Note: Descriptions are shown in the official language in which they were submitted.
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(~-AMINOACID COMPOUNDS USEFUL FOR INHIBITING
(~-AMYLOID PEPTIDE RELEASE AND/OR ITS SYNTHESIS
This invention relates to (3-aminoacid containing
compounds which inhibit (3-amyloid peptide release and/or its
synthesis and are useful in treating Alzheimer's disease.
BACKGROUND OF THE INVENTION
Alzheimer's Disease is a degenerative brain disorder
characterized clinically by progressive loss of memory,
cognition, reasoning, judgment and emotional stability that
gradually leads to profound mental deterioration and
ultimately death. Alzheimer's disease is a very common
cause of progressive mental failure (dementia) in aged
humans and is believed to represent the fourth most common
medical cause of death in the United States. Alzheimer's
disease has been observed in races and ethnic groups
worldwide and presents a major present and future public
health problem. The disease is currently estimated to
affect about two to three million individuals in the United
States alone. Alzheimer's disease is at present incurable.
No treatment that effectively prevents Alzheimer's disease
or reverses its symptoms and course is currently known.
The brains of individuals with Alzheimer's disease
exhibit characteristic lesions termed senile (or amyloid)
plaques, amyloid angiopathy (amyloid deposits in blood
vessels) and neurofibrillary tangles. Large numbers of
these lesions, particularly amyloid plaques and
neurofibrillary tangles, are generally found in several
areas of the human brain important for memory and cognitive
function in patients with Alzheimer's disease. Smaller
numbers of these lesions in a more restrictive anatomical
distribution are also found in the brains of most aged
humans who do not have clinical Alzheimer's disease.
Amyloid plaques and amyloid angiopathy also characterize the
brains of individuals with Trisomy 21 (Down's Syndrome) and
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Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch
Type (HCHWA-D). At present, a definitive diagnosis of
Alzheimer's disease usually requires observing the
aforementioned lesions in the brain tissue of patients who
have died with the disease or, rarely, in small biopsied
samples of brain tissue taken during an invasive
neurosurgical procedure.
The principal chemical constituent of the amyloid
plaques and vascular amyloid deposits (amyloid angiopathy)
characteristic of Alzheimer's disease and the other
disorders mentioned above is an approximately 4.2 kilodalton
(kD) protein of about 39-43 amino acids designated the (3-
amyloid peptide ((3AP) or sometimes A(3, A(3P or (3/A4. (3-
Amyloid peptide was first purified and a partial amino acid
sequence was provided by Glenner, et al.Biochem. Biophys.
Res. Commun., 120:885-890, (1984). The isolation procedure
and the sequence data for the first 28 amino acids are
described in U.S. Patent No. 4,666,8292.
Molecular biological and protein chemical analyzes have
shown that the (3-amyloid peptide is a small fragment of a
much larger precursor protein termed the amyloid precursor
protein (APP), that is normally produced by cells in many
tissues of various animals, including humans. Knowledge of
the structure of the gene encoding APP has demonstrated that
(3-amyloid peptide arises as a peptide fragment that is
cleaved from APP by protease enzyme(s). The precise
biochemical mechanism by which the (3-amyloid peptide
fragment is cleaved from APP and subsequently deposited as
amyloid plaques in the cerebral tissue and in the walls of
the cerebral and meningeal blood vessels is currently
unknown.
Several lines of evidence indicate that progressive
cerebral deposition of (3-amyloid peptide plays a seminal
role in the pathogenesis of Alzheimer's disease and can
precede cognitive symptoms by years or decades. See, for
example, Selkoe, Neuron, 6:487-498 (1991). The most
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important line of evidence is the discovery that missense
DNA mutations at amino acid 717 of the 770-amino acid
isoform of APP can be found in affected members but not
unaffected members of several families with a genetically
determined (familial) form of Alzheimer's disease (Goate, et
al., Nature, 349:704-706 (1990); Chartier Harlan, et al.,
Nature, 353:844-846 (1989); Murrell, et al., Science,
254:97-99 (1991)) and is referred to as the Swedish variant.
A double mutation changing lysine595-methionine596 to
asparagine595-leucine596 (with reference to the 695 isoform)
found in a Swedish family was reported in 1992 (Mullan, et
al., Nature Genet., 1:345-347 (1992)). Genetic linkage
analyses have demonstrated that these mutations, as well as
certain other mutations in the APP gene, are the specific
molecular cause of Alzheimer°s disease in the affected
members of such families. In addition, a mutation at amino
acid 693 of the 770-amino acid isoform of APP has been
identified as the cause of the (3-amyloid peptide deposition
disease, HCHWA-D, and a change from alanine to glycine at
amino acid 692 appears to cause a phenotype that resembles
Alzheimer's disease is some patients but HCHWA-D in others.
The discovery of these and other mutations in APP in
genetically based cases of Alzheimer's disease prove that
alteration of APP and subsequent deposition of its (3-amyloid
peptide fragment can cause Alzheimer's disease.
Despite the progress which has been made in
understanding the underlying mechanisms of Alzheimer's
disease and other (3-amyloid peptide related diseases, there
remains a need to develop methods and compositions for
treatment of the disease(s). Ideally, the treatment methods
would advantageously be based on drugs which are capable of
inhibiting (3-amyloid peptide release and/or its synthesis in
V1V0.
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SUNINlP~RY OF THE INVENTION
This invention provides (3-aminoacid containing
compounds of formula I:
O R4
R~\ W
H H
n3 O
formula I
wherein
R1 is selected from the group consisting of alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted
alkyl, substituted alkenyl, substituted alkynyl,
substituted cycloalkyl, substituted cycloalkenyl, aryl,
heteroaryl and heterocyclic;
RZ is selected from the group consisting of hydrogen,
alkyl, cycloalkyl, and aryl;
R3 is selected from the group consisting of hydrogen,
alkyl, cycloalkyl, and aryl;
R4 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, aryl, alkaryl, substituted
alkaryl, cycloalkyl, and substituted cycloalkyl;
Z is represented by the formula -CX'X"-
wherein
X' is selected from the group consisting of
hydrogen, hydroxy, and fluoro,
X" is selected from the group consisting of
hydrogen, hydroxy, and fluoro, or
X' and X" together form an oxo group;
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W is selected from the group consisting of
-OR5 and -NR6R~
wherein
RS is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, aryl,
cycloalkyl, substituted cycloalkyl, and alkaryl;
R6 is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, aryl,
cycloalkyl, substituted cycloalkyl, and alkaryl;
R~ is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, aryl,
cycloalkyl, substituted cycloalkyl, and alkaryl;
or
R6 and R~ together with the nitrogen to which they
are attached form a 3 to 7 membered cyclic group
or a 6 to 7 membered group containing a heteroatom
selected from the group consisting of O, S, NR8
wherein R8 is selected from the group consisting
of hydrogen, alkyl, aryl, and alkaryl;
and the pharmaceutically acceptable salts thereof.
This invention also provides for novel pharmaceutical
compositions comprising a compound of the formula I and a
pharmaceutically acceptable diluent.
Additionally, this invention
provides a method for inhibiting (3-amyloid peptide
release and/or its synthesis in a cell which method
comprises administering to such a cell an amount of a
compound or a mixture of compounds of formula I above
effective in inhibiting the cellular release and/or
synthesis of (3-amyloid peptide.
Because the in vivo generation of (3-amyloid peptide is
associated with the pathogenesis of Alzheimer's disease the
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compounds of formula I can also be employed in conjunction
with a pharmaceutical composition to prophylactically and/or
therapeutically prevent and/or treat Alzheimer's disease.
Accordingly, the present invention provides a prophylactic
method for preventing the onset of Alzheimer's disease in a
patient at risk for developing Alzheimer's disease which
method comprises administering to said patient a
pharmaceutical composition comprising a pharmaceutically
inert carrier and an effective amount of a compound or a
mixture of compounds of formula I above.
The present invention also provides a therapeutic
method for treating a patient with Alzheimer's disease in
order to inhibit further deterioration in the condition of
that patient which method comprises administering to said
patient a pharmaceutical composition comprising a
pharmaceutically inert carrier and an effective amount of a
compound or a mixture of compounds of formula I above.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the following terms have the meanings
indicated:
The term "(3-amyloid peptide" refers to a 39-43 amino
acid peptide having a molecular weight of about 4.2 kD,
which peptide is substantially homologous to the form of the
protein described by Glenner, et al. Biochem. Biophys. Res.
Commun. (1984) 120:885-890, including mutations and post-
translational modifications of the normal (3-amyloid peptide.
In whatever form, the (3-amyloid peptide is an approximate
39-43 amino acid fragment of a large membrane-spanning
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glycoprotein, referred to as the (3-amyloid precursor protein
(APP). Its 43-amino acid sequence is:
1
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
11
Glu Val His His Gln LysLeu Val Phe Phe
21
Ala Glu Asp Val Gly SerAsn Lys Gly Ala
31
Ile Ile Gly Leu Met ValGly Gly Val Val
41
Ile Ala Thr (SEQ NO:1)
ID
or a is substantially homologous
sequence
which
thereto.
"Alkyl" refers to monovalent alkyl groups preferably
having from 1 to 20 carbon atoms and more preferably 1 to 6
carbon atoms. This term is exemplified by groups such as
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,
sec-butyl, n-pentyl, n-hexyl, and the like. It is
understood that the term alkyl includes C1-C4 alkyl.
"C1-C4 alkyl" refers to monovalent alkyl groups
preferably having from 1 to 4 carbon atoms. This term is
exemplified by groups such as methyl, ethyl, n-propyl, iso-
propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.
"Substituted alkyl" refers to an alkyl group,
preferably of from 1 to 10 carbon atoms, having from 1 to 5
substituents, and preferably 1 to 3 substituents, selected
from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyacylamino,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, vitro, -SO-alkyl,
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-SO-substituted alkyl, -SO-aryl,-SO-heteroaryl, -SOz-alkyl,
-SOZ-substituted alkyl, -SOz-aryl and -SO2-heteroaryl.
"Alkenylene" refers to divalent alkenylene groups
preferably having from 2 to 10 carbon atoms and more
preferably 2 to 6 carbon atoms. This term is exemplified by
groups such as ethenylene (-CH=CH-), the propenylene isomers
(e. g., -CHZCH=CH- and -C(CH3)=CH-) and the like.
