U.S. patent application number 13/833231 was filed with the patent office on 2013-08-08 for novel compounds, use and preparation thereof.
This patent application is currently assigned to AKINION PHARMACEUTICALS AB. The applicant listed for this patent is AKINION PHARMACEUTICALS AB. Invention is credited to Annika Jenmalm Jensen, Fredrik Lehmann, Bjorn M. Nilsson, Erik Nordling, Vendela Parrow.
Application Number | 20130203774 13/833231 |
Document ID | / |
Family ID | 40527384 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203774 |
Kind Code |
A1 |
Jensen; Annika Jenmalm ; et
al. |
August 8, 2013 |
NOVEL COMPOUNDS, USE AND PREPARATION THEREOF
Abstract
The present invention relates to compounds of the general
formula (I) wherein R.sup.1, R.sup.2 and R.sup.3 are as defined
herein, which can act as inhibitors of protein kinases, specially
the Fms-like tyrosine kinase 3 (FLT3). The invention also relates
to the use of the compounds in therapy, pharmaceutical compositions
comprising the compounds and the use of the compounds for the
preparation of a medicament for the prophylaxis and treatment of
hematological malignancies, such as AML, MLL, T-ALL, B-ALL and
CMML, myeloproliferative disorders, other proliferative disorders
like cancer, autoimmune disorders and skin disorders like psoriasis
and atopic dermatitis.
Inventors: |
Jensen; Annika Jenmalm;
(Uppsala, SE) ; Nordling; Erik; (Danderyd, SE)
; Lehmann; Fredrik; (Uppsala, SE) ; Nilsson; Bjorn
M.; (Uppsala, SE) ; Parrow; Vendela; (Uppsala,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AKINION PHARMACEUTICALS AB; |
Stockholm |
|
SE |
|
|
Assignee: |
AKINION PHARMACEUTICALS AB
Stockholm
SE
|
Family ID: |
40527384 |
Appl. No.: |
13/833231 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12865359 |
Dec 17, 2010 |
8436171 |
|
|
PCT/EP2009/050931 |
Jan 28, 2009 |
|
|
|
13833231 |
|
|
|
|
61123039 |
Apr 4, 2008 |
|
|
|
Current U.S.
Class: |
514/255.05 ;
514/255.06; 544/336; 544/405 |
Current CPC
Class: |
A61P 7/00 20180101; C07D
409/14 20130101; C07D 405/14 20130101; C07D 417/14 20130101; A61K
31/497 20130101; C07D 401/04 20130101; A61P 35/02 20180101; A61P
17/06 20180101; A61P 17/00 20180101; A61P 43/00 20180101; A61P
35/00 20180101; A61P 37/08 20180101; C07D 401/14 20130101; A61P
37/02 20180101; A61P 37/06 20180101; C07D 241/20 20130101; A61K
31/4965 20130101; C07D 403/12 20130101 |
Class at
Publication: |
514/255.05 ;
544/405; 544/336; 514/255.06 |
International
Class: |
A61K 31/497 20060101
A61K031/497; C07D 241/20 20060101 C07D241/20; A61K 31/4965 20060101
A61K031/4965; C07D 401/14 20060101 C07D401/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2008 |
SE |
0800250-3 |
May 21, 2008 |
SE |
0801185-0 |
Claims
1-24. (canceled)
25. A compound which is:
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-5-amine,
N-[6-(2-fluoropyridin-4-yl)pyrazin-2-yl]-1H-indol-5-amine,
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-6-amine,
N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine,
2-methyl-N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine,
4-[6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide,
4-{6-[(trans-4-hydroxycyclohexyl)amino]pyrazin-2-yl}benzamide,
N3-1H-indol-5-yl-5-pyridin-3-ylpyrazine-2,3-diamine,
5-(2-chloropyridin-4-yl)-N3-1H-indol-5-ylpyrazine-2,3-diamine,
N3-(2-methyl-1H-indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine,
N3-(2-methyl-1H-indol-5-yl)-5-pyridin-3-ylpyrazine-2,3-diamine,
N3-1H-indol-4-yl-5-pyridin-4-ylpyrazine-2,3-diamine,
N3-1H-indol-5-yl-5-(3-thienyl)pyrazine-2,3-diamine,
5-(3-furyl)-N3-1H-indol-5-ylpyrazine-2,3-diamine,
5-(3-fluorophenyl)-N3-1H-indol-5-ylpyrazine-2,3-diamine,
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide,
4-{5-amino-6-{(2-methyl-1H-indol-5-yl)amino}pyrazin-2-yl}benzamide,
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzamid-
e,
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-cyanoethyl)benzamid-
e, 4-[5-amino-6-(1H-indol-4-ylamino)pyrazin-2-yl]benzamide,
trans-4-[(3-amino-6-pyridin-4-ylpyrazin-2-yl)amino]cyclohexanol,
N3-1H-indazol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine,
4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzam-
ide, and 4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]benzamide,
or a pharmacologically acceptable salt thereof.
26. A pharmaceutical formulation comprising a compound according to
claim 25.
27. A compound according to claim 25 for use in therapy.
28. A method for the treatment of one or more conditions selected
from the group consisting of haematological malignancies,
myeloproliferative disorders, cancer, autoimmune disorders, and
skin disorders, said method comprising administering a
therapeutically effective amount of the compound according to claim
25 to a human in need thereof.
29. The method of claim 28, wherein the condition is a
haematological malignancy.
30. The method of claim 29, wherein the haematological malignancy
is selected from the group consisting of acute myeloic leukemia
(AML), mixed lineage leukemia (MLL), T-cell type acute lymphocytic
leukemia (T-ALL), B-cell type acute lymphocytic leukemia (B-ALL)
and chronic myelo-monocytic leukemia (CMML).
31. The method of claim 30, wherein the haematological malignancy
is acute myeloic leukemia (AML).
32. A pharmaceutical composition comprising a compound according to
claim 25, in conjunction with another molecularly targeted
agent.
33. The pharmaceutical composition according to claim 32, wherein
the molecularly targeted agent is a conventional cytotoxic agent,
or a compound used in postchemotherapy, stem-cell directed
maintenance therapy and in MLL-rearranged infant acute
lymphoblastic leukemia.
34. A method for the treatment of haematological malignancies, said
method comprising administering a therapeutically effective amount
of the compound as defined in claim 25 and another molecularly
targeted agent to a human in need thereof, wherein the compound
according to claim 25 is administered simultaneously or
sequentially with said molecularly targeted agent.
35. The method of claim 34 wherein the molecularly targeted agent
is a conventional cytotoxic agent, or a compound used in
postchemotherapy, stem-cell directed maintenance therapy and in
MLL-rearranged infant acute lymphoblastic leukemia.
36. The method of claim 34, wherein the haematological disorder is
acute myeloic leukemia (AML).
37. The method of claim 28, wherein the condition is a skin
disorder.
38. The method of claim 37, wherein the skin disorder is psoriasis
or atopic dermatitis.
39. The method of claim 38, wherein the skin condition is
psoriasis.
40. The method of claim 38, wherein the skin condition is atopic
dermatitis.
Description
TECHNICAL FIELD
[0001] The present invention relates to pyrazine compounds that act
as inhibitors of protein kinases, specially the Fms-like tyrosine
kinase 3 (FLT3). The invention further relates to pharmaceutical
compositions comprising these compounds, and to the use of the
compounds for the preparation of a medicament for the treatment of
hematological malignancies like AML, MLL, T-ALL, B-ALL and CMML,
myeloproliferative disorders, other proliferative disorders like
cancer, autoimmune disorders and skin disorders like psoriasis and
atopic dermatitis.
BACKGROUND ART
[0002] Protein kinases are involved in the regulation of cellular
metabolism, proliferation, differentiation and survival. Protein
kinases phosphorylate proteins on serine/threonine or tyrosine
residues. Activation of one class of kinase typically leads to
activation of more than one signaling pathway through signaling
crosstalk. The receptor tyrosine kinases (RTKs) are a major type of
cell-surface receptors, where the intracellular part of the
receptor has a kinase domain. The activating ligands are
peptide/protein hormones, like the FL-ligand, Vascular Endothelial
Growth factor (VEGF), Epidermal Growth factor (EGF), Fibroblast
growth factor (FGF), nerve growth factor (NGF), platelet-derived
growth factor (PDGF), insulin, etc. Binding of a ligand to the
extracellular domain of an RTK results in receptor dimerisation and
a conformational change that activates the kinase site on the
intracellular domain. The kinase activity leads to a
signal-transduction cascade by phosphorylation of other proteins
that regulates cellular physiology and patterns of gene expression
(for a review see Schlessinger, J. (2000) Cell 103: 211-225; and
Blume-Jensen P. & Hunter T. (2001) Nature 411: 355-365). The
intracellular signaling proteins activated in the signaling cascade
can be other kinases and/or proteins involved in transcription and
translation. There are several families of intracellular kinases.
The Janus kinase (JAK) family of tyrosine kinases (JAK1, 2, 3, and
Thy1) are activated through interaction with other proteins (see
O'Shea, J. J. et al. (2002) Cell 109 (Suppl.) 121-131 and
references therein). Serine/threonine kinases like the protein
kinase C (PKC) family of isozymes and the mitogen activated kinases
(MAP-kinase family) are also involved in the regulation of cell
survival, proliferation and differentiation. The PKC-isozymes are
activated by calcium, and diacylglycerol is an allosteric activator
of some of the members of the PKC family (alpha beta gamma).
Intracellular kinases interact with other proteins and are often
translocated to other compartments upon activation (see Manning, G.
et al. (2002) Science 298: 1912-1934; Martin. P. M. & Hussaini
I. M. (2005) Expert Opin. Ther. Targets 9(2) 299-313 and references
therein). Membrane association can be regulated by myristoylation,
as in the case of PKC isozymes. Nuclear association has been
described for several different classes of kinases. MAP-kinases are
activated by other proteins and capable of translocating to the
nucleus, where proteins involved in transcription and regulators of
cell-cycle and differentiation becomes phosphorylated.
[0003] During normal development and differentiation both kinase
activation and deactivation is tightly regulated. Oncogenic
mutations, leading to constitutively active kinases, can transform
normal cells to cancer cells. An activating mutation can be the
result of a chromosome translocation giving rise to a fusion
protein, for example as in chronic myeloic leukemia where the
ABL-tyrosine kinase domain is fused to the BCR protein (for a
review see Ostman, A. (2007) Helix Review Series Oncology 2: 2-9;
and Deininger, M. et al. (2005) Blood 105: 2640-2653).
