Note: Descriptions are shown in the official language in which they were submitted.
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Methods For Treating Neurodegenerative Disorders
Using Asparlyl Protease Inhibitors
GOVERNMENT RIGHTS
This invention was made with Government support under Grant (Contract)
Nos. RO1 GM53696 and ROl GM50353 awarded by the National Institutes of Health.
The Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the most common form of both senile and
presenile dementia in the world and is recognized clinically as relentlessly
progressive
dementia that presents with increasing loss of memory, intellectual function
and
disturbances in speech (Merritt, 1979, A Textbook of Neurology, 6th edition,
484-489
Lea & Febiger, Philadelphia). The disease itself usually has a slow and
insidious
progress that affects both sexes equally, worldwide. It begins with mildly
inappropriate
behavior, uncritical statements, irritability, a tendency towards grandiosity,
euphoria and
deteriorating performance at work; it progresses through deterioration in
operational
judgement, loss of insight, depression and loss of recent memory; it ends in
severe
disorientation and confusion, apraxia of gait, generalized rigidity and
incontinence
(Gilroy and Meyer, 1979, Medical Neurology, 175-179 MacMillan Publishing Co.).
Alzheimer's disease afflicts an estimated 4 million human beings in the United
States
alone at a cost of 35 billion dollars a year (Hay and Ernst, Am. J. Public
Health,
77:1169-1175 ( 1987)) . It is found in 10 % of the population over the age of
65 and 47
of the population over the age of 85 (Evans, et al. , JAMA, 262:2551-2556 (
1989)) . In
addition, the disease is found at much lower levels in the younger age groups,
usually
beginning at about 30 years of age and even rarely in late childhood (Adams
and Victor,
Principles of Neurology, 401-407 ( 1977)) .
Proteases and, in particular, aspartyl proteases have been implicated in
diseases, such as Alzheimer's Disease, that are characterized by the
accumulation of
amyloid plaques. Amyloidogenic A(3 peptides (A~i) are the principle component
of the
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amyloid plaques that accumulate intracellularly and extracellularly in the
neuritic plaques
in the brain in AD. A/3 is a 4.5 kD protein, about 40-42 amino acids long,
that is
derived from the C-terminus of amyloid precursor protein (APP). APP is a
membrane-
spanning glycoprotein that, in the normal processing pathway, is cleaved
inside the A;~
protein to produce a-sAPP, a secreted form of APP. Formation of alpha a-sAPP
precludes formation of A~i. It has been proposed that A(3 accumulates by
virtue of
abnormal processing of APP, so that compounds that inhibit the activity of the
enzymes
responsible for A(3 production are desirable (see, e. g. , Wagner, et al. ,
Biotech. Report,
106-107 (1994/1995); and Selkoe, TINS, 16:403-409 (1993)).
In addition to the accumulation of amyloid plaques, neurons in AD brains
exhibit specific alterations in T, a family of phosphoproteins that bind
tubulin
(Weingarten, et al., Proc. Natl. Acad. Sci. USA, 72:1858-1862 (1975); and
Williams and
Detrich, Biochemistry, 18:2499-2503 (1979)), and stabilize microtubules (Goode
and
Feinstein, J. Cell Biol. , 124:769-782 ( 1994)) . In these brains, T proteins
adopt an
altered form and comprise the dominant component of abnormal cytosketal fibers
known
as paired helical filaments (PHFs) (see, Kosik, et al. , Proc. Natl. Acad.
Sci. USA,
83:4044-4088 (1986); Lee, et al., Science, 251:675-678 (1991); and Mann, et
al.,
Neuropathol. Appl. Neurobiol., 13:123-139 (1987)). Molecular dissection of
PHFs has
revealed two specific alterations in T. First, PHF-T proteins maintain an
excessively
phosphorylated state throughout postmortem intervals (Matsuo, et al. , Neuron,
13 : 989-
1002 (1994)). Second, after treatment of PHFs with reducing agents and
detergents, the
remaining filaments contain truncated forms of T (Nieto, et al. , Biochem.
Biophys. Res.
Commun. , 154:660-667 (1988); Nieto, et al. , J. Neurosci. , 37:163-170
(1990); and
Wischik, et al., Proc. Natl. Acad. Sci. USA, 85:4506-4510 (1988)). These
results
suggest that modifications in the posttranslational processing of T contribute
to the
formation of PHFs. It has been proposed that z-fragments accumulate by viriure
of
abnormal processing of T by proteases (see, Bednarski and Lynch, J. Neurochem.
,
67(5):1845-1855 (1996)). As such, compounds that inhibit the acitivity of the
enzymes
responsible for T-fragment production are desirable.
Because proteases are implicated in Alzheimer's Disease and in numerous
other disorders, there remains a need in the art for the development of potent
and
specific inhibitors of these enzymes. Quite surprisingly, the present
invention fulfills this
and other need.
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SUl~fil~iARY O~' THE I~TVENTIOl~i
The present invention relates to (i) non-peptide aspartyl protease inhibitors;
(ii) methods for modulating the processing of an amyloid precursor protein
(APP); (iii)
methods for modulating the processing of a tau protein (T-protein); and (iv)
methods for
treating neurodegenerative diseases.
In one aspect, the present invention provides a method for modulating the
processing of an amyloid precursor protein (APP), the method comprising
contacting a
composition containing the APP with an aspartyl protease inhibitor having the
general
formula:
H
R3 11 N R2
O O
Rs
(I)
In Formula I, R,, RZ and R3 are members independently selected from the
group consisting of alkyl, substituted alkyl, aryl, substituted aryl,
arylalkyl, substituted
arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted
heteroaryl,
heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted
heterocycles,
heterocyclicalkyl and substituted heterocyclicalkyl.
In Formula I, RS and R6 are independently selected from the group
consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted
aryl, arylalkyl,
substituted arylalkyl, aryloxyalkyl and substituted aryloxyalkyl. In an
alternative
embodiment, RS and R6 and the carbons to which they are bound, join to form an
optionally substituted 9- or 10-ring atom carbocyclic or heterocyclic fused
ring system.
Typical 9- or 10-atom fused ring systems include, but are not limited to,
napthalyl, 1,3-
benzodioxolyl, 2,3-benzofuranyl, 1,4-benzodioxanyl, benzimidazoyl,
benzothiazolyl etc.
Within the scope of the above Formula I, certain embodiments are
preferred. In Formula I, one preferred embodiment is that in which Rl is a
functional
group including, but not limited to, substituted arylalkyl, substituted aryl,
substituted
alkyl and substituted heterocyclic groups. Examples of such functional groups
include,
but are not limited to, the following:
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CZ
i I ~- i I y-. ~ I y- .
o w ; ~ ~ , ~ ~ >
,I NH-. ,I ~- Iw
H3C \ : ~ Me
Another preferred embodiment is that in which RZ is a functional group
including, but not limited to, substituted alkyl, heterocyclic and substituted
heterocyclic
groups. Examples of such functional groups include, but are not limited to,
the
following:
Cl HC-
/O
/ N~~- / \ / \
w \ . ; w
0
0
/ ~ ~C~-' HN H3C-N
N
\ ; ;
O O O
C1 C1
C1 / ~- / O~~- /
Cl \ a Cl \ Cl ~ \ Cl
In one embodiment, R2 is a functional group other than a nitrogen-bonded
cyclic a-amino
acid or ester thereof.
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Also preferred is the embodiment in which R3 is a functional group
including, but not limited to, substituted alkyl and substituted aryl groups.
E;camples of
such functional groups include, but are not limited to, the following:
Cl HC/
/ I ~ / O~Cf~ / / /
\ Cl . Cl \ CI , \ \ ~ Me0 \ Cl
O OMc
H3 / O.~ Cl / O.-,/ H3 / O~~ H3 /
\ ( \ I .\
C1 ~ ~~ '
/
\ o
\ I \ I \ I \
o-~-; ~_~-, ~o ~ , ;
c~
/ o rr~cHz- / o~~- ~ / oy-
\ ~, \ / ' Q
sr o
0
~ : and 'O y
Me0
OMs
5 In another preferred embodiment, R5 and R6 and'the carbons to which they
are bound join to form an optionally substituted napthalene ring. In other
preferred
embodiments, RS and R6 are both hydrogen or RS is hydrogen and R6 is a meta or
para
substituent.
In a particularly preferred embodiment, the aspartyl protease inhibitor is
selected from the group consisting of:
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o--~
0
c~ / o
/ I H N" CH3
N~N
O
IOI ~ O
I / and
o--~
0
(
ci ~ o
/ I H N CH3
H
N~N
N
IIH
0 ~ O
I / .
The modulation of APP can be demonstrated in a variety of ways. For
example, aspartyl protease inhibitors can be evaluated for the ability to
modulate
generation of A/3 or a-sAPP. In one preferred embodiment, the formation of A(3
is
decreased compared to the amount formed in the absence of the aspartyl
protease
inhibitor. In another preferred embodiment, formation of a-sAPP is increased
compared
to the amount formed in the absence of the asparty protease inhibitor. In one
embodiment, the composition is a body fluid. In a preferred embodiment, the
body fluid
is cerebral spinal fluid (CSF).
In another aspect, the present invention provides a method for modulating
the processing of a tau-protein (T-protein), the method comprising contacting
a
composition containing the T-protein with an aspartyl protease inhibitor of
Formula I.
The modulation of z-protein can be demonstrated in a variety of ways. For
example,
aspartyl protease inhibitors can be evaluated for the ability to modulate
generation of T-
fragments. In one preferred embodiment, the formation of T-fragments is
decreased
compared to the amount formed in the absence of the aspartyl protease
inhibitor. In one
embodiment, the composition is a body fluid. In a preferred embodiment, the
body fluid
is cerebral spinal fluid (CSF)
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In yet another aspect, the present invention provides a method of treating a
neurodegenerative disorder, the method comprising: administering to a mammal a
therapeutically effective amount of an aspartyl protease inhibitor of Formula
I and a
pharmaceutically acceptable carrier or excipient. In one embodiment, the
neurodegenerative disorder is characterized by the accumulation of amyloid
plaques. In
another embodiment, the neurodegenerative disorder is characterized by the
accumulation
of z-fragments. As such, the aspartyl protease inhibitors of the present
invention can be
used to treat all amyloid-pathology related diseases and all tau pathology-
related diseases.
Examples of such neurodegenerative diseases include, but are not limited to,
the
following: Alzheimer's disease, Parkinson's disease, cognition deficits, Downs
Syndrome, cerebral hemorrhage with amyloidosis, dementia (e. g. , dementia
pugilistica)
and head trauma.
Other features, objects and advantages of the invention and its preferred
embodiments will become apparent from the detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrate isostere-based inhibitor design.
FIG. 2 illustrates components employed to prepare the libraries targeting
cathepsin D. The same disconnections provide scaffold 2. Isocyanates and
sulfonyl
chlorides, which can be used to incorporate R2 and R3, provide ureas and
sulfonamides,
respectively.
FIG. 3 illustrates the used of BUILDERopt in designing the combinatorial
library: (a) Modeling the Scaffold. Coordinates and Pl-P3 conformations of the
pepstatin
inhibitor were used as the starting geometry for hydroxyethylamine scaffold.
Methyl
groups were placed at each of the scaffold's Rl-R4 positions. (b) Scaffold
Conformation.
A conformational search about the three torsion angles of the scaffold yielded
4
conformational families. A benzyl sidechain (Bn) was added to each of these
families at
the R4 position. (c) Evaluating library components. The program
BUILDERopt performed a limited conformational search on all possible
components at
each variable position (Rl-R3) on each family, and scored the components by
their
potential interaction with cathepsin D. The top scoring candidates for each
family were
merged.
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FIGS. 4A-4C illustrate the components used to prepare the Directed
Library. Directed library components are labeled with a letter code. EHA is
defined as
R,=E; R2=H; and R3=A.
FIGS. 5A-SC illustrates the components used to prepare the Diverse
Library. Diverse library components are labeled by lower case letter code as
for the
directed library. In FIG. SA, the t-butyl ester of Rl = i was used in the
coupling
reaction. In FIG. 5C, the Boc protected amine of R3 = d was used in the
coupling
reaction. These protecting groups are removed during TFA:H20 cleavage.
FIGS. 6A-6C illustrates the components in each of the clusters (see
Experimental Design) that contained the most active sidechains, Rl. = E, F; R2
= F, H;
R3 = A, D. J. Thirty-nine compounds incorporating these sidechains were
synthesized
on resin as described previously, EFD, EHD, FFD, FHD, KFD, KHD, LFD, LHD,
MFD, MHD, NFD, NHD, OFD, OHD, PFD, PHD, QFD, QHD, RFD, RHD, SFD,
SHD, TFD, THD, UFD, UHD, VFD, VHD, EHA, EHJ, EHK, EHL, EHM, EHN,
EHO, EHP, EHQ, EHR, EHS. The compounds were assayed at 333 nM, 100 nM and
33 nM in high-throughput screening. The most active compounds were synthesized
on
large scale and the K; values were determined (Table 3).
FIG. 7 illustrates structural diversity being introduced via Grignard
addition to solid support-bound « - alkoxy pyrrolidine amide.
FIG. 8 illustrates synthesis of solid phase aspartyl protease inhibitor
synthesis.
FIG. 9 illustrates components to generate library diversity in a 204
compound library.
FIG. 10 illustrates that the cathepsin D inhibitor, i. e. , CELS-172, by
itself, did not detectably change the concentration of either the tau fragment
or the APP
fragment, but it did block most, if not all, of the increases in the tau and
APP fragments
produced by ZDAP.
FIG. 11 illustrates that like CELS-172, the cathepsin D inhibitor EA-1, by
itself, did not detectably change the concentration of either the
phosphorylated tans
fragment, but it exhibited a much higher blocking effect than CELS-172.
FIG. 12 illustrates the structures of three inhibitors used in the
experiments set forth in Example III, all of which have molecular weights of
650-800
Daltons and Ki's for cathepsin D of between 1-15 nM.
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FIG. 13 illustrates the morphological and physiological effects of cathepsin
inhibitors. Semi-thin sections through the cell body layer of field CA1 of
cultured
hippocampal slices given no treatment (A), a 6-day exposure to an inhibitor of
cathepsins
B and L (B), or a-6 day exposure to an inhibitor of cathepsin D (C). Note the
presence
in (B) of large numbers of small, dense bodies that in some cases are
clustered into
torpedo shaped expansions (arrows) . These effects are sufficiently robust to
be easily
detected by naive observers. The proliferation and expansions (meganeurites)
are not
found in (C) and there are no obvious morphological differences between this
slice and
the control. Synaptic responses recorded in field CA1 after 6 days of
treatment with the
cathepsin D inhibitor (EA-1) are shown in (D). EPSCs collected with whole cell
clamp
from the pyramidal cell bodies (i) have a rapid onset after stimulation
(arrow) of the
Schaffer-commissural fibers, are well developed, and have a waveform typically
seen in
slices tested at room temperature. Field EPSPs in the apical dendrites (ii)
are recorded
without spikes or afterpotentials. As in previous reports, the amplitude of
extracellular
monosynaptic responses is smaller in cultured slices than is the case for
acute slices.
