Note: Descriptions are shown in the official language in which they were submitted.
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Solvay Pharmaceuticals GmbH
30173 Hannover
Amidomethyl-substituted 1-(carboxyalkyl)-cyclopentylcarbonylamino-benzazepine-
N
acetic acid derivatives, process and intermediate products for their
preparation and
medicaments containing these compounds
The present invention relates to novel amido methyl-substituted 1-
(carboxyalkyl)-
cyclopentylcarbonylamino-benzazepine-N-acetic acid derivatives which are
useful e.g.
for the prophylaxis and/or treatment of cardiovascular conditions or diseases,
especially
cardiac insufficiency, in particular congestive heart failure; hypertension,
including sec-
ondary forms of hypertension such as essential hypertension, renal
hypertension and/or
pulmonary hypertension and/or for the prophylaxis and/or treatment of sexual
dysfunc-
tion and/or for the prophylaxis and/or treatment of adverse conditions
associated with
apoptosis, and also to medicaments containing these compounds. Furthermore,
the in-
vention relates to a process for the preparation of the novel amidomethyl-
substituted
benzazepine-N-acetic acid derivatives and intermediate products of this
process.
Sexual dysfunction (SD) is a significant clinical problem which can affect
both
males and females. The causes of SD may be both organic as well as
psychological.
Organic aspects of SD are typically caused by underlying vascular diseases,
such as
those associated with hypertension or diabetes mellitus, by prescription
medication
and/or by psychiatric disease such as depression. Physiological factors
include fear, per-
formance anxiety and interpersonal conflict. SD impairs sexual performance,
diminishes
selfesteem and disrupts personal relationships thereby inducing personal
distress.
Apoptosis is closely involved in morphogenesis and histogenesis in the develop-
ment process, maintenance of homeostasis, and bio-defense, and it is cell
death having
an important role in maintaining individual lives. When the death process
regulated by
genes is congenially or postnatally hindered, apoptosis is excessively induced
or inhib-
ited to cause functional disorders in various organs, and thus diseases. Drugs
showing
an apoptosis inhibitory activity can be used as agents for the prophylaxis and
treatment
of diseases which are thought to be mediated by promotion of apoptosis.
Cardiovascular-active benzazepine-, benzoxazepine- and benzothiazepine-N-
acetic
acid derivatives having a marked inhibitory action on the enzyme neutral
endopeptidase
(= NEP) are already known from specification EP 0 733 642 A1 (= US 5,677,297).
In
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addition, the compounds described therein also have lesser properties which
inhibit en-
dothelin-converting enzyme (= ECE). Further favourable pharmacological
properties of
compounds falling within the structural scope of EP 0 733 642 A1 are known
from docu-
ments EP 0 830 863 A1 (= US 5,783,53), WO 00148601 A1 (= US 6,482,820) and WO
01/03699 A1 (=US-2003-0040512-A1).
Phosphonic acid substituted benzazepinone-N-acidic acid derivatives with a
combined inhibitory effect on NEP and ECE are disclosed in document EP 0 916
679 A1
(= US 5,952,327).
Pharmaceutical preparations are known from specification WO 02/094176 A2
which contain compounds having an advantageous combinatory action which
inhibits the
metalloprotease enzymes NEP and IGS5 and have, inter alia, cardiovascular-
active
properties. Suitable compounds for such combination preparations are also
compounds
which fall within the scope of specifications EP 0 733 642 A1 and EP 0 916 679
A1. The
enzyme IGSS, as it is to be understood in the context of this invention, and
its physio-
logical role in connection with cardiovascular diseases, is known per se from
the specif i-
cation WO 01/36610 A1. The aforementioned enzyme IGS5 is also known as "human
soluble endopeptidase" (= hSEP).
From document WO 99/55726 A1 it is known, that certain thiol inhibitors of ECE
are
useful for treating or inhibiting i.a. erectile dysfunction.
Document EP 1 097 719 A1 discloses the use of NEP inhibitors for the treatment
of
female sexual dysfunction (= FSD).
Publication WO 02/06492 A1 discloses i.a. antibodies against and inhibitors of
a
specific polypeptide having soluble secreted endopeptidase (= SEP) activity.
In patent application US 20030045449 it is described that matrix-
metalloprotease
inhibitors are useful for the treatment of neurodegenerative diseases.
Problems associ-
ated with that invention are first that matrix-metalloprotease inhibitors
comprise a broad
group of protease inhibitors, and second that according to the said
application the
metalloproteases must be used in a pharmaceutical composition also containing
a N-
NOS inhibitor.
Published patent application US 2002/0013307 teaches the use of vasopeptidase
inhibitors to treat or slow the progression of cognitive dysfunction and to
treat and/or
prevent dementia.
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M. Sumitomo et al. (see Clinical Cancer Research 10 (2004) 260-266) do
describe
the chemosensitization of androgen-independent prostate cancer with NEP.
It was an object of the present invention to provide novel active substances
having
a combined action profile inhibiting the enzymes NEP, hSEP and ECE which are
i.a. suit-
able for the prophylaxis and/or treatment of cardiovascular conditions or
diseases,
especially cardiac insufficiency, in particular congestive heart failure;
hypertension, in-
cluding secondary forms of hypertension such as essential hypertension, renal
hyperten-
sion and/or pulmonary hypertension; andlor for the prophylaxis and/or
treatment of sex-
ual dysfunction and/or for the prophylaxis and/or treatment of adverse
conditions associ-
ated with apoptosis.
It has now surprisingly been found that a group according tv the invention of
novel
amidomethyl-substituted 1-(carboxyalkyl)-cyclopentylcarbonylamino-benzazepine-
N-
acetic acid derivatives is distinguished by an action profile which inhibits
the enzymes
NEP and hSEP, and to a certain extent also ECE, and therefore appears suitable
for the
prophylaxis and/or treatment of cardiovascular conditions or diseases,
especially cardiac
insufficiency, in particular congestive heart failure; hypertension, including
secondary
forms of hypertension such as essential hypertension, renal hypertension
and/or pulmo-
nary hypertension; and/or or for the prophylaxis and/or treatment of sexual
dysfunction
and/or for the prophylaxis andlor treatment of adverse conditions associated
with apop-
tosis.
The subject of the invention is novel amidomethyl-substituted 1-(carboxyalkyl)-
cyclopentylcarbonylamino-benzazepine-N-acetic acid derivatives of the general
formula I,
O
R3
\N
R2 / H
N I
R'OOC
O
O
COOR4
wherein
R' is hydrogen or a group forming a biolabile ester,
R2 is hydrogen, C~~-alkyl or C~~,-hydroxyalkyl, the hydroxyl group of which is
optionally
esterified with C2~-alkanoyl or an amino acid residue, and
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R3 is C~~-alkyl; C~~-alkoxy-C,~-alkyl; C~~-hydroxyalkyl, which is optionally
substituted
by a second hydroxyl group and the hydroxyl groups of which are each
optionally
esterified with C2-a-alkanoyl or an amino acid residue; (C~a-alkyl)2amino-C,_6-
alkyl;
C~~-cycloalkyl; Cue,-cycloalkyl-C,~-alkyl; phenyl-C,~-alkyl, the phenyl group
of which
is optionally substituted 1-2 times by C,~-alkyl, C,~-alkoxy and/or halogen;
naphthyl-C~_4-alkyl; C~s-oxoalkyl; phenylcarbonylmethyl, the phenyl group of
which
is optionally substituted 1-2 times by C,~-alkyl, C,~-alkoxy and/or halogen,
or 2-
oxoazepanyl, or
R2 and R3 together are C~~-alkylene, the methylene groups of which are
optionally re-
placed 1-2 times by carbonyl, nitrogen, oxygen and/or sulphur and which are op-
tionally substituted once by hydroxy, which is optionally esterified with C2.~-
alkanoyl
or an amino acid residue; C,_4-alkyl; C~~-hydroxyalkyl, the hydroxyl group of
which
is optionally esterified with C2~-alkanoyl or an amino acid residue; phenyl or
benzyl,
and
R4 is hydrogen or a group forming a biolabile ester,
and physiologically compatible salts of acids of Formula I and/or
physiologically compati-
ble acid addition salts of compounds of Formula I. Furthermore, a subject of
the inven-
tion is medicaments containing the compounds of Formula I. Even further, a
subject of
the invention is a process for the preparation of the compounds of Formula I
and inter-
mediate products of this process.
Where in the compounds of Formula I or in other compounds described within the
context of the present invention substituents are or contain C,~-alkyl, these
may each be
straight-chain or branched. Where substituents in compounds of Formula I stand
for
halogen, fluorine, chlorine or bromine are suitable. Chlorine is preferred.
Where substitu-
ents contain CZ~-alkanoyl, this may be straight-chain or branched. Acetyl is
preferred as
C2_4-alkanoyl.
Where in the compounds of Formula I hydroxyl groups are esterified with amino
acid residues, these amino acid residues may be derived from natural or non-
natural, a-
or ~-amino acids. Suitable amino acids which can be used are for example
selected from
the group cosisting of alanine, 2-aminohexanoic acid (= norleucine), 2-
aminopentanoic
acid (= norvaline), arginine, asparagine, aspartic acid, cysteine, 3,4-
dihydroxy-
phenylalanine (= dopa), glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine,
lysine, methionine, ornithine (= 2,5-diaminovaleric acid), 5-oxo-2-
pyrrolidinecarbonic acid
(= pyroglutamic acid), phenylalanine, proline, serine, threonine, thyronine,
tryptophan,
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tyrosine and valine. Preferred are amino acid residues which are derived from
alanine,
asparagine, glutamine, glycine, isoleucine, leucine, lysine, ornithine,
phenylalanine,
proline and valine.
The compounds of Formula I represent dicarboxylic acid derivatives optionally
es-
terified with groups forming biolabile esters. The biolabile esters of Formula
I as a rule
represent administerable precursors (_ "prodrugs") of the free acids. Then,
monoesters
or diesters of the compounds of Formula I may occur. Depending on the form of
admini-
stration, the biolabile esters or the free acids are preferred, the latter
being suitable in
particular for intravenous (= i.v.) administration.
Groups which can be cleaved under physiological conditions in vivo, releasing
bioavailable derivatives of the compounds of Formula I, are suitable as groups
forming
biolabile esters R' and R4. Suitable examples of this are C,~-alkyl groups, in
particular
methyl, ethyl, n-propyl and isopropyl; C,~-alkyloxy-C,~-alkyloxy-C,_4-alkyl
groups, in par-
ticular methoxyethoxymethyl; C~~-cycloalkyl groups, in particular cyclohexyl;
C~~_
cycloalkyl-C,~-alkyl groups, in particular cyclopropylmethyl; N,N-di-(C~-
alkyl)amino-C,_s-
alkyl groups; phenyl or phenyl-C,~-alkyl groups optionally substituted in the
phenyl ring
once or twice by halogen, C,_4-alkyl or C,_4-alkoxy or by a C,~-alkylene chain
bonded to
two adjacent carbon atoms; dioxolanylmethyl groups optionally substituted in
the di-
oxolane ring by C,~-alkyl; C2_s-alkanoyloxy-C,~-alkyl groups optionally
substituted at the
oxy-C,~-alkyl group by C,~-alkyl; double esters like 1-[[(C,~-
alkyl)carbonyl]oxy]C,_4-alkyl
esters, e.g. (RS)-1-[[(isopropyl)carbonyl]oxy]ethyl or (RS)-1-
[((ethyl)carbonyl]oxy]-2-
methylpropyl (for preparation see e.g. F.W. Sum et al., Bioorg. Med. Chem.
Lett. 9
(1999) 1921-1926 or Y. Yoshimura et al., The Journal of Antibiotics 39/9
(1986) 1329-
1342 ); carbonate esters like 1-[[(Cø,-cycloalkyloxy)carbonyl]oxy] C,~-alkyl
esters, pref-
erably (RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl (= cilexetil; for
preparation see e.g. K.
Kubo et al., J. Med. Chem. 36 (1993) 2343-2349, cited as °Kubo et al."
hereinafter)) or
2-oxo-1,3-dioxolan-4-yl- C,~-alkyl esters which optionally contain a double
bond in the
dioxolan ring, preferably 5-methyl-2-oxo-1,3-dioxolen-4-yl-methyl (=
medoxomil, for
preparation see e.g. Kubo et al.) or 2-oxo-1,3-dioxolan-4-yl-methyl (_
(methyl)ethylene-
carbonate) . Where the group forming a biolabile ester represents an
optionally substi-
tuted phenyl-C,~-alkyl group, this may contain an alkylene chain with 1 to 3,
preferably 1,
carbon atoms and preferably stands for optionally substituted benzyl, in
particular for 2-
chlorobenzyl or 4-chlorobenzyl. Where the group forming a biolabile ester
represents an
optionally substituted phenyl group, the phenyl ring of which is substituted
by a lower
alkylene chain, this may contain 3 to 4, preferably 3, carbon atoms and in
particular be
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indanyl. Where the group forming a biolabile ester represents an optionally
substituted
G2_s-alkanoyloxy-C,.~-alkyl group, the C2_s-alkanoyl group may be straight-
chain or
branched.
R' preferably has the meanings hydrogen, ethyl, methoxyethoxymethyl, (RS)-1-
[[(isopropyl)carbonyl]oxy]ethyl, (RS)-1-[[(ethyl)carbonyl]oxy]-2-methylpropyl,
(RS)-1-
[[(cyclohexyloxy)carbonyl]oxy]ethyl, 5-methyl-2-oxo-1,3-dioxolen-4-yl-methyl,
2-oxo-1,3-
dioxolan-4-yl-methyl or (RS)-1-[[(ethoxy)carbonyl]oxy]ethyl.
R2 preferably has the meanings hydrogen, methyl, ethyl, 2-hydroxyethyl or 3-
hydroxypropyl, each hydroxyl group optionally being esterified with C2~-
alkanoyl or an
amino acid residue.
Where R3 has the meaning (Coy-alkyl)2amino-C,_s-alkyl, one or two C~-alkyl
groups
can independently of each other be present. More specifically, "(Co..4-
alkyl)2amino-C,_s-
alkyl" expressly comprises the meanings "(Co)2-alkylamino-C,_s-alkyl",
"(Co)(C,~)-alkyl-
amino-C,_s-alkyl° and "(C,~)2-alkylamino-C,_s-alkyl". "(Co)2-alkylamino-
C,_s-alkyl" is meant
to denominate an unsubstituted primary (_ -NH2) amino group bonded to C,_6-
alkyl(en);
"(Co)(C,~,~alkylamino-C,_s-alkyl" is meant to denominate a secondary amino
group mono-
substituted by (C,.~)-alkyl and bonded to C,_s-alkyl(en); "(C,~)2-alkylamino-
C,_s-alkyl" is
meant to denominate a tertiary amino group disubstituted by (C,~)-alkyl and
bonded to
C,_s-alkyl(en). R3 preferably has the meanings isopropyl; methoxyethyl; 2-
hydroxyethyl or
3-hydroxypropyl, each hydroxyl group optionally being esterified with C2~-
alkanoyl or an
amino acid residue; 3-acetyloxy-n-propyl; cyclopropylmethyl; 2-methoxybenzyl,
4-
methoxybenzyl; 4-methoxyphenylethyl; 2,4 -dimethoxybenzyl; 1-naphthylmethyl; 3-
oxo-
1,1-dimethylbutyl; phenyl-2-oxoethyl; 2-(4-methoxyphenyl)-2-oxoethyl; 3-(2-
oxoaze-
panyl); (Ca4-alkyl)2amino-C,_s-alkyl, in particular dimethylamino-n-propyl,
(methyl)amino-
ethyl, amino-n-propyl, amino-n-butyl or amino-n-pentyl.
Where R2 and R3 together are Ca.ralkylene, the methylene groups of which are
op-
tionally replaced or optionally substituted, optionally in each case
morpholine; piperidine;
4-ketopiperidine; 4-hydroxypiperidine, optionally being esterified with C2_a-
alkanoyl or an
amino acid residue at the hydroxyl group; piperazine or pyrrolidine is
preferred.