[0004] During normal hematopoesis, FLT3 is active at the myeloblast
stage, but the FLT3 activity is then switched off upon normal
hematopoetic differentiation to mature blood cells (Gilliand, D. G,
& Griffin, J. D. (2002) Blood 100: 1532-1542; Weisel, K. C. et
al. (2007) Ann. N.Y. Acad. Sci. 1106: 190-196). In acute myeloic
leukemia, (AML), the FLT3 expression is high in the majority of
patients (70-90%) (Carow, C. E. et al. (1996) Blood 87 (3):
1089-1096; and Rosnet, O. et al (1993) Crit. Rev. Oncogenesis 4:
595-613). Furthermore, the FLT3 kinase activity is upregulated in
one third of the patients due to an internal tandem duplication in
the juxtamembrane position (FLT3-ITD), resulting in a ligand
independent receptor dimerization and a constitutively active
kinase. FLT3-ITD is a prognostic marker, with a statistically
significant reduction in survival in the patient population
harboring the mutation, specially if both alleles are affected.
There are also activating point mutations (FLT3-PM) of FLT3
described in AML patients. These activating mutations can be found
in the activation loop of the kinase domain (AL-mutations) or in
the juxtamembrane domain (JM-mutations). For a review see Carow, C.
E. et al. (1996) Blood 87 (3): 1089-1096; Tickenbrock, L. et al.
(2006) Expert Opin. Emerging Drugs 11(1): 153-165; Anjali S. &
Advani, A. S. (2005) Current Pharmaceutical Design 11: 3449-3457;
Lee B. H. et al. (2007) Cancer Cell 12: 367-380); Stam, R. W. et
al. (2005) Blood 106(7): 2484-2490; and references therein. In
addition FLT3-ITD or FLT3-PM has been found in subsets of patients
with other lymphoid or myeloid malignancies such as MLL, T-ALL and
CMML, and a high FLT3-activity has been described in B-ALL (for a
review see Lee, B. H. et al. (2007) Cancer Cell 12: 367-380.
[0005] However, FLT3 activity is part of the normal hematopoesis.
If the proliferation of immature blast cells in the bone marrow is
dysregulated, by an overstimulation of kinases like FLT3, this
might result in a depletion of other hematopoetic cells. Blast
cells then enter the bloodstream, instead of mature differentiated
cells. The acute leukemic state results in anemia and neutropenia.
Thus, blocking unfavorable kinase activity could reduce the
proliferation of blast cells, and reduce the leukemic state.
Several FLT3 kinase inhibitor has been tested in models of AML and
in clinical indications where FLT3 is involved (Cheng, Y. &
Paz, K. (2008) IDrugs 11(1): 46-56; Kiyoi, H. et al. (2007) Clin.
Cancer Res. 13(15): 4575-4582; Roboz, G. J. et al. (2006) Leukemia
20: 952-957; Tse, K-F. et al. (2002) Leukemia 16: 2027-2036; Smith,
B. D. et al. (2004) Blood 103: 3669-3676; Knapper, S. et al. (2006)
Blood 108 (10): 3494-3503; and Furukawa, Y. et al. (2007) Leukemia
21: 1005-1014). The AML cell-line MV4-11 carries the FLT3-ITD. This
cell-line is very sensitive in viability/proliferation assays to
inhibitors of FLT3 activity. However, in ex-vivo patient cells
there is also crosstalk between the signaling pathways, molecules
activated downstream of the FLT3 receptor can also be activated by
other kinases. Knapper et al 2006 showed that even though the
autophosphorylation of FLT3 was down-regulated in patient cells
after exposure to FLT3 inhibitors, the phosphorylation state of the
down-stream effectors STAT and ERK were not diminished, possibly
due to dysregulation of other signaling pathways apart from
FLT3-phosphorylation.
[0006] The activity of FLT3 and other RTK is regulated by
autophosphorylation and internalisation, the phosphorylation of the
receptor is then removed by specific phosphatases that are also
subject to regulation. A dysregulation of the internalization
process and the dephosphorylation of the phosphatases could also
have an impact on the RTK-activity and thus alter viability and
proliferation of cells. As there are several orders of regulation,
a kinase inhibitor needs to have a certain profile regarding its
target specificity and mode of action to effectively inhibit
proliferation and viability in cancer or a proliferative
disorder.
DISCLOSURE OF THE INVENTION
[0007] This invention relates generally to certain pyrazine
compounds that can act as inhibitors of the receptor tyrosine
kinase FLT3 and related pharmaceutical compositions and
methods.
[0008] While not wishing to be bound by theory, it is believed that
the compounds described herein can be used, e.g., for the treatment
or prevention of haematological malignancies, such as acute myeloic
leukemia (AML); mixed lineage leukemia (MLL); T-cell type acute
lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic
leukemia (B-ALL); chronic myelomonocytic leukemia (CMML);
myeloproliferative disorders; other proliferative disorders, such
as cancer; autoimmune disorders; and skin disorders, such as
psoriasis and atopic dermatitis.
[0009] The compounds can further be used in conjunction with
molecularly targeted agent, such as a conventional cytotoxic agent,
or a compound used in postchemotherapy, stem-cell-directed
maintenance therapy and in MLL-rearranged infant acute
lymphoblastic leukaemia.
[0010] In a first aspect, this invention provides a compound of the
Formula (I) and the geometrical isomers, racemates, tautomers and
optical isomers thereof, as well as the pharmaceutically acceptable
salts, hydrates, N-oxides and physiologically hydrolysable and
acceptable esters and any prodrug forms thereof:
##STR00001##
wherein: [0011] R.sup.1 is selected from a group consisting of:
[0012] (a) indolylethyl, [0013] (b) cyclohexyl, [0014] (c)
hydroxycyclohexyl, [0015] (d) 1,3-benzothiazolyl, [0016] (e)
C.sub.1-3-alkyl-1,3-benzothazolyl, [0017] (f) benzothienyl, [0018]
(g) indolyl, [0019] (h) indazolyl, [0020] (i)
C.sub.1-3-alkylindolyl, [0021] (j) carboxylndolyl, [0022] (k)
C.sub.1-3-alkoxycarbonylindolyl, [0023] (l) carbamoylindolyl,
[0024] (m) 4-methylpiperazin-1-ylcarbonylindolyl, [0025] (n)
carboxymethylindolyl, [0026] (o) acetylaminophenyl, and [0027] (p)
C.sub.1-3-alkylbenzimidazolyl; [0028] R.sup.2 is selected from a
group consisting of: [0029] (a) pyridinyl, [0030] (b)
fluoropyridinyl, [0031] (c) chloropyridinyl, [0032] (d)
C.sub.1-3-alkoxypyridinyl, [0033] (e) thienyl, [0034] (f) furyl,
[0035] (g) phenyl, [0036] (h) fluorophenyl, [0037] (i)
hydroxyphenyl, [0038] (j) cyanophenyl, [0039] (k)
hydroxymethylphenyl, [0040] (l) aminophenyl, [0041] (m)
carbamoylphenyl, [0042] (n) C.sub.1-3-alkylaminocarbonylphenyl,
[0043] (o) dimethylaminocarbonylphenyl, [0044] (p)
(C.sub.1-2-alkoxy-C.sub.2-3-alkylaminocarbonyl)phenyl, [0045] (q)
(cyano-C.sub.2-3-alkylaminocarbonyl)phenyl, [0046] (r)
(dimethylamino-C.sub.2-3-alkylaminocarbonyl)phenyl, [0047] (s)
N-methoxy-N-methylaminocarbonylphenyl, [0048] (t)
morpholin-4-ylcarbonylphenyl, [0049] (u)
piperidin-1-ylcarbonylphenyl, and [0050] (v) quinolinyl; [0051]
R.sup.3 is hydrogen or NH.sub.2; with the proviso that the compound
is not: [0052]
4-(6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamide; [0053]
N'-(1H-indol-5-yl)-5-(quinolin-5-yl)pyrazine-2,3-diamine; [0054]
5-(3-aminophenyl)-N'-(1H-indol-5-yl)pyrazine-2,3-diamine; [0055]
3-[5-amino-6-(1H-indol-5-ylamino)pyrazinyl]phenol; [0056]
4-[5-amino-6-(1H-indol-5-ylamino)pyrazinyl]phenol; or [0057]
1-methyl-N-[6-(2-pyridinyl)pyrazinyl]-1H-benzimidazol-2-amine.
[0058] A preferred group of compounds of the invention are
compounds of Formula (I) wherein R.sup.3 is H, forming compounds of
Formula (Ia):
##STR00002##
wherein: [0059] R.sup.1 is selected from a group consisting of:
[0060] (a) hydroxycyclohexyl, [0061] (b)
C.sub.1-3-alkyl-1,3-benzothiazol-5-yl, [0062] (c)
1,3-benzothiazolyl, [0063] (d) benzothienyl, [0064] (e) indolyl,
[0065] (f) C.sub.1-3-alkylindol-5-yl, [0066] (g) carboxylndolyl,
[0067] (h) C.sub.1-3-alkoxycarbonylindolyl; and [0068] R.sup.2 is
selected from a group consisting of: [0069] (a) pyridinyl [0070]
(b) fluoro-pyridinyl and [0071] (c) carbamoylphenyl
[0072] A more preferred group of compounds of Formula (Ia) are
those wherein [0073] R.sup.1 is selected from a group consisting
of: [0074] (a) 4-hydroxycyclohexyl, [0075] (b)
2-methyl-1,3-benzothiazol-5-yl [0076] (c) 1,3-benzothiazol-5-yl
[0077] (d) indol-5-yl and [0078] (e) indol-6-yl and [0079] R.sup.2
is selected from a group consisting of: [0080] (a) 4-pyridinyl,
[0081] (b) 2-fluoro-4-pyridinyl and [0082] (c)
4-carbamoylphenyl.
[0083] Preferred compounds of Formula (Ia) are: [0084]
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-5-amine, [0085]
N-[6-(2-fluoropyridin-4-yl)pyrazin-2-yl]-1H-indol-5-amine, [0086]
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-6-amine, [0087]
N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine, [0088]
2-methyl-N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine,
[0089] 4-[6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide, and [0090]
4-{6-[(4-hydroxycyclohexyl)amino]pyrazin-2-yl}benzamide.