IPSCs were well developed in treated slices (iii) as can be seen in the
Schaffer-
commissural responses collected with the membrane potential set to -SOmV. A
negative
going EPSC recorded at -70mV is also shown.
FIG. 14 illustrates the effects of cathepsin inhibitors on concentrations of
phosphorylated tau fragments. Cultured slices were incubated for 6 days with
an inhibitor
of cathepsins B and L (ZPAD), an inhibitor of cathepsin D, or both. Western
blots were
then prepared from slice homogenates using an antibody against the
hyperphosphorylated
tau found in human neurofibrillary tangles. The top panels show immunostaining
in the
25-35 kDa region of the blots. ZPAD increased the concentrations of
phosphorylated
bands in this region over the levels found in controls. The cathepsin D
inhibitors CEL-5
(A, lane 4) and EA-1 (B, lane 3) had no detectable effect on concentrations of
the
peptide. Slices treated with ZPAD and a cathepsin D inhibitor (A, lanes 5 and
6;B, lane
~ tended to have greater concentrations than controls (A and B, lane 1) but
clearly not to
the level found with ZPAD alone (A, lanes 2 and 3;B, lane 2). The bottom
panels
summarize analysis of AT8 staining from five separate experiments with all
values
expressed as percent of yoked controls. *, P < 0.05; **, P < 0.01; error bars,
standard
errors. C. Western blots showing the native tau proteins probed by tau 1 and
AT8
antibodies. Lane 1, control; lane 2, incubated with ZPAD; Lane 3, incubated
with EA-1;
Lane 4, incubated with EA-1 and ZPAD.
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FIG. 15 illustrates the time course and dose dependency for suppression of
phosphorylated tau fragments by a cathepsin D inhibitor. (A) . Cultured
hippocampal
slices were incubated for 2, 4, or 6 days with the cathepsin B/L inhibitor-
ZPAD, the
cathepsin D inhibitor-EA-1, or both. Western blot analyses for phosphorylated
tau
5 fragments were carried out at the end of the incubation with densitometric
values
expressed as percent of concentrations in yoked controls. ZPAD induced
increases were
detectable after 48 hrs and continued to grow thereafter. The cathepsin D
inhibitor had
no apparent effect but blocked the increases produced by ZPAD at all time
points. (B).
Slices were incubated with ZPAD, EA-1, or ZPAD plus the indicated
concentrations of
10 EA-1 for six days. The cathepsin D inhibitor had no detectable effects on
concentrations
of phosphorylated tau fragments at the concentrations tested. A dose of 1 ~.M
caused a
sizeable decrease in the effect of ZPAD while 5 ~,M completely suppressed it.
*,
P < 0.05; **, P < 0.01; error bars, standard errors.
FIG. 16 illustrates the effects of cathepsin inhibitors on tau and cathepsin
D isoforms. Slices were incubated with ZPAD, EA-1, or both for 6 days after
which
Western blots were used to assess the concentrations of the target proteins
with tau 1
antibodies (A), or anti-cathepsin D antisera (B). Densitometeic values were
expressed as
percent change from the concentrations in yoked control slices. (A). ZPAD
caused
sizeable reductions in four unphosphorylated isoforms of native tau; EA-1 was
without
effect itself and did not block the changes produced by ZPAD. ZPAD also
generated a
large increase in a 29 kDa tau fragment; this was completely blocked by EA-1.
(B) .
ZPAD resulted in modest increases in procathepsin D and larger increases in
the active,
heavy chain variant of the protease. EA-1 suppressed the second of these
effects.
DETAILED DESCRIPTION OF THE INVNETION
AND PREFERRED EMBODIMENTS
The present invention relates to (i) non-peptide aspartyl protease inhibitors;
(ii) methods for modulating the processing of an amyloid precursor protein
(APP); (iii)
methods for modulating the processing of a tau protein (T-protein); and (iv)
methods for
treating neurodegenerative diseases.
A. Definitions
The term "independently selected" is used herein to indicate that the three
R groups, i. e. , R1, RZ and R3, can be identical or different (e.g. , Rl, RZ
and R3 may all
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be substituted alkyls or R1 and RZ may be a substituted alkyl and R3 may be an
aryl,
etc. ) .
The term "alkyl" is used herein to refer to a branched or unbranched,
saturated or unsaturated, monovalent hydrocarbon radical having from 1-12
carbons and
preferably, from 1-6 carbons. When the alkyl group has from 1-6 carbon atoms,
it is
referred to as a "lower alkyl. " Suitable alkyl radicals include, for example,
methyl,
ethyl, n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl, i-butyl
(or 2-
methylpropyl), etc. As used herein, the term encompasses "substituted alkyls.
"
"Substituted alkyl" refers to alkyl as just described including one or more
functional groups such as lower alkyl, aryl, substituted aryl, acyl, halogen
(i.e.,
alkylhalos, e. g. , CF3), hydroxy, amino, alkoxy, alkylamino, acylamino,
thioamido,
acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and
unsaturated
cyclic hydrocarbons, heterocycles and the like. These groups may be attached
to any
carbon of the alkyl moiety. Additionally, these groups may be pendent from, or
integral
to, the alkyl chain.
The term "aryl" is used herein to refer to an aromatic substituent which
may be a single aromatic ring or multiple aromatic rings which are fused
together, linked
covalently, or linked to a common group such as a methylene or ethylene
moiety. The
common linking group may also be a carbonyl as in benzophenone. The aromatic
rings)
may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among
others. The term "aryl" encompasses "arylalkyl."
The term "arylalkyl" is used herein to refer to a subset of "aryl" in which
the aryl group is attached to the nucleus shown in Formula 1 by an alkyl group
as
defined herein.
"Substituted aryl" refers to aryl as just described including one or more
functional groups such as lower alkyl, acyl, halogen, alkylhalos (e. g. ,
CF3), hydroxy,
amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto and both
saturated
and unsaturated cyclic hydrocarbons, optionally substituted with one or more
heteroatoms, which are fused to the aromatic ring(s), linked covalently or
linked to a
common group such as a methylene or ethylene moiety. The linking group may
also be
a carbonyl such as in cyclohexyl phenyl ketone. The term "substituted aryl"
encompasses "substituted arylalkyl. "
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"Substituted arylalkyl" defines a subset of "substituted aryl" wherein the
substituted aryl group is attached to the nucleus shown in Formula 1 by an
alkyl group as
defined herein.
The term "acyl" is used to describe a ketone substituent, -C(O)R, where
R is alkyl or substituted alkyl, aryl or substituted aryl as defined herein.
The term "halogen" is used herein to refer to fluorine, bromine, chlorine
and iodine atoms.
The term "hydroxy" is used herein to refer to the group -OH.
The term "amino" is used to describe primary amines, R-NH2.
The term "alkoxy" is used herein to refer to the -OR group, where R is a
lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or
substituted
arylalkyl wherein the alkyl, aryl, substituted aryl, arylalkyl and substituted
arylalkyl
groups are as described herein. Suitable alkoxy radicals include, for example,
methoxy,
ethoxy, phenoxy, substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.
The term "alkylamino" denotes secondary and tertiary amines wherein the
alkyl groups may be either the same or different and are as described herein
for "alkyl
groups. "
As used herein, the term "acylamino" describes substituents of the general
formula RC(O)NR', wherein R' is a lower alkyl group and R represents the
nucleus
shown in Formula 1 or an alkyl group, as defined herein, attached to the
nucleus.
The term "acyloxy" is used herein to describe an organic radical derived
from an organic acid by the removal of the acidic hydrogen. Simple acyloxy
groups
include, for example, acetoxy, and higher homologues derived from carboxylic
acids
such as ethanoic, propanoic, butanoic, etc. The acyloxy moiety may be oriented
as
either a forward or reverse ester (i.e., RC(O)OR' or R'OC(O)R, respectively,
wherein R
comprises the portion of the ester attached either directly or through an
intermediate
hydrocarbon chain to the nucleus shown in claim 1).
As used herein, the term "aryloxy" denotes aromatic groups which are
linked to the nucleus shown in FIG. 1 directly through an oxygen atom. This
term
encompasses "substituted aryloxy" moieties in which the aromatic group is
substituted as
described above for "substituted aryl. "
As used herein "aryloxyalkyl" defines aromatic groups attached, through
an oxygen atom to an alkyl group, as defined herein. The alkyl group is
attached to the
nucleus shown in FIG. 1. The term "aryloxyalkyl" encompasses "substituted
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aryloxyalkyl" moieties in which the aromatic group is substituted as described
for
"substituted aryl. "
As used herein, the term "mercapto" defines moieties of the general
structure R-S-R' wherein R and R' are the same or different and are alkyl,
aryl or
heterocyclic as described herein.
The term "saturated cyclic hydrocarbon" denotes groups such as the
cyclopropyl, cyclobutyl, cyclopentyl, ete. , and substituted analogues of
these structures.
These cyclic hydrocarbons can be single- or mufti-ring structures.
The term "unsaturated cyclic hydrocarbon" is used to describe a
monovalent non-aromatic group with at least one double bond, such as
cyclopentene,
cyclohexene, etc. and substituted analogues thereof. These cyclic hydrocarbons
can be
single- or mufti-ring structures.
The term "heteroaryl" as used herein refers to aromatic rings in which one
or more carbon atoms of the aromatic rings) are substituted by a heteroatom
such as
nitrogen, oxygen or sulfur. Heteroaryl refers to structures which may be a
single
aromatic ring, multiple aromatic ring(s), or one or more aromatic rings
coupled to one or
more non-aromatic ring(s). In structures having multiple rings, the rings
can'be fused
together, linked covalently, or linked to a common group such as a methylene
or
ethylene moiety. The common linking group may also be a carbonyl as in phenyl
pyridyl ketone. As used herein, rings such as thiophene, pyridine, isoxazole,
phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues of these
rings are
defined by the term "heteroaryl. "
"Heteroarylalkyl" defines a subset of "heteroaryl" wherein an alkyl group,
as defined herein, links the heteroaryl group to the nucleus shown in FIG. 1.
"Substituted heteroaryl" refers to heteroaryl as just described wherein the
heteroaryl nucleus is substituted with one or more functional groups such as
lower alkyl,
acyl, halogen, alkylhalos (e.g., CF3), hydroxy, amino, alkoxy, alkylamino,
acylamino,
acyloxy, mercapto, etc. Thus, substituted analogues of heteroaromatic rings
such as
thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or
benzo-fused
analogues of these rings are defined by the term "substituted heteroaryl. "
"Substituted heteroarylalkyl" refers to a subset of "substituted heteroaryl"
as described above in which an alkyl group, as defined herein, links the
heteroaryl group
to the nucleus shown in FIG. 1.
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The term "heterocyclic" is used herein to describe a monovalent saturated
or unsaturated non-aromatic group having a single ring or multiple condensed
rings from
1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or
oxygen
within the ring. Such heterocycles are, for example, tetrahydrofuran,
morpholine,
piperidine, pyrrolidine, etc.
The term "substituted heterocyclic" as used herein describes a subset of
"heterocyclic" wherein the heterocycle nucleus is substituted with one or more
functional
groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy,
amino,
alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
The term "heterocyclicalkyl" defines a subset of "heterocyclic" wherein an
alkyl group, as defined herein, links the heterocyclic group to the nucleus
shown in FIG.
1.
The term "optionally substituted napthylene ring" describes a naphthalene
ring which may be unsubstituted or may be substituted with one or more
functional
groups including lower alkyl, halogen, acyl, hydroxy, amino, alkoxy,
alkylamino,
acylamino, acyloxy or aryl.
The term "substituted heterocyclicalkyl" defines a subset of "heterocyclic
alkyl" wherein the heterocyclic nucleus is substituted with one or more
functional groups
such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy, amino,
alkoxy,
alkylamino, acylamino, acyloxy, mercapto, etc.
The term "amyloid precursor protein" or "APP" is used herein to refer to
the progenitor of deposited amyloidogenic A(3 peptides (A~i) found in senile
plaques in
patients with diseases, such as Alzheimer's disease (AD), that are
characterized by such
deposition. a-sAPP is an alternative cleavage product of APP; its formation
precludes
formation of A(3.
The term "contacting" is used herein interchangeably with the following:
combined with, added to, mixed with, passed over, incubated with, flowed over,
etc.
Moreover, the aspartyl protease inhibitors of present invention can be
"administered" by
any conventional method such as, for example, parenteral, oral, topical and
inhalation
routes as described herein.
"An amount sufficient" or "an effective amount" is that amount of a given
aspartyl protease inhibitor which exhibits the binding/inhibitory activity of
interest or,
which provides either a subjective relief of a symptoms) or an objectively
identifiable
improvement as noted by the clinician or other qualified observer.
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B. Non peptide Aspartyl Protease Inhzbztors
The present invention relates to the identification of a number of
small-molecule compounds which are capable of binding to and inhibiting
aspartyl
proteases and, in particular, cathepsin D employing a combined combinatorial
library
5 (see, e.g., Thompson, et al.; Chemical Reviews, 96, 55~-600 (1996)) and
structure based
design approach (see, e.g., Kuntz, LD., Science, 257, 1078-1082 (1992)). The
libraries
of potential aspartyl protease inhibitors (e.g., cathepsin D inhibitors) were
based upon
the display of functionality about the hydroxyethylamine scaffold illustrated
in FIG. 1.
For the initial libraries, the Pl sidechain (R4) was held constant as a benzyl
substituent
10 based upon X-ray crystallographic data of cathepsin D complexed with the
peptide-based
natural product pepstatin as reported by Erickson (Baldwin, et al., Proc.
Natl. Acad. Sci.
USA, 90, 6796-6800 (1993)). As illustrated in FIG. 2, diversity was introduced
at three
positions: a primary amine introduced the Rl substituent, and acylating agents
serve to
introduce the RZ and R3 substituents. Once prepared, the libraries were
screened to
15 identify compounds capable of binding to and inhibiting aspartyl proteases
and, in
particular, cathepsin D. Thereafter, a second generation library was prepared
in an
effort to further explore variants of the most active compounds. Thus, by
combining a
structure-based design and a combinatorial library approach, non-peptidic
compounds
capable of inhibiting aspartyl proteases and, in particular, cathepsin D have
now been
identified.