R4 preferably has the meanings hydrogen, C,.a-alkyl, p-methoxybenzyl, N,N-di-
(Co~-
alkyl)amino-C,_s-alkyl, (RS)-1-[[(isopropyl)carbonyl]oxy]ethyl, (RS)-1-
[[(ethyl)carbonyl]-
oxy]-2-methylpropyl, (RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl, 5-methyl-2-
oxo-1,3-
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dioxolen-4-yl-methyl, 2-oxo-1,3-dioxolan-4-yl-methyl or (RS)-1-
[[(ethoxy)carbonyl]oxy]-
ethyl.
Particularly preferred compounds of Formula I are selected from the group
consist-
ing of
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4.-[isopropyl(methyl)amino]-4-oxobutanoic acid
(32);
2-{[1-({(1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-(dimethylamino)-4-oxobutanoic acid
(54);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-(diethylamino)-4-oxobutanoic acid
(55);
2-{[1-({(1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}car-
bonyl)cyclopentyl]methyl}-4-[(2-hydroxyethyl)(methyl)amino]-4-oxobutanoic acid
(43);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}car-
bonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)(methyl)amino]-4-oxobutanoic
acid (56);
2-{[1-({(1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-(4-hydroxypiperidin-1-yl)-4-oxobutanoic acid
(57);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-oxo-4-[4-(L-valyloxy)piperidin-1-yl]butanoic
acid (68);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-morpholin-4-yl-4-oxobutanoic acid (66);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-oxo-4-(4-oxopiperidin-1-yl)butanoic acid (45);
4-[bis(2-hydroxyethyl)amino]-2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic acid (58);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}car-
bonyl)cyclopentyl]methyl}-4-{ethyl[3-(ethylamino)propyl]amino}-4-oxobutanoic
acid
(52);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-[[2-(dimethylamino)ethyl](methyl)amino]-4-
oxobutanoic
acid (59);
4-[(3-aminopropyl)(ethyl~mino]-2-{[1-({(1-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic acid (67),
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2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-{methyl(2-(methylamino)ethyl]amino}-4-
oxobutanoic
acid (68);
4-[(4-aminobutyl)(methyl)amino]-2-{(1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-
1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic acid (75);
4-[(4-aminobutyl)(ethyl )amino]-2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyf]methyl}-4-oxobutanoic acid (76);
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-{methyl[3-(methylamino)propyl]amino}-4-
oxobutanoic
acid (77) and
4-[(5-aminopentyl)(methyl)amino]-2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-
1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic acid (78),
together with their biolabile esters and physiologically compatible salts of
acids of these
compounds of Formula I and/or physiologically compatible acid addition salts
of these
compounds of Formula I.
According to the invention, the novel compounds of Formula I and their salts
are
obtained by reacting a compound of the general formula II,
O
HO
H \
N II
R~°~ OOC
O N
O
~COOR4o'
wherein R'°' and R4°', independently of each other, are each an
acid-protecting group,
with a compound of the general formula III,
R3
R2-NH III
wherein RZ and R3 have the above meanings,
where R2 andlor R3 contain free hydroxyl groups, if desired these are reacted
with a com-
pound of the general formula IV,
C~_3-C(O)-X Iv
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9
wherein X stands for a leaving group, or with an amino acid derivative
protected by a
suitable protective group,
where R'°' and/or R4°' do not represent desired groups forming a
biolabile ester and/or
where R2 and/or R3 comprise protective groups in any present amino acid
residue, these
are cleaved off in succession in the resulting compounds simultaneously or
individually in
any desired sequence and if desired the acid functions released in each case
are con-
verted into biolabile ester groups,
and if desired resulting acids of Formula I are converted into their
physiologically com-
patible salts, or salts of the acids of Formula I are converted into the free
acids and/or
bases of Formula I are converted into their acid addition salts or acid
addition salts are
converted into free bases of Formula I.
Suitable physiologically compatible salts of acids of Formula I are in each
case al-
kali metal, alkaline-earth metal or ammonium salts thereof, for example
sodium, potas-
sium or calcium salts thereof, physiologically compatible, pharmacologically
neutral or-
ganic salts thereof with amines such as for example ammonia, diethylamine,
tent. bu-
tylamine, N-methylglucamine, choline, or with amino acids such as for example
arginine.
Where in compounds of Formula I the substituents R2 andlor R3 contain basic
groups, in
particular nitrogen, the compounds of Formula I may also occur in the form of
acid addi-
tion salts. Physiologically compatible acid addition salts of compounds of
Formula I are
their conventional salts with inorganic acids, for example sulphuric acid,
phosphoric acid
or hydrohalic acids, preferably hydrochloric acid, or with organic acids, for
example lower
aliphatic monocarboxylic, dicarboxylic or tricarboxylic acids such as malefic
acid, fumaric
acid, tartaric acid, citric acid, or with sulphonic acids, for example lower
alkanesulphonic
acids such as methanesulphonic acid.
Conventional protective groups for protecting carboxylic acid functions may be
se-
lected as acid-protecting groups R'°' and R°°', which can
then be cleaved off again using
known methods. Suitable protective groups for carboxylic acids are known, for
example,
from McOmie, "Protective Groups in Organic Chemistry", Plenum Press (cited as
°McOmie° hereinafter), and Greene, Wuts, "Protective Groups in
Organic Synthesis",
Wiley Interscience Publication (cited as "Greene" hereinafter), each in the
most recent
edition. Groups forming a biolabile ester may also be used as acid-protecting
groups.
The compounds obtained upon reaction of compounds of Formula II with compounds
of
Formula III in these cases already represent esters of Formula I according to
the inven-
tion.
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Suitable acid-protecting groups R'°' and R4°' are in particular
those groups which
can be selectively cleaved or selectively introduced independently of each
other. Exam-
ples of acid-protecting groups which are cleavable under different conditions,
which may
also represent groups forming biolabile esters, are: unbranched lower alkyl
groups such
as ethyl, which can be cleaved off relatively easily under basic conditions;
branched
lower alkyl groups such as tert. butyl, which can be cleaved off easily by
acids such as
trifluoroacetic acid; phenylmethyl groups optionally substituted in the phenyl
ring such as
benzyl, which can easily be cleaved off by hydrogenolysis or alternatively
under basic
conditions; phenylmethyl groups substituted one or more times in the phenyl
ring by
lower alkoxy, such as p-methoxybenzyl, which are cleaved relatively easily
under oxida-
tive conditions, for example under the action of 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (= DDQ) or ceric ammonium nitrate; or the known. silicon-
containing pro-
tective groups which can easily be cleaved by fluoride ions. The person
skilled in the art
is familiar with selecting suitable protective groups to obtain a desired
substitution pat-
tern.
The compounds of Formula I contain two chiral carbon atoms, namely the carbon
atom bearing the amide side chain in position 3 of the benzazepine skeleton (=
Cb*) and
the carbon atom bearing the radical "-COOR'" (= Ca*). The compounds can thus
be pre-
sent in a total of four stereoisomeric forms. The present invention comprises
both the
mixtures of stereoisomers and enantiomers, and also the isomerically pure
compounds
of Formula I. Isomerically pure compounds of Formula I are preferred.
Particularly pre-
ferred are compounds of Formula I wherein the carbon atom bearing the amide
side
chain in position 3 of the benzazepine skeleton is in the "S" configuration.
With respect
to the chiral carbon atom "*Ca" bearing the radical "-COOR'", the
configuration of the
compounds of Formula I which is preferred according to the invention in the
context of
this invention is provisionally assigned the configuration designation "rel1"
(see the
experimental part). It can be derived by analogous observations of suitable
compounds
of known configuration that the preferred configuration "rel1" at the chiral
centre "*Ca" is
probably likewise the "S" configuration.
The reaction of the acids of Formula II with the amines of Formula III can be
carried
out according to conventional methods for the formation of amide groups by
aminoacyla-
tion. The carboxylic acids of Formula II or their reactive derivatives may be
used as acy-
lation agents. In particular mixed acid anhydrides and acid halides are
suitable reactive
derivatives. Thus for example acid chlorides or acid bromides of the acids of
Formula II
or mixed esters of the acids of Formula II with organic sulphonic acids, for
example with
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11
lower-alkanesulphonic acids optionally substituted by halogen, such as
methanesul-
phonic acid or trifluoromethanesulphonic acid, or with aromatic sulphonic
acids such as
benzenesulphonic acids or with benzenesulphonic acids substituted by lower
alkyl or
halogen, e.g. toluenesulphonic acids or bromobenzenesulphonic acids, can be
used.
The acylation may take place in an organic solvent which is inert under the
reaction con-
ditions at temperatures between -20°C and room temperature (= RT).
Suitable solvents
are halogenated hydrocarbons such as dichloromethane or aromatic hydrocarbons
such
as benzene or toluene or cyclic ethers such as tetrahydrofuran (= THF) or
dioxane or
mixtures of these solvents.
The acylation can expediently, in particular if a mixed anhydride of the acids
of
Formula II with a sulphonic acid is used as acylation agent, be carried out in
the pres-
ence of an acid-binding reagent. Suitable acid-binding agents are for example
organic
bases which are soluble in the reaction mixture such as tertiary nitrogen
bases, for ex-
ample tert.-lower alkylamines and pyridines such as triethylamine,
tripropylamine,
N-methylmorpholine, pyridine, 4-dimethylaminopyridine, 4-diethylaminopyridine
or
4-pyrrolidinopyridine. Organic bases used in excess can also serve as solvents
at the
same time.
If the acids of Formula II themselves are used as acylation agents, the
reaction of
the amino compounds of Formula III with the carboxylic acids of Formula II can
expedi-
ently also be carried out in the presence of a coupling reagent known e.g.
from peptide
chemistry as being suitable for amide formation. Examples of coupling reagents
which
promote amide formation with the free acids by reacting with the acid in situ,
forming a
reactive acid derivative, are in particular: ethyl chloroformate,
alkylcarbodiimides, e.g.
cycloalkylcarbodiimides such as dicyclohexylcarbodiimide or N-(3-
dimethylaminopropyl)-
N'-ethylcarbodiimide (= EDC), carbonyldiimidazole and N-lower alkyl-2-
halopyridinium
salts, in particular halides or toluenesulphonates. The reaction in the
presence of a cou-
pling reagent can be carried out expediently at temperatures of -30° to
+50°C in solvents
such as halogenated hydrocarbons and/or aromatic solvents and optionally in
the pres-
ence of an acid-binding amine described above.
In the compounds obtained by reacting the compounds of Formula II with the com-
pounds of Formula III, wherein R2 and/or R3 contain free hydroxyl groups,
these may if
desired be reacted in known manner with a compound of Formula IV. In compounds
of
Formula IV, the leaving group X stands for example for halogen, preferably for
chlorine.
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12
In the compounds obtained by reacting the compounds of Formula II with the com-
pounds of Formula III, wherein R2 and/or R3 contain free hydroxyl groups,
these may if
desired be reacted in known manner with an amino acid derivative protected by
a suit-
able protective group. Suitable protective groups for amino acids together
with methods
of introducing them or selectively cleaving them are known in the art, e.g.
from McOmie
or from Greene. Suitably protected amino acid derivatives are either
commercially avail-
able or can be prepared in a known manner.
The protective groups R'°' a nd R'°', provided that they do not
represent any de-
sired groups forming a biolabile ester, andlor the protective groups which may
be present
in any present amino acid moiety in R2 and/or R3, can be cleaved in known
manner and
if desired selectively from the compounds obtained by reacting the compounds
of For-
mula II with the compounds of Formula III.
Compounds of Formula I may be isolated from the reaction mixture and if neces-
sary purified in known manner, for example by high-performance liquid
chromatography
(= HPLC).
The starting compounds of Formula II are novel compounds which are suitable as
intermediate products for the preparation of novel active substances, for
example for the
preparation of the compounds of Formula I. The compounds of Formula II can be
pre-
pared by reacting compounds of the general formula V,
O
R50
* OH V
R'°' OOC
O
wherein R5 is an acid-protecting group and R'°' has the above meaning,
with compounds
of the general formula VI,
HZN
* I VI
N
O
~COOR~°'
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13
wherein R4°' has the above meaning, and subsequently cleaving off the
acid-protecting
groups R5 again in known manner. The reaction can be carried out in a manner
known
for aminoacylations, for example corresponding to the manner indicated above
for the
reaction of compounds of Formula II with compounds of Formula III. To avoid
undesir-
able secondary reactions, it may be advantageous to cleave the acid-protecting
groups
R5 by means of a method which does not operate in alkaline medium and
consequently
to select correspondingly suitable acid-protecting groups R5.
The amines of Formula ifl are known per se or can be prepared in known manner
from known compounds.
The reactive acid derivatives of Formula IV are known per se or can be
prepared in
known manner from known compounds. These are straight-chain or branched C,~-
carboxylic acid derivatives.
Compounds of Formula V can be prepared by reacting acrylic ester derivatives
of
the general formula VII,
O
R50
VII
R'°'OOC
wherein R'°' and RS have the above meanings, with
cyclopentanecarboxylic acid. The
reaction can take place in known manner under the conditions of a Michael
condensation
in an organic solvent which is inert under the reaction conditions by reaction
of the
cyclopentanecarboxylic acid with a strong base capable of forming the dianion
of the
cyclopentanecarboxylic acid and subsequent reaction with the acrylic ester
derivative of
Formula VII. Suitable solvents are ethers, in particular cyclic ethers such as
THF. Suit-
able strong bases are non-nucleophilic organic alkali metal amides or alkali
metal lower
alkyls such as lithium diisopropylamide or n-butyllithium. Expediently, the
cyclopentane-
carboxylic acid is reacted in THF with two equivalents of n-butyllithium and
the reaction
mixture is then reacted further with the compound of Formula VII. The reaction
tempera-
ture may be between -80° and 0°C.
Compounds of Formula VI are known, for example from the specification
EP 0 733 642 A1, and can be prepared in the form of their racemates or
alternatively in
isomerically pure form according to the methods described therein or methods
analogous
thereto.
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14
Compounds of Formula VII can be prepared by esterifying compounds of the gen-
eral formula VIII,
O
R50
VIII
HOOC
wherein R5 represents an acid-protecting group, in known manner with a desired
alcohol.
Compounds of Formula VIII can for example obtained by reacting itaconic acid
an-
hydride under known conditions which open the anhydride group with a reagent
capable
of formation of the acid-protecting group R5 such as a correspondingly
substituted alco-
hol.
In the reactions described above, the chiral centres in the starting compounds
of
Formula V and of Formula VI are not changed, so that depending on the type of
starting
compounds finally isomerically pure compounds of Formula I or isomer mixtures
can be
obtained. For the preparation of stereochemically uniform compounds of Formula
I, ex-
pediently stereochemically uniform compounds of Formula V are reacted with
stereo-
chemically uniform compounds of Formula VI. If an enantiomerically pure
compound of
Formula V is reacted with a racemic compound of Formula VI or a racemic
compound of
Formula V with an enantiomerically pure compound of Formula VI, in each case a
mix-
ture of two diastereomers is obtained, which if desired can be separated at
the stage of
the compounds of Formula II or at the stage of the compounds of Formula I in
known
manner. The reaction of racemic compounds of Formula V with racemic compounds
of
Formula VI yields corresponding mixtures of four isomers, which can be
separated if de-
sired in known manner, for example by HPLC separation on possibly chiral
separating
materials.
The compounds of Formula V have a chiral centre at the carbon atom bearing the
radical "-COOK'°'" and are obtained upon synthesis from acrylic ester
derivatives of
Formula VII in the form of their racemates. The optically active compounds can
in princi-
ple be obtained from the racemic mixtures in a manner known per se, e.g. by
chroma-
tographic separation on chiral separating materials or by reaction with
suitable optically
active bases, e.g. a-methylbenzylamine, cinchonidine or pseudoephedrine, and
subse-
quent separation into their optical antipodes by fractional crystallisation of
the salts ob-
tained.
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The compounds of Formula I and their pharmacologically compatible salts are
dis-
tinguished by advantageous pharmacological properties. In particular, the
substances
inhibit the enzyme NEP. NEP is an enzyme which breaks down endogenous
natriuretic
peptides, e.g. atrial natriuretic peptide (= ANP). Due to their inhibitory
action on the NEP
activity, the substances are capable of improving the biological activity and
useful life of
the natriuretic peptides which can be attacked by NEP, in particular ANP, and
are there-
fore suitable for the treatment of pathological conditions which are
beneficially influenced
by the action of such hormones, above all of cardiovascular diseases,
especially cardiac
insufficiency, in particular congestive heart failure.