[0091] A preferred group of compounds of the invention are
compounds of Formula (I) wherein R.sup.3 is NH.sub.2 forming
compounds of Formula (Ib)
##STR00003##
wherein: [0092] R.sup.1 is selected from a group consisting of
[0093] (a) indolethyl, [0094] (b) cyclohexyl, [0095] (c)
hydroxycyclohexyl, [0096] (d) C.sub.1-3-alkyl-1,3-benzothiazolyl,
[0097] (e) benzothienyl, [0098] (f) indolyl, [0099] (g) indazolyl,
[0100] (h) C.sub.1-3-alkylindol-5-yl, and [0101] (i)
carbamoylindolyl; [0102] R.sup.2 is selected from a group
consisting of: [0103] (a) pyridinyl, [0104] (b) chloropyridinyl,
[0105] (c) fluoropyridinyl, [0106] (d) C.sub.1-3-alkoxypyridinyl,
[0107] (e) thienyl, [0108] (f) furyl, [0109] (g) phenyl, [0110] (h)
fluorophenyl, [0111] (i) hydroxyphenyl, [0112] (j) cyanophenyl,
[0113] (k) hydroxymethylphenyl, [0114] (l) aminophenyl, [0115] (m)
carbamoylphenyl, [0116] (n) C.sub.1-3-alkylaminocarbonylphenyl,
[0117] (o) dimethylaminocarbonylphenyl, [0118] (p)
(C.sub.1-2-alkoxy-C.sub.2-3-alkylaminocarbonyl)phenyl, [0119] (q)
cyano-C.sub.2-3-alkylaminocarbonyl)phenyl, [0120] (r)
(dimethylamino-C.sub.2-3-alkylaminocarbonyl)phenyl, and [0121] (s)
(N-methoxy-N-methylaminocarbonylphenyl [0122] (t)
(piperidin-1-ylcarbonyl)phenyl, [0123] (u)
(morpholin-4-ylcarbonyl)phenyl, [0124] (v) quinolinyl.
[0125] A more preferred group of compounds of Formula (Ib) are
those wherein [0126] R.sup.1 is selected from a group consisting
of: [0127] (a) 2-(indol-3-yl)ethyl [0128] (b) 4-hydroxycyclohexyl,
[0129] (c) indol-5-yl, [0130] (d) indol-4-yl, [0131] (e)
indazol-5-yl, and [0132] (f) 2-methylindol-5-yl; and [0133] R.sup.2
is selected from a group consisting of: [0134] (a) 3-pyridinyl,
[0135] (b) 4-pyridinyl, [0136] (c) 2-chloropyridin-4-yl, [0137] (d)
3-thienyl, [0138] (e) 3-furyl, [0139] (f) 3-fluorophenyl, [0140]
(g) 3-hydroxyphenyl, [0141] (h) 4-cyanophenyl, [0142] (i)
4-aminophenyl, [0143] (j) 4-carbamoylphenyl, [0144] (k)
3-carbamoylphenyl, [0145] (l) 4-dimethylaminocarbonylphenyl, [0146]
(m) 4-[(2-methoxyethyl)aminocarbonyl]phenyl,
[0147] Preferred compounds of Formula (Ib) are: [0148]
N3-1H-indol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine, [0149]
N3-1H-indol-5-yl-5-pyridin-3-ylpyrazine-2,3-diamine, [0150]
5-(2-chloropyridin-4-yl)-N3-1H-indol-5-ylpyrazine-2,3-diamine,
[0151]
N3-(2-methyl-1H-indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine,
[0152]
N3-(2-methyl-1H-indol-5-yl)-5-pyridin-3-ylpyrazine-2,3-diamine,
[0153] N3-1H-indol-4-yl-5-pyridin-4-ylpyrazine-2,3-diamine, [0154]
N3-1H-indol-5-yl-5-(3-thienyl)pyrazine-2,3-diamine, [0155]
5-(3-furyl)-N3-1H-indol-5-ylpyrazine-2,3-diamine, [0156]
N3-1H-indol-5-yl-5-phenylpyrazine-2,3-diamine, [0157]
5-(3-fluorophenyl)-N3-1H-indol-5-ylpyrazine-2,3-diamine, [0158]
3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide, [0159]
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide, [0160]
4-{5-amino-6-[(2-methyl-1H-indol-5-yl)amino]pyrazin-2-yl}benzamide,
[0161]
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)b-
enzamide, [0162]
4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-cyanoethyl)benzamide,
[0163] 4-[5-amino-6-(1H-indol-4-ylamino)pyrazin-2-yl]benzamide,
[0164]
N3-[2-(1H-indol-3-yl)ethyl]-5-pyridin-4-ylpyrazine-2,3-diamine,
[0165]
N3-[2-(1H-indol-3-yl)ethyl]-5-pyridin-3-ylpyrazine-2,3-diamine,
[0166]
4-(5-amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamide,
[0167]
4-(5-amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)-N,N-dime-
thylbenzamide, [0168]
5-(4-aminophenyl)-N3-[2-(1H-indol-3-yl)ethyl]pyrazine-2,3-diamine,
[0169]
trans-4-[(3-amino-6-pyridin-4-ylpyrazin-2-yl)amino]cyclohexanol,
[0170] 3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]phenol, [0171]
N3-1H-indazol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine, [0172]
4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzam-
ide, and [0173]
4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]benzamide.
[0174] In one aspect, the present invention relates to a compound
of Formula (I) for use in therapy, especially for use in the
treatment or prophylaxis of a FLT3 related disorder. Examples of
FLT3 related disorders include acute myeloic leukemia (AML); mixed
lineage leukemia (MLL); T-cell type acute lymphocytic leukemia
(T-ALL); B-cell type acute lymphocytic leukemia (B-ALL); chronic
myelomonocytic leukemia (CMML). The present invention also relates
to a compound of Formula (I) for use in the treatment or
prophylaxis of hematological disorders related to dysregulated
kinase activity such as myeloproliferative disorders; other
proliferative disorders, such as cancer; autoimmune disorders; and
skin disorders, such as psoriasis and atopic dermatitis.
[0175] In another aspect, the present invention relates to a
pharmaceutical formulation comprising a compound of Formula (I) as
active ingredient, in combination with a pharmaceutically
acceptable diluent or carrier, especially for use in the treatment
or prophylaxis of a FLT3 related disorder.
[0176] In one aspect, the present invention relates to a method for
treating a human or animal subject suffering from a FLT3 related
disorder. In a further aspect, the present invention relates to a
method for treating a human or animal subject suffering from
haematological malignancies such as acute myeloic leukemia (AML);
mixed lineage leukemia (MLL); T-cell type acute lymphocytic
leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL);
chronic myelomonocytic leukemia (CMML), and other hematological
disorders like myeloproliferative disorders; other proliferative
disorders, such as cancer; autoimmune disorders; and skin
disorders, such as psoriasis and atopic dermatitis. The method can
include administering to a subject (e.g., a human or an animal,
dog, cat, horse, cow) in need thereof an effective amount of one or
more compounds of Formula (I), their salts, or compositions
containing the compounds or salts.
[0177] Methods delineated herein include those wherein the subject
is identified as in need of a particular stated treatment.
Identifying a subject in need of such treatment can be in the
judgment of a subject or a health care professional and can be
subjective (e.g. opinion) or objective (e.g. measurable by a test
or diagnostic method).
[0178] In other aspects, the invention provides a method of
treating a subject suffering from or susceptible to a FLT3 related
disorder or disease, comprising administering to said subject in
need thereof, an effective amount of a compound of Formula I or
pharmaceutical composition thereof, such that said subject is
treated for said disorder or disease.
[0179] In a further aspect, this invention relates to the use of a
compound of formula (I) (e.g., as a medicament) for the treatment
of a disease, disorder, or condition related to undesired activity
of FLT3 kinase as described herein.
[0180] In another aspect, this invention relates to the use of a
compound of formula (I) in the manufacture of a medicament
containing a compound of formula I for the treatment of a disease,
disorder, or condition related to undesired activity of FLT3 kinase
as described herein.
[0181] One aspect of the present invention provides a
pharmaceutical composition comprising an effective amount of a
combination of an inhibitor of the receptor tyrosine kinase FLT3
according to formula (I) and another molecularly targeted agent,
preferably a conventional cytotoxic agent, or a compound used in
postchemotherapy, stem-cell-directed maintenance therapy and in
MLL-rearranged infant acute lymphoblastic leukaemia; and optionally
a pharmaceutically acceptable carrier.
[0182] Another aspect of the invention provides a method of
preventing or treating haematological malignancies,
myeloproliferative disorder, other proliferative disorders,
autoimmune disorders and skin disorders, comprising administering
to a human or animal subject in need thereof an inhibitor of the
receptor tyrosine kinase FLT3 according to formula (T)
simultaneously or sequentially with another molecularly targeted
agent, preferably a conventional cytotoxic agent, or a compound
used in postchemotherapy, stem-cell-directed maintenance therapy
and in MLL-rearranged infant acute lymphoblastic leukaemia; in
sufficient amounts to provide a therapeutic effect.
[0183] Still another aspect of the invention provides the use of an
inhibitor of the receptor tyrosine kinase FLT3 according to formula
(I) together with another molecularly targeted agent, such as a
conventional cytotoxic agent, or a compound used in
postchemotherapy, stem-cell-directed maintenance therapy and in
MLL-rearranged infant acute lymphoblastic leukaemia; for the
manufacture of a medicament for the treatment of haematological
malignancies, myeloproliferative disorder, other proliferative
disorders, autoimmune disorders and skin disorders.
[0184] Another aspect of the invention provides a process for
preparing a pharmaceutical composition, wherein an inhibitor of the
receptor tyrosine kinase FLT3 according to formula (I) and another
molecularly targeted agent, such as a conventional cytotoxic agent,
or a compound used in postchemotherapy, stem-cell-directed
maintenance therapy and in MLL-rearranged infant acute
lymphoblastic leukaemia; in a combined therapeutic amount are
intimately mixed with a pharmaceutically acceptable carrier.
[0185] Yet another aspect of the invention provides a product
containing an inhibitor of the receptor tyrosine kinase FLT3
according to formula (I) further comprising another molecularly
targeted agent, such as a conventional cytotoxic agent, or a
compound used in postchemotherapy, stem-cell-directed maintenance
therapy and in MLL-rearranged infant acute lymphoblastic leukaemia;
as a combined preparation for simultaneous, separate or sequential
use in therapy of haematological malignancies, myeloproliferative
disorder, other proliferative disorders, autoimmune disorders and
skin disorders.