"Substituted alkenylene" refers to an alkenylene group,
preferably of from 2 to 10 carbon atoms, having from 1 to 3
substituents selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkoxy, substituted cycloalkoxyl, acyl, acylamino,
acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl,
heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -
SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl,
-SOz-substituted alkyl, -S02-aryl, and -SOz-heteroaryl.
Additionally, such substituted alkylene groups include those
where 2 substituents on the alkylene group are fused to form
one or more cycloalkyl, aryl, heterocyclic or heteroaryl
groups fused to the alkylene group.
"Alkaryl" refers to -alkylene-aryl groups preferably
having from 1 to 8 carbon atoms in the alkylene moiety and
from 6 to 10 carbon atoms in the aryl moiety. Such alkaryl
groups are exemplified by benzyl, phenethyl and the like.
"Alkoxy" refers to the group "alkyl-0-"~ Preferred
alkoxy groups include, by way of example, methoxy, ethoxy,
n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,
n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
"Substituted alkoxy" refers to the group "substituted
alkyl-O-" where substituted alkyl is as defined above.
"Alkenyl" refers to alkenyl groups preferably having
from 2 to 10 carbon atoms and more preferably 2 to 6 carbon
atoms and having at least 1 and preferably from 1-2 sites of
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alkenyl unsaturation. Preferred alkenyl groups include
ethenyl(-CH=CHZ), n-propenyl (-CH2CH=CHZ), iso-propenyl
( -C ( CH3 ) =CHZ ) , and the 1 ike .
°'Substituted alkenyl" refers to an alkenyl group as
defined above having from 1 to 3 substituents selected from
the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted
cycloalkoxyl, acyl, acylamino, acyloxy, amino, substituted
amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl,
carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy,
substituted thioalkoxy, aryl, heteroaryl, heterocyclic,
heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO-
aryl, -SO-heteroaryl, -S02-alkyl, -SOz-substituted alkyl, -
S02-aryl, and -SO2-heteroaryl.
"Alkynyl" refers to alkynyl groups preferably having
from 2 to 10 carbon atoms and more preferably 2 to 6 carbon
atoms and having at least 1 and preferably from 1-2 sites of
alkynyl unsaturation. Preferred alkynyl groups include
ethynyl, propargyl, and the like.
"Substituted alkynyl" refers to an alkynyl group as
defined above having from 1 to 3 substituents selected from
the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted
cycloalkoxyl, aryl, acylamino, acyloxy, amino, substituted
amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl,
carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy,
substituted thioalkoxy, aryl, heteroaryl, heterocyclic,
heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO-
aryl, -SO-heteroaryl, -502-alkyl, -S02-substituted alkyl,
-SOZ-aryl, and -SOZ-heteroaryl.
"Acyl" refers to the groups alkyl-C(O)-, substituted
alkyl-C(O)-, cycloalkyl-C(0)-, substituted cycloalkyl-C(O)-,
aryl-C(O)-, heteroaryl-C(0)- and heterocyclic-C(0)- where
alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
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"Acylamino" refers to the group -C(O)NRR where each R
is independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,
heterocyclic and where both R groups are joined to form a
heterocyclic group, wherein alkyl, substituted alkyl, aryl,
heteroaryl and heterocyclic are as defined herein.
"Substituted amino" refers to the group -N(R)2 where
each R is independently selected from the group consisting
of hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl,
substituted cycloalkyl, and where both R groups are joined
to form a heterocyclic group. As is readily apparent to
those skilled in the art, when both R groups are hydrogen, -
N(R)2 is an amino group. Examples of substituted amino
groups include, by way of illustration, mono- and di-
alkylamino, mono- and di-(substituted alkyl)amino, mono- and
di-arylamino, mono- and di-heteroarylamino, mono- and di-
heterocyclic amino, and unsymmetric di-substituted amines
having different substituents selected from alkyl,
substituted alkyl, aryl, and the like.
The term "blocking group" or "protecting group" refers
to any group which prevents undesired reactions from
occurring at the protected functionality and which may be
removed by conventional chemical and/or enzymatic
procedures. Selection and use of protecting groups is well
understood and appreciated in the art. For example see,
Protecting Groups in Organic Synthesis, Theodora Greene (1St
and 2nd Editions, Wiley-Interscience). A protecting group
may also be a covalently attached to a solid support as is
well known and appreciated in the art of peptide synthesis
and combinatorial chemistry.
"Aminoacyl" refers to the group -NRC(0)R where each R
is independently hydrogen, alkyl, substituted alkyl, aryl,
heteroaryl, or heterocyclic wherein alkyl, substituted
alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
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"Aminoacyloxy" refers to the group -NRC(O)OR where each
R is independently hydrogen, alkyl, substituted alkyl, aryl,
heteroaryl, or heterocyclic wherein alkyl, substituted
alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
"Acyloxy" refers to the groups alkyl-C(O)0-,
substituted alkyl-C(O)O-, cycloalkyl-C(0)0-, substituted
cycloalkyl-C(0)-, aryl-C(O)O-, heteroaryl-C(O)0-, and
heterocyclic-C(O)O- wherein alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and
heterocyclic are as defined herein.
"Aryl'° refers to an unsaturated aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring
(e. g., phenyl) or multiple condensed (fused) rings (e. g.,
naphthyl or anthryl). Preferred aryls include phenyl,
naphthyl and the like.
Unless otherwise constrained by the definition for the
aryl substituent, such aryl groups can optionally be
substituted with from 1 to 5 substituents selected from the
group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy,
alkenyl, alkynyl, substituted alkyl, substituted alkoxy,
substituted alkenyl, substituted alkynyl, amino, substituted
amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido,
carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl,
heterocyclic, aminoacyloxy, oxyacylamino, thioalkoxy,
substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-
alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl,
-SOz-alkyl, -SOZ-substituted alkyl, -SO2-aryl,
-S02-heteroaryl and trihalomethyl. Preferred substituents
include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,
and thioalkoxy.
"Aryloxy" refers to the group aryl-O- wherein the aryl
group is as defined above including optionally substituted
aryl groups as also defined above.
"Carboxyalkyl" refers to the groups "-C(O)Oalkyl" and
"-C(O)O-substituted alkyl" where alkyl is as defined above.
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"Cycloalkyl" refers to cyclic alkyl groups of from 3 to
12 carbon atoms having a single cyclic ring or multiple
condensed rings. Such cycloalkyl groups include, by way of
example, single ring structures such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclooctyl, and the like, or
multiple ring structures such as quininclidine, adamantanyl,
and the like.
"Substituted cycloalkyl" refers to cycloalkyl groups
having from 1 to 5 (preferably 1 to 3) substituents selected
from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyacylamino,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl,
-SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl,
-SO2-substituted alkyl, -SOz-aryl, and -SOz-heteroaryl.
"Cycloalkenyl" refers to cyclic alkenyl groups of from
4 to 8 carbon atoms having a single cyclic ring and at least
one point of internal unsaturation. Examples of suitable
cycloalkenyl groups include, for instance, cyclobut-2-enyl,
cyclopent-3-enyl, cyclooct-3-enyl and the like.
"Substituted cycloalkenyl" refers to cycloalkenyl
groups having from 1 to 5 substituents selected from the
group consisting of alkoxy, substituted alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, acylamino, acyloxy, amino, substituted
amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,
halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted
alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -SOZ-
substituted alkyl, -SOZ-aryl, and -S02-heteroaryl.
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"Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo and preferably is either fluoro or chloro.
"Heteroaryl" refers to an aromatic group of from 1 to
15 carbon atoms and 1 to 4 heteroatoms selected from oxygen,
nitrogen and sulfur within at least one ring (if there is
more than one ring).
Unless otherwise constrained by the definition for the
heteroaryl substituent, such heteroaryl groups can be
optionally substituted with 1 to 5 substituents selected
from the group consisting of acyloxy, hydroxy, acyl, alkyl,
alkoxy, alkenyl, alkynyl, substituted alkyl, substituted
alkoxy, substituted alkenyl, substituted alkynyl, amino,
substituted amino, aminoacyl, acylamino, alkaryl, aryl,
aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro,
heteroaryl, heterocyclic, aminoacyloxy, oxyacylamino,
thioalkoxy, substituted thioalkoxy, thioaryloxy,
thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-
aryl, -SO-heteroaryl, -SOZ-alkyl, -S02-substituted alkyl, -
S02-aryl, -S02-heteroaryl and trihalomethyl. Such heteroaryl
groups can have a single ring (e.g., pyridyl or furyl) or
multiple condensed rings (e.g., indolizinyl or
benzothienyl). Preferred heteroaryls include pyridyl,
pyrrolyl and furyl.
"Heteroaryloxy" refers to the group "-O-heteroaryl".
"Heterocycle" or "heterocyclic" refers to a monovalent
saturated or unsaturated group having a single ring or
multiple condensed rings, from 1 to 15 carbon atoms and from
1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen
within the ring.
Unless otherwise constrained by the definition for the
heterocyclic substituent, such heterocyclic groups can be
optionally substituted with 1 to 5 substituents selected
from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, aryl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyacylamino,
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cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl,
-SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl,
-SOZ-substituted alkyl, -SOZ-aryl, and -SOZ-heteroaryl. Such
heterocyclic groups can have a single ring or multiple
condensed rings. Preferred heterocyclics include
morpholino, piperidinyl, and the like.
Examples of heterocycles and heteroaryls include, but
are not limited to, pyrrole, furan, imidazole, pyrazole,
pyridine, pyrazine, pyrimidine, pyridazine, indolizine,
isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthylpyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline, phenanthridine, acridine, phenanthroline,
isothiazole, phenazine, isoxazole, phenoxazine,
phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, morpholino, piperidinyl,
tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen
containing heterocycles.
"Heterocyclooxy" refers to the group "-O-heterocycle".
"Oxyacylamino" refers to the group -OC(O)NRR where each
R is independently hydrogen, alkyl, substituted alkyl, aryl,
heteroaryl, or heterocyclic wherein alkyl, substituted
alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
"Thioalkoxy" refers to the group -S-alkyl.
"Substituted thioalkoxy" refers to the group -S-
substituted alkyl.
"Thioaryloxy" refers to the group aryl-S- wherein the
aryl group is as defined above including optionally
substituted aryl groups also defined above.