In congestive heart failure, a peripheral vascular resistance which is
increased by
reflex occurs due to a disease-induced reduced ejection fraction of the heart.
This means
that the myocardium has to start pumping against an increased afterload. This
leads in a
vicious circle to increased strain on the heart and makes the situation even
worse. The
increase in the peripheral resistance is mediated, inter alia, by the
vasoactive peptide
endothelin (= ET-1 ). Endothelin is the most powerful currently-known
endogenous vaso-
constrictor substance and is produced from the precursor big endothelin (= Big-
ET-1).
According to what is currently known, various enzymes collaborate in the
conversion of
Big-ET-1 to ET-1, inter alia the enzymes ECE and hSEP (see on this point e.g.
WO
02/094176).
In congestive heart failure, as a result of the decreased cardiac output and
the in-
crease in peripheral resistance, back-pressure phenomena of the blood occur in
the pul-
monary circulation and the heart itself. As a result, an increased wall
tension of the heart
muscle occurs in the area of the auricles and chambers. In such a situation,
the heart
functions as an endocrine organ and secretes, inter alia, ANP into the
bloodstream. Due
to its marked vasodilatory and natriureticldiuretic activity, ANP brings about
both a reduc-
tion in the peripheral resistance and a decrease in the circulating blood
volume. The
consequence is a marked pre- and afterload decrease. This constitutes an
endogenous
cardioprotective mechanism. This positive endogenous mechanism is limited in
that ANP
only has a very short half-life in the plasma. The reason for this is that the
hormone is
very rapidly broken down by NEP. .
The compounds according to the invention reduce the production of endothelin
by
inhibiting the ECE activity and additionally inhibiting the hSEP activity and
thus counter-
act an increase in the peripheral resistance, which consequently results in
relieving myo-
cardial strain. Results hitherto furthermore suggest that the substances
according to the
invention by inhibiting the NEP activity result in higher ANP levels and an
extended dura-
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16
tion of action of ANP. This should result in intensification of the ANP-
mediated endoge-
nous mechanism of cardioprotective action and impart to the substances of
Formula I
high effectiveness with respect to intensification of the diuretic/natriuretic
ANP-induced
activities.
NEP is involved not only in the breakdown of ANP but also in the breakdown of
en-
dothelin. It follows from this that pure NEP inhibition in addition to the
desired increase in
the ANP levels would also lead to an unfavourable increase in the endothelia
levels. For
this reason, a mixed profile of NEP, hSEP and a certain proportion of ECE
inhibition
should be regarded as particularly beneficial, since it prevents both the
breakdown of the
natriuretiddiuretic ANP (by NEP blockade), and simultaneously inhibits the
formation of
endothelia (by hSEP and ECE inhibition). As a result, a positive influence can
be brought
to bear on the adverse attendant effect of pure NEP inhibitors (namely
undesirable in-
crease in the endothelia levels).
The combined action profile of compounds of Formula I as inhibitors of NEP,
hSEP
and, to a lesser extent, also of ECE, makes the compounds according to the
invention
appear particularly suitable for the prophylaxis and/or treatment of
pathological condi-
tions like conditions or diseases such as cardiovascular conditions or
diseases, espe-
cially cardiac insufficiency, including acute heart failure and chronic heart
failure and in
particular congestive heart failure; but also hypertension, including
secondary forms of
hypertension such as essential hypertension, renal hypertension and/or
pulmonary hy-
pertension; heart failure, angina pectoris, cardiac arrhythmias, myocardial
infarction, pe-
rioperative myocardial infarction, poor prognosis myocardial infarction,
cardiac hypertro-
phy, congestive cardiomyopathy, hypertrophic obstructive cardiomyopathy,
hypertrophic
non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocarditis,
pericarditis
and/or endocarditis la rger mammals, particularly humans. The compounds of
Formula I
may also be used beneficially in the prophylaxis or treatment of damage to the
heart, in
particular to the myocardium, induced by cardiotoxic doses of medicaments, in
particular
of cytostatic agents, preferably of cytostatic antibiotics or chemicals;
angina abdominal is,
cerebral ischaemias, peripheral vascular disease, subarachnoid haemorrhage,
chronic
obstructive pulmonary disease (COPD), asthma, renal disease (renal failure),
atheroscle-
rosis, and pain in cases of colorectal or prostatic carcinoma in larger
mammals, particu-
larly humans.
What is striking is the surprisingly good effectiveness of the compounds of
Formula
I after i.v. administration with regard to their blood pressure-regulating
action, in particular
their hypotensive action.
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17
Description of the pharmacological test methods
The example numbers quoted relate to the preparation examples described be-
low.
1. In-vitro investigation of the NEP inhibitory action of the substances
To demonstrate the inhibitory action of the substances according to the
invention on
NEP, the inhibitory action of the substances on the hydrolytic breakdown of
the polypep-
tide Mca-Asp-Ile-Ala-Trp-Phe-Dpa-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-Gly-Leu-Gly-
COOH
occurring as a result of the enzymatic activity of NEP was investigated in a
standard test
in vitro. In this test, the measure of the inhibitory activity of the
substances which was
determined was their ICS value. The ICS value of a test substance having
enzyme-
inhibitory activity is that concentration of the test substance at which 50%
of the enzy-
matic activity of the NEP is blocked.
Test buffer: 100 mM Tris pH 7.0, 250 mM NaCI
Enzyme: soluble, human recombinant NEP
Prof. Crine, University of Montreal, Canada
stock solution: 100 Ngiml in 20 mM Tris pH 7.0,
Working solution: Stock solution with test buffer diluted to 2 Ng/ml
Substrate: Mca*-Asp-Ile-Ala-Trp-Phe-Dpa**-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-
Gly-Leu-Gly-COOH; a fluorescence-quenched Big-ET-1 analogon,
i.e. a substrate of metalloproteases which is detectable via the fluo-
rescence signal, in particular of NEP and ECE-1. The fluorescence
of the MCA fluorophore is initially quenched by the presence of the
"quencher" Dpa.
*Mca = (7-methoxycoumarin-4-yl)
**Dpa = (3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)
from Polypeptide Laboratories, Wolfenbuttel, Germany
Stock solution: 100 NM in test buffer
Test substances: All the substances were dissolved in DMSO (10 mM) and diluted
to
the concentration to be tested with test buffer.
70 NI test buffer, 10 NI enzyme working solution and 10 NI test substance
solution were
mixed in an Eppendorf vessel and preincubated at 37°C for 15 minutes (=
min.). Then 10
NI substrate stock solution was added and the test batch was incubated for 60
minutes at
37°C. The enzymatic reaction was then ended by 5-minutes' heating to
95°C. After cen-
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18
trifugation (Heraeus Biofuge B, 3 min.), the liquid supernatant was
investigated by HPLC
in accordance with the following specifications.
The substrate was separated from cleavage products by means of reversed-phase
HPLC (CC 125/4 Nucleosil 300/5 C,8 RP column with CC 8/4 Nucleosil 100/5 C18
pre-
column, from Macherey-Nagel, Duren, Germany). For this, 60 NI of the test
mixture was
injected into the HPLC sample injection point and the column was then eluted
at a flow
rate of 1 mllmin with the following gradient:
Mobile Phase A: 100% H20 + 0.5M H3POa pH 2.0
Mobile Phase B: 100% acetonitrile + 0.5M H3P04
0 -2 min. 20% B 8 -10 min. 60 - 90% B
2 -6 min. 20 - 60% B 10 -13 min. 90% B
6 -8 min. 60% B 13 -15 min. 90 - 20% B
All the peptides were detected by absorption at 214 nm and by fluorescence
with an ex-
citation wavelength of 328 nm and an emission wavelength of 393 nm.
Upon enzymatic cleavage of the peptide, the fluorophore (= Mca) and the
quencher end
up in different peptide fragments, which reduces the effectiveness of the
quench. This
results in an increase in fluorescence. The increasing fluorescence signal
(corresponds
to the surface, A) of the HPLC peak of the peptide with the non-quenched Mca
fluoro-
phore is used for the further calculations. This signal was compared for
samples with (_
~nhib) and without (= A~°~,) test substance of Formula I, and the value
"% inhibition" was
calculated on the basis of the respective peak areas according to the
following formula:
inhibitl0n = 100*(1-A;~h~blA~"m)
All the samples were measured in duplicates and average values were calculated
there-
from. A standard inhibitor (10 nM thiorphan) and a solvent control (0.1% DMSO)
were
likewise measured as quality controls on each run.
In this test model the test substances of Formula I listed in Table 1 below
had the IC~
values given below:
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19
Table 1: NEP-inhibiting action of the test substances in vitro
Exam 1e ICS NEP
No.
3 17.9
37
16 20.0
17 <1
19 18.2
12.9
21 16.9
23 10.6
24 11.1
15.5
27 7.8
31 4.0
32 13.8
43 3.2
59 12
63 9
76 10.0
2. In vitro investigation of the hSEP-inhibitory action of the substances
To demonstrate the inhibitory action of the substances according to the
invention on
hSEP, the inhibitory action of the substances on the hydrolytic breakdown of
the poly-
peptide Mca-Asp-Ile-Ala-Trp-Phe-Dpa-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-Gly-Leu-
Gly-
COOH occurring as a result of the enzymatic activity of the hSEP was
investigated in a
standard test in vitro. In this test, the measure of the inhibitory activity
of the substances
which was determined was their ICS value. The ICS value of a test substance
having
enzyme-inhibitory activity is that concentration of the test substance at
which 50% of the
enzymatic activity of the hSEP is blocked.
Test buffer: 100 mM Tris pH 7.0, 250 mM NaCI
Enzyme: His6-tagged hSEP ectodomain
from Innogenetics, Ghent, Belgium
Stock solution: 53 mg/ml in 20 mM HEPES pH 7.2, 5% glycerol,
0.005% Tween20, 100 mM NaCI, purity >99%
Working solution: stock solution with test buffer diluted to 10 mg/ml
Substrate: Mca-Asp-Ile-Ala-Trp-Phe-Dpa-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-Gly-
Leu-Gly-COOH; fluorescence-quenched Big-ET-1 analogon.
Stock solution: 100 NM in test buffer
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from Polypeptide Laboratories, Wolfenbiittel, Germany
Test substances: All the substances were dissolved in DMSO (10 mM) and diluted
to
the concentration to be tested with test buffer.
The test and the HPLC procedure were carried out analogously to the manner set
forth
above for determining the in vitro inhibitory action of the test substances on
NEP. 10 nM
phosphoramidon served as standard inhibitor in the HPLC procedure.
In this test model the test substances of Formula I listed in Table 2 below
had the ICS
values given below:
Table 2: hSEP-inhibiting action of the test substances in vitro
Exam 1e No. ICS hSEP
2 21.4
3 7.8
10 25.3
16 15.0
17 24.0
19 9.5
20 36.3
23 17.3
24 27.0
3.4
27 26.8
28 11.9
31 12.3
43 2.9
56 2.5
59 4.0
76 4.0
3. In vivo investigation of the inhibitory action of the substances on the
formation of ET-1
from Bid-ET-1 in rats
To demonstrate the inhibitory action of the substances according to the
invention on the
formation of ET-1 from Big-ET-1, the inhibitory action of the test substances
on the hy-
drolytic breakdown of Big-ET-1 to ET-1 occurring as a result of the enzymatic
activity of
ECE and related enzymes such as hSEP was investigated in a standard test in
vivo. ET-
1 is an endogenous strongly vasoconstrictor substance. An increase in the ET-1
level
results in an increase in blood pressure. Upon infusion of Big-ET-1, an
increase in blood
pressure takes place to the extent that ET-1 is produced therefrom by enzyme-
catalysed
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21
cleavage of Big-ET-1. As a measurement of the enzyme-inhibiting action of the
sub-
stances, their inhibitory action on the increase in blood pressure induced by
infusion of
Big-ET-1 was determined.
Rats (Sprague-Dawley, CRLD = Charles River) were anaesthetised with 1 ml/kg
Rompun
/ Ketavet 1:1. A pressure transducer (Statham) was inserted into the carotid
artery to
measure blood pressure. One jugular vein was cannulated for administering the
sub-
stance, and the other for administering Big-ET-1. After a 20-minute rest
phase, the rats
were administered the corresponding test substance of Formula I in a
concentration as a
rule of 10 Nmol/kg, or a vehicle. Five minutes later, 0.5 nmollkg Big-ET-1 was
infused
over a period of one minute. The systolic (SAP = systolic arterial pressure)
and the dia-
stolic (DAP = diastolic arterial pressure) blood pressure and the heart rate
were each
measured before administration of the substance or before administration of
Big-ET and
in each case every five minutes over a period of 30 minutes after Big-ET
administration
using the pressure transducer in known manner. The maximum Big-ET-induced
increase
in blood pressure and the maximum lowering of heart rate were calculated from
the
measured values as the difference between the value measured at the moment of
maximum development of the Big-ET action (typically after 5 min.) and the
value meas-
ured before Big-ET infusion. Furthermore, the integral of the blood pressure
curve under
the influence of Big-ET-1 was determined over 30 minutes (AUC = area under the
curve).
The AUC value provides information about the entire extent and duration of the
Big-ET
action or the reduction thereof by substances; the AUC value can therefore -
in addition
to the maximum Big-ET action - provide additional information about the effect
of the
substances, for example in the event that the substances e.g. do not, or only
slightly,
influence the maximum Big-ET action, but considerably accelerate the subsiding
of this
action.
The percentage inhibition of the maximum Big-ET-1 effect on the systolic
arterial blood
pressure (SAP) after i.v. administration of the test substances compared with
administra-
tion of a vehicle is set forth in Table 3 below:
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22
Table 3: In vivo investigation of the antihypertensive properties of the test
substances.
Example No. % substance-related inhibition
of the maxi-
mum Bi -ET effect on SAP vs. control
2 - 53
3 - 94
4 - 95
8 - 113
14 - 59 3 mol
16 - 45
17 - 46
20 - 67
21 - 43
23 - 40
24 - 54
26 - 53
29 _ 4g I
32 - 52
34 - 78
35 - 63
38 - 48 3 mol
44 -75
59 -98
67 -109
68 -108
75 -52 3 mol
76 I -g3
The compounds of Formula I also do exhibit ECE-inhibitory properties to a
certain
extent. The ECE-inhibitory properties of the substances of Formula I can be
demon-
strated in a standard test in vitro.
The compounds of Formula I are dually acting compounds which are capable of in-
hibiting NEP and hSEP and are also suited for prophylaxis and/or treatment of
SD.
In the clinic, SD disorders have been divided into female sexual dysfunction
(FSD)
disorders and male sexual dysfunction (MSD) disorders (see Melman, A. &
Gingell, J. C.
(1999). The epidemiology and pathophysiology of erectile dysfunction. J
Urology 161 : 5-
11, hereinafter cited as °Melman et al. 1999"). The dually acting
compounds of the inven-
tion which are capable of inhibiting NEP and hSEP, in particular the compounds
of For-
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23
mula I, are particularly beneficial for the prophylaxis and/or treatment of
MSD (e. g. male
erectile dysfunction-MED). A further advantage of the compounds of Formula I
in this
indication is a certain ECE inhibitory share at their profile of action.
MSD is generally associated with erectile dysfunction, also known as male
erectile
dysfunction (= MED) (see Genet, A. E. et al (1994), Male erectile dysfunction
assessment
and treatment options. C07Sp. They. 20: 669-673) hereinafer cited as
°Benet et al.
1994"). MED is defined as: "... the inability to achieve and/or maintain a
penile erection
for satisfactory sexual performance (see NIH Consensus Development Panel on
Impo-
tence (1993). NIH Consensus Conference Impotence. JA. M. A. 270: 83) ...". It
has been
estimated that the prevalence of erectile dysfunction (= ED) of all degrees
(minimal,
moderate and complete impotence) is 52% in men 40 to 70 years old, with higher
rates
in those older than 70 (Melman et al. 1999). The condition has a significant
negative im-
pact on the quality of life of the patient and their partner, often resulting
in increased
anxiety and tension which leads to depression and low self esteem. Whereas two
dec-
ades ago, MED was primarily considered to be a psychological disorder (Benet
et al.