[0186] Another aspect of the present invention is a process for the
preparation of a compound according to formula (I) of the invention
comprising reacting 2-amino-3,5-dibromo-pyrazin and the appropriate
amine followed by a Suzuki coupling. More specifically, the process
for the preparation of a compound according to formula (I) of the
invention comprising one or more of the following steps:
2-amino-3,5-dibromo-pyrazin (3 equiv) and the appropriate amine is
dissolved in 4 mL water and the resulting mixture heated to
195.degree. C. for 1 hour. Water and ethyl acetate is added and the
phases separated. The water phase is extracted once more with ethyl
acetate. The combined organic phases is washed (water and brine)
and concentrated to yield a crude mixture of product and unreacted
amine or alcohol. This crude mixture is used without further
purification or characterization in the subsequent Suzuki reaction
which is performed according to typical Suzuki protocols published
in the literature.
[0187] The chemicals used in the synthetic routes delineated herein
may include, for example, solvents, reagents, catalysts, and
protecting group and deprotecting group reagents. The methods
described above may also additionally include steps, either before
or after the steps described specifically herein, to add or remove
suitable protecting groups in order to ultimately allow synthesis
of the compounds. In addition, various synthetic steps may be
performed in an alternate sequence or order to give the desired
compounds. Synthetic chemistry transformations useful in
synthesizing applicable compounds are known in the art and include,
for example, those described in R. Larock, Comprehensive Organic
Transformations, VCH Publishers (1989); L. Fieser and M. Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995) and subsequent
editions thereof.
[0188] Methods for carrying out the reactions described above are
well known to those skilled in the art. The necessary starting
materials for preparing the compounds of formula (I) are either
known or may be prepared in analogy with the preparation of known
compounds. The compounds of formula (I) may possess one or more
chiral carbon atoms, and they may therefore be obtained in the form
of optical isomers, e.g. as a pure enantiomer, or as a mixture of
enantiomers (racemate) or as a mixture containing diastereomers.
The separation of mixtures of optical isomers to obtain pure
enantiomers is well known in the art and may, for example, be
achieved by fractional crystallization of salts with optically
active (chiral) acids or by chromatographic separation on chiral
columns. All isomeric forms possible (pure enantiomers,
diastereomers, tautomers, racemic mixtures and unequal mixtures of
two enantiomers) for the compounds delineated are within the scope
of the invention. When the compounds described herein contain
olefinic double bonds of geometric asymmetry, it is intended to
include both trans and cis (E and Z) geometric isomers.
[0189] The compounds of the formula (I) may be used as such or,
where appropriate, as pharmacologically acceptable salts (acid or
base addition salts) thereof. The pharmacologically acceptable
addition salts mentioned above are meant to comprise the
therapeutically active non-toxic acid and base addition salt forms
that the compounds are able to form. Compounds that have basic
properties can be converted to their pharmaceutically acceptable
acid addition salts by treating the base form with an appropriate
acid. Exemplary acids include inorganic acids, such as hydrogen
chloride, hydrogen bromide, hydrogen iodide, sulphuric acid,
phosphoric acid; and organic acids such as formic acid, acetic
acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic
acid, glycolic acid, maleic acid, malonic acid, oxalic acid,
benzenesulphonic acid, toluenesulphonic acid, methanesulphonic
acid, trifluoroacetic acid, fumaric acid, succinic acid, malic
acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic
acid, pamoic acid, benzoic acid, ascorbic acid and the like.
Exemplary base addition salt forms are the sodium, potassium,
calcium salts, and salts with pharmaceutically acceptable amines
such as, for example, ammonia, alkylamines, benzathine, and amino
acids, such as, e.g. arginine and lysine. The term addition salt as
used herein also comprises solvates which the compounds and salts
thereof are able to form, such as, for example, hydrates,
alcoholates and the like.
[0190] For clinical use, the compounds of the invention are
formulated into pharmaceutical formulations for oral, rectal,
parenteral or other mode of administration. Pharmaceutical
formulations are usually prepared by mixing the active substance,
or a pharmaceutically acceptable salt thereof, with conventional
pharmaceutical excipients. Examples of excipients are water,
gelatin, gum arabicum, lactose, microcrystalline cellulose, starch,
sodium starch glycolate, calcium hydrogen phosphate, magnesium
stearate, talcum, colloidal silicon dioxide, and the like. Such
formulations may also contain other pharmacologically active
agents, and conventional additives, such as stabilizers, wetting
agents, emulsifiers, flavouring agents, buffers, and the like.
Usually, the amount of active compounds is between 0.1-95% by
weight of the preparation, preferably between 0.2-20% by weight in
preparations for parenteral use and more preferably between 1-50%
by weight in preparations for oral administration.
[0191] The formulations can be further prepared by known methods
such as granulation, compression, microencapsulation, spray
coating, etc. The formulations may be prepared by conventional
methods in the dosage form of tablets, capsules, granules, powders,
syrups, suspensions, suppositories or injections. Liquid
formulations may be prepared by dissolving or suspending the active
substance in water or other suitable vehicles. Tablets and granules
may be coated in a conventional manner.
[0192] The dose level and frequency of dosage of the specific
compound will vary depending on to a variety of factors including
the potency of the specific compound employed, the metabolic
stability and length of action of that compound, the patient's age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity
of the condition to be treated, and the patient undergoing therapy.
The daily dosage may, for example, range from about 0.001 mg to
about 100 mg per kilo of body weight, administered singly or
multiply in doses, e.g. from about 0.01 mg to about 1000 mg each.
Normally, such a dosage is given orally but parenteral
administration may also be chosen.
DEFINITIONS
[0193] The following definitions shall apply throughout the
specification and the appended claims.
[0194] The terms "FLT3 related disorder", and "disorder or
condition related to undesired activity of FLT3", have been used
interchangeably herein to denote any disorder or symptom wherein
the FLT3 is involved in the process or presentation of the disorder
or the symptom. The FLT3 related disorders thus e.g. include, but
are not limited to, haematological malignancies, such as acute
myeloic leukemia (AML); mixed lineage leukemia (MLL); T-cell type
acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic
leukemia (B-ALL) and chronic myelomonocytic leukemia (CMML).
[0195] Unless otherwise stated or indicated, the term
"C.sub.1-6-alkyl" denotes a straight or branched alkyl group having
from 1 to 6 carbon atoms. Examples of said C.sub.1-6-alkyl include
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,
t-butyl and straight- and branched-chain pentyl and hexyl. For
parts of the range "C.sub.1-6-alkyl" all subgroups thereof are
contemplated such as C.sub.1-5-alkyl, C.sub.1-4-alkyl,
C.sub.1-3-alkyl, C.sub.1-2-alkyl, C.sub.2-6-alkyl, C.sub.2-5-alkyl,
C.sub.2-4-alkyl, C.sub.2-3-alkyl, C.sub.3-6-alkyl, C.sub.4-5-alkyl,
etc. Likewise, "aryl-C.sub.1-6-alkyl" means a C.sub.1-6-alkyl group
substituted by an aryl group. Examples include benzyl,
2-phenylethyl, 1-phenylethyl and 1-naphthylmethyl.
[0196] Unless otherwise stated or indicated, the term
"C.sub.1-3-alkoxy" denotes a straight or branched alkoxy group
having from 1 to 3 carbon atoms. Examples of said C.sub.1-3-alkoxy
include methoxy, ethoxy, n-propoxy, iso-propoxy. For parts of the
range "C.sub.1-3-alkoxy" all subgroups thereof are contemplated
such as C.sub.1-2-alkoxy and C.sub.2-3-alkoxy.
[0197] Unless otherwise stated or indicated, the term
"C.sub.1-3-alkoxy-carbonyl" denotes a straight or branched alkoxy
group having from 1 to 3 carbon atoms connected to an carbonyl
group. Examples of said C.sub.1-3-alkoxy-carbonyl include
methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl. For parts of
the range "C.sub.1-3-alkoxy-carbonyl" all subgroups thereof are
contemplated such as C.sub.1-2-alkoxy-carbonyl and
C.sub.2-3-alkoxycarbonyl.
[0198] "Pharmaceutically acceptable" means being useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic and neither biologically nor otherwise undesirable and
includes being useful for veterinary use as well as human
pharmaceutical use.
[0199] "Treatment" as used herein includes prophylaxis of the named
disorder or condition, or amelioration or elimination of the
disorder once it has been established.
[0200] "An effective amount" refers to an amount of a compound that
confers a therapeutic effect on the treated subject. The
therapeutic effect may be objective (i.e., measurable by some test
or marker) or subjective (i.e., subject gives an indication of or
feels an effect). The term "prodrug forms" means a
pharmacologically acceptable derivative, such as an ester or an
amide, which derivative is biotransformed in the body to form the
active drug. Reference is made to Goodman and Gilman's, The
Pharmacological basis of Therapeutics, 8.sup.th ed., Mc-craw-Hill,
Int. Ed. 1992, "Biotransformation of Drugs", p. 13-15; and "The
Organic Chemistry of Drug Design and Drug Action" by Richard B.
Silverman. Chapter 8, p 352. (Academic Press, Inc. 1992. ISBN
0-12-643730-0).
[0201] Combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds. The term "stable" as used herein, refers to
compounds which possess stability sufficient to allow manufacture
and which maintains the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein (e.g.,
therapeutic administration to a subject for the treatment of a FLT3
related disorder or disease (including those delineated herein),
e.g. haematological malignancies, such as acute myeloic leukemia
(AML); mixed lineage leukemia (MLL); T-cell type acute lymphocytic
leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL)
and chronic myelomonocytic leukemia (CMML)).
[0202] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0203] The invention will now be further illustrated by the
following non-limiting Examples. The specific examples below are to
be construed as merely illustrative, and not limitative of the
remainder of the disclosure in any way whatsoever. Without further
elaboration, it is believed that one skilled in the art can, based
on the description herein, utilize the present invention to its
fullest extent. All publications cited herein are hereby
incorporated by reference in their entirety.
[0204] The structures depicted herein, may contain certain --NH--,
--NH.sub.2 (amino) and --OH (hydroxyl) groups where the
corresponding hydrogen atom(s) do not explicitly appear; however
they are to be read as --NH--, --NH.sub.2 or --OH as the case may
be.
