Note: Descriptions are shown in the official language in which they were submitted.
CA 02412954 2002-12-30
WO 97/30072 PGTIL1S97/02930 -
PEPTIDOMIMETIC INHIBITORS OF CATHEPSIN D
AND PLASMEPSINS I AND II
FIELD Ol~ THE INVENTION
The invention relates to linear and cyclic inhibitor compounds of
cathepsin D and plasmepsins I and II, and the use of these compounds for the
prevention and treatment of diseases, and the like.
BACKGROUND OF THE INVENTION
Human cathepsin D is an intracellular aspartic protease normally found
in lysozymes of all cells. The main physiological action of cathepsin D is
degradation
of cellular and phagocytosed proteins. Cathepsin D has also been implicated in
a
number of diseases. Elevated levels of cathepsin D have been correlated with
poor
prognosis in breast cancer. It has also been correlated with increase cell
invasion and
increased risk of metastasis, as well as shorter relapse-free survival. (See
Rochefort,
H., ~emin. Canser Biol. 1: 153 (1990) and Tandon, A. K. et al. N. Eng. J. Med.
322, 297 (1990)). The increased level of secretion of cathepsin D in breast
cancer
cells is due to both overexpression of the gene and altered processing of the
protein.
High levels of cathepsin D and other proteases, such as collagenase, produced
in the
vicinity of the growing tumor may degrade the extracellular matrix and thereby
promote the escape of cancer cells to the lymphatic and circulatory systems
and
enhance the invasion of new tissues. (Liotta L. A., Scientific American Feb:
54
(1992); and Liotta L. A. and Stetier-Stevenson W.G., Cancer Biol. 1: 99
(1990)).
Most deaths incurred from cancer are due to its metastatic spread to secondary
organs,
therefore an inhibitor of metastasis would have widespread therapeutic use.
Cathepsin D is also believed to be associated with degenerative brain
changes, such as those associated with Alzheimer's disease. Cathepsin D is
associated
with cleavage of amyloid-(3-protein precursor, (Cataldo, A.M. et al., Proc.
Natl.
A~~d. ci. 87: 3861 ( 1990)) including a mutant precursor that enhances amvloid
protein production in transfected cells (Ladror, U.S., et al. J. Biol. Chem.
269: 18422
CA 02412954 2002-12-30
WO 97/30072 PCTlUS97~2930
-2-
(I994)). The amyloid-/3-protein that results from proteolysis of the amyloid-a-
protein
precursor leads to senile plaque formation in brains and may be responsible
for
Alzheimer's disease. Recently elevated levels of cathepsin D have been found
in
cerebrospinal fluid in Alzheimer's patients (Schwager, A.L., et al. J.
Neurochem. 64:
443 (1995).
There is little known about substrate specificity and specific inhibitors
for cathepsin D (Agarwal, N.S. and Rich, D.H., J. Med. Chem. 29: 2519 (1986);
Jupp, R.A. et al., Biochem. J. 265: 871 (1990); Scarborough, P.E. et al., Prot
in
science 2: 264 (1993); Baldwin, E.T. et al., Proc. Natl. Acad. Sci. 90: 6796
( 1993)).
Accordingly, there is a need for the synthesis of compounds which are
specific for the inhibition of the activity of cathepsin D and which may be
used in the
treatment of metastatic disease and Alzheimer's disease.
A number of cathepsin D inhibitors have been reported (Lin, T. Y. and
Williams, H.R., J. Biol. Chem. 25: 11875 (1970)). Agarwal and Rich reported
the
design and synthesis of cathepsin D inhibitors wherein the scissile dipeptide
unit in a
substrate sequence was replaced with a statine (Sta) residue or by a
phenylstatine (Pst)
unit. (Agarwal, N.S. and Rich, D.H., J. Med. Chem. 29: 2519 (1986)). Further,
Agarwal and Rich evaluated the inhibition of cathepsin D by various analogues
of
pepstatin, finding that the fragment spanning P, to P', is necessary for the
maximum
inhibition of bovine cathepsin D.
U.S. Patent No. 4,746,648 describes peptide derivatives, modeled on
the basis of pepstatin, which inhibit renin and acid protease. Tamburini et
al., EP 0
569 777 A2 relates to the use of cathepsin D inhibitors for Alzheimer's
disease.
Patents relating to cyclic peptide inhibitors of renin include Boger et al.,
U.S. Patent
No. 4.489.099 and Watkins, U.S. Patent No. 4,906,613.
Cyclizing peptidornimetics can increase binding to a target enzyme due
to preorganization into the desired conformation. Additionally, such
macrocycles
offer increased stability against proteolytic cleavage. An area where this
approach has
been extensively explored is that of renin inhibitors, where various cycles
connecting
CA 02412954 2002-12-30
wo 9~r~oo~ rc'r~rs9~roZ93o -
-3-
- different positions were introduced. The most successful approach was
cyclizing from
the P, to P', position to generate a series of potent, orally bioavailable
renin inhibitors
(Weber, A.E. et al. J. Med. Chem. 34: 2692 (1991); Dhanoa, D.S. et al. et. Le
t.
33: 1725 (1992); Weber, A.E. et al. J. MediChem. 35: 3755 (1992); Yang, L, et
al.
Te . 34: 7035 (1993)). Cyclization of P, to P~ also gave potent renin
inhibitors
(Sham, H.L. et al. J. Chem. Six. Chem. Commun. 66b (1990); Thaisrivvngs, S. et
al. ~ Med. Chem. 34: 1276 (1991)). Some other cycles have been studied
(Szewczuk, Z. et al. Int. J. Pelt. Prot. Res. 40: 233 (1992); Sham, H.L. et
al. ~
,yied. Chem. 31: 284 (1988); Dutta, A.S. et al. J. Med. Chem. 33: 2552 and
2560
( 1990)), but the only example of which we are aware of a PZ to P~3 bridge in
an
aspartyl protease inhibitor is a simple disulfide (Boger,1. Peptides 1983, pp.
569-578,
Proceedings of the 8th American Peptide Symposium).
The cyclic compounds described herein differ significantly from this
prior art. Many of the compounds incorporate a P~ to P ; cycle where the
scissile
bond isostere is pan of the ring. Since the macrocycle spans a large portion
of the
binding cleft, activity is retained after truncation to remove the exocyclic
backbone
extension. This has the dual advantages of decreasing molecular weight and
removing
the residues most subject to metabolic cleavage.
Modeling studies reveal that the active sites of the aspartic
hemoglvbinases (Plasmepsins I and II) from Plasmodium falciparurn are highly
homologous with that of human cathepsin D. Goldberg, et al. isolated and
characterized plasmepsins I and II, aspartic proteases responsible for the
initial
cleavage of hemoglobin that occurs inside the protozoan Plasmodium's digestive
vacuole (Goldberg, D.E. et al., J.~p. Med. 173: 961 (1991), Hill, J. et al.
FEES
Let rs 352: 155 (1994)). Protozoans of the genus Plasmodium are the causative
agents of malaria. The cleavage of hemoglobin by plasmepsins I and II occurs
at sites
within the hemoglobin sequence that are conserved in human hemoglobins. _
These
cleavage events are essential for the conformational breakdown of hemoglobin
that
enables its subsequent cleavage by a series of other proteolytic enzymes. The
digested
hemoglobin is a primary nutrition source for the malarial parasite, which
cannot grow
CA 02412954 2002-12-30
4
in the absence of functional hemoglobinases. Goldberg has demonstrated
that inhibitors of plasmepsins can kill the parasite in a cell culture of
infected
human erythrocytes (Goldberg, D.E. et al., EMBO J. 13: 306 (1994),
Gluzman, I.Y. et al. J. Clin. Invest. 93: 1602 (1994)).
Therefore, there is a need for cathepsin D inhibitors in the treatment of
Alzheimer's disease, cancer, and for aiding in the further elucidation of the
roles of cathepsin D in human diseases and a need for plasmepsin inhibitors
to treat malaria.
It is an object of an aspect of the present invention to provide cathepsin
1o D and plasmepsin I and II inhibitors for use in the treatment of metastatic
disease, for use in the inhibition of cleavage of ~i-amyloid precursors and
for
the prevention of progressive neurological dysfunction in Alzheimer's disease,
as well as for the prevention of the growth of Plasmodium parasites including
Plasmodium falciparum, the most deadly cause of malaria.
SUMMARY OF THE INVENTION
The present invention relates to cathepsin D and plasmepsin I and II
inhibitor compounds and their se as pharmaceutically active agents. The
2o present invention further provides for uses of these inhibitors for the
prevention and treatment of diseases, such as, for example, cancer,
Alzheimer's disease, and malaria.
Specifically, the present invention provides for novel linear compounds
as well as branched and cyclic analogs of these compounds which are
inhibitors of cathepsin D and which exhibit inhibitory potency against
plasmepsins I and II. Further, the present invention relates to novel related
cyclic compounds which are cathepsin D and plasmepsin I and II inhibitors.
The present invention also provides for pharmaceutical compositions
comprising effective amounts of at least one of the present inhibitors for use
in
3o the prevention and treatment of diseases, such as cancer, Alzheimer's
disease, and malaria.
CA 02412954 2002-12-30
Specifically, the present invention provides for pharmaceutical
compositions which decrease the levels of activity of cathepsin D present in a
subject, thereby inhibiting cancer cell invasion and metastasis.
The present invention also provides for pharmaceutical compositions
comprising at least one of the present inhibitors for the treatment and
prevention of Alzheimer's disease which decrease the occurrence of cleavage
of amyloid-~-protein precursors and senile plaque formation in a subject.
