Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-sulfonam ides and their use as
pharmaceuticals
The present invention relates to N-[4-(1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-
sulfonamides of the formula I,
R1
(R2), N
0
Ar i/
11101
// N
0 H
wherein Ar, R1, R2 and n have the meanings indicated below. The compounds of
the
formula I are valuable pharmacologically active compounds which modulate
protein
kinase activity, specifically the activity of serum and glucocorticoid
regulated kinase
(SGK), in particular of serum and glucocorticoid regulated kinase isoform 1
(SGK-1,
SGK1), and are suitable for the treatment of diseases in which SGK activity is
inappropriate, for example degenerative joint disorders or inflammatory
processes
such as osteoarthritis or rheumatism. The invention furthermore relates to
processes
for the preparation of the compounds of the formula I, their use as
pharmaceuticals,
and pharmaceutical compositions comprising them.
Due to their physiologic importance, variety, and ubiquity, protein kinases
have
become one of the most important and widely-studied family of enzymes in
biochemical and medical research. Currently, there are about 500 different
known
protein kinases. However, because three to four percent of the human genome is
a
code for the formation of protein kinases, there may be many thousands of
distinct
and separate kinases in the human body. Protein kinases serve to catalyze the
phosphorylation of an amino acid side chain in various proteins by the
transfer of the
gamma-phosphate of the ATP-Mg2+ complex to said amino acid side chain. These
enzymes control the majority of the signaling processes inside cells, thereby
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governing cell function, growth, differentiation and destruction (apoptosis)
through
reversible phosphorylation of the hydroxyl groups of serine, threonine and
tyrosine
residues in proteins. Studies have shown that protein kinases are key
regulators of
many cell functions, including signal transduction, transcriptional
regulation, cell
motility, and cell division. Several oncogenes have also been shown to encode
protein kinases, suggesting that kinases play a role in oncogenesis. These
processes
are highly regulated, often by complex intermeshed pathways where each kinase
will
itself be regulated by one or more kinases. Consequently, aberrant or
inappropriate
protein kinase activity can contribute to the rise of disease states
associated with
such aberrant kinase activity.
The protein kinase family of enzymes is typically classified into two main
subfamilies,
protein tyrosine kinases, which phosphorylate tyrosine residues, and protein
serine/threonine kinases (PSTK), which phosphorylate serine and threonine
residues. The PSTK subfamily includes cyclic AMP- and cyclic GMP-dependent
protein kinases, calcium- and phospholipid-dependent protein kinase, calcium-
and
calmodulin-dependent protein kinases, casein kinases, cell division cycle
protein
kinases and others. These kinases are usually cytoplasmic or associated with
the
particulate fractions of cells, possibly by anchoring proteins. Aberrant
protein
serine/threonine kinase activity has been implicated or is suspected in a
number of
pathologies such as rheumatoid arthritis, psoriasis, septic shock, bone loss,
many
cancers and other proliferative diseases. Accordingly, serine/threonine
kinases and
their associated signal transduction pathways are important targets for drug
design.
Serum and glucocorticoid regulated kinases, also designated as
serum/glucocorticoid
regulated kinase, serum and glucocorticoid induced kinase, serum and
glucocorticoid
inducible kinase or serum and glucocorticoid dependent kinase, form a family
of
serine/threonine kinases. Currently three members are known, designated as SGK-
1,
SGK-2 and SGK-3. SGK-3 is also described under the name SGKL (SGK-like), and
CISK. SGK-1 was described in 1993 for the first time as an "immediate early
genes"
in a rat mammary cancer cell line (Webster et al., 1993a; Webster et al.,
1993b). At
the protein level the three isoforms show a homology of at least 80% in their
catalytic
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domain. SGK-1 is expressed in almost all tissues that have been tested so far,
but
the amounts of mRNA expressed vary greatly depending on the nature of the
studied
tissue type (Gonzalez-Robayna et al., 1999; Waldegger et al., 1999; Alliston
et al.,
2000; Klingel et al., 2000; Lang et al., 2000; Loffing et al., 2001; Fillon et
al., 2002;
Warntges et al., 2002a). In addition, SGK-1 mRNA is found in several embryonic
tissues. During mouse embryogenesis, the SGK-1 mRNA shows development-
dynamic changes in specific tissues of the embryo (decidua, yolk sac, otic
vesicle),
and is detectable during the organogenesis in lung buds, brain, heart, liver,
thymus,
etc. (Lee et al., 2001). SGK-2 is expressed with greatest abundance in
epithelial
tissues, such as in the kidney, liver, pancreas, and specific areas of the
brain
(Kobayashi et al., 1999). SGK-3 was detected in all tested tissues and is
especially
found in the adult heart and spleen (Kobayashi et al., 1999; Liu et al.,
2000).
A distinguishing feature of SGK to many other kinases is based on the
stringent
stimulus-dependent regulation of transcription, cellular localization and
enzymatic
activation (Firestone et al., 2003) of the molecule. In order to induce and
activate
SGK-1, a variety of stimuli are known. These include mineralocorticoids
(Brennan
and Fuller, 2000; Shigaev et al., 2000; Bhargava et al., 2001), gonadotropins
(Richards et al., 1995; Gonzalez-Robayna et al., 2000), 1,25(OH)2D3 (Akutsu et
al.,
2001), p53, osmotic, hypotonic and cellular volume changes (Waldegger et al.,
1997;
Klingel et al., 2000; Waldegger et al., 2000; Rozansky et al., 2002; Warntges
et al.,
2002a), cytokines such as GM-CSF and TNF-alpha (Cooper et al., 2001) or by TGF-
beta (Kumar et al., 1999; Waldegger et al.; 1999; Lang et al., 2000). In
further
growth-dependent signaling pathways SGK is induced by serum (Webster et al.,
1993a), insulin and IGF-1 (Kobayashi and Cohen, 1999; Park et al., 1999;
Perrotti et
al., 2001), FSH (Alliston et al., 1997), Fibroblast and Platelet-derived
growth factor
(Davies et al., 2000), activators of the Erk signaling cascade (Hayashi et
al., 2001)
and TPA (Mizuno and Nishida, 2001). SGK-1 is also known to be activated in
pathological changes such as ischemic brain injury (Imaizumi et al., 1994),
viral
hepatitis (Fillon et al., 2002), pulmonary fibrosis (Warntges et al., 2002b)
or cardiac
fibrosis (Funder 2001).
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In order to be converted into its functional form, SGK-1 requires activation
by
phosphorylation. This is mediated by a signaling cascade involving the
phosphatidylinositol 3 (PI-3) kinase and phosphoinositide 3-dependent kinases
PDK1
and PDK2. The activation of SGK-1 through the PI-3 kinase signaling pathway is
known to be a response to insulin, IGF and growth factors. For activation the
phosphorylation of two amino acid residues is necessary, threonine256 on the T-
loop
and serine422 at the hydrophobic motif of the protein. Phosphorylation at
threonine256
is mediated by PDK1, phosphorylation at serine422 should be catalyzed by a
putative
PDK2, which is not yet known (Kobayashi and Cohen, 1999; Park et al., 1999;
Biondi
et al., 2001).
For the function of SGK, there are a series of studies that show regulatory
influence
of SGK-1, SGK-2 and SGK-3 on cell membrane channels. It was shown that the
epithelial Na + channel (ENaC), the main transporter for the mineralocorticoid-
regulated Na + reabsorption in the renal tubule, is a target of SGK-1, SGK-2
and SGK-
3 (Alvarez de la Rosa et al., 1999; BOhmer et al., 2000; Wagner et al, 2001;
Wang et
al., 2001; Faletti et al., 2002; Friedrich et al., 2003). The interaction of
ENaC and
SGK is not by direct phosphorylation (Lang et al., 2000), but due to the
inactivation of
the ubiquitin ligase Nedd4-2 (Debonneville et al., 2001; Snyder et al., 2002)
as a
result of phosphorylation by SGK. As a result, the amount and residence time
of
ENaC in the cell membrane is increased (Staub et al., 1997; Alvarez de la Rosa
et
al., 1999; Wagner et al., 2001). It has also been shown in a number of
experiments
that ROMK1 is a molecular target of SGK. However, ROMK1 is not directly
regulated
by SGK, but needs the "Na+/H+ exchange regulating factor 2" (NHERF2) as an
intermediary molecule (Shenolikar and Weinmann, 2001; Yun, 2003). The same
mechanism applies to another target molecule of SGK, the Na4YH+ transporter
NHE3
(Yun et al., 2002). In addition it has also been shown in experiments on
Xenopus
oocytes that SGK influences the Kv1.3 channel-dependent K+ current (Gamper et
al.,
2002; Warntges et al., 2002a). It was also reported that SGK regulates the
amino
acid transporter SN1 and 42F/LAT (Wagner et al., 2000; BOhmer et al., 2003a,
b).
SGK-1 has also been shown to play a role in cell proliferation and electrolyte
homeostasis (Loffing et al., 2006; McCormick et al., 2005; Vallon et al.,
2005; Vallon
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and Lang, 2005; Lang et al., 2003). SGK-1 is thought to regulate several
cellular
mechanisms that contribute to disease states. For example, SGK-1 has been
shown
to mediate fibronectin formation in diabetic nephropathy (Feng et al., 2005).
SGK1
has also been shown to mediate insulin, IGF-1, and aldosterone-induced Na+
5 retention in renal and cardiovascular disease (McCormick et al., 2005;
ValIon et al.,
2005; Lang et al., 2003). In addition, SGK-1 has been shown to be involved in
inducing the transcription and procoagulation activity of tissue factor (TF)
(BelAiba et
al., 2006), and in regulating IGF-1-mediated cell proliferation (Henke et al.,
2004).
Osteoarthritis (OA) is one of the most common degenerative joint diseases and
leads
in an advanced stage to a loss of joint function. During the chronic course of
illness,
there is a destruction of the articular cartilage down to the underlying bone
tissue,
which makes a joint replacement surgery in affected patients necessary. In
addition
to the destruction of the cartilage, pathological changes in the synovial
membrane
and the ligaments can also be observed. The disease is temporarily accompanied
by
inflammatory processes like in rheumatoid arthritis, but differs from it. The
exact
causes of the disease are still unknown, however, several factors come into
question,
such as metabolic changes, mechanical stress, genetic disorders or joint
injuries.
Regardless of the original trigger, the degradation of articular cartilage
occurs as a
common pathological feature of OA. A key feature of the pathological condition
of OA
is the proteolytic cleavage of collagens and proteoglycans. Simultaneously a
number
of other processes occur such as anabolic repair mechanisms redifferentiation
of the
cells or cell death. The precise molecular mechanisms underlying these
processes
are still poorly understood.
The healthy functioning of the adult cartilage is created by its unique
biomechanical
properties, providing both the resistance against high pressure as well as the
necessary elasticity of the tissue. The decisive factor is the special
organization of
the cartilage tissue. Unlike most other tissues, the cartilage cells are not
in direct
contact but are embedded separately from each other in an extracellular matrix
(ECM). The macromolecules of this ECM guarantee the viability of the articular
cartilage and joints. The basic structure of the ECM consists of a network
that is
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formed by fibrils of collagen types II, IX and Xl. Proteoglycans, mainly
aggrecan, are
embedded in the ECM producing an extremely high osmotic water binding
capacity.
The water pressure generated in connection with the properties of the collagen
backbone guarantee the specific properties of the cartilage. A main feature of
the
pathogenesis of OA is the loss of the ECM of the cartilage and the articular
cartilage
tissue. The function of the affected joint is restricted by or lost by this
mechanism. In
addition, various symptomatic parameters such as pain appear during
symptomatic
progression of the disease. Current treatments for osteoarthritis are limited
mostly to
the alleviation of symptomatic complaints. A causal therapy based on drugs,
which
leads to the decrease of cartilage degeneration, is not possible to current
knowledge.
Therefore, there is a considerable need for novel drugs for the prevention
and/or
therapy of osteoarthritis.
It has been shown, through comparative gene expression analysis of samples of
total-cellular RNA from healthy and degenerated/degenerating cartilage that
SGK-1
is expressed in degenerated/degenerating osteoarthritic cartilage, while it is
not
detectable in healthy articular cartilage (Bartnik et al., 2006). Moreover,
further
experiments gave evidence of the causal implication of SGK in the pathogenesis
of
degenerative cartilage changes (Bartnik et al., 2006). As a conclusion of
these
studies, SGK-1 is specifically involved in pathological conditions of the
cartilage, for
example in the context of rheumatoid arthritis or osteoarthritis, in
particular in the
context of osteoarthritis, and thus represents a key molecule inducing
cartilage
degradative processes. Due to the high homology between the SGK family
members,
it is assumed that this also applies to the SGK-2 and SGK-3.
The identification of these relationships allows the discovery of drugs for
the
prevention or therapy of degenerative cartilage changes by determining the
effect of
potential drugs on the activity of SGK and / or the levels of SGK by known
test
methods. The causal implication of SGK in the pathogenesis of degenerative
joint
disease allows a focused search for therapeutic agents that target regulatory
mechanisms for the restoration of normal cell physiology of cartilage. In the
joints of
mouse embryos SGK-1 mRNA was detected specifically in hypertrophic
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chondrocytes but not in proliferative cells. The role of SGK-1 in this model
of skeletal
development and endochondral ossification shows that the natural occurrence of
SGK-1 in cartilage is not associated with the synthesis and maintenance of
cartilage,
but exerts its function in the conversion (hypertrophy) and degradation. The
expression of SGK-1 in osteoarthritic cartilage is thus a process that causes
or
promotes the pathology of OA. Due to its regulatory properties SGK-1 could be
a key
molecule for the induction of early pathological changes in cartilage as well
as for the
later degradative activities. Therefore, SGK-1 is a very relevant target for
the
pharmacological intervention in osteoarthritis.
To specifically study the function of SGK-1 during differentiation of
cartilage, human
SGK-1 was overexpressed in murine ATDC5 cells. In these experiments, it was
clearly demonstrated that overexpression of SGK-1 causes inhibition of
cartilage
synthesis. Both the amount of Alcian blue stained proteoglycan as well as
aggrecan
m RNA was significantly reduced. A kinase deficient SGK-1 form, however, had
no
negative effect on these parameters. Regarding the effect of SGK-1 in OA
diseased
articular cartilage, several conclusions can be drawn from these experiments.
On the
one hand, SGK-1 expressing chondrocytes are no longer able to synthesize
sufficient
extracellular matrix such as proteoglycans, which are essential for the
function of the
tissue. On the other hand, the cartilage cells are inhibited to compensate
for, or
repair, degradation processes by increasing the expression of genes such as
aggrecan. Therefore a function of SGK-1 as a potential cause and central
factor of
OA pathology is confirmed. SGK-1 thus represents a highly relevant target
molecule
for the development of novel drugs for the treatment of degenerative cartilage
changes, especially osteoarthritis.
In view of the relevance of SGK-1 for various physiological processes outlined
above,
inhibitors of SGK-1 such as the compounds of the present invention can be used
in
the treatment, including therapy and prophylaxis, of various disease states in
which
SGK-1 activity plays a role or which are associated with an inappropriate SGK-
1
activity, or in which an inhibition, regulation or modulation of signal
transduction by
SGK-1 is desired by the physician, for example degenerative joint disorders
and
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degenerative cartilage changes including osteoarthritis, osteoarthrosis,
rheumatoid
arthritis, spondylosis, chondrolysis following joint trauma and prolonged
joint
immobilization after meniscus or patella injuries or ligament tears,
connective tissue
disorders such as collagenoses, periodontal disorders, wound-healing
disturbances,
diabetes including diabetes mellitus, diabetic nephropathy, diabetic
neuropathy,
diabetic angiopathy and microangiopathy, obesity, metabolic syndrome
(dyslipidaemia), systemic and pulmonary hypertension, cerebral infarctions,
cardiovascular diseases including cardiac fibrosis after myocardial
infarction, cardiac
hypertrophy and heart failure, arteriosclerosis, renal diseases including
glomerulosclerosis, nephrosclerosis, nephritis, nephropathy and electrolyte
excretion
disorder, and any type of fibrosis and inflammatory processes including liver
cirrhosis, lung fibrosis, fibrosing pancreatitis, rheumatism, arthritis, gout,
Crohn's
disease, chronic bronchitis, radiation fibrosis, sclerodermatitis, cystic
fibrosis, scar
formation and Alzheimer's disease. Inhibitors of SGK-1 such as the compounds
of
the present invention can also be used in the treatment of pain including
acute pain
like pain following injuries, post-operative pain, pain in association with an
acute
attack of gout and acute pain following jaw-bone surgery interventions, and
chronic
pain like pain associated with chronic musculoskeletal diseases, back pain,
pain
associated with osteoarthritis or rheumatoid arthritis, pain associated with
inflammation, amputation pain, pain associated with multiple sclerosis, pain
associated with neuritis, pain associated with carcinomas and sarcomas, pain
associated with AIDS, pain associated with chemotherapy, trigeminus neuralgia,
headache, migraine cephalalgia, neuropathic pains, post-herpes zoster
neuralgia.
Inhibitors of SGK-1 such as the compounds of the present invention can also be
used in tumor therapy for inhibiting the growth of tumor cells and tumor
metastases.
Inhibitors of SGK-1 such as the compounds of the present invention can also be
used for the treatment of chronic disorders of the locomotor system such as
inflammatory, immunologically or metabolically related acute and chronic
arthritides,
arthropathies, myalgias and disturbances of bone metabolism. Further,
inhibitors of
SGK-1 such as the compounds of the present invention can be used in the
treatment
of peptic ulcers, especially in forms that are triggered by stress, in the
treatment of
tinnitus, in the treatment of bacterial infections and in anti-infective
therapy, for
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increasing the learning ability and attention, for counteracting cellular
aging and
stress and thus increasing life expectancy and fitness in the elderly, in
states of
neuronal excitability including epilepsy, in the treatment of glaucoma or
cataracts,
and in the treatment of coagulopathies including dysfibrinogenaemia,
hypoproconvertinaemia, haemophilia B, Stuart-Prower defect, prothrombin
complex
deficiency, consumption coagulopathy, fibrinolysis, immunokoagulopathy or
complex
coagulopathies.
Further details about the physiological role of SGK can be found, for example,
in the
mentioned literature articles, the particulars of which are as follows.
Akutsu,N., Lin. R., Bastien.Y., Bestawros,A., Enepekides,D.J., Black,M.J., and
White,J.H. (2001). Regulation of gene Expression by 1alpha,25-dihydroxyvitamin
D3
and Its analog EB1089 under growth-inhibitory conditions in squamous Carcinoma
Cells. Mol Endocrinol, 15, 1127-1139.
Alliston,T.N., Maiyar,A.C., Buse, P., Firestone,G.L., and Richards,J.S.
(1997). Follicle
stimulating hormone-regulated expression of serum/glucocorticoid-inducible
kinase in
rat ovarian granulosa cells: a functional role for the Sp1 family in Promoter
activity.
Mol Endocrinol, 11, 1934-1949.
Alliston,T.N., Gonzalez-Robayna,J.J., Buse, P., Firestone,G.L., and
Richards,J.S.
(2000). Expression and localization of serum/glucocorticoid-induced kinase in
the rat
ovary: relation to follicular growth and differentiation. Endocrinology, 141,
385-395.
Alvarez,d.I.R., Zhang,P., Naray-Fejes-Toth,A., Fejes-Toth,G., and Canessa,C.M.
(1999). The serum and glucocorticoid kinase sgk increases the abundance of
epithelial sodium Channels in the plasma membrane of Xenopus oocytes. J Biol
Chem, 274, 37834-37839.
Bartnik,E., Aigner,T., Dietz,U., and Brimmer,A., (2006). The use of a
Serum/Glucocorticoid-regulated Kinase. WO 03/061130.
BelAiba,R.S., Djordjevic,T., Bonello,S., Artunc,F., Lang, F., Hess,J., and
Gbrlach,A.
(2006). The serum- and glucocorticoid-inducible kinase Sgk-1 is involved in
pulmonary vascular remodeling: role in redox-sensitive regulation of tissue
factor by
thrombin. Circ Res 98(6), 828-836.
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Klingel,K., Warntges,S., Bock,J., Wagner,C.A., Sauter,M., Waldegger,S.,
Kandolf,R.,
and Lang,F. (2000). Expression of cell volume-regulated kinase h-sgk in
pancreatic
tissue. Am J Physiol Gastrointest Liver Physiol, 279, G998-G1002.
Bhargava,A., Fullerton,M.J., Myles, K., Purdy,T.M., Funder,J.W., Pearce, D.,
and
5 Cole,T.J. (2001). The serumand glucocorticoid-induced kinase is a
physiological
mediator of aldosterone action. Endocrinology, 142, 1587-1594.
Biondi,R.M., Kieloch,A., Currie,R.A., Deak,M., and Alessi,D.R. (2001). The PIF-
binding pocket in PDK1 is essential for activation of 56K and SGK, but not
PKB.
EMBO J, 20, 4380-4390.
10 Boehmer,C., Okur,F., Setiawan,I., Broer,S., and Lang,F. (2003a).
Properties and
regulation of glutamine transporter SN1 by protein kinases SGK and PKB.
Biochem
Biophys Res Commun, 306, 156-162.
Boehmer,C., Wilhelm,V., Palmada,M., Wallisch,S., Henke,G., Brinkmeier,H.,
Cohen,P., Pieske,B., and Lang,F. (2003b). Serum and glucocorticoid inducible
kinases in the regulation of the cardiac sodium Channel SCN5A. Cardiovasc Res,
57,
1079-1084.
Boehmer,C, Wagner,C.A, Beck,S., Moschen,I., Melzig,J., Werner,A., Lin,J.T.,
Lang,F., and Wehner,F. (2000). The shrinkage-activated Na(+) conductance of
rat
hepatocytes and its possible correlation to rENaC. Cell Physiol Biochem, 10,
187-
194.
Brennan,F.E. and Fuller,P.J. (2000). Rapid upregulation of serum and
glucocorticoidregulated kinase (sgk) gene expression by corticosteroids in
vivo. Mol
Cell Endocrinol, 166, 129-136.
Cooper,M.S., Bujalska,I., Rabbitt,E., Walker,E.A, Bland, R., Sheppard,M.C,
Hewison,M., and Stewart,P.M. (2001). Modulation of 11beta-hydroxysteroid
dehydrogenase isozymes by proinflammatory cytokines in osteoblasts: an
autocrine
switch from glucocorticoid inactivation to activation. J Bone Miner Res, 16,
1037-
1044.
Davies,S.P., Reddy,H., Caivano,M., and Cohen,P. (2000). Specificity and
mechanism of action of some commonly used protein kinase inhibitors. Biochem
J,
351, 95-105.
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Debonneville,C, Flores,S.Y., Kamynina,E., Plant,P.J., Tauxe,C, Thomas,M.A.,
Munster,C., Chraibi,A., Pratt,J.H., Horisberger,J.D., Pearce, D., Loffing,J.,
and
Staub,O. (2001). Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+)
Channel cell surface expression. EMBO J, 20, 7052-7059.
Faletti,C.J., Perrotti,N., Taylor,S.L, and Blazer-Yost,B.L. (2002). sgk: an
essential
convergence point for peptide and Steroid hormone regulation of ENaCmediated
Na+
transport. Am J Physiol Cell Physiol, 282, C494-0500.
Feng,Y., Wang,Q., Wang,Y., Yard,B., Lang,F. (2005). SGK1-mediated fibronectin
formation in diabetic nephropathy. Cell Physiol Biochem, 16(4-6), 237-244.
Fillon,S., Klingel,K., Warntges,S., Sauter,M., Gabrysch,S., Pestel,S.,
Tanneur,V.,
Waldegger,S., Zipfel,A., Viebahn,R., Haussinger,D., Broer,S., Kandolf,R., and
Lang,F. (2002). Expression of the serine/threonine kinase hSGK1 in chronic
viral
hepatitis. Cell Physiol Biochem, 12, 47-54.
Firestone,G.L., Giampaolo,J.R., and O'Keeffe,B.A. (2003). Stimulus-dependent
regulation of serum and glucocortieoid inducible protein kinase (SGK)
transcription,
subcellular localization and enzymatic activity. Cell Physiol Biochem, 13, 1-
12.
Friedrich,B., Feng,Y., Cohen, P., Risler,T., Vandewalle,A., Broer,S., Wang,J.,
Pearce,D., Lang,F. (2003). The serine/threonine kinases SGK2 and SGK3 are
potent
stimulators of the epithlial Na(+) Channel alpha, beta, gamma-EnaC. Pflugers
Arch,
445(6), 693-696.
Funder,J. (2001 ). Mineralocorticoids and cardiac fibrosis: the decade in
review. Clin
Exp Pharmacol Physiol, 28, 1002-1006.
Gamper,N., Fillon,S., Huber,S.M., Feng,Y., Kobayashi,T., Cohen,P., and Lang,F.
(2002). IGF-1 up-regulates K+ Channels via P13-kinase, PDK1 and SGK1. Pflugers
Arch, 443, 625-634.
Gonzalez-Robayna,I.J., Alliston,T.N., Buse, P., Firestone,G.L., and
Richards,J.S.
(1999). Functional and subcellular changes in the A-kinase-signaling pathway:
relation to aromatase and Sgk expression during the transition of granulosa
cells to
luteal cells. Mol Endocrinol, 13, 1318-1337.
Gonzalez-Robayna,I.J., Falender,A.E., Ochsner,S., Firestone,G.L., and
Richards,J.S.
(2000). Follicle-Stimulating hormone (FSH) stimulates phosphorylation and
activation
of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase
(Sgk):
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evidence for A kinase-independent signaling by FSH in granulosa cells. Mol
Endocrinol, 14, 1283-1300.
Hayashi,M., Tapping,R.I., Chao,T.H., Lo,J.F., King,C.C., Yang,Y., Lee,J.D.
(2001 ).
BMK1 mediates growth factor-induced cell proliferation through direct cellular
activation of serum and glucocorticoid-inducible kinase. J Biol Chem 276(12),
8631-
8634.
Henke,G., Maier,G., Wallisch,S., Boehmer,C., Lang,F. (2004). Regulation of the
voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum
and
glucocorticoid inducible kinase SGK1. J Cell Physiol, 199(2), 194-199.
Imaizumi,K., Tsuda,M., Wanaka,A., Tohyama,M., and Takagi,T. (1994).
Differential
expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in
rat
brain after CNS injury. Brain Res Mol Brain Res, 26, 189-196.
Kobayashi,T. and Cohen,P. (1999). Activation of serumand glucocorticoid-
regulated
protein kinase by agonists that activate phosphatidylinositide 3-kinase is
mediated by
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Kobayashi,T., Deak,M., Morrice,N., and Cohen,P. (1999). Characterization of
the
structure and regulation of two novel isoforms of serumand
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Kumar,J.M., Brooks,D.P., Olson,B.A., and Laping,N.J. (1999). Sgk, a putative
serine/threonine kinase, is differentially expressed in the kidney of diabetic
mice and
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Lang,F., Henke,G., Embark,H.M., Waldegger,S., Palmada,M., BOhmer,C., Vallon,V.
