Note: Descriptions are shown in the official language in which they were submitted.
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Pyrazolof 1 5-alpyrimidin-7-yl-amine derivatives for use in the treatment of
protein kinase
dependent diseases
Summary of the Invention
The invention relates to the use of pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives in the
treatment of protein kinase dependent diseases, or for the manufacture of
pharmaceutical
compositions for use in the treatment of said diseases, methods of use of
pyrazolo[1,5a]pyrimidin-7-yl amine derivatives in the treatment of said
diseases,
pharmaceutical preparations comprising pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives for
the treatment of said diseases, novel pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives,
processes for the manufacture of the novel pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives
and pharmaceutical preparations, the use or methods of use of the
pyrazolo[1,5a]pyrimidin-
7-yl amine derivatives as mentioned above, and/or these
pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives for use in the treatment of the animal or human body.
Background of the Invention:
Pyrazolo[1,5-a]pyrimidin-7-yl-amine derivatives have been reported in the
literature as
ligands of benzodiazepine receptors (e.g., S. Selleri et al., Bioorg. Med.
Chem 7 (12), 2705-
11 (1999)), antagonists of the corticotropin releasing factor (EP 1097709),
angiotensin II
receptor antagonists (e. g., S. Takeshi et al., Japn. Pharm. Bull. 47 (7), 928-
38 (1999)),
monoxide synthetase inhibitors (JP 10101671), analgesics (WO 9535298),
fungicides (EP
071792) or anti-inflammatory reagents (WO 9218504).
We have now found that the pyrazolo[1,5-a]pyrimidin-7-ylaminene residue can be
also be
used as a template for the design of potent kinase inhibitors.
In view of the large number of protein kinase inhibitors and the multitude of
proliferative and
other protein kinase-related diseases, there is an ever-existing need to
provide novel classes
of compounds that are useful as protein kinase inhibitors and thus in the
treatment of related
diseases.
What is desirable from the point of view of possible treatments of
proliferative diseases is to
have a plethora of compound classes each tailored to specific protein kinases
or protein
kinase classes, thus allowing to come to specific treatments. Therefore, a
strong need
exists to find new classes of compounds allowing for such specific inhibitory
effects.
Summary of the Invention
The class of pyrazolo[1,5a]pyrimidin-7-yl amine compounds described herein,
especially
novel compounds falling under this class, has surprisingly been found to have
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pharmaceutically advantageous properties, allowing for the inhibition of
specific types or
classes or groups of kinases, especially c-Abl, Bcr-Abl, c-Kit, c-Raf, Flt-1,
Flt-3, KDR, Her-1,
PDGFR-kinase, c-Src, RET-receptor kinase, FGF-R1, FGF-R2, FGF-R3, FGF-R4,
Ephrin
receptor kinases (e. g., EphB2 kinase, EphB4 kinase and related Eph kinases),
casein
kinases (CK-1, CK-2, G-CK), Pak, ALK, ZAP70, Jak1, Jak2, Axl, Cdkl, cdk4,
cdk5, Met,
FAK, Pyk2, Syk, Insulin receptor kinase, Tie-2 or constitutively activating
mutations of
kinases (activating kinases) such as of Bcr-Abl, c-Kit, c-Raf, Flt-3, FGF-R3,
PDGF-receptors,
RET, and Met. The class of pyrazolo[1,5a]pyrimidin-7-yl amine compounds
described herein
further inhibit mutants of said kinases. In addition to this established
activity, the
pyrazolo(1,5a]pyrimidin-7-yl amine derivatives have the advantage in that
their backbone
allows for a plethora of substitution patterns that offer a broad possibility
to achieve a fine
tuning for specific interaction with the binding site of the targeted kinase
or kinases, thus
opening a new perspective and providing kinase inhibitors of various degrees
of specificity.
In view of these activities, the compounds can be used for the treatment of
diseases related
to especially aberrant or excessive activity of such types of kinases,
especially those
mentioned.
Detailed Description of the Invention
In one embodiment, the invention relates to the use of a compound of the
formula (I):
NHz
N~N ~ Rz
R~ /~ i
_N R3
A (I)
wherein:
RZ is H; substituted or unsubstituted aryl; substituted or unsubstituted
heteroaryl; substituted
or unsubstituted aliphatic residue; a functional group; or a substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl or substituted or unsubstituted
aliphatic residue which
is connected by one connecting group or atom to the pyrazolo[1,5a]pyrimidinyl
ring;
R3 can be H, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl,
substituted or unsubstituted aliphatic residue, a functional group, or a
substituted or
unsubstituted aliphatic residue which may be connected by a connecting group
or atom to
the pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
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heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl or substituted or unsubstituted heteroaryl; and
R~ is H, halogen or lower alkyl,
or pharmaceutically acceptable salts thereof for treating a protein kinase
dependent disease.
A preferred embodiment is the use of a compound according to the above,
wherein:
R2 is H; lower alkyl; cycloalkyl; benzyl; benzo thienyl, indyl substituted by
lower alkyl, pyridyl
or thiazolyl optionally substituted by lower alkyl; unsubstituted phenyl or
phenyl substituted
by one or two substituents chosen from the group consisting of; halo, hydroxy,
alkoxy,
benzyloxy, cycloalkyl, amino, acetyl amino, lower alkyl sulfonamide and
benzene
sulfonamide substituted by one or two halo;
R3 is H; lower alkyl optionally substituted by halo; phenyl, pyridyl, or
oxazolyl;
A is
(a) H; halo; benzothienyl; pyridyl; methyl piperazinyl phenoxyl; indolyl
substituted with lower
alkyl;
(b) phenyl which is unsubstituted or substituted with one or more of the
substituents chosen
from the group consisting of; mono-, di- or tri-lower alkoxy, di-lower
alkylaminyl, morpholinyl
which is optionally di-substituted by alkyl,
piperazinyl which is substituted with one or more of the substituents chosen
from the group
consisting of lower alkyl, lower alkoxy, lower alkyl piperazinyl,
pyrrolidinyl, dialkyl aminyl and
lower alkanol; and
R, is H,
or pharmaceutically acceptable salts thereof for treating a protein kinase
dependent disease.
A protein kinase dependent disease is preferably one that depends on c-Abl,
Bcr-Abl, c-Kit,
c-Raf, Flt-1, Flt-3, Her-1, KDR, PDGFR-kinase, c-Src, RET-receptor kinase, FGF-
R1, FGF-
R2, FGF-R3, FGF-R4, Ephrin receptor kinases (e. g., EphB2 kinase, EphB4 kinase
and
related Eph kinases), casein kinases (CK-1, CK-2, G-CK), Pak, ALK, ZAP70,
Jak1, Jak2,
Axl, Cdk1, cdk4, cdk5, Met, FAK, Pyk2, Syk, Insulin receptor kinase, Tie-2 or
costitutively
activating mutations of kinases (activating kinases) such as of Bcr-Abl, c-
Kit, c-Raf, Flt-3,
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FGF-R3, PDGF-receptors, RET, and Met and (especially aberrantly highly
expressed or
activated) kinase-dependent disease or disease dependent on the activation of
the kinase
pathways, or a disease dependent on any two or more of the kinases just
mentioned.
A protein kinase dependent disease is more preferably one that depends on c-
abl, Flt-3,
KDR, c-Src, RET, EphB4, c-kit, cdk1, FGFR-1, c-raf, Her-1, Ins-R orTek.
Most preferably, the disease to be treated is a proliferative disease,
preferably a benign or
especially malignant tumor, more preferably carcinoma of the brain, kidney,
liver, adrenal
gland, bladder, breast, stomach (especially gastric tumors), ovaries, colon,
rectum, prostate,
pancreas, lung, vagina, thyroid, sarcoma, glioblastomas, multiple myeloma or
gastrointestinal cancer, especially colon carcinoma or colorectal adenoma, or
a tumor of the
neck and head, an epidermal hyperproliferation, especially psoriasis, prostate
hyperplasia, a
neoplasia, especially of epithelial character, preferably mammary carcinoma,
or a leukemia.
In a further embodiment, the disease to be treated is a disease which is
triggered by
persistent angiogenesis, such as psoriasis; Kaposi's sarcoma; restenosis,
e.g., stent-
induced restenosis; endometriosis; Crohn's disease; Hodgkin's disease;
leukemia; arthritis,
such as rheumatoid arthritis; hemangioma; angiofibroma; eye diseases, such as
diabetic
retinopathy and neovascular glaucoma; renal diseases, such as
glomerulonephritis; diabetic
nephropathy; malignant nephrosclerosis; thrombotic microangiopathic syndromes;
transplant
rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the
liver; mesangial
cell-proliferative diseases; arteriosclerosis; injuries of the nerve tissue.
The compounds of the present invention can also be used for inhibiting the re-
occlusion of
vessels after balloon catheter treatment, for use in vascular prosthetics or
after inserting
mechanical devices for holding vessels open, such as, e.g., stents, as
immunosuppressants,
as an aid in scar-free wound healing, and for treating age spots and contact
dermatitis.
In a further embodiment, the invention relates to a compound of formula (I):
NH2
N~N ~ R2
i
~N R3
a (I)
wherein:
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R2 is H; substituted or unsubstituted aryl; substituted or unsubstituted
heteroarjrl; an aliphatic
residue; a functional group; or a substituted or unsubstituted aryl,
substituted or
unsubstituted heteroaryl or aliphatic residue which is connected by one
connecting group or
atom to the pyrazolo[1,5a]pyrimidinyl ring;
R3 can be H, substituted or unsubstituted aryl, heteroaryl, an aliphatic
residue, a functional
group, or an aliphatic residue which may be connected by a connecting group or
atom to the
pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring, and provided that both R2 and A cannot both be unsubstituted phenyl;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl or heteroaryl; and
R~ is H, halogen or lower alkyl,
or a pharmaceutically acceptable salt thereof.
A preferred embodiment is a compound according to the above, wherein;
R2 is H; lower alkyl; cycloalkyl; benzyl; benzo thienyl, indyl substituted by
lower alkyl, pyridyl
or thiazolyl optionally substituted by lower alkyl; unsubstituted phenyl or
phenyl substituted
by one or two substituents chosen from the group consisting of; halo, hydroxy,
alkoxy,
benzyloxy, cycloalkyl, amino, acetyl amino, lower alkyl sulfonamide and
benzene
sulfonamide substituted by one or two halo;
R3 is H; lower alkyl optionally substituted by halo; phenyl, pyridyl, or
oxazolyl;
A is
(a) H; halo; benzothienyl; pyridyl; methyl piperazinyl phenoxyl; indolyl
substituted with lower
alkyl;
(b) phenyl which is unsubstituted or substituted with one or more of the
substituents chosen
from the group consisting of; mono-, di- or tri-lower alkoxy, di-lower
alkylaminyl, morpholinyl
which is optionally di-substituted by alkyl,
piperazinyl which is substituted with one or more of the substituents chosen
from the group
consisting of lower alkyl, lower alkoxy, lower alkyl piperazinyl,
pyrrolidinyl, dialkyl aminyl and
lower alkanol; and
R~ is H;
and provided that both R2 and A cannot both be unsubstituted phenyl.
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Most preferably, the compound is selected from the group consisting of:
3-{7-Amino-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(3-benzyloxy-phenyl)-3-[4-(4-methyl,-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-yl)-
phenol;
6-(3-Methoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3,5-Dimethoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(4-Chloro-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-phenyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
5-Methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-6-phenyl-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-Methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-phenyl-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
N-{4-[7-Amino-3-(4-dimethylamino-phenyl)-pyrazolo[1,5-a]pyrimidin-6-yl]-
phenyl}-2,3-
dichloro-benzenesulfonamide;
4-Chloro-benzenesulfonic acid 4-(7-amino-3-(4-dimethylamino-phenyl)-
pyrazolo[1,5-
a]pyrimidin-6-yl]-phenyl ester;
6-(4-Methoxy-phenyl)-5-methyl-3-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
3-(4-Methoxy-phenyl)-5-methyl-6-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
6-(4-Bromo-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-(4-Bromo-phenyl)-5-methyl-3-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
6-(2,6-Dichloro-phenyl)-3-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
3-(3-Methoxy-phenyl)-6-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
3-Bromo-5-phenyl-pyrazolo(1,5-a]pyrimidin-7-ylamine;
6-Benzo[b]thiophen-3-yl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
3-(4-Bromo-phenyl)-5-phenyl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
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3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-thiophen-3-yl-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
3-Benzo[b]thiophen-3-yl-6-(3-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-Benzo-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-(3-Methoxy-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(1-Methyl-1 H-indol-3-yl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-(4-Methoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(2-Methoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[ 1, 5-
a]pyrim idin-7-
ylamine;
6-(3-Methoxy-phenyl)-3-pyridin-3-yl-pyrazolo[1, 5-a]pyrimidin-7-ylam ine;
3-{7-Amino-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(3-Benzyloxy-phenyl)-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
3-{7-Amino-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-6-yl}-
phenol;
6-(2-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
2-{7-Amino-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(4-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-py.razolo[1,5-
a]pyrimidin-7-
ylamine;
4-{7-Amino-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(2-Benzyloxy-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
2-{7-Amino-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(4-Benzyloxy-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
4-{7-Amino-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-phenol;
6-(2-Benzyloxy-phenyl)-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
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2-{7-Amino-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-6-yl}-
phenol;
6-(4-Benzyloxy-phenyl)-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
4-{7-Amino-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-6-yl)-
phenol;
6-(3-Benzyloxy-phenyl)-3-[1-methyl-1 H-indol-3-yl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
3-[7-Amino-3-(1-methyl-1 H-indol-3-yl)-pyrazolo[1,5-a]pyrimidin-6-yl]-phenol;
3-[7-Amino-3-pyridin-3-yl-pyrazolo[1,5-a]pyrimidin-6-yl]-phenol;
6-(3-Benzyloxy-phenyl)-3-(2-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
3-[7-Amino-3-(2-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-6-yl]-phenol;
3-[3-(4-Methyl-piperazin-1-yl)-phenyl]-6-thiophen-3-yl-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
3-(2-M ethoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-6-thiophen-3-yl-pyrazolo[1,
5-a]pyrimidin-7-
ylamine;
3-[4-(4-M ethyl-piperazin-1-yl)-phenyl]-6-pyridin-4-yl-pyrazolo[1, 5-
a]pyrimidin-7-ylam ine;
6-(3-Amino-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Amino-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(2-Amino-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
3-[4-(4-M ethyl-piperazin-1-yl)-phenyl]-6-(4-methyl-thiazol-2-yl)-pyrazolo[1,
5-a]pyrimidin-7-
ylamine;
6-Benzo[b]thiophen-3-yl-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-Benzo[b]thiophen-3-yl-3-[4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
3-(3-Methoxy-phenyl)-6-thiophen-3-yl-pyrazolo[1,5-a]pyrimidin-7-ylamine;
6-(3-Benzyloxy-phenyl)-3-(3-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
3-[7-Amino-3-(3-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-6-yl]-phenol;
(4-{7-Amino-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl)-phenyl)-
carbamic acid ethyl ester
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6-(3-Chloro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-
7-ylamine;
6-(3-Chloro-phenyl)-5-methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-3-[2-methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-5-methyl-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-3-[2-methoxy-4-(4-methyl-piperazin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
3-{7-Amino-3-[2-methoxy-4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-6-yl)-
phenol;
6-(2-Chloro-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(2-Chloro-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(4-Fluoro-phenyl)-5-methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-
7-ylamine;
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-py
razolo[1,5-a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-5-methyl-3-{3-[4-(1-methyl-piperidin-4-yl)-piperazin-1-yl]-
phenyl}-
pyrazolo[1,5-a]pyrimidin-7-ylamine;
6-(3-Chloro-4-fluoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-
phenyl]pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-4-fluoro-phenyl)-5-methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(3-Bromo-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3-Bromo-benzyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazo1o[1, 5-
a]pyrim idin-7-
ylamine;
6-(3-Bromo-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-5-methyl-3-(3-morpholin-4-yl-phenyl)-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-3-[3-((2R,6S)-2,6-dimethyl-morpholin-4-yl)-phenyl]-5-
methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine;
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2-(4-{3-[7-Amino-6-(3-chloro-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-3-yl]-
phenyl}-
piperazin-1-yl)-ethanol;
6-Benzyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-fluoromethyl-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-
7-ylamine;
6-(3-Chloro-4-fluoro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Chloro-4-fluoro-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(4-Fluoro-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
2-(4-{3-[7-Amino-6-(4-fl uoro-phenyl)-5-methyl-pyrazolo[ 1, 5-a]pyrimidin-3-
yl]-phenyl}-
piperazin-1-yl)-ethanol;
6-(3,4-Difluoro-phenyl)-5=methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3,4-Difluoro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
2-(4-{3-[7-Amino-6-(3-chloro-4-fluoro-phenyl)-5-methyl-,pyrazolo[1,5-a]
pyrimidin-3-yl]-
phenyl}-piperazin-1-yl)-ethanol;
2-(4-~3-[7-Amino-6-(3,4-difluoro-phenyl)-5-methyl-pyrazolo[ 1, 5-a]pyrim idin-
3-yl]-phenyl}-
piperazin-1-yl)-ethanol;
6-(3-Chloro-phenyl)-5-methyl-3-[3-(4-pyrrolidin-1-yl-piperidin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-pyrrolidin-1-yl-piperidin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-3-[3-(4-diethylamino-piperidin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
3-[3-(4-Diethylamino-piperidin-1-yl)-phenyl]-6-(4-fluoro-phenyl)-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-4-oxy-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-1,4-dioxy-piperazin-1-yl)-phenyl]-
pyrazolo[1, 5-
a]pyrimidin-7-ylamine;
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6-(3-Chloro-phenyl)-3-[3-(4-dimethylamino-piperidin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3,4-Difluoro-phenyl)-3-[3-(4-dimethylamino-piperidin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3-Chloro-phenyl)-5-methyl-3-(3,4,5-trimethoxy-phenyl)-pyrazolo[1,5-
a]pyrimidin-7-ylamine;
6-(3,4-Difluoro-phenyl)-5-methyl-3-(3,4,5-trimethoxy-phenyl)-pyrazolo[1,5-
a]pyrimidin-7-
ylamine;
6-(3-Chloro-phenyl)-3-(3-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine;
6-[7-Amino-3-(3,4-dimethoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-6-yl]-pyridin-2-
ol;
6-Benzyl-3-(3,4-dimethoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-ylamine; and
3-(3,4-Dimethoxy-phenyl)-6-(3-fluoro-benzyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine.
Yet another embodiment is the use of a compound according to the above in the
preparation
of a pharmaceutical composition.
Yet another embodiment is a pharmaceutical composition comprising a compound
according
to the above.
The pharmaceutical composition preferably comprises a compound according to
the above
and an acceptable pharmaceutical carrier.
In another embodiment, there is provided the use of a compound according to
the above in
the preparation of a pharmaceutical compositions for use in the treatment of a
kinase
dependent disease.
A further embodiment is a process to prepare a compound according to the above
comprising:
(a) reacting a nitrite, A-CH2-C=N, with ethyl formate in the presence of an
organic solvent to
form a substituted 3-oxo-propionitrile,
(b) condensing the substituted 3-oxo-propionitriles of step (a) with hydrazine
monohydrate in
an organic solvent to form a 2H-pyrazol-3-ylamine of formula (III):
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~ ~NH
R~
~NHZ
A
(d) formylating a substituted nitrite in the presence of ethanolate and formic
acid ethyl ester
to prepare a 3-oxo-propionitrile of formula (II):
N~\ R2
Rs
(II)
(c) condensing the 3-oxo- propionitrile of formula (II) and the 2H-pyrazol-3-
ylamines of
formula (III) in the presence of an organic solvent to form a compound of
formula (I).
The invention in particular relates to pyrazolo[1,5a]pyrimidin-7-yl amine
compounds of the
formula (I):
NH2
N~N ~ R2
R~ l ~ i
~N R3
A (I)
wherein:
R2 is H; substituted or unsubstituted aryl; substituted or unsubstituted
heteroaryl; substituted
or unsubstituted aliphatic residue; a functional group; or a substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl or substituted or unsubstituted
aliphatic residue which
is connected by one connecting group or atom to the pyrazolo[1,5a]pyrimidinyl
ring;
R3 is H, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted
or unsubstituted aliphatic residue, a functional group, or an aliphatic
residue which may be
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connected by a connecting group or atom to the pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
R~ is H, halogen or lower alkyl,
or pharmaceutically acceptable salts thereof,
in the treatment of protein kinase (especially tyrosine protein kinase)
dependent diseases or
for the manufacture of pharmaceutical compositions for use in the treatment of
said disea-
ses, methods of use of compounds of formula (I) in the treatment of said
diseases, or
pharmaceutical preparations comprising compounds of formula (I) for the
treatment of said
diseases.
The present invention is especially related to a compound of formula (I)
wherein R2 is H;
substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;
substituted or
unsubstituted aliphatic residue; a functional group; or a substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl or substituted or unsubstituted
aliphatic residue which
is connected by one connecting group or atom to the pyrazolo[1,5a]pyrimidinyl
ring;
R3 is H, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted
or unsubstituted aliphatic residue, a functional group, or an aliphatic
residue which may be
connected by a connecting group or atom to the pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring, and provided that R2 and A cannot both be unsubstituted phenyl;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl or heteroaryl; and
R, is H, halogen or lower alkyl,or pharmaceutically acceptable salts thereof,
in the treatment of protein kinase (especially tyrosine protein kinase)
dependent diseases or
for the manufacture of pharmaceutical compositions for use in the treatment of
said disea-
ses, methods of use of compounds of formula (I) in the treatment of said
diseases,
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pharmaceutical preparations comprising compounds of formula (I) for the
treatment of said
diseases, compounds of formula (I) for use in the treatment of said diseases.
The present invention also relates to a method of treating kinase dependent
diseases
comprising administering pyrazolo[1,5a]pyrimidin-7-yl amine compounds of the
formula (I) to
a warm-blooded animal, especially a human. The present invention also relates
to
pharmaceutical preparations comprising an pyrazolo[1,5a]pyrimidin-7-yl amine
compound of
the formula (I), especially for the treatment of a kinase dependent disease,
novel
pyrazolo[1,5a]pyrimidin-7-yl amine compounds of the formula (I), a process for
the
manufacture of the pyrazolo[1,5a]pyrimidin-7-yl amine compounds of the formula
(I), and
novel starting materials and intermediates for their manufacture. The present
invention also
relates to use of a compound of formula 1 in the manufacture of a
pharmaceutical
preparation for the treatment of a kinase dependent disease.
The general terms used hereinbefore and hereinafter preferably have within the
context of
this disclosure the following meanings, unless otherwise indicated:
"Aryl" is an aromatic radical having 6 to 14 carbon atoms, especially phenyl,
naphthyl,
indenyl, azulenyl, or anthryl, and is unsubstituted or substituted by one or
more, preferably
one or two substituents, wherein the substituents are selected from any of the
functional
groups defined below, and including: lower halo, alkyl, substituted alkyl,
halo lower alkyl e.g.
trifluoromethyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower alkoxy,
hydroxy, another
aryl, etherified or esterified hydroxy, amino, mono- or disubstituted amino,
amino lower alkyl,
amino lower alkoxy; acetyl amino; amidino, halogen, nitro, cyano, cyano lower
alkyl, carboxy,
esterified carboxy especially lower alkoxy carbonyl, e.g. methoxy carbonyl, n-
propoxy
carbonyl or iso-propoxy carbonyl, alkanoyl, benzoyl, carbamoyl, N-mono- or N,N-
disubstituted carbamoyl, carbamates, alkyl carbamic acid esters, amidino,
guanidino, urea,
ureido, mercapto, sulfo, lower alkylthio, sulfoamino, sulfonamide,
benzosulfonamide,
sulfonate, phenyl, benzyl, phenoxy, benzyloxy, phenylthio, phenyl-lower
alkytthio,
alkylphenylthio, lower alkylsulfinyl, phenylsulfinyl, phenyl-tower
alkylsulfinyl,
alkylphenylsulfinyl, lower alkanesulfonyl, phenylsulfonyl, phenyl-lower
alkylsulfonyl,
alkylphenylsulfonyl, halogen-lower, alkylmercapto, halogen-lower
alkylsulfonyl, such as
especially trifluoromethane sulfonyl, dihydroxybora (-B(OH)2), heterocyclyl,
and lower
alkylene dioxy bound at adjacent C-atoms of the ring, such as methylene dioxy,
phosphono
(-P(=O)(OH)2), hydroxy-lower alkoxy phosphoryl or di-lower alkoxyphosphoryl,
carbamoyl,
mono- or di-lower alkylcarbamoyl, mono- or di-(hydroxy-lower alkyl)-carbamoyl,
or-NR4R5,
wherein R4 and R5 can be the same or different and are independently H; lower
alkyl (e.g.
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methyl, ethyl or propyl); or R4 and R5 together with the N atom form a 3- to 8-
membered
heterocyclic ring containing 1-4 nitrogen, oxygen or sulfur atoms (e.g.
piperazinyl, lower
alkyl-piperazinyl, azetidinyl, pyrrolidinyl, piperidino, morpholinyl,
imidazoliny~.
Aryl is more preferably phenyl which is either unsubstituted or independently
substituted by
one or two substituents selected from a solubilizing group selected from the
group consisting
of: halo (such as CI, Br or F); hydroxy; lower alkyl (such as C~-C3 lower
alkyl or methyl); aryl
(such as phenyl or benzyl); amino; amino lower alkyl (such as dimethylamino);
acetyl amino;
amino lower alkoxy (such as ethoxyamine); substituted lower alkyl (such as
fluror ethyl);
alkoxy (such as methoxy or benzyloxy where the benzyl ring may be substituted
or
unsubstituted, such as 3, 4 -dichlorobenzyloxy); sulfoamino; substituted or
unsubstituted
sulfonamide (such as benzo sulfonamide, chlorobenzene sulfonamide or 2,3-
dichloro
benzene sulfonamide); substituted or unsubstituted sulfonate (such as chloro-
phenyl
sulfonate); substituted urea (such as 3-trifluoro-methyl-phenyl urea or 4-
morpholin-4-yl-3-
triflurormethyl-phenyl-urea); alkyl carbamic acid ester or carbamates (such as
ethyl-N-
phenyl-carbamate) or -NR4R5, wherein R4 and R5 can be the same or different
and are
independently H; lower alkyl (e.g. methyl, ethyl or propyl); or R4 and R5
together with the N
atom form a 3- to 8-membered heterocyclic ring containing 1-4 nitrogen, oxygen
or sulfur
atoms (e.g. piperazinyl, lower alkyl-piperazinyl, pyridyl, indolyl,
thiophenyl, thiazolyl,
morpholinyl n-methyl piperazinyl, benzothiophenyl, azetidinyl, pyrrolidinyl,
piperidino or
imidazolinyl) where when R4 and R5 together with the N form an heterocyclic
ring, said ring
may be substituted with 1, 2 or more of any of the substituents described
herein, preferably
piperazinyl, pyrrolidinyl, alkyl such as methyl, or hydroxy alkyl such as
ethanyl. Examples of
the heteroring formed by R4 and R5 together with the N include morpholinyl,
which can be
unsubstituted or substituted with methyl or dimethyl; piperazinyl which can be
unsubstituted
or substituted with 1, 2 or 3 substituents prefereably methyl, oxy or ethanol;
or piperadinyl
which can be unsubstituted or substituted with 1, 2 or 3 substituents
prefereably pyrrolidinyl,
amine, alkyl amine, methyl amine, dialkyl amine, dimethylamine or
diethylamine;
A heteroaryl group is preferably monocyclic, but may be bi- or tri-cyclic, and
comprises 3-24,
preferably 4-16 ring atoms, wherein at least one or more, preferably one to
four ring carbons
are replaced by a heteroatom selected from O, N or S. Preferably the
heteroaryl group is
selected from pyridyl, indolyl, pyrimidyl, pyrazolyl, oxazolyl, thiophenyl,
benzothiophenyl, 2H-
pyrrolyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, purinyl,
pyrazinyl,
pyridazinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinoxalyl,
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quinazolinyl, quinnolinyl, indolizinyl, 3H-indolyl, isoindolyl, isoxazolyl,
thiazolyl, isothiazolyl,
triazolyl, tetrazolyl, furazanyl and benzo[d]pyrazol.
More preferably the heteroaryl group is selected from the group consisting of
pyridyl, indolyl,
pyrimidyl, pyrazolyl, oxazolyl, thiophenyl or benzothiophenyl.
The heteroaryl group may be unsubstituted or substituted by one or more
substituents
selected from the group defined above as substituents for aryl, most
preferably by hydroxy,
halogen, lower alkyl, such as methyl or lower alkoxy, such as methoxy or
ethoxy.