"Thioheteroaryloxy" refers to the group heteroaryl-S
wherein the heteroaryl group is as defined above including
optionally substituted aryl groups as also defined above.
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Compounds in which R6 and R~ together with the nitrogen
to which they are attached form a 3 to 7 membered cyclic
group 3 to 7 membered cyclic group, refers to groups such as
a pyrrolido, piperidino, azepino, and the like or when R6
and R~ together with the nitrogen to which they are attached
form a 6 to 7 membered group containing a heteroatom
selected from the group consisting of 0, S, NRg wherein R8
refers to groups such as morpholino, thiomorpholino,
piperazino, 4-methylpiperazino, 4-ethylpiperazino, 4-phenyl
piperazino, 4-benzylpiperazino, and the like.
The term "pharmaceutically-acceptable addition salt"
refers to an acid addition salt.
The compound of formula I and the intermediates
described herein form pharmaceutically acceptable acid
addition salts with a wide variety of organic and inorganic
acids and include the physiologically acceptable salts which
are often used in pharmaceutical chemistry. Such salts are
also part of this invention. A pharmaceutically-acceptable
addition salt is formed from a pharmaceutically-acceptable
acid as is well known in the art. Such salts include the
pharmaceutically acceptable salts listed in Journal of
Pharmaceutical Science, 66, 2-19 (1977) which are known to
the skilled artisan. Typical inorganic acids used to form
such salts include hydrochloric, hydrobromic, hydriodic,
nitric, sulfuric, phosphoric, hypophosphoric,
metaphosphoric, pyrophosphoric, and the like. Salts derived
from organic acids, such as aliphatic mono and dicarboxylic
acids, phenyl substituted alkanoic acids, hydroxyalkanoic
and hydroxyalkandioic acids, aromatic acids, aliphatic and
aromatic sulfonic acids, may also be used. Such
pharmaceutically acceptable salts thus include acetate,
phenylacetate, trifluoroacetate, acrylate, ascorbate,
benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, methylbenzoate, o-acetoxybenzoate,
naphthalene-2-benzoate, bromide, isobutyrate,
phenylbutyrate, a-hydroxybutyrate, butyne-1,4-dicarboxylate,
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hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate,
citrate, formate, fumarate, glycollate, heptanoate,
hippurate, lactate, malate, maleate, hydroxymaleate,
malonate, mandelate, mesylate, nicotinate, isonicotinate,
nitrate, oxalate, phthalate, teraphthalate, propiolate,
propionate, phenylpropionate, salicylate, sebacate,
succinate, suberate, benzenesulfonate, p-
bromobenzenesulfonate, chlorobenzenesulfonate,
ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-
toluenesulfonate, xylenesulfonate, tartarate, and the like.
The term "ee" or "enantiomeric excess" refers to the
percent by which one enantiomer, E1, is in excess in a
mixture of both enantiomers (E1 + E2), as calculated by the
equation {(E1 - E2) . (E1 + E2)} x 1000 = ee.
As is appreciated by the skilled person, compounds of
formula I exist as stereoisomers. The present invention
relates to the stereoisomers of the compounds of formula I.
Herein, the Cahn-Prelog-Ingold designations of (R)- and (S)-
and the designations of L- and D- for stereochemistry
relative to the isomers of glyceraldehyde are used to refer
to specific isomers where designated.
The specific isomers of the compounds of formula I can
by prepared by stereospecific synthesis. The compounds of
formula I and the starting materials for their preparation
can be resolved and recovered by techniques known in the
art, such as, chromatography on chiral stationary phases,
and fractional recrystallization of addition salts formed by
reagents used for that purpose. Useful methods of resolving
and recovering specific stereoisomers are known in the art
and described in Stereochemistry of Organic Compounds, E.L.
Eliel and S.H. Wilen (Wiley-Interscience 1994),
Enantiomers, Racemates, and Resolutions, J. Jacques, A.
Collet, and S.H. Wilen (Wiley-Interscience 1981), and
European Patent Application No. EP-A-838448, published April
29, 1998.
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It is to be understood that the invention extends to
each of the isomeric forms of the compounds of the present
invention including the geometric, diastereomeric,
enantiomeric, and racemic forms of the compound of formula
I.
As with any group of pharmaceutically active compounds,
some groups are preferred in their end use application.
Preferred embodiments of the present invention are given
below:
Compounds in. which R1 is alkyl or aryl are preferred.
Compounds in which R1 is C1-C4 alkyl are more preferred,
with isopropyl being most preferred.
Compounds in which R1 is phenyl substituted with from 1
to 3 substituents selected from the group consisting of
hydrogen, alkyl, alkoxy, and halo are more preferred with
3,5-difluorophenyl being most preferred.
Compounds in which Rz and R3 are hydrogen, alkyl,
cycloalkyly, or aryl are preferred.
Compounds in which one of RZ or R3 are hydrogen are
preferred.
Compounds in which Rz or R3 are C1-C4 alkyl are more
preferred, with methyl being most preferred.
Compounds in which RZ or R3 are phenyl substituted with
from 1 to 3 substituents selected from the group consisting
of hydrogen, alkyl, alkoxy, and halo are more preferred with
phenyl being most preferred.
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Compounds in which R4 is selected from the group
consisting of hydrogen, alkyl, aryl, cycloalkyl, and alkaryl
are preferred, with aryl being more preferred and phenyl
being most preferred.
Compounds in which Z is -CH2- or -CH(OH)- are
preferred.
Compounds in which W is -ORS in which RS is selected
from the group consisting of alkyl, substituted alkyl,
cycloalkyl, and substituted cycloalkyl are preferred.
Compounds in which W is -ORS in which RS is selected
from the group consisting of alkyl and cycloalkyl are more
preferred.
Compounds in which W is -ORS in which R5 is C1-C4 alkyl
being more preferred and methyl, isopropyl,. sec-butyl,
isobutyl, t-butyl being most preferred.
Compounds in which W is -NR6R~ in which R6 is hydrogen,
alkyl, or cycloalkyl; and R~ is hydrogen, alkyl, or alkaryl
are preferred.
2 5 Compounds in whi ch W i s -NR6R~ in whi ch R6 and R~
together with the nitrogen to which they are attached form a
3 to 7 membered cyclic group in which the cyclic group is
pyrrolidino or piperidino are preferred.
Compounds in which W is -NR6R~ in which R6 and R?
together with the nitrogen to which they are attached form a
6 to 7 membered group containing a heteroatom selected from
the group consisting of O, S, NR8 wherein R8 is selected
from the group consisting of hydrogen, alkyl, aryl, and
alkaryl in which the cyclic group is morpholino, piperazino,
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4-methylpiperazino, 4-phenyl piperazino, 4-benzylpiperazino,
are preferred.
The compounds of formula I are prepared as described in
Reaction Scheme A.1 and A.2 below. Reaction Scheme A.1 and
A.2, all substituents, unless otherwise indicated, are as
previously defined. Reaction Scheme A.1 and A.2 all
reagents are well known and appreciated in the art.
Reaction Scheme A.1
Rz O Ra Rz O Ra
Pg~ +. W, -~ Pg W.
~OH ~N ~ \N
HzN step a H H
(1) 3 (2) O R3 (3) O
step b
O
R~
OH
O Rz O R Rz O Ra
X, X" ( 5 )
W.
R~ N N W H2N N
H H
X' X" step c R O
R3 0 3 (4)
formula I
Reaction Scheme A.1, step a, depicts the coupling reaction
of an appropriate amino-protected (3-amino acid of formula
(1) and an appropriate compound of formula (2). Appropriate
amino-protected (3-amino acids are ones in which RZ and R3
are as desired in the final product of formula I and readily
available to the person skilled in the art and can be
prepared as described herein. An appropriate compound of
formula (1) may also have the stereochemistry that is
desired in the final compound of formula I. An appropriate
compound of formula (2) is one in which W' is W as desired
in the final compound of formula I. Alternately, an
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appropriate compound of formula (2) is one in which is a
protecting group which after removal and esterification or
amidation give rise to W as desired in the final product of
formula I. An appropriate compound of formula (2) may also
have the stereochemistry that is desired in the final
compound of formula I. Compounds of formula (2) are readily
available to the skilled person. For example, an
appropriate compound of formula (2) is one in which W° is W
as desired in the final compound is readily prepared by
esterification or amidation of amino protected amino acids,
followed by deprotection to give a compound of formula (2).
The coupling reaction depicted in Reaction Scheme A.1,
step a, involves a reaction which is conventionally
conducted for peptide synthesis and synthetic methods used
therein can also be employed. For example, well known
coupling reagents such as carbodiimides with or without the
use of well known additives such as N-hydroxysuccinimide, 1-
hydroxybenzotriazole, etc. can be used to facilitate
coupling. The reaction is conventionally conducted in an
inert aprotic polar diluent such as dimethylformamide,
dichloromethane, chloroform, acetonitrile, tetrahydrofuran
and the like. Alternatively, the acid halide of compound
(1) can be employed in the reaction and, when so employed,
it is typically employed in the presence of a suitable base
to scavenge the acid generated during the reaction.
Suitable bases include, by way of example, triethylamine,
N,N-diisopropylethylamine, N-methylmorpholine and the like.
The xeaction is preferably conducted at from about 0-°C
to about 60°-C until reaction completion which typically
occurs within 1 to about 24 hours. Upon reaction
completion, the product.of formula (3) is recovered by
conventional methods including precipitation,
chromatography, filtration and the like or alternatively is
deprotected to the corresponding amine of formula (4)
without purification and/or isolation other than
conventional work-up (e. g., aqueous extraction, etc.).
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Reaction Scheme A.1, step b, depicts the deprotection
of a compound of formula (3) to give a compound or formula
(4). Such deprotections of amino protecting groups is well
known and appreciated in the art.
Reaction Scheme A.1, step c, depicts the coupling
reaction of an appropriate compound of formula (5),
R1CX'X"C(0)-OH, and a compound of formula (4). Appropriate
compounds of formula (5) are compounds in which R1, X' and
X" are as desired in the final product of formula I and are
well known in the art and available as described herein. An
appropriate compound of formula (5) may also have the
stereochemistry that is desired in the final compound of
formula I. The coupling reaction depicted in step c is
carried out using the acid of formula (5) or the acid halide
derived therefrom, in a manner similar to those taught in
Reaction Scheme A.1, step a.