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Paulmichl,M., Gerke,V., Risler,T., Gamba,G., Capasso,G., Kandolf,R.,
Hebert,S.C.,
Massry,S.G., and Broer,S. (2000). Deranged transcriptional regulation of cell-
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Lee,E., Lein,E.S., Firestone,G.L. (2001 ). Tissue-specific expression of the
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McCormick,J.A., Bhalla,V., Pao,A.C., Pearce,D. (2005). SGK1: a rapid
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Yun,C.C. (2003). Concerted roles of SGK1 and the Na+/H+ exchanger regulatory
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The identification of small compounds that specifically inhibit, regulate or
modulate
signal transduction by SGK, is therefore desirable and an object of the
present
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invention. But besides being effective SGK inhibitors, it is desirable that
such
inhibitors also have further advantageous properties, for example high
bioavailability,
stability in plasma and liver, and selectivity versus other kinases or
receptors whose
inhibition or activation is not intended. Thus, it is an object of the present
invention to
provide SGK inhibitors which effectively inhibit an aberrant activity of SGK
in a
pathological context and which have further advantageous properties, for
example
high bioavailability, stability in plasma and liver, and selectivity versus
other kinases
and receptors which are not intended to be influenced in an agonistic or
antagonistic
manner. This object is achieved by providing the novel compounds of the
formula I
which exhibit excellent SGK-1 inhibitory activity and are favorable agents
with high
bioavailability and stability in plasma and liver.
Thus, a subject of the present invention are the compounds of the formula I,
in any of
their stereoisomeric forms or a mixture of stereoisomeric forms in any ratio,
and the
pharmaceutically acceptable salts thereof,
R1
(R2), N
0
Ar i/
11101
// N
0 H
wherein
Ar is selected from the series consisting of phenyl and a 5-membered or 6-
membered monocyclic aromatic heterocycle comprising 1, 2 or 3 identical or
different
ring heteroatoms selected from the series consisting of nitrogen, oxygen and
sulfur
and bonded via a ring carbon atom, which are all unsubstituted or substituted
by one
or more identical or different substituents R10;
n is selected from the series consisting of 0, 1 and 2;
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R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14, -C(0)-N(R16)-R17, -CN, (Ci -C4)-
alkyl and -(Ci-C4)-alkyl-O-R18;
R2 is selected from the series consisting of halogen, -(Ci-C4)-alkyl,
and -CN;
R10 is selected from the series consisting of halogen, (Ci-C4)-alkyl, (C3-C7)-
cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, -0-(C3-C7)-cycloalkyl,
-N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-
C(0)-R22, -NO2, -C(0)-N(R23)-R24 and -CN,
and two groups R10 bonded to adjacent ring carbon atoms in Ar, together with
the
carbon atoms carrying them, can form a 5-membered to 8-membered unsaturated
ring which comprises 0, 1 or 2 identical or different ring heteroatoms
selected from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one or more identical or different substituents selected from
the series
consisting of halogen, -(Ci-C4)-alkyl, -0-(Ci-C4)-alkyl and -CN;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen, (Ci-C4)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
or R11 and R12, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 7-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R11 and R12, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl;
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R14 and R15 are independently of one another selected from the series
consisting of
(Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -
(Ci-C4)-
alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het, wherein phenyl and Het all are
unsubstituted
or substituted by one or more identical or different substituents R30;
R16 is selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl;
R17 is selected from the series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-
cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl,
Het and
-(Ci-C4)-alkyl-Het, wherein phenyl and Het all are unsubstituted or
substituted by one
or more identical or different substituents R30,
or R16 and R17, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 7-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R16 and R17, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R18 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R19 is selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl;
R20 is selected from the series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-
cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl,
Het and
-(Ci-C4)-alkyl-Het, wherein phenyl and Het all are unsubstituted or
substituted by one
or more identical or different substituents R30,
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or R19 and R20, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 7-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R19 and R20, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R21 is selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl;
R22 is selected from the series consisting of (Ci-C4)-alkyl, (C3-C7)-
cycloalkyl
and -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl;
R23 and R24 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl;
R30 is selected from the series consisting of halogen, (Ci-C4)-alkyl,
and -CN;
Het is a monocyclic, 4-membered to 7-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 or 2 identical or different ring
heteroatoms
selected from the series consisting of nitrogen, oxygen and sulfur, and which
is
bonded via a ring carbon atom;
wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents.
If structural elements such as groups, substituents or numbers, for example,
can
occur several times in the compounds of the formula I, they are all
independent of
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each other and can in each case have any of the indicated meanings, and they
can
in each case be identical to or different from any other such element. In a
dialkylamino group, for example, the alkyl groups can be identical or
different.
5 Alkyl groups, i.e. saturated hydrocarbon residues, can be linear
(straight-chain) or
branched. This also applies if these groups are substituted or are part of
another
group, for example an -0-alkyl group (alkyloxy group, alkoxy group) or an HO-
substituted alkyl group (-alkyl-OH, hydroxyalkyl group). Depending on the
respective
definition, the number of carbon atoms in an alkyl group can be 1, 2, 3, 4, 5,
6, 7 or 8,
10 1, 2, 3, 4, 5 or 6, or 1, 2, 3 or 4, or 1, 2 or 3, or 1 or 2, or 1.
Examples of alkyl are
methyl, ethyl, propyl including n-propyl and isopropyl, butyl including n-
butyl, sec-
butyl, isobutyl and tert-butyl, pentyl including n-pentyl, 1-methylbutyl,
isopentyl,
neopentyl and tert-pentyl, hexyl including n-hexyl, 2,2,-dimethylhexyl, 3,3-
dimethylbutyl, 2-methylpentyl, 3-methylpentyl and isohexyl, heptyl including n-
heptyl,
15 and octyl including n-octyl. Examples of -0-alkyl groups are methoxy,
ethoxy, n-
propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy.
A substituted alkyl group can be substituted in any positions, provided that
the
respective compound is sufficiently stable and is suitable as a pharmaceutical
active
20 compound. The prerequisite that a specific group and a compound of the
formula I
are sufficiently stable and suitable as a pharmaceutical active compound,
applies in
general with respect to the definitions of all groups in the compounds of the
formula I.
An alkyl group which, independently of any other substituents, can be
substituted by
one or more fluorine substituents, can be unsubstituted by fluorine
substituents, i.e.
not carry fluorine substituents, or substituted, for example by 1, 2, 3, 4, 5,
6, 7, 8, 9,
10 or 11 fluorine substituents, or by 1, 2, 3, 4 or 5 fluorine substituents,
or by 1, 2 or 3
fluorine substituents, which can be located in any positions. For example, in
a fluoro-
substituted alkyl group one or more methyl groups can carry three fluorine
substituents each and be present as trifluoromethyl groups, and/or one or more
methylene groups (CH2) can carry two fluorine substituents each and be present
as
difluoromethylene groups. The explanations with respect to the substitution of
a
group by fluorine also apply if the group additionally carries other
substituents and/or
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is part of another group, for example of an -0-alkyl group. Examples of fluoro-
substituted alkyl groups are -CF3 (trifluoromethyl), -CHF2, -
CHF-CF3, -CHF-
CHF2, -CHF-CH2F, -CH2-CF3, -CH2-CHF2, -CH2-CH2F, -CF2-CF3, -CF2_CHF2, -CF2-
CH2F, -CH2-CHF-CF3, -CH2-CHF-CHF2, -CH2-CHF-CH2F, -CH2-CH2-CF3, -CH2-CH2-
CHF2, -CH2-CH2-CH2F, -CH2-CF2-CF3, -CH2-CF2-CHF2, -CH2-CF2-CH2F, -CHF-CHF-
CF3, -CHF-CHF-CHF2, -CHF-CHF-CH2F, -CHF-CH2-CF3, -CHF-cH2-CHF2, -CHF-
CH2-CH2F, -CHF-CF2-CF3, -CHF-CF2-CHF2, -CHF-CF2-CH2F, -CF2_CHF-CF3, -CF2-
CHF-CHF2, -CF2-CHF-CH2F, -CF2-CH2-CF3, -CF2-CH2-CHF2, -CF2-CH2-CH2F, -CF2-
CF2-CF3, -CF2-CF2-CHF2 or -CF2_CF2-CH2F. Examples of fluoro-substituted -0-
alkyl
groups are trifluoromethoxy (-0CF3), 2,2,2-trifluoroethoxy, pentafluoroethoxy
and
3,3,3-trifluoropropoxy. With respect to all groups or substituents in the
compounds of
the formula I which can be an alkyl group which can generally contain one or
more
fluorine substituents, as an example of groups or substituents containing
fluorine-
substituted alkyl which may be included in the definition of the group or
substituent,
the group CF3 (trifluoromethyl), or a respective group such as CF3-O-, may be
mentioned.
The above explanations with respect to alkyl groups apply correspondingly to
alkyl
groups which in the definition of a group in the compounds of the formula I
are
bonded to two adjacent groups, or linked to two groups, and may be regarded as
divalent alkyl groups (alkanediyl groups), like in the case of the alkyl part
of a
substituted alkyl group. Thus, such groups can also be linear or branched, the
bonds
to the adjacent groups can be located in any positions and can start from the
same
carbon atom or from different carbon atoms, and they can be unsubstituted or
substituted by fluorine substituents independently of any other substituents.
Examples of such divalent alkyl groups are -CH2-, -CH2-CH2-, -CH2-CH2-CH2-,
-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-
CH2-, -CH(CH3)-, -C(CH3)2-, -CH(CH3)-CH2-, -CH2-CH(CH3)-, -C(CH3)2-CH2-7
-CH2-C(CH3)2-. Examples of fluoro-substituted alkanediyl groups, which can
contain
1, 2, 3, 4, 5 or 6 fluorine substituents, or 1, 2, 3 or 4 fluorine
substituents, or 1 or 2
fluorine substituents, for example, are -CF2-, -CHF-, -CHF-CHF2-, -CHF-CHF-, -
CH2-
CF2-, -CH2-CHF-, -CF2-CF2-, -CF2-CHF-, -CH2-CHF-CF2-, -CH2-CHF-CHF-, -CH2-
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CH2-CF2-, -CH2-CH2-CHF, -CH2-CF2-CF2-, -CH2-CF2-CHF-, -CHF-CHF-CF2-, -CHF-
CHF-CHF-, -CHF-CH2-CF2-, -CHF-CH2-CHF-, -CHF-CF2-CF2-, -CHF-CF2-CHF-,
-CF2-CHF-CF2-, -CF2-CHF-CHF-, -CF2-CH2-CF2-, -CF2-CH2-CHF-, -CF2-CF2-CF2-, or
-CF2-CF2-CHF.
The number of ring carbon atoms in a (C3-C7)-cycloalkyl group can be 3, 4, 5,
6 or 7.
Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl
and
cycloheptyl. Cycloalkyl which, independently of any other substituents, can be
substituted by one or more (Ci-C4)-alkyl substituents, can be unsubstituted by
alkyl
substituents, i.e. not carry alkyl substituents, or substituted, for example
by 1, 2, 3 or
4 identical or different (Ci-C4)-alkyl substituents, for example by methyl
groups, which
substituents can be located in any positions. Examples of such alkyl-
substituted
cycloalkyl groups are 1-methylcyclopropyl, 2,2-dimethylcyclopropyl, 1-
methylcyclopentyl, 2,3-dimethylcyclopentyl, 1-methylcyclohexyl, 4-
methylcyclohexyl,
4-isopropylcyclohexyl, 4-tert-butylcyclohexyl, 3,3,5,5-tetramethylcyclohexyl.
Cycloalkyl groups which, independently of any other substituents, can be
substituted
by one or more fluorine substituents, can be unsubstituted by fluorine
substituents,
i.e. not carry fluorine substituents, or substituted, for example by 1, 2, 3,
4, 5, 6, 7, 8,
9, 10 or 11 fluorine substituents, or by 1, 2, 3, 4, 5 or 6 fluorine
substituents, or by 1,
2 or 3 fluorine substituents. The fluorine substituents can be located in any
positions
of the cycloalkyl group and can also be located in an alkyl substituent.
Examples of
fluoro-substituted cycloalkyl groups are 1-fluorocyclopropyl, 2,2-
difluorocyclopropyl,
3,3-difluorocyclobutyl, 1-fluorocyclohexyl, 4,4-difluorocyclohexyl,
3,3,4,4,5,5-
hexafluorocyclohexyl. Cycloalkyl groups can also be substituted simultaneously
by
fluorine and alkyl. Examples of the group -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl
are
cyclopropylmethyl-, cyclobutylmethyl-, cyclopentylmethyl-, cyclohexylmethyl-,
cycloheptylmethyl-, 1-cyclopropylethyl-, 2-cyclopropylethyl-, 1-
cyclobutylethyl-, 2-
cyclobutylethyl-, 1-cyclopentylethyl-, 2-cyclopentylethyl-, 1-cyclohexylethyl-
, 2-
cyclohexylethyl-, 1-cycloheptylethyl-, 2-cycloheptylethyl-. In one embodiment
of the
invention, a -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl group in any one or more
occurrences of
such a group, independently of any other occurrences, is a -(Ci-C2)-alkyl-(C3-
C7)-
cycloalkyl group, in another embodiment a -CH2-(C3-C7)-cycloalkyl group. In
the
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group -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, and likewise in all other groups, the
terminal
hyphen denotes the free bond via which the group is bonded, and thus indicates
via
which subgroup a group composed of subgroups is bonded.
In substituted phenyl groups, including phenyl groups representing Ar, the
substituents can be located in any positions. In monosubstituted phenyl
groups, the
substituent can be located in position 2, in position 3 or in position 4. In
disubstituted
phenyl groups, the substituents can be located in positions 2 and 3, in
positions 2
and 4, in positions 2 and 5, in positions 2 and 6, in positions 3 and 4, or in
positions 3
and 5. In trisubstituted phenyl groups, the substituents can be located in
positions 2,
3 and 4, in positions 2, 3 and 5, in positions 2, 3 and 6, in positions 2, 4
and 5, in
positions 2, 4 and 6, or in positions 3, 4 and 5. If a phenyl group carries
four
substituents, some of which can be fluorine atoms, for example, the
substituents can
be located in positions 2, 3, 4 and 5, in positions 2, 3, 4 and 6, or in
positions 2, 3, 5
and 6. If a polysubstituted phenyl group or any other polysubstituted group
carries
different substituents, each substituent can be located in any suitable
position, and
the present invention comprises all positional isomers. The number of
substituents in
a substituted phenyl group can be 1, 2, 3, 4 or 5. In one embodiment of the
invention,
the number of substituents in a substituted phenyl group, like the number of
substituents in any other substituted group which can carry one or more
substituents,
for example the group Het, is 1, 2, 3 or 4, in another embodiment 1, 2 or 3,
in another
embodiment 1 or 2, in another embodiment 1, where the number of substituents
in
any occurrence of such a substituted group is independent of the number of
substituents in other occurrences.
In heterocyclic groups, including the group Het, heterocycles representing Ar
and
other heterocyclic rings which can be present in the compounds of the formula
I,
such as rings formed by two group together with the atom or atoms carrying
them,
the hetero ring members can be present in any combination and located in any
suitable ring positions, provided that the resulting group and the compound of
the
formula I are suitable and sufficiently stable as a pharmaceutical active
compound. In
one embodiment of the invention, two oxygen atoms in any heterocyclic ring in
the
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compounds of the formula I cannot be present in adjacent ring positions. In
another
embodiment of the invention, two hetero ring members selected from the series
consisting of oxygen atoms and sulfur atoms cannot be present in adjacent ring
positions in any heterocyclic ring in the compounds of the formula I. In
another
embodiment of the invention, two hetero ring members selected from the series
consisting of nitrogen atoms carrying an exocyclic group like a hydrogen atom
or a
substituent, sulfur atoms and oxygen atoms cannot be present in adjacent ring
positions in any heterocyclic ring in the compounds of the formula I. The
choice of
hetero ring members in an aromatic heterocyclic ring is limited by the
prerequisite
that the ring is aromatic, i.e. it comprises a cyclic system of six
delocalized pi
electrons. Monocyclic aromatic heterocycles are 5-membered or 6-membered rings
and, in the case of a 5-membered ring, comprise one ring heteroatom selected
from
the series consisting of oxygen, sulfur and nitrogen, wherein this ring
nitrogen carries
an exocyclic group like a hydrogen atom or a substituent, and optionally one
or more
further ring nitrogen atoms, and, in the case of a 6-membered ring, comprise
one or
more nitrogen atoms as ring heteroatoms, but no oxygen atoms and sulfur atoms
as
ring heteroatoms. Heterocyclic groups in the compounds of the formula I are
bonded
via a ring carbon atom or a ring nitrogen atom, as specified in the definition
of the
respective group, where a heterocyclic group can be bonded via any suitable
carbon
atom or nitrogen atom, respectively, in the ring. In substituted heterocyclic
groups,
the substituents can be located in any positions.
The number of ring heteroatoms which can be present in a heterocyclic group in
the
compounds of the formula I, the number of ring members which can be present,
and
the degree of saturation, i.e. whether the heterocyclic group is saturated and
does
not contain a double bond within the ring, or whether it is partially
unsaturated and
contains one or more, for example one or two, double bonds within the ring but
is not
aromatic, or whether it is aromatic and thus contains two double bonds within
the ring
in the case of a 5-membered monocyclic aromatic heterocycle and three double
bonds within the ring in the case of a 6-membered monocyclic aromatic
heterocycle,
is specified in the definitions of the individual groups in the compounds of
the formula
I. As examples of heterocyclic ring systems, from which heterocyclic groups in
the
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compounds of the formula I including the bicyclic heterocyclic ring system
which can
result in case two groups R10 bonded to adjacent ring carbon atoms in Ar
together
with the carbon atoms carrying them form a ring, can be derived, and from any
one
or more of which any of the heterocyclic groups in the compounds of the
formula I is
5 selected in one embodiment of the invention, provided that the ring
system is
comprised by the definition of the group, oxetane, thietane, azetidine, furan,
tetrahydrofuran, thiophene, tetrahydrothiophene, pyrrole, pyrroline,
pyrrolidine, 1,3-
dioxole, 1,3-dioxolane, isoxazole ([1,2]oxazole), isoxazoline, isoxazolidine,
oxazole
([1,3]oxazole), oxazoline, oxazolidine, isothiazole ([1,2]thiazole),
isothiazoline,
10 isothiazolidine, thiazole ([1,3]thiazole), thiazoline, thiazolidine,
pyrazole, pyrazoline,
pyrazolidine, imidazole, imidazoline, imidazolidine, [1,2,3]triazole,
[1,2,4]triazole,
[1,2,4]oxadiazole, [1,3,4]oxadiazole, 1,2,5-oxadiazole, [1,2,4]thiadiazole,
pyran,
tetrahydropyran, thiopyran, tetrahydrothiopyran, 2,3-dihydro[1,4]dioxine, 1,4-
dioxane,
pyridine, 1,2,5,6-tetrahydropyridine, piperidine, morpholine, thiomorpholine,
15 piperazine, pyridazine, pyrimidine, pyrazine, [1,2,4]triazine, oxepane,
thiepane,
azepane, [1,3]diazepane, [1,4]diazepane, [1,4]oxazepane, [1,4]thiazepane,
benzofuran, isobenzofuran, benzothiophene (benzo[b]thiophene), 1H-indole, 2,3-
dihydro-1H-indole, 2H-isoindole, benzo[1,3]dioxole, benzoxazole, benzthiazole,
1H-
benzim idazole, chroman, isochroman, thiochroman, benzo[1,4]dioxane, 3,4-
dihydro-
20 2H-benzo[1,4]oxazine, 3,4-dihydro-2H-benzo[1,4]thiazine, quinoline,
5,6,7,8-
tetrahydroquinoline, isoquinoline, 5,6,7,8-tetrahydroisoquinoline, cinnoline,
quinazoline, quinoxaline, phthalazine, [1,8]naphthyridine and 3,4-dihydro-2H-
benzo[b][1,4]dioxepine, which latter ring system may also be named as 3,4-
dihydro-
2H-1,5-benzodioxepine, may be mentioned, which can all be unsubstituted or
25 substituted in any suitable positions as specified in the definition of
the respective
group in the compounds of the formula I, wherein the given degree of
unsaturation is
by way of example only, and in the individual groups also ring systems with a
higher
or lower degree of saturation, or hydrogenation, or of unsaturation can be
present as
specified in the definition of the group. Ring sulfur atoms, in particular in
saturated
and partially unsaturated heterocycles, can generally carry one or two oxo
groups,
i.e. doubly bonded oxygen atoms, and in such heterocycles, besides a ring
sulfur
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atom, also an S(0) group (S(=0)) and an S(0)2 group (S(=0)2) can be present as
hetero ring member.
As mentioned, the heterocyclic groups can be bonded via any suitable ring atom
as
specified in the definition of the respective group in the compound of the
formula I.
For example, among others can an oxetane and a thietane ring be bonded via
positions 2 and 3, an azetidine ring via positions 1, 2 and 3, a furan ring, a
tetrahydrofuran ring, a thiophene ring and a tetrahydrothiophene via positions
2 and
3, a pyrrole ring and a pyrrolidine ring via positions 1, 2 and 3, an
isoxazole ring and
an isothiazole ring via positions 3, 4 and 5, a pyrazole ring via positions 1,
3, 4 and 5,
an oxazole ring and a thiazole ring via positions 2, 4 and 5, an imidazole
ring and an
imidazolidine ring via positions 1, 2, 4 and 5, a tetrahydropyran and a
tetrahydrothiopyran ring via positions 2, 3 and 4, a 1,4-dioxane ring via
position 2, a
pyridine ring via positions 2, 3 and 4, a piperidine ring via positions 1, 2,
3 and 4, a
morpholine ring and a thiomorpholine ring via positions 2, 3 and 4, a
piperazine ring
via positions 1 and 2, a pyrimidine ring via positions 2, 4 and 5, a pyrazine
ring via
position 2, an azepane ring via positions 1, 2, 3 and 4, a benzofuran ring and
a
benzothiophene ring via positions 2, 3, 4, 5, 6 and 7, a 1H-indole ring and a
2,3-
dihydro-1H-indole ring via positions 1, 2, 3, 4, 5, 6 and 7, a
benzo[1,3]dioxole ring via
positions 4, 5, 6 and 7, a benzoxazole ring and a benzthiazole ring via
positions 2, 4,
5, 6 and 7, a 1H-benzimidazole ring via positions 1, 2, 4, 5, 6 and 7, a
benzo[1,4]dioxane ring via positions 5, 6, 7 and 8, a quinoline ring via
positions 2, 3,
4, 5, 6, 7 and 8, a 5,6,7,8-tetrahydroquinoline via positions 2, 3 and 4, an
isoquinoline
ring via positions 1, 3, 4, 5, 6, 7 and 8, a 5,6,7,8-tetrahydroisoquinoline
ring via
positions 1, 3 and 4, for example, wherein the resulting residues of the
heterocyclic
groups can all be unsubstituted or substituted in any suitable positions as
specified in
the definition of the respective group in the compounds of the formula I.
Halogen is fluorine, chlorine, bromine or iodine. In one embodiment of the
invention,
halogen is in any of its occurrences fluorine, chlorine or bromine, in another
embodiment fluorine or chlorine, in another embodiment fluorine, in another
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embodiment chlorine, where all occurrences of halogen are independent of each
other.
The present invention comprises all stereoisomeric forms of the compounds of
the
formula I, for example all enantiomers and diastereomers including cis/trans
isomers.
The invention likewise comprises mixtures of two or more stereoisomeric forms,
for
example mixtures of enantiomers and/or diastereomers including cis/trans
isomers,
in all ratios. Asymmetric centers contained in the compounds of the formula I
can all
independently of each other have S configuration or R configuration. The
invention
relates to enantiomers, both the levorotatory and the dextrorotatory antipode,
in
enantiomerically pure form and essentially enantiomerically pure form, and in
the
form of their racemate, i.e. a mixture of the two enantiomers in molar ratio
of 1:1, and
in the form of mixtures of the two enantiomers in all ratios. The invention
likewise
relates to diastereomers in the form of pure and essentially pure
diastereomers and
in the form of mixtures of two or more diastereomers in all ratios. The
invention also
comprises all cis/trans isomers of the compounds of the formula I in pure form
and
essentially pure form, and in the form of mixtures of the cis isomer and the
trans
isomer in all ratios. Cis/trans isomerism can occur in substituted rings. The
preparation of individual stereoisomers, if desired, can be carried out by
resolution of
a mixture according to customary methods, for example, by chromatography or
crystallization, or by use of stereochemically uniform starting compounds in
the
synthesis, or by stereoselective reactions. Optionally, before a separation of
stereoisomers a derivatization can be carried out. The separation of a mixture
of
stereoisomers can be carried out at the stage of the compound of the formula I
or at
the stage of an intermediate in the course of the synthesis. For example, in
the case
of a compound of the formula I containing an asymmetric center the individual
enantiomers can be prepared by preparing the racemate of the compound of the
formula I and resolving it into the enantiomers by high pressure liquid
chromatography on a chiral phase according to standard procedures, or
resolving the
racemate of any intermediate in the course of its synthesis by such
chromatography
or by crystallization of a salt thereof with an optically active amine or acid
and
converting the enantiomers of the intermediate into the enantiomeric forms of
the
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final compound of the formula I, or by performing an enantioselective reaction
in the
course of the synthesis. The invention also comprises all tautomeric forms of
the
compounds of the formula I.
Besides the free compounds of the formula I, i.e. compounds in which acidic
and
basic groups are not present in the form of a salt, the present invention
comprises
also the physiologically or toxicologically acceptable salts of the compounds
of the
formula I, especially their pharmaceutically acceptable salts, which can be
formed on
one or more acidic or basic groups in the compounds of the formula I, for
example on
basic heterocyclic moieties. The compounds of the formula I may thus be
deprotonated on an acidic group by an inorganic or organic base and be used,
for
example, in the form of the alkali metal salts. Compounds of the formula I
comprising
at least one basic group may also be prepared and used in the form of their
acid
addition salts, for example in the form of pharmaceutically acceptable salts
with
inorganic acids and organic acids, such as salts with hydrochloric acid and
thus be
present in the form of the hydrochlorides, for example. Salts can in general
be
prepared from acidic and basic compounds of the formula I by reaction with an
acid
or base in a solvent or diluent according to customary procedures. If the
compounds
of the formula I simultaneously contain an acidic and a basic group in the
molecule,
the invention also includes internal salts (betaines, zwitterions) in addition
to the salt
forms mentioned. The present invention also comprises all salts of the
compounds of
the formula I which, because of low physiological tolerability, are not
directly suitable
for use as a pharmaceutical, but are suitable as intermediates for chemical
reactions
or for the preparation of physiologically acceptable salts, for example by
means of
anion exchange or cation exchange.
