Note: Descriptions are shown in the official language in which they were submitted.
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l Y
1
NOVEL HETEROCYCLICALLY SUBSTITUTED AMIDES WITH
CYSTEINE PROTEASE-INHIBITING EFFECT
The present invention relates to novel:amides which are
inhibitors of enzymes, especially cysteine proteases
such as calpain (= calcium dependant cysteine
proteases) and its isoenzymes and cathepsins, for
example B and L.
Calpains are intracellular proteolytic enzymes from the
group of cysteine proteases and are found in many
cells. Calpains are activated by an increase in the
calcium concentration, a distinction being made between
calpain I or ~-calpain, which is activated by ~-molar
concentrations of calcium ions, and calpain II or
m-calpain, which is activated by m-molar concentrations
of calcium ions (P. Johnson, Int. J. Biochem. 1990,
22(8), 811-22). Further calpain isoenzymes have now
been postulated too (K. Suzuki et al., Biol. Chem.
Hoppe-Seyler, 1995, 376(9), 523-9).
It is suspected that calpains play an important part in
various physiological processes. These include
cleavages of regulatory proteins such as protein kinase
C, cytoskeletal proteins such as MAP 2 and spectrin,
muscle proteins, protein degradation in rheumatoid
arthritis, proteins in the activation of platelets,
neuropeptide metabolism, proteins in mitosis and others
which are listed in M.J. Barrett et al., Life Sci.
1991, 48, 1659-69 and K.K. Wang et al., Trends in
Pharmacol. Sci., 1994, 15, 412-9.
Elevated calpain levels have been measured in various
pathophysiological processes, for example: ischemia of
the heart (e.g. myocardial infarct), of the kidney or
of the central nervous system (e. g. stroke),
inflammations, muscular dystrophies, cataracts of the
eyes, injuries to the central nervous system (e. g.
trauma), Alzheimer's disease etc. (see K.K. Wang,
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above). It is suspected that there is a connection
between these disorders and elevated and persistent
intracellular calcium levels. This results in
overactivation of calcium-dependent processes, which
are then no longer subject to physiological control.
Accordingly, overactivation of calpains may also induce
pathophysiological processes.
It has therefore been postulated that inhibitors of
calpain enzymes may be useful for treating these
disorders. Various investigations have confirmed this.
Thus, Seung-Chyul Hong et al., Stroke 1994, 25(3),
663-9 and R.T. Bartus et al., Neurological Res. 1995,
17, 249-58 have shown a neuroprotective effect of
calpain inhibitors in acute neurodegenerative disorders
or ischemias like those occurring after a stroke.
Likewise, calpain inhibitors improved the recovery of
the memory deficits and neuromotor disturbances
occurring after experimental brain trauma (K. E. Saatman
et al. Proc. Natl. Acad. Sci. USA, 1996, 93, 3428-
3433). C.L. Edelstein et al., Proc. Natl. Acad. Sci.
USA, 1995, 92, 7662-6, found a protective effect of
calpain inhibitors on kidneys damaged by hypoxia.
Yoshida, Ken Ischi et al., Jap. Circ. J. 1995, 59(1),
40-8, were able to show beneficial effects of calpain
inhibitors after cardiac damage produced by ischemia or
reperfusion. Since the release of the ~-AP4 protein is
inhibited by calpain inhibitors, a potential
therapeutic use for Alzheimer's disease has been
proposed (J. Higaki et al., Neuron, 1995, 14, 651-59).
The release of interleukin-la is likewise inhibited by
calpain inhibitors (N. Watanabe et al., Cytokine 1994,
6(6), 597-601). It has further been found that calpain
inhibitors have cytotoxic effects on tumor cells
(E. Shiba et al. 20th Meeting Int. Ass. Breast Cancer
Res., Sendai Jp, 1994, 25-28 Sept., Int. J. Oncol.
5 (Suppl.), 1994, 381).
1 Y
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Further possible uses of calpain inhibitors are
detailed in K.K. Wang, Trends in Pharmacol. Sci., 1994,
15, 412-8.
Calpain inhibitors have already been described in the
literature. However, these are predominantly either
irreversible or peptide inhibitors. Irreversible
inhibitors are usually alkylating substances and have
the disadvantage that they react nonselectively or are
unstable in the body. Thus, these inhibitors often show
unwanted side effects such as toxicity, and are
accordingly of limited use or unusable. The
irreversible inhibitors can be said to include, for
example, the epoxides E 64 (E. B. McGowan et al.,
Biochem. Biophys. Res. Commun. 1989, 158, 432-5), a-
halo ketones (H. Angliker et al., J. Med. Chem. 1992,
35, 216-20) or disulfides (R. Matsueda et al., Chem.
Lett. 1990, 191-194).
Many known reversible inhibitors of cysteine proteases
such as calpain are peptide aldehydes, in particular
dipeptide and tripepide [sic] aldehydes such as, for
example, Z-Val-Phe-H (MDL 28170) (S. Mehdi, Tends [sic]
in Biol. Sci. 1991, 16, 150-3). Under physiological
conditions, peptide aldehydes have the disadvantage
that, owing to the high reactivity, they are often
unstable, may be rapidly metabolized and are prone to
nonspecific reactions which may cause toxic effects
(J.A. Fehrentz and B. Castro, Synthesis 1983, 676-78.
JP 08183771 (CA 1996, 605307) and EP 520336 have
described aldehydes derived from 4-piperidinoylamides
[sic] and 1-carbonylpiperidino-4-ylamides [sic] as
calpain inhibitors. However, the aldehydes which are
claimed herein and are derived from amides of the
general structure I with heteroaromatic substituents
have not previously been described.
Peptide ketone derivatives are likewise inhibitors of
cysteine proteases, in particular calpains. Thus, for
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example, ketone derivatives where the keto group is
activated by an electron-attracting group such as CF3
are known to be inhibitors of serine proteases. In the
case of cysteine proteases, derivatives with ketones
activated by CF3 or similar groups have little or no
activity (M. R. Angelastro et al., J. Med. Chem. 1990,
33, 11-13). Surprisingly, to date only ketone
derivatives in which, on the one hand, leaving groups
in the a position cause irreversible inhibition and, on
the other hand, the keto group is activated by a
carboxylic acid derivative have been found to be
effective inhibitors of calpain (see M.R. Angelastro et
al., see above; WO 92/11850; WO 92,12140; WO 94/00095
and WO 95/00535). However, only peptide derivatives of
these keto amides and keto esters have been described
as effective (Zhaozhao Li et al., J. Med. Chem. 1993,
36, 3472-80; S.L. Harbenson et al., J. Med. Chem. 1994,
37, 2918-29 and see above M.R. Angelastro et al.).
Ketobenzamides have already been described in the
literature. Thus, the keto ester PhCO-Abu-COOCHZCH3 has
been described in WO 91/09801, WO 94/00095 and
92/11850. The analogous phenyl derivative
Ph-CONH-CH(CH2Ph)-CO-COCOOCH3 was, however, found to be
only a weak calpain inhibitor in M.R. Angelastro et
al. , J. Med. Chem. 1990, 33, 11-13 . This derivative is
also described in J.P. Burkhardt, Tetrahedron Lett.,
1988, 3433-36. The significance of the substituted
benzamides has, however, never been investigated to
date.
In a number of therapies, such as [lacuna] stroke, the
active ingredients are administered intravenously, for
example as infusion solution. To do this it is
necessary to have available substances, in this case
calpain inhibitors, which have adequate solubility in
water so that an infusion solution can be prepared.