Further, the present invention provides for pharmaceutical
compositions which inhibit plasmepsin I or II or both and prevent hemoglobin
degradation and are thus useful in the treatment of malaria.
In accordance with one embodiment, the present invention provides a
compound of formula (I):
O RZ OH 0 R~- O R6 (I)
H H
RNH ~ N N NHR~
H H S
R~ O R3 . O R; O
in which R, R~ = represent hydrogen, alkoxycarbonyl, aralkoxycarbonyl,
alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,
cycloalkylalkanoyl, aralkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyl,
aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylalkanoyl, heterocyclylalkoxycarbonyl, heteroaralkanoyl,
heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaroyl, alkyl, alkenyl,
cycloalkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl,
aminocarbonyl, aminoalkanoyl, mono- and disubstituted aminocarbonyl and
mono- and disubstituted aminoalkyl, alkoxyalkyl, alkylthioalkyl, mono- and
disubstituted aminoalkanoyl radicals wherein the substituents are selected
from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heteroaralkyl,
heterocycloalkyl, heterocycloalkylalkyl radicals, or where said aminoalkanoyl
radical is disubstituted, said substituents along with the nitrogen atom to
which they are attached form a heterocycloalkyl or heteroaryl radical;
R~, R2, R4, R5, Rs = optionally substituted lower alkyl, lower cycloalkyl,
aryl,
CA 02412954 2002-12-30
5a
aralkyl and heteroaryl;
R3 = optionally substituted lower alkyl, lower alkoxy, lower alkylthio, mono
or
di-lower alkyl amino, aralkyl, aralkoxy, aralkylthio, aralkylamino,
cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalkylamino;
and
wherein R2 and R5 or R~ and R3 are connected by an optionally substituted
bridging moiety comprised of a stable combination of C, N, O, or S atoms.
In accordance with a further embodiment, the present invention provides a
compound
which
is
selected
from
the
group
consisting
of
(1 Iva-Val-Val-Sta-Val-Leu-Gly-NH2(SEQ. ID. NO.
) 1 );
(2) Iva-Gln-Val-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
2);
(3) Iva-Lys-Val-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
3);
(4) Tba-Val-Val-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
4);
(5) Iva-Val-Ile-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
5);
(6) Iva-Val-Leu-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
6);
(7) Tba-Val-Val-Pst-Val-Leu-Gly-NH2(SEQ. ID. NO.
7);
(8) Tba-Val-Cys-Pst-Val-Leu-Gly-NH2(SEQ. ID. NO.
8);
(9) Tba-Val-Glu-Pst-Val-Leu-Gly-NH2(SEQ. ID. NO.
9);
(10)Tba-Val-Asp-Pst-Val-Leu-Gly-NH2(SEQ. ID. NO.
10);
(11 Iva-Val-Val-Sta-Ala-Leu-Gly-NH2(SEQ. ID. NO.
) 11 );
(12)Tba-Val-Cys-Pst-Val-Cys-Gly-NH2(SEQ. ID. N0.12);
NH CO
.,,,~
OH
COVaINH CONH ~COVaINH COGIyNHz
(SEQ. ID. NO. 28);
CA 02412954 2002-12-30
5b
CO NH
_, .. ,,,
H
COVaiNH CONH COVaINH COGiyNHi
s
(SEQ. ID. NO. 29);
NH
CO
~/
OH
COVaINH CONH ~~COVaINH COGIyNH~
(SEQ. ID. NO. 30);
NH CO
OH
COVaINH CONH ~ COVaINH COGIyNHi
a
(SEQ. ID. NO. 3I);
CA 02412954 2002-12-30
5c
....,''' S'~.'CH2 -CHZ~
S
OH
COVaINH CONH ~COVaINH COGI NH
Y z
(SEQ. ID. NO. 32);
S',.. CH=-CHZ-C HZ ~
S
H
COVaINH CONH COVaINH COGIyNH~
(SEQ. ID. NO. 33);
S/.CHZ-CH=-CHZ-CHz ~S
H
COVaINH CONH COVaINH COGIyNH~
(SEQ. ID. NO. 34);
CA 02412954 2002-12-30
5d
...~~ s s
off
~ COVeINH - CONH ~COVaINH COGIyNHZ
(SEQ. ID. IVO. 3S);
.,.aa
off
CONH ~ COVaINH COVaILeuGIyNH~
1
CONH
(SEQ. ID. NO. 36);
~,,.~a
OH
CONH ~ COVaINH .,,,/~,~~ COVaILeuGIyNHZ
r
i
CONH
(SEQ. ID. NO. 37).
In accordance with a further embodiment, the present invention provides
CA 02412954 2002-12-30
5e
a compound of formula II:
Rz off o
H
N
RNH ~ v -NHR~
II
o Rs
in which R, R~ = represent hydrogen, alkoxycarbonyl, aralkoxycarbonyl,
alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,
cycloalkylalkanoyl, aralkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyl,
aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylalkanoyl, heterocyclylalkoxycarbonyl, heteroaralkanoyl,
heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaroyl, alkyl, alkenyl,
cycloalkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl,
aminocarbonyl, aminoalkanoyl, mono- and disubstituted aminocarbonyl and
mono- and disubstituted aminoalkyl, alkoxyalkyl, akylthioalkyl, mono- and
disubstituted aminoalkanoyl radicals wherein the substituents are selected
from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heteroaralkyl,
heterocycloalkyl, heterocycloalkylalkyl radicals, or where said aminoalkanoyl
radical is disubstituted, said substituents along with the nitrogen atom to
which they are attached form a heterocycloalkyl or heteroaryl radical;
R2, = optionally substituted lower alkyl, lower cycloalkyl, aryl, aralkyl and
heteroaryl;
R3 = optionally substituted lower alkyl, lower alkoxy, lower alkylthio, mono
or
di-lower alkyl amino, aralkyl, aralkoxy, aralkylthio, aralkylamino,
cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalkylamino;
with the proviso that NHR~ does not comprise or form an alpha-amino acid.
In accordance with a further embodiment, the present invention
provides a compound of formula III:
CA 02412954 2002-12-30
5f
OH 0 R,
RNH NHR~
N
H III
O R~ O
in which R, R~ = represent hydrogen, alkoxycarbonyl, aralkoxycarbonyl,
alkylcarbonyl, cycloalkykarbonyl, cycloalkylalkoxycarbonyl,
cycloalkylalkanoyl,
aralkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyi, aryloxyalkanoyl,
heterocyclylcarbonyl, heterocyclyloxycarbonyl, heterocyclylalkanoyl,
heterocyclylalkoxycarbonyl, heteroaralkanoyl, heteroaralkoxycarbonyl,
heteroaryloxycarbonyl, heteroaroyl, alkyl, alkenyl, cycloalkyl, aryl, aralkyl,
aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl, aminocarbonyl, aminoalkanoyl,
hydroxyalkanoyl, alkenoyl, alkoxyalkanoyl, alkylthioalkanoyl,
arylthioalkanoyl,
alkoxycarbonylalkanoyl, hydroxycarbonylalkanoyl, aryloxycarbonylalkanoyl,
heteroaryloxycarbonylalkanoyl, aminocarbonylalkanoyl, mono- and
disubstituted aminocarbonylalkanoyl, alkylthioalkoxycarbonyl,
arylthioalkoxycarbonyl, cycloalkylthioalkoxycarbonyl,
heteroarylthioalkoxycarbonyl, heterocyclylalkylthioalkoxycarbonyl,
cycloalkyloxycarbonyl, hydroxyalkoxycarbonyl, alkoxyalkoxycarbonyl,
aryloxyalkoxycarbonyl, heteroaryloxyalkoxycarbonyl,
cycloalkyloxyalkoxycycarbonyl, heterocyclylalkyloxyalkoxycarbonyl,
cycloalkyloxyalkanoyl, heterocyclyloxyalkanoyl, heteroaryloxyalkanoyl,
cycloalkylthioalkanoyl, heterocyclylalkylthioalkanoyl, heteroarylthioalkanoyl,
aralkenoyl, mono- and disubstituted aminoalkoxycarbonyl, mono-and
disubstituted aminocarbonyl and mono- and disubstituted aminoalkyl,
alkoxyalkyl, alkylthioalkyl, mono- and disubstituted aminoalkanoyl radicals
wherein the substituents are selected from alkyl, aryl, aralkyl, cycloalkyl,
cycloalkylalkyl, heteroaryl, heteroaralkyl, heterocycloalkyl,
heterocycloalkylalkyl radicals, or where said aminoalkanoyl radical is
disubstituted, said substituents along with the nitrogen atom to which they
are
attached form a heterocycloakyl or heteroaryl radical;
CA 02412954 2002-12-30
5g
R4 = optionally substituted lower alkyl. lower cycloalkyl, aryl, aralkyl and
heteroaryl;
R3 = optionally substituted lower alkyl, lower alkoxy, lower alkylthio, mono
or
di-lower alkyl amino, aralkyl, aralkoxy, aralkylthio, aralkylamino,
cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalkylamino;
with the proviso that R does not comprise an alpha-amino acid.
In accordance with a further embodiment, the present invention provides
a compound which is selected from the group consisting of
i
CONH r :OValNH
OH
(SEQ. ID. NO. 18);
N
COVaINH COAIaLeuNH
N v . 2
H OH
(SEQ. ID. NO. 19);
CA 02412954 2002-12-30
5h
COVaINH ~ ~COAIaLeuNH=
OH
(SEQ. ID. NO. 20);
~N~
COVaINH NHS
OH
(SEQ. ID. NO. 21);
\ \
N COVatNH COAtaLeuNH2
OH
(SEQ. ID. NO. 22);
CA 02412954 2002-12-30
5i
~N~ COVaINH :OAtaLeuNHz
OH
(sEQ. ID. No. 2~);
0
~COVaiNH t COAIaLtuNHz
s
OH
ISEQ. ID. NO. 24);
i
COVaiYaINH COAIaLeuNHz
OH
(SEQ. ID. NO. 25);
CA 02412954 2002-12-30
5j
l
w
0
~ COVatVaINH COAIaLeuNHz
a
ON
(SEQ. ID. NO. 26);
~0
COVatVatNH ;OVaILeuNH ~'~./N~"/
OH
(SfiQ. ID. NO. 2~.