In one embodiment of the invention, an aromatic heterocycle representing the
group
Ar comprises 1 or 2 identical or different ring heteroatoms, in another
embodiment 1
or 2 identical or different ring heteroatoms which are selected from the
series
consisting of nitrogen and sulfur. In another embodiment, an aromatic
heterocycle
representing Ar is a 5-membered heterocycle which comprises 1 or 2 identical
or
different ring heteroatoms which are selected from the series consisting of
nitrogen
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and sulfur, or it is a 6-membered heterocycle which comprises 1 or 2 ring
heteroatoms which are nitrogen atoms, in another embodiment it is a 5-membered
heterocycle which comprises 1 or 2 identical or different ring heteroatoms
which are
selected from the series consisting of nitrogen and sulfur. In another
embodiment, an
aromatic heterocycle representing Ar is selected from the series consisting of
thiophene, thiazole, pyrazole, imidazole, pyridine, pyridazine, pyrimidine and
pyrazine, in another embodiment from the series consisting of thiophene,
thiazole,
pyrazole, imidazole and pyridine, in another embodiment from the series
consisting
of thiophene, thiazole, pyrazole and imidazole, in another embodiment from the
series consisting of thiophene and pyrazole, in another embodiment it is
thiophene,
and in another embodiment it is pyrazole, which heterocycles are all
unsubstituted or
substituted by one or more substituents R10. In one embodiment of the
invention, Ar
is phenyl which is unsubstituted or substituted by one or more identical or
different
substituents R10, in another embodiment Ar is phenyl which is substituted by
one or
more identical or different substituents R10, in another embodiment Ar is a 5-
membered or 6-membered aromatic heterocycle which is unsubstituted or
substituted
by one or more identical or different substituents R10, and in another
embodiment Ar
is a 5-membered or 6-membered aromatic heterocycle which is substituted by one
or
more identical or different substituents R10. In one embodiment of the
invention, the
number of identical or different substituents R10 which can be present in the
group
Ar is 1, 2, 3 or 4, in another embodiment it is 1, 2 or 3, in another
embodiment it is 1
or 2, in another embodiment it is 1, in another embodiment it 2, 3 or 4, in
another
embodiment it is 2 or 3, in another embodiment it is 3, in another embodiment
it is 2.
In one embodiment, Ar is substituted by one or more identical or different
substituents R10.
In one embodiment of the invention, the number n is selected from the series
consisting of 0 and 1, in another embodiment from the series consisting of 1
and 2, in
another embodiment it is 1, in another embodiment it is O.
In one embodiment of the invention, R1 is selected from the series consisting
of
hydrogen, -N(R11)-R12, -N(R13)-C(0)-R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-
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R14, (Ci-C4)-alkyl and -(Ci-C4)-alkyl-O-R18, in another embodiment from the
series
consisting of hydrogen, -N(R11)-R12, -N(R13)-C(0)-R14, -N(R13)-S(0)2-
R15, -N(R13)-C(0)-NH-R14, -C(0)-N(R16)-R17, -CN and (Ci-C4)-alkyl, in another
embodiment from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-C(0)-
5 R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14 and (Ci-C4)-alkyl, in another
embodiment from the series consisting of hydrogen, -C(0)-N(R16)-R17, -CN, (Ci-
C4)-alkyl and -(Ci-C4)-alkyl-O-R18, in another embodiment from the series
consisting
of hydrogen and (Ci-C4)-alkyl, in another embodiment from the series
consisting
of -N(R11)-R12, -N(R13)-C(0)-R14, -N(R13)-S(0)2-R15 and -N(R13)-C(0)-NH-R14,
10 in another embodiment from the series consisting of -N(R11)-R12 and -
N(R13)-C(0)-
R14, in another embodiment it is -N(R11)-R12, and in another embodiment R1 is
selected from the series consisting of -N(R11)-R12 and (Ci-C4)-alkyl. In one
embodiment, a (Ci-C4)-alkyl group representing R1 is (Ci-C2)-alkyl, in another
embodiment it is methyl.
In one embodiment of the invention, R2 is selected from the series consisting
of
halogen, (Ci-C4)-alkyl and -CN, in another embodiment from the series
consisting of
halogen and (Ci-C4)-alkyl, in another embodiment from the series consisting of
halogen and -CN, in another embodiment from the series consisting of halogen.
In
one embodiment, a (Ci-C4)-alkyl group present in R2 is a methyl group. In one
embodiment, halogen representing R2 is selected from the series consisting of
fluorine and chlorine, in another embodiment it is fluorine. Ring carbon atoms
in the
divalent phenyl group depicted in formula I which have a free binding site,
i.e. are not
bonded to adjacent groups in formula I, and which do not carry a group R2,
carry
hydrogen atoms, as does likewise the carbon atom in position 5 of the
pyrazolo[3,4-b]pyrazine ring system depicted in formula I. Thus, in case the
number n
is 0 and hence no group R2 is present, all four carbon atoms in the ring
positions of
the divalent phenyl group depicted in formula I which in formula l' are
designated as
positions 2', 3', 5' and 6', carry hydrogen atoms. In case the number n is 1
and hence
one group R2 is present, one of the four carbon atoms in the ring positions of
the
divalent phenyl group depicted in formula I which in formula l' are designated
as 2',
3', 5' and 6', carries the group R2 and the other three said carbon atoms
carry
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31
hydrogen atoms. In case the number n is 2 and hence two groups R2 are present,
two of the four carbon atoms in the ring positions of the divalent phenyl
group
depicted in formula I which in formula l' are designated as positions 2', 3',
5' and 6',
carry the groups R2 and the other two said carbon atoms carry hydrogen atoms.
4 R1
5 3
(R2)n 5,
/N 2
0 61 N
Ar
6 H 1
7
3
// N '
0 H 2'
Groups R2 can be present in any positions of the divalent phenyl group
depicted in
formula I which have a free binding site. If one group R2 is present, in one
embodiment of the invention the group R2 is present in the position which is
formula
l' is designated as position 2', which is equivalent to position 6', and in
another
embodiment it is present in the position which in formula l' is designated as
position
3', which is equivalent to position 5'. If two groups R2 are present, in one
embodiment of the invention the groups R2 are present in the positions which
in
formula l' are designated as positions 2' and 3', in another embodiment in the
positions which in formula l' are designated as positions 2' and 5', in
another
embodiment in the positions which in formula l' are designated as positions 2'
and 6',
in another embodiment in the positions which in formula l' are designated as
positions 3' and 5'.
If two groups R10 bonded to adjacent ring carbon atoms in Ar together with the
ring
carbon atoms carrying them form a 5-membered to 8-membered ring, this ring is
at
least mono-unsaturated, i.e., the resulting ring contains at least one double
bond
within the ring, which double bond is present between the said two adjacent
ring
carbon in the aromatic ring Ar which are common to the ring Ar and the ring
formed
by the two groups R10, and because of the rules of nomenclature for fused
rings is
regarded as a double bond present in both rings. The ring formed by two groups
R10
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together with the carbon atoms carrying them can contain 1, 2 or 3 double
bonds
within the ring. In one embodiment, the formed ring contains 1 or 2 double
bonds, in
another embodiment 1 double bond within the ring. In the case of a 6-membered
carbocyclic or heterocyclic ring or a 5-membered heterocyclic ring the formed
ring
can be aromatic and, together with the aromatic ring Ar, form a bicyclic
aromatic ring
system, for example a naphthalene ring system, a quinoline ring system, an
isoquinoline ring system or a benzothiophene ring system. The case that two
groups
R10 bonded to adjacent ring carbon atoms in Ar together with the carbon atoms
carrying them form a 5-membered to 8-membered unsaturated ring, can in other
terms be regarded as two groups R10 together forming a divalent residue
comprising
a chain of 3 to 6 atoms of which 0, 1 or 2 are identical or different
heteroatoms
selected from the series consisting of nitrogen, oxygen and sulfur, the
terminal atoms
of which are bonded to the two adjacent ring carbon atoms in Ar. Examples of
such
divalent residues, from any one or more of which two groups R10 bonded to
adjacent
ring carbon atoms in Ar are selected in one embodiment of the invention, are
the
residues -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH=CH-
CH=CH-, -N=CH-CH=CH-, -CH=CH-CH=N-, -CH=N-CH=CH-, -CH=CH-N=CH-,-0-
CH2-CH2-,-CH2-CH2-0-, -0-CH2-0-, -0-CH2-CH2-0-, -0-CH2-CH2-CH2-0-, -0-CF12-
CH2-CH2-CH2-0-,-S-CH=CH-, -CH=CH-S-, =CH-S-CH=-, -N=CH-S-, -S-CH=N-,
-N=CH-O-, -0-CH=N-, -NH-CH2-CH2-0-, -0-CH2-CH2-NH-, -S-CH2-CH2-NH- and
-NH-CH2-CH2-S-, which can all be substituted by substituents selected from the
series consisting of halogen, (Ci-C4)-alkyl- -0-(Ci-C4)-alkyl and -CN, and can
thus
also be present, for example, as the divalent residues -0-CF2-0-, -0-C(CH3)2-0-
, -S-
C(C1)=CH-, -CH=C(CI)-S-, -N(CH3)-CH2-CH2-0-, -0-CH2-CH2-N(CH3)-, -S-CH2-CH2-
N(CH3)- and -N(CH3)-CH2-CH2-S-. In one embodiment of the invention, the ring
heteroatoms which are optionally present in a ring formed by two groups R10
bonded
to adjacent ring carbon atoms in Ar together with the carbon atoms carrying
them,
are selected from the series consisting of nitrogen and oxygen, in another
embodiment from the series consisting of oxygen and sulfur, and in another
embodiment they are oxygen atoms. In one embodiment of the invention, the ring
which can be formed by two groups R10 bonded to adjacent ring carbon atoms in
Ar
together with the ring carbon atoms carrying them, is a 5-membered to 7-
membered,
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in another embodiment a 5-membered to 6-membered, in another embodiment a 6-
membered to 7-membered, in another embodiment a 5-membered, in another
embodiment a 6-membered ring, in another embodiment a 7-membered ring. In one
embodiment of the invention, the ring which can be formed by two groups R10
bonded to adjacent carbon atoms in Ar together with the carbon atoms carrying
them, comprises 0 ring heteroatoms, i.e. it is a carbocyclic ring, and in
another
embodiment it comprises 1 or 2 identical or different ring heteroatoms. In one
embodiment of the invention, the number of substituents which can be present
in a
ring formed by two groups R10 bonded to adjacent ring carbon atoms in Ar
together
with the carbon atoms carrying them, is 1, 2, 3 or 4, in another embodiment 1,
2 or 3,
in another embodiment 1 or 2, in another embodiment 1, in another embodiment
it is
O. In one embodiment of the invention, substituents which can be present in a
ring
formed by two groups R10 bonded to adjacent ring carbon atoms in Ar together
with
the carbon atoms carrying them, are selected from the series consisting of
halogen,
(Ci-C4)-alkyl and -CN, in another embodiment from the series consisting of
halogen
and (Ci-C4)-alkyl, and in another embodiment are substituents in such a ring
bonded
to a ring nitrogen atom selected from the series consisting of (Ci-C4)-alkyl.
In one embodiment of the invention, R10 is selected from the series consisting
of
halogen, (Ci-C4)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl,
-0-(C3-C7)-cycloalkyl, -N(R19)-R20,
-N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN, in another embodiment
from the series consisting of halogen, (Ci-C4)-alkyl, -N(R19)-R20,
-N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2, -C(0)-N(R23)-R24 and -CN, in
another embodiment from the series consisting of halogen, (Ci-C4)-alkyl,
alkyl, -N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN, in
another embodiment from the series consisting of halogen, (Ci-C4)-alkyl,
alkyl, -N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN, in another embodiment from
the series consisting of halogen, (Ci-C4)-alkyl, -N(R19)-R20,
-N(R21)-C(0)-R22 and -CN, in another embodiment from the series consisting of
halogen, (Ci-C4)-alkyl,
-N(R21)-C(0)-R22, -NO2 and -CN, in another
embodiment from the series consisting of halogen, (Ci-C4)-alkyl,
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-N(R21)-C(0)-R22 and -CN, in another embodiment from the series consisting of
halogen, (Ci-C4)-alkyl, -
NO2 and -CN, in another embodiment from
the series consisting of halogen, (Ci-C4)-alkyl, -0-(Ci-C4)-alkyl and -CN, in
another
embodiment from the series consisting of halogen, (Ci-C4)-alkyl and -CN, in
another
embodiment from the series consisting of halogen and (Ci-C4)-alkyl, and in
another
embodiment from the series consisting of halogen, and in all these embodiment
two
groups R10 bonded to adjacent ring carbon atoms in Ar, together with the
carbon
atoms carrying them, can form a 5-membered to 8-membered unsaturated ring
which
comprises 0, 1 or 2 identical or different ring heteroatoms selected from the
series
consisting of nitrogen, oxygen and sulfur, and which is unsubstituted or
substituted
by one or more identical or different substituents selected from the series
consisting
of halogen, -(Ci-C4)-alkyl, -0-(Ci-C4)-alkyl and -CN.
In one embodiment, R10 is selected from the series consisting of halogen, (Ci-
C.4.)-
alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, -0-(C3-
C7)-cycloalkyl, -
N(R19)-R20, -N(R21 )-N(R1 9)-
R20, -N(R21)-C(0)-R22, -NO2, -C(0)-N(R23)-R24 and -CN, in another embodiment
from the series consisting of halogen, (Ci-C4)-alkyl, (C3-C7)-cycloalkyl, -(Ci-
C4)-alkyl-
(C3-C7)-cycloalkyl, -
0-(C3-C7)-cycloalkyl, -0-(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl-, -N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN,
in
another embodiment from the series consisting of halogen, (Ci-C4)-alkyl,
alkyl, -N(R19)-R20, -N(R21 )-N(R19)-R20, -N(R21 )-C(0)-R22, -NO2, -C(0)-N(R23)-
R24 and -CN, in another embodiment from the series consisting of halogen, (Ci-
C4)-
alkyl, -0-(Ci-C4)-alkyl, -N(R19)-R20, -N(R21 )-N(R19)-R20, -N(R21 )-C(0)-R22, -
NO2
and -CN, in another embodiment from the series consisting of halogen, (Ci-C4)-
alkyl,
-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN, in another
embodiment from the series consisting of halogen, (Ci-C4)-alkyl,
-N(R19)-R20, -N(R21)-C(0)-R22 and -CN, in another embodiment from the series
consisting of halogen, (Ci-C4)-alkyl, -
N(R21)-C(0)-R22, -NO2 and
-CN, in another embodiment from the series consisting of halogen, (Ci-C4)-
alkyl, -0-
-N(R21)-C(0)-R22 and -CN, in another embodiment from the series
consisting of halogen, (Ci-C4)-alkyl, -NO2 and -CN, in another
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embodiment from the series consisting of halogen, (Ci-C4)-alkyl, -0-(Ci-C4)-
alkyl and
-CN, in another embodiment from the series consisting of halogen, (Ci-C4)-
alkyl and
-CN, in another embodiment from the series consisting of halogen and (Ci-C4)-
alkyl,
and in another embodiment from the series consisting of halogen.
5
In one embodiment, substituents R10 which are bonded to a ring nitrogen atom
in Ar,
such as in the case of a pyrrole, pyrazole or imidazole ring representing Ar,
are
selected from the series consisting of (Ci-C4)-alkyl, (C3-C7)-cycloalkyl, -(Ci-
C4)-alkyl-
(C3-C7)-cycloalkyl and -C(0)-N(R23)-R24, in another embodiment from the series
10 consisting of (Ci-C4)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-alkyl-(C3-
C7)-cycloalkyl, in
another embodiment from the series consisting of (Ci-C4)-alkyl.
In one embodiment of the invention, a (Ci-C4)-alkyl group which represents R10
or is
present in the group -0-(Ci-C4)-alkyl representing R10, is a (Ci-C3)-alkyl
group, in
15 another embodiment a (Ci-C2)-alkyl group, in another embodiment a methyl
group,
where all these alkyl groups can optionally be substituted by fluorine
substituents as
applies to alkyl groups in general, and also occur as a trifluoromethyl group,
for
example. In one embodiment of the invention, a (C3-C7)-cycloalkyl group which
represents R10 or is present in a group R10, is a (C3-C6)-cycloalkyl group, in
another
20 embodiment a (C3-C4)-cycloalkyl group, in another embodiment a
cyclopropyl group.
In on embodiment of the invention, the total number of -NO2 (nitro) groups
representing R10 in a compound of the formula I is not greater than 2, in
another
embodiment it is not greater than 1.
25 Examples of groups Ar including the optional substituents R10 on Ar,
from any one or
more of which Ar is selected in one embodiment of the invention, are 2,3-
dichloro-
phenyl, 2,5-dichloro-phenyl, 5-chloro-2-hydrazino-phenyl, 5-chloro-2-cyano-
phenyl,
2-cyano-5-methyl-phenyl, 2-fluoro-5-methyl-phenyl, 2-chloro-5-methoxy-phenyl,
2,5-
dichloro-thiophen-3-yl, 8-chloro-3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl, 5-
chloro-
30 1,3-dimethyl-pyrazol-4-yl, naphthalen-1-yl, 2,4,6-trichloro-phenyl, 5-
chloro-2-fluoro-
phenyl, 2,4,5-trifluoro-phenyl), 2,4,5-trichloro-phenyl, 5-chloro-2,4-difluoro-
phenyl,
2,3,4-trichloro-phenyl, 2,3,4-trifluoro-phenyl, 2-chloro-4-trifluoromethyl-
phenyl, 5-
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cyano-2-fluoro-phenyl, 2-cyano-5-methoxy-phenyl, 2-cyano-5-fluoro-phenyl, 2-
fluoro-
5-methoxy-phenyl, 4-acetylamino-2-methyl-phenyl, 2-methyl-5-nitro-phenyl, and
2-
nitro-4-trifluoromethyl-phenyl.
The monocyclic heterocycle which can be formed by the groups R11 and R12
together with the nitrogen atom carrying them, which heterocycle is thus
bonded via
a ring nitrogen atom, can be 4-membered, 5-membered, 6-membered or 7-
membered. In one embodiment of the invention, the heterocycle formed by the
groups R11 and R12 together with the nitrogen atom carrying them, is 4-
membered
to 6-membered, in another embodiment it is 5-membered or 6-membered, in
another
embodiment it is 6-membered. In one embodiment, the further ring heteroatom
which
is optionally present in a heterocycle formed by the groups R11 and R12
together
with the nitrogen atom carrying them, is selected from the series consisting
of
nitrogen and oxygen, in another embodiment it is a nitrogen atom, and in
another
embodiment it is an oxygen atom, and in another embodiment no further ring
heteroatom is present. In one embodiment of the invention, the number of
substituents selected from the series consisting of fluorine and (Ci-C4)-
alkyl, which
can be present in a ring formed by the groups R11 and R12 together with the
nitrogen atom carrying them, is 1, 2 or 3, in another embodiment 1 or 2, in
another
embodiment 1. In one embodiment of the invention, substituents which can be
present in a ring formed by the groups R11 and R12 together with the nitrogen
atom
carrying them, are fluorine substituents, and in another embodiment they are
(Ci-C4)-
alkyl substituents, for example methyl substituents, and in another embodiment
are
substituents in such a ring bonded to a ring nitrogen atom selected from the
series
consisting of (Ci-C4)-alkyl. Examples of heterocyclic groups, from any one or
more of
which the heterocyclic groups formed by the groups R11 and R12 together with
the
nitrogen atom carrying them is selected in one embodiment of the invention,
are
azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-
4-yl, and 4-
methylpiperazin-1-yl.
In one embodiment of the invention, one of the groups R11 and R12 is selected
from
the series consisting of hydrogen and (Ci-C4)-alkyl, and the other is selected
from the
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series consisting of hydrogen, (Ci-C4)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
C7)-cycloalkyl, in another embodiment R11 and R12 are independently of one
another selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl, in another embodiment from the series consisting of hydrogen and
(Ci-
C4)-alkyl, in another embodiment from the series consisting of (Ci-C4)-alkyl,
and in
another embodiment they are hydrogen, i.e., in this latter embodiment the
group -N(R11)-R12 representing R1 is the group -NH2 (amino), or in all these
embodiments R11 and R12, together with the nitrogen atom carrying them, form a
monocyclic, 4-membered to 7-membered, saturated heterocycle which, in addition
to
the nitrogen atom carrying R11 and R12, comprises 0 or 1 further ring
heteroatom
selected from the series consisting of nitrogen, oxygen and sulfur, and which
is
unsubstituted or substituted by one ore more identical or different
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl.
In one embodiment of the invention, one of the groups R11 and R12 is selected
from
the series consisting of hydrogen and (Ci-C4)-alkyl, and the other is selected
from the
series consisting of hydrogen, (Ci-C4)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
C7)-cycloalkyl, In another embodiment, R11 and R12 are independently of one
another selected from the series consisting of hydrogen, (Ci-C4)-alkyl and (C3-
C7)-
cycloalkyl, in another embodiment from the series consisting of hydrogen and
(Ci-
C4)-alkyl, in another embodiment from the series consisting of (Ci-C4)-alkyl,
and in
another embodiment R11 and R12 are hydrogen.
In one embodiment of the invention, R13 is selected from the series consisting
of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the series consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, and in another embodiment R13 is hydrogen.
In one embodiment of the invention, R14 and R15 are independently of one
another
selected from the series consisting of (C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-
alkyl-
phenyl, Het and -(Ci-C4)-alkyl-Het, in anther embodiment from the series
consisting
of (C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl and Het, in another
embodiment
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from the series consisting of (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-
alkyl-(C3-C7)-
cycloalkyl, -(Ci-C4)-alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another
embodiment from
the series consisting of phenyl and Het, and in another embodiment are one or
both
of R14 and R15 independently of one another selected from the series
consisting of
(Ci-C8)-alkyl, in another embodiment from the series consisting of (C3-C7)-
cycloalkyl,
in another embodiment from the series consisting of -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
in another embodiment from the series consisting of phenyl, in another
embodiment
from the series consisting of -(Ci-C4)-alkyl-phenyl, in another embodiment
from the
series consisting of Het, and in another embodiment from the series consisting
of -(Ci-C4)-alkyl-Het, wherein in all these embodiments phenyl and Het all are
unsubstituted or substituted by one or more identical or different
substituents R30.
The explanations given above with respect to a monocyclic ring which can be
formed
by R11 and R12 together with the nitrogen atom carrying them, and the
embodiments specified above with respect to this ring, apply correspondingly
to the
monocyclic ring which can be formed by R16 and R17 together with the nitrogen
atom carrying them. For example, the ring which can be formed by the groups
R16
and R17 together with the nitrogen atom carrying them, which heterocycle is
thus
bonded via a ring nitrogen atom, can be 4-membered, 5-membered, 6-membered or
7-membered. In one embodiment, the further ring heteroatom which is optionally
present in a heterocycle formed by the groups R16 and R17 together with the
nitrogen atom carrying them, is selected from the series consisting of
nitrogen and
oxygen, in another embodiment it is a nitrogen atom, and in another embodiment
it is
an oxygen atom, and in another embodiment no further ring heteroatom is
present. In
one embodiment of the invention, substituents in a ring formed by the groups
R16
and R17 together with the nitrogen atom carrying them, which are bonded to a
ring
nitrogen atom, are, selected from the series consisting of (Ci-C4)-alkyl.
Examples of
heterocyclic groups, from any one or more of which the heterocyclic groups
formed
by the groups R16 and R17 together with the nitrogen atom carrying them is
selected
in one embodiment of the invention, likewise are azetidin-1-yl, pyrrolidin-1-
yl,
piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, and 4-methylpiperazin-1-
yl.
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In one embodiment of the invention, R16 is selected from the series consisting
of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the series consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, and in another embodiment R16 is hydrogen, and in one
embodiment R17 is selected from the series consisting of hydrogen, (Ci-C8)-
alkyl,
(C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-
phenyl, Het
and -(Ci-C4)-alkyl-Het, in another embodiment from the series consisting of
hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment from the series
consisting
of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
phenyl, -(Ci-C4)-alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment
from the
series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
C7)-cycloalkyl, in another embodiment from the series consisting of hydrogen,
(Ci-
C8)-alkyl and (C3-C7)-cycloalkyl, in another embodiment from the series
consisting of
hydrogen and (Ci-C8)-alkyl, in another embodiment from the series consisting
of (Ci-
C8)-alkyl, and in another embodiment R17 is hydrogen, wherein phenyl and Het
all
are unsubstituted or substituted by one or more identical or different
substituents
R30, or R16 and R17, together with the nitrogen atom carrying them, form in
these
embodiments a monocyclic, 4-membered to 7-membered, saturated heterocycle
which, in addition to the nitrogen atom carrying R16 and R17, comprises 0 or 1
further ring heteroatom selected from the series consisting of nitrogen,
oxygen and
sulfur, and which is unsubstituted or substituted by one ore more identical or
different
substituents selected from the series consisting of fluorine and (Ci-C4)-
alkyl.
In another embodiment of the invention, R16 is selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the series consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, and in another embodiment R16 is hydrogen, and in one
embodiment R17 is selected from the series consisting of hydrogen, (Ci-C8)-
alkyl,
(C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-
phenyl, Het
and -(Ci-C4)-alkyl-Het, in another embodiment from the series consisting of
hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
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alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment from the series
consisting
of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
phenyl, -(Ci-C4)-alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment
from the
series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
5 C7)-cycloalkyl, in another embodiment from the series consisting of
hydrogen, (Ci-
C8)-alkyl and (C3-C7)-cycloalkyl, in another embodiment from the series
consisting of
hydrogen and (Ci-C8)-alkyl, in another embodiment from the series consisting
of (Ci-
C8)-alkyl, and in another embodiment R17 is hydrogen, wherein phenyl and Het
all
are unsubstituted or substituted by one or more identical or different
substituents
10 R30.
In one embodiment of the invention, a (Ci-C8)-alkyl group representing R17 is
(Ci-
C4)-alkyl, in another embodiment (Ci-C3)-alkyl, in another embodiment (Ci-C2)-
alkyl,
in another embodiment methyl.
In one embodiment of the invention, R18 is selected from the series consisting
of
hydrogen and (Ci-C2)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, in another embodiment R18 is hydrogen, in another
embodiment R18 is selected from the series consisting (Ci-C4)-alkyl, in
another
embodiment from the series consisting of (Ci-C2)-alkyl, and in another
embodiment
R18 is methyl.
The explanations given above with respect to a monocyclic ring which can be
formed
by R11 and R12 together with the nitrogen atom carrying them, and the
embodiments specified above with respect to this ring, apply correspondingly
to the
monocyclic ring which can be formed by R19 and R20 together with the nitrogen
atom carrying them. For example, the ring which can be formed by the groups
R19
and R20 together with the nitrogen atom carrying them, which heterocycle is
thus
bonded via a ring nitrogen atom, can be 4-membered, 5-membered, 6-membered or
7-membered. In one embodiment, the further ring heteroatom which is optionally
present in a heterocycle formed by the groups R19 and R20 together with the
nitrogen atom carrying them, is selected from the series consisting of
nitrogen and
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oxygen, in another embodiment it is a nitrogen atom, and in another embodiment
it is
an oxygen atom, and in another embodiment no further ring heteroatom is
present. In
one embodiment of the invention, substituents in a ring formed by the groups
R19
and R20 together with the nitrogen atom carrying them, which are bonded to a
ring
nitrogen atom, are, selected from the series consisting of (Ci-C4)-alkyl.