Methods
[0205] .sup.1H Nuclear magnetic resonance (NMR) and .sup.13C NMR
were recorded on a Bruker Advance DPX 400 spectrometer at 400.1 MHz
and 100.6 MHz, respectively. All spectra were recorded using
residual solvent or tetramethylsilane (TMS) as internal standard.
Low-resolution electrospray ionization mass spectra (LRESIMS) were
obtained using an Agilent MSD mass spectrometer or a Waters ZQ mass
spectrometer. High-resolution electrospray ionization mass spectra
(HRESIMS) were obtained on an Agilent LC/MSD TOF connected to an
Agilent 1100 LC-system, Ion Source: ESI, Ion polarity: pos, Data:
profile mode, Scan range: 100-1100 Da, MS parameters: Fragmentor
215V, Skimmer 560V och OCT RF (octpole rods) 250 V.; Reference
Masses 121.050873 and 922.009798 (Agilent reference Mix); LC: A 15
mM ammonium acetate; B 100 MeCN; flow 400 .mu.L/min isocratic.
Flash chromatography was performed on Merck silica gel 60 (230-400
mesh). Microwave irradiations were carried out using the Smith
Creator or Optimizer (Personal Chemistry) using 0.5-2 mL or 2-5 mL
Smith Process vials fitted with aluminum caps and septa. The
compounds were automatically named using ACD/NAME 6.0 (Advanced
Chemistry Development, Inc., Toronto, Canada).
[0206] Analytical LCMS data were obtained with:
System A: Agilent MSD mass spectrometer; Agilent 1100 system; ACE 3
C8 column (50.times.3.0 mm); Water containing 0.1% TFA and
acetonitrile were used as mobile phases at a flow rate of 1 mL/min
with gradient times of 3.0 min (gradient 10-97% acetonitrile); or
System B: Agilent MSD mass spectrometer; Agilent 1100 system; YMC
ODS-AQ column (33.times.3.0 mm); Water containing 0.1% TFA and
acetonitrile were used as mobile phases at a flow rate of 1 mL/min
with gradient times of 3.0 min (gradient 10-97% acetonitrile); or
System C: Waters ZQ mass spectrometer; Waters 996 PDA detector (DAD
215-395 nm); ACE C8 (3 .mu.m) column (30.times.3.0 mm) (from ACT);
Water containing 10 mM ammonium acetate (pH=7) and acetonitrile
were used as mobile phases at a flow rate of 1 mL/min with gradient
times of 3.2 min (gradient 5-100% acetonitrile).
[0207] Preparative HPLC was performed on Gilson system equipped
with:
System D: ACE C8 5 .mu.m (21.2.times.50 mm) column. Water
containing 0.1% TFA and acetonitrile were used as mobile phases at
a flow rate of 25 mL/min with gradient times of 6 min.; or System
E: XTerra Prep MS C18 5 .mu.m (19.times.50 mm) column. Water
containing 50 mM NH.sub.4HCO.sub.3 (pH=10) and acetonitrile were
used as mobile phases at a flow rate of 25 mL/min with gradient
times of 6 min; or Xterra MS C18 5 .mu.m (30.times.100 mm) column.
Water containing 50 mM NH.sub.4HCO.sub.3 (pH=10) and acetonitrile
were used as mobile phases at a flow rate of 40 mL/min with
gradient times of 8.5 min; or System F: YMC ODS-AQ 10 .mu.M
(30.times.150 mm) column. Water containing 0.1% TFA and
acetonitrile were used as mobile phases at a flow rate of 45 mL/min
with gradient times of 8.5 min.
[0208] The following abbreviations have been used: [0209] DMSO
means dimethyl sulphoxide, [0210] HPLC means high performance
liquid chromatography, [0211] TFA means trifluoroacetic acid.
[0212] HRMS means high resolution mass spectrometry
EXAMPLES
Procedure A
General procedure for S.sub.NAr on 2-amino-3,5-dibromo-pyrazine
[0213] 2-Amino-3,5-dibromo-pyrazine, triethylamine (3 equiv) and
the appropiate amine or alcohol (3 equiv) were dissolved in 4 mL
water and the resulting mixture was heated to 195.degree. C. for 1
hour. Water and ethyl acetate were added and the phases separated.
The water phase was extracted once more with ethyl acetate. The
combined organic phases were washed (water and brine) and
concentrated to yield a crude mixture of product and unreacted
amine or alcohol. This crude mixture was used without further
purification or characterization in the subsequent Suzuki
reaction.
Procedure B
General Procedure for Suzuki Coupling
[0214] A mixture of the pyrazinyl bromide from procedure A (1
equiv), the appropriate boronic acid (1 equiv), K.sub.2CO.sub.3 (3
equiv) and Pd(dppf)Cl.sub.2*CH.sub.2Cl.sub.2 (0.1 equiv) in 4 mL
dioxane/water (4:1) was heated to 150.degree. C. for 15 min. The
mixture was filtered through a small plug of silica and
concentrated. The crude product was purified by preparative HPLC
(ACE C8 column; mobile phase: 0.1% TFA-CH.sub.3CN) to give the
title compound as a white solid in the form of its corresponding
trifluoroacetate salt.
Intermediate 1
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine
[0215] Using procedure A: 2-Amino-3,5-dibromo-pyrazine (100 mg) and
5-aminoindole (200 mg) yielded 150 mg of a 1:1 mixture of
5-aminoindole and the desired product MS m/z 303 [M+H].sup.+ which
was used without further purification or characterization.
Example 1
##STR00004##
[0216] N3-1H-Indol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine,
trifluoroacetate
[0217] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (20 mg) and
4-pyridyl-boronic acid (12 mg) yielded 1.7 mg of the title
compound. MS m/z 303 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. ppm 6.48 (d, J=3.01 Hz, 1H) 7.26-7.33 (m, 1H) 7.33-7.50 (m,
2H) 7.92 (d, J=1.25 Hz, 1H) 8.37 (s, 1H) 8.45 (d, J=7.03 Hz, 2H)
8.64 (d, J=7.03 Hz, 2H).
Example 2
##STR00005##
[0218] N3-1H-Indol-5-yl-5-pyridin-3-ylpyrazine-2,3-diamine,
trifluoroacetate
[0219] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (20 mg) and
3-pyridyl-boronic acid (12 mg) yielded 1.3 mg of the title
compound. HRMS calcd for C.sub.17H.sub.14N.sub.6: 302.1280, found:
302.1279. .sup.1H NMR (400 MHz, CD.sub.3OD): 6.49 (d, 1H, J=4 Hz),
7.29-7.46 (m, 3H), 7.88-7.94 (m, 2H), 8.02 (s, 1H), 8.64 (d, 1H,
J=8 Hz), 8.84 (d, 1H, J=8 Hz), 9.19 (s, 1H).
Example 3
##STR00006##
[0220] 4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide,
trifluoroacetate
[0221] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (20 mg) and
4-benzamide boronic acid (16 mg) yielded 0.9 mg of the title
compound. HRMS calcd for C.sub.19H.sub.16N.sub.6O: 344.1386, found:
344.1381. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 6.49 (d, 1H,
J=4 Hz), 7.30 (d, 1H, J=4 Hz), 7.42-7.47 (m, 2H), 7.81 (s, 1H),
7.94 (d, 2H, J=8 Hz), 8.01-8.06 (m, 3H).
Example 4
##STR00007##
[0222]
5-(2-Chloropyridin-4-yl)-N3-1H-indol-5-ylpyrazine-2,3-diamine,
trifluoroacetate
[0223] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (20 mg) and
2-chloropyridin-4-yl boronic acid (20 mg) yielded 4.0 mg of the
title compound.
Example 5
##STR00008##
[0224]
4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)be-
nzamide, trifluoroacetate
[0225] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (25 mg) and
[4-[[(2-methoxyethyl)amino]carbonyl]phenyl]boronic acid (27 mg)
yielded 4.2 mg of the title compound. MS m/z 403 [M+H].sup.+.
Example 6
##STR00009##
[0226]
4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-cyanoethyl)benz-
amide, trifluoroacetate
[0227] Using procedure B:
5-Bromo-N3-1H-indol-5-yl-pyrazine-2,3-diamine (25 mg) and
[4-(2-cyanoethylaminocarbonyl)phenyl]boronic acid (27 mg) yielded
3.2 mg of the title compound. MS m/z 398 [M+H].sup.+. NMR (500 MHz,
DMSO-d.sub.6) .delta. ppm 2.78 (t, J=6.70 Hz, 2H) 3.51 (q, J=6.09
Hz, 2H) 6.42 (s, 1H) 7.27-7.47 (m, 3H) 7.76-8.18 (m, 6H) 8.38 (s,
1H) 8.63-9.11 (m, 1H) 10.98 (s, 1H).
Intermediate 2
5-Bromo-N3-1H-indol-4-yl-pyrazine-2,3-diamine
[0228] Using procedure A: 2-Amino-3,5-dibromo-pyrazine (300 mg) and
4-aminoindole (470 mg) yielded 700 mg of a 1:1 mixture of
4-aminoindole and the desired product MS m/z 303 [M+H].sup.+ which
was used without further purification or characterization.
Example 7
##STR00010##
[0229] N3-1H-Indol-4-yl-5-pyridin-4-ylpyrazine-2,3-diamine,
trifluoroacetate
[0230] Using procedure B:
5-Bromo-N3-1H-indol-4-yl-pyrazine-2,3-diamine (25 mg) and
4-pyridinyl boronic acid (15 mg) yielded 1.2 mg of the title
compound. HRMS calcd for C.sub.17H.sub.14N.sub.6: 302.1280, found:
302.1278. .sup.1H NMR (400 MHz, CD.sub.3OD) ppm 6.41 (d, J=3 Hz,
1H) 7.19 (d, J=7 Hz, 1H) 7.21-7.32 (m, 2H) 7.38 (d, J=7 Hz, 1H)
8.33 (d, J=6 Hz, 2H) 8.44 (s, 1H) 8.57 (d, J=6 Hz, 2H).
Example 8
##STR00011##
[0231] 4-[5-Amino-6-(1H-indol-4-ylamino)pyrazin-2-yl]benzamide,
trifluoroacetate
[0232] Using procedure B:
5-Bromo-N3-1H-indol-4-yl-pyrazine-2,3-diamine (25 mg) and
4-benzamide boronic acid (20 mg) yielded 1.1 mg of the title
compound. HRMS calcd for C.sub.19H.sub.16N.sub.60: 344.1386, found:
344.1384. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 6.50 (d, J=2
Hz, 1H) 7.20 (t, J=7 Hz, 1H) 7.28 (d, J=3 Hz, 1H) 7.33 (d, J=8 Hz,
1H) 7.49 (d, J=7 Hz, 1H) 7.82-7.96 (m, 5H).