In accordance with a further embodiment, the present invention provides
a compound of formula II:
RZ H OH O
N
RNH ~ ~ -NHA~
B
O R~
in which R, R~ = represent hydrogen, alkoxycarbonyl, aralkoxycarbonyl,
alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,
cycloalkylalkanoyl, aralkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyl,
aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylalkanoyl, heterocyclylalkoxycarbonyl, heteroaralkanoyl,
CA 02412954 2002-12-30
5k
heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaroyl, alkyl, alkenyl,
cycloalkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl,
aminocarbonyl, aminoalkanoyl, mono- and disubstituted aminocarbonyl and
mono- and disubstituted aminoalkyl, alkoxyalkyl, alkylthioalkyl, mono- and
disubstituted aminoalkanoyl radicals wherein the substituents are selected
from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heteroaralkyl,
heterocycloalkyl, heterocycloalkylalkyl radicals, or where said aminoalkanoyl
radical is disubstituted, said substituents along with the nitrogen atom to
which they are attached form a heterocycloalkyl or heteroaryl radical;
R2,= optionally substituted lower alkyl, lower cycloalkyl, aryl, aralkyl and
heteroaryl;
R3 = optionally substituted lower alkyl, lower alkoxy, lower alkylthio, mono
or
di-lower alkyl amino, aralkyl, aralkoxy, aralkylthio, aralkylamino,
cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalkylamino;
and
wherein R and R3 are connected by an optionally substituted bridging moiety
comprised of a stable combination of C, N, O, or S atoms.
These and other features of the invention will be better understood
through the following detailed description of the invention. The scope of the
invention is limited only through the claims appended hereto.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds of formula (I):
O R~ OH 0 R4- O Re
RNN _ N _ N NHR~
H ~ v H ~ H
R~ O R3 . O R5 O
in which R, R~ = represent hydrogen, alkoxycarbonyl, aralkoxycarbonyl,
alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,
cycloalkylalkanoyl, aralkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyl,
CA 02412954 2002-12-30
51
aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylalkanoyl, heterocyclylalkoxycarbonyl, heteroaralkanoyl,
heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaroyl, alkyl, alkenyl,
cycloalkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl,
aminocarbonyl, aminoalkanoyl, mono- and disubstituted aminocarbonyl and
mono- and di-substituted aminoalkyl, alkoxyalkyl,
CA 02412954 2002-12-30
WO 97!3007Z PCf/US97/02930 , -
-6-
- - akylthioalkyl, mono- and disubstituted aminoalkanoyl radicals wherein the
substituents
are selected from alkyl, aryl, aralkyl, cycloalkyi, cycloalkylalkyl,
heteroaryl,
heteroaralkyl, heterocycloalkyl, heterocycloalkylalkyl radicals, or where said
aminoalkanoyl radical is disubstituted, said substituents along with the
nitrogen atom
to which they are attached form a heterocycloalkyl or heteroaryl radical;
R,, R2, R,, R5, R.6 = the side chain of a residue of an amino acid remaining
after
formation of a peptide linkage and include optionally substituted lower alkyl,
lower
cycloalkyl, aryl, aralkyl and heteroaryl;
R3 = optionally substituted lower alkyl, lower alkoxy, lower alkylthio, mono
or di-
lower alkyl amino, aralkyl, aralkoxy, aralkylthio, aralkylamino,
cycloalkylalkyl.
cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalkylamino. Also encompassed
are
compounds where RZ and RS or R, and R3 are connected by a bridging moiety
having
4-14 atoms comprised of any stable combination of C, N, O, or S. This chain
may
be optionally substituted by halo, hydroxy, amino, lower alkyl, lower alkoxy,
lower
alkylthio, mono or di-lower alkyl amino, oxo, thiono, alkylimino, mono- or
dialkylmethylidene, COR3. The bridging group may also be unsaturated so as to
include the residues of alkenes, imines, alkynes and alienes. Furthermore, any
part
of the bridging moiety may comprise part of an optionally substituted
aromatic,
heteroaromatic, or cycloalkyl ring. Amino acids from which the residues
containing
R, . R,, R,, RS and R6 can be derived include glycine, alanine, valine,
leucine,
isoleucine, serine, threonine, cysteine, methionine, aspartic acid,
asparagine, glutamic
acid, glutamine, lysine, hydroxylysine, arginine, phenylalanine, tyrosine,
tryptophan,
and histidine. Useful substituents and the optional substituted R,, Rz, R,, R5
and R6
moieties include the functional groups which are attached to the above named
aminoacids.
The present invention further relates to compounds of the Formula (I1):
p? OH O
H
N NHR~
RNH ~ II
O Rs
CA 02412954 2002-12-30
WO 97130072 PCTIITS97I02930
wherein R, RZ, Rj, R, are defined as in formula I, or of Formula (III):
RZ OH O Ra
H
N NHR~ III
RNH
H
O Ra O
in which R. R,, R3, R" R, are defined as in formula I.
The present invention also relates to cyclic compounds of formula tIV):
/" A
IV
R Z
X~
'R
OH
where X = CWNR, CWOR, S(O)nNR, P(O)(Q)NR; W = O, S, NR; n = 0, I. '_':
Q = R, OR, N(R)z
Y = NRCW, NRS(O)~, NRP(O)(Q)
Z = CRR' , NR
A = a bridging group having 2-15 atoms comprised of any stable combination of
C.
N, O, or S. This bridging group may itself be bridged by one or more chains
comprised of C, N, O, or S atoms so as to generate additional rings of 3-~
atoms.
The bridging groups may be optionally substituted by OH. NH,, halo, optionally
substituted lower alkyl, lower alkoxy, lower alkylthio, mono or di-lower alkyl
amino, -
oxo, thiono, alkylimino, mono- or dialkylmethylidene, COR3. The bridging
moiety
may also be unsaturated so as to contain portions which are the residues of
alkene.
imine, alkvne and allene groups. Furthermore, any part of the bridging moiety
may
comprise pan of an optionally substituted aromatic, heteroaromatic, alicvclic.
or
heterocyclic ring.
R = is as defined hereinabove
CA 02412954 2002-12-30
' 'WO 97130072 PCT/US97/02930
-s-
- R', RZ = H, halo, optionally substituted lower alkyl, lower alkoxy, lower
alkylthto.
mono or di-lower alkyl amino
R3 = is as defined hereinabove
Also encompassed within the scope of the invention are compounds of
formula IVa where IVa has the structure shown for IV and X, Y = NRCW, NRS(O)~,
NRP(O)(Q); Z = CRR', NR, O, S and all other definitions are as above.
Additionally contemplated are compounds of the formula I Vb where IVb
has the structure shown for IV, where X = OCWNR, SCWNR; Y = NRCW.
NRS(O)n, NRP(O)(Q); Z = CRR', NR and all other definitions are as above.
The present invention also relates to cyclic compounds of formula ( V ):
a-'~
Y R2 Z
R ~ '
X
R
OH Rs
where X = CWNR, S(O)~NR, P(0)(Q)NR; W = O, S, NR; n = 0. 1, '_';
Q = R, OR, N(R)~ -
Y = NRCW, NRS(O)~, NRP(O)(Q)
Z = CRR'. NR
A = is as defined hereinabove
R = is as defined hereinabove.
R', R~ and Ra = H, halo, optionally substituted lower alkyl, lower alkoxy.
lower
alkylthio, mono or di-lower alkyl amino;
Also encompassed within the present invention are compounds of
formula Va where Va has the structure shown for V and where X, Y = NRCW.
NRS(O)e, NRP(O)(Q); Z = CRR', NR. 0. S and all other dennitions are as above.
Additionally encompassed are compounds of formula Vb where V'b has
the structure shown for V and where ~ = OCWNR. SCWNR: ~' - :~RCV~.
NRS(O)n, NRP(O)(Q): Z = CRR'. NR and all other definitions are as above.
CA 02412954 2002-12-30
' WO 97130072 PCTItTS97/02930
-9 -
As utilized herein, the term "alkyl", alone or in combination, means a
straight-chain or branched-chain alkyl radical containing from 1 to about t8,
preferably from 1 to about 10, carbon atoms. Examples of such radicals include
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, ten-butyl,
amyl.
isoamyl, hexyl, octyl and the Like. The term "alkenyl", alone or in
combination,
means a straight-chain or branched-chain hydrocarbon radical having one or
more
double bonds and containing from 2 to about 18 carbon atoms preferably from 2
to
about 10 carbon atoms. Examples of suitable alkenyl radicals include ethenvl,
propenyl, allyl, 1,3-butadienyl and the like. The term "atkynyl", alone or in
combination, means a straight-chain hydrocarbon or branched chain radical
having one
or more triple bonds and containing from 2 to about 14 carbon atoms. Examples
of
alkynyl radicals include ethynyl, propynyl, propargyl, butynyl and the like.