Examples of
heterocyclic groups, from any one or more of which the heterocyclic groups
formed
by the groups R19 and R20 together with the nitrogen atom carrying them is
selected
in one embodiment of the invention, likewise are azetidin-1-yl, pyrrolidin-1-
yl,
piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, and 4-methylpiperazin-1-
yl.
In one embodiment of the invention, R19 is selected from the series consisting
of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the serious consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, and in another embodiment R19 is hydrogen, and in one
embodiment R20 is selected from the series consisting of hydrogen, (Ci-C8)-
alkyl,
(C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-
phenyl, Het
and -(Ci-C4)-alkyl-Het, in another embodiment from the series consisting of
hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment from the series
consisting
of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
phenyl, -(Ci-C4)-alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment
from the
series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
C7)-cycloalkyl, in another embodiment from the series consisting of hydrogen,
(Ci-
C8)-alkyl and (C3-C7)-cycloalkyl, in another embodiment from the series
consisting of
hydrogen and (Ci-C8)-alkyl, in another embodiment from the series consisting
of (Ci-
C8)-alkyl, and in another embodiment R20 is hydrogen, wherein phenyl and Het
all
are unsubstituted or substituted by one or more identical or different
substituents
R30, or R19 and R20, together with the nitrogen atom carrying them, form in
these
embodiment a monocyclic, 4-membered to 7-membered, saturated heterocycle
which, in addition to the nitrogen atom carrying R19 and R20, comprises 0 or 1
further ring heteroatom selected from the series consisting of nitrogen,
oxygen and
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sulfur, and which is unsubstituted or substituted by one ore more identical or
different
substituents selected from the series consisting of fluorine and (Ci-C4)-
alkyl.
In another embodiment of the invention, R19 is selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the serious consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, and in another embodiment R19 is hydrogen, and in one
embodiment R20 is selected from the series consisting of hydrogen, (Ci-C8)-
alkyl,
(C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-
phenyl, Het
and -(Ci-C4)-alkyl-Het, in another embodiment from the series consisting of
hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment from the series
consisting
of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl, -(Ci-C4)-alkyl-(C3-C7)-
cycloalkyl,
phenyl, -(Ci-C4)-alkyl-phenyl and -(Ci-C4)-alkyl-Het, in another embodiment
from the
series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-cycloalkyl and -(Ci-C4)-
alkyl-(C3-
C7)-cycloalkyl, in another embodiment from the series consisting of hydrogen,
(Ci-
C8)-alkyl and (C3-C7)-cycloalkyl, in another embodiment from the series
consisting of
hydrogen and (Ci-C8)-alkyl, in another embodiment from the series consisting
of (Ci-
C8)-alkyl, and in another embodiment R20 is hydrogen, wherein phenyl and Het
all
are unsubstituted or substituted by one or more identical or different
substituents
R30.
In one embodiment of the invention, a (Ci-C8)-alkyl group representing R20 is
(Ci-
C4)-alkyl, in another embodiment (Ci-C3)-alkyl, in another embodiment (Ci-C2)-
alkyl,
in another embodiment methyl.
In one embodiment of the invention, R21 is selected from the series consisting
of
hydrogen and (Ci-C4)-alkyl, in another embodiment from the series consisting
of
hydrogen and (Ci-C3)-alkyl, in another embodiment from the series consisting
of
hydrogen and methyl, in another embodiment R21 is hydrogen, and in another
embodiment R21 is methyl.
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In one embodiment of the invention R22 is selected from the series consisting
of (Ci-
C4)-alkyl, (C3-C6)-cycloalkyl and -(Ci-C2)-alkyl-(C3-C6)-cycloalkyl, in
another
embodiment from the series consisting of (Ci-C4)-alkyl and (C3-C6)-cycloalkyl,
in
another embodiment from the series consisting of (Ci-C4)-alkyl and -(Ci-C2)-
alkyl-
(C3-C6)-cycloalkyl, in another embodiment from the series consisting of (Ci-
C4)-alkyl,
in another embodiment from the series consisting of (Ci-C3)-alkyl, and in
another
embodiment R22 is methyl.
In one embodiment of the invention, R23 and R24 are independently of one
another
selected from the series consisting of hydrogen and (Ci-C3)-alkyl, in another
embodiment from the series consisting of hydrogen and (Ci-C2)-alkyl, in
another
embodiment from the series consisting of hydrogen and methyl, and in another
embodiment R23 and R24 are hydrogen.
In one embodiment of the invention, R30 is in any of its occurrences,
independently
of its other occurrences, selected from the series consisting of halogen, (Ci-
C4)-alkyl
and -CN; in another embodiment from the series consisting of halogen, (Ci-C4)-
alkyl
and -0-(Ci-C4)-alkyl, in another embodiment from the series consisting of
halogen
and (Ci-C4)-alkyl, in another embodiment from the series consisting of halogen
and -CN, in another embodiment from the series consisting of halogen. In one
embodiment, a group R30 which is bonded to ring nitrogen atom in a group Het,
is
selected from the series consisting of (Ci-C4)-alkyl. In one embodiment, a (Ci-
C4)-
alkyl group representing R30 or occurring in R30 is in any occurrence of R30,
independently of other occurrences, selected from (Ci-C3)-alkyl, in another
embodiment from (Ci-C2)-alkyl, and in another embodiment it is methyl.
The monocyclic group Het can be 4-membered, 5-membered, 6-membered or 7-
membered. In one embodiment of the invention, Het is in any of its
occurrences,
independently of its other occurrences, 4-membered, 5-membered or 6-membered,
in another embodiment 5-membered or 6-membered, in another embodiment 5-
membered, in another embodiment 6-membered, in another embodiment 5-
membered, 6-membered or 7-membered. In one embodiment, Het is in any of its
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occurrences, independently of its other occurrences, a saturated or partially
saturated heterocycle, in another embodiment a saturated heterocycle, in
another
embodiment a saturated or aromatic heterocycle, in another embodiment an
aromatic
heterocycle. In one embodiment, the ring heteroatoms in a heterocycle Het
which is
saturated or partially unsaturated, are selected from the series consisting of
nitrogen
and oxygen, in another embodiment from the series consisting of oxygen and
sulfur.
In one embodiment, the ring heteroatoms in a heterocycle Het which is
aromatic, are
selected from the series consisting of nitrogen and sulfur. In one embodiment,
Het
comprises in any of its occurrences, independently of its other occurrences, 1
ring
heteroatom selected from the series consisting of nitrogen, oxygen and sulfur.
Examples of groups, from any one or more which Het is in any of its
occurrences,
independently of any other occurrence, selected in one embodiment of the
invention,
are oxetanyl including oxetan-2-yland oxetan-3-yl, tetrahydrofuranyl including
tetrahydrofuran-2-yland tetrahydrofuran-3-yl, tetrahydropyranyl including
tetrahydropyran-2-yl, tetrahydropyran-3-yland tetrahydropyran-4-yl, oxepanyl
including oxepan-2-yl, oxepan-3-yland oxepan-4-yl, azetidinyl including
azetidin-2-y1
and azetidin-3-yl, pyrrolidinyl including pyrrolidin-2-yland pyrrolidin-3-yl,
piperidinyl
including piperidin-2-yl, piperidin-3-yland piperidin-4-yl, azepanyl including
azepan-2-
yl, azepan-3-yland azepan-4-yl, morpholinyl including morpholin-2-yland
morpholin-
3-yl, thiomorpholinyl including thiomorpholin-2-yland thiomorpholin-3-yl,
piperazinyl
including piperazin-2-yl, furanyl including furan-2-yland furan-3-yl,
thiophenyl
(thienyl) including thiophen-2-yland thiophen-3-yl, pyrrolyl including pyrrol-
2-yland
pyrrol-3-yl, isoxazolyl including isoxazol-3-yl, isoxazol-4-yland isoxazol-5-
yl, oxazolyl
including oxazol-2-yl, oxazol-4-yland oxazol-5-yl, thiazolyl including thiazol-
2-yl,
thiazol-4-yland thiazol-5-yl, pyrazolyl including pyrazol-3-yl, pyrazol-4-
yland pyrazol-
5-yl, imidazolyl including imidazol-2-yl, imidazol-4-yland imidazol-5-yl,
pyridinyl
(pyridyl) including pyridin-2-yl, pyridin-3-yland pyridin-4-yl, pyrazinyl
including
pyrazin-2-yl.
A subject of the invention are all compounds of the formula I wherein any one
or
more structural elements such as groups, residues, substituents and numbers
are
defined as in any of the specified embodiments or definitions of the elements,
or
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have one or more of the specific meanings which are mentioned herein as
examples
of elements, wherein all combinations of one or more definitions of compounds
or
elements and/or specified embodiments and/or specific meanings of elements are
a
subject of the present invention. Also with respect to all such compounds of
the
5 formula I, all their stereoisomeric forms and mixtures of stereoisomeric
forms in any
ratio, and their pharmaceutically acceptable salts are a subject of the
present
invention.
As an example of compounds of the invention which with respect to any
structural
10 elements are defined as in specified embodiments of the invention or
definitions of
such elements, compounds of the formula I may be mentioned, wherein
Ar is selected from the series consisting of phenyl and a 5-membered or 6-
membered monocyclic aromatic heterocycle comprising 1 or 2 identical or
different
15 ring heteroatoms selected from the series consisting of nitrogen, oxygen
and sulfur
and bonded via a ring carbon atom, which are all unsubstituted or substituted
by one
or more identical or different substituents R10;
n is selected from the series consisting of 0, 1 and 2;
R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14, (Ci-C4)-alkyl and -(Ci-C4)-alkyl-
0-
R18;
R2 is selected from the series consisting of halogen, -(Ci-C4)-alkyl and -CN;
R10 is selected from the series consisting of halogen, (Ci-C4)-alkyl,
-N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2, -C(0)-N(R23)-R24
and -CN,
and two groups R10 bonded to adjacent ring carbon atoms in Ar, together with
the
carbon atoms carrying them, can form a 5-membered to 7-membered unsaturated
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ring which comprises 0, 1 or 2 identical or different ring heteroatoms
selected from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one or more identical or different substituents selected from
the series
consisting of halogen, -(Ci-C4)-alkyl and -CN;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl,
or R11 and R12, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 6-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R11 and R12, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R14 and R15 are independently of one another selected from the series
consisting of
(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het,
wherein
phenyl and Het all are unsubstituted or substituted by one or more identical
or
different substituents R30;
R18 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R19 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R20 is selected from the series consisting of hydrogen, (Ci-C8)-alkyl, (C3-C7)-
cycloalkyl and -(Ci-C4)-alkyl-(C3-C7)-cycloalkyl;
or R19 and R20, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 6-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R19 and R20, comprises 0 or 1 further ring heteroatom selected
from
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47
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R21 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R22 is selected from the series consisting of (Ci-C4)-alkyl and (C3-C7)-
cycloalkyl;
R23 and R24 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl;
R30 is selected from the series consisting of halogen, (Ci-C4)-alkyl and -CN;
Het is a monocyclic, 4-membered to 7-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 or 2 identical or different ring
heteroatoms
selected from the series consisting of nitrogen, oxygen and sulfur, and which
is
bonded via a ring carbon atom;
wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents;
in any of their stereoisomeric forms or a mixture of stereoisomeric forms in
any ratio,
and the pharmaceutically acceptable salt thereof.
As another such example, compounds of the formula I may be mentioned, wherein
Ar is selected from the series consisting of phenyl and a 5-membered or 6-
membered monocyclic aromatic heterocycle comprising 1 or 2 identical or
different
ring heteroatoms selected from the series consisting of nitrogen, oxygen and
sulfur
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48
and bonded via a ring carbon atom, which are all unsubstituted or substituted
by one
or more identical or different substituents R10;
n is selected from the series consisting of 0 and 1;
R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14 and (Ci-C4)-alkyl;
R2 is selected from the series consisting of halogen and -(Ci-C4)-alkyl;
R10 is selected from the series consisting of halogen, (Ci-C4)-alkyl, -0-(Ci-
C4)-alkyl,
-N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN,
and two groups R10 bonded to adjacent ring carbon atoms in Ar, together with
the
carbon atoms carrying them, can form a 5-membered to 7-membered unsaturated
ring which comprises 0, 1 or 2 identical or different ring heteroatoms
selected from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one or more identical or different substituents selected from
the series
consisting of halogen and -(Ci-C4)-alkyl;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl,
or R11 and R12, together with the nitrogen atom carrying them, form a
monocyclic,
5-membered or 6-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R11 and R12, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
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R14 and R15 are independently of one another selected from the series
consisting of
(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het,
wherein
phenyl and Het all are unsubstituted or substituted by one or more identical
or
different substituents R30;
R19 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R20 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
or R19 and R20, together with the nitrogen atom carrying them, form a
monocyclic,
4-membered to 6-membered, saturated heterocycle which, in addition to the
nitrogen
atom carrying R19 and R20, comprises 0 or 1 further ring heteroatom selected
from
the series consisting of nitrogen, oxygen and sulfur, and which is
unsubstituted or
substituted by one ore more identical or different substituents selected from
the
series consisting of fluorine and (Ci-C4)-alkyl;
R21 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R22 is selected from the series consisting of (Ci-C4)-alkyl;
R30 is selected from the series consisting of halogen, (Ci-C4)-alkyl and -CN;
Het is a monocyclic, 5-membered or 6-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 or 2 identical or different ring
heteroatoms
selected from the series consisting of nitrogen, oxygen and sulfur, and which
is
bonded via a ring carbon atom;
wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents;
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in any of their stereoisomeric forms or a mixture of stereoisomeric forms in
any ratio,
and the pharmaceutically acceptable salt thereof.
5 As another such example, compounds of the formula I may be mentioned,
wherein
Ar is phenyl which is unsubstituted or substituted by one or more identical or
different
substituents R10;
10 n is selected from the series consisting of 0 and 1;
R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14 and (Ci-C4)-alkyl;
15 R2 is selected from the series consisting of halogen and -(Ci-C4)-alkyl;
R10 is selected from the series consisting of halogen, (Ci-C4)-alkyl,
-N(R19)-R20, -N(R21)-N(R19)-R20, -N(R21)-C(0)-R22, -NO2 and -CN,
20 and two groups R10 bonded to adjacent ring carbon atoms in Ar, together
with the
carbon atoms carrying them, can form a 5-membered to 7-membered unsaturated
ring which comprises 0, 1 or 2 oxygen atoms as ring heteroatoms, and which is
unsubstituted or substituted by one or more identical or different
substituents
selected from the series consisting of halogen and -(Ci-C4)-alkyl;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R14 and R15 are independently of one another selected from the series
consisting of
(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het,
wherein
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phenyl and Het all are unsubstituted or substituted by one or more identical
or
different substituents R30;
R19 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R20 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R21 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R22 is selected from the series consisting of (Ci-C4)-alkyl;
R30 is selected from the series consisting of halogen and (Ci-C4)-alkyl;
Het is a monocyclic, 5-membered or 6-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 ring heteroatom selected from the
series
consisting of nitrogen, oxygen and sulfur, and which is bonded via a ring
carbon
atom;
wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents;
in any of their stereoisomeric forms or a mixture of stereoisomeric forms in
any ratio,
and the pharmaceutically acceptable salt thereof.
As another such example, compounds of the formula I may be mentioned, wherein
Ar is selected from the series consisting of 2,3-dichloro-phenyl, 2,5-dichloro-
phenyl,
5-chloro-2-hydrazino-phenyl, 5-chloro-2-cyano-phenyl, 2-cyano-5-methyl-phenyl,
2-
fluoro-5-methyl-phenyl, 2-chloro-5-methoxy-phenyl, 2,5-dichloro-thiophen-3-yl,
8-
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chloro-3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl, 5-chloro-1,3-dimethyl-
pyrazol-4-yl,
naphthalen-1-yl, 2,4,6-trichloro-phenyl, 5-chloro-2-fluoro-phenyl, 2,4,5-
trifluoro-
phenyl, 2,4,5-trichloro-phenyl, 5-chloro-2,4-difluoro-phenyl, 2,3,4-trichloro-
phenyl,
2,3,4-trifluoro-phenyl, 2-chloro-4-trifluoromethyl-phenyl, 5-cyano-2-fluoro-
phenyl, 2-
cyano-5-methoxy-phenyl, 2-cyano-5-fluoro-phenyl, 2-fluoro-5-methoxy-phenyl, 4-
acetylam ino-2-methyl-phenyl, 2-methyl-5-nitro-phenyl, and 2-nitro-4-
trifluoromethyl-
phenyl;
n is selected from the series consisting of 0 and 1;
R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14 and (Ci-C4)-alkyl;
R2 is selected from the series consisting of halogen and -(Ci-C4)-alkyl;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
R14 and R15 are independently of one another selected from the series
consisting of
(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het,
wherein
phenyl and Het all are unsubstituted or substituted by one or more identical
or
different substituents R30;
R30 is selected from the series consisting of halogen and (Ci-C4)-alkyl;
Het is a monocyclic, 5-membered or 6-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 ring heteroatom selected from the
series
consisting of nitrogen, oxygen and sulfur, and which is bonded via a ring
carbon
atom;
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wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents;
in any of their stereoisomeric forms or a mixture of stereoisomeric forms in
any ratio,
and the pharmaceutically acceptable salt thereof.
As another such example, compounds of the formula I may be mentioned, wherein
Ar is selected from the series consisting of 2,3-dichloro-phenyl, 2,5-dichloro-
phenyl,
5-chloro-2-hydrazino-phenyl, 5-chloro-2-cyano-phenyl, 2-cyano-5-methyl-phenyl,
2-
fluoro-5-methyl-phenyl, 2-chloro-5-methoxy-phenyl, 2,5-dichloro-thiophen-3-yl,
8-
chloro-3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl, 5-chloro-1,3-dimethyl-
pyrazol-4-yl,
naphthalen-1-yl, 5-cyano-2-fluoro-phenyl, 2-cyano-5-methoxy-phenyl, 2-cyano-5-
fluoro-phenyl, 2-fluoro-5-methoxy-phenyl, 4-acetylamino-2-methyl-phenyl, 2-
methyl-
5-nitro-phenyl, and 2-nitro-4-trifluoromethyl-phenyl;
n is selected from the series consisting of 0 and 1;
R1 is selected from the series consisting of hydrogen, -N(R11)-R12, -N(R13)-
C(0)-
R14, -N(R13)-S(0)2-R15, -N(R13)-C(0)-NH-R14 and (Ci-C4)-alkyl;
R2 is selected from the series consisting of halogen and -(Ci-C4)-alkyl;
R11 and R12 are independently of one another selected from the series
consisting of
hydrogen and (Ci-C4)-alkyl;
R13 is selected from the series consisting of hydrogen and (Ci-C4)-alkyl;
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R14 and R15 are independently of one another selected from the series
consisting of
(C3-C7)-cycloalkyl, phenyl, -(Ci-C4)-alkyl-phenyl, Het and -(Ci-C4)-alkyl-Het,
wherein
phenyl and Het all are unsubstituted or substituted by one or more identical
or
different substituents R30;
R30 is selected from the series consisting of halogen and (Ci-C4)-alkyl;
Het is a monocyclic, 5-membered or 6-membered, saturated, partially
unsaturated or
aromatic heterocycle which comprises 1 ring heteroatom selected from the
series
consisting of nitrogen, oxygen and sulfur, and which is bonded via a ring
carbon
atom;
wherein all cycloalkyl groups can be substituted by one or more identical
substituents
selected from the series consisting of fluorine and (Ci-C4)-alkyl;
wherein all alkyl groups, independently of any other substituents which can be
present on an alkyl group, can be substituted by one ore more fluorine
substituents;
in any of their stereoisomeric forms or a mixture of stereoisomeric forms in
any ratio,
and the pharmaceutically acceptable salt thereof.
A subject of the invention also is a compound of the formula I which is
selected from
any of the specific compounds of the formula I which are disclosed herein, or
is any
one of the specific compounds of the formula I which are disclosed herein,
irrespective thereof whether they are disclosed as a free compound and/or as a
specific salt, or a pharmaceutically acceptable salt thereof, wherein the
compound of
the formula I is a subject of the invention in any of its stereoisomeric forms
or a
mixture of stereoisomeric forms in any ratio. For example, a subject of the
invention
is a compound of the formula I which is selected from the series consisting
of:
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,3-dichloro-
benzenesulfonamide,
2,5-dichloro-N-[4-(1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]benzenesulfonam ide,
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2, 5-dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2,3-dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-
benzenesulfonamide,
5 N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,5-dichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-hydrazino-
benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-pheny1]-5-chloro-2-
fluoro-
10 benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-pheny1]-2,5-dichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-pheny1]-2,3-dichloro-
benzenesulfonam ide,
15 2,5-dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2, 3-dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
5-chloro-2-fluoro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
20 yl)phenyl]benzenesulfonam ide,
5-chloro-2-cyano-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2-cyano-5-methyl-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
25 2-fluoro-5-methyl-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2-chloro-5-methoxy-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-cyano-5-methyl-
30 benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-chloro-5-methoxy-
benzenesulfonamide,
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N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)pheny1]-2-fluoro-5-methyl-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-y1)-pheny1]-5-chloro-2-cyano-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)pheny1]-2,5-dichloro-thiophene-3-
sulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)pheny1]-8-chloro-3,4-dihydro-2H-
benzo[b][1,4]dioxepine-7-sulfonamide,
N-[4-(3-am ino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)phenyl]-5-chloro-1,3-dimethyl-
pyrazole-
4-sulfonamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]cyclopropanecarboxamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]tetrahydropyran-4-carboxam ide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]piperidine-4-carboxamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]cyclopentanecarboxam ide,
2,3-dichloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-
pyrazolo[3,4-1D]pyrazin-3-yl]benzamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]cyclohexanecarboxamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-y1]-2-phenyl-acetamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
1D]pyrazin-
3-yl]thiophene-3-carboxamide,
4-chloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-
pyrazolo[3,4-
1D]pyrazin-3-yl]benzamide,
N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)phenyl]naphthalene-1-
sulfonamide,
N-[4-(3-amino-1H-pyrazolo[3,4-1D]pyrazin-6-yl)pheny1]-2,4,6-trichloro-
benzenesulfonam ide,
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N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-fluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,4,5-trifluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,4,5-trichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2,4-difluoro-
benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,3,4-trichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,3,4-trifluoro-
benzenesulfonam ide),
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-chloro-4-
trifluoromethyl-
benzenesulfonamide,
5-chloro-N-[443-[(5-chloro-2,4-difluoro-phenyl)sulfonylam ino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-2,4-difluoro-benzenesulfonamide,
5-chloro-N-[443-[(5-chloro-1,3-dimethyl-pyrazol-4-yl)sulfonylamino]-1H-
pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-1,3-dimethyl-pyrazole-4-sulfonam ide,
2,4,5-trifluoro-N-[443-[(2,4,5-trifluorophenyl)sulfonylam ino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]phenyl]benzenesulfonamide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-5-cyano-2-fluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-cyano-5-methoxy-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-cyano-5-fluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-fluoro-5-methoxy-
benzenesulfonamide,
14644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-3-
yI]-3-(3-pyridyl)urea,
1-(4-chloropheny1)-34644-[(2-fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]urea,
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2-chloro-N-[443-[[2-chloro-4-trifluoromethyl-phenyl]sulfonylamino]-1H-
pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-4-trifluoromethyl-benzenesulfonamide,
N-[644-(1-naphthylsulfonylam ino)phenyI]-1H-pyrazolo[3,4-b]pyrazin-3-
yl]naphthalene-1-sulfonam ide,
2,4,6-trichloro-N-[443-[(2,4,6-trichlorophenyl)sulfonylamino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]phenyl]benzenesulfonamide,
N-[3-methyl-4-[[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]sulfamoyl]phenyl]acetam ide,
2-methyl-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-n itro-
benzenesulfonamide, and
N-[4-(3-methyl-1H-pyrazolo[3, 4-b]pyrazin-6-yl)phenyI]-2-n itro-4-trifluorom
ethyl-
benzenesulfonam ide,
or which is any one of these compounds, and its pharmaceutically acceptable
salts.
Another subject of the invention is a compound of the formula l which is
selected
from the series consisting of:
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,3-dichloro-
benzenesulfonam ide,
2,5-dichloro-N-[4-(1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]benzenesulfonam ide,
2,5-d ich loro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2,3-dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-yI)-phenyl]-
benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,5-dichloro-
benzenesulfonamide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-hydrazino-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-phenyl]-5-chloro-2-
fluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-phenyl]-2,5-dichloro-
benzenesulfonam ide,
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N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-pheny1]-2,3-dichloro-
benzenesulfonam ide,
2, 5-dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2, 3-dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
5-chloro-2-fluoro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
5-chloro-2-cyano-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2-cyano-5-methyl-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2-fluoro-5-methyl-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
2-chloro-5-methoxy-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-cyano-5-methyl-
benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-chloro-5-methoxy-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-fluoro-5-methyl-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-5-chloro-2-cyano-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,5-dichloro-thiophene-3-
sulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-8-chloro-3,4-dihydro-2H-
benzo[b][1,4]dioxepine-7-sulfonamide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-chloro-1,3-dimethyl-
pyrazole-
4-sulfonamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
3-yl]cyclopropanecarboxamide,
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N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
3-yl]tetrahydropyran-4-carboxam ide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
3-yl]piperidine-4-carboxamide,
5 N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
3-yl]cyclopentanecarboxam ide,
2,3-dichloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]benzamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
10 3-yl]cyclohexanecarboxamide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
b]pyrazin-
3-y1]-2-phenyl-acetam ide,
N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-pyrazolo[3,4-
b]pyrazin-
3-yl]thiophene-3-carboxamide,
15 4-chloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-
pyrazolo[3,4-
b]pyrazin-3-yl]benzamide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]naphthalene-1-sulfonamide,
5-chloro-N-[443-[(5-chloro-2,4-difluoro-phenyl)sulfonylam ino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-2,4-difluoro-benzenesulfonamide,
20 5-chloro-N-[443-[(5-chloro-1,3-dimethyl-pyrazol-4-yl)sulfonylamino]-1H-
pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-1,3-dimethyl-pyrazole-4-sulfonam ide,
2,4,5-trifluoro-N-[443-[(2,4,5-trifluorophenyl)sulfonylamino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]phenyl]benzenesulfonamide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-5-cyano-2-fluoro-
25 benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-cyano-5-methoxy-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-cyano-5-fluoro-
benzenesulfonam ide,
30 N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-2-fluoro-5-methoxy-
benzenesulfonam ide,
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14644-[(2-fluoro-5-methyl-phenyl)sulfonylam ino]pheny1]-1H-pyrazolo[3,4-
b]pyrazin-3-
y1]-3-(3-pyridyl)urea,
1-(4-chloropheny1)-34644-[(2-fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]urea,
2-chloro-N-[443-[[2-chloro-4-trifluoromethyl-phenyl]sulfonylamino]-1H-
pyrazolo[3,4-
b]pyrazin-6-yl]pheny1]-4-trifluoromethyl-benzenesulfonamide,
N-[644-(1-naphthylsulfonylam ino)phenyI]-1H-pyrazolo[3,4-b]pyrazin-3-
yl]naphthalene-1-sulfonam ide,
2,4,6-trichloro-N-[443-[(2,4,6-trichlorophenyl)sulfonylam ino]-1H-pyrazolo[3,4-
b]pyrazin-6-yl]phenyl]benzenesulfonamide,
N-[3-methyl-4-[[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]sulfamoyl]phenyl]acetam ide,
2-methyl-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-nitro-
benzenesulfonamide, and
N-[4-(3-methyl-1H-pyrazolo[3, 4-b]pyrazin-6-yl)phenyI]-2-n itro-4-trifluorom
ethyl-
benzenesulfonam ide,
or which is any one of these compounds, and its pharmaceutically acceptable
salts.