Reaction Scheme A.1, optional step d, not shown, an
acid addition salt is formed using a pharmaceutically-
acceptable acid. The formation of acid addition salts is
well known and appreciated in the art.
Reaction Scheme A.2
Rz O
O O R2 O
\ R R
H2N ~ OPg ~ OH step a ' H OPg
(6) R3 X. X.. (5) X, X.. (7) R3
step b
Ra
O R2 O
O Rz O RQ H2N
) R
O ~ ~\
R~ N N E H OH
H H s t ep c X, X., R
X, Xn Rs O ( 8 ) s
formula I
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Reaction Scheme A.2, step a, depicts the coupling
reaction of an appropriate carboxy-protected (3-amino acid of
formula (6) and an appropriate compound of formula (5), as
described above, to give a compound of formula (7).
Appropriate carboxy-protected (3-amino acids are ones in
which Rz and R3 are as desired in the final product of
formula I and readily available to the person skilled in the
art and can be prepared as described herein. An appropriate
compound of formula (6) may also have the stereochemistry
that is desired in the final compound of formula I. This
coupling reaction is carried out using the acid of formula
(5) or the acid halide derived therefrom, in a manner
similar to those taught in Reaction Scheme A.1, step a.
Reaction Scheme A.2, step b, depicts the deprotection
of a compound of formula (7) to give a compound or formula
(8). Such deprotections of carboxy protecting groups is
well known and appreciated in the art.
Reaction Scheme A.2, step c, depicts the coupling
reaction of an appropriate compound of formula (2), as
described above, and a compound of formula (8). Appropriate
compounds of formula (2) are the coupling reaction depicted
in step c are taught in Reaction Scheme A.1, step a.
Reaction Scheme A.2, optional step d, not shown, an
acid addition salt is formed using a pharmaceutically-
acceptable acid. The formation of acid addition salts is
well known and appreciated in the art.
The following synthetic and biological examples are
offered to illustrate this invention and are not to be
construed in any way as limiting the scope of this
invention.
In the examples below, the following abbreviations have
the following meanings. If an abbreviation is not defined,
it has its generally accepted meaning.
BEMP refers to 2-tert-butylimino-2-diethylamino-1,3-
dimethylperhydro-1,3,2-diazaphosphorine; Boc refers to t-
butoxycarbonyl; BOP refers to benzotriazol-1-yloxy-
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tris(dimethylamino)phosphonium
hexafluorophosphate; bd refers to broad doublet; bs refers
to broad singlet; d refers to doublet; dd refers to doublet
of doublets; DIC refers to diisopropyl
carbodiimide; DMF refers to dimethylformamide; DMAP refers
to 4-dimethylaminopyridine; DMSO refers to
dimethylsulfoxide; EDC refers to ethyl-1-(3-
dimethyaminopropyl)carbodiimide; eq. or eqv. refer to
equivalents; EtOAc refers to ethyl acetate; g refers to
grams; h refers to hours; HOBT refers to 1-
hydroxybenzotriazole hydrate; Hunig's base refers to N,N-
diisopropylethylamine; L refers to liter; m refers to
multiplet; M refers to molar; max refers to
maximum; meq refers to milliequivalent; mg refers to
milligram; mL refers to milliliter; mm refers to
millimeter; mmol refers to millimole; MOC refers to
methoxyoxycarbonyl; N refers to normal; N/A refers to not
available; ng refers to nanogram; nm refers to
nanometers; OD refers to optical density; PEPC refers to 1-
(3-(1-pyrrolidinyl)propyl)-3-ethylcarbodiimide; PP-
HOBT refers to piperidine-piperidine-1-
hydroxybenzotrizole; psi refers to pounds per square
inch; Ph refers to phenyl; q refers to
quartet; quint. refers to quintet; rpm refers to rotations
per minute; s refers to singlet; t refers to
triplet; TFA refers to trifluoroacetic acid; THF refers to
tetrahydrofuran; tlc refers to thin layer
chromatography; ~,L refers to microliter; UV refers to ultra-
violet.
In the examples below, all temperatures are in degrees
Celsius (unless otherwise indicated). The compounds set
forth in the examples below were prepared using the
following general procedures as indicated.
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GENERAL PROCEDURE A
First EDC Coupling Procedure
To a 1:1 mixture of the corresponding acid and the
corresponding amine in dichloromethane at 0°-C was added 1.5
equivalents triethylamine, followed by 2.0 equivalents
hydroxybenzotriazole monohydrate and then 1.25 equivalents
of ethyl-3-(3-dimethylamino)propyl carbodiimide ~ HC1. The
reaction mixture was stirred overnight at room temperature
and then transferred to a separatory funnel. The mixture
was washed with water, saturated aqueous NaHC03, 1N HC1 and
saturated aqueous NaCl, and then dried over MgS04. The
resulting solution was stripped free of solvent on a rotary
evaporator to yield the crude product.
GENERAL PROCEDURE B
Second EDC Coupling Procedure
A mixture of the corresponding acid (1 eqv), N-1-
hydroxybenzotriazole (1.6 eqv), the corresponding amine (1
eqv), N-methylmorpholine (3 eqv) and dichloromethane (or DMF
for insoluble substrates) was cooled in an ice-water bath
and stirred until a clear solution was obtained. EDC (1.3
eqv) was then added to the reaction mixture. The cooling
bath was then allowed to warm to ambient temperature over 1-
2 h and the reaction mixture was stirred overnight. The
reaction mixture was then evaporated to dryness under
vacuum. To the residue was added 20% aqueous potassium
carbonate and the mixture was shaken thoroughly and then
allowed to stand until the oily product solidified
(overnight if necessary). The solid product was then
collected by filtration, washed thoroughly with 20o aqueous
potassium carbonate, water, 10o HCl, and water to give the
product, usually in pure state. No racemization was
observed.
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GENERAL PROCEDURE C
Third EDC Coupling Procedure
The acid was dissolved in methylene chloride. The
amine (1 eq.), N-methylmorpholine (5 eq.) and
hydroxybenzotriazole monohydrate (1.2 eq.) were added in
sequence. A cooling bath was applied to the round bottomed
flask until the solution reached 0-°C. At that time, 1.2 eq.
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride was added. The solution was allowed to stir
overnight and come to room temperature under nitrogen
pressure. The reaction mixture was worked up by washing the
organic phase with saturated aqueous sodium carbonate, 0.1M
citric acid, and brine before drying with sodium sulfate.
The solvents were then removed to yield crude product.
GENERAL PROCEDURE D
Fourth EDC Coupling Procedure
A round bottom flask was charged with the corresponding
carboxylic acid (1.0 eq.), hydroxybenzotriazole hydrate (1.1
eq.) and the corresponding amine (1.0 eq.) in THF under
nitrogen atmosphere. An appropriate amount (1.1 eq for free
amines and 2.2 eq. for hydrochloride amine salts) of base,
such as Hunig's base was added to the well stirred mixture
followed by EDC (1.1 eq.). After stirring from 4 to 17
hours at room temperature the solvent was removed at reduced
pressure, the residue taken up in ethyl acetate (or similar
solvent) and water, washed with saturated aqueous sodium
bicarbonate solution, 1 N HCl, brine, dried over anhydrous
sodium sulfate and the solvent removed at reduced pressure
to provide the product.
GENERAL PROCEDURE E
BOP Coupling Procedure
To a stirred solution of acid (2 mmol) in DMF, cooled
in an ice-water bath, was added BOP (2.4 mmol) and N-
methylmorpholine (6 mmol). The reaction mixture was stirred
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for 50 min. and then a solution of amine (2 mmol) in DMF
cooled at 0°-C was added. The cooling bath was allowed to
warm to ambient temperature over 1-2 h and the reaction
mixture was then stirred overnight. A 20o aqueous potassium
carbonate solution (60 mL) was added and this mixture shaken
thoroughly. No solid formed. The mixture was then washed
with ethyl acetate (150 mL) and evaporated to dryness under
vacuum to give a white solid. Water (50 mL) was then added
and this mixture shaken thoroughly. The precipitate that
formed was collected by filtration, then washed thoroughly
with water, followed by 1 mL of diethyl ether to give the
product.
GENERAL PROCEDURE F
Coupling of an Acid Chloride with an Amine
To a stirred solution of amine (4.6 mmol) in 5 ml of
pyridine was added 4.6 mmol of the acid chloride. The
mixture was stirred for 3.5 h to 48 h, dissolved in 100 mL
of diethyl ether, washed with 10o HCl three times, brine
once, 20o potassium carbonate once and brine once. The
solution was dried over magnesium sulfate, filtered, and
evaporated to yield the product. Other amino acid esters
may also be employed in this procedure.
GENERAL PROCEDURE G
Coupling of an Acid with an Amine
A solution of the acid (3.3 mmol) and 1,1'-
carbodiimidazole (CDI) in 20 mL THF was stirred for 2 h.
amine hydrochloride (3.6 mmol) was added, followed by 1.5 mL
(10.8 mmol) of triethylamine. The reaction mixture was
stirred overnight. The reaction mixture was dissolved in
100 mL of diethyl ether, washed with 10% HC1 three times,
brine once, 20% potassium carbonate once and brine once.
The solution was dried over magnesium sulfate, filtered, and
evaporated to yield the product. Other amino acid esters
may also be employed in this procedure.
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GENERAL PROCEDURE H
Fifth EDC Coupling Procedure
In a round bottom flask was added an acid (1.1 eq.) in
THF, an amine hydrochloride (1.0 eq.), 1-
hydroxybenzotriazole hydrate (1.1 eq.), N,N-
diisopropylethylamine (2.1 eq.), followed by 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
(1.1 eq.). The reaction mixture stirred at room temperature
for 10-20 hours under an atmosphere of nitrogen. The
mixture was diluted with EtOAc and washed with 0.1 M HC1 (1
x 10 mL), saturated NaHC03 (1 x 10 mL), H20 (1 x 10 mL), and
brine and dried over MgS04. The drying agent was removed by
filtration and the filtrate was concentrated in vacuo. The
residue was purified by flash column chromatography on
silica gel followed by trituration from EtOAc and hexanes.