The term
"alkoxy", alone or in combination, means an alkyl ether radical wherein the
term alkyl
is as defined above. Examples of suitable alkyl ether radicals include
methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy. sec-butoxy, tert-butoxy
and the
like. The term "cycloalkyl", alone or in combination, means a saturated or
partially
saturated monocyclic, bicyclic or tricyciic alkyl radical wherein each cyclic
moiety
contains from about 3 to about 8 carbon atoms. Examples of such cycloalkyl
radicals
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. The
term
"cycloalkylalkyl" means an alkyl radical as defined above which is substituted
by a
cycloalkyl radical as defined above. Examples of cycloalkylalkyl radicals
include
cyclopropylmethyl, cyclopropylethyl, cyclohexylmethyl, cyclohexylethyk and the
like.
The term "aryl", alone or in combination, means an aromatic monocycle or
bicyclic
or tricyclic such as phenyl, naphthyl, or anthracenyl which optionally carries
one or
more substituents selected from alkyl, alkoxy, halogen, hydroxy, amino, vitro.
cyano,
haloalkyl and the like; such as phenyl, p-tolyl, 4-ethoxyphenyl, 4-(tent-
butoxy~ phenyl,
4-fiuorophenyl, 4-chlorophenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl. and
the
like. The term "aralkyl", alone or in combination, means an alkyl radical as
det~ned
above in which one hydrogen atom is replaced by an aryl radical as defined
above.
such as benzyl, 2-phenylethyl and the like. The term "aryloxy" means a radical
of the
CA 02412954 2002-12-30
WO 97130072 PCT/US97102930
- 10-
- - formula aryl-O- in which the term aryl has the significance given above.
The term
"alkanoyl", alone or in combination, means an acyl radical derived from an
alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl,
valeryl.
4-methylvaleryl, and the like. The heterocyclyl portion of a heterocyclyl-
containing
group is a saturated or partially unsaturated monocyclic, bicyclic or
tricyclic
heterocyle which contains one or more hetero atoms selected from nitrogen,
oxygen
and sulphur, which is optionally substituted on one or more carbon atoms by
halogen,
alkyl, alkoxy, oxo, and the like, andlor on a secondary nitrogen atom by
alkyl.
aralkoxycarbonyl, alkanoyl, aryl or aralkyl or on a tertiary nitrogen atom by
oxido
and which is attached via a carbon atom. The heteroaryl group is an aromatic
monocyclic, bicyclic, or tricyclic heterocycle which contains the hetero atoms
and is
optionally substituted as defined above with respect to the definition of
heterocyclyl.
Examples of such heterocyclyl and heteroaryl groups are pyrrolidinyl,
piperidinyl,
piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, imidazolyl (e.g. imidazol
4-yl,
1-benzyloxycarbonylimidazol-4-yl, etc.), pyrazolyl, pyridyl, pyrazinyl,
pyrimidinyl.
furyl, thienyl, triazolyl, oxazolyl, thiazolyl, indolyl (e.g. 2-indolyl),
quinolinyl,
(e.g., 3-quinolinyl, 2-quinolinyl, etc.), isoquinolinyl (e.g., l,'_',3,4-
tetrahydro-1-oxo-
isoquinolinyl, etc.), quinoxalinyl, (3-carbolinyl, - benzofuranyl, 1-, 2-, ~-
or 5-
benzimidazolyl, and the like. The term "aminoalkanoyl" means an acyl group
derived
from an amino-substituted alkanecarboxylic acid wherein the amino group can be
a
primary, secondary or tertiary amino group containing substituents selected
from
hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyi radicals and the
like. The
term "halogen" means fluorine, chlorine, bromine or iodine. The term
"haloalkyl"
means an alkyl radical having the significance as defined above wherein one or
more
hydrogens are replaced with a halogen. Examples of such haloalkyl radicals
include
chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl,
1,1,1-
trifluoroethyl and the like.
The possible optional substituents mentioned in the hereinabove generic
description include at least one alkyl, cycloalkyl, aryl, aralkyl, alkaryl.
heteroay I.
alkoxy, halogen, hydroxy, amino, vitro, cyano, haloalkyl wherein the optional
CA 02412954 2002-12-30
WO 97130072 PCT/US97102930
substituents may also be optionally substituted and the radicals which are
optionally
substituted may be singly or multiply substituted with the same or different
optional
substituents.
The compounds provided by the present invention are advantageous as
demonstrated by their activity. Furthermore, the structures of the cyclic
compounds
of the present invention potentially provide protection of the compounds from
enzyme
degradation. The low molecular weight and few peptide bonds in these analogs
may
also contribute to improved bioavailability.
Specific, but non-limiting examples of peptides of Formula (I) useful
in the present invention include the following:
K, cachepsin D (nbi)
(1) Iva-Val-Val-Sta-Val-Leu-Gly-NHZ (SEQ. ID. NO. 1); 0.1
(2) Iva-Gln-Val-Sta-Ala-Leu-Gly-NHZ (SEQ. ID. NO. 2); 0.35
(3) Iva-Lys-Val-Sta-Ala-Leu-Gly-NHZ (SEQ. ID. NO. 3); 3.9
(4) Tba-Val-Val-Sta-Ala-Leu-Gly-NHZ (SEQ. ID. NO. 4); 0.04
(5) Iva-Val-Ile-Sta-Ala-Leu-Gly-NHZ (SEQ. ID. NO. 5); 0.04
(6) Iva-Val-Leu-Sta-Ala-Leu-Gly-NHZ (SEQ. ID. NO. 6); 0.5
(7) Tba-Val-Val-Pst-Val-Leu-Gly-NHZ (SEQ. ID. NO. 7): 0.015
(8) Tba-VaI-Cys-Pst-Val-Leu-Gly-NH, (SEQ. ID. NO. 8); 0.25
(9) Tba-Val-Glu-Pst-Val-Leu-Gly-NH, (SEQ. ID. NO. 9); 1.25
(10) Tba-Val-Asp-Pst-Val-L,eu-Gly-NHZ (SEQ. ID. NO. 10); ~.1
(11) Iva-Val-Val-Sta-Ala-Leu-Gly-NHS (SEQ. ID. NO. 11); 0.03
(12) Tba-Val-Cys-Pst-Val-Cys-Gly-NH,(SEQ.ID.NO.I2). 0.66
which has a PMI K; = 32 nM PMII K; = 1.7 nM.
Iva = isovaleryl
Tba = t-butylacetyl
Specific, but not limiting examples of peptides of Formula (II_) of the
present invention include the following:
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-12-
COVaIVaINH CONH~
OH
which has a K; of 800 nM, (SEQ. ID. NO. 13);
COVatVaiNH
CONH
OH
which has a Ki of 12 nM, (SEQ. ID. N0. 14);
~ COVaiNH CONH
OH
which has a K; of 1600 nM, (SEQ. ID. NO. 15);
COVaIVaINH CONH OH
OH
which has an ICso of 600 nlvi. !SEQ. ID. NO. 16);
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-13-
~O
COVatVsINH CONH~N~
OH
which has a K; of 1834 nM (SEQ.ID.NO. 17);
Additional Examples of Cathepsin D Inhibitors within Formula II are shown
below
based on the formula:
R O R'HN O
~H O
NH
N
H
CA 02412954 2002-12-30
WO 97!3007Z PCTIUS97/02930
- 14-
R R' MW Ki (nM)
(CH~)4CH3 (CH~6CH3 503 24 _
(CH~ZSPr (CH~ZSPr 539 75
(CH~ZSCHZPh (CH=)2SCHrPh 635 5
(CHZ)ZSCH2Ph (CHi)3SCHZPh 649 17
(CH~,SCH2Ph (CH~3SCH2Ph 663 4.0
O(CH~3-4-Pyr (CHZ)3SCH,Ph 634 249
(CHZ),SCH,Ph (CHz)6CH, 597 b.2
(CH2)ZSCHIPh (CHZ)6CH3 583 7.7
(CH2)4CH3 (CHZ)3SBn 569 6.1
(CHz)ZCOzH n-C,H,s 505 41
(CHZ)2COzMe n-C,H,S 519 178
(CH2)ZCOIVHZ n-C,H,S 504 479
(CH2)3C02H n-C,H,S 519 22
(CHZ)3CO~Me n-C,H,S S33 112
CHZ-p-Ph-Ph n-C,H,S 599 16
(E)-3- n-C,H,s 579 45
CH =CHbenzodioxolane
Ph = phenyl, pyr = pyridyl and Bn = benzyl
Specific, but not limiting examples of compounds of Formula (III)
include:
CONH COYaINH
~H
which has an K; of 400 nM, (SEQ. ID. NO. 18);
CA 02412954 2002-12-30
PCT/US97102930
WO 97130072
- 15-
OVaINH COAIaLeuNH
C 2
N
H OH
which has an ICso of 500 nM, (SEQ. ID. NO. 19);
COVaINH COAIaLeuNHZ
OH
which has a K; of 8 nM, (SEQ. ID. NO. 20);
~N~
NH LeuNH2
COVaI
OH
which has an ICso of 1000 nM, (~Fn m ntn. 21~:
/
N COVaINH NHZ
OH
which has a K; of 21 nM, (SEQ. ID. NO. 22);
/ COVaINH COAIaLeuNHz
N
OH
which has a K; of 20 nM, (SEQ. ID. NO. 23);
CA 02412954 2002-12-30
PCTIUS97102930
WO 97!30072
- 16-
O
~COVafNH COAIaLeuNHZ
OH
which has an ICS of 20 nM, (SEQ. ID. NO. 24);
COVaiVaINH COAIaLeuNH2
OH
which has a K; of 0.18 nM, (SEQ. ID. NO. 25);
O
~COVaIVaINH COAIaLeuNH2
OH
which has a K; of 0.24 nM, (SEQ. ID. NO. 26):
\ ~O
COVaIVaINH COValteuNH ~./N~
OH
which has a K; of 0.14 nM, (SEQ. ID. NO. 27).