Intermediate 3
5-Bromo-N3-(2-methyl-1H-indol-5-yl)-pyrazine-2,3-diamine
[0233] Using procedure A: 2-Amino-3,5-dibromo-pyrazine (300 mg) and
5-amino-2-methyl-indole (520 mg) yielded 400 mg of a 1:1 mixture of
and 5-amino-2-methyl-indole and the desired product MS m/z 319
[M+H].sup.+ which was used without further purification or
characterization.
Example 9
##STR00012##
[0234]
N3-(2-Methyl-1H-indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine,
trifluoroacetate
[0235] Using procedure B:
5-Bromo-N3-(2-methyl-1H-indol-5-yl)-pyrazine-2,3-diamine (26 mg)
and 4-pyridinyl boronic acid (14 mg) yielded 3.0 mg of the title
compound. HRMS calcd for C.sub.18H.sub.16N.sub.6: 316.1436, found:
316.1437. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 2.45 (s,
3H) 7.16-7.47 (m, 3H) 7.76 (s, 1H) 8.35 (s, 1H) 8.45 (d, J=6 Hz,
2H) 8.65 (d, J=6 Hz, 2H).
Example 10
##STR00013##
[0236]
N3-(2-Methyl-1H-indol-5-yl)-5-pyridin-3-ylpyrazine-2,3-diamine,
trifluoroacetate
[0237] Using procedure B:
5-Bromo-N3-(2-methyl-1H-indol-5-yl)-pyrazine-2,3-diamine (26 mg)
and 3-pyridinyl boronic acid (14 mg) yielded 3.4 mg of the title
compound. HRMS calcd for C.sub.18H.sub.16N.sub.6: 316.1436, found:
316.1434.
Example 11
##STR00014##
[0238]
4-{5-Amino-6-[(2-methyl-1H-indol-5-yl)amino]pyrazin-2-yl}benzamide,
trifluoroacetate
[0239] Using procedure B:
5-Bromo-N3-(2-methyl-1H-indol-5-yl)-pyrazine-2,3-diamine (26 mg)
and 4-benzamide boronic acid (19 mg) yielded 2.2 mg of the title
compound. HRMS calcd for C.sub.20H.sub.18N.sub.6O: 358.1542, found:
358.1542. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.46 (s, 3H)
7.15-7.48 (m, 3H) 7.77 (s, 1H) 7.90 (s, 1H) 7.91-7.96 (m, 2H)
8.00-8.12 (m, 2H).
Intermediate 4
5-Bromo-N3-(1H-indazol-5-yl)-pyrazine-2,3-diamine
[0240] Using procedure A: 2-Amino-3,5-dibromo-pyrazine (300 mg) and
5-amino-indazole (470 mg) yielded 320 mg of a 1:3 mixture of
5-amino-indazole and the desired product MS m/z 306 [M+H].sup.+
which was used without further purification or
characterization.
Example 12
##STR00015##
[0241] N3-1H-Indazol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine,
trifluoroacetate
[0242] Using procedure B:
5-Bromo-N3-(1H-indazol-5-yl)-pyrazine-2,3-diamine (15 mg) and
4-pyridyl boronic acid (9 mg) yielded 1.3 mg of the title compound.
HRMS calcd for C.sub.16H.sub.13N.sub.7: 303.1232, found:
303.1231.
Example 13
##STR00016##
[0243] 4-[5-Amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]benzamide,
trifluoroacetate
[0244] Using procedure B:
5-Bromo-N3-(1H-indazol-5-yl)-pyrazine-2,3-diamine (15 mg) and
4-benzamide boronic acid (12 mg) yielded 1.5 mg of the title
compound. HRMS calcd for C.sub.18H.sub.15N.sub.7O: 345.1338, found:
345.1335.
Example 14
##STR00017##
[0245]
4-[5-Amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)-
benzamide, trifluoroacetate
[0246] Using procedure B:
5-Bromo-N3-(1H-indazol-5-yl)-pyrazine-2,3-diamine (15 mg) and
[4-[[(2-methoxyethyl)amino]carbonyl]phenyl]boronic acid (16 mg)
yielded 2.5 mg of the title compound. HRMS calcd for
C.sub.21H.sub.21N.sub.7O.sub.2: 403.1757, found: 403.1751.
Intermediate 5
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine
[0247] Using procedure A: 2-Amino-3,5-dibromo-pyrazine (300 mg) and
tryptamine (570 mg) yielded 600 mg of a 1:1 mixture of tryptamine
and the desired product MS m/z 333 [M+H].sup.+ which was used
without further purification or characterization.
Example 15
##STR00018##
[0248]
N3-[2-(1H-Indol-3-yl)ethyl]-5-pyridin-4-ylpyrazine-2,3-diamine,
trifluoroacetate
[0249] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 4-pyridinyl boronic acid (14 mg) yielded 2.4 mg of the title
compound. HRMS calcd for C.sub.19H.sub.18N.sub.6: 330.1593, found:
330.1602 .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.20 (t, J=7
Hz, 2H) 3.95 (t, J=7 Hz, 2H) 6.92-7.19 (m, 3H) 7.32 (d, J=8 Hz, 1H)
7.58 (d, J=8 Hz, 1H) 8.18 (s, 1H) 8.41 (d, J=6 Hz, 2H) 8.62 (d, J=6
Hz, 2H).
Example 16
##STR00019##
[0250]
N3-[2-(1H-Indol-3-yl)ethyl]-5-pyridin-3-ylpyrazine-2,3-diamine,
trifluoroacetate
[0251] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 3-pyridinyl boronic acid (14 mg) yielded 2.7 mg of the title
compound. HRMS calcd for C.sub.19H.sub.18N.sub.6: 330.1593, found:
330.1602. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.20 (t,
J=7 Hz, 2H) 3.99 (t, J=7 Hz, 2H) 6.94-7.18 (m, 3H) 7.30 (d, J=8 Hz,
1H) 7.58 (d, J=7 Hz, 1H) 7.80 (s, 1H) 7.83-7.94 (m, 1H) 8.66 (d,
J=5 Hz, 1H) 8.77 (d, J=8 Hz, 1H) 9.14 (s, 1H).
Example 17
##STR00020##
[0252]
5-(2-Fluoropyridin-4-yl)-N3-[2-(1H-indol-3-yl)ethyl]pyrazine-2,3-di-
amine, trifluoroacetate
[0253] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 2-fluoro-4-pyridinyl boronic acid (16 mg) yielded 3.1 mg of the
title compound.
[0254] HRMS calcd for C.sub.19H.sub.17FN.sub.6: 348.1499, found:
348.1505. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.21 (t,
J=7 Hz, 2H) 3.99 (t, J=7 Hz, 2H) 6.92-7.20 (m, 3H) 7.33 (d, J=8 Hz,
1H) 7.57 (d, J=7 Hz, 1H) 7.79 (s, 1H) 8.14 (dd, J=7, 5 Hz, 1H) 8.46
(d, J=5 Hz, 1H) 8.59 (d, J=4 Hz, 1H).
Example 18
##STR00021##
[0255]
5-(4-Aminophenyl)-N3-[2-(1H-indol-3-yl)ethyl]pyrazine-2,3-diamine,
trifluoroacetate
[0256] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 4-aminophenyl boronic acid (15 mg) yielded 4.4 mg of the title
compound. HRMS calcd for C.sub.20H.sub.20N.sub.6: 344.1749, found:
344.1748. .sup.1H NMR (400 MHz, CD.sub.3OD) 8 ppm 3.22 (t, J=7 Hz,
2H) 3.98 (t, J=7 Hz, 2H) 6.94-7.05 (m, 1H) 7.08-7.16 (m, 2H)
7.17-7.25 (m, 2H) 7.35 (d, J=8 Hz, 1H) 7.51 (s, 1H) 7.58 (d, J=7
Hz, 1H) 7.87-8.03 (m, 2H).
Example 19
##STR00022##
[0257]
4-(5-Amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)-N,N-dimet-
hylbenzamide, trifluoroacetate
[0258] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 4-N,N-dimethylbenzamide boronic acid (22 mg) yielded 3.0 mg of
the title compound. MS m/z 437 [M+H].sup.+. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 3.06 (s, 3H) 3.15 (s, 3H) 3.22 (t, J=7 Hz, 2H)
3.99 (t, J=7 Hz, 2H) 6.96-7.05 (m, 1H) 7.06-7.16 (m, 2H) 7.34 (d,
J=8 Hz, 1H) 7.48-7.55 (m, 2H) 7.58 (d, J=7 Hz, 1H) 7.61 (s, 1H)
7.96-8.05 (m, 2H).
Example 20
##STR00023##
[0259]
4-(5-Amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamide,
trifluoroacetate
[0260] Using procedure B:
5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]-pyrazine-2,3-diamine (25 mg)
and 4-benzamide boronic acid (19 mg) yielded 4.8 mg of the title
compound. HRMS calcd for C.sub.21H.sub.20N.sub.6O: 372.1699, found:
372.1696. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.23 (t, J=7
Hz, 2H) 3.99 (t, J=7 Hz, 2H) 7.02 (t, J=7 Hz, 1H) 7.07-7.17 (m, 2H)
7.35 (d, J=8 Hz, 1H) 7.58 (d, J=8 Hz, 1H) 7.64 (s, 1H) 7.94-8.00
(m, 2H) 8.00-8.08 (m, 2H).
Example 21
##STR00024##
[0261] 4-(6-{[2-(1H-Indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamide,
trifluoroacetate
[0262] Tryptamine (100 mg), 2,6-dichloropyrazine (100 mg) and
triethylamine (135 mg) were mixed in 4 mL acetonitrile and heated
to 150.degree. C. for 1 h. Aqueous saturated NaHCO.sub.3 and
dichloromethane were added to the reaction mixture and the phases
were separated. The water phase was extracted with dichloromethane.