GENERAL PROCEDURE I
Sixth EDC Coupling Procedure
To a solution or suspension of the amine or amine
hydrochloride (1.0 eq.) in THF (0.05-0.1 M) under N2 at 0°-C
was added the carboxylic acid (1.0-1.1 eq.),
hydroxybenzotriazole monohydrate (1.1-1.15 eq.), Hunig's
base (1.1 eq. for free amines and 1.1-2.3 eq. for
hydrochloride amine salts), followed by 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.1-
1.15 eq.). The cooling bath was removed and the mixture
allowed to warm to room temperature for 10-24 hours. The
solution or mixture was diluted with EtOAc, in a 3-5 volume
multiple of the initial THF volume, and washed with 0.1-1.0
M aq. HC1 (1 or 2x), dilute NaHC03 (1 or 2x), and brine
(1x). Then, the organic phase was dried over either MgS04
or Na2S04, filtered, concentrated to provide the crude
product, which was either further purified or utilized
without further purification.
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GENERAL PROCEDURE J
EEDQ Coupling Procedure
To a solution of the amine in THF (1.0 eq., 0.05-0.08
M, final molarity) under NZ at room temperature was added
the N-t-Boc protected (3-amino acid (1.1 eq., either as a
solid or in THF via cannula), followed by EEDQ (Aldrich, 1.1
eq.). The pale yellow solution was stirred at.room
temperature for 16-16.5 hours, then diluted with EtOAc (in a
3-5 volume multiple of the initial THF volume), and washed
with 1M aq. HCl (2x), dilute aq. NaHC03 (2x), and brine
(1x). The organic phase was dried over either Na2S04 or
MgS04, filtered, and concentrated.
GENERAL PROCEDURE K
Boc Protection of Amino Acids
The amine was stirred in 2 eq. of 1. ON sodium hydroxide
at 0°C. 1.1 eq. of Boc-anhydride in dioxane was added
dropwise via addition funnel. After addition was complete,
the reaction was allowed to warm to room temperature, and
stirring continued for a minimum of 17 hours. 5o KHSO4
solution was added to acidify, and the mixture extracted
with dichloromethane and ethyl acetate. The organics were
combined, dried, and concentrated.
GENERAL PROCEDURE L
BOC Removal Procedure
A stream of anhydrous HCl gas was passed through a
stirred solution of the N-t-Boc protected amine in 1,4-
dioxane (0.03-0.09 M), chilled in a ice bath to ~10°-C under
N2, for 10-15 minutes. The solution was capped, the cooling
bath removed, and the solution was allowed to warm to room
temperature with stirring for 2-8 hours, monitoring by TLC
for the consumption of starting material. The solution was
concentrated (and in some instances dissolved in CHZC12 then
re-concentrated and placed in vacuum oven at 60-70°-C to
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remove most of the residual dioxane) and used without
further purification.
PREPARATION 1
Synthesis of N-methyl-N-(L-phenylglycinyl)-aminocyclohexane
Following General Procedure J, and using
benzyloxycarbonyl-L-phenylglycine (Aldrich) and
cyclohexylmethyl amine (Aldrich), N-methyl-N-(N'-
benzyloxycarbonyl-L-phenylglycinyl)-aminocyclohexane was
prepared. Cz3Hz8N2O3 (MW=380.49); mass spectroscopy (MH+)
381. OR: 7~,=589 nm, c=1o in MeOH: specific rotation 78.04.
1H NMR data was as follows: 1H NMR (400 MHz, CD30D) ~ 7.37-
7.22 (10H m), 8 5.59 (0.5H s), 8 5.49 (0.5H s), b 5.2-4.99
(2H m), 8 4.28-4.25 (0.5H m) 8 3.61-3.58 (0.5H m), ~ 2.75
(1.5H s), 8 2.72 (1.5H s), 8 1.82-0.83 (10H m).
N-methyl-N-(N'-benzyloxycarbonyl-L-phenylglycinyl)-
aminocyclohexane, was suspended in MeOH at room temperature
under a nitrogen atmosphere. 5o Pd/C, 25 mg catalyst/1 mmol
substrate was added under nitrogen, and the reaction mixture
was stirred under a hydrogen atmosphere (balloon) for 17
hours. The mixture was filtered through Celite, and the
solvents removed in vacuo. The residue was purified via
radial chromatography to prepare the title compound.
ClSHzzNzO (MW=246.36) ; mass spectroscopy (MH+) 247.
OR: ~,=589 nm, c=0.9o in MeOH: specific rotation=74.41. 1H
NMR data was as follows: 1H NMR (400 MHz, CD30D) 8 7.35-7.24
(5H m), 8 4.80 (0.5H s), b 4.71 (0.5H s), 8 4.35-4.30 (0.5 H
m), 8 3.61-3.47 (0.5H m), 8 2.71 (1.5H s), 8 2.69 (1.5H s), 8
1.29-0.80 (10H m).
PREPARATION 2
Synthesis of (R/S)-N-Boc-(3-methyl-(3-alanine
Following General Procedure K and using D,L-3-
aminobutyric acid (Aldrich), the title compound was
prepared. C9H1~N04 (MW=203.24); mass spectroscopy (MH+) 204.
1H NMR data as follows: 1H NMR (400 MHz, CD30D) b 3.9 (1H q,
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J=3.9 Hz), 8 2.5 (1H dd, J=6.9, 16.1 Hz), 8 2.3 (1H dd,
J=7.3, 15.2 Hz), 8 1.4 (9H s), b 1.1 (3H d, J=6.8 Hz).
PREPARATION 3
Synthesis of S-(+)-3,5-Difluoromandelic Acid
To a solution of 3,5-difluorobenzaldehyde (Aldrich) in
CH2C12 (100 mL) was added ZnCl2 (6.7 g, 21.1 mmol) to form a
slurry. Trimethysilyl cyanide (21.0 g, 211.2 mmol)
dissolved in CH2Clz (100 mL) was slowly added to the slurry
at 0°-C. The resulting solution was stirred at room
temperature for 4 h. The reaction mixture was then diluted
with water and the organic layer separated. The combined
organic layers were concentrated to a residue. The residue
was dissolved with MeOH (200 mL) at 0°-C and anhydrous HCl
gas was bubbled into the solution for 10 min. After
stirring at room temperature for 18 h, the solution was
concentrated to a solid. The solid was dissolved in
CHzCl2/H20 and the aqueous portion extracted with CH2Clz.
The combined organics were washed with brine, dried over
anhydrous MgS04 and concentrated to give methyl (~)-3,5-
difluoromandelate as a solid (37.4 g, 87.6%), mp = 77-78°-C.
1H NMR (300 MHz, CDC13): S = 6.97 (dd, J = 9.6 Hz, J = 1.79
Hz, 2H), 6.74 (dt, J = 8.82, J = 2.28 Hz, 1H), 5.14 (d, J =
4.64 Hz, 1H), 3.78 (s, 3H), 3.54 (d, J = 5.1 Hz, 1H).
Methyl (~)-3,5-difluoromandelate was separated via
preparative chiral HPLC to give methyl S-(+)-3,5-
difluoromandelate as a white solid having a melting point of
70-71-°C. C4HaF203 (MW = 202.17); mass spectroscopy found
(M+NH4+) 220Ø Anal. Calcd. for C4H8F203: C, 53.47; H,
3.99. Found: C, 53.40; H, 3.89.
A solution of methyl S-(+)-3,5-difluoromandelate (1
eq.) in 74o aqueous THF was cooled to 0°-C and treated with
lithium hydroxide. After 40 minutes at 0°-C the reaction was
complete by TLC. The contents were transferred to a
separatory funnel and partitioned between CHzCl2 and
saturated aqueous NaHC03. The aqueous layer was acidified
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with 0.5 N NaHS04 and extracted thrice with ethyl acetate.
The combined extracts were washed with brine, dried over
Na2S04, filtered, and concentrated to a white solid having a
melting point of 119-122°-C. The 1H NMR was consistent with
known 3,5-difluoromandelic acid.
F~'XZ1MDT.F 1
Synthesis of N-Methyl-N-(N'-(N"-((S)-3,5-difluorophenyl-oc
hydroxyacetyl)-(R)-(3-methyl-(3-alaninyl)-L-phenylglycinyl)
aminocyclohexane and N-Methyl-N-(N'-(N"-((S)-3,5
difluorophenyl-oc-hydroxyacetyl)-(S)-(3-methyl-(3-alaninyl)-L-
phenylglycinyl)-aminocyclohexane
F
N
O
Following General Procedure J and using (R/S)-N-Boc-(3-
methyl-(3-alanine and N-methyl-N-(L-phenylglycinyl)-
aminocyclohexane N-methyl-N-(N'-(N " -Boc-(R/S)-(3-methyl-(3-
alaninyl)-L-phenylglycinyl)-aminocyclohexane was prepared.
2O C24H3~N3O4 (MW=431.58) ; mass spectroscopy (MH+) 432. 1H NMR
data as follows:lH NMR (400 MHz, CD30D) b 7.36-7.28 (5H m),
8 5.82 (0.5H s), 8 5.75 (0.5H s), 8 4.35-4.30 (0.5H m), 8
3.91-3.80 (1H m), 8 3.65-3.58 (0.5H m), S 2.76 (1.5H s), cS
2.74 (1.5H s), 8 2.42-2.20 (2H m), 8 1.96-0.60 (22H m).
Following General Procedure L using no cooling bath and
using N-methyl-N-(N'-((R/S)-N " -Boc-(3-methyl-(3-alaninyl)-L-
phenylglycinyl)-aminocyclohexane, N-methyl-N-(N'-((R/S)-(3-
methyl-(3-alaninyl)-L-phenylglycinyl)-aminocyclohexane
hydrochloride was prepared. C19Hz9N3O2 (MW=331.46) ; mass
spectroscopy (MH+) 332. 1H NMR data as follows: 1H NMR (400
MHz, CD30D) 8 7.36-7.33 (5H m), b 5.82 (0.5H s), b 5.73 (0.5H
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s), 8 4.20-4.15 (0.5 H m), 8 3.70-3.65 (0.5 H m), cS 3.59
3.45 (1H m), 8 2.81-2.40 (5H m), 8 1.95-0.60 (13H m).
Following General Procedure D using (S)-3,5-
difluoromandelic acid and N-methyl-N-(N'-((R/S)-(3-methyl-(3-
alaninyl)-L-phenylglycinyl)-aminocyclohexane hydrochloride
the title compounds were prepared. The isomers were
separated via reverse phase HPLC, using Vydac C18 column
(0.46 x 25cm), W@214 nm, gradient of 5o to 70% (over
45minutes) of 0 . 1 o TFA/CH3CN in 0 . 1 o TFA/H20.