Many of the described calpain inhibitors have, however,
the disadvantage that they have only low or no
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solubility in water and thus are unsuitable for
intravenous administration. Active ingredients of this
type can be administered only with ancillary substances
intended to confer solubility in water (cf. R.T. Bartus
et al. J. Cereb. Blood Flow Metab. 1994, 14, 537-544).
These ancillary substances, for example polyethylene
glycol, often have side effects, however, or are even
incompatible. A non-peptide calpain inhibitor which is
soluble in water without ancillary substances would
thus be a great advantage. No such inhibitor has been
described to date, and it would thus be novel.
Substituted non-peptide aldehydes, keto carboxylic
esters and keto amide derivatives were described in the
present invention. These compounds are novel and
surprisingly show the possibility of obtaining potent
non-peptide inhibitors of cysteine proteases, such as,
for example, calpain, by incorporating rigid structural
fragments. In addition, all the present compounds of
the general formula I have at least one aliphatic amine
radical and are thus able to bind [sic] salts with
acids. A large number of these substances are soluble
in water in a 0.5~ strength solution at pH 0.4-5 and
thus the show the required profile for intravenous
administration as is necessary, for example, for stroke
therapy.
The present invention relates to amides of the general
formula I
~R2) R~
n Rs
R~ ~A~
I
Ray CH2)~ O
and their tautomeric and isomeric forms, possible
enantiomeric and diastereomeric forms, and possible
physiologically tolerated salts, in which the variables
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have the following meanings:
R1 can be hydrogen, C1-C6-alkyl, branched and
unbranched, phenyl, naphthyl, quinolyl, pyridyl,
pyrimidyl, pyrazyl, pyridazyl, quinazolyl,
quinoxalyl, thienyl, benzothienyl, benzofuranyl,
furanyl and indolyl, it being possible for the
rings also to be substituted by up to 3 R6
radicals, and
R2 are hydrogen, C1-C6-alkyl, branched or unbranched,
0-C1-C6-alkyl, branched or unbranched,
Cz-C6-alkenyl, C2-C6-alkynyl, C1-C6-alkyl-phenyl,
Cz-C6-alkenyl-phenyl, C2-C6-alkynyl-phenyl, OH, C1,
F, Br, I, CF3, NOz, NHZ, CN, COOH, COO-C1-CQ-alkyl,
NHCO-Cl-C4-alkyl , NHCO-phenyl , CONHR9 ,
NHS02-C1-C4-alkyl, NHSOz-phenyl, SOZ-Cl-C4-alkyl and
S02-phenyl , and
R3 can be NR~RB or a ring such as
-N N~p~ ~N~R' . -N_ - ) -N O N-R~
U
\ . I \ . -N ~ \ . ~ \
N
R4 is -C1-C6-alkyl, branched or unbranched, which may
also carry a phenyl, pyridyl or naphthyl ring
which is in turn substituted by a maximum of two
R6 radicals, and
R5 is hydrogen, COORll and CO-Z in which Z is NR12Ri3
and
R'
-NVN_R~ . -N~R' ,-N\ ' ) and
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R6 is hydrogen, C1-C4-alkyl, branched or unbranched,
-O-C1-C4-alkyl, OH, C1, F, Br, I, CF3, N02, NHz, CN,
COOH, COO-C1-C4-alkyl, -NHCO-C1-C4-alkyl,
-NHCO-phenyl , -NHS02-C1-C4-alkyl , -NHS02-phenyl ,
-S02-Cl-C4-alkyl and -SOZ-phenyl, and
R' is hydrogen, C1-C6-alkyl, linear or branched, and
which may be substituted by a phenyl ring which
itself may also be substituted by one or two Rlo
radicals, and
R8 is hydrogen, C1-C6-alkyl, linear or branched, which
may be substituted by a phenyl ring which may
itself also be substituted by one or two Rlo
radicals, and
R9 is hydrogen, C1-C6-alkyl, branched or unbranched,
which may also carry a substituent R16, or phenyl,
pyridyl, pyrimidyl, pyridazyl, pyrazinyl, pyrazyl,
naphthyl, quinolyl, imidazolyl, which may also
carry one or two substituents R14, and
R1° can be hydrogen, C1-C4-alkyl, branched or
unbranched, -O-C1-C4-alkyl, OH, C1, F, Br, I, CF3,
N02, NH2, CN, COOH, COO-C1-C4-alkyl,
-NHCO-Cl-CQ-alkyl, -NHCO-phenyl, -NHS02-C1-C4-alkyl,
-NHSOZ-phenyl , -S02-C1-CQ-alkyl and -SOZ-phenyl
Rll is hydrogen, C1-C6-alkyl, linear or branched, and
which may be substituted by a phenyl ring which
may itself also be substituted by one or two Rlo
radicals, and
R12 is hydrogen, C1-C6-alkyl, branched and unbranched,
and
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R~
_N~ _p~ ~N~pa -N~ -N
. .
_N~ N~p~ .(~-N
[SlCj
R13 is hydrogen, C1-C6-alkyl, branched or unbranched,
which may also be substituted by a phenyl ring
which may also carry an R1° radical, and by
[ lacuna]
and
R14 is hydrogen, C1-C6-alkyl, branched or unbranched,
0-Cl-C6-alkyl, branched or unbranched, OH, Cl, F,
Br, I, CF3, NO2, NHz, CN, COOH, COO-C1-CQ-alkyl, or
two R14 radicals may represent a bridge OC (R15) 20,
and
R15 is hydrogen, C1-C6-alkyl, branched and unbranched,
and
R16 can be a phenyl, pyridyl, pyrimidyl, pyridazyl,
pyrazinyl, pyrazyl, pyrrolyl, naphthyl, quinolyl,
imidazolyl ring, which may also carry one or two
substituents R6, and
A is -(CHZ)m-. -(CH2)m-0-(CH2)o-. -(CH2)o-S-(CHz)m-,
- ( CH2 ) o-SO- ( CHy ) m- , - ( CHz ) o-S02- ( CH2 ) m- . -CH=CH- ,
2 5 -C=C- , -CO-CH=CH- , - ( CHz ) o-CO- ( CHZ ) m- ,
- ( CH2 ) m-NHCO- ( CHZ ) o- . - ( CHz ) m-CONH- ( CHZ ) o- ,
- ( CH2 ) m-NHSOZ- ( CHZ ) o- . -NH-CO-CH=CH- ,
- ( CH2 ) m-S02NH- ( CHZ ) o- , -CH=CH-CONH- and
(R~. O ~,~ O (R'.
and
' x
o ' o
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0
[sic]
R1-A together are also
[lacuna]
and
B is phenyl, pyridine, pyrimidine, pyrazine,
imidazole and thiazole and
x is 1, 2 or 3, and
n is a number 0, 1 or 2, and
m, o is, independently of one another, a number 0, 1,
2, 3 or 4.
The compounds of the formula I can be employed as
racemates, as enantiomerically pure compounds or as
diastereomers. If enantiomerically pure compounds are
required, these can be obtained, for example, by
carrying out a classical racemate resolution with the
compounds of the formula I or their intermediates using
a suitable optically active base or acid. On the other
hand, the enantiomeric compounds can likewise be
prepared by using commercially purchasable compounds,
for example optically active amino acids such as
phenylalanine, tryptophan and tyrosine.