1994), it is now known that for the majority of patients there is an
underlying organic
cause. As a result, much progress has been made in identifying the mechanism
of nor-
mal penile erection and the pathophysiology of MED.
When the dually acting compounds capable of inhibiting NEP and hSEP of the in-
vention, in particular the compounds of Formula I, are used in the therapy of
FSD, ther-
apy of female sexual arousal disorder (= FSAD) is preferred.
FSD is best defined as the difficulty or inability of a woman to find
satisfaction in
sexual expression. FSD is a collective term for several diverse female sexual
disorders
(Leiblum, S. R. (1998). Definition and classification of female sexual
disorders. Int. J.
Impotence Res., 10, S104-S106 ; German, J. R., German, L.8~ Goldstein, I.
(1999). Fe-
male sexual dysfunction: Incidence, pathophysiology, evaluations and treatment
options.
Urology, 54,385-391.). The woman may have lack of desire, difficulty with
arousal or or-
gasm, pain with intercourse or a combination of these problems. Several types
of dis-
ease, medications, injuries or psychological problems can cause FSD.
Treatments in
development are targeted to treat specific subtypes of FSD, predominantly
desire and
arousal disorders. The categories of FSD are best defined by contrasting them
to the
phases of normal female sexual response: desire, arousal and orgasm (Leiblum,
S. R.
(1998). Definition and classification of female sexual disorders. Int. J.
Impotence Res.,
10, S104-S106).
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24
Desire or libido is the drive for sexual expression. Its manifestations often
include
sexual thoughts either when in the company of an interested partner or when
exposed to
other erotic stimuli.
Arousal is the vascular response to sexual stimulation, an important component
of
which is genital engorgement and includes increased vaginal lubrication,
elongation of
the vagina and increased genital sensation/sensitivity.
Orgasm is the release of sexual tension that has culminated during arousal.
hence, FSD occurs when a woman has an inadequate or unsatisfactory response in
any
of these phases, usually desire, arousal or orgasm.
FSD categories include hypoactive sexual desire disorder, sexual arousal
disorder,
orgasmic disorders and sexual pain disorders.
Although the compounds of the invention will improve the genital response to
sex-
ual stimulation (as in female sexual arousal disorder), in doing so they may
also improve
the associated pain, distress and discomfort associated with intercourse and
so treat
other female sexual disorders. Thus, in accordance with a particular aspect of
the inven-
tion, there is provided use of a compound of the invention in the preparation
of a me-
dicament for the treatment or prophylaxis of hypoactive sexual desire
disorder, sexual
arousal disorder, orgasmic disorder and sexual pain disorder, more preferably
for the
treatment or prophylaxis of sexual arousal disorder, orgasmic disorder, and
sexual pain
disorder, and preferably in the treatment or prophylaxis of sexual arousal
disorder. Hy-
poactive sexual desire disorder is present if a woman has no or little desire
to be sexual,
and has no or few sexual thoughts or fantasies. This type of FSD can be caused
by low
testosterone levels, due either to natural menopause or to surgical menopause.
Other
causes include illness, medications, fatigue, depression and anxiety.
FSAD is characterised by inadequate genital response to sexual stimulation.
The
genitalia do not undergo the engorgement that characterises normal sexual
arousal. The
vaginal walls are poorly lubricated, so that intercourse is painful. Orgasms
may be im-
peded. Arousal disorder can be caused by reduced oestrogen at menopause or
after
childbirth and during lactation, as well as by illnesses, with vascular
components such as
diabetes and atherosclerosis. Other causes result from treatment with
diuretics, antihis-
tamines, antidepressants e. g. selective serotonin re-uptake inhibitors (=
SSRIs) or anti-
hypertensive agents.
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Sexual pain disorders (includes dyspareunia and vaginismus) is characterised
by
pain resulting from penetration and may be caused by medications which reduce
lubrica-
tion, endometriosis, pelvic inflammatory disease, inflammatory bowel disease
or urinary
tract problems. The prevalence of FSD is difficult to gauge because the term
covers sev-
eral types of problem, some of which are difficult to measure, and because the
interest in
treating FSD is relatively recent.
Many women's sexual problems are associated either directly with the female
age-
ing process or with chronic illnesses such as diabetes and hypertension.
Because FSD
consists of several subtypes that express symptoms in separate phases of the
sexual
response cycle, there is not a single therapy.
Current treatment of FSD focuses principally on psychological or relationship
is-
sues. Treatment of FSD is gradually evolving as more clinical and basic
science studies
are dedicated to the investigation of this medical problem. Female sexual
complaints are
not all psychological in pathophysiology, especially for those individuals who
may have a
component of vasculogenic dysfunction (e. g. FSAD) contributing to the overall
female
sexual complaint. There are at present no drugs licensed for the treatment of
FSD. Em-
pirical drug therapy includes oestrogen administration (topically or as
hormone replace-
ment therapy), androgens or mood-altering drugs such as buspirone or
trazodone. These
treatment options are often unsatisfactory due to low efficacy or unacceptable
side ef-
fects. Since interest is relatively recent in treating FSD pharmacologically,
therapy con-
sists of the following: psychological counselling, over-the-counter sexual
lubricants, and
investigational candidates, including drugs approved for other conditions.
These medica-
tions consist of hormonal agents, either testosterone or combinations of
oestrogen and
testosterone and more recently vascular drugs, that have proved effective in
MED. None
of these agents has yet been demonstrated to be effective in treating FSD.
The Diagnostic and Statistical Manual (DSM) IV of the American Psychiatric
Association defines FSAD as being: "... a persistent or recurrent inability to
attain or to
maintain until completion of the sexual activity adequate lubrication-swelling
response of
sexual excitement. The disturbance must cause marked distress or interpersonal
diffi-
culty....". The arousal response consists of vasocongestion in the pelvis,
vaginal lubrica-
tion and expansion and swelling of the external genitalia. The disturbance
causes
marked distress and/or interpersonal difficulty. Studies investigating sexual
dysfunction in
couples reveals that up to 76% of women have complaints of sexual dysfunction
and that
30-50% of women in the USA experience FSD (Barman, J. R., Barman, L. A.,
Werbin, T.
J. et al. (1999). Female sexual dysfunction: Anatomy, physiology, evaluation
and treat-
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26
ment options, Curr Opin Urology, 9,563-568). FSAD is a highly prevalent sexual
disorder
affecting pre-, peri-and post-menopausal (hormone replacement therapy (HRT))
women.
It is associated with concomitant disorders such as depression, cardiovascular
diseases,
diabetes and urogenital disorders. The primary consequences of FSAD are lack
of en-
gorgement/swelling, lack of lubrication and lack of pleasurable genital
sensation. The
secondary consequences of FSAD are reduced sexual desire, pain during
intercourse
and difficulty in achieving an orgasm. It has recently been hypothesised that
there is a
vascular basis for at least a proportion of patients with symptoms of FSAD
(Goldstein et
al., Int. J. Impot. Res., 10, S84-S90, 1998) with animal data supporting this
view (Park et
al., Int. J. Impot. Res., 9,27-37,1997).
It is known that inhibitors of SEP enhance pelvic nerve-stimulated and
vasoactive
intestinal peptide (= VIP)-induced increases in vaginal and clitoral blood
flow. It is also
known that SEP inhibitors enhance VIP and nerve-mediated relaxations of the
isolated
vagina wall. Thus the present invention is advantageous as it helps provide a
means for
restoring a normal sexual arousal response-namely increased genital blood flow
leading
to vaginal, clitoral and labial engorgement. This will result in increased
vaginal lubrication
via plasma transudation, increased vaginal compliance and increased genital
sensitivity.
Hence, the present invention provides a means to restore, or potentiate, the
normal sex-
ual arousal response. By female genitalia herein it is meant :"The genital
organs consist
of an internal and external group. The internal organs are situated within the
pelvis and
consist of ovaries, the uterine tubes, uterus and the vagina. The external
organs are su-
perficial to the urogenital diaphragm and below the pelvic arch. They comprise
the mons
pubis, the labia majora and minora pudendi, the clitoris, the vestibule, the
bulb of the
vestibule, and the greater vestibular glands" (Gray's Anatomy, C. D. Clemente,
13th
American Edition). R. J. Levin teaches that, because "... male and female
genitalia de-
velop embryologically from the common tissue anlagen, [that] male and female
genital
structures are argued to be homologues of one another. Thus the clitoris is
the penile
homologue and the labia homologues of the scrotal sac...." (Levin, R. J.
(1991), Exp.
Clin. Efzdocrinol., 98,6169).
With regard to MSD, in particular to MED, penile erection is a haemodynamic
event
which is dependent upon the balance of contraction and relaxation of the
corpus caver-
nosal smooth muscle and vasculature of the penis (see Lerner, S. E. et al
{1993). A re-
view of erectile dysfunction: new insights and more questions. J. Urology 149:
1246-
1255). Corpus cavemosal smooth muscle is also referred to herein as corporal
smooth
muscle or in the plural sense corpus cavernosa. Relaxation of the corpus
cavernosal
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27
smooth muscle leads to an increased blood flow into the trabecular spaces of
the corpus
cavernosa, causing them to expand against the surrounding tunics and compress
the
draining veins. This produces a vast elevation in cavernosal blood pressure
which results
in an erection (see Naylor, A. M. (1998). Endogenous neurotransmitters
mediating penile
erection. Br.J.Urology 81: 424-431), hereinafter cited as "Naylor, 1998"). The
changes
that occur during the erectile process are complex and require a high degree
of co-
ordinated control involving the peripheral and central nervous systems, and
the endo-
crine system (Naylor, 1998). Corporal smooth muscle contraction is modulated
by sym-
pathetic noradrenergic innerusticn via activation of postsynaptic a-
adrenoceptors. MED
may be associated with an increase in the endogenous smooth muscle tone of the
cor-
pus cavernosum. However, the process of corporal smooth muscle relaxation is
medi-
ated partly by non-adrenergic, non-cholinergic (= NANC) neurotransmission.
There are a
number of other NANC neurotransmitters found in the penis, other than nitric
oxide (_
NO), such as calcitonin gene related peptide (= CGRP) and VIP. The main
relaxing fac-
tor responsible for mediating this relaxation is NO, which is synthesised from
L-arginine
by nitric oxide synthase (= NOS) (see e.g. Taub, H. C. et al (1993).
Relationship between
contraction and relaxation in human and rabbit corpus cavernosum. Urology 42:
698-
704). It is thought that reducing corporal smooth muscle tone may aid NO to
induce re-
laxation of the corpus cavernosum. During sexual arousal in the male, NO is
released
from neurones and the endothelium and binds to and activates soluble guanylate
cyclase
(sGC) located in the smooth muscle cells and endothelium, leading to an
elevation in
intracellular cyclic guanosine 3', 5'-monophosphate (cGMP) levels. This rise
in cGMP
leads to a relaxation of the corpus cavernosum due to a reduction in the
intracellular cal-
cium concentration ([Ca2+] i), via unknown mechanisms thought to involve
protein kinase
G activation (possibly due to activation of Ca2+ pumps and Ca2+-activated K+-
channels).
Recently it has been shown that c-type natriuretic peptide (= CNP) may also
play a
role in MED, acting at the membrane-bound guanylyl cyclase B (= GC-B) which is
ex-
pressed in human corpus cavernosum tissue. Stimulation of GC-B leads to an
increase
in intracellular cGMP and, consequently, smooth muscle relaxation. PDES-
inhibitors, e.g.
sildenafil increase intracellular cGMP in corpus cavernosum tissue by
inhibiting its break-
down. PDES-inhibitors are inactive in the absence of a stimulator of cGMP
formation,
e.g. in the absence of NO. This finding suggests that the basal (unstimulated)
rate of
cGMP formation in the corpus cavernosum is rather low, so that inhibition of
cGMP
breakdown by PDES inhibitors is not sufficient for an erectile response
without concomit-
tant stimulation of guanylyl cyclase. Increasing the concentration of CNP
leads to ele-
vated intracellular cGMP concentration, by an increase in cGMP formation.
Conse-
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28
quently, elevating the CNP concentration in the corpus cavernosum will
presumably have
similar effects as inhibiting PDES. Due to their different mechanisms of
action, i.e. in-
creasing formation of cGMP vs. inhibition of its breakdown, the approaches of
inhibiting
PDES or the breakdown of CNP, respectively are deemed to be additive thus
making it a
reasonable assumption that a combination of these two mechanisms of action
will be
particularly effective in patients who do not respond to the administration of
PDES inhibi-
tors alone.
VIP positive nerve fibres have been found in the trabecular meshwork of the
corpus
cavernosum, suggesting a role of VIP release in penile erection. Effects of
VIP are
thought to be mediated via increases in cAMP and are thus complementary to
those of
cGMP-elevating agents. In patients with ED an intracavernosal injection of VIP
(com-
bined with the a-adrenoceptor antagonist phentolamine) was found to be a safe
and ef-
fective treatment, with a response rate of 67% (erections sufficient for
sexual inter-
course).
The endopeptidases NEP and hSEP both degrade CNP and VIP and thereby limit
the effects of CNP and VIP on cavernosal smooth muscle. Inhibition of CNP and
VIP
breakdown will lead to increased availability of these vasorelaxing factors
thereby
increasing blood flow to the corpus cavernosum which finally should result in
improved
erectile function. Support can be found for this from experimental data in
rabbits, show-
ing a significant increase in intracavernosal pressure and female genital
blood flow after
application of an NEP-inhibitor (see document WO 02/079143). Furthermore, a
gene
(SMR1 ) encoding a pro-peptide of the endogenous NEP-inhibitor sialorphine was
found
(see User H.M., Zelner D.J., McKenna K.E., McVary K.T. (2003). Microan-ay
analysis and
description of SMR1 gene in rat penis in a post-radical prostatectomy model of
erectile
dysfunction. J Uro1.;170(1 ):298-301 ) to be markedly downregulated (> 80-
fold) in a rat
model of neurogenic erectile dysfunction suggesting that in this disease NEP
activity may
be enhanced and contribute to the development of erectile dysfunction.
Description of the pharmacological test method
The example numbers quoted relate to the preparation examples described below.
The inhibition of the enzymatic breakdown of CNP and VIP by the compounds used
ac-
cording to the invention was measured in an enzymatic in vitro assay according
to the
following protocol:
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29
Enrymes: a) hSEP (sol hu)(his)6; or: His6-tagged hSEP ectodomain.
stock solution: 53 Ng/ml in 20 mM HEPES pH 7.2, 5% glycerol, 0.005%
Tween20, 100 mM NaCI, purity >99%
working solution: stock solution diluted with assay buffer to 5 Ng/ml
Supplier: Innogenetics, Ghent, Belgium. Preparation and purification of
the protein were performed as described in WO 02/094176.
b) NEP (prepared from pig kidney cortex)
stock solution: 120 Ng/ml in 20 mM bisTris, purity >95%
working solution: stock solution diluted with assay buffer to 5 Ng/ml
Supplier: Dr. Philippe Crine, Univ. of Montreal, Canada
Substrates: a) VIP
b) CNP (32-53)
stock solution: 100 NM in assay buffer
Supplier: Bachem, Weil am Rhein, Germany
Assay buffer: 100 mM Tris pH 7.0, 250 mM NaCI
All test compounds were dissolved in DMSO at 10 mM and further diluted with
assay
buffer.
Activity Assay procedure
80 NI of assay buffer, 10 NI of enzyme working solution (NEP or hSEP) and 10
NI of pep-
tide stock solution (VIP or CNP) were mixed in an Eppendorf vial and incubated
for 120
min. at 37 °C. The enzymatic reaction was subsequently terminated by
heating to 95 °C
for 5 min. After centrifugation (Heraeus Biofuge B, 3 min) the supernatant was
subjected
to HPLC.
Inhibition Assay procedure
70 NI of assay buffer, 10 NI of enzyme working solution (NEP or hSEP) and 10
NI of a
test compound solution were mixed in an Eppendorf vial and preincubated at 37
°C for
15 minutes. Then, 10 NI of peptide stock solution (VIP or CNP) was added and
the reac-
tion mixture was incubated at 37 °C for 60 min. to allow enzymatic
hydrolysis. The enzy-
matic reaction was subsequently terminated by heating to 95 °C for 5
min. After centrifu-
gation
(Heraeus Biofuge B, 3 min) the supernatant was subjected to HPLC.
