U.S. patent application number 13/893347 was filed with the patent office on 2013-09-26 for substituted piperidines.
This patent application is currently assigned to BAYER INTELLECTUAL PROPERTY GmbH. The applicant listed for this patent is BAYER INTELLECTUAL PROPERTY GmbH. Invention is credited to Eckhard BENDER, Anja BUCHMULLER, Yolanda CANCHO GRANDE, Christoph GERDES, Kersten Matthias GERICKE, Mark Jean GNOTH, Dirk HEIMBACH, Mario JESKE, Susanne ROHRIG, Katfa ZIMMERMANN.
Application Number | 20130252948 13/893347 |
Document ID | / |
Family ID | 42537882 |
Filed Date | 2013-09-26 |
United States Patent
Application |
20130252948 |
Kind Code |
A1 |
HEIMBACH; Dirk ; et
al. |
September 26, 2013 |
Substituted piperidines
Abstract
The invention relates to novel substituted piperidines, to
processes for preparation thereof, to the use thereof for treatment
and/or prophylaxis of diseases and to the use thereof for
production of medicaments for treatment and/or prophylaxis of
diseases, especially of cardiovascular diseases and tumour
diseases.
Inventors: |
HEIMBACH; Dirk; (Dusseldorf,
DE) ; ROHRIG; Susanne; (Hilden, DE) ; CANCHO
GRANDE; Yolanda; (Leverkusen, DE) ; BENDER;
Eckhard; (Langenfeld, DE) ; ZIMMERMANN; Katfa;
(Dusseldorf, DE) ; BUCHMULLER; Anja; (Essen,
DE) ; GERDES; Christoph; (Koln, DE) ; GNOTH;
Mark Jean; (Mettmann, DE) ; GERICKE; Kersten
Matthias; (Wuppertal, DE) ; JESKE; Mario;
(Solingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER INTELLECTUAL PROPERTY GmbH |
Monheim |
|
DE |
|
|
Assignee: |
BAYER INTELLECTUAL PROPERTY
GmbH
Monheim
DE
|
Family ID: |
42537882 |
Appl. No.: |
13/893347 |
Filed: |
May 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13322593 |
Feb 17, 2012 |
8440657 |
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PCT/EP2010/003024 |
May 18, 2010 |
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13893347 |
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Current U.S.
Class: |
514/227.8 |
Current CPC
Class: |
A61P 7/02 20180101; A61P
9/14 20180101; A61P 35/04 20180101; A61P 9/10 20180101; A61P 9/00
20180101; A61P 9/06 20180101; A61P 9/12 20180101; C07D 417/14
20130101; A61P 11/00 20180101; A61P 7/06 20180101; C07D 413/04
20130101; A61P 35/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/227.8 |
International
Class: |
C07D 413/04 20060101
C07D413/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2009 |
DE |
102009022894.2 |
Claims
1-14. (canceled)
15. A method of prophylaxis of thromboembolic disorders in humans
and animals comprising administering to the human or animal an
anticoagulatory amount of a compound of formula (I): ##STR00158##
in which R.sup.1 is trifluoromethyl, 1,1-difluoroethyl,
2,2-difluoroethyl, 2,2,2-trifluoroethyl, difluoromethoxy,
trifluoromethoxy or ethyl, R.sup.2 is 2-hydroxyeth-1-yl,
2-methoxyeth-1-yl, 2-ethoxyeth-1-yl, cyclopropyl or
1-methoxycycloprop-1-yl, R.sup.3 is a group of the formula
##STR00159## where * is the point of attachment to the carbonyl
group, or a salt thereof.
16. The method of claim 15, wherein in the compound of formula (I),
R.sup.1 is trifluoromethyl, 2,2,2-trifluoroethyl, trifluoromethoxy
or ethyl, R.sup.2 is 2-methoxyeth-1-yl, cyclopropyl or
1-methoxycycloprop-1-yl, R.sup.3 is a group of the formula
##STR00160## where * is the point of attachment to the carbonyl
group.
17. The method of claim 15, wherein in the compound of formula (I),
R.sup.1 is trifluoromethoxy. R.sup.2 is 2-methoxyeth-1-yl or
cyclopropyl, and R.sup.3 is a group of the formula ##STR00161##
where * is the point of attachment to the carbonyl group.
18. The method of claim 15, wherein in the compound of formula (I)
R.sup.1 is trifluoromethoxy, R.sup.2 is cyclopropyl, and R.sup.3 is
a group of the formula ##STR00162## where * is the point of
attachment to the carbonyl group.
19. The method of claim 15, wherein the phenyl substituent and the
1,2,4-oxadiazol-5-yl substituent of the compound of formula (I)
bonded to the piperidine ring are in cis positions to one
another.
20. The method of claim 15, wherein the carbon atom to which the
phenyl substituent is bonded in the compound of formula (I) is in
an S configuration and the carbon atom to which the
1,2,4-oxadiazol-5-yl substituent is bonded is also in an S
configuration.
21. The method of claim 15, wherein the compound of formula (I) is
has the formula: ##STR00163##
22. The method of claim 21, wherein the compound is a cis
stereoisomer.
23. The method of claim 21, wherein the compound of formula (I) is
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]pip-
eridin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [cis isomer],
or a salt thereof.
24. The method of claim 15, wherein the compound of formula (I) is
administered as a pharmaceutical composition, comprising the
compound of formula (I) and an inert nontoxic pharmaceutically
acceptable excipient.
25. The method of claim 15, wherein the thromboembolic disorder is
selected from the group consisting of: ST-segment elevation
myocardial infarction (STEMI) and non-ST-segment elevation
myocardial infarction (non-STEMI), stable angina pectoris, unstable
angina pectoris, reocclusions and restenoses after coronary
interventions such as angioplasty, stent implantations or
aortocoronary bypass, peripheral arterial occlusion diseases,
pulmonary embolisms, deep venous thromboses and renal vein
thromboses, transitory ischaemic attacks and also thrombotic and
thromboembolic stroke.
26. The method of claim 15, wherein the condition is a cardiogenic
thromboembolism selected from the group consisting of brain
ischaemias, stroke and systemic thromboembolisms and ischaemias; an
acute, intermittent or persistent cardiac arrhythmia
27. The method of claim 15, wherein the condition is a
thromboembolic complication connected with microangiopathic
haemolytic anaemias, extracorporeal circulation.
28. A method of prophylaxis of a pulmonary disorder comprising
administering to a patient in need thereof a therapeutically
effective, anticoagulatory, amount of a compound of formula (I):
##STR00164## in which R.sup.1 is trifluoromethyl,
1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl,
difluoromethoxy, trifluoromethoxy or ethyl, R.sup.2 is
2-hydroxyeth-1-yl, 2-methoxyeth-1-yl, 2-ethoxyeth-1-yl, cyclopropyl
or 1-methoxycycloprop-1-yl, R.sup.3 is a group of the formula
##STR00165## where * is the point of attachment to the carbonyl
group, or a salt thereof.
29. The method of claim 28, wherein the compound of formula (I) is
administered as a pharmaceutical composition, comprising the
compound of formula (I) and an inert nontoxic pharmaceutically
acceptable excipient.
30. The method of claim 28, wherein the pulmonary disorder is
selected from the group consisting of pulmonary fibrosis; pulmonary
hypertension, including pulmonary arterial hypertension, and
disorders characterized by pulmonary occlusion; and an inflammatory
pulmonary disorder.
Description
[0001] The invention relates to novel substituted piperidines, to
processes for preparation thereof, to the use thereof for treatment
and/or prophylaxis of diseases and to the use thereof for
production of medicaments for treatment and/or prophylaxis of
diseases, especially of cardiovascular diseases and tumour
diseases.
[0002] Thrombocytes (blood platelets) are a significant factor both
in physiological haemostasis and in thromboembolic disorders. In
the arterial system in particular, platelets are of central
importance in the complex interaction between blood components and
the wall of the vessel. Unwanted platelet activation may, as a
result of formation of platelet-rich thrombi, lead to
thromboembolic disorders and thrombotic complications with
life-threatening conditions.
[0003] One of the most potent platelet activators is the blood
coagulation protease thrombin, which is formed at injured blood
vessel walls and which, in addition to fibrin formation, leads to
the activation of platelets, endothelial cells and mesenchymal
cells (Vu T K H, Hung D T, Wheaton V I, Coughlin S R, Cell 1991,
64, 1057-1068). In platelets in vitro and in animal models,
thrombin inhibitors inhibit platelet aggregation and the formation
of platelet-rich thrombi. In man, arterial thromboses can be
prevented or treated successfully with inhibitors of platelet
function and thrombin inhibitors (Bhatt D L, Topol E J, Nat. Rev.
Drug Discov. 2003, 2, 15-28). Accordingly, there is a high
probability that antagonists of thrombin action on blood platelets
reduce the formation of thrombi and the occurrence of clinical
sequelae such as myocardial infarction and stroke. Other cellular
effects of thrombin, for example on endothelial and smooth muscle
cells of vessels, leukocytes and fibroblasts, are possibly
responsible for inflammatory and proliferative disorders.
[0004] At least some of the cellular effects of thrombin are
mediated via a family of G-protein-coupled receptors (Protease
Activated Receptors, PARs), the prototype of which is the PAR-1
receptor. PAR-1 is activated by bindung of thrombin and proteolytic
cleavage of its extracellular N-terminus. The proteolysis exposes a
new N-terminus having the amino acid sequence SFLLRN . . . , which,
as agonist ("tethered ligand"), leads to intramolecular receptor
activation and transmission of intracellular signals. Peptides
derived from the tethered ligand sequence can be used as agonists
of the receptor and, on platelets, lead to activation and
aggregation. Other proteases are likewise capable of activating
PAR-1; these proteases include, for example, plasmin, factor VIIa,
factor Xa, trypsin, activated protein C (aPC), tryptase, cathepsin
G, proteinase 3, granzyme A, elastase and matrix metalloprotease 1
(MMP-1).
[0005] In contrast to the inhibition of protease activity of
thrombin with direct thrombin inhibitors, blockade of PAR-1 should
result in an inhibition of platelet activation without reduction in
the coagulability of the blood (anticoagulation).
[0006] Antibodies and other selective PAR-1 antagonists inhibit the
thrombin-induced aggregation of platelets in vitro at low to medium
thrombin concentrations (Kahn M L, Nakanishi-Matsui M, Shapiro M J,
Ishihara H, Coughlin S R, J. Clin. Invest. 1999, 103, 879-887). A
further thrombin receptor with possible significance for the
pathophysiology of thrombotic processes, PAR-4, was identified on
human and animal platelets. In experimental thromboses in animals
having a PAR expression pattern comparable to humans, PAR-1
antagonists reduce the formation of platelet-rich thrombi (Derian C
K, Damiano B P, Addo M F, Darrow A L, D'Andrea M R, Nedelman M,
Zhang H-C, Maryanoff B E, Andrade-Gordon P, J. Pharmacol. Exp.
Ther. 2003, 304, 855-861).
[0007] In the last few years, a large number of substances have
been examined for their platelet function-inhibiting action; but
only a few platelet function inhibitors have been found to be
useful in practice. There is therefore a need for pharmaceuticals
which specifically inhibit an increased platelet reaction without
significantly increasing the risk of bleeding, thus reducing the
risk of thromboembolic complications.
[0008] Effects of thrombin which are mediated via the PAR-1
receptor influence the progression of the disease during and after
coronary artery bypass graft (CABG) and other operations, and in
particular operations with extracorporeal circulation (for example
heart-lung machine). During the course of the operation, there may
be bleeding complications owing to pre- or intraoperative
medication with coagulation-inhibiting and/or platelet-inhibiting
substances. For this reason, for example, medication with
clopidogrel has to be interrupted several days prior to a CABG.
Moreover, as mentioned, disseminated intravascular coagulation or
consumption coagulopathy (DIC) may develop (for example owing to
the extended contact between blood and synthetic surfaces during
extracorporeal circulation or blood transfusions), which in turn
can lead to bleeding complications. At a later stage, there is
frequently restenosis of the venous or arterial bypasses grafted
(which may even result in occlusion) owing to thrombosis,
intimafibrosis, arteriosclerosis, angina pectoris, myocardial
infarction, heart failure, arrhythmias, transitory ischaemic attack
(TIA) and/or stroke.
[0009] In man, the PAR-1 receptor is also expressed in other cells
including, for example, endothelial cells, smooth muscle cells and
tumour cells. Malignant tumour disorders (cancer) have a high
incidence and are generally associated with high mortality. Current
treatments achieve full remission in only a fraction of patients
and are typically associated with severe side effects. There is
therefore a high demand for more effective and safer therapies. The
PAR-1 receptor contributes to cancer generation, growth,
invasiveness and metastasis. Moreover, PAR-1 expressed on
endothelial cells mediates signals resulting in vascular growth
("angiogenesis"), a process which is vital for enabling tumour
growth beyond about 1 mm.sup.3 Angiogenesis also contributes to the
genesis or worsening of other disorders including, for example,
haematopoetic cancer disorders, macular degeneration, which leads
to blindness, and diabetic retinopathy, inflammatory disorders,
such as rheumatoid arthritis and colitis.
[0010] Sepsis (or septicaemia) is a common disorder with high
mortality. Initial symptoms of sepsis are typically unspecific (for
example fever, reduced general state of health); however, during
further progression there may be generalized activation of the
coagulation system ("disseminated intravascular coagulation" or
"consumption coagulopathy" (DIC)) with the formation of
microthrombi in various organs and secondary bleeding
complications. DIC may also occur independently of sepsis, for
example during operations or associated with tumour disorders.
[0011] Treatment of sepsis consists firstly in the rigorous
elimination of the infectious cause, for example by operative focal
reconstruction and antibiosis. Secondly, it consists in temporary
intensive medical support of the affected organ systems. Treatments
of the different stages of this disease have been described, for
example, in the following publication (Dellinger et al., Crit. Care
Med. 2004, 32, 858-873). There are no proven effective treatments
for DIC.
[0012] It is therefore an object of the present invention to
provide novel PAR-1 antagonists for the treatment of disorders, for
example cardiovascular disorders and thromboembolic disorders, and
also tumour disorders in humans and animals.
[0013] WO 2006/012226, WO 2006/020598, WO 2007/038138, WO
2007/130898, WO 2007/101270 and US 2006/0004049 describe
structurally similar piperidines as 11-.beta. HSD1 inhibitors for
treatment of, inter alia, diabetes, thromboembolic disorders and
stroke.
[0014] The invention provides compounds of the formula
##STR00001##
in which [0015] R.sup.1 is trifluoromethyl, 1,1-difluoroethyl,
2,2-difluoroethyl, 2,2,2-trifluoroethyl, difluoromethoxy,
trifluoromethoxy or ethyl, [0016] R.sup.2 is 2-hydroxyeth-1-yl,
2-methoxyeth-1-yl, 2-ethoxyeth-1-yl, cyclopropyl or
1-methoxycycloprop-1-yl, [0017] R.sup.3 is a group of the
formula
[0017] ##STR00002## [0018] where [0019] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0020] Inventive compounds are the compounds of the formula (I) and
their salts, solvates and solvates of the salts; the compounds,
encompassed by formula (I), of the formulae mentioned below and
their salts, solvates and solvates of the salts, and the compounds
encompassed by the formula (I), cited below as working examples,
and their salts, solvates and solvates of the salts if the
compounds, encompassed by the formula (I), cited below are not
already salts, solvates and solvates of the salts.
[0021] Depending on their structure, the inventive compounds may
exist in stereoisomeric forms (enantiomers, diastereomers).
Accordingly, the invention encompasses the enantiomers or
diastereomers and their respective mixtures. From such mixtures of
enantiomers and/or diastereomers, it is possible to isolate the
stereoisomerically uniform constituents in a known manner.
[0022] If the inventive compounds can occur in tautomeric forms,
the present invention encompasses all tautomeric forms.
[0023] In the context of the present invention, preferred salts are
physiologically acceptable salts of the inventive compounds. Also
encompassed, however, are salts which for their part are not
suitable for pharmaceutical applications, but which can be used,
for example, for isolating or purifying the inventive
compounds.
[0024] Physiologically acceptable salts of the inventive compounds
include acid addition salts of mineral acids, carboxylic acids and
sulphonic acids, for example salts of hydrochloric acid,
hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic
acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic
acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic
acid, propionic acid, lactic acid, tartaric acid, malic acid,
citric acid, fumaric acid, maleic acid and benzoic acid.
[0025] Physiologically acceptable salts of the inventive compounds
also include salts of customary bases, such as, by way of example
and with preference, alkali metal salts (for example sodium salts
and potassium salts), alkaline earth metal salts (for example
calcium salts and magnesium salts) and ammonium salts, derived from
ammonia or organic amines having 1 to 16 carbon atoms, such as, by
way of example and with preference, ethylamine, diethylamine,
triethylamine, ethyldiisopropylamine, monoethanolamine,
diethanolamine, triethanolamine, dicyclohexylamine,
dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine,
arginine, lysine, ethylenediamine, N-methylpiperidine and
choline.
[0026] In the context of the invention, solvates refer to those
forms of the inventive compounds which, in solid or liquid state,
form a complex by coordination with solvent molecules. Hydrates are
a specific form of the solvates in which the coordination is with
water.
[0027] Moreover, the present invention also encompasses prodrugs of
the inventive compounds. The term "prodrugs" includes compounds
which for their part may be biologically active or inactive but
which, during the time they spend in the body, are converted to
inventive compounds (for example metabolically or
hydrolytically).
[0028] In the formula of the group which may be R.sup.3, the end
point of the line marked by * does not represent a carbon atom or a
CH.sub.2 group, but is part of the bond to the atom to which
R.sup.3 is attached.
[0029] Preference is given to compounds of the formula (I) in which
[0030] R.sup.1 is trifluoromethyl, 1,1-difluoroethyl,
2,2-difluoroethyl, 2,2,2-trifluoroethyl, difluoromethoxy,
trifluoromethoxy or ethyl, [0031] R.sup.2 is 2-methoxyeth-1-yl,
cyclopropyl or 1-methoxycycloprop-1-yl, [0032] R.sup.3 is a group
of the formula
[0032] ##STR00003## [0033] where [0034] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0035] Preference is also given to compounds of the formula (I) in
which [0036] R.sup.1 is trifluoromethyl, 2,2,2-trifluoroethyl,
trifluoromethoxy or ethyl, [0037] R.sup.2 is 2-methoxyeth-1-yl,
cyclopropyl or 1-methoxycycloprop-1-yl, [0038] R.sup.3 is a group
of the formula
[0038] ##STR00004## [0039] where [0040] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0041] Preference is also given to compounds of the formula (I) in
which [0042] R.sup.1 is trifluoromethyl, 2,2,2-trifluoroethyl or
trifluoromethoxy, [0043] R.sup.2 is cyclopropyl or
1-methoxycycloprop-1-yl, [0044] R.sup.3 is a group of the
formula
[0044] ##STR00005## [0045] where [0046] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0047] Preference is also given to compounds of the formula (I) in
which [0048] R.sup.1 is trifluoromethyl, trifluoromethoxy or ethyl,
[0049] R.sup.2 fur 2-hydroxyeth-1-yl, 2-methoxyeth-1-yl,
2-ethoxyeth-1-yl or cyclopropyl, [0050] R.sup.3 is a group of the
formula
[0050] ##STR00006## [0051] where [0052] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0053] Preference is also given to compounds of the formula (I) in
which [0054] R.sup.1 is trifluoromethyl or ethyl, [0055] R.sup.2 is
2-hydroxyeth-1-yl, 2-methoxyeth-1-yl or cyclopropyl, [0056] R.sup.3
is a group of the formula
[0056] ##STR00007## [0057] where [0058] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0059] Preference is also given to compounds of the formula (I) in
which [0060] R.sup.1 is trifluoromethyl or ethyl, [0061] R.sup.2 is
2-methoxyeth-1-yl, [0062] R.sup.3 is a group of the formula
[0062] ##STR00008## [0063] where * is the point of attachment to
the carbonyl group, and their salts, their solvates and the
solvates of their salts.
[0064] Preference is also given to compounds of the formula (I) in
which [0065] R.sup.1 is trifluoromethyl or ethyl, [0066] R.sup.2 is
2-methoxyeth-1-yl, [0067] R.sup.3 is a group of the formula
[0067] ##STR00009## [0068] where * is the point of attachment to
the carbonyl group, and their salts, their solvates and the
solvates of their salts.
[0069] Preference is given to compounds of the formula (I) in which
[0070] R.sup.1 is trifluoromethoxy, [0071] R.sup.2 is
2-methoxyeth-1-yl or cyclopropyl, [0072] R.sup.3 is a group of the
formula
[0072] ##STR00010## [0073] where [0074] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0075] Preference is given to compounds of the formula (I) in which
[0076] R.sup.1 is trifluoromethoxy, [0077] R.sup.2 is cyclopropyl,
[0078] R.sup.3 is a group of the formula
[0078] ##STR00011## [0079] where [0080] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0081] Preference is given to compounds of the formula (I) in which
[0082] R.sup.1 is trifluoromethoxy, [0083] R.sup.2 is
2-methoxyeth-1-yl or cyclopropyl, [0084] R.sup.3 is a group of the
formula
[0084] ##STR00012## [0085] where [0086] * is the point of
attachment to the carbonyl group, and their salts, their solvates
and the solvates of their salts.
[0087] Preference is also given to compounds of the formula (I) in
which the phenyl substituent and 1,2,4-oxadiazol-5-yl substituent,
which are bonded to the piperidine ring, are in cis positions to
one another.
[0088] Preference is also given to compounds of the formula (I) in
which the carbon atom to which the phenyl substituent is bonded has
S configuration and the carbon atom to which the
1,2,4-oxadiazol-5-yl substituent is bonded likewise has S
configuration.
[0089] Preference is also given to compounds of the formula (I) in
which R.sup.1 is trifluoromethyl.
[0090] Preference is also given to compounds of the formula (I) in
which R.sup.1 is trifluoromethoxy.
[0091] Preference is also given to compounds of the formula (I) in
which R.sup.1 is ethyl.
[0092] Preference is also given to compounds of the formula (I) in
which R.sup.2 is 2-methoxyeth-1-yl.
[0093] Preference is also given to compounds of the formula (I) in
which R.sup.2 is cyclopropyl.
[0094] Preference is also given to compounds of the formula (I) in
which R.sup.2 is 1-methoxycycloprop-1-yl.
[0095] Preference is also given to compounds of the formula (I) in
which
[0096] R.sup.3 is a group of the formula
##STR00013## [0097] where [0098] * is the point of attachment to
the carbonyl group. Preference is also given to compounds of the
formula (I) in which [0099] R.sup.3 is a group of the formula
[0099] ##STR00014## [0100] where [0101] * is the point of
attachment to the carbonyl group.
[0102] The individual radical definitions specified in the
respective combinations or preferred combinations of radicals are,
independently of the respective combinations of the radicals
specified, also replaced as desired by radical definitions of other
combinations.
[0103] Very particular preference is given to combinations of two
or more of the preferred ranges mentioned above.
[0104] The invention further provides a process for preparing the
compounds of the formula (I), or their salts, their solvates or the
solvates of their salts, wherein
[A] compounds of the formula
##STR00015##
in which R.sup.1 and R.sup.2 are each as defined above are reacted
with compounds of the formula
##STR00016##
in which R.sup.3 is as defined above and X.sup.1 is halogen,
preferably bromine or chlorine, or hydroxyl or 4-nitrophenoxy, or
[B] compounds of the formula (II) are reacted in the first stage
with 4-nitrophenyl chloroformate and in the second stage with
compounds of the formula
R.sup.3--H (IV)
in which R.sup.3 is as defined above, or [C] compounds of the
formula
##STR00017##
in which R.sup.1 and R.sup.3 are each as defined above are reacted
with compounds of the formula
##STR00018##
in which R.sup.2 is as defined above, or [D] compounds of the
formula
##STR00019##
in which R.sup.1 and R.sup.2 are each as defined above are reacted
with 0.8 to 1.1 equivalents of meta-chloroperbenzoic acid to give
compounds of the formula
##STR00020##
in which R.sup.1 and R.sup.2 are each as defined above or [E]
compounds of the formula (Ia) are reacted with 2.0 to 3.0
equivalents of meta-chloroperbenzoic acid to give compounds of the
formula
##STR00021##
in which R.sup.1 and R.sup.2 are each as defined above.
[0105] The compounds of the formulae (Ia), (Ib) and (Ic) are a
subset of the compounds of the formula (I).
[0106] When X.sup.1 is halogen, the reaction according to method
[A] is generally effected in inert solvents, optionally in the
presence of a base, preferably in a temperature range of
-30.degree. C. to 50.degree. C. at standard pressure.
[0107] Inert solvents are, for example, tetrahydrofuran, methylene
chloride, pyridine, dioxane or dimethylformamide, preference being
given to methylene chloride.
[0108] Bases are, for example, triethylamine, diisopropylethylamine
or N-methylmorpholine, preference being given to triethylamine or
diisopropylethylamine
[0109] When X.sup.1 is hydroxyl, the reaction according to method
[A] is generally effected in inert solvents, in the presence of a
dehydrating reagent, optionally in the presence of a base,
preferably in a temperature range of -30.degree. C. to 50.degree.
C. at standard pressure.
[0110] Inert solvents are, for example, halohydrocarbons such as
dichloromethane or trichloromethane, hydrocarbons such as benzene,
nitromethane, dioxane, dimethylformamide or acetonitrile. It is
equally possible to use mixtures of the solvents. Particular
preference is given to dichloromethane or dimethylformamide.
[0111] Suitable dehydrating reagents in this context are, for
example, carbodiimides, for example N,N'-diethyl-, N,N'-dipropyl-,
N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide,
N-(3-dimethylamino-isopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC), N-cyclohexylcarbodiimide-N'-propyloxymethylpolystyrene
(PS-carbodiimide), or carbonyl compounds such as
carbonyldiimidazole, or 1,2-oxazolium compounds such as
2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or
2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino
compounds such as 2-ethoxy-1-ethoxy-carbonyl-1,2-dihydroquinoline,
or propanephosphonic anhydride, or isobutyl chloroformate, or
bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride or
benzotriazolyloxytri(dimethylamino)phosphonium hexafluorophosphate,
or O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU),
2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU) or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), or 1-hydroxy-benzotriazole (HOBt), or
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP), or N-hydroxysuccinimide, or mixtures of
these, with bases.
[0112] Bases are, for example, alkali metal carbonates, for example
sodium carbonate or potassium carbonate, or sodium
hydrogencarbonate or potassium hydrogencarbonate, or organic bases
such as trialkylamines, for example triethylamine,
N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine or
diisopropylethylamine.
[0113] Preferably, the condensation is effected with HATU or with
EDC in the presence of HOBt.
[0114] When X.sup.1 is 4-nitrophenoxy, the reaction according to
method [A] is generally effected in inert solvents, optionally in
the presence of a base, optionally in a microwave, preferably in a
temperature range of 50.degree. C. to 200.degree. C. at standard
pressure to 5 bar.
[0115] Inert solvents are, for example, N-methylpyrrolidone,
dioxane or dimethylformamide, preference being given to
N-methylpyrrolidone.
[0116] Bases are, for example, triethylamine, diisopropylethylamine
or N-methylmorpholine, preference being given to triethylamine or
diisopropylethylamine
[0117] The compounds of the formula (III) are known or can be
synthesized by known processes from the appropriate starting
compounds.
[0118] The reaction of the first stage according to method [B] is
generally effected in inert solvents, in the presence of a base,
preferably in a temperature range of 0.degree. C. to 50.degree. C.
at standard pressure.
[0119] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane, carbon tetrachloride or
1,2-dichloroethane, preference being given to methylene
chloride.
[0120] Bases are, for example, organic bases such as
trialkylamines, for example triethylamine, N-methyl-morpholine,
N-methylpiperidine, 4-dimethylaminopyridine or
diisopropylethylamine, preference being given to triethylamine.
[0121] The reaction of the second stage according to method [B] is
generally effected in inert solvents, in the presence of a base,
optionally in a microwave, preferably in a temperature range of
50.degree. C. to 200.degree. C. at standard pressure to 5 bar.
[0122] Inert solvents are, for example, dimethyl sulphoxide,
dimethylformamide or N-methylpyrrolidone, preference being given to
dimethylformamide.
[0123] Bases are, for example, alkali metal carbonates, for example
sodium carbonate or potassium carbonate, preference being given to
potassium carbonate.
[0124] The compounds of the formula (IV) are known or can be
synthesized by known processes from the appropriate starting
compounds.
[0125] The reaction according to method [C] is generally effected
in inert solvents, in the presence of a dehydrating reagent,
optionally in the presence of a base, preferably in a temperature
range from room temperature up to reflux of the solvents at
standard pressure.