Accordingly, in one embodiment, the present invention provides
compounds having the general formula:
OH Rl
H
R3\ /N N\ /R2
I~OI ~ I~IO
R6W
(I)
In Formula I, Rl, RZ and R3 are members independently selected from the
group consisting of alkyl, substituted alkyl, aryl, substituted aryl,
arylalkyl, substituted
arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted
heteroaryl,
heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted
heterocycles,
heterocyclicalkyl and substituted heterocyclicalkyl.
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16
In Formula I, RS and R6 are independently selected from the group
consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted
aryl, arylalkyl,
substituted arylalkyl, aryloxyalkyl and substituted aryloxyalkyl. In an
alternative
embodiment, R5 and R6 and the carbons to which they are bound, join to form an
~ optionally substituted 9- or 10-ring atom carbocyclic or heterocyclic fused
ring system.
Typical 9- or 10-atom fused ring systems include, but are not limited to,
napthalyl, 1,3-
benzodioxolyl, 2,3-benzofuranyl, 1,4-benzodioxanyl, benzimidazoyl,
benzothiazolyl etc.
Within the scope of the above Formula I, certain embodiments are
preferred. In Formula I, one preferred embodiment is that in which Rl is a
functional
group including, but not limited to, substituted arylalkyl, substituted aryl,
substituted
alkyl and substituted heterocyclic groups. Examples of such functional groups
include,
but are not limited to, the following:
a
~~k / ~ ~~ / ~ ~~
o \ : ~ \ . ~ \
/ I rrH
\ . H3C \ : and Me /
Another preferred embodiment is that in which R2 is a functional group
including, but not limited to, substituted alkyl, heterocyclic and substituted
heterocyclic
groups. Examples of such functional groups include, but are not limited to,
the
following:
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17
C1 tiC-
O
/ ~ \
/ \ / ~~
> ;
0
0
- HN H3C-N
N
> ;
O
O O
C1 C1
~- / O~~-
Cl \ ~ Cl \ Cl a \ Cl
~z-
O
In one embodiment, R2 is a functional group other than a nitrogen-bonded
cyclic a-amino
acid or ester thereof.
Also preferred is the embodiment in which R3 is a functional group
including, but not limited to, substituted alkyl and substituted aryl groups.
Examples of
such functional groups include, but are not limited to, the following:
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18
CI HC
/ I ~ / O~(~ / / /
Cl . Cl ~ Cl . ~ ~ . Me0 ~ Cl
O OMe
/ O'~ H3 / O\~ ~ /
CI > CF3~ '
/)
0
'o-c~- , o-c~-, ~-cx~-, H9c v ' ;
c~
o rr~c~- / °y - ~ / o
/ ~ ~ ~
. , ,
c~t ~ - c~t
0
- o /
v
Me0 \ ~ ~ / : and 'O
\O
OMs
In another preferred embodiment, RS and R6 and the carbons to which they
are bound join to form an optionally substituted napthalene ring. In other
preferred
embodiments, RS and R6 are both hydrogen or RS is hydrogen and R6 is a meta or
para
substituent.
In Formula I, the benzyl ring may be replaced by the substituent R4 (see
below) . In this embodiment, R4 can be a member selected from the group
consisting of
alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl,
aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted heteroaryl,
heteroarylalkyl,
substituted heteroarylalkyl, heterocycles, substituted heterocycles,
heterocyclicalkyl and
substituted heterocyclicalkyl.
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19
H OH Rl
R3 N~N Rz
O R4 O
It will be readily apparent to those of skill in the art that depending on the
substituents, the compounds of Formula I can be a racemic mixture (mixtures of
diastereomers or enantiomers) or as stereochemically distinct compounds.
However, in a
preferred embodiment, the compounds of the present invention have the
following
stereochemistry:
H OH Rl
R3 N ~~ N R2
O ~ O
R5
R6 /
Formula I
Tables I and II set forth compounds in accordance with the present
invention that are particularly preferred. The compounds in Table I and
throughout this
specification are often referred to by code numbers, which are used for
convenience
only, and are strictly arbitrary for purposes of this invention.
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Table Z Exemplar Aspartyl Protease Inhibitors
Compound Protease Binding Compounds
Code No. Formula
EAA
o--~
0
I
/
0
H off c1 ~ c~
O N N N O I /
O ~ O Q
i I/
EFA
0
I
/
o a
o H ox
N N I / Q
0
I
/
EHA
o--~
0
I~
/
0 0
g OH
O~N~N~N~N
'' \
O \ O O
I/
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21
FAA
a
I
/ a
0
H OH a \ a
O N N N O I /
0 \ O G1
I/
I
i
FFA
a
I\
/ a
o a
O H OH I \
N~N a
O O
I
FHA
a
I\
/ a
0 0
H OH
N N N N I
O \ 0 O
I /
EHB
o-~
0
I
/
a o
H OH
i
/ / N~N~N I
a O ~ O O
I I
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22
EFD
0
I
a
H off I \ a
~O~N~N~~~~a
a IoI ~ IIo
I /
EHD
0
I
a
a o
g OH
O tt N v v N n - N / I
a O ~ O O
I/
EEF
0
I
/
~N / I a H OH ~ I \ O\
~N~N \ / O/
O ~ O
I /
EHF
o-~
0
\
I
/
/ a o
g OH
\ I N N N I
O I \ O O
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23
FHF
a
I
/ a
o~ / a o
I H OH
~N~N~N ~
jIl O I \ O O
EFH
o-~
0
I
/
0
/ \ H off I \
N~N~N~~~~
O O ~ IIO
I/
EHH
/
/ \ 0 0
g OA
N~N~N~N
O
O I \ O O
FFH
a
\.
I
/ a
/ \ ° a
H off I \
N~N N~~~Q
O IOI ~ O
I/
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FAH
a
I
/ a
/ \ ° ox a \ a
- I N~N~N~O I /
0 0 ~ o a
I /
EFI
o--\
0
I/
~--o
° ~ H OH ~ a
I / N\/uN ~ / a
0 ~ O
I/
°~\
O
I
/
EHI °
0 0
I' w1 H ox
\C~N~N~N
'' \
O ~ O °
I/
EAJ
o--\
0
I
oMa /
Mao / I Q H Ox CI I ~ a
\ N~N~O /
O ~ O a
I /
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EFJ
o--~
' o
I
oM~ /
Me0 / I a OH I ~ a
N~N~~I
a
o ~ o
I /
EGJ
o-~
I
oMs /
Me0 / I Cl H OH a I
N~N \ /
O ~ O C1
I~
EHJ
o--~
0
I
OMe /
Me0 / a O
OH
\ ~ N N N I
O ~ \ O O
FHJ
a
I
oru / a
Me0 / a O
I g OH
N~N~N / I
' \
O I \ O O
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2b
EHO
0
I
/
0
g OH
_ ~ /
II N
O I ~ O O
FHO
a
I
/ Q .
O
off
N
II O
O ~ O
I /
EHM i ~ o
/
0
H off
'N N' ~ ' N
~
v
O O
OO IW
EHR
o-~
0
I
/
O
g OH
'N N\ ~ / N
~
v
I~ O
O
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27
EHS
o-~
0
I
/
Me0 / Br Ox O
H
Me0 ~ I N I
O I \ O O
UHD
M<
I
/
O
g OH
II N II N ~ I
Q O ~ \ O O
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28
TABLE 11.
cH,
o"'.\ o'
0
\ \
/ / ~C.o I /
0 - \ I o
/ H H I H H NH
I o ~ \ / > o /
\ ~N N' ~ 'N H3C~ \ N N
l~p~f I \ I~OI v O O ( \ O
O~ > O-~-
O O
I\ I\
CS / CI /
O
/ H \ / / OH N
\ I O N N N : \ I O N N .
O \ O O O \ O
/ O~ / O/CH3
H~C~
/ O
~O /
H3C~0 \ I , >
O
O--~
H~C~ O
\ I O / I . CH7
H3 C.0 \
O
~3
O--~
/ I
/ ~N~CA.s
I _ N Hj and
\ OO
O
O-~~
C1
H
/I
\ ~N
l~ ~fH
O
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29
The compounds of the present invention can be synthesized in a variety of
ways, using conventional synthetic chemistry techniques. Typically, the
compounds of
the present invention are prepared according to the reaction scheme set forth
in FIG. 2,
wherein R,, R2 and R3 are as defined above. The use of appropriate organic
solvents,
temperature and time conditions for running the reactions are within the level
of skill in
the art. Reactions of this type are generally described by E.K. Kick and J.A.
Ellman, J.
Med. Chem. 38, 1427-1430 (1990, the teachings of which are hereby incorporated
by
reference.
C. Uses For the Asparlyl Protease Inhibitors
The compounds of the present invention have been found to be potent
inhibitors of aspartyl proteases and, in particular, cathepsin D. As such, the
present
invention contemplates using the compounds of the present invention to inhibit
cathepsin
D, either in vivo or in vitro. In one embodiment, the present invention
provides a
method of inhibiting cathepsin D, the method comprising contacting cathepsin D
with an
aspariyl protease inhibitor having the general formula:
OH R,
O
Rs
(I)
In the above formula, R,, R2 and R3 are members independently selected from
the group
consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,
substituted
arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted
heteroaryl,
heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted
heterocycles,
heterocyclicalkyl and substituted heterocyclicalkyl. The prior discussions
pertaining to
Rl, R2 and R3 and their preferred embodiments are fully applicable to the
aspartyl
protease inhibitors used in this method of the present invention and, thus,
will not be
repeated with respect to this particular method. RS and R6 are as defined
above.
In another embodiment, the present invention provides a method of
inhibiting protein processing by cathepsin D in living cells, the method
comprising
contacting the cells with an effective amount of a compound having the general
formula:
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(I)
The prior discussions pertaining to Rl, RZ R3 RS and R6 and their preferred
embodiments
are fully applicable to the aspartyl protease inhibitors used in this method
of the present
invention and, thus, will not be repeated with respect to this particular
method.
5 Compounds capable of inhibiting cathepsin D can readily be identified
using the assays described herein which measure a change in the hydrolysis of
a peptide
substrate. More particularly, a fluorometric high through-put assay for
activity toward
human liver cathepsin D (Calbiochem) can be used to screen the compounds of
the
present invention for their ability to inhibit cathepsin D. This assay was
previously
10 described by G. A. Kraft, et al., Methods Enzymol. 241, 70-86 (1994), the
teachings of
which are incorporated herein by reference. Moreover, the peptide substrate
(Ac-Glu-
Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NHZ) used
in
the assay has been previously reported (Km = 6 ~,M) (E. T. Baldwin, et al. ,
Proc. Natl.
Acad. Sci., U.S.A. 90, 6796-6800 (1993)). Generally, the reactants are mixed,
the
15 reaction is allowed to proceed for a specific period of time and the
fluorescence of the
reaction products is monitored to determine the extent to which the peptide
substrate has
been cleaved. Compounds found to exhibit inhibitory activity towards cathepsin
D using
the foregoing assay can be synthesized on a larger scale and a more detailed
kinetic
analaysis can be carried out using an assay similar to that set forth in Table
IV, infra,
20 and described in greater detail by G. A. Kraft, et al., Methods Enzymol.
241, 70-86
( 1994) . As such, following the methods of the present invention, compounds
can be
readily synthesized and screened to identify compounds that inhibit cathepsin
D.
As explained above, the aspartyl protease inhibitors of the present
invention modulate the processing of numerous proteins, such as amyloid
precursor
25 protein (APP), involved in diseases. In a presently preferred embodiment,
the aspartyl
proteases of the present invention are used to modulate the processing of APP.
As such,
in yet another embodiment, the present invention provides a method for
modulating the
processing of an amyloid precursor protein (APP), the method comprising
contacting a
OH R,
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31
composition containing the APP with an aspartyl protease inhibitor having the
general
formula:
O
Rs
(I)
The prior discussions pertaining to Rl, RZ R3 RS and R6 and their preferred
embodiments
S are fully applicable to the aspartyl protease inhibitors used in this method
of the present
invention and, thus, will not be repeated with respect to this particular
method.
The modulation of APP can be demonstrated in a variety of ways. For
example, aspartyl protease inhibitors can be evaluated for the ability to
modulate
generation of A(3 or a-sAPP. In one preferred embodiment, the formation of A/3
is
decreased compared to the amount formed in the absence of the aspartyl
protease
inhibitor. In another preferred embodiment, formation of a-sAPP is increased
compared
to the amount formed in the absence of the asparty protease inhibitor. In one
embodiment, the composition is a body fluid. In a preferred embodiment, the
body fluid
is cerebral spinal fluid (CSF). Numerous in vitro and in vivo animal models
can be used
to screen a given aspartyl protease inhibitor for its ability to modulate APP
processing.
Exemplar assays are set forth below, in the Example Section and in, for
example,
Hoffman, et al. , Neuroscience Letters, 250:75-78 ( 1998); Bahr, et al. ,
Experimental
Neurology, 129:81-94 (1994); and U.S. Patent No. 5,872,101, the teachings of
which are
incorporated herein by reference. In addition, it will be readily apparent to
those of skill
in the art that a number of commercially available tests can be used to detect
A~3 in a
composition (e. g. , CSF) . For instance, the ADmark Assay, which is
commercially
available from Athena Neurosciences, Inc. , can be used to detect A(3 in CSF.
1. In vitro assays
The aspariyl protease inhibitors provided herein yield a positive result in
one or more in vitro assays that assess the effects of test compounds on
processing of
APP. In particular, in vitro assay systems for identifying such compounds are
provided
herein. These assays evaluate the effects of a test compound on processing of
APP and
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32
use cultured human glioblastoma cell lines that have been transfected with DNA
encoding
either a wild-type 695 amino acid isoform of APP or a mutein of APP that
contains
changes (in this case two or three amino acid changes have been made) that
appear to
make the molecule more susceptible to proteolytic cleavage that results in
increased
production of A/3 (see, e. g. , Mullan, et al. , Nature Genet. , 1: 345-347 (
1992)) .
In performing these assays, a test compound is added to the culture
medium and, after a selected period of time, the culture medium and/or cell
lysates are
analyzed using immunochemical assays to detect the relative amounts of A~3,
total soluble
APP (sAPP), a portion of sAPP designated a-sAPP, and C-terminal fragments of
APP.