In one embodiment of the invention, the compounds of the formula I are defined
as
above in their generic definition or in any of the more specific definitions
or
embodiments, with the proviso that the compound of the formula I is not one of
the
following compounds:
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,4,6-trichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-fluoro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,4,5-trifluoro-
benzenesulfonam ide,
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,4,5-trichloro-
benzenesulfonamide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2,4-difluoro-
benzenesulfonam ide,
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N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,3,4-trichloro-
benzenesulfonam ide,
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,3,4-trifluoro-
benzenesulfonam ide, and
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-chloro-4-
trifluoromethyl-
benzenesulfonam ide,
and in another embodiment the excluded compounds are excluded as the free
compounds, i.e. they are not excluded in the form of a salt with an acid or
base.
In another embodiment of the invention, the compounds of the formula I are
defined
as above in their generic definition or in any of the more specific
definitions or
embodiments, with the proviso that the compound of the formula I is not a
compound
in which simultaneously the group Ar is a phenyl group which is substituted by
three
identical or different halogen substituents, n is 0, and R1 is the group -NH2
(amino),
and the proviso that the compound of the formula I is not one of the following
compounds:
N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-fluoro-
benzenesulfonamide, and
N-[4-(3-am ino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-chloro-4-
trifluoromethyl-
benzenesulfonamide,
and in another embodiment the excluded compounds are excluded as the free
compounds, i.e. they are not excluded in the form of a salt with an acid or
base.
In another embodiment of the invention, the compounds of the formula I are
defined
as above in their generic definition or in any of the more specific
definitions or
embodiments, with the proviso that the compound of the formula I is not a
compound
in which simultaneously the group Ar is selected from the series consisting of
2,4,6-
trichloro-phenyl, 5-chloro-2-fluoro-phenyl, 2,4,5-trichloro-phenyl, 2,4,5-tri-
fluoro-
phenyl, 5-chloro-2,4-difluoro-phenyl, 2,3,4-trichloro-phenyl, 2,3,4-trifluoro-
phenyl and
2-chloro-4-trifluoromethyl-phenyl, n is 0, and R1 is the group -NH2 (amino),
and in
another embodiment the excluded compounds are excluded as the free compounds,
i.e. they are not excluded in the form of a salt with an acid or base.
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Another subject of the present invention are processes for the preparation of
the
compounds of the formula I which are outlined below and by which the compounds
of
the formula I and intermediates and occurring in the course of their
synthesis, and
salts thereof, are obtainable. The compounds of the formula I can be prepared
by
utilizing procedures and techniques which per se are known to a person skilled
in the
art. In general, 1H-pyrazolo[3,4-b]pyrazine compounds of the formula I can be
prepared, for example, in the course of a convergent synthesis, by linking two
or
more fragments which can be derived retrosynthetically from the formula I.
More
specifically, suitably substituted starting 1H-pyrazolo[3,4-b]pyrazine
derivatives can
be employed as building blocks in the preparation of the compounds of formula
I,
which can be synthesized from suitable precursor compounds, which allow the
introduction of a variety of substituents into the various positions of the 1H-
pyrazolo[3,4-b]pyrazine system and which can be chemically modified further in
order
to finally arrive at the compound of the formula I having the desired
substituent
pattern. For the synthesis of 1H-pyrazolo[3,4-b]pyrazines, use can also be
made of
procedures and transformations which are described in the literature with
respect to
indazoles. As reviews in which numerous details and literature references on
the
chemistry of indazoles and on synthetic procedures for their preparation can
be
found, J. Eiguero in Comprehensive Heterocyclic Chemistry II, Eds. A.
Katritzky, Ch.
Rees, E. Scriven, Elsevier 1996, Vol. 3; W. Stadlbauer in Houben-Weyl,
Methoden
der Organischen Chem ie (Methods of Organic Chemistry), Georg Thieme Verlag,
Stuttgart, Germany 1994, Vol. E8b, Hetarene; W. Stadlbauer in Houben-Weyl,
Science of Synthesis, Georg Thieme Verlag, Stuttgart, Germany 2002, Vol. 12.2,
227-324, may be mentioned. The starting materials employed in the synthesis of
the
compounds of the formula I are commercially available or can be prepared
according
to procedures, or in analogy to procedures, described in the literature or
herein. As
examples of literature articles relating to synthetic procedures and
transformations
which can be used in the synthesis of the compounds of the formula I, the
following
may be mentioned:
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Brown et al., Bioorg. Med. Chem. Lett. 2010, 20, 679; Knochel et al., Chem.
Commun. 2009, 37, 5615; which relate to the formation of 1H-pyrazolo[3,4-
b]pyrazines from 2-acy1-3-chloro-pyrazines and hydrazine
US 2010/0029653; which relates to the formation of 1H-pyrazolo[3,4-b]pyrazines
from 2-alkyny1-3-chloro-pyrazines and hydrazine
Hajos et al., J. Org. Chem. 2008, 73, 3900; Maitte et al., J. Heterocycl.
Chem. 1983,
20, 1645; which relate to the formation of 1H-pyrazolo[3,4-b]pyrazines from 2-
acyl-
pyrazines and hydrazines
Stanovnik et al., Heterocycles 1982, 19, 339; Tisler et al., Monatshefte fOr
Chem ie,
1982, 113, 731; which relate to the formation of 3-acylamino-1H-pyrazolo[3,4-
b]pyrazines from 3-amino-241,2,4]oxadiazol-2-yl-pyrazines in the presence of a
base
Stanovnik et al., Heterocycles 1982, 19, 339; Tisler et al. Monatshefte fOr
Chem ie
1982, 113, 731; Augustynowicz-Kopec et al., Farmaco 2005, 60, 513; Otomasu et
al.,
Chem. Pharm. Bull. 1984, 32, 3361 ;which relate to the formation 3-amino-1H-
pyrazolo[3,4-b]pyrazines from 2-cyano-3-chloro-pyrazines and hydrazines
Guarneri et al., J. Heterocycl. Chem. 1986, 23, 585, which relates to the
transformation of 1H-6-oxa-1,2,4,7-tetraaza-inden-5-ones into 1H-pyrazolo[3,4-
b]pyrazines
US 2005/0070542; Sio et al., Farmaco Sci. 1982, 37, 116; Andaluz et al., J.
Heterocycl. Chem. 1989, 26, 949; Hofmann et al., Journal fuer Praktische
Chemie
1990, 332, 584; Townsend et al., Tetrahedron Lett. 2004, 45, 4105; which
relate to
the formation of 1H-pyrazolo[3,4-b]pyrazines by reaction of diamino-pyrazoles
and
amino-nitro-pyrazoles with 2-keto-carboxylic acids and 1,2-dicarbonyl
compounds.
In one synthetic approach for the preparation of compounds of the formula I, a
compound of the formula 11 and a compound of the formula 111 are reacted to
give a
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compound of the formula IV, which can already be the final compound of the
formula
I, or which is converted into the desired final compound of the formula I.
R1
R1
G1 VNNII
G3 (R2),
11101
G3
(R2), G4
G5 le G4 IV
5
More specifically, in particular in case the group R1 in the compound of the
formula I
is hydrogen or an optionally substituted alkyl group, according to this
approach a
compound of the formula II is obtained by reacting a compound of the formula V
with
1 0 a hydrazine of the formula VI, the obained compound of the formula II
and a
compound of the formula III are reacted to give a compound of the formula IV,
and
the compound of the formula IV converted into the compound of the formula I.
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R1 R1
Ncl H2N-NH-G3
1 1 N
G1 N G2 VI G1 71\r---NI
\
G3
V II
R1
N --
G5
Si
(R2),
1 ---.-*-----
N
I 4-
NN
\
G3 4 _____ 0 (R2),
G4 G4
IV III
In an alternative approach, a compound of the formula IV can be obtained by
first
reacting a compound of the formula V with a compound of the formula III to
give a
compound of the formula VII, and then reacting the compound of the formula VII
with
a hydrazine of the formula VI.
G5
R1 IS (R2), R1
G4
N
N (R2), 0 H2N-NH-G3
0
G1 N G2 1
NG2
IV
III 11101 VI
G4
V VII
1 0 In
another synthetic approach for the preparation of compounds of the formula I,
in
particular in case of compounds in which the group R1 is bonded via a nitrogen
atom
to the 1 H-pyrazolo[3,4-b]pyrazine ring system, specifically in case of the
preparation
of compounds in which R1 is an amino group, a compound of the formula X is
obtained by reacting a compound of the formula VIII with a hydrazine of the
formula
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VI, and the obtained compound of the formula IX and a compound of the formula
III
are reacted to give a compound of the formula X, which can already be the
final
compound of the formula I, or which is converted into the desired final
compound of
the formula I..
NH2
,N, ,CN H2N-NH-G3
NG2
G1 VI G1
G3
VIII IX
NH2 G5
(R2),
(R2),
I
G3
G
G4 4
X
In an alternative approach, a compound of the formula X can be obtained by
first
reacting a compound of the formula VIII with a compound of the formula III to
give a
1 0 compound of the formula XI, and then reacting the compound of the
formula XI with a
hydrazine of the formula VI.
G5
e
N G4
,CN (R2) N CN , H2N-NH-G3
G1 NG2
N G2 X
1110 VI
G4
VIII XI
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The groups R1 and R2 and the number n in the compounds of the formulae II,
III, IV,
V, VII, X and XI are defined as in the compounds of the formula I, and
additionally
can functional groups be present in protected form or in the form of a
precursor group
which is subsequently converted into the final group. The group G1 in the
compounds of the formulae II, V, VIII and IX is a leaving group which can be
replaced
in a Suzuki-type reaction or Stille-type reaction, such as a halogen atom, in
particular
bromine or chlorine, or a sulfonyloxy group, in particular
trifluoromethanesulfonyloxy,
methanesulfonyloxy, benzenesulfonyloxy or tosyloxy (4-
methylbenzenesulfonyloxy).
The group G2 in the compounds of formulae V, VII, VIII and XI can be identical
to or
1 0 different from the group G1 and is a leaving group, such as a halogen
atom, in
particular bromine or chlorine, or a sulfonyloxy group, in particular
trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy or
tosyloxy.
The group G3 in the compounds of formulae II, IV, VI, IX and X can be
hydrogen,
and in this case the compound of the formula VI thus be hydrazine, or it can
be a
1 5 protecting group which is suitable for protecting a ring nitrogen atom
in the 1 H-
pyrazolo[3,4-b]pyrazine ring system or similar ring systems such as the
pyrazole ring
system, for example, like a tetrahydropyran-2-y1 group, a tert-butoxycarbonyl
group,
an ethoxycarbonyl group, a benzyl group or a substituted benzyl group like a 4-
methoxybenzyl group or a 2,5-dimethoxybenzyl group. The group G4 in the
20 compounds of formulae III, IV, VII, X and XI can already be the desired
final
sulfonamide group of the formula Ar-S(0)2-NH-, in which Ar is defined as in
the
compounds of the formula I and additionally can functional groups be present
in
protected form or in the form of a precursor group which is subsequently
converted
into the final group. G4 can also be a group which can be converted into the
desired
25 final sulfonamide group of the formula Ar-S(0)2-NH- at an appropriate
stage of the
synthesis, for example in the compounds of the formulae IV and X, such as a
precursor group like a nitro group which can be reduced to an amino group, or
a
protected amino group like a tert-butoxycarbonylamino group or a
benzyloxycarbonylamino group which can be deprotected to an amino group, or a
30 free amino group, and the amino group then be converted into the group
Ar-S(0)2-NH- by reaction with a sulfonyl chloride under standard conditions.
The
group G5 in the compounds of formula 111 is a trialkylstannyl group, for
example a
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tri((Ci-C4)-alkyl)stannyl group, or a boronic acid group (-B(OH)2) or a
boronic acid
ester group or cyclic boronic acid ester group, for example a -B(0-(Ci-C4)-
alky1)2
group or a 4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-ylgroup, in particular a
boronic
acid group or a boronic acid ester group or cyclic boronic acid ester group,
which
allows performing a Suzuki-type reaction or Stille-type reaction for coupling
the
compounds of the formulae II, V, VIII and IX with the compounds of the formula
III.
The starting compounds in the synthesis of the compounds of the formula I can
also
be employed, and the intermediates obtained and/or employed, in the form of
salts,
for example acid addition salts in case of basic compounds. The intermediates
can
also be present in another tautomeric form, for example in the case of the
compounds of the formulae II or IX in which G3 is hydrogen, which can be
present in
the form of the respective 2H-pyrazolo[3,4-b]pyrazine derivatives in which the
mobile
hydrogen atom, which in the compound of the formula II is bonded to the ring
nitrogen atom in position 1 of the pyrazolo[3,4-b]pyrazine ring system, is
bonded to
the ring nitrogen atom in position 2 of the pyrazolo[3,4-b]pyrazine ring
system.
The reaction of compounds of the formulae V, VII, VIII and XI with a hydrazine
of the
formula VI is generally carried out in a protic or aprotic solvent such as
water, an
alcohol like methanol, ethanol, trifluoroethanol, n-propanol, isopropanol,
butanol,
isobutanol, tert-butanol, 2-methylbutan-2-ol, 3-methyl-3-pentanol, 3-ethyl-3-
pentanol,
a hydrocarbon like benzene, toluene, xylene, mesitylene, a nitrile like
acetonitrile, an
ether like tetrahydrofuran or diglyme (di(2-methoxyethyl) ether), an amide
like
dimethylformamide, N-methylpyrrolidinone, dimethylacetamide, a sulfoxide like
dimethylsulfoxide, or an amine like pyridine, or in a mixture of solvents, at
temperatures from about 20 C to about 200 C, for example at temperatures
from
about 80 C to about 120 C. The reaction time generally is from about 30
minutes to
about 48 hours, for example from about 5 hours to about 16 hours, depending on
the
particulars of the specific case and the chosen temperature range. Instead of
using
conventional heating, the reaction can also be carried out in a microwave oven
utilizing microwave radiation at temperatures from about 60 C to about 200
C, for
example at temperatures from about 80 C to about 120 C. In such case, the
reaction time generally is from about 5 minutes to about 12 hours, for example
from
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about 10 minutes to about 3 hours, depending on the particulars of the
specific case
and the chosen temperature range. The compound of the formula VI can be
employed in free form, i.e., not in the form of a salt, for example in the
form of a
solution in a solvent like ethanol or isopropanol, or in the form of an acid
addition salt,
5 for example in the form of a salt with hydrochloric acid. In case a salt
is employed, it
can be transformed into the free form prior to the reaction or in situ with an
organic or
inorganic base such as an amine like triethylamine, ethyldiisopropylamine, N-
methylmorpholine or 1,8-diazabicyclo[5.4.0]unde-7-ene, an alkoxide like sodium
methoxide, sodium ethoxide, potassium methoxide, potassium tert-butoxide, an
10 amide like lithium diisopropylamide or sodium amide, or an alkali metal
carbonate like
sodium carbonate, potassium carbonate or cesium carbonate, for example.
The reaction of compounds of the formulae II, V, VIII and IX with a compound
of the
formula III in which G5 is a boronic acid group or a boronic acidester group
or cyclic
15 boronic acid ester group, is a Suzuki-type reaction, and is generally
carried out in the
presence of catalytic palladium compound, for example a palladium(II) salt
like
palladium(II) acetate or palladium(II) chloride, which can be employed in the
presence of a phosphine such as 1,1'-bis(diphenylphosphino)ferrocene,
tricyclohexylphosphine or triphenylphosphine, or a palladium complex such as
20 tetrakis(triphenylphosphine)palladium(0), 1,1'-
bis(diphenylphosphino)ferrocene-
palladium(II)dichloride, palladium(0) bis(tri-tert-butylphosphin) or
bis(triphenylphosphine)palladium(II) chloride, and favorably in the presence
of a
base, for example an alkali metal carbonate or alkali metal phosphate like
cesium
carbonate, sodium carbonate or tripotassium phosphate, in an inert solvent,
such as
25 a hydrocarbon like benzene, toluene or xylene, or an ether like
tetrahydrofuran
(THF), dioxane or 1,2-dimethoxyethane (DME), or water, or a mixture of
solvents, at
temperatures from about 20 C to about 200 C, for example at temperatures
from
about 80 C to about 120 C. The reaction time generally is from about 30
minutes to
about 48 hours, for example from 30 minutes to about 16 hours, depending on
30 particulars of the specific case and the chosen temperature range.
Except for the use
of water as solvent, these explanations on the Suzuki-type reactions
substantially
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apply also to reactions with compounds of the formula III in which G5 is a
trialkylstannyl group, i.e. Stille-type reactions.
Further, in order to obtain the desired 1H-pyrazolo[3,4-b]pyrazine compound of
the
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72
G. Gudelsky, J. Nash, J. Med. Chem, 1994, 37, 4347; P. Lam, C. Clark, S.
Saubern,
J. Adams, M. Winters, D. Chan, A. Combs, Tetrahedron Lett., 1998, 39, 2941; D.
Chan, K. Monaco, R. Wang, M. Winters, Tetrahedron Lett. 1998, 39, 2933; V.
Farina,
V. Krishnamurthy, W. Scott, The Stille Reaction, Wiley, 1994; F. Qing et al.,
J. Chem.
Soc. Perkin Trans. I 1997, 3053; S. Buchwald et al. J. Am. Chem. Soc. 2001,
123,
7727; S. Kang et al. Synlett 2002, 3, 427; S. Buchwald et al., Organic Lett.
2002, 4,
581; T. Fuchikami et al., Tetrahedron Lett. 1991, 32, 91; Q. Chen et al.,
Tetrahedron
Lett. 1991, 32, 7689; M. R. Netherton, G. C. Fu, Topics in Organometallic
Chemistry
2005, 14, 85-108; A. F. Littke, G. F. Fu, Angew. Chem. Int. Ed. 2002, 41, 4176-
4211;
A. R. Muci, S. L. Buchwald, Topics in Current Chemistry 2002, 219, 131-209,
for
example. Nitro groups can be reduced to amino groups with various reducing
agents,
such as sulfides, dithionites, complex hydrides or by catalytic hydrogenation.
A
reduction of a nitro group may also be carried out simultaneously with a
reaction
performed on another functional group, for example when reacting a group like
a
cyano group with hydrogen sulfide or when hydrogenating a group. Amino groups
can then be modified according to standard procedures, for example alkylated
by
reaction with optionally substituted alkyl halogenides like chlorides,
bromides or
iodides or sulfonyloxy compounds like tosyloxy, mesyloxy or
trifluoromethylsulfonyloxy compounds, preferably in the presence of a base
like
potassium carbonate, cesium carbonate, sodium hydride or potassium tert-
butoxide,
or by reductive amination of carbonyl compounds, or acylated by reaction with
activated carboxylic acid derivatives such as acid chlorides, anhydrides,
activated
esters or others or by reaction with carboxylic acids in the presence of an
activating
agent, or sulfonylated by reaction with sulfonyl chlorides. Ester groups can
be
hydrolyzed to the corresponding carboxylic acids which after activation can
then be
reacted with amines under standard conditions. Furthermore, ester or acid
groups
can be reduced to the corresponding alcohols by many standard procedures, and
the
resulting hydroxy compounds alkylated. Ether groups, for example benzyloxy
groups
or other easily cleavable ether groups, can be cleaved to give hydroxy groups
which
can then be reacted with a variety of agents, for example etherification
agents or
activating agents allowing replacement of the hydroxy group by other groups. A
hydroxy group can also be converted into a leaving group and reacted with
various
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73
reaction partners under the well-known conditions of the Mitsunobu reaction
(O.
Mitsunobu, Synthesis 1981, 1), or by further procedures (cf. A. Tunoori, D.
Dutta, G.
Gunda, Tetrahedron Lett. 39 (1998) 8751; J. Pelletier, S. Kincaid, Tetrahedron
Lett.
41 (2000) 797; D. L.Hughes, R. A.Reamer, J. J.Bergan, E. J. J.Grabowski, J.
Am.
Chem. Soc. 110 (1998) 6487; D. J. Camp, I. D. Jenkins, J. Org. Chem. 54 (1989)
3045; D. Crich, H. Dyker, R. J. Harris, J. Org. Chem. 54 (1989) 257).
The mentioned reactions for the conversion of functional groups are, in
general,
extensively described in textbooks of organic chemistry like M. Smith, J.
March,
March's Advanced Organic Chemistry, Wiley-VCH, 2001, and in Houben-Weyl,
"Methoden der Organischen Chemie" (Methods of Organic Chemistry), Georg
Thieme Verlag, Stuttgart, Germany; "Organic Reactions", John Wiley & Sons, New
York; R. C. Larock, " Comprehensive Organic Transformations", Wiley-VCH, 2nd
ed
(1999); B. Trost, I. Fleming (eds.) Comprehensive Organic Synthesis,
Pergamon,1991; A. Katritzky, C. Rees, E. Scriven Comprehensive Heterocyclic
Chemistry II, Elsevier Science, 1996; for example, in which details on the
reactions
and primary source literature can be found. Due to the fact that in the
present case
the functional groups occur in 1H-pyrazolo[3,4-b]pyrazine compounds, it may in
certain cases become necessary to specifically adapt reaction conditions or
choose
specific reagents from a variety of reagents that can in principle be employed
in a
conversion reaction, or otherwise take specific measures for achieving a
desired
conversion, for example to use protection group techniques, as applies in
general
and is known to the person skilled in the art.
In the course of the preparation of the compounds of the formula I it can
generally be
advantageous or necessary in order to reduce or prevent undesired reactions or
side
reactions in the respective synthesis steps, to block functional groups
temporarily by
protecting groups suited to the specific synthesis problem, or to have them
present,
or introduce them, in the form of precursor groups, and later convert them
into the
desired functional groups. Such strategies are well known to a person skilled
in the
art and are described, for example, in Greene and Wuts, Protective Groups in
Organic Synthesis, Wiley, 1991, or P. Kocienski, Protecting Groups, Thieme
1994.
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Examples of precursor groups are cyano groups and nitro groups. The cyano
group
can, in a later step, be transformed by hydrolysis into carboxylic acid
derivatives or
by reduction into aminomethyl groups. Nitro groups can be transformed by
reduction
like catalytic hydrogenation into amino groups. Examples of protective groups
which
may be mentioned, are benzyl protective groups, for example benzyl ethers of
hydroxy compounds and benzyl esters of carboxylic acids, from which the benzyl
group can be removed by catalytic hydrogenation in the presence of a palladium
catalyst, tert-butyl protective groups, for example tert-butyl esters of
carboxylic acids,
from which the tert-butyl group can be removed by treatment with
trifluoroacetic acid,
acyl protective groups, for example ester and amides of hydroxy compounds and
amino compounds, which can be cleaved again by acidic or basic hydrolysis, or
alkoxycarbonyl protective groups, for example tert-butoxycarbonyl derivatives
of
amino compounds, which can be cleaved again by treatment with trifluoroacetic
acid.
Compounds of the formula I can also be prepared by solid phase techniques. In
such
a synthetic approach, the solid phase may also be regarded as having the
meaning
of a protecting group, and cleavage from the solid phase as removal of the
protective
group. The use of such techniques is known to a person skilled in the art (cf.
Burgess
K (Ed.), Solid Phase Organic Synthesis, New York, Wiley, 2000). For example, a
phenolic hydroxy group can be attached to a trityl-polystyrene resin, which
serves as
a protecting group, and the molecule cleaved from the resin by treatment with
trifluoroacetic acid or another acid at a later stage of the synthesis.
As is usual and applies to all reactions performed in the course of the
synthesis of a
compound of the formula I, appropriate details of the conditions applied in a
specific
preparation process, including the solvent, a base or acid, the temperature,
the order
of addition, the molar ratios and other parameters, are routinely chosen by
the skilled
person in view of the characteristics of the starting compounds and the target
compound and the other particularities of the specific case. As is also known
by the
skilled person, not all processes described herein will in the same way be
suitable for
the preparation of all compounds of the formula I and their intermediates, and
adaptations have to be made. In all processes for the preparation of the
compounds
of the formula I, workup of the reaction mixture and the purification of the
product is
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performed according to customary methods known to the skilled person which
include, for example, quenching of a reaction mixture with water, adjustment
of a
certain pH, precipitation, extraction, drying, concentration, crystallization,
distillation
and chromatography. As further examples of methods applicable in the synthesis
of
5 the compounds of the formula I, microwave assistance for speeding-up,
facilitating or
enabling reactions, as described by P. Lidstrom, J. Tierney, B. Wathey, J.
Westman,
Tetrahedron, 57(2001), 9225, for example, may be mentioned, and modern
separation techniques like preparative high pressure liquid chromatography
(HPLC),
which can be used for separating mixtures of positional isomers which may
occur in
10 any reactions. Also for the characterization of the products customary
methods are
used such as NMR, IR and mass spectroscopy.
Another subject of the present invention are the novel starting compounds and
intermediates occurring in the synthesis of the compounds of the formula I,
including
15 the compounds of the formulae II, III, IV, V, VII, VIII, IX, X and XI,
wherein the groups
R1, R2, G1, G2, G3, G4 and G5 and the number n are defined as above, in any of
their stereoisomeric forms or a mixture of stereoisomeric forms in any ratio,
and their
salts, and their use as synthetic intermediates or starting compounds. All
general
explanations, specifications of embodiments and definitions of numbers and
groups
20 given above with respect to the compounds of the formula I apply
correspondingly to
the said intermediates and starting compounds. A subject of the invention are
in
particular the novel specific starting compounds and intermediates described
herein.
Independently thereof whether they are described as a free compound and/or as
a
specific salt, they are a subject of the invention both in the form of the
free
25 compounds and in the form of their salts, and if a specific salt is
described,
additionally in the form of this specific salt.
The compounds of the present invention are SGK inhibitors, which are capable
of
inhibiting an exaggerated, or inappropriate, activity of SGK in pathological
conditions
30 and are therefore suitable for the prophylaxis and therapy of the
diseases mentioned
above and below. In particular, they are highly active inhibitors of the SGK-1
enzyme.