[0126] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane or 1,2-dichloroethane, ethers
such as dioxane, tetrahydrofuran or 1,2-dimethoxyethane, or other
solvents such as acetone, dimethylformamide, dimethylacetamide,
2-butanone or acetonitrile. It is equally possible to use mixtures
of the solvents. Preference is given to dimethylformamide or a
mixture of dioxane and dimethylformamide.
[0127] Suitable dehydrating reagents in this context are, for
example, carbodiimides, for example N,N'-diethyl-, N,N'-dipropyl-,
N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide,
N-(3-dimethylamino-isopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC), N-cyclohexylcarbodiimide-N'-propyloxymethylpolystyrene
(PS-carbodiimide), or carbonyl compounds such as
carbonyldiimida-zole, or 1,2-oxazolium compounds such as
2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or
2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino
compounds such as 2-ethoxy-1-ethoxy-carbonyl-1,2-dihydroquinoline,
or propanephosphonic anhydride, or isobutyl chloroformate, or
bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride or
benzotriazolyloxytri(dimethylamino)phosphonium hexafluorophosphate,
or O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU),
2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU) or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), or 1-hydroxy-benzotriazole (HOBt), or
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP), or
benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate (PYBOP), or N-hydroxysuccinimide, or mixtures
of these with bases.
[0128] Bases are, for example, alkali metal carbonates, for example
sodium carbonate or potassium carbonate, or sodium
hydrogencarbonate or potassium hydrogencarbonate, or organic bases
such as trialkylamines, for example triethylamine,
N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine or
diisopropylethylamine, preference being given to
diisopropylethylamine.
[0129] Preferably, the condensation is carried out with HATU in the
presence of diisopropylethylamine or alternatively only with
carbonyldiimidazole.
[0130] The compounds of the formula (VI) are known or can be
synthesized by known processes from the appropriate starting
compounds.
[0131] The reaction according to method [D] is generally performed
in inert solvents, preferably in a temperature range from room
temperature up to reflux of the solvents at standard pressure.
[0132] meta-Chloroperbenzoic acid is preferably used in an amount
of 0.9 to 1.0 equivalent.
[0133] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane or 1,2-dichloroethane.
Preference is given to methylene chloride.
[0134] The reaction according to method [E] is generally effected
in inert solvents, preferably in a temperature range from room
temperature up to reflux of the solvents at standard pressure.
[0135] meta-Chloroperbenzoic acid is preferably used in an amount
of 2.3 to 2.6 equivalents, more preferably in an amount of 2.5
equivalents.
[0136] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane or 1,2-dichloroethane.
Preference is given to methylene chloride.
[0137] The compounds of the formula (II) are known or can be
prepared by reacting compounds of the formula
##STR00022##
in which R.sup.1 is as defined above in the first stage with
compounds of the formula (VI) and in the second stage with an
acid.
[0138] The first stage reaction is effected as described for
process [C]
[0139] The second stage reaction is generally effected in inert
solvents, preferably in a temperature range from room temperature
to 60.degree. C. at standard pressure.
[0140] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane, carbon tetrachloride or
1,2-dichloroethane, or ethers such as tetrahydrofuran or dioxane,
preference being given to methylene chloride.
[0141] Bases are, for example, trifluoroacetic acid or hydrogen
chloride in dioxane, preference being given to trifluoroacetic
acid.
[0142] The compounds of the formula (VII) are known or can be
prepared by reacting compounds of the formula
##STR00023##
in which R.sup.1 is as defined above and R.sup.4 is methyl or
ethyl, in the first stage with di-tert-butyl dicarboxylate and in
the second stage with a base.
[0143] The first stage reaction is generally effected in inert
solvents, in the presence of a base, preferably in a temperature
range from room temperature to 50.degree. C. at standard
pressure.
[0144] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane, carbon tetrachloride or
1,2-dichloroethane, preference being given to methylene
chloride.
[0145] Bases are, for example, triethylamine, diisopropylethylamine
or N-methylmorpholine, preference being given to triethylamine or
diisopropylethylamine
[0146] The second stage reaction is generally effected in inert
solvents, in the presence of a base, preferably in a temperature
range from room temperature up to reflux of the solvents at
standard pressure.
[0147] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane, tetrachloromethane or
1,2-dichloroethane, alcohols such as methanol or ethanol, ethers
such as diethyl ether, methyl tert-butyl ether,
1,2-dimethoxyethane, dioxane or tetrahydrofuran, or other solvents
such as dimethylformamide, dimethylacetamide, acetonitrile or
pyridine, or mixtures of solvents, or mixtures of solvent with
water, preference being given to methanol or methanol with one
equivalent of water, or a mixture of tetrahydrofuran and water.
[0148] Bases are, for example, alkali metal hydroxides such as
sodium, lithium or potassium hydroxide, or alkali metal carbonates
such as caesium carbonate, sodium or potassium carbonate, or
alkoxides such as potassium or sodium tert-butoxide, preference
being given to lithium hydroxide or potassium tert-butoxide.
[0149] The compounds of the formula (VIII) are known or can be
prepared by hydrogenating compounds of the formula
##STR00024##
in which R.sup.1 and R.sup.4 are each as defined above.
[0150] The hydrogenation is generally effected with a reducing
agent in inert solvents, optionally with addition of acid such as
mineral acids and carboxylic acids, preferably acetic acid,
preferably in a temperature range from room temperature up to
reflux of the solvents and in a pressure range from standard
pressure to 100 bar, preferably at standard pressure or at 50-80
bar.
[0151] A preferred reducing agent is hydrogen with palladium on
activated carbon, with rhodium on activated carbon, with ruthenium
on activated carbon or mixed catalysts thereof, or hydrogen with
palladium on alumina or with rhodium on alumina, or hydrogen with
palladium on activated carbon and platinum(IV) oxide, preference
being given to hydrogen with palladium on activated carbon or with
rhodium on activated carbon or hydrogen with palladium on activated
carbon and platinum(IV) oxide. It is also possible to hydrogenate
under pressure with hydrogen and platinum(IV) oxide alone.
[0152] Inert solvents are, for example, alcohols such as methanol,
ethanol, n-propanol, isopropanol, n-butanol or tert-butanol, or
concentrated acetic acid or methanol with addition of concentrated
hydrochloric acid, preference being given to methanol or ethanol or
concentrated acetic acid or methanol with addition of concentrated
hydrochloric acid.
[0153] The compounds of the formula (IX) are known or can be
prepared by reacting compounds of the formula
##STR00025##
in which R.sup.4 is as defined above with compounds of the
formula
##STR00026##
in which R.sup.1 is as defined above.
[0154] The reaction is generally effected in inert solvents, in the
presence of a catalyst, if appropriate in the presence of an
additional reagent, preferably in a temperature range from room
temperature up to reflux of the solvent at standard pressure.
[0155] Inert solvents are, for example, ethers such as dioxane,
tetrahydrofuran or 1,2-dimethoxyethane, hydrocarbons such as
benzene, xylene or toluene, or other solvents such as nitrobenzene,
dimethylformamide, dimethylacetamide, dimethyl sulphoxide or
N-methylpyrrolidone; if appropriate, some water is added to these
solvents. Preference is given to toluene with water or to a mixture
of 1,2-dimethoxyethane, dimethylformamide and water.
[0156] Catalysts are, for example, palladium catalysts customary
for Suzuki reaction conditions, preference being given to catalysts
such as dichlorobis(triphenylphosphine)palladium,
tetrakistriphenylphosphinepalladium(0), palladium(II) acetate or
bis(diphenylphosphine-ferrocenyl)palladium(II) chloride, for
example.
[0157] Additional reagents are, for example, potassium acetate,
caesium, potassium or sodium carbonate, barium hydroxide, potassium
tert-butoxide, caesium fluoride, potassium fluoride or potassium
phosphate, or mixtures thereof, preference being given to potassium
fluoride or sodium carbonate, or a mixture of potassium fluoride
and potassium carbonate.
[0158] The compounds of the formulae (X), (XI) and (XIII) are known
or can be synthesized by known processes from the appropriate
starting compounds.
[0159] The compounds of the formula (V) are known or can be
prepared by reacting compounds of the formula
##STR00027##
in which R.sup.1 and R.sup.3 are each as defined above and R.sup.4
is methyl or ethyl, with a base.
[0160] The reaction is generally effected in inert solvents, in the
presence of a base, preferably in a temperature range from room
temperature up to reflux of the solvents at standard pressure.
[0161] Inert solvents are, for example, halohydrocarbons such as
methylene chloride, trichloromethane, tetrachloromethane or
1,2-dichloroethane, alcohols such as methanol or ethanol, ethers
such as diethyl ether, methyl tert-butyl ether,
1,2-dimethoxyethane, dioxane or tetrahydrofuran, or other solvents
such as dimethylformamide, dimethylacetamide, acetonitrile or
pyridine, or mixtures of solvents, or mixtures of solvent with
water, preference being given to methanol or methanol with one
equivalent of water, or a mixture of tetrahydrofuran and water.
[0162] Bases are, for example, alkali metal hydroxides such as
sodium, lithium or potassium hydroxide, or alkali metal carbonates
such as caesium carbonate, sodium or potassium carbonate, or
alkoxides such as potassium or sodium tert-butoxide, preference
being given to lithium hydroxide or potassium tert-butoxide.
[0163] The compounds of the formula (XII) are known or can be
prepared by reacting compounds of the formula (VIII) with compounds
of the formula (III).
[0164] The reaction is effected as described for method [A].
[0165] In an alternative method, the compounds of the formula (XII)
can be prepared by reacting compounds of the formula (VIII) in the
first stage with 4-nitrophenyl chloroformate and in the second
stage with compounds of the formula (IV).
[0166] The reaction is effected as described for method [B].
[0167] The preparation of the compounds of the formula (I) can be
illustrated by the synthesis scheme below.
##STR00028##
[0168] The inventive compounds have an unforeseeable, valuable
spectrum of pharmacological and pharmacokinetic activity. They are
selective antagonists of the PAR-1 receptor acting in particular as
platelet aggregation inhibitors, as inhibitors of endothelial cell
activation, as inhibitors of smooth muscle cell proliferation and
as inhibitors of tumour growth. For some of the diseases mentioned,
for example cardiovascular diseases with high thromboembolic risk,
permanent protection by PAR-1 antagonism with simultaneously simple
management of medication is of great significance. The PAR-1
antagonists of the present invention exhibit long-lasting action
after single oral administration, i.e. an action which lasts at
least 16 hours.
[0169] Accordingly, they are suitable for use as medicaments for
treatment and/or prophylaxis of diseases in man and animals.
[0170] The present invention further provides for the use of the
inventive compounds for treatment and/or prophylaxis of disorders,
preferably of thromboembolic disorders and/or thromboembolic
complications.
[0171] "Thromboembolic disorders" in the context of the present
invention include in particular disorders such as ST-segment
elevation myocardial infarction (STEMI) and non-ST-segment
elevation myocardial infarction (non-STEMI), stable angina
pectoris, unstable angina pectoris, reocclusions and restenoses
after coronary interventions such as angioplasty, stent
implantations or aortocoronary bypass, peripheral arterial
occlusion diseases, pulmonary embolisms, deep venous thromboses and
renal vein thromboses, transitory ischaemic attacks and also
thrombotic and thromboembolic stroke.
[0172] The substances are therefore also suitable for prevention
and treatment of cardiogenic thrombo-embolisms, for example brain
ischaemias, stroke and systemic thromboembolisms and ischaemias, in
patients with acute, intermittent or persistent cardiac
arrhythmias, for example atrial fibrillation, and those undergoing
cardioversion, and also in patients with heart valve disorders or
with intravasal objects, for example artificial heart valves,
catheters, intraaortic balloon counterpulsation and pacemaker
probes.
[0173] Thromboembolic complications are also encountered in
connection with microangiopathic haemolytic anaemias,
extracorporeal circulation, for example haemodialysis,
haemofiltration, ventricular assist devices and artificial hearts,
and also heart valve prostheses.
[0174] Moreover, the inventive compounds are also used for
influencing wound healing, for the prophylaxis and/or treatment of
atherosclerotic vascular disorders and inflammatory disorders such
as rheumatic disorders of the locomotive system, coronary heart
diseases, of heart failure, of hypertension, of inflammatory
disorders, for example asthma, COPD, inflammatory pulmonary
disorders, glomerulonephritis and inflammatory intestinal
disorders, and additionally also for the prophylaxis and/or
treatment of Alzheimer's disease, autoimmune disorders, Crohn's
disease and ulcerative colitis.
[0175] Moreover, the inventive compounds can be used for inhibiting
tumour growth and the formation of metastases, for
microangiopathies, age-related macular degeneration, diabetic
retinopathy, diabetic nephropathy and other microvascular
disorders, and also for the prevention and treatment of
thromboembolic complications, for example venous thromboembolisms,
for tumour patients, in particular those undergoing major surgical
interventions or chemo- or radiotherapy.
[0176] The inventive compounds are additionally suitable for
treatment of cancer. Cancers include: carcinomas (including breast
cancer, hepatocellular carcinomas, lung cancer, colorectal cancer,
cancer of the colon and melanomas), lymphomas (for example
non-Hodgkin's lymphomas and mycosis fungoides), leukaemias,
sarcomas, mesotheliomas, brain cancer (for example gliomas),
germinomas (for example testicular cancer and ovarian cancer),
choriocarcinomas, renal cancer, cancer of the pancreas, thyroid
cancer, head and neck cancer, endometrial cancer, cervical cancer,
bladder cancer, stomach cancer and multiple myeloma.
[0177] Moreover, PAR-1 expressed on endothelial cells mediates
signals resulting in vascular growth ("angiogenesis"), a process
which is vital for enabling tumour growth beyond about 1
mm.sup.3.
[0178] Induction of angiogenesis is also relevant for other
disorders; these include disorders of the rheumatic type (for
example rheumatoid arthritis), pulmonary disorders (for example
pulmonary fibrosis, pulmonary hypertension, in particular pulmonary
arterial hypertension, disorders characterized by pulmonary
occlusion), arteriosclerosis, plaque rupture, diabetic retinopathy
and wet macular degeneration.
[0179] In addition, the inventive compounds are suitable for
treatment of sepsis. Sepsis (or septicaemia) is a common disorder
with high mortality. Initial symptoms of sepsis are typically
unspecific (for example fever, reduced general state of health);
however, during further progression there may be a general
activation of the coagulation system ("disseminated intravascular
coagulation" or "consumption coagulopathy"; referred to hereinafter
as "DIC") with the formation of microthrombi in various organs and
secondary bleeding complications. Moreover, there may be
endothelial damage with increased permeability of the vessels and
diffusion of fluid and proteins into the extravasal space. Later,
there may be organ dysfunction or organ failure (for example kidney
failure, liver failure, respiratory failure, deficits of the
central nervous system and heart/circulatory failure) and even
multiple organ failure. In principle, this may affect any organ;
the most frequently encountered organ dysfunctions and organ
failures are those of the lung, the kidney, the cardiovascular
system, the coagulation system, the central nervous system, the
endocrine glands and the liver. Sepsis may be associated with an
"acute respiratory distress syndrome" (referred to hereinafter as
ARDS). ARDS may also occur independently of sepsis. "Septic shock"
refers to the occurrence of hypotension, which requires treatment,
and promotes further organ damage and is associated with a
worsening of the prognosis.
[0180] Pathogens may be bacteria (gram-negative and gram-positive),
fungi, viruses and/or eukaryotes. The site of entry or primary
infection may be pneumonia, an infection of the urinary tract or
peritonitis, for example. The infection may, but need not
necessarily, be associated with bacteriaemia.
[0181] Sepsis is defined as the presence of an infection and a
"systemic inflammatory response syndrome" (referred to hereinafter
as "SIRS"). SIRS occurs during infections, but also during other
states such as injuries, burns, shock, operations, ischaemia,
pancreatitis, reanimation or tumours. The definition of the
ACCP/SCCM Consensus Conference Committee of 1992 (Crit. Care Med.
1992, 20, 864-874) describes the symptoms required for the
diagnosis "SIRS" and measurement parameters (inter alia a change in
body temperature, increased heart rate, breathing difficulties and
altered blood profile). The later (2001) SCCM/ESICM/ACCP/ATS/SIS
International Sepsis Definitions Conference essentially maintained
the criteria, but fine-tuned details (Levy et al., Crit. Care Med.
2003, 31, 1250-1256).
[0182] DIC and SIRS may occur during sepsis, but also as a result
of operations, tumour disorders, burns or other injuries. In the
case of DIC, there is a massive activation of the coagulation
system at the surface of damaged endothelial cells, the surfaces of
foreign bodies or injured extravascular tissue. As a consequence,
there is coagulation in small vessels of various organs with
hypoxia and subsequent organ dysfunction. A secondary effect is the
consumption of coagulation factors (for example factor X,
prothrombin, fibrinogen) and platelets, which reduces the
coagulability of the blood and may result in heavy bleeding.
[0183] In addition, the inventive compounds can also be used for
preventing coagulation ex vivo, for example for preserving blood
and plasma products, for cleaning/pretreating catheters and other
medical auxiliaries and instruments, including extracorporeal
circulation, for coating synthetic surfaces of medical auxiliaries
and instruments used in vivo or ex vivo or for platelet-containing
biological samples.
[0184] The present invention further provides for the use of the
inventive compounds for coating medical instruments and implants,
for example catheters, prostheses, stents or artificial heart
valves. In this case, the inventive compounds can be firmly
attached to the surface or, for local action, be released over a
certain period of time from a carrier coating into the immediate
environment.
[0185] The present invention further provides for the use of the
inventive compounds for treatment and/or prophylaxis of disorders,
in particular the disorders mentioned above.
[0186] The present invention further provides for the use of the
inventive compounds for producing a medicament for treatment and/or
prophylaxis of disorders, in particular the disorders mentioned
above.
[0187] The present invention further provides a method for
treatment and/or prophylaxis of disorders, in particular the
disorders mentioned above, using a therapeutically effective amount
of an inventive compound.
[0188] The present invention further provides medicaments
comprising an inventive compound and one or more further active
ingredients, in particular for treatment and/or prophylaxis of the
disorders mentioned above. Active ingredients suitable for
combinations are, by way of example and with preference:
calcium channel blockers, for example amlodipine besilate (for
example Norvasc.RTM.), felodipine, diltiazem, verapamil,
nifedipine, nicardipine, nisoldipine and bepridil; iomerizine;
statins, for example atorvastatin, fluvastatin, lovastatin,
pitavastatin, pravastatin, rosuvastatin and simvastatin;
cholesterol resorption inhibitors, for example ezetimibe and
AZD4121; cholesteryl ester transfer protein ("CETP") inhibitors,
for example torcetrapib; low molecular weight heparins, for example
dalteparin sodium, ardeparin, certoparin, enoxaparin, parnaparin,
tinzaparin, reviparin and nadroparin; further anticoagulants, for
example warfarin, marcumar, fondaparinux; antiarrhythmics, for
example dofetilide, ibutilide, metoprolol, metoprolol tartrate,
propranolol, atenolol, ajmaline, disopyramide, prajmaline,
procainamide, quinidine, sparteine, aprindine, lidocaine,
mexiletine, tocamide, encamide, flecamide, lorcamide, moricizine,
propafenone, acebutolol, pindolol, amiodarone, bretylium tosylate,
bunaftine, sotalol, adenosine, atropine and digoxin;
alpha-adrenergic agonists, for example doxazosin mesylate,
terazoson and prazosin; beta-adrenergic blockers, for example
carvedilol, propranolol, timolol, nadolol, atenolol, metoprolol,
bisoprolol, nebivolol, betaxolol, acebutolol and bisoprolol;
aldosterone antagonists, for example eplerenone and spironolactone;
angiotensin-converting enzyme inhibitors ("ACE inhibitors"), for
example moexipril, quinapril hydrochloride, ramipril, lisinopril,
benazepril hydrochloride, enalapril, captopril, spirapril,
perindopril, fosinopril and trandolapril; angiotensin II receptor
blockers ("ARBs"), for example olmesartan-medoxomil, candesartan,
valsartan, telmisartan, irbesartan, losartan and eprosartan;
endothelin antagonists, for example tezosentan, bosentan and
sitaxsentan-sodium; inhibitors of neutral endopeptidase, for
example candoxatril and ecadotril; phosphodiesterase inhibitors,
for example milrinone, theophylline, vinpocetine, EHNA
(erythro-9-(2-hydroxy-3-nonyl)adenine), sildenafil, vardenafil and
tadalafil; fibrinolytics, for example reteplase, alteplase and
tenecteplase; GP IIb/IIIa antagonists, for example integrillin,
abciximab and tirofiban; direct thrombin inhibitors, for example
AZD0837, argatroban, bivalirudin and dabigatran; indirect thrombin
inhibitors, for example odiparcil; direct and indirect factor Xa
inhibitors, for example fondaparinux-sodium, apixaban, razaxaban,
rivaroxaban (BAY 59-7939), KFA-1982, DX-9065a, AVE3247, otamixaban
(XRP0673), AVE6324, SAR377142, idraparinux, SSR126517, DB-772d,
DT-831j, YM-150, 813893, LY517717 and DU-1766; direct and indirect
factor Xa/IIa inhibitors, for example enoxaparin-sodium, AVE5026,
SSR128428, SSR128429 and BIBT-986 (Tanogitran);
lipoprotein-associated phospholipase A2 ("LpPLA2") modulators;
diuretics, for example chlorthalidone, ethacrynic acid, furosemide,
amiloride, chlorothiazide, hydrochlorothiazide, methylclothiazide
and benzthiazide; nitrates, for example isosorbide 5-mononitrate;
thromboxane antagonists, for example seratrodast, picotamide and
ramatroban; platelet aggregation inhibitors, for example
clopidogrel, tiklopidin, cilostazol, aspirin, abciximab, limaprost,
eptifibatide and CT-50547; cyclooxygenase inhibitors, for example
meloxicam, rofecoxib and celecoxib; B-type natriuretic peptides,
for example nesiritide and ularitide; NV1FGF modulators, for
example XRP0038; HT1B/5-HT2A antagonists, for example SL65.0472;
guanylate cyclase activators, for example ataciguat (HMR1766),
HMR1069, riociguat and cinaciguat; e-NOS transcription enhancers,
for example AVE9488 and AVE3085; antiatherogenic substances, for
example AGI-1067: CPU inhibitors, for example AZD9684; renin
inhibitors, for example aliskirin and VNP489; inhibitors of
adenosine diphosphate-induced platelet aggregation, for example
clopidogrel, ticlopidine, prasugrel, AZD6140, ticagrelor and
elinogrel; NHE-1 inhibitors, for example AVE4454 and AVE4890.
[0189] Antibiotic therapy: various antibiotics or antifungal
medicament combinations are suitable, either as calculated therapy
(before a microbial assessment has been made) or as specific
therapy; fluid therapy, for example crystalloid or colloidal
fluids; vasopressors, for example norepinephrine, dopamine or
vasopressin; inotropic therapy, for example dobutamine;
corticosteroids, for example hydrocortisone, or fludrocortisone;
recombinant human activated protein C, Xigris; blood products, for
example erythrocyte concentrates, platelet concentrates,
erythropoietin or fresh frozen plasma; assisted ventilation in
sepsis-induced acute lung injury (ALI) or acute respiratory
distress syndrome (ARDS), for example permissive hypercapnia, low
tidal volumes; sedation: for example diazepam, lorazepam, midazolam
or propofol. Opioids: for example fentanyl, hydromorphone,
morphine, meperidine or remifentanil. NSAIDs: for example
ketorolac, ibuprofen or acetaminophen. Neuromuscular blockade: for
example pancuronium; glucose control, for example insulin, glucose;
renal replacement therapies, for example continuous veno-venous
haemofiltration or intermittent haemodialysis. Low-dose dopamine
for renal protection; anticoagulants, for example for thrombosis
prophylaxis or for renal replacement therapies, for example
unfractionated heparins, low molecular weight heparins,
heparinoids, hirudin, bivalirudin or argatroban; bicarbonate
therapy; stress ulcer prophylaxis, for example H2 receptor
inhibitors, antacids.
[0190] Medicaments for proliferative disorders: uracil,
chlormethine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil, pipobroman, triethylenemelamine,
triethylenethiophosphoramine, busulfan, carmustine, lomustine,
streptozocin, dacarbazine, methotrexate, 5-fluorouracil,
floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine,
fludarabine phosphate, pentostatin, vinblastine, vincristine,
vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin,
epirubicin, idarubicin, paclitaxel, mithramycin, deoxycoformycin,
mitomycin-C, L-asparaginase, interferons, etoposide, teniposide,
17.alpha.-ethynylestradiol, diethylstilbestrol, testosterone,
prednisone, fluoxymesterone, dromostanolone propionate,
testolactone, megestrol acetate, tamoxifen, methylprednisolone,
methyltestosterone, prednisolone, triamcinolone, chlorotrianisene,
hydroxyprogesterone, aminoglutethimide, estranrustine,
medroxyprogesterone acetate, leuprolide, flutamide, toremifene,
goserelin, cisplatin, carboplatin, hydroxyurea, amsacrine,
procarbazine, mitotane, mitoxantrone, levamisole, navelbene,
anastrazole, letrazole, capecitabine, reloxafine, droloxafine,
hexamethylmelamine, oxaliplatin (Eloxatin), Iressa (gefmitib,
Zd1839), XELODA.RTM. (capecitabine), Tarceva.RTM. (erlotinib),
Azacitidine (5-azacytidine; 5-AzaC), temozolomide (Temodar.RTM.),
gemcitabine (e.g. GEMZAR.RTM. (gemcitabine HCl)), vasostatin or a
combination of two or more of the above.
[0191] The present invention further provides a method for
preventing the coagulation of blood in vitro, in particular in
banked blood or biological samples containing platelets, which is
characterized in that an anticoagulatory amount of the inventive
compound is added.
[0192] The inventive compounds can act systemically and/or locally.
For this purpose, they can be administered in a suitable way, for
example by the oral, parenteral, pulmonal, nasal, sublingual,
lingual, buccal, rectal, dermal, transdermal, conjunctival, otic
route, or as implant or stent.
[0193] The inventive compounds can be administered in
administration forms suitable for these administration routes.
[0194] Suitable administration forms for oral administration are
those which function according to the prior art and deliver the
inventive compounds rapidly and/or in modified fashion, and which
contain the inventive compounds in crystalline and/or amorphized
and/or dissolved form, for example tablets (uncoated or coated
tablets, for example having enteric coatings or coatings which are
insoluble or dissolve with a delay and control the release of the
inventive compound), tablets which disintegrate rapidly in the
mouth, or films/wafers, films/lyophilizates, capsules (for example
hard or soft gelatin capsules), sugar-coated tablets, granules,
pellets, powders, emulsions, suspensions, aerosols or
solutions.
[0195] Parenteral administration can take place with avoidance of
an absorption step (e.g. intravenous, intraarterial, intracardiac,
intraspinal or intralumbar) or with inclusion of an absorption
(e.g. intramuscular, subcutaneous, intracutaneous, percutaneous or
intraperitoneal). Administration forms suitable for parenteral
administration include preparations for injection and infusion in
the form of solutions, suspensions, emulsions, lyophilizates or
sterile powders.
[0196] Oral administration is preferred.
[0197] Suitable for the other administration routes are, for
example, pharmaceutical forms for inhalation (inter alia powder
inhalers, nebulizers), nasal drops, solutions or sprays; tablets
for lingual, sublingual or buccal administration, films/wafers or
capsules, suppositories, preparations for the ears or eyes, vaginal
capsules, aqueous suspensions (lotions, shaking mixtures),
lipophilic suspensions, ointments, creams, transdermal therapeutic
systems (e.g. patches), milk, pastes, foams, dusting powders,
implants or stents.
[0198] The inventive compounds can be converted to the
administration forms mentioned. This can be done in a manner known
per se by mixing with inert, nontoxic, pharmaceutically suitable
excipients. These excipients include carriers (for example
microcrystalline cellulose, lactose, mannitol), solvents (e.g.
liquid polyethylene glycols), emulsifiers and dispersants or
wetting agents (for example sodium dodecylsulphate, polyoxysorbitan
oleate), binders (for example polyvinylpyrrolidone), synthetic and
natural polymers (for example albumin), stabilizers (e.g.
antioxidants for example ascorbic acid), colours (e.g. inorganic
pigments for example iron oxides) and masking flavours and/or
odours.
[0199] The present invention further provides medicaments
comprising at least one inventive compound, preferably together
with one or more inert nontoxic pharmaceutically suitable
auxiliaries, and their use for the purposes mentioned above.