In particular, the culture medium and cell lysates are analyzed by
.immunoblotting
coupled with laser scanning densitometry and ELISAs using several different
antibodies.
A positive test occurs when: (1) there is a decrease in the approximately
equal to 4-kDa
amyloid (3-protein (A(3) in the medium relative to control cultures (4-kDa
assay); and/or
(2) the relative amount of sAPP in the medium increases; and/or (3) there is a
decrease
in the amount of C-terminal amyloidogenic fragments larger than 9 kDa and
smaller than
22 kDa in the cell lysate as a result of differential processing; and/or (4)
there is an
increase in the amount of a-sAPP in the medium relative to control cultures.
Control
cultures can be cultures that have not been contacted with the compound. The
A(3 assay
is done using cells (e. g. , HGB 717/Swed) that have been transfected with DNA
encoding
the mutein APP; the other assays are performed using cells, such as HGB695
cells, that
have been transfected with DNA encoding a wild-type APP.
2. The amount of a-sAPP and the ratio of a-sAPP to total sAPP in
cerebral spinal fluid (CSF) as an indicator of Alzheimer's Disease
(AD) and the effectiveness of therapeutic intervention
The relative amount of a-sAPP and the ratio of a-sAPP to total sAPP in
CSF are known to be useful markers in the detection of neurodegenerative
disorders
characterized by cerebral deposition of amyloid (e. g. , AD) and in monitoring
the
progression of such disease. Furthermore, assay systems incorporating these
markers
can be used in monitoring therapeutic intervention of these diseases.
The amount of a-sAPP and the ratio of a-sAPP to total sAPP in CSF
samples can be used as an indicator of Alzheimer's Disease and other
neurodegenerative
disorders. For purposes herein, this amount and/or the ratio can also be used
to assess
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33
the effectiveness of compounds provided herein in treating Alzheimer's Disease
and
neurodegenerative disorders.
It has been found that patients with suspected Alzheimer's disease (as
diagnosed by other indicia, or confirmed by autopsy) have a statistically
significant lower
ratio of a-sAPP to total sAPP in CSF and also have statistically significant
lower levels
of a-sAPP. Therefore, by comparison with non-Alzheimer's disease controls or
by
existence of a ratio lower than a predetermined standard, based, for example,
on
averages in samples from large numbers of unafflicted individuals, or an
amount of a-
sAPP lower than a predetermined standard, Alzheimer's disease or, depending
upon
other indications, another neurodegenerative disease is indicated.
Compounds, such as the aspartyl protease inhibitors provided herein, that
alter this ratio or the level of a-sAPP closer to that of individuals who do
not have a
neurodegenerative disorder characterized by the cerebral deposition of amyloid
are
considered useful for treating these disorders.
3. In Vivo Assays
The ability of compounds to modulate processing of APP can also be
evaulated using in vivo assays (See, e. g. , Lamb, et al. , Nature Genet. ,
5:22-29 ( 1993);
Pearson, et al. Proc. Natl. Acad. Sci. U.S.A. 90:10578-10582 (1993); Kowall,
et al.,
Proc. Natl. Acad. Sci. U.S.A., 88:7247-7251 (1991)). Compounds can be
administered
through a canula implanted in the cranium of a rat or other suitable test
animal. After a
predetermined period of administration the rats are sacrificed. The hippocampi
are
evaluated in immunoblot assays or other suitable assays to determine the
relative level of
a-sAPP and C-terminal fragments of APP compared to untreated control animals.
Aspariyl protease inhibitors that result in relative increases in the amount
of a-sAPP are
selected.
In still another embodiment, the present invention provides a method for
modulating the processing of a tau-protein (T-protein), the method comprising
contacting
a composition containing the T-protein with an aspartyl protease inhibitor
having the
general formula:
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34
(I)
The prior discussions pertaining to RI, R2 R3 RS and R6 and their preferred
embodiments
are fully applicable to the aspartyl protease inhibitors used in this method
of the present
invention and, thus, will not be repeated with respect to this particular
method.
The modulation of T-protein can be demonstrated iwa variety of ways. For
example, aspartyl protease inhibitors can be evaluated for the ability to
modulate
generation of T-fragments. In one preferred embodiment, the formation of T-
fragments is
decreased compared to the amount formed in the absence of the aspartyl
protease
inhibitor. In one embodiment, the composition is a body fluid. In a preferred
embodiment, the body fluid is cerebral spinal fluid (CSF). Numerous in vitro
and in
vivo animal models can be used to screen a given aspartyl protease inhibitor
for its ability
to modulate the processing of T-protein. Exemplar assays are set forth below,
in the
Example Section and in, for example, Bednarski and Lynch, J. Neurochem. ,
67(5):1845-
1855 (1996); and U.S. Patent No. 5,492,812, the teachings of which are
incorporated
herein by reference. In addition, it will be readily apparent to those of
skill in the art
that a number of commercially available tests can be used to detect T-
fragments in a
composition (e. g. , CSF) . For instance, the ADmark Assay, which is
commercially
available from Athena Neurosciences, Inc., can be used to detect T-proteins in
CSF.
1. Partial Purification of T and Proteolytic Assays with Cathepsin D
and Test Compound
T-proteins can be partially purified from rat brain by using a modified
version of the method reported by Lindwall and Cole, J. Biol. Chem. ,
259:12241-12245
(1984). Brain tissue ( ~20 g) is homogenized in buffer A (20 mM MES, 80 mM
NaCI,
2mM EGTA, 0.1 mM EDTA, 1mM MgCl2, 1 mM 2-mercaptoethanol, pH 6.75) that
additionally contains 0.1 mM GTP. Following configuration for 25 min at 30,000
g (all
centrifugation steps described in this method occurred at 4 ° C), the
supernatant is made to
% ammonium sulfate and kept on ice for 30 minutes. The slurry is centrifuged
for 20
OH R,
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minutes at 10,000 g; supernatant is saved, made to 45 % ammonium sulfate, and
incubated on ice for 30 minutes. After centrifugation for 20 minutes at 15,000
g, the
pellet is resuspended in --- 4 mL of buffer A and made to 2.5 % perchloric
acid.
Following a 15 minute incubation on ice, the slurry is centrifuged for 15
minutes at
5 15,000 g. The supernatant is made to 20% trichloroacetic acid, ice for 25
minutes and
centrifuged for 15 minutes at 15,000 g. The pellet is resuspended in 95 %
ethanol and
dried under vacuum.
To measure the ability of the aspartyl protease inhibitors of the present
invention to modulate the processing, e.g., degradation, of partially purified
T by
10 cathepsin D, protease, test compound and substrate are combined and
incubated at 37 ° C
for various durations. Partially purified T is first resuspended in assay
buffer (50 mM
citric acid/sodium citrate buffer, pH 4.0). Reactions are initiated by the
addition of
human liver cathepsin D (1 U; Calbiochem, San Diego, Ca, U.S.A.) and test
compound
to 0.1 mg of T and terminated by removing aliquots at the designated time,
adding SDS
15 and 2-mercaptoethanol, and boiling for 5 minutes. One unit of cathepsin D
is defined as
the amount of enzyme that generates an increase in absorbance (at 280 nm) of
1.0 per
hour when co-incubated with hemoglobin in 10 % trichloroacetic acid. The
specific
activity of the enzyme is 300 U/mg of protein, and its purity is greater than
98 % by
SDS-PAGE.
2. Assay for the Proteolysis of T in Conical Homogenates by
Exogenous Cathepsin D and Test Compounds
Brains from 3-month-old Sprague-Dawley rats are removed and dissected
in artificial cerebrospinal fluid (124 mM NaCI, 20 mM glucose, 5mM HEPES, 3 mM
KCI, 1.25 mM KHZP04, 2.8 mM MgS04, 2 mM CaCl2, mM NaHC03, 0.5 mM
ascorbate, Ph 7.4). Frontal cortices are homogenized (Teflon to glass, 10
strokes) in 7
mM HEPES buffer, pH 7.35, additionally containing 135 mM NaCI, 2mM EDTA, 2mM
EGTA, and 2.0 ~,M Okadaic acid. Slurries are centrifuged at 1,000 g for five
minutes at
4°C. The supernatant is collected, sonicated, and subjected to two
freeze/thaw cycles.
Proteolytic assays are conducted by co-incubating 0.1 mg of the
supernatant described above with 0.35 U of human liver cathepsin D and the
test
compound. The enzyme-to-substrate ratio should be about 1:86 (wt/wt). The
reaction is
allowed proceeded at constant pH for 5 hours at 37°C and is terminated
by adding SDS
and 2-mercaptoethanol and boiling the samples for five minutes.
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36
As explained above, aspartyl proteases, e.g., cathepsin D, are enzymes
that plays an important role in protein metabolism, catabolism and antigen
processing.
As a result of their ability to inhibit aspartyl proteases, the compounds of
the present
invention can be used for a number of therapeutic applications. As such, in
yet another
embodiment, the present invention provides a method of treating a
neurodegenerative
disorder, the method comprising: administering to a mammal a therapeutically
effective
amount of an aspartyl protease inhibitor and a pharmaceutically acceptable
carrier or
excipient, the aspariyl protease inhibitor having the general formula:
0 0
(I)
The prior discussions pertaining to Rl, R2, R3 RS and R6and their preferred
embodiments
are fully applicable to the aspartyl protease inhibitors used in this method
of the present
invention and, thus, will not be repeated with respect to this particular
method.
In one embodiment, the neurodegenerative disorder is characterized by the
accumulation of amyloid plaques. In another embodiment, the neurodegenerative
disorder is characterized by the accumulation of T-fragments. As such, the
aspartyl
protease inhibitors of the present invention can be used to treat all amyloid-
pathology
related diseases and all tau pathology-related diseases. Examples of such
neurodegenerative diseases include, but are not limited to, the following:
Alzheimer's
disease, Parkinson's disease, cognition deficits, Downs Syndrome, cerebral
hemorrhage
with amyloidosis, dementia (e.g., dementia pugilistica) and head trauma.
The compounds, i. e. , aspartyl protease inhibitors, of the present invention
can be incorporated into a variety of formulations for therapeutic
administration. More
particularly, the compounds of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate, pharmaceutically
acceptable carriers or diluents, and may be formulated into preparations in
solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,
granules,
ointments, solutions, suppositories, injections, inhalants and aerosols. As
such,
administration of the compounds can be achieved in various ways, including
oral, buccal,
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37
rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal,
etc. ,
administration. Suitable formulations for use in the present invention are
found in
Remington's Pharmaceutical Sciences (Mack Publishing Company, Philadelphia,
PA,
17th ed. ( 1980), which is incorporated herein by reference. In addition, for
a brief
review of methods for drug delivery, see, Larger, Science 249:127-1533 (1990),
which
is incorporated herein by reference.
The compounds of the present invention can be administered alone, in
combination with each other, or they can be used in combination with other
known
compounds (e. g. , other protease inhibitors) . In pharmaceutical dosage
forms, the
compounds may be administered in the form of their pharmaceutically acceptable
salts,
or they may also be used alone or in appropriate association, as well as in
combination
with other pharmaceutically active compounds. The following methods and
excipients
are merely exemplary and are in no way limiting. It should be noted that since
the
compounds of the present invention are non-peptidic in nature, they tend to
have better
pharmacokinetic properties (e. g. , better oral availability and increased
circulating half-
lives) than compounds that are peptidic in nature.
For oral preparations, the compounds can be used alone or in combination
with appropriate additives to make tablets, powders, granules or capsules, for
example,
with conventional additives, such as lactose, mannitol, corn starch or potato
starch; with
binders, such as crystalline cellulose, cellulose derivatives, acacia, corn
starch or
gelatins; with disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium stearate;
and if
desired, with diluents, buffering agents, moistening agents, preservatives and
flavoring
agents.
The compounds can be formulated into preparations for injections by
dissolving, suspending or emulsifying them in an aqueous or nonaqueous
solvent, such as
vegetable or other similar oils, synthetic aliphatic acid glycerides, esters
of higher
aliphatic acids or propylene glycol; and if desired, with conventional
additives such as
solubilizers, isotonic agents, suspending agents, emulsifying agents,
stabilizers and
preservatives.
The compounds can be utilized in aerosol formulation to be administered
via inhalation. The compounds of the present invention can be formulated into
pressurized acceptable propellants such as dichlorodifluoromethane, propane,
nitrogen
and the like.
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38
Furthermore, the compounds can be made into suppositories by mixing
with a variety of bases such as emulsifying bases or water-soluble bases. The
compounds
of the present invention can be administered rectally via a suppository. The
suppository
can include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which
melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs,
and suspensions may be provided wherein each dosage unit, for example,
teaspoonful,
tablespoonful, tablet or suppository, contains a predetermined amount of the
composition
containing one or more compounds of the present invention. Similarly, unit
dosage
forms for injection or intravenous administration may comprise the compound of
the
present invention in a composition as a solution in sterile water, normal
saline or another
pharmaceutically acceptable carrier.
The term "unit dosage form, " as used herein, refers to physically discrete
units suitable as unitary dosages for human and animal subjects, each unit
containing a
predetermined quantity of compounds of the present invention calculated in an
amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable
diluent, carrier or vehicle. The specifications for the novel unit dosage
forms of the
present invention depend on the particular compound employed and the effect to
be
achieved, and the pharmacodynamics associated with each compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or diluents, are readily available to the public. Moreover,
pharmaceutically
acceptable auxiliary substances, such as pH adjusting and buffering agents,
tonicity
adjusting agents, stabilizers, wetting agents and the like, are readily
available to the
public.
Preferred formulations of the compounds are oral preparations, particularly
capsules or tablets containing each from about 10 milligrams up to about 1000
milligrams
of active ingredient. The compounds are formulated in a variety of
physiologically
compatible matrixes or solvents suitable for ingestion or injection.
The invention will be described in greater detail by way of specific
examples. The following examples are offered for illustrative purposes, and
are not
intended to limit the invention in any manner. Those of skill in the art will
readily
recognize a variety of noncritical parameters which can be changed or modified
to yield
essentially the same results.
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39
EXA~1~IPLES
Z EXAMPLE I
A. Specific Approach
One powerful strategy to target an enzyme class is to incorporate a stable
mimetic or isostere of the transition state or of an intermediate of the
enzyme-catalyzed
reaction (R. A. Wiley, et al., Med. Res. Rev. 13, 327-384 (1993)). The
libraries for
potential cathepsin D inhibitors are based upon the well-known
hydroxyethylamine
isostere (see, FIG. 1). For the initial libraries, the P, side chain (R4) is
held constant as
the benzyl substituent, based on the X-ray crystallographic structure of
cathepsin D
complexed with the natural peptide inhibitor pepstatin (E. T. Baldwin, et al.