For separating the remaining substrate from the cleavage products, a reversed
phase
HPLC technique with a CC 125/4 Nucleosil 300/5 C,8 RP column and a CC 8/4
Nucleosil
100/5 C18 precolumn (Macherey-Nagel, Duren, Germany) was used. 60 NI of the
reac-
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tion samples were injected into the HPLC and the column was eluted at a flow
rate of 1
ml/min with the following gradient:
Solution A: 100% H20 + 0.5M H3P04 pH 2.0
Solution B: 100% acetonitrile + 0.5M H3P04
0-2 min: 5 % B 8-10 min: 90 % B
2-7 min: 5-50 % B 10-12 min: 90-5 % B
7-8 min: 50-90 % B
All peptides were detected by absorbance at 214 nm (UV spectroscopy).
The percentage (_ %) of hydrolysis was calculated on the basis of the peak
area of the
undegraded peptide for an enzyme containing sample Y in correlation to a
sample con-
taining the same concentration of peptide without enzyme (blank) by the
following equa-
tion:
hydrolysis= 100*(blank-Y)
Basis of the calculation of % inhibition is the peak area of the undegraded
peptide (VIP
or CNP) for an inhibitor containing sample X in comparison to samples
containing only
peptide (blank) or peptide and enzyme without inhibitor (control) according to
the follow-
ing equation:
inhib= 100*(X-control)/(blank-control)
All samples were run in duplicate and mean values were used. A solvent control
(0.1
DMSO) was added to each assay run.
CNP and VIP were cleaved by NEP and hSEP in vitro. Breakdown of both peptides
was
faster with hSEP than with NEP, as is shown in table 4 below:
Table 4: Breakdown rates of VIP and CNP by NEP or hSEP
breakdown of CNP breakdown of VIP
hSEP NEP hSEP NEP
degradation at 46 % 39 % 36 % 28
t=2 h
The test compounds according to the invention were able to prevent degradation
of CNP
and VIP by both NEP and SEP. In this test model the test substances of Formula
I listed
in Table 5 below had the ICS values given below:
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31
Table 5: Prevention of degradation of CNP and VIP by the test compounds
breakdown of CNP breakdown of VIP
inhibition of
hSEP NEP hSEP NEP
breakdown by example
IC~ (nM) IC~ (nM) IC~ (nM) IC~ (nM)
no.
4 1.0 1 0.1 3.1 3.1
The compounds of Formula I are also suited for the prophylaxis and/or
treatment of
adverse conditions associated with apoptosis.
Said conditions are for instance: neuro-degenerative disorders such as e.g.
ischemic stroke, cerebral ischemia, traumatic brain injury, acute disseminated
encepha-
lomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild
cognitive im-
pairment, Alzheimer's disease, Pick's disease, senile dementia, progressive
supranu-
clear palsy, subcortical dementias, Wilson disease, multiple infarct disease,
arterioscle-
rotic dementia, AIDS associated dementia, cerebellar degeneration,
spinocerebellar de-
generation syndromes, Friedreichs ataxia, ataxia telangiectasia, epilepsy
related brain
damage, spinal cord injury, restless legs syndrome, Huntington's disease and
Parkin-
son's disease, striatonigral degeneration, cerebral vasculitis, mitochondria)
encephalo-
myopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies,
lysosomal storage
disorders with central nervous system involvement, leukodystrophies, urea
cycle defect
disorders, hepatic encephalopathies, renal encephalopathies, metabolic ence-
phalopathies, porphyria, bacterial or viral meningitis and
meningoencephalitis, prion dis-
eases, poisonings with neurotoxic compounds, Guillain Barre syndrome, chronic
inflam-
matory neuropathies, polymyositis, dermatomyositis, radiation-induced brain
damage;
gastrointestinal disorders like irritable bowel disease and inflammatory bowel
diseases,
Crohn's disease and ulcerative colitis, coeliac disease, Helicobacter pylori
gastritis and
other infectious gastritides, necrotizing enterocolitis, pseudomembranous
enterocolitis,
radiation-induced enterocolitis, lymphocytic gastritis, graft-versus-host
disease, acute
and chronic pancreatitis; hepatic diseases such as e.g. alcoholic hepatitis,
viral hepati-
tis, metabolic hepatitis, autoimmune hepatitis, radiation-induced hepatitis,
liver cirrhosis,
hemolytic uremic syndrome, glomerulonephritis, lupus nephritis, viral diseases
such as
fulminant hepatitis: joint-diseases such as trauma and osteoarthritis; immuno-
suppression or immunodeficiency, in particular autoimmune diseases like
idiopathic
inflammatory myopathy, chronic neutropenia, thrombotic thrombocytopenic
purpura,
rheumatoid arthritis, idiopathic thrombocytopenic purpura, autoimmune
haemolytic syn-
CA 02539895 2006-03-22
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32
dromes, antiphospholipid antibody syndromes, myocarditis, multiple sclerosis
and its
diagnostic sub-classifications relapsing-remitting multiple sclerosis,
secondary progres-
sive multiple sclerosis, primary progressive multiple sclerosis, progressive
relapsing mul-
tiple sclerosis, acute multiple sclerosis, benign relapsing multiple sclerosis
or asympto-
matic multiple sclerosis, neuromyelitis optics (Devic's syndrome), lymphocytic
hypophysi-
tis, Grave's disease, Addison's disease, hypoparathyroidism, type 1 diabetes,
systemic
lupus erythematodes, pemphigus vulgaris, bullous pemphigoid, psoriatic
arthritis, endo-
metriosis, autoimmune orchitis, autoimmune erectile dysfunction, sarcoidosis,
Wegener's
granulomatosis, autoimmune deafness, Sjogren's disease, autoimmune
uveoretinitis,
interstitial cystitis, Goodpasture's syndrome and fibromyalgia;
myelodysplasias such as
aplastic anemia; dermatological diseases including pemphigous vulgaris,
dermatomy-
ositis, atopic dermatitis, Henoch-Schonlein purpura, acne, systemic sclerosis,
sebor-
rhoeic keratosis, cutaneous mastocytosis, chronic proliferative dermatitis,
dyskeratosis,
scleroderma, interstitial granulomatous dermatitis, psoriasis, bacterial
infections of the
skin, dermatomycoses, lepra, cutaneous leishmaniasis, vitil igo, toxic
epidermal necroly-
sis, Steven Johnson syndrome, sebaceous adenoma, alopecia, photodamage of the
skin, lichen sclerosus, acute cutaneous wounds, incontinentia pigmenti,
thermal damage
of the skin, exanthematous pustulosis, lichenoid dermatosis, cutaneous
allergic vascu-
litis, cytotoxic dermatitis; diseases of the inner ear such as e.g. acoustic
trauma-
induced auditory hair cell death and hearing loss, aminoglycoside induced
auditory hair
cell death and hearing loss, ototoxic drug-induced hearing loss, perilymphatic
fistula,
cholesteatoma, cochlear or vestibular ischemia, Meniere's disease, radiation-
induced
hearing loss, hearing loss induced by bacterial or viral infections and
idiopathic hearing
loss; transplantation: graft-versus-host disease, acute and chronic rejection
of heart-,
lung-, kidney-, skin-, corneal-, bone marrow- or liver-transplants; wound
healing and
tissue rejection.
The usefulness of the amidomethyl-substituted 1-(carboxyalkyl)-cydopentylcar-
bonylamino-benzazepine-N-acetic acid derivatives of Formula I for the
prophylaxis and
treatment of said adverse conditions associated with apoptosis can be
demonstrated in
suitable animal models predictive of anti-apoptotic activity.
Description of the pharmacological test methods
The example numbers quoted relate to the preparation examples described below.
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33
1. Traumatic brain iniurv: Delayed apoptotic neuronal death
Contusing device. The contusing device consisted of a stainless steel tube, 40
cm
in length, perforated at 1 cm intervals to prevent air compression in the
tube. Adult Wi-
star rats, 230-270 g, were anesthetized with chloral hydrate, 400 mglkg i.p.,
a craniotomy
over the right hemisphere was made, the device guiding a falling weight onto
the footpla-
te resting upon the surface of the dura was placed perpendicular to the
surface of the
skull, and a force of 380 g x cm produced by a 20 g weight was selected to
produce
brain contusion. A maximum of 2.5 mm depression of the brain surface was
allowed to
avoid mechanical puncture of the dura. The center of the footplate was
stereotaxically
positioned 1.5 mm posterior and 2.5 mm lateral to the bregma. The rats
underwent per-
fusion fixation 3 days after brain injury with a solution containing 4%
paraformaldehyde in
phosphate buffer.
Intracerebroventricular injections: Compounds were administered intracerebro-
ventricularly (= i.c.v.) by means of a Hamilton syringe in a volume of 5-15
NI. Injections
were performed over 5 min, 15 min - 8 hrs after trauma using the following
stereotaxic
coordinates: AP= -0.5 mm, L= - 2 mm and V= -5.5 in relation to bregma
(Swanson, L.
W. (1992) Brain Maps: Structure of the Rat Brain, Elsevier, Amsterdam).
Morphometric analysis in hippocampus. The damage in the hippocampal CA3
subfield was determined stereologically at 5 different rostrocaudal levels
extending from
10.21 to 11.21 mm (Swanson, L. W. (1992) Brain Maps: Structure of the Rat
Brain, Else-
vier, Amsterdam) and throughout its mediolateral axis three days after
traumatic injury.
To quantitatively assess neuronal loss in the hippocampus, stereological
disector techni-
que (Cruz-Orive, L. M. & Weibel, E. R. (1990) Am. J. Physiol. 258, L148-L156)
was used
to estimate numerical density (Nv) of pyramidal neurons. An unbiased counting
frame
(0.05 mm x 0.05 mm; disector height 0.01 mm) and a high-aperture objective
(x40) were
used for sampling. Normal neurons were identified by the presence of the
typical nuclei
with clear nucleoplasm and distinct nucleolus surrounded by cytoplasm
containing Nissl
substance. The border between CA2 and CA3 subfields was considered as the
point
where the looser arrangement of large pyramidal cells goes into densely packed
pyrami-
dal cells of the subfield CA3. An arbitrary line connecting the lateral ends
of the dentate
granule cell layers was considered a junction between subfields CA3 and CA4.
In this test model the test substance of Example 3 elicited a dose-dependent
neu-
roprotective effect. A neuroprotective effect was still evident when the test
substance of
Example 3 was administered i.c.v. up to 8 hrs after trauma:
CA 02539895 2006-03-22
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34
Dose response of the neuroprotective effect of the test substance of Example 3
when administered i.c.v. 15 min after trauma to adult Wistar rats was
measured. Neuro-
nal densities were determined in the CA3 hippocampal subfield as described in
the me-
thods. Densities of CA3 neurons t Standard Error of Measurement (= SEM) in 6
stereo-
tactic levels in the left non-traumatized side of vehicle treated rats and the
traumatized
right side of vehicle treated rats and in rats treated with the test substance
of Example 3
were measured and the results listed in table 6 below.
In all of the following tables the numbers (°n") indicate the number of
rats per group,
where applicable.
Table 6: Neuronal densities CA3 hippocampus, cells x 103/mm3
Stereo-Vehicle Vehicle Compound Compound Compound
tacticleft; right; of of of
level (n=10) (n=10) Ex. 3, 3Ng; Ex. 3, 10Ng;Ex. 3, 30Ng
(n=10) (n=10) (n=10)
10.21 159.003.6291.2017.6098.4014.39 108.6014.30108.4013.15
10.41 158.203.0387.2018.1789.0015.05 108.6015.34105.20f5.76
10.61 157.2012.8866.8017.6872.8016.01 111.4017.0994.20f5.10
10.81 159.6012.9956.8015.9684.2016.47 112.0016.4283.207.10
11.01 152.402.9951.406.8986.0017.44 111.407.11 80.207.45
11.21 151.602.4771.6018.2295.406.96 119.2013.7090.0019.24
Injection of vehicle resulted in the decrease of neuronal densities in the CA3
hippo-
campus up to 35 % of control values, while injection of 3, 10 or 30 Ng of the
test sub-
stance of Example 3 partially prevented hippocampal neuronal loss, with the
dose of
Ng being most effective. Analysis of variance ("ANOVA") revealed that there
was a
significant protective effect of treatment on neuronal loss in the CA3
hippocampus for all
three tested doses of the test substance of Example 3 (P<0.001; n = 10 per
group).
ANOVA also revealed that the dose of 10 Ng conferred significantly better
neuroprotecti-
on than the doses of 3 Ng or 30 Ng.
The time window of the neuroprotective effect of test substance of Example 3
when
administered i.c.v. 2, 4 or 8 hrs after trauma to adult Wistar rats was
measured. Neuronal
densities were determined in the CA3 hippocampal subfield as described in the
methods.
Densities of CA3 neurons t SEM in 6 stereotactic levels in the traumatized
right side of
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rats treated with either vehicle or the test compound of Example 3 were
measured and
the results listed in table 7 below.
Table 7: Neuronal densities CA3 hippocampus, cells x 103/mm3
Stereotac-Vehicle Compound Compound Compound
tic levelright; of of of
(n=8) Ex. 3, 2 Ex. 3, 4 Ex. 3, 8
hrs; hrs; hrs;
(n=8) (n=8) (n=8)
10.21 55.2115.8172.3014.80 72.2015.70 62.0014.90
10.41 50.657.3068.1016.30 65.908.80 53.0D6.44
10.61 49.3518.7660.8015.60 63.0016.30 53.006.00
10.81 51.21 60.2019.40 60.50110.50 52.5014.48
7.97
11.01 54.80f10.3063.00111.7062.20113.50 61.8014.48
11.21 60.0013.0067.70114.0066.30115.90 65.904.90
Injection of vehicle resulted in decrease of neuronal densities in the CA3
hippo-
campus up to 35 % of control values. Intracerebroventricular injection of 10
Ng of the test
substance of Example 3 partially prevented hippocampal neuronal loss. ANOVA
revea-
led that there was a significant effect of treatment with of the test
substance of Example
3 on neuronal loss in the CA3 hippocampus for all three time points (P<0.001
at 2 and 4
hrs, P<0.01 for 8 hrs).
2. Adriamycin toxicity: Determination of anti-apoptotic activity
Wistar rats, weighing 200-250 g, were anesthetized with chloral hydrate,
400 mg/kg, and Alzet osmotic minipumps (2ML1), were implanted subcutaneously
(= s.c.). The pumps had been filled with either vehicle or solution containing
compounds
of the invention at the appropriate concentration and primed prior to
implantation. Ani-
mals subsequently received adriamycin at three equal daily doses of 5 mg/kg
i.p. on
days 1, 2 and 3. Rats were euthanized 5 days after the first injection of
adriamycin and
transcardially perfused with a solution containing 4% paraformaldehyde in
phosphate
buffer. The heart, liver and kidneys were subsequently removed and embedded in
para-
fin.
TUNEL staining: For terminal deoxynucleotide transferase-mediated dUTP nick
end-label (TUNEL) based histological analysis, organs were post-fixed for 5
days at 4 °C
and paraffin-embedded. TUNEL staining was performed on 10 Nm thick paraffin
sections
using the ApopTag Peroxidase kit (S 7100, Oncor Appligene, Heidelberg,
Germany)
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36
according to the manufacturer's instructions. Briefly, after pretreatment with
proteinase K
and quenching of endogenous peroxidase, sections were incubated in
equilibration buf-
fer followed by working strength TdT enzyme (incorporating digoxigenin labeled
dUTP
nucleotides to free 3'-OH DNA termini), (1 hr, 37 °C). Sections were
incubated in
stop/wash buffer (30 min, 37 °C), then with anti-digoxigenin-peroxidase
conjugate
(30 min) followed by DAB substrate (Sigma, Deisenhofen, Germany) and lightly
counterstained with methylgreen.
In this test model the test substance of Example 4 conferred significant
protection
against adriamycin toxicity in the heart, liver and kidney in that it
significantly reduced the
densities of TUNEL positive cells in the three organs. This effect was dose-
dependent
with the dose of 100 mg/kg and day being the most effective:
Wistar rats were administered adriamycin at the cumulative dose of 15 mg/kg
i.p.