[0200] In the case of parenteral administration, it has generally
been found to be advantageous to administer amounts of about 5 to
250 mg every 24 hours to achieve effective results. In the case of
oral administration the amount is about 5 to 100 mg every 24
hours.
[0201] It may nevertheless be necessary where appropriate to
deviate from the stated amounts, in particular as a function of the
body weight, route of administration, individual response to the
active ingredient, nature of the preparation and time or interval
over which administration takes place.
[0202] The percentages in the tests and examples which follow are,
unless indicated otherwise, percentages by weight; parts are parts
by weight. Solvent ratios, dilution ratios and concentration data
for the liquid/liquid solutions are each based on volume. "w/v"
means "weight/volume". For example, "10% w/v" means: 100 ml of
solution or suspension comprise 10 g of substance.
A) EXAMPLES
Abbreviations
[0203] approx. approximately [0204] CDI carbonyldiimidazole [0205]
d day(s), doublet (in NMR) [0206] TLC thin-layer chromatography
[0207] DCI direct chemical ionization (in MS) [0208] dd double
doublet (in NMR) [0209] DMAP 4-dimethylaminopyridine [0210] DMF
N,N-dimethylformamide [0211] DMSO dimethyl sulphoxide [0212] DPPA
diphenyl phosphorazidate [0213] DSC disuccinimidyl carbonate [0214]
eq. equivalent(s) [0215] ESI electrospray ionization (in MS) [0216]
h hour(s) [0217] HATU
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0218] HPLC high-pressure, high-performance
liquid chromatography [0219] LC-MS liquid chromatography-coupled
mass spectroscopy [0220] LDA lithium diisopropylamide [0221] m
multiplet (in NMR) [0222] min minute(s) [0223] MS mass spectroscopy
[0224] NMR nuclear magnetic resonance spectroscopy [0225] PYBOP
benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate [0226] q quartet (in NMR) [0227] RP reversed
phase (in HPLC) [0228] RT room temperature [0229] R.sub.t retention
time (in HPLC) [0230] singlet (in NMR) [0231] t triplet (in NMR)
[0232] THF tetrahydrofuran
HPLC Methods:
[0233] Method 1A:
[0234] Instrument: HP 1100 with DAD detection; column: Kromasil 100
RP-18, 60 mm.times.2.1 mm, 3.5 .mu.m; eluent A: 5 ml of perchloric
acid (70%)/1 of water, eluent B: acetonitrile; gradient: 0 min 2%
B.fwdarw.0.5 min 2% B.fwdarw.4.5 min 90% B.fwdarw.6.5 min 90%
B.fwdarw.6.7 min 2% B.fwdarw.7.5 min 2% B; flow rate: 0.75 ml/min;
column temperature: 30.degree. C.; UV detection: 210 nm.
LC-MS Methods:
[0235] Method 1B:
[0236] MS instrument type: Micromass ZQ; HPLC instrument type: HP
1100 Series; UV DAD; column: Phenomenex Gemini 3.mu., 30
mm.times.3.0 mm; eluent A: 1 l of water+0.5 ml of 50% formic acid,
eluent B: 1 l of acetonitrile+0.5 ml of 50% formic acid; gradient:
0.0 min 90% A.fwdarw.2.5 min 30% A.fwdarw.3.0 min 5% A.fwdarw.4.5
min 5% A; flow rate: 0.0 min 1 ml/min, 2.5 min/3.0 min/4.5 min 2
ml/min; oven: 50.degree. C.; UV detection: 210 nm.
[0237] Method 2B:
[0238] Instrument: Micromass QuattroPremier with Waters UPLC
Acquity; column: Thermo Hypersil GOLD 1.9.mu., 50 mm.times.1 mm;
eluent A: 1 l of water+0.5 ml of 50% formic acid, eluent B: 1 l of
acetonitrile+0.5 ml of 50% formic acid; gradient: 0.0 min 90%
A.fwdarw.0.1 min 90% A.fwdarw.1.5 min 10% A.fwdarw.2.2 min 10% A;
oven: 50.degree. C.; flow rate: 0.33 ml/min; UV detection: 210
nm.
[0239] Method 3B:
[0240] MS instrument type: Micromass ZQ; HPLC instrument type:
Waters Alliance 2795; column: Phenomenex Synergi 2.5.mu. MAX-RP
100A Mercury, 20 mm.times.4 mm; eluent A: 1 l of water+0.5 ml of
50% formic acid, eluent B: 1 l of acetonitrile+0.5 ml of 50% formic
acid; gradient: 0.0 min 90% A.fwdarw.0.1 min 90% A.fwdarw.3.0 min
5% A.fwdarw.4.0 min 5% A.fwdarw.4.01 min 90% A; flow rate: 2
ml/min; oven: 50.degree. C.; UV detection: 210 nm.
[0241] Method 4B:
[0242] Instrument: Micromass Quattro Micro MS with HPLC Agilent
series 1100; column: Thermo Hypersil GOLD 3.mu. 20 mm.times.4 mm;
eluent A: 1 l of water+0.5 ml of 50% formic acid, eluent B: 1 l of
acetonitrile+0.5 ml of 50% formic acid; gradient: 0.0 min 100%
A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A.fwdarw.4.01 min 100%
A.fwdarw.5.00 min 100% A; oven: 50.degree. C.; flow rate: 2 ml/min;
UV detection: 210 nm.
[0243] Method 5B:
[0244] Instrument: Waters ACQUITY SQD UPLC System; column: Waters
Acquity UPLC HSS T3 1.8.mu. 50 mm.times.1 mm; eluent A: 1 l of
water+0.25 ml of 99% formic acid, eluent B: 1 l of
acetonitrile+0.25 ml of 99% formic acid; gradient: 0.0 min 90%
A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; oven: 50.degree. C.;
flow rate: 0.40 ml/min; UV detection: 210-400 nm.
[0245] Method 6B:
[0246] MS instrument type: Waters (Micromass) Quattro Micro; HPLC
instrument type: Agilent 1100 series; column: Thermo Hypersil GOLD
3.mu. 20 mm.times.4 mm; eluent A: 1 l of water+0.5 ml of 50% formic
acid, eluent B: 1 l of acetonitrile+0.5 ml of 50% formic acid;
gradient: 0.0 min 100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10%
A.fwdarw.4.01 min 100% A; (flow rate: 2.5 ml/min).fwdarw.5.00 min
100% A; oven: 50.degree. C.; flow rate: 2 ml/min; UV detection: 210
nm.
[0247] Method 7B:
[0248] MS instrument type: Waters ZQ; HPLC instrument type: Agilent
1100 Series; UV DAD; column: Thermo Hypersil GOLD 3.mu. 20
mm.times.4 mm; eluent A: 1 l of water+0.5 ml of 50% formic acid,
eluent B: 1 l of acetonitrile+0.5 ml of 50% formic acid; gradient:
0.0 min 100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A oven:
55.degree. C.; flow rate: 2 ml/min; UV detection: 210 nm.
Preparative Separation of Enantiomers:
[0249] Method 1D:
[0250] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.20 mm,
eluent: isohexane/isopropanol 25:75; flow rate: 15 ml/min,
temperature: 45.degree. C.; UV detection: 220 nm.
[0251] Method 2D:
[0252] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: methanol/acetonitrile 25:75; flow rate: 15 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0253] Method 3D:
[0254] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: methanol/acetonitrile 50:50; flow rate: 15 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0255] Method 4D:
[0256] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: tert-butyl methyl ether/methanol 50:50; flow rate: 15
ml/min; temperature: 30.degree. C.; UV detection: 220 nm.
[0257] Method 5D:
[0258] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: methanol/acetonitrile 25:75; flow rate: 15 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0259] Method 6D:
[0260] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.20 mm,
eluent: isohexane/ethanol 25:75; flow rate: 15 ml/min, temperature:
45.degree. C.; UV detection: 220 nm.
[0261] Method 7D:
[0262] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.20 mm,
eluent: ethanol 100%; flow rate: 15 ml/min, temperature: 45.degree.
C.; UV detection: 220 nm.
[0263] Method 8D:
[0264] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.20 mm,
eluent: isohexane/isopropanol 30:70; flow rate: 15 ml/min,
temperature: 45.degree. C.; UV detection: 220 nm.
[0265] Method 9D:
[0266] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: acetonitrile/methanol 70:30; flow rate: 15 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0267] Method 10D:
[0268] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.20 mm,
eluent: acetonitrile/methanol 70:30; flow rate: 20 ml/min,
temperature: 35.degree. C.; UV detection: 210 nm.
[0269] Method 11D:
[0270] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.20 mm,
eluent: isohexane/ethanol 70:30; flow rate: 15 ml/min, temperature:
40.degree. C.; UV detection: 220 nm.
Analytical Separation of Enantiomers:
[0271] Method 1E:
[0272] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.4 mm;
eluent: isopropanol/isohexane: 75:25; flow rate: 1 ml/min;
temperature: 45.degree. C.; UV detection: 220 nm.
[0273] Method 2E:
[0274] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.4.6 mm;
eluent: isohexane/isopropanol 25:75+0.2% trifluoroacetic acid+1%
water; flow rate: 1 ml/min; temperature: 45.degree. C.; UV
detection: 235 nm.
[0275] Method 3E:
[0276] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: acetonitrile/methanol 75:25; flow rate: 1 ml/min;
temperature: 25.degree. C.; UV detection: 220 nm.
[0277] Method 4E:
[0278] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: acetonitrile/methanol 50:50; flow rate: 1 ml/min;
temperature: 25.degree. C.; UV detection: 220 nm.
[0279] Method 5E:
[0280] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: tert-butyl methyl ether/methanol 50:50; flow rate: 1
ml/min; temperature: 25.degree. C.; UV detection: 220 nm.
[0281] Method 6E:
[0282] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.4.6 mm,
eluent: ethanol 100%; flow rate: 1 ml/min, temperature: 45.degree.
C.; UV detection: 220 nm.
[0283] Method 7E:
[0284] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.4.6 mm,
eluent: isohexane/isopropanol 30:70; flow rate: 1 ml/min,
temperature: 45.degree. C.; UV detection: 220 nm.
[0285] Method 8E:
[0286] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: acetonitrile/methanol 70:30; flow rate: 15 ml/min,
temperature: 25.degree. C.; UV detection: 220 nm.
[0287] Method 9E:
[0288] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: acetonitrile/methanol 70:30; flow rate: 15 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0289] Method 10E:
[0290] Phase: Daicel Chiralpak IA, 5 .mu.m 250 mm.times.4.6 mm,
eluent: acetonitrile/methanol 70:30; flow rate: 1 ml/min,
temperature: 30.degree. C.; UV detection: 220 nm.
[0291] Method 11E:
[0292] Phase: Daicel Chiralpak AD-H, 5 .mu.m 250 mm.times.4.6 mm,
eluent: isohexane/ethanol 25:75+0.2% trifluoroacetic acid+1% water;
flow rate: 1 ml/min, temperature: 45.degree. C.; UV detection: 220
nm.
[0293] GC-MS Methods:
[0294] Method 1F:
[0295] Instrument: Micromass GCT, GC6890; column: Restek RTX-35, 15
m.times.200 .mu.m.times.0.33 .mu.m; constant flow rate with helium:
0.88 ml/min; oven: 70.degree. C.; inlet: 250.degree. C.; gradient:
70.degree. C., 30.degree. C./min.fwdarw.310.degree. C. (hold for 3
min)
[0296] The microwave reactor used was a "single mode" instrument of
the Emrys.TM. Optimizer type.
Starting Compounds
General Method 1A: N'-Hydroxyimidamide Formation
[0297] A solution of the appropriate nitrile (1.0 eq) in ethanol
(1.2 ml/mmol) is admixed at RT with hydroxylammonium chloride (1.5
eq.) and triethylamine (1.2 eq.). The reaction mixture is stirred
at room temperature overnight. For workup, the ethanol is removed
under reduced pressure, saturated aqueous sodium hydrogencarbonate
solution is added and the reaction mixture is extracted with ethyl
acetate. The organic phase is dried over sodium sulphate and
concentrated. The residue is reacted without further
purification.
General Method 2A: N'-Hydroxyimidamide Formation
[0298] A solution of the appropriate nitrile (1.0 eq) in a mixture
of ethanol (1.9 ml/mmol) and water (0.5 ml/mmol) is admixed at RT
with hydroxylammonium chloride (1.08 eq.) and sodium hydroxide
(1.12 eq.). The reaction mixture is stirred at room temperature for
16 hours. For workup, the reaction mixture is concentrated under
reduced pressure, admixed with dichloromethane and filtered. The
filtrate is concentrated under reduced pressure and the residue is
reacted without further purification.
General Method 3A: Suzuki Coupling
[0299] A mixture of the appropriate bromopyridine in toluene (1.8
ml/mmol) is admixed under argon and at RT with
tetrakis(triphenylphosphine)palladium (0.02 eq.), with a solution
of the appropriate arylboronic acid (1.2 eq.) in ethanol (0.5
ml/mmol) and with a solution of potassium fluoride (2.0 eq.) in
water (0.2 ml/mmol). The reaction mixture is stirred under reflux
for several hours until the conversion is substantially complete.
After addition of ethyl acetate and phase separation, the organic
phase is washed once with water and once with saturated aqueous
sodium chloride solution, dried (magnesium sulphate), filtered and
concentrated under reduced pressure. The crude product is purified
by flash chromatography (silica gel 60, eluent:
dichloromethane/methanol mixtures).
General Method 4A: Hydrogenation of the Pyridine
[0300] A solution of the pyridine in ethanol (9 ml/mmol) is admixed
under argon with palladium on activated carbon (moistened with
approx. 50% water, 0.3 g/mmol), and the mixture is hydrogenated at
60.degree. C. in a 50 bar hydrogen atmosphere overnight. The
catalyst is then filtered off through a filter layer and washed
repeatedly with ethanol. The combined filtrates are concentrated
under reduced pressure.
General Method 5A: Methyl Ester Hydrolysis/Epimerization
[0301] At RT, potassium tert-butoxide (10 eq.) is added to a
solution of the appropriate methyl ester (1.0 eq.) in methanol
(35-40 ml/mmol). The mixture is stirred at 60.degree. C. overnight.
If the conversion is incomplete, water (1.0 eq.) is added and the
mixture is stirred at 60.degree. C. until the conversion is
complete. For workup, the methanol is removed under reduced
pressure, the residue is admixed with water and the mixture is
acidified (pH 1) with aqueous 1 N hydrochloric acid solution. The
mixture is extracted with ethyl acetate and the organic phase is
dried with magnesium sulphate, filtered and concentrated under
reduced pressure.
General Method 6A: Oxadiazole Formation
[0302] A solution of the appropriate piperidine-3-carboxylic acid
in dimethylformamide (10-20 ml/mmol) is admixed under argon at RT
with HATU (1.2 eq.), N,N-diisopropylethylamine (2.2 eq.) and the
appropriate N-hydroxyimidamide (1.1 eq.). The reaction mixture is
stirred at RT until the formation of the intermediate is complete
and then stirred further at 120.degree. C. until the desired
product has formed from this intermediate. The reaction mixture is
then purified by means of preparative HPLC.
Example 1A
N'-Hydroxy-3-methoxypropanimidamide
##STR00029##
[0304] According to General Method 1A, 20.0 g (235.0 mmol) of
3-methoxypropionitrile were reacted. Yield: 18.1 g (49% of theory,
purity 74%)
[0305] HPLC (Method 1A): R.sub.t=0.35 min; MS (ESIpos): m/z=119
[M+H].sup.+.
Example 2A
3-Ethoxy-N'-hydroxypropanimidamide
##STR00030##
[0307] According to General Method 2A, 5.0 g (50.4 mmol) of
3-ethoxypropionitrile were reacted. Yield: 0.6 g (8% of theory,
purity 90%)
[0308] HPLC (Method 1A): R.sub.t=0.60 min; MS (ESIpos): m/z=133
[M+H].sup.+.
Example 3A
N'-Hydroxycyclopropanecarboximidamide
##STR00031##
[0310] According to General Method 2A, 7.2 g (107.3 mmol) of
cyclopropanecarbonitrile were reacted. Yield: 4.8 g (44% of
theory)
[0311] LC-MS (Method 2B): R.sub.t=0.16 min; MS (ESIpos): m/z=101
[M+H].sup.+.
Example 4A
Methyl 5-(4-ethylphenyl)pyridine-3-carboxylate
##STR00032##
[0313] According to General Method 3A, 32 g (148 mmol) of methyl
5-bromonicotinate and 27 g (178 mmol, 1.2 eq.) of
4-ethylphenylboronic acid were reacted. Yield: 24 g (64% of
theory)
[0314] LC-MS (Method 3B): R.sub.t=2.03 min; MS (ESIpos): m/z=242
[M+H].sup.+.
[0315] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=9.13 (d, 1H),
9.05 (d, 1H), 8.45 (t, 1H), 7.72 (d, 2H), 7.38 (d, 2H), 3.93 (s,
3H), 2.68 (q, 2H), 1.22 (t, 3H).
Example 5A
Methyl 5-(4-ethylphenyl)piperidine-3-carboxylate [racemic cis/trans
isomer mixture]
##STR00033##
[0317] According to General Method 4A, 24 g (94 mmol) of methyl
5-(4-ethylphenyl)pyridine-3-carboxylate were hydrogenated. Yield:
20 g (77% of theory)
[0318] LC-MS (Method 4B): R.sub.t=1.43 min; MS (ESIpos): m/z=248
[M+H].sup.+.
Example 6A
[0319] Methyl
5-(4-ethylphenyl)-1-(thiomorpholin-4-ylcarbonyl)piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00034##
[0320] 5.00 g (12.1 mmol) of 3-methyl 1-(4-nitrophenyl)
5-(4-ethylphenyl)piperidine-1,3-dicarboxylate (Example 30A), 3.57 g
(36.4 mmol) of thiomorpholine and 5.03 g (36.4 mmol) of potassium
carbonate were added to 76 ml of DMF and heated in 5 portions at
150.degree. C. for 1.5 h in a single-mode microwave (Emrys
Optimizer). For workup, the reaction solutions were combined and
filtered, and the residue was purified by means of preparative
HPLC. Yield: 3.07 g (67% of theory)
[0321] LC-MS (Method 5B): R.sub.t=1.16 and 1.18 min (cis/trans
isomers); MS (ESIpos): m/z=377 [M+H].sup.+.
Example 7A
5-(4-Ethylphenyl)-1-(thiomorpholin-4-ylcarbonyl)piperidine-3-carboxylic
acid [racemic cis isomer]
##STR00035##
[0323] According to General Method 5A, 3.00 g (7.97 mmol) of the
compound from Example 6A and 8.94 g (79.7 mmol) of potassium
tert-butoxide were reacted. The reaction led selectively to the cis
isomer. Yield: 2.74 g (93% of theory)
[0324] LC-MS (Method 5B): R.sub.t=1.04 min; MS (ESIpos): m/z=363
[M+H].sup.+.
Example 8A
{3-(4-Ethylphenyl)-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]piperidin-1--
yl}(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00036##
[0326] According to General Method 6A, 300 mg (0.828 mmol) of the
compound from Example 7A and 134 mg (0.910 mmol) of
N'-hydroxy-3-methoxypropanimidamide were reacted Yield: 185 mg (49%
of theory)
[0327] LC-MS (Method 5B): R.sub.t=1.22 min; MS (ESIpos): m/z=445
[M+H].sup.+.
Example 9A
[3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-(4-ethylphenyl)piperidin-1-yl](t-
hiomorpholin-4-yl)-methanone [racemic cis isomer]
##STR00037##
[0329] According to General Method 6A, 300 mg (0.828 mmol) of the
compound from Example 7A and 91 mg (0.91 mmol) of
N-hydroxycyclopropanecarboximidamide were reacted Yield: 141 mg
(40% of theory)
[0330] LC-MS (Method 5B): R.sub.t=1.32 min; MS (ESIpos): m/z=427
[M+H].sup.+.
[0331] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.22 (d, 2H),
7.15 (d, 2H), 3.92 (d, 1H), 3.52 (d, 1H), 3.44 (br. s., 4H),
3.38-3.31 (m, 1H), 3.03-2.79 (m, 3H), 2.63-2.55 (m, 6H), 2.25 (d,
1H), 2.10 (td, 1H), 1.91 (q, 1H), 1.16 (t, 3H), 1.09-1.01 (m, 2H),
0.92-0.85 (m, 2H).
Example 10A
{3-(4-Ethylphenyl)-5-[3-(2-hydroxyethyl)-1,2,4-oxadiazol-5-yl]piperidin-1--
yl}(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00038##
[0333] According to General Method 6A, 300 mg (0.828 mmol) of the
compound from Example 7A and 112 mg (1.08 mmol) of
N',3-dihydroxypropanimidamide [Graham A. Showell et al., J. Med.
Chem., 1991, 34, 1086-1094] were reacted. Yield: 248 mg (66% of
theory)
[0334] LC-MS (Method 5B): R.sub.t=2.22 min; MS (ESIpos): m/z=431
[M+H].sup.+.
Example 11A
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-(4-ethylphenyl)piperidin-1-y-
l}(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00039##
[0336] According to General Method 6A, 600 mg (1.655 mmol) of the
compound from Example 7A and 355 mg (approx. 2.152 mmol) of
3-Ethoxy-N'-hydroxypropanimidamide were reacted Yield: 389 mg (49%
of theory)
[0337] LC-MS (Method 6B): R.sub.t=2.61 min; MS (ESIpos): m/z=459
[M+H].sup.+.
[0338] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.16 (d, 2H), 3.95 (d, 1H), 3.71 (t, 2H), 3.54 (d, 1H), 3.48-3.34
(m, 7H), 3.08-2.81 (m, 5H), 2.63-2.55 (m, 6H), 2.29 (d, 1H), 1.95
(q, 1H), 1.16 (t, 3H), 1.07 (t, 3H).
Example 12A
Methyl 5-[4-(trifluoromethyl)phenyl]pyridine-3-carboxylate
##STR00040##
[0340] According to General Method 3A, 28 g (132 mmol) of methyl
5-bromonicotinate and 30 g (158 mmol, 1.2 eq.) of
4-trifluoromethylphenylboronic acid were reacted. Yield: 32 g (85%
of theory)
[0341] LC-MS (Method 4B): R.sub.t=2.27 min; MS (ESIpos): m/z=282
[M+H].sup.+.
Example 13A
Methyl 5-[4-(trifluoromethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00041##
[0343] 32 g (112 mmol) of methyl
5-[4-(trifluoromethyl)phenyl]pyridine-3-carboxylate (Example 12A)
were hydrogenated according to General Method 4A. Yield: 26 g (82%
of theory)
[0344] LC-MS (Method 1B): R.sub.t=1.35 and 1.41 min (cis/trans
isomers); MS (ESIpos): m/z=288 [M+H].+-..
[0345] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=9.22 (d, 1H),
9.14 (d, 1H), 8.57 (t, 1H), 8.06 (d, 2H), 7.89 (d, 2H), 3.94 (s,
3H).
Example 14A
3-Methyl 1-(4-nitrophenyl)
5-[4-(trifluoromethyl)phenyl]piperidine-1,3-dicarboxylate [racemic
cis/trans isomer mixture]
##STR00042##
[0347] 20.0 g (69.6 mmol) of methyl
5-[4-(trifluoromethyl)phenyl]piperidine-3-carboxylate (Example 13A)
were dissolved in 1.0 l of dichloromethane, and admixed at
0.degree. C. with 14.1 g (139 mmol) of triethylamine. Subsequently,
14.0 g (69.6 mmol) of 4-nitrophenyl chlorocarbonate were added
dropwise. The reaction mixture was stirred at 0.degree. C. for 2 h
and then at RT for 16 h. For workup, the mixture was washed with
saturated aqueous sodium hydrogencarbonate solution. The organic
phase was dried over magnesium sulphate, filtered and concentrated
under reduced pressure. This gave 31.3 g of crude product, which
was reacted without any further purification steps.
[0348] LC-MS (Method 3B): R.sub.t=2.44 min and 2.48 min (cis/trans
isomers); MS (ESIpos): m/z=453 [M+H].+-..
Example 15A
[0349] Methyl
1-(thiomorpholin-4-ylcarbonyl)-5-[4-(trifluoromethyl)phenyl]piperidine-3--
carboxylate [racemic cis/trans isomer mixture]
##STR00043##
[0350] 10.0 g (22.1 mmol) of 3-methyl
1-(4-nitrophenyl)-5-[4-(trifluoromethyl)phenyl]piperidine-1,3-dicarboxyla-
te, 6.84 g (66.3 mmol) of thiomorpholine and 9.17 g (66.3 mmol) of
potassium carbonate were added to 150 ml of DMF and heated in 10
portions at 150.degree. C. for 1 h in a single-mode microwave
(Emrys Optimizer). For workup, the reaction solutions were combined
and filtered, and the residue was purified by means of preparative
HPLC. Yield: 5.16 g (55% of theory)
[0351] LC-MS (Method 5B): R.sub.t=1.13 and 1.16 min (cis/trans
isomers); MS (ESIpos): m/z=417 [M+H].+-..
Example 16A
1-(Thiomorpholin-4-ylcarbonyl)-5-[4-(trifluoromethyl)phenyl]piperidine-3-c-
arboxylic acid [racemic cis isomer]
##STR00044##
[0353] According to General Method 5A, 5.16 g (12.4 mmol) of the
compound from Example 15A and 13.9 g (124 mmol) of potassium
tert-butoxide were reacted. The reaction led selectively to the cis
isomer. Yield: 4.90 g (98% of theory)
[0354] LC-MS (Method 5B): R.sub.t=1.04 min; MS (ESIpos): m/z=403
[M+H].sup.+.
Example 17A
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethyl)phenyl]piper-
idin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00045##
[0356] According to General Method 6A, 600 mg (1.491 mmol) of the
compound from Example 16A and 164 mg (1.640 mmol) of
N-hydroxycyclopropanecarboximidamide were reacted Yield: 352 mg
(47% of theory)
[0357] LC-MS (Method 5B): R.sub.t=1.28 min; MS (ESIpos): m/z=467
[M+H].sup.+.
[0358] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.56 (d, 2H), 3.92 (d, 1H), 3.57 (d, 1H), 3.45 (br. s., 4H),
3.40-3.34 (m, 1H), 3.08-2.95 (m, 3H), 2.59 (br. s., 4H), 2.30 (d,
1H), 2.16-2.07 (m, 1H), 2.04-1.91 (m, 1H), 1.10-1.01 (m, 2H),
0.92-0.85 (m, 2H).
Example 18A
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00046##
[0360] According to General Method 6A, 600 mg (1.491 mmol) of the
compound from Example 16A and 242 mg (1.640 mmol) of
N'-hydroxy-3-methoxypropanimidamide were reacted Yield: 350 mg (46%
of theory)
[0361] LC-MS (Method 5B): R.sub.t=1.18 min; MS (ESIpos): m/z=485
[M+H].sup.+.
[0362] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.70 (d, 2H), 7.57
(d, 2H), 3.95 (d, 1H), 3.68 (t, 2H), 3.58 (d, 1H), 3.51-3.36 (m,
5H), 3.23 (s, 3H), 3.13-2.96 (m, 3H), 2.94 (t, 2H), 2.60 (br. s.,
4H), 2.33 (br. d., 1H), 2.10-1.95 (m, 1H).
Example 19A
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]p-
iperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00047##
[0364] According to General Method 6A, 600 mg (1.491 mmol) of the
compound from Example 16A and 320 mg (approx. 1.983 mmol) of
3-ethoxy-N'-hydroxypropanimidamide were reacted Yield: 343 mg (46%
of theory)
[0365] LC-MS (Method 6B): R.sub.t=2.57 min; MS (ESIpos): m/z=499
[M+H].sup.+.
[0366] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.57 (d, 2H), 3.96 (d, 1H), 3.71 (t, 2H), 3.58 (d, 1H), 3.49-3.37
(m, 7H), 3.11-2.97 (m, 3H), 2.93 (t, 2H), 2.60 (br. s., 4H), 2.34
(br. d., 1H), 2.02 (q, 1H), 1.07 (t, 3H).
Example 20A
{3-[3-(2-Hydroxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl}-
piperidin-1-yl]-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00048##
[0368] According to General Method 6A, 600 mg (1.491 mmol) of the
compound from Example 16A and 201 mg (1.938 mmol) of
N',3-dihydroxypropanimidamide were reacted Yield: 494 mg (68% of
theory)
[0369] LC-MS (Method 5B): R.sub.t=1.04 min; MS (ESIpos): m/z=471
[M+H].sup.+.