Aliphatic as used herein refers to any non-aromatic carbon based residue.
Examples of
aliphatic residues include substituted or unsubstituted alkyl, cycloalkyl,
alkenyl and alkynyl.
Alkyl includes lower alkyl preferably alkyl with up to 7 carbon atoms,
preferably from 1 to and
including 5, and is linear or branched; preferably, lower alkyl is pentyl,
such as n-pentyl,
butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such as n-
propyl or isopropyl,
ethyl or methyl. Preferably lower alkyl is methyl, propyl or tent-butyl.
A cycloalkyl group is preferably cyclopentyl, cyclohexyl or cycloheptyl, and
may be unsubsti-
tuted or substituted by one or more, especially one or two, substituents
selected from the
group defined above as substituents for aryl, most preferably by lower alkyl
such as methyl,
lower alkoxy such as methoxy or ethoxy, or hydroxy.
Alkenyl and alkynyl preferably have up to 7 carbon atoms, preferably from 1 to
and including
5, and can be linear or branched.
Alkyl, cycloalkyl, alkenyl and alkynyl can be substituted or unsubstituted,
and when
substituted may be with up to 3 substituents including other alkyl,
cycloalkyl, alkenyl, alkynyl,
any of the substituents defined above for aryl or any of the functional groups
defined below.
Halo or halogen is preferably fluoro, chloro, bromo or iodo, most preferably
fluoro, chloro or
bromo.
The term "connecting atom or group" as used herein includes alkyl, (such as -
CH2-); oxy -O
keto -CO-; thio -S-; sulfonyl -SO2-; sulfoxides -SO-; amines -NH- or-NR-;
carboxylic acid;
alcohol; esters (-COO-); amides (- -CONK-, -CONHR'-); sulfonamides (, -SOzNH-,
-SOZNR'-
); sulfones (-SOZ-); sulfoxides (-SO-); amino-group; ureas ( -NH-CO-NH-, -NR-
CO-NH-, -NH-
CO-NR-, -NR-CO-NR-); ethers (-O-); carbamates (-NH-CO-O-, -NR-CO-O-); or
inverse
amides sulfonamides and esters (-NH-CO-, -NR-CO-, -NH-S02-, -NR-SOZ-, -OOC-).
The term "functional group" as used herein includes: carboxylic acid;
hydroxyl; halogen;
cyano (-CN); ethers (-OR); ketones (-CO-R); esters (-COOR); amides (-CONH2, -
CONHR, -
CONRR'); thioethers (-SR); sulfonamides (-S02NH2, -S02NHR, -S02NRR'); sulfones
(-SOZ-
R); sulfoxides (-SO-R); amines (-NHR, NR'R); ureas (-NH-CO-NH2, -NH-CO-NHR);
ethers (-
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O-R); halogens; carbamates (-NH-CO-OR); aldehyde-function (-CHO); then also
inverse
amides; sulfonamides and esters (-NH-CO-R, -NH-SOz-R, -OOC-R);
R and R' are the same are different and may be H or are any aliphatic, aryl or
heteroaryl
moiety as defined above.
Where the plural form is used for compounds, salts, pharmaceutical
preparations, diseases
and the like, this is intended to mean also a single compound, salt, or the
like.
Salts are especially the pharmaceutically acceptable salts of compounds of
formula I.
Such salts are formed, for example, as acid addition salts, preferably with
organic or inor-
ganic acids, from compounds of formula (I) with a basic nitrogen atom,
especially the phar-
maceutically acceptable salts. Suitable inorganic acids are, for example,
halogen acids, such
as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic
acids are, for
example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example
acetic acid, pro-
pionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid,
lactic acid, fumaric
acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid,
malic acid, tartaric
acid, citric acid, amino acids, such as glutamic acid or aspartic acid,
malefic acid, hydroxy-
maleic acid, methylmaleic acid, cyclohexanecarboxylic acid,
adamantanecarboxylic acid,
benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid,
phenylacetic acid, mandelic
acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic
acid, ethane-
1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5-
naphthalene-
disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid,
ethylsulfuric acid,
dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-
propyl-sulfamic
acid, or other organic protonic acids, such as ascorbic acid.
In the presence of negatively charged radicals, such as carboxy or sulfo,
salts may also be
formed with bases, e.g. metal or ammonium salts, such as alkali metal or
alkaline earth me-
tal salts, for example sodium, potassium, magnesium or calcium salts, or
ammonium salts
with ammonia or suitable organic amines, such as tertiary monoamines, for
example triethyl-
amine or tri(2-hydroxyethyl)amine, or heterocyclic bases, for example N-ethyl-
piperidine or
N,N'-dimethylpiperazine.
When a basic group and an acid group are present in the same molecule, a
compound of
formula (I) may also form internal salts.
For isolation or purification purposes it is also possible to use
pharmaceutically unacceptable
salts, for example picrates or perchlorates. For therapeutic use, only
pharmaceutically
acceptable salts or free compounds are employed (where applicable in the form
of pharma-
ceutical preparations), and these are therefore preferred.
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In view of the close relationship between the compounds in free form and those
in the form
of their salts, including those salts that can be used as intermediates, for
example in the
purification or identification of the compounds, tautomers or tautomeric
mixtures and their
salts, any reference to the compounds hereinbefore and hereinafter especially
the
compounds of the formula I, is to be understood as referring also to the
corresponding
tautomers of these compounds, especially of compounds of the formula I,
tautomeric mix-
tures of these compounds, especially of compounds of the formula I, or salts
of any of these,
as appropriate and expedient and if not mentioned otherwise.
Where "a compound ..., a tautomer thereof; or a salt thereof' or the like is
mentioned, this
means "a compound ..., a tautomer thereof, or a salt of the compound or the
tautomer".
Any asymmetric carbon atom may be present in the (R)-, (S)- or (R,S)-
configuration, pre-
ferably in the (R)- or (S)-configuration. Substituents at a ring at atoms with
saturated bonds
may, if possible, be present in cis- (= Z-) or trans (= E-) form. The
compounds may thus be
present as mixtures of isomers or preferably as pure isomers, preferably as
enantiomer-pure
diastereomers or pure enantiomers.
The present invention also relates to pro-drugs of a compound of formula (I)
that convert in
vivo to the compound of formula (I) as such. Any reference to a compound of
formula (I) is
therefore to be understood as referring also to the corresponding pro-drugs of
the compound
of formula (I), as appropriate and expedient.
The compounds of formula (I) have valuable pharmacological properties and are
useful in
the treatment of kinase dependent diseases, e.g., as drugs to treat
proliferative diseases.
The term "treatment of tyrosine protein kinase dependent diseases" refers to
the prophylac-
tic or preferably therapeutic (including palliative and/or curing) treatment
of said diseases,
especially of the diseases mentioned below.
Where subsequently the term "USE" is mentioned, this includes any one or more
of the fol-
lowing embodiments of the invention, respectively: the use in the treatment of
(especially ty-
rosine) protein kinase dependent diseases, the use for the manufacture of
pharmaceutical
compositions for use in the treatment of said diseases, methods of use of
pyrazolo[1,5a]pyrimidin-7-yl amine derivatives in the treatment of said
diseases,
pharmaceutical preparations comprising pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives for
the treatment of said diseases, and pyrazolo[1,5a]pyrimidin-7-yl amine
derivatives for use in
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the treatment of said diseases, as appropriate and expedient, if not stated
otherwise. In
particular, diseases to be treated and are thus preferred for USE of a
compound of formula
(I) are selected from (especially tyrosine) protein kinase dependent
("dependent" meaning
also "supported", not only "solely dependent") diseases mentioned below,
especially
corresponding proliferative diseases, more especially diseases that depend on
c-Abl, Bcr-
Abl, c-Kit, c-Raf, Flt-1, Flt-3, KDR, Her-1, PDGFR-kinase, c-Src, RET-receptor
kinase, FGF-
R1, FGF-R2, FGF-R3, FGF-R4, Ephrin receptor kinases (e. g., EphB2 kinase,
EphB4 kinase
and related Eph kinases), casein kinases (CK-1, CK-2, G-CK), Pak, ALK, ZAP70,
Jak1,
Jak2, Axl, Cdk1, cdk4, cdk5, Met, FAK, Pyk2, Syk, Insulin receptor kinase, Tie-
2 or
constitutively activating mutations of kinases (activating kinases) such as of
Bcr-Abl, c-Kit, c-
Raf, Flt-3, FGF-R3, PDGF-receptors, RET, and Met, (hereinafter "said kinases")
can
therefore be used in the treatment of kinase dependent diseases, especially
diseases
depending on said kinases and (especially aberrantly highly-expressed or
constitutively
activated) said kinase-dependent disease or disease dependent on the
activation of the said
kinase pathways or any combination of two or more of the mentioned kinases.
Most preferred is use of a compound of formula (I) for treating diseases
dependant upon c-
abl, Flt-3, KDR, c-Src, RET, EphB4, c-kit, cdk1, FGFR-1, c-raf, Her-1, Ins-R
and Tek, and
use of a compound of formula (I) as an inhibitor of c-abl, Flt-3, KDR, c-Src,
RET, EphB4, c-
kit, FGFR-1, c-raf, cdk1, Her-1, Ins-R and Tek.
There are also experiments to demonstrate the antitumor activity of compounds
of the
formula (I) in vivo.
The compounds of formula (I) have valuable pharmacological properties and are
useful in
the treatment of protein kinase dependent diseases, e.g., as drugs to treat
proliferative
diseases.
The inhibition of RET is measured as follows: The baculovirus donor vector pFB-
GSTX3
is used to generate a recombinant baculovirus that expresses the amino acid
region 658-
1072 (Swiss prot No. Q9BTB0) of the intra-cytoplasmic kinase domain of human
RET-
Men2A which corresponds to the wild-type kinase domain of RET (wtRET) and RET-
Men2B,
which differs from the wtRET by the activating mutation in the activation loop
M918T. The
coding sequences for the cytoplasmic domain of wtRET and RET-Men2B are
amplified by
PCR from the plasmids pBABEpuro RET-Men2A and pBABEpuro RET-Men2B. The
amplified DNA fragments and the pFB-GSTX3 vector are made compatible for
ligation by
digestion with Sall and Kpnl. Ligation of these DNA fragments results in the
baculovirus
donor plasmid pFB-GX3-RET-Men2A and pFB-GX3-RET-Men2B, respectively.
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Production of virus: Transfer vectors containing the kinase domains are
transfected into the
DH10Bac cell line (GIBCO) and plated on selective agar plates. Colonies
without insertion
of the fusion sequence into the viral genome (carried by the bacteria) are
blue. Single, white
colonies are picked and viral DNA (bacmid) are isolated from the bacteria by
standard
plasmid purification procedures. Sf9 cells or Sf21 (American Type Culture
Collection) cells
are then transfected in 25 cm2 flasks with the viral DNA using Cellfectin
reagent.
Determination of small scale protein expression in Sf9 cells: Virus-containing
media is
collected from the transfected cell culture and used for infection to increase
its titer. Virus-
containing media obtained after two rounds of infection is used for large-
scale protein
expression. For large-scale protein expression 100 cmz round tissue culture
plates are
seeded with 5 x 10' cellsiplate and infected with 1 mL of virus-containing
media
(approximately 5 MOIs). After 3 days, the cells are scraped off the plate and
centrifuged at
500 rpm for 5 minutes. Cell pellets from 10-20, 100 cm2 plates, are re-
suspended in 50 mL
of ice-cold lysis buffer (25 mM tris-HCI, pH 7.5, 2 mM EDTA, 1 % NP-40, 1 mM
DTT, 1 mM P
MSF). The cells are stirred on ice for 15 minutes and then centrifuged at
5,000 rpms for 20
minutes.
Purification of GST tagged proteins: The centrifuged cell lysate is loaded
onto a 2 mL
glutathione-sepharose column (Pharmacia) and is washed 3 x with 10 mL of 25 mM
tris-HCI,
pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCI. The GST-tagged proteins are then
eluted
by 10 applications (1 mL each) of 25 mM tris-HCI, pH 7.5, 10 mM reduced-
glutathione, 100
mM NaCI, 1 mM DTT, 10% glycerol and stored at -70°C.
Measure of enzyme activity: Tyrosine protein kinase assays with either
purified GST-wtRET
or GST-RET-Men2B protein are carried out in a final volume of 30 pL containing
15 ng of
either GST-wtRET or GST-RET-Men2B protein, 20 mM tris-HCI, pH 7.5, 1 mM MnCl2,
10
mM MgCl2, 1 mM DTT, 3 pglmL poly(GIu,Tyr) 4:1, 1% DMSO, 2.0 pM ATP (y-[33P]-
ATP 0.1
pCi). The activity is assayed in the presence or absence of inhibitors, by
measuring the
incorporation of 33P from [y33P~ ATP into poly(GIu,Tyr) 4:1. The assay is
carried out in 96-
well plates at ambient temperature for 15 minutes under conditions described
below and
terminated by the addition of 20 pL of 125 mM EDTA. Subsequently, 40 pL of the
reaction
mixture are transferred onto Immobilon-PVDF membrane (Millipore) previously
soaked for 5
minutes with methanol, rinsed with water, then soaked for 5 minutes with 0.5%
H3P04 and
mounted on vacuum manifold with disconnected vacuum source. After spotting all
samples,
vacuum is connected and each well-rinsed with 200 pL 0.5% H3P04. Membranes are
removed and washed 4 x on a shaker with 1.0% H3P04, once with ethanol.
Membranes are
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counted after drying at ambient temperature, mounting in Packard TopCount 96-
well frame,
and addition of 10 NUwell of Microscint TM (Packard). ICSO values are
calculated by linear
regression analysis of the percentage inhibition of each compound in
duplicate, at 4
concentrations (usually 0.01, 0.1, 1 and 10 pM). One unit of protein kinase
activity is defined
as 1 nmole of 33P ATP transferred from [y33P] ATP to the substrate
protein/minute/mg of
protein at 37°C.
ICSO calculations
input 3 x 4 pL stopped assay on Immobilon membrane, not washed
background (3 wells) assay with H20 instead of enzyme
positive control (4 wells) 3% DMSO instead of compound
bath control (1 well) no reaction mix
ICSO values are calculated by logarithmic regression analysis of the
percentage inhibition of
each compound at 4 concentrations (usually 3- or 10-fold dilution series
starting at 10 pM).
In each experiment, the actual inhibition by reference compound is used for
normalization of
ICSO values to the basis of an average value of the reference inhibitor:
Normalized ICSO = measured ICSO average ref. ICSO / measured ref. ICSo
Example: Reference inhibitor in experiment 0.4 pM, average 0.3 pM
Test compound in experiment 1.0 pM, normalization: 0.3/0.4 = 0.75 NM
For example, staurosporine or a synthetic staurosporine derivative are used as
reference
compounds.
Using this protocol, the compounds of the formula (I) are found to show ICSO
values for RET
inhibition in the range from 0.005-100 pM, preferably in the range from 0.01-2
pM.
The efficacy of the compounds of the invention as inhibitors of c-Abl protein-
tyrosine
kinase activity can be demonstrated as follows: An in vitro enzyme assay is
performed in
96-well plates as a filter binding assay as described by Geissler et al. in
Cancer Res. 1992;
52:4492-4498, with the following modifications. The His-tagged kinase domain
of c-Abl is
cloned and expressed in the baculovirus/Sf9 system as described by Bhat et al.
in
J.BioLChem. 1997; 272:16170-16175. A protein of 37 kD (c-Abl kinase) is
purified by a two-
step procedure over a Cobalt metal chelate column followed by an anion
exchange column
with a yield of 1-2 mg/L of Sf9 cells (Bhat et al., reference cited). The
purity of the c-Abl
kinase is >90% as judged by SDS-PAGE after Coomassie blue staining. The assay
contains
(total volume of 30 pL): c-Abl kinase (50 ng), 20 mM Tris~HCl, pH 7.5, 10 mM
MgClz, 10 pM
Na3V04, 1 mM DTT and 0.06 pCi/assay [~/33 P]-ATP (5 pM ATP) using 30 pg/mL
poly-
AIa,GIu,Lys,Tyr-6:2:5:1 (Poly-AEKY, Sigma P1152) in the presence of 1 % DMSO.
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._....._... _._.._ 22
Reactions are terminated by adding 10 pL of 250 mM EDTA and 30 NL of the
reaction
mixture is transferred onto Immobilon-PVDF membrane (Millipore, Bedford, MA,
USA)
previously soaked for 5 min with methanol, rinsed with water, then soaked for
5 min with 0.5
H3PO4 and mounted on vacuum manifold with disconnected vacuum source. After
spot-
ting all samples, vacuum is connected and each well rinsed with 200 pL 0.5 %
H3P04. Mem-
branes are removed and washed on a shaker with 0.5 % H3P04 (4 times) and once
with
ethanol. Membranes are counted after drying at ambient temperature, mounting
in Packard
TopCount 96-well frame, and addition of 10 NUwell of Microscint TM (Packard).
Using this test system, compounds of the formula I show ICSO values of
inhibition for c-Abl
inhibition in the range of 0.002 to 100 ~M, usually between 0.002 and 5 pM.
The efficacy of the compounds of the invention as inhibitors of KDR protein-
tyrosine
kinase activity can be demonstrated as follows: The inhibition of VEGF-induced
receptor
autophosphorylation can be confirmed with a further in vitro experiments in
cells such as
transfected CHO cells, which permanently express human VEGF-R2 receptor (KDR),
are
seeded in complete culture medium (with 10% fetal calf serum = FCS) in 6-well
cell-culture
plates and incubated at 37°C under 5% C02 until they show about 80%
confluency. The
compounds to be tested are then diluted in culture medium (without FCS, with
0.1 % bovine
serum albumin) and added to the cells. (Controls comprise medium without test
compounds). After two hours' incubation at 37°C, recombinant VEGF is
added; the final
VEGF concentration is 20 ngiml). After a further five minutes incubation at
37°C, the cells
are washed twice with ice-cold PBS (phosphate-buffered saline) and immediately
lysed in
100 p1 lysis buffer per well. The lysates are then centrifuged to remove the
cell nuclei, and
the protein concentrations of the supernatants are determined using a
commercial protein
assay (BIORAD). The lysates can then either be immediately used or, if
necessary, stored at
-20°C.
A sandwich ELISA is carried out to measure the VEGF-R2 phosphorylation: a
monoclonal
antibody to VEGF-R2 (for example Mab 1495.12.14; ProQinase, Freiburg, Germany)
is
immobilized on black ELISA plates (OptiPIateTM HTRF-96 from Packard). The
plates are
then washed and the remaining free protein-binding sites are saturated with 3%
TopBlock~
(Juro, Cat. # TB232010) in phosphate buffered saline with Tween 20~
(polyoxyethylen(20)-
sorbitane monolaurate, ICI/Uniquema) (PBST). The cell lysates (20 Ng protein
per well) are
then incubated in these plates overnight at 4°C together with an
antiphosphotyrosine
antibody coupled with alkaline phosphatase (PY20:AP from Zymed). The (plates
are washed
again and the) binding of the antiphosphotyrosine antibody to the captured
phosphorylated
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receptor is then demonstrated using a luminescent AP substrate (CDP-Star,
ready to use,
with Emerald II; Applied Biosystems). The luminescence is measured in a
Packard Top
Count Microplate Scintillation Counter. The difference between the signal of
the positive
control (stimulated with VEGF) and that of the negative control (not
stimulated with VEGF)
corresponds to VEGF-induced VEGF-R2 phosphorylation (= 100 %). The activity of
the
tested substances is calculated as percent inhibition of VEGF-induced VEGF-R2
phosphorylation, wherein the concentration of substance that induces half the
maximum
inhibition is defined as the ICSO (inhibitory dose for 50% inhibition).
Compounds of the
formula I here show an ICSO in the range of 0.005 to 20 ~M, preferably between
0.005 and 1
~.M for KDR inhibition.
FIt3 kinase inhibition is determined as follows: The baculovirus donor vector
pFbacG01
(GIBCO) is used to generate a recombinant baculovirus expressing the amino
acid region
amino acids 563-993 of the cytoplasmic kinase domain of human Flt-3. The
coding sequen-
ce for the cytoplasmic domain of Flt-3 is amplified by PCR from human c-DNA
libraries
(Clontech). The amplified DNA fragments and the pFbacG01 vector are made
compatible for
ligation by digestion with BamH1 and Hindlll. Ligation of these DNA fragments
results in the
baculovirus donor plasmid Flt-3(1.1). The production of the viruses, the
expression of pro-
teins in Sf9 cells and the purification of the GST-fused proteins are
performed as follows:
Production of virus: Transfer vector (pFbacG01-Flt-3) containing the Flt-3
kinase domain is
transfected into the DH10Bac cell line (GIBCO) and the transfected cells are
plated on selec-
tive agar plates. Colonies without insertion of the fusion sequence into the
viral genome (car-
ried by the bacteria) are blue. Single white colonies are picked and viral DNA
(bacmid) is iso-
lated from the bacteria by standard plasmid purification procedures. Sf9 or
Sf21 cells (Ameri-
can Type Culture Collection) are then transfected in flasks with the viral DNA
using Cellfectin
reagent.
Determination of small scale protein expression in Sf9 cells: Virus containing
media is collec-
ted from the transfected cell culture and used for infection to increase its
titre. Virus contai-
ning media obtained after two rounds of infection is used for large-scale
protein expression.
For large-scale protein expression 100 cm2 round tissue culture plates are
seeded with 5 x
10' cells/plate and infected with 1 mL of virus-containing media (approx. 5
MOIs). After 3
days the cells are scraped off the plate and centrifuged at 500 rpm for 5 min.
Cell pellets
from 10-20, 100 cm2 plates, are resuspended in 50 mL of ice-cold lysis buffer
(25 mMTris-
HCI, pH 7.5, 2mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF). The cells are stirred
on ice
for 15 min and then centrifuged at 5000 rpms for 20 min.
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Purification of GST tagged proteins: The centrifuged cell lysate is loaded
onto a 2 mL gluta-
thione-sepharose column (Pharmacia) and washed three times with 10 mL of 25 mM
Tris-
HCI, pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCI. The GST-tagged protein is then
eluted
by 10 applications (1 mL each) of 25 mM Tris-HCI, pH 7.5, 10 mM reduced-
glutathione, 100
mM NaCI, 1 mM DTT, 10 % Glycerol and stored at -70°C.
Measurement of enzyme activity: Tyrosine protein kinase assays with purified
GST-Flt-3 are
carried out in a final volume of 30 pL containing 200-1800 ng of enzyme
protein (depending
on the specific activity), 20 mM Tris-HCI, pH 7.6, 3 mM MnCl2, 3 mM MgCh, 1 mM
DTT, 10
pM Na3V04, 3 wg/mL poly(GIu,Tyr) 4:1, 1 % DMSO, 8.0 p.M ATP and 0.1 ~Ci [y33
P] ATP).
The activity is assayed in the presence or absence of inhibitors, by measuring
the incorpo-
ration of 33P from [y33P] ATP into the poly(GIu,Tyr) substrate. The assay (30
p,L) is carried
out in 96-well plates at ambient temperature for 20 min under conditions
described below
and terminated by the addition of 20 p,L of 125 mM EDTA. Subsequently, 40 wL
of the
reaction mixture is transferred onto Immobilon-PVDF membrane (Millipore,
Bedford, MA,
USA) previously soaked for 5 min with methanol, rinsed with water, then soaked
for 5 min
with 0.5 % H3P04 and mounted on vacuum manifold with disconnected vacuum
source. After
spotting all samples, vacuum is connected and each well rinsed with 200 p.L
0.5 % H3P04.
Membranes are removed and washed 4 x on a shaker with 1.0 % H3P04, once with
ethanol.
Membranes are counted after drying at ambient temperature, mounting in Packard
TopCount 96-well frame, and addition of 10 p.Llwell of Microscint TM
(Packard). ICSO values
are calculated by linear regression analysis of the percentage inhibition of
each compound in
duplicate, at four concentrations (usually 0.01, 0.1, 1 and 10 p,M). One unit
of protein kinase
activity is defined as 1 nmole of 33P ATP transferred from [y33P] ATP to the
substrate protein
per minute per mg of protein at 37 °C. The compounds of the formula I
show ICSO values for
Flt-3 inhibition in the range between 0.01 and 100 pM, preferably between 0.05
and 10 wM.
The compounds of formula I also inhibit other tyrosine protein kinases such as
especially the
c-Src kinase, c-Kit, VEGF-R and/or FGFR; all of which play a part in growth
regulation and
transformation in animal, especially mammal cells, including human cells. An
appropriate
assay is described in Andrejauskas-Buchdunger et al., Cancer Res. 52, 5353-8
(1992).
Using this test system, compounds of the formula I show ICSo values for
inhibition of c-Src in
the range of 0.005 to 100 p.M, usually between 0.005 and 5 ~M. Compounds of
formula I
also show ICSO values for c-kit inhibition in the range of 0.005 to 10 pM,
usually between
0.005 and 5 p,M; and for inhibition of FGFR-1, up to 95% inhibition at 10 p,M.
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The inhibition of IGF-1 R and Ins-R can be determined as follows: The
baculovirus donor
vector pfbgx3lGFIRcd is used to generate a recombinant baculovirus that
expresses the
amino acid region 950-1337 of the mature peptide cytoplasmic domain of the
human IGF-IR.
To generate the cDNA fragment encoding the amino acid region 919-1343 of the
intra-
cytoplasmic kinase domain of the human insulin receptor, pCShInsR is used. The
fragments
of the human IGF-IR and Ins-R are cloned, expressed and small-scale purified
as a factor
Xa-cleavable glutathione-S-transferase (GST)-fusion protein using the Bac-to-
BacTM system
(GIBCO BRL) of recombinant baculovirus generation. Virus containing media iss
collected
from the transfected cell culture and used for infection to increase its
titer. Virus containing
media obtained after two rounds of infection iss used for large-scale protein
expression. Cell
extracts are prepared and loaded onto a glutathione-Sepharose (Pharmacia)
column. After
washing, the GST-tagged proteins are then eluted with a glutathione-containing
buffer.
Purified protein is stored at -70°C in elution buffer. Tyrosine protein
kinase assays with
purified GST-IGF-1 R and GST-Ins-R are carried in a final volume of 30 p1
containing 20 mM
Tris-HCI, pH 7:6, 10 mM MgCl2, 0.01 mM Na3V04, 1 % DMSO, 1 mM DTT, 3 pg/ml
poly(GIu,Tyr) 4:1 and 10 pM ATP (y-[33P]-ATP 0.1 NCi). The assay is performed
in 96-well
plates at ambient temperature for 20 min and terminated by addition of 25 p1
0.05 M EDTA
pH 7Ø An aliquot of 40 p1 is spotted with a multichannel dispenser on
Whatman P81
membranes mounted in a Millipore Microtiter filter manifold connected to a low
vacuum
source. After elimination of liquid, the membrane is transferred to a sequence
of 4 washing
baths containing 0.5% H3P04 and one with EtOH (shaking incubation for 10 min
each),
dried, mounted onto a Hewlett Packard TopCount manifold added 10 NI
Microscint~ and
counted. Compounds of formula (I) show up to 90% inhibition of Ins-R at
10,OOOnM,
preferably between 60-90% inhibition.
The inhibition of Tek can be determined as follows: The procedure of the
expression,
purification and assay these kinases has been described. Fabbro et al.,
Pharmacol. Ther.
82(2-3) 293-301 (1999). In brief, the glutathione S-transferase (GST) gene
from the pAcG1
vector (Pharmingen) is excised with EcoRV and EcoRl and inserted into the
cloning site of
the Fast-Bac baculoviral vector (GIBCO) creating a 5530 by vector with N-
terminal cloning
sites derived from the pAcG1 fusion vector (FBGO). The C-terminal cloning site
may be any
cloning site (from the Fast-Bac vector) downstream of the N-terminal cloning
site used. N-
terminally GST-fused (pAcG1, Pharmingen) KDR, Flt-1, Flk-1, Tek and PDGFR-[3
kinase
domains are obtained from ProQinase, Freiburg, Germany. Tek is recloned into
the FBG1
vector by EcoRl excision and ligation into EcoRl digested FBG1 (FBG1-Tek). The
coding
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sequences for the whole cytoplasmic domain of c-Kit (aa 544-976) and c-Fms (aa
538-972)
are amplified by PCR from human uterus and from human bone marrow cDNA
libraries
(Clontech), respectively. The amplified DNA fragments are fused to GST by
cloning them
into FBG1 as BamHl-EcoRl insertions, to yield FBG1-c-Kit and FBG1-c-Fms. Tek
is recloned
into the FBGO transfer vector by EcoRl excision and ligation into EcoRl
digested FBGO
(FBG-Tie2/Tek). FGFR-1 and c-met kinase domains are obtained by PCR from human
A431
cells. N-terminal primers contain an overhanging EcoRl site, while C-terminal
primers
contain a Xhol site to aid cloning into the transfer vectors. After digestion
of both the PCR
fragments and FBGO the cleavage products are gel-purified and ligated together
to form the
kinase constructs (FBG-Met, FBG-FGFR-1).