, Proc. Natl.
Acad. Sci., U.S.A. 90, 6796-6800 (1993)), and upon inhibition constants of
peptide-
based inhibitors (R. A. Jupp, et al., Biochem. J. 265, 871-878 (1990); N. S.
Agarwal,
etc., J. Med. Chem. 29, 2519-2524 (1986)).
In a pilot study both S and R epimers at the hydroxyl carbon (see,
structures l and 2 of FIG. 1) were prepared since both diastereomers have been
found in
potent inhibitors of other aspartic acid proteases (R. A. Wiley, et al. , Med.
Res. Rev.
13, 327-384 (1993)). Because inhibition at 1 ~,M was only found with compounds
of
scaffold 1 in the pilot study, further syntheses of libraries toward cathepsin
D used only
scaffold 1. Computer modeling (see below) predicted that structure 1 (FIG. 1)
would
provide the most potent inhibitors. Diversity is introduced in three
positions: a primary
amine for the R, substituent and acylating agents for the RZ and R3
substituents (FIG. 2) .
The optimization of the synthesis sequence was previously reported (E. K.
Kick, J. A.
Ellman, J. Med. Chem. 38, 1427-1430 (1995)).
The library synthesis was designed to use commercially available
compounds for incorporation of the functionality at R,, R2, and R3. Exhaustive
combination of available materials would provide a library of over 10 billion
compounds.
To reduce these possibilities in a sensible way, version 93.2 of the Available
Chemical
Directory (ACD) from MDL Information Systems (San Leandro, CA) was used to
search
for all amines, carboxylic acids, sulfonyl chlorides and isocyanates with MW <
275
daltons. Compounds with functionality obviously incompatible with the
synthesis were
eliminated. The resulting list included approximately 700 amines and 1900
acylating
agents. However, this list still provided access to more than 1 billion
compounds.
Clearly, additional selection criteria were required, and a computational
screening
process was turned to in an effort to enhance selection.
CA 02367112 2001-09-24
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B. IJirected Library Desi.2n
The structure-based design process began with coordinates for pepstatin in
a complex with cathepsin D (E. T. Baldwin, et al., Proc. Natl. Acad. Sci.,
U.S.A. 90,
6796-6800 (1993)). The scaffold is identical to pepstatin on the P,-P3 side,
but differs on
5 the B1.-P3. side and cannot form the same hydrogen bonds with the enzyme
(FIG. 3A).
Thus, the pepstatin positions for the Pi-P3 side were used and the three
scaffold torsion
angles on the P,.-P3. side were systemically rotated. Each rotation was
followed by
energy minimization within the cathepsin D active site, using the AMBER (S. J.
Weiner,
et al. , J. Am. Chem. Soc. 106, 765-784 ( 1984)) force field in Sybyl, a
molecular
10 modeling software package from Tripos Associates (St. Louis, MO). During
minimization, the enzyme was kept rigid, but full flexibility of the scaffold
was allowed.
Both S and R epimers, structures 1 and 2, were modeled using methyl groups for
each of
the R,-R4 groups. The conformational energies of the R epimers were generally
ca. 2
kcal higher than for S epimers, leading to the prediction that the S epimers
would bind
15 more tightly than the R epimers. All minimized conformations of S epimers
within a 2
kcal/mol range were collected and clustered into four families based on
geometric
similarity (FIG. 3B) . A benzyl group was added to each family at the R4
position. The
processed list of compounds for the ACD was passed through Sybyl to obtain
Gasteiger
and Marsili partial atomic charges for each component (J. Gasteiger, et al. ,
Tetrahedron
20 Lett 36, 3219 (1980); J. Gasteiger, M. Marsili, Organ. Magn. Reson. 15, 353
(1981)).
To reduce the computational time for searching the components, compounds with
more
than 4 torsional bonds were identified and removed. A new feature of the
BUILDER
molecular modeling program (R. A. Lewis, et al., J. Mol. Graphics 10, 66-78
(1992);
D. C. Roe, and Kuntz, LD., JCAMD 9, 269-282 (1995)), called BUILDERopt (D. C.
25 Roe, Dissertation, University of California, San Francisco (1995)), was
used to position
each of the R,, R2, and R3 components onto the scaffold and to perform a full
conformational search for the torsion angles of the substituent at 15 degree
increments.
In order to reduce the combinatoric problem, the R,, R2, and R3 components
were
examined independently, but a probability-based clash grid was constructed to
identify Ri
30 and RZ conformations that might overlap. For example, if an Rl conformation
clashed
with more than 50 % of the R2 components, that conformation was discarded.
Each
rotation was then examined for intramolecular clashes with the scaffold and
overlap with
cathepsin D. Each accepted conformation was rigid-body minimized (D. A.
Gschwend,
et al., J. Compt-Aided Drug Design 10, 123-132 (1996)) and scored with a force-
field
CA 02367112 2001-09-24
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41
grid (E. C. Meng, et al., J. Comput. Chem. 13, 50~-524 (1992)). The total
computer
time required to evaluate all torsion angles for all sidechains attached to
four different
scaffold conformations was 16 hours on a Silicon Graphics Iris 84400. The
fifty best
scoring components for all families were merged for each of the three variable
positions,
and sorted by overall lowest score. Components with cost above $35/gm were
removed,
leaving 34, 35, and 41 components at R,, R2 and R3, respectively. Each
remaining
component was structurally fingerprinted (Daylight Clustering Toolkit,
Daylight
Chemical Information Systems, Inc., Santa Fe, NM) and hierarchically clustered
(similarity cutoff = 0.63) (H. C. Romesburg, Cluster Analysis For Researchers
(Lifetime Learning Publications, Belmont, CA, 1984)) using the Tanimoto
similarity
metric (P. Willett, Similarity and Clustering in Chemical Information Systems
(John
Wiley & Sons, New York, NY, 1987); P. Willett, et al., J. Chem. If. Comput.
Sci. 26,
109-118 (1986)). For RI, Rz, and R3, the ten best scoring components from
unique
clusters were selected for the directed library.
C. Diverse Library Design
A diverse library, which was set at the same size as the directed library,
was prepared to provide a "hit" rate when structure-based methods were not
employed.
The diverse library was designed to maximize the variety of functional groups
and
structural motifs of the library components. The sidechains for this library
were selected
by clustering the original list of components based on their similarity to
each other.
Components were clustered with the Jarvis-Patrick algorithm (R. A. Jarvis, et
al., IEEE
Comput C22, 1025-1034 (1973)) using the Daylight connectivity measure of
similarity
(Daylight Clustering Toolkit, Daylight Chemical Information Systems, Inc. ,
Santa Fe,
NM) and a binary Tanimoto metric (P. Willett, Similarity and Clustering in
Chemical
Information Systems (John Wiley & Sons, New York, NY, 1987); P. Willett, et
al., J.
Chem. If. Comput. Sci. 26, 109-118 (1986)). In the Jarvis-Patrick method, two
compounds are placed in the same cluster if they: 1) are neighbors of one
another, and
2) share at least p neighbors from a list of q nearest neighbors, where p and
q are
adjustable parameters. The compound nearest the cluster centroid was chosen as
the
cluster representative.
The Rl (amine) components were clustered directly as the primary amines.
The RZ and R3 acylating agents were each attached to a portion of the scaffold
before
clustering to yield the proper chemical context at the linkage site. The first
round of
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42
clustering yielded 47, 1~4, and 162 clusters using p/q = 4/11, p/q = 4/12, and
p/q =
4/12 for R,, RZ, and R3, respectively. The representative R2 and R3 components
were
clustered a second time (p/q = 4/7 for RZ and p/q = 4/7 for R3), resulting in
23 R~ and
35 R3 components. It is noted that it is not practical to condense a large
number of
compounds into an arbitrarily small number of clusters because the cluster
membership
can become very diverse. Final selection of ten compounds from each list was
based
upon: size, cost, availability and synthetic feasibility. Additionally, a
balance of
functional groups for each set of sidechains was sought. A comparison of the
directed
and diverse libraries (FIGS. 4 and 5~ shows the much greater range of
functionality
spanned in the diverse library.
D. Library Synthesis and Screening
The directed and diverse libraries ( 1000 compounds each) were prepared
using diastereomer 1 of the hydroxyethylamine scaffold with the components
used in
library syntheses shown in FIGS. 4 and S, respectively. Because the pilot
study with R
and S epimers only showed activity at 1 ~,M inhibitor concentration for the S
epimers,,
only the S epimers of the directed and diverse library were synthesized. All
libraries
were synthesized in a spatially separate format, and were screened in a high-
throughput
fluorometric assay for inhibitory activity against cathepsin D (G. A. Krafft,
et al.,
Methods Enzymol. 241, 70-86 (1994))
1. Library Synthesis
The optimization of the solid-phase synthesis sequence to prepare the
hydroxyethylamine inhibitors and the demonstration of reaction generality was
previously
reported by E. K. Kick and J. A. Ellman (J. Med. Chem. 38, 1427-1430 (1995)).
Further testing was performed to establish that the different functionality to
be displayed
at Rl, RZ and R3 would be successfully incorporated into the potential
inhibitors. First,
all the amines and acylating agents to be incorporated in both the diverse and
directed
libraries were treated with trifluoroacetic acid for 2 h at room temperature
to ensure
stability to the support-cleavage conditions, by far the harshest reaction
conditions in the
synthesis sequence. Second, components that might pose difficulties on
chemical or
steric grounds were evaluated by trial syntheses. Five amines and four
carboxylic acids
that did not provide the expected final compound in high yields or purity were
discarded.
The following amines and acylating agents were successfully tested in the
synthesis
CA 02367112 2001-09-24
WO 00/56335 PCT/US00/07804
43
sequence: R1 = B, C, E, F, a, e, h, i, j; RZ = B, C, D, E, H, a, e, f; R3 = A,
D E H,
a, b, e, g, h, i (FIGS. 4 and 5). The remaining components were assumed to be
compatible with the synthesis sequence.
The library synthesis was performed on polystyrene beads (20-40 mesh).
The library was synthesized in a spatially separate array using a 96-well
filter apparatus.
Transfer of the resin to the individual wells was performed using an isopycnic
mixture of
N,N dimethylformamide (DMF) and 1,2-dichloroethane. Incorporation of Rl was
carried
out using 1.0 M free amine in N methylpyrrolidinone (NMP) at 80°C for
36 h.
Incorporation of RZ was carried out using stock solutions of 0.3 M carboxylic
acid, 0.3
M benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBOP),
0.3 M 7-aza-1-hydroxybenzotriazole (HOAt), and 0.9 M iPr2EtN in NMP overnight.
The coupling reactions were performed twice to ensure that complete coupling
had
occurred. After azide reduction with SnCl2, PhSH and Et3N, incorporation of R3
was
carried out as reported above for R2. Carboxylic acid RZ = E was coupled using
0-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU)
instead
PyBOP due to formation of a precipitate under the standard coupling procedure.
The
isocyanate RZ = b was coupled at 0.3 M in NMP overnight, and the sulfonyl
chlorides
RZ = a and R3 = c were coupled at 0.3 M in NMP that was 0.9 M in iPrZEtN.
Cleavage of the material from the support was achieved by subjecting the resin
to 95:5
trifluoroacetic acid: H20 for 30 min. The cleavage mixture was removed from
the resin
via filtration, followed by rinsing the resin and concentration of the
filtrates using a
Jouan 10.10 centrifugation concentrator. Toluene was added to form an
azeotrope with
trifluoroacetic acid during the concentration step. After concentration, the
libraries were
stored at -20° C.
Compounds from each library, picked by random number generation, were
analyzed by mass spectrometry in a matrix of a-cyano cinnamic acid on a
Perseptive
Biosystems MALDI instrument. For the diverse library the expected molecular
ion
peaks were observed for 46 of 49 compounds (poor ionization was obtained for
the other
three) . Molecular ion peaks were obtained for 44 of 49 compounds from the
directed
library. In addition, the synthesis has been validated by the reasonable
correlation of the
approximate ICSO values of the crude material from the libraries with purified
material
that was synthesized on large scale for a number of compounds (see, Table IV,
infra).
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44
2. Screening of the Libraries for Compounds Having Inhibitory
Activity Against Cathepsin D
Briefly, a fluorometric high through-put assay for activity toward human
S liver cathepsin D (Calbiochem) was performed in 96-well microtiter plates
(G. A. Krafft,
et al., Methods Enzymol. 241, 70-86 (1994)). The peptide substrate (Ac-Glu-
Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NHZ) used
in
the assay has been previously reported (K,r, = 6 ~,M) (E. T. Baldwin, et al. ,
Proc. Natl.
Acad. Sci., U.S.A. 90, 6796-6800 (1993)). The assay was performed in DYNATECH
Microfluor fluorescence microtiter plates, and readings were taken on a Perkin-
Elmer
LS-SOB with an attached 96-well plate reader. The excitation wavelength was
340 nm.
A 340 nm interference filter (Hoya, U-340) for excitation and a 430 nm cut-off
filter for
emission were used. For the microtiter-based assays, the substrate
concentration was 5
~,M and the cathepsin D concentration was 9 nM in a 0.1 M formic acid buffer
(pH =
3 .7) . DMSO ( 10 % ) was used to ensure complete dissolution of the
inhibitors. The
fluorescent unit readings were taken at three time points within the linear
region of the
substrate cleavage, and percent activity of the enzyme was determined by
comparing the
change of fluorescent units (FU) for each well to the average change in FU for
six
control wells without inhibitor. Each library was screened at approximately 1
~,M
inhibitor with the concentration based on the assumption that 50 % of the
theoretical yield
was obtained for each inhibitor. All wells that showed < 50 % cathepsin D
activity were
screened at subsequent three-fold dilutions. All active compounds that showed
< 60
enzyme activity in 1 ~,M or lower inhibitor concentrations were assayed in
duplicate).
E. Assay Results
At approximately 1 ~,M of crude compound, the directed library yielded 67
compounds that inhibited cathepsin D activity >_ 50 % (G. A. Krafft, et al.,
Methods
Enzymol. 241, 70-86 (1994)). Further dilution of 333 nM and 100 nM inhibitor
concentrations afforded 23 and 7 compounds, respectively, that inhibited
cathepsin D
activity >_ 50% (see, Table III). The data for the diverse library are also in
Table III,
infra. There are many uncertainties that can influence the results of a high-
throughput
fluorescence assay, including the purity of each compound, the concentration
of the
compounds, and the experimental errors associated with the microtiter
fluorescence
assay. From repetitive experiments, these errors were estimated to be
approximately
30 % , expressed as enzyme activity.