Example 21A
Methyl 5-[4-(trifluoromethoxy)phenyl]pyridine-3-carboxylate
##STR00049##
[0371] According to General Method 3A, 23 g (105 mmol) of methyl
5-bromonicotinate and 26 g (126 mmol, 1.2 eq.) of
4-trifluoromethoxyphenylboronic acid were reacted. Yield: 14 g (41%
of theory)
[0372] LC-MS (Method 1B): R.sub.t=2.44 min; MS (ESIpos): m/z=298
[M+H].sup.+.
[0373] Alternative Synthesis:
[0374] A solution of 26 g (121 mmol) of methyl 5-bromonicotinate in
toluene (220 ml) was admixed under argon at RT with 2.8 g (2.4
mmol) of tetrakis(triphenylphosphine)palladium, and then a solution
of 30 g (146 mmol) of 4-trifluoromethoxyphenylboronic acid in
ethanol (58 ml) was added. After adding 14 g (243 mmol) of
potassium fluoride in water (58 ml), the mixture was stirred under
reflux overnight, a further 0.70 g (0.61 mmol) of
tetrakis(triphenylphosphine)palladium was added, and the mixture
was stirred under reflux for a further 24 h. After adding another
1.4 g (1.2 mmol) of tetrakis(triphenylphosphine)palladium, the
mixture was stirred under reflux for 20 h, and the reaction
solution was admixed with ethyl acetate and washed with water and
saturated aqueous sodium chloride solution. The organic phase was
dried over magnesium sulphate, filtered and concentrated under
reduced pressure. The residue was purified by means of column
chromatography (silica gel, cyclohexane/dichloromethane
1:1.fwdarw.dichloromethane). Yield: 31 g (86% of theory)
[0375] LC-MS (Method 4B): R.sub.t=2.32 min; MS (ESIpos): m/z=298
[M+H].sup.+;
[0376] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=9.17 (d, 1H),
9.10 (d, 1H), 8.51 (t, 1H), 7.95 (d, 2H), 7.52 (d, 2H), 3.94 (s,
3H).
Example 22A
Methyl 5-[4-(trifluoromethoxy)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00050##
[0378] 14 g (45 mmol) of methyl
5-[4-(trifluoromethoxy)phenyl]pyridine-3-carboxylate in ethanol
(500 ml) were admixed with 17 g of moistened palladium/carbon
catalyst (10% palladium, 50% water), and then hydrogenated at
60.degree. C. and a 50 bar hydrogen atmosphere overnight. The
reaction solution was filtered, the filter residue was washed with
ethanol and the filtrate was concentrated under reduced pressure.
The residue was purified by means of column chromatography (silica
gel, dichloromethane/methanol 600:1.fwdarw.10:1). Yield: 8 g (59%
of theory)
[0379] LC-MS (Method 1B): R.sub.t=1.29 min and 1.33 min (cis/trans
isomers); MS (ESIpos): m/z=304 [M+H].sup.+;
[0380] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.43-7.35 (m,
4H), 7.31-7.25 (m, 4H), 3.60 (s, 3H), 3.40-3.21 (m, 5H), 3.16 (d,
1H), 3.01-2.89 (m, 3H), 2.88-2.78 (m, 2H), 2.78-2.65 (m, 4H), 2.17
(d, 1H), 2.09 (d, 1H), 1.82 (td, 1H), 1.68 (q, 1H), approx. 1:1.3
mixture of the cis/trans isomers, two protons hidden.
Example 23A
3-Methyl
1-(4-nitrophenyl)-5-[4-(trifluoromethoxy)phenyl]piperidine-1,3-di-
carboxylate [racemic cis/trans isomer mixture]
##STR00051##
[0382] At 0.degree. C., 5.32 g (26.4 mmol) of 4-nitrophenyl
chloroformate were added slowly to 8.0 g (26.4 mmol) of methyl
5-(4-(trifluoromethoxy)phenyl)piperidine-3-carboxylate (Example
22A) and 5.34 g (26.3 mmol) of triethylamine in 666 ml of
dichloromethane. The mixture was stirred at RT for 2 h. For workup,
the reaction mixture was washed first with saturated aqueous sodium
hydrogencarbonate solution, then with water. The organic phase was
dried over sodium sulphate and concentrated under reduced pressure.
The residue was purified by means of flash chromatography on silica
gel (eluent: cyclohexane/ethyl acetate 1:2->1:1). Yield: 7.32 g
(54% of theory)
[0383] LC-MS (Method 3B): R.sub.t=2.47 min; MS (ESIpos): m/z=469
[M+H].sup.+.
Example 24A
Methyl
1-(thiomorpholin-4-ylcarbonyl)-5-[4-(trifluoromethoxy)phenyl]piperi-
dine-3-carboxylate [racemic cis/trans isomer mixture]
##STR00052##
[0385] 12.0 g (25.1 mmol) of 3-methyl
1-(4-nitrophenyl)-5-[4-(trifluoromethoxy)phenyl]piperidine-1,3-dicarboxyl-
ate, 7.77 g (75.3 mmol) of thiomorpholine and 10.4 g (75.3 mmol) of
potassium carbonate were added to 180 ml of DMF and heated in 12
portions at 150.degree. C. for 2 h in a single-mode microwave
(Emrys Optimizer). For workup, the reaction solutions were combined
and filtered, and the residue was purified by means of preparative
HPLC. Yield: 7.88 g (73% of theory)
[0386] LC-MS (Method 5B): R.sub.t=1.16 and 1.18 min (cis/trans
isomers); MS (ESIpos): m/z=433 [M+H].+-..
[0387] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.46-7.39 (m,
4H), 7.32 (d, 4H), 3.84 (dd, 2H), 3.64 (s, 3H), 3.63 (s, 3H),
3.55-3.34 (m, 10H), 3.09 (dd, 1H), 3.06-2.96 (m, 1H), 2.92-2.81 (m,
6H), 2.76-2.67 (m, 1H), 2.65-2.56 (m, 7H), 2.25-2.10 (m, 2H),
1.95-1.84 (m, 1H), 1.76 (q, 1H), approx. 1:1 mixture of the
cis/trans isomers.
Example 25A
1-(Thiomorpholin-4-ylcarbonyl)-5-[4-(trifluoromethoxy)phenyl]piperidine-3--
carboxylic acid [racemic cis/trans isomer mixture]
##STR00053##
[0389] 20.4 g (182 mmol) of potassium tert-butoxide were added at
RT to a solution of 7.85 g (18.2 mmol) of the compound from Example
24A in methanol (650 ml). The mixture was stirred at 60.degree. C.
overnight. For workup, the methanol was removed under reduced
pressure, the residue was admixed with water and the mixture was
acidified (pH 1) with aqueous 1 N hydrochloric acid solution. The
mixture was extracted with ethyl acetate, and the organic phase was
dried with magnesium sulphate, filtered and concentrated under
reduced pressure. The reaction led to an 85:15 cis/trans isomer
mixture. Yield: 7.70 g (99% of theory)
[0390] LC-MS (Method 5B): R.sub.t=1.03 (trans isomer) and 1.04 min
(cis isomer); MS (ESIpos): m/z=419 [M+H].sup.+;
[0391] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=12.44 (br. s.,
1H), 7.47-7.39 (m, 2H), 7.31 (d, 2H), 3.79 (d, 1H), 3.56-3.48 (m,
1H), 3.46-3.37 (m, 4H), 2.91-2.73 (m, 3H), 2.63-2.55 (m, 5H), 2.14
(d, 1H), 1.81-1.66 (m, 1H).
Example 26A
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl-
]piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00054##
[0393] According to General Method 6A, 600 mg (1.43 mmol) of the
compound from Example 25A and 232 mg (1.58 mmol) of
N'-hydroxy-3-methoxypropanimidamide were reacted Yield: 398 mg (53%
of theory)
[0394] LC-MS (Method 5B): R.sub.t=1.21 min; MS (ESIpos): m/z=501
[M+H].sup.+.
[0395] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.47 (d, 2H),
7.33 (d, 2H), 3.95 (d, 1H), 3.68 (t, 2H), 3.56 (d, 1H), 3.50-3.35
(m, 5H), 3.23 (s, 3H), 3.08-2.86 (m, 5H), 2.60 (br. s., 4H), 2.32
(d, 1H), 1.97 (q, 3H).
Example 27A
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]pipe-
ridin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00055##
[0397] According to General Method 6A, 300 mg (0.717 mmol) of the
compound from Example 25A and 79 mg (0.789 mmol) of
N-hydroxycyclopropanecarboximidamide were reacted Yield: 135 mg
(39% of theory)
[0398] LC-MS (Method 2B): R.sub.t=1.44 min; MS (ESIpos): m/z=483
[M+H].sup.+.
Alternative Synthesis:
[0399] 600 mg (1.43 mmol) of the compound from Example 25A in
dimethylformamide (29.0 ml) were admixed at RT with 654 mg (1.72
mmol) of HATU and 0.55 ml (498 mg, 3.16 mmol) of
N,N-diisopropylethylamine, and the mixture was stirred for 30 min
Subsequently, 158 mg (1.58 mmol) of
N'-hydroxycyclopropanecarboximidamide were added and the mixture
was stirred at RT overnight. The reaction solution was heated to
120.degree. C. and stirred at this temperature for 1 h. The
reaction solution was subsequently purified directly by means of
preparative HPLC. Yield: 315 mg (45% of theory)
[0400] LC-MS (Method 5B): R.sub.t=1.30 min; MS (ESIpos): m/z=483
[M+H].sup.+;
[0401] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.46 (d, 2H), 7.33
(d, 2H), 3.91 (d, 1H), 3.55 (d, 1H), 3.45 (br. s., 4H), 3.39-3.32
(m, 1H), 3.05-2.91 (m, 3H), 2.59 (br. s., 4H), 2.28 (d, 1H),
2.17-2.08 (m, 1H), 1.93 (q, 1H), 1.10-1.02 (m, 2H), 0.92-0.84 (m,
2H).
Example 28A
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl]-
piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00056##
[0403] According to General Method 6A, 600 mg (1.434 mmol) of the
compound from Example 25A and 307 mg (approx. 1.864 mmol) of
3-ethoxy-N'-hydroxypropanimidamide were reacted Yield: 403 mg (55%
of theory)
[0404] LC-MS (Method 6B): R.sub.t=2.61 min; MS (ESIpos): m/z=515
[M+H].sup.+.
[0405] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.47 (d, 2H), 7.33
(d, 2H), 3.95 (d, 1H), 3.71 (t, 2H), 3.56 (d, 1H), 3.50-3.35 (t,
7H), 3.10-2.88 (m, 5H), 2.60 (br. s., 4H), 2.32 (d, 1H), 2.02-1.92
(m, 1H), 1.07 (t, 3H).
Example 29A
{3-[3-(2-Hydroxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl-
]piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00057##
[0407] According to General Method 6A, 1.00 g (2.390 mmol) of the
compound from Example 25A and 323 mg (3.107 mmol) of
N',3-dihydroxypropanimidamide were reacted Yield: 848 mg (69% of
theory)
[0408] LC-MS (Method 6B): R.sub.t=2.26 min; MS (ESIpos): m/z=487
[M+H].sup.+.
Example 30A
3-Methyl 1-(4-nitrophenyl)
5-(4-ethylphenyl)piperidine-1,3-dicarboxylate [racemic cis/trans
isomer mixture]
##STR00058##
[0410] 3.0 g (12.1 mmol) of the compound from Example 5A were
initially charged in 30 ml of dichloromethane, cooled to 0.degree.
C. and admixed with 3.4 ml (2.4 g, 12.1 mmol) of triethylamine and
2.4 g (12.1 mmol) of 4-nitrophenyl chloroformate. The reaction
mixture was allowed to warm up slowly to RT and stirred at RT for
16 h. The mixture was washed several times with water, dried over
sodium sulphate, filtered and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel
(eluent dichloromethane.fwdarw.dichloromethane/methanol 100:2).
Yield: 4.7 g (83% of theory, purity 89%)
[0411] HPLC (Method 1A): R.sub.t=4.94 min and 5.00 min (cis/trans
isomer); MS (ESIpos): m/z=413 [M+H].sup.+.
Example 31A
4-Nitrophenyl thiomorpholine-4-carboxylate
##STR00059##
[0413] 7.7 g (74.4 mmol) of thiomorpholine were initially charged
in 100 ml of dichloromethane and, while cooling with an ice bath,
admixed with 20.7 ml (15.1 g, 148.8 mmol) of triethylamine. 10.0 g
(49.6 mmol) of 4-nitrophenyl chloroformate were added in portions.
The reaction mixture was stirred at RT for one hour, and admixed
with water and ethyl acetate. The organic phase was removed, washed
with 1 N hydrochloric acid and saturated aqueous sodium chloride
solution, dried over sodium sulphate, filtered and concentrated
under reduced pressure. Yield: 13.2 g (99% of theory)
[0414] LC-MS (Method 5B): R.sub.t=0.98 min; MS (ESIpos): m/z=269
[M+H].sup.+.
[0415] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=8.28 (d, 2H),
7.46 (d, 2H), 3.86 (br. s., 2H), 3.72 (br. s., 2H), 2.71 (br. d.,
4H).
Example 32A
4-Nitrophenyl thiomorpholine-4-carboxylate 1-oxide
##STR00060##
[0417] 13.1 g (49.0 mmol) of 4-nitrophenyl
thiomorpholine-4-carboxylate were initially charged in 135 ml of
dichloromethane and admixed at 0.degree. C. with 7.6 g (44.1 mmol)
of m-chloroperbenzoic acid in portions. The mixture was stirred at
RT for two hours, water was added and the organic phase was
removed. The organic phase was washed rapidly with saturated
aqueous sodium hydrogencarbonate solution, filtered and
concentrated under reduced pressure. The crude product was purified
by means of preparative HPLC. Yield: 7.8 g (56% of theory)
[0418] LC-MS (Method 5B): R.sub.t=0.69 min; MS (ESIpos): m/z=285
[M+H].sup.+.
[0419] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=8.30 (d, 2H),
7.49 (d, 2H), 4.20-3.70 (m, 4H), 3.03 (dt, 2H), 2.85 (d, 2H).
Example 33A
[5-(Methoxycarbonyl)pyridin-3-yl]boronic acid hydrochloride
##STR00061##
[0421] 17.6 g (81.4 mmol) of methyl 5-bromonicotinate were
initially charged in 375 ml of DMF under argon and admixed with
26.9 g (105.8 mmol) of
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane, 3.0 g
(3.6 mmol) of tris(dibenzylideneacetone)dipalladium(0), 1.8 g (6.5
mmol) of tricyclohexylphosphine and 32.0 mmol (325.9 mmol) of
potassium acetate. The reaction mixture was stirred at 100.degree.
C. for 20 h. Subsequently, the solvent was removed under reduced
pressure, the residue was admixed with 40 ml of water and 140 ml of
tert-butyl methyl ether, and the organic phase was removed. The
aqueous phase was extracted three times with 80 ml each time of
tert-butyl methyl ether. The combined organic extracts were washed
with saturated aqueous sodium chloride solution, dried over
magnesium sulphate, filtered and concentrated. The residue was
taken up in 360 ml of methanol and admixed with 36 ml of
concentrated hydrochloric acid. The reaction mixture was heated to
reflux for 22 h and then stirred at RT for 12 h. About half of the
solvent was removed under reduced pressure, and the solution was
filtered and concentrated further under reduced pressure. The oily
residue was recrystallized twice from acetone, and the residue was
taken up in 10 ml of acetone and admixed with 100 ml of tert-butyl
methyl ether. After 16 h, the precipitate formed was removed from
the solution. This precipitate was stirred in 50 ml of acetone and
left to stand at RT for 5 weeks, and the solution was removed
again. The solutions were combined, concentrated and dissolved in
50 ml of tert-butyl methyl ether. The mixture was left to stand at
RT for 5 weeks and then the precipitate was removed. The
precipitate was washed three times with tert-butyl methyl ether and
dried in a drying cabinet under reduced pressure.
[0422] LC-MS (Method 4B): R.sub.t=0.91 min; MS (ESIpos): m/z=182
[M+H].sup.+.
Example 34A
Methyl 5-[4-(difluoromethoxy)phenyl]nicotinate
##STR00062##
[0424] 10.0 g (44.8 mmol) of 4-(difluoromethoxy)bromobenzene were
reacted according to General Method 3A with 14.6 g (67.3 mmol) of
[5-(methoxycarbonyl)pyridin-3-yl]boronic acid hydrochloride. The
release of the hydrochloride was achieved by additional addition of
6.80 g (49.3 mmol) of potassium carbonate. Yield: 8.6 g (67% of
theory)
[0425] LC-MS (Method 2B): R.sub.t=1.15 min; MS (ESIpos): m/z=280
[M+H].sup.+.
Example 35A
[0426] Methyl 5-[4-(difluoromethoxy)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00063##
[0427] A solution of 8.6 g (30.9 mmol) of methyl
5-[4-(difluoromethoxy)phenyl]nicotinate in concentrated acetic acid
(112 ml) was admixed with 841 mg of palladium/carbon (10%
palladium) and 1.12 g of platinum(IV) oxide. This was followed by
hydrogenation under a hydrogen atmosphere at standard pressure for
24 h. The reaction solution was concentrated under reduced
pressure. The residue was taken up in water, acidified (pH 1) with
1 N hydrochloric acid, extracted with diethyl ether, then basified
(pH>10) with saturated aqueous sodium hydrogencarbonate solution
and extracted repeatedly with ethyl acetate. The combined filtrates
were dried over sodium sulphate, filtered and concentrated under
reduced pressure. Yield: 6.6 g (74% of theory)
[0428] LC-MS (Method 5B): R.sub.t=0.65 min and 0.66 min (cis/trans
isomers); MS (ESIpos): m/z=286 [M+H].sup.+.
Example 36A
[0429] Methyl
5-[4-(difluoromethoxy)phenyl]-1-[(1,1-dioxidothiomorpholin-4-yl)carbonyl]-
piperidine-3-carboxylate [cis/trans isomer mixture]
##STR00064##
[0430] 2.2 g (7.7 mmol) of methyl
5-[4-(difluoromethoxy)phenyl]piperidine-3-carboxylate were
dissolved in 14 ml of N-methylpyrrolidone, and admixed with 4.0 ml
(3.0 g, 23.0 mmol) of N,N-diisopropylethylamine and 3.5 g (11.5
mmol) of 4-nitrophenyl thiomorpholine-4-carboxylate 1,1-dioxide.
The reaction mixture was converted in a microwave at 180.degree. C.
for seven minutes. Subsequently, water and ethyl acetate were
added, and the aqueous phase was removed and extracted repeatedly
with ethyl acetate. The combined organic extracts were washed with
water and saturated aqueous sodium chloride solution, dried over
sodium sulphate, filtered and concentrated under reduced pressure.
The residue was taken up in diethyl ether and filtered, and the
filtrate was purified by means of preparative HPLC. Yield: 2.0 g
(51% of theory)
[0431] LC-MS (Method 5B): R.sub.t=0.92 min and 0.94 min (cis/trans
isomers); MS (ESIpos): m/z=447 [M+H].sup.+.
Example 37A
5-[4-(Difluoromethoxy)phenyl]-1-[(1,1-dioxidothiomorpholin-4-yl)carbonyl]p-
iperidine-3-carboxylic acid [racemic cis isomer mixture]
##STR00065##
[0433] According to General Method 4A, 2.7 g (6.1 mmol) of methyl
5-[4-(difluoromethoxy)phenyl]-1-[(1,1-dioxidothiomorpholin-4-yl)carbonyl]-
piperidine-3-carboxylate were reacted with 6.9 g (61.3 mmol) of
potassium tert-butoxide. Yield: 2.1 g (77% of theory)
[0434] LC-MS (Method 5B): R.sub.t=0.82 min; MS (ESIpos): m/z=433
[M+H].sup.+.
Example 38A
[0435] Methyl
5-[4-(difluoromethoxy)phenyl]-1-[(1-oxidothiomorpholin-4-yl)carbonyl]pipe-
ridine-3-carboxylate [cis/trans isomer mixture]
##STR00066##
[0436] 2.2 g (7.7 mmol) of methyl
5-[4-(difluoromethoxy)phenyl]piperidine-3-carboxylate were
dissolved in 14 ml of N-methylpyrrolidone, and admixed with 4.0 ml
(3.0 g, 23.0 mmol) of N,N-diisopropylethylamine and 3.3 g (11.5
mmol) of 4-nitrophenyl thiomorpholine-4-carboxylate 1-oxide. The
reaction mixture was converted in a microwave at 180.degree. C. for
seven minutes. Subsequently, water and ethyl acetate were added,
and the aqueous phase was removed and extracted repeatedly with
ethyl acetate. The combined organic extracts were washed with water
and saturated aqueous sodium chloride solution, dried over sodium
sulphate, filtered and concentrated under reduced pressure. The
residue was purified by means of preparative HPLC. Yield: 2.2 g
(59% of theory)
[0437] LC-MS (Method 5B): R.sub.t=0.90 min and 0.92 min (cis/trans
isomers); MS (ESIpos): m/z=431 [M+H].sup.+.
Example 39A
5-[4-(Difluoromethoxy)phenyl]-1-[(1-oxidothiomorpholin-4-yl)carbonyl]piper-
idine-3-carboxylic acid [racemic cis isomer mixture]
##STR00067##
[0439] According to General Method 4A, 2.7 g (6.3 mmol) of methyl
5-[4-(difluoromethoxy)phenyl]-1-[(1-oxidothiomorpholin-4-yl)carbonyl]pipe-
ridine-3-carboxylate were reacted with 7.1 g (63.3 mmol) of
potassium tert-butoxide. The reaction mixture was concentrated
under reduced pressure, and the residue was suspended in water and
acidified with concentrated hydrochloric acid. The precipitate was
filtered off, washed with water and dried under reduced pressure.
Yield: 1.1 g (34% of theory)
[0440] LC-MS (Method 5B): R.sub.t=0.75 min; MS (ESIpos): m/z=417
[M+H].sup.+.
Example 40A
1-Bromo-4-(2,2,2-trifluoroethyl)benzene
##STR00068##
[0442] A solution of 25.0 g (100 mmol) of 4-bromobenzyl bromide in
1-methyl-2-pyrrolidone (121 ml) was admixed at RT with 4.95 g (26.0
mmol) of copper(I) iodide and 37.5 g (195 mmol) of methyl
2,2-difluoro-2-(fluorosulphonyl)acetate. The mixture was heated to
80.degree. C. and then stirred overnight. The reaction solution was
added to water and extracted with diethyl ether, and the organic
phase was dried over sodium sulphate. After filtering and
concentrating the organic phase under reduced pressure, the residue
was purified by means of column chromatography (silica gel,
cyclohexane/ethyl acetate 20:1). Yield: 16.1 g (67% of theory)
[0443] GC-MS (Method 1F): R.sub.t=2.66 min; MS (ESIpos): m/z=240
[M+H].sup.+.
Example 41A
Methyl 5-[4-(2,2,2-trifluoroethyl)phenyl]nicotinate
##STR00069##
[0445] A solution of 8.00 g (33.5 mmol) of the compound from
Example 40A in toluene (304 ml) was admixed under argon at RT with
10.9 g (50.2 mmol) of the compound from Example 33A in ethanol (100
ml) and 5.10 g (36.8 mmol) of potassium carbonate. After stirring
for 10 min, 3.87 g (3.35 mmol) of
tetrakis(triphenylphosphine)palladium and then 5.83 g (100 mmol) of
potassium fluoride in water (64 ml) were added. The mixture was
stirred under reflux for 8 h, and the reaction solution was cooled
and diluted with ethyl acetate. The reaction solution was washed in
water, and the organic phase was dried over magnesium sulphate,
filtered and concentrated under reduced pressure. The residue was
purified by means of column chromatography (silica gel,
dichloromethane/methanol 100:1.fwdarw.80:1). Yield: 9.20 g (69% of
theory, purity 75%)
[0446] LC-MS (Method 5B): R.sub.t=1.06 min; MS (ESIpos): m/z=296
[M+H].sup.+.
Example 42A
Methyl 5-[4-(2,2,2-trifluoroethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00070##
[0448] A solution of 9.20 g (23.4 mmol) of the compound from
Example 41A in concentrated acetic acid (192 ml) was admixed with
1.94 g of palladium/carbon (10% palladium) and 2.23 g of
platinum(IV) oxide. This was followed by hydrogenation under a
hydrogen atmosphere at standard pressure for 6 h, then addition of
another 1.00 g of palladium/carbon (10% palladium) and 2.00 g of
platinum(IV) oxide, and hydrogenation under a hydrogen atmosphere
at standard pressure overnight. Subsequently, a further 1.00 g of
palladium/carbon (10% palladium) and 3.00 g of platinum(IV) oxide
were added, and hydrogenation was effected under a hydrogen
atmosphere at standard pressure for a further 24 h. The reaction
solution was filtered through Celite, the filter residue was washed
with methanol/water and the combined filtrates were concentrated
under reduced pressure. The residue was taken up in dichloromethane
and then washed with a 1 N aqueous sodium carbonate solution. The
organic phase was dried over sodium sulphate, filtered and
concentrated under reduced pressure. Yield: 6.64 g (85% of theory,
purity 90%)
[0449] LC-MS (Method 2B): R.sub.t=0.83 and 0.84 min (cis/trans
isomers); MS (ESIpos): m/z=302 [M+H].sup.+.
Example 43A
3-Methyl 1-(4-nitrophenyl)
5-[4-(2,2,2-trifluoroethyl)phenyl]piperidine-1,3-dicarboxylate
[racemic cis/trans isomer mixture]
##STR00071##
[0451] A solution of 6.62 g (19.8 mmol, purity 90%) of the compound
from Example 42A in dichloromethane (211 ml) was admixed with 9.65
ml (7.00 g, 69.2 mmol) of triethylamine and then admixed at
0.degree. C. with 3.99 g (19.8 mmol) of 4-nitrophenyl
chloroformate. The mixture was warmed to RT and stirred for 1 h.
The reaction solution was washed with saturated aqueous sodium
hydrogencarbonate solution and water, and the organic phase was
dried over magnesium sulphate, filtered and concentrated under
reduced pressure. Yield: 10.3 g (91% of theory, purity 81%)
[0452] LC-MS (Method 2B): R.sub.t=1.40 and 1.42 min (cis/trans
isomers); MS (ESIpos): m/z=467 [M+H].sup.+.
Example 44A
Methyl
1-(thiomorpholin-4-ylcarbonyl)-5-[4-(2,2,2-trifluoroethyl)phenyl]pi-
peridine-3-carboxylate [racemic cis/trans isomer mixture]
##STR00072##
[0454] A solution of 10.3 g (17.9 mmol, purity 81%) of the compound
from Example 43A in 1-methyl-2-pyrrolidone (65 ml) was admixed with
12.6 ml (13.7 g, 132 mmol) of thiomorpholine and 11.5 ml (8.56 g,
66.2 mmol) of N,N-diisopropylethylamine and then heated in 5
portions in a single-mode microwave (Emrys Optimizer) at
150.degree. C. for 1 h. For workup, the reaction solutions were
combined and purified directly by means of preparative HPLC. Yield:
5.63 g (71% of theory)
[0455] LC-MS (Method 5B): R.sub.t=1.13 and 1.16 min (cis/trans
isomers); MS (ESIpos): m/z=431 [M+H].+-..
Example 45A
1-(Thiomorpholin-4-ylcarbonyl)-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-
e-3-carboxylic acid [racemic cis isomer]
##STR00073##
[0457] 7.74 g (69.0 mmol) of potassium tert-butoxide were added at
RT to a solution of 2.97 g (6.90 mmol) of the compound from Example
44A in methanol (83 ml). The mixture was stirred at 60.degree. C.
overnight. For workup, the methanol was removed under reduced
pressure, the residue was admixed with water and the mixture was
acidified (pH 1) with aqueous 1 N hydrochloric acid solution. The
mixture was extracted with ethyl acetate, and the organic phase was
dried with magnesium sulphate, filtered and concentrated under
reduced pressure. Yield: 2.61 g (76% of theory, purity 84%)
[0458] LC-MS (Method 5B): R.sub.t=1.02 min; MS (ESIpos): m/z=417
[M+H].sup.+.
Example 46A
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00074##
[0460] According to General Method 6A, 300 mg (0.720 mmol) of the
compound from Example 45A and 79.3 mg (0.792 mmol) of
N'-hydroxycyclopropanecarboximidamide were reacted Yield: 160 mg
(45% of theory)
[0461] LC-MS (Method 5B): R.sub.t=1.23 min; MS (ESIpos): m/z=481
[M+H].sup.+.
[0462] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.32 (s, 4H),
3.92 (d, 1H), 3.68-3.52 (m, 3H), 3.44 (br. s., 4H), 3.39-3.33 (m,
1H), 3.03-2.85 (m, 3H), 2.59 (br. s., 5H), 2.28 (d, 1H), 2.16-2.06
(m, 1H), 1.92 (q, 1H), 1.10-1.01 (m, 2H), 0.92-0.85 (m, 2H).