Viruses for each of the kinases are made according to the protocol supplied by
GIBCO. In
brief, transfer vectors containing the kinase domains are transfected into the
DH10Bac cell
line (GIBCO), plated on agar plates containing the recommended concentrations
of Blue-
Gal, IPTG, Kanamycin, Tetracycline, and Gentamycin. Colonies without insertion
of the
fusion sequence into the viral genome (carried by the bacteria) are blue. A
single white
colony is usually picked and viral DNA (bacmid) isolated from the bacteria by
standard
plasmid mini prep procedures. Sf9 cells or High Five cells (GIBCO) are then
transfected in
25 cm2 flasks with the viral DNA using the Cellfectin reagent and protocol
supplied with the
Bac-to-Bac kit (GIBCO). Virus containing media is collected from the
transfected cell culture
and used for infection to increase its titer. Virus containing media obtained
after two rounds
of infection is used for large-scale protein expression. For large-scale
protein expression
100 cm2 round tissue culture plates are seeded with 5x10' cells/plate and
infected with 1 ml
of virus-containing media (about 5 MOIs). After 3 days the cells are scraped
off the plate and
centrifuged at 500 rpm for 5 min.
Cell pellets from 10-20, 100 cm2 plates, are resuspended in 50 ml of ice-cold
lysis buffer
(25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF). The cells
are
stirred on ice for 15 min and then centrifuged at 5000 rpms for 20 min. The
supernatant is
loaded onto a 2 ml glutathione-sepharose column and washed three times with 10
ml of
25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCI. The GST-tagged
proteins
are then eluted by 10 applications. (1 ml each) of 25 mM Tris-HCI, pH 7.5, 10
mM reduced-
glutathione, 100 mM NaCI, 1 mlVl DTT, 10% Glycerol and stored at -70°C.
The assays (30 p1) contain 200-1800 ng of enzyme protein (depending on the
specific
activity), 20 mM Tris-HCI, pH 7.6, 3 mM MnCl2, 3 mM MgCIZ, 1 mM DTT, 10 pM
Na3V04,
3 pg/ml poly(GIu,Tyr) 4:1, 8 pM ATP (y-[33P]-ATP 0.1 pCi). Reactions are
incubated for
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20 min at ambient temperature and then stopped by addition of 25 p1 0.25 M
EbTA (pH 7.0).
An aliquot of 40 p1 is spotted with a multichannel dispenser on Immobilon P
membranes
mounted in a Millipore Microtiter filter manifold connected to a low vacuum
source. After
elimination of liquid, the membrane is transferred to a sequence of 4 washing
baths
containing 0.5% H3P04 and one with EtOH (shaking incubation for 10 min each),
dried,
mounted onto a Hewlett Packard TopCount manifold added 10 p1 Microscint~ and
counted.
Compounds of formula (I) show IC50 values, calculated by linear regression
analysis, for Tek
inhibition of about 0.1-100p.M.
The inhibition of Cdk1 can be determined as follows: Cdk1/cycB: Cdk1/cycB are
obtained from ProQinase, Freiburg, Germany. Starfish oocytes are induced to
enter M phase
of the cell cycle with 10 ~M 1-methyladenine and frozen in liquid nitrogen and
stored at -
80°C. When required, the oocytes are homogenized and centrifuged as
described (Arion et
al., Cell 55: 371-378 (1988) and Rialet et al., Anticancer Res. 11: 1581-1590
(1991)).
Cdk1/cycB kinase is purified on pgcKSns-sepharose beads and eluted with
recombinant
human pgcKSns as described (Azzi et al., Eur. J. Biochem. 203: 353-360.
(1992)). Briefly, the
supernatant from oocytes is equilibrated for 30 min at 4°C under
constant rotation with the
p9cKSns-sepharose beads. The beads are extensively washed and active cdk1/cycB
kinase is
eluted with purified p9CKShs (3 mg/ml). The activity of Cdk1/cycB is measured
as described
(Arion et al., Cell 55: 371-378 (1988), Meijer et al., EMBO J. 1989; 8: 2275-
2282 and Meijer
et al., EMBO J. 1991; 8: 2275-2282). The assay is carried with slight
modifications in 96-well
plates at ambient temperature for 20 min. The final volume of 30 p1 contains
0.1-0.3U of
Cdk1/cycB, 1 mg/ml histone H1 as a substrate, 60 mM ~i-glycerophosphate, 30 mM
nitrophenylphosphate, 25 mM MOPS, 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 0.1 mM
Na3V04, 15 pM ATP and 0.1 p,Ci y-33P-ATP (75 pM, 8800 cpm/pmole). The reaction
is
terminated by addition of 25 p1 0.05 M EDTA pH 7Ø An aliquot of 40 p1 is
spotted with a
multichannel dispenser on Immobilon P membranes mounted in a Millipore
Microtiter filter
manifold connected to a low vacuum source. After elimination of liquid, the
membrane is
transferred to a sequence of 4 washing baths containing 0.5% H3P04 and one
with EtOH
(shaking incubation for 10 min each), dried, mounted onto a Hewlett Packard
TopCount
manifold added 10 p1 Microscint~ and counted. Compounds of formula (I) show up
to 100%
Cdk1 inhibition at 10,OOOnM.
The inhibition of c-Raf-1 can be determined as follows: Production of
recombinant c-Raf-
1 protein, is obtained by triple infection of Sf21 cells with GST-c-Raf-1
recombinant
baculovirus together with v-Src and v-Ras recombinant baculoviruses that are
required for
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active c-Raf-1 kinase production (Williams et al., PNAS 1992; 89: 2922-
2926).~Active Ras (v-
Ras) is required to recruit c-Raf-1 to the cell membrane and v-Src to
phosphorylate c-Raf-1
to fully activate it (Williams et al., PNAS 1992; 89: 2922-2926). Cells were
seeded at
2.5 x 10'cells per 150 mm dish and allowed to attach to a 150 mm dish for 1 hr
at RT. Media
(SF90011 containing 10 % FBS) is aspirated and recombinant baculovirus; GST-C-
Raf-1, v-
Ras and v-Src are added at MOI of 3.0, 2.5 and 2.5 receptively in a total
volume of 4-5 mL.
Cells are incubated for 1 hr at RT and then 15 mL of medium is added. Infected
cells are
incubated for 48-72 hr at 27°C. Infected Sf21 cells are scraped and
collected into a 50 mL
tube and centrifuged for 10 min at 4°C at 1100 g in a Sorvall
centrifuge. The cell pellet is
washed once with ice cold PBS and lysed with 0.6 mL lysis buffer per 2.5 x 10'
cells.
Complete lysis of cells is achieved after 10 min on ice with occasional
pipetting. The cell
lysates are centrifuged for 10 min at 4°C at 14,500 g in a Sorvall
centrifuge with SS-34 rotor
and the supernatant is transferred to a fresh tube and stored at -80°C.
c-Raf-1 is purified
from cell lysates using 100 uL of packed Glutathione-Sepharose 4B beads
equilibrated in
ice cold PBS per 2.5 x 10' cells. GST-c-Raf-1 was allowed to bind to the beads
at 4°C for 1hr
with rocking. Bound GST-c-Raf-1 with beads was transferred to a column. The
column is
washed once with lysis buffer and twice with ice cold Tris buffered saline.
Ice cold elution
buffer is added and column flow is stopped to allow the free glutathione to
disrupt the
interaction of GST-c-Raf-1 with glutathione sepharose beads. Fractions (1 mL)
are collected
into pre-chilled tubes. Each tube contains 10 % glycerol (final concentration)
to maintain
kinase activity during freeze thaw cycles. Purified fractions of GST-c-Raf-1
kinase protein
are stored at -80°C.
IKB was used as substrate for the c-Raf-1 kinase. IxB is expressed in bacteria
as a His-
tagged protein (cloned and kindly provided by Dr. Eder; ABM, Novartis, Basel).
BL21 LysS
bacteria containing the IKB plasmid are grown to an ODsoo of 0.6 in LB medium
then induced
to express the kb with IPTG (final concentration of 1 mM) for 3 hrs at
37° C and then
bacteria are lysed by sonication (microtip limit setting for 3 times at 1 min
each in sonication
buffer [50 mM Tris pH 8.0, 1 mM DTT, 1 mM EDTA] and centrifuged at 10,000 g
for 15 min.
The supernatant is mixed with ammonium sulfate to give a final concentration
of 30 %. This
mixture is rocked for 15 min at 4 °C then spun at 10,000 g for 15 min.
The pellet is
resuspended in binding buffer (Novagen) containing 10 mM BSA. This solution is
applied to
Ni-agarose (Novagen) and washed according to the Novagen manual. IKB is eluted
from the
column using elution buffer (0.4 M imidazole, 0.2 M NaCI, 8 mM Tris pH 7.9).
Fractions
containing protein are dialysed in 50 mM Tris pH 8, 1 mM DTT.
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The activity of c-Raf-1 protein kinase is assayed in the presence or absence
of inhibitors, by
measuring the incorporation of 33P from [y33P] ATP into IB. The assay is
carried out in 96-
well plates at ambient temperature for 60 min. It contains (total volume of 30
p1): c-rafl1
kinase (400 ng), 25 mM Tris~HCl, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 10 pM Na3V04,
1 mM
DTT and 0.3 pCi/assay ~Y33 P]_ATP (10 pM ATP) using 600 ng IB in the presence
of 1
DMSO. Reactions are terminated by adding 10 pL of 250 mM EDTA and 30 pL of the
reaction mixture is transferred onto Immobilon-PVDF membrane (Millipore,
Bedford, MA,
USA) previously soaked for 5 min with methanol, rinsed with water, then soaked
for 5 min
with 0.5 % H3P04 and mounted on vacuum manifold with disconnected vacuum
source. After
spotting all samples, vacuum is connected and each well rinsed with 200 pL 0.5
% H3P04.
Membranes are removed and washed 4 x on a shaker with 0.5 % H3P04, once with
ethanol.
Membranes are counted after drying at ambient temperature, mounting in Packard
TopCount 96-well frame, and addition of 10 pL/well of Microscint TM (Packard).
Compounds of formula (I) show c-Raf-1 inhibition in the range between 0.1-50
wM,
preferably between 0.1 and 10 ~,M.
Experiments to demonstrate the antitumor activity of compounds of the formula
(I) in
vivo: For example, in order to test whether a compound of the formula (I),
e.g. that of
Example 1 given below, inhibits VEGF-mediated angiogenesis in vivo, its effect
on the
angiogenic response induced by VEGF in a growth factor imlant model in mice is
tested: A
porous Teflon chamber (volume 0.5 mL) is filled with 0.8 % w/v agar containing
heparin (20
units/ml) with or without growth factor (2 ~g/ml human VEGF) is implanted
subcutaneously
on the dorsal flank of C57/C6 mice. The mice are treated with the test
compound (e.g. 25,
50 or 100 mg/kg p.o. once daily) or vehicle starting on the day of
implantation of the
chamber and continuing for 4 days after. At the end of the treatment, the mice
are killed, and
the chambers are removed. The vascularized tissue growing around the chamber
is carefully
removed and weighed, and the blood content is assessed by measuring the
hemoglobin
content of the tissue (Drabkins method; Sigma, Deisenhofen, Germany). It has
been shown
previously that these growth factors induce dose-dependent increases in weight
and blood
content of this tissue growing (characterized histologically to contain
fibroblasts and small
blood vessels) around the chambers and that this response is blocked by
antibodies that
specifically neutralize VEGF (see Wood JM et al., Cancer Res. 60(8), 2178-
2189, (2000);
and Schlaeppi et al., J. Cacner Res. Clin. Oncol. 125, 336-342, (1999)). With
this model,
inhibition can be shown in the case of compounds of the formula (I).
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Synthetic Procedure
Compounds of formula (I) are prepared analogously to the procedure described
by Alicade,
E; De Mendoza, J; Garcia-Marquina, JM; Almera, C; J. Heterocycl. Chem. 11, 423
(1974) by:
(a) reacting a nitrite, A-CH2-C=N, with ethyl formate in the presence of an
organic solvent to
form a substituted 3-oxo-propionitrile,
(b) condensing the substituted 3-oxo-propionitriles of step (a) with hydrazine
monohydrate in
an organic solvent to form a 2H-pyrazol-3-ylamine of formula (III):
~ ~NH
R~
~NHZ
A
(d) formylating a substituted nitrite in the presence of ethanolate and formic
acid ethyl ester
to prepare a 3-oxo-propionitrile of formula (II):
\\ R2
Ra
(II)
(c) condensing the 3-oxo- propionitrile of formula (II) and the 2H-pyrazol-3-
ylamines of
formula (III) in the presence of an organic solvent to form a compound of
formula (I).
Specifically,compounds of formula (I) are prepared by condensing 3-oxo-
propionitriles (II)
and the corresponding 2H-pyrazol-3-ylamines (III) in the presence of ethanolic
HCI (Figure
2). The 2H-pyrazol-3-ylamines (III) are prepared by condensing hydrazine
monohydrate with
the corresponding 3-oxo-propionitriles dissolved in an organic solvent, such
as EtOH,
dioxane or AcOH and heated at elevated temperatures (preferably at 100
°C) for several
hours. The preferred procedure for preparing the pyrazolo moiety of the title
compounds was
stirring the hydrazine monohydrate with the corresponding 3-oxo-propionitriles
in acetic acid
at 100 °C for 2-3 h followed by addition of aqueous HCI and further
refluxing the reaction
mixture for further 20 min. In case where R1 is not H, the corresponding
substituted
hydrazines are used. The 3-oxo-propionitriles (I) and (II) are synthesized
from the
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corresponding nitrites by classical formylation reaction using freshly
prepared sodium
ethanolate and formic acid ethyl ester (refluxing for 1 h in EtOH).
Alternatively, instead of
performing the condensation reactions with the 3-oxo-propionitiles, the
corresponding 3,3-
dialkoxy-propionitiles (in analogy to the procedure described by Seneci, P.,
Nicola, M.,
Inglesi, M., Vanotti, E., Resnati, G. Synth. Commun. 29 (2), 311-341 (1999))
or 3-
dimethylamino-acrylonitriles can be used.
\\ R NHa
2
R / ~NH II R NON \ R2
/ O R3 ~ // ~
NHZ NI _R3
A EtOH, ethanolic HCI, reflux ,q
Hydrazine monohydrate (in case of R1 = H),
1) AcOH, 100 °C, 2-3 h
2) aqueous HCI, reflux, 20 min
O
H
i ~N
/ /
A A
IV NaOEt, HCOOEt,
EtOH, reflux, 1 h
Figure 2.
Alternatively, compounds of formula (I) can be prepared by first synthesizing
the
pyrazolo[1,5-a]pyrimidin-7-ylamine core scaffold carrying a corresponding
functional groups
(X, see Figure 3) where residues A, R2, or R3, respectively, can be introduced
by known
reactions as indicated in Figure 3.
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NHa
X = functional group for further N~N \ X
chemical modifications R~ /
i
N R3
A
NHZ
NON \ Rz
R' li
'N R3
A
NHZ
N~N \ R2
NHZ R~ /
N~ Rz N X
/ N \~ A
R1 i
N R~
X
Figure 3.
wherein R~, R~, R3, and X are as defined for compounds of the formula I,
and, if desired, after reaction (a), (b) or (c), transforming an obtainable
compound of formula
(I) into a different compound of formula I; transforming a salt of an
obtainable compound of
formula (I) into the free compound or a different salt or an obtainable free
compound of
formula (I) into a salt; and/or separating an obtainable mixture of isomers of
compounds of
formula (I) into the individual isomers;
where for all reactions mentioned functional groups in the starting materials
that shall not
take part in the reaction are, if required, present in protected form by
readily removable pro-
tecting groups, and any protecting groups are subsequently removed.
The following reaction conditions are preferred, respectively:
Within the scope of this text, only a readily removable group that is not a
constituent of the
particular desired end product of formula (I) is designated a "protecting
group", unless the
context indicates otherwise. The protection of functional groups by such
protecting groups,
the protecting groups themselves, and their cleavage reactions are described
for example in
standard reference works, such as J. F. W. McOmie, "Protective Groups in
Organic Che-
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mistry", Plenum Press, London and New York 1973, in T. W. Greene and P. G. M.
Wuts,
"Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999,
in "The Pep-
tides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London
and New
York 1981, in "Methoden der organischen Chemie" (Methods of Organic
Chemistry), Houben
Weyl, 4th edition, Volume 1511, Georg Thieme Verlag, Stuttgart 1974, in H.-D.
Jakubke and
H. Jeschkeit, "Aminosauren, Peptide, Proteine" (Amino acids, Peptides,
Proteins), Verlag
Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann,
"Chemie der
Kohlenhydrate: Monosaccharide and Derivate" (Chemistry of Carbohydrates:
Monosaccha-
rides and Derivatives), Georg Thieme Verlag, Stuttgart 1974. A characteristic
of protecting
groups is that they can be removed readily (i.e. without the occurrence of
undesired secon-
dary reactions) for example by solvolysis, reduction, photolysis or
alternatively under physio-
logical conditions (e.g. by enzymatic cleavage).
Salts of compounds of formula (I) having at least one salt-forming group may
be prepared in
a manner known per se. For example, salts of compounds of formula (I) having
acid groups
may be formed, for example, by treating the compounds with metal compounds,
such as
alkali metal salts of suitable organic carboxylic acids, e.g. the sodium salt
of 2-ethylhexanoic
acid, with organic alkali metal or alkaline earth metal compounds, such as the
corresponding
hydroxides, carbonates or hydrogen carbonates, such as sodium or potassium
hydroxide,
carbonate or hydrogen carbonate, with corresponding calcium compounds or with
ammonia
or a suitable organic amine, stoichiometric amounts or only a small excess of
the salt-
forming agent preferably being used. Acid addition salts of compounds of
formula (I) are
obtained in customary manner, e.g. by treating the compounds with an acid or a
suitable
anion exchange reagent. Internal salts of compounds of formula (I) containing
acid and basic
salt-forming groups, e.g. a free carboxy group and a free amino group, may be
formed, e.g.
by the neutralisation of salts, such as acid addition salts, to the
isoelectric point, e.g. with
weak bases, or by treatment with ion exchangers.
Salts can be converted in customary manner into the free compounds; metal and
ammonium
salts can be converted, for example, by treatment with suitable acids, and
acid addition salts,
for example, by treatment with a suitable basic agent.
Mixtures of isomers obtainable according to the invention can be separated in
a manner
known per se into the individual isomers; diastereoisomers can be separated,
for example,
by partitioning between polyphasic solvent mixtures, recrystallisation and/or
chromatographic
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separation, for example over silica gel or by e.g. medium pressure liquid
chromatography
over a reversed phase column, and racemates can be separated, for example, by
the forma-
tion of salts with optically pure salt-forming reagents and separation of the
mixture of dia-
stereoisomers so obtainable, for example by means of fractional
crystallisation, or by chro-
matography over optically active column materials.
Intermediates and final products can be worked up and/or purified according to
standard
methods, e.g. using chromatographic methods, distribution methods, (re-)
crystallization, and
the like.
General process conditions
The following applies in general to all processes mentioned hereinbefore and
hereinafter,
while reaction conditions specifically mentioned above or below are preferred:
All the above-mentioned process steps can be carried out under reaction
conditions that are
known per se, preferably those mentioned specifically, in the absence or,
customarily, in the
presence of solvents or diluents, preferably solvents or diluents that are
inert towards the re-
agents used and dissolve them, in the absence or presence of catalysts,
condensation or
neutralizing agents, for example ion exchangers, such as cation exchangers,
e.g. in the H+
form, depending on the nature of the reaction and/or of the reactants at
reduced, normal or
elevated temperature, for example in a temperature range of from about -100
°C to about
190°C, preferably from approximately -80°C to approximately
150°C, for example at from -80
to -60°C, at room temperature, at from -20 to 40°C or at reflux
temperature, under atmos-
pheric pressure or in a closed vessel, where appropriate under pressure,
and/or in an inert
atmosphere, for example under an argon or nitrogen atmosphere.
At all stages of the reactions, mixtures of isomers that are formed can be
separated into the
individual isomers, for example diastereoisomers or enantiomers, or into any
desired mix-
tures of isomers, for example racemates or mixtures of diastereoisomers, for
example ana-
logously to the methods described under "Additional process steps".
The solvents from which those solvents that are suitable for any particular
reaction may be
selected include those mentioned specifically or, for example, water, esters,
such as lower
alkyl-lower alkanoates, far example ethyl acetate, ethers, such as aliphatic
ethers, for
example diethyl ether, or cyclic ethers, for example tetrahydrofurane or
dioxane, liquid
aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol,
ethanol or
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1- or 2-propanol, nitrites, such as acetonitrile, halogenated hydrocarbons,
such as methylene
chloride or chloroform, acid amides, such as dimethylformamide or dimethyl
acetamide, ba-
ses, such as heterocyclic nitrogen bases, for example pyridine or N-
methylpyrrolidin-2-one,
carboxylic acid anhydrides, such as lower alkanoic acid anhydrides, for
example acetic an-
hydride, cyclic, linear or branched hydrocarbons, such as cyclohexane, hexane
or isopen-
tane, or mixtures of those solvents, for example aqueous solutions, unless
otherwise indica-
ted in the description of the processes. Such solvent mixtures may also be
used in working
up, for example by chromatography or partitioning.
The compounds, including their salts, may also be obtained in the form of
hydrates, or their
crystals may, for example, include the solvent used for crystallization.
Different crystalline
forms may be present.
The invention relates also to those forms of the process in which a compound
obtainable as
intermediate at any stage of the process is used as starting material and the
remaining pro-
cess steps are carried out, or in which a starting material is formed under
the reaction condi-
tions or is used in the form of a derivative, for example in protected form or
in the form of a
salt, or a compound obtainable by the process according to the invention is
produced under
the process conditions and processed further in situ. In the process of the
present invention
those starting materials are preferably used which result in new compounds of
formula (I)
described at the beginning as being especially valuable. Special preference is
given to reac-
tion conditions that are identical or analogous to those mentioned in the
Examples.
Preferred embodiments according to the invention:
In the following preferred embodiments, general expression can be replaced by
the cor-
responding more specific definitions provided above and below, thus yielding
stronger
preferred embodiments of the invention.
Preferred is the USE of compounds of the formula I, tautomers thereof or
pharmaceutically
acceptable salts thereof, where the tyrosine protein kinase dependent disease
to be treated
is a proliferative disease depending on any one or more of of the following
tyrosine protein
kinases: especially c-Abl, Bcr-Abl, c-Kit, c-Raf, Flt-1, Flt-3, KDR, Her-1,
PDGFR-kinase, c-
Src, RET-receptor kinase, FGF-R1, FGF-R2, FGF-R3, FGF-R4, Ephrin receptor
kinases (e.
g., EphB2 kinase, EphB4 kinase and related Eph kinases), casein kinases (CK-1,
CK-2, G-
CK), Pak, ALK, ZAP70, Jak1, Jak2, Axl, Cdk1, cdk4, cdk5, Met, FAK, Pyk2, Syk,
Insulin
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receptor kinase, Tie-2 or constitutively activating mutations of kinases
(activating kinases)
such as of Bcr-Abl, c-Kit, cdk1, c-Raf, Flt-3, FGF-R3, PDGF-receptors, RET,
and Met.
More preferably, compounds of formula (I) may be used to treat a proliferative
disease
depending on the following kinases: c-abl, Flt-3, KDR, c-Src, RET, EphB4, c-
kit, cdk1,
FGFR-1, c-raf, Her-1, Ins-R and Tek.
The invention relates especially to a compound of the formula (I),
NH2
N~N ~ R2
R1 /
i i
N R3
A (I)
wherein:
R2 is H; substituted or unsubstituted aryl; substituted or unsubstituted
heteroaryl; substituted
or unsubstituted aliphatic residue; a functional group; or a substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl or substituted or unsubstituted
aliphatic residue which
is connected by one connecting group or atom to the pyrazolo[1,5a]pyrimidinyl
ring;
R3 can be H, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl,
substituted or unsubstituted aliphatic residue, a functional group, or an
aliphatic residue
which may be connected by a connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl or substituted or unsubstituted heteroaryl; and
R~ is H, halogen or lower alkyl,
or pharmaceutically acceptable salts thereof,
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and use of compounds of formula (I) in the treatment of kinase dependent
diseases or for
the manufacture of pharmaceutical preparations for the treatment of kinase
dependent
diseases.
The invention further relates to a compound of the formula (I),
NH2
N~N ~ R2
R~ /~ i
~N R3
A (I)
wherein:
RZ is H; substituted or unsubstituted aryl; substituted or unsubstituted
heteroaryl; substituted
or unsubstituted aliphatic residue; a functional group; or a substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl or substituted or unsubstituted
aliphatic residue which
is connected by one connecting group or atom to the pyrazolo[1,5a]pyrimidinyl
ring;
R3 can be H, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl,
substituted or unsubstituted aliphatic residue, a functional group, or a
substituted or
unsubstituted aliphatic residue which may be connected by a connecting group
or atom to
the pyrazolo[1,5a]pyrimidinyl ring,
at least one of R2 or R3 is substituted or unsubstituted aryl; substituted or
unsubstituted
heteroaryl; or a substituted or unsubstituted heteroaryl or substituted or
unsubstituted aryl
residue which is connected by one connecting group or atom to the
pyrazolo[1,5a]pyrimidinyl
ring;
and provided that R2 and A cannot both be unsubstituted phenyl;
A is H, halogen (such as bromo), an aliphatic moiety, a functional group,
substituted or
unsubstituted aryl or heteroaryl; and
R, is H, halogen or lower alkyl,
or pharmaceutically acceptable salts thereof,
and use of compounds of formula (I) in the treatment of kinase dependent
diseases or for
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the manufacture of pharmaceutical preparations for the treatment of kinase
dependent
diseases.
More preferred is a compound of the formula (I), wherein
the connecting atom or group is selected from the group consisting of: alkyl,
(such as -CH2-
); oxy -O-; keto -CO-; thio -S-; sulfonyl -SO2-; sulfoxides -SO-; amines -NH-
or -NR-;
carboxylic acid; alcohol; esters (-COO-); amides (- -CONK-, -CONHR'-);
sulfonamides ( -
SOZNH-, -SOZNR'-); (-S03-); sulfoxides (-SO-); amino-group; ureas ( -NH-CO-NH-
, -NR-CO-
NH-, -NH-CO-NR-, -NR-CO-NR-); ethers (-O-); carbamates (-NH-CO-O-, -NR-CO-O-);
or
inverse amides sulfonamides and esters (-NH-CO-, -NR-CO-, -NH-S02-, -NR-SO2-, -
OOC-);
with alkyl, (such as -CH2-); oxy -O-; keto -CO-; sulfonyl -SOZ-; sulfonamides
(-SOZNH-, -
S02NR'-); (-S03-); and ureas ( -NH-CO-NH-, -NR-CO-NH-, -NH-CO-NR-, -NR-CO-NR-)
being especially preferred,
and the functional group is selected from the group consisting of: carboxylic
acid; hydroxyl;
halogens; cyano (-CN); ethers (-OR); ketones (-CO-R); esters (-COOR); amides (-
CONH2, -
CONHR, -CONRR'); thioethers (-SR); sulfonamides (-SO~NH2, -S02NHR, -SO~NRR');
sulfones (-S02-R); sulfoxides (-SO-R); amines (-NHR, NR'R); ureas (-NH-CO-NH2,
-NH-CO-
NHR); ethers (-O-R); halogens; carbamates (-NH-CO-OR); aldehyde-function (-
CHO); then
also inverse amides; sulfonamides and esters (-NH-CO-R, -NH-S02-R, -OOC-R);
with
halogens; hydroxyl; ethers (-OR); amides (-CONH2, -CONHR, -CONRR');
sulfonamides (-
S02NH2, -S02NHR, -S02NRR'); amines (-NHR, NR'R); and ureas (-NH-CO-NHS, -NH-CO-
NHR); being especially preferred,
or a pharmaceutically acceptable salt thereof, as such or especially for use
in the
preparation of a pharmaceutical composition, or for use in the diagnostic or
therapeutic
treatment of a warm-blooded animal, especially a human.
