U.S. patent application number 10/574536 was filed with the patent office on 2007-01-18 for certain substituted spirocyclic lactams and use thereof as pharmaceuticals.
Invention is credited to Yves Auberson, Ralf Glatthar, Rhys Salter, Oliver Simic, Marina Tintelnot-Blomley.
Application Number | 20070015781 10/574536 |
Document ID | / |
Family ID | 29415468 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070015781 |
Kind Code |
A1 |
Auberson; Yves ; et
al. |
January 18, 2007 |
Certain substituted spirocyclic lactams and use thereof as
pharmaceuticals
Abstract
The present invention relates to novel
2-(6-oxo-1,7-diaza-spiro[4.4]non-7-yl)-propionamides of the formula
##STR1## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, m and p are as defined in the specification, to their
preparation, to their use as pharmaceuticals and to pharmaceutical
compositions containing them.
Inventors: |
Auberson; Yves; (Allschwil,
CH) ; Glatthar; Ralf; (Bad Sackingen, DE) ;
Salter; Rhys; (Basel, CH) ; Simic; Oliver;
(Basel, CH) ; Tintelnot-Blomley; Marina;
(Maulburg, DE) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
29415468 |
Appl. No.: |
10/574536 |
Filed: |
October 4, 2004 |
PCT Filed: |
October 4, 2004 |
PCT NO: |
PCT/EP04/11054 |
371 Date: |
March 31, 2006 |
Current U.S.
Class: |
514/278 ;
514/409; 546/16; 548/410 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/28 20180101; A61P 35/04 20180101; C07D 487/10 20130101;
A61P 9/00 20180101 |
Class at
Publication: |
514/278 ;
514/409; 546/016; 548/410 |
International
Class: |
A61K 31/4747 20070101
A61K031/4747; A61K 31/407 20070101 A61K031/407; C07D 471/10
20070101 C07D471/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2003 |
GB |
0323204.8 |
Claims
1. A compound of the formula ##STR5## wherein R.sub.1 is hydrogen
or (C.sub.1-4)alkyl, R.sub.2 is optionally substituted
(C.sub.1-8)alkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, aryl or heteroaryl, R.sub.3
is --CH(R.sub.e)C(.dbd.O)N(R.sub.a)R.sub.b or
--(CH.sub.2).sub.kN(R.sub.c)R.sub.d, wherein k is 0, 1 or 2,
R.sub.a, R.sub.b, R.sub.c and R.sub.d, independently, are hydrogen
or an optionally substituted (C.sub.1-8)alkyl,
(C.sub.5-9)bicycloalkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, aryl, aryl(C.sub.1-4)alkyl,
heteroaryl, heteroaryl(C.sub.1-4)alkyl, 4-chromanyl,
1,2,3,4-tetrahydro-quinolin-4-yl,
1,2,3,4-tetrahydro-naphthalen-1-yl, thiochroman-4-yl-1,1-dioxide,
4-isochromanyl, 1,2,3,4-tetrahydro-isoquinolin-4-yl,
thioisochroman-4-yl-1,1-dioxide,
1,1-dioxo-1,2,3,4-tetrahydro-1lambda*6*-benzo[e][1,2]thiazin-4-yl,
1,1-dioxo-3,4-dihydro-1H-1lambda*6*-benzo[c][1,2]oxathiin-4-yl,
2,2-dioxo-1,2,3,4-tetrahydro-2lambda*6*-benzo[c][1,2]thiazin-4-yl
or 2,2-dioxo-3,4-dihydro-2H-2lambda*6*-benzo[e][1,2]oxathiin-4-yl
group, or R.sub.a and R.sub.b, or R.sub.c and R.sub.d, together
with the nitrogen to which they are attached, form an optionally
substituted pyrrolidinyl, piperidinyl, morpholinyl or piperazinyl
group, and R.sub.e is (C.sub.1-8)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl, (C.sub.3-7)cycloalkyl or
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, R.sub.4 is hydrogen or an
optionally substituted (C.sub.1-8)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, (C.sub.2-6)alkenyl,
(C.sub.2-6)alkynyl, (C.sub.3-7)cycloalkoxy(C.sub.1-4)alkyl or aryl
group, R.sub.5 is hydrogen or optionally substituted
(C.sub.1-4)alkyl, R.sub.6 is hydrogen, hydroxy or halogen, and m
and p, independently, are 1 or 2, in free base form or in acid
addition salt form.
2. A process for the preparation of a compound as defined in claim
1 of the formula I, in free base form or in acid addition salt
form, comprising the steps of acylating a compound of the formula
##STR6## wherein R.sub.1, R.sub.2 and R.sub.3 are as defined for
the formula I, with an acid of the formula ##STR7## wherein
R.sub.4, R.sub.5, R.sub.6, m and p are as defined for the formula
I, or an activated form, such as an ester or an acid halogenide,
thereof and recovering the so obtainable compound of the formula I
in free base form or in acid addition salt form.
3. A compound according to claim 1, in free base form or in
pharmaceutically acceptable acid addition salt form, for use as a
pharmaceutical.
4. A compound according to claim 1, in free base form or in
pharmaceutically acceptable acid addition salt form, for use in the
treatment of neurological or vascular disorders related to
beta-amyloid generation and/or aggregation.
5. A pharmaceutical composition comprising a compound as claimed in
claim 1, in free base form or in pharmaceutically acceptable acid
addition salt form, as active ingredient and a pharmaceutical
carrier or diluent.
6. The use of a compound as claimed in claim 1, in free base form
or in pharmaceutically acceptable acid addition salt form, as a
pharmaceutical for the treatment of neurological or vascular
disorders related to beta-amyloid generation and/or
aggregation.
7. The use of a compound as claimed in claim 1, in free base form
or in pharmaceutically acceptable acid addition salt form, for the
manufacture of a medicament for the treatment of neurological or
vascular disorders related to beta-amyloid generation and/or
aggregation.
8. A method for the treatment of neurological or vascular disorders
related to beta-amyloid generation and/or aggregation in a subject
in need of such treatment, which comprises administering to such
subject a therapeutically effective amount of a compound as claimed
in claim 1, in free base form or in pharmaceutically acceptable
acid addition salt form.
9. A combination comprising a therapeutically effective amount of a
compound as claimed in claim 1, in free base form or in
pharmaceutically acceptable acid addition salt form, and a second
drug substance, for simultaneous or sequential administration.
10. The use of a compound as claimed in claim 1, in free base form
or in pharmaceutically acceptable acid addition salt form, as
histopathological labeling agent, imaging agent and/or biomarker
for the selective labeling of the beta-secretase cleaving enzyme
BACE.
Description
[0001] The present invention relates to novel
2-(6-oxo-1,7-diaza-spiro[4.4]non-7-yl)-propionamides, to their
preparation, to their use as pharmaceuticals and to pharmaceutical
compositions containing them.