The invention also relates to compounds which are
mesomers or tautomers of compounds of the formula I,
for example those in which the aldehyde or keto group
in formula I is in the form of an enol tautomer.
The invention further relates to the physiologically
tolerated salts of the compounds I which can be
obtained by reacting compounds I with a suitable acid
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or base. Suitable acids and bases are listed, for
example, in Fortschritte der Arzneimittelforschung,
1966, Birkhauser Verlag, Vol. 10, pp. 224-285. These
include, for example, hydrochloric acid, citric acid,
tartaric acid, lactic acid, phosphoric acid,
methanesulfonic acid, acetic acid, formic acid, malefic
acid, fumaric acid etc., and sodium hydroxide, lithium
hydroxide, potassium hydroxide and tris.
The amides I according to the invention can be prepared
in various ways which has [sic] been outlined in the
synthesis scheme.
Synthesis scheme
Heterocyclic carboxylic acids II are linked to suitable
amino alcohols III to give the corresponding amides IV.
Conventional peptide coupling methods are used for
this, as detailed either in C.R. [sic] Larock,
Comprehensive Organic Transformations, VCH Publisher,
1989, page 972 et seq., or in Houben-Weyl, Methoden der
organischen Chemie, 4th edition, E5, Chapter V. It is
preferred to use "activated" acid derivatives of II,
with the acid group COOH being converted into a group
COL. L is a leaving group such as, for example, C1,
imidazole and N-hydroxybenzotriazole. This activated
acid is then reacted with amines to give the amides IV.
The reaction takes place in anhydrous inert solvents
such as methylene chloride, tetrahydrofuran and
dimethylformamide at temperatures from -20 to +25°C.
These alcohol derivatives IV can be oxidized to the
aldehyde derivatives I according to the invention.
Various conventional oxidation reactions can be used
for this (see C.R. [sic] Larock, Comprehensive Organic
Transformations, VCH Publisher, 1989, page 604 et seq.)
such as, for example, Swern and Swern-analogous
oxidations (T. T. Tidwell, Synthesis, 1990, 857-70),
sodium hypochloride [sic]/TEMPO (S. L. Harbenson et al.,
see above) or Dess-Martin (J. Org. Chem. 1983, 48,
4155). Preferably used for this are inert aprotic
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solvents such as dimethylformamide, tetrahydrofuran or
methylene chloride with oxidizing agents such as
DMSO/py x S03 or DMSO/oxalyl chloride at temperatures
from -50 to +25°C, depending on the method (see above
literature).
Alternatively, the carboxylic acid II can be reacted
with amino hydroxamic acid derivatives VI to give
benzamides VII. The reaction in this case is carried
out in the same way as for preparing IV. The hydroxamic
derivatives VI can be obtained from the protected amino
acids V by reaction with a hydroxylamine. An amide
preparation process already described is also used in
this case. Elimination of the protective group X, for
example Boc, takes place in a normal way, for example
with trifluoroacetic acid. The amide hydroxamic acids
VII obtained in this way can be converted by reduction
into the aldehydes I according to the invention. The
reducing agent used for this is, for example, lithium
aluminum hydride at temperatures from -60 to 0°C in
inert solvents such as tetrahydrofuran or ether.
Carboxylic acids or acid derivatives such as esters IX
(P = COOR', COSR') can also be prepared in analogy to
the last process and can likewise be converted by
reduction into the aldehydes I according to the
invention. These processes are listed in R.C. Larock,
Comprehensive Organic Transformations, VCH Publisher,
1989, pages 619-26.
The amides I according to the invention, which have
heterocyclic substituents and have a keto amide or keto
ester group, can be prepared in various ways which have
been outlined in synthesis schemes 2 and 3.
The carboxylic esters IIa are converted, where
appropriate, with acids or bases such as lithium
hydroxide, sodium hydroxide or potassium hydroxide in
aqueous medium or in mixtures of water and organic
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solvents such as alcohols or tetrahydrofuran at room
temperature or elevated temperatures, such as 25-100°C,
into the acids II.
These acids II are linked to an a-amino acid derivative
using customary conditions which are listed, for
example, in Houben-Weyl, Methoden der organischen
Chemie, 4th edition, E5, Chapter V, and C.R. [sic]
Larock, Comprehensive Organic Transformations, VCH
Publisher, 1989, Ch. 9.
For example, the carboxylic acids II are converted into
the "activated" acid derivatives IIb - Y-COL, where L
is a leaving group such as C1, imidazole and
N-hydroxybenzotriazole, and then converted into the
derivative XI by adding an amino acid derivative
HZN-CH (R3 ) -COOR. This reaction takes place in anhydrous
inert solvents such as methylene chloride,
tetrahydrofuran and dimethylformamide at temperatures
from -20 to +25°C.
Scheme 1
OR' R'-A_ OH
----1 /~ B
~R~. ~ ~ O (R~~ ~ O
C o C
pa Ra
R~-A 1
R A 1 ~ _ -CONH~
~p -~ ~p~ ~ ~ COOH
~R=In ~ I
C 1m
C XI
Ra
Ra
R,_A R~_A'' O
B - CONH ~ ~ ~p~ ~ ~ ~ ~ Rs
~R~n I p O
C I
C I.
C = R3-(C"2)X
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The derivatives XI, which are usually esters, are
converted into the keto carboxylic acids XII by
hydrolysis analogous to that described above. The keto
esters I' are prepared in a Dakin-West-analogous
reaction using a method of ZhaoZhao Li et al. , J. Med.
Chem., 1993, 36, 3472-80. This entails a [sic]
carboxylic acids such as XII being reacted with oxalic
monoester chloride at elevated temperature (50-100°C)
in solvents such as, for example, tetrahydrofuran, and
the product obtained in this way then being reacted
with bases such as sodium ethanolate in ethanol at
temperatures of 25-80°C to give the keto ester I'
according to the invention. The keto esters I' can be
hydrolyzed as described above for example to keto
carboxylic acids according to the invention.
The reaction to give keto benzamides I' likewise takes
place in analogy to the method of ZhaoZhao Li et al.
(see above). The keto group in I' is protected by
adding 1,2-ethanedithiol with Lewis acid catalysis,
such as, for example, boron trifluoride etherate, in
inert solvents such as methylene chloride at room
temperature, resulting in a dithiane. These derivatives
are reacted with amines R3-H in polar solvents such as
alcohols at temperatures of 0-80°C, resulting in the
keto amides I (R4 = Z or NR'R8) .
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Scheme 2
R, p.
tR9~ o
COX ~ ~ B _ ~N oOX
$ ~ ~ R~~A
R~.A~ ~ O OH ~C O
II C ~ XIV
( X . O.allcyl )
R~
\$ -CONH O~$ (X.RS)
R'-A ~~
0
C ~'
1
0
(R~a ~ O O~cidatim \ $ -~H
' ~ $ -CONH ~ -' pt . /~ ~ ~ O pa
R -A
° C I'
C xv
C = R3 - (CHz)X -
An alternative method is depicted in scheme 2. The keto
carboxylic acids II are reacted with amino hydroxy
carboxylic acid derivatives XIII (for preparation of
XIII, see S.L. Harbenson et al., J. Med. Chem. 1994,
37, 2918-29 or J.P. Burkhardt et al. Tetrahedron Lett.