Example 47A
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(2,2,2-trifluoroethyl)ph-
enyl]piperidin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00075##
[0464] According to General Method 6A, 300 mg (0.720 mmol) of the
compound from Example 45A and 93.6 mg (0.792 mmol) of
N'-hydroxy-3-methoxypropanimidamide were reacted Yield: 231 mg (63%
of theory, approx. 15% trans isomer)
[0465] LC-MS (Method 5B): R.sub.t=1.14 min; MS (ESIpos): m/z=499
[M+H].sup.+.
Example 48A
1-Bromo-4-(1,1-difluoroethyl)benzene
##STR00076##
[0467] A solution of 10.0 g (50.2 mmol) of 4-bromoacetophenone in
tetrahydrofuran (20 ml) was admixed with 50.0 ml (151 mmol, 50% in
tetrahydrofuran) of bis(2-methoxyethyl)aminosulphur trifluoride
(Deoxofluor) and 3 drops of methanol, and then stirred under reflux
for 4 days. The reaction mixture was cautiously added dropwise to a
mixture of saturated aqueous sodium hydrogencarbonate solution and
ice (1:1) and then extracted with diethyl ether. The organic phase
was dried over sodium sulphate, filtered and concentrated under
reduced pressure. The residue was purified by means of column
chromatography (silica gel, petroleum ether/dichloromethane 3:1).
Yield: 8.46 g (76% of theory)
[0468] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.70 (d, 2H), 7.52
(d, 2H), 1.96 (t, 3H).
Example 49A
Methyl 5-[4-(1,1-difluoroethyl)phenyl]nicotinate
##STR00077##
[0470] A solution of 2.98 g (13.3 mmol) of the compound from
Example 48A in toluene (25.0 ml) was admixed under argon at RT with
3.62 g (16.7 mmol) of the compound from Example 33A in ethanol (8.4
ml) and 2.03 g (14.7 mmol) of potassium carbonate. After stirring
for 10 min, 1.54 g (1.34 mmol) of
tetrakis(triphenylphosphine)palladium and then 2.33 g (40.0 mmol)
of potassium fluoride in water (5.8 ml) were added. The mixture was
stirred under reflux for 8 h, and the reaction solution was cooled
and diluted with ethyl acetate. The reaction solution was washed in
water, and the organic phase was dried over magnesium sulphate,
filtered and concentrated under reduced pressure. The residue was
purified by means of column chromatography (silica gel,
dichloromethane/methanol 100:1.fwdarw.80:1). Yield: 2.62 g (69% of
theory, 4:1 mixture of methyl and ethyl ester)
[0471] LC-MS (Method 2B): R.sub.t=1.20 min (methyl ester) and 1.28
min (ethyl ester); MS (ESIpos): m/z=278 [M+H]+(methyl ester) and
292 [M+H]+(ethyl ester).
Example 50A
Methyl 5-[4-(1,1-difluoroethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00078##
[0473] A solution of 2.30 g (8.30 mmol) of the compound from
Example 49A in methanol (52 ml) and concentrated hydrogen chloride
solution (6.5 ml) was admixed with 1.05 g of palladium/carbon (10%
palladium) and 1.92 g of platinum(IV) oxide and then hydrogenated
overnight in a hydrogen atmosphere at standard pressure. The
reaction solution was filtered through Celite, the filter residue
was washed with methanol/water and the combined filtrates were
concentrated under reduced pressure. The residue was taken up in
dichloromethane and then washed with a 1 N aqueous sodium carbonate
solution. The organic phase was dried over sodium sulphate,
filtered and concentrated under reduced pressure. Yield: 2.30 g
(81% of theory, purity 82%)
[0474] LC-MS (Method 2B): R.sub.t=0.80 and 0.81 min (cis/trans
isomers); MS (ESIpos): m/z=284 [M+H].+-..
Example 51A
3-Methyl 1-(4-nitrophenyl)
5-[4-(1,1-difluoroethyl)phenyl]piperidine-1,3-dicarboxylate
[racemic cis/trans isomer mixture]
##STR00079##
[0476] A solution of 1.30 g (3.78 mmol, purity 82%) of the compound
from Example 50A in dichloromethane (44 ml) was admixed with 1.84
ml (1.34 g, 13.2 mmol) of triethylamine and then admixed at
0.degree. C. with 762 mg (3.78 mmol) of 4-nitrophenyl
chloroformate. The mixture was warmed to RT and stirred for 2 days.
The reaction solution was washed with saturated aqueous sodium
hydrogencarbonate solution and water, and the organic phase was
dried over magnesium sulphate, filtered and concentrated under
reduced pressure. Yield: 1.93 g (92% of theory, purity 81%, 2:1
mixture of methyl and ethyl ester)
[0477] LC-MS (Method 5B): R.sub.t=2.58 min and 2.61 (methyl ester,
cis/trans isomers) and 2.68 and 2.70 (ethyl ester, cis/trans
isomers); MS (ESIpos): m/z=278 [M+H]+(methyl ester) and 292
[M+H]+(ethyl ester).
Example 52A
Methyl
5-[4-(1,1-difluoroethyl)phenyl]-1-(thiomorpholin-4-ylcarbonyl)piper-
idine-3-carboxylate [racemic cis/trans isomer mixture]
##STR00080##
[0479] A solution of 1.94 g (3.50 mmol, purity 81%) of the compound
from Example 51A in 1-methyl-2-pyrrolidone (18 ml) was admixed with
1.99 ml (2.17 g, 21.0 mmol) of thiomorpholine and 1.83 ml (1.36 g,
10.5 mmol) of N,N-diisopropylethylamine and then heated in 3
portions in a single-mode microwave (Emrys Optimizer) at
150.degree. C. for 45 min For workup, the reaction solutions were
combined and purified directly by means of preparative HPLC. Yield:
530 mg (34% of theory)
[0480] LC-MS (Method 5B): R.sub.t=2.28 and 2.35 min (cis/trans
isomers); MS (ESIpos): m/z=413 [M+H].sup.+.
Example 53A
5-[4-(1,1-Difluoroethyl)phenyl]-1-(thiomorpholin-4-ylcarbonyl)piperidine-3-
-carboxylic acid [racemic cis/trans isomer mixture]
##STR00081##
[0482] 1.44 g (12.8 mmol) of potassium tert-butoxide were added at
RT to a solution of 528 mg (1.28 mmol) of the compound from Example
52A in 15 ml of methanol. The mixture was stirred at 60.degree. C.
overnight. For workup, the methanol was removed under reduced
pressure, the residue was admixed with water and the mixture was
acidified (pH 1) with aqueous 1 N hydrochloric acid solution. The
mixture was extracted with ethyl acetate, and the organic phase was
dried with magnesium sulphate, filtered and concentrated under
reduced pressure. Yield: 471 mg (91% of theory, 2:1 cis/trans
isomer mixture)
[0483] LC-MS (Method 5B): R.sub.t=0.99 and 1.01 min; MS (ESIpos):
m/z=399 [M+H].sup.+.
Example 54A
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(thiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00082##
[0485] According to General Method 6A, 150 mg (0.376 mmol) of the
compound from Example 53A and 41.5 mg (0.414 mmol) of
N'-hydroxycyclopropanecarboximidamide were reacted Yield: 77.9 mg
(44% of theory)
[0486] LC-MS (Method 2B): R.sub.t=1.37 min; MS (ESIpos): m/z=463
[M+H].sup.+.
Example 55A
Methyl 5-[4-(2-hydroxyethyl)phenyl]nicotinate
##STR00083##
[0488] According to General Method 3A, 6.00 g (29.8 mmol) of
2-(4-bromophenyl)ethanol and 19.6 g (74.6 mmol) of methyl
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate were
reacted. Yield: 6.12 g (74% of theory)
[0489] LC-MS (Method 2B): R.sub.t=0.86 min; MS (ESIpos): m/z=258
[M+H].sup.+.
Example 56A
Methyl 5-[4-(2-hydroxyethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00084##
[0491] A solution of 5.40 g (19.6 mmol) of the compound from
Example 55A in concentrated acetic acid (124 ml) was admixed with
1.00 g of palladium/carbon (10% palladium) and 1.00 g of
platinum(IV) oxide. This was followed by hydrogenation under a
hydrogen atmosphere at standard pressure for 6 h, then addition of
another 1.00 g of palladium/carbon (10% palladium) and 1.00 g of
platinum(IV) oxide, and hydrogenation under a hydrogen atmosphere
at standard pressure overnight. This was followed by hydrogenation
in a Parr apparatus under a 3 bar hydrogen atmosphere for a further
2 h. The reaction solution was filtered through Celite, the filter
residue was washed with methanol/water and the combined filtrates
were concentrated under reduced pressure. The residue was
codistilled repeatedly with toluene and then dried under high
vacuum. Yield: 6.63 g (56% of theory, purity 44%)
[0492] LC-MS (Method 2B): R.sub.t=0.36 min and 0.40 min (cis/trans
isomers); MS (ESIpos): m/z=264 [M+H].sup.+.
Example 57A
[0493] Methyl
1-acetyl-5-[4-(2-hydroxyethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00085##
[0494] A solution of 5.58 g (9.33 mmol, purity 44%) of the compound
from Example 56A in dichloromethane (80 ml) was admixed with 2.60
ml (1.89 g, 18.7 mmol) of triethylamine and then cooled to
0.degree. C. At this temperature, 0.33 ml (0.37 g, 4.67 mmol) of
acetyl chloride was added dropwise and the mixture was stirred for
2 h. A further 0.13 ml (0.15 g, 1.86 mmol) of acetyl chloride was
added and the mixture was stirred for 1 h. Subsequently, the
reaction solution was washed with aqueous 1 N hydrochloric acid,
and the organic phase was dried over magnesium sulphate, filtered
and concentrated under reduced pressure. The residue was purified
by means of column chromatography (silica gel,
dichloromethane/methanol 30:1), and the crude product obtained was
purified once more by means of preparative HPLC. Yield: 1.17 g (41%
of theory)
[0495] LC-MS (Method 5B): R.sub.t=0.72 min and 0.74 min (cis/trans
isomers); MS (ESIpos): m/z=306 [M+H].sup.+.
Example 58A
[0496] Methyl
1-acetyl-5-[4-(2,2-difluoroethyl)phenyl]piperidine-3-carboxylate
[racemic cis/trans isomer mixture]
##STR00086##
[0497] A solution of 631 mg (2.05 mmol) of the compound from
Example 57A in dichloromethane (20.8 ml) was admixed with 1.45 ml
(1.60 g, 20.5 mmol) of dimethyl sulphoxide and 1.78 ml (1.32 g,
10.2 mmol) of N,N-diisopropylamine. Subsequently, 1.30 g (8.18
mmol) of sulphur trioxide-pyridine complex were added at
-20.degree. C. and the mixture was stirred overnight, in the course
of which it was warmed slowly to RT. The reaction solution was
diluted with dichloromethane, and the organic phase was washed with
water, dried over magnesium sulphate, filtered and concentrated
under reduced pressure. The crude product (778 mg) was subsequently
initially charged in dichloromethane (5.2 ml) and admixed dropwise
at RT with 0.50 ml (615 mg, 3.81 mmol) of diethylaminosulphur
trifluoride (DAST). The mixture was stirred at RT for 4 h and then
the reaction was ended by cautiously adding 2 N aqueous sodium
carbonate solution. After phase separation, the organic phase was
dried over magnesium sulphate, filtered and concentrated under
reduced pressure. The crude product was purified by means of
preparative HPLC. Yield: 120 mg (24% of theory, purity 61%, approx.
2:1 cis/trans isomer mixture)
[0498] LC-MS (Method 2B): R.sub.t=1.07 min and 1.09 min (cis/trans
isomers); MS (ESIpos): m/z=326 [M+H].sup.+.
Example 59A
1-Acetyl-5-[4-(2,2-difluoroethyl)phenyl]piperidine-3-carboxylic
acid [racemic cis isomer]
##STR00087##
[0500] 410 mg (3.65 mmol) of potassium tert-butoxide were added at
RT to a solution of 205 mg (0.365 mmol, purity 61%) of the compound
from Example 58A in methanol (6.9 ml). The mixture was stirred at
60.degree. C. overnight. For workup, the methanol was removed under
reduced pressure, the residue was admixed with water and the
mixture was acidified (pH 1) with aqueous 1 N hydrochloric acid
solution. The mixture was extracted with ethyl acetate, and the
organic phase was dried with magnesium sulphate, filtered and
concentrated under reduced pressure. Yield: 153 mg (67% of theory,
purity 50%)
[0501] LC-MS (Method 5B): R.sub.t=1.74 min; MS (ESIpos): m/z=312
[M+H].sup.+.
Example 60A
1-{3-[4-(2,2-Difluoroethyl)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-
-yl]piperidin-1-yl}-ethanone [racemic cis isomer]
##STR00088##
[0503] According to General Method 6A, 153 mg (0.270 mmol, purity
50%) of the compound from Example 59A and 41.3 mg (0.297 mmol,
purity 85%) of N'-hydroxy-3-methoxypropanimidamide were reacted
Yield: 46.6 mg (32% of theory, purity 72%)
[0504] LC-MS (Method 2B): R.sub.t=1.07 min; MS (ESIpos): m/z=394
[M+H].sup.+.
Example 61A
3-[4-(2,2-Difluoroethyl)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]piperidine [racemic cis isomer]
##STR00089##
[0506] A solution of 45.0 mg (0.083 mmol, purity 72%) of the
compound from Example 60A in ethanol (10.0 ml) was admixed with 69
.mu.l (15 mg, 0.42 mmol) of concentrated hydrochloric acid.
Subsequently, the mixture was stirred under reflux for 24 h, and
the reaction solution was diluted with water and washed with
diethyl ether. The aqueous phase was alkalized and extracted with
dichloromethane. The organic phase was dried over magnesium
sulphate, filtered and concentrated under reduced pressure. Yield:
36.3 mg (87% of theory, purity 70%)
[0507] LC-MS (Method 5B): R.sub.t=0.71 min; MS (ESIpos): m/z=352
[M+H].sup.+.
Example 62A
4-Nitrophenyl
3-[4-(2,2-difluoroethyl)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-y-
l]piperidine-1-carboxylate [racemic cis isomer]
##STR00090##
[0509] A solution of 36.3 mg (0.061 mmol, purity 70%) of the
compound from Example 61A in dichloromethane (2.0 ml) was admixed
with 0.03 ml (21.6 mg, 0.21 mmol) of triethylamine and then 12.3 mg
(0.061 mmol) of 4-nitrophenyl chloroformate were added at RT. The
mixture was stirred at RT for 2 h and then the reaction solution
was washed with saturated aqueous sodium hydrogencarbonate solution
and water, and the organic phase was dried over magnesium sulphate,
filtered and concentrated under reduced pressure. Yield: 56.2 mg
(91% of theory, purity 60%)
[0510] LC-MS (Method 4B): R.sub.t=2.56 min; MS (ESIpos): m/z=517
[M+H].sup.+.
Example 63A
{3-[4-(2,2-Difluoroethyl)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-y-
l]piperidin-1-yl}(thiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00091##
[0512] A solution of 56.0 mg (0.065 mmol, purity 60%) of the
compound from Example 62A in 1-methyl-2-pyrrolidone (2.0 ml) was
admixed with 37.0 pi (40.0 mg, 0.390 mmol) of thiomorpholine and
34.0 pi (25.0 mg, 0.195 mmol) of N,N-diisopropylethylamine and then
heated in a single-mode microwave (Emrys Optimizer) at 150.degree.
C. for 30 min For workup, the reaction solutions were combined and
purified directly by means of preparative HPLC. Yield: 14.7 mg (47%
of theory)
[0513] LC-MS (Method 5B): R.sub.t=1.09 min; MS (ESIpos): m/z=481
[M+H].sup.+.
Example 64A
N-Hydroxy-1-methoxycyclopropanecarboximidamide
##STR00092##
[0515] 100 mg (1.03 mmol) of 1-methoxycyclopropanecarboxamide [L.
N. Owen, H. M. Babatunde Somade, J. Chem. Soc. 1947, 1030-1034] in
tetrahydrofuran (22.7 ml) was admixed with 1.51 g (6.08 mmol) of
methyl N-(triethylammoniumsulphonyl)carbamate (Burgess reagent) and
then stirred at 60.degree. C. for 1.5 h. The reaction mixture was
admixed with dichloromethane and water, and the organic phase was
dried over magnesium sulphate, filtered and concentrated under
reduced pressure (637 mg of crude product). 100 mg of the crude
product were initially charged in ethanol (1.2 ml), admixed with
107 mg (1.55 mmol) of hydroxylammonium chloride and 0.17 ml (125
mg, 1.24 mg) of triethylamine and then stirred under reflux
overnight. The reaction solution was concentrated under reduced
pressure, and the residue was admixed with saturated aqueous sodium
chloride solution and then extracted with dichloromethane. The
organic phase was dried over magnesium sulphate, filtered and
concentrated under reduced pressure. The residue was subsequently
stirred with ethyl acetate, the insoluble salts were filtered off
and the filtrate was concentrated under reduced pressure. Yield:
32.3 mg (23% of theory)
[0516] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=9.09 (br. s.,
1H), 5.39 (br. s., 2H), 3.15 (s, 3H), 0.81 (d, 4H).
Example 65A
{3-[3-(1-Methoxycyclopropyl)-1,2,4-oxadiazol-5-yl]-5-[4-(2,2,2-trifluoroet-
hyl)phenyl]piperidin-1-yl}(thiomorpholin-4-yl)methanone [racemic
cis isomer]
##STR00093##
[0518] According to General Method 6A, 93.1 mg (0.224 mmol) of the
compound from Example 45A and 32.0 mg (0.246 mmol) of
N'-hydroxy-1-methoxycyclopropanecarboximidamide from Example 64A
were reacted. Yield: 31.1 mg (27% of theory)
[0519] LC-MS (Method 5B): R.sub.t=1.22 min; MS (ESIpos): m/z=511
[M+H].sup.+.
[0520] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.33 (s, 4H),
3.94 (d, 1H), 3.69-3.52 (m, 3H), 3.48-3.42 (m, 4H), 3.41-3.34 (m,
4H), 3.06-2.85 (m, 3H), 2.63-2.57 (m, 4H), 2.32-2.25 (m, 1H),
2.02-1.88 (m, 1H), 1.34-1.28 (m, 2H), 1.19-1.11 (m, 2H).
Example 66A
4-Nitrophenyl thiomorpholine-4-carboxylate 1,1-dioxide
##STR00094##
[0522] 17.0 g (99.2 mmol) of thiomorpholine 1,1-dioxide
hydrochloride were initially charged in 100 ml of dichloromethane
and, while cooling with an ice bath, admixed with 20.7 ml (15.1 g,
148.8 mmol) of triethylamine 10.0 g (49.6 mmol) of 4-nitrophenyl
chloroformate were added in portions. The reaction mixture was
stirred at RT for 30 minutes, admixed with water and ethyl acetate
and then filtered. The residue was dried under high vacuum. Yield:
12.4 g (83% of theory)
[0523] LC-MS (Method 5B): R.sub.t=0.75 min; MS (ESIpos): m/z=301
[M+H].sup.+.
[0524] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=8.34-8.28 (m, 2H),
7.55-7.50 (m, 2H), 4.01 (br. s., 2H), 3.87 (br. s., 2H), 3.37 (br.
s., 2H), 3.28 (br. s., 2H).
Working Examples
General Method 1: Sulphoxide Formation
[0525] A solution of the appropriate thioether (1.0 eq) in
dichloromethane (approx. 20-40 ml/mmol) is admixed at RT with 50%
meta-chloroperoxybenzoic acid (0.9-1.0 eq.). The reaction mixture
is stirred at room temperature for 30 min The solvent is removed
under reduced pressure and the residue is purified by means of
preparative HPLC.
General Method 2: Sulphone Formation
[0526] A solution of the appropriate thioether (1.0 eq) in
dichloromethane (approx. 20-40 ml/mmol) is admixed at RT with 50%
meta-chloroperoxybenzoic acid (2.5 eq.). The reaction mixture is
stirred at room temperature for 30 min. The solvent is removed
under reduced pressure and the residue is purified by means of
preparative HPLC.
Example 1
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl-
]piperidin-1-yl}-(oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00095##
[0528] According to General Method 1, 100 mg (0.200 mmol) of the
compound from Example 26A were reacted. Yield: 90 mg (87% of
theory)
[0529] LC-MS (Method 5B): R.sub.t=0.97 min; MS (ESIpos): m/z=517
[M+H].sup.+;
[0530] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.48 (d, 2H),
7.33 (d, 3H), 3.99 (d, 1H), 3.69-3.66 (m, 3H), 3.65-3.58 (m, 4H),
3.57-3.48 (m, 3H), 3.23 (s, 3H), 3.09-2.88 (m, 7H), 2.75-2.66 (m,
3H), 2.35-2.28 (m, 1H), 2.00 (m, 1H).
Example 2
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]pipe-
ridin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00096##
[0532] According to General Method 1, 100 mg (0.207 mmol) of the
compound from Example 27A were reacted. Yield: 95 mg (91% of
theory)
[0533] LC-MS (Method 5B): R.sub.t=1.05 min; MS (ESIpos): m/z=499
[M+H].sup.+;
[0534] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.47 (d, 2H),
7.33 (d, 2H), 3.95 (d, 1H), 3.67-3.48 (m, 5H), 3.05-2.85 (m, 5H),
2.75-2.65 (m, 2H), 2.28 (d, 1H), 2.14-2.08 (m, 1H), 1.94 (q, 1H),
1.09-1.01 (m, 2H), 0.92-0.84 (m, 2H).
Example 3
[3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-(4-ethylphenyl)piperidin-1-yl](1-
-oxidothiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00097##
[0536] According to General Method 1, 55 mg (0.130 mmol) of the
compound from Example 9A were reacted. Yield: 43 mg (75% of
theory)
[0537] LC-MS (Method 5B): R.sub.t=1.06 min; MS (ESIpos): m/z=443
[M+H].sup.+;
[0538] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.24 (d, 2H),
7.17 (d, 2H), 3.96 (d, 2H), 3.67-3.46 (m, 5H), 3.045-2.85 (m, 5H),
2.74-2.66 (m, 2H), 2.57 (q, 2H), 2.25 (d, 1H), 2.14-2.06 (m, 1H),
1.91 (q, 1H), 1.16 (t, 3H), 1.08-1.02 (m, 2H), 0.91-0.86 (m,
2H.)
Example 4
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-(4-ethylphenyl)piperidin-1-y-
l}(1-oxidothiomorpholin-4-yl)methanone [racemic cis isomer]
##STR00098##
[0540] According to General Method 1, 50 mg (0.109 mmol) of the
compound from Example 11A were reacted. Yield: 17 mg (32% of
theory)
[0541] LC-MS (Method 5B): R.sub.t=1.01 min; MS (ESIpos): m/z=475
[M+H].sup.+;
[0542] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.17 (d, 2H), 3.98 (d, 2H), 3.71 (t, 2H), 3.67-3.47 (m, 4H), 3.43
(q, 1H), 3.07-2.89 (m, 3H), 2.75-2.66 (m, 3H), 2.57 (q, 3H), 1.95
(q, 1H), 1.24 (br s, 1H), 1.16 (t, 3H), 1.07 (t, 3H).
Example 5
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-(4-ethylphenyl)piperidin-1-y-
l}(1-oxidothiomorpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00099##
[0544] Enantiomer separation of the racemate from Example 4
according to Method 6D gave 37.7 mg of the title compound from
Example 5 and 20.0 mg of the title compound from Example 6.
[0545] LC-MS (Method 5B): R.sub.t=1.01 min; MS (ESIpos): m/z=475
[M+H].sup.+;
[0546] HPLC (Method 1E): R.sub.t=6.48 min, >99.5% ee;
[0547] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.17 (d, 2H), 3.98 (d, 2H), 3.71 (t, 2H), 3.67-3.47 (m, 4H), 3.43
(q, 1H), 3.07-2.89 (m, 3H), 2.75-2.66 (m, 3H), 2.57 (q, 3H), 1.95
(q, 1H), 1.24 (br s, 1H), 1.16 (t, 3H), 1.07 (t, 3H).
Example 6
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-(4-ethylphenyl)piperidin-1-y-
l}(1-oxidothiomorpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00100##
[0549] Enantiomer separation of the racemate from Example 4
according to Method 6D gave 37.7 mg of the title compound from
Example 5 and 20.0 mg of the title compound from Example 6.
[0550] LC-MS (Method 5B): R.sub.t=1.01 min; MS (ESIpos): m/z=475
[M+H].sup.+;
[0551] HPLC (Method 1E): R.sub.t=7.27 min, >99.5% ee;
[0552] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.17 (d, 2H), 3.98 (d, 2H), 3.71 (t, 2H), 3.67-3.47 (m, 4H), 3.43
(q, 1H), 3.07-2.89 (m, 3H), 2.75-2.66 (m, 3H), 2.57 (q, 3H), 1.95
(q, 1H), 1.24 (br s, 1H), 1.16 (t, 3H), 1.07 (t, 3H).
Example 7
{3-[3-(2-Hydroxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00101##
[0554] According to General Method 1, 80 mg (0.170 mmol) of the
compound from Example 20A were reacted. Yield: 77 mg (91% of
theory)
[0555] LC-MS (Method 5B): R.sub.t=0.85 min; MS (ESIpos): m/z=487
[M+H].sup.+;
[0556] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.77 (t, 1H), 3.99 (d, 1H), 3.74 (q, 2H), 3.68-3.59
(m, 3H), 3.57-3.48 (m, 2H), 3.48-3.38 (m, 1H), 3.12-3.01 (m, 3H),
2.98-2.86 (m 2H), 2.85-2.80 (m, 2H), 1.55 (q, 1H).
Example 8
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(trifluoromethoxy)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00102##
[0558] According to General Method 2, 150 mg (0.300 mmol) of the
compound from Example 26A were reacted. Enantiomer separation of
the racemate according to Method 1D gave 65.0 mg of the title
compound from Example 8 and 72.0 mg of the title compound from
Example 9.
[0559] LC-MS (Method 5B): R.sub.t=1.04 min; MS (ESIpos): m/z=533
[M+H].sup.+;
[0560] HPLC (Method 2E): R.sub.t=15.64 min, >99.5% ee;
[0561] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.48 (d, 2H),
7.33 (d, 2H), 4.03 (d, 1H), 3.70-3.58 (m, 7H), 3.45-3.35 (m, 1H),
3.23 (s, 3H), 3.21-3.15 (m, 4H), 3.12-2.90 (m, 5H), 2.33 (d, 1H),
1.97 (q, 1H).
Example 9
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(trifluoromethoxy)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00103##
[0563] According to General Method 2, 150 mg (0.300 mmol) of the
compound from Example 26A were reacted. Enantiomer separation of
the racemate according to Method 1D gave 65.0 mg of the title
compound from Example 8 and 72.0 mg of the title compound from
Example 9.
[0564] LC-MS (Method 5B): R.sub.t=1.04 min; MS (ESIpos): m/z=533
[M+H].sup.+;
[0565] HPLC (Method 2E): R.sub.t=42.42 min, >99.5% ee;
[0566] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.48 (d, 2H),
7.33 (d, 2H), 4.03 (d, 1H), 3.70-3.58 (m, 7H), 3.45-3.35 (m, 1H),
3.23 (s, 3H), 3.21-3.15 (m, 4H), 3.12-2.90 (m, 5H), 2.33 (d, 1H),
1.97 (q, 1H).
Example 10
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]pipe-
ridin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00104##
[0568] 136 mg (0.281 mmol) of
{3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]-pi-
peridin-1-yl}(thiomorpholin-4-yl)methanone [racemic cis isomer]
(Example 27A) in dichloromethane (11.6 ml) were admixed at RT with
243 mg (0.703 mmol) of meta-chloroperbenzoic acid and then stirred
for 30 min The reaction solution was concentrated under reduced
pressure, and the residue was taken up in acetonitrile and purified
by means of preparative HPLC. Enantiomer separation of 136 mg of
the racemate according to Method 2D gave 61.7 mg of the title
compound from Example 10 and 59.6 mg of the title compound from
Example 11.
[0569] LC-MS (Method 5B): R.sub.t=1.12 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0570] HPLC (Method 3E): R.sub.t=4.26 min, >99.05% ee;
[0571] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.47 (d, 2H),
7.33 (d, 2H), 3.99 (d, 1H), 3.67-3.56 (m, 5H), 3.40-3.33 (m, 1H),
3.20-3.14 (m, 4H), 3.08-2.96 (m, 3H), 2.28 (d, 1H), 2.14-2.06 (m,
1H), 1.94 (q, 1H), 1.08-1.03 (m, 2H), 0.91-0.86 (m, 2H).