They are selective SGK-1 inhibitors inasmuch as they do not substantially
inhibit or
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promote the activity of other enzymes and receptors whose activation or
inhibition is
not desired. The activity of the compounds of the formula I can be determined,
for
example, in the assays described below or in other in vitro, ex vivo or in
vivo assays
known to the person skilled in the art. For example, the ability of the
compounds to
inhibit the SGK enzyme may be measured by methods similar to those described
in
D. Perrin et al., Expert Opin. Drug Discov. (2010) 5, 51-63, and by the assay
described below. With respect to SGK-1 inhibitory activity, one embodiment of
the
invention comprises compounds which have an 1050 value of < 1 pM, in another
embodiment of < 0.1 pM, in another embodiment of < 0.01 pM, for SGK-1
inhibition
as determined in the assay described below, and which in a further embodiment
do
not substantially influence the activity of other enzymes and receptors whose
inhibition or activation is not desired. The ability of the compounds to
inhibit the SGK-
1 mediated glycogen synthase kinase 3beta (GSK3beta) phosphorylation in a
cellular
setting may be measured by methods similar to those described by H. Sakoda et
al.
J. Biol. Chem. 2003, 278, 25802-25807, and by the method described below. The
ability of the compounds to inhibit SGK1 dependent activation of epithelial
Na+
channel (ENaC) currents in cell monolayers may be measured by methods similar
to
those described by D. Alvarez de la Rosa et al., Am. J. Physiol. Cell Physiol.
284:404-414, 2003, D. Alvarez de la Rosa et al.; J. Gen. Physiol. 2004 Oct;
124(4):395-407, and by the assay described below. The inappropriate SGK-1
activity
referred to herein is any SGK-1 activity that deviates from the expected
normal SGK-
1 activity. Inappropriate SGK-1 activity may take the form of, for example, an
abnormal increase in activity, or an aberration in the timing and/or control
of SGK-1
activity. Such inappropriate activity may result then, for example, from
overexpression or mutation of the protein kinase leading to inappropriate or
uncontrolled activation. As SGK-1 inhibitors, the compounds of the formula 1
and their
pharmaceutically acceptable salts are generally suitable for the prophylaxis
and/or
therapy of conditions in which the inappropriate activity of SGK-1 enzyme
plays a
role or has an undesired extent, or which can favorably be influenced by
inhibiting the
SGK-1 enzyme or decreasing the activity, or for the prevention, alleviation or
cure of
which an inhibition of SGK-1 or a decrease in the activity is desired by the
physician.
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Because of their pharmacological properties, the compounds of the present
invention
are suitable for the treatment of all disorders in the progression of which an
enhanced activity of SGK enzyme is involved. These include the indications
described in the introduction. The invention relates in particular to the use
of a
compound of the formula I or a pharmaceutically acceptable salt thereof for
the
treatment of degenerative joint disorders and degenerative cartilage changes
including osteoarthritis, osteoarthrosis, rheumatoid arthritis, spondylosis,
chondrolysis following joint trauma and prolonged joint immobilization after
meniscus
or patella injuries or ligament tears, connective tissue disorders such as
collagenoses, periodontal disorders, wound-healing disturbances, diabetes
including
diabetes mellitus, diabetic nephropathy, diabetic neuropathy, diabetic
angiopathy and
microangiopathy, obesity, metabolic syndrome (dyslipidaemia), systemic and
pulmonary hypertension, cerebral infarctions, cardiovascular diseases
including
cardiac fibrosis after myocardial infarction, cardiac hypertrophy and heart
failure,
arteriosclerosis, renal diseases including glomerulosclerosis,
nephrosclerosis,
nephritis, nephropathy and electrolyte excretion disorder, any type of
fibrosis and
inflammatory processes including liver cirrhosis, lung fibrosis, fibrosing
pancreatitis,
rheumatism, arthritis, gout, Crohn's disease, chronic bronchitis, radiation
fibrosis,
sclerodermatitis, cystic fibrosis, scar formation, Alzheimer's disease, pain
including
acute pain like pain following injuries, post-operative pain, pain in
association with an
acute attack of gout and acute pain following jaw-bone surgery interventions,
and
chronic pain like pain associated with chronic musculoskeletal diseases, back
pain,
pain associated with osteoarthritis or rheumatoid arthritis, pain associated
with
inflammation, amputation pain, pain associated with multiple sclerosis, pain
associated with neuritis, pain associated with carcinomas and sarcomas, pain
associated with AIDS, pain associated with chemotherapy, trigeminus neuralgia,
headache, migraine cephalalgia, neuropathic pains, post-herpes zoster
neuralgia,
chronic disorders of the locomotor system such as inflammatory,
immunologically or
metabolically related acute and chronic arthritides, arthropathies, myalgias
and
disturbances of bone metabolism, peptic ulcers, especially in forms that are
triggered
by stress, tinnitus, bacterial infections, glaucoma, cataracts, coagulopathies
including
dysfibrinogenaemia, hypoproconvertinaemia, haemophilia B, Stuart-Prower
defect,
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prothrombin complex deficiency, consumption coagulopathy, fibrinolysis,
immunokoagulopathy or complex coagulopathies, and to the use in tumor therapy
including inhibition of tumor growth and tumor metastases, the use in anti-
infective
therapy, the use for increasing the learning ability and attention, the use
for
counteracting cellular aging and stress and thus increasing life expectancy
and
fitness in the elderly, and the use in states of neuronal excitability
including epilepsy.
The treatment of diseases is to be understood herein as generally meaning both
the
therapy of existing pathological changes or malfunctions of the organism or of
existing symptoms with the aim of relief, alleviation or cure, and the
prophylaxis or
prevention of pathological changes or malfunctions of the organism or of
symptoms
in humans or animals which are susceptible thereto and are in need of such a
prophylaxis or prevention, with the aim of a prevention or suppression of
their
occurrence or of an attenuation in the case of their occurrence. For example,
in
patients who on account of their disease history are susceptible to myocardial
infarction, by means of the prophylactic or preventive medicinal treatment the
occurrence or re-occurrence of a myocardial infarct can be prevented or its
extent
and sequelae decreased. The treatment of diseases can occur both in acute
cases
and in chronic cases.
The compounds of the formula I and their pharmaceutically acceptable salts can
therefore be used in animals, in particular in mammals and specifically in
humans, as
a pharmaceutical or medicament on their own, in mixtures with one another, or
in the
form of pharmaceutical compositions. A subject of the present invention also
are the
compounds of the formula I and their pharmaceutically acceptable salts for use
as a
pharmaceutical. A subject of the present invention also are pharmaceutical
compositions and medicaments which comprise at least one compound of the
formula I and/or a pharmaceutically acceptable salt thereof as an active
ingredient, in
an effective dose for the desired use, and a pharmaceutically acceptable
carrier, i.e.
one or more pharmaceutically innocuous, or nonhazardous, vehicles and/or
excipients, and optionally one or more other pharmaceutical active compounds.
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A subject of the present invention also are the compounds of the formula I and
their
pharmaceutically acceptable salts for use in the treatment of the diseases
mentioned
above or below, including the treatment of any one of the mentioned diseases,
for
example the treatment of degenerative joint disorders, degenerative cartilage
changes, diabetes, cardiovascular diseases, fibrosis, inflammatory processes,
pain,
tumors or cerebral infarctions, wherein treatment of diseases comprises their
therapy
and prophylaxis as mentioned above, or for use as an inhibitor of serum and
glucocorticoid regulated kinase (SGK). A subject of the present invention also
are the
use of the compounds of the formula I and their pharmaceutically acceptable
salts for
the manufacture of a medicament for the treatment of the diseases mentioned
above
or below, including the treatment of any one of the mentioned diseases, for
example
inhibitor the treatment of degenerative joint disorders, degenerative
cartilage
changes, diabetes, cardiovascular diseases, fibrosis, inflammatory processes,
pain,
tumors or cerebral infarctions, wherein treatment of diseases comprises their
therapy
and prophylaxis as mentioned above, or a medicament for inhibition of serum
and
glucocorticoid regulated kinase (SGK). A subject of the present invention also
are
methods for the treatment of the diseases mentioned above or below, including
the
treatment of any one of the mentioned diseases, for example the treatment of
degenerative joint disorders, degenerative cartilage changes, diabetes,
cardiovascular diseases, fibrosis, inflammatory processes, pain, tumors or
cerebral
infarctions, wherein treatment of diseases comprises their therapy and
prophylaxis as
mentioned above, and a method for inhibiting serum and glucocorticoid
regulated
kinase (SGK), which comprise administering an efficacious amount of at least
one
compound of the formula I and/or a pharmaceutically acceptable salt thereof to
a
human or an animal which is in need thereof.
The compounds of the formula I and their pharmaceutically acceptable salts,
and
pharmaceutical compositions and medicaments comprising them, can be
administered enterally, for example by oral or rectal administration in the
form of pills,
tablets, lacquered tablets, coated tablets, granules, hard and soft gelatin
capsules,
solutions, syrups, emulsions, suspensions, aerosol mixtures or suppositories,
or
parenterally. Parenteral administration can be carried out, for example,
intravenously,
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intraarticularly, intraperitoneally, intramuscularly or subcutaneously, in the
form of
injection solutions or infusion solutions, microcapsules, implants or rods, or
percutaneously, transdermally or topically, for example in the form of
ointments,
solutions or tinctures, or in other ways, for example in the form of aerosols
or nasal
5 sprays. The preferred form of administration depends on the particulars
of the
specific case.
Pharmaceutical formulations adapted for transdermal administration can be
administered as plasters for extended, close contact with the epidermis of the
10 recipient. For topical administration, formulations such as ointments,
creams,
suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils can
be used. For the treatment of the eye or other external tissue, for example
mouth and
skin, suitable formulations are topical ointments or creams, for example. In
the case
of ointments, the active ingredient can be employed either with a paraffinic
or a
15 water-miscible cream base. Alternatively, the active ingredient can be
formulated to
give a cream with an oil-in-water cream base or a water-in-oil base.
Pharmaceutical
formulations adapted for topical application to the eye include eye drops, in
which the
active ingredient is dissolved or suspended in a suitable carrier, in
particular an
aqueous solvent.
The pharmaceutical compositions according to the invention are prepared in a
manner known per se and familiar to the person skilled in the art by admixing
one ore
more pharmaceutically acceptable inert inorganic and/or organic vehicles and
excipients with one or more compounds of the formula I and/or pharmaceutically
acceptable salts thereof, and bringing them into a suitable form for dosage
and
administration, which can then be used in human medicine or veterinary
medicine.
For the production of pills, tablets, coated tablets and hard gelatin capsules
it is
possible to use, for example, lactose, cornstarch or derivatives thereof,
talc, stearic
acid or its salts. For the production of gelatin capsules and suppositories
fats, waxes,
semisolid and liquid polyols, natural or hardened oils, for example, can be
used. For
the production of solutions, for example injection solutions, or of emulsions
or syrups
water, saline, alcohols, glycerol, polyols, sucrose, invert sugar, glucose,
vegetable
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oils, for example, can be used, and for the production of microcapsules,
implants or
rods copolymers of glycolic acid and lactic acid, for example, can be used.
The
pharmaceutical compositions normally contain from about 0.5 % to 90 % by
weight of
the compounds of the formula I and/or their pharmaceutically acceptable salts.
The
amount of the active ingredient of the formula I and/or its pharmaceutically
acceptable salts in the pharmaceutical compositions normally is from about 0.5
mg to
about 1000 mg, preferably from about 1 mg to about 500 mg per unit dose.
Depending on the kind of the pharmaceutical composition and other particulars
of the
specific case, the amount may deviate from the indicated ones
In addition to the active ingredients of the formula I and/or their
pharmaceutically
acceptable salts and to vehicles, or carrier substances, the pharmaceutical
compositions can contain excipients, or auxiliaries or additives, such as, for
example,
fillers, disintegrants, binders, lubricants, wetting agents, stabilizers,
emulsifiers,
preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners,
diluents,
buffer substances, solvents, solubilizers, agents for achieving a depot
effect, salts for
altering the osmotic pressure, coating agents or antioxidants. They can also
contain
two or more compounds of the formula I, and/or their pharmaceutically
acceptable
salts. In case a pharmaceutical composition contains two or more compounds of
the
formula I, the selection of the individual compounds can aim at a specific
overall
pharmacological profile of the pharmaceutical composition. For example, a
highly
potent compound with a shorter duration of action may be combined with a long-
acting compound of lower potency. The flexibility permitted with respect to
the choice
of substituents in the compounds of the formula I allows a great deal of
control over
the biological and physico-chemical properties of the compounds and thus
allows the
selection of such desired compounds.
When using the compounds of the formula I, the dose can vary within wide
limits and,
as is customary and is known to the physician, is to be suited to the
individual
conditions in each individual case. It depends, for example, on the specific
compound
employed, on the nature and severity of the disease to be treated, on the mode
and
the schedule of administration, or on whether an acute or chronic condition is
treated
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or whether prophylaxis is carried out. An appropriate dosage can be
established
using clinical approaches known to the person skilled in the art. In general,
the daily
dose for achieving the desired results in an adult weighing about 75 kg is
from about
0.01 mg/kg to about 100 mg/kg, preferably from about 0.1 mg/kg to about 50
mg/kg,
in particular from about 0.1 mg/kg to about 10 mg/kg, in each case in mg per
kg of
body weight. The daily dose can be divided, in particular in the case of the
administration of relatively large amounts, into several, for example 2, 3 or
4, part
administrations. As usual, depending on individual behavior it may be
necessary to
deviate upwards or downwards from the daily dose indicated.
The compounds of the present invention are also useful as standard or
reference
compounds, for example as a quality standard or control, in tests or assays
involving
the inhibition of the SGK enzyme. For such use, for example in pharmaceutical
research involving the SGK enzyme, the compounds may be provided in a
commercial kit. For example, a compound of the present invention can be used
as a
reference in an assay to compare its known activity to a compound with an
unknown
activity. Furthermore, the compounds of the formula I can be used as synthesis
intermediates for the preparation of other compounds, in particular of other
pharmaceutical active compounds, which may be obtained from the compounds of
the formula I by introduction of substituents or modification of functional
groups, for
example.
The following examples illustrate the present invention.
Examples
When in the final step of the synthesis of an example compound an acid such as
trifluoroacetic acid or acetic acid was used, for example when trifluoroacetic
acid was
employed to remove an acid-labile protecting group containing a tert-butyl
group, or
when a compound was purified by chromatography using an eluent which contained
such an acid, in some cases, depending on the work-up procedure, for example
the
details of a freeze-drying process, the compound was obtained partially or
completely
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in the form of a salt of the acid used, for example in the form of the salt
with acetic
acid salt or trifluoroacetic acid salt. In the names of the example compounds
and the
structural formulae such contained trifluoroacetic acid or acetic acid is not
specified.
The prepared compounds were in general characterized by spectroscopic data and
chromatographic data, in particular mass spectra (MS) and/or nuclear magnetic
resonance (NMR) spectra. In the NMR characterization, the chemical shift 8 (in
ppm),
the number of hydrogen atoms (H), the coupling constant J (in Hz) and the
multiplicity (s: singlet, d: doublet, dd: double doublet, t: triplet, m:
multiplet; br: broad)
of the peaks are given. In the MS characterization, the mass number (m/e) of
the
peak of the molecular ion (M) or of a related ion such as the ion (M+1), i.e.
the
protonated molecular ion (M+H), or the ion (M-1), which was formed depending
on
the ionization method used, is given. Generally, the ionization method was
electrospray ionization (ES I+ or ES-).
Abbreviations
DCM Dichloromethane
dioxane [1,4]Dioxane
DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
Et0Ac Ethyl acetate
iPrOH Isopropanol
MeCN Acetonitrile
RT Room temperature (20 C to 25 C)
TFA Trifluoroacetic acid
Example 1: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,3-dichloro-
benzenesulfonamide
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CI 0
CI S
=\HI _ NNH2
N¨
FI
,N
(i) 2,3-Dichloro-N44-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-y1)-phenyl]-
benzenesulfonamide
(ii) 2,3-Dichloro-N-[4-(6-chloro-5-cyano-pyrazin-2-
yl)phenyl]benzenesulfonamide
20 2,3-Dichloro-N44-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-y1)-phenyl]-
benzenesulfonamide (5.78 g) was added to a reaction vessel containing a
magnetic
stirring bar together with 3,5-dichloro-pyrazine-2-carbonitrile (2.35 g), 1,1'-
bis(diphenylphosphino)ferrocene-palladium(11) dichloride (Pd(dppf)2Cl2) (791
mg) and
cesium carbonate (13.2 g), followed by 100 ml dioxane and 10 ml water, and the
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oil. Purification by flash chromatography on silica gel using a mixture of
Et0Ac and
heptane as the eluent afforded 2,3-dichloro-N44-(6-chloro-5-cyano-pyrazin-2y1)-
phenyl]-benzenesulfonamide as a light brown foam after evaporation of the
solvents
under reduced pressure. Yield: 4.32 g (73%).
5 MS (ES-): m/e = 436.0 (M-H).
(iii) N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,3-dichloro-
benzenesulfonamide
2,3-Dichloro-N44-(6-chloro-5-cyano-pyrazin-2y1)-phenyl]-benzenesulfonamide
(1.0 g)
10 was suspended in a mixture of 5 ml iPrOH and 5 ml 35% hydrazine in water
at RT
and heated to 120 C by microwave irradiation for 20 min under stirring in a
sealed
vessel. The reaction mixture was left to cool to RT. The precipitate was
filtered off
and washed with water to give the title compound as a yellow solid after
drying under
vacuum. Yield: 536 mg (54%).
15 1H-NMR (DMSO-d6): 8 (ppm) = 5.67 (br s, 2H), 7.26 (d, J = 8.8 Hz, 2H),
7.58 (t, J =
8.0 Hz, 1H), 7.93 (dd, J = 1.4, 8.0 Hz, 1H), 8.06 (d, J = 8.8 Hz, 2H), 8.11
(dd, J = 1.5,
8.0 Hz, 1H), 8.86 (s, 1H), 12.30 (s,1H).
MS (ES+): m/e = 435.2 (M+H), chloro pattern.
20 Example 2: 2,5-Dichloro-N-[4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide hydrochloride
CI 0
0
S'
1401 \FIN 40
N=c 11\1
25 (i) 2,5-Dichloro-N44-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-y1)-
phenyl]-
benzenesulfonamide
To a solution of 10 g of 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yI)-
phenylamine
in 100 ml DCM and 4 ml pyridine, 11.6 g of 2,5-dichloro-benzenesulfonyl
chloride
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were added, and the reaction mixture was stirred for 16 h at RT. Then, the
solvents
were removed under reduced pressure and the crude product was purified by
chromatography on silica gel eluting with a gradient of n-heptane/Et0Ac. The
fractions containing the product were combined and the solvent evaporated
under
reduced pressure. Yield: 17.9 g.
(ii) 2,5-Dichloro-N44-(6-chloro-5-formyl-pyrazin-2-y1)-phenyl]-
benzenesulfonamide
A solution of 100 mg of 3,5-dichloro-pyrazine-2-carbaldehyde, 241 mg 2,5-
dichloro-
N44-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-y1)-phenyl]-benzenesulfonamide
and
552 mg of cesium carbonate in 3.4 ml of dioxane and 0.6 ml of water was purged
with argon. Then, 33 mg of 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)
dichloride were added and the reaction mixture was heated to 100 C. After 40
min,
the reaction mixture was cooled to RT and diluted with water. After filtration
through a
chem elut cartridge by eluting with Et0Ac, the solvents were removed under
reduced pressure. The crude product was purified by chromatography on silica
gel
eluting with a gradient of n-heptane/Et0Ac and finally methanol. The fractions
containing the product were combined and the solvent evaporated under reduced
pressure. Yield: 90 mg.
(iii) 2,5-Dichloro-N-[4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
To a solution of 90 mg of 2,5-dichloro-N44-(6-chloro-5-formyl-pyrazin-2-y1)-
phenyl]-
benzenesulfonamide in 0.7 ml isopropanol 0.7 ml of a hydrazine solution (35%
in
isopropanol) were added and the reaction mixture was heated for 20 min to 120
C
by using microwave irradiation (Biotage lnitiatorTM apparatus). The reaction
mixture
was cooled to RT and diluted with acetic acid (20%). The precipitated product
was
collected by filtration and purified by preparative HPLC (C18 reversed phase
column,
elution with a water/MeCN gradient with 0.1 A TFA). The fractions containing
the
product were lyophilized to yield the title compound in the form of its salt
with
trifluoroacetic acid as a solid, which was dissolved in 1 ml of a
water/acetonitrile
mixture. 0.5 ml of a 1 M aqueous hydrochloric acid was added and the solution
was
again lyophilized to yield the title compound in the form of 2,5-dichloro-N-[4-
(1H-
pyrazolo[3,4-b]pyrazin-6-yl)phenyl]benzenesulfonamide hydrochloride. Yield:
5.2 mg.
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MS(ES+): m/e = 420.2 (M+H), chloro pattern.
Example 3: 2,5-Dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide hydrochloride
CI 0
0
Sc
NH
,N
CI
(i) 2,5-Dichloro-N42-fluoro-4-(4,4,5,5-tetramethy1-1,3,2-dioxaborolan-2-
yl)phenyl]benzenesulfonamide
To a solution of 1.5 g of 2-fluoro-4-(4,4,5,5-tetramethy1-1,3,2-dioxaborolan-2-
yl)aniline in 17 ml DCM and 0.5 ml pyridine, 1.5 g of 2,5-dichloro-
benzenesulfonyl
chloride were added, and the reaction mixture was stirred for 16 h at RT. Then
the
solvents were removed under reduced pressure and the crude product was
purified
by chromatography on silica gel eluting with a gradient of n-heptane/Et0Ac.
The
fractions containing the product were combined and the solvent evaporated
under
reduced pressure. Yield: 2.2 g.
(ii) 2,5-Dichloro-N-[4-(6-chloro-5-formyl-pyrazin-2-yI)-2-fluoro-
phenyl]benzenesulfonamide
A solution of 100 mg of 3,5-dichloro-pyrazine-2-carbaldehyde, 252 mg 2,5-
dichloro-
N42-fluoro-4-(4,4,5,5-tetramethy1-1,3,2-dioxaborolan-2-
yl)phenyl]benzenesulfonamide and 552 mg of cesium carbonate in 3.4 ml of
dioxane
and 0.6 ml of water was purged with argon. Then, 33 mg of 1,1'-
bis(diphenylphosphino)ferrocene-palladium(11)dichloride were added and the
reaction
mixture was heated to 100 C. After 6 h the reaction mixture was cooled to RT
and
diluted with water. After filtration through a chem elut cartridge by eluting
with
Et0Ac the solvents were removed under reduced pressure. The crude product was
purified by chromatography on silica gel eluting with a gradient of n-
heptane/Et0Ac
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and finally methanol. The fractions containing the product were combined and
the
solvent evaporated under reduced pressure. Yield: 180 mg.
(iii) 2,5-Dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
To a solution of 120 mg of 2,5-dichloro-N-[4-(6-chloro-5-formyl-pyrazin-2-yI)-
2-fluoro-
phenyl]benzenesulfonamide in 0.9 ml isopropanol 0.9 ml of a hydrazine solution
(35% in isopropanol) were added and the reaction mixture was heated for 20 min
to
120 C by using microwave irradiation (Biotage lnitiatorTM apparatus). The
reaction
mixture was cooled to RT and diluted with acetic acid (20%). The precipitated
product was collected by filtration. The crude product was purified by
preparative
HPLC (C18 reversed phase column, elution with a water/MeCN gradient with 0.1 A
TFA). The fractions containing the product were lyophilized to yield the pure
product
in the form of its salt with trifluoroacetic acid as a solid, which was
dissolved in 1 ml
of a water/acetonitrile mixture. 0.5 ml of a 1 M aqueous hydrochloric acid was
added
and the solution was again lyophilized to yield the title compound in the form
of 2,5-
dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]benzenesulfonam
ide
hydrochloride. Yield: 2.2 mg.
MS(ES+): m/e = 438.2 (M+H), chloro pattern.
Example 4: 2,3-Dichloro-N-[2-fluoro-4-(1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-
benzenesulfonamide
CI 0
Cl S'
=1\11
N¨
NI,N
The title compound was prepared by adapting the procedures described in
example
3, employing 2,3-dichlorobenzenesulfonyl chloride used instead of 2,5-dichloro-
benzenesulfonyl chloride.
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MS (ES+): m/e = 438.1 (M+H), chloro pattern.
Example 5: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,5-dichloro-
benzenesulfonamide
CI 0
c
S 40, N
NH2
N¨
CI
NFI
The title compound was prepared in 22% yield according to the procedure
described
in example 1, employing 2,5-dichloro-benzenesulfonyl chloride instead of 2,3-
dichloro-benzenesulfonyl chloride as starting material. The following
modification was
made. The crude reaction mixture was evaporated to dryness, redissolved in DMF
and purified by preparative HPLC (C18 reversed phase column, elution with a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
1H-NMR (DMSO-d6): 8 (ppm) = 7.28 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.6 Hz,
1H),
7.75 (dd, J = 2.5, 8.6 Hz, 1H), 8.08 (d, J = 2.5 Hz, 1H), 8.09 (d, J = 8.8 Hz,
2H), 8.89
(s, 1H), 11.13 (s,1H).
MS (ES+): m/e = 434.9 (M+H), chloro pattern.
Example 6: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-phenyl]-5-
chloro-
2-fluoro-benzenesulfonamide
F
Sc
CI
NNH2
N¨
F H
N
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The title compound was prepared in 6% yield according to the procedure
described
in example 1, employing 5-chloro-2-fluoro-benzenesulfonyl chloride instead of
2,3-
dichloro-benzenesulfonyl chloride and 2-fluoro-4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-y1)-phenylamine instead of 4-(4,4,5,5-tetramethyl-
5 [1,3,2]dioxaborolan-2-yI)-phenylamine as starting material. The following
modification
was made. The crude reaction mixture was evaporated to dryness, redissolved in
DMF and purified by preparative HPLC (C18 reversed phase column, elution with
a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
10 MS (ES+): m/e = 437.0 (M+H), chloro pattern.
Example 7: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-pheny1]-2,5-
dichloro-benzenesulfonamide
CI 0
Sc 40, z N
NH2
N¨
N,N
15 CI
The title compound was prepared in 5% yield according to the procedures
described
in example 1, employing 2,5-dichloro-benzenesulfonyl chloride instead of 2,3-
dichloro-benzenesulfonyl chloride and 2-fluoro-4-(4,4,5,5-tetramethyl-
20 [1,3,2]dioxaborolan-2-yI)-phenylamine instead of 4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-y1)-phenylamine as starting material. The following
modification
was made. The crude reaction mixture was evaporated to dryness, redissolved in
DMF and purified by preparative HPLC (C18 reversed phase column, elution with
a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
25 lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
MS (ES+): m/e = 452.9 (M+H), chloro pattern.
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Example 8: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-chloro-2-
hydrazino-benzenesulfonamide
H2N,
NH 0
= S(N =N
NH2
N¨
NFI
CI
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-5-chloro-2-fluoro-benzenesulfonamide.
MS (ES+): m/e = 431.0 (M+H), chloro pattern.