[0002] More particularly the invention relates to compounds of the
formula ##STR2## wherein [0003] R.sub.1 is hydrogen or
(C.sub.1-4)alkyl, [0004] R.sub.2 is optionally substituted
(C.sub.1-8)alkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, aryl or heteroaryl, [0005]
R.sub.3 is --CH(R.sub.e)C(.dbd.O)N(R.sub.a)R.sub.b or
--(CH.sub.2).sub.kN(R.sub.c)R.sub.d, wherein [0006] k is 0, 1 or 2,
[0007] R.sub.a, R.sub.b, R.sub.a and R.sub.d, independently, are
hydrogen or an optionally substituted (C.sub.1-8)alkyl,
(C.sub.5-9)bicycloalkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, aryl, aryl(C.sub.1-4)alkyl,
heteroaryl, heteroaryl(C.sub.1-4)alkyl, 4-chromanyl,
1,2,3,4-tetrahydro-quinolin-4-yl,
1,2,3,4-tetrahydro-naphthalen-1-yl, thiochroman-4-yl-1,1-dioxide,
4-isochromanyl, 1,2,3,4-tetrahydro-isoquinolin-4-yl,
thioisochroman-4-yl-1,1-dioxide,
1,1-dioxo-1,2,3,4-tetrahydro-1lambda*6*-benzo[e][1,2]thiazin-4-yl,
1,1-dioxo-3,4-dihydro-1H-1lambda*6*-benzo[c][1,2]oxathiin-4-yl,
2,2-dioxo-1,2,3,4-tetrahydro-2lambda*6*-benzo[c][1,2]thiazin-4-yl
or 2,2-dioxo-3,4-dihydro-2H-2lambda*6*-benzo[e][1,2]oxathiin-4-yl
group, or [0008] R.sub.a and R.sub.b, or R.sub.c and R.sub.d,
together with the nitrogen to which they are attached, form an
optionally substituted pyrrolidinyl, piperidinyl, morpholinyl or
piperazinyl group, and [0009] R.sub.e is (C.sub.1-8)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl, (C.sub.3-7)cycloalkyl or
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, [0010] R.sub.4 is hydrogen
or an optionally substituted (C.sub.1-8)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl, (C.sub.3-7)cycloalkyl,
(C.sub.3-7)cycloalkyl(C.sub.1-4)alkyl, (C.sub.2-6)alkenyl,
(C.sub.2-6)alkynyl, (C.sub.3-7)cycloalkoxy(C.sub.1-4)alkyl or aryl
group, [0011] R.sub.5 is hydrogen or optionally substituted
(C.sub.1-4)alkyl, [0012] R.sub.6 is hydrogen, hydroxy or halogen,
and [0013] m and p, independently, are 1 or 2, [0014] in free base
form or in acid addition salt form.
[0015] On account of the asymmetrical carbon atoms present in the
compounds of the formula I and their salts, the compounds may exist
in optically active form or in the form of mixtures of optical
isomers, e.g. in the form of racemic mixtures. All optical isomers
and their mixtures, including the racemic mixtures, are part of the
present invention.
[0016] Further, the compounds of the formula I and their salts may
contain a radioisotope, such as tritium, .sup.14C, .sup.11C or
.sup.18F. All radiolabeled compounds and their use as biomarkers,
in vitro or in vivo imaging agents, or in biochemical assays, e.g.
binding assays, are part of the present invention.
[0017] Substituents on the above defined non-aromatic groups are
selected from hydroxy, halogen, carbamoyl, carboxy,
hydroxy(C.sub.1-4)alkyl, (C.sub.1-4)alkoxy,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkoxy, (C.sub.1-4)alkylsulfanyl,
(C.sub.1-4)alkoxycarbonyl, (C.sub.1-4)alkylcarbonyloxy,
(C.sub.1-4)alkylcarbonyl, (C.sub.1-4)alkylsulfonyl, cyano, oxo,
(C.sub.3-7)cycloalkyl, hetero(C.sub.3-7)cycloalkyl, optionally
substituted aryl or optionally substituted heteroaryl.
(C.sub.3-7)cycloalkyl or hetero-(C.sub.3-7)cycloalkyl groups can
also be fused with an additional (C.sub.3-7)cycloalkyl,
hetero(C.sub.3-7)cycloalkyl, or an aromatic or heteroaromatic
ring.
[0018] Substituents on above defined aromatic or heteroaromatic
groups are selected from halogen, hydroxy, cyano, nitro,
trifluoromethyl, benzyloxy, phenoxy, SO.sub.2NH.sub.2,
NHSO.sub.2(C.sub.1-3) alkyl, carboxy, (C.sub.1-4)alkyloxycarbonyl,
carbamoyl, (C.sub.1-4)alkylcarbamoyl, (C.sub.1-4)alkylsulfonyl,
(C.sub.1-4)alkylcarbonyloxy, (C.sub.1-4)alkylcarbonyl,
(C.sub.1-6)alkyl, (C.sub.1-4)alkoxy, hydroxy(C.sub.1-4)alkyl, aryl,
heteroaryl or an optionally substituted amino group.
[0019] Substitutents on amino or carbamoyl groups can be one or two
groups selected from (C.sub.1-4)alkyl,
(C.sub.1-4)alkoxy(C.sub.1-4)alkyl, (C.sub.1-4)alkoxycarbonyl,
aryl(C.sub.1-4)alkyloxycarbonyl or
heteroaryl(C.sub.1-4)alkyloxycarbonyl.
[0020] Aryl is an aromatic 6-membered ring optionally mono-, di- or
tri-substituted by, independently, hydroxy, cyano, trifluoromethyl,
halogen, carboxy, (C.sub.1-4)alkyloxycarbonyl,
(C.sub.1-4)alkylcarbamoyl, (C.sub.1-4)alkylsulfonyl, (C.sub.1-4)
alkylcarbonyloxy, (C.sub.1-4)alkylcarbonylamino,
(C.sub.1-4)alkylcarbonyl, (C.sub.1-6)alkyl, (C.sub.1-4)alkoxy or
hydroxy(C.sub.1-4)alkyl. Aryl groups can also be fused with a
(C.sub.3-7)cycloalkyl, hetero(C.sub.3-7)cycloalkyl or additional
aromatic or heteroaromatic ring (e.g. to form a naphthyl,
quinolinyl or indolyl group).
[0021] Heteroaryl is an aromatic 5- or 6-membered ring, in which 1,
2 or 3 atoms are heteroatoms independently selected from O, N and
S. Heteroaryl is, for example, 1-methyl-1H-pyrrol-2-yl or
1H-imidazol-2-yl. It can also be fused with a cycloalkyl or
additional aromatic or heteroaromatic ring (e.g. to form a
quinolinyl or indolyl group).
[0022] Halogen denotes fluorine, bromine, chlorine or iodine.
[0023] Any alkyl, alkenyl, alkynyl or alkoxy group is straight or
branched.
[0024] Unless defined otherwise, carbon containing groups and
molecules contain 1 to 8, preferably 1 to 6, more preferably 1 to
4, carbon atoms.