The test substance of Example 4 was administered s.c. at the doses of 20, 50
or
100 mg/kg and day by means of Alzet osmotic minipumps over 5 days. Animals
were
euthanized and transcardially perfused 5 days after the first injection of
adriamycin and
the heart, kidney and liver were processed for TUNEL staining. Densities of
TUNEL posi-
tive cells were determined as described in the methods. Results for each organ
(heart,
liver, kidney) were measured as mean densities of TUNEL positive cells t SEM
for the
control groups and the different test groups (20, 50 or 100 mg/kg and day of
test com-
pound of Example 4) and listed in table 8 below.
Table 8: TUNEL positive cells/mm3 X 102
Heart Liver Kid ney
Adriamycin (n=24)5.417 t 10.420 t 9.438 t 0.198
0.146 0.275
+ Compound of 4.350 t 8.750 t 0.301**7.900 t 0.306***
Ex. 4; 0.248***
20mg/kg (n=10)
+ Compound of 3.700 t 8.250 t 7.850 t 0.587**
Ex. 4; 0.260*** 0.271***
50 mg/kg (n=10)
+ Compound of 3.550 t 7.450 t 6.300 f 0.260***
Ex. 4; 0.157*** 0.329***
100 mg/kg (n=10)
The test substance of Example 4 dose-dependently decreased the cytotoxic
effect
of adriamycin in all three organs. Comparisons between groups were performed
by
means of Student's t test (**P<0.01; ***P<0.001 compared to vehicle treated
rats).
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37
The present invention also provides a method of treating or preventing
cardiovas-
cular disorders or diseases and/or treatment of adverse conditions associated
with apop-
tosis in mammals and humans comprising administering to a subject in need
thereof an
effective amount of a compound of Formula I.
The present invention further provides a method of treating or preventing
sexual
dysfunction in mammals and humans comprising administering to a subject in
need
thereof an effective amount of a dually acting compound capable of inhibiting
NEP and
hSEP, in particular of a compound of Formula I, according to the invention.
The compounds of Formula I may be administered in conventional pharmaceutical
compositions. The doses to be used may vary individually and will naturally
vary accord-
ing to the type of condition to be treated and the substance used. In general,
however,
medicinal forms with an active substance content of 0.2 to 500 mg, in
particular 10 to
200 mg, active substance per individual dose are suitable for administration
to humans
and larger mammals. The agents of the present invention may also be
administered by
intravenous infusion, at a dose which is likely to range from 0.001-10
mg/kg/hr. The
above dosages are exemplary of the average case. The compounds may be
contained
according to the invention, together with conventional pharmaceutical
auxiliaries and/or
excipients, in solid or liquid pharmaceutical compositions. Examples of solid
pharmaceu-
tical compositions a re compositions which can be administered orally, such as
tablets,
coated tablets, capsules, powders or granules, or alternatively suppositories.
These
pharmaceutical compositions may contain conventional pharmaceutical inorganic
and/or
organic excipients, such as talcum, lactose or starch, in addition to
conventional pharma-
ceutical auxiliaries, for example lubricants or tablet disintegrating agents.
Liquid pharma-
ceutical compositions such as suspensions or emulsions of the active
substances may
contain the usual diluents such as water, oils and/or suspension agents such
as polyeth-
ylene glycols and the like. Other auxiliaries may additionally be added, such
as preserva-
tives, taste correctives and the like.
The active substances may be mixed and formulated with the pharmaceutical
auxil-
iaries and/or excipients in known manner. For the preparation of solid
medicament forms,
the active substances may for example be mixed with the auxiliaries and/or
excipients in
conventional manner and may be wet or dry granulated. The granules or powder
may be
poured directly into capsules or be pressed into tablet cores in conventional
manner.
These may be coated in known manner if desired.
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38
The following examples are intended to explain the invention further, without
limit-
ing its scope.
The mass spectra were measured using the following method:
HPLC-MS: AP1100 Quadrupol mass spectrometer (PE Applied Biosystems)
coupled to a LC200 pump (PE). Electrospray ionisation, positive
mode. Scan range m/z 100 to 1000. Software MassChrom 1.2.
Xterra~ column (4.6 mm x 50 mm, 2.5 pm).
Solvent system: Water (10 mM ammonium acetate, pH 5) and acetonitrile, linear
gradient from 5% acetonitrile to 95% in 10 min.
Example 1:
Ethyl 2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-
1-
benzazepin-3-yl]amino)carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyrate
O
N H w
O
~O O
O //
O
A) 91.9 ml benzyl alcohol was added to 99.07 g itaconic acid anhydride and the
mix-
ture was stirred for 8 hours (= h) at 65°C. The crystals produced on
cooling were
made into a slurry with 35 ml of a mixture of n-hexane/diethyl ether 2:1 (v/v)
and fil-
tered off from the solvent. The resulting crude product was dissolved in 150
ml di-
ethyl ether in warm conditions and crystallised again by addition of 80 ml n-
hexane.
The combined mother lyes were reduced, recrystallised corresponding to the
above
method and the crystals obtained were finally added to the main quantity. 120
g 2-
[(2-benzyloxy)-2-oxoethyl]acrylic acid was obtained which was used directly
for the
next reaction without further purification, 'H-NMR (CDCI3): 7.35, m, [5];
6.47, s, [1];
5.83, s, [1]; 5.15, s, [2]; 3.40, s, [2] ppm.
B) 100 g of the 2-[(2-benzyloxy)-2-oxoethyl]acrylic acid obtained above was
sus-
pended in 100 ml methyl-tert. butylether (= MTBE) and 0.5 ml pyridine was
added
thereto. 47 ml thionyl chloride was added dropwise thereto and the resulting
mix-
ture was heated for 1.5 h under reflux cooling to boiling. After cooling to
RT, it was
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39
evaporated approximately to dryness at reduced pressure. The resulting residue
was dissolved in 50 ml dichloromethane and added dropwise at 0 - 5°C to
a receiv-
ing solution consisting of 16 ml ethanol and 36.5 ml triethylamine in 150 ml
di-
chloromethane. Once addition had ended, stirring was continued for 1 h at
approx.
0°C. Then it was washed in succession twice with 250 ml water each
time, once
with 100 ml dilute aqueous sodium bicarbonate solution and finally once with
satu-
rated aqueous common salt solution. The organic phase was dried over sodium
sulphate and evaporated as far as possible under reduced pressure.
Distillation of
the resulting residue at 0.015 mbar and 150°C yielded 56.3 g 2-
methylenesuccinic
acid-4-benzylester-1-ethylester, which was used without further purification
or char-
acterisation directly for the next reaction.
C) 118 ml diisopropylamine was dissolved in 3 I dry tetrahydrofuran (= THF)
under
nitrogen atmosphere and the solution was cooled to 0°C. 340 ml of a 2.5
M solution
of n-butyllithium in n-hexane was added to this receiving solution and
stirring was
continued for another 45 minutes at 0°C once the addition had ended.
Then a solu-
tion of 45 g cyclopentanecarboxylic acid in 100 ml dry THF was dropped into
the
resulting mixture at 0 - 5°C and the mixture was then stirred for 2 h
at 0°C. It was
cooled to -80°C and a solution of 72.6 g of a 2-methylenesuccinic acid-
4-
benzylester-1-ethylester as obtained above (total quantity from several
batches) in
100 ml THF was added dropwise thereto. It was stirred for 2 h at -75°C
and then
1.5 I of a 2N aqueous hydrochloric acid was added. After thawing and phase
sepa-
ration, the aqueous phase was extracted twice with ethyl acetate (= EA), the
or-
ganic phases were combined and dried over sodium sulphate. The solvent was
evaporated at reduced pressure and volatile substances were separated off by
dis-
tillation at 0.02 mbar and 140°C. Chromatography of the residue
remaining after
distillation on silica gel (mobile phase: EAIn-hexane 1:6 to 1:7 v/v) yielded
22.8 g 1-
[4-(benzyloxy)-2-(ethoxycarbonyl)-4-oxobutyl]cyclopentanecarboxylic acid; 'H-
NMR
(CDCI3): 7.33, m, [5]; 5.10, s, [2]; 4.04, m, [2]; 2.88, m, [1]; 2.80-2.48,AB-
Q., [2];
2.2-2.1, m, [2]; 1.7-1.4, m, [6]; 1.20, tr, [3].
D) 49.5 g of a 1-[4-(benzyloxy)-2-(ethoxycarbonyl)-4-
oxobutyl]cyclopentanecarboxylic
acid as obtained above (total quantity from several batches) was dissolved in
435
ml dichloromethane. 39.5 g tent. butyl-[(3S)-3-amino-2-oxo-2,3,4,5-tetrahydro)-
1 H
benzazepin-1-yl]acetate (for production see EP 0 733 642 A1 ), 18.3 g
hydroxyben-
zotriazole and 60 ml morpholine were added to this receiving solution. Then 52
g
EDCxHCI was added to the resulting mixture in one portion and stirring was
carried
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out overnight at RT. Then the solvent was evaporated at reduced pressure and
the
remaining residue was taken up in 750 ml of EA. The organic phase was washed
in
succession twice with 100 ml 2N aqueous hydrochloric acid each time; twice
with
100 ml water each time and once with 100 ml saturated aqueous common salt solu-
tion and dried over sodium sulphate. Evaporation of the solvent at reduced
pres-
sure and drying of the remaining residue in an oil pump vacuum (5x102 mbar)
yielded 87.9 g 2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-
1H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}succinic acid-4-benzyl-
ester-1-ethylester as yellowish oil, which was used without further
purification or
characterisation for the subsequent reaction.
E) 87.9 g of the 2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-
1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}succinic acid-4-
benzylester-1-
ethylester obtained above was dissolved in 600 ml ethylacetate (= EA) and 20 g
palladium on activated carbon (= Pd/C) was added thereto. It was hydrogenated
for
2 h at a hydrogen pressure of 1 bar and the reaction mixture was then filtered
over
Cellite. The filter cake was subsequently washed with 1.5 I EA and the
combined
organic phases were very largely evaporated at reduced pressure. The residue
was
taken up in 500 ml EA/cyclohexane (1:1, v/v) and extracted twice with 200 ml
semi-
saturated Na2C03 s olution each time. The aqueous phase was acidulated with
conc. KHS04 solution and extracted 3 times with 200 ml EA each time. After
drying
over sodium sulphate, it was evaporated under reduced pressure. Drying of the
re-
maining residue in an oil pump vacuum yielded 71 g 3-{[1-({((3S)-1-(2-tert.
butoxy-2-
oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}carbonyl)cyclopen-
tyl]methyl}-4-methoxy-4-oxobutyric acid as white foam, 'H-NMR (CDCI3): 7.31 -
7.17, m, [3]; 7.11,d, [0.5]; 7.08,d, [0.5]; 6.81,d, [0.5]; 6.73, d, [0.5].
The intermediate product obtained in this case can if desired be separated
into its
diastereomerically pure constituents by preparative high-performance liquid
chro-
matography (= HPLC). 70 g of the intermediate product obtained above was sepa-
rated off using the method set forth below:
Column: LC80-1, 23.4x8 cm; stationary phase: 740 g ChiraIpakAD, 20 N; mobile
phase: heptanelisopropanol (85:15); UV detection; cycle time: 45 minutes;
Analysis: stationary phase: Chiralpak AD, 20 N; mobile phase:
heptanelisopropanol
9:1 (v/v), flow rate: 2ml/min; cycle time: 15 minutes.
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41
With a retention time of 11.6 min., there was obtained 30 g of the first
stereoi-
somer, which was assigned the designation "rel1" in relation to the chiral
centre
"*Ca" bearing the group -COOR', as (3"rel1")-3-{[1-({[(3S)-1-(2-tert. butoxy-2-
oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-yl]amino}carbonyl)cydopen-
tyl]methyl}-4-ethoxy-4-oxobutyric acid, 'H-NMR (CDCI3): 7.31-7.18, m, [3];
7.09, d,
[1]; 6.74, d, [1]; 4.53, 4.48, 4.37, 4.32, AB-Q., [2]; 4.48, m, [1]; 4.11, m,
[1].
With a retention time of 6.5 min., there was obtained 33 g of the second
stereoi-
somer, which was assigned the designation "rel2" in relation to the chiral
centre
"*Ce" bearing the group "-COOR'", as (3"rel2")-3-{[1-({[(3S)-1-(2-tert. butoxy-
2-
oxoethyl)-2-oxo-2, 3,4,5-tetrahydro-1 H-1-benzazepin-3-yl]amino}carbonyl
)cydopen-
tyl]ethyl}-4-ethoxy-4-oxobutyric acid, 'H-NMR (CDCI3): 7.31 - 7.17, m, [3];
7.11, d,
[2]; 6.$1, d, [1]; 4.60, 4.56, 4.35, 4.31, AB-Q. [2]; 4.48, m, [1]; 4.10, m,
[1]; [a]o = -
136° (1% in methanol).
F) 4 g of the 3-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-ethoxy-4-oxobutyric acid
ob-
tained above was dissolved in 15 ml dichloromethane. After this receiving
solution
had been cooled to 0°C, 1.12 ml triethylamine and 0.77 ml ethyl
chloroformate were
added slowly dropwise thereto in succession and the mixture was stirred for 30
min-
utes at 0°C. Then 0.94 ml of isopropylamine was added thereto and
stirring was
continued for a further 3 h at 0°C. The solvent was largely evaporated
at reduced
pressure and the remaining residue was taken up in 100 ml EA. The organic
phase
was washed in succession once each with 50 ml saturated aqueous KHSOa sol u-
tion and with saturated aqueous common salt solution, dried over sodium
sulphate
and the solvent was very largely evaporated at reduced pressure. Drying of the
re-
maining residue in an oil pump vacuum yielded 4.29 g of the title compound as
yel-
lowish oil, MS: [M+H]': 586; m/z: 530; 484; 425.
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42
Example 2:
2-{[(3S)-1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-oxobutyric acid
O
H w
O
OH O
O
OH
9.97 g of an ethyl 2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H
1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyrate
as obtained above under 1E) was dissolved in 200 ml of a water/ethanol mixture
(1:1 v/v)
and 6.64 g solid NaOH was added thereto with stirring. Stirring was continued
over night,
the solvent was then very largely evaporated at reduced pressure and the
remaining
residue was taken up in 100 ml of EA. The aqueous phase was neutralised with
satu-
rated aqueous KHS04 solution and extracted three times with EA. The combined
organic
phases were washed with 100 ml saturated aqueous common salt solution and
dried
over sodium sulphate. Evaporation of the solvent at reduced pressure and
drying of the
remaining residue in an oil pump vacuum yielded 5.59 g of the title compound.
ExamQe 3:
(2"rel 1 ")-2-{[(3S)-1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-
benzazepi n-3-
yl]amino}-carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-oxobutyric acid
H w
O
rel1 N O
OH O
O
OH
400 mg of the diastereomer mixture obtained above in Example 2) was separated
by
HPLC in accordance with the procedure set forth below:
Column: LC80-1, 25x8 cm; stationary phase: ChiraIpakAD, 20 N; mobile phase:
hep-
tane/isopropanol 85:15 (v/v) + 0.1% v/v trifluoroacetic acid (= TFA); UV
detection; flow
rate: 1 ml/min.; cycle time: 15 minutes;
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43
Analysis: column: DAICEL Chiralpak AD; length: 250 mm; diameter: 4.6 mm;
mobile
phase: n-heptane 800 ml, 2-propanol 200 ml, TFA 2 ml; flow rate: 0.8 ml/min.;
analysis
time: 30 minutes.
With a retention time of 13.5 min., there was obtained under these conditions
130 mg of
the first stereoisomer (= title compound), which was assigned the designation
"rel1" in
relation to the chiral centre "*Ca' bearing the group "-COOR'", as white
solid, which pre-
cipitated from EE; 'H-NMR (methanol): 7.37 - 7.2, m, [4]; 4.76, 4.71, 4.43,
4.38, AB-Q.;
4.4, m, [1]; 3.90, m, [1]; 3.40, m, [1]; 2.22 - 2.60, m, [2]; 2.48- 2.0, m,
[12]; 1.10, d, [6];
[a]p = -90° (0.5% in methanol); Mp.:145°C.
With a retention time of 16.2 min., there was obtained under these conditions
the second
stereoisomer, which was assigned the designation "rel2" in relation to the
chiral centre
"*Ca" bearing the group "-COOR'".