Specific examples of cyclic compounds within the scope of the present
invention
include: (Formula I:)
CA 02412954 2002-12-30
WO 97r30072 PGT/US97102930
- I7-
NH CO
,,,'~r
OH
COVaINH CONH ~~~ COVaINH COGIyNHz
which has a K; of 1.4 nM, (SEQ. ID. NO. 28);
CO NH
,,,,,'
OH
COVaINH CONH ~~/~,,.COVaINH COGIyNH2
i
which has a K; of 2.3 nM, (SEQ. ID. NO. 29);
NN
CO
OH
COVaINH CONH ~~,~.COVaINH COGIyNHz
which has a K; of I2 nM, (SEQ. ID. NO. 30);
NH CO
OH
COVaINH CONH ~~/~COVaINH COGIyNH2
which has a K; value of 100 nM, (SEQ. ID. NO. 31);
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WO 97/30072 PGT/US97IOZ930 ' -
- 18-
/.CH2 CHz.,,
S S
OH
COVaINH CONH ~~COVaINH COGIyNHZ
i
which has a K; value of 1.9 nM, (SEQ. ID. NO. 32);
S~CHZ-CH2-CHz~s
,,,...
OH
COVaINH CONH ~~,/~,COVaiNH COGIyNHz
which has a K; value of 1.5 nM, (SEQ. ID. NO. 33);
~CHT-CHI-CHZ-CHZ \
S S
OH
COVaINH CONH ~~,,~COVaINH COGIyNH2
which has a K; value of 0.1 nM, (SEQ. ID. NO. 34);
,~~,,o g S
OH
COVaINH CONH ~~,/~COVaINH COGIyNH2
which has a K; value of 150 nM, (SEQ. ID. NO. 35);
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WO 97/30072 PCT/t1S97/02930
- 19-
.,,,,,. _
OH
CONH ~. COVaINH COVatl.euGlyNH2
CONH
which has a K; value of 0.26 nM, (SEQ. ID. NO. 36);
,~,,.a
OH
CONH ~ COVaINH COVaIteuGIyNH2
CONH
which has a K; value of 2.5 nM, (SEQ. ID. NO. 37);
Formula II:
CONH
CO O
NH
S \,.'''' ~CONH'~N~
OH
which has an ICso of 10,000 nM (SEQ.ID.NO. 38);
Formula IV:
~(CH2ly
C~O NH
NH CO
..
CONH
OH
which has a K; value of 4 nM; (SEQ.ID.NO. 39);
CA 02412954 2002-12-30
WO 971300T1 PCT/US97/02930
-20-
~(CHz)y
C~O NH
NH CO
I ~ ..... I
CONH ~~. \
OH
which has a K; value of 21 nM; (SEQ.ID.NO. 40);
~(CH~~~
CO NH
NH CO
f
..... J ....,
'''' CONH '''
OH
which has a K; value of 85 nM; (SEQ.ID.NO. 41);
~(CHZj~~
C~O NH
NH CO
J....
CONH
OH
which has a K; value of 22 nM; (SEQ.ID. NO. 42);
~(CH~i~
C~O NH
NH CO
v...,
~''"~~ ~ CONH
OH
which has a K; value of 10 nM; (SEQ.ID.NO. 43);
CA 02412954 2002-12-30
WO 97!3007Z PCT/US97102930
-21 -
~(CH~~~
C~O NH
NH CO
., .
CONH ~~'''
OH
which has a K; value of 20 nM; (SEQ.ID.NO. 44);
~(CH2)~~
C~O \N H
NH CO
_..
CONH
OH
which has a Ki value of 140 nM. (SEQ.ID.NO. 45).
Additional Examples of Cathepsin D Inhibitors within Formula IV are shown
below
based on the following formula:
A
(CH2~~ OH CH
CONH ~ CONH (
~,~ ONH
R'
CA 02412954 2002-12-30
WO 97/300TZ PCT/US97/02930
-22-
R R' m n A Ki
I~1
PhCHz D-iPr 11 - - -
PhCH2 iBu 11 - - 20
PhCH2 Me 11 - - 46
PhCH2 Et 11 - - 22
PhCH2 3-PyrCH2 11 - - 38
PhCH2 CH3CH(OCHzPh 11 - - 460
)CHZ
PhCH2 CH3CH(OH)CH~ 1I - - 300
PhCH2 tBu 11 - - 18
PhCH2 Ph 11 - - 11
p-(PhCH20)- iPr 11 - - 41
PhCHz
p-(OH)PhCHz iPr 11 - - 53, 45,
27
. p- iPr 11 - - 8.3
(OMe)PhCH2
p-(OEt)- iPr I I - _ - 8.1
PhCH2
p-(OiPr)- iPr 11 - - 7.4
PhCH2
p-(3PyrCH20)- iPr 11 - - 81
PhCHz
p- iPr 11 - - 13
bie0(CHZ)20-
PhCH2
(F5-Ph)CH2 iPr 1I - - 90
(m,p- iPr 11 - -
CIzPh)CHZ
(mp-FZPh)CHZ iPr 11 - - -
p-F-PhCH2 iPr 11 - - -
HSCHZ iPr I1 - - 410
CA 02412954 2002-12-30
PCTIITS97/02930 -
. ~ WO 97130072
-23-
- g g~ m a A Ki
(x'17
_
MeSCH2 iPr 11 - - 150,
183
EtSCHZ iPr 11 - _ 84
PrSCH2 iPr 11 - - 90
CH2=CHCHZ iPr 11 - - 140
SCH2
BuSCHz iPr 11 - - -
iBuSCH2 iPr 11 - - -
PhCH2SCHz iPr 11 - - 68
PhCH2CH2 iPr 11 - - 1600
2-naph-CH2 iPr 11 - - 1.1
1-naph-CH2 iPr 11 - - 121
4-thiazolyl- iPr 11 - - 186
CHZ
45
3-indolyl-CHZ iPr 11 -
10 O 36
PhCH2 iPr
10
PhCH2 - O
Pr 2
-
iPr 2 2 S(CHZ)3S 1900
PhCHZ
iPr 2 2 S(CHZ)4S S73
PhCH2
iPr 2 2 S(CHZ)SS 53
PhCH2
iPr 2 2 S(CHZ)6S 19
PhCH2
iPr 2 2 S(CHZ),S 56
PhCH2
iPr 2 2 SCHZCH =CHCHZS 380
PhCHZ (cis)
iPr 2 2 SCHZCH=CHCHZS 320
PhCH2 (traps)
iPr 2 2 SCHZCCCHZS 65
PhCHz 2 2 SCHZp-Ph-CHZS 9 ~
8
PhCHz iPr
iPr 2 2 SCHZm-Ph-CHZS
PhCH2
iPr 2 2 SCHZo-Ph-CHZS
PhCH2
CA 02412954 2002-12-30
WO 9'7/30072 PCT/US97~2930 ~ -
- 24 -
R R' m n A Ki
I~l
PhCHz iPr 2 2 SCHZp-(Cl)4Ph- 0.89
CHZS
PhCH2 iPr 11 - replace amide NH 63
by O
naph. = naphthyl
The compounds of the present invention may exist in a free, i.e.
unprotected, or protected form. The protected form herein refers to compounds
wherein one or more reactive groups, e.g. amino groups or -OH groups, are
substituted by a protecting group. Suitable protecting groups are any of those
known
in the art, such as acetyl, benzyloxycarbonyl and t-butoxycarbonyl.
The compounds of the present invention, whether they are in a free or
protected form, may exist as salts or as complexes. Acid addition salts may be
formed with organic acids, polymeric acids, and inorganic acids, for example.
Such
acid addition salt forms include inter alia the hydrochlorides and acetates.
Complexes
are herein defined as compounds of known type, formed on addition of inorganic
substances, such as inorganic salts or hydroxides such as Ca- and Zn- salts,
and/or on
addition of polymeric organic substances.
The present invention further provides methods and compositions for
preventing or treating diseases. Particular non-limiting examples of diseases
include
cancer including for example breast cancer, Alzheimer's disease, and malaria.
Specifically, this invention provides for the use of the compounds and
compositions of the present invention to inhibit the activities of cathepsin D
and
piasmepsins I and II for treating and preventing diseases such as cancer,
Alzheimer's
disease, and malaria. The present invention also provides pharmaceutical
compositions comprising the same.
The present invention further provides methods of preventing or treating
a disease by the administration of a therapeutically effective amount of a
cathepsin D
or plasmepsin I or II inhibitor compound.
CA 02412954 2002-12-30
wo s~r~oo~rz rc~rrtrs9~ro293o -
-25-
More particularly, the present invention provides methods of treating
cancer by administration of a therapeutically effective amount of at least one
cathepsin
D inhibitor described herein which, for example, inhibits the invasion and
metastasis
of cancerous cells.
In addition, the present invention provides methods of treating
Alzheimer's disease by administration of a therapeutically effective amount of
at least
one cathepsin D inhibitor described herein which, for example, inhibits the
formation
of senile plaques.
In addition, the present invention provides methods of treating malaria
by administration of a therapeutically effective amount of a plasmepsin I or
II inhibitor
described herein which, for example, inhibits the degradation of hemoglobin by
the
malarial intracellular parasite.
The present invention also provides methods of preventing or treating
diseases by the administration of a therapeutically effective amount of at
least one
compound of the present invention in combination with chemotherapeutic agents,
toxins, or irradiation. Examples of chemotherapeutic agents are known to those
skilled in the art and include, but are not limited to, bleomycin, mitomycin,
cyclophosphamide, doxorubicin, paclitaxel, and cisplatin.