[0025] In a preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.1 is hydrogen.
[0026] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.2 is aryl, preferably phenyl, more
preferably unsubstituted phenyl.
[0027] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.3 is
--CH(R.sub.e)C(.dbd.O)N(R.sub.a)R.sub.b or
--(CH.sub.2).sub.kN(R.sub.c)R.sub.d, wherein k is 0; R.sub.a is
hydrogen; R.sub.b is (C.sub.1-8)alkyl or (C.sub.5-9)bicycloalkyl
such as bicycloheptyl; R.sub.c is hydrogen; R.sub.d is optionally
substituted aryl(C.sub.1-4)alkyl, preferably benzyl substituted in
the phenyl ring by (C.sub.1-4)alkyl, or is optionally substituted
(C.sub.3-7)cycloalkyl, preferably cyclopropyl substituted by phenyl
optionally substituted by halogen, such as bromine, or is
4-chromanyl optionally substituted, preferably by halogen and/or
(C.sub.1-4)alkyl; and R.sub.e is (C.sub.1-4)alkyl.
[0028] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.4 is (C.sub.1-8)alkyl, or is
(C.sub.1-8)alkyl substituted, preferably mono-substituted, by
(C.sub.3-7)cycloalkyl, preferably cyclopropyl, by halogen, such as
fluorine, by (C.sub.1-4)alkoxy, or by hydroxy, or is
(C.sub.2-6)alkenyl optionally substituted, preferably
mono-substituted, by hydroxy, or is (C.sub.2-6)alkynyl, or is aryl,
preferably phenyl, preferably unsubstituted phenyl.
[0029] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.4 is 2,2,3,3-tetratritiopropyl.
[0030] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.5 is (C.sub.1-8)alkyl, preferably
(C.sub.1-4)alkyl.
[0031] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which R.sub.6 is hydrogen or halogen, preferably
hydrogen or fluorine.
[0032] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which m is 1.
[0033] In another preferred embodiment, the invention relates to a
compound of the formula I, in free base form or in acid addition
salt form, in which p is 1.
[0034] In a further aspect, the invention relates to a process for
the preparation of the compounds of the formula I and their salts,
comprising the steps of acylating a compound of the formula
##STR3## wherein R.sub.1, R.sub.2 and R.sub.3 are as defined above
for the formula I, with an acid of the formula ##STR4## wherein
R.sub.4, R.sub.5, R.sub.6, m and p are as defined above for the
formula I, or an activated form, such as an ester or an acid
halogenide, thereof and recovering the so obtainable compound of
the formula I in free base form or in acid addition salt form.
[0035] The reaction can be effected according to conventional
methods, for example as described in the examples.
[0036] The compounds of the formula I can also be produced by
further conventional processes, e.g. as described in the
examples.
[0037] The starting materials of the formulae II and III are known
or may be prepared according to conventional procedures starting
from known compounds, for example as described in the examples.
[0038] The working-up of the reaction mixtures and the purification
of the compounds thus obtainable may be carried out in accordance
with known procedures.
[0039] Acid addition salts may be produced from the free bases in
known manner, and vice-versa.
[0040] Compounds of the formula I and their pharmaceutically
acceptable acid addition salts, hereinafter referred to as agents
of the invention, exhibit valuable pharmacological properties when
tested in vitro and in animals, and are therefore useful as
pharmaceuticals.
[0041] The agents of the invention are inhibitors of aspartic
proteases and can be used for the treatment of disorders involving
processing by such enzymes. Particularly they inhibit
beta-secretase and as such inhibit the generation of beta-amyloid
and the subsequent aggregation into oligomers and fibrils.
[0042] In addition, the agents of the invention exhibit valuable
properties as histopathological labeling agents, imaging agents
and/or biomarkers, hereinafter "markers", for the selective
labeling of BACE (beta-secretase cleaving enzyme).
[0043] More particularly the agents of the invention are useful as
markers for labeling BACE in vitro or in vivo (see Examples 9 and
10).
[0044] The agents of the invention are therefore useful, for
instance, for determining the levels of active site occupancy of a
drug acting at BACE, or for diagnostic purposes for diseases
resulting from a dysfunction of BACE-related processes, and for
monitoring the effectiveness of pharmacotherapies of such
diseases.
[0045] In accordance with the above, the present invention also
provides an agent of the invention for use as a marker for
neuroimaging.
[0046] In a further aspect, the present invention provides a
composition for labeling brain and peripheral structures involving
BACE in vivo and in vitro comprising an agent of the invention.
[0047] In still a further aspect, the present invention provides a
method for labeling brain and peripheral structures involving BACE
in vitro or in vivo, which comprises contacting brain or peripheral
tissue with an agent of the invention.
[0048] The method of the invention may comprise a further step
aimed at determining whether the agent of the invention labeled the
target structure. Said further step may be effected by observing
the target structure using autoradiography, positron emission
tomography (PET), or any device allowing detection of radioactive
radiations.
Test 1: Inhibition of Human BACE
[0049] Recombinant BACE (extracellular domain, expressed in
baculovirus and purified using standard methods) at 6 nM
concentration is incubated with the test compound at various
concentrations for 1 hour at room temperature in 100 mM acetate
buffer, pH 4.5, containing 0.1% CHAPS. Synthetic peptide substrate
Mca-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP) is added to a
final concentration of 3 .mu.M and the increase in fluorescence is
recorded at excitation of 325 nm and emission at 400 nm in a
microplate spectro-fluorimeter for 20 minutes in 1-minute
intervals. IC.sub.50 values are calculated from percentage of
inhibition of BACE-activity as a function of the test compound
concentration.
Test 2: Inhibition of Human BACE-2
[0050] Recombinant BACE-2 (extracellular domain, expressed in
baculovirus and purified using standard methods) at 2.5 nM
concentrations is incubated with the test compound at various
concentrations for 1 hour at room temperature in 100 mM acetate
buffer, pH 4.5, containing 0.1% CHAPS. Synthetic peptide substrate
Mca-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP) is added to a
final concentration of 3 .mu.M and the increase in fluorescence is
recorded at excitation of 325 nm and emission at 400 nm in a
microplate spectro-fluorimeter for 20 minutes in 1-minute
intervals. IC.sub.50 values are calculated from percentage of
inhibition of BACE-2-activity as a function of the test compound
concentration.
Test 3: Inhibition of Human Cathepsin D
[0051] Recombinant cathepsin D (expressed as procathepsin D in
baculovirus, purified using standard methods and activated by
incubation in sodium formate buffer pH 3.7) is incubated with the
test compound at various concentrations for 1 hour at room
temperature in 100 mM sodium formate buffer, pH 3.1. Synthetic
peptide substrate
Mca-Gly-Lys-Pro-lle-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D-Arg-NH.sub.2 is
added to a final concentration of 2 .mu.M and the increase in
fluorescence is recorded at excitation of 325 nm and emission at
400 nm in a microplate spectro-fluorimeter for 20 minutes in
1-minute intervals. IC.sub.50 values are calculated from percentage
of inhibition of cathepsin D-activity as a function of the test
compound concentration.