Example 9: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-2-fluoro-phenyl]-2,3-
dichloro-benzenesulfonamide
CI 0
CI is S(
F
_N H2
N¨
H
NN
The title compound was prepared in 4% yield according to the procedure
described
in example 1, employing 2-fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-
yI)-
phenylamine instead of 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yI)-
phenylamine
as starting material. The following modification was made. The crude reaction
mixture was evaporated to dryness, redissolved in DMF and purified by
preparative
HPLC (C18 reversed phase column, elution with a water/MeCN gradient with 0.1 A
TFA). The fractions containing the product were lyophilized to yield the title
compound in the form of its salt with trifluoroacetic acid.
MS (ES+): m/e = 452.9 (M+H), chloro pattern.
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Example 10: 2,3-Dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
CI 0
CI S
= 1\11 _CH3
N¨
N,N
(i) 1-(3,5-Dichloro-pyrazin-2-yI)-ethanol
3,5-Dichloro-pyrazine-2-carbaldehyde (5.0 g) was dissolved in dry
tetrahydrofuran
(100 ml) in a reaction vessel equipped with a magnetic stirring bar under an
argon
atmosphere. The solution was cooled on an ice-bath before slow addition of
10.3 ml
methylmagnesium bromide solution (3M in tetrahydrofuran), keeping the internal
temperature in the reaction vessel below 5 C. After the addition the cooling
bath was
removed and the reaction mixture stirred for another 10 min. Then the reaction
mixture was quenched with a saturated aqueous sodium bicarbonate solution (100
ml) and extracted with Et0Ac (3 x 200 ml). The combined aqueous phases were
dried over sodium sulfate, filtered and evaporated to afford 1-(3,5-dichloro-
pyrazin-2-
y1)-ethanol as a dark brown oil. Yield: 5.23g (96%).
(ii) 1-(3,5-Dichloro-pyrazin-2-yI)-ethanone
5 g of 1-(3,5-dichloro-pyrazin-2-yI)-ethanol obtained in step (i) were
dissolved in dry
DCM (100 ml) at RT in a reaction vessel containing a magnetic stirring bar and
80.7
ml Dess-Martin periodinane (1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxo1-
3-(1H)-
one) solution (15% in DCM), and the mixture stirred for 30 min before the
reaction
was quenched with a saturated aqueous sodium bicarbonate solution (100 ml) and
extracted with Et0Ac (3 x 200 ml). The combined organic phases were dried over
sodium sulfate, filtered and evaporated to afford the crude product as a brown
oil.
Purification by flash chromatography on silica gel using a mixture of Et0Ac
and
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heptane as the eluent afforded 1-(3,5-dichloro-pyrazin-2-y1)-ethanone as a
colorless
oil after evaporation of the solvents under reduced pressure. Yield: 1.9 g
(38%).
(iii) N44-(5-Acety1-6-chloro-pyrazin-2-y1)-pheny1]-2,3-dichloro-
benzenesulfonamide
1-(3,5-Dichloro-pyrazin-2-y1)-ethanone (200 mg) and 2,3-dichloro-N44-(4,4,5,5-
tetramethyl-[1,3,2]dioxaborolan-2-y1)-phenyl]-benzenesulfonamide (448.3 mg),
prepared as in example 1, was added to a reaction vessel containing a magnetic
stirring bar together with 1,1'-bis(diphenylphosphino)ferrocene-
palladium(I1)dichloride
(61 mg) and cesium carbonate (1.0 g), followed by 9 ml dioxane and 1 ml water,
and
the mixture heated to 100 C under stirring. After 2 h the reaction mixture was
cooled
to RT and quenched with a saturated aqueous sodium bicarbonate solution (30
ml)
and extracted with Et0Ac (3 x 30 ml). The combined organic phases were dried
over
sodium sulfate, filtered and evaporated to afford the crude product as a brown
oil-
Purification by flash chromatography on silica gel using a mixture of Et0Ac
and
heptane as the eluent afforded N44-(5-acety1-6-chloro-pyrazin-2-y1)-pheny1]-
2,3-
dichloro-benzenesulfonamide as a colorless solid after evaporation of the
solvents
under reduced pressure. Yield: 230 mg (48%).
(iv) 2,3-Dichloro-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
N44-(5-acety1-6-chloro-pyrazin-2-y1)-pheny1]-2,3-dichloro-benzenesulfonamide
(230
mg) was suspended in a mixture of 2 ml iPrOH and 2 ml 35% hydrazine in water
at
RT and heated to 120 C by microwave irradiation (Biotage lnitiatorTM
apparatus) for
20 min under stirring in a sealed vessel. The reaction mixture was left to
cool to RT,
quenched with a saturated aqueous sodium bicarbonate solution (10 ml) and
extracted with Et0Ac (3 x 30 ml). The combined organic phases were dried over
sodium sulfate, filtered and evaporated to afford the crude product.
Purification by
recrystallisation from an acetone-water mixture afforded the title compound as
a pale
yellow solid after drying under vacuum. Yield: 81.6 mg (38%).
1H-NMR (DMSO-d6): 8 (ppm) = 2.54 (s, 3H), 7.28 (d, J = 8.8 Hz, 2H), 7.59 (t, J
= 8.0
Hz, 1H), 7.94 (dd, J = 1.5, 8.1 Hz, 1H), 8.10 (d, J = 8.8 Hz, 2H), 8.13 (dd, J
= 1.5, 8.0
Hz, 1H), 9.07 (s, 1H), 11.18 (s, 1H), 13.57 (s,1H).
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MS (ES+): m/e = 434.0 (M+H), chloro pattern.
Example 11: 2,5-Dichloro-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
CI 0
c
S 40,
/CH3
N=<
CI
NFI
The title compound was prepared in 5% yield according to the procedure
described
in example 10, employing 2,5-dichloro-benzenesulfonyl chloride instead of 2,3-
dichloro-benzenesulfonyl chloride as starting material. The following
modification was
made. The crude reaction mixture was evaporated to dryness, redissolved in DMF
and purified by preparative HPLC (C18 reversed phase column, elution with a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
1H-NMR (DMSO-d6): 8 (ppm) = 2.55 (s, 3H), 7.29 (d, J = 8.8 Hz, 2H), 7.70 (d, J
= 8.5
Hz, 1H), 7.76 (dd, J = 2.5, 8.5 Hz, 1H), 8.09 (d, J = 2.5 Hz, 1H), 8.12 (d, J
= 8.8 Hz,
2H), 9.08 (s, 1H), 11.17 (s, 1H), 13.57 (br,1H).
MS (ES+): m/e = 434.1(M+H), chloro pattern.
Example 12: 5-Chloro-2-fluoro-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
F 0
c
S 40,
/CH3
N=<
CI
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The title compound was prepared in 9% yield according to the procedure
described
in example 10, employing 5-chloro-2-fluoro-benzenesulfonyl chloride instead of
2,3-
dichloro-benzenesulfonyl chloride as starting material. The following
modification was
made. The crude reaction mixture was evaporated to dryness, redissolved in DMF
5 and purified by preparative HPLC (C18 reversed phase column, elution with
a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
1H-NMR (DMSO-d6): 8 (ppm) = 2.55 (s, 3H), 7.31 (d, J = 8.6 Hz, 2H), 7.53 (dd,
J =
8.6, 9.2 Hz, 1H), 7.80 (m, 1H), 7.89 (dd, J = 2.7, 6.0 Hz, 1H), 8.14 (d, J =
8.6 Hz, 2H),
10 9.09 (s, 1H), 11.15 (s, 1H), 13.58 (br,1H).
MS (ES+): m/e = 418.1 (M+H), chloro pattern.
Example 13: 5-Chloro-2-cyano-N-[4-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
CN 0
CH3
N¨
CI NFI
(i) [4-(5-Acetyl-6-chloro-pyrazin-2-y1)-phenyI]-carbamic acid tert-butyl ester
1-(3,5-Dichloro-pyrazin-2-yI)-ethanone (2.2 g), prepared as described in
example 10,
and (4-tert-butoxycarbonyl-aminophenyl)boronic acid (2.7 g), was added to a
reaction
vessel containing a magnetic stirring bar together with 1,1'-
bis(diphenylphosphino)ferrocene-palladium(11) dichloride (674 mg) and cesium
carbonate (11.2 g), followed by 100 ml dioxane and 10 ml water, and the
mixture
heated to 100 C under stirring. After 1h the reaction mixture was cooled to RT
and
quenched with a saturated aqueous sodium bicarbonate solution (50 ml) and
extracted with Et0Ac (3 x 100 ml). The combined organic phases were dried over
sodium sulfate, filtered and evaporated to afford the crude product as a dark
brown
oil. Purification by flash chromatography on silica gel using a mixture of
Et0Ac and
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heptane as the eluent afforded [4-(5-acetyl-6-chloro-pyrazin-2-yI)-phenyl]-
carbamic
acid tert-butyl ester as a colorless solid after evaporation of the solvents
under
reduced pressure. Yield: 2.44g (61 A) mg.
(ii) [4-(3-Methyl-1H-pyrazolo[3,4-b]pyrazin-6-yI)-phenyl]-carbamic acid tert-
butyl ester
[4-(5-Acetyl-6-chloro-pyrazin-2-yI)-phenyl]-carbamic acid tert-butyl ester
(2.18 g) was
suspended in a mixture of 21 ml iPrOH and 21 ml 35% hydrazine in water at RT
and
heated to 120 C by microwave irradiation for 20 min under stirring in a sealed
vessel.
The reaction mixture was left to cool to RT, quenched with a saturated aqueous
sodium bicarbonate solution (10 ml) and extracted with Et0Ac (3 x 30 ml). The
combined organic phases were dried over sodium sulfate, filtered and
evaporated to
afford the crude product. Purification by trituration in boiling Et0Ac and
subsequent
filtration afforded [4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-yI)-phenyl]-
carbamic acid
tert-butyl ester as a yellow solid. Yield: 1.42 g (70%).
(iii) 5-Chloro-2-cyano-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
To a reaction vessel containing a magnetic stirring bar and 179 mg [4-(3-
methyl-1H-
pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-carbamic acid tert-butyl ester was added
3 ml 4N
hydrogen chloride in dioxane solution, and the mixture stirred at RT. After 2
h the
reaction mixture was evaporated to dryness under reduced pressure and the
residue
redissolved in 3 ml pyridine, and 131 mg 5-chloro-2-cyano-benzenesulfonyl
chloride
was added and the mixture heated to 100 C in a sealed vessel. After 30 min the
reaction mixture was cooled and evaporated to dryness, redissolved in DMF and
purified by preparative HPLC (C18 reversed phase column, elution with a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
Yield: 24 mg (10%).
1H-NMR (DMSO-d6): 8 (ppm) = 2.55 (s, 3H), 7.31 (d, J = 8.8 Hz, 2H), 7.97 (dd,
J =
2.2, 8.3 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 8.16 (d,
J = 8.8
Hz, 2H), 9.10 (s, 1H), 11.25 (s, 1H), 13.58 (br,1H).
MS (ES+): m/e = 425.2(M+H), chloro pattern.
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Example 14: 2-Cyano-5-methyl-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
CN 0
= Sc
/CH3
N ==<
NFI
CH3
The title compound was prepared in 21% yield according to the procedure
described
in example 13, employing 2-cyano-5-methyl-benzenesulfonyl chloride instead of
5-
chloro-2-cyano-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 2.47 (s, 3H), 2.55 (s, 3H), 7.29 (d, J = 8.7 Hz,
2H),
7.32 (d, J = 8.6 Hz, 1H), 7.46-7.51 (m, 1H), 7.72 (dd, J = 2.1, 7.1 Hz, 1H),
8.10 (d, J =
8.7 Hz, 2H), 9.07 (s, 1H), 10.93 (s, 1H), 13.55 (br,1H).
MS (ES+): m/e = 405.3 (M+H).
Example 15: 2-Fluoro-5-methyl-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
F
Sc
i NFI
/CH3
N=<
CH3
The title compound was prepared in 14% yield according to the procedure
described
in example 13, employing 2-fluoro-5-methyl-benzenesulfonyl chloride instead of
5-
chloro-2-cyano-benzenesulfonyl chloride as starting material.
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1H-NMR (DMSO-d6): 8 (ppm) = 2.34 (s, 3H), 2.55 (s, 3H), 7.31 (d, J = 8.6 Hz,
2H),
7.53 (dd, J = 8.6, 9.2 Hz, 1H), 7.80 (m, 1H), 7.89 (dd, J = 2.7, 6.0 Hz, 1H),
8.14 (d, J
= 8.6 Hz, 2H), 9.09 (s, 1H), 11.15 (s, 1H), 13.58 (br,1H).
MS (ES+): m/e = 398.2 (M+H).
Example 16: 2-Chloro-5-methoxy-N-[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]benzenesulfonamide
CI 0
S\:1 40, /CH3
N=<
OCH3
The title compound was prepared in 14% yield according to the procedure
described
in example 13, employing 2-chloro-5-methoxy-benzenesulfonyl chloride instead
of 5-
chloro-2-cyano-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 2.54 (s, 3H), 3.82 (s, 3H), 7.21 (dd, J = 3.1, 8.7
Hz,
1H), 7.29 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 3.1 Hz,
1H), 8.10
(d, J = 8.8 Hz, 2H), 9.07 (s, 1H), 10.99 (s, 1H), 13.55 (br,1H).
MS (ES+): m/e = 430.2 (M+H), chloro pattern.
Example 17: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-cyano-5-
methyl-
benzenesulfonamide
CN 0
Ii F1
S NH2
N=<
CH3
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(i) [4-(6-Chloro-5-cyano-pyrazin-2-y1)-pheny1]-carbamic acid tert-butyl ester
(4-tert-Butoxycarbonyl-aminophenyl)boronic acid pinacol ester (8.26 g) was
added to
a reaction vessel containing a magnetic stirring bar together with 3,5-
dichloro-
pyrazine-2-carbonitrile (5.0 g), 1,1'-bis(diphenylphosphino)ferrocene-
palladium(11)
dichloride (1.68 g) and cesium carbonate (28.1 g), followed by 100 ml dioxane
and
ml water, and the mixture heated to 100 C under stirring. After 1h the
reaction
mixture was cooled to RT and quenched with a saturated aqueous sodium
bicarbonate solution (100 ml) and extracted with Et0Ac (3 x 200 ml). The
combined
organic phases were dried over sodium sulfate, filtered and evaporated to
afford the
10 crude product as a brown oil which was purified by flash chromatography
on silica gel
using a mixture of Et0Ac and heptane as the eluent. The obtained product was
recrystallized from methyl tert-butyl ether to afford [4-(6-chloro-5-cyano-
pyrazin-2-y1)-
pheny1]-carbamic acid tert-butyl ester as a pale yellow solid after drying
under
vacuum. Yield: 6.92 g (73%).
(ii) [4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-carbamic acid tert-
butyl ester
[4-(6-Chloro-5-cyano-pyrazin-2-y1)-pheny1]-carbamic acid tert-butyl ester (1.0
g) was
suspended in a mixture of 10 ml iPrOH and 10 ml 35% hydrazine in water at RT
and
heated to 120 C by microwave irradiation for 70 min under stirring in a sealed
vessel.
The reaction mixture was left to cool to RT, and the precipitate was filtered
off and
washed with water to afford [4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-
pheny1]-
carbamic acid tert-butyl ester as a yellow solid after drying under vacuum.
Yield: 815
mg (83%).
(iii) N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-cyano-5-methyl-
benzenesulfonamide
To a reaction vessel containing a magnetic stirring bar and 180 mg [4-(3-amino-
1H-
pyrazolo[3,4-b]pyrazin-6-y1)-pheny1]-carbamic acid tert-butyl ester was added
3 ml 4N
hydrogen chloride in dioxane solution and the mixture stirred at RT. After 2 h
the
reaction mixture was evaporated to dryness under reduced pressure and the
residue
redissolved in 3 ml pyridine and 131 mg 2-cyano-5-methyl-benzenesulfonyl
chloride
was added and the mixture heated to 100 C in a sealed vessel. After 1 h the
reaction
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mixture was cooled and evaporated to dryness, redissolved in DMF and purified
by
preparative HPLC (C18 reversed phase column, elution with a water/MeCN
gradient
with 0.1 A TFA). The fractions containing the product were lyophilized to
yield the title
compound in the form or its salt with trifluoroacetic acid salt. Yield: 58 mg
(20%).
MS (ES+): m/e = 406.2 (M+H).
Example 18: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-chloro-5-
methoxy-benzenesulfonamide
CI 0
S\'N2
N
¨
OCH3
The title compound was prepared in 22% yield according to the procedure
described
in example 17, employing 2-chloro-5-methoxy-benzenesulfonyl chloride instead
of 2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 3.82 (s, 3H), 7.22 (dd, J = 3.0, 8.7 Hz, 1H), 7.27
(d, J
= 8.8 Hz, 2H), 7.55 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 3.1 Hz, 1H), 8.07 (d, J
= 8.8 Hz,
2H), 8.87 (s, 1H), 10.98 (s, 1H), 13.52 (br,1H).
MS (ES+): m/e = 431.1 (M+H), chloro pattern.
Example 19: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-fluoro-5-
methyl-
benzenesulfonamide
F
Sc \ NH2
N¨
CH3
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To a solution of 500 mg of 6-(4-amino-phenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
ylamine
hydrochloride and 397 mg of 2-fluoro-5-methyl-benzenesulfonyl chloride in 4 ml
DCM, 0.16 ml pyridine were added and the reaction mixture was stirred for 16 h
at
RT. Then, the solvents were removed under reduced pressure and the crude
product
was purified by chromatography on silica gel eluting with a gradient of n-
heptane/Et0Ac. The fractions containing the product were combined and the
solvent
evaporated under reduced pressure. Yield: 220 mg.
MS (ES+): m/e = 399.2 (M+H).
Example 20: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-5-chloro-2-
cyano-benzenesulfonamide
CN 0
ScN
N¨
N H2,N
CI
The title compound was prepared in according to the procedure described in
example 17, employing 5-chloro-2-cyano-benzenesulfonyl chloride instead of 2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
MS (ES): m/e = 426.1 (M+H), chloro pattern.
Example 21: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,5-dichloro-
thiophene-3-sulfonamide
Cl 0
S , N
\N 110 NH2
N¨
N,N
CI
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The title compound was prepared by adapting the procedures described in
example
19, employing 2,5-dichlorothiophene-3-sulfonyl chloride instead of 2-fluoro-5-
methyl-
benzenesulfonyl chloride.
1H-NMR (DMSO-d6): 8 (ppm) = 7.30 (d, J = 8.8 Hz, 2H), 7.41 (s, 1H), 8.14 (d, J
= 8.8
Hz, 2H), 8.91 (s, 1H), 11.07 (s, 1H), 12.3 (br,1H).
MS (ES+): m/e = 441.1 (M+H), chloro pattern.
Example 22: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-8-chloro-3,4-
dihydro-2H-benzo[b][1,4]dioxepine-7-sulfonamide
CI 0
S'
NFI
N H2
,N
0 lel H N¨
The title compound was prepared by adapting the procedures described in
example
19, employing 8-chloro-3,4-dihydro-2H-benzo[b][1,4]dioxepine-7-sulfonyl
chloride
instead of 2-fluoro-5-methyl-benzenesulfonyl chloride.
1H-NMR (DMSO-d6): 8 (ppm) = 2.13 (t, J = 5.6 Hz, 2H), 4.20 (t, J = 5.6 Hz,
2H), 4.25
(t, J = 5.6 Hz, 2H), 7.21 (s, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.60 (s, 1H),
8.07 (d, J = 8.8
Hz, 2H), 8.87 (s, 1H), 10.89 (s, 1H), 12.33 (br,1H).
MS (ES+): m/e = 473.2 (M+H), chloro pattern.
Example 23: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-chloro-1,3-
dimethyl-pyrazole-4-sulfonamide
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))CI 0\sc)
H3C-N \N 1100NH2
H
N=<
CH3 N,N
The title compound was prepared by adapting the procedures described in
example
19, employing 5-chloro-1,3-dimethyl-pyrazole-4-sulfonyl chloride instead of 2-
fluoro-
5-methyl-benzenesulfonyl chloride.
1H-NMR (DMSO-d6): 8 (ppm) = 2.30 (s, 3H), 3.72 (s, 3H), 7.25 (d, J = 8.8 Hz,
2H),
8.09 (d, J = 8.8 Hz, 2H), 8.90 (s, 1H), 10.81 (s, 1H), 12.30 (br,1H).
MS (ES+): m/e = 419.1 (M+H), chloro pattern.
Example 24: 2,3-Dichloro-N-[644-[(2-fluoro-5-methyl-
phenyl)sulfonylamino]pheny1]-
1H-pyrazolo[3,4-b]pyrazin-3-yl]benzam ide
F 0
H S Cl c N
ClNFI
N¨ 0
CH3
(i) N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-fluoro-5-methyl-
benzenesulfonamide
To a solution of 500 mg of 6-(4-amino-phenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
ylamine
hydrochloride and 397 mg of 2-fluoro-5-methyl-benzenesulfonyl chloride in 4 ml
DCM, 0.16 ml pyridine were added and the reaction mixture was stirred for 16 h
at
RT. Then, the solvents were removed under reduced pressure and the crude
product
was purified by chromatography on silica gel eluting with a gradient of n-
heptane/Et0Ac. The fractions containing the product were combined and the
solvent
evaporated under reduced pressure. Yield: 220 mg.
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(ii) 2,3-Dichloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]benzamide
To a solution of 70 mg of N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-
2-
fluoro-5-methyl-benzenesulfonamide in 0.5 ml pyridine, 13 mg of 2,3-dichloro-
benzoyl chloride were added and the reaction mixture was stirred for 16 h at
RT.
Then, the reaction mixture was diluted with water and filtered through a chem
elut
cartridge by eluting with Et0Ac. After removal of the solvents under reduced
pressure the crude product was purified by preparative HPLC (C18 reversed
phase
column, elution with a water/MeCN gradient with 0.1 A TFA). The fractions
containing
the product were lyophilized to yield the title compound in the form of its
salt with
trifluoroacetic acid. Yield: 5 mg.
1H-NMR (DMSO-d6): 8 (ppm) = 2.33 (s, 3H), 7.29 (d, J = 8.8 Hz, 2H), 7.32 (m,
1H),
7.48 (m, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.73 (m, 1H), 7.80 (d, J = 8.8 Hz,
1H), 7.98 (d,
J = 8.8 Hz, 2H), 9.29 (s, 1H), 10.95 (s, 1H), 12.40 (br,1H).
MS (ES+): m/e = 571.3 (M+H), chloro pattern.
Example 25: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]tetrahydropyran-4-carboxamide
0
F 0
0
Sc N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing tetrahydropyran-4-carbonyl chloride instead of 2,3-dichloro-
benzoyl
chloride.
MS (ES+): m/e = 511.2 (M+H).
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Example 26: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]piperidine-4-carboxamide
NH
F 0
S(N N
N¨ N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing piperidine-4-carbonyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 510.3 (M+H).
Example 27: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]cyclopentanecarboxamide
F 0
Sc = N
N
N¨ N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing cyclopentanecarbonyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES-): m/e = 493.4 (M-H).
Example 28: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]cyclopropanecarboxamide
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F 0
0
N
N¨ N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing cyclopropanecarbonyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 467.3 (M+H).
Example 29: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]cyclohexanecarboxamide
F 0
0
H =
N
N¨ NJN
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing cyclohexanecarbonyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 509.4 (M+H).
Example 30: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-y1]-2-phenyl-acetamide
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F
Sc N
n
N N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing 2-phenylacetyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 517.4 (M+H).
Example 31: N-[644-[(2-Fluoro-5-methyl-phenyl)sulfonylam ino]phenyI]-1H-
pyrazolo[3,4-b]pyrazin-3-yl]thiophene-3-carboxamide
F 0
0
S'
\N 1100
N=<\
N,N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing thiophene-3-carbonyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 509.3 (M+H).
Example 32: 4-Chloro-N-[644-[(2-fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-
1H-
pyrazolo[3,4-b]pyrazin-3-yl]benzamide
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F 0\\O H
CI
,
Sc N 1110
N
N¨
N,N
CH3
The title compound was prepared by adapting the procedures described in
example
24, employing 4-chloro-benzoyl chloride instead of 2,3-dichloro-benzoyl
chloride.
MS (ES+): m/e = 537.3 (M+H), chloro pattern.
Example 33: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]naphthalene-1-
sulfonamide
0 n
=
S(N
2
N¨
NN
The title compound was prepared by adapting the procedures described in
example
19, employing naphthalene-1-sulfonyl chloride instead of 2-fluoro-5-methyl-
benzenesulfonyl chloride.
MS (ES+): m/e = 417.2 (M+H).
Example 34: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,4,6-
trichloro-
benzenesulfonamide
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CI 0
S'
\ NH2
N=<
CI =
CI
N,N
The title compound was prepared by adapting the procedures described in
example
19, employing 2,4,6-trichlorobenzenesulfonamide instead of 2-fluoro-5-methyl-
benzenesulfonyl chloride.
MS (ES+): m/e = 470.1 (M+H), chloro pattern.
Example 35: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-chloro-2-
fluoro-
benzenesulfonamide
F 0
\2
N¨ \N
CI
NFI
The title compound was prepared by adapting the procedures described in
example
1, employing 5-chloro-2-fluoro-benzenesulfonyl chloride instead of 2,3-
dichloro-
benzenesulfonyl chloride as starting material. The following modification was
made.
The crude reaction mixture was evaporated to dryness, redissolved in DMF and
purified by preparative HPLC (C18 reversed phase column, elution with a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
1H-NMR (DMSO-d6): 8 (ppm) = 7.27 (d, J = 8.8 Hz, 2H), 7.55 (t, J = 8.7 Hz,
1H), 7.81
(m, 1H), 7.88 (m, 1H), 8.10 (d, J = 8.8 Hz, 2H), 8.91 (s, 1H), 11.10 (s, 1H).
MS (ES+): m/e = 419.0 (M+H), chloro pattern.
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Example 36: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-chloro-4-
trifluoromethyl-benzenesulfonamide
CI 0
F3C S' / \N NH2
N=<
5
The title compound was prepared by adapting the procedures described in
example
19, employing 2-chloro-4-trifluoromethyl-benzenesulfonyl chloride instead of 2-
fluoro-
5-methyl-benzenesulfonyl chloride.
MS (ES+): m/e = 469.2 (M+H), chloro pattern.
Example 37: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,4,5-
trifluoro-
benzenesulfonamide
o
F S\' , N
2
=
The title compound was prepared by adapting the procedures described in
example
19, employing 2,4,5-trifluoro-benzenesulfonyl chloride instead of 2-fluoro-5-
methyl-
benzenesulfonyl chloride.
MS (ES+): m/e = 421.1 (M+H).
Example 38: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,4,5-
trichloro-
benzenesulfonamide
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1 1 1
O
CI
\N = /NH2
N=<CI CI
N,N
The title compound was prepared by adapting the procedures described in
example
19, employing 2,4,5-trichloro-benzenesulfonyl chloride instead of 2-fluoro-5-
methyl-
benzenesulfonyl chloride.
1H-NMR (DMSO-d6): 8 (ppm) = 7.28 (d, J = 8.6 Hz, 2H), 8.09 (d, J = 8.6 Hz,
2H), 8.24
(s, 1H), 8.87 (s, 1H), 11.18 (s, 1H), 12.30 (br,1H).