Isomer 1: CZ~H33F2N3O4 (MW=501.58) ; mass spectroscopy (MH+)
502. 1H NMR (400 MHz, CD30D) ~ 7.34-7.28 (5H m), S 7.03 (2H
d, J=8.3 Hz), 8 6.81-6.79 (1H m), 8 5.81 (0.5H s), 8 5.71
(0.5H s), 8 4.93-4.90 (1H m), b 4.33-4.25 (0.5H m), b 4.17
(1H q, J=6.3 Hz), 8 3.62-3.57 (0.5H m), 8 2.76 (1.5H s), 8
2.72 (1.5H s), 8 2.45-2.32 (2H m), 8 1.80-0.63 (13H m).
Isomer 2: CZ~H33FzN3O4 (MW=501.58); mass spectroscopy (MH+)
502. 1H NMR data as follows: 1H NMR (400 MHz, CD30D) b 7.29-
7.24 (5H m), 8 6.97 (2H d, J=6.4 Hz), 8 6.78-6.72 (1H m), 8
5.78 (0.5H s), b 5.69 (0.5H s), b 4.87 (1H s), 8 4.38-4.21
(0.5H m), 8 4.16 (1H q, J=6.8 Hz), 8 3.60-3.50 (0.5H m),
2.72 (1.5H s), 8 2.68 (1.5H s), 8 2.42-2.26 (2H m), 8 1.76-
0.58 (13H m).
L'YTMD7.L' 7
Synthesis of N-methyl-N-(N'-(N"-((S)-2-Hydroxy- 3-
methylbutyrl)-(R)-(3-methyl-(3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane and N-methyl-N-(N'-(N"-((S)-2-Hydroxy-3-
methylbutyrl)-(S)-(3-methyl-(3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane
N \
N Y a H
H I IIO
O
O O
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Following General Procedure D using (S)-2-Hydroxy-3-
methylbutyric acid (Aldrich) and N-methyl-N-(N'-((R/S)-(3-
methyl-(3-alaninyl)-L-phenylglycinyl)-aminocyclohexane
hydrochloride the title compounds were prepared. The
isomers were separated via reverse phase HPLC, using Vydac
C1$ column (0.46 x 25cm), UV@214 nm, gradient of 5% to 700
(over 45 minutes) of 0.1% TFA/CH3CN in 0.1o TFA/H20.
Isomer 1: Cz4H3~N3O4 (MW=431.58) ; mass spectroscopy (MH+)
432. 1H NMR data as follows: 1H NMR (400 MHz, CD30D) 8 7.35-
7.28 (5H m), 8 5.81 (0.5H s), 8 5.73 (0.5H s), 8 4.36-4.32
(0.5H m), b 4.23 (1H q, J=6.8 Hz), 8 3.72 (1H m), 8 3.62-
3.55 (0.5H m), 8 2.75 (1.5H s), 8 2.73 (1.5H s), 8 2.47 (1H
dd, J=6.3, 13.1 Hz), ~ 2.33 (1H dd, J=6.3, 14.1 Hz), 8 1.99-
1.93 (1H m), b 1.80-0.62 (19H m).
Isomer 2 : C24H3~N3O4 (MW=431 . 58 ) ; mass spectroscopy (MH+)
432. 1H NMR data as follows: 1H NMR (400 MHz, CD30D)
7.35-7.29 (5H m), 8 5.83 (0.5H s), 8 5.75 (0.5H s), 8 4.38-
4.20 (1.5H m), b 3.73 (1H d, J=3.4 Hz), 8 3.60-3.54 (0.5H
m), b 2.76 (1.5H s), 8 2.73 (1.5H s), 8 2.46-2.29 (2H m), 8
2.10-1.95 (1H m), 8 1.80-0.61 (19H m).
PREPARATRION 4
Synthesis of (R/S)-N-Boc-a-methyl-(3-alanine.
Following General Method K and using D,L-3-
aminoisobutyric acid (Aldrich), the title compound was
prepared. C9H1~N04 (MW=203.24); mass spectroscopy (MH+) 204.
1H NMR data as follows: 1H NMR (400 MHz, CD30D) 8 3.21 (1H
dd, J=7.3, 13.6 Hz), 8 3.01 (1H dd, J=6.3, 13.1 Hz), 8 2.55
(1H q, J=6.8 Hz), 8 1.36 (9H s), 8 1.08 (3H d, J=7.3 Hz).
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T:'YTMDT.L' ~
Synthesis of N-methyl-N-(N'-(N"-((S)-3,5-difluorophenyl-a-
hydroxyacetyl)-(R/S)-a-methyl-(3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane
F
Following General Procedure J and using (R/S)-N-Boc-a-
methyl-(3-alanine and N-methyl-N-(L-phenylglycinyl)-
aminocyclohexane N-methyl-N-(N'-(N " -Boc-(R/S)-a-methyl-(3-
alaninyl)-L-phenylglycinyl)-aminocyclohexane was prepared.
C2qH3~N3O4 (MW=431 . 58 ) ; mass spectroscopy (MH+) 432 . Anal .
Calcd. for CZqH3~N3O4: C 66.79, H 8.64, N 9.74. Found: C
66.39, H 8.36, N 9.44.
Following General Procedure L, using no cooling bath,
and using N-methyl-N-(N'-((R/S)-N " -Boc-a-methyl-(3-
alaninyl)-L-phenylglycinyl)-aminocyclohexane, N-methyl-N-
(N°-((R/S)-a-methyl-(3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane hydrochloride was prepared. Cl9HZgN3O2
(MW=331.46); mass spectroscopy (MH+) 332. 1H NMR data as
follows: 1H NMR (400 MHz, CD30D) 8 7.40-7.33 (5H m), b 5.77
(0.5H s), 8 5.67 (0.5H s), 8 4.40-4.30 (1H m), b 3.62-3.53
(1H m), b 3.20-2.92 (2H m), 8 2.78 (1.5H s), 8 2.75 (1.5H
s), 8 1.90-0.60 (13H m).
Following General Procedure D using (S)-3,5-
difluoromandelic acid and N-methyl-N-(N'-((R/S)-a-methyl-(3-
alaninyl)-L-phenylglycinyl)-aminocyclohexane hydrochloride
the title compound was prepared as a mixture of
diastereomers . C2~H33FzN3O4 (MW=501 . 58 ) ; mass spectroscopy
(MH+) 502. 1H NMR data as follows: 1H NMR (400 MHz, CD30D)
7.38-7.30 (5H m), 8 7.10 (2H, d, J=8.3Hz), 8 6.83-6.78 (1H,
m), 8 5.81 (0.5H, s), 8 5.70 (0.5H, s), S 5.02-5.01 (1H, m),
8 4.39-4.36 (0.5H, m), 8 3.67-3.58 (0.5H, m), 8 3.33-3.26
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(2H, m), 8 2.79 (1.5H, s), b 2.73 (1.5H, s), 8 2.63-2.56
(1H, m), 8 1.87-0.81 (13H, m).
~~raNrnr.~ n
Synthesis of N-methyl-N-(N'-(N"-((S)-2-Hydroxy-3-
methylbutyrl)-(R)-a-methyl-(3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane and N-methyl-N-(N'-(N"-((S)-2-Hydroxy-3-
methylbutyrl)-(S)-CC-methyl-~3-alaninyl)-L-phenylglycinyl)-
aminocyclohexane
N \
N N
HO H
0
O O
Following General Procedure D using (S)-2-Hydroxy-3-
methylbutyric acid (Aldrich) and N-methyl-N-(N'-((R/S)-oc-
methyl-(3-alaninyl)-L-phenylglycinyl)-aminocyclohexane
hydrochloride the title compounds were prepared. The
isomers were separated via reverse phase HPLC, using Vydac
C18 column (0.46 x 25cm), UV@214 nm, gradient of 5o to 700
(over 45 minutes) of 0.1o TFA/CH3CN in 0.1o TFA/H20.
Isomer 1: C24H3~N3O4 (MW=431.58) ; mass spectroscopy (MH+) 432
1H NMR data as follows: 1H NMR (400 MHz, CD30D) 8 7.35-7.29
(5H m), 8 5.82 (0.5H s), 8 5.72 (0.5H s), b 4.29-4.22 (0.5H
m), 8 3.73-3.70 (1H m), cS 3.64-3.58 (0.5H m), b 3.34-3.15
(2H m), 8 2.77 (1.5H s), 8 2.73 (1.5H s), cS 2.65-2.62 (1H
m), 8 1.96-1.93 (1H m), b 1.80-0.76 (19H m).
Isomer 2: C24H3~N3O4 (MW=431.58) ; mass spectroscopy (MH+)
432. 1H NMR data as follows: 1H NMR (400 MHz, CD30D) 8 7.35-
7.29 (5H m), 8 5.81 (0.5H s), 8 5.73 (0.5H s), 8 4.29-4.22
(0.5H m), b 3.78-3.72 (1H m), 8 3.63-3.58 (0.5H m), 8 3.34-
3.24 (2H m), 8 2.77 (1.5H s), ~ 2.73 (1.5H s), b 2.64-2.61
(1H m), cS 2.03-1.99 (1H m), 8 1.81-0.76 (19H m).
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Example Bio-1
Cellular Screen for the Detection of Inhibitors of (3-Amyloid
Production
Numerous compounds of formula I above were assayed for
their ability to inhibit (3-amyloid production in a cell line
possessing the Swedish mutation. This screening assay
employed cells (K293 - human kidney cell line) which were
stably transfected with the gene for amyloid precursor
protein 751 (APP751) containing the double mutation
Lys651Met652 to Asn651Leu652 (APP751 numbering) in the
manner described in International Patent Application
Publication No. 94/105698 and Citron, et al., Nature,
360:672-674 (1992). This mutation is commonly called the
Swedish mutation and the cells, designated as "293 751 SWE",
were plated in Corning 96-well plates at 2-4 x 104 cells per
well in Dulbecco's minimal essential media (Sigma, St.
Louis, MO) plus 10% fetal bovine serum. Cell number is
important in order to achieve (3-amyloid ELISA results within
the linear range of the assay (--0.2 to 2.5 ng per mL).