Test 4: Inhibition of Cellular Release of Amyloid Peptide 1-40
[0052] Chinese hamster ovary cells are transfected with the gene
for amyloid precursor protein. Cells are plated at a density of
8000 cells/well in a 96-well microtiter plate and cultivated for 24
hours in DMEM cell culture medium containing 10% FCS. The test
compound is added to the cells at various concentrations, and cells
are cultivated for 24 hours in the presence of the test compound.
The supernatants are collected, and the concentration of amyloid
peptide 1-40 is determined using sandwich ELISA. The potency of the
compound is calculated from the percentage of inhibition of amyloid
peptide release as a function of the test compound
concentration.
[0053] In at least one of the above-indicated tests, the agents of
the invention show activity at concentrations below 20 .mu.M.
[0054] The agents of the invention are therefore useful e.g. for
the treatment and/or prevention of neurological and vascular
disorders related to beta-amyloid generation and/or aggregation,
such as neurodegenerative diseases like Alzheimer's disease, Down's
Syndrome, memory and cognitive impairment, dementia, amyloid
neuropathies, brain inflammation, nerve and brain trauma, vascular
amyloidosis, or cerebral haemorrhage with amyloidosis.
[0055] Some of the agents of the invention also inhibit BACE2
(beta-site APP-cleaving enzyme 2) or Cathepsin D, close homologues
of the pepsin-type aspartyl proteases. Due to the correlation of
BACE2 and CathD expression with a more tumorigenic and metastatic
potential of tumor cells, such inhibitors are useful for the
suppression of the metastasis process associated with tumor
cells.
[0056] For the above-mentioned indications, the appropriate dosage
will of course vary depending upon, for example, the compound
employed, the host, the mode of administration and the nature and
severity of the condition being treated. However, in general,
satisfactory results in animals are indicated to be obtained at a
daily dosage of from about 0.1 to about 100, preferably from about
1 to about 50, mg/kg of animal body weight. In larger mammals, for
example humans, an indicated daily dosage is in the range from
about 10 to about 2000, preferably from about 10 to about 200, mg
of an agent of the invention conveniently administered, for
example, in divided doses up to four times a day or in sustained
release form.
[0057] The agent of the invention may be administered by any
conventional route, in particular enterally, preferably orally, for
example in the form of tablets or capsules, or parenterally, for
example in the form of injectable solutions or suspensions.
[0058] In accordance with the foregoing, the present invention also
provides an agent of the invention, for use as a pharmaceutical,
e.g. for the treatment of neurological or vascular disorders
related to beta-amyloid generation and/or aggregation.
[0059] The present invention furthermore provides a pharmaceutical
composition comprising an agent of the invention in association
with at least one pharmaceutical carrier or diluent. Such
compositions may be manufactured in conventional manner. Unit
dosage forms contain, for example, from about 1 to about 1000,
preferably from about 1 to about 500, mg of an agent of the
invention.
[0060] The agents of the invention can be administered alone or in
combination with other pharmaceutical agents effective in the
treatment of conditions mentioned above.
[0061] The pharmaceutical combination may be in the form of a unit
dosage form, whereby each unit dosage will comprise a predetermined
amount of the two components, in admixture with suitable
pharmaceutical carriers or diluents. Alternatively, the combination
may be in form of a package containing the two components
separately, e.g. a pack or dispenser-device adapted for the
concomitant or separate administration of the two active agents,
wherein these agents are separately arranged.
[0062] Moreover the present invention provides the use of an agent
of the invention, for the manufacture of a medicament for the
treatment of any neurological or vascular disorders related to
beta-amyloid generation and/or aggregation.
[0063] In still a further aspect, the present invention provides a
method for the treatment of any neurological or vascular disorders
related to beta-amyloid generation and/or aggregation, in a subject
in need of such treatment, which comprises administering to such
subject a therapeutically effective amount of an agent of the
invention.
[0064] The following Examples illustrate the invention.
Abbreviations:
BOC tert-butoxycarbonyl
BOP benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate
DAST (Diethylamino)sulfur trifluoride
DCM dichloromethane
DMF N,N-dimethylformamide
DMPU N,N'-dimethylpropyleneurea
EDC HCl 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide
hydrochloride
EtOAc ethylacetate
h hours
HCl hydrochloric acid
HOBt hydroxybenzotriazole
HPLC high pressure liquid chromatography
LAH lithium aluminum hydride
min minutes
Mp melting point
MS mass spectroscopy
Rf retention factor (TLC)
rt room temperature
TBAF tetrabutylammonium fluoride
TBME tert-butyl methyl ether
TBTU
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetrafluoroborat-
e
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin-layer chromatography
EXAMPLE 1
4-(S)-hydroxy-5-(S)-[2-(S)-(1-isobutyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non--
7-yl)-propionylamino]-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
[0065] 113 mg (0.4 mmol) of
2-(S)-(1-isobutyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic
acid methyl ester are dissolved in 18 ml of THF and treated with 2
mL of a 0.5 N aqueous LiOH solution and stirred at room temperature
for four hours. The reaction mixture is cooled to 5.degree. C. and
treated with an aqueous 1N HCl solution until a pH of 4 is reached.
The solution is submitted to two consecutive azeotropic
evaporations with 80 mL toluene and the residue dried under high
vacuum, then taken up in 20 mL dichloromethane and stirred at room
temperature for twenty hours with 117 mg (0.4 mmol)
5-(S)-amino-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide, 85 mg (0.44 mmol) EDC.HCl, 54 mg (0.4 mmol) HOBt and
0.17 mL triethylamine (1.2 mmol). The reaction mixture is quenched
with 10 mL ice-cold saturated aqueous sodium bicarbonate solution,
then extracted twice with dichloromethane. The combined organic
phases are evaporated and the residue is column chromatographed
(silica gel, TBME/EtOAc/EtOH 49:59:2) to yield after evaporation of
the pure fractions the desired product as a colorless resin.
MS(El+): 453 (M+1)
[0066] The starting materials can be prepared as described
hereafter:
a)
2-(S)-(1-isobutyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic
acid methyl ester
[0067] 260 mg (0.8 mmol)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester is stirred in 3 mL of a 4N solution
HCl in dioxane for three hours at room temperature, then
evaporated. The residue is taken up in toluene and evaporated to
dryness (twice), then taken up in 6 mL methanol, treated with 0.145
mL (1.6 mmol) isobutyraldehyde, 300 mg 3 .ANG. powdered molecular
sieve and 100 mg (1.6 mmol) sodium cyanoborohydride and stirred
overnight at room temperature. The reaction mixture is treated with
4 mL saturated aqueous ammonium chloride solution and after 10
minutes with 8 mL saturated aqueous sodium bicarbonate, then
extracted with EtOAc. The combined organic phases are evaporated
and the residue column chromatographed (silica gel, EtOAc) to yield
after evaporation of the corresponding fractions the desired
product as a colorless oil.