1988, 29, 3433-3436) using customary peptide coupling
methods (see above, Houben-Weyl), resulting in amides
XIV. These alcohol derivatives XIV can be oxidized to
the keto carboxylic acid derivatives I according to the
invention. It is possible to use for this various
customary oxidation reactions (see C.R. [sic] Larock,
Comprehensive Organic Transformations, VCH Publisher,
[lacuna] page 604 et seq. ) such as, for example, Swern
and Swern-analogous oxidations, preferably dimethyl
sulfoxide/pyridine-sulfur trioxide complex in solvents
such as methylene chloride or tetrahydrofuran, where
appropriate with the addition of dimethyl sulfoxide, at
room temperature or temperatures from -50 to 25°C
(T. T. Tidwell, Synthesis 1990, 857-70) or sodium
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hypochloride [sic]/TEMPO (S.L. Harbenson et al., see
above).
In the case of a-hydroxy esters XIV (X - O-alkyl),
these can be hydrolyzed to carboxylic acids XV using
methods analogous to those above, but preferably using
lithium hydroxide in water/tetrahydrofuran mixtures at
room temperature. Other esters or amides XVI are
prepared by reaction with alcohols or amines under
coupling conditions described above. The alcohol
derivative XVI can be oxidized to give keto carboxylic
acid derivatives I according to the invention.
The preparation of the carboxylic esters II had already
been described for some instances, or it takes place by
usual chemical methods.
Compounds in which X is a bond are prepared by
conventional aromatic coupling, for example Suzuki
coupling with boric acid derivatives and halides with
palladium catalysis or copper-catalyzed coupling of
aromatic halides. The alkyl-bridged radicals
(X = -(CHZ)m-) can be prepared by reducing the analogous
ketones or by alkylating the organolithium, e.g. ortho-
phenyloxazolidines, or other organometallic compounds
(cf. I.M. Dordor et al., J. Chem. Soc. Perkins Trans.
I, 1984, 1247-52).
Ether-bridged derivatives are prepared by alkylating
the corresponding alcohols or phenols with halides.
The sulfoxides and sulfones can be obtained by
oxidizing the corresponding thioethers.
Alkene- and alkyne-bridged compounds are prepared, for
example, by the Heck reaction from aromatic halides and
corresponding alkenes and alkynes (cf. I. Sakamoto et
al., Chem. Pharm. Bull., 1986, 34, 2754-59).
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The chalcones are produced by condensing acetophenones
with aldehydes and can, where appropriate, be converted
into the analogous alkyl derivatives by hydrogenation.
Amides and sulfonamides are prepared from the amines
and acid derivatives in analogy to the methods
described above.
The dialkylaminoalkyl substituents are obtained by
reductive amination of the aldehyde derivatives with
the appropriate amines in the presence of boron
hydrides such as the BH3/pyridine complex or or [sic]
NaBH3CN (A. F. Abdel-Magid, C.A. Maryanoff, K.G. Carson,
Tetrahedron Lett. 10990 [sic], 31, 5595; A.E. Moormann,
Synth. Commun. 1993, 23, 789).
The amides I with heterocyclic substituents of the
present invention are inhibitors of cysteine proteases,
especially cysteine proteases such as calpains I and II
and cathepsins B and L.
The inhibitory effect of the amides I with heterocyclic
substituents has been determined using enzyme assays
known from the literature, determining as criterion of
effect a concentration of the inhibitor at which 50~ of
the enzyme activity is inhibited (= ICSO) . The amides I
were measured in this way for their inhibitory effect
on calpain I, calpain II and cathepsin B.
Cathepsin 8 assay
The inhibition of cathepsin B was determined by a
method analogous to that of S. Hasnain et al., J. Biol.
Chem., 1993, 268, 235-40.
2 ~tl of an inhibitor solution prepared from inhibitor
and DMSO ( final concentrations : 100 EtM to 0 . O1 E.tM) are
added to 88 ~1 of cathepsin B (cathepsin B from human
liver (Calbiochem), diluted to 5 units in 500 ~,M
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buffer). This mixture is preincubated at room
temperature (25°C) for 60 minutes and then the reaction
is started by adding 10 ~1 of 10 mM Z-Arg-Arg-pNA (in
buffer with 10~ DMSO). The reaction is followed in a
microtiter plate reader at 405 nM [sic] for 30 minutes.
The ICsos are then determined from the maximum
gradients.
Calpaia I aad II assay
The testing of the inhibitory properties of calpain
inhibitors takes place in buffer with 50 mM tris-HC1,
pH 7.5; 0.1 M NaCl; 1 mM dithiotreithol [sic]; 0.11 mM
CaCl2, using the fluorogenic calpain substrate
Suc-Leu-Tyr-AMC (25 mM dissolved in DMSO, Bachem/
Switzerland). Human ~-calpain is isolated from
erythrocytes, and enzyme with a purity > 95~, assessed
by SDS-PAGE, Western blot analysis and N-terminal
sequencing, is obtained after several chromatographic
steps (DEAE-Sepharose, phenyl-Sepharose, Superdex 200
and blue Sepharose). The fluorescence of the cleavage
product 7-amino-4-methylcoumarin (AMC) is followed in a
Spex Fluorolog fluorimeter at ~.ex - 380 nm and ~,em -
460 nm. The cleavage of the substrate is linear in a
measurement range of 60 min., and the autocatalytic
activity of calpain is low, if the tests are carried
out at temperatures of 12°C. The inhibitors and the
calpain substrate are added to the test mixture as DMSO
solutions, and the final concentration of DMSO ought
not to exceed 2$.
In a test mixture, 10 ~1 of substrate (250 )tM final)
and then 10 ~1 of ~-calpain (2 ~g/ml final, i.e. 18 nM)
are added to a 1 ml cuvette containing buffer. The
calpain-mediated cleavage of the substrate is measured
for 15 - 20 min. Then 10 ~1 of inhibitor (50-100 EtM
solution in DMSO) are added and the inhibition of
cleavage is measured for a further 40 min.
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K; values are determined using the classical equation
for reversible inhibition:
(Methods in Enzymology, )
Ki - I(v0/vi)-1; where I - inhibitor concentration,
v0 = initial rate before addition of the inhibitor;
vi = reaction rate at equilibrium.
The rate is calculated from v - AMC liberation/time,
i.e. height/time.
Calpain is an intracellular cysteine protease. Calpain
inhibitors must pass through the cell membrane in order
to prevent intracellular proteins from being broken
down by calpain. Some known calpain inhibitors, such
as, for example, E 64 and leupeptin, cross cell
membranes only poorly and accordingly show only a poor
effect on cells, although they are good calpain
inhibitors. The aim is to find compounds better able to
cross membranes. Human platelets are used to
demonstrate the ability of calpain inhibitors to cross
membranes.
Calpain-mediated breakdown of tyrosine kinase pp60src
in platelets
Tyrosine kinase pp60src is cleaved by calpain after
activation of platelets. This has been investigated in
detail by Oda et al. in J. Biol. Chem., 1993, Vol. 268,
12603-12608. This revealed that the cleavage of pp60src
can be prevented by calpeptin, a calpain inhibitor. The
cellular efficacy of our substances was tested based on
this publication. Fresh, citrated, human blood was
centrifuged at 200 g for 15 min. The platelet-rich
plasma was pooled and diluted 1:1 with platelet buffer
(platelet buffer: 68 mM NaCl, 2.7 mM KC1, 0.5 mM
MgClz x 6 HzO, 0.24 mM NaH2P04 x H20, 12 mM NaHC03,
5.6 mM glucose, 1 mM EDTA, pH 7.4). After a
centrifugation step and washing step with platelet
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buffer, the platelets were adjusted to 10' cells/ml.