Especially preferred is a compound of the formula (I), wherein
A is H; a halo (such as Br); or aryl (such as phenyl or benzyl) or
heterocyclyl (such as
pyridinyl, indolyl or benzothiophenyl),
wherein the aryl or heterocyclyl may be substituted or unsubstituted with up
to 4, preferably
up to 2 substituents, wherein the substituents are the same or different and
are
independently selected from halo (such as CI or Br); hydroxy; amino; amino
lower alkyl (such
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as dimethylamino); amino lower alkoxy (such as ethoxyamine); lower alkyl (such
as methyl);
lower alkoxy (such as methoxy); substituted or unsubstituted sulfonamide (such
as benzo
sulfonamide, chlorobenzene sulfonamide or dichloro benzene sulfonamide);
carbamates;
R4R5, wherein R4 and R5 can be the same or different and are independently H;
lower alkyl
(e.g. methyl, ethyl or propyl); or R4 and R5 together with the N atom form a 3-
to 8-membered
heterocyclic ring containing 1-4 nitrogen, oxygen or sulfur atoms (e.g.
piperazinyl or lower
alkyl piperazinyl) where when R4 and RS together with the N form an
heterocyclic ring, said
ring may be substituted with 1, 2 or more of any of the substituents described
herein,
preferably piperazinyl, pyrrolidinyl, alkyl such as methyl, or hydroxy alkyl
such as ethanyl.
Examples of the heteroring formed by R4 and R5 together with the N include
morpholinyl,
which can be unsubstituted or substituted with methyl or dimethyl; piperazinyl
which can be
unsubstituted or substituted with 1, 2 or 3 substituents prefereably methyl,
oxy or ethanol;
or piperadinyl which can be unsubstituted or substituted with 1, 2 or 3
substituents
prefereably pyrrolidinyl, amine, alkyl amine, methyl amine, dialkyl amine,
dimethylamine or
diethylamine;
Rz is H, C~-C3 lower alkyl (such as methyl) or aryl (such as phenyl or benzyl)
or heterocyclyl
(such as pyridyl, indolyl, thiophenyl, thiazolyl or benzothiophenyl), wherein
the aryl or
heterocyclyl may be substituted or unsubstituted with up to 4, preferably up
to 2 substituents,
wherein the substituents are the same or different and are independently
selected from halo
(such as CI, F or Br); hydroxy; amino; amino lower alkyl; C~-C3 lower alkyl;
alkoxy (such as
methoxy and benzyloxy where the benzyl ring may be substituted or
unsubstituted, such as
3, 4 -dichlorobenzyloxy); sulfoamino; substituted or unsubstituted
benzosulfonamide (such
as 2, 3-dichloroberizene sulfonamide); substituted or unsubstituted sulfonate
(such as
chloro-phenyl sulfonate); substituted or unsubstituted ureas (such as 3-
trifluoro-methyl-
phenyl urea or 4-morpholin-4-yl-3-triflurormethyl-phenyl-urea) or carbamates
(such as ethyl-
N-phenyl carbamate);
R3 is H; C~-C3 alkyl, methyl; phenyl; pyridinyl or oxaz-5-yl;
or a pharmaceutically acceptable salt thereof, as such or especially for use
in the
preparation of a pharmaceutical composition, or for use in the diagnostic or
therapeutic
treatment of a warm-blooded animal, especially a human.
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Especially preferred is the use of a compound of formula (I), or a
pharmaceutically
acceptable salt thereof, in the manufacture of a pharmaceutical preparation
for the treatment
of a kinase dependent disease.
Also preferred is a compound of the formula (I), or a pharmaceutically
acceptable salt
thereof, as shown above for use in the treatment of a kinase dependent
disease, especially
one depending on said kinases and (especially aberrantly highly expressed or
constitutively
activated) said kinases-dependent disease or disease dependent on the
activation of the
said kinases pathways or disease dependent on any two or more of said kinases.
In a broader sense of the invention, a kinase dependant disease may be a
proliferative
disease including a hyperproliferative condition, such as leukemias,
hyperplasias, fibrosis
(especially pulmonary, but also other types of fibrosis, such as renal
fibrosis), angiogenesis,
psoriasis, atherosclerosis and smooth muscle proliferation in the blood
vessels, such as
stenosis or restenosis following angioplasty.
Very preferred is a method of treating a kinase dependent disease comprising
administering
a compound of formula (I), where the disease to be treated is a proliferative
disease,
preferably a benign or especially malignant tumor, more preferably carcinoma
of the brain,
kidney, liver, adrenal gland, bladder, breast, stomach (especially gastric
tumors), ovaries,
colon, rectum, prostate, pancreas, lung (especially SCLC), vagina, thyroid,
sarcoma,
glioblastomas, multiple myeloma or gastrointestinal cancer, especially colon
carcinoma or
colorectal adenoma, or a tumor of the neck and head, an epidermal
hyperproliferation,
especially psoriasis, prostate hyperplasia, a neoplasia, especially of
epithelial character,
preferably mammary carcinoma, or a leukemia. Also for the treatment of
atherosclerosis,
thrombosis, psoriasis, scleroderma and fibrosis.
Compounds of formula (I) are able to bring about the regression of tumors and
to prevent
the formation of tumor metastases and the growth of (also micro)metastases. In
addition
they can be used in epidermal hyperproliferation (e.g. psoriasis), in prostate
hyperplasia, and
in the treatment of neoplasias, especially of epithelial character, for
example mammary
carcinoma. It is also possible to use the compounds of formula (I) in the
treatment of
diseases of the immune system insofar as several or, especially, individual
tyrosine protein
kinases are involved; furthermore, the compounds of formula (I) can be used
also in the
treatment of diseases of the central or peripheral nervous system where signal
transmission
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by at least one tyrosine protein kinase, especially selected from those
mentioned specifically,
is involved.
KDR inhibitors are thus especially appropriate for the therapy of diseases
related to VEGF
receptor tyrosine kinase overexpression. Among these diseases, especially
retinopathies,
age-related macula degeneration, psoriasis, haemangioblastoma, haemangioma,
arteriosclerosis, inflammatory diseases, such as rheumatoid or rheumatic
inflammatory
diseases, especially arthritis, such as rheumatoid arthritis, or other chronic
inflammatory
disorders, such as chronic asthma, arterial or post-transplantational
atherosclerosis,
endometriosis, and especially neoplastic diseases, for example so-called solid
tumors
(especially cancers of the gastrointestinal tract, the pancreas, breast,
stomach, cervix,
bladder, kidney, prostate, ovaries, endometrium, lung, brain, melanoma,
Kaposi's sarcoma,
squamous cell carcinoma of head and neck, malignant pleural mesotherioma,
lymphoma or
multiple myeloma) and liquid tumors (e.g. leukemias) are especially important.
FIt3 (FMD-like tyrosine kinase) is especially expressed in hematopoietic
progenitor cells and
in progenitors of the lymphoid and myeloid series. Aberrant expression of the
FIt3 gene has
been documented in both adult and childhood leukemias including AML (acute
myelogenous
leukemia), AML with trilineage myelodysplasia (AML/TMDS), ALL (acute
lymphoblastic leu-
kemia), CML (chronic myelogenous leukemia) and myelodysplastic syndrome (MDS),
which
are therefore the preferred diseases to be treated with compounds of the
formula I. Activa-
ting mutations in FIt3 have been found in approximately 25 to 30 % of patients
with AML.
Thus there is accumulating evidence for the role of FIt3 in human leukemias,
and the
pyrazolo[1,5a]pyrimidin-7-yl amine derivatives useful according to the
invention, especially
the compounds of the formula I, as FIt3 inhibitors are especially of use in
the therapy of this
type of diseases (see Tse et al., Leukemia 15(7), 1001-1010 (2001); Tomoki et
al., Cancer
Chemother. Pharmacol. 48 (Suppl. 1), S27-S30 (2001); Birkenkamp et al.,
Leukemia 15(12),
1923-1921 (2001 ); Kelly et al., Neoplasia 99(1 ), 310-318 (2002)).
In chronic myelogeous leukemia (CML), a reciprocally balanced chromosomal
translocation
in hematopoietic stem cells (HSCs) produces the BCR-ABL hybrid gene. The
latter encodes
the oncogenic Bcr-Abl fusion protein. Whereas ABL encodes a tightly regulated
protein tyro-
sine kinase, which plays a fundamental role in regulating cell proliferation,
adherence and
apoptosis, the BCR-ABL fusion gene encodes as constitutively activated kinase,
which trans-
forms HSCs to produce a phenotype exhibiting deregulated clonal proliferation,
reduced ca-
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pacity to adhere to the bone marrow stroma and a reduces apoptotic response to
mutagenic
stimuli, which enable it to accumulate progressively more malignant
transformations. The re-
sulting granulocytes fail to develop into mature lymphocytes and are released
into the circu-
lation, leading to a deficiency in the mature cells and increased
susceptibility to infection.
ATP-competitive inhibitors ~of Bcr-Abl have been described which prevent the
kinase from ac-
tivating mitogenic and anti-apoptotic pathways (e.g. P-3 kinase and STATS),
leading to the
death of the BCR-ABL phenotype cells and thereby providing an effective
therapy against
CML. The pyrazolo[1,5a]pyrimidin-7-yl amine derivatives useful according to
the present
invention, especially the compounds of formula I, as Bcr-Abl inhibitors are
thus especially
appropriate for the therapy of diseases related to its overexpression,
especially leukemias,
such as leukemias, e.g. CML or ALL.
The compounds of formula (I) which inhibit the tyrosine kinase activity of the
EGF-R or of the
other protein tyrosine kinases mentioned are therefore useful, for example, in
the treatment
of benign or malignant tumors. The compounds of formula (I) are e.g. able to
simultaneously
inhibit the growth of tumors with deregulated EGF-R and/or ErbB-2 activity as
well as to
inhibit the vascularisation of solid tumors triggered by VEGF. This combined
activity leads to
an improved antitumor effect (see also WO 02/41882). Moreover, the use of a
dual inhibitor
reduces the risk of drug-drug interactions and further reduces the total drug
load as
compared to a combination therapy. The compounds of formula (I) are capable of
slowing
down tumor growth or effecting tumor regression and of preventing the
formation of tumor
metastases and the growth of micrometastases. They can be used especially in
the case of
epidermal hyperproliferation (psoriasis), in the treatment of solid cancers
like for example
non-small cell lung cancer, squameous carcinoma (head and neck), breast,
gastric, ovarian,
colon and prostate cancers as well as gliomas and in the treatment of
leukemias, such as
especially acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In
addition,
the compounds of formula (I) can be used in the treatment of those disorders
of the immune
system in which several or, especially, individual protein tyrosine kinases
and/or
(furthermore) serine/threonine protein kinases are involved; the compounds of
formula (I)
can also be used in the treatment of those disorders of the central or
peripheral nervous
system in which signal transmission by several or, especially, a single
protein tyrosine
kinase(s) and/or (furthermore) serine/threonine protein kinase(s) is/are
involved.
Angiogenesis is regarded as an absolute prerequisite for those tumors which
grow beyond a
maximum diameter of about 1-2 mm; up to this limit, oxygen and nutrients may
be supplied
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to the tumor cells by diffusion. Every tumor, regardless of its origin and its
cause, is thus
dependent on angiogenesis for its growth after it has reached a certain size.
Three principal mechanisms play an important part in the activity of
angiogenesis inhibitors
against tumors: 1) Inhibition of the growth of vessels, especially
capillaries, into avascular
resting tumors, with the result that there is no net tumor growth owing to the
balance that is
achieved between apoptosis and proliferation; 2) Prevention of the migration
of tumor cells
owing to the absence of blood flow to and from tumors; and 3) Inhibition of
endothelial cell
proliferation, thus avoiding the paracrine growth-stimulating effect exerted
on the
surrounding tissue by the endothelial cells which normally line the vessels.
The present invention can also be used to prevent or treat diseases that are
triggered by
persistent angiogenesis, such as psoriasis; Kaposi's sarcoma; restenosis,
e.g., stent-
induced restenosis; endometriosis; Crohn's disease; Hodgkin's disease;
leukemia; arthritis,
such as rheumatoid arthritis; hemangioma; angiofibroma; eye diseases, such as
diabetic
retinopathy and neovascular glaucoma; renal diseases, such as
glomerulonephritis; diabetic
nephropathy; malignant nephrosclerosis; thrombotic microangiopathic syndromes;
transplant
rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the
liver; mesangial
cell-proliferative diseases; arteriosclerosis; injuries of the nerve tissue;
and for inhibiting the
re-occlusion of vessels after balloon catheter treatment, for use in vascular
prosthetics or
after inserting mechanical devices for holding vessels open, such as, e.g.,
stents, as
immunosuppressants, as an aid in scar-free wound healing, and for treating age
spots and
contact dermatitis.
Most preferred is the use in accordance with the present invention of a
compound of the
formula (I), or a pharmaceutically acceptable salt thereof, as exemplified
hereinbelow under
'Examples'.
Pharmaceutical Compositions
The invention relates also to pharmaceutical compositions comprising a
compound of
formula (I), to their use in the therapeutic (in a broader aspect of the
invention also
prophylactic) treatment or a method of treatment of a kinase dependent
disease, especially
the preferred diseases mentioned above, to the compounds for said use and to
the
preparation of pharmaceutical preparations, especially for said uses.
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The present invention also relates to pro-drugs of a compound of formula (I)
that convert in
vivo to the compound of formula (I) as such. Any reference to a compound of
formula (I) is
therefore to be understood as referring also to the corresponding pro-drugs of
the compound
of formula (I), as appropriate and expedient.
The pharmacologically acceptable compounds of the present invention may be
used, for
example, for the preparation of pharmaceutical compositions that comprise an
effective
amount of a compound of the formula (I), or a pharmaceutically acceptable salt
thereof, as
active ingredient together or in admixture with a significant amount of one or
more inorganic
or organic, solid or liquid, pharmaceutically acceptable carriers.
The invention relates also to a pharmaceutical composition that is suitable
for administration
to a warm-blooded animal, especially a human (or to cells or cell lines
derived from a warm-
blooded animal, especially a human, e.g. lymphocytes), for the treatment or,
in a broader
aspect of the invention, prevention of (= prophylaxis against) a disease that
responds to
inhibition of kinase activity, comprising an amount of a compound of formula
(I) or a
pharmaceutically acceptable salt thereof, which is effective for said
inhibition, especially the
in, together with at least one pharmaceutically acceptable carrier.
The pharmaceutical compositions according to the invention are those for
enteral, such as
nasal, rectal or oral, or parenteral, such as intramuscular or intravenous,
administration to
warm-blooded animals (especially a human), that comprise an effective dose of
the
pharmacologically active ingredient, alone or together with a significant
amount of a
pharmaceutically acceptable carrier. The dose of the active ingredient depends
on the
species of warm-blooded animal, the body weight, the age and the individual
condition,
individual pharmacokinetic data, the disease to be treated and the mode of
administration.
The invention relates also to a method of treatment for a disease that
responds to inhibition
of a kinase; which comprises administering an (against the mentioned disease)
prophylactically or especially therapeutically effective amount of a compound
of formula
(I)according to the invention, especially to a warm-blooded animal, for
example a human,
that, on account of one of the mentioned diseases, requires such treatment.
The dose of a compound of the formula (I) or a pharmaceutically acceptable
salt thereof to
be administered to warm-blooded animals, for example humans of approximately
70 kg body
weight, is preferably from approximately 3 mg to approximately 10 g, more
preferably from
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approximately 10 mg to approximately 1.5 g, most preferably from about 100 mg
to about
1000 mg /person/day, divided preferably into 1-3 single doses which may, for
example, be of
the same size. Usually, children receive half of the adult dose.
The pharmaceutical compositions comprise from approximately 1 % to
approximately 95%,
preferably from approximately 20% to approximately 90%, active ingredient.
Pharmaceutical
compositions according to the invention may be, for example, in unit dose
form, such as in
the form of ampoules, vials, suppositories, dragees, tablets or capsules.
The pharmaceutical compositions of the present invention are prepared in a
manner known
per se, for example by means of conventional dissolving, lyophilizing, mixing,
granulating or
confectioning processes.
Solutions of the active ingredient, and also suspensions, and especially
isotonic aqueous
solutions or suspensions, are preferably used, it being possible, for example
in the case of
lyophilized compositions that comprise the active ingredient alone or together
with a carrier,
for example mannitol, for such solutions or suspensions to be produced prior
to use. The
pharmaceutical compositions may, be sterilized and/or may comprise excipients,
for example
preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers,
salts for regulating
the osmotic pressure andlor buffers, and are prepared in a manner known per
se, for
example by means of conventional dissolving or lyophilizing processes. The
said solutions
or suspensions may comprise viscosity-increasing substances, such as sodium
carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone
or gelatin.
Suspensions in oil comprise as the oil component the vegetable, synthetic or
semi-synthetic
oils customary for injection purposes. There may be mentioned as such
especially liquid
fatty acid esters that contain as the acid component a long-chained fatty acid
having from 8-
22, especially from 12-22, carbon atoms, for example lauric acid, tridecylic
acid, myristic
acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic
acid, behenic
acid or corresponding unsaturated acids, for example oleic acid, elaidic acid,
erucic acid,
brasidic acid or linoleic acid, if desired with the addition of antioxidants,
for example vitamin
E, ~3-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of
those fatty
acid esters has a maximum of 6 carbon atoms and is a mono- or poly-hydroxy,
for example
a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol,
butanol or
pentanol or the isomers thereof, but especially glycol and glycerol. The
following examples
of fatty acid esters are therefore to be mentioned: ethyl oleate, isopropyl
myristate, isopropyl
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palmitate, "Labrafil M 2375" (polyoxyethylene glycerol trioleate, Gattefosse,
Paris), "Miglyol
812" (triglyceride of saturated fatty acids with a chain length of C8 to C12,
Huls AG,
Germany), but especially vegetable oils, such as cottonseed oil, almond oil,
olive oil, castor
oil, sesame oil, soybean oil and more especially groundnut oil.
The injection compositions are prepared in customary manner under sterile
conditions; the
same applies also to introducing the compositions into ampoules or vials and
sealing the
containers.
Pharmaceutical compositions for oral administration can be obtained by
combining the active
ingredient with solid carriers, if desired granulating a resulting mixture,
and processing the
mixture, if desired or necessary, after the addition of appropriate
excipients, into tablets,
dragee cores or capsules. It is also possible for them to be incorporated into
plastics carriers
that allow the active ingredients to diffuse or be released in measured
amounts.
Suitable carriers are especially fillers, such as sugars, for example lactose,
saccharose,
mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for
example
tricalcium phosphate or calcium hydrogen phosphate, and binders, such as
starch pastes
using for example corn, wheat, rice or potato starch, gelatin, tragacanth,
methylcellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone,
and/or, if desired, disintegrators, such as the above-mentioned starches,
and/or
carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or
a salt thereof,
such as sodium alginate. Excipients are especially flow conditioners and
lubricants, for
example silicic acid, talc, stearic acid or salts thereof, such as magnesium
or calcium
stearate, and/or polyethylene glycol. Dragee cores are provided with suitable,
optionally
enteric, coatings, there being used, inter alia, concentrated sugar solutions
which may
comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or
titanium dioxide,
or coating solutions in suitable organic solvents, or, for the preparation of
enteric coatings,
solutions of suitable cellulose preparations, such as ethylcellulose phthalate
or
hydroxypropylmethylcellulose phthalate. Capsules are dry-filled capsules made
of gelatin
and soft sealed capsules made of gelatin and a plasticizes, such as glycerol
or sorbitol. The
dry-filled capsules may comprise the active ingredient in the form of
granules, for example
with fillers, such as lactose, binders, such as starches, and/or glidants,
such as talc or
magnesium stearate, and if desired with stabilizers. In soft capsules the
active ingredient is
preferably dissolved or suspended in suitable oily excipients, such as fatty
oils, paraffin oil or
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liquid polyethylene glycols, it being possible also for stabilizers and/or
antibacterial agents to
be added. Dyes or pigments may be added to the tablets or dragee coatings or
the capsule
casings, for example for identification purposes or to indicate different
doses of active
ingredient.
Combinations
A compound of the formula (I) may also be used to advantage in combination
with other
antiproliferative agents. Such antiproliferative agents include, but are not
limited to aro-
matase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II
inhibitors;
microtubule active agents; alkylating agents; histone deacetylase inhibitors;
compounds
which induce cell differentiation processes; cyclooxygenase inhibitors; MMP
inhibitors;
mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds
targeting/decreasing a protein or lipid kinase activity and further anti-
angiogenic compounds;
compounds which target, decrease or inhibit the activity of a protein or lipid
phosphatase;
gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors;
bisphosphonates; biological response modifiers; antiproliferative antibodies;
heparanase
inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors;
proteasome inhibitors;
agents used in the treatment of hematologic malignancies; compounds which
target,
decrease or inhibit the activity of Flt-3; Hsp90 inhibitors; temozolomide
(TEMODAL~); and
leucovorin.
The term "aromatase inhibitor" as used herein relates to a compound which
inhibits the
estrogen production, i.e. the conversion of the substrates androstenedione and
testosterone
to estrone and estradiol, respectively. The term includes, but is not limited
to steroids,
especially atamestane, exemestane and formestane and, in particular, non-
steroids,
especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane,
testolactone,
ketokonazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane can
be admi-
nistered, e.g., in the form as it is marketed, e.g. under the trademark
AROMASIN. Form-
estane can be administered, e.g., in the form as it is marketed, e.g. under
the trademark
LENTARON. Fadrozole can be administered, e.g., in the form as it is marketed,
e.g. under
the trademark AFEMA. Anastrozole can be administered, e.g., in the form as it
is marketed,
e.g. under the trademark ARIMIDEX. Letrozole can be administered, e.g., in the
form as it is
marketed, e.g. under the trademark FEMARA or FEMAR. Aminoglutethimide can be
administered, e.g., in the form as it is marketed, e.g. under the trademark
ORIMETEN. A
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combination of the invention comprising a chemotherapeutic agent which is an
aromatase
inhibitor is particularly useful for the treatment of hormone receptor
positive tumors, e.g.
breast tumors.
The term "antiestrogen" as used herein relates to a compound which antagonizes
the effect
of estrogens at the estrogen receptor level. The term includes, but is not
limited to
tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen can
be admi-
nistered, e.g., in the form as it is marketed, e.g. under the trademark
NOLVADEX. Ralo-
xifene hydrochloride can be administered, e.g., in the form as it is marketed,
e.g. under the
trademark EVISTA. Fulvestrant can be formulated as disclosed in US 4,659,516
or it can be
administered, e.g., in the form as it is marketed, e.g. under the trademark
FASLODEX. A
combination of the invention comprising a chemotherapeutic agent which is an
antiestrogen
is particularly useful for the treatment of estrogen receptor positive tumors,
e.g. breast
tumors.
The term "anti-androgen" as used herein relates to any substance which is
capable of in-
hibiting the biological effects .of androgenic hormones and includes, but is
not limited to,
bicalutamide (CASODEX), which can be formulated, e.g. as disclosed in US
4,636,505.
The term "gonadorelin agonist" as used herein includes, but is not limited to
abarelix, go-
serelin and goserelin acetate. Goserelin is disclosed in US 4,100,274 and can
be admi-
nistered, e.g., in the form as it is marketed, e.g. under the trademark
ZOLADEX. Abarelix
can be formulated, e.g. as disclosed in US 5,843,901.
The term "topoisomerase I inhibitor" as used herein includes, but is not
limited to topotecan,
gimatecan, irinotecan, camptothecian and its analogues, 9-nitrocamptothecin
and the
macromolecular camptothecin conjugate PNU-166148 (compound A1 in W099/ 17804).
Irinotecan can be administered, e.g. in the form as it is marketed, e.g. under
the trademark
CAMPTOSAR. Topotecan can be administered, e.g., in the form as it is marketed,
e.g.
under the trademark HYCAMTIN.
The term "topoisomerase II inhibitor" as used herein includes, but is not
limited to the an-
thracyclines such as doxorubicin (including liposomal formulation, e.g.
CAELYX), dauno-
rubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones
mitoxantrone and lo-
soxantrone, and the podophillotoxines etoposide and teniposide. Etoposide can
be ad-
ministered, e.g. in the form as it is marketed, e.g. under the trademark
ETOPOPHOS.
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Teniposide can be administered, e.g. in the form as it is marketed, e.g. under
the trademark
VM 26-BRISTOL. Doxorubicin can be administered, e.g. in the form as it is
marketed, e.g.
under the trademark ADRIBLASTIN or ADRIAMYCIN. Epirubicin can be administered,
e.g. in
the form as it is marketed, e.g. under the trademark FARMORUBICIN. Idarubicin
can be
administered, e.g. in the form as it is marketed, e.g. under the trademark
ZAVEDOS.
Mitoxantrone can be administered, e.g. in the form as it is marketed, e.g.
under the
trademark NOVANTRON.
The term "microtubule active agent" relates to microtubule stabilizing,
microtubule destabi-
lizing agents and microtublin polymerization inhibitors including, but not
limited to taxanes,
e.g. paclitaxel and docetaxel, vinca alkaloids, e.g., vinblastine, especially
vinblastine sulfate,
vincristine especially vincristine sulfate, and vinorelbine, discodermolides,
cochicine and
epothilones and derivatives thereof, e.g. epothilone B or D or derivatives
thereof. Paclitaxel
may be administered e.g. in the form as it is marketed, e.g. TAXOL. Docetaxel
can be
administered, e.g., in the form as it is marketed, e.g. under the trademark
TAXOTERE.
Vinblastine sulfate can be administered, e.g., in the form as it is marketed,
e.g. under the
trademark VINBLASTIN R.P. Vincristine sulfate can be administered, e.g., in
the form as it is
marketed, e.g. under the trademark FARMISTIN. Discodermolide can be obtained,
e.g., as
disclosed in US 5,010,099: Also included are Epothilone derivatives which are
disclosed in
WO 98/10121, US 6,194,181, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461
and WO 00/31247. Especially preferred are Epothilone A and/or B.
The term "alkylating agent" as used herein includes, but is not limited to,
cyclophosphamide,
ifosfamide, melphalan or nitrosourea (BCNU or Gliadel). Cyclophosphamide can
be
administered, e.g., in the form as it is marketed, e.g. under the trademark
CYCLOSTIN.
Ifosfamide can be administered, e.g., in the form as it is marketed, e.g.
under the trademark
HOLOXAN.
The term "histone deacetylase inhibitors" or "HDAC inhibitors" relates to
compounds which
inhibit the histone deacetylase and which possess antiproliferative activity.
This includes
compounds disclosed in WO 02/22577, especially N-hydroxy-3-[4-[[(2-
hydroxyethyl)[2-(1 H-
indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-
(2-methyl-1 H-
indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide and pharmaceutically
acceptable
salts thereof. It further especially includes Suberoylanilide hydroxamic acid
(SAHA).
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The term "antineoplastic antimetabolite" includes, but is not limited to, 5-
Fluorouracil or 5-FU,
capecitabine, gemcitabine, DNA demethylating agents, such as 5-azacytidine and
decitabine, methotrexate and edatrexate, and folic acid antagonists such as
pemetrexed.
Capecitabine can be administered, e.g., in the form as it is marketed, e.g.
under the
trademark XELODA. Gemcitabine can be administered, e.g., in the form as it is
marketed,
e.g. under the trademark GEMZAR. Also included is the monoclonal antibody
trastuzumab
which can be administered, e.g., in the form as it is marketed, e.g. under the
trademark
HERCEPTIN.
The term "platin compound" as used herein includes, but is not limited to,
carboplatin, cis-
platin, cisplatinum and oxaliplatin. Carboplatin can be administered, e.g., in
the form as it is
marketed, e.g. under the trademark CARBOPLAT. Oxaliplatin can be administered,
e.g., in
the form as it is marketed, e.g. under the trademark ELOXATIN.