Example 4:
{(3S)-3-[({1-[(2"rel1")-2-ethoxycarbonyl)-4.-(isopropylamino)-4-
oxobutyl]cyclopentyl}- car-
bonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-1-yl}acetic acid
O
N H w
O
~O rel1 O
O
OH
4.29 g ethyl (2"rel1")-2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetrahydro-
1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxo-
butyrate (prepared analogously to Example 1, but with the (3"rel1")-3-{[1-
({[(3S)-1-(2-tert.
butoxy-2-oxoethyl~2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-3-
yl]amino}carbonyl)cyclo-
pentyl]methyl}-4-ethoxy-4-oxobutyric acid obtained by HPLC separation being
used as
intermediate product of stage 1 E), was dissolved in 30 ml dichloromethane and
17 ml of
TFA was added. The mixture was left to stand overnight and the solvent and
excess TFA
were evaporated at reduced pressure. The remaining residue was taken up in 100
ml EA
and the organic phase was washed with water until it became pH-neutral. The
organic
phase was dried over sodium sulphate and then the solvent was very largely
evaporated
at reduced pressure. 30 ml toluene in each case was added twice to the residue
and the
mixture was again evaporated at reduced pressure. Drying of the remaining
residue in an
oil pump vacuum yielded 2.8 g of the title compound as white foam; 'H-NMR
(CDC13):
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44
7.33, m, [4]; 6.82, d, [1]; 5.86, d, [1]; 4.64, m, [1]; 4.54, 4.50, 4.46,
4.42, AB-Q.; 3.20, m,
[1]; 1.23, [3]; 1.09, [6]; [a]o: -155° (1% in methanol).
Example 5:
Ethyl (2"rel1")-2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-
4-
oxobutyrate
O
HON
O
O ~e11 O
~N O
O
O
4.2 g (3"rel1")-3-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-ethoxy-4-oxobutyric acid
(prepara-
tion of the diastereomer mixture in accordance with Example 1 E) and
subsequent sepa-
ration of the diastereomers by means of HPLC) was dissolved in 30 ml
dichloromethane.
1.17 ml 3-amino-1-propanol, 235 mg dimethylaminopyridine and 1.61 g EDC were
added
to this receiving solution with stirring. After 1 h, the mixture was largely
evaporated at
reduced pressure, the remaining residue was taken up in 100 ml EA and the
organic
phase was shaken out twice with 30 ml dilute aqueous KHS04 solution each time.
The
organic phase was washed twice more with 30 ml saturated aqueous common salt
solu-
tion each time, dried over sodium sulphate and the solvent was then largely
evaporated
at reduced pressure. Drying of the remaining residue in an oil pump vacuum
yielded 4 g
of the title compound as white foam resin, MS: [M+H]+: 602; m/z: 546, 500,
425; 'H-NMR
(CDCI3): 7.32 - 7.18, m, [3]; 7.12, d, [2]; 6.63, d, [1]; 6.49, tr, [1]; 4.57,
4.63, 4.34, 4.30,
AB-Q. [2]; 4.51, m, [1]; 4.11, m, [2]; 3.57, tr, [2].
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Example 6:
Ethyl (2"rel1")-4-{[3-(acetyloxy)propyl]amino}-2-{[1-({[(3S)-1-(2-tert. butoxy-
2-oxoethyl)-2-
oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-
oxo butyrate
O O
~O~N H w
O N
rel1
~O O
O
1 g of the ethyl (2"rel1")-2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetra-
hydro-1 H-1-benzazepin-3-yl]amino]}carbonyl)cyclopentyl]methyl}-4.-[(3-
hydroxypropyl)-
amino-4-oxobutyrate obtained above in Example 5 was dissolved in 20 ml
dichloro-
methane and 340 NI acetyl chloride was added thereto. After 90 minutes, the
solvent was
largely evaporated at reduced pressure and the remaining residue was taken up
in 20 ml
EA and washed with 10 ml of a dilute aqueous sodium bicarbonate solution. Then
it was
dried over magnesium sulphate, the solvent was largely evaporated at reduced
pressure
and the remaining residue was chromatographed on silica gel (mobile phase:
EA/n-
hexane 7:3 v/v). Drying the product fractions in an oil pump vacuum (5x10-2
mbar)
yielded 920 g of the title compound as colourless oil; MS: [M+H]: 644; m/z:
588, 542,
482, 425.
Example 7:
{(3"rel1 ")-3-[({1-[(2S)-4-{[3-(acetyloxy)propyl]amino}-2-(ethoxycarbonyl)-4-
oxobutyl]cyclopentyl}carbonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-
1-yl}
acetic acid
0 0
~O~N H w
O
rel1
~O O
O
OH
929 mg of the ethyl (2"rel1")-4-{[3-(acetyloxy)propyl]amino}-2-{(1-({[(3S)-1-
(2-tert. butoxy-
2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-yl]amino}carbonyl)-
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cyclopentyl]methyl}-4-oxobutyrate obtained above in Example 6 was dissolved in
10 ml
dichloromethane and 2.2 ml TFA was added thereto. The mixture was left to
stand over-
night, the solvent was then largely evaporated at reduced pressure and the
remaining
residue was taken up in 30 ml EA. The organic phase was washed with water
until it be-
came pH neutral, was again largely evaporated at reduced pressure and the
remaining
residue was fumed off twice with 10 ml toluene each time. 750 mg of the title
compound
was obtained as a white foam resin, MS: [M+H]: 588; m/z: 542, 482, 425; 'H-NMR
(CDCI3): 7.33- 7.14, m, [4]; 6.67, d, [1]; 6.59, tr, [1]; 4.69, 4.64, 4.35,
4.30, AB-Q., [2];
4.63, m, [1]; 4.17, m, [1]; 4.09, q, [2]; 3.33, m, [1]; 3.15, m, (2].
Example 8:
((3Sr3-{[( 1-{(2"rel1 ")-2-ethoxycarbonyl)-4-[(3-hydroxypropyl)amino]-4-
oxobutyl]cyclo
pentyl}-carbonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-1-yl} acetic
acid
O
HON
O
rell
~O O
O
OH
580 mg ethyl (2"rel1")-2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetrahydro-
1 H-1-benzazepin-3-yl]amino]}carbonyl)cyclopentyl]methyl}-4-[(3-
hydroxypropyl)amino-4-
oxobutyrate (for preparation see Example 5) was reacted with TFA in accordance
with
the method set forth above in Example 4. After purification of the resulting
crude product
by column chromatography (stationary phase: silica gel; mobile phase:
EA/methanol 9:1
(v/v)), 240 mg of the title compound was obtained as colourless resin, 'H-NMR
(CDCI3):
7.34 - 7.15, m, [4]; 6.76, tr, (1 ]; 6.61, d, [1 ]; 4.75, 4.71, 4.20, 4.16, AB-
Q., [2]; 4.57, m, [1 ];
4.09, q, [2]; MS: [M+H]+: 546; [a]o =-112.5° (1% in methanol).
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Example 9:
2-{[1-({[(3S)-1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-4-oxobutyric acid
O
HON H
O
OH O
O
OH
6.43 g ethyl (2S)-2-{[1-({((3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepi n-3-yl]amino}carbonyl)cyclopentyl] methyl}-4-[(3-hydroxypropyl)a mi
no]-4-oxo-
butyrate (for preparation see Example 5) was dissolved in 140 ml of a 1:1
(v/v) mixture of
water and ethanol, and 4.28 g solid NaOH was added thereto with stirring.
After 15 h, the
solvent was evaporated at reduced pressure, the residue was taken up in 100 ml
EA and
washed once with 50 ml aqueous KHS04 solution. The aqueous phase was extracted
twice with 30 ml EA each time. The combined organic phases were washed twice
with 30
ml aqueous common salt solution each time and dried over sodium sulphate.
Evapora-
tion of the solvent yielded 5.41 g of the title compound.
Example 10:
(2"rel 1 ")-2-{[1-({[(3S)-1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-
benzazepin-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-4-oxobutyric
acid
O
HON H
O
OH cell O
O
OH
800 mg of the isomer mixture obtained above in Example 9 was separated by
prepara-
tive HPLC in accordance with the procedure set forth below:
Stationary phase: Nucleosil 100-10; column: 250 mm long, 20 mm diameter; flow
rate:
8 ml/min.; mobile phase: n-heptane (800 ml), 2-propanol (200 ml), TFA (1 ml).
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Analysis: stationary phase: EC 250/4 Nucleosil 100-10; column 250 ml long, 4
mm di-
ameter, flow rate: 1.5 ml/min.; mobile phase: n-heptane (800 ml), 2-propanol
(200 ml),
TFA (1 ml).
With a retention time of 7.89 min., there was obtained under these conditions
200 mg of
the first stereoisomer (= title compound), which was assigned the designation
"rel1" in
relation to the chiral centre "*C8" bearing the group "-COOR'", 'H-NMR
(CD30D): 7.38,
m, [4]; 4.78, 4.73, 4.43, 4.38, AB-Q., [2]; 4.41, m, [1]; 3.93, m, [1]; 3.56,
tr [2]; 3.40, m,
[1]; 3.31, m, [1]; 3.22, m, [2]; 2.78, m, [1]; 2.65, m, [1].
With a retention time of 4.47 min., there was obtained under these conditions
the second
stereoisomer, which was assigned the designation "rel2" in relation to the
chiral centre
"*Ca" bearing the group "-COOR'".
Example 11:
2-{[1-({[(3S)-1-(2-ethoxy-2-oxoethyl )-2-oxo-2, 3,4,5-tetrahydro-1 H-1-
benzazepi n-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-4-oxobutyric
acid
O
HON H
O
OH O
O
O
800 mg of the 2-{[1-({[(3S)-1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-
3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-4-
oxobutyric acid
(isomer mixture) obtained above according to Example 9 was dissolved in 15 ml
dimethyl
formamide (= DMF). 302.5 mg Cs2C03 and 169 mg ethyl bromide were added to this
receiving solution at RT with stirring. After stirring overnight, it was
diluted with 42 ml wa-
ter and 21 ml dichloromethane and the aqueous phase was extracted with
dichloro-
methane. The solvent was largely evaporated at reduced pressure and the
remaining
residue was chromatographed (stationary phase: silica gel, mobile phase: EA
(100%) to
EE/MeOH 7:3 (v/v)). Drying the product fractions in an oil pump vacuum (5x10-2
mbar)
yielded 241 g of the title compound as foam resin, MS: [M+H]+: 546; m/z: 453,
425, 379;
1H-NMR (CDCI3): 7.34 - 7.1, m, [4]; 4.82, 4.77, 4.34, 4.29, AB-Q-. [2]; 3.62,
m, [2]; 3.37,
m, [3].
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49
Example 12:
2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-
yl]amino}-carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-oxobutyric acid
O
N H w
O
OH O
O //
O
2,6 g ethyl 2-{[(3S)-1-({[1-(2-tent. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H 1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyrate
(for preparation see Example 1 ) was dissolved in 52 ml of ethanol. A solution
of 710 mg
solid NaOH in 52 ml water was added to this receiving solution. After 30
minutes, it was
acidulated with dilute aqueous KHSOa solution to approximately pH 2 and the
aqueous
phase was extracted three times with 50 ml EA each time. The combined organic
phases
were dried over magnesium sulphate, the solvent was largely evaporated at
reduced
pressure and the remaining residue was chromatographed on silica gel (mobile
phase:
EA/cyclohexane 2:1 vlv). Drying the product fractions in an oil pump vacuum
(5x10-2
mbar) yielded 2.2 g of the title compound as white foam resin, MS: [M+H]+:
558; m/z:
502, 425, 397, 323.
Example 13:
4-chlorobenzyl-2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}-carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyrate
O
N H w
CI / O
\ I O O ~N O
O ~~
O
300 mg of the 2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H 1-
benzazepin-3-yl]amino}-carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyric
acid obtained above was dissolved in 5 ml dichloromethane. 33 mg of
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4-dimethylaminopyridine (= DMAP), 85 mg 4-chlorobenzyl alcohol and 124 mg
EDCxHCI
were added thereto and stirring was then carried out overnight. The mixture
was diluted
with 5 ml dichloromethane and the organic phase was washed in succession once
each
with 2 ml dilute aqueous KHSOa solution and with saturated aqueous common salt
solu-
tion. The organic phase was dried over magnesium sulphate, the solvent was
largely
evaporated to dryness at reduced pressure and the remaining residue was
chromatogra-
phed on silica gel (mobile phase: EA/cyclohexane 3:2 v/v). Drying the product
fractions in
an oil pump vacuum (5x10-2 mbar) yielded 320 g of the title compound as white
foam;
MS: [M+H]+: 682/684; m/z: 626/628, 576, 484, 425.
Example 14:
{(3S)-3-[({1-[2-{[(4-ch lorobenzyl)oxy]carbonyl}-4-(isopropyla mi no~4-
oxobutyl]cyclopentyl}carbonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-
1-yl}
acetic acid
O
N H w
CI / O
O p ~N O
OH
318 g of the 4-chlorobenzyl-2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetra-
hydro-1 H-1-benzazepin-3-yl]amino}-carbonyl)cyclopentyl]methyl}-4-
(isopropylamino)-4-
oxobutyrate obtained above was dissolved in 11 ml dichloromethane, 1.08 ml TFA
was
added thereto and the mixture was stirred overnight. Then the solvent was
largely evapo-
rated at reduced pressure, the remaining residue was taken up in 10 ml EA and
the or-
ganic phase was washed with water until it became pH-neutral. Then the solvent
was
evaporated again at reduced pressure and the remaining residue was fumed off
once
with 5 ml of toluene. 305 mg of the title compound was obtained as a white
foam resin,
MS: [M+H]+: 626/628; m/z: 657, 484, 425.
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Example 15:
(2-methoxyethoxy)methyl-2-{[1-({[(3S)-1-(2-Pert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-
tetrahydro-1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-
(isopropylamino)-
4-oxobutyric acid
O
N H w
O N
w0~0~0 O
O
O
300 mg 2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-
1-benz-
azepin-3-yl]amino}-carbonyl)cyclopentyl]methyl}-4-(isopropylamino)-4-
oxobutyric acid
(for preparation see Example 12) was dissolved in 5 ml dichloromethane. 33 mg
DMAP,
74 NI methoxyethoxymethyl chloride and 90 NI triethylamine were added to this
receiving
solution. The reaction mixture was stirred overnight, then diluted with 5 ml
dichloro-
methane and the organic phase was washed in succession once each with 3 ml
dilute
aqueous KHS04 solution and saturated aqueous common salt solution. The organic
phase was dried over magnesium sulphate, the solvent was largely evaporated at
re-
duced pressure and the remaining residue was chromatographed on silica gel
(mobile
phase: EAlcyclohexane 2:1 v/v). Drying the product fractions in an oil pump
vacuum
yielded 191 g of the title compound, MS: [M+H]+: 646; m/z: 590, 540, 484, 425.
Example 44:
Ethyl (2"rel1")-2-{[1-({[(3S)-1-(2-ethoxy-2-oxoethyl~2-oxo-2,3,4,5-tetrahydro-
1H-1-
benzazepi n-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[(3-hydroxypropyl)amino]-
4-
oxobutyrate
N
o N 1 ,
~O rel1 O
O
O
140 mg {(3S)-3-[({1-[(2"rel1")-2-ethoxycarbonyl)-4-(isopropylamino)-4-
oxobutyl]cyGo-
pentyl}-carbonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl}acetic
acid (for
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52
preparation see Example 4) was dissolved in 3 ml ethanol, 5 drops of conc.
sulphuric
acid were added thereto and the mixture was stirred for 2 days at RT. Then the
solvent
was largely removed at reduced pressure and the remaining residue was taken up
in 5
ml EA. The organic phase was washed twice with 2 ml aqueous NaHS04 solution
each
time. After drying over sodium sulphate, the solvent was distilled off at
reduced pressure
and the residue was chromatographed on silica gel (mobile phase:
EA/cyclohexane 8:2
(v/v)). 46 mg of the title compound was obtained as a white foam; MS: [M+H]+:
574; m/z:
528, 323; 'H-NMR (CDCI3): 7.33 - 7.11, m, [4]; 6.69, m, [1]; 6.44, m, [1];
4.79, 4.75, 4.34,
4.30, AB-Q-. (2]; 4.48, m, [1].