MS (ES+): 283 (M+1)
b)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-
-carboxylic acid tert-butyl ester and
7-(1-Methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4nonane-1-ca-
rboxylic acid tert-butyl ester
[0068] 4.3 g (12 mmol)
2-[2-(1-methoxycarbonyl-ethylamino)-(S)-ethyl]-pyrrolidine-1,2-dicarboxyl-
ic acid 1-tert-butyl ester 2-methyl ester are dissolved in 60 mL
xylene and heated to 150.degree. C. for two hours. The reaction
mixture is evaporated and the residue column chromatographed
(silica gel, EtOAc/petroleum ether 3:2) to yield 1.85 g (46%)
7-(1-methoxycarbonyl-(S)-ethyl)
6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic acid tert-butyl
ester and 1.8 g (45%)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester. The absolute stereochemistry is
confirmed by X-ray of a sample recrystallized in
diisopropylether.
c)
2-[2-(1-methoxycarbonyl-(S)-ethylamino)-ethyl]-pyrrolidine-1,2-dicarbox-
ylic acid 1-tert-butyl ester 2-methyl ester
[0069] 271 mg (1 mmol) 2-(2-oxo-ethyl)-pyrrolidine-1,2-dicarboxylic
acid 1-tert-butyl ester 2-methyl ester and 154 mg (1.1 mmol)
L-alanine methyl ester hydrochloride are suspended in 10 mL toluene
and treated with 0.073 mL (1 eq.) triethylamine. The reaction
mixture is stirred 10
a)
2-(S)-(1-isobutyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic
acid methyl ester
[0070] 260 mg (0.8 mmol)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester is stirred in 3 mL of a 4N solution
HCl in dioxane for three hours at room temperature, then
evaporated. The residue is taken up in toluene and evaporated to
dryness (twice), then taken up in 6 mL methanol, treated with 0.145
mL (1.6 mmol) isobutyraldehyde, 300 mg 3 .ANG. powdered molecular
sieve and 100 mg (1.6 mmol) sodium cyanoborohydride and stirred
overnight at room temperature. The reaction mixture is treated with
4 mL saturated aqueous ammonium chloride solution and after 10
minutes with 8 mL saturated aqueous sodium bicarbonate, then
extracted with EtOAc. The combined organic phases are evaporated
and the residue column chromatographed (silica gel, EtOAc) to yield
after evaporation of the corresponding fractions the desired
product as a colorless oil.
MS (ES+): 283 (M+1)
b)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-
-carboxylic acid tert-butyl ester and
7-(1-Methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester
[0071] 4.3 g (12 mmol)
2-[2-(1-methoxycarbonyl-ethylamino)(S)-ethyl]-pyrrolidine-1,2-dicarboxyli-
c acid 1-tert-butyl ester 2-methyl ester are dissolved in 60 mL
xylene and heated to 150.degree. C. for two hours. The reaction
mixture is evaporated and the residue column chromatographed
(silica gel, EtOAc/petroleum ether 3:2) to yield 1.85 g (46%)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester and 1.8 g (45%)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester. The absolute stereochemistry is
confirmed by X-ray of a sample recrystallized in
diisopropylether.
c)
2-[2-(1-methoxycarbonyl-(S)-ethylamino)-ethyl]-pyrrolidine-1,2-dicarbox-
ylic acid 1-tert-butyl ester 2-methyl ester
[0072] 271 mg (1 mmol) 2-(2-oxo-ethyl)-pyrrolidine-1,2-dicarboxylic
acid 1-tert-butyl ester 2-methyl ester and 154 mg (1.1 mmol)
L-alanine methyl ester hydrochloride are suspended in 10 mL toluene
and treated with 0.073 mL (1 eq.) triethylamine. The reaction
mixture is stirred 10 minutes at room temperature and slowly
evaporated in a rotary evaporator. The residue is taken up in 15 mL
acetonitrile and 95 mg (1.5 mmol) sodium cyanoborohydride in 2 mL
methanol added dropwise. Upon completion of the reaction (TLC,
EtOAc/petroleum ether 4:1), the reaction mixture is evaporated and
the residue taken up in ethyl acetate and treated with an ice-cold,
saturated aqueous ammonium chloride solution, then extracted with
ethyl acetate and saturated aqueous sodium bicarbonate. The
combined organic phases are dried over sodium sulfate, filtered and
evaporated to yield the crude desired product as a thick oil.
MS(El+): 359 (M+1)
Rf(EtOAc): 0.22
d) 5-(S)-amino-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
[0073] 32 mg (0.1 mmol)
[1-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-(S)-phenyl-ethyl]-car-
bamic acid tert-butyl ester are stirred at room temperature for two
hours in 1 mL of a 4N HCl solution in dioxane. The reaction mixture
is evaporated to dryness, the residue taken up in toluene and
evaporated to dryness twice before drying under high vacuum. The
residue is taken up in 1 mL (excess) butylamine and stirred at
25.degree. C. overnight, then evaporated and the residue extracted
twice with ethyl acetate and saturated sodium bicarbonate. The
combined organic phases are evaporated, the crude desired product
obtained quantitatively as a colorless resin and used without
further purification.
MS(El+): 293 (M+1).