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Table II1. Number of Compounds with >_ 50% Inhibition of Cathepsin D in
Library Screen°
Library
[Inhibitor] ~ Directed Diverse #
100 nM ~ 7 * 1 ~
330 nM ~ 23 j- 3~
10 1 ,~M ~ 67 26
10 ~cM ~ 11/95
a Inhibitors of cathepsin D at respective concentrations: *EAA, EFA, EHA, EHD,
EHI,
15 EHJ, FHA. An additional six compounds provided 40-50% inhibition of
cathepsin D.
fiEAA, EFA, EHA, FAA, FFA, FHA, EHB, EFD, EHD, EEF, EHF, FHF, Ei~H,
EHH, FAH, FFH, EFI, EHI, EAJ, EFJ, EGJ, EHJ, FHJ. An additional thirty
compounds provided 40-50% inhibition of cathepsin D. $One hundred compounds
were
selected by random number generation for testing at 10 ~M. Five compounds were
20 highly fluorescent at these concentrations, so that accurate assay data
could not be
obtained in these cases. ~fbb, ~[fba, fbb, fcb. Four compounds (fca, fdb, fib,
hhb)
provided 40-50 % inhibition of cathepsin D; with the experimental error in the
assay, this
activity is similar to the activity for the three that are listed. #The
diverse library was
not tested at 10 ~cM.
25 In order to obtain accurate inhibition constants (K) several of the
compounds most likely to be potent inhibitors based on the library screening
were
synthesized on a larger scale, purified by chromatography, and characterized
by NMR
and mass spectrometry. The K; values were calculated from ICSO determinations
(see,
Table IV). From the compounds that were fully characterized, one compound was
30 obtained from the directed library with a K; below 100 nM, whereas the
diverse library
contained inhibitors that were 3-4 times less potent.
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46
Table IV Inhibition Constants for a Number of the Compounds ?'hat Are Potent
Inhibitors°
Cpd Code Scaffold K;{nM)
EHD 1 73 9
EHD 2 > 5000
EHJ 1 111 t 8
EHA 1 131 12
EFA 1 171 25 '
FHA 1 231 ~ 31
fbb 1 356 31
fdb 1 595 66
a The cathepsin D assay for "hits" from the directed and diverse libraries was
performed in a quartz cuvette with a Perkin-Elmer LS-SOB spectrometer. The
substrate
concentration was 2.5 ~cM and the cathepsin D concentration was 10 nM.
Inhibition
constants (K;) were determined from ICSO values taken from plots of V;/Vo
versus
inhibitor concentration, where Vo is the velocity in absence of the inhibitor
and V; is the
velocity with inhibitor: Since the substrate concentration is significantly
below km, the
ICso values were converted to K; by the equation K; = (ICSO - E212), where EL
= enzyme
concentration (S. Cha, et al., Biochem. Pharmacol., 24, 2187-2197 (1975)).
F. (i) Second Generation Library
In the design of the directed library, derivatives with a high level of
structural similarity were selected against by applying a clustering algorithm
to the
highest scoring components (see Directed Library Design). These clusters were
re-
examined to explore the important structural elements of the most active
compounds from
the directed library. In particular, a small second generation library from
the clusters for
the Rl, RZ and R3 positions that provided the most active compounds was
synthesized and
screened (see, FIG. 6). At 1 ~,M, 92% of the compounds screened inhibited
cathepsin D
>_ 50 % , and 18 % of the compounds at 100 -nM inhibited cathepsiri D >_ 50 %
.
Inhibition constants were determined for selected compounds (see, Table V),
providing
several potent inhibitors (K; <_ 15 nM) of cathepsin D.
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47
Table V. Second Generation Assay (see, FIG. 6)°
Cpd. Code Scaffold ICSO (nM) K;(nM)
EHO 1 19 2 15
EHO 2 > 5000
~O 1 18 2 14
EHM 1 14 2 9
EHR 1 20 2 15
EHS 1 64 6 59
UHD 1 229 44 224
a Assay conditions are reported in Table IV .
F. (ii) Additional Compounds
Known aspartyl protease inhibitors have both (R) and (,S~ stereocenters
about the hydroxyl group in Formula I. Employing a-alkoxy chelation and non-
chelation
controlled reductions, the following synthetic strategy demonstrates acyclic
diastereocontrol on solid support providing access to either desired
diastereomer. By
exploring different functional groups for RS and R6 and selecting the Rl, R2,
and R3
substituents providing the most potent Cathepsin D inhibitors, additional low
nanomolar
Cathepsin D inhibitors were discovered.
Structural diversity may be derived through Grignard addition to a solid
support-bound a-alkoxy pyrrolidine amide 3 (see, FIG. 7) . The source of
diversity is
derived from aromatic and alkyl Grignard reagents. The Grignard reagents that
are not
commercially available can be synthesized using activated magnesium turnings,
or a
magnesium anthracene THF complex and the corresponding aromatic and alkyl
halides.
Grignard reagents are a suitable source to introduce diversity in the Pl site
of potential
aspartyl protease inhibitors, since the S1 protease surface tends to be
hydrophobic. The
resulting ketone is reduced using chelation and non-chelation conditions to
provide the
desired diastereomer. After several functional group manipulations, known
azido-
nosylate intermediate 2 is derived and carried through the previously reported
synthesis
to obtain potential aspartyl protease inhibitor 1 (see E.K. Kick, J.A. Ellman,
J. Med.
Chem. 38, 1427-1430 (1995)) (see, FIG. 7).
The pyrrolidine amide 4 prepared in 3 steps in an overall 76 % yield from
commercially available methyl (s)-(-)-2,2-dimethyl-1,3-dioxolane-4-
carboxylate, was
coupled to benzyloxybenzyl bromide resin 5 using sodium hydride,
tetrabutylammonium
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48
iodide, and catalytic 18-Crown-6 in THF for 2 hours at 45 ° C (see,
FIG. 8). Bromide
resin 5 was derived from carbon tetrabromide, triphenylphosphine, and
commercially
available Wang resin.
Grignard addition in THF at O ° C to support-bound pyrrolidine
amide 6
followed by « -alkoxy chelation controlled reduction of the resulting ketone
using zinc
borohydride in diethyl ether at -20°C afforded secondary alcohol 7 in a
85:15
diastereomeric mixture with the major diastereomer shown (see, FIG. 8). A
small
portion of secondary alcohol 7 was cleaved from the support to provide the
corresponding triol product which was converted to the corresponding
triacetate using
acetic anhydride and DMAP (Dimethyl amino pyridine). Diastereoselectivity was
determined from GC analysis of the corresponding triacetates. No over
alkylation from
the Grignard addition was detected for all components used in the library.
Secondary alcohol 7 was converted to azide 8 through the formation of a
secondary nosylate using 4-nitrobenzenesulfonyl chloride and 4-
pyrrolidinopyridine in
chloroform followed by azide displacement with sodium azide in N, N
dimethylformamide
at 50°C. The p-methoxy trityl protecting group was selectively removed
using 1 % p-
toluenesulfonic acid in methylene chloride. Nosylation of the primary alcohol
with 4-
nitrobenzenesulfonyl chloride and pyridine in chloroform provided azido-
nosylate 9.
Amine displacement in N methylpyrrolidinone (NMP) at 80°C followed
by
acylation with the desired carboxylic acid, benzotriazole-1-yl-oxy-tris-
pyrrolidino-
phosphonium hexafluorophosphate (PyBOP), aza-1-hydroxybenzotriazole (HOAt) or
isocyanate in NMP afforded intermediate 10 with the Pl, R,, and R2 sites of
diversity in
place. Reduction of the azide with tin(II) chloride, thiophenol, and
triethylamine
followed by acylation with the R3 carboxylic acid, PyBOP, and HOAt, and
lastly,
cleavage from the support using a trifluoroacetic acid:methylene chloride
(90:10) mixture
provided the desired potential aspartyl protease inhibitor la.
A library of 204 compounds was derived from the components in FIG. 9.
The most potent inhibitors of Cathepsin D were synthesized on a larger scale,
purified,
and biologically assayed to determine K; values as detailed in Table VI.
Overall yields
of these scaled-up inhibitors ranged from 46-48 % for the entire 12 step solid-
phase
synthesis as determined by the mass balance of desired product after column
chromatography purification.
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49
Table V1. Inhibition constants for selected compounds (1~~
Code K; Overall Yield
Inhibitor (P, R, R2 R3) (nM) ( 12 steps)
Kbcf 1.9~0.2 46%
Gbcf 2.6 ~0.2 48 %
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Obcf 2. 6 ~ 0.2 48 %
Qbcf 6.7 ~0.7 46 %
Synthesis of inhibitors
Several of the most potent compounds were synthesized on an average of
5 115 milligram scale on the solid support following the aforementioned
method. These
compounds were purified by column chromatography and characterized by 'H NMR
and
elemental analysis. Overall yields of the compounds were based on the entire
12 step
solid-phase synthesis and determined by the mass balance of desired product
after column
chromatography purification. The characterization data are listed with the
corresponding
10 compound code. The 1H NMR data is reported for the major amide rotomer of
the
major diastereomer for each compound.
Kbcf. (57 mg, 46 % ) 'H NMR (400 MHz, CDC13) d 2.65 (m, 2H), 2.88 (apparent t,
J
= 7.7, 2H), 3.01 (apparent t, J = 6.9, 2H), 3.24 (m, 1H), 3.47 (m, 2H), 3.83 -
3.96
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51
(m, 4H), 3.85 (s, 3H), 3.89 (s, 3H), 4.34 (apparent q, J = 8.3, 1H), 4.66 (br.
s, 1H),
6.71 (d, J = 9.2, 1H), 6.84 (dd, J = 1.7, 8.0, 1H), 6.93 - 7.00 (m, 5H), 7.05
(m, 1H),
7.05 (s, 1H), 7.07 (s, 1H), 7.16 (dd, J = 2.1, 8.1, 1H), 7.23 - 7.30 (m, 3H),
7.34 (d, J
= 2.1, 1H), 7.71 (dd, J = 3.1, 5.4, 2H), 7.83 (dd, J = 3.1, 5.4, 2H). Anal.
calc'd for
C~H4oN308C12Br1: C, 59.41; H, 4.53; N, 4.72. Found: C, 59.22; H, 4.76; N,
4.52.
Gbcf. (48 mg, 48 % ) 'H NMR (400 MHz, CDC13) d 2.62 (apparent t, J = 7.5, 2H),
2.82 (apparent t, J = 7.6, 2H), 3.18 - 3.25 (m, 3H), 3.40 - 3.47 (m, 2H), 3.57
(s, 3H),
3.85 (s, 3H), 3.91 - 3.96 (m, 4H), 4.47 (apparent q, J = 8.4, 1H), 4.76 (br.
s, 1H),
6.69 (s, 1H), 6.92 (d, J = 8.2, 1H), 6.95 (s, 1H), 7.04 (dd, J = 2.1, 8.2,
1H), 7.29 (d,
J = 2.1, 1H), 7.40 - 7.45 (m, 3H), 7.68 (dd, J = 3.0, 5.5, 2H), 7.71 - 7.80
(m, 6H).
Anal. calc'd for C42H38N3O,C12Br1: C, 59.52; H, 4.52; N, 4.96. Found: C,
59.63; H,
4.67; N, 4.69.
Obcf. (55 mg, 48 % ) 1H NMR (400 MHz, CDC13) d 2.65 (m, 2H), 2.85 (apparent t,
J
= 7.3, 2H), 3.C8 (apparent t, J = 6.7, 2H), 3.23 (m, 1H), 3.44 (m, 1H), 3.57
(m, 1H),
3.75 (s, 3H), 3.86 (s, 3H), 3.94 (m, 4H), 4.39 (apparent q, J = 8.3, 1H), 4.73
(br. s,
1H), 6.78 (d, J = 9.2, 1H), 6.93 (s, 1H), 6.97 (s, 1H), 7.02 (d, J = 8.2, 1H),
7.10
(dd, J = 2.1, 8.2, 1H), 7.30 (d, J = 2.1, 1H), 7.36 - 7.42 (m, 5H), 7.51 -
7.54 (m,
4H), 7.68 (dd, J = 3.0, 5.4, 2H), 7.81 (dd, J = 3.0, 5.4, 2H). Anal. calc'd
for
G~H4oN30.,C12Br1: C, 60.49; H, 4.62; N, 4.81. Found: C, 60.23; H, 4.86; N,
4.58.
Qbcf. (55 mg, 46%) 1H NMR (400 MHz, CDC13) d 2.64 (m, 2H), 2.86 (apparent t, J
= 7.1, 2H), 2.96 (m, 2H), 3.20 (m, 1H), 3.46 (m, 1H), 3.54 (m, 1H), 3.78 (m,
2H),
3.82 (s, 3H), 3.86 (s, 3H), 3.91 (m, 2H), 4.31 (apparent q, J = 8.5, 1H), 4.73
(br. s,
1H), 6.73 (d, J = 9.3, 1H), 6.85 (s, 1H), 6.96 (s, 1H), 7.03 (d, J = 8.3, 1H),
7.14 (m,
2H), 7.16 (dd, J = 2.2, 8.3, 1H), 7.32 (d, J = 2.2, 1H), 7.37 - 7.41 (m, 2H),
7.70
(dd, J = 3.0, 5.5, 2H), 7.80 (dd, J = 3.0, 5.5, 2H). Anal. calc'd for
C38H35N3O,C12Br2: C, 52.08; H, 4.03; N, 4.79. Found: C, 52.28; H, 4.09; N,
4.60.
G. Results
Novel low nanomolar inhibitors of cathepsin D were identified rapidly
using combinatorial chemistry coupled with two different computational
strategies. The
diverse and directed libraries together yielded over 90 compounds active at 1
~cM and 26
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52
active in the submicromolar range. The "hit rate" for activity at 1 ~,M is 6-7
~o for the
directed library and 2-3 % for the diverse library. Even though both the
directed and
diverse libraries are based on the "active" epimer of the scaffold, the
results from the
directed library are clearly superior. At all concentrations < 1 ~cM, there
were more
"hits" in the directed library than the diverse library. The most potent
inhibitors from
the directed library are 3-4 fold better than those in the diverse library. It
is clear from
the results that the number and quality of the active compounds can be
increased by
using relevant information about the target.
A strength of the structure-based procedure is that it leads directly to
testable geometrical hypotheses. In this study there are three hypotheses: 1)
S epimers
are predicted to bind better than the R epimers; 2) there are two
energetically reasonable
scaffold conformations (family 1 +2, family 3 +4), which place R groups into
different
pockets; 3) all the inhibitors are assumed to bind in approximately the same
orientation
as pepstatin.