The combined organic phases were washed with brine and
concentrated. The crude intermediate,
6-chloro-N-[2-(1H-indol-3-yl)ethyl]pyrazin-2-amine,
(4-aminocarbonylphenyl)boronic acid (121 mg), K.sub.2CO.sub.3 (275
mg) and Pd(tetrakis(triphenylphosphine)) (38 mg) were dissolved in
4 mL dioxane and 1 mL H.sub.2O and the reaction mixture was heated
to 100.degree. C. over night. 1M NaOH.sub.(aq) and dichloromethane
were added to the mixture and the phases separated. The water phase
was extracted with dichloromethane. The combined organic phases
were washed with brine and concentrated. The crude product was
purified by preparative HPLC (ACE C8 column; mobile phase: 0.1%
TFA-CH.sub.3CN) to give the title compound (85 mg) as a white solid
in the form of its corresponding trifluoroacetate salt. HRMS calcd
for C.sub.21H.sub.19N.sub.5O: 357.1590, found: 357.1585. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 3.03 (t, J=7 Hz, 2H) 3.68 (t,
J=7 Hz, 2H) 6.99-7.01 (m, 1H) 7.05-7.15 (m, 1H) 7.22 (d, J=2 Hz,
1H) 7.36 (d, J=8 Hz, 1H) 7.44 (s, 1H) 7.62 (d, J=7 Hz, 1H) 7.94 (s,
1H) 7.98 (d, J=8 Hz, 2H) 8.14 (d, J=8 Hz, 2H) 8.33 (s, 1H) 10.84
(s, 1H).
Example 22
##STR00025##
[0263] 4-[6-(1H-Indol-5-ylamino)pyrazin-2-yl]benzamide,
trifluoroacetate
[0264] 5-Aminoindole (100 mg), 2,6-dichloropyrazine (100 mg) and
triethylamine (135 mg) were mixed in 4 mL acetonitrile and heated
to 150.degree. C. for 1 h. Aqueous saturated NaHCO.sub.3 and
dichloromethane were added to the reaction mixture and the phases
were separated. The water phase was extracted with dichloromethane.
The combined organic phases were washed with brine and
concentrated. The crude intermediate,
6-chloro-N-(1H-indol-5-yl)pyrazin-2-amine,
(4-aminocarbonylphenyl)boronic acid (121 mg), K.sub.2CO.sub.3 (275
mg) and Pd(tetrakis(triphenylphosphine)) (38 mg) were dissolved in
4 mL dioxane and 1 mL H.sub.2O and the reaction mixture was heated
to 100.degree. C. over night. 1M NaOH.sub.(aq) and dichloromethane
were added to the mixture and the phases separated. The water phase
was extracted with dichloromethane. The combined organic phases
were washed with brine and concentrated. The crude product was
purified by preparative HPLC (ACE C8 column; mobile phase: 0.1%
TFA-CH.sub.3CN) to give the title compound (85 mg) as a white solid
in the form of its corresponding trifluoroacetate salt. HRMS calcd
for C.sub.19H.sub.13N.sub.5O: 329.1277, found: 329.1279. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 7.27 (d, J=3.01 Hz, 1H) 7.31-7.37
(m, 1H) 7.40-7.43 (m, 1H) 7.47-7.51 (m, 1H) 7.85-7.93 (m, 1H)
7.95-8.08 (m, 3H) 8.19-8.26 (m, 2H) 8.38 (s, 1H).
Example 23
##STR00026##
[0265]
5-(3-fluorophenyl)-N.about.3.about.-1H-indol-5-ylpyrazine-2,3-diami-
ne
[0266] Was acquired from BioFocus DPI: HRMS calcd for C18H14FN5:
319.123324, found mass: 319.123684. MS m/z 320 [M+H].sup.+.
Example 14
##STR00027##
[0267] 5
5-(3-furyl)-N.about.3.about.-1H-indol-5-ylpyrazine-2,3-diamine
[0268] Was acquired from BioFocus DPI: HRMS calcd for C16H13N5O:
291.112010, found mass: 291.112130. MS m/z 292 [M+H].sup.+.
Example 25
##STR00028##
[0269] 3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide
[0270] Was acquired from BioFocus DPI: HRMS calcd for C19H16N6O:
344.138559, found mass: 344.138509. MS m/z 345 [M+H].sup.+.
Example 26
##STR00029##
[0271]
N.about.3.about.-1H-indol-5-yl-5-(3-thienyl)pyrazine-2,3-diamine
[0272] Was acquired from BioFocus DPI: HRMS calcd for C16H13N5S:
307.089166, found mass: 307.089106. MS m/z 308 [M+H].sup.+.
Example 27
##STR00030##
[0273] 3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]phenol
[0274] Was acquired from BioFocus DPI: HRMS calcd for C18H15N5O:
317.127660, found mass: 317.127990. MS m/z 318 [M+H].sup.+.
Example 28
##STR00031##
[0275]
4-{6-[(trans-4-Hydroxycyclohexyl)amino]pyrazin-2-yl}benzamide,
trifluoroacetate
[0276] 2,6-Dichloropyrazine (500 mg), trans-4-amino-cyclohexanol
(380 mg) and triethylamine (500 mg) were dissolved in 4 mL
acetonitrile/1 mL water and the reaction mixture was heated to
150.degree. C. for 15 min. Water and dichloromethane were added to
the mixture and the phases were separated. The water phase was
extracted once more with dichloromethane. The combined organic
phases were washed (water and brine) and evaporated to yield 750 mg
of intermediate
6-chloro-N-(trans-4-hydroxycyclohexyl)pyrazin-2-amine with 85%
purity. A portion of this material (30 mg), potassium carbonate (55
mg), 4-benzamide boronic acid (26 mg) and
Pd(tetrakis(triphenylphosphine)) (5 mg) were dissolved in 4 mL
dioxane and 1 mL H.sub.2O and the reaction mixture was heated to
100.degree. C. over night. 1M NaOH.sub.(aq) and dichloromethane
were added to the mixture and the phases separated. The water phase
was extracted with dichloromethane. The combined organic phases
were washed with brine and concentrated. The crude product was
purified by preparative HPLC (ACE C8 column; mobile phase: 0.1%
TFA-CH.sub.3CN) to give the title compound (5.0 mg) as a white
solid in the form of its corresponding trifluoroacetate salt. HRMS
calcd for C.sub.17H.sub.20N.sub.4O.sub.2: 312.1586, found:
312.1585.
Example 29
##STR00032##
[0277]
trans-4-[(3-Amino-6-pyridin-4-ylpyrazin-2-yl)amino]cyclohexanol
[0278] A suspension of 2,6-dibromo-3-aminopyrazine (6.44 g, 0.0255
mol), K.sub.2CO.sub.3 (6.9 g, 0.05 mol) and
trans-4-amino-cyclohexanol (HCl salt) (7.55 g, 0.05 mol) in
H.sub.2O (10.0 mL) was heated under reflux for 72 h (a homogenous
solution is rapidly formed and after ca 30 h a solid is slowly
precipitated. The mixture was cooled and the insoluble solid
collected and washed with water to afford 4.336 g (59%) of
intermediate
trans-4-[(3-amino-6-bromopyrazin-2-yl)amino]cyclohexanol. To a
solution of the crude material (4.336 g, 0.0151 mol),
4-pyridylboronic acid (1.84 g, 0.0151 mol)
tetrakis(triphenylphosphine)palladium(0) (870 mg, 0.7 mmol; 5 mol
%) in PhMe (200 mL) were added aqueous 2M sodium carbonate (40 ml),
and ethanol (40 mL). The mixture was heated at reflux overnight.
The mixture was concentrated by evaporation and an insoluble dark
coloured solid collected by filtration. This material was then
dissolved in MeOH and flash chromatographed over silica EtOAc-MeOH
(9:1) to give a pale yellow solid (2.2 g). Further elution with
EtOAc-MeOH (7:1) gave an additional crop of pale yellow solid (930
mg) which was quite heavily contaminated with silica. Both crops of
solid were combined and purified by preparative HPLC (ACE C8
column; mobile phase: 0.1% TFA-CH.sub.3CN) to afford 2.2 g of the
title product. HPLC purity 100%; .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.33-1.40 (m, 4H), 1.89-1.92 (m, 2H),
2.01-2.04 (m, 2H), 3.47-3.49 (m, 1H), 3.93-3.97 (m, 1H), 8.29 (s,
1H), 8.41 (d, 2H, J=5.0 Hz), 8.80 (d, 2H, J=5.0 Hz); MS
(API-ES/Positive); m/z: 286 (M+H).sup.+.
Example 30
##STR00033##
[0279] N-(6-Pyridin-4-ylpyrazin-2-yl)-1H-indol-5-amine
[0280] A mixture of 2,6-dichloropyrazine (0.845 g, 5.67 mmol),
5-aminoindole (0.5 g, 3.78 mmol), BINAP (0.051 g, 0.0831 mmol),
sodium tertiary butoxide (0.51 g, 5.29 mmol) and palladium acetate
(0.0186 g, 0.0831 mmol) in toluene (25 mL) was heated at 85.degree.
C. for 22 h under nitrogen. CH.sub.2Cl.sub.2 was added, the
reaction mixture was filtered through Celite, and the solvent was
evaporated. The residue was purified by column chromatography (5%
methanol in CH.sub.2Cl.sub.2 as eluent) to give 0.180 g (13%) of
intermediate N-(6-chloro-pyrazin-2-yl)-1H-indol-5-amine. .sup.1H
NMR (CD.sub.3OD) .delta. 7.97 (s, 1H), 7.84 (s, 1H), 7.77 (s, 1H),
7.42-7.39 (d, J=8.63 Hz, 1H), 7.28-7.20 (m, 2H), 6.47-6.46 (d,
J=2.83 Hz, 1H); MS (API-ES/Positive); m/z: 245 (M+H).sup.+.
[0281] A mixture of N-(6-chloro-pyrazin-2-yl)-1H-indol-5-amine
(0.030 g, 0.123 mmol), pyridine-4-boronic acid (0.018 g, 0.147
mmol), sodium carbonate (0.067 g, 0.615 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.007 g, 0.006 mmol) in
DME:water (3:2, 5 mL) was heated at reflux for 20 h. The reaction
mixture was concentrated under reduced pressure and the residue
obtained was extracted with dichloromethane. The organic layer was
washed with water, brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to yield
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-5-amine (0.011 g, 31%) as a
yellow solid. .sup.1H NMR (CD.sub.3OD) .delta. 8.70-8.68 (d, J=6.17
Hz, 2H), 8.47 (s, 1H), 8.15 (s, 1H), 8.14 (d, J=1.50 Hz, 2H) 7.96
(d, J=1.70 Hz, 1H), 7.45-7.43 (d, J=8.64 Hz, 1H), 7.37-7.35 (dd,
J=10.46, 1.83 Hz, 1H), 7.29-7.28 (d, J=3.06 Hz, 1H), 6.49-6.48 (d,
J=3.02 Hz, 1H); MS (API-ES/Positive); m/z: 288 (M+H).sup.+.