[0074] The following compounds can be obtained by a similar
procedure:
EXAMPLE 1a
5-(S)-[2-(S)-(1-cyclopropylmethyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-
-propionylamino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 541 (M+1)
EXAMPLE 1b
5-(S)-[2-(S)-(1-propyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionyla-
mino-]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 529 (M+1)
EXAMPLE 1c
5-(S)-[2-(S)-(1-phenyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionyla-
mino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 591 (M+1)
EXAMPLE 1d
5-(S)-[2-(S)-(1-phenyl-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-7-yl)-propionyla-
mino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 591 (M+1)
EXAMPLE 1e
4-(S)-hydroxy-2-(R)-methyl-5-(S)-[2-(S)-(6-oxo-1-propyl-1,7-diaza-spiro-(S-
)-[4.4]non-7-yl)-propionylamino]-6-phenyl-hexanoic acid
(2,2-dimethyl-propyl)-amide
MS(El+): 543 (M+1)
EXAMPLE 1f
5-(S)-[2-(S)-(1-cyclopropylmethyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-
-propionylamino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
bicyclo[2.2.1]hept-exo-2-(R,S)-ylamide
MS(El+): 579 (M+1)
EXAMPLE 1g
5-(S)-{2-(S)-[1-(2,2-dimethyl-propyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-
-yl]-propionylamino}-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic
acid butylamide
MS(El+): 557 (M+1)
EXAMPLE 1h
4-(S)-hydroxy-5-(S)-{2-(S)-[1-(3-methoxy-propyl)-6-oxo-1,7-diaza-spiro-(S)-
-[4.4]non-7-yl-]-propionylamino}-2-(R)-methyl-6-phenyl-hexanoic
acid butylamide
MS(El+): 559 (M+1)
EXAMPLE 1i
5-(S)-[2-(S)-(1-cyclopropylmethyl-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-7-yl)-
-propionylamino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
bicyclo[2.2.1]hept-exo-2-(R,S)-ylamide
MS(El+): 579 (M+1)
EXAMPLE 1j
4-(S)-hydroxy-5-(S)-{2-(S)-[1-(3-methoxy-propyl)-6-oxo-1,7-diaza-spiro-(R)-
-[4.4]non-7-yl]-propionylamino}-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 559 (M+1)
EXAMPLE 1k
5-(S)-2-(S)-(1-propyl-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-7-yl)-propionylam-
ino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 529 (M+1)
EXAMPLE 1l
4-(S)-hydroxy-5-(S)-[2-(S)-(1-(2-fluoroethyl)-6-oxo-1,7-diaza-spiro-(S)-[4-
.4]non-7-yl)-propionylamino]-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 533 (M+1)
EXAMPLE 2
5-(S)-[2-(S)-(1-allyl-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-7-yl)-propionylam-
ino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
[0075] 52 mg (0.1 mmol)
4-(S)-hydroxy-2-(R)-methyl-5-(S)-[2-(S)-(6-oxo-1,7-diaza-spiro-(R)-[4.4]n-
on-7-yl)-propionylamino]-6-phenyl-hexanoic acid butyl amide
hydrochloride, 28 mg potassium carbonate and 0.01 mL allyl bromide
are stirred at room temperature for 65 hours in 3 mL DMF, then
extracted with EtOAc and brine (twice). The combined organic phases
are dried over sodium sulfate, evaporated and column
chromatographed to yield the desired product as a light-colored
resin.
MS(El+): 527 (M+1)
[0076] The starting materials can be prepared as described
hereafter:
a)
4-(S)-hydroxy-2-(R)-methyl-5-(S)-[2-(S)-(6-oxo-1,7-diaza-spiro-(R)-[4.4-
]non-7-yl)-propionylamino]-6-phenyl-hexanoic acid butyl amide
hydrochloride
[0077] 170 mg (0.33 mmol)
7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phenyl-e-
thylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester are dissolved in 1.5 mL (excess) butylamine
and heated to 65.degree. C. under argon for two hours. The reaction
mixture is evaporated, the residue taken up in toluene and
evaporated to dryness, redissolved in 5 mL isopropanol, treated
with 1 mL of a 6N HCl solution in isopropanol and stirred at room
temperature for four hours, then evaporated, taken up in toluene
and evaporated again to yield the desired product, which is used
without further purification.
MS (El+): 487 (M+1)
b)
7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phenyl-
-ethylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester and
7-{1-(S)-[1-(R)-(4-(S)-methyl-5-oxo-tetrahydro-furan-2-(R)-yl)-2-phenyl-e-
thylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester
[0078] 326 mg (1 mmol)
7-(1-(S)-methoxycarbonyl-ethyl)-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester are dissolved in 15 mL THF, cooled
to 10.degree. C. and treated with 7 mL (1.05 eq) of a 0.15 N THF
solution of LiOH. After two hours stirring at room temperature, a 1
N aqueous HCl solution is added until a pH of 4 was reached, and
the reaction mixture evaporated. The residue is taken up in
toluene, evaporated to dryness and dried under high vacuum, then
taken up in 20 mL dichloromethane and stirred for 18 hours after
addition of 230 mg racemic
5-(1-amino-2-phenyl-ethyl)-3-methyl-dihydro-furan-2-one, 135 mg
HOBt (1 mmol), 208 mg EDC.HCl (1.1 mmol) and 0.031 mL triethylamine
(2.25 mmol). The reaction mixture is extracted with EtOAc and
saturated aqueous sodium bicarbonate, the combined organic
fractions are washed with brine, evaporated and column
chromatographed (silica gel, EtOAc/diisopropylether 4:1) to yield
7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phenyl-e-
thylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester and
7-{1-(S)-[1-(R)-(4-(S)-methyl-5-oxo-tetrahydro-furan-2-(R)-yl)-2-phenyl-e-
thylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester is white solids in respectively 33 and 30%
yields.
[0079] The absolute stereochemistry is confirmed by comparison with
optically pure material made from
5-(S)-1-(S)-amino-2-phenyl-ethyl)-3-(R)-methyl-dihydro-furan-2-one.
MS(El+): 514 (M+1)
[0080] The following compounds can be obtained by a similar
procedure:
EXAMPLE 2a
5-(R)-[2-(S)-(1-allyl-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-7-yl)-propionylam-
ino]-4-(R)-hydroxy-2-(S)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 527 (M+1)
EXAMPLE 2b
5-(R)-[2-(S)-(1-allyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionylam-
ino]-4-(R)-hydroxy-2-(S)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 527 (M+1)
EXAMPLE 2c
5-(S)-[2-(S)-(1-allyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionylam-
ino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 527 (M+1)
EXAMPLE 3
4-(S)-hydroxy-5-(S)-{2-(S)-[1-(4-hydroxy-butyl)-6-oxo-1,7-diaza-spiro-(R)--
[4.4]non-7-yl]-propionylamino}-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
[0081] 30 mg (0.06 mmol)
4-(7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-phenyl-ethylcar-
bamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-1-yl)-butyric
acid methyl ester are stirred overnight at room temperature in 1 mL
butylamine. The reaction mixture is evaporated to dryness, taken up
in toluene and evaporated again, and the residue dissolved in THF,
cooled to 5.degree. C. and treated with 3 mg (2 eq.) lithium
borohydride. After stirring for two hours at room temperature, the
reaction mixture is cooled below 10.degree. C., quenched with 2 mL
of saturated aqueous ammonium chloride and 2 mL of saturated
aqueous sodium bicarbonate and stirred an additional 10 minutes
before extraction with EtOAc (twice). The combined organic phases
are dried over sodium sulfate, evaporated, and the residue column
chromatographed (silica gel, DCM/EtOH/ammonia 90:10:0.05) to yield
the desired product as a light-colored resin.
MS(El+): 559 (M+1)
[0082] The starting materials can be prepared as described
hereafter:
a)
4-(7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phe-
nyl-ethylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-1-yl)-butyri-
c acid methyl ester
[0083] 30 mg (0.06 mmol)
4-(7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-pheny-
l-ethylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-1-yl)-but-2-en-
oic acid methyl ester are stirred in THF under hydrogen for two
hours in the presence of a catalytic amount of 10% Pd/C, then
filtered through celite and evaporated to yield 30 mg desired
product, which is used without further purification.