Example 11
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]pipe-
ridin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00105##
[0573] 136 mg (0.281 mmol) of
{3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethoxy)phenyl]-pi-
peridin-1-yl}(thiomorpholin-4-yl)methanone [racemic cis isomer]
(Example 27A) in dichloromethane (11.6 ml) were admixed at RT with
243 mg (0.703 mmol) of meta-chloroperbenzoic acid and then stirred
for 30 min. The reaction solution was concentrated under reduced
pressure, and the residue was taken up in acetonitrile and purified
by means of preparative HPLC. Enantiomer separation of 136 mg of
the racemate according to Method 2D gave 61.7 mg of the title
compound from Example 10 and 59.6 mg of the title compound from
Example 11.
[0574] LC-MS (Method 5B): R.sub.t=1.12 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0575] HPLC (Method 3E): R.sub.t=5.68 min, >99.0% ee;
[0576] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.47 (d, 2H),
7.33 (d, 2H), 3.99 (d, 1H), 3.67-3.56 (m, 5H), 3.40-3.33 (m, 1H),
3.20-3.14 (m, 4H), 3.08-2.96 (m, 3H), 2.28 (d, 1H), 2.14-2.06 (m,
1H), 1.94 (q, 1H), 1.08-1.03 (m, 2H), 0.91-0.86 (m, 2H).
Example 12
(1,1-Dioxidothiomorpholin-4-yl){3-(4-ethylphenyl)-5-[3-(2-methoxyethyl)-1,-
2,4-oxadiazol-5-yl]-piperidin-1-yl}methanone [enantiomerically pure
cis isomer]
##STR00106##
[0578] According to General Method 2, 77.0 mg (0.173 mmol) of the
compound from Example 8A were reacted. Enantiomer separation of
74.9 mg of the racemate according to Method 3D gave 36.0 mg of the
title compound from Example 12 and 35.0 mg of the title compound
from Example 13.
[0579] LC-MS (Method 2B): R.sub.t=1.17 min; MS (ESIpos): m/z=477
[M+H].sup.+;
[0580] HPLC (Method 4E): R.sub.t=5.49 min, >99.0% ee;
[0581] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.17 (d, 2H), 4.03 (d, 1H), 3.73-3.56 (m, 7H), 3.46-3.35 (m, 1H),
3.23 (s, 3H), 3.17 (br s, 4H), 3.06 (t, 1H), 3.01-2.83 (m, 4H),
2.58 (d, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.16 (t, 3H).
Example 13
(1,1-Dioxidothiomorpholin-4-yl){3-(4-ethylphenyl)-5-[3-(2-methoxyethyl)-1,-
2,4-oxadiazol-5-yl]-piperidin-1-yl}methanone [enantiomerically pure
cis isomer]
##STR00107##
[0583] According to General Method 2, 77.0 mg (0.173 mmol) of the
compound from Example 8A were reacted. Enantiomer separation of
74.9 mg of the racemate according to Method 3D gave 36.0 mg of the
title compound from Example 12 and 35.0 mg of the title compound
from Example 13.
[0584] LC-MS (Method 2B): R.sub.t=1.17 min; MS (ESIpos): m/z=477
[M+H].sup.+;
[0585] HPLC (Method 4E): R.sub.t=12.07 min, >99.0% ee;
[0586] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.17 (d, 2H), 4.03 (d, 1H), 3.73-3.56 (m, 7H), 3.46-3.35 (m, 1H),
3.23 (s, 3H), 3.17 (br s, 4H), 3.06 (t, 1H), 3.01-2.83 (m, 4H),
2.58 (d, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.16 (t, 3H).
Example 14
[3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-(4-ethylphenyl)piperidin-1-yl](1-
,1-dioxidothiomorpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00108##
[0588] According to General Method 2, 55 mg (0.130 mmol) of the
compound from Example 9A were reacted. Enantiomer separation of
53.3 mg of the racemate according to Method 4D gave 23.0 mg of the
title compound from Example 14 and 23.0 mg of the title compound
from Example 15.
[0589] LC-MS (Method 2B): R.sub.t=1.30 min; MS (ESIpos): m/z=459
[M+H].sup.+;
[0590] HPLC (Method 5E): R.sub.t=8.89 min, >99.0% ee;
[0591] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.16 (d, 2H), 3.99 (d, 1H), 3.67-3.55 (m, 5H), 3.39-3.32 (m, 1H),
3.17 (br s, 4H), 3.07-2.91 (m, 2H), 2.91-2.81 (m, 1H), 2.62-2.55
(m, 2H), 2.26 (d, 1H), 2.16-2.08 (m, 1H), 1.91 (q, 1H), 1.16 (t,
3H), 1.10-1.02 (m, 2H), 0.92-0.85 (m, 2H).
Example 15
[3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-(4-ethylphenyl)piperidin-1-yl](1-
,1-dioxidothiomorpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00109##
[0593] According to General Method 2, 55.5 mg (0.130 mmol) of the
compound from Example 9A were reacted. Enantiomer separation of
53.3 mg of the racemate according to Method 4D gave 23.0 mg of the
title compound from Example 14 and 23.0 mg of the title compound
from Example 15.
[0594] LC-MS (Method 2B): R.sub.t=1.27 min; MS (ESIpos): m/z=459
[M+H].sup.+;
[0595] HPLC (Method 5E): R.sub.t=12.06 min, >99.0% ee;
[0596] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (d, 2H),
7.16 (d, 2H), 3.99 (d, 1H), 3.67-3.55 (m, 5H), 3.39-3.32 (m, 1H),
3.17 (br s, 4H), 3.07-2.91 (m, 2H), 2.91-2.81 (m, 1H), 2.62-2.55
(m, 2H), 2.26 (d, 1H), 2.16-2.08 (m, 1H), 1.91 (q, 1H), 1.16 (t,
3H), 1.10-1.02 (m, 2H), 0.92-0.85 (m, 2H).
Example 16
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethyl)phenyl]piper-
idin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00110##
[0598] According to General Method 2, 269 mg (0.578 mmol) of the
compound from Example 17A were reacted. Enantiomer separation of
292 mg of the racemate according to Method 1D gave 57.8 mg of the
title compound from Example 16 and 99.7 mg of the title compound
from Example 17.
[0599] LC-MS (Method 2B): R.sub.t=1.26 min; MS (ESIpos): m/z=499
[M+H].sup.+;
[0600] HPLC (Method 1E): R.sub.t=10.53 min, >99.0% ee;
[0601] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.57 (d, 2H), 4.00 (d, 1H), 3.67 (d, 1H), 3.61 (br s, 4H),
3.44-3.33 (m, 1H), 3.17 (br s, 4H), 3.12-2.98 (m, 3H), 2.31 (d,
1H), 2.11 (dt, 1H), 1.98 (q, 1H), 1.12-1.00 (m, 2H), 0.95-0.84 (m,
2H).
Example 17
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethyl)phenyl]piper-
idin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00111##
[0603] According to General Method 2, 269 mg (0.578 mmol) of the
compound from Example 17A were reacted. Enantiomer separation of
292 mg of the racemate according to Method 1D gave 57.8 mg of the
title compound from Example 16 and 99.7 mg of the title compound
from Example 17.
[0604] LC-MS (Method 2B): R.sub.t=1.26 min; MS (ESIpos): m/z=499
[M+H].sup.+;
[0605] HPLC (Method 1E): R.sub.t=16.04 min, >99.0% ee;
[0606] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.57 (d, 2H), 4.00 (d, 1H), 3.67 (d, 1H), 3.61 (br s, 4H),
3.44-3.33 (m, 1H), 3.17 (br s, 4H), 3.12-2.98 (m, 3H), 2.31 (d,
1H), 2.11 (dt, 1H), 1.98 (q, 1H), 1.12-1.00 (m, 2H), 0.95-0.84 (m,
2H).
Example 18
(1,1-Dioxidothiomorpholin-4-yl)
{3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl-
]piperidin-1-yl}methanone [enantiomerically pure cis isomer]
##STR00112##
[0608] According to General Method 2, 317 mg (0.616 mmol) of the
compound from Example 28A were reacted. Enantiomer separation of
294 mg of the racemate according to Method 2D gave 132 mg of the
title compound from Example 18 and 129 mg of the title compound
from Example 19.
[0609] LC-MS (Method 6B): R.sub.t=2.34 min; MS (ESIpos): m/z=547
[M+H].sup.+;
[0610] HPLC (Method 3E): R.sub.t=4.60 min, >99.0% ee;
[0611] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.48 (d, 2H),
7.33 (d, 2H), 4.03 (d, 1H), 3.71 (t, 2H), 3.68-3.56 (m, 5H), 3.43
(q, 3H), 3.17 (br s, 4H), 3.07 (t, 1H), 3.01 (d, 2H), 2.93 (t, 2H),
2.32 (d, 1H), 2.05-1.91 (m, 1H), 1.07 (t, 3H).
Example 19
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-
-5-[4-(trifluoromethoxy)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00113##
[0613] According to General Method 2, 317 mg (0.616 mmol) of the
compound from Example 28A were reacted. Enantiomer separation of
294 mg of the racemate according to Method 2D gave 132 mg of the
title compound from Example 18 and 129 mg of the title compound
from Example 19.
[0614] LC-MS (Method 6B): R.sub.t=2.34 min; MS (ESIpos): m/z=547
[M+H].sup.+;
[0615] HPLC (Method 3E): R.sub.t=11.53 min, >99.0% ee;
[0616] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.48 (d, 2H),
7.33 (d, 2H), 4.03 (d, 1H), 3.71 (t, 2H), 3.68-3.56 (m, 5H), 3.43
(q, 3H), 3.17 (br s, 4H), 3.07 (t, 1H), 3.01 (d, 2H), 2.93 (t, 2H),
2.32 (d, 1H), 2.05-1.91 (m, 1H), 1.07 (t, 3H).
Example 20
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(trifluoromethyl)phenyl]piper-
idin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00114##
[0618] According to General Method 1, 50.0 mg (0.107 mmol) of the
compound from Example 17A were reacted. Yield: 4.3 mg (8% of
theory)
[0619] LC-MS (Method 5B): R.sub.t=1.05 min; MS (ESIpos): m/z=483
[M+H].sup.+.
Example 21
{3-[3-(2-Hydroxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethoxy)phenyl-
]piperidin-1-yl}-(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00115##
[0621] According to General Method 1, 100.0 mg (0.206 mmol) of the
compound from Example 29A were reacted. Yield: 105.2 mg (99% of
theory)
[0622] LC-MS (Method 5B): R.sub.t=0.89 min; MS (ESIpos): m/z=503
[M+H].sup.+.
Example 22
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]p-
iperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00116##
[0624] According to General Method 1, 50.0 mg (0.100 mmol) of the
compound from Example 19A were reacted. Yield: 50.2 mg (92% of
theory)
[0625] LC-MS (Method 5B): R.sub.t=1.02 min; MS (ESIpos): m/z=515
[M+H].sup.+;
Example 23
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]p-
iperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00117##
[0627] Enantiomer separation of 50.2 mg of the racemate from
Example 22 according to Method 1D gave 25.5 mg of the title
compound from Example 23 and 22.4 mg of the title compound from
Example 24.
[0628] LC-MS (Method 2B): R.sub.t=1.14 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0629] HPLC (Method 2E): R.sub.t=7.51 min, >99.0% ee;
[0630] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.00 (d, 1H), 3.71 (t, 2H), 3.63 (d, 3H), 3.57-3.48
(m, 2H), 3.43 (q, 3H), 3.14-3.00 (m, 3H), 2.93 (t, 4H), 2.77-2.65
(m, 2H), 2.35 (d, 1H), 2.10-1.95 (m, 1H), 1.06 (m, 3H).
Example 24
{3-[3-(2-Ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]p-
iperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00118##
[0632] Enantiomer separation of 50.2 mg of the racemate from
Example 22 according to Method 1D gave 25.5 mg of the title
compound from Example 23 and 22.4 mg of the title compound from
Example 24.
[0633] LC-MS (Method 2B): R.sub.t=1.14 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0634] HPLC (Method 2E): R.sub.t=13.93 min, >99.0% ee;
[0635] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.00 (d, 1H), 3.71 (t, 2H), 3.63 (d, 3H), 3.57-3.48
(m, 2H), 3.43 (q, 3H), 3.14-3.00 (m, 3H), 2.93 (t, 4H), 2.77-2.65
(m, 2H), 2.35 (d, 1H), 2.10-1.95 (m, 1H), 1.06 (m, 3H).
Example 25
{3-(4-Ethylphenyl)-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]piperidin-1--
yl}(1-oxidothio-morpholin-4-yl)methanone [racemic cis isomer]
##STR00119##
[0637] According to General Method 1, 77.0 mg (0.173 mmol) of the
compound from Example 8A were reacted. Yield: 63.2 mg (79% of
theory)
[0638] LC-MS (Method 5B): R.sub.t=0.97 min; MS (ESIpos): m/z=461
[M+H].sup.+;
[0639] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (m, 2H),
7.17 (m, 2H), 3.99 (d, 1H), 3.72-3.46 (m, 7H), 3.46-3.34 (m, 2H),
3.09-2.98 (m, 1H), 2.97-2.83 (m, 6H), 2.65 (br s, 1H), 2.76-2.64
(m, 2H), 2.58 (d, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.16 (t, 3H);
one proton hidden.
Example 26
{3-(4-Ethylphenyl)-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]piperidin-1--
yl}(1-oxidothio-morpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00120##
[0641] Enantiomer separation of 63.2 mg of the racemate from
Example 25 according to Method 6D gave 17.8 mg of the title
compound from Example 26 and 18.7 mg of the title compound from
Example 27.
[0642] LC-MS (Method 5B): R.sub.t=0.97 min; MS (ESIpos): m/z=461
[M+H].sup.+;
[0643] HPLC (Method 2E): R.sub.t=6.48 min, >99.0% ee;
[0644] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (m, 2H),
7.17 (m, 2H), 3.99 (d, 1H), 3.72-3.46 (m, 7H), 3.46-3.34 (m, 2H),
3.09-2.98 (m, 1H), 2.97-2.83 (m, 6H), 2.65 (br s, 1H), 2.76-2.64
(m, 2H), 2.58 (d, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.16 (t, 3H);
one proton hidden.
Example 27
{3-(4-Ethylphenyl)-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]piperidin-1--
yl}(1-oxidothio-morpholin-4-yl)methanone [enantiomerically pure cis
isomer]
##STR00121##
[0646] Enantiomer separation of 63.2 mg of the racemate from
Example 25 according to Method 6D gave 17.8 mg of the title
compound from Example 26 and 18.7 mg of the title compound from
Example 27.
[0647] LC-MS (Method 5B): R.sub.t=0.97 min; MS (ESIpos): m/z=461
[M+H].sup.+;
[0648] HPLC (Method 2E): R.sub.t=7.97 min, >99.0% ee;
[0649] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (m, 2H),
7.17 (m, 2H), 3.99 (d, 1H), 3.72-3.46 (m, 7H), 3.46-3.34 (m, 2H),
3.09-2.98 (m, 1H), 2.97-2.83 (m, 6H), 2.65 (br s, 1H), 2.76-2.64
(m, 2H), 2.58 (d, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.16 (t, 3H);
one proton hidden.
Example 28
(1,1-Dioxidothiomorpholin-4-yl)
{3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl-
]piperidin-1-yl}methanone [enantiomerically pure cis isomer]
##STR00122##
[0651] According to General Method 2, 269 mg (0.556 mmol) of the
compound from Example 18A were reacted. Enantiomer separation of
the racemate according to Method 5D gave 126 mg of the title
compound from Example 28 and 122 mg of the title compound from
Example 29.
[0652] LC-MS (Method 6B): R.sub.t=2.18 min; MS (ESIpos): m/z=517
[M+H].sup.+;
[0653] HPLC (Method 2E): R.sub.t=4.98 min, >99.0% ee;
[0654] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.03 (d, 1H), 3.68 (t, 3H), 3.62 (br s, 4H),
3.49-3.38 (m, 1H), 3.23 (s, 3H), 3.18 (br s, 4H), 3.14-3.02 (m,
3H), 2.94 (t, 2H), 2.35 (d, 1H), 2.02 (d, 1H).
Example 29
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(trifluoromethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00123##
[0656] According to General Method 2, 269 mg (0.556 mmol) of the
compound from Example 18A were reacted. Enantiomer separation of
the racemate according to Method 5D gave 126 mg of the title
compound from Example 28 and 122 mg of the title compound from
Example 29.
[0657] LC-MS (Method 6B): R.sub.t=2.18 min; MS (ESIpos): m/z=517
[M+H].sup.+;
[0658] HPLC (Method 2E): R.sub.t=15.96 min, >99.0% ee;
[0659] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.03 (d, 1H), 3.68 (t, 3H), 3.62 (br s, 4H),
3.49-3.38 (m, 1H), 3.23 (s, 3H), 3.18 (br s, 4H), 3.14-3.02 (m,
3H), 2.94 (t, 2H), 2.35 (d, 1H), 2.02 (d, 1H).
Example 30
(1,1-Dioxidothiomorpholin-4-yl)
{3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}methanone [enantiomerically pure cis isomer]
##STR00124##
[0661] According to General Method 2, 259 mg (0.519 mmol) of the
compound from Example 19A were reacted. Enantiomer separation of
252 mg of the racemate according to Method 2D gave 104 mg of the
title compound from Example 30 and 91.0 mg of the title compound
from Example 31.
[0662] LC-MS (Method 6B): R.sub.t=2.29 min; MS (ESIpos): m/z=531
[M+H].sup.+;
[0663] HPLC (Method 3E): R.sub.t=4.92 min, >99.0% ee;
[0664] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.71 (d, 2H),
7.58 (d, 2H), 4.03 (d, 1H), 3.75-3.68 (m, 3H), 3.62 (br s, 4H),
3.43 (q, 3H), 3.18 (br s, 4H), 3.14-3.01 (m, 3H), 2.93 (t, 2H),
2.35 (d, 1H), 2.12-1.95 (m, 1H), 1.07 (t, 3H).
Example 31
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-
-5-[4-(trifluoromethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00125##
[0666] According to General Method 2, 259 mg (0.519 mmol) of the
compound from Example 19A were reacted. Enantiomer separation of
252 mg of the racemate according to Method 2D gave 104 mg of the
title compound from Example 30 and 91.0 mg of the title compound
from Example 31.
[0667] LC-MS (Method 6B): R.sub.t=2.29 min; MS (ESIpos): m/z=531
[M+H].sup.+;
[0668] HPLC (Method 3E): R.sub.t=13.63 min, >99.0% ee;
[0669] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.71 (d, 2H),
7.58 (d, 2H), 4.03 (d, 1H), 3.75-3.68 (m, 3H), 3.62 (br s, 4H),
3.43 (q, 3H), 3.18 (br s, 4H), 3.14-3.01 (m, 3H), 2.93 (t, 2H),
2.35 (d, 1H), 2.12-1.95 (m, 1H), 1.07 (t, 3H).
Example 32
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-hydroxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(trifluoromethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00126##
[0671] According to General Method 2, 100 mg (0.213 mmol) of the
compound from Example 20A were reacted. Enantiomer separation of
97.4 mg of the racemate according to Method 2D gave 33.9 mg of the
title compound from Example 32 and 35.0 mg of the title compound
from Example 33.
[0672] LC-MS (Method 5B): R.sub.t=0.91 min; MS (ESIpos): m/z=503
[M+H].sup.+;
[0673] HPLC (Method 3E): R.sub.t=4.75 min, >99.0% ee;
[0674] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.71 (d, 2H),
7.58 (d, 2H), 4.77 (t, 1H), 4.03 (d, 1H), 3.74 (q, 2H), 3.68 (d,
1H), 3.62 (br s, 4H), 3.47-3.37 (m, 1H), 3.18 (br s, 4H), 3.13-3.00
(m, 3H), 2.82 (t, 2-H), 2.35 (d, 1H), 2.10-1.94 (m, 1H).
Example 33
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-hydroxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(trifluoromethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00127##
[0676] According to General Method 2, 100 mg (0.213 mmol) of the
compound from Example 20A were reacted. Enantiomer separation of
97.4 mg of the racemate according to Method 2D gave 33.9 mg of the
title compound from Example 32 and 35.0 mg of the title compound
from Example 33.
[0677] LC-MS (Method 5B): R.sub.t=0.91 min; MS (ESIpos): m/z=503
[M+H].sup.+;
[0678] HPLC (Method 3E): R.sub.t=8.97 min, >99.0% ee;
[0679] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.71 (d, 2H),
7.58 (d, 2H), 4.77 (t, 1H), 4.03 (d, 1H), 3.74 (q, 2H), 3.68 (d,
1H), 3.62 (br s, 4H), 3.47-3.37 (m, 1H), 3.18 (br s, 4H), 3.13-3.00
(m, 3H), 2.82 (t, 2-H), 2.35 (d, 1H), 2.10-1.94 (m, 1H).
Example 34
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-
-5-(4-ethylphenyl)-piperidin-1-yl}methanone [enantiomerically pure
cis isomer]
##STR00128##
[0681] According to General Method 2, 303 mg (0.661 mmol) of the
compound from Example 11A were reacted. Enantiomer separation of
297 mg of the racemate according to Method 2D gave 139 mg of the
title compound from Example 34 and 117 mg of the title compound
from Example 35.
[0682] LC-MS (Method 2B): R.sub.t=1.24 min; MS (ESIpos): m/z=491
[M+H].sup.+;
[0683] HPLC (Method 3E): R.sub.t=4.81 min, >99.0% ee;
[0684] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.24 (m, 2H),
7.17 (m, 2H), 4.03 (d, 1H), 3.71 (t, 2H), 3.67-3.57 (m, 5H),
3.49-3.35 (m, 3H), 3.17 (br s, 4H), 3.06 (t, 1H), 2.98-2.86 (m,
3H), 2.62-2.55 (m, 3H), 2.30 (d, 2H), 1.95 (q, 1H), 1.16 (t, 3H),
1.07 (t, 3H).
Example 35
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-ethoxyethyl)-1,2,4-oxadiazol-5-yl]-
-5-(4-ethylphenyl)-piperidin-1-yl}methanone [enantiomerically pure
cis isomer]
##STR00129##
[0686] According to General Method 2, 303 mg (0.661 mmol) of the
compound from Example 11A were reacted. Enantiomer separation of
297 mg of the racemate according to Method 2D gave 139 mg of the
title compound from Example 34 and 117 mg of the title compound
from Example 35.
[0687] LC-MS (Method 2B): R.sub.t=1.24 min; MS (ESIpos): m/z=491
[M+H].sup.+;
[0688] HPLC (Method 3E): R.sub.t=6.80 min, >99.0% ee;
[0689] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.24 (m, 2H),
7.17 (m, 2H), 4.03 (d, 1H), 3.71 (t, 2H), 3.67-3.57 (m, 5H),
3.49-3.35 (m, 3H), 3.17 (br s, 4H), 3.06 (t, 1H), 2.98-2.86 (m,
3H), 2.62-2.55 (m, 3H), 2.30 (d, 2H), 1.95 (q, 1H), 1.16 (t, 3H),
1.07 (t, 3H).
Example 36
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00130##
[0691] According to General Method 1, 196 mg (0.405 mmol) of the
compound from Example 18A were reacted. Yield: 194 mg (96% of
theory)
[0692] LC-MS (Method 2B): R.sub.t=1.08 min; MS (ESIpos): m/z=501
[M+H].sup.+;
[0693] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.00 (d, 1H), 3.73-3.58 (m, 5H), 3.57-3.48 (m, 2H),
3.48-3.39 (m, 1H), 3.13-2.99 (m, 3H), 2.97-2.84 (m, 4H), 2.77-2.65
(m, 2H), 2.35 (d, 1H), 2.03 (q, 1H).
Example 37
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00131##
[0695] Enantiomer separation of 194 mg of the racemate from Example
36 according to Method 1D gave 81.1 mg of the title compound from
Example 37 and 78.5 mg of the title compound from Example 38.
[0696] LC-MS (Method 2B): R.sub.t=1.08 min; MS (ESIpos): m/z=501
[M+H].sup.+;
[0697] HPLC (Method 1E): R.sub.t=8.45 min, >99.0% ee;
[0698] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.00 (d, 1H), 3.73-3.58 (m, 5H), 3.57-3.48 (m, 2H),
3.48-3.39 (m, 1H), 3.13-2.99 (m, 3H), 2.97-2.84 (m, 4H), 2.77-2.65
(m, 2H), 2.35 (d, 1H), 2.03 (q, 1H).
Example 38
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(trifluoromethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00132##
[0700] Enantiomer separation of 194 mg of the racemate from Example
36 according to Method 1D gave 81.1 mg of the title compound from
Example 37 and 78.5 mg of the title compound from Example 38.
[0701] LC-MS (Method 2B): R.sub.t=1.08 min; MS (ESIpos): m/z=501
[M+H].sup.+;
[0702] HPLC (Method 1E): R.sub.t=18.94 min, >99.0% ee;
[0703] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.70 (d, 2H),
7.58 (d, 2H), 4.00 (d, 1H), 3.73-3.58 (m, 5H), 3.57-3.48 (m, 2H),
3.48-3.39 (m, 1H), 3.13-2.99 (m, 3H), 2.97-2.84 (m, 4H), 2.77-2.65
(m, 2H), 2.35 (d, 1H), 2.03 (q, 1H).
Example 39
{3-[4-(2,2-Difluoroethyl)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-y-
l]piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [racemic
cis isomer]
##STR00133##
[0705] 14.7 mg (0.031 mmol) of the compound from Example 63A were
reacted according to General Method 2 with 26.3 mg (0.076 mmol) of
meta-chloroperbenzoic acid. Yield: 9.5 mg (60% of theory)
[0706] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.33 (d, 2H),
7.26 (m, 2H), 6.23 (tt, 1H), 4.03 (d, 1H), 3.71-3.56 (m, 7H),
3.46-3.36 (m, 1H), 3.23 (s, 3H), 3.21-3.13 (m, 5H), 3.12-2.87 (m,
6H), 2.31 (d, 1H), 1.97 (q, 1H).
Example 40
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00134##
[0708] 78.0 mg (0.162 mmol) of the compound from Example 46A were
reacted according to General Method 1 with 50.4 mg (0.146 mmol) of
meta-chloroperbenzoic acid. Yield: 76.2 mg (88% of theory)
[0709] LC-MS (Method 5B): R.sub.t=1.02 min; MS (ESIpos): m/z=497
[M+H].sup.+.
Example 41
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00135##
[0711] Enantiomer separation of 76.2 mg of the racemate from
Example 40 according to Method 7D gave 29.1 mg of the title
compound from Example 41 (Enantiomer 1) and 28.9 mg of the title
compound from Example 42 (Enantiomer 2).
[0712] LC-MS (Method 7B): R.sub.t=2.18 min; MS (ESIpos): m/z=497
[M+H].sup.+;
[0713] HPLC (Method 6E): R.sub.t=13.2 min, >99.0% ee;
[0714] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 3.96 (d, 1H), 3.68-3.47 (m, 7H), 3.41-3.33 (m, 1H), 3.07-2.85
(m, 5H), 2.74-2.66 (m, 2H), 2.28 (d, 1H), 2.16-2.08 (m, 1H), 1.93
(q, 1H), 1.08-1.02 (m, 2H), 0.92-0.85 (m, 2H).
Example 42
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00136##
[0716] Enantiomer separation of 76.2 mg of the racemate from
Example 40 according to Method 7D gave 29.1 mg of the title
compound from Example 41 (Enantiomer 1) and 28.9 mg of the title
compound from Example 42 (Enantiomer 2).
[0717] LC-MS (Method 7B): R.sub.t=2.18 min; MS (ESIpos): m/z=497
[M+H].sup.+;
[0718] HPLC (Method 6E): R.sub.t=16.4 min, >99.0% ee;
[0719] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 3.96 (d, 1H), 3.68-3.47 (m, 7H), 3.41-3.33 (m, 1H), 3.07-2.85
(m, 5H), 2.74-2.66 (m, 2H), 2.28 (d, 1H), 2.16-2.08 (m, 1H), 1.93
(q, 1H), 1.08-1.02 (m, 2H), 0.92-0.85 (m, 2H).
Example 43
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [racemic
cis isomer]
##STR00137##
[0721] 78.0 mg (0.162 mmol) of the compound from Example 46A were
reacted according to General Method 2 with 140 mg (0.146 mmol) of
meta-chloroperbenzoic acid. Yield: 87.5 mg (100% of theory)
[0722] LC-MS (Method 2B): R.sub.t=1.25 min; MS (ESIpos): m/z=513
[M+H].sup.+.