The first hypothesis was directly tested in pilot experiments where no
inhibitors based upon the R epimer had activity at 1 ~,M. In addition, the R
epimer of
one of the most potent compounds had a K; no better than S ~,M while the K; of
the S
epimer was 15 nM (see, Table V). This conclusion and the inhibitor
orientations in the
cathepsin D complex will be examined crystallographically.
Using the methodology described herein, active compounds can be
identified and then the activity is optimized. The optimization criteria can
include
improved potency, selectivity, pharmacokinetic properties, or reduced
toxicity. Each of
these issues appears amenable to library design. For example, compounds with
five-six
fold improved potencies were rapidly identified by synthesizing and screening
a small
second generation library that explored variants of the most active compounds.
The success of the directed library in finding potent inhibitors
demonstrates the power of coupling combinatorial libraries with structure-
based design.
Combinatorial libraries allow a larger area of molecular space to be explored
with the
functionality selected by the structure-based design, removing the need to
identify in
advance a single "best" target. Similarly, computational methods allow rapid
examination of extremely large virtual regimes > 10'° compounds) and
focus the
chemical efforts into productive regimes.
It addition to the above methods, additional methods for synthesizing the
aspartyl protease inhibitors of the present invention are disclosed in U.S.
Patent
CA 02367112 2001-09-24
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53
Application Serial No. , entitled "Nanomolar, Non-Peptide Inhibitors of
Plasmepsin," filed on March 24, 1995 and bearing Attorney Docket No. 023072-
0085320, the teachings of which are incorporated herein by reference.
IZ EXAMPLE II
A. Assays
1. Preparation and Maintenance of Entorhinohippocampal Slice
Cultures
Organotypic entorhinohippocampal cultures were prepared using the
technique of Stoppini, et al., J. Neurosci. Methods, 37, 173-182 (1991).
Briefly, the
caudal pole of the cerebral hemisphere containing the entorhinal cortex and
hippocampus
were harvested from brains of 6-7 days old Sprague-Dawley rat pups under
sterile
condition. 400 ~.m horizontal entorhinohippocampal sections cut vertical to
the long axes
of hippocampus were obtained using a McIlwain tissue chopper in a cutting
medium
consisting of MEM (with Earle's salts, Gibco), 25 mM HEPES, 10 mM Tris Base,
10
mM Glucose, and 3 mM MgCl2 (pH 7.2). Brain tissue explants were then planted
onto
30 mm cell culture inserts (Illicell-CM, Millipore, Bedford, MA) that were
placed in 6
well culture trays with 1 mL of growth medium (MEM with Hank's salts, Gibco,
20
horse serum, 3 mM glutamine, 25 mM HEPES, 5 mM NaHC03, 25 mM glucose, 0.5
mM ascorbate, 2 mM CaCl2, 2.5 mM MgCl2, 0.5 mg/L insulin, and penicillin, pH
7.2;
Bi, et al., J. Comp. Neuro., 401, 382-394 (1998). The cultures were incubated
at 35°C
with a 5 % C02-enriched atmosphere and fed every other day until use.
After 10-14 days in vitro, organotypic cultures were incubated with growth
medium containing either 20 ~,M N-CBZ-L-phenylalanyl-L-alanine-
diazomethylketone
(ZPAD; BACHEM Bioscience, Torrance, CA), a selective inhibitor of cathepsins B
and
L (Shaw and Dean, 1980), in 0.01 % DMSO, 20 ~cM chloroquine (Sigma) or vehicle
alone for days as specified. To test the effect of EA-1 on the generation of
hyperphosphorylated tau fragments found in neurofibrillary tangles in
Alzheimer's
disease and other tau pathology-related diseases, 1 ~,M of EA-1 or 10 ~cM of
CELS-172
were applied alone or together with 20 ~,M ZPAD.
2. Immunoblotting
For western blot, entorhinohippocampal explants were collected and
sonicated in 10 mM Tris-HCl buffer (pH 7.4) containing 0.32 M sucrose, 2 mM
EDTA,
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54
2 mM EGTA, and 0.1 mM leupeptin. Aliquots of homogenate (80-100 ~cg
protein/lane)
were diluted with equal amounts of 2x sample buffer [ lx sample buffer
consists of 2 %
sodium dodecyl sulphate (SDS), 50 mM Tris-HCl (pH 6.8), 10 % 2-
mercaptoethanol,
% glycerol and 0.1 % Bromophenol Blue] . After heating to 90-100 ° C
for 5 min,
5 proteins were subjected to SDS-PAGE performed according to the method of
Laemmli
( 1970) using 10 % polyacrylamide gel; and then transferred on to
nitrocellulose
membranes as described by Towbin, et al. , Proc. Natl. Acad. Sci. USA, 76,
4350-4354
( 1979) . Nitrocellulose membranes were first incubated in 3 % gelatin in Tris-
buffered
saline (TBS) for 1 hour at room temperature, followed by incubation with 1 %
gelatin in
10 TBS with 0.5 % Tween 20 (TTBS) containing antibodies that recognize either
the
phosphorylated tau protein (ATB; 1:500) or unphosphorylated tau protein (tau
1, PC1
C6; 1:100, Boehringer Mannheim) at room temperature overnight. After two
washes
with TTBS for 5 min, membranes were incubated with alkaline phosphatase
conjugated
anti-mouse IgG ( 1:3000; BioRad) for 2 hr at RT, then visualized with solution
containing
nitroblue tetrazolium and 5-brow-4-chlor-3-indolyl-phosphate toluidine salt
(BioRad) in
DMF according to the manufacturer's instruction. Immunoblots were scanned, and
the
digitized images were quantitatively analyzed by densitometry using the NIH
Image
analysis system program.
B. Results
1. Effect of CELS-172
Four groups of cultured entorhinohippocampal slices were maintained for
14 days and then exposed to one of the following treatments for six additional
days: (1)
control medium; (2) a selective inhibitor ('ZPAD' at 20 ~,M) of cathepsins B
and L; (3)
a selective inhibitor ('CELS-172' at 10 ~cM) of cathepsin D; (4) ZPAD combined
with
CELS-172. Following this, the slices were homogenized and samples processed
for
immunoblotting. Antibodies against phosphorylated tau or the carboxyterminal
region of
the amyloid precursor protein (anti-C APP~3_69s) were used. Previous studies
showed
that ZPAD alone increases the concentrations of a 27 kDa phosphorylated tau
fragment
and a 29 kDa APP fragment. Both results were confirmed as shown in the
accompanying figure. The cathepsin D inhibitor by itself did not detectably
change the
concentrations of either antigen (see, FIG. 10). It did, however, block most,
if not all,
of the increases in tau and APP fragments produced by ZPAD; densiometric
values for
CA 02367112 2001-09-24
WO 00/56335 PCT/US00/07804
5~
the combined treatment were close to control values and clearly reduced from
those for
ZPAD alone.
2. Effect of EA-1
Another four groups of cultured entorhinohippocampal slices were
maintained for 14 days and used to test the effect of EA-1: (1) control
medium; (2) 20
~,M of ZPAD; (3) a new selective inhibitor (EA-1 at 1 ~,M) of cathepsin D; (4)
ZPAD
combined with EA-1. Following this, the slices ere homogenized and samples
processed
for immunoblotting. Like CELS-172, EA-1 by itself did not detectably change
the
concentrations of hyperphosphorylated tau fragments (see, FIG. 11); however,
it
exhibited a much higher blocking effect than CELS-172. It is noted that EA-1
and
CELS-172 have the following structures, respectively:
o--~
0
ci ~ o
H N CHs
N~N
O~ ,
O ~ O
and
0
ct ~ o
H N CH3
H
N~N
H
O ~ O
/ .
IIL EXAMPLE Ill
A. Assays and Abbreviations
1. Abbreviations used
ACSF, artificial cerebrospinal fluid; EDTA, ethylenediaminetetraacetic
acid; EGTA, ethyleneglycol bis ((3-amino-ethylether) N,N,N',N'-tetraacetic
acid; PBS,
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56
phosphate-buffered saline; SDS, sodium dodecyl sulphate; TBS, Tris-buffered
saline;
ZPAD, N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone.
2. Preparation and maintenance of hippocampal slice cultures
Organotypic hippocampal cultures were prepared using the technique of
Stoppini; et al. (J. Neurosci. Meth., 37:173-182 (1991)). Briefly, hippocampi
were
harvested from brains of 9-11 days old Sprague-Dawley rat pups under sterile
condition.
Horizontal sections vertical to the long axis of hippocampus were cut at 400
~,m and
collected in a cutting medium consisting of MEM (with Earle's salts, Gibco),
25 mM
HEPES, 10 mM Tris Base, 10 mM Glucose, and 3 mM MgCl2 (pH 7.2). Slices were
positioned onto 30 mm cell culture inserts (Millicell-CM, Millipore, Bedford,
MA) that
were placed in 6 well culture trays with 1 ml of growth medium (MEM with
Hank's
salts, Gibco, 20 % horse serum, 3 mM glutamine, 25 mM HEPES, 5 mM NaHC03, 25
mM glucose, 0.5 mM ascorbate, 2 mM CaCl2, 2.5 mM MgCl2, 0.5 mg/1 insulin, and
penicillin, pH 7.2; 9). The cultures were incubated at 35 °C with a 5%
C02-enriched
atmosphere with medium changed every other day until use. Incubations were
carried
out for 14 days before the start of experiments. This period is sufficient for
the slices to
take on a variety of adult characteristics (Bahr, J Neurosci Res., 42:294-305
(1995); and
Muller, et al., Dev. Brain Res., 71:93-100 (1993)).
Experiments were carried out using a yoked design in which one of the six
culture trays was always used as a control and values for experimentally
treated slices
were expressed as percents of the same plate control. Hippocampal slice
cultures were
exposed to medium containing one of three cathepsin D inhibitors (see, below)
or to
'ZPAD' (N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone), a selective
inhibitor of
cathepsins B and L (Green, et al., J. Biol. Chem., 256:1923-1928 (1981);
Richardson, et
al., J. Cell Biol., 107:2097-2107 (1988); and Shaw, et al., Biochem. J.,
186:385-390
(1980)). ZPAD was used at 20 ~,M, and both ZPAD and cathepsin D inhibitors
were
dissolved first in dimethyl sulfoxide (DMSO), then diluted to the
concentrations needed
using culture media. Equal amount of DMSO ( < 0.1 % ) was also applied to
control
slices.
3. Recording and stimulation
Physiology experiments were performed on hippocampal slices kept in
vitro for 2 weeks followed by being incubated with cathepsin inhibitors for an
additional
CA 02367112 2001-09-24
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57
six days. The slices were placed in a submersion chamber containing artificial
cerebrospinal fluid (ACSF) and maintained at room temperature. The flow rate
of ACSF
through the recording chamber was 1.2 ml/min. Electrodes were positioned 120
min
after the slices had been placed in the chamber. Patch-clamp recordings were
made from
pyramidal neurons in the stratum pyramidale of area CA1. The recording
pipettes had
resistances of 3-5 MS2. Holding potentials were -70 mV . Currents were
recorded using a
patch amplifier with a 4-pole low-pass Bessel filter at 2 kHz and digitized at
10 kHz.
Field EPSPs were simultaneously recorded in stratum radiatum using low
resistance (2-6
MSI) microelectrodes. All experiments involved stimulation of the Schaffer
collateral/commissural afferents at 0.033 Hz using a bipolar stimulating
electrode placed
in stratum radiatum and were performed at room temperature.
4. Western blot analysis
Hippocampal slices were collected and sonicated in 10 mM Tris-HCl
buffer (pH 7.4) containing 0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, and 0.1 mM
leupeptin. Aliquots of homogenate (80-100 ,ug protein/lane) were diluted with
equal
amount of 2x sample buffer [lx sample buffer consists 2% sodium dodecyl
sulphate
(SDS), 50 mM Tris-HCl (pH 6.8), 10% 2 mercaptoethanol, 10% glycerol and 0.1
bromophenol blue. After heated to 90-100 °C for 5 min, proteins were
subjected to
SDS-PAGE performed according to the method of Laemmli (Nature, 227:680-685
( 1970)) using 10 % polyacrylamide gel; and then transferred on to
nitrocellulose
membranes as described by Towbin, et al. (Proc. Natl. Acad. Sci. USA, 76:4350-
4354
(1979)). Nitrocellulose membranes were first incubated in 3 % gelatin in Tris-
buffered
saline (TBS) for 1 hr at room temperature, followed by incubation with 1 %
gelatin in
TBS with 0.5 % Tween 20 (TTBS) containing antibodies that recognize either the
phosphorylated tau protein (ATB; 1:500; Innogenetics, Belgium),
unphosphorylated tau
protein (tau 1, PC1 C6; 1 : 100; Boehringer Mannheim, Indianapolis, IN), or
anti-
cathepsin D antibodies ( 1:100; Oncogene Science, Cambridge, MA) at room
temperature
overnight. After two washes with TTBS for 5 min, membranes were incubated with
alkaline phosphatase conjugated anti-mouse IgG (1:3000; BioRad) for 2 hrs at
room
temperature, then visualized with solution containing nitroblue tetrazolium
and 5-bromo-
4-chloro-3-indolyl-phosphate toluidine salt (BioRad) in dimethyl formamide
according to
the manufacture's instruction. Immunoblots were scanned and the digitized
images were
quantitatively analyzed by densitometry using the NIH Image analysis system
program.
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58
B. Results
Prior work showed that ZPAD and related drugs cause cultured slices to
develop several characteristic features of the aged human brain including
lysosomal
hyperplasia (Bednarski, et al., J Neurosci., 17:4006-21 (1997); Bi, et al.,
Exp Neurol.,
158:312-327 (1999); Yong, et al., Exp. Neurol., 157:150-160 (1999)),
disruptions in the
transport of hypothalamic releasing factors (Bi, et al. , J Comp Neurol.,
401:382-94
(1998)), meganeurites (Bednarski, et al:, J Neurosci., 17:4006-21 (1997); Bi,
et al.,
supra (1999); Yong, et al., supra (1999)), amyloidogenic peptides (Bahr, et
al., Exp
Neurol., 129:1-14 (1994)), hyperphosphorylated tau fragments (Bednarski, et
al., J
Neurochem., 67:1846-1855 (1996); Bi, et al., supra (1999)), and early stage
versions of
plaques and tangles (Bi, et al., supra (1999)). Importantly, certain of these
features
occur in regional patterns resembling those found in the aged human brain. No
obvious
changes in synaptic responses have been found after incubating hippocampal
slices with
ZPAD for 6-8 days (Fan and Lynch unpublished observation). Whether and to what
degree cathepsin D inhibitors block the formation of hyperphosphorylated tau
fragments
was tested in slices exposed to both ZPAD and the inhibitors. Incubations
continued for
2, 4, or 6 days after which the slices were tested for synaptic physiology or
processed
for histological or biochemical assays.