The term "compounds targeting/decreasing a protein or lipid kinase activity;
or a protein or
lipid phosphatase activity; or further anti-angiogenic compounds" as used
herein includes,
but is not limited to: protein tyrosine kinase and/or serine andlor threonine
kinase inhibitors
or lipid kinase inhibitors, e.g.:
a) compounds targeting, decreasing or inhibiting the activity of the platelet-
derived growth
factor-receptors (PDGFR), such as compounds which target, decrease or inhibit
the activity
of PDGFR, especially compounds which inhibit the PDGF receptor, e.g. a N-
phenyl-2-
pyrimidine-amine derivative, e.g. imatinib, SU101,. SU6668, and GFB-111;
b) compounds targeting, decreasing or inhibiting the activity of the
fibroblast growth factor-
receptors (FGFR);
c) compounds targeting, decreasing or inhibiting the activity of the insulin-
like growth factor
receptor I(IGF-IR), such as compounds which target, decrease or inhibit the
activity of IGF-
IR, especially compounds which inhibit the IGF-IR receptor, such as those
compounds
disclosed in WO 02/092599;
d) compounds targeting, decreasing or inhibiting the activity of the Trk
receptor tyrosine
kinase family;
e) compounds targeting, decreasing or inhibiting the activity of the Axl
receptor tyrosine
kinase family;
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f) compounds targeting, decreasing or inhibiting the activity of the c-Met
receptor;
g) compounds targeting, decreasing or inhibiting the activity of the Kit/SCFR
receptor
tyrosine kinase;
h) compounds targeting, decreasing or inhibiting the activity of the C-kit
receptor tyrosine
kinases - (part of the PDGFR family), such as compounds which target, decrease
or inhibit
the activity of the c-Kit receptor tyrosine kinase family, especially
compounds which inhibit
the c-Kit receptor, e.g imatinib;
i) compounds targeting, decreasing or inhibiting the activity of members of
the c-Abl family
and their gene-fusion products (e.g. BCR-Abl kinase), such as compounds which
target
decrease or inhibit the activity of c-Abl family members and their gene fusion
products, e.g.
a N-phenyl-2-pyrimidine-amine derivative, e.g. imatinib; PD180970; AG957; NSC
680410; or
PD173955 from ParkeDavis;
j) compounds targeting, decreasing or inhibiting the activity of members of
the protein
kinase C (PKC) and Raf family of serine/threonine kinases, members of the MEK,
SRC,
JAK, FAK, PDK and Ras/MAPK family members, or PI(3) kinase family, or of the
PI(3)-
kinase-related kinase family, and/or members of the cyclin-dependent kinase
family (CDK)
and are especially those staurosporine derivatives disclosed in US 5,093,330,
e.g.
midostaurin; examples of further compounds include e.g. UCN-01, safingol, BAY
43-9006,
Bryostatin 1, Perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis
3521;
LY333531/LY379196; isochinoline compounds such as those disclosed in WO
00/09495;
FTIs; PD184352 or QAN697( a P13K inhibitor);
k) compounds targeting, decreasing or inhibiting the activity of protein-
tyrosine kinase
inhibitors, such as compounds which target, decrease or inhibit the activity
of protein-
tyrosine kinase inhibitors include imatinib mesylate (GLEEVEC) or tyrphostin.
A tyrphostin is
preferably a low molecular weight (Mr < 1500) compound, or a pharmaceutically
acceptable
salt thereof, especially a compound selected from the benzylidenemalonitrile
class or the S-
arylbenzenemalonirile or bisubstrate quinoline class of compounds, more
especially any
compound selected from the group consisting of Tyrphostin A23/RG-50810; AG 99;
Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44;
Tyrphostin
B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and
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adaphostin (4-([(2,5-dihydroxyphenyl)methyljamino)-benzoic acid adamantyl
ester; NSC
680410, adaphostin); and
I) compounds targeting, decreasing or inhibiting the activity of the epidermal
growth factor
family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or
heterodimers),
such as compounds which target, decrease or inhibit the activity of the
epidermal growth
factor receptor family are especially compounds, proteins or antibodies which
inhibit
members of the EGF receptor tyrosine kinase family, e.g. EGF receptor, ErbB2,
ErbB3 and
ErbB4 or bind to EGF or EGF related ligands, and are in particular those
compounds,
proteins or monoclonal antibodies generically and specifically disclosed in WO
97/02266,
e.g. the compound of ex. 39, or in EP 0 564 409, WO 99/03854, EP 0520722, EP 0
566 226,
EP 0 787 722, EP 0 837 063, US 5,747,498, WO 98110767, WO 97130034, WO
97/49688,
WO 97/38983 and, especially, WO 96/30347 (e.g. compound known as CP 358774),
WO
96/33980 (e.g. compound ZD 1839) and WO 95/03283 (e.g. compound ZM105180);
e.g.
trastuzumab (HERCEPTIN), cetuximab, Iressa, Tarceva, OSI-774, CI-1033, EIfB-
569, GW-
2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7;6.3, and 7H-pyrrolo-[2,3-
d]pyrimidine
derivatives which are disclosed in WO 03/013541.
Further anti-angiogenic compounds include compounds having another mechanism
for their
activity, e.g. unrelated to protein or lipid kinase inhibition e.g.
thalidomide (THALOMID) and
TN P-470.
Compounds which target, decrease or inhibit the activity of a protein or lipid
phosphatase are
e.g. inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25, e.g. okadaic
acid or a
derivative thereof.
Compounds which induce cell differentiation processes are e.g. retinoic acid,
a- y- or 8-
tocopherol or a-'y- or &--tocdtrienol.
The term cyclooxygenase inhibitor as used herein includes, but is not limited
to, e.g. Cox-2
inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives,
such as
celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a 5-alkyl-2-
arylaminophenylacetic acid, e.g. 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl
acetic acid,
lumiracoxib.
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The term "bisphosphonates" as used herein includes, but is not limited to,
etridonic,
clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and
zoledronic acid.
"Etridonic acid" can be administered, e.g., in the form as it is marketed,
e.g. under the
trademark DIDRONEL. "Clodronic acid" can be administered, e.g., in the form as
it is
marketed, e.g. under the trademark BONEFOS. "Tiludronic acid" can be
administered, e.g.,
in the form as it is marketed, e.g. under the trademark SKELID. "Pamidronic
acid" can be
administered, e.g. in the form as it is marketed, e.g. under the trademark
AREDIATM.
"Alendronic acid" can be administered, e.g., in the form as it is marketed,
e.g. under the
trademark FOSAMAX. "Ibandronic acid" can be administered, e.g., in the form as
it is
marketed, e.g. under the trademark BONDRANAT. "Risedronic acid" can be
administered,
e.g., in the form as it is marketed, e.g. under the trademark ACTONEL.
"Zoledronic acid" can
be administered, e.g. in the form as it is marketed, e.g. under the trademark
ZOMETA.
The term "mTOR inhibitors" relates to compounds which inhibit the mammalian
target of
rapamycin (mTOR) and which possess antiproliferative activity such as
sirolimus
(Rapamune~), everolimus (CerticanTM), CCI-779 and ABT578.
The term "heparanase inhibitor" as used herein refers to compounds which
target, decrease
or inhibit heparin sulphate degradation. The term includes, but is not limited
to, PI-88.
The term " biological response modifier" as used herein refers to. a
lymphokine or
interferons, e.g. interferon y.
The term "inhibitor of Ras oncogenic isoforms", e.g. H-Ras, K-Ras, or N-Ras,
as used herein
refers to compounds which target, decrease or inhibit the oncogenic activity
of Ras e.g. a
"farnesyl transferase inhibitor" e.g. L-744832, DK8G557 or P115777
(Zarnestra).
The term "telomerase inhibitor" as used herein refers to compounds which
target, decrease
or inhibit the activity of telomerase. Compounds which target, decrease or
inhibit the activity
of telomerase are especially compounds whicf~ inhibit the telomerase receptor,
e.g.
telomestatin.
The term "methionine aminopeptidase inhibitor" as used herein refers to
compounds which
target, decrease or inhibit the activity of methionine aminopeptidase.
Compounds which
target, decrease or inhibit the activity of methionine aminopeptidase are e.g.
bengamide or a
derivative thereof.
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The term "proteasome inhibitor" as used herein refers to compounds which
target, decrease
or inhibit the activity of the proteasome. Compounds which target, decrease or
inhibit the
activity of the proteasome include e.g. PS-341 and MLN 341.
The term "matrix metalloproteinase inhibitor" or ("MMP inhibitor") as used
herein includes,
but is not limited to collagen peptidomimetic and nonpeptidomimetic
inhibitors, tetracycline
derivatives, e.g. hydroxamate peptidomimetic inhibitor batimastat and its
orally bioavailable
analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551) BMS-
279251, BAY 12-9566, TAA211, MM1270B or AAJ996.
The term "agents used in the treatment of hematologic malignancies" as used
herein
includes, but is not limited to FMS-like tyrosine kinase inhibitors e.g.
compounds targeting,
decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors
(Flt-3R); interferon,
1-b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors e.g.
compounds
which target, decrease or inhibit anaplastic lymphoma kinase.
Compounds which target, decrease or inhibit the activity of FMS-like tyrosine
kinase
receptors (Flt-3R) are especially compounds, proteins or antibodies which
inhibit members of
the Flt-3R receptor kinase family, e.g.PKC412, midostaurin, a staurosporine
derivative,
SU11248 and MLN518.
The term "HSP90 inhibitors" as used herein includes, but is not limited to,
compounds
targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90;
degrading,
targeting, decreasing or inhibiting the HSP90 client proteins via the
ubiquitin proteasome
pathway. Compounds targeting, decreasing or inhibiting the intrinsic ATPase
activity of
HSP90 are especially compounds, proteins or antibodies which inhibit the
ATPase activity of
HSP90 e.g,17-allylamino,l7-demethoxygeldanamycin (17AAG), a geldanamycin
derivative;
other geldanamycin related compounds; radicicol and HDAC inhibitors.
The term "antiproliferative antibodies" as used herein includes, but is not
limited to
trastuzumab (HerceptinTM), Trastuzumab-DM1, erlotinib (TarcevaTM), bevacizumab
(AvastinTM), rituximab (Rituxan~), PR064553 (anti-CD40) and 2C4 Antibody. By
antibodies
is meant e.g. intact monoclonal antibodies, polyclonal antibodies,
multispecific antibodies
formed from at least 2 intact antibodies, and antibodies fragments so long as
they exhibit the
desired biological activity. .
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For the treatment of acute myeloid leukemia (AML), compounds of formula (I)
can be used in
combination with standard leukemia therapies, especially in combination with
therapies used
for the treatment of AML. In particular, compounds of formula (I) can be
administered in
combination with e.g. farnesyl transferase inhibitors and/or other drugs
useful for the
treatment of AML, such as Daunorubicin, Adriamycin, Ara-C, VP-16, Teniposide,
Mitoxantrone, Idarubicin, Carboplatinum and PKC412.
The term "antileukemic compounds" includes, for example, Ara-C, a pyrimidine
analog,
which is the 2'-alpha-hydroxy ribose (arabinoside) derivative of
deoxycytidine. Also included
is the purine analog of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine
phosphate.
Compounds which target, decrease or inhibit activity of histone deacetylase
(HDAC)
inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA)
inhibit the
activity of the enzymes known as histone deacetylases. Specific HDAC
inhibitors include
MS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compounds disclosed
in
US 6,552,065, in particular, N-hydroxy-3-[4-[[[2-(2-methyl-1 H-indol-3-yl)-
ethyl]-
amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt
thereof and
N-hydroxy-3-[4-[(2-hydroxyethyl)(2-(1 H-indol-3-yl)ethyl]-amino]methyl]phenyl]-
2E-2-
propenamide, or a pharmaceutically acceptable salt thereof, especially the
lactate salt.
Compounds which target, decrease or inhibit the activity of serine/theronine
mTOR kinase
are especially compounds, proteins or antibodies which inhibit members of the
mTOR kinase
family e.g. RAD, RAD001, CCI-779, ABT578, SAR543, rapamycin and derivatives
thereof;
AP23573 from Ariad; everolimus (CERTICAN); and sirolimus.
Somatostatin receptor antagonists as used herein refers to agents which
target, treat or
inhibit the somatostatin receptor such as octreoride, and SOM230.
Tumor cell damaging approaches refer to approaches such as ionizing radiation.
The term
"ionizing radiation" referred to above and hereinafter means ionizing
radiation that occurs as
either electromagnetic rays (such as X-rays and gamma rays) or particles (such
as alpha
and beta particles). Ionizing radiation is provided in, but not limited to,
radiation therapy and
is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in
Principles and
Practice of Oncology, Devita et al., Eds., 4th Edition, Vol. 1, pp. 248-275
(1993).
The term EDG binders as used herein refers a class of immunosuppressants that
modulates
lymphocyte recirculation, such as FTY720.
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CERTICAN (everolimus, RAD) an investigational novel proliferation signal
inhibitor that
prevents proliferation of T-cells and vascular smooth muscle cells.
The term ribonucleotide reductase inhibitors refers to pyrimidine or puring
nucleoside
analogs including, but not limited to, fludarabine and/or cytosine arabinoside
(ara-C),
6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in
combination with
ara-C against ALL) and/or pentostatin. Ribonucleotide reductase inhibitors are
especially
hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives, such as PL-1, PL-
2, PL-3,
PL-4, PL-5, PL-6, PL-7 or PL-8 mentioned in Nandy et al., Acta Oncologica,
Vol. 33, No. 8,
pp. 953-961 (1994).
The term "S-adenosylmethionine decarboxylase inhibitors" as used herein
includes, but is
not limited to the compounds disclosed in US 5,461,076.
Also included are in particular those compounds, proteins or monoclonal
antibodies of VEGF
disclosed in WO 98/35958, e.g. 1-(4-chloroanilino)-4-(4-
pyridylmethyl)phthalazine or a
pharmaceutically acceptable salt thereof, e.g. the succinate, or in WO
00/09495,
WO 00/27820, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0 769 947; those as
described by Prewett et al, Cancer Res, Vol. 59, pp. 5209-5218 (1999); Yuan et
al.,
Proc Natl Acad Sci U S A, Vol. 93, pp. 14765-14770 (1996); Zhu et al., Cancer
Res, Vol. 58,
pp. 3209-3214 (1998); and Mordenti et al., Toxicol Pathol, Vol. 27, No. 1, pp.
14-21 (1999);
in WO 00/37502 and WO 94/10202; ANGIOSTATIN, described by O'Reilly et al.,
Cell,
Vol. 79, pp. 315-328 (1994); ENDOSTATIN, described by O'Reilly et al., Cell,
Vol. 88,
pp. 277-285 (1997); anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668;
bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, e.g.
rhuMAb and
RHUFab, VEGF aptamer e.g. Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2
IgG1
antibody, Angiozyme (RPI 4610) and Avastan.
Photodynamic therapy as used herein refers to therapy which uses certain
chemicals known
as photosensitizing agents to treat or prevent cancers. Examples of
photodynamic therapy
includes treatment with agents, such as e.g. VISUDYNE and porfimer sodium.
Angiostatic steroids as used herein refers to agents which block or inhibit
angiogenesis,
such as, e.g., anecortave, triamcinolone. hydrocortisone, 11-a-
epihydrocotisol, cortexolone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone,
estrone and
dexamethasone.
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Implants containing corticosteroids refers to agents, such as e.g.
fluocinolone,
dexamethasone.
Other chemotherapeutic agents include, but are not limited to, plant
alkaloids, hormonal
agents and antagonists; biological response modifiers, preferably lymphokines
or
interferons; antisense oligonucleotides or oligonucleotide derivatives; or
miscellaneous
agents or agents with other or unknown mechanism of action.
The structure of the active agents identified by code nos., generic or trade
names may be
taken from the actual edition of the standard compendium "The Merck Index" or
from
databases, e.g. Patents International (e.g. IMS World Publications).
The above-mentioned compounds, which can be used in combination with a
compound of
the formula (I), can be prepared and administered as described in the art such
as in the
documents cited above.
A compound of the formula (I) may also be used to advantage in combination
with known
therapeutic processes, e.g., the administration of hormones or especially
radiation.
A compound of formula (I) may in particular be used as a radiosensitizer,
especially for the
treatment of tumors which exhibit poor sensitivity to radiotherapy.
By "combination", there is meant either a fixed combination in one dosage unit
form, or a kit
of parts for the combined administration where a compound of the formula (I)
and a
combination partner may be administered independently at the same time or
separately
within time intervals that especially allow that the combination partners show
a cooperative,
e.g. synergistic, effect, or any combination thereof.
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EXAMPLES
The following examples serve to illustrate the invention without limiting the
scope thereof:
Abbreviations
DMSO dimethylsulfoxide
ES-MS electrospray mass spectrometry
EtOAc Ethyl Acetate
HPLC high-pressure liquid chromatography
mL mililiter(s)
NMR nuclear magnetic resonance
RT room temperature
AtRET HPLC retention time in minutes (method A)
BtRET HPLC retention time in .minutes (method B)
CtRET HPLC retention time in minutes (method C)
DtRET HPLC retention time in minutes (method D)
TFA trifluoroacetic acid
THF tetrahydrofuran
TMSCI Trimethylsilyl chloride
Where no temperatures are given, the reaction takes place at ambient (room)
temperature.
Ratios of solvents, e.g., in eluents or solvent mixtures, are given in volume
by volume
(~/~).
Syntheses
Flash chromatography is performed by using silica gel (Merck; 40-63 pm). For
thin
layer chromatograhy, pre-coated silica gel (Merck 60 F254) plates are used.
Detection of
the components is made by UV light (254 nm). HPLC is performed on an Agilent
HP 1100
using a Nucleosil 100-3 C~8 HD 125 x 4.0 mm column [1 mL/min.; 20-100% NeCN /
0.1
TFA in 7 minutes) (Method A); SpectraSystem SP8800/UV2000 using a Nucleosil
100-5 C~$
AB 250 x 4.6 mm column (2 mL/min.; 2-100% MeCN / 0.1 % TFA in 10 minutes)
(Method B);
using a Chromalith Speed ROD RP18 50-4.6 mm column (Merck) (2 mL/min.; 2-100%
MeCN / 0.1 % TFA in 2 minutes) (Method C); or a C8 2.1-50 mm 3 pm column
(Waters)
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(2 mL/min.; 5-95% MeCN / 0.1% TFA in 2 minutes) (Method D). 'H-NMR
measurements are
performed on a Varian Gemini 400 or a Bruker DRX 500 spectrometer using
tetraethylsilane
as internal standard. Chemical shifts are expressed in ppm downfield from
tetraethylsilane
and coupling constants (J) are expressed in Hertz (Hz). Electrospray mass
spectra are
obtained with a Fisons Instruments VG Platform II. Melting points are measured
with a
Buchi 510 melting point apparatus. Commercially-available solvents and
chemicals are used
for syntheses.
Analytical HPLC conditions:
System 1
Linear gradient 20-100% CH3CN (0.1 %TFA) and HZO (0.1 % TFA) in 7min + 2min
100%
CH3CN (0.1 %TFA); detection at 215 nm, flow rate 1 mL/min at 30°C.
Column: Nucleosil 100-
3 C18HD (125 x 4mm).
System 2
Linear gradient 2-100% CH3CN (0.1 %TFA) and HBO (0.1 % TFA) in 7min + 2min
100%
CH3CN (0.1 %TFA); detection at 215 nm, flow rate 1 mUmin at 30°C.
Column: Nucleosil 100-
3 C18HD (125 x 4mm).
Example 1
3-[7-Am i no-3-[4-(4-methyl-pi perazi n-1-yl)-phenyls-pyrazolo[1,5-a~ pyrim id
i n-6-yl}-phenol
6-(3-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine (Stage 1.1) (25 mg, 0.051 mmol) dissolved in THF (6 mL) is
hydrogenated in the
presence of Pd/C (10% Engelhard 4505, 6 mg) for 13 hours. After filtration and
evaporating
the solvent under reduced pressure, the residue is flash chromatographed
(silica gel, CH2CIZ
l MeOH / NH3 = 95:5:0.1) to give compound of Example 1 as white solid (14 mg,
0.035
mmol; 70%): ES-MS: M+H = 401.1, Rf (CH2C12 / MeOH / NH3 = 90:10:0.1) = 0.33,
HPLC:
p'tRet = 2.77 minutes.
'H-NMR (400 MHz, DMSO-ds): 9.59 (s, 1 H, OH), 8.58/8.18 (s/s, 1 H/1 H,
pyrazolopyrimidinyl), 8.01 (d, 9.0 Hz, 2H, phenyl), 7.48 (s, 2H, NH2), 7.32
(t, 8.5 Hz, 1 H,
phenyl-OH), 6.99 (d, 9.0 Hz, 2H, phenyl), 6.96 (d, 8.5 Hz, 1 H, phenyl-OH),
6.93 (s, 1 H,
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phenyl OH), 6.80 (d, 8.5 Hz, 1 H, phenyl OH), 3.17/2.48 (m/m, 4H/4H,
piperazinyl),
2.24 (s, 3H, CH3).
Stage 1.16-(3-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-7-ylamine
4-(4-(4-methyl-piperazin-1-yl)-phenyl)-2H-pyrazol-3-ylamine (Stage 1.2) (100
mg, 0.388
mmol), 2-(3-benzyloxy-phenyl)-3-oxo-propionitrile (Stage 1.3) (98 mg, 0.388
mmol), HCI (2.5
mM in EtOH; 1,55 mmol, 0.9 mL) dissolved in EtOH (1 mL) are stirred for 17
hours at RT.
After adding H20 (4 mL) and K2C03 (250 mg), the reaction mixture is extracted
with CH~CIz
(20 mL, 2 x). The combined organic phases are washed with H20 (10 mL), dried
(Na2S04),
concentrated under reduced pressure and flash chromatographed (silica gel, 2.5
x 15 cm,
CHZCI2 l MeOH = 9:1 ) to give compound of Stage 1.1 as white solid (60 mg,
0.122 mmol;
32%); ES-MS: M+H = 491.0, Rf (CH2Ch / MeOH / NH3 = 90:10:0.1 ) = 0.42; HPLC:
AtRet =
4.69 minutes.
'H-NMR (400 MHz, DMSO-ds): 8.79/8.21 (s/s, 1 H/1 H, pyrazolopyrimidinyl), 8.03
(d, 9.0 Hz,
2H, phenyl), 7.53 (s, 2H, NHS), 7.44 (m, 5H, benzyl), 7.32 (t, 8.5 Hz, 1 H,
phenyl OH), 7.29
(s, 1 H, phenyl OH), 7.13 (d, 8.5 Hz, 1 H, phenyl-OH), 7.06 (d, 8.5 Hz, 1 H,
phenyl-OH), 6.97
(d, 9.0 Hz, 2H, phenyl), 5.19 (s, 2H, benzyl), 3.17/2.48 (m/m, 4H/4H,
piperazinyl), 2.24 (s,
3H, CH3).
Stare 1.2 4-(4-(4-Methyl-piperazin-1-yl)-phenyl)-2H-pyrazol-3-ylamine
2-[4-(4-Methyl-piperazin-1-yl)-phenyl]-3-oxo-propionitrile (Stage 1.4) (370
mg, 1.52 mmol),
hydrazine monohydrate (4.185 mL, 3.8 mmol) dissolved in AcOH are stirred at
98°C for 3
hours. After cooling down to RT, H2O (8 mL) and concentrated HCI (0.8 mL) are
added and
the reaction mixture is stirred under reflux for 20 minutes. After cooling
down to RT, the
reaction mixture is adjusted to alkaline pH by slowly adding NH3 (25%).
Precipitating
material is filtered-off and kept for further purification. The reaction
solution is extracted with
CH2CI2 (50 mL, 3 x), dried (Na2S04) and concentrated under reduced pressure.
Precipitated
and extracted material is combined and flash chromatographed (silica gel, 3.0
x 18 cm,
CH2CI2 / MeOH / NH3 = 9:1:01) to give compound of Stage 1.2 as white solid
(277 mg,
1.08 mmol; 71%); ES-MS: M+H = 258.1, Rf (CHZCI2 / MeOH / NH3 = 90:10:0.1) =
0.28;
HPLC: At~et = 4.33 minutes.
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'H-NMR (400 MHz, DMSO-ds): 11.55 (s/broad, 1 H, NH), 7.55 (s, 1 H, pyrolyl),
7.35 (d, 9.0
Hz, 2H, phenyl), 6.91 (d; 9.0 Hz, 2H, phenyl), 4.55 (s/broad, 2H, NHZ),
3.10/2.46 (m/m,
4H/4H, piperazinyl), 2.23 (s, 3H, CH3).
Stage 7.3 2-(3-Benzyloxy-phenyl)-3-oxo-propionitrile
Na (260 mg, 11.3 mmol) is dissolved in absolute EtOH (11 mL) under Ar during
20 minutes.
After adding (3-benzylox-phenyl)-acetonitrile (1.9 g, 8.68 mmol) and ethyl
formate (1.05 mL,
13.0 mmol), the reaction mixture is stirred under reflux for 2 hours. After
evaporating the
solvent under reduced pressure, adding HBO (20 mL), and adjusting to pH = 4.0
by adding
AcOH, the reaction suspension is extracted with CHZCIZ (30 mL, 2 x). The
combined organic
phases are washed with H20 (10 mL), dried (Na2SO4), concentrated under reduced
pressure
and flash chromatographed (silica gel, 4.5 x 25 cm, CH~CI2 / MeOH = 98:2) to
give
compound of Stage 1.3 as white solid (780 mg, 3.11 mmol; 36%); ES-MS: M-H =
250.0, Rf
(CH2C12 / MeOH = 95:5) = 0.49; HPLC: AtRer = 6.07 minutes.
'H-NMR (400 MHz, DMSO-ds): 7.45-7.25/6.98-6.88 (m/m, 8 H, aryl), 5.09 (s, 2H,
CHI), 3.98
(s, 2H, CH2).
Stage 7.4 2-[4-(4-Methyl-piperazin-1-yl)-phenyl]-3-oxo-propionitrile
Na 160 mg (7.0 mmol) is dissolved in absolute EtOH (6 mL) under Ar during 10
minutes.
After adding [4-(4-methyl-piperazin-1-yl)-phenyl]-acetonitrile (Stage 1.5) (1
g, 4.64 mmol)
and ethyl formate (0.56 mL, 7.0 mmol), the reaction mixture is stirred under
reflux for 1 hour.
After washing the reaction pulp with ether (50 mL, 3 x), the solid residue is
dissolved in H20
(60 mL) and adjusted to pH = 3.9 by adding AcOH. The aqueous solution is
extracted with
CH2C12 (50 mL, 3 x). The combined organic phases are washed with H20 (50 mL).
Both
aqueous phases are combined and lyophilized. The resulting residue is
crystallized from
MeOH / CH2CI2 to give compound of Stage 1.4 as white crystals (721 mg, 3.0
mmol; 64%);
ES-MS: M+H = 244.1; HPLC: AtRef= 2.43 minutes.
'H-NMR (400 MHz, DMSO-ds): the compound forms a tautomeric equilibrium in
solution:
7.87/7.77 (s/s, 1 H, CH=/CM-OH), 7.53/7.17 (d/d, 9.0 Hz, 2H, phenyl),
7.84/7.82 (d/d, 9.0 Hz,
2H, phenyl), 3.10 (m, 4H, piperazinyl), 2.57/2.51 (m/m, 4H, piperazinyl),
2.29/2.26 (s, 3H,
CH3).
Sta ec~1.5 [4-(4-Methyl-piperazin-1-yl)-phenyl]-acetonitrile
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(4-Bromo-phenyl)-acetonitrile (5 g, 25.5 mmol), 1-methyl-piperazine (3.4 mL,
30.6 mmol),
K2C03 (7.68 g, 35.7 mmol), Pd(Ac0)2 (280 mg, 1.275 mmol), 2-(di-tert-
butylphosphino)-
biphenyl (1.14 g, 3.825 mmol) dissolved in 1,2-dimethoxyethane (70 mL) are
stirred underAr
at 85°C for 20 hours. After adding H20 (100 mL), the reaction mixture
is extracted with
CH2Clz (100 mL, 3 x). The combined organic phases are washed with H20 (100
mL), dried
(Na2S04), concentrated under reduced pressure and flash chromatographed
(silica gel, 4.5 x
34 cm, CH2C12 / MeOH = 95:5) to give compound of Stage 1.5 as white solid (2.8
g, 13
mmol; 51 %); ES-MS: M+H = 216.1; Rf (CH2C12 / MeOH = 9:1 ) = 0.47; HPLC: AtRer
= 2.24
minutes.
'H-NMR (400 MHz, DMSO-ds): 7.14/6.91 (d/d, 9.5 Hz, 2H/2H, phenyl), 7.53 (s,
2H, NH2),
7.44 (m, 5H, benzyl), 7.32 (t, 8.5 Hz, 1 H, phenyl OH), 7.29 (s, 1 H, phenyl
OH), 3.84 (s, 2H,
benzyl), 3.09/2.42 (t/t, 5.0 Hz, 4H/4H, piperazinyl), 2.18 (s, 3H, CH3).
Example 2
6-(3-Methoxy-phenyl)-3-[4-(4-methyl-pi perazin-1-yl)-phenyl]-pyrazolo[1,5-a]
pyrim idi n-7-
ylamine
6-(3-Methoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine is synthesized by condensation of compound of Stage 1.2 and 2-(3-
methoxy-
phenyl)-3-oxo-propionitrile (Stage 2.1) analogously to the preparation of
compound of
Example 1. Yield: 48%, solid powder; ES-MS: M+H = 415.1; HPLC: AtRet = 3.45
minutes.