MS (ES+): m/e = 469.0 (M+H), chloro pattern.
Example 39: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-5-chloro-2,4-
difluoro-benzenesulfonamide
0\\O
\N =
/NH2
N=<
N,N
The title compound was prepared by adapting the procedures described in
example
19, employing 5-chloro-2,4-difluoro-benzenesulfonyl chloride instead of 2-
fluoro-5-
methyl-benzenesulfonyl chloride.
1H-NMR (DMSO-d6): 8 (ppm) = 7.29 (d, J = 8.6 Hz, 2H), 7.84 (t, J = 9.4 Hz,
1H), 8.10
(d, J = 8.8 Hz, 2H), 8.90 (s, 1H), 11.14 (s, 1H), 12.30 (br,1H).
MS (ES+): m/e = 437.1 (M+H), chloro pattern.
Example 40: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,3,4-
trichloro-
benzenesulfonamide)
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CI 0
CI
NH2
N=<
CI
N,N
The title compound was prepared by adapting the procedures described in
example
19, employing 2,3,4-trichloro-benzenesulfonyl chloride instead of 2-fluoro-5-
methyl-
5 benzenesulfonyl chloride.
MS (ES+): m/e = 470.9 (M+H), chloro pattern.
Example 41: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2,3,4-
trifluoro-
benzenesulfonamide
F 0
F S(
==NH2
=<NH
The title compound was prepared by adapting the procedures described in
example
19, employing 2,3,4-trifluoro-benzenesulfonyl chloride instead of 2-fluoro-5-
methyl-
benzenesulfonyl chloride.
MS (ES+): m/e = 421.2 (M+H).
Example 42: 5-Chloro-N-[443-[(5-chloro-2,4-difluoro-phenyl)sulfonylamino]-1H-
pyrazolo[3,4-b]pyrazin-6-yl]pheny1]-2,4-difluoro-benzenesulfonamide
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F
F
0
S\'N CI
=r\j,N
CI
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-5-chloro-2,4-difluoro-benzenesulfonamide.
MS (ES+): m/e = 647.1 (M+H), chloro pattern.
Example 43: 5-Chloro-N-[443-[(5-chloro-1,3-dimethyl-pyrazol-4-
y1)sulfonylamino]-1H-
pyrazolo[3,4-b]pyrazin-6-yl]pheny1]-1,3-dimethyl-pyrazole-4-sulfonamide
H3C N
CH3 0\\
// CI
I-13CN CI ,11\1
The title product was isolated as a by-product in the synthesis of 5-chloro-
1,3-
dimethyl-1H-pyrazole-4-sulfonic acid [4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-
y1)-
phenyl]-amide.
MS (ES+): m/e = 611.1 (M+H), chloro pattern.
Example 44: 2,4,5-Trifluoro-N-[443-[(2,4,5-trifluorophenyl)sulfonylamino]-1H-
pyrazolo[3,4-b]pyrazin-6-yl]phenyl]benzenesulfonamide
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H F
F
S\' N
N,
1_1 104 /0
F N ,N
\ 0
NI
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-2,4,5-trifluoro-benzenesulfonamide.
MS (ES+): m/e = 615.1 (M+H).
Example 45: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-5-cyano-2-
fluoro-
benzenesulfonamide
F 0
Sc /N H2
N=<
CN
The title compound was prepared in 11 A yield according to the procedure
described
in example 17, employing 5-cyano-2-fluoro-benzenesulfonyl chloride instead of
2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 5.70 (br s, 3H), 7.29 (d, J = 8.7 Hz, 2H), 7.71
(m, 1H),
8.07 (d, J = 8.7 Hz, 2H), 8.25 (m, 1H), 8.40 (dd, J = 1.9, 6.5 Hz, 1H), 8.88
(s, 1H),
11.22 (s, 1H), 12.32 (s,1H).
MS (ES+): m/e = 410.2 (M+H).
Example 46: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-2-cyano-5-
methoxy-benzenesulfonamide
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CN 0
/40 1100
N=< jr\\I
OCH3
The title compound was prepared in 15% yield according to the procedure
described
in example 17, employing 2-cyano-5-methoxy-benzenesulfonyl chloride instead of
2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 3.89 (s, 3H), 5.70 (br s, 3H), 7.29 (d, J = 8.8
Hz, 2H),
7.36 (dd, J = 2.5, 8.6 Hz, 1H), 7.54 (d, J = 2.5 Hz, 1H), 8.02 (d, J = 8.6 Hz,
1H), 8.10
(d, J = 8.8 Hz, 2H), 8.88 (s, 1H), 11.13 (s, 1H), 12.32 (s,1H).
MS (ES+): m/e = 422.3 (M+H).
Example 47: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-2-cyano-5-
fluoro-
benzenesulfonamide
CN 0
Sc /NH2
N=<
The title compound was prepared in 15% yield according to the procedure
described
in example 17, employing 2-cyano-5-fluoro-benzenesulfonyl chloride instead of
2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 5.71 (br s, 3H), 7.29 (d, J = 8.7 Hz, 2H), 7.76
(m, 1H),
7.96 (dd, J = 2.6, 8.2 Hz, 1H), 8.11 (d, J = 8.7 Hz, 2H), 8.22 (dd, J = 5.1,
8.6 Hz, 1H),
8.89 (s, 1H), 11.27 (s, 1H), 12.33 (s,1H).
MS (ES+): m/e = 410.2 (M+H).
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Example 48: N-[4-(3-Amino-1H-pyrazolo[3,4-b]pyrazin-6-y1)-phenyl]-2-fluoro-5-
methoxy-benzenesulfonamide
F
= S'
\N 1100NH2
N=c jr1\1
OCH3
The title compound was prepared in 11 A yield according to the procedure
described
in example 17, employing 2-fluoro-5-methoxy-benzenesulfonyl chloride instead
of 2-
cyano-5-methyl-benzenesulfonyl chloride as starting material.
1H-NMR (DMSO-d6): 8 (ppm) = 3.79 (s, 3H), 5.68 (br s, 3H), 7.21-7.26 (m, 1H),
7.28
(d, J = 8.7 Hz, 2H), 7.32-7.39 (m, 2H), 8.06 (d, J = 8.7 Hz, 2H), 8.87 (s,
1H), 10.97 (s,
1H), 12.30 (s,1H).
MS (ES+): m/e = 415.2 (M+H).
Example 49: 14644-[(2-Fluoro-5-methyl-phenyl)sulfonylamino]pheny1]-1H-
pyrazolo[3,4-b]pyrazin-3-y1]-3-(3-pyridy1)-urea
F 0
S' n
N=<
NH
,N
CH3
To a solution of 25 mg of N-[4-(3-amino-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-
2-
fluoro-5-methyl-benzenesulfonamide in 0.5 ml dioxane, 9 mg of 3-isocyanato-
pyridine
and 7 mg 1,3-dimethylimidazolidin-2-one were added and the reaction mixture
was
stirred for 16 h at RT. Then the reaction mixture was concentrated under
reduced
pressure and the crude product was purified by preparative HPLC (C18 reversed
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phase column, elution with a water/MeCN gradient with 0.1 A TFA). The
fractions
containing the product were lyophilized to yield the title compound in the
form of its
salt with trifluoroacetic acid. Yield: 2 mg.
MS (ES+): m/e = 519.3 (M+H).
Example 50: 1-(4-Chloropheny1)-34644-[(2-fluoro-5-methyl-
phenyl)sulfonylamino]pheny1]-1H-pyrazolo[3,4-b]pyrazin-3-y1]-urea
F
,0
Sc N\\
N=Cli
NH Cl
CH3
The title compound was prepared by adapting the procedures described in
example
49, employing 1-chloro-4-isocyanato-benzene instead of 3-isocyanato-pyridine.
MS (ES+): m/e = 552.2 (M+H), chloro pattern.
Example 51: 2-Chloro-N-[443-[[2-chloro-4-trifluoromethyl-phenyl]sulfonylamino]-
1H-
pyrazolo[3,4-b]pyrazin-6-yl]pheny1]-4-trifluoromethyl-benzenesulfonamide
CF3
Cl o Cl
,0
F3C =
S\'N =
N= \I
,N
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2-chloro-4-trifluoromethyl-
benzenesulfonamide.
MS (ES+): m/e = 711.1 (M+H), chloro pattern.
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Example 52: N-[644-(1-Naphthylsulfonylamino)pheny1]-1H-pyrazolo[3,4-b]pyrazin-
3-
yl]naphthalene-1-sulfonamide
0 ,_,
H 411
1110
N¨IN 0
401
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-yl)phenyl]naphthalene-1-sulfonamide.
MS (ES+): m/e = 607.3 (M+H).
Example 53: 2,4,6-Trichloro-N-[443-[(2,4,6-trichlorophenyl)sulfonylamino]-1H-
pyrazolo[3,4-b]pyrazin-6-yl]phenyl]benzenesulfonamide
Cl
Cl 0 Cl =
Cl = C
S
11 , N
N,0S,
N_ \ Cl
I,N
The title product was isolated as a by-product in the synthesis of N-[4-(3-
amino-1H-
pyrazolo[3,4-b]pyrazin-6-yl)pheny1]-2,4,6-trichloro-benzenesulfonamide.
MS (ES+): m/e = 710.1 (M+H), chloro pattern.
Example 54: N-[3-Methyl-4-[[4-(3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-
yl)phenyl]sulfamoyl]phenyl]acetamide
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CH3 0
CH3 401 S =c
N¨
O
N,N
FI
The title compound was prepared in 1 A yield according to the procedure
described
in example 10, employing 4-acetylamino-2-methyl-benzenesulfonyl chloride
instead
of 2,3-dichloro-benzenesulfonyl chloride as starting material. The following
modification was made. The crude reaction mixture was evaporated to dryness,
redissolved in DMF and purified by preparative HPLC (C18 reversed phase
column,
elution with a water/MeCN gradient with 0.1 A TFA). The fractions containing
the
product were lyophilized to yield the title compound in the form of its salt
with
trifluoroacetic acid.
MS (ES+): m/e = 437.2 (M+H).
Example 55: 2-Methyl-N44-(3-methy1-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]-5-
nitro-
benzenesulfonamide
CH3 0
\\
S\'N = N
CH3
NFI
NO2
The title compound was prepared in 18% yield according to the procedure
described
in example 10, employing 2-methyl-5-nitro-benzenesulfonyl chloride instead of
2,3-
dichloro-benzenesulfonyl chloride as starting material. The following
modification as
made. The crude reaction mixture was evaporated to dryness, redissolved in DMF
and purified by preparative HPLC (C18 reversed phase column, elution with a
water/MeCN gradient with 0.1 A TFA). The fractions containing the product were
lyophilized to yield the title compound in the form of its salt with
trifluoroacetic acid.
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MS (ES+): m/e = 425.2 (M+H).
Example 56: N-[4-(3-Methyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)phenyI]-2-nitro-4-
trifluoromethyl-benzenesulfonamide
NO2 0
\\
S\'N z N
F3C N¨
N CH3,N
The title compound was prepared in 5% yield according to the procedure
described
in example 10, employing 2-nitro-4-trifluoromethyl-benzenesulfonyl chloride
instead
of 2,3-dichloro-benzenesulfonyl chloride as starting material. The following
modification was made. The crude reaction mixture was evaporated to dryness,
redissolved in DMF and purified by preparative HPLC (C18 reversed phase
column,
elution with a water/MeCN gradient with 0.1 A TFA). The fractions containing
the
product were lyophilized to yield the title compound in the form of its salt
with
trifluoroacetic acid.
MS (ES+): m/e = 479.1 (M+H).
Pharmacological testing
The ability of the compounds of the invention to inhibit SGK-1 was assessed in
an
enzymatic activity assay by determining their effect on the ability of the
isolated SGK
enzyme to catalyze the transfer of phosphate from ATP to serine/threonine
residues
in a labeled substrate peptide, and in cellular assays by determining their
effect on
cellular function. In one of the cellular assays, the SGK-1 dependent
phosphorylation
of glycogen synthase kinase 3beta (GSK3beta) in U205 cells was measured, in
another one, a functional electrophysiological assay, the SGK-1 dependent
activation
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of epithelial Na + channel (ENaC) currents in A6 cell monolayers, and in
another one
chondrocyte hypertrophic differentiation in mouse chondrogenic ATDC5 cells.
A) Enzymatic activity assay
The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-
1) inhibitory activity in a substrate phosphorylation assay designed to
measure the
ability of the isolated enzyme to catalyze the transfer of phosphate from ATP
to
serine/threonine residues in a fluorescein-labeled substrate peptide, using
recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol,
Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide
substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated
substrate peptide and non-phosphorylated substrate peptide were separated with
caliper life science's lab-chip technology based on a micro fluidics method.
All fluid
flow was established on the chip by applying a vacuum of a few psi to the
waste well
transporting fluid from various sources through interconnecting channels.
Because
the phosphoryl group is doubly negatively charged, under the pressure-driven
hydrodynamic flow and the voltage-driven flow within the electric field, the
fluorescent
labeled peptide substrate and its phosphorylation product appear at different
times in
the detection window to the detection point. Substrate turnover can thus be
determined as the ratio of the product peak area and the sum of substrate peak
area
and product peak area.
The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCI (pH
7.4),
5 mM MgC12, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The
enzyme was pre-incubated with the test compound for 30 min at 24 C. The kinase
reaction was initiated by addtion of the substrate mixture containing the
peptide
substrate (final concentration 1 pM) and ATP (final concentration 10 pM).
After 60
min incubation at 37 C, the enzyme reaction was terminated by adding a buffer
containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
For the determination of the compound dose response, a 10 mM DMSO stock
solution was diluted and tested in a ten-point, three-fold dilution series run
in
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duplicate beginning at 30 pM final concentration. Data were analyzed using a
four-
parameter curve fit with a fixed minimum and maximum experimentally defined as
the average positive and negative controls on each plate. 1050 values (in pM
(micromol/liter)) for inhibition of SGK-1 determined in this assay are given
in Table 1.
Table 1. 1050 values for inhibition of SGK-1 enzymatic activity by example
compounds
Example no. 1050 [PM] Example no. 1050 [PM]
1 0.003 12 0.004
2 0.439 13 0.003
3 0.496 14 0.003
4 0.419 15 0.002
5 0.005 16 0.002
6 0.002 35 0.001
7 0.002 42 0.413
8 0.196 43 1.050
9 0.003 45 0.047
0.013 46 0.002
11 0.002
10 B) Determination of the effect on SGK-1 dependent phosphorylation of
GSK3beta in
U2OS cells
It has been shown that glycogen synthase kinase 3beta (GSK3beta) is a
phosphorylation target of SGK-1 (Sakoda,H., Gotoh,Y., Katagiri,H.,
Kurokawa,M.,
Ono, H., Onishi,Y., Anai,M., Ogihara,T., Fujishiro,M., Fukushima,Y., Abe,M.,
Shojima,N., Kikuchi,M., Oka,Y., Hirai,H., Asano,T.; Differing roles of Akt and
serum-
and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and
oncogenic activity. J. Biol. Chem. 278 (2003), 25802-25807). The ability of
the
compounds of the invention to inhibit the enzymatic activity of serum and
glucocorticoid-regulated Kinase 1 (SGK-1) was determined in a cellular assay
which
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measures the SGK-1 dependent phosphorylation of GSK3beta in U2OS cells (ATCC
HTB-96) overexpressing recombinant SGK-1 and GSK3beta after transfection with
recombinant BacMam viruses.
U205 cells were cultured in 1:1 Dulbecco modified Eagle medium / Ham's F12 and
10% heat inactivated fetal calf serum (FCS Gold) at 37 C, 7% CO2 and 95%
relative
humidity. Cells were harvested and mixed with BacMam virus containing
expression
constructs for human SGK-1 (amino acids S61 - L431 with serine 422 replaced by
aspartate) at an MOI (multiplicity of infection) of 50 and BacMam virus
containing
expression constructs for human GSK3beta at an MOI of 125. Cell suspension
mixed
with BacMam viruses was seeded in 96 well pCLEAR plates (Greiner) at 3x104
cells
per well in 250 pL medium. To reduce background phosphorylation of GSK3beta by
AKT, 1 pL of a selective Akt-inhibitor was added (final concentration 2 pM). 1
pL of a
solution of the test compound at 250 x final concentration was added. Cells
are
incubated at 37 C, 7% CO2 and 95% relative humidity. After 6 h, medium was
aspirated and 50 pl of fixation solution (3.7% paraformaldehyde in phosphate
buffered saline (PBS)) was added for 10 min. After removing the fixation
solution,
cells were permeabilised by adding 200 pl PBT (0.2% Triton X-100 in PBS) per
well
for 5 min. After removing PBT, cells were blocked by adding 200 pl of blocking
solution (1% bovine serum albumine in PBS) per well. Blocking solution was
removed and 50 pl of primary antibody (rabbit anti-phospho-GSK-3beta (Ser9),
and
mouse anti-GSK-3beta) were added for lh. After washing the cells 3 times with
PBS,
50 pl of secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG, and Alexa
Fluor
488 goat anti-mouse IgG) were added and incubated for 1 h in the dark. After
washing the cells 3 times with PBS, 200 pl of PBS were added. Fluorescence
signals
were measured with the ImageXpress MICRO (Molecular Devices). IC50 values were
calculated using the ratio of phosphorylated GSK3beta to total GSK3beta to
compensate for unspecific effects and are given in Table 2.
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Table 2. IC50 values for inhibition of SGK-1 dependent phosphorylation of
GSK3beta
in U2OS cells by example compounds
Example no. IC50 [P1V1] Example no. IC50 [P1V1]
1 1.4 14 2.9
0.63 15 2.4
6 2.1 16 1.5
7 2.6 35 0.69
12 1.5
5 C) Functional electrophysiological assay for determination of SGK-1
dependent
activation of ENaC-currents in A6 cell monolayers
SGK-1 is up-regulated in A6 cells in response to induction of a hypoosmotic
shock
(Alvarez de la Rosa et al.; J. Gen. Physiol. 124 (2004), 395-407). As a
consequence
of SGK-1 induction, ENaC function in the plasma membrane is upregulated and
the
effect of SGK-1 inhibitors on functional ENaC surface expression can be
investigated
with Ussing chamber technology.
Materials and methods for Ussing chamber measurement of A6 cells: The renal
Xenopus laevis cell line A6 (Rafferty, K. A.; Mass culture of amphibia cells:
methods
and observations concerning stability of cell type. In: Biology of Amphibian
Tumors,
edited by M. Mizell. New York: Springer-Verlag, 1969, p. 52-81) was used for
the
experiments. Cells were grown in cell culture flasks (Nunc) at 28 C in a
humidified
atmosphere with 4% CO2. The culture medium contained a 7:3 mixture of
Leibovitz's
L-15 (Sigma-Aldrich), / Coon's (Sigma-Aldrich) media supplemented with 10%
fetal
bovine serum (PAA), 20 % sterile water, 25 mM NaHCO3 (Sigma-Aldrich), 100 U/ml
penicillin (PAA) and 100 pg/ml streptomycin (PAA). The osmolality of the
medium
was 270 mOsml/kg H20). Cells were detached with accutase (PAA) and seeded for
electrophysiological measurements into transwell filter inserts (polyester 0.4
pm pore
size, Corning) at a density of 0.4x106 cells/filter. Cells were cultivated for
7-10 days,
and confluent A6 cell monolayers were identified by repetitive resistance
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measurements in cell culture medium using an EVOM2 ohmmeter (World Precision
Instruments). Monolayers with a resistance of >10 kOhm were considered
confluent.
Filters with confluent A6 cells were transferred into a continuously perfused
Ussing-
chamber, and electrophysiological parameters were measured under open circuit
conditions using a transepithelial clamp amplifier (EP Design). Short circuit
current
(l'sc) was calculated by Ohm's law. The Ringer-solutions for Ussing chamber
experiments contained NaCI: 122 mmo1/1(isoosmotic = 260 mOsml/kg H20) or 82
mmo1/1(hypoosmotic = 180 mOsml/kg H20); KHCO3: 2.5 mmo1/1; CaCl2: 1 mmo1/1;
MgC12: 1 mmo1/1; glucose: 5 mmo1/1. The pH was adjusted to 8.2. All
measurements
were done at room temperature. Am iloride, an inhibitor of epithelial Na +
channel
(ENaC)-dependent ion transport, was employed at a concentration of 25 pM.
To evaluate the effects of SGK inhibitors on ENaC-mediated transepithelial
currents,
A9 monolayers were first equilibrated for 5 min with isoosmotic Ringer-
solution from
both the luminal and basolateral side of the cell layer. Am iloride was
applied to the
luminal site to establish the basal ENaC-dependent current (I'scbasal). Cell
layers were
then perfused from the basolateral side for 10 min with compounds in isotonic
buffer
or control isotonic buffer. SGK signaling leading to increased ENaC activity
and
subsequent increase in l'sc was stimulated by application of hypoosmotic
Ringer-
solution for 45 min to both sides of the A6 cell layer. ENaC-dependent l'sc
after the
hypoosmotic shock (I'schypc,) was determined by application of amiloride at
the end of
the experiment. Total changes of am iloride-sensitive Isc was calculated as
Al'sc =
l'schypo - l'scbasai. The experimental protocol allows detecting and excluding
compounds with an intrinsic effect on ENaC, however, there was no direct
effect on
ENaC by the compounds under investigation. The inhibition of Al'sc by the test
compounds was determined relative to the Al'sc measured with control
monolayers
which were not treated with the test compound. IC50 value were determined by
fitting
the data to the general dose-response equation
For the compound of example 1, in this test an IC50 value of 2.1 pM was
determined.
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D) Determination of the effect on chondrocyte hypertrophic differentiation in
mouse
chondrogenic ATDC5 cells
The ATDC5 cell assay was used as in vitro model to determine the effects of
the
compounds of the invention on chondrocyte hypertrophic differentiation by
monitoring
the expression levels of collagen type X (Coll 0a1) as specific marker of
chondrocyte
hypertrophy.
Background: ATDC5 cells are a clonal mouse embryonic cell line derived from
multipotent AT805 teratocarcinoma cells (Atsumi T, Miwa Y, Kimata K, lkawa Y.;
A
chondrogenic cell line derived from a differentiating culture of AT805
teratocarcinoma
cells. Cell Differ. Dev. 30 (1990), 109-116). The cells can undergo insulin-
dependent
chondrogenic cell differentiation entailing distinct differentiation stages
starting from
an undifferentiated, subconfluent stage, a condensation stage, a cartilage
nodule
formation stage and a calcification stage within 45 days of in vitro culture.
Chondrogenic differentiation can be shown by measuring the expression of the
cartilage main collagen (Col2a1) and aggrecan (AGC1) and glycosaminoglycan-
staining with Alcian Blue within two weeks after insulin-triggered
differentiation, and
hypertrophic differentiation can be monitored by the expression of collagen
type X
(Coll 0a1), a specific marker of chondrocyte hypertrophy within 21 days of in
vitro
culture. (Shukunami C, Shigeno C, Atsumi T, lshizeki K, Suzuki F, Hiraki Y.;
Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in
vitro:
differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-
related peptide receptor. J. Cell. Biol. 133 (1996):457-468). Growth factor
BMP-2 is
known to stimulate cell differentiation and can stimulate early and late-phase
ATDC5
differentiation (Shukunami C, Ohta Y, Sakuda M, Hiraki Y.; Sequential
progression of
the differentiation program by bone morphogenetic protein-2 in chondrogenic
cell line
ATDC5. Exp. Cell Res. 241 (1998), 1-11). Thyroid hormone triiodothyronine (T3)
promotes hypertrophic differentiation of growth plate chondrocytes (Robson H,
Siebler T, Stevens DA, Shalet SM, Williams GR; Thyroid hormone acts directly
on
growth plate chondrocytes to promote hypertrophic differentiation and inhibit
clonal
expansion and cell proliferation. Endocrinology. 141 (2000):3887-3897).
Addition of
BMP2 and T3 can accelerate ATDC5 hypertrophic differentiation leading to the
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strong induction Coll0a1 expression between 10-14 days. SGK-inhibitors were
added to differentiating ATDC5 cells for 14 days and Coll0a1 gene expression
was
quantified to determine effects on chondrocyte hypertrophic differentiation.
Cell assay description: ATDC5 cells were maintained in 300cm2 tissue culture
flasks
in DMEM/Ham's F12 + 5% FCS supplemented with 10 pg/ml human transferrin, 30
nM sodium selenite, 50 pg/ml kanamycin and grown at 37 C in 5% CO2 in 95% air.
To initiate cell differentiation, 9.9 x 104 cells were plated in 24 well
plates and grown
for 2 days. Medium was exchanged with DMEM/Ham's F12 + 5% FCS supplemented
with 10 pg/ml human transferrin, 30 nM sodium selenite, 50 pg/ml ascorbic acid
and
1 pg/ml BMP2. The assay was run in triplicates, compounds were added in 10%
DMSO, and medium changed every 2-3 days including supplementation of
compound. At day 7 after initiation of cell differentiation, 1 pM T3 was used
as
additional supplement in the cell culture.
After two weeks of cell culture, RNA was isolated and converted to cDNA for
determination of gene expression by quantitative real-time PCR. Cells were
lysed in
600 pl of RLT-buffer (Qiagen) and total RNA was isolated using the RNA-easy
Mini
RNA isolation Kit (Qiagen) which was run on a Qiacube system (Qiagen)
according
to the supplier's instructions. RNA was isolated in 30 pl of pure water and
the RNA
content measured by UV-spectroscopy (Nanodrop, Peqlab). For cDNA synthesis 50
ng total RNA was reverse transcribed using the High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Product Number 4368813) according to
the
manufacturers instructions. Briefly, a 20 pl reaction was set up, containing 4
mM
dNTPs, random primers, RNAse inhibitor and 1p1 MultiScribe reverse
transcriptase
and incubated for 10 min at 25 C, 120 min at 37 C, 5 min at 85 C.
Quantitative Real-Time PCR: Taqman Fast PCR reaction was performed in a 20 pl
volume using Taqman Fast Advanced Master Mix (Applied Biosystems, product
number 4444965) and Taqman Gene expression assays for RPL37a (Applied
Biosystems, product number Mm01253851_g1) as housekeeping gene and Coll0a1
(Applied Biosystems, product number Mm00487041_m1) for Collagen type X
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expression. Briefly, 2 pl of the cDNA-reaction was combined with 10 pl 2x
Taqman
Fast Advanced Master Mix, 1 pl of Taqman Gene Expression Assay containing
primers and 5'-Fam-labelled minor groove binding Taqman probe according to the
manufacturers instructions in fast thermal cycling 96 well plates. 40
amplification
rounds were run in a Viaa7 Real Time PCR System (Applied Biosystems), with 1
sec
at 95 C for denaturing and 20 sec at 60 C for annealing/extension.
Fluoresecence
data were collected and converted to Ct-Values and expressed values were
calculated based on the comparative Ct method (Nat. Protoc. 3 (2008), 1101-
1108);
Analyzing real-time PCR data by the comparative C(T) method).
For the compound of example 6, in this test for the inhibition of collagen
type X
expression an 1050 value of 0.559 pM was determined.