The three inhibitors used in the below experiments had molecular weights
of 650-800 Da and Ki's for cathepsin D between 1-15 nM (see, FIG. 12). They
were
products of a synthesis program in which the crystal structure of cathepsin D
complexed
with the peptide-based natural product pepstatin served as a model with which
to select
building blocks for a combinatorial library. Equivalent energy conformations
of a
(hydroxyethyl) amine scaffold were grouped into families and computational
methods
(Lewis, et al., J. Mol. Graph., 10:66-78, 106 (1992); Roe, Application and
development
of tools for structure-based drug design, University of California, San
Francisco, USA
(1995a); Roe, et al., J. Comput. Aided Mol. Des., 9:269-82 (1995b)) used to
position
Rl-3 moieties onto the scaffolds. Conformations with overlapping Rl-R2 groups
were
eliminated to reduce the combinatorial problem. A library of 1000 compounds
was
prepared with parallel synthesis and screened with a fluorometric assay for
activity
against cathepsin D (Krafft, et al., Methods Enzymol., 241:70-86 (1994)). The
clusters
for the R1-3 positions that generated the most active compounds were then used
to build
a small, second generation library (Kick, et al. , Chemistry & Biology., 4:297-
307
(1997)). Lower molecular weight compounds with better likelihood of membrane
CA 02367112 2001-09-24
WO 00/56335 PCT/CTS00/07804
59
penetration were identified using additional small, optimization libraries
(Hague, et al. ,
Nled. Chem., 42:1428-1440 (1999); Lee, et al., J. Am. Chem. Soc., 120:9735
(1998)).
These compounds were prepared in quantities sufficient for use with cultured
slices.
Six-day incubations with the inhibitor 'EA-1', in marked contrast to
ZPAD or chloroquine, did not produce detectable increases in the number of
lysosomes,
as can be seen in semi-thin sections through hippocampal field CAl (see,
FIGS.13A, B
and C) . Blockade of cathepsin D thus does not reduce protein breakdown to a
degree
sufficient for triggering lysosomal hyperplasia. This accords with evidence
that the
enzyme participates in limited proteolysis of biologically active proteins
rather than in
bulk degradation and that cathepsin D (-/-) mice are viable well into post-
natal life
(Saftig, et al., J. Biol. Chem., 271:27241-27244 (1996)).
Carboxy-terminal fragments of the amyloid precursor protein are a
characteristic feature of slices treated with ZPAD, chloroquine, or exogenous
amyloid
(Bahr, et al., Exp Neurol., 129:1-14 (1994); Bahr, et al., J Comp Neurol.,
397:139-147
(1998)). The cathepsin D inhibitors did not induce these peptides (not shown).
The
compounds were also without evident effect on inhibitory and excitatory
synaptic
currents, extracellular field potentials, or post-synaptic responses to
repetitive stimulation
(FIG. 13D) . In all, the new inhibitors are selective in that they do not
elicit anatomical
and biochemical changes found with inhibitors of cathepsins B and L, or with
more
generalized lysosomotropic agents, and do not influence sensitive
physiological indices.
Antibodies (e.g., 'AT8'; Goedert, et al., Proc. Natl. Acad. Sci. USA,
90:5066-70 (1993); Greenberg, et al., Proc. Natl. Acad. Sci. USA, 87:5827-31
(1990))
against hyperphosphorylated tau or paired helical filaments in human brain
variably label
a 29 kDa band in western blots from adult rat brains (Bednarski, et al., J
Neurochem.,
67:1846-1855 (1996)) or 'mature' cultured slices (Bi, et al., supra (1999)).
Antibodies
against native tau recognize this peptide to a lesser degree than they do tau
itself ; ATB,
conversely, labels the 29 kDa peptide much more intensely than it does native
tau (Bi, et
al., supra (1999)). Moreover, the interaction of native tau and the 29 kDa
peptide with
tau 1 antibody is eliminated by phosphatase inhibitors. These results
established that the
29 kDa band consists mainly of hyperphosphorylated tau fragments ('tau 29').
Immunoblots probed with AT8 confirmed earlier reports that a 6-day treatment
of
hippocampal cultures with inhibitors of cathepsins B and L causes a marked
increase in
the concentration of tau 29 (FIG. 14A, upper panels; lanes 1 vs 2/3). Six-day
incubations with the cathepsin D inhibitor CELS at 5 ~,M (lane 4) had little
if any effect
CA 02367112 2001-09-24
WO 00/56335 PCT/US00/07804
in this regard. They did, however, markedly reduce the increase in tau-29
produced by
ZPAD (lanes 4,5). The bottom segment of FiG. 14A summarizes results from 5
separate experiments, each involving 6-8 slices per treatment condition. As
shown,
ZPAD by itself caused a 104 ~ 18 % (mean ~ s. e.m. ) increase in the
5 hyperphosphorylated tau fragment, a value which was reduced to 34 ~ 18 % in
the
presence of the cathepsin D inhibitor. Note that concentrations of the
fragment in slices
treated with CELS by itself were the same as those in control slices. The
difference
between the two conditions (ZPAD vs. ZPAD + CELS) was in the predicted
direction
and statistically significant. Results similar to these were obtained with two
small,
10 structurally distinct non-peptide cathepsin D inhibitors. FIG. 14B
summarizes the results
for EA-1. This compound again had no detectable effects on tau 29
concentrations (lane
3 of FIG. 14B), but virtually eliminated the increase caused by ZPAD (compare
lanes 2
and 4). Densitometric measurements for 5 experiments (FIG. 14B, bottom)
confirmed
that fragment levels were not statistically different from control in slices
exposed to
15 ZPAD and the cathepsin D inhibitor. Incubation with ZPAD and cathepsin D
inhibitors
did not cause detectable changes in native tau when probed with AT8 (FIG.
14C), while
a marked decrease was revealed with tau 1 antibodies (FIG. 14C; see, the
following
section for detailed analysis).
FIG. 15 describes the time and dose dependencies of the interactions
20 between cathepsin inhibitors. ZPAD induced increases in the phosphorylated
tau
fragment appeared at 48 hrs -- the earliest time point tested -- and increased
steadily
thereafter (FIG. 15A). The effect of the cathepsin D inhibitor was evident
from the first
measurement and resulted in a complete blockade of the ZPAD-elicited changes
by 96
hrs. The inhibitor EA-1 had dose dependent effects in slices treated with ZPAD
for 6
25 days (FIG. 15B); threshold concentration appeared to lie between 0.05 (no
detectable
effect) and 1.0 ~cM (41 % reduction in ZPAD induced fragments) . Note that the
cathepsin D inhibitor by itself had no effect on tau 29 concentrations at any
time point or
dosage.
Increases in tau fragments are accompanied by measurable decreases in the
30 concentration of native tau, as shown in the experiment summarized in FIG.
16A. An
antibody against native (unphosphorylated) tau was used to measure the effects
of six-day
treatments with ZPAD on the concentrations of four known tau isoforms. As
described
in FIG. 16, tau concentrations in the experimental slices (solid bars) were
reduced by an
average of 19-32 % from yoked controls (open bars) (p < 0.01, ttest, 2-tail,
n=8) and this
CA 02367112 2001-09-24
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61
was accompanied by a large increase (101 % ) in the weakly labeled 29 kDa
fragments.
The inhibitor EA-1 blocked the latter effect, but not the former. Conversion
of cathepsin
D into active forms may involve autocatalysis (Corner, Biochem. J., 263:601-
604
(1989); Corner, et al., Biochem., 28:3530-3533 (1989); and Hasilik, et al.,
Eur. J.
Biochem., 125:317-321 (1982)). If so, then the inhibitors used here could
indirectly
block the formation of tau fragments by preventing the increases in lysosomal
and
cytoplasmic catliepsin D that develop within hours of chemically induced
lysosomal
dysfunction (Bednarski, et al., supra (1998); Hoffman, et al., Neurosci.
Lett., 250:75-78
( 1998)) . To test whether cathepsin D inhibitors have any effect on the
biosynthesis and
maturation of cathepsin D, cultured hippocampal slices were treated with
inhibitor alone
or inhibitor plus ZPAD. EA-1 by itself did not detectably alter the levels of
cathepsin D
isoforms at concentrations from 50 nM to 5 ~,M (FIG. 16B). When hippocampal
cultures were incubated with ZPAD and cathepsin D inhibitor, the levels of
procathepsin
D and single chain cathepsin D were similar to those observed in cultures
treated with
ZPAD alone. However, the increase in heavy chain isoform was substantially
reduced
(70 % to 15 % , FIG. 16) in the presence of EA-1
C. Discussion
The above data constitute the first results on the effects of transiently and
selectively suppressing cathepsin D in mature brain tissue. The novel
inhibitors did not
induce a robust lysosomal hyperplasia, an effect typically seen with
pharmacological or
genetic disturbances of intra-lysosomal functioning (Bednarski, et al., J
Neurosci.,
17:4006-21 (1997); Bi, et al., supra (1999); Braak, et al., Acta Neuropathol.,
46:79-83
(1979); Purpura, et al., Brain Res., 116:1-21 (1976); Yong, et al., supra
(1999)). The
compounds did not cause evident physiological changes over the time courses
tested and
leave unchanged biochemical measures sensitive to cathepsins B/L inhibitors or
to the
broad-spectrum inhibitor chloroquine. It appears, then, that inhibition of
cathepsin D to
a degree sufficient to block specific biochemical reactions (below) has
discrete
consequences and, in general, is well tolerated by brain tissue for at least
several days.
The findings also provide a direct test of the hypothesis that the rapid
formation of hyperphosphorylated tau fragments occurring in association with
lysosomal
dysfunction is due to cathepsin D, or cathepsin D-like aspartyl proteases.
Three distinct
inhibitors produced near complete suppression of the increases that normally
follow
pharmacologically induced lysosomal dysfunction. The blocking effects were in
evidence
CA 02367112 2001-09-24
WO 00/56335 PCT/US00/07804
62
from the first appearance of tau fragmentation and had threshold
concentrations in the
sub-micromolar range. That the inhibitors did not reduce baseline levels may
indicate
that the fragments have a long half life, a point of possible significance
with regard to
the production of tangles. The differential effects of cathepsin D inhibitors
on ZPAD-
induced tau 29 vs basal level tau 29 demonstrate that the blocking effect is
not due to
modification of the antigenic epitopes by these non-peptidic compounds.
Cathepsin D
inhibitors markedly reduced the formation of tau 29, but did not reverse
decreases in
native tau, suggesting that cathepsin D is not solely responsible for the
breakdown of tau
protein that occurs following pharmacologically induced lysosomal dysfunction.
Inhibition of cathepsin B and L increases procathepsin D and its
maturation into the active, two-chain form (composed of heavy and light chain)
within
lysosomes, as described earlier (Bednarski, et al. , supra ( 1998); Hoffman,
et al. , supra
(1998)) and confirmed here. These events, in common with other circumstances
involving lysosomal impairments (Nakamura, et al., Neurosci. Lett., 97:215-220
(1989);
Nakanishi, et al. , J. Neurochem., 68:739-739 (1997); Nakanishi, et al. , Exp.
Neurol.,
126:119-128 (1994)) are accompanied by lealrage of active cathepsin D into the
cytoplasm (Bednarski, et al. , NeuroReport, 9:2089-2094 ( 1998); Nakamura, et
al. , supra
1989; Nakanishi, et al., supra (1997); Nakanishi, et al., supra (1994)),
reductions in the
concentration of unphosphorylated tau proteins, and increases in
hyperphosphorylated tau
fragments. The cathepsin D inhibitors did not significantly affect the
increases in pro- or
single chain forms of cathepsin D, but blocked that for the heavy chain. This
strongly
suggests that autocatalysis plays an important role in the maturation of
cathepsin D, as
previously proposed (Corner, supra (1989); Hasilik, et al., supra (1982)), at
least under
conditions in which biosynthesis is accelerated. Blockade of heavy chain
formation
would presumably reduce the active cathepsin D available for leakage and thus
for the
neutral pH cleavage of tau at amino acids 200-257, the event that results in
the 29 kDa
product (Bednarski, et al. , supra ( 1996); Kenessey, et al. , J. Neurochem. ,
69:2026-
2038 (1997)).
The potent and selective effects described above indicate that the inhibitors
have therapeutic value. With longer incubation periods, hyperphosphorylated
tau
fragments in cultured slices assemble into structures having the appearance,
size, and
epitopes of early stage neurofibrillary tangles in human brain (Bi, et al.,
supra (1999)).
Accordingly, there is reason to expect that blocking their formation would
slow the
production of a primary component of AD. Beyond this, extra-lysosomal
cathepsin D is
CA 02367112 2001-09-24
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63
one of a collection (see, Murphy, et al., J. Biol. Chem., 274:11914-11923
(1999)) of
enzymes that may generate Beta-amyloid (Austen, et al., Biomed. Pept Proteins
Nucleic
Acids, 1:243-6 (1995); Chevallier, et al., Brain Res., 750:11-9 (1997);
Dreyer, et al.,
Eur. J. Biochem., 224:265-271 (1994); Estus, et al. , Ann N Y Acad Sci.,
674:148-148
( 1992); Mackay, et al. , Eur. J. Biochem., 244:414-425 ( 1997)) and has
recently been
linked to apoptosis (Isahara, et al., Neuroscience, 91:233-49 (1999); Levy-
Strumpf, et
al. , Oncogene, 17:3331-3340 (1998); Ohsawa, et al. , Arch. Histol. Cytol.,
61:395-403
(1998); Roberg, et al., Am. J. Pathol., 152:1151-1156 (1988); Shibata, et al.,
supra
( 1998)) .
In view of the foregoing results, in a preferred embodiment of the present
invention, the cathepsin D inhibitor is a compound selected from the group
consisting of
CELS-A, CELS-G and EA-1, the structures of which are set forth in FIG. 12.
It is to be understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent to those
of skill in
the art upon reading the above description. The scope of the inventio~t
should, therefore,
be determined not with reference to the above description, but should instead
be
determined with reference to the appended claims, along with the full scope of
equivalents to which such claims are entitled. The disclosures of all articles
and
references, including patent applications and publications, are incorporated
herein by
reference for all purpose.