In one embodiment of the invention, the compounds of the present
invention are administered in a pharmaceutically acceptable carrier. A
pharmaceutically acceptable carrier encompasses any of the standard
pharmaceutical
carriers such as sterile solution, tablets, coated tablets and capsules. Such
carriers
may typically contain excipients such as starch, milk, sugar, certain types of
clay,
gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other
known
excipients. Such carriers may also include flavor and color additives and
other
ingredients. ,
In the practice of the methods of this invention, the amount of the
enzyme inhibitor incorporated in the composition may vary widely. Methods for
determining the precise amount depend upon the subject being treated, the
specific
pharmaceutical carrier, the route of administration being employed, the
frequency with
CA 02412954 2002-12-30
wo ~r~oon rc~r~s9~roz93o . -
-26-
which the compound is to be administered, and whether the composition is
administered in conjunction with a chemotherapeutic agent and/or irradiation
treatment.
The present invention further provides a method of treating a subject
afflicted with a tumor which comprises contacting the tumor with an amount of
one
of the cathepsin D inhibitors described herein which is administered to the
subject
previous to, simultaneous to, or subsequent to, administration of a
chemotherapeutic
agent or to an amount of irradiation effective to treat the tumor. The
administration
of the composition may be effected by any of the well known methods, including
but
not limited to, oral, intravenous, intramuscular, and subcutaneous
administration.
Plasmepsin I and II are enzymes which are required for specific
degradation of hemoglobin by the malarial intracellular parasite. Due to the
high
active site similarity between cathepsin D and plasmepsins, some of the
cathepsin D
inhibitors of the present invention are useful in preventing the growth of
Plasmodium
,f'alclparum, a causative agent of malaria. Non-limiting examples of compounds
which
exhibit plasmepsin I or II (aspartic hemoglobinases from Plasmodium
falciparum)
inhibitory activity include the following:
(Formula I):
COVaIVaINH COAtat-euGIyNH2
OH
which has a PMI K; = 1.2 nM PMII K; = 0.1 nM (SEQ. ID. NO. 46); .
CA 02412954 2002-12-30
rcrrt~s9~roz93o -
wo 9~r~oon
-27-
..,a~'' _
OH
CONH ~ COVaiNH COVaILeuGIyNH2
CONH
which has a PMI K;= 22 nM PMII K; = 0.2 nM (SEQ. ID. NO. 36);
COVaINH CON COAIaLeuGIyNH2
OH
which has a PMI K; = 7 nM PMII K; = 3 nM (SEQ. ID. NO. 47);
(Formula III):
N COVaINH
Ohi
which has a PMI K; = 16 nM PMII K; = 0.9 nm (SEQ.ID.NO. 22).
CA 02412954 2002-12-30
WO 97130072 PCTIITS97I02930
-28-
S~1THF~IS:
LINEAR INHIBITORS:
The linear peptide inhibitors of the present invention were synthesized
by the solid phase method using the Fmoc group for a-amino protection and acid
labile groups for side chain protection of trifunctional amino acids e.g.
[FmocCys(Trt)OH, FmocAsp(OtBu)OH and FmocGlu(OtBu)OH]. The aminomethyl
polystyrene resin (Bachem California) was modified with acid labile linker
Fmoc-2,4-
dimethoxy-4'-(carboxymethoxy)-benzhydrylamine(Bachem Bioscience, Inc.) to a
fnal
substitution of 0.3-0.5 mmol/g. The Fmoc protected amino acids were coupled as
HOBt esters, DIC {diisopropylcarbodiimide) was used for activation, and a 20 %
solution of piperidine in DMF (dimethyl formamide) for deprotection in each
step.
The Fmoc protected derivatives of statine and 3-hydroxy-4-amino-5-phenyl
pentanoic
acid (AHPPA or phenylstatine) were prepared according to the modified
described
procedure of Jouin, P. et al. J. Chem. Soc. Perl~,.n. Trans. 1, 1177 (1987).
The final
cleavage of inhibitors from the resin was accomplished by treating with TFA
{trifluoroacetic acid) containing 5 % of water (and 3 % of triethyl silane in
the case of
cysteine containing inhibitors). All the inhibitors were purified by reverse
phase high
performance liquid chromatography (HPLC) (column VYDAC C-.18 2.5 X 25 cm)
using water-acetonitrile mixtures containing 0.05 % TFA in a gradient elution.
Purity
of all compounds was checked with analytical reverse phase HPLC (column CYDAC
C-18 0.4 X 25 cm). All compounds gave correct molecular peaks in the mass
spectrum (SIMS).
CYCLIC ANALOGS:
Preparation of Cyclic Disulfides.
The dibenzylated dithiol ( 100-500 mg) was dissolved in 150 ml of
refluxing anhydrous liquid ammonia. Sodium metal was then added in small
pieces
to the solution until it remained blue. The addition was finished when the
blue color
persisted for 3 minutes. The mixture was then decolorized with a crystal of
NH~CI
CA 02412954 2002-12-30
' WO 9~I30072 PCT/US97I02930
-29-
. - and ammonia was evaporated. The solid residue was suspended in 10% aq.
KHS04
and the precipitated dithiol was filtered off. Yield is usually close to 100 %
.
The dithiol (30 mg) was dissolved in 30 ml of anhydrous degassed
DMF containing 10 eq. of diisopropylethylamine (DIEA). A solution of 1.1 eq.
of
a,w-dihaloalkane in 5 ml of DMF was then added dropwise during 12 hours to the
above solution. The mixture was left to stand for an additional 12 hours at
room
temperature. DMF was evaporated under reduced pressure and the obtained solid
residue was washed with 5% aq. NaHC03 and ether. Yield 70-90% of crude cyclic
inhibitor. The product was purified by preparative HPLC for the purpose of I
determination.
Other Cyclic ~naloes
Compounds related to Seq. ID number 42 but lacking the Ile can be
prepared by condensing an N-protected statine analog with an w-amino ester,
hydrolyzing the ester, removing the N-protecting group of the statine, and
cyclizing
to form the Iactam. Alternatively an ester of statine can be condensed with an
N-
protected w-amino acid to provide the N-acylated statine which can be
deprotected and
cyclized to give the lactam.
Analogs such as in compounds of formula IV where a carboxamide is
replaced with a sulfonamide or a phosphonamide may be prepared in an analogous
way to the carboxamides, but in place of typical peptide coupling conditions,
more
vigorous activation of the sulfonic or phosphoric acid such as by preparing
the
sulfonyl or phosphoryl chloride may be required.
The amides described above can be replaced by hydrazides by using a
suitably C-protected w-hydrazino acid and condensing it with a suitably
activated
carboxylic or related acid derivative.
The oxygen of the carbonyl containing derivatives such as carboxamides
may be replaced by sulfur by using such common thionating agents as P,SS or
Lawesson's reagent. The resulting thiocarbonyls can be converted into imino
CA 02412954 2002-12-30
WO 97130072 PCT/US97102930 .
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derivatives by treating with amines, in some cases after prior activation of
the
thiocarbonyl by, for example, alkylation.
~R~~ING GROUPS
It is recognized that the c~-amino acid may have up to 4 of its carbon
atoms replaced by heteroatoms such as O, N, or S. These amino acids may be
prepared by using methods known to those skilled in the art. For example a
shorter
chain w-halo acid derivative can be reacted with an N-protected amino alcohol,
aminoalkanethiol, or diamine to provide an m-amino acid with an O, S, or N
beta to
the amino terminus. Similarly an w-hydroxy acid could be derivatized as, for
example, a sulfonate, and a similar displacement reaction run. Alternatively a
suitably
protected shorter chain w-amino, hydroxy, or sulfhydryl acid could be
derivatized, for
example, by reaction with a suitably N-protected amino alkyl halide or
sulfonate.
Similarly it is recognized that replacing a sulfonate or halide in the above
alkylations
by an epoxide would allow formation of chains with hydroxyl substituents. It
should
be noted that chains containing heteroatoms need not be synthesized only by
using
alkylation type chemistry. Chains containing an amide bond can be synthesized,
for
example, by reacting an amino acid with another amino acid using standard
peptide
coupling conditions to generate the w-amino acid required. Furthermore an O-
protected c~-amino acid could be reacted with a phosgene equivalent to provide
an
isocyanate which could then be reacted with an N-protected amino alcohol or
diamine
to provide a urethane or urea-substituted chain.
It is recognized that the chain need not be built up separately, but may
be formed by attaching pieces of the chain to the amino and carboxy ends of
the
molecule and then connecting them in some way. This approach is exemplified in
Seq. ID numbers 32-34, where the two cysteine substituents are connected by a
dihalide .to form the desired bridge compounds. Similarly Seq. ID. NO. 28 can
be
prepared by cyclization of a compound with an amino alkyl substituent at one
end and
a carboxy alkyl substituent at the other to give the chain containing an amide
functionality.
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PCTlUS9710Z930
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It should be noted that chains where carbon atoms have been replaced
by heteroatorns are amenable to further chemistry which can also lead to
active
compounds. For example sulfides can be converted by peracids to sulfoxides or
sulfones. Secondary amines can be alkylated or acylated.
Chains containing oxo or hydroxy substitution can also be elaborated.
For example oxo groups may be condensed with amines, hydrazines,
hydroxylamine,
alkoxyamines, phosphorous pentasulfide, diethylamino sulfur trifluoride
(DAST),
alkylidene triphenyl phosphoranes or the like to provide imines, hydrazones,
oximes,
thiones, difluoromethylenes, alkylidenes and the like. Reduction provides
alcohols,
or methylenes. Alcohols may be converted into halides for example by
phosphorous
trihalide, oxidized to oxo compounds, acylated to give esters or alkylated to
give
ethers.