'H-NMR (400 MHz, DMSO-d6j: 8.59/8.23 (s/s, 1H/1H, pyrazolopyrimidinyl), 8.06
(d, 9.0 Hz,
2H, phenyl), 7.55 (s, 2H, NHS), 7.43 (t, 8.5 Hz, 1 H, phenyl OMe), 7.10 (d,
8.5 Hz, 1 H, phenyl
OMe), 7.08 (s, 1 H, phenyl-OMe), 6.80 (d, 8.5 Hz, 1 H, phenyl-OMe), 6.98 (d,
9.0 Hz, 2H,
phenyl), 3.83 (s, 3H, CH3-O), 3.16/2.47 (m/m, 4H/4H, piperazinyl), 2.25 (s,
3H, CH3).
Sta ~~ 2-(3-Methoxy-phenyl)-3-oxo-propionitrile
2-(3-Methoxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation
of compound of Stage 1.3: Yield: 76%; white powder; ES-MS: M-H = 174.0; HPLC:
'°tRet =
4.75 minutes.
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'H-NMR (400 MHz, DMSO-ds): the compound forms a tautomeric equilibrium in
solution: 8.09/7.67 (s/s, 1 H, CH=/CH-OH), 7.38-7.23 (m, 2H, phenyl), 7.01-
6.97 (m, 1 H,
phenyl), 6.88-6.79 (m, 1 H, phenyl), 3.74 (s/broad, 3H, CH3-O).
Example 3
6-(3,5-Dimethoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5
a]pyrimidin-7-ylamine
6-(3,5-Dimethoxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-7-
ylamine is synthesized by condensation of compound of Stage 1.2 and 2-(3,5-
dimethoxy-
phenyl)-3-oxo-propionitrile (Stage 3.1) analogously to the preparation of
compound of
Example 1. Yield: 44%, solid powder; ES-MS: M+H = 445.0; HPLC: AtRer = 3.77
minutes.
'H-NMR (400 MHz, DMSO-ds): 8.59/8.23 (s/s, 1 H/1 H, pyrazolopyrimidinyl), 8.06
(d, 9.0 Hz,
2H, phenyl), 7.55 (s, 2H, NH2), 7.43 (t, 8.5 Hz, 1 H, phenyl-OMe), 7.10 (d,
8.4 Hz, 1 H, phenyl
OMe), 7.57 (s, 2H, NH2), 7.01 (d, 9.0 Hz, 2H, phenyl), 6.89 (s, 2H, phenyl-
OMe), 6.54 (s, 1 H,
phenyl-OMe), 3.83 (s, 6H, CH3-O), 3.16/2.47 (m/m, 4H/4H, piperazinyl), 2.24
(s, 3H, N-CH3).
Stage 3.1 2-(3,5-Dimethoxy-phenyl)-3-oxo-propionitrile
2-(3,5-Dimethoxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3. Yield: 48%; white powder; ES-MS: M+H = 206.0; HPLC:
AtRet = 4.79
minutes.
'H-NMR (400 MHz, DMSO-ds): the compound forms a tautomeric equilibrium in
solution:
8.11/7.68 (s/s, 1 H, CH=/CH-OH), 6.85/6.54 (s/s, 2H, phenyl), 6.44/6.38 (s/s,
1 H, phenyl),
3.74 (s/broad, 6H, CH3-O).
Example 4
6-(3-Benzyloxy-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-
a]pyrimidin-
7-ylamine
Prepared by the step disclosed in Stage 1.1.
Examples 5-69
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The following Examples enlisted on Table 1 are synthesized analogously to the
preparation
of Example 1. The syntheses of intermediates for the preparation of compounds
of
Examples 5-69 which are not commercially available are described in the text
below Table 1.
In cases where the title compounds carry a free amino group (Examples 52 -
54), the final
products are generated from their corresponding nitro-function carrying
precursors by
hydrogenation in the presence of Pd/C (10 %) in THF/MeOH during several hours.
NHZ
R2
NwN \
i /
~N R3
Table 1. H
Nb. A R2 R3 Analytical
Data
4-(4-Methyl-piperazin-4-Chlorophenyl H ES-MS [M+1]+
1-
yl)phenyl = 419.0/421.0;
HPLC AtRet
=
3.71 minutes
6 4-(4-Methyl-piperazin-1-yl)3-Chlorophenyl H ES-MS [M+1]+
phenyl = 419.0/421.0;
HPLC AtRet
=
3.92 minutes
7 4-(4-Methyl-piperazin-1-yl)Phenyl H ES-MS [M+1]+
phenyl = 385.1;
PLC AtRet
=
3.31 minutes
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Nb. A R2 R3 Analytical
Data
8 4-(4-Methyl-piperazin-1-yl)Phenyl Methyl ES-MS [M+1]+
phenyl = 399.1;
HPLC P'tRet
=
3.34 minutes
9 4-(4-Methyl-piperazin-1-yl)Methyl Phenyl ES-MS [M+1]+
phenyl = 399.0;
HPLC AtRet
=
. 3.36 minutes
4-dimethyl amino ~~ ~~ H m.p. 143-
phenyl
H o 146C;
N ~s ~ ~
0
Rt (CH2CIa
/
MeOH =
98:2): 0.32
ES-MS [M+1
]+
= 552.8;
HPLC AtRet
=
4.72 minutes
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Nb. A R2 R3 Analytical
Data
11 4-dimethyl amino o H Cp. 186-188
phenyl \ ~ ~~
I %
o
Rt (CH~CI2
/
MeOH =
98:2): 0.50;
ES-MS [M+1
]+
= 519.8;
H P LC AtRet
=
5.27 minutes
12 Phenyl 4-Methoxyphenyl Methyl ES-MS [M+1
]+
= 331.1;
HPLC BtRet
=
6.4 minutes
13 4-Methoxy-phenyl Phenyl Methyl ES-MS [M+1
]+
= 331.1;
HPLC BtRet
=
6.3 minutes
14 4-Methoxy-phenyl 4-Bromophenyl Methyl ES-MS [M+1
]+
= 408.9/410.9;
HPLC BtRet
=
7.1 minutes
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Nb. A R2 R3 Analytical
Data
15 Phenyl 4-Bromophenyl Methyl ES-MS (M+1]+
= 378.9/380.9;
HPLC BtRet
=
7.0 minutes
16 Phenyl 2,6-DichlorophenylH ES-MS [M+1]+
= 354.9/356.9;
HPLC BtRet
=
7.9 minutes
17 3-Methoxy-phenyl Phenyl H ES-MS [M+1
]+
= 317.1;
H P LC BtRet
=
6.8 minutes
18 Br H Phenyl ES-MS [M+1]+
= 288.9/290.9;
HPLC BtRet
=
6.3 minutes
19 4-(4-Methyl-piperazin-1-yl) H ES-MS [M+1]+
phenyl I ~ ~ = 441.0;
S
HPLC AtRet
=
1.91 minutes
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Nb. A R2 R3 Analytical
Data
20 4-Bromo-phenyl H Phenyl ES-MS [M+1]+
= 367.0;
HPLC AtRet
=
2.56 minutes
21 4-(4-Methyl-piperazin-1-yl) H ES-MS [M+1]+
phenyl ~ ~ = 391.1;
H P LC ''~tRet
=
1.49 minutes
22 3-Methoxyphenyl H ES-MS [M+1
]+
= 373.2;
HPLC ~tRet
=
2.22 minutes
23 4-(4-Methyl-piperazin-1-yl)Benzyl H ES-MS [M+1]+
phenyl = 399.2;
H P LC ~tRet
=
1.79 minutes
24 3-(4-Methyl-piperazin-1-yl)3-Methoxyphenyl H ES-MS [M+1]+
phenyl = 415.2;
HPLC ~tRet
=
1.82 minutes
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Nb. A R2 R3 Analytical
Data
25 4-(4-Methyl-piperazin-1-yl) H ES-MS [M+1]+
phenyl I \ ~ = 438.2;
/ ,
HPLC ~tRet
=
1.91 minutes
26 4-(4-Methyl-piperazin-1-yl)4-Methoxyphenyl H ES-MS [M+1]+
phenyl = 415.2;
H P LC ~tRet
=
2.04 minutes
27 4-(4-Methyl-piperazin-1-yl)2-Methoxyphenyl H ES-MS [M+1]+
phenyl = 415.2;
HPLC ~tRet
=
1.75 minutes
28 Pyridin-3-yl 3-Methoxyphenyl H ES-MS [M+1
]+
= 318.6;
HPLC ~tRet
=
1.84 minutes
29 3-(4-Methyl-piperazin-1-yl)3-Hydroxyphenyl H ES-MS [M+1J+
phenyl = 401.6;
HPLC AtRet
=
1.78 minutes
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Nb. A R2 R3 Analytical
Data
30 2-Methoxy-5-(4-Methyl-3-BenzyloxyphenylH ES-MS [M+1
]+
piperazin-1-yl)phenyl = 521.3;
HPLC ~tRet
=
2.07 minutes
31 2-Methoxy-5-(4-Methyl-3-Hydroxyphenyl H ES-MS [M+1]+
piperazin-1-yl)phenyl = 431.7;
HPLC ~tRet
=
1.66 minutes
32 4-(4-Methyl-piperazin-1-yl)2-BenzyloxyphenylH ES-MS [M+1]+
phenyl = 491.2;
HPLC ~tRet
=
1.77 minutes
33 4-(4-Methyl-piperazin-1-yl)2-Hydroxyphenyl H ES-MS [M+1]+
phenyl = 401.2;
HPLC tRet
=
1.37 minutes
34 4-(4-Methyl-piperazin-1-yl)4-BenzyloxyphenylH ES-MS [M+1]+
phenyl = 491.2;
HPLC ~tRer
=
1.85 minutes
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Nb. A R2 R3 Analytical
Data
35 4-(4-Methyl-piperazin-1-yl)4-Hydroxyphenyl H ES-MS [M+1]+
phenyl
= 401.2;
HPLC tRet
=
1.32 minutes
36 3-(4-Methyl-piperazin-1-yl)2-BenzyloxyphenylH ES-MS [M+1]+
phenyl = 491.3;
HPLC '4tRet
=
2.02 minutes
37 3-(4-Methyl-piperazin-1-yl)2-Hydroxyphenyl H ES-MS [M+1]+
phenyl = 401.3;
HPLC AtRet
=
1.71 minutes
38 3-(4-Methyl-piperazin-1-yl)4-BenzyloxyphenylH ES-MS [M+1]+
phenyl = 491.3;
HPLC ~tRet
=
2.05 minutes
39 3-(4-Methyl-piperazin-1-yl)4-Hydroxyphenyl H ES-MS [M+1]+
phenyl = 401.3;
HPLC ~tRet
=
1.70 minutes
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Nb. A R2 R3 Analytical
Data
40 2-Methoxy-5-(4-Methyl-2-BenzyloxyphenylH ES-MS [M+1
]+
piperazin-1-yl)phenyl = 521.3;
HPLC ~tRer
=
1.99 minutes
41 2-Methoxy-5-(4-Methyl-2-Hydroxyphenyl H ES-MS [M+1
]+
piperazin-1-yl)phenyl = 431.3;
HPLC ~tRet
=
1.70 minutes
42 2-Methoxy-5-(4-Methyl-4-BenzyloxyphenylH ES-MS [M+1]+
piperazin-1-yl)phenyl = 521.3;
HPLC CtRet
=
2.05 minutes
43 2-Methoxy-5-(4-Methyl-4-Hydroxyphenyl H ES-MS [M+1
]+
piperazin-1-yl)phenyl = 431.3;
HPLC ~tRet
=
1.68 minutes
44 1-methyl-1H-indol-3-yl3-BenzyloxyphenylH ES-MS [M+1]+
= 446.2;
HPLC ~tRet
=
2.39 minutes
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Nb. A R2 R3 Analytical
Data
45 1-methyl-1H-indol-3-yl3-Hydroxyphenyl H ES-MS [M+1]+
= 356.6;
HPLC ~tRet
=
2.06 minutes
46 3-Pyridyl 3-Hydroxyphenyl H ES-MS [M+1
]+
= 304.1;
HPLC ~tRet
=
1.72 minutes
47 2-methoxy phenyl 3-BenzyloxyphenylH ES-MS [M+1]+
= 423.2;
HPLC ctRet
=
2.10 minutes
48 2-methoxy phenyl 3-Hydroxyphenyl H ES-MS [M+1]+
= 333.2;
HPLC ~tRet
=
1.98 minutes
49 3-(4-Methyl-piperazin-1-yl)s H ES-MS [M+1]+
.
phenyl ~ / = 391.1;
HPLC tRet
=
1.56 minutes
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Nb. A R2 R3 Analytical
Data
50 2-Methoxy-5-(4-Methyl-s H ES-MS [M+1
]+
piperazin-1-yl)phenyl~ / = 421..1;
HPLC tRet
=
1.49 minutes
51 4-(4-Methyl-piperazin-1-yl)~N H ES-MS [M+1]+
phenyl ~ / = 386.2;
HPLC ~tRet
=
0.44 minutes
52 3-(4-Methyl-piperazin-1-yl)~ H ES-MS [M+1]+
phenyl ~ / = 400.2;
NH2
HPLC ~tRet
=
1.57 minutes
53 4-(4-Methyl-piperazin-1-yl)~ H ES-MS [M+1]+
phenyl ~ / = 400.0;
NH2
HPLC tRet
=
1.75 minutes
54 4-(4-Methyl-piperazin-1-yl)~ H ES-MS [M+1]+
phenyl
= 400.2;
NHZ HPLC tRet
=
1.40 minutes
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Nb. A R2 R3 Analytical
Data
55 4-(4-Methyl-piperazin-1-yl)4-methyl thiazol-2-ylH ES-MS [M+1]+
phenyl = 405.6;
HPLC ~tRet
=
2.11 minutes
56 2-Methoxy-5-(4-Methyl-s H ES-MS [M+1]+
piperazin-1-yl)phenylI ~ \ = 471.5;
HPLC CtRet
=
1.80 minutes
57 4-methoxy phenyl s H ES-MS [M+1]+
I = 373.7;
HPLC p'tRet
=
2.24 minutes
58 3-methoxy phenyl s H ES-MS [M+1]+
= 323.1;
H P LC ~tRet
=
2.09 minutes
59 3-methoxy phenyl ~ H ES-MS [M+1]+
= 423.2;
~
p HPLC
tRet =
2.38 minutes
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Nb. A R2 R3 Analytical
Data
60 3-methoxy phenyl ~ H ES-MS [M+1]+
I = 333.6;
.
OH
H P LC CtRet
=
2.02 minutes
61 4-(4-Methyl-piperazin-1-yl)~ H ES-MS
phenyl
/ [M+11+ _
448.2;
HN~ /j
S
o/ ~ H P LC :
~tRet =
1.62 minutes
62 4-(4-Methyl-piperazin-1-yl)~ H ES-MS
phenyl I i [M+1 ]+
_
HN~S j 558.2;
o~
~
HPLC :
t,qet =
1.87 minutes
63 4-(4-Methyl-piperazin-1-yl)~ H ES-MS
phenyl I [M+1 ]+
-_
442.1;
HN Me
HPLC : tRet
=
1.39 minutes
64 3-(4-Methyl-piperazin-1-yl)~ ~ I F H ES-MS
0
( +
~
~
phenyl ~ ~ N: [M+1 ]
~~ -_
o
558.4;
HPLC : ~tRet
=
2.OOminutes
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Nb. A R2 R3 Analytical
Data
65 3-(4-Methyl-piperazin-1-yl)~ o H ES-MS
phenyl ~ ~ ~ [M+1]+-_
N
442.6;
HPLC : CtRet
=
1.70minutes
66 4-(4-Methyl-piperazin-1-yl)s H ES-MS
~~~ +
phenyl [M+1]
_
N
391.5;
HPLC : ~tRet
=
1.79 minutes
67 4-(4-Methyl-piperazin-1-yl)~ H ES-MS
phenyl ~ , ~S~ [M+1]+
H \ 47824;
HPLC : ~tRet
=
1.76 minutes
68 4-(4-Methyl-piperazin-1-yl)~ ~ ~ F H ES-MS
0
~ +
~
s
phenyl . . ~ N% _
v [M+1 ]
H
558.2;
~ HPLC : tRet
=
1.94 minutes
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Nb. A R2 R3 Analytical
Data
69 4-(4-Methyl-piperazin-1-yl) ~ ~ H ES-MS
phenyl ~ , ~ [M+1 ]+ -_
H 442.2;
HPLC : ~tRet =
1.62 minutes
Stage 5.1: 2-(4-Chloro-phenyl)-3-oxo-propionitrile
2-(4-Chloro-phenyl)-3-oxo-propionitrile is prepared analogously to the
preparation of
compound of Stage 1.3: 89%; ES-MS [M-1]-= 177.9/179.9; HPLC'~tRet= 5.67
minutes.
Stage 6.1: 2-(3-Chloro-phenyl)-3-oxo-propionitrife
2-(3-Chloro-phenyl)-3-oxo-propionitrile is prepared analogously to the
preparation of
compound of Stage 1.3: 89%; ES-MS [M-1 ]- = 177.9/179.9; HPLC AtRer = 5.60
minutes.
Stage 8.1 3-Oxo-2-phenyl-butyronitrile
3-Oxo-2-phenyl-butyronitrile is prepared analogously to the preparation of
compound of
Stage 1.3: 62%, white crystals, m.p. >215°C; ES-ES-MS M-H = 157.9, Rf
(hexane / AcOEt =
1:1 ) = 0.57.
'H-NMR (400 MHz, DMSO-ds): 7.84 (d, 9.0 Hz, 2H), 7.04 (t, 9.0 Hz, 2H), 6.68
(t, 9.0 Hz,
1 H), 3.21 (s/broad, 1 H, CH), 2.03 (s, 3H, CH3).
Sta ec~9.1 2-Methyl-3-oxo-3-phenyl-propionitrile
2-Methyl-3-oxo-3-phenyl-propionitrile is prepared analogously to the procedure
of Yoo et al.,
Tetrahedron Lett., Vol. 43, No. 27, pp. 4813-4815 (2002). 2-Bromo-
propionitrile (0.965 mL,
11.05 mmol) and In-powder (975 mg, 8.5 mmol) are stirred under Ar in THF (15
mL) for 1
hour. After adding benzoylnitrile (735 mg, 5.6 mmol) during 2 minutes, the
reaction mixture
is stirred at 60°C in a microwave ofen (Emrys optimizer, personal
chemistry, Sweden) for 30
minutes. After filtration over Hyflo and washing with THF (5 mL), the reaction
solution is
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concentrated under reduced pressure and partitioned between ether (150 mL) and
phosphate buffer (pH = 7, 150 mL). After separation of the organic phase, the
aqueous
phase is extracted with ether (150 mL). The combined organic phases are washed
with
brine (30 mL), dried (Na2S04), concentrated under reduced pressure and flash
chromatography (silica gel, 2 x 18 cm, hexane / AcOEt = 3:1) to compound of
Stage 9.1 as
slightly yellowish oil (300 mg, 1.9 mmol; 34%); ES-MS: M-H = 157.9; Rf (hexane
/ AcOEt =
1:1) = 0.60.
'H-NMR (400 MHz, DMSO-d6): 8.06 (d, 8.5 Hz, 2H), 7.74 (t, 8.5 Hz, 1H), 7.62
(t, 8.5 Hz, 2H),
5.17 (q, 8.5 Hz, 1 H, CH), 1.52 (s, 3H, CH3).
The compound of Example 10 is synthesized analogously to the preparation of
compound of Stage 1.1 by condensing 2,3-dichloro-N-[4-(cyano-formyl-methyl)-
phenyl]-
benzenesulfonamide (Stage 10.1) and 4-(4-dimethylamino-phenyl)-2H-pyrazol-3-
ylamine
(Stage 10.3).
Stage 10.1 2,3-Dichloro-N-[4-(cyano-formyl-methyl)-phenyl]-benzenesulfonamide
Under an atmosphere of N2 is added portion-wise freshly-cut pieces of sodium
(2.3 g total,
100 mmol) to EtOH abs. (230 mL) within 15 minutes which is a slightly
exothermic (up to
43°C). After all sodium is dissolved (ca. 1 hour) 2,3-dichloro-N-(4-
cyanomethyl-phenyl)-
benzene-sulfonamide (Stage 10.2) (26.27 g, 77 mmol) and formic acid ethyl
ester (11.2 mL,
139 mmol) is added to the colorless solution at RT. The mixture is heated to
reflux for 2
hours. After cooling to RT, the solvent is removed under reduced pressure and
the residue
dissolved in H20 (20 mL), followed by addition of AcOH (200 mL; pH 4). The
aqueous layer
is extracted with CH2C12 (2 x, 500 mL), the combined organics are washed with
H20 and
dried over Na2S04. Purification is. done by repeated chromatography (silica
gel, EtOAc and
CHzCl2 l MeOH = 98:2) to obtain 2,3-dichloro-N [4-(cyano-formyl-methyl)-
phenyl]-
benzenesulfonamide (233 mg, 1 % yield) as beige crystals: m.p. 88-
102°C; (CHzCIz / MeOH
= 95:5): 0.22; ES-MS [M+1 ]+ = 368; HPLC BtRer = 5.61 minutes.
Stage 10.2 2,3-Dichloro-N-(4-cyanomethyl-phenyl)-benzenesulfonamide
To the solution of 4-aminobenzylcyanide (12 g, 90.8 mmol) in pyridine (11 mL)
at RT, a
solution of 2,3-dichlorobenzene-sulphonylchloride (22.29 g, 90.8 mmol) in THF
(80 mL) is
added within 20 minutes. 'The reaction is stirred at reflux for 2 hours. After
cooling, the
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solvent is removed under reduced pressure and the remaining solid suspended in
10% HCI
(200 mL). The crude crystalline product is filtered-off, washed with H20 and
dried at 60°C.
Final purification is done by suspending the crude compound in MeOH (250 mL),
heating to
reflux, filtration and drying. 2,3-Dichloro-N-(4-cyanomethyl-phenyl)-
benzenesulfonamide
(26.54 g, 86%) is obtained as orange crystals: m.p: 202-206°C; (CH2CI2
/ MeOH 98:2): 0.54;
ES-MS [M-1 ]- = 338.8; HPLC BtRe~ = 5.85 minutes.
Stage 70.3 4-(4-Dimethylamino-phenyl)-2H-pyrazol-3-ylamine
4-(4-Dimethylamino-phenyl)-2H-pyrazol-3-ylamine is prepared from 2-(4-
dimethylamino-
phenyl)-3-oxo-propionitrile (Stage 10.4) and hydrazine hydrate as described in
U.S. Patent
No. 2,989,539 (20.6.61; Anderson and Reiff; Example 18). 4-(4-Dimethylamino-
phenyl)-2H-
pyrazol-3-ylamine : m.p. 173-176°C; (CH~CIz l MeOH / NH3 = 90:10:1):
0.37; ES-MS [M+1]+
= 203; HPLC BtRet = 1.40 minutes.
Stage 70.4 2-(4-Dimethylamino-phenyl)-3-oxo-propionitrile
2-(4-Dimethylamino-phenyl)-3-oxo-propionitrile is prepared from (4-
dimethylamino-phenyl)-
acetonitrile, ethyl formate and sodium as described in U.S. Patent No.
2,989,539 (Example
18).
2-(4-Dimethylamino-phenyl)-3-oxo-propionitrile: m.p. 175-178°C; ES-MS
[M+1]+ = 189;
HPLC BtRet = 2.00 minutes.
The compound of Example 11 is prepared analogously to the synthesis of the
compound of
Example 10 using 4-(4-dimethylamino-phenyl)-2H-pyrazol-3-ylamine (Stage 10.3)
and 4-
chloro-benzenesulfonic acid 4-(cyano-formyl-methyl)-phenyl ester (Stage 11.1).
Stage 77.7 4-Chloro-benzenesulfonic acid 4-(cyano-formyl-methyl)-phenyl
ester
4-Chloro-benzenesulfonic acid 4-(cyano-formyl-methyl)-phenyl ester is prepared
as
described in Example 10 (Stage 10.1), using commercially-available 4-
(cyanomethyl)phenyl-
4-chlorobenzene-1-sulfonate instead.
4-Chloro-benzenesulfonic acid 4-(cyano-formyl-methyl)-phenyl ester (162 mg);
yellowish
solid; (CH2CI2 / MeOH = 95:2): 0.32; ES-MS [M+1 ]+ = 335; HPLC BtRer = 6.23
minutes.
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Stage 12.1 2-(4-Methoxy-phenyl)-3-oxo-butyronitrile
2-(4-Methoxy-phenyl)-3-oxo-butyronitrile is prepared as described by Smith,
Breen, Hajek
and Awang, J. Org. Chem., Vol. 35, No. 7, pp. 2215-2221 (1970).
Stage 14.1 2-(4-Bromo-phenyl)-3-oxo-butyronitrile
2-(4-Bromo-phenyl)-3-oxo-butyronitrile is synthesized according to the
procedure of Rau,
Ger. Offen., DE 3001266 (1980).
Stage 16.1 1,2-(2,6-Dichloro-phenyl)-3-oxo-propionitrile
1,2-(2,6-Dichloro-phenyl)-3-oxo-propionitrile is prepared as described by
Menzer, Lankau
and Unverferth, Ger. Otfen., DE 19521822 (1996).
Stae~e 17.1 4-(3-Methoxy-phenyl)-2H-pyrazol-3-ylamine
4-(3-Methoxy-phenyl)-2H-pyrazol-3-ylamine and Stage 22.2 are prepared as
described by
Bruni et al., Heterocyclic. Chem., Vol. 32, No. 1, pp. 291-298 (1995).
Stage 19.1 2-Benzo[b]thiophen-3-yl-3-oxo-propionitrile
2-Benzo[b]thiophen-3-yl-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 56%; white powder; ES-MS: M-H = 123.9; HPLC:
AtRer = 2.20
minutes.
'H-NMR (300 MHz, DMSO-d6): 12.0 (s/broad, 1H), 8.00-7.70 (m, 3H), 7.45-7.35
(m, 2H).
Stage 21.1 3-Oxo-2-thiophen-3-yl-propionitrile
3-Oxo-2-thiophen-3-yl-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 51 %; white powder, ES-MS: M-H = 112.9; HPLC:
AtRet =
2.03 minutes.
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz, DMSO-
ds):
7.95/7.55 (s/s, 1 H, CH=/CH-OH), 7.55-7.50 (m, 2H), 7.30-7.20 (m, 1 H).
Stage 22.1 4-Benzo[b]thiophen-3-yl-1 H-pyrazol-3-ylamine
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4-Benzo[b]thiophen-3-yl-1H pyrazol-3-ylamine is synthesized analogously to the
preparation
of compound of Stage 1.2: Yield: 80%; white powder; ES-MS: M+H = 216Ø
'H-NMR (300 MHz, DMSO-ds): 12.0 (s/broad, 1H), 8.00-7.80 (m, 2H), 7.75
(s/broad, 1H),
7.60 (s/broad, 1 H), 7.40-7.30 (m, 2H).
Stage 22.2 (2-(3-Methoxy-phenyl)-3-oxo-propionitrile)
(2-(3-Methoxy-phenyl)-3-oxo-propionitrile) is prepared as described by Bruni
et al.,
Heterocyclic. Chem., Vol. 32, No. 1, pp. 291-298 (1995).
Stage 23.7 2-Formyl-3-phenyl-propionitrile
2-Formyl-3-phenyl-propionitrile is synthesized analogously to the preparation
of compound of
Stage 1.3: Yield: 77%; oil; ES-MS: M-H = 158Ø
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz, DMSO-
ds):
7.40-7.15 (m, 5H), 2.85-2.75 (m, 2H).
Stage 24.7 4 [3-(4-Methyl-piperazin-1-yl)-phenyl]-acetonitrile
4 [3-(4-Methyl-piperazin-1-yl)-phenyl]-acetonitrile is synthesized analogously
to the
preparation of compound of Stage 1.5: Yield: 55 %; brown solid; ES-MS: M+H =
216.7;
HPLC: ~tRet = 1.65 minutes.
'H-NMR (300 MHz, CDCI3): 7.30-7.25 (m, 1 H), 6.90-6.82 (m, 2H), 6.80-6.75 (m,
1 H), 3.70 (s,
2H), 3.25-3.15 (m, 4H), 2.60-2.50 (m, 4H), 2.35 (s, 3H).
Stage 24.2 2-[3-(4-Methyl-piperazin-1-yl)-phenyl]-3-oxo-propionitrile
2-[3-(4-Methyl-piperazin-1-yl)-phenyl]-3-oxo-propionitrile is synthesized
analogously to the
preparation of compound of Stage 1.3: Yield: 100%; brown solid; ES-MS: M+H =
244.1;
HPLC: ~tRet = 1.67 minutes.