The human platelets were isolated at RT.
In the assay mixture, isolated platelets (2 x 106) were
preincubated with various concentrations of inhibitors
(dissolved in DMSO) at 37°C for 5 min. The platelets
were then activated with 1 EtM ionophore A23187 and 5 mM
CaCl2. After incubation for 5 min., the platelets were
briefly centrifuged at 13,000 rpm, and the pellet was
taken up SDS sample buffer (SDS sample buffer: 20 mM
Tris-HC1, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 mM PMSF,
5 ~.g/ml leupeptin, 10 ~.g/ml pepstatin, 10~ glycerol and
1~ SDS). The proteins were fractionated in a 12~ gel,
and pp60src and its 52 kDa and 47 kDa cleavage products
were identified by Western blotting. The polyclonal
rabbit antibody used, anti-cys-src (pp60'-src) ~ was
purchased from Biomol Feinchemikalien (Hamburg). This
primary antibody was detected using a second,
HRP-coupled goat antibody (Boehringer Mannheim, FRG).
The Western blotting was carried out by known methods.
The cleavage of pp60src was quantified by densitometry,
using as controls unactivated (control 1: no cleavage)
and ionophore- and calcium-treated platelets
(control 2: corresponds to 100 cleavage). The EDSo
corresponds to the concentration of inhibitor at which
the intensity of the color reaction is reduced by 50~.
Glutamate-induced cell death in cortical neurones
The test was carried out as in Choi D.W., Maulucci-
Gedde M.A. and Kriegstein A.R., "Glutamate neuro-
toxicity in cortical cell culture". J. Neurosci. 1989,
7, 357-368.
The cortex halves were dissected out of 15-day old
mouse embryos and the single cells were obtained
enzymatically (trypsin). These cells (glia and cortical
neurones) are seeded out in 24-well plates. After three
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days (laminin-coated plates) or seven days (ornithine-
coated plates), the mitosis treatment is carried out
with FDU (5-fluoro-2-deoxyuridines [sic]). 15 days
after preparation of the cells, cell death is induced
by adding glutamate (15 minutes). After removal of
glutamate, the calpain inhibitors are added. 24 hours
later, the cell damage is estimated by determining
lactate dehydrogenase (LDH) in the cell culture
supernatant.
It is postulated that calpain is also involved in
apoptotic cell death (M.K.T. Squier et al., J. Cell.
Physiol. 1994, 159, 229-237; T. Patel et al. Faseb
Journal 1996, 590, 587-597). For this reason, in
another model, cell death was induced in a human cell
line with calcium in the presence of a calcium
ionophore. Calpain inhibitors must get inside the cell
and inhibit calpain there in order to prevent the
induced cell death.
Calcium-mediated cell death is NT2 cells
Cell death can be induced in the human cell line NT2 by
calcium in the presence of the ionophore A 23187.
105 cells/well were plated out in microtiter plates
20 hours before the test. After this period, the cells
were incubated with various concentrations of
inhibitors in the presence of 2.5 E.iM ionophore and 5 mM
calcium. 0.05 ml of XTT (Cell Proliferation Kit II,
Boehringer Mannheim) was added to the reaction mixture
after 5 hours. The optical density was determined
approximately 17 hours later, in accordance with the
manufacturer's information, in an SLT Easy Reader
EAR 400. The optical density at which half the cells
have died is calculated from the two controls with
cells without inhibitors incubated in the absence and
presence of ionophore.
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Elevated glutamate activities occur in a number of
neurological disorders of psychological disturbances
and lead to states of overexcitation or toxic effects
in the central nervous system (CNS). The effects of
glutamate are mediated by various receptors. Two of
these receptors are classified, in accordance with the
specific agonists, as NNmA receptor and AMPA receptor.
Antagonists to these glutamate-mediated effects can
thus be employed for treating these disorders, in
particular for therapeutic use for neurodegenerative
disorders such as Huntington's chorea and Parkinson's
disease, neurotoxic impairments after hypoxia, anoxia,
ischemia and after lesions like those occurring after
stroke and trauma, or else as antiepileptics (cf.
Arzneim. Forschung 1990, 40, 511-514; TIPS, 1990, 11,
334-338; Drugs of the Future 1989, 14, 1059-1071). De
[sic]
Protection from cerebral overexcitatioa by excitatory
amino acids (I~NlDA and AMPA antagonism in mice)
Intracerebral administration of excitatory amino acids
(EAA) induces such drastic overexcitation that it leads
to convulsions and death of the animals (mice) within a
short time. These signs can be inhibited by systemic,
e.g. intraperitoneal, administration of centrally
acting substances (EAA antagonists). Since excessive
activation of EAA receptors in the central nervous
system plays a significant part in the pathogenesis of
various neurological disorders, it is possible to infer
from the detected EAA antagonism in vivo that the
substances may have therapeutic uses for such CNS
disorders. As a measure of the efficacy of the
substances, an EDSO was determined, at which 50~ of the
animals are free of signs, owing to the previous i.p.
administration of the measured substance, by a fixed
dose of either NNmA or AMPA.
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The amides I with heterocyclic substituents are
inhibitors of cysteine derivatives [sic] such as
calpain I and II and cathepsin B and L, and can thus be
used to control diseases associated with an elevated
activity of calpain enzymes or cathepsin enzymes. The
present amides I can accordingly be used to treat
neurodegenerative disorders occurring after ischemia,
trauma, subarachnoid hemorrhages and stroke, and
neurodegenerative disorders such as multi-infarct
dementia, Alzheimer's disease, Huntington's disease and
epilepsies and, in addition, to treat damage to the
heart after cardiac ischemia, damage to the kidneys
after renal ischemia, skeletal muscle damage, muscular
dystrophies, damage caused by proliferation of smooth
muscle cells, coronary vasospasms, cerebral vasospasms,
cataracts of the eyes, restenosis of the blood vessels
after angioplasty. In addition, the amides I may be
useful in the chemotherapy of tumors and metastasis
thereof and for treating disorders in which an elevated
interleukin-1 level occurs, such as inflammation and
rheumatic disorders.
The pharmaceutical preparations according to the
invention comprise a therapeutically effective amount
of the compounds I in addition to conventional
pharmaceutical ancillary substances.
The active ingredients can be present in the usual
concentrations for local external use, for example in
dusting powders, ointments or sprays. As a rule, the
active ingredients are present in an amount of from
0.001 to 1~ by weight, preferably 0.001 to 0.1~ by
weight.
For internal use, the preparations are administered in
single doses. From 0.1 to 100 mg are given per kg of
body weight in a single dose. The preparation may be
administered in one or more doses each day, depending
on the nature and severity of the disorders.
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The pharmaceutical preparations according to the
invention comprise, apart from the active ingredient,
the customary excipients and diluents appropriate for
the required mode of administration. For local external
use it is possible to use pharmaceutical ancillary
substances such as ethanol, isopropanol, ethoxylated
castor oil, ethoxylated hydrogenated castor oil,
polyacrylic acid, polyethylene glycol, polyethylene
glyco [sic] stearate, ethoxylated fatty alcohols,
liquid paraffin, petrolatum and wool fat. Suitable
examples for internal use are lactose, propylene
glycol, ethanol, starch, talc and polyvinylpyrrolidone.
