolin-induced cyclic AMP accumulation caused by glutamate, they are
considered to be Group II/III antagonists.
Results:
Group IIIIII' '~T Group IIIIII~ : A
Carrtpound ~ 3 , - ECso (M~ EC~a (~~
Agonist Antagonist.
Compound 7 No - Yes 1.4 x
10-
Compound 8 No - yes 1.0 x
10-
Compound 16 Yes 10 x 10-"'No -
Compound 22 No - No 8.7 x
10-1"
Compound 26 Yes 1.6 x 10-"Yes -
acid Compound Yes $.7 x 10-"Yes -
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Phosphatidylinositol Assay
Rationale:
Group I metabotropic glutamate receptors (mGluRs) are positively coupled on
inositol phosphate metabolism. Agonists at these receptors lead to an increase
in
intracellular free inositol phosphates, while antagonists inhibit the increase
in
intracellular free inositol phosphate induced by standard agonists (i.e.
ACPD).
Method:
1o Cross-chopped slices were prepared from neonatal Sprague Dawley rat tissue
(age:
p7-pl4). The slices were pre-labelled with [3H] myo-inositol. Following pre-
labelling, the slices were incubated with the test compounds and standard
(known
Group I agonists i.e. ACPD) for a period of 45 minutes. The incubation was
terminated by the addition of chloroform/methanol/HCl (100:200:2). The
resulting
mixture was separated into two phases by the addition of chloroform and
distilled
water. The aqueous fraction was applied to ion exchange columns, and inositol
phosphates were eluted using 800 mM Ammonium Formate/100 mM Formic Acid.
The eluent was then analyzed using liquid scintillation counting. The amount
of
inositol phosphate accumulation was expressed as a percentage of that induced
by
2o ACPD.
Results Ihtefp~etation:
If the drugs cause an increase in intracellular free inositol phosphate
accumulation,
they are considered to be Group I agonists. If they inhibit the increase in
intracellular free inositol phosphate accumulation induced by ACPD, they are
considered to be Group II antagonists.
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Results:
G~o~p Group T
Compound 1 ~C~~ {M) ECSO
Axatagonist(M)
Agonist
Compound 7 No - Yes 2.2 x
10-
Compound 8 No - Yes 9.4 x
10-
Compound 16 No - Yes 1.0 x
10-'
Compound 22 No - No -
Compound 26 No - Yes 8.8 x
10-
acid Compound No - Yes 8.4 x
27 10-'~'
The invention being thus described, it will be obvious that the same may be
varied in many
ways. Such variations are not to be regarded as a departure from the spirit
and scope of the
invention, and all such modifications as would be obvious to one skilled in
the art are
intended to be included within the scope of the following claims.
57