Stage 24.3 4-[3-(4-Methyl-piperazin-1-yl)-phenyl]-1 H pyrazol-3-ylamine
4-[3-(4-Methyl-piperazin-1-yl)-phenyl]-1 H-pyrazol-3-ylamine is synthesized
analogously
to the preparation of compound of Stage 1.2: Yield: 36%; yellow foam; ES-MS:
M+H =
258.2; HPLC: ~tRet = 1.46 minutes:
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'H-NMR (300 MHz, CDCI3): 7.45 (s, 1 H), 7.30-7.25 (m, 1 H), 7.05-7.00 (m, 1
H),
6.95-6.90 (m, 1 H), 6.85-6.80 (m, 1 H), 4.00 (s/broad, 2H), 3.30-3.20 (m, 4H),
2.65-2.58
(m, 4H), 2.35 (s, 3H).
Sta ec~ 25.? 2-(1-Methyl-1H-indol-3-yl)-3-oxo-propionitrile
2-(1-Methyl-1H-indol-3-yl)-3-oxo-propionitrile is synthesized analogously to
the
preparation of compound of Stage 1.3: Yield: 59%; oil; ES-MS: M+H =199.1.
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz,
CDC13):
8.00/7.95 (s/s, 1 H), 7.60-7.20 (m, 5H), 3.75 (s, 3H).
Sta. eg 26.1 2-(4-Methoxy-phenyl)-3-oxo-propionitrile
2-(4-Methoxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 80%; white solid; ES-MS: M-H =174.3.
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz, DMSO-
ds):
7.80/7.58(s/s, 1 H), 7.55-7.50 (m, 1 H), 7.30-7.20 (m, 1 H), 6.90-6.80 (m,
2H), 3.73/3.70 (s/s,
3H).
Stae~e 27.1 2-(2-Methoxy-phenyl)-3-oxo-propionitrile
2-(2-Methoxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 40%; brown oil; ES-MS: M-H = 174.3; HPLC CtRer =
2.01
minutes.
Stage 28.1 3-Oxo-2-pyridin-3-yl-propionitrile
3-Oxo-2-pyridin-3-yl-propionitrile is synthesized analogously to the
preparation of compound
of Stage 1.3: Yield: 71 %; brown solid; ES-MS: M+H =147.2; HPLC ~tRef = 1.31
minutes.
Stage 28.2 4-Pyridin-3-yl-1H-pyrazol-3-ylamine
4-Pyridin-3-yl-1H-pyrazol-3-ylamine is synthesized analogously to the
preparation of
compound of Stage 1.2: Yield: 68%; brown solid; ES-MS: M+H =161.2; HPLC ~tRet
=
0.50 minutes.
Stage 30.1 [2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-acetonitrile
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[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-acetonitrile is synthesized
analogously to the
preparation of compound of Stage 1.5: Yield: 51 %; brown solid; ES-MS: M+H =
246.6;
HPLC: ~tRet = 1.72 minutes.
'H-NMR (300 MHz, CDCI3): 7.00-6.95 (m, 1H), 6.85-6.75 (m, 2H), 3.80 (s, 3H),
3.65 (s, 2H),
3.15-3.05 (m, 4H), 2.60-2.55 (m, 4H), 2.35 (s, 3H).
Stage 30.2 2-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-3-oxo-
propionitrile
2-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-3-oxo-propionitrile is
synthesized
analogously to the preparation of. compound of Stage 1.4: Yield: 100%; brown
solid; ES-MS:
M+H = 274.1; HPLC: ~tRet = 1.62 minutes.
Sta a 30.3 4-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-2H-pyrazol-3-
ylamine
4-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-2H-pyrazol-3-ylamine is
synthesized
analogously to the preparation of compound of Stage 1,2: Yield: 32%; brown
solid; ES-MS:
M+H = 288.2; HPLC: ~tRet = 1.46 minutes.
'H-NMR (300 MHz, CDC13): 7.50 (s, 1 H), 7.00-6.95 (m, 1 H), 6.90-6.80 (m, 2H),
3.80 (s, 3H),
3.20-3.10 (m, 4H), 2.65-2.55 (m, 4H), 2.35 (s, 3H).
Stage 32.7 2-(2-Benzyloxy-phenyl)-3-oxo-propionitrile
2-(2-Benzyloxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 85%; white solid; ES-MS: M+H = 252.6; HPLC:
~tRet = 2.35
minutes.
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz, DMSO-
ds):
11.6/7.78 (s, 1 H), 7.55-7.45 (m, 2H), 7.40-7.20 (m, 5H), 7.15-7.05 (m, 1 H),
7.00-6.90 (m,
1 H), 5.15 (s, 2H).
Stage 34.7 2-(4-Benzyloxy-phenyl)-3-oxo-propionitrile
2-(4-Benzyloxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 95%; white solid; ES-MS: M-H = 250.3; HPLC:
~tRet =
2.41 minutes.
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The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz,~DMSO-
ds):
12.0/11.7 (s, 1 H), 7.90-7.80 and 7.60-7.50 (m, 1 H), 7.40-7.25 (m, 6H), 7.05-
6.95 (m, 2H),
5.10 (s, 2H).
Sta eg 44.1 4-(1-Methyl-1H-indol-3-yl)-2H-pyrazol-3-ylamine
4-(1-Methyl-1 H-indol-3-yl)-2H-pyrazol-3-ylamine is synthesized analogously to
the
preparation of compound of Stage 1.2: Yield: 10%; brown foam; ES-MS: M+H =
213.2;
HPLC: °tRet = 1.66 minutes.
'H-NMR (300 MHz, DMSO-d6): 7.70 (d, 1 H), 7.60 (s, 1 H), 7.35 (d, 1 H), 7.30-
7.25 (m, 1 H),
7.20-7.10 (m, 2H), 3.80 (s, 3H).
Stage 47.1 2-(2-Methoxy-phenyl)-3-oxo-propionitrile
2-(2-Methoxy-phenyl)-3-oxo-propionitrile is synthesized analogously to the
preparation of
compound of Stage 1.3: Yield: 59%; white solid; ES-MS: M+H =175.3; HPLC: ~tRet
=
2.01 minutes.
Sta_ ec~ 47.2 4-(2-Methoxy-phenyl)-2H pyrazol-3-ylamine
4-(2-Methoxy-phenyl)-2H-pyrazol-3-ylamine is synthesized analogously to the
preparation of
compound of Stage 1.2: Yield: 35%; white solid; ES-MS: M+H =190.1; HPLC:
~tRet= 1.40
minutes.
'H-NMR (300 MHz, DMSO-ds): 11.5 (bs, _1 H), 7.50 (bs, 1 H), 7.30 (bs, 1 H),
7.20-7.05 (m,
1 H), 7.00-6.85 (m, 2H), 4.30 (bs, 2H), 3.75 (s, 3H).
Stage 51.1 3-Oxo-2-pyridin-4-yl-propionitrile
3-Oxo-2-pyridin-4-yl-propionitrile is synthesized analogously to the
preparation of compound
of Stage 1.3: Yield: 59%; orange solid; ES-MS: M+H = 147.2; HPLC: ~tRet = 1.00
minute.
The compound forms a tautomeric equilibrium in solution: 'H-NMR (300 MHz, DMSO-
ds):
13.1/9.60 (bs, 1 H), 9.10 (bs, 1 H), 8.20-8.00 (m, 2H), 7.95-7.80 (m, 1 H).
Stage 52.1 (~-3-Dimethylamino-2-(3-nitro-phenyl)-acrylonitrile
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(3-Nitro-phenyl)-acetonitrile (1.51 g, 9.31 mmol), dimethoxymethyl-dimethyl-
amine (6.2 mL,
46.5 mmol) in xylene (30 mL) are stirred at reflux for 1 hour. After adding
hexane (20 mL),
the reaction mixture is cooled at 0°C. Precipitating material is
filtered-off to give compound
of Stage 52.1 as brown solid (1.76 g, 8.19 mmol; 88%); ES-MS: M+H = 218.1;
HPLC: ~tRet =
2.24 minutes.
'H-NMR (300 MHz, DMSO-ds): 8.10-8.05 (m, 1 H), 7.90-7.85 (m, 1 H), 7.75-7.72
(m, 1 H),
7.70 (s, 1 H), 7.65-7.60 (m, 1 H), 3.30 (s, 6H).
Stage 52.2 3-[3-(4-Methyl-piperazin-1-yl)-phenyl]-6-(3-nitro-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
4-[3-(4-Methyl-piperazin-1-yl)-phenyl]-1H-pyrazol-3-ylamine (Stage 24.3) (305
mg,
1.18 mmol), (~-3-dimethylamino-2-(3-nitro-phenyl)-acrylonitrile (Stage 52.1)
(335 mg,
1.54 mmol) dissolved in AcOH (10 mL) and BuOH (10 mL) are stirred at reflux
for 16 hours.
After adding saturated NaHC03 aqueous solution, the reaction mixture is
extracted with
EtOAc (50 mL, 2 x). The combined organic phases are washed with H20 (10 mL),
dried
(Na~S04), concentrated under reduced pressure and flash chromatographed
(silica gel, 2.5 x
15 cm, CHZCIZ / MeOH = 9:1 ) to give compound of Stage 52.2 as orange solid
(224 mg,
0.52 mmol; 44%); ES-MS: M+H = 430.1; HPLC: ~tRet = 1.91 minutes.
'H-NMR (300 MHz, DMSO-ds): 8.70 (s, 1 H), 8.35-8.30 (m, 1 H), 8.25 (s, 1 H),
8.22-8.18 (m,
1 H), 7.98-7.95 (m, 1 H), 7.90 (bs, 2H), 7.80-7.70 (m, 2H), 7.65-7.60 (m, 1
H), 7.25-7.18 (m,
1 H), 6.80-6.75 (m, 1 H), 3.20-3.10 (m, 4H), 2.50-2.40 (m, 4H), 2.20 (s, 3H).
Stage 53.1 3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-(3-nitro-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-(3-nitro-phenyl)-pyrazolo[1,5-
a]pyrimidin-7-ylamine is
synthesized analogously to the preparation of compound of Stage 52.2: Yield:
30%; red
solid; ES-MS: M+H = 430Ø
'H-NMR (300 MHz, DMSO-ds): 8.60 (s, 1 H), 8.35-8.30 (m, 1 H), 8.22 (s, 1 H),
8.20-8.10 (m,
1 H), 8.00 (d, 2H, J=7.9Hz), 7.95-7.90 (m, 1 H), 7.85 (bs, 2H), 7.80-7.75 (m,
1 H), 6.95 (d, 1 H,
J=7.9Hz), 3.20-3.10 (m, 4H), 2.50-2.40 (m, 4H), 2.20 (s, 3H).
Stage 54.1 (~-3-Dimethylamino-2-(2-nitro-phenyl)-acrylonitrile
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(~-3-Dimethylamino-2-(2-nitro-phenyl)-acrylonitrile is synthesized analogously
to the
preparation of compound of Stage 52.1: Yield: 97%; brown solid.
'H-NMR (300 MHz, DMSO-ds): 7.82-7.78 (m, 1H), 7.62-7.55 (m, 1H), 7.45-7.35 (m,
2H),
7.20 (s, 1 H), 3.15 (s, 6H).
Stage 54.2 3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-(2-nitro-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
3-[4-(4-Methyl-piperazin-1-yl)-phenyl]-6-(2-nitro-phenyl)-pyrazolo[1,5-
a]pyrimidin-7-ylamine is
synthesized analogously to the preparation of compound of Stage 55.2: brown
solidl; ES-
MS: M+H = 430.0; HPLC: °tRer = 1:61 minutes.
'H-NMR (300 MHz, DMSO-d6): 8.55 (s, 1H), 8.20-8.15 (m, 1H), 8.05-7.95 (m, 3H),
7.82-7.60
(m, 5H), 7.00-6.95 (m, 2H), 3.15-3.05 (m, 4H), 2.45-2.40 (m, 4H), 2.20 (s,
3H).
Stage 55.7 (E7-3-Dimethylamino-2-(4-methyl-thiazol-2-yl)-acrylonitrile
(E7-3-Dimethylamino-2-(4-methyl-thiazol-2-yl)-acrylonitrile is synthesized
analogously to the
preparation of compound of Stage 52.1: Yield: 74%; black solid; ES-MS: M+H =
194.2;
HPLC: ~tRet = 1.57 minutes.
'H-NMR (300 MHz, DMSO-ds): 7.76 (s, 1H), 6.60 (s, 1H), 3.25 (bs, 6H), 2.35 (s,
3H).
Example 61
3-{7-Am i no-2-methyl-3-[4-(4-methyl-piperazin-1-yl)phenyl]-pyrazolo[1,5-a~
pyrimid i n-6-
yl}-phenol
3-(7-Amino-2-methyl-3-[4-(4-methyl-piperazin-1-yl)phenyl]-pyrazolo[1,5-
a]pyrimidin-6-yl}-
phenol is synthesized analogously to the preparation of Example 1 by using
methyl
hydrazine instead of hydrazine when the pyrazole ring is formed: ES-MS: M+H =
415.2;
HPLC: °tRer = 1.45 minutes.
'H-NMR (300 MHz, DMSO-d6): 9.53 (s, 1H, OH), 2.56 (s, 3H CH3), 2.24 (s, 3H
CH3).
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Example 62
(4-[7-Amino-3-[4-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-6-
yl}-
phenyl)-carbamic acid ethyl ester
4-(4-(4-Methyl-piperazin-1-yl)-phenyl)-2H-pyrazol-3-ylamine (Stage 1.2) (200
mg, 0.81 mmol)
and [4-(2-cyano-1-formyl-ethyl)-phenyl]-carbamic acid ethyl ester (Stage 62.1)
(275 mg, 0.04
mmol) dissolved in EtOH (4 mL) and ethanolic HCI (1.6 mL, 2.5 N) are stirred
under reflux
for 17 hours under Ar. After adding H20 (4 mL) and KZC03 (250 mg), the
reaction mixture is
extracted with CH2CI2 (20 mL, 2 x). The combined organic phases are washed
with Hz0 (10
mL), dried (Na~S04), concentrated under reduced pressure and flash
chromatographed
(silica gel, 2.5 x 15 cm, CH2CIz / MeOH / NH3 = 95:5:0.5) to give compound of
Example 62
as white solid (58 mg, 0.123 mmol; 15%); ES-MS: M+H = 472.0; Rf (CH~Ch / MeOH
/ NH3 =
90:10:0.1 ) = 0.42; HPLC: AtRet = 4.26 minutes.
'H-NMR (400 MHz, DMSO-d6): 8.75/8.58 (s/s, 1 H/1 H, pyrazolopyrimidinyl), 8.03
(d, 9.0 Hz,
2H, phenyl), 7.61 (d, 9 Hz, 2H, phenyl), 7.53 (s, 2H, NH2), 7.46 (d, 9 Hz, 2H,
phenyl), 7.00
(d, 9 Hz, 2H, phenyl), 4.17 (q, 7.5 Hz, 2H, CH2-Ethyl), 3.1712.48 (m/m, 4H/4H,
piperazinyl),
2.24 (t, 7.5 Hz, 3H, CH3).
Stage 62a.1 [4-(Cyano-1-formyl-methyl)-phenyl]-carbamic acid ethyl ester
[4-(Cyano-methyl)-phenyl]-carbamic acid benzyl ester (Stage 62a.2) (1 g, 3.76
mmol) is
formylated in analogy to the preparation of Stage 1.3 giving the corresponding
carbamic acid
ethyl ester (thereby also transforming the benzyl ester function into the
ethyl ester function):
colorless crystals (654 mg, 2.66 mmol, 70%). ES-MS: M+H = 233Ø
'H-NMR (400 MHz, DMSO-ds): 4.12 (q/broad, 7.5 Hz, 2H, CH2-Ethyl), 1.23
(t/broad, 7.5 Hz,
3H, CH3-Ethyl).
Stage 62.2 [4-(Cyano-methyl)-phenyl]-carbamic acid benzyl ester
(4-Amino-phenyl)-acetonitrile (2 g, 15.1 mmol) and dibenzyl dicarbonate (4.33
g, 15.1 mmol)
dissolved in dioxane (16 mL) are stirred for 1 hour at RT. After evaporating
the solvent, the
product is isolated by flash chromatography (silica gel, 4.5 x 25 cm, CH2CI2 /
MeOH = 99:1):
white solid (3.82 g, 14.4 mmol; 95%); ES-MS: M-H = 265.0; Rf (CH2Clz / MeOH =
95:5) _
0.49; HPLC: AtRer = 6.32 minutes.
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'H-NMR (400 MHz, DMSO-ds): 9.82 (s, 1 H, NH), 7.51 - 7.35 (m, 7H, aryl), 7.26
(d, 8.5 Hz,
2H, aryl), 5.15 (s, 2H, CH2), 3.95 (s, 2H, CH2).
(Z-)3-Dimethylamino-2-thiazol-4-yl-acrylonitrile
(Z-)3-Dimethylamino-2-thiazol-4-yl-acrylonitrile is synthesized analogously to
the preparation
ofcompound of Stage 52.1: ES-MS [M+1]+ = 180.1; HPLC : ~tRet = 1.91 minutes
Compounds 61, 62, 64, 67, and 68 carrying sulfonamide and acetylamide
functions
(compounds 63, 65 and 69) are prepared by reacting the amino precursor with
the
corresponding sulfonic acid chloride or acetic acid anhydride in the presence
of pyridine.
Examples 70 and 71
The compounds in Table 2 and Table 3 are prepared according to Example 1.
R2
R3
Table 2 - Example 70
Nb. R' R2 R3
A 4-(4-Methyl-piperazin-1-yl) \ H
y
~s
B 4-(-O-(CH~)2-NH2) 3-Hydroxyphenyl H
C 4-(-O-(CHZ)2-NH2) H 4-pyridinyl
D 4-(4-Methyl-piperazin-1-yl) ~ NH2 H
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N b. R' R2 R3
E 4-(4-Methyl-piperazin-1-yl) ~ ~ F F H
\ WF
~i
F 4-(4-Methyl-piperazin-1-yl)/2-CI ~ H
OH
G 4-Dimethylaminyl F H ° c~ c~ H
Non
\ /
/ o
H 4-(4-Methyl-piperazin-1-yl) H ° c~ c~ H
~N. N
is \ l
/ o
I 4-Dimethylaminyl c~ H
\ ° \ / ci
K 4-(4-Methyl-piperazin-1-yl) c~ H
° \ / ci
L 4-(4-Methyl-piperazin-1-yl) o\ ~~ H
~ ~ o \ /
M 4-(4-Methyl-piperazin-1-yl) H 4-Pyridinyl
N 4-(4-Methyl-piperazin-1-yl) ~ N H
/ /Me
O
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N b. R' ~ R2 R3
O 4-(4-Methyl-piperazin-1-yl) ~ N H
OH
R 4-(4-Methyl-piperazin-1-yl) ~ O~e H
N
i
S 4-(4-Methyl-piperazin-1-yl) ~ OH H
iN
V 4-(4-Methyl-piperazin-1-yl) H
/N
O
W 4-(4-Methyl-piperazin-1-yl)/2- ~ H
Methoxy
/ O~IIe
X 4-(4-Methyl-piperazin-1-yl)/2- ~ H
Methoxy
OH
Y 4-(4-Methyl-piperazin=1-yl) ~ H
/ / a
O
Z 4-(4-Methyl-piperazin-1-yl) ~ H
I /
OH
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N b. R' R2 R3
z1 3-(4-Methyl-piperazin-1-yl) ~ N H
/ /Me
O
z2 3-(4-Methyl-piperazin-1-yl) ~ N H
I~
OH
z3 3-(4-Methyl-piperazin-1-yl) ~ O~Me H
N
z4 3-(4-Methyl-piperazin-1-yl) ~ OH H
I ~N
z5 3-(4-Methyl-piperazin-1-yl) 1 H
/N
O
Z6 4-(4-Methyl-piperazin-1-yl) ~ N F F H
~~ ,
i
0
Z7 3-(4-Methyl-piperazin-1-yl) ~ CH3
OH
Z8 3-(4-Methyl-piperazin-1-yl) ~~ ~~ H
N' ~o _
is \ l
0
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N b. R' R2 R3
Z9 4-(4-Methyl-piperazin-1-yl) ~ H
~N
O
z10 4-(4-Methyl-piperazin-1-yl) ~ N H
(/
OH
z11 3-(4-Methyl-piperazin-1-yl) ~ H
~N
O
z12 3-(4-Methyl-piperazin-1-yl) ~ N H
/
OH
z13 5-(4-Methyl-piperazin-1-yl)/2- ~ H
methoxy
~N
O
z14 5-(4-Methyl-piperazin-1-yl) /2- ~ N H
methoxy
/ OH
z15 4-(4-Methyl-piperazin-1-yl)/2- ~ H
methoxy
~~N
O
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Nb. R' R2 R3
z16 4-(4-Methyl-piperazin-1-yl)~ N H
/2-
methoxy
OH
z17 4-(4-Methyl-piperazin-1-yl)Br H
z13 3-(4-Methyl-piperazin-1-yl)~ H
~s/
/
Z19 4-(4-Methyl-piperazin-1-yl)/2-3-Benzyloxyphenyl H
Methoxy
z20 4-(4-Methyl-piperazin-1-yl)/2-3-Hydroxyphenyl H
Methoxy
z21 3-(4-Methyl-piperazin-1-yl)3-Chlorophenyl Me
z22 4-(4-Methyl-piperazin-1-yl)/2-3-Chlorophenyl Me
methoxy
Z23 5-(4-Methyl-piperazin-1-yl)/2-3-Chlorophenyl Me
methoxy
NHz
NON \ Rz
i /
N . R3
A
Table 3 - Example 71
Nb. A R2 R3
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A Br H 4-Pyridinyl
B ,H H 4-Pyridinyl
C Br ~ N H
I / /Me
O
D Br ~ N H
OH
E Br ~ O~Me H
N
i
F Br ~ OH H
G H H ,., F F H
N N ~ F
~i
H Pyridin-4-yl ~ H
/ /e
O
Example 72
6-(3-Chloro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-
phenyl]-pyrazolo[1,5-a]pyrimidin-7-ylamine
4-[3-(4-Methyl-piperazin-1-yl)-phenyl]-1H-pyrazol-3-ylamine (Stage 72.2) (1.29
g, 5 mmol), is
dissolved in EtOH (25 mL), followed by the addition of 2-(3-Chloro-phenyl)-3-
oxo-butyronitrile
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(Stage 72.3) (0.97 g, 5 mmol) and HCI (1.25 M in EtOH; 20 mmol, 16 mL) at RT.
The
yellowish solution is refluxed under stirring for 20h. After cooling to RT,
H20 (80 mL) is
added as well as I<ZC03 (2.5 g) to render the mixture basic. The aequeous
layer is extracted
with CHZCI2 (200 mL, 2 x). The combined organic phases are washed with H20 (50
mL, 2x),
dried (Na~S04), concentrated under reduced pressure and chromatographed
(silica gel,
120g RediSep, ISCO Sg-100 CHZCI2 / MeOH /NH3 = 95:5:0,1) to obtain the title
compound
72 as white crystals (1.03 g, 2.38mmol; 48%); mp. 110-115°C;
MS(ESI+):m/z= 433 (M+H)+;
HPLC: At~et = 3.72 minutes (System1).
Stare 72.7 : 2-(3-Chloro-phenyl)-3-oxo-butyronitrile.
355 ml of ethanol is heated to 55°C under N2. To this solution is added
sodium (3.91 g; 0.17
mol) within 30 min. and stirred for 1.5 h until all metal is dissolved. 3-
Chlorobenzyl cyanide
(15.31 g; 0.1 mol) and ethyl acetate (28.53 mL; 0.29 mol) are added to the
colorless solution,
followed by stirring under reflux for 5 h. After completion of the reaction,
the yellow mixture is
cooled to rt. and evaporated under reduced pressure. The crude material is
taken up into
water (200 mL) and neutralized by addition of 25 g of citric acid. The aqueous
layer is
extracted with CH2CI2 (2x 250 mL). The combined organic phases are washed with
H20 (2 x
150 mL,), dried (Na2S04), concentrated under reduced pressure and
chromatographed
(silica gel, 1 kg, Merck 60 (0.040 -0.063), eluting with EtOAc/Hexanes 1:1 )
to obtain the title
compound 72.1 as yellowish crystals (9.7 g, 0.05 mol; 50%); mp. 92-97
°C; MS(ESI+):m/z=
302.9 (M+H)+; HPLC: AtRet = 5.67 minutes (System1).
Stage 72.2 : 4-[3-(4-Methyl-piperazin-1-yl)-phenyl]-1 H-pyrazol-3-ylamine
The title compound is prepared as described in example 24; Stage 24.1 - 24.3
Example 73
6-(3-C hloro-phenyl)-5-methyl-3-[4-(4-methyl-pi perazi n-1-yl)-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[4-(4-
Methyl-piperazin-
1-yl)-phenyl]-2H-pyrazol-3-ylamine (Example 1; Stage 1.2) and 2-(3-Chloro-
phenyl)-3-oxo-
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butyronitrile (Example 73, Stage 73.1 ) instead. Beige crystals; mp. 113-
115°C;
MS(ESI+):m/z= 433 (M+H)+; HPLC: AtRet = 3.56 minutes (System1).
Example 74
6-(3-Chloro-phenyl)-3-[2-methoxy-5-(4-methyl-piperazi n-1-yl)-phenyl]-5
methyl-pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[2-Methoxy-
5-(4-methyl-
piperazin-1-yl)-phenyl]-2H-pyrazol-3-ylamine and 2-(3-Chloro-phenyl)-3-oxo-
butyronitrile
(Example 72, Stage 72.1) instead . Beige crystals; mp. 116-121°C;
MS(ESI+):m/z= 463
(M+H)+; HPLC: AtRet = 3.68 minutes (System1).
Stage 74.1: 4-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-2H-pyrazol-3-
ylamine
The title compound is prepared as described in example 1, (Stage 1.2; Stage
1.4 and 1.5);
using 5-Bromo-2-methoxy-phenylacetonitrile and N-methylpiperazine instead.
Yellowish
foam; MS(ESI+):m/z= 288.2 (M+H)+; HPLC: AtRet = 3.53 minutes (System2).
Example 75
6-(3-Chloro-phenyl)-3-[2-methoxy-4-(4-methyl-pi perazi n-1-yl)-phenyl]-5-
methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[2-Methoxy-
4-(4-methyl-
piperazin-1-yl)-phenyl]-2H-pyrazol-3-ylamine and 2-(3-Chloro-phenyl)-3-oxo-
butyronitrile
(Example 72, Stage 72.1) instead . Beige crystals; mp. 215-217°C;
MS(ESI+):m/z= 463
(M+H)+; HPLC: AtRet = 3.63 minutes (Systeml).
Stage 75.1: 4-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-2H-pyrazol-3-
ylamine
The title compound is prepared as described in example 1, (Stage 1.2; Stage
1.4 and 1.5);
using 4-Bromo-2-methoxy-phenylacetonitrile and N-methylpiperazine instead.
Green-brown
crystals; mp. 173.7-178.1 °C; MS(ESI+):mlz= 288.1 (M+H)+; HPLC: AtRet =
3.40 minutes
(System2).
Example 76
3-~7-Am i no-3-[2-methoxy-4-(4-methyl-piperazi n-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-6-yl}-phenol
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The title compound is prepared by dissolving 6-(3-Benzyloxy-phenyl)-3-[2-
methoxy-4-(4-
methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-7-ylamine in methanol
and subjecting
it to catalytic hydrogenation in the presence of Pd/C as described in example
1. : Beige
crystals; mp. 217-220°C; MS(ESI+):m/z= 431.0 (M+H)+; HPLC: AtRet = 2.65
minutes
(System1 ).
Stage 76.1: 6-(3-Benzyloxy-phenyl)-3-[2-methoxy-4-(4-methyl-piperazin-1-yl)-
phenyl]-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 1, (Stage 1.2; Stage
1.4 and 1.5);
using 4-Bromo-2-methoxy-phenylacetonitrile and N-methylpiperazine instead.
Yellowish
solid; MS(ESI+):m/z= 521 (M+H)+; HPLC: AtRec = 4.38 minutes (System 1 ).
Example 77
6-(2-Chloro-phenyl)-3-[4-(4-methyl-piperazin-1-yl)-phenyl~-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[4-(4-
Methyl-piperazin-
1-yl)-phenyl]-2H-pyrazol-3-ylamine and (Z)-2-(2-Chloro-phenyl)-3-dimethylamino-
acrylonitrile
instead. Yellow solid; mp. 197-200°C; MS(ESI+):m/z= 419 (M+H)+; HPLC:
AcRet = 3.33
minutes (System1).