Example 44
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00138##
[0724] Enantiomer separation of 87.5 mg of the racemate from
Example 43 according to Method 8D gave 29.1 mg of the title
compound from Example 44 (Enantiomer 1) and 30.7 mg of the title
compound from Example 45 (Enantiomer 2).
[0725] LC-MS (Method 7B): R.sub.t=2.34 min; MS (ESIpos): m/z=513
[M+H].sup.+;
[0726] HPLC (Method 7E): R.sub.t=9.86 min, 99.0% ee;
[0727] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 4.00 (d, 1H), 3.67-3.56 (m, 7H), 3.17 (br. s., 4H), 3.07-2.87
(m, 3H), 2.28 (d, 1H), 2.16-2.08 (m, 1H), 1.93 (q, 1H), 1.09-1.02
(m, 2H), 0.92-0.85 (m, 3H), one proton hidden.
Example 45
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(2,2,2-trifluoroethyl)phenyl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00139##
[0729] Enantiomer separation of 87.5 mg of the racemate from
Example 43 according to Method 8D gave 29.1 mg of the title
compound from Example 44 (Enantiomer 1) and 30.7 mg of the title
compound from Example 45 (Enantiomer 2).
[0730] LC-MS (Method 7B): R.sub.t=2.34 min; MS (ESIpos): m/z=513
[M+H].sup.+;
[0731] HPLC (Method 7E): R.sub.t=10.9 min, 97.5% ee;
[0732] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 4.00 (d, 1H), 3.67-3.56 (m, 7H), 3.17 (br. s., 4H), 3.07-2.87
(m, 3H), 2.28 (d, 1H), 2.16-2.08 (m, 1H), 1.93 (q, 1H), 1.09-1.02
(m, 2H), 0.92-0.85 (m, 3H), one proton hidden.
Example 46
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(2,2,2-trifluoroethyl)ph-
enyl]piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00140##
[0734] 113 mg (0.227 mmol) of the compound from Example 47A were
reacted according to General Method 1 with 70.4 mg (0.204 mmol) of
meta-chloroperbenzoic acid. Enantiomer separation of 108 mg of the
racemate according to Method 9D gave 37.1 mg of the title compound
from Example 46 (Enantiomer 1) and 41.8 mg of the title compound
from Example 47 (Enantiomer 2).
[0735] LC-MS (Method 5B): R.sub.t=0.96 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0736] HPLC (Method 8E): R.sub.t=5.48 min, >99.0% ee;
[0737] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.38-7.29 (m,
4H), 4.00 (d, 1H), 3.72-3.48 (m, 9H), 3.46-3.37 (m, 1H), 3.23 (s,
3H), 3.10-2.85 (m, 7H), 2.76-2.65 (m, 3H), 2.32 (d, 1H), 1.98 (q,
1H).
Example 47
{3-[3-(2-Methoxyethyl)-1,2,4-oxadiazol-5-yl]-5-[4-(2,2,2-trifluoroethyl)ph-
enyl]piperidin-1-yl}(1-oxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00141##
[0739] 113 mg (0.227 mmol) of the compound from Example 47A were
reacted according to General Method 1 with 70.4 mg (0.204 mmol) of
meta-chloroperbenzoic acid. Enantiomer separation of 108 mg of the
racemate according to Method 9D gave 37.1 mg of the title compound
from Example 46 (Enantiomer 1) and 41.8 mg of the title compound
from Example 47 (Enantiomer 2).
[0740] LC-MS (Method 5B): R.sub.t=0.96 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0741] HPLC (Method 8E): R.sub.t=7.15 min, >99.0% ee;
[0742] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.38-7.29 (m, 4H),
4.00 (d, 1H), 3.72-3.48 (m, 9H), 3.46-3.37 (m, 1H), 3.23 (s, 3H),
3.10-2.85 (m, 7H), 2.76-2.65 (m, 3H), 2.32 (d, 1H), 1.98 (q,
1H).
Example 48
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00142##
[0744] 113 mg (0.227 mmol) of the compound from Example 47A were
reacted according to General Method 2 with 196 mg (0.567 mmol) of
meta-chloroperbenzoic acid. Enantiomer separation of 121 mg of the
racemate according to Method 9D gave 34.4 mg of the title compound
from Example 48 (Enantiomer 1) and 29.2 mg of the title compound
from Example 49 (Enantiomer 2).
[0745] LC-MS (Method 7B): R.sub.t=2.17 min; MS (ESIpos): m/z=531
[M+H].sup.+;
[0746] HPLC (Method 8E): R.sub.t=4.34 min, >99.0% ee;
[0747] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.39-7.29 (m,
4H), 4.03 (d, 1H), 3.72-3.56 (m, 10H), 3.46-3.36 (m, 1H), 3.23 (s,
3H), 3.18 (br. s., 4H), 3.11-2.90 (m, 5H), 2.33-2.27 (m, 1H), 1.97
(q, 1H).
Example 49
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl-
]-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00143##
[0749] 113 mg (0.227 mmol) of the compound from Example 47A were
reacted according to General Method 2 with 196 mg (0.567 mmol) of
meta-chloroperbenzoic acid. Enantiomer separation of 121 mg of the
racemate according to Method 9D gave 34.4 mg of the title compound
from Example 48 (Enantiomer 1) and 29.2 mg of the title compound
from Example 49 (Enantiomer 2).
[0750] LC-MS (Method 7B): R.sub.t=2.18 min; MS (ESIpos): m/z=531
[M+H].sup.+;
[0751] HPLC (Method 8E): R.sub.t=7.86 min, >99.0% ee;
[0752] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.39-7.29 (m,
4H), 4.03 (d, 1H), 3.72-3.56 (m, 10H), 3.46-3.36 (m, 1H), 3.23 (s,
3H), 3.18 (br. s., 4H), 3.11-2.90 (m, 5H), 2.33-2.27 (m, 1H), 1.97
(q, 1H).
Example 50
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00144##
[0754] 34.1 mg (0.074 mmol) of the compound from Example 54A were
reacted according to General Method 1 with 22.9 mg (0.066 mmol) of
meta-chloroperbenzoic acid. Yield: 39.7 mg (100% of theory).
[0755] LC-MS (Method 5B): R.sub.t=0.99 min; MS (ESIpos): m/z=479
[M+H].sup.+.
Example 51
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(1-oxidothiomorpholin-4-yl)methanone [enantiomerically
pure cis isomer]
##STR00145##
[0757] Enantiomer separation of 35.5 mg of the racemate from
Example 50 according to Method 9D gave 12.0 mg of the title
compound from Example 51 (Enantiomer 1) and 14.0 mg of the title
compound from Example 52 (Enantiomer 2).
[0758] LC-MS (Method 5B): R.sub.t=1.00 min; MS (ESIpos): m/z=479
[M+H].sup.+;
[0759] HPLC (Method 9E): R.sub.t=5.27 min, >99.0% ee;
[0760] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.53 (d, 2H),
7.45 (d, 2H), 3.96 (d, 1H), 3.71-3.46 (m, 5H), 3.42-3.35 (m, 1H),
3.09-2.84 (m, 5H), 2.71 (d, 2H), 2.29 (d, 1H), 2.16-2.08 (m, 1H),
2.02-1.90 (m, 4H), 1.10-1.02 (m, 2H), 0.93-0.85 (m, 2H).
Example 52
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(1-oxido-thiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00146##
[0762] Enantiomer separation of 35.5 mg of the racemate from
Example 50 according to Method 9D gave 12.0 mg of the title
compound from Example 51 (Enantiomer 1) and 14.0 mg of the title
compound from Example 52 (Enantiomer 2).
[0763] LC-MS (Method 5B): R.sub.t=1.00 min; MS (ESIpos): m/z=479
[M+H].sup.+;
[0764] HPLC (Method 9E): R.sub.t=6.78 min, >99.0% ee;
[0765] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.53 (d, 2H),
7.45 (d, 2H), 3.96 (d, 1H), 3.71-3.46 (m, 5H), 3.42-3.35 (m, 1H),
3.09-2.84 (m, 5H), 2.71 (d, 2H), 2.29 (d, 1H), 2.16-2.08 (m, 1H),
2.02-1.90 (m, 4H), 1.10-1.02 (m, 2H), 0.93-0.85 (m, 2H).
Example 53
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00147##
[0767] 34.1 mg (0.074 mmol) of the compound from Example 54A were
reacted according to General Method 2 with 63.6 mg (0.184 mmol) of
meta-chloroperbenzoic acid. Yield: 37.1 mg (99% of theory)
[0768] LC-MS (Method 5B): R.sub.t=1.06 min; MS (ESIpos): m/z=495
[M+H].sup.+;
[0769] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.52 (d, 2H),
7.44 (d, 2H), 4.00 (d, 1H), 3.69-3.56 (m, 5H), 3.41-3.34 (m, 1H),
3.17 (br. s., 4H), 3.10-2.95 (m, 3H), 2.28 (d, 1H), 2.17-2.07 (m,
1H), 2.03-1.89 (m, 4H), 1.10-1.01 (m, 2H), 0.94-0.85 (m, 2H).
Example 54
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00148##
[0771] Enantiomer separation of 37.1 mg of the racemate from
Example 53 according to Method 9D gave 13.0 mg of the title
compound from Example 54 (Enantiomer 1) and 14.0 mg of the title
compound from Example 55 (Enantiomer 2).
[0772] HPLC (Method 9E): R.sub.t=5.81 min, >99.0% ee;
[0773] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.52 (d, 2H),
7.44 (d, 2H), 4.00 (d, 1H), 3.69-3.56 (m, 5H), 3.41-3.34 (m, 1H),
3.17 (br. s., 4H), 3.10-2.95 (m, 3H), 2.28 (d, 1H), 2.17-2.07 (m,
1H), 2.03-1.89 (m, 4H), 1.10-1.01 (m, 2H), 0.94-0.85 (m, 2H).
Example 55
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(1,1-difluoroethyl)phenyl]pip-
eridin-1-yl}-(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00149##
[0775] Enantiomer separation of 37.1 mg of the racemate from
Example 53 according to Method 9D gave 13.0 mg of the title
compound from Example 54 (Enantiomer 1) and 14.0 mg of the title
compound from Example 55 (Enantiomer 2).
[0776] HPLC (Method 9E): R.sub.t=9.63 min, >99.0% ee;
[0777] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.52 (d, 2H),
7.44 (d, 2H), 4.00 (d, 1H), 3.69-3.56 (m, 5H), 3.41-3.34 (m, 1H),
3.17 (br. s., 4H), 3.10-2.95 (m, 3H), 2.28 (d, 1H), 2.17-2.07 (m,
1H), 2.03-1.89 (m, 4H), 1.10-1.01 (m, 2H), 0.94-0.85 (m, 2H).
Example 56
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(difluoromethoxy)phenyl]piper-
idin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00150##
[0779] 100 mg (0.23 mmol) of the compound from Example 37A and 46
mg (0.46 mmol) of N-hydroxycyclopropanecarboximidamide were
initially charged in 0.8 ml of DMF and reacted with 132 mg (0.35
mmol) of HATU and 0.12 ml (90 mg, 0.69 mmol) of
N,N-diisopropylethylamine. The reaction mixture was stirred at RT
for 15 minutes and then partitioned between water and ethyl
acetate. The organic phase was washed repeatedly with water, dried
over sodium sulphate and concentrated under reduced pressure. The
residue was taken up in 3.0 ml of DMF and converted in a microwave
at 180.degree. C. for 2 minutes. The reaction mixture was purified
by means of preparative HPLC. Yield: 46 mg (37% of theory)
[0780] LC-MS (Method 2B): R.sub.t=1.20 min; MS (ESIpos): m/z=497
[M+H].sup.+;
[0781] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.43-7.30 (m,
2H), 7.14 (d, 2H), 4.09 (q, 1H), 3.99 (br. d, 1H), 3.63 (br. d.,
1H), 3.40-3.33 (m, 1H), 3.33-3.28 (m, 4H), 3.22-3.10 (m, 4H),
3.08-2.88 (m, 3H), 2.28 (br. d, 1H), 2.16-2.07 (m, 1H), 2.00-1.87
(m, 1H), 1.12-0.99 (m, 2H), 0.94-0.84 (m, 2H).
Example 57
{3-[4-(Difluoromethoxy)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone [racemic
cis isomer]
##STR00151##
[0783] 300 mg (0.69 mmol) of the compound from Example 37A and 246
mg (2.08 mmol) of N'-hydroxy-3-methoxypropanimidamide were
initially charged in 2.6 ml of DMF and reacted with 396 mg (1.0
mmol) of HATU and 0.36 ml (269 mg, 2.1 mmol) of
N,N-diisopropylethylamine. The reaction mixture was stirred at RT
for 15 minutes and then partitioned between water and ethyl
acetate. The organic phase was washed repeatedly with water, dried
over sodium sulphate and concentrated under reduced pressure. The
residue was dissolved in 2.0 ml of DMF and converted in a microwave
at 180.degree. C. for 2 minutes. The reaction mixture was purified
by means of preparative
[0784] HPLC. Yield: 141 mg (38% of theory)
[0785] LC-MS (Method 2B): R.sub.t=1.10 min; MS (ESIpos): m/z=515
[M+H].sup.+;
[0786] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 6=7.43-7.33 (m, 2H),
7.15 (d, 2H), 4.11-3.99 (m, 2H), 3.71-3.64 (m, 3H), 3.64-3.55 (m,
4H), 3.46-3.35 (m, 1H), 3.35-3.30 (m, 4H), 3.18 (br. s, 3H),
3.14-2.90 (m, 5H), 2.30 (br. d, 1H), 2.03-1.92 (m, 1H).
Example 58
{3-[4-(Difluoromethoxy)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00152##
[0788] The enantiomer separation of 117 mg of the racemate from
Example 57 according to Method 10D gave 43 mg of the compound from
Example 58 (Enantiomer 1) and 38 mg of the compound from Example 59
(Enantiomer 2).
[0789] HPLC (Method 10E): R.sub.t=4.17 min, >99.0% ee;
[0790] LC-MS (Method 2B): R.sub.t=1.10 min; MS (ESIpos): m/z=515
[M+H].sup.+.
Example 59
{3-[4-(Difluoromethoxy)phenyl]-5-[3-(2-methoxyethyl)-1,2,4-oxadiazol-5-yl]-
piperidin-1-yl}(1,1-dioxidothiomorpholin-4-yl)methanone
[enantiomerically pure cis isomer]
##STR00153##
[0792] The enantiomer separation of 117 mg of the racemate from
Example 57 according to Method 10D gave 43 mg of the compound from
Example 58 (Enantiomer 1) and 38 mg of the compound from Example 59
(Enantiomer 2).
[0793] HPLC (Method 10E): R.sub.t=9.24 min, >99.0% ee;
[0794] LC-MS (Method 2B): R.sub.t=1.10 min; MS (ESIpos): m/z=515
[M+H].sup.+.
Example 60
{3-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-5-[4-(difluoromethoxy)phenyl]piper-
idin-1-yl}(1-oxidothiomorpholin-4-yl)methanone [racemic cis
isomer]
##STR00154##
[0796] 200 mg (0.48 mmol) of the compound from Example 39A and 96
mg (0.96 mmol) of N'-hydroxycyclopropanecarboximidamide were
initially charged in 1.8 ml of DMF and reacted with 274 mg (0.72
mmol) of HATU and 0.25 ml (186 mg, 1.44 mmol) of
N,N-diisopropylethylamine. The reaction mixture was stirred at RT
for 15 minutes and then partitioned between water and ethyl
acetate. The organic phase was washed repeatedly with water, dried
over sodium sulphate and concentrated under reduced pressure. The
residue was dissolved in 2.0 ml of DMF and converted in a microwave
at 180.degree. C. for 2 minutes. The reaction mixture was purified
by means of preparative HPLC. Yield: 25 mg (10% of theory)
[0797] LC-MS (Method 2B): R.sub.t=1.12 min; MS (ESIpos): m/z=481
[M+H].sup.+;
[0798] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.42-7.35 (m,
2H), 7.14 (d, 2H), 4.01-3.87 (m, 1H), 3.69-3.45 (m, 5H), 3.42-3.34
(m, 1H), 3.07-2.85 (m, 5H), 2.70 (br. d, 2H), 2.34-2.23 (m, 1H),
2.15-2.07 (m, 1H), 1.99-1.88 (m, 1H), 1.12-1.01 (m, 2H), 0.94-0.85
(m, 2H).
Example 61
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(1-methoxycyclopropyl)-1,2,4-oxadiazo-
l-5-yl]-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-1-yl}methanone
[racemic cis isomer]
##STR00155##
[0800] 29.0 mg (0.162 mmol) of the compound from Example 65A were
reacted according to General Method 2 with 49.0 mg (0.142 mmol) of
meta-chloroperbenzoic acid. Yield: 31.2 mg (95% of theory)
[0801] LC-MS (Method 5B): R.sub.t=1.06 min; MS (ESIpos): m/z=543
[M+H].sup.+;
[0802] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 4.02 (d, 1H), 3.68-3.55 (q, 7H), 3.38 (s, 3H), 3.17 (br. s.,
4H), 3.10-2.88 (m, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.34-1.28 (m,
2H), 1.20-1.12 (m, 2H).
Example 62
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(1-methoxycyclopropyl)-1,2,4-oxadiazo-
l-5-yl]-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00156##
[0804] Enantiomer separation of 31.2 mg of the racemate from
Example 61 according to Method 11D gave 12.0 mg of the title
compound from Example 62 (Enantiomer 1) and 12.0 mg of the title
compound from Example 63 (Enantiomer 2).
[0805] LC-MS (Method 2B): R.sub.t=1.26 min; MS (ESIpos): m/z=543
[M+H].sup.+;
[0806] HPLC (Method 11E): R.sub.t=17.9 min, >99.0% ee;
[0807] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 4.02 (d, 1H), 3.68-3.55 (q, 7H), 3.38 (s, 3H), 3.17 (br. s.,
4H), 3.10-2.88 (m, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.34-1.28 (m,
2H), 1.20-1.12 (m, 2H).
Example 63
(1,1-Dioxidothiomorpholin-4-yl){3-[3-(1-methoxycyclopropyl)-1,2,4-oxadiazo-
l-5-yl]-5-[4-(2,2,2-trifluoroethyl)phenyl]piperidin-1-yl}methanone
[enantiomerically pure cis isomer]
##STR00157##
[0809] Enantiomer separation of 31.2 mg of the racemate from
Example 61 according to Method 11D gave 12.0 mg of the title
compound from Example 62 (Enantiomer 1) and 12.0 mg of the title
compound from Example 63 (Enantiomer 2).
[0810] LC-MS (Method 2B): R.sub.t=1.26 min; MS (ESIpos): m/z=543
[M+H].sup.+;
[0811] HPLC (Method 11E): R.sub.t=29.2 min, >99.0% ee;
[0812] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37-7.29 (m,
4H), 4.02 (d, 1H), 3.68-3.55 (q, 7H), 3.38 (s, 3H), 3.17 (br. s.,
4H), 3.10-2.88 (m, 3H), 2.30 (d, 1H), 1.95 (q, 1H), 1.34-1.28 (m,
2H), 1.20-1.12 (m, 2H).
B) ASSESSMENT OF PHYSIOLOGICAL ACTIVITY
Abbreviations:
[0813] BSA bovine serum albumin
DMEM Dulbecco's Modified Eagle Medium
[0814] EGTA ethylene glycol
bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid FCS fetal calf serum
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
[3H]haTRAP tritiated high affinity thrombin receptor activating
peptide PRP platelet-rich plasma
[0815] The suitability of the inventive compounds for treating
thromboembolic disorders can be demonstrated in the following assay
systems:
1.) In Vitro Assays
1.a) Cellular Functional In Vitro Test
[0816] A recombinant cell line is used to identify agonists of the
human protease activated receptor 1 (PAR1) and to quantify the
activity of the substances described herein. The cell is originally
derived from a human embryonal kidney cell (HEK293; ATCC: American
Type Culture Collection, Manassas, Va. 20108, USA). The test cell
line constitutively expresses a modified form of the
calcium-sensitive photoprotein aequorin which, after reconstitution
with the cofactor coelenterazine, emits light when the free calcium
concentration in the inner mitochondrial compartment is increased
(Rizzuto R, Simpson A W, Brini M, Pozzan T.; Nature 1992, 358,
325-327). Additionally, the cell stably expresses the endogenous
human PAR1 receptor and the endogenous purinergic receptor P2Y2.
The resulting PAR1 test cell responds to stimulation of the
endogenous PAR1 or P2Y2 receptor with an intracellular release of
calcium ions, which can be quantified through the resulting
aequorin luminescence with a suitable luminometer (Milligan G,
Marshall F, Rees S, Trends in Pharmacological Sciences 1996, 17,
235-237).
[0817] For the testing of the substance specificity, the effect
thereof after activation of the endogenous PAR1 receptor is
compared with the effect after activation of the endogenous
purinergic P2Y2 receptor which utilizes the same intracellular
signal path.
[0818] Test Procedure:
[0819] The cells are plated out two days (48 hours) before the test
in culture medium (DMEM F12, supplemented with 10% FCS, 2 mM
glutamine, 20 mM HEPES, 1.4 mM pyruvate, 0.1 mg/ml gentamycin,
0.15% sodium bicarbonate; BioWhittaker Cat. #BE04-687Q; B-4800
Verviers, Belgium) in 384-well microtitre plates and kept in a cell
incubator (96% atmospheric humidity, 5% v/v CO.sub.2, 37.degree.
C.). On the day of the test, the culture medium is replaced by a
Tyrode's solution (in mM: 140 sodium chloride, 5 potassium
chloride, 1 magnesium chloride, 2 calcium chloride, 20 glucose, 20
HEPES), which additionally contains the cofactor coelenterazine (25
.mu.M) and glutathione (4 mM), and the microtitre plate is then
incubated for a further 3-4 hours. The test substances are then
pipetted onto the microtitre plate, and 5 minutes after the
transfer of the test substances into the wells of the microtitre
plate the plate is transferred into the luminometer, a PAR1 agonist
concentration which corresponds to EC.sub.50 is added and the
resulting light signal is immediately measured in the luminometer.
To distinguish an antagonist substance action from a toxic action,
the endogenous purinergic receptor is immediately subsequently
activated with agonist (ATP, final concentration 10 .mu.M) and the
resulting light signal is measured. The results are shown in Table
A:
TABLE-US-00001 TABLE A Example No. IC.sub.50 [nM] 1 43 8 33 10 8.0
15 5.1 20 23 31 32 52 4.7 54 4.3 61 15.7
1.b) PAR-1 Receptor Binding Assay
[0820] Platelet membranes are incubated with 12 nM [3H]haTRAP and
test substance in different concentrations in a buffer (50 mM Tris
pH 7.5, 10 mM magnesium chloride, 1 mM EGTA, 0.1% BSA) at room
temperature for 80 mM Then the mixture is transferred to a filter
plate and washed twice with buffer. After addition of scintillation
liquid, the radioactivity on the filter is measured in a beta
counter.
1.c) Platelet Aggregation in Plasma
[0821] Platelet aggregation is determined using blood from healthy
volunteers of both genders, who had not received any thrombocyte
aggregation-influencing medication for the last ten days. The blood
is taken up into monovettes (Sarstedt, Numbrecht, Germany) which
contain, as anticoagulant, sodium citrate 3.8% (1 part citrate+9
parts blood). To obtain platelet-rich plasma, the citrated whole
blood is centrifuged at 140 g for 20 min.
[0822] For the aggregation measurements, aliquots of the
platelet-rich plasma with increasing concentrations of test
substance are incubated at 37.degree. C. for 10 min. Subsequently,
aggregation is triggered by addition of a thrombin receptor agonist
(TRAP6, SFLLRN) in an aggregometer and determined at 37.degree. C.
by means of the turbidimetry method according to Born (Born, G. V.
R., Cross M. J., The Aggregation of Blood Platelets; J. Physiol.
1963, 168, 178-195). The SFLLRN concentration leading to maximum
aggregation is, if appropriate, determined individually for each
donor.
[0823] To calculate the inhibitory effect, the maximum increase in
light transmission (amplitude of the aggregation curve in %) is
determined within 5 minutes after addition of the agonist in the
presence and absence of test substance, and the inhibition is
calculated. The inhibition curves are used to calculate the
concentration which inhibits aggregation by 50%. The results are
shown in Table B:
TABLE-US-00002 TABLE B Example No. IC.sub.50 [nM] 8 0.29 10 0.49 13
0.17 52 0.58
1.d) Platelet Aggregation in Buffer
[0824] Platelet aggregation is determined using blood from healthy
volunteers of both genders, who had not received any thrombocyte
aggregation-influencing medication for the last ten days. The blood
is taken up into monovettes (Sarstedt, Numbrecht, Germany) which
contain, as anticoagulant, sodium citrate 3.8% (1 part citrate+9
parts blood). To obtain platelet-rich plasma, the citrated whole
blood is centrifuged at 140 g for 20 mM One quarter of the volume
of ACD buffer (44.8 mM sodium citrate, 20.9 mM citric acid, 74.1 mM
glucose and 4 mM potassium chloride) is added to the PRP, and the
mixture is centrifuged at 1000 g for 10 minutes. The platelet
pellet is resuspended in wash buffer and centrifuged at 1000 g for
10 minutes. The platelets are resuspended in incubation buffer and
adjusted to 200 000 cells/W. Prior to the start of the test,
calcium chloride and magnesium chloride, final concentration in
each case 2 mM (2M stock solution, dilution 1:1000), are added.
Note: in the case of ADP-induced aggregation, only calcium chloride
is added. The following agonists can be used:
TRAP6-trifluoroacetate salt, collagen, human .alpha.-thrombin and
U-46619. For each donor, the concentration of the agonist is
tested.
[0825] Test Procedure:
[0826] 96-well microtitre plates are used. The test substance is
diluted in DMSO, and 2 .mu.l per well is initially charged. 178
.mu.l of platelet suspension are added, and the mixture is
preincubated at room temperature for 10 minutes. 20 .mu.l of
agonist are added, and the measurement in the Spectramax, OD 405
nm, is started immediately. Kinetics are determined in 11
measurements of 1 minute each. Between the measurements, the
mixture is shaken for 55 seconds.
1.e) Platelet Aggregation in Fibrinogen-Depleted Plasma
[0827] To determine platelet aggregation, blood of healthy
volunteers of both genders, who had not received any thrombocyte
aggregation-influencing medication for the last ten days, is used.
The blood is taken up into monovettes (Sarstedt, Numbrecht,
Germany) which contain, as anticoagulant, sodium citrate 3.8% (1
part citrate+9 parts blood).
1.e) Preparation of Fibrinogen-Depleted Plasma:
[0828] To obtain low-platelet plasma, the citrated whole blood is
centrifuged at 140 g for 20 min. The low-platelet plasma is admixed
in a ratio of 1:25 with reptilase (Roche Diagnostic, Germany) and
inverted cautiously. This is followed by 10 mM of incubation at
37.degree. C. in a water bath, followed directly by 10 mM of
incubation on ice. The plasma/reptilase mixture is centrifuged at
1300 g for 15 mM, and the supernatant (fibrinogen-depleted plasma)
is obtained.
[0829] Platelet Isolation:
[0830] To obtain platelet-rich plasma, the citrated whole blood is
centrifuged at 140 g for 20 min. One quarter of the volume of ACD
buffer (44.8 mM sodium citrate, 20.9 mM citric acid, 74.1 mM
glucose and 4 mM potassium chloride) is added to the PRP, and the
mixture is centrifuged at 1300 g for 10 minutes. The platelet
pellet is resuspended in wash buffer and centrifuged at 1300 g for
10 minutes. The platelets are resuspended in incubation buffer and
adjusted to 400 000 cells/4 and calcium chloride solution is added
to a final concentration of 5 mM (dilution 1/200).
[0831] For the aggregation measurements, aliquots (98 .mu.l of
fibrinogen-depleted plasma and 80 .mu.l of platelet suspension) are
incubated with increasing concentrations of test substance at RT
for 10 mM Subsequently, aggregation is triggered by addition of
human alpha thrombin in an aggregometer and determined at
37.degree. C. by means of the turbidimetry method according to Born
(Born, G. V. R., Cross M. J., The Aggregation of Blood Platelets;
J. Physiol. 1963, 168, 178-195). The alpha thrombin concentration
which just leads to the maximum aggregation is determined
individually for each donor.
[0832] To calculate the inhibitory activity, the increase in the
maximum light transmission (amplitude of the aggregation curve in
%) is determined within 5 minutes after addition of the agonist in
the presence and absence of test substance, and the inhibition is
calculated. The inhibition curves are used to calculate the
concentration which inhibits aggregation by 50%.