The chain could also incorporate unsaturation such as one or more
double or triple bonds. These can be generated by elaboration of saturated
analogs,
such as, for example, by elimination of an alcohol derivative such as a
xanthate ester,
or a halide. Alternatively the unsaturation can be present in one of the
precursors for
the chain. For example an unsaturated fatty acid could be converted to an w-
amino
unsaturated acid, for example, via the w-bromo derivative. This material could
then
be used to form the ring as described for the saturated analogs. It should be
noted
that compounds containing unsaturation in the ring are amenable to further
transformation. Cycloadditions such as Diels-Alder reactions, 1,3-dipolar
additions,
and 2+2 cycloadditions lead to bridged 6-,5-, and 4-membered ring systems.
Three
membered rings can be generated by carbene reactions, epoxidations with, for
example, peracids, and aziridinations. Alternatively the ring can be part of
the w-
amino acid before attachment to the core. Thus, for example, w-aminoalkyl
benzoic
acids could be utilized to form the chain.
Carbamates, thiocarbamates and ureas at Y may be generated by
reacting the C-protected amino acid core with a phosgene equivalent such as
trichloroacetyl chloride followed by a mono N-protected w-diamine, w-amino
alcohol.
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or c.~-amino thiol. Similarly sulfamides can be generated by replacing the
phosgene
equivalent by sulfuryl chloride, or an equivalent. _
Compounds of formula IVa may be generated by replacing the amino
acid core in IV by a diamine or equivalent. One way this can be accomplished
is by
taking a suitably N-protected amino acid, forming an active ester, displacing
by NaN3
to form the acyl azide, and then heating to form the isocyanate. Reaction with
mono
N-protected c~-diamines, c~-amino alcohols, or m-amino thiols as above
provides
carbamates, thiocarbamates and areas at X. Alternatively, the isocyanate can
be
hydrolyzed to give the amine which can be acylated to 'form amide derivatives.
Similarly, sulfonylation can provide sulfonyl analogs.
Compounds of formula IVb replace the amine at X in IVa by oxygen
or sulfur. These compounds can be synthesized, for example, by using
phenylalaninal
as the starting material. Conversion of the aldehyde to the epoxide may be
accomplished either directly with an appropriate ylide, or indirectly via
Wittig
olefination followed by epoxidation, for example, with a peracid. The epoxide
can
then be reacted with a suitable alcohol or thiol to provide a diol or mercapto
alcohol
respectively. Acylation of these moieties, with for example a suitable
isocyanate
provides the X functionality described in IVb. -
Compounds of formula V may be prepared in a similar fashion as those
for formula IV but using an optionally a-alkylated homostatine core rather
than a
statine core. Homostatine analogs can be synthesized, for example, by
activating the
carboxylic acid of a protected statine analog, reacting with diazomethane to
generate
the diazoketone, and then subjecting it to Wolff rearrangement using thermal,
photochemical, or metal-catalyzed conditions. The resulting carboxylic acid
can then
be elaborated as in formula IV.
Compounds of formula Va have the amide of X in formula V reversed.
Such compounds are available from derivatives of statine. Thus a statinal
derivative
can be reductively aminated to give the required precursor amine which can
then be
acylated, sulfonylated, or phosphorylated as for previously described
examples.
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Alternatively the amine could be synthesized by reduction of a statine amide
or
reaction of an activated statinol derivative with an amine equivaient. -
Sirnilarly, compounds of formula Vb can be derived from statinol
analogs by acylation on oxygen with, for example, an isocyanate derivative.
Alternatively, the alcohol can be activated as, for example, a sulfonate
derivative and
then reacted with a thiol to give the sulfur analogs.
The Examples herein are meant to exemplify the various aspects of
carrying out the invention and are not intended to limit the scope of the
invention in
any way.
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EXAMPLE 1
The synthesis of this inhibitor is represented in Scheme A below:
tSuOCOt~t COON + ~C~. ~Ctt~y". Co00~ ~. ~t OtEI,
t~OCD~fl1 . G01W ~(Ci~i~,r ~ r'
Ts~tl. hl0et OtEA
T~Ai~! C~!!!-(C~Sr~~ ~' ~ ~ 01!
QII
tauoeo~ ~ r~ 3 ca~~ ccx~"- ~o~, ""°"
o~
(~lflT ~ _. ~~ Q~ OIIf
ON
~tO~O~t C0~1
CO
",J
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The various compounds shown in scheme A can be prepared as follows:
12-aminododecanoic acid methylester hydrochloride (11 _
To the suspension of 12-aminododecanoic acid (2. I5 g; 10 mmol) in 2,2-
dimethoxypropane (I00 ml) was added 36% aqueous HCl (10 ml) and the mixture
was
treated in an ultrasonic bath until all solid material was dissolved. The
mixture was
then left to stand overnight, concentrated to dryness, dissolved in 40 ml of
methanol
and precipitated with approximately 500 ml of dry ether to give 2.4 g of 1
(90.5 %
yield).
2- ut 'n 1 - inod d i i me h 1e er 2
Boc-Ile-NH-(CHZ)"-COOCH3
Boc (t-butyloxycarbonyl)-Ile(isoleucine)-OH. 1/2H20 (384 mg; 1.6 mmol),
HC1.HZN-
(CH2)"-COOCH3 (1) (398 mg; 1.5 mmol), HOST (N-hydroxybenzotriazole) (260 mg;
1.7 mmol) and TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate)) (546 mg; 1.7 mmol) were dissolved in DMF (5 ml). DIEA
(diisopropylethylamine) (860 ~.1; 5 mmol) was added to the above solution and
the
mixture was left to stand overnight. Five drops of N-aminoethylmorpholine were
then
added to the solution. After 30 minutes DMF was removed in vacuo and the solid
residue suspended in 10% aqueous KHS04 (20 ml). The precipitate was filtered
off,
washed with water, 5 % aqueous NaHC03, water again, and then dried in a vacuum
desiccator to give 630 mg of 2 (95 °Io yield).
12-l t-Butvloxvcarbo~v~~henvlstatinvl-isoleuc_invil-aminododecanoic acid
methvlester
Boc-Pst-Ile-NH-(CH,~"-COOCH3
Boc-Ile-(CH2)"-COOCH3 (2) (384 mg; 0.8 mmol) was dissolved in TFA
(trifluoroacetic acid) (3 ml). After 30 minutes of standing at room
temperature, the
TFA was removed in vacuo, the oily residue was concentrated three more times
with
ether to remove the remaining TFA, and then dissolved in 5 ml of DMF. Boc-Pst-
OH
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PCTJUS97/02930 . '
-36-
(247 mg; 0.8 mmol), TBTU (289 mg; 0.9 mmol) and DIEA (1030 ~cl; 6 mmol) were
added to the above solution and the mixture was left to stand overnight.
Workup as
for 2 yielded 492 mg of 3 (97%).
'n 1-i 1 a i 1 oic i 4
Boc-Pst-Ile-NH-(CHZ)"-COON
Boc-Pst-Ile-NH-(CHZ)I,-COOCH3 (3) (380 mg; 0.6 mmol) was dissolved in a
mixture
of 1,4 dioxane (5 ml) and methanol (1 ml) and combined with 1N aqueous
solution
of NaOH (I.5 mI). The reaction was complete in approximately two hours. The
organic solvents were evaporated and the remaining aqueous slurry was
acidified with
10% aqueous KHS04 (10 ml). The precipitate was filtered off, washed with
water,
and dried in a vacuum desiccator to yield 353 mg of 4 (95 % yield).
C~~cLl2-aminododec~oyl-phenylstatinyl-isoleucinyl (5l
-CO-Pst-Ile-NH-(CHZ)"-
Boc-Pst-Ile-NH-(CH2)"-COON (4) (248 mg; 0.4 mmol) was deprotected with TFA
as described for 3 and dissolved in DMF (20 ml). This solution was added
dropwise
to a stirred solution of DPPA {diphenyl phosphorylazide) ( 1.1 g; 4 mmol) and
DIEA
(1.38 ml; 8 mmol) in DMF (100 ml) over the course of 17 hours via syringe
pump.
The mixture was then stirred for an additional 3 hours, evaporated to dryness
and
triturated with ether. The precipitated product was filtered off, washed with
ether,
% aqueous NaHC03, and water and then dried in a vacuum desiccator to provide
160
mg of crude 5 (80% yield). For the purpose of biological testing, the product
was
purified by RP-HPLC.
SIMS MS M+Na+524
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EXAMPLE 2
An alternate sequence to a cyclic inhibitor is represented in Scheme B below:
?'eTtJ. N01L OQV
coop ~a ~, DI!
J
rarer. ~oe~. dtA~ ~uoeow~.to~sh~
011
1. TfA
tauOCOIAI.( !
_. O~PA~ OIEA~Ii~
CD !W
CD
9
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t-,iBu~y~oxycarbonyl- henvlstatinyl valine methylester (61
Boc-Pst-Val-OCH3
Boc-Pst-OH (309 mg; 1 mmol), HC1.H-ValOCH3 {184 mg; 1.1 mmol) and TBTU
(385 mg; 1.2 mmol) were dissolved in 5 ml of DMF and DIEA (688 ~cl; 4 mmol)
was
addexi to the solution. The mixture was left to stand overnight and then
evaporated
to dryness. The oily residue was dissolved in ethylacetate and the
ethylacetate
solution was washed with 10% aqueous KHSO,, water, 5% aqueous NAHCO3, and
water again, dried over MgSO,, and concentrated to give 420 mg of 6.