Stage 77.7: (Z)-2-(2-Chloro-phenyl)-3-dimethylamino-acrylonitrile.
N,N-Dimethylformamide-dimethylacetal (9.06 mL; 64.3 mMol) and 2-
chlorobenzylcyanide
(1.95 g; 12.86 mMol) is heated under stirring to 100°C under an
atmosphere of Argon. After
cooling to rt, the mixture is concentrated under reduced pressure and purified
by and
chromatography (silica gel, 120g RediSep, ISCO Sg-100, eluting with
EtOAc/hexanes 1:1)
to obtain the title compound as yellow thick oil (2.44 g, 11.8mmol; 92%);
MS(ESI+):m/z=
207 (M+H)+; TLC (EtOAc/hexanes 1:1 ) Rf = 0.38.
Example 78
6-(2-Chloro-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,
5-a]pyrimidin-7-ylamine
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The title compound is prepared as described in example 72; using (Z)-2-(2-
Chloro-phenyl)-3-
dimethylamino-acrylonitrile (Example 77, Stage 77.1) instead. Yellowish
crystals; mp. 200-
203°C; MS(ESI+):m/z= 419.0 (M+H)+; HPLC: AtRet = 3.65 minutes
(System1).
Example 79
6-(4-Fluoro-phenyl)-5-methyl-3-[4-(4-methyl-pi perazi n-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[4-(4-
Methyl-piperazin-
1-yl)-phenyl]-2H-pyrazol-3-ylamine and 2-(4-Fluoro-phenyl)-3-oxo-butyronitrile
instead. White
crystals; mp. 289-291 °C; MS(ESI+):m/z= 417.1 (M+H)+; HPLC: AtRet =
3.21 minutes
(System1 ).
Stae~e 79.7: 2-(4-Fluoro-phenyl)-3-oxo-butyronitrile.
The title compound is prepared as described for example 72, Stage 72.1 using
(4-Fluoro-
phenyl)-acetonitrile instead. Beige crystals; mp. 77-83°C;
MS(ESI+):m/z= 176.9 (M+H)+;
HPLC: AtRet = 5.15 minutes (System1).
Example 80
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-py
razolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 2-(4-Fluoro-
phenyl)-3-
oxo-butyronitrile (Example 79, Stage 79.1) instead. White crystals; mp. 204-
206°C;
MS(ESI+):mlz= 417.1 (M+H)+; HPLC: AtRet = 3.34 minutes (System1).
Example 81
6-(3-Chloro-phenyl)-5-methyl-3-{3-[4-(1-methyl-piperidin-4-yl)-piperazin-1-yl]-
phenyl~
pyrazolo[1,5-a]pyrimidin-7-ylamine
r
The title compound is prepared as described in example 72; using 4-{3-[4-(1-
Methyl-
piperidin-4-yl)-piperazin-1-yl]-phenyl)-2H-pyrazol-3-ylamine instead. Beige
crystals; mp. 180-
185 °C; MS(ESI+):m/z= 516.0 (M+H)+; HPLC: AtRet = 4.96 minutes
(System1).
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Stage 81.1: 4-(3-[4-(1-Methyl-piperidin-4-yl)-piperazin-1-yl]-phenyl}-2H-
pyrazol-3-ylamine.
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-Brorno-phenyl)-acetonitrile and 1-(1-Methyl-piperidin-4-yl)-
piperazine instead.
Yellowish crystals; mp. 213-220 °C; MS(ESI+): m/z= 341.18 (M+H)+; HPLC:
AtRei = 3.57
minutes (System1).
Example 82
6-(3-Chloro-4-fluoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)
phenyl~pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 2-(3-Chloro-4-
fluoro-
phenyl)-3-oxo-butyronitrile instead. White crystals; mp. 224-226°C;
MS(ESI+):m/z= 451
(M+H)+; HPLC: AtRet = 3.86 minutes (System1).
Stage 82.1: 2-(3-Chloro-4-fluoro-phenyl)-3-oxo-butyronitrile
The title compound is prepared as described for example 72, Stage 72.1 using
(3-Chloro-4-
fluoro-phenyl)-acetonitrile instead. White crystals; mp. 133-134°C;
MS(ESI ):m/z= 209.9 (M-
H) ; HPLC: AtRet = 5.79 minutes (System1).
Example 83
6-(3-Chloro-4-fluoro-phenyl)-5-methyl-3-[4-(4-methyl-piperazin-1-yl)-phenyl]
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described for example 72, using 4-[4-(4-
Methyl-piperazin-
1-yl)-phenyl]-2H-pyrazol-3-ylamine and 2-(3-Chloro-4-fluoro-phenyl)-3-oxo-
butyronitrile
(Example 82; stage 82.1) instead. White crystals; mp. 264-265°C;
MS(ESI+):m/z= 451
(M+H)+; HPLC: AtRet = 3.72 minutes (System1).
Example 84
6-(3-Bromo-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a~pyrimidin-7-ylamine
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The title compound is prepared as described for example 72, Stage 72.1 using 2-
(3-Bromo-
phenyl)-3-oxo-butyronitrile instead. White crystals; mp. 107-113°C;
MS(ESI+):m/z= 477
(M+H)+; HPLC: AtRet = 4.90 minutes (System1).
Stage 84. 7: 2-(3-Bromo-phenyl)-3-oxo-butyronitrile.
The title compound is prepared as described for example 72, Stage 72.1 using
(3-Bromo-
phenyl)-acetonitrile instead. White crystals; mp. 96-100°C; MS(ESI
):m/z= 235.9 (M-H) ;
HPLC: AtRet = 5.76 minutes (System1).
Example 85
6-(3-Bromo-benzyl)-3-[3-(4-methyl-pi perazin-1-yl)-phenyl]
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 3-(3-Bromo-
phenyl)-2-
formyl-propionitrile instead. White crystals; mp. 170-171 °C;
MS(ESI+):m/z= 477.0 (M+H)+;
HPLC: AtRet = 3.84 minutes (System1).
Stage 85.7: 3-(3-Bromo-phenyl)-2-formyl-propionitrile.
3-(3-Bromophenyl)propionitrile (0:703 mL; 4.66 mMol) and ethyl formate (1.499
mL; 18.64
mMol) are dissolved in THF anhydrous (12.5 mL) followed by the addition of NaH
(60% in
mineral oil; 670 mg) at rt. After 17 h at rt, additional NaH (448 mg) and
ethyl formate (0.765
mL) is added. Since this results in a strong exothermic reaction, additional
solvent is added
(15 mL of THF). After completion (3 days), the reaction mixture is cooled to
0°C, treated with
a few little ice cubes, followed by addition of 6N HCI (3 mL) to acidify the
mixture. _After
addition of water (50 mL), the mixture is extracted with EtOAc (3x 100 mL).
The combined
organic phases are washed with HZO (50 mL, 2x), brine, dried (Na2SO4),
concentrated under
reduced pressure and chromatographed (silica gel, 40g RediSep, ISCO Sg-100,
eluting with
EtOAclhexanes 1:1 ) to obtain the title compound as a brownish oil (220 mg;
20%); MS(ESI-)
:m/z= 235.9 (M-H)-; TLC EtOAc/hexanes 1:1) Rf = 0.28.
Example 86
6-(3-Bromo-phenyl)-3-[3-(4-methyl-piperazin-1-yl)-phenyl]
pyrazolo[1,5-a]pyrimidin-7-ylamine
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. -102-
The title compound is prepared as described in example 72; using (Z)-2-(3-
Bromo-phenyl)-3-
dimethylamino-acrylonitrile instead. White crystals; mp. 195.3-197.2°C;
MS(ESI+):m/z= 463.0
(M+H)+; HPLC: AtRet = 4.05 minutes (System1).
Stage 86.1: (Z)-2-(3-Bromo-phenyl)-3-dimethylamino-acrylonitrile is prepared
as described in
example 77, Stage 77.1.: Gold brown crystals; mp. 102-105 °C;
MS(ESI+):m/z= 251.0
(M+H)+; HPLC: AtRet = 6.45 minutes (System1).
Example 87
6-(3-Chloro-phenyl)-5-methyl-3-(3-morpholin-4-yl-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3-
Morpholin-4-yl-
phenyl)-2H-pyrazol-3-ylamine instead. Off-white crystals; mp. 165-167
°C; MS(ESI+):m/z=
420 (M+H)+; HPLC: AtRet = 4.49 minutes (System1).
Sta eg 87.1: 4-(3-Morpholin-4-yl-phenyl)-2H-pyrazol-3-ylamine
The title compound is prepared as described in example 1, (Stage 1.2; Stage
1.4 and 1.5);
using (3-Bromo-phenyl)-acetonitrile and morpholine instead. Off-white
crystals; mp. 166-168
°C; MS(ESI+):m/z= 245.1 (M+H)+; HPLC: AtRet = 1.79 minutes (System1).
Example 88
6-(3-Chloro-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine
The title compound is prepared as described in example 72; using 4-(4-Methoxy-
phenyl)-
2H-pyrazol-3-ylamine instead. White crystals; mp. 171-172 °C;
MS(ESI+):m/z= 365 (M+H)+;
HPLC: AtRet = 4.96 minutes (Systeml).
Stage 88.1: 4-(4-Methoxy-phenyl)-2H-pyrazol-3-ylamine
The title compound is prepared as described in example 1, (Stage 1.4 and 1.2);
using (4-
methoxy-phenyl)-acetonitrile instead. White crystals; mp. 198-201 °C;
MS(ESI+):m/z= 190
(M+H)+; HPLC: AtRec = 2.85 minutes (System1).
Example 89
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6-(3-Chloro-phenyl)-3-[3-((2R,6S)-2,6-dimethyl-morpholin-4-yl)-phenyl]-5-
methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[3-((2R,6S)-
2,6-
Dimethyl-morpholin-4-yl)-phenyl]-2H-pyrazol-3-ylamine instead. White crystals;
mp. 165-167
°C; MS(ESI+):m/z= 448 (M+H)+; HPLC: AtRet = 5.14 minutes (SYSTEM1).
Stage 89.7: 4-[3-((2R,6S)-2,6-Dimethyl-morpholin-4-yl)-phenyl]-2H-pyrazol-3-
ylamine
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-Bromo-phenyl)-acetonitrile and (2R,6S)-2,6-Dimethyl-morpholine
instead. White
crystals; mp. 158-160 °C; MS(ESI+):m/z= 273.1 (M+H)+; HPLC: AtRet =
3.02 minutes
(System 1 ).
' Example 90
2-(4-{3-[7-Amino-6-(3-chloro-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-3-yl]-
phenyl~-
piperazin-1-yl)-ethanol
The title compound is prepared as described in example 72; using 2-{4-[3-(5-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperazin-1-yl)-ethanol instead. Off-white crystals; mp.
108-116 °C;
MS(ESI+):m/z= 463 (M+H)+; HPLC: AtRet = 3.62 minutes (System1).
Stage 90.7: 2-{4-[3-(5-Amino-1 H-pyrazol-4-yl)-phenyl]-piperazin-1-yl)-ethanol
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-Bromo-phenyl)-acetonitrile and 2-Piperazin-1-yl-ethanol instead.
Yellowish foam;
mp. 40-48°C; MS(ESI+):m/z= 288.1 (M+H)+; HPLC: AtRet = 3.45 minutes
(System1).
Example 91
6-Benzyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-pyrazolo[1,5-a]pyrimidin-7-
ylamine
The title compound is prepared as described in example 72; using 2-Formyl-3-
phenyl-
propionitrile (Example 23; Stage 23.1) instead. Yellowish crystals; mp. 72-
75°C;
MS(ESI+):m/z= 399.1 (M+H)+; HPLC: AtRet = 3.30 minutes (System1).
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Example 92
6-(3-Chloro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-fluoromethyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4-
Dimethoxy-phenyl)-
2H-pyrazol-3-ylamine (Example 93; Stage 93.1 ) and 2-(3-Chloro-phenyl)-4-
fluoro-3-oxo-
butyronitrile instead. Yellow crystals; mp. 228-230°C; MS(ESI+):m/z=
413 (M+H)+; HPLC:
AtRet = 6.65 minutes (System1).
Stage 92.1: 2-(3-Chloro-phenyl)-4-fluoro-3-oxo-butyronitrile
The title compound is prepared as described for example 72, Stage 72.1 using
fluoro-acetic
acid ethyl ester instead. Beige crystals; mp. 90-96°C; MS(ESI ):m/z=
209.9 (M-H) ; HPLC:
AtRet = 5.66 minutes (System1).
Example 93
6-(3-Chloro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4-
Dimethoxy-
phenyl)-2H-pyrazol-3-ylamine instead. Off-white solid; mp. 223-226 °C;
MS(ESI+):m/z= 395.0
(M+H)+; HPLC: AtRec = 4.69 minutes (System1).
Sta a 9e~3.1.~ 4-(3,4-Dimethoxy-phenyl)-2H-pyrazol-3-ylamine
The title compound is prepared as described in example 1, (Stage 1.4 and 1.2);
using (3,4-
Dimethoxy-phenyl)-acetonitrile instead. White crystals; mp. 143-146 °C;
MS(ESI+):m/z=
220.1 (M+H)+; HPLC: AtRet = 2.28 minutes (System1).
Example 94
6-(3-Chloro-4-fluoro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4-
Dimethoxy-
phenyl)-2H-pyrazol-3-ylamine (Example 93; Stage 93.1) and 2-(3-Chloro-4-fluoro-
phenyl)-3-
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oxo-butyronitrile (Example 82; stage 82.1) instead. Off-white solid; mp. 235-
238 °C;
MS(ESI+):m/z= 413.0 (M+H)+; HPLC: AtRet = 4.83 minutes (System1).
Examale 95
6-(3-Chloro-4-fluoro-phenyl)-3-(4-methoxy-phenyl)-5-methyl
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(4-Methoxy-
phenyl)-
2H-pyrazol-3-ylamine (Example 88; Stage 88.1 ) and 2-(3-Chloro-4-fluoro-
phenyl)-3-oxo-
butyronitrile (Example 82; stage 82.1) instead. White crystals; mp. 224-227
°C;
MS(ESI+):m/z= 383 (M+H)+; HPLC: AtRet = 5.08 minutes (System1).
Example 96
6-(4-Fluoro-phenyl)-3-(4-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a] pyrimidin-7-
ylam i ne
The title compound is prepared as described in example 72; using 4-(4-Methoxy-
phenyl)-
2H-pyrazol-3-ylamine (Example 88, Stage 88.1) and 2-(4-Fluoro-phenyl)-3-oxo-
butyronitrile
(Example 79; Stage 79.1) instead. White crystals; mp. 243-244°C;
MS(ESI+):m/z= 349,1
(M+H)+; HPLC: AtRet = 4.56 minutes (System1).
Example 97
2-(4-~3-[7-Amino-6-(4-fluoro-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-3-yl]-
phenyl}-piperazin-1-yl)-ethanol
The title compound is prepared as described in example 72; using 2-{4-[3-(5-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperazin-1-yl}-ethanol (Example 90, Stage 90.1) and 2-
(4-Fluoro-
phenyl)-3-oxo-butyronitrile (Example 79; Stage 79.1) instead. Off-white
crystals; mp. 209-
212°C; MS(ESI+):m/z= 447.1 (M+H)+; HPLC: AtRet = 3.24 minutes
(System1).
Example 98
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6-(3,4-Difl uoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 2-(3,4-
difluoro-phenyl)-3-
oxo-butyronitrile instead. White solid; mp. 216-219°C; MS(ESI+):m/z=
435 (M+H)+; HPLC:
AtRet = 3.30 minutes (SYSTEM1).
Stage 98.7: 2-(3,4-Difluoro-phenyl)-3-oxo-butyronitrile
The title compound is prepared as described in example 1, (Stage 1.4 and 1.2);
using (3,4-
difluoro-phenyl)-acetonitrile instead. White crystals; mp. 147-152 °C;
MS(ESI+):m/z= 195
(M+H)+; HPLC: AtRet = 5.39 minutes (System1).
Example 99
6-(3,4-Difluoro-phenyl)-3-(3,4-dimethoxy-phenyl)-5-methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4-
Dimethoxy-phenyl)-
2H-pyrazol-3-ylamine (Example 93; Stage 93.1) and 2-(3,4-difluoro-phenyl)-3-
oxo-
butyronitrile (Example 98; Stage 98.1) instead. Off-white solid; mp. 230-
235°C;
MS(ESI+):m/z= 397.0 (M+H)+; HPLC: AtRet = 4.53 minutes (System1).
Example 100
2-(4- f 3-[7-Am i no-6-(3-chloro-4-fluoro-phenyl)-5-methyl-pyrazolo[1,5-a]
pyrimidin-3-yl]-phenyl]~-piperazin-1-yl)-ethanol
The title compound is prepared as described in example 72; using 2-{4-[3-(5-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperazin-1-yl)-ethanol (Example 90, Stage 90.1) and 2-
(3-Chloro-4-
fluoro-phenyl)-3-oxo-butyronitrile (Example 82; stage 82.1) instead. Off-white
crystals; mp.
104-107 °C; MS(ESI+):m/z= 481 (M+H)+; HPLC: AtRet = 4.00 minutes
(System1).
Example 101
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2-(4-{3-[7-Amino-6-(3,4-difluo.ro-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-3-
yl]-
phenyl}-piperazin-1-yl)-ethanol
The title compound is prepared as described in example 72; using 2-{4-[3-(5-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperazin-1-yl}-ethanol (Example 90, Stage 90.1) and 2-
(3,4-difluoro-
phenyl)-3-oxo-butyronitrile (Example 98; Stage 98.1 ) instead. Off-white
crystals; mp. 172-
174 °C; MS(ESI+):m/z= 465 (M+H)+; HPLC: AtRet = 3.71 minutes (System1
).
Example 102
6-(3-Ch loro-phenyl)-5-methyl-3-[3-(4-pyrrol idi n-1-yl-piperidi n-1-yl)-
phenyl]-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[3-(4-
Pyrrolidin-1-yl-
piperidin-1-yl)-phenyl]-1 H-pyrazol-3-ylamine instead. Yellow crystals; mp.
188-193 °C;
MS(ESI+):m/z= 487.0 (M+H)+; HPLC: AtRet = 4.21 minutes (System1 ).
Stage 102.1: 4-[3-(4-Pyrrolidin-1-yl-piperidin-1-yl)-phenyl]-1 H-pyrazol-3-
ylamine
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-Bromo-phenyl)-acetonitrile and 4-Pyrrolidin-1-yl-piperidine instead.
Yellow crystals;
mp. 214-216 °C; MS(ESI+):m/z= 312.1 (M+H)+; HPLC: AtRet = 3.71 minutes
(System1).
Example 103
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-pyrrolidin-1-yl-piperidin-1-yl)
phenyl]-pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-[3-(4-
Pyrrolidin-1-yl-
piperidin-1-yl)-phenyl]-1H-pyrazol-3-ylamine (Example 102; Stage 102.1) and 2-
(4-Fluoro-
phenyl)-3-oxo-butyronitrile (Example 79; Stage 79.1) instead. White crystals;
mp. 244-
249°C; MS(ESI+):m/z=471.0 (M+H)+; HPLC: AtRet= 3.82 minutes (System1).
Example 104
6-(3-Chloro-phenyl)-3-[3-(4-diethylamino-piperidin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-
a]pyrimidin-7-ylamine
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The title compound is prepared as described in example 72; using {1-[3-(3-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperidin-4-yl}-diethyl-amine instead. White crystals;
mp. 163-168°C;
MS(ESI+):m/z= 489.0 (M+H)+; HPLC: AtRet = 4.02 minutes (Systeml).
Stage 704.7: {1-[3-(3-Amino-1H-pyrazol-4-yl)-phenyl]-piperidin-4-yl}-diethyl-
amine
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-bromo-phenyl)-acetonitrile and diethyl-piperidin-4-yl-amine instead.
Beige solid,
amorphous; MS(ESI+):m/z= 314.2 (M+H)+; HPLC: AtRet = 3.75 minutes (System1).
Example 105
3-[3-(4-Diethylamino-piperidin-1-yl)-phenyl]-6-(4-fluoro-phenyl)-5-methyl
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using {1-[3-(3-
Amino-1H-
pyrazol-4-yl)-phenyl]-piperidin-4-yl}-diethyl-amine (Example 104, Stage 104.1)
and 2-(4-
Fluoro-phenyl)-3-oxo-butyronitrile (Example 79; Stage 79.1) instead. White
crystals; mp.
208-210 °C; MS(ESI+):m/z= 473.1 (M+H)+; HPLC: AtRet = 3.63 minutes
(System1).
Example 106
6-(4-FI uoro-phenyl)-5-methyl-3-[3-(4-methyl-4-oxy-piperazi n-1-yl)-phenyl]-
pyrazolo[1,5-
a]pyrimidin-7-ylamine
6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-phenyl]-
pyrazolo[1,5-a]pyrimidin-
7-ylamine (Example 80) (50mg; 0.12 mMol) is dissolved in CH2CI2 (10 mL) and at
0°C
treated with 3-chloroperbenzoic acid (31.1 mg; 0.126 mMol) for 1 h, followed
by stirring at rt
for 2h. After removald of the solvent under reduced pressure, the crude
mixture is purified by
chromatography (silica gel, 12g RediSep, ISCO Sg-100 CHZCIz / MeOH /NH3 =
80:20:1) to
obtain the title compound as beige crystals (44 mg); mp. 210-223°C;
MS(ESI+):m/z= 449
(M+H)+; HPLC: AtRet = 3.31 minutes (System1).
Example 107
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6-(4-Fluoro-phenyl)-5-methyl-3-[3-(4-methyl-1,4-d f oxy-piperazi n-1-yl)-
phenyl]
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is isolated from the same reaction described in Example
106: beige
crystals (20 mg); mp. 161-169°C; MS(ESI+):m/z= 433 (M+H)+; HPLC: :
AtRet = 3.89 minutes
(System 1 ).
Example 108
6-(3-Chloro-phenyl)-3-[3-(4-dimethylamino-piperidin-1-yl)-phenyl]-5-methyl
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using {1-[3-(5-
Amino-1 H-
pyrazol-4-yl)-phenyl]-piperidin-4-yl}-dimethyl-amine instead.
Stage 108.1 {1-[3-(5-Amino-1 H-pyrazol-4-yl)-phenyl]-piperidin-4-yl}-dimethyl-
amine
The title compound is prepared as described in example 1, (Stage 1.2 and 1.4
and 1.5);
using (3-bromo-phenyl)-acetonitrile and dimethyl-piperidin-4-yl-amine instead.
Example 109
6-(3,4-Difluoro-phenyl)-3-[3-(4-dimethylamino-piperidin-1-yl)-phenyl]-5-methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using {1-[3-(5-
Amino-1H-
pyrazol-4-yl)-phenyl]-piperidin-4-yl}-dimethyl-amine (Example 108; Stage
108.1) and 2-(3,4-
difluoro-phenyl)-3-oxo-butyronitrile (Example 98; Stage 98.1 ) instead.
Example 110
6-(3-Chloro-phenyl)-5-methyl-3-(3,4,5-trimethoxy-phenyl)
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4,5-
trimethoxy-
phenyl)-2H-pyrazol-3-ylamine instead.
Sfaae 110.1: 4-(3,4,5-Trimethoxy-phenyl)-2H-pyrazol-3-ylamine
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The title compound is prepared as described in example 1, (Stage 1.4 and 1.2);
using
(3,4,5-trimethoxy-phenyl)-acetonitrile instead.
Example 111
6-(3,4-Difluoro-phenyl)-5-methyl-3-(3,4,5-trimethoxy-phenyl)-
pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 72; using 4-(3,4,5-
trimethoxy-
phenyl)-2H-pyrazol-3-ylamine (Example 110; Stage 110.1) and 2-(3,4-difluoro-
phenyl)-3-oxo-
butyronitrile (Example 98; Stage 98.1) instead.
Example 112
6-(3-Chloro-phenyl)-3-(3-methoxy-phenyl)-5-methyl-pyrazolo[1,5-a]pyrimidin-7-
ylamine
The title compound is prepared as described in example 72; using 4-(3-Methoxy-
phenyl)-
2H-pyrazol-3-ylamine instead.
Stage 112.1: 4-(3-Methoxy-phenyl)-2H-pyrazol-3-ylamine
The title compound is prepared as described in example 1, (Stage 1.4 and 1.2);
using (3-
methoxy-phenyl)-acetonitrile instead.
Example 113
6-[7-Amino-3-(3,4-dimethoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-6-yl]-pyridin-2-
of
The title compound is prepared as described in example 1; using 2-(6-Hydroxy-
pyridin-2-yl)-
3-oxo-propionitrile and 4-(3,4-Dimethoxy-phenyl)-2H-pyrazol-3-ylamine (Example
93; Stage
96.1 ) instead.
Example 114
6-Benzyl-3-(3,4-dimethoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-7-ylamine
The title compound is prepared as described in example 93; using 4-(3,4-
Dimethoxy-
phenyl)-2H-pyrazol-3-ylamine (Example 93; Stage 93.1) instead.
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Example 115
3-(3,4-Dimethoxy-phenyl)-6-(3-fluoro-benzyl)-pyrazolo[1,5-a]pyrimidin-7-
ylamine
The title compound is prepared as described in example 114; using 2-(3-Fluoro-
benzyl)-3-
oxo-propionitrile instead.
Example 116
Tablets 1 comprising compounds of the formula (I)
Tablets, comprising, as active ingredient, 50 mg of any one of the compounds
of formula (I)
mentioned in the preceding Examples 1-115 of the following composition are
prepared using
routine methods:
Composition:
Active Ingredient 50 mg
Wheat starch 60 mg
Lactose 50 mg
Colloidal silica 5 mg
Talcum 9 mg
Magnesium stearate 1 mg
175 mg
Manufacture: The active ingredient is combined with part of the wheat starch,
the lactose
and the colloidal silica and the mixture pressed through a sieve. A further
part of the wheat
starch is mixed with the 5-fold amount of water on a water bath to form a
paste and the
mixture made first is kneaded with this paste until a weakly plastic mass is
formed.
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The dry granules are pressed through a sieve having a mesh size of 3 mm, mixed
with a
pre-sieved mixture (1 mm sieve) of the remaining corn starch, magnesium
stearate and
talcum and compressed to form slightly biconvex tablets.
Example 117
Tablets 2 comprising compounds of the formula (I)
Tablets, comprising, as active ingredient, 100 mg of any one of the compounds
of formula (I)
of Examples 1-115 are prepared with the following composition, following
standard
procedures:
Composition:
Active Ingredient 100 mg
Crystalline lactose 240 mg
Avicel 80 mg
PVPPXL 20 mg
Aerosil 2 mg
Magnesium stearate 5 mg
447 mg
Manufacture: The active ingredient is mixed with the carrier materials and
compressed by
means of a tabletting machine (Korsch EKO, Stempeldurchmesser 10 mm).
Example 118
Capsules
Capsules, comprising, as active ingredient, 100 mg of any one of the compounds
of formula
(I) given in Examples 1-115, of the following composition are prepared
according to standard
procedures:
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Comaosition:
Active Ingredient 100 mg
Avicel 200 mg
PVPPXL 15 mg
Aerosil 2 mg
Magnesium stearate 1.5 mg
318.5 mg
Manufacturing is done by mixing the components and filling them into hard
gelatine
capsules, size 1.
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Example 119
Kinase inhibition by compounds of the present invention
Activity determinations of compounds of the preceding examples, using the
testing method
described above, with the following test compounds of formula (I) exhibit
activity for the
following kinases shown in Table 4 (An "x" indicates activity for that
kinase). "Activity" as
used herein is defined as having ICSO values for kinase inhibition of 10pM or
less than:
Table 4:
Ex. c-ablFlt-3KDR s-ScrRET c-kit Cdk1 Ins-R
1 x - x - x x - X
2 _ _ _ _ _ _ X _
7 _ _ _ x _ _ _ _
16 - - - - - - - X
18 - - - - - - X X
19 x - - x _ _ _ _
21 - - - - - x - -
24 - _ _ _ _ x _ _
27 - - - x - - - -
29 - - x - x - - -
35 x - - - x - - -
37 - - - - - - X -
39 - x - - x - - -
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Ex. c-ablFlt-3KDR s-ScrRET c-kit Cdk1 Ins-R
45 - _ _ _ _ _ _ _
46 x - x - - - X -
48 x - - x x - - -
49 - x _ _ _ _ _ _
60 x - - - - - X -
61 - - x - - - - -
61a - - - - x - - -