Example 31
##STR00034##
[0282]
N-[6-(2-Fluoropyridin-4-yl)pyrazin-2-yl]-1H-indol-5-amine
[0283] A mixture of N-(6-chloro-pyrazin-2-yl)-1H-indol-5-amine
(0.05 g, 0.205 mmol), 2-fluoropyridine-4-boronic acid (0.057 g, 0.4
mmol), sodium carbonate (0.112 g, 1.025 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.012 g, 0.01 mmol) in
DME:water (3:2, 3 mL) was heated at reflux for 20 h. The reaction
mixture was concentrated under reduced pressure and the residue
obtained was extracted with dichloromethane. The organic layer was
washed with water, brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to yield
the title compound (0.015 g, 24%) as a yellow solid. .sup.1H NMR
(CD.sub.3OD) .delta. 8.47 (s, 1H), 8.35-8.33 (d, J=5.29 Hz, 1H),
8.16 (s, 1H), 8.01-8.00 (d, J=5.13 Hz, 1H), 7.94 (s, 1H), 7.78 (s,
1H), 7.45-7.43 (d, J=8.64 Hz, 1H), 7.35-7.33 (dd, J=10.33, 1.72 Hz,
1H), 7.29 (d, J=2.95 Hz, 1H), 6.48-6.47 (d, J=2.58 Hz, 1H).; MS
(API-ES/Positive); m/z: 306 (M+H).sup.+.
Example 32
##STR00035##
[0284] N-(6-Pyridin-4-ylpyrazin-2-yl)-1H-indol-6-amine
[0285] A mixture of 2,6-dichloropyrazine (0.150 g, 1.006 mmol),
6-aminoindole (0.200 g, 1.51 mmol), BINAP (0.0137 g, 0.02215 mmol),
sodium tertiary butoxide (0.136 g, 1.409 mmol) and palladium
acetate (0.005 g, 0.02215 mmol) in toluene (8 mL) was heated at
85.degree. C. for 16 h under nitrogen. CH.sub.2Cl.sub.2 was added,
the reaction mixture was filtered through Celite, and the solvent
was evaporated. The residue was purified by column chromatography
(5% methanol in CH.sub.2Cl.sub.2 as eluent) to give 0.070 g (33%)
of intermediate (6-chloropyrazin-2-yl)-(1H-indol-6-yl)-amine. NMR
(CDCl.sub.3) .delta. 8.36 (brs, 1H, NH), 8.08 (s, 1H), 7.92 (s,
1H), 7.64-7.59 (m, 2H), 7.23 (s, 1H), 7.01-6.98 (d, J=8.37 Hz, 1H),
6.87 (s, 1H, NH), 6.56 (s, 1H); MS (API-ES/Positive); m/z: 245
(M+H).sup.+.
[0286] A mixture of (6-chloro-pyrazin-2-yl)-(1H-indol-6-yl)-amine
(0.070 g, 0.2868 mmol), pyridine-4-boronic acid (0.042 g, 0.344
mmol), sodium carbonate (0.150 g, 1.43 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.0165 g, 0.0143 mmol) in
DME:water (3:2, 5 mL) was heated at reflux for 20 h. The reaction
mixture was concentrated under reduced pressure and the residue
obtained was extracted with dichloromethane. The organic layer was
washed with water, brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to yield
N-(6-pyridin-4-ylpyrazin-2-yl)-1H-indol-6-amine (0.030 g, 36.5%) as
a yellow solid. .sup.1H NMR (CD.sub.3OD) .delta. 8.73-8.72 (d,
J=5.68 Hz, 2H), 8.51 (s, 1H), 8.23-8.20 (m, 4H), 7.56-7.54 (d,
J=8.46 Hz, 1H), 7.22 (d, J=2.99 Hz, 1H), 7.14 (dd, J=10.18, 1.74
Hz, 1H), 6.45 (d, J=2.75 Hz, 1H); MS (API-ES/Positive); m/z: 288
(M+H).sub.+.
Example 33
##STR00036##
[0287]
2-Methyl-N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine
[0288] A mixture of 2,6-dichloropyrazine (0.150 g, 1.006 mmol),
5-amino-2-methylbenzothiazole (0.250 g, 1.51 mmol), BINAP (0.0137
g, 0.02215 mmol), sodium tertiary butoxide (0.136 g, 1.409 mmol)
and palladium acetate (0.005 g, 0.02215 mmol) in toluene (8 mL) was
heated at 85.degree. C. for 16 h under nitrogen. CH.sub.2Cl.sub.2
was added, the reaction mixture was filtered through Celite, and
the solvent was evaporated. The residue was purified by column
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to give
0.180 g (65%) of intermediate
(6-chloro-pyrazin-2-yl)-(2-methyl-benzothiazol-5-yl)-amine. .sup.1H
NMR (CDCl.sub.3) .delta. 7.59-7.54 (d, J=15.6 Hz, 2H), 7.38 (s,
1H), 7.19-7.16 (d, J=8.65 Hz, 1H), 6.98-6.95 (d, J=8.41 Hz, 1H),
6.71 (brs, 1H, NH), 2.31 (s, 3H, CH.sub.3); MS (API-ES/Positive);
m/z: 277 (M+H).sup.+. A mixture of
(6-chloro-pyrazin-2-yl)-(2-methyl-benzothiazol-5-yl)-amine (0.075
g, 0.271 mmol), pyridine-4-boronic acid (0.040 g, 0.326 mmol),
sodium carbonate (0.143 g, 1.35 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.0156 g, 0.0135 mmol) in
DME:water (3:2, 5 mL) was heated at reflux for 20 h. The reaction
mixture was concentrated under reduced pressure and the residue
obtained was extracted with dichloromethane. The organic layer was
washed with water, brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to yield
2-methyl-N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine
(0.075 g, 86.5%) as a yellow solid. .sup.1H NMR (CD.sub.3OD)
.delta. 8.82 (m, 3H), 8.72 (s, 1H), 8.45-8.44 (m, 2H), 8.35 (s,
1H), 7.92-7.90 (d, J=8.67 Hz, 1H), 7.61-7.59 (dd, J=10.63, 1.94 Hz,
1H), 2.89 (s, 3H, CH.sub.3); MS (API-ES/Positive); m/z: 320
(M+H).sup.+.
Example 34
##STR00037##
[0289] N-(6-Pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine
[0290] A mixture of 2,6-dichloropyrazine (0.150 g, 1.006 mmol),
5-amino-benzothiazole (0.151 g, 1.006 mmol), BINAP (0.0137 g,
0.02215 mmol), sodium tertiary butoxide (0.136 g, 1.409 mmol) and
palladium acetate (0.005 g, 0.02215 mmol) in toluene (8 mL) was
heated at 85.degree. C. for 16 h under nitrogen. CH.sub.2Cl.sub.2
was added, the reaction mixture was filtered through Celite, and
the solvent was evaporated. The residue was purified by column
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to give
0.140 g (53%) of intermediate
benzothiazol-5-yl-(6-chloro-pyrazin-2-yl)-amine. .sup.1H NMR
(CDCl.sub.3) .delta. 10.12 (s, 1H), 9.38 (s, 1H), 8.59 (s, 1H),
8.22 (s, 1H), 8.11-8.08 (d, J=8.67 Hz, 1H), 8.02 (s, 1H), 7.6-7.57
(d, J=8.67 Hz, 1H); MS (API-ES/Positive); m/z: 263 (M+H).sup.+.
[0291] A mixture of benzothiazol-5-yl-(6-chloro-pyrazin-2-yl)-amine
(0.06 g, 0.228 mmol), pyridine-4-boronic acid (0.043 g, 0.342
mmol), sodium carbonate (0.124 g, 1.14 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.013 g, 0.0114 mmol) in
DME:water (3:2, 5 mL) was heated at reflux for 22 h. The reaction
mixture was concentrated under reduced pressure and the residue
obtained was extracted with dichloromethane. The organic layer was
washed with water, brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash
chromatography (5% methanol in CH.sub.2Cl.sub.2 as eluent) to yield
N-(6-pyridin-4-ylpyrazin-2-yl)-1,3-benzothiazol-5-amine (0.035 g,
50%) as a yellow solid. .sup.1H NMR (CD.sub.3OD) .delta. 9.31 (s,
1H), 8.98 (d, J=2.02 Hz, 1H), 8.75-8.74 (d, J=5.31 Hz, 2H), 8.64
(s, 1H), 8.31 (s, 1H), 8.24-8.22 (d, J=5.99 Hz, 2H), 8.06-8.04 (d,
J=8.77 Hz, 1H), 7.74-7.71 (dd, J=10.73, 2.01 Hz, 1H); MS
(API-ES/Positive); m/z: 306 (M+H).sup.+.
Biological Methods
[0292] The ability of a compound of the invention to inhibit FLT3
can be determined using in vitro and in vivo assays known in the
art. Several in vitro kinase assays for FLT3 inhibition have been
described in the literature using cloned kinase domain and
measuring phosphorylation of a substrate peptide. In addition,
cell-lines expressing FLT3 have been used to measure the effect on
viability and proliferation in a cellular assay.
Enzyme Inhibition Assay
[0293] The compounds according to the invention were evaluated for
their inhibition of FLT3 by the following method:
In Vitro FLT3 Kinase Assay
[0294] An enzyme inhibition assay for the tyrosine kinase domain of
FLT3 was established using a fluorescence polarization technique,
Immobilized Metal Ion Affinity-Based Fluorescence Polarization
(IMAP) from Molecular Devices.
[0295] Briefly: kinase activity is measured by incubating a
fluorescent peptide substrate with the kinase domain. After
completion of the kinase reaction a binding buffer is added. Upon
phosphorylation of the substrate, the fluorescent peptide gains the
ability to bind to a metal-coated nanoparticle. When the substrate
is bound to the nanoparticle, the rotational is speed of the
peptide is reduced, and thus the fluorescence polarization (fp)
becomes high. Compounds inhibiting the kinase activity of the
enzyme will result in a low degree of phosphorylated substrate and
a low fp-signal.
Reagents
[0296] IMAP Buffer kit with Progressive Binding System (Molecular
Devices, #