11-t-ButylcLxyc bony!-aminoundecanoyl-phen !y statin'yl valine methyl ester 7)
Boc-NH-(C Hz), o-CO-Pst-V al-OCH3
Boc-Pst-Val-OCH3 (420 mg; 1 mmol) was deprotected with TFA as described for 3.
After addition of ether, a precipitate of TFA.H-Pst-Val-OCH3 (349 mg; 0.8
mmol)
was obtained. This was dissolved in 5 ml of DMF together with Boc-NH-(CHZ),o-
COOH (241 mg; 0.8 mmol), TBTU (289 mg; 0.9 mmol) and DIEA (516 ~cl; 3 mmol).
After 10 hours of standing at room temperature, DMF was removed in vacuo, the
residue dissolved in ethylacetate and worked up as described above for 6 to
yield 480
mg of oily 7.
11-t-Butxlox~carbony_l-aminoundecano rLI-,phenylstatinyl valine l8)
Boc-NH-(CHz) 1 o-CO-P st-V aIOH
Hydrolysis of crude Boc-NH(CHZ),o-CO-Pst-ValOCH3 (7) (480 mg; 0.8 mmol) as
described above for 4 provided 435 mg of oily 8.
Cyclo-11-aminoundecanovl-nhenylstatinxl-valine (91
-CO-Pst-Val-NH-(CHZ),p
Boc-NH-(CHZ),o-CO-Pst-VaIOH (8) was deprotected and cyclized using the
procedure
described for 5. Yield 260 mg (55 % from Boc-Pst-OH used in 6) of crude solid
9.
For the purpose of biological testing the product 9 was purified by RP-HPLC.
SIMS
MS h4H+474
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Example 3 Solid phase synthesis of a cyclic inhibitor.
A 1
0
F~ COC
w
t) Pf~rtdn~
Z)~n~. OIC. H09t
f 'L,%
0
~H coo
NQ~
1I PIpI~IdIM I
=1 le~C~t-0CQ0
0
~~"~~tt 000
?~A
C0~1 COOEI
i
a~~, au 10
/"'_ ~~ n
HN
CO
H
_I J
cowl
ow
(SEQ. ID. N0.48)
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Solid ~hgs_e Synthesis of TFA.H?N-ICH~" ~~Ol stV~l-OH l10)
FmocVal-WANG-resin (1g, 0.37 mmol) was swollen in DMF, washed
with DMF (3x2 min), deprotected with 20% piperidine in DMF (1x2 min; 1x20 min)
and washed ~ again with DMF (Sx2 min). FmocPstOH (319 mg, 0.74 mmol) was then
coupled using DIC (diisopropylcarbodiimide) ( 101 mg, 0. 8 mmol) and HOBt (
122 mg,
0.8 mmol) for activation. The resulting resin was then deprotected and washed
as
described above. The solution of BocNH-(CHZ)"-COO-pC6H,-NOZ (452 mg, 1 mmol)
and DIEA (190 ~cl, 1.1 mmol) in 5 ml of DMF was added and the mixture was
shaken
overnight. The resin was washed with DMF (5x2 min), 20% piperidine in DMF (2x2
min), DMF (3x2 min), pyridine-DCM (1:1) (3x2 min) and DCM (dichloromethane)
(5x2 min). The product was then cleaved off the resin with 95 % TFA (2x30 min)
and
the resin was washed with DCM (5x2 min). The combined solutions were
evaporated
to dryness. The oily residue (200 mg) was used directly for the next reaction.
Synthesis of ( 11 ) lcvclization)
TFA.HZN-(CHZ)1,-OCOPstVal-OH(10)was cyclized using theprocedure
described for 5. Yield 40 mg (21.5 % based on the resin substitution) of crude
11.
For the purpose of biological testing the product was purified by RP-HPLC.
Product
was characterized by SIMS-MS, M+H+ = 504, M+Na+ = 526.
Compound 11 has a K; against cathepsin D of 3.6 nM.
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Example 4 Synthesis of a cyclic urea.
Synthesis of Inhibitor 15 _
;OOH DPP4. Dle~
90~100°C
QtEA
t8u ~ TF~H~N CONH ~ (Cf~y~~~ COOCH~
t8u0 CONH - (Ctl~t,~ COOCH~ NCH. M~OH
13
CONH ~ (Cii~~~ COOH
t8tt0 Z DPPI~, OtEJI.DMF
14
(CH~~~
CO ~HN
NN CO
'.
NhICONH
OH 1~
(SEQ.ID.N0.49)
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Svnth~sis of 13
Boc-Pst(acetonide)OH (850 mg, 2.44 mmol) was dissolved in 13 ml of
toluene and approx. 2-3 ml of toluene was distilled off to dry the solution.
DPPA (I
g, 3.66 mmol) and DIEA (0.84 ml, 4.88 mmol) were added and the mixture was
heated to 90-100°C for 2 hours. A solution of TFA.H-VaINH-(CHZ)"-COOMe
prepared from I.2 g (2.8 mmol) Boc-VaINH(CH~"C02Me and DIEA (1.46 ml, 8.5
mmol) in 3 ml of dry toluene was added to the reaction mixture, the mixture
was
heated for an additional 30 min and then left to stand overnight .at room
temperature.
The toluene was removed in vacuo, the residue dissolved in ethyl acetate and
washed
subsequently with 10% aq. KHSO,, brine, 5 % NaHC03, brine, and finally dried
with
MgS04. The ethyl acetate was then removed in vacuo and the oily residue
purified
by flash chromatography in hexane-ethyl acetate (3:2). Yield 873 mg, 53 % of
13.
Product was characterized by SIMS-MS, M+H* = 675.
Synthesis of I4
13 (850 mg, 1.26 mmol) was hydrolyzed with NaOH according to the
procedure described above for 4. Yield 850 mg (- 100%) of oily 14, that was
used
directly for the next reaction.
Synthesis of 15 ~Cycli~ation~
This compound was prepared using the procedure described above for
S. Yield 438 mg (69% calculated from 13). Product was characterized by SIMS-
MS,
M+H* = 503. ' Compound 1S has a K; against cathepsin D of 45 nM.
Inhibition Assays
Kinetic measurements. Fluorogenic substrates Ac-Glu-Glu(EDANS)-Lys-Pro-Iie-
Cys-Phe-Phe-Arg-Leu-GIy-Lys(DABCYL)-Glu-NH2 and Ac-Glu-Glu(EDANS)-Lys-
Pro-Ile-Cys-Phe-Leu-Arg-Leu-Gly-Lys(DABCYL)-Giu-NH2 were used for measuring
the activity of cathepsin D and plasmepsin 2 correspondingly. Typically, 485
~cl of
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- 50 mM Gly-HC 1 buffer, pH 3.5 (in the case of cathepsin D), or 100 mM sodium
acetate buffer, pH 5.0 (in the case of plasmepsin 2), was mixed with 5 ~cI of
DMSO
and 5 ~.1 of titrated protease (final concentration 0.2-10 nM) and incubated 3
min at
37°C. The reaction was initiated by the addition of 5 ~.1 of substrate
stock solution in
DMSO. Increase in fluorescence intensity at the emission maximum of 487 nm
(excitation wavelength was 349 nm) was monitored as a function of time using
an
Aminco Bowman-2 luminescence spectrometer (SLM Instruments, Inc.).
Plasmepsin 1 assays were run similarly to plasmepsin 2, using the
fluorogenic substrate DABCYL-Gaba-Glu-Arg-Met-Phe-Leu-Ser-Pro-Gaba-
Glu(EDANS)-NH2.
The initial rate of hydrolysis was calculated by a second degree
polynomial fit using SLM AB2 2.0 operating software. Kinetic parameters were
determined by nonlinear regression fitting of initial rate versus substrate
concentration
data to the Michaelis-Menten equation using the program Enzfitter version 1.05
(Leatherbarrow, R.J. 1987. Enzfitter, a program for non-linear regression
analysis.
Elsevier Scientific, New York).
For inhibition studies inhibitors were prepared as stock solutions at
different concentrations in DMSO. In a typical experiment 485 ~d of the
appropriate
buffer was mixed with 5 ~1 of inhibitor stock solution and S ~.1 of titrated
protease
(final concentration 0.2-10 nM) and preincubated 3 min at 37°C. The
reaction was
initiated by the addition of 5 ~1 of substrate stock solution in DMSO. For
data
analysis the mathematical model for tight-binding inhibitors (Williams, J.W.,
and
Morrison, J.F. Methods~nz, mil. 63: 437 (1979)) was used. The data were fitted
_
by nonlinear regression analysis to the equation
V =Vd2Et({[K;( 1 +S/K"~ +hF~,]z+4K;( 1 +S/K"~E,} "z-[K;( 1 +S!K"~ +I,-
~l)
with the program Enzfitter (version 1.05), where V and Vo are initial
velocities with
and without inhibitor, K~, is a Michaelis-Menten constant and S, E, and I, are
the
concentrations of substrate, active enzyme and inhibitor respectively.
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Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be
obvious to those skilled in the art that certain changes and modifications may
be
practiced without departing from the spirit and scope thereof as described in
the
specification and as defined in the appended claims.
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REFERENCES
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2. Baldwin E.T. et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6796, (1993).
3. Boger, J. Peptides 1983 pp. 569-578, Proceedings of the 8th American
Peptide Symposium.
4. Cataldo A.M. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3861, (1990).
5. Dhanaa, D.S. et al., Tet. Lett. 33: 1725 (1992).
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WO 97/30072 PCT/LJS97/02930
- 46 -
26. Tandon A.K. et al., N. Eng. J. used. 322, 297, (1990).
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