It is also possible for antioxidants such as tocopherol
and butylated hydroxyanisole, and butylated hydroxy-
toluene, flavor-improving additives, stabilizers,
emulsifiers and lubricants to be present.
The substances which are present in the preparation in
addition to the active ingredient, and the substances
used in producing the pharmaceutical preparations, are
toxicologically acceptable and compatible with the
active ingredient in each case. The pharmaceutical
preparations are produced in a conventional way, for
example by mixing the active ingredient with other
[sic] customary excipients and diluents.
The pharmaceutical preparations can be administered in
various ways, for example orally, parenterally, such as
intravenously by infusion, subcutaneously, intra-
peritoneally and topically. Thus, possible
presentations are tablets, emulsions, solutions for
infusion and injection, pastes, ointments, gels,
creams, lotions, dusting powders and sprays.
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Examples
Example 1
2-((4-Pheaylpiperazia-1-yl)methyl)beazoic acid N-(3-
pheaylpropaa-1-al-2-yl)amide
a) Methyl 2-(4-phenyl-1-piperazinylmethyl)benzoate
10.0 g of methyl 2-chloromethylbenzoate, 15 g of
potassium carbonate, 8.8 g of N-phenylpiperazine
and a spatula-tip of 18-crown-6 in 200 ml of DMF
were heated at 100°C for 5 h and then stirred at
room temperature for 60 h. The excess potassium
carbonate was filtered off, the filtrate was
concentrated, and the residue was partitioned
between water and ethyl acetate. Drying of the
organic phase over magnesium sulfate and removal
of the solvent resulted in 16.8 g (1000 of the
product.
b) 2-(4-phenyl-1-piperazinylmethyl)benzoic acid
16.8 g of intermediate la were introduced into
150 ml of THF, and 1.7 g of LiOH in 150 ml of
water were added at room temperature. The cloudy
solution was clarified by adding 10 ml of MeOH.
The reaction mixture was stirred at room
temperature for 12 h and hydrolyzed with an
equimolar amount of 1 M HC1. The reaction mixture
was evaporated to dryness, and the residue was
taken up in methanol/toluene. Removal of the
solvent resulted in 15.2 g (86~) of the product,
which still contained salt.
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c) 2-((4-Phenylpiperazin-1-yl)methyl)benzoic acid N-
(3-phenylpropan-1-ol-2-yl)amide
3.0 g of intermediate lb and 3 ml of triethylamine
were introduced into 50 ml of DMF. 5 g of sodium
sulfate were added and the mixture was stirred for
30 min. 1.5 g of phenylalaninol, 1.4 g of HOBT and
2.I g of EDC were successively added at 0°C, and
the mixture was stirred at room temperature
overnight. The reaction mixture was poured into
distilled water, made alkaline with NaHC03,
saturated with NaCl and extracted three times with
100 ml of methylene chloride. The organic phases
were washed twice with water and dried over
magnesium sulfate. Removal of the solvent resulted
in 2.5 g (59~) of the product.
d) 2-((4-Phenylpiperazin-1-yl)methyl)benzoic acid N-
(3-phenylpropan-1-al-2-yl)amide
2.3 g of intermediate 1c were introduced into
50 ml of DMSO in the presence of 2.4 g of
triethylamine, and 2.5 g of S03/pyridine complex
were added. The mixture was stirred at room
temperature overnight. The mixture was poured into
250 ml of distilled water, made alkaline with
NaHC03, saturated with NaCl and extracted with
100 ml of methylene chloride, and the organic
phase was dried over magnesium sulfate. After
removal of the solvent, the residue was dissolved
in THF, and the hydrochloride was precipitated
with HC1 in dioxane. The precipitate was filtered
off with suction and washed several times with
ether, resulting in 1.9 g (71~) of the product.
1H-NMR (d6-DMSO): 8 = 2.9 (2H), 3.0-3.3 (8H),
4.1-4.5 (2H), 4.7 (1H), 6.8-7.7 (14H), 9.3 (1H),
9.8 (1H) ppm.
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Example 2
2-((4-Benzylpiperazin-1-yl)methyl)beazoic acid N-(3-
phenylpropan-1-al-2-yl)amide
a) Methyl 2-((4-benzyl-1-piperazinyl)methyl)benzoate
[sic]
10.0 g of methyl 2-chlorobenzoate and 9.6 g of
N-benzylpiperazine were reacted in 200 ml of DMF
in the presence of 15 g of potassium carbonate at
100°C in analogy to Example 1a, resulting in
17.6 g (1000 of the product.
b) 2-((4-Benzyl-1-piperazinyl)methyl)benzoic [sic]
acid
17.5 g of intermediate 2a in 150 ml of THF were
hydrolyzed with 1.6 g of LiOH in 150 ml of water
in analogy to Example lb, resulting in 9.1 g (54~)
of the product.
c) 2-((4-Benzylpiperazin-1-yl)methyl)benzoic acid N-
(3-phenylpropan-1-ol-2-yl)arnide
3.0 g of intermediate 2b were reacted in 60 ml of
DMF with 3 ml of triethylamine, 1.5 g of
phenylalaninol, 1.3 g of HOBT and 2.0 g of EDC in
analogy to Example 1c, resulting in 2.0 g (46~) of
the product.
d) 2-((4-Benzylpiperazin-1-yl)methyl)benzoic acid N-
(3-phenylpropan-1-al-2-yl)amide
1.5 g of intermediate 2c were oxidized in 40 ml of
DMSO with 1.9 g of S03/pyridine complex in 20 ml
of DMSO in the presence of 2.3 ml of triethylamine
in analogy to Example 1d, resulting in 0.4 g (21~)
of the product in the form of the fumarate.
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1H-NMR (d6-DMSO): b = 2.1-2.3 (8H), 2.9-3.0 (1H),
3.3-3.6 (6H), 4.5 (1H), 6.6 (2H), 7.1-7.7 (14H),
9.7 (1H), 10.3 (1H) ppm.
Example 3
2-((4-Benzylpiperazin-1-yl)methyl)benzoic acid N-(1-
carbamoyl-1-oxo-3-phenylpropan-2-yl)amide
a) 2-((4-Benzylpiperazin-1-yl)methyl)benzoic acid N-
(1-carbamoyl-1-ol-3-phenylpropan-2-yl)amide
1.5 g of intermediate 2b were reacted in 40 ml of
DMF with 0.7 ml of triethylamine, 1.0 g of
3-amino-2-hydroxy-4-phenylbutyramide hydro-
chloride, 0.6 g of HOBT and 0.9 g of EDC in
analogy to Example lc, resulting in 0.8 g (380) of
the product.
b) 2-((4-Benzylpiperazin-1-yl)methyl)benzoic acid N
(1-carbamoyl-1-oxo-3-phenylpropan-2-yl)amide
0.7 g of intermediate 3a were oxidized in 20 ml of
DMSO with 0.7 g of S03/pyridine complex in the
presence of 0.8 g of triethylamine in analogy to
Example ld, resulting in 0.1 g (180) of the
product in the form of the free base.
1H-NMR (d6-DMSO): 8 = 2.3 (4H), 2.8-3.5 (8H), 5.3
(1H) , 6. 7-7. 5 (16H) , 7. 8 (1H) , 8. 1 (1H) , 10.3 (1H)
ppm.