1.f) Stimulation of Washed Platelets and Analysis in Flow
Cytometry
[0833] Isolation of Washed Platelets:
[0834] Human whole blood is obtained by venipuncture from voluntary
donors and transferred into monovettes (Sarstedt, Numbrecht,
Germany) containing sodium citrate as anticoagulant (1 part sodium
citrate 3.8%+9 parts whole blood). The monovettes are centrifuged
at 90.degree. rotations per minute and 4.degree. C. for a period of
20 minutes (Heraeus Instruments, Germany; Megafuge LORS). The
platelet-rich plasma is cautiously removed and transferred into a
50 ml Falcon tube. ACD buffer (44 mM sodium citrate, 20.9 mM citric
acid, 74.1 mM glucose) is then added to the plasma. The volume of
the ACD buffer corresponds to one quarter of the plasma volume.
Centrifuging at 2500 rpm and 4.degree. C. for ten minutes sediments
the platelets. Thereafter, the supernatant is cautiously decanted
off and discarded. The precipitated platelets are first cautiously
resuspended in one millilitre of wash buffer (113 mM sodium
chloride, 4 mM disodium hydrogenphosphate, 24 mM sodium
dihydrogenphosphate, 4 mM potassium chloride, 0.2 mM ethylene
glycol bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid, 0.1% glucose)
and then made up with wash buffer to a volume which corresponds to
that of the amount of plasma. The wash procedure is repeated. The
platelets are precipitated by another ten-minute centrifugation at
2500 rpm and 4.degree. C. and then carefully resuspended in one
millilitre of incubation buffer (134 mM sodium chloride, 12 mM
sodium hydrogencarbonate, 2.9 mM potassium chloride, 0.34 mM sodium
dihydrogencarbonate, 5 mM HEPES, 5 mM glucose, 2 mM calcium
chloride and 2 mM magnesium chloride) and adjusted with incubation
buffer to a concentration of 300 000 platelets per .mu.l.
[0835] Staining and Stimulation of the Human Platelets with Human
.alpha.-Thrombin in the Presence or Absence of a PAR-1
Antagonist:
[0836] The platelet suspension is preincubated with the substance
to be tested or the appropriate solvent at 37.degree. C. for 10
minutes (Eppendorf, Germany; Thermomixer Comfort). Platelet
activation is triggered by addition of the agonist (0.5 .mu.M or 1
.mu.M .alpha.-thrombin; Kordia, the Netherlands, 3281 NIH units/mg;
or 30 .mu.g/ml of thrombin receptor activating peptide (TRAP6);
Bachem, Switzerland) at 37.degree. and with shaking at 500 rpm. At
each of 0, 1, 2.5, 5, 10 and 15 minutes, one aliquot of 50 .mu.l is
removed and transferred into one millilitre of singly concentrated
CellFix.TM. solution (Becton Dickinson Immunocytometry Systems,
USA). To fix the cells, they are incubated in the dark at 4.degree.
C. for 30 minutes. The platelets are precipitated by centrifuging
at 600 g and 4.degree. C. for ten minutes. The supernatant is
discarded and the platelets are resuspended in 400 .mu.l
CellWash.TM. (Becton Dickinson Immunocytometry Systems, USA). One
aliquot of 100 .mu.l is transferred to a new FACS tube. 1 .mu.l of
the platelet-identifying antibody and 1 .mu.l of the activation
state-detecting antibody are made up to a volume of 100 .mu.l with
CellWash.TM.. This antibody solution is then added to the platelet
suspension and incubated in the dark at 4.degree. C. for 20
minutes. After staining, the mixture volume is increased by
addition of a further 400 .mu.l of CellWash.TM.
[0837] The platelets are identified using a fluorescein
isothiocyanate-conjugated antibody directed against human
glycoprotein IIb (CD41) (Immunotech Coulter, France; Cat. No.
0649). With the aid of the phycoerythrin-conjugated antibody
directed against human glycoprotein P-selectin (Immunotech Coulter,
France; Cat. No. 1759), it is possible to determine the activation
state of the platelets. P-selectin (CD62P) is localized in the
.alpha.-granules of resting platelets. However, following in vitro
or in vivo stimulation, it is translocalized to the external plasma
membrane.
[0838] Flow Cytometry and Data Evaluation:
[0839] The samples are analysed in the FACSCalibur.TM. Flow
Cytometry System instrument from Becton Dickinson Immunocytometry
Systems, USA, and evaluated and graphically represented with the
aid of the CellQuest software, Version 3.3 (Becton Dickinson
Immunocytometry Systems, USA). The degree of platelet activation is
determined by the percentage of CD62P-positive platelets
(CD41-positive events). From each sample, 10 000 CD41-positive
events are counted.
[0840] The inhibitory effect of the substances to be tested is
calculated via the reduction in platelet activation, which relates
to the activation by the agonist.
1.g) Platelet Aggregation Measurement Using the Parallel-Plate Flow
Chamber
[0841] Platelet aggregation is determined using blood from healthy
volunteers of both genders, who had not received any thrombocyte
aggregation-influencing medication for the last ten days. The blood
is taken up into monovettes (Sarstedt, Numbrecht, Germany) which
contain, as anticoagulant, sodium citrate 3.8% (1 part citrate+9
parts blood). To obtain platelet-rich plasma, the citrated whole
blood is centrifuged at 140 g for 20 mM One quarter of the volume
of ACD buffer (44.8 mM sodium citrate, 20.9 mM citric acid, 74.1 mM
glucose and 4 mM potassium chloride) is added to the PRP, and the
mixture is centrifuged at 1000 g for 10 minutes. The platelet
pellet is resuspended in wash buffer and centrifuged at 1000 g for
10 minutes. For the perfusion study, a mixture of 40% erythrocytes
and 60% washed platelets (200 000/.mu.l) is prepared and suspended
in HEPES-tyrode buffer. Platelet aggregation under flow conditions
is measured using the parallel-plate flow chamber (B. Nieswandt et
al., EMBO J. 2001, 20, 2120-2130; C. Weeterings, Arterioscler
Thromb. Vasc. Biol. 2006, 26, 670-675; J J Sixma, Thromb. Res.
1998, 92, 43-46). Glass slides are wetted with 100 .mu.l of a
solution of human .alpha.-thrombin (dissolved in Tris buffer) at
4.degree. C. overnight (.alpha.-thrombin in different
concentrations, for example 10 to 50 .mu.g/ml) and finally blocked
using 2% BSA.
[0842] Reconstituted blood is passed over the thrombin-wetted glass
slides at a constant flow rate (for example a shear rate of
300/second) for 5 minutes and observed and recorded using a
microscope video system. The inhibitory effect of the substances to
be tested is determined morphometrically via the reduction in
platelet aggregate formation. Alternatively, the inhibition of the
platelet activation can be determined by flow cytometry, for
example via p-selectin expression (CD62p) (see Method 1.f).
1.h) Platelet Aggregation and Activation Measurement Using the
Parallel-Plate Flow Chamber (Anticoagulated Blood, Collagen)
[0843] Platelet activation under flow conditions is determined
using blood from healthy volunteers of both genders, who had not
received any thrombocyte aggregation-influencing medication for the
last ten days. The blood is taken up into monovettes (Sarstedt, N
mbrecht, Germany) which contain, as anticoagulant, sodium citrate
3.8% (1 part citrate+9 parts blood).
[0844] The measurement of platelet activation is carried out using
the parallel-plate flow chamber (B. Nieswandt et al., EMBO J. 2001,
20, 2120-2130; C. Weeterings, Arterioscler Thromb. Vasc. Biol.
2006, 26, 670-675; J J Sixma, Thromb. Res. 1998, 92, 43-46). Glass
slides are wetted with 20 .mu.l of collagen suspension (collagen
reagent: Horm, Nycomed) at 4.degree. C. overnight (type I collagen
in different concentrations, e.g. 1-10 ng/slide) and finally
blocked using 2% BSA.
[0845] To prevent fibrin clot formation, citrated whole blood is
admixed with Pefabloc FG (Pentapharm, final concentration 3 mM)
and, by addition of CaCl.sub.2 solution (final Ca.sup.++
concentration 5 mM), passed over the collagen-coated glass slides
at a constant flow rate (for example a shear rate of 1000/second)
for 5 minutes and observed and recorded using a microscope video
system. The inhibitory effect of the substances to be tested is
determined morphometrically via the reduction in platelet aggregate
formation. Alternatively, the inhibition of the platelet activation
can be determined by flow cytometry, for example via p-selectin
expression (CD62p) (see Method 1.0.
1.i) Platelet Aggregation and Activation Measurement Using the
Parallel-Plate Flow Chamber (Nonanticoagulated Blood, Collagen)
[0846] Platelet activation under flow conditions is determined
using blood from healthy volunteers of both genders, who had not
received any thrombocyte aggregation-influencing medication for the
last ten days. The blood is taken up into neutral monovettes
(Sarstedt, Numbrecht, Germany) which do not contain any
anticoagulant, and immediately admixed with Pefabloc FG
(Pentapharm, final concentration 3 mM) to prevent fibrin clot
formation. Test substances dissolved in DMSO are added
simultaneously with Pefablock FG and introduced without further
incubation into the parallel-plate flow chamber. The measurement of
platelet activation is conducted by morphometry or flow cytometry
in the collagen-coated parallel-plate flow chamber, as described in
Method 1.h).
2.) Ex Vivo Assay
2.a) Platelet Aggregation (Primates, Guinea Pigs)
[0847] Awake or anaesthetized guinea pigs or primates are treated
orally, intravenously or intraperitoneally with test substances in
suitable formulations. As a control, other guinea pigs or primates
are treated in an identical manner with the corresponding vehicle.
Depending on the mode of application, blood is obtained from the
deeply anaesthetized animals by puncture of the heart or of the
aorta for different times. The blood is transferred into monovettes
(Sarstedt, Numbrecht, Germany) which, as anticoagulant, contain
sodium citrate 3.8% (1 part citrate solution+9 parts blood). To
obtain platelet-rich plasma, the citrated whole blood is
centrifuged at 140 g for 20 min.
[0848] Aggregation is triggered by addition of a thrombin receptor
agonist (TRAP6, SFLLRN, 50 .mu.g/ml; in each experiment, the
concentration is determined for each animal species) in an
aggregometer and determined using the turbidimetry method according
to Born (Born, G. V. R., Cross M. J., The Aggregation of Blood
Platelets; J. Physiol. 1963, 168, 178-195) at 37.degree. C.
[0849] To measure the aggregation, the maximum increase in the
light transmission (amplitude of the aggregation curve in %) is
determined 5 minutes after addition of the agonist. The inhibitory
effect of the administered test substances in the treated animals
is calculated via the reduction in aggregation, based on the mean
of the control animals.
[0850] In addition to measurement of aggregation, the inhibition of
platelet activation can be determined by flow cytometry, for
example via p-selectin expression (CD62p) (see Method 1.f).
2.b) Platelet Aggregation and Activation Measurement in the
Parallel-Plate Flow Chamber (Primates)
[0851] Awake or anaesthetized primates are treated orally,
intravenously or intraperitoneally with test substances in suitable
formulations. As a control, other animals are treated in an
identical manner with the corresponding vehicle. According to the
mode of administration, blood is obtained from the animals by
venipuncture for different times. The blood is transferred into
monovettes (Sarstedt, Numbrecht, Germany) which, as anticoagulant,
contain sodium citrate 3.8% (1 part citrate solution+9 parts
blood). Alternatively, nonanticoagulated blood can be taken with
neutral monovettes (Sarstedt). In both cases, the blood is admixed
with Pefabloc FG (Pentapharm, final concentration 3 mM) to prevent
fibrin clot formation.
[0852] Citrated whole blood is recalcified before the measurement
by adding CaCl.sub.2 solution (final Ca.sup.++ concentration 5 mM).
Nonanticoagulated blood is introduced directly into the
parallel-plate flow chamber for analysis. The measurement of
platelet activation is conducted by morphometry or flow cytometry
in the collagen-coated parallel-plate flow chamber, as described in
Method 1.h).
3.) In Vivo Assays
3.a) Thrombosis Model
[0853] The inventive compounds can be studied in thrombosis models
in suitable animal species in which thrombin-induced platelet
aggregation is mediated via the PAR-1 receptor. Suitable animal
species are guinea pigs and, in particular, primates (cf.: Lindahl,
A. K., Scarborough, R. M., Naughton, M. A., Harker, L. A., Hanson,
S. R., Thromb Haemost 1993, 69, 1196; Cook J J, Sitko G R, Bednar
B, Condra C, Mellott M J, Feng D-M, Nutt R F, Shager J A, Gould R
J, Connolly T M, Circulation 1995, 91, 2961-2971; Kogushi M,
Kobayashi H, Matsuoka T, Suzuki S, Kawahara T, Kajiwara A,
Hishinuma I, Circulation 2003, 108 Suppl. 17, IV-280; Derian C K,
Damiano B P, Addo M F, Darrow A L, D'Andrea M R, Nedelman M, Zhang
H-C, Maryanoff B E, Andrade-Gordon P, J. Pharmacol. Exp. Ther.
2003, 304, 855-861). Alternatively, it is possible to use guinea
pigs which have been pretreated with inhibitors of PAR-3 and/or
PAR-4 (Leger A J et al., Circulation 2006, 113, 1244-1254), or
transgenic PAR-3- and/or PAR-4-knockdown guinea pigs.
3.b) Impaired Coagulation and Organ Dysfunction in the Case of
Disseminated Intravasal Coagulation (DIC)
[0854] The inventive compounds can be studied in models of DIC
and/or sepsis in suitable animal species. Suitable animal species
are guinea pigs and, in particular, primates, and for the study of
endothelium-mediated effects also mice and rats (cf.: Kogushi M,
Kobayashi H, Matsuoka T, Suzuki S, Kawahara T, Kajiwara A,
Hishinuma I, Circulation 2003, 108 Suppl. 17, IV-280; Derian C K,
Damiano B P, Addo M F, Darrow A L, D'Andrea M R, Nedelman M, Zhang
H-C, Maryanoff B E, Andrade-Gordon P, J. Pharmacol. Exp. Ther.
2003, 304, 855-861; Kaneider N C et al., Nat Immunol, 2007, 8,
1303-12; Camerer E et al., Blood, 2006, 107, 3912-21; Riewald M et
al., J Biol Chem, 2005, 280, 19808-14.). Alternatively, it is
possible to use guinea pigs which have been pretreated with
inhibitors of PAR-3 and/or PAR-4 (Leger A J et al., Circulation
2006, 113, 1244-1254), or transgenic PAR-3- and/or PAR-4-knockdown
guinea pigs.
3.b.1) Thrombin-Antithrombin Complexes
[0855] Thrombin-antithrombin complexes (referred to hereinafter as
"TAT") are a measure of the thrombin formed endogenously by
coagulation activation. TATs are determined via an ELISA assay
(Enzygnost TAT micro, Dade-Behring). Plasma is obtained from
citrated blood by centrifugation. 50 .mu.l of TAT sample buffer are
added to 50 .mu.l of plasma, shaken briefly and incubated at room
temperature for 15 min The samples are filtered with suction, and
the well is washed 3 times with wash buffer (300 .mu.l/well).
Between the wash steps, the plate is tapped to remove any residual
wash buffer. Conjugate solution (100 .mu.l) is added and the
mixture is incubated at room temperature for 15 min The samples are
filtered with suction, and the well is washed 3 times with wash
buffer (300 .mu.l/well). The chromogenic substrate (100 .mu.l/well)
is then added, the mixture is incubated in the dark at room
temperature for 30 min, stop solution (100 .mu.l/well) is added,
and the development of colour at 492 nm is measured (Safire plate
reader).
3.b.2) Parameters of Organ Dysfunction
[0856] Various parameters are determined, which allow conclusions
to be drawn with respect to the functional restriction of various
internal organs owing to the administration of LPS, and the
therapeutic effect of test substances can be assessed. Citrated
blood or, if appropriate, lithium heparin blood, is centrifuged,
and the plasma is used to determine the parameters. Typically, the
following parameters are determined: creatinine, urea, aspartate
aminotransferase (AST), alanine aminotransferase (ALT), total
bilirubin, lactate dehydrogenase (LDH), total protein, total
albumin and fibrinogen. The values give information regarding
kidney function, liver function, cardiovascular function and
vascular function.
3.b.3) Parameters of Inflammation
[0857] The extent of the inflammatory reaction triggered by
endotoxin can be demonstrated by the increase in inflammation
mediators, for example interleukins (1, 6, 8 and 10), tumour
necrosis factor alpha or monocyte chemoattractant protein-1, in the
plasma. ELISAs or the Luminex system may be used for this
purpose.
3.c) Antitumour Activity
[0858] The inventive compounds can be tested in models of cancer,
for example in the human breast cancer model in immunodeficient
mice (cf.: S. Even-Ram et. al., Nature Medicine, 1988, 4,
909-914).
3.d) Antiangiogenetic Activity
[0859] The inventive compounds can be tested in in vitro and in
vivo models of angiogenesis (cf.: Caunt et al., Journal of
Thrombosis and Haemostasis, 2003, 10, 2097-2102; Haralabopoulos et
al., Am J Physiol, 1997, C239-C245; Tsopanoglou et al., JBC, 1999,
274, 23969-23976; Zania et al., JPET, 2006, 318, 246-254).
3.e) Blood Pressure- and Pulse-Modulating Activity
[0860] The inventive compounds can be tested in in vivo models for
their action on arterial blood pressure and heart rate. To this
end, rats (for example Wistar) are provided with implantable
radiotelemetry units, and an electronic data acquisition and
storage system (Data Sciences, MN, USA) consisting of a chronically
implantable transducer/transmitter unit in combination with a
liquid-filled catheter is employed. The transmitter is implanted
into the peritoneal cavity, and the sensor catheter is positioned
in the descending aorta. The inventive compounds can be
administered (for example orally or intravenously). Prior to the
treatment, the mean arterial blood pressure and the heart rate of
the untreated and treated animals are measured, and it is ensured
that they are in the range of about 131-142 mmHg and 279-321
beats/minute. PAR-1-activating peptide (SFLLRN; for example doses
between 0.1 and 5 mg/kg) is administered intravenously. Blood
pressure and heart rate are measured at different time intervals
and periods with and without PAR-1-activating peptide and with and
without an inventive compound (cf.: Cicala C et al., The FASEB
Journal, 2001, 15, 1433-5; Stasch J P et al., British Journal of
Pharmacology 2002, 135, 344-355).
3.f) Thrombosis Model
[0861] A further in vivo thrombosis assay which is suitable for
determining the efficacy of the compounds of the present invention
is described in Tucker E I, Marzec U M, White T C, Hurst S, Rugonyi
S, McCarty O J T, Gailani D, Gruber A, Hanson SR: Prevention of
vascular graft occlusion and thrombus-associated thrombin
generation by inhibition of factor XI. Blood 2009, 113,
936-944.
4.) Determination of the Solubility
Preparation of the Starting Solution (Original Solution)
[0862] At least 1.5 mg of the test substance are weighed out
accurately into a wide-mouth 10 mm screw V-vial (from Glastechnik
Grafenroda GmbH, Art. No. 8004-WM-H/V15.mu.) with fitting screw cap
and septum, DMSO is added to a concentration of 50 mg/ml and the
vial is vortexed for 30 minutes.
Preparation of the Calibration Solutions:
[0863] The pipetting steps necessary are effected in 1.2 ml 96-deep
well plates (DWP) with the aid of a liquid-handling robot. The
solvent used is a mixture of acetonitrile/water 8:2.
[0864] Preparation of the Starting Solution of Calibration
Solutions (Stock Solution):
[0865] 833 .mu.l of the solvent mixture are added to 10 .mu.l of
the original solution (concentration=600 .mu.g/ml), and the mixture
is homogenized. 1:100 dilutions in separate DWPs are prepared from
each test substance, and these are homogenized in turn.
[0866] Calibration Solution 5 (600 ng/ml):
[0867] 270 .mu.l of the solvent mixture are added to 30 .mu.l of
the stock solution, and the mixture is homogenized.
[0868] Calibration Solution 4 (60 ng/ml):
[0869] 270 .mu.l of the solvent mixture are added to 30 .mu.l of
the calibration solution 5, and the mixture is homogenized.
[0870] Calibration Solution 3 (12 ng/ml):
[0871] 400 .mu.l of the solvent mixture are added to 100 .mu.l of
the calibration solution 4, and the mixture is homogenized.
[0872] Calibration Solution 2 (12 ng/ml):
[0873] 270 .mu.l of the solvent mixture are added to 30 .mu.l of
the calibration solution 3, and the mixture is homogenized.
[0874] Calibration Solution 1 (0.6 ng/ml):
[0875] 150 .mu.l of the solvent mixture are added to 150 .mu.l of
the calibration solution 2, and the mixture is homogenized.
Preparation of the Sample Solutions:
[0876] The pipetting steps necessary are effected in 1.2 ml 96-well
DWPs with the aid of a liquid-handling robot. 1000 .mu.l of PBS
buffer pH 6.5 are added to 10.1 .mu.l of the stock solution. (PBS
buffer pH 6.5: 61.86 g of sodium chloride, 39.54 g of sodium
dihydrogenphosphate and 83.35 g of 1 N aqueous sodium hydroxide
solution are weighed out into a 1 litre standard flask and made up
to the mark with water, and the mixture is stirred for about 1
hour. 500 ml of this solution are introduced into a 5 litre
measuring flask and made up to the mark with water. The pH is
adjusted to 6.5 using 1 N aqueous sodium hydroxide solution.)
Procedure:
[0877] The pipetting steps necessary are effected in 1.2 ml 96-well
DWPs with the aid of a liquid-handling robot. The sample solutions
prepared in this manner are shaken at 1400 rpm and at 20.degree. C.
using a variable temperature shaker for 24 hours. 180 .mu.l are
removed from each of these solutions and transferred into Beckman
Polyallomer centrifuge tubes. These solutions are centrifuged at
about 223 000.times.g for 1 hour. From each sample solution, 100
.mu.l of the supernatant are removed and diluted 1:10 and 1:1000
with PBS buffer 6.5.
Analysis:
[0878] The samples are analysed by means of HPLC/MS-MS. The test
compound is quantified by means of a five-point calibration curve.
The solubility is expressed in mg/l. Analysis sequence: 1) blank
(solvent mixture); 2) calibration solution 0.6 ng/ml; 3)
calibration solution 1.2 ng/ml; 4) calibration solution 12 ng/ml;
5) calibration solution 60 ng/ml; 6) calibration solution 600
ng/ml; 7) blank (solvent mixture); 8) sample solution 1:1000; 9)
sample solution 1:10.
HPLC/MS-MS Method:
[0879] HPLC: Agilent 1100, quat. pump (G1311A), autosampler CTC HTS
PAL, degasser (G1322A) and column thermostat (G1316A); column:
Oasis HLB 20 mm.times.2.1 mm, 25.mu.; temperature: 40.degree. C.;
eluent A: water+0.5 ml of formic acid/1; eluent B: acetonitrile+0.5
ml of formic acid/1; flow rate: 2.5 ml/min; stop time 1.5 min;
gradient: 0 min 95% A, 5% B; ramp: 0-0.5 min 5% A, 95% B; 0.5-0.84
min 5% A, 95% B; ramp: 0.84-0.85 min 95% A, 5% B; 0.85-1.5 min 95%
A, 5% B.
[0880] MS/MS: WATERS Quattro Micro Tandem MS/MS; Z-Spray API
interface; HPLC-MS inlet splitter 1:20; measurement in the ESI
mode.
5.) In Vitro Clearance Determinations with Hepatocytes
[0881] Incubations with fresh primary hepatocytes are carried out
at 37.degree. C. in a total volume of 1.5 ml with a modified
Janus.RTM. robot (Perkin Elmer) while shaking The incubations
typically contain 1 million living liver cells/ml, approx 1 .mu.M
substrate and 0.05 M potassium phosphate buffer (pH=7.4). The final
acetonitrile concentration in the incubation is 1%.
[0882] Aliquots of 125 .mu.l are withdrawn from the incubations
after 2, 10, 20, 30, 50, 70 and 90 min and transferred into 96-well
filter plates (0.45 .mu.m low-binding hydrophilic PTFE; Millipore:
MultiScreen Solvinert). Each of these contain 250 .mu.l of
acetonitrile to stop the reaction. After the centrifugation, the
filtrates are analysed by MS/MS (typically API 3000).
[0883] The in vitro clearances are calculated from the half-lives
of the substance degradation, using the following equation:
CL'.sub.intrinsic [ml/(minkg)]=(0.693/in vitro t1/2[min])(liver
weight [g liver/kg bodyweight])(cell count[1.110 8]/liver weight
[g])/(cell count[110 6]/incubation volume [ml])
[0884] The CL.sub.blood is calculated without taking into account
the free fraction ("nonrestricted well stirred model") by the
following equation:
CL.sub.blood well-stirred [l/(hkg)]=(Q.sub.H
[l/(hkg)]CL'.sub.intrinsic [l/(hkg)])/(Q.sub.H
[l/(hkg)]+CL'.sub.intrinsic [l/(hkg)])
[0885] The species-specific extrapolation factors used for the
calculation are summarized in the following table:
TABLE-US-00003 male/female Mouse Mouse Rat Dog Cyno Man m f m/f m/f
f m/f Cell number/g 110 110 110 110 110 110 liver [10.sup.6 cells]
Liver[g]/ 50 43 32 39 30 21 kg bodyweight Liver blood 5.4 5.4 4.2
2.1 2.5 1.3 flow [l/(h kg)]
[0886] Fmax values which state the maximum possible
bioavailability--based on the hepatic extraction--are calculated as
follows:
F.sub.max well-stirred [%]=(1-(CL.sub.blood well-stirred
[l/(hkg)]/Q.sub.H [l/(hkg)]))100
6.) Determination of In Vivo Pharmacokinetics
[0887] To determine the in vivo pharmacokinetics, the test
substances are dissolved in different formulation media (e.g.
plasma, ethanol, DMSO, PEG400, etc.) or mixtures of these
solubilizers, and administed intravenously or perorally to mice,
rats, dogs or monkeys. Intravenous administration is effected
either as a bole or as an infusion. The doses administered are in
the range from 0.1 to 5 mg/kg. Blood samples are taken by means of
a catheter or as sacrifice plasma at different times over a period
of up to 26 h. In addition, some organ, tissue and urine samples
are also obtained. The substances are determined quantitatively in
the test samples by means of calibration samples which are
established in the particular matrix. Proteins present in the
samples are removed by precipitation with acetonitrile or methanol.
Subsequently, the samples are separated by means of HPLC on a 2300
HTLC system (Cohesive Technologies, Franklin, Mass., USA) or
Agilent 1200 (Boblingen, Germany) using reverse-phase columns The
HPLC system is coupled via a turbo ion spray interface to an API
3000 or 4000 triple quadropole mass spectrometer (Applied
Biosystems, Darmstadt, Germany). The plot of plasma concentration
against time is evaluated using a validated kinetics evaluation
program.
C) WORKING EXAMPLES OF PHARMACEUTICAL COMPOSITIONS
[0888] The inventive substances can be converted in pharmaceutical
preparations as follows:
Tablet:
Composition:
[0889] 100 mg of the compound of Example 1, 50 mg of lactose
(monohydrate), 50 mg of maize starch, 10 mg of polyvinylpyrrolidone
(PVP 25) (from BASF, Germany) and 2 mg of magnesium stearate.
[0890] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12
mm
Production:
[0891] The mixture of the compound of Example 1, lactose and starch
is granulated with a 5% solution (m/m) of the PVP in water. The
granules are dried and then mixed with the magnesium stearate for 5
min. This mixture is compressed in a conventional tablet press (see
above for tablet format).
Oral Suspension:
Composition:
[0892] 1000 mg of the compound of Example 1, 1000 mg of ethanol
(96%), 400 mg of Rhodigel (xanthan gum) (from FMC, USA) and 99 g of
water.
[0893] A single dose of 100 mg of the inventive compound
corresponds to 10 ml of oral suspension.
Production:
[0894] The Rhodigel is suspended in ethanol, and the compound of
Example 1 is added to the suspension. The water is added while
stirring. The mixture is stirred for approx. 6 h until the Rhodigel
has finished swelling.
Intravenously Administrable Solution:
Composition:
[0895] 1 mg of the compound of Example 1, 15 g of polyethylene
glycol 400 and 250 g of water for injections.
Production:
[0896] The compound of Example 1 is dissolved together with
polyethylene glycol 400 by stirring in the water. The solution is
sterile-filtered (pore diameter 0.22 .mu.m) and dispensed under
aseptic conditions into heat-sterilized infusion bottles. The
latter are closed with infusion stoppers and crimped caps.
* * * * *