MS(El+): 514 (M+1)
b)
4-(7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phe-
nyl-ethylcarbamoyl]-ethyl}-6-oxo-1,7-diaza-spiro-(R)-[4.4]non-1-yl)-but-2--
enoic acid methyl ester
[0084] 130 mg (0.25 mmol)
7-{1-(S)-[1-(S)-(4-(R)-methyl-5-oxo-tetrahydro-furan-2-(S)-yl)-2-phenyl-e-
thylcarbamoyl]-ethyl}-oxo-1,7-diaza-spiro-(R)-[4.4]nonane-1-carboxylic
acid tert-butyl ester are dissolved in 2 mL of 4N HCl in dioxane.
The reaction mixture is evaporated after 90 minutes, taken up in
toluene and evaporated to dryness. The residue is taken up in
dichloromethane and stirred at room temperature for 18 hours in the
presence of 92 mg (1 eq.) tetrabutylammonium iodide, 0.03 mL
trans-4-bromobut-2-enoic acid methyl ester and 0.09 mL (2 eq.)
diisopropylethylamine. The reaction mixture is extracted with
dichloromethane and aqueous bicarbonate (twice), the combined
organic phases evaporated and the residue column chromatographed
(silica gel, EtOAc) to yield the desired product as a slightly
brownish resin.
MS(El+): 512 (M+1)
[0085] The following compound can be obtained by a similar
procedure:
EXAMPLE 3a
4-(S)-hydroxy-5-(S)-{2-(S)-[1-(4-hydroxy-butyl)-6-oxo-1,7-diaza-spiro-(S)--
[4.4]non-7-yl]-propionylamino}-2-(R)-methyl-6-phenyl-hexanoic acid
butylamide
MS(El+): 559 (M+1)
EXAMPLE 4
4-hydroxy-5-{2-[1-(4-hydroxy-but-2-enyl)-6-oxo-1,7-diaza-spiro[4.4]non-7-y-
l]-propionylamino}-2-methyl-6-phenyl-hexanoic acid butylamide
MS(El+): 557 (M+1)
is obtained in a similar manner to example 1, but using
2-[1-(4-hydroxy-but-2-enyl)-6-oxo-1,7-diaza-spiro[4.4]non-7-yl]-propionic
acid methyl ester in step 1a.
[0086] The starting material can be prepared as described
hereafter:
2-[1-(4-hydroxy-but-2-enyl)-6-oxo-1,7-diaza-spiro[4.4]non-7-yl]-propionic
acid methyl ester
[0087] 82 mg (0.25 mmol)
(S,S)-7-(1-methoxycarbonyl-ethyl)-6-oxo-1,7-diaza-spiro[4.4]nonane-1-carb-
oxylic acid tert-butyl ester are stirred at room temperature for
three hours in 1 mL of a 4N HCl dioxane solution, evaporated, then
taken up in toluene and evaporated again (twice). The residue is
taken up in 2 mL dichloromethane and stirred at room temperature
for 65 hours in the presence of 74 mg (0.2 mmol) tetrabutylammonium
iodide, 0.035 mL (0.2 mmol) diisopropylethylamine and 30 mg (0.2
mmol) 4-bromo-but-2-en-1-ol. The reaction mixture is extracted with
EtOAc and saturated aqueous sodium bicarbonate, the combined
organic phases washed with brine, evaporated to dryness and the
residue column chromatographed (slica gel, EtOAc/EtOH 9:1) to yield
the desired product as a thick liquid.
MS(El+): 297 (M+1)
EXAMPLE 5
5-(S)-[2-(S)-(3-(S)-fluoro-6-oxo-1-propyl-1,7-diaza-spiro-(S)-[4.4]non-7-y-
l)-propionylamino]-4-(S)-hydroxy-2-(R)-methyl-6-phenyl-hexanoic
acid butylamide
[0088] This compound can be synthesized as described in Example 1,
starting from
(1R,4S)-4-fluoro-2-(2-oxo-ethyl)-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester instead of
2-(2-oxo-ethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 2-methyl ester.
MS(El+): 547 (M+1)
[0089] The starting materials can be made as follows:
a) (2R,4S)-4-fluoro-2-(2-oxo-ethyl)-pyrrolidine-1,2-dicarboxylic
acid 1-tert-butyl ester 2-methyl ester
[0090] 1 g (3.5 mmol)
(2R,4S)-2-allyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester is dissolved in 60 mL
dichloromethane/methanol 1:1, cooled to -78.degree. C., flushed
with oxygen for two minutes then treated with a flux of ozone until
the solution turns light blue. The solution is allowed to warm up
to room temperature after addition of 1 g (1.1 eq.)
triphenylphosphine, stirred an additional 5 h and column
chromatographed (silica gel, TBME/petroleum ether 3:2) to yield 870
mg of the desired product as a clear oil.
MS (El+): 290 (M+1).
b) (2R,4S)-2-allyl-fluoro-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester
[0091] 0.39 mL DAST (1.2 eq) are dissolved in 30 mL
dichloromethane, cooled to -78.degree. C. and treated over 10 min.
with a dropwise addition of 10 mL dichloromethane containing 1.8 g
(6.3 mmol) (2R,4R)-2-allyl-4-hydroxy-pyrrolidine-1,2-dicarboxylic
acid 1-tert-butyl ester 2-methyl ester. The solution is allowed to
reach room temperature under stirring, over 2 h. After cooling
below 5.degree. C. the reaction mixture is treated with an ice-cold
saturated aqueous sodium carbonate solution and extrated with
dichloromethane. The combined extracts are washed with brine and
dried over sodium sulphate, evaporated and the residue column
chromatographed (silica gel, TBME/petroleum ether 1:1) to yield 1 g
desired product as a colorless thick oil.
MS(El+): 288 (M+1)
c) (2R,4R)-2-allyl-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester
[0092] 3.3 g (8.27 mmol)
(2R,4R)-(2-allyl-4-(tert-butyl-dimethyl-silanyloxy)-pyrrolidine-1,2-dicar-
boxylic acid 1-tert-butyl ester 2-methyl ester are dissolved in 60
mL THF, cooled below 5.degree. C. and treated with 8.68 mL (1.05
eq) 1 M TBAF in THF under stirring. The reaction mixture is allowed
to slowly reach room temperature, while being stirred another 4 h.
Ice and AcOEt are added, the mixture washed with brine twice, and
the organic phase evaporated to yield a crude product which is
column chromatographed (silica gel, TBME/petroleum ether 1:1) to
yield 1.8 g desired product as a colorless, thick oil, which is
used as such in the next step.
EXAMPLE 6
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-((-
S)-6-oxo-1-propyl-1,7-diaza-spiro[4.4]non-7-yl)-propionamide
[0093] A solution of 100 mg
(2R,3S)-3-amino-1-(3-isopropyl-benzylamino)-4-phenyl-butan-2-ol
dihydrochloride, 79 mg
(S-2-((S)-6-oxo-1-propyl-1,7-diaza-spiro[4.4]non-7-yl)-propionic
acid, 102 mg TBTU and 0.171 mL N-methyl morpholine in 5 mL
CH.sub.2Cl.sub.2 is stirred for 5 h at ambient temperature. The
solution is diluted with DCM and subsequently washed with
bicarbonate, brine, 0.1N HCl and bicarbonate. After drying with
MgSO.sub.4 all volatiles are evaporated in vacuo and the product is
purified by column chromatography (silica gel, DCM/MeOH 95:5) to
give 53 mg (37%) of the desired product.