Example 4
2-(4-((3-Methylphenyl)piperazin-1-yl)methyl)benzoic
acid N-(1-carbamoyl-1-oxo-3-phenylpropan-2-yl)amide
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a) Methyl 2-(4-((3-methylphenyl)-1-piperazinyl)-
methyl)benzoate [sic]
4.0 g of methyl 2-chloromethylbenzoate and 4.4 g
of 3-methylphenylpiperazine were heated in 200 ml
of DMF in the presence of 4.5 g of potassium
carbonate at 140°C for 3 h. The reaction mixture
was poured into water and extracted three times
with ethyl acetate. The combined organic phases
were washed three times with saturated brine,
dried over magnesium sulfate and concentrated,
resulting in 6.5 g (92~) of the product.
b) 2-(4-((3-Methylphenyl)-1-piperazinyl)methyl)-
benzoic [sic] acid
5.9 g of intermediate 4a were dissolved in 75 ml
of THF and hydrolyzed with 0.9 g of LiOH in 75 ml
of water in analogy to Example 1b, resulting in
2.9 g (51~) of the product.
c) 2-(4-((3-Methylphenyl)piperazin-1-yl)methyl)-
benzoic acid N-(1-carbamoyl-1-ol-3-phenylpropan-2-
yl)amide
1.8 g of intermediate 4b were introduced into
50 ml of DMF in the presence of 2.7 ml of
triethylamine, and 0.8 g of HOBT, 1.3 g of
3-amino-2-hydroxy-4-phenylbutyramide hydrochloride
and 1.2 g of EDC were successively added, in
analogy to Example lc, resulting in 1.4 g (50$) of
the product.
d) 2-(4-((3-Methylphenyl)piperazin-1-yl)methyl)-
benzoic acid N-(1-carbamoyl-1-oxo-3-phenylpropan-
2-yl)amide
1.2 g of intermediate 4c were dissolved in 30 ml
of DMSO and oxidized with 1.6 g of S03/pyridine
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complex in the presence of 1.5 ml of triethylamine
in analogy to Example ld, resulting in 1.0 g (83~)
of the product.
MS : m/e = 484 (M+)
Examples 5 and 6 were synthesized in analogy to
Example 1.
Example 5
3-((9-Phenylpiperazin-1-yl)methyl)benzoic acid N-(3-
phenylpropan-1-al-2-yl)amide fumarate
1H-NMR (d6-DMSO): S = 2.5 (4H), 2.9 (1H), 3.2 (4H), 3.3
(1H), 3.7 (2H), 4.5 (1H), 6.6 (2H), 6.75 (1H), 6.9
(2H), 7.2 (2H), 7.2-7.3 (5H), 7.45 (1H), 7.55 (1H),
7.75 (1H), 7.8 (2H), 8.9 (1H), 9.7 (1H) ppm.
Example 6
3-((4-(2-tert-Butyl-4-trifluoromethylpyrimidin-6-yl)-
homopiperazin-1-yl)methyl)benzoic acid N-(3-phenyl-
propan-1-al-2-yl)amide
MS: m/e = 568 (M++1)
Example 7
4-(N-(3,4-Dioxomethylene)benzyl-N-methylaminomethyl)-
benzoic acid N-(3-phenylpropan-1-al-2-yl)amide
a) 4-(N-(3,4-Dioxomethylene)benzyl-N-methylamino-
methyl)benzoic acid
11.5 g of N-(3,4-dioxomethylene)benzyl-N-
methylamine and 15.5 g of triethylamine were
introduced into [lacuna], and 15.0 g of
4-bromomethylbenzoic acid in 100 ml of THF were
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added. The reaction mixture was briefly heated to
reflux and then stirred at room temperature for
15 h. After filtering off the salts, the mother
liquor was concentrated, and the residue was
dissolved in ethyl acetate and washed with water.
The aqueous phase was made alkaline and extracted
several times with ethyl acetate, resulting in
6.6 g (32~) of the product as a white solid.
b) 4-(N-(3,4-Dioxomethylene)benzyl-N-methylamino-
methyl)benzoic acid N-(3-phenylpropan-1-ol-2-yl)-
amide
4.4 g of intermediate 5a [sic] were introduced
into 50 ml of DMF in the presence of 2.9 g of
triethylamine, and 1.8 g of HOBT, 2.0 g of
phenylalaninol and 2.8 g of EDC were successively
added, in analogy to Example 1c, resulting in
2.3 g (400) of the product.
c) 4-(N-(3,4-Dioxomethylene)benzyl-N-methylamino-
methyl)benzoic acid N-(3-phenylpropan-1-al-2-yl)-
amide
2.0 g of intermediate 5b [sic] were dissolved in
60 ml of DMSO and oxidized with 2.1 g of
S03/pyridine complex in the presence of 1.8 ml of
triethylamine in analogy to Example ld, resulting
in 1.3 g (68~) of the product.
1H-NMR (CF3COOD) : b = 2. 9 (3H) , 3.2 (2H) , 4 .3-4. 9
(5H), 6.1 (2H), 6.6 (1H), 6.9 (3H), 7.2-7.4 (5H),
7 . 8 (2H) , 8.25 (2H) ppm.
MS: m/e = 430 (M+)
Examples 8-28 were prepared in analogy to Example 7.
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Exa~le 8
4-(N-Benzyl-N-methylaminomethyl)benzoic acid N-(3-
phenylpropan-1-al-2-yl)amide
1H-NMR (CF3COOD): b = 2.9 (3H), 3.2 (2H), 4.3-5.0 (5H),
6.7 (1H), 7.25-7.5 (8H), ?.55 (2H), 7.8 (2H), 8.2 (2H)
ppm.
MS: m/e = 386 (M+)
Example 9
4-(N-(4-Methoxy)benzyl-N-methylaminomethyl)benzoic acid
N-(3-phenylpropan-1-al-2-yl)amide
1H-NMR (CF3COOD): S = 2.9 (3H), 3.3 (ZH), 4.0 (3H),
4.3-4.9 (5H), 6.7 (1H), 7.1-7.4 (7H), 7.5 (2H), 7.8
(2H), 8.2 (2H) ppm.
MS: m/e = 416 (M+)
Exa~le 10
4-(N-Benzyl-N-methylaminomethyl)benzoic acid N-(3-
butan-1-al-2-yl)amide
1H-NNgt (CF3COOD) : 8 = 1. 1 (3H) , 1.6 (2H) , 2.0 (2H) , 2.9
(3H), 4.3-4.5 (3H), 4.7 (1H), 4.8 (1H), 6.6 (1H),
7.3-7.6 (5H), 7.8 (2H), 8.3 (2H) ppm.
MS: m/e = 338 (M')
Example li
4-(N-(3,4-Dioxomethylene)benzyl-N-methylaminomethyl)-
benzoic acid N-(3-butan-1-al-2-yl)amide
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1H-NMR (CF3COOD): b = 1.1 (3H), 1.6 (2H), 1.9 (2H), 2.9
(3H), 4.25-4.6 (4H), 4.75 (1H), 6.1 (2H), 6.6 (1H), 6.9
(3H), 7.8 (2H), 8.3 (2H) ppm.
MS: m/e = 382 (M+)
Example 12
4-(N-(4-Methoxy)benzyl-N-methylaminomethyl)benzoic acid
N-(3-butan-1-al-2-yl)amide
MS: m/e = 368 (M')
Example 13
4-(N-(3,4-Dioxomethylene)benzyl-N-methylaminomethyl)-
benzoic acid N-(3-cyclohexylpropan-1-al-2-yl)amide
1H-NMR (CF3COOD): 8 = 1.0-2.0 (13H), 2.9 (3H), 4.3-4.9
(4H), 6.1 (2H), 6.6 (1H), 6.9 (3H), 7.8 (2H), 8.3 (2H)
ppm.