Following overnight incubation at 37°-C in an incubator
equilibrated with 10% carbon dioxide, media were removed and
replaced with 200 ~L of a compound of formula I (drug)
containing media per well for a two hour pretreatment period
and cells were incubated as above. Drug stocks were
prepared in 100% dimethyl sulfoxide such that at the final
drug concentration used in the treatment, the concentration
of dimethyl sulfoxide did not exceed 0.5o and, in fact,
usually equaled 0.10.
At the end of the pretreatment period, the media were
again removed and replaced with fresh drug containing media
as above and cells were incubated for an additional two
hours. After treatment, plates were centrifuged in a
Beckman GPR at 1200 rpm for five minutes at room temperature
to pellet cellular debris from the conditioned media. From
each well, 100 ~,L of conditioned media or appropriate
dilutions thereof were transferred into an ELISA plate
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precoated with antibody 266 (P. Seubert, Nature (1992)
359:325-327) against amino acids 13-28 of ~3-amyloid peptide
as described in International Patent Application Publication
No. 94/105698 and stored at 4°-C overnight. An ELISA assay
employing labeled antibody 3D6 (P. Seubert, Nature (1992)
359:325-327) against amino acids 1-5 of (3-amyloid peptide
was run the next day to measure the amount of (3-amyloid
peptide produced.
Cytotoxic effects of the compounds were measured by a
modification of the method of Hansen, et a1.13. To the
cells remaining in the tissue culture plate was added 25 ~L
of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Sigma, St. Louis, MO) stock solution (5
mg/mL) to a final concentration of 1 mg/mL. Cells were
incubated at 37°-C for one hour, and cellular activity was
stopped by the addition of an equal volume of MTT lysis
buffer (20% w/v sodium dodecylsulfate in 500
dimethylformamide, pH 4.7). Complete extraction was
achieved by overnight shaking at room temperature. The
difference in the OD562~, and the OD6so,-u" was measured in a
Molecular Device's WmaX microplate reader as an indicator of
the cellular viability.
The results of the (3-amyloid peptide ELISA were fit to
a standard curve and expressed as ng/mL (3-amyloid peptide.
In order to normalize for cytotoxicity, these results were
divided by the MTT results and expressed as a percentage of
the results from a drug free control. The test compounds
were assayed for ~3-amyloid peptide production inhibition
activity in cells using this assay.
Example Bio-2
In Vivo Suppression of (3-Amyloid Release and/or Synthesis
This example illustrates how the compounds of this invention
could be tested for in vivo suppression of (3-amyloid release
and/or synthesis. For these experiments, 3 to 4 month old
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PDAPP mice are used (Games et al., (1995) Nature 373:523
527). Depending upon which compound is being tested, the
compound is usually formulated at between 1 and 10 mg/mL.
Because of the low solubility factors of the compounds, they
may be formulated with various vehicles, such as corn oil
(Safeway, South San Francisco, CA); 10o ethanol in corn oil;
2-hydroxypropyl-(3-cyclodextrin (Research Biochemicals
International, Natick MA); and carboxy-methyl-cellulose
(Sigma Chemical Co., St. Louis MO).
The mice are dosed subcutaneously with a 26 gauge
needle and 3 hours later the animals are euthanized via C02
narcosis and blood is taken by cardiac puncture using a 1 cc
25G 5/8" tuberculin syringe/needle coated with solution of
0.5 M EDTA, pH 8Ø The blood is placed in a Becton-
Dickinson vacutainer tube containing EDTA and spun down for
15 minutes at 1500 xg at 5-°C. The brains of the mice are
then removed and the cortex and hippocampus are dissected
out and placed on ice.
1. Brain Assay
To prepare hippocampal and cortical tissue for enzyme-
linked immunosorbent assays (ELISAs) each brain region is
homogenized in 10 volumes of ice cold guanidine buffer (5.0
M guanidine-HCl, 50 mM Tris-HC1, pH 8.0) using a Kontes
motorized pestle (Fisher, Pittsburgh PA). The homogenates
are gently rocked on a rotating platform for three to four
hours at room temperature and stored at -20°-C prior to
quantitation of ~3-amyloid.
The brain homogenates are diluted 1:10 with ice-cold
casein buffer (0.250 casein, phosphate buffered saline
(PBS), 0.05% sodium azide, 20 ~g/ml aprotinin, 5 mM EDTA, pH
8.0, 10 ~g/ml leupeptin), thereby reducing the final
concentration of guanidine to 0.5 M, before centrifugation
at 16,000 xg for 20 minutes at 4-°C. Samples are further
diluted, if necessary, to achieve an optimal range for the
ELISA measurements by the addition of casein buffer with 0.5
M guanidine hydrochloride added. The (3-amyloid standards
CA 02388750 2002-05-07
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(1-40 or 1-42 amino acids) were prepared such that the final
composition equaled 0.5 M guanidine in the presence of 0.10
bovine serum albumin (BSA).
The total (3-amyloid sandwich ELISA, quantitating both
(3-amyloid (aa 1-40) and (3-amyloid (aa 1-42) consists of two
monoclonal antibodies (mAb) to (3-amyloid. The capture
antibody, 266 (P. Seubert, Nature (1992) 359:325-327), is
specific to amino acids 13 - 28 of (3-amyloid. The antibody
3D6 (Johnson-Wood et al., PNAS USA (1997) 94:1550-1555),
which is specific to amino acids 1 - 5 of (3-amyloid, is
biotinylated and served as the reporter antibody in the
assay. The 3D6 biotinylation procedure employs the
manufacturer's (Pierce, Rockford IL) protocol for NHS-biotin
labeling of immunoglobulins except that 100 mM sodium
bicarbonate, pH 8.5 buffer is used. The 3D6 antibody does
not recognize secreted amyloid precursor protein (APP) or
full-length APP but detects only (3-amyloid species with an
amino terminal aspartic acid. The assay has a lower limit
of sensitivity of --50 pg/ml (11 pM) and shows no cross-
reactivity to the endogenous murine (3-amyloid peptide at
concentrations up to 1 ng/ml.
The configuration of the sandwich ELISA quantitating
the level of (3-amyloid (aa 1-42) employs the mAb 21F12
(Johnson-Wood et al., PNAS USA (1997) 94:1550-1555) (which
recognizes amino acids 33-42 of (3-amyloid) as the capture
antibody. Biotinylated 3D6 is also the reporter antibody in
this assay which has a lower limit of sensitivity of 125
pg/ml (28 pM).
The 266 and 21F12 capture mAbs are coated at 10 ~g/ml
into 96 well immunoassay plates (Costar, Cambridge MA)
overnight at room temperature. The plates are then
aspirated and blocked with 0.250 human serum albumin in PBS
buffer for at least 1 hour at room temperature, then stored
desiccated at 4°-C until use. The plates are dehydrated with
wash buffer (Tris-buffered saline, 0.050 Tween 20) prior to
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use. The samples and standards are added to the plates and
incubated overnight at 4-°C. The plates are washed ~ 3 times
with wash buffer between each step of the assay. The
biotinylated 3D6, diluted to 0.5 ~g/ml in casein incubation
buffer (0.25% casein, PBS, 0.050 Tween 20, pH 7.4) is
incubated in the well for 1 hour at room temperature.
Avidin-HRP (Vector, Burlingame CA) diluted 1:4000 in casein
incubation buffer is added to the wells for 1 hour at room
temperature. The colorimetric substrate, Slow TMB-ELISA
(Pierce, Cambridge MA), is added and allowed to react for 15
minutes, after which the enzymatic reaction is stopped with
addition of 2 N H2S04. Reaction product is quantified using
a Molecular Devices Vmax (Molecular Devices, Menlo Park CA)
measuring the difference in absorbance at 450 nm and 650 nm.
2. Blood Assay
The EDTA plasma is diluted 1:1 in specimen diluent (0.2 gm/1
sodium phosphate~H20 (monobasic), 2.16 gm/1 sodium
phosphate~7 H20 (dibasic), 0.5gm/1 thimerosal, 8.5 gm/1
sodium chloride, 0.5 ml Triton X-405, 6.0 g/1 globulin-free
bovine serum albumin; and water). The samples and standards
in specimen diluent are assayed using the total (3-amyloid
assay (266 capture/3D6 reporter) described above for the
brain assay except the specimen diluent was used instead of
the casein diluents described.
Formulations other than those described above can also
be used for oral delivery and intravenous delivery to a
mammal. For oral delivery, the compound can be mixed with
either 1000 corn oil or, alternatively, in a solution
containing 80% corn oil, 19.5% oleic acid and 0.50 labrafil.
The compound can be mixed with the above solutions in
concentrations ranging from 1 mg/mL to 10 mg/mL. The
compound in solution is preferably administered orally to
the mammal at a dose volume of 5 mL/kg of body weight. For
IV delivery, the compound is preferably mixed with a
solution of 3% ethanol, 3o solutol HS-15 and 94% saline.
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The compound is preferably mixed with the above solution in
concentrations ranging from 0.25 mg/mL to 5 mg/mL. The
compound in solution is preferably administered by IV to the
mammal at a dose volume of 2 mL/kg of body weight.
Formulations other than those described above can also
be used for oral delivery and intravenous delivery to a
mammal. For oral delivery, the compound can be mixed with
either 1000 corn oil or, alternatively, in a solution
containing 80o corn oil, 19.5% oleic acid and 0.50 labrafil.
The compound can be mixed with the above solutions in
concentrations ranging from 1 mg/mL to 10 mg/mL. The
compound in solution is preferably administered orally to
the mammal at a dose volume of 5 mL/kg of body weight. For
IV delivery, the compound is preferably mixed with a
solution of 3o ethanol, 3o solutol HS-15 and 94% saline.
The compound is preferably mixed with the above solution in
concentrations ranging from 0.25 mg/mL to 5 mg/mL. The
compound in solution is preferably administered by IV to the
mammal at a dose volume of 2 mL/kg of body weight.
When employed as pharmaceuticals, 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.