The compounds of Formula I listed in Table 9 below can also be prepared
according to
the processes described in the examples above or according to processes
analogous
thereto:
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Table 9: Further compounds of Formula I
Ex. R' R2 R3 R4 Config.Config.[M+H]+
NO. Ca* Cb*
16 H H methox eth H rac S 518
I
17 H H 3-(2-oxoazepanyl)H rac S 571
18 eth I - CHp H rac S 558
2-O-
CH2
2-
19 H - CH2 H rel1 S
2-O-
CH2
2-
20 H H 4-methoxyphenyl-H rac S 608
2-oxoeth I
21 H H 3-oxo-1,1- H rac S 558
dimeth Ibut
I
22 H H hen I-2-oxoethH rac S 578
I
23 H H c clo ro ImethH rac S 514
I
24 H H 4-metho benz H rac S 580
I
25 H H 4-methoxyphenyl-H rac S 594
eth I
26 H H 2-methox benz H rac S 580
I
27 H H ben I H rac S 550
28 H H meth I H rac S 474
29 ethyl H 2-(4-methoxy- H rac S 636
hen I -2-oxoeth
I
30 eth I H metho eth I H rel1 S 546
31 H H 2-metho Benz H rac S 580
I
32 H meth iso ro I H rac S 516
I
3,4-dimethoxy- 638
33 eth I H ben I H rac S
34 eth I H c clo ro I H rac S 528
35 Et H 2-h drox eth H rac S 532
I
36 Et H 4-metho benz H rac S 608
I
37 Et H 1-na hth ImethH rac S 628
I
38 Et H 4-methoxyphenyl-H rac S 622
eth I
39 isopropylH isopropyl H rac S 544
40 n-but H iso ro I H rac S 558
I
41 H H isopropyl methoxy-~c S 590
ethoxy-
meth
I
42 2-chloro-H isopropyl H rac S 627
benz
I
43 H meth 2-h drox eth H rac S 518
I I
44 see above
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54
45 H - CH2 H rac S 542
2-CO-
CH2
2-
46 Et - CH2 H rac S 570
2-CO-
CH2
2-
47 Et - CH2 H rac S 647
2-N
Bn
- CH2
2-
48 Et - CH2 H rac S 574
2-S-
CH2
r
49 H - CH2 H rac S 514
4-
50 H - CHZ CH CH2-OH -CH2-H rac S 558
3-
51 -CHr(CHOH)- 548
H meth CH20H H rac S
I
52 H eth - CH2 3-NH-C2HsH rac S 573
I
53 Et 2- 2-hydroxyethylH rac S 576
hydroxy-
eth
I
54 H meth meth I H rac S 4gg
I
55 H eth eth I H rac S 516
I
56 H meth 3-h dro ro H rac S 532
I I
57 H - CH2 H rac S 544
2-CH
OH
- CH2
2-
58 H 2- 2-hydroxyethylH rac S 548
hydroxy-
eth
I
59 H meth - CH2 2-N CH3 H rel1 S 545
I 2
60 H meth - CH2 3-N CH3 H rac S 559
I 2
61 Et -(CH2)2-CH(-O-valine)-(CH2)2- H rac S
671
62 Et methyl -(CH2)3-O-valineH rac S 659
63 H meth iso ro I H rel1 S 516
I
64 H methyl -(CH2)g-N(CHg)2H rel1 S 559
65 H methyl -(CH2)3-NH2 H rac S 531
66 H -(CH2)-O-(CH2)2 H rac S 530
67 H ethyl -(CH2)3-NH2 H rac S 545
',
68 H methyl -(CH2)2-NH(CH3)H rac S 531
75 H methyl -(CH2)4-NH2 H rac S 545
76 H ethyl -(CH2)4-NH2 H rac S 559
77 H methyl -(CH2)3-NH(CH3)H rac S 545
78 H methyl -(CH2)5-NH2 H rac S
79 H ethyl -(CH2)5-NH2 H rac S
Table 9, continued; rac = racemic; Bn = benzyl
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Example 69:
Tert. butyl 2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H 1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-(4-hydroxypiperidin-1-yl)-
4-
oxobutanoate
O
HO--( _N H w
~O N
O O
O O
A) 100 g of 2-((2-benzyloxy)-2-oxoethyl]acrylic acid (for production see
example 1A)
was reacted with 47 ml of thionyl chloride, 43 ml of tert. butanol and 110 ml
of pyri-
dine according to the procedure described in example 1B) to yield 69.8 g of 2-
methylenesuccinic acid-4-benzylester-1-tert. butylester, [M+H]+: 277.
B) 29.6 g of 2-methylenesuccinic acid-4-benzylester-1-tert. butylester as
obtained
above was reacted with 41.4 ml of diisopropylamine, 200 ml of a 1.6 M solution
of
n-butyllithium in n-hexane and 12 ml of cyclopentanecarboxylic acid according
to
the procedure described in example 1C) to yield 24.5 g of 1-[4-(benzyloxy)-2-
(tert.
butoxycarbonyl)-4-oxobutyl]cydopentanecarboxylic acid.
C) 15.8 g of 1-[4-(benzyloxy)-2-(tent. butoxycarbonyl)-4-
oxobutyl]cyclopentanecar-
boxylic acid as obtained above was reacted with 11.75 g of tert. butyl-[(3S)-3-
amino-2-oxo-2,3,4,5-tetrahydro)-1H-benzazepin-1-yl]acetate (for production see
EP
0 733 642 A1 ) according to the procedure described in example 1 D) to yield
21 g of
2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzaze-
pin-3-yl]amino}carbonyl)cyclopentyl]methyl}succinic acid-4-benzylester-1-tert.
butyl-
ester.
D) 21 g of 2-{[(3S)-1-({[1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cydopentyl]methyl}succinic acid-4-benzylester-1-
tert. butylester as obtained above was treated with 6 g of palladium on
activated
carbon and hydrated for 12 h and a hydrogen pressure of 1.3 bar according to
the
procedure described in example 1E) to yield 10 g of 4-tert. butoxy-3-{[1-
({[(3S)-1-(2-
tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-
yl]amino}car-
bonyl)cyclopentyl]methyl}-4-oxobutanoic acid; MS: [M+H]+: 573; m/z: 517, 461;
'H-
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56
NMR (CDC13): 7.31-7.17, m, [3]; 7.10, m, [1]; 6.80, d, [0.5]; 6.72, d, [0.5];
4.60-4.30,
m, [3]; 3.30, m, [0.5]; 3.17, m, [0.5].
E) 1.11g of 4-tert. butoxy-3-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetra-
hydro-1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic
acid
as obtained above was dissolved in 7.8 ml of dichloromethane and 300N1 of
triethylamine was added. After cooling to 0 °C in an ice bath, 222N1 of
ethylchloro-
formate was added dropwise to this receiving solution. The mixture was allowed
to
stir for 30 minutes, then 216 mg of 4-hydroxypiperidine was added and the
mixture
was stirred over night. The mixture was diluted with EA and washed with
aqueous
KHS04 - solution and with brine. Drying of the organic layer over magnesium
sul-
phate and co lumn chromatography on silica gel (liquid phase: EAlcyclohexane
1:1 (v/v) changed to pure EA changed to EA/methanol 4:1 (v/v)) yielded 550 mg
of
the title compound as a white foam, MS: [M+H]+: 656; m/z: 425, 397, 323.
Example 70
2-{[1-({[1-(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}-
carbonyl)cyclopentyl]methyl}-4-oxo-4-[4-(L-valyloxy)piperidin-1-yl]butanoic
acid
O O
NH O--( N H w
~/O
OH O
O
OH
A) 548 mg of tert. butyl 2-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-
2,3,4,5-
tetrahydro-1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-(4-
hydroxy-
piperidin-1-yl)-4-oxobutanoate as obtained in example 67 was dissolved in 3 ml
of
dichloromethane. Then 51 mg of DMAP, 182 mg of BOC-L-valine and 176 mg of
EDC were added. After stirring for 3 h the mixture was diluted with EA and con-
secutively washed with aqueous KHS04 solution and with brine. Drying of the or-
ganic layer over magnesium sulphate and column chromatography on silica gel
(liq-
uid phase: EA/cyclohexane 1:1(v/v) changed to pure EA) yielded 551 mg of 1-(4-
tert. butoxy-3-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1 H-1-
benzazepi n-3-yl]amino}carbonyl)cydo pentyl] methyl}-4-oxobutanoyl)piperidin-4-
yl-N-
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57
4-yl-N-(tert. butoxycarbonyl)-L-valinate, MS: [M+H]+: 855; m/z: 699, 643, 625,
425,
397, 323, 235.
B) 551 mg of 1-(4-tert. butoxy-3-{[1-({[(3S)-1-(2-tert. butoxy-2-oxoethyl)-2-
oxo-2,3,4,5-
tetrahydro-1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-
oxobutano-
yl)piperidin-4-yl-N-(tert. butoxycarbonyl)-L-valinate as obtained above was di
s-
solved in 14 ml of dichloromethane and 1.49 ml of trifluoroacetic acid was
added to
this receiving solution. After stirring over night the solvent and excess of
acid were
evaporated at reduced pressure. EA was added to the remaining residue and the
organic layer was washed with an aqueous saturated sodium bicarbonate solution
until a pH of 4 was reached. The aqueous layer was then extracted thrice with
EA
and the combined organic layers were dried over magnesium sulphate.
Evaporation
of the solvent at reduced pressure and subsequent drying of the remaining
residue
in an oil pump vacuum yielded 310 mg of the title compound as a white foam,
MS:
[M+H]: 643; m/z: 425, 397, 323.
Example 71
Tert. butyl 2-{[1-({[(3S)-1-(2-ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-
1-benz-
azepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[isopropyl(methyl)amino]-4-
oxo-
butanoate
O
/N H
O N
O O
O
A) 20 g of 1-[4-(benzyloxy)-2-(tert. butoxycarbonyl)-4-
oxobutyl]cyclopentanecarboxylic
acid (for preparation see example 69 B)) was reacted with 13.4 g of ethyl-
[(3S)-3-
amino-2-oxo-2,3,4,5-tetrahydro)-1H-benzazepin-1-yl]acetate (preparation analo-
gous to methods described in EP 0 733 642 A1 ) according to the procedure de-
scribed in example 1D) to yield 28.6 g of 4-benzyl-1-tert. butyl-2-{[1-({((3S)-
1-(2-
ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}carbonyl)-
cyclopentyl]methyl}succinate.
B) 28.6 g of 4-benzyl-1-tert. butyl-2-{[1-({[(3S)-1-(2-ethoxy-2-oxoethyl)-2-
oxo-2,3,4,5-
tetrahydro-1H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}succinate as
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58
obtained above was treated with 5 g of palladium on activated carbon and
hydrated
for 4.5 h and a hydrogen pressure of 2.3 bar according to the procedure
described
in example 1 E) to yield 16 g of 4-tert. butoxy-3-{[1-({[(3S)-1-(2-ethoxy-2-
oxoethyl)-2-
oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}carbonyl)cyclopentyl]methyl}-4-
oxobutanoic acid, [M+H]+; 545; m/z: 489.
C) 3 g of 4-tert. butoxy-3-{[1-({[(3S~1-(2-ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-
1H-1-benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-oxobutanoic acid as
obtained above was reacted with 859 NI methylisopropylamine according to the
procedure described in example 1F) to yield 1.6 g of the title compound as a
white
foam, MS: [M+H]+: 600; m/z: 544.
Example 72
2-{[1-({[(3S)-1-(2-Ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-
benzazepin-3-yl]-
amino}carbonyl)cyclopentyl]methyl}-4-[isopropyl(methyl)amino]-4-oxobutanoic
acid
/N H
O
OH O
O
O
507 mg of tert. butyl 2-{[1-({((3S)-1-(2-ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[isopropyl(methyl)amino]-
4-oxo-
butanoate as obtained in example 69 was dissolved in 18 ml of dichloromethane
and
1.95 ml of trifluoroacetic acid was added to this receiving solution. After
stirring over
night the solvent and excess of acid were evaporated at reduced pressure. EA
was
added to the remaining residue and the organic layer was washed with an
aqueous satu-
rated sodium bicarbonate solution, until a pH of 5 of the aqueous layer was
reached.
The organic layer was then dried over magnesium sulphate. Drying of the
organic layer
over magnesium sulphate and column chromatography on silica gel (liquid phase:
EA/cyclohexane 1:1(vlv) changed to pure EA) yielded 430 mg of the title
compound as a
white foam, MS: (M+H]+: 544.
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59
Example 73
1-[(Ethoxycarbonyl)oxy]ethyl 2-{[1-({[(3S)-1-(2-ethoxy-2-oxoethyl)-2-oxo-
2,3,4,5-tetra-
hydro-1 H-1-benzazepin-3-yl]amino}carbonyl)cyclopentylJmethyl}-4-
[isopropyl(methyl~
amino]-4-oxobutanoate
O
/N H
O N
~O O O O N O
O ~ O
107 mg of 2-{[1-({[(3S)-1-(2-Ethoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H 1-
benz-
azepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[isopropyl(methyl)amino]-4-
oxobutanoic
acid (for preparation see example 72) was dissolved in 1 ml of DMF. Then 83 NI
of
triethylamine, 20 mg of solid K2C03 and 85N1 of chloroethylethylcarbonate was
added.
After stirring over night the mixture was diluted with EA and consecutively
washed with
an aqueous KHSOa solution and with brine. Drying of the organic layer over
magnesium
sulphate and column chromatography on silica gel (liquid phase: EA/cyclohexane
1:1 (v/v)) yielded 41 mg of the title compound as a white foam, MS: (M+H]+:
660; m/z:
526, 449, 310, 253.
Example 74
1-[(Ethoxycarbonyl)oxy]ethyl 2-{[1-({[(3S)-1-(2-{1-
[(ethoxycarbonyl)oxy]ethoxy}-2-oxo-
ethyl)-2-oxo-2,3,4,5-tetrahydro-1 H-1-benzazepin-3-
yl]amino}carbonyl)cyclopentyl]-
methyl}-4-[isopropyl(methyl)amino]-4-oxobutanoate
O
/N H
O N
~O O O O N O O
1~ 1' 0 0
o ~ o
500 mg of ethyl 2-{[1-({[(3S)-1-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-
tetrahydro-1H-1-
benzazepin-3-yl]amino}carbonyl)cyclopentyl]methyl}-4-[isopropyl(methyl)amino]-
4-oxo-
butanoate (see example 32, synthesis analogous to example 2) was dissolved in
10 ml
of DMF. Then 312 NI of chloroethylethylcarbonate, 758 mg of solid Cs2C03 and
80 mg of
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solid potassium iodide were added. After stirring for 5 h at 60 °C the
mixture was d fluted
with EA and was then twice washed with water. Drying of the organic layer over
magne-
sium sulphate and column chromatography on silica gel (liquid phase:
cyclohexane,
changed to EA/cyclohexane 1:1(v/v)) yielded 360 mg of the title compound as a
white oil,
MS: [M+H]+: 748; m/z: 614, 480.
Example I:
Capsules containing {(3S)-3-[({1-[(2"rel1")-2-ethoxycarbonyl)-4-
(isopropylamino)-4-
oxobutyl]cyclopentyl}-carbonyl)amino]-2-oxo-2,3,4,5-tetrahydro-1 H-1-
benzazepin-1-
yl}acetic acid:
Capsules with the following composition per capsule were produced:
{(3S)-3-[({1-[(2"rel1 ")-2-ethoxycarbonyl)-4-(isopropyl-amino)-
4-oxobutyl]cyclopentyl}-carbonyl)amino]-2-oxo-2,3,4,5-
tetrahydro-1H-1-benzazepin-1-yl} -acetic acid 20 mg
Corn starch 60 mg
Lactose 300 mg
EA q.s.
The active substance, the corn starch and the lactose were processed into a
homogene-
ous pasty mixture using EA. The paste was ground and the resulting granules
were
placed on a suitable tray and dried at 45°C in order to remove the
solvent. The dried
granules were passed through a crusher and mixed in a mixer with the further
following
auxiliaries:
Talcum 5 mg
Magnesium stearate 5 mg
Corn starch 9 mg
and then poured into 400 mg capsules (= capsule size 0).