MS-ESl+: 549 [M+]
Rf: 0.28 (CH.sub.2Cl.sub.2/MeOH=9/1)
[0094] The starting material can be prepared as described
hereafter:
a)
(2R,3S)-3-amino-1-(3-isopropyl-benzylamino)-4-phenyl-butan-2-ol-dihydro-
chloride
[0095] A solution of 700 mg (2.7 mmol)
tert-butyl(S--(R,R)(-)-(1-oxiranyl-2-phenylethyl)-carbamate and 470
mg (3.3 mmol) 3-iso-propylbenzylamine in 10 ml EtOH is heated for
15 h at 50.degree. C. Evaporation of the solvent and purification
by column chromatography (silica gel, DCM/MeOH 9:1) afforded 820 mg
of
[(1S,2R)-1-benzyl-2-hydroxy-3-(3-iso-propyl-benzylamino)-propyl]-carbamic
acid tert-butyl ester as a colourless solid. This material is
dissolved in 10 ml 4N HCl in dioxane, stirred for 2 h at ambient
temperature and all volatiles removed in vacuo to give 643 mg
desired compound.
[0096] The following compounds can be obtained by a similar
procedure:
EXAMPLE 6a
(S)-N-[(1S,2R)-1-benzyl-3-(6-bromo-2,2-dimethyl-chroman-4-ylamino)-2-hydro-
xy-propyl]-2-((S)-1-cyclopropylmethyl-6-oxo-1,7-diaza-spiro[4.4]non-7-yl)--
propionamide
MS(El+): 667, 669 (M+1)
EXAMPLE 6b
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-((-
S)-1-cyclopropylmethyl-3-(S)-fluoro-6-oxo-1,7-diaza-spiro-[4.4]non-7-yl)-p-
ropionamide
MS(El+): 579 (M+1)
EXAMPLE 6c
(S)-N-[(1S,
2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-((S)-1-propy-
l-3-(S)-fluoro-6-oxo-1,7-diaza-spiro-[4.4]non-7-yl)-propionamide
MS(El+): 567 (M+1)
EXAMPLE 6d
(S)-N-{(1S,2R)-1-benzyl-3-[1-(3-bromo-phenyl)-cyclopropylamino]-2-hydroxy--
propyl}-2-((S)-1-cyclopropylmethyl-3-(S)-fluoro-6-oxo-1,7-diaza-spiro[4.4]-
non-7-yl)-propionamide
MS(El+): 641, 643 (M+1)
EXAMPLE 6e
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-[(-
S)-1-(2-fluoro-ethyl)-6-oxo-1,7-diaza-spiro[4.4]non-7-yl]-propionamide
MS(El+): 553 (M+1)
EXAMPLE 6f
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-((-
S)-1-cyclopropylmethyl-6-oxo-1,7-diaza-spiro[4.4]non-7-yl)-propionamide
MS(El+): 561 (M+1)
EXAMPLE 7
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-propyl]-2-((-
S)-6-oxo-1-prop-2-ynyl-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionamide
[0097] This compound can be prepared as the compound of Example 6,
but starting from
2-(6-oxo-1-prop-2-ynyl-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic
acid methyl ester instead of
2-(S)-(1-isobutyl-6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic
acid methyl ester.
[0098] The starting material can be prepared as follows:
7a)
2-(S)-(6-oxo-1-prop-2-ynyl-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propioni-
c acid methyl ester
[0099] 163 mg (0.5 mmol)
7-(1-methoxycarbonyl-(S)-ethyl)-6-oxo-1,7-diaza-spiro-(S)-[4.4]nonane-1-c-
arboxylic acid tert-butyl ester are dissolved in 1.5 mL 4N HCl in
dioxane and stirred for 3 h at room temperature. The reaction
mixture is evaporated, the residue taken up in 15 mL
ethanol/toluene 1:2 and evaporated (twice), to yield
2-(S)-(6-oxo-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propionic acid
methyl ester hydrochloride, which is dissolved in 10 mL DMF,
treated with 815 mg (5 eq.) cesium carbonate and 0.048 mL (1.25 eq)
prop-2-ynyl bromide. The reaction mixture is stirred at room
temperature for 18 h, then extracted (AcOEt/water), washed with
brine and the combined organic fractions evaporated. The residue is
column chromatographed (silica gel, TBME) to yield 78 mg desired
product.
MS(El+): 265 (M+1)
EXAMPLE 8
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropylbenzylamino)propyl]-2-((S)-
-6-oxo-1-(2,2,3,3-tetratritiopropyl)-1,7-diaza-spiro[4,4]non-7-yl)-propion-
amide
[0100]
(S)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-isopropyl-benzylamino)-prop-
yl]-2-((S)-6-oxo-1-prop-2-ynyl-1,7-diaza-spiro-(S)-[4.4]non-7-yl)-propiona-
mide was reduced under 0.9 atmosphere tritium gas on 10% Pd/C for 2
h. The desired compound was obtained after HPLC purification of the
reaction mixture (column: Nucleosil 100-5 C18 HD, 5 .mu.m,
250.times.4.9 mm; eluent: H.sub.2O/MeCN and 0.1% trifluoroacetic
acid; gradient: 9:1 to 1:1 over 20 minutes) and formulation in
ethanol to a final concentration of 9.9 microgram/mL (specific
activity 68.6 MBq/mL).
HPLC: RT=12.87 min
ME (ES+): 557 (M+1)
EXAMPLE 9
In Vitro Autoradiography
[0101] The tissue of interest is cut in 10 micrometer thick slices
for receptor autoradiography with a microtome cryostat and
thaw-mounted on silane-coated microscope slides (Vectabond).
Sections are air-dried. Preincubation in buffer (50 mM Tris pH 7.4;
2 mM EGTA, 5 mM MgCl2, 0.1 mM bacitracin, 0.2% bovine serum
albumine) is for 10 min at air temperature. Ligand binding is done
for 1 hr at room temperature in buffer supplemented with 10 nM
tritiated compound (specific activity 3.8 TBq/mmol). Non-specific
binding is determined in the presence of 10 microM cold compound.
Three consecutive washes in buffer and distilled water are followed
by air-drying and autoradiography (exposure to Biomax MR film
(Eastman Kodak Company) for 4 weeks).
EXAMPLE 10
Ex Vivo Autoradiography
[0102] The tritiated compound is administrated e.g. i.v. with an
appropriate formulation, the animal sacrificed at the time point of
desired observation, and the tissue of interest is analyzed for
instance as described in Example 9.
* * * * *