This invention also includes pharmaceutical
compositions which contain, as the active ingredient, one or
more of the compounds of formula I above associated with
pharmaceutically acceptable carriers. In making the
compositions of this invention, the active ingredient is
usually mixed with an excipient, diluted by an excipient or
enclosed within such a carrier which can be in the form of a
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capsule, sachet, paper or other container. When the
excipient serves as a diluent, it can be a solid, semi-
solid, or liquid material, which acts as a vehicle, carrier
or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges,
sachets, cachets, elixirs, suspensions, emulsions,
solutions, syrups, aerosols (as a solid or in a liquid
medium), ointments containing, for example, up to 10% by
weight of the active compound, soft and hard gelatin
capsules, suppositories, sterile injectable solutions, and
sterile packaged powders.
In preparing a formulation, it may be necessary to mill
the active
compound to provide the appropriate particle size prior
to combining with the other ingredients. If the active
compound is substantially insoluble, it ordinarily is milled
to a particle size of less than 200 mesh. If the active
compound is substantially water soluble, the particle size
is normally adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents
such as talc, magnesium stearate, and mineral oil; wetting
agents; emulsifying and suspending agents; preserving agents
such as methyl- and propylhydroxy-benzoates; sweetening
agents; and flavoring agents. The compositions of the
invention can be formulated so as to provide quick,
sustained or delayed release of the active ingredient after
administration to the patient by employing procedures known
in the art.
The compositions are preferably formulated in a unit
dosage form, each dosage containing from about 5 to about
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100 mg, more usually about 10 to about 30 mg, of the active
ingredient. The term "unit dosage forms" 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. Preferably, the
compound of formula I above is employed at no more than
about 20 weight percent of the pharmaceutical composition,
more preferably no more than about 15 weight percent, with
the balance being pharmaceutically inert carrier(s).
The active compound is effective over a wide dosage
range and is generally administered in a pharmaceutically
effective amount. It, will be understood, however, 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, the severity of the patient's symptoms,
and the like.
For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition
containing a homogeneous mixture of a compound of the
present invention. When referring to these preformulation
compositions as homogeneous, it is meant that the active
ingredient is dispersed evenly throughout the composition so
that the composition may be readily subdivided into equally
effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into
unit dosage forms of the type described above containing
from, for example, 0.1 to about 500 mg of the active
ingredient of the present invention.
The tablets or pills of the present invention may be
coated or otherwise compounded to provide a dosage form
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affording the advantage of prolonged action. For example,
the tablet or pill can comprise an inner dosage and an outer
dosage component, the latter being in the form of an
envelope over the former. The two components can separated
by enteric layer which serves to resist disintegration in
the stomach and permit the inner component to pass intact
into the duodenum or to be delayed in release. A variety of
materials can be used for such enteric layers or coatings,
such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac,
cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the
present invention may be incorporated for administration
orally or by injection include aqueous solutions suitably
flavored syrups, aqueous or oil suspensions, and flavored
emulsions with edible oils such as cottonseed oil, sesame
oil, coconut oil, or peanut oil, as well as elixirs and
similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable,
aqueous or organic solvents, or mixtures thereof, and
powders. The liquid or solid compositions may contain
suitable pharmaceutically acceptable excipients as described
supra. Preferably the compositions are administered by the
oral or nasal respiratory route for local or systemic
effect. Compositions in preferably pharmaceutically
acceptable solvents may be nebulized by use of inert gases.
Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached
to a face masks tent, or intermittent positive pressure
breathing machine. Solution, suspension, or powder
compositions may be administered, preferably orally or
nasally, from devices which deliver the formulation in an
appropriate manner.
The following formulation examples illustrate the
pharmaceutical compositions of the present invention.
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Formulation Example 1
Hard gelatin capsules containing the following ingredients
are prepared:
Quantity
Ingredient (mg/capsule)
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard
gelatin capsules in 340 mg quantities.
Formulation Example 2
A tablet formula is prepared using the ingredients below:
Quantity
Ingredient (mg/tablet)
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
The components are blended and compressed to form
tablets, each weighing 240 mg.
Formulation Example 3
A dry powder inhaler formulation is prepared containing the
following components:
Ingredient Weight o
Active Ingredient 5
Lactose 95
The active ingredient is mixed with the lactose and the
mixture is added to a dry powder inhaling appliance.
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Formulation Example 4
Tablets, each containing 30 mg of active ingredient,
are prepared as follows:
Quantity
Ingredient (mg/tablet)
Active Ingredient 30.0 mg
Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone
(as 10o solution in sterile water) 4.0 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1.0 mg
Total 120 mg
The active ingredient, starch and cellulose are passed
through a No. 20 mesh U.S. sieve and mixed thoroughly. The
solution of polyvinyl-pyrrolidone is mixed with the
resultant powders, which are then passed through a 16 mesh
U.S. sieve. The granules so produced are dried at 50°- to
60°-C and passed through a 16 mesh U.S. sieve. The sodium
carboxymethyl starch, magnesium stearate, and talc,
previously passed through a No. 30 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 Example 5
Capsules, each containing 40 mg of medicament are made as
follows:
Quantity
Ingredient (mg/capsule)
Active Ingredient 40.0 mg
Starch 109.0 mg
Magnesium stearate 1.0 mg
Total 150.0 mg
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The active ingredient, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled
into hard gelatin capsules in 150 mg quantities.
Formulation Example 6
Suppositories, each containing 25 mg of active ingredient
are made as follows:
Ingredient Amount
Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
The active ingredient is passed through a No. 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The
mixture is then poured into a suppository mold of nominal
2.0 g capacity and allowed to cool.
Formulation Example 7
Suspensions, each containing 50 mg of medicament
per 5.0 mL
dose are made as follows:
Ingredient Amount
Active Ingredient 50.0 mg
Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (110)
Microcrystalline cellulose (890) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v.
Purified water to 5.0 mL
The active ingredient, sucrose and xanthan
gum are blended,
passed through a No. 10 mesh U.S. sieve, and then mixed with
a previously made solution of the microcrystalline
cellulose
and sodium carboxymethyl cellulose in water. The sodium
benzoate, flavor, and color are diluted with some of the
water and added with stirring. Sufficient water
is then
added to produce the required volume.
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Formulation Example 8
Quantity
Ingredient (mg/capsule)
Active Ingredient 15.0 mg
Starch 407.0 mg
Magnesium stearate 3.0 mg
Total 425.0 mg
The active ingredient, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled
into hard gelatin capsules in 560 mg quantities.
Formulation Example 9
A subcutaneous formulation may be prepared as follows:
Ingredient Quantity
Active Ingredient 1.0 mg
corn oil 1 mL
(Depending on the solubility of the active ingredient in
corn oil, up to about 5.0 mg or more of the active
ingredient may be employed in this formulation, if desired).
Formulation Example 10
A topical formulation may be prepared as follows:
Ingredient Quantity
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 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.
Another preferred.formulation employed in the methods
of the present invention employs transdermal delivery
devices ("patches"). Such transdermal patches may be used
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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 agents is well known in the art.
See, e.g., U.S. Patent 5,023,252, issued June 11, 1991,
herein incorporated by reference. Such patches may be
constructed for continuous, pulsatile, or on demand delivery
of pharmaceutical agents.
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. Patent 5,011,472 which is herein
incorporated by reference.
Indirect techniques, which are generally preferred,
usually involve formulating the compositions to provide for
drug latentiation by the conversion of hydrophilic drugs
into lipid-soluble drugs. 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 which can transiently open
the blood-brain barrier.
Other suitable formulations for use in the present
invention can be found in Remington's Pharmaceutical
Sciences, Mace Publishing Company, Philadelphia, PA, 17th
ed. (1985).
The compounds and pharmaceutical compositions of the
invention are useful in inhibiting (3-amyloid peptide release
and/or its synthesis, and, accordingly, have utility in
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diagnosing and treating Alzheimer's disease in mammals
including humans.
As noted above, the compounds described herein are
suitable for use in a variety of drug delivery systems
described above. Additionally, in order to enhance the in
vivo serum half-life of the administered compound, the
compounds may be encapsulated, introduced into the lumen of
liposomes, prepared as a colloid, or other conventional
techniques may be employed which provide an extended serum
half-life of the compounds. A variety of methods are
available for preparing liposomes, as described in, e.g.,
Szoka, et al., U.S. Patent Nos. 4,235,871, 4,501,728 and
4,837,028 each of which is incorporated herein by reference.
The amount of compound administered to the patient will
.vary depending upon what is being administered, the purpose
of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration, and the
like. In therapeutic applications, compositions are
administered to a patient already suffering from Alzheimer's
disease in an amount sufficient to at least partially arrest
further onset of the symptoms of the disease and its
complications. An amount adequate to accomplish this is
defined as "therapeutically effective dose." Amounts
effective for this use will depend on the judgment of the
attending clinician depending upon factors such as the
degree or severity of Alzheimer's disease in the patient,
the age, weight and general condition of the patient, and
the like. Preferably, for use as therapeutics, the
compounds described herein are administered at dosages
ranging from about 1 to about 500 mg/kg/day.
In prophylactic applications, compositions are
administered to a patient at risk of developing Alzheimer's
disease (determined for example by genetic screening or
familial trait) in an amount sufficient to inhibit the onset
of symptoms of the disease. An amount adequate to
accomplish this is defined as "prophylactically effective
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dose." Amounts effective for this use will depend on the
judgment of the attending clinician depending upon factors
such as the age, weight and general condition of the
patient, and the like. Preferably, for use as
prophylactics, the compounds described herein are
administered at dosages ranging from about 1 to about 500
mg/kg/day.
As noted above, the compounds administered to a patient
are in the form of pharmaceutical compositions described
above. These compositions may be sterilized by conventional
sterilization techniques, or may be sterile filtered. The
resulting aqueous solutions may be packaged for use as is,
or lyophilized, the lyophilized preparation being combined
with a sterile aqueous carrier prior to administration. The
pH of the compound preparations typically will be between 3
and 11, more preferably from 5 to 9 and most preferably from
7 and 8. It will be understood that use of certain of the
foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
The compounds described herein are also suitable for
use in the administration of the compounds to a cell for
diagnostic and drug discovery purposes. Specifically, the
compounds may be used in the diagnosis of cells releasing
and/or synthesizing (3-amyloid peptide. In addition the
compounds described herein are useful for the measurement
and evaluation of the activity of other candidate drugs on
the inhibition of the cellular release and/or synthesis of
(3-amyloid peptide.
From the foregoing description, various modifications
and changes in the composition and method will occur to
those skilled in the art. All such modifications coming
within the scope of the appended claims are intended to be
included therein.