MS: m/e = 436 (M;)
Example 14
4-(N-(4-Henzyl-N-methylaminomethyl)benzoic acid N-(3-
cyclohexylpropan-1-al-2-yl)amide
1H-NMR (d6-DMSO): b = 1.0-1.8 (13H), 2.1 (3H), 3.4 (2H),
3.5 (2H), 4.3 (1H), 7.1-7.4 (5H), 7.5 (2H), 7.8 (2H),
8.8 (1H), 9.5 (1H) ppm.
Example 15
4-(N-(4-Methoxy)benzyl-N-methylaminomethyl)benzoic acid
N-(3-cyclohexylpropan-1-al-2-yl)amide
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1H-NMFt (CDC13): 8 = 1.0-1.8 (13H), 2.1 (3H), 3.4 (2H),
3.5 (2H), 3.7 (3H), 4.3 (1H), 6.8 (2H), 7.25 (2H), 7.5
(2H), 7.9 (2H), 8.8 (1H), 9.5 (1H) ppm.
Example 16
4-((2-Pheaylpyrrolid-1-yl)methyl)beazoic acid N-(3-
cyclohexylpropaa-1-al-2-yl)amide
MS: m/e = 420 (M'')
Example 17
4-((2-Phenylpyrrolid-1-yl)methyl)beazoic acid N-(3-
butan-1-al-2-yl)amide
MS: m/e = 364 (M'")
Example 18
4-((2-Phenylpyrrolid-1-yl)methyl)benzoic acid N-(3-
phenylpropan-1-al-2-yl)amide
MS: m/e = 412 (M+)
Example 19
4-((1,2,3,4-Dihydroquinolia-1-yl)methyl)beazoic acid N-
(3-cyclohexylpropan-1-al-2-yl)amide
1H-NMR (CDC13):S 1.0-1.9 (13H), 2.0 (2H), 2.8 (2H),
=
3.3 (2H), 4.5 (2H),4.8 (1H), 6.4 (1H), 6.5 (2H), 7.0
(2H), 7.4 (2H),7.8 (2H), 9.7 (1H) ppm.
MS: m/e = 404 (Mi)
CA 02328720 2000-10-13
0050/48969 - 34 -
Example 20
4-((1,2,3,4-Dihydroquinolin-1-yl)methyl)benzoic acid N-
(3-phenylpropan-1-al-2-yl)amide
1H-NMR (d6-DMSO): b = 1.9 (2H), 2.75 (2H), 2.9 (1H), 3.3
(1H) , 3.4 (2H) , 4.4 (1H) , 4.5 (2H) , 6.3 (2H) , 6.8 (2H) ,
7.1-7.25 (5H), 7.3 (2H), 7.7 (2H), 8.8 (1H), 9.5 (1H)
ppm.
MS: m/e = 398 (M+)
Example 21
4-((1,2,3,4-Dihydroguinolin-1-yl)methyl)benzoic acid N-
(3-butan-1-al-2-yl)amide
1H-NMR (ds-DMSO) : b = 0.9 (3H) , 1.2-2.0 (6H) , 2.7 (2H) ,
3.3 (2H), 4.2 (1H), 4.5 (2H), 6.4 (2H), 6.8 (2H), 7.3
(2H), 7.8 (2H), 8.8 (1H), 9.5 (1H) ppm.
MS: m/e = 350 (M+)
Example 22
4-((1,2,3,4-Dihydroisoquinolin-2-yl)methyl)benzoic acid
N-(3-cyclohexylpropan-1-al-2-yl)amide
1H-NMR (d6-DMSO): S = 0.9-1.8 (13H), 2.7-2.9 (4H), 3.6
(2H), 3.75 (2H), 4.4 (1H), 6.9-7.1 (4H), 7.4 (2H), 7.8
(2H), 8.8 (1H), 9.5 (1H) ppm.
MS: m/e = 404 (M+)
Example 23
4-((1,2,3,4-Dihydroisoquinolin-2-yl)methyl)benzoic acid
N-(3-phenylpropan-1-al-2-yl)amide
CA 02328720 2000-10-13
0050/48969 - 35 -
1H-NMR (d6-DMSO): 8 = 2.7 (2H), 2.8 (2H), 2.9 (1H), 3.2
(1H), 3.5 (2H), 3.7 (2H), 4.5 (1H), 6.9-7.1 (4H),
7.2-7.3 (5H), 7.5 (2H), 7.75 (2H), 8.8 (1H), 9.5 (1H)
ppm.
MS: m/e = 398 (M+)
Example 24
4-((1,2,3,4-Dihydroisoquinolin-2-yl)methyl)benzoic acid
N-(3-butan-1-al-2-yl)amide hydrochloride
1H-NMR (d6-DMSO): b = 0.9 (3H), 1.2-2.0 (4H), 3.0 (1H),
3.3 (2H), 3.6 (1H), 4.1-4.6 (5H), 7.2 (4H), 7.8 (2H),
8.0 (2H), 9.0 (1H), 9.5 (1H), 11.75 (1H) ppm.
Example 25
4-((6,7-Dimethoxy-1,2,3,4-dihydroisoquinolin-2-yl)-
methyl)benzoic acid N-(3-cyclohexylpropan-1-al-2-yl)-
amide
1H-NMR (d6-DMSO) : 8 = 0.9-1.9 (13H) , 2.7 (4H) , 3.4 (2H) ,
3.6 (3H), 3.65 (2H), 3.7 (3H), 4.3 (1H), 6.5 (1H), 6.6
(1H), 7.5 (2H), 7.8 (2H), 8.8 (1H), 9.5 (1H) ppm.
MS: m/e = 464 (M+)
Example 26
4-((6,7-Dimethoxy-1,2,3,4-dihydroisoquinolin-2-yl)-
methyl)benzoic acid N-(3-phenylpropan-1-al-2-yl)amide
1H-NMR (ds-DMSO) : S = 2.7 (4H) , 2.9 (1H) , 3.25 (1H) , 3.6
(6H), 3.7 (2H), 4.5 (1H), 6.6 (1H), 6.7 (1H), 7.2-7.3
(5H), 7.4 (2H), 7.8 (2H), 8.9 (1H), 9.6 (1H) ppm.
MS: m/e = 458 (M+)
CA 02328720 2000-10-13
0050/48969 - 36 -
Example 27
4-((6,7-Dimethoxy-1,2,3,4-dihydroisoguinolin-2-yl)-
methyl)benzoic acid N-(3-butan-1-al-2-yl)amide
1H-NMR (ds-DMSO): 8 = 0.9 (3H), 1.4 (2H), 1.5-1.8 (2H),
2.7 (4H), 3.4 (2H), 3.7 (3H), 3.75 (3H), 3.8 (2H), 4.3
(1H) , 6.6 (1H) , 6.7 (1H) , 7.4 (2H) , 7.8 (2H) , 8.8 (1H) ,
9.5 (1H) ppm.
MS: m/e = 410 (M+)
Example 28
2-((1,2,3,4-Dihydroquinolin-1-yl)methyl)benzoic acid N-
(3-butan-1-al-2-yl)amide
MS: m/e = 441 (M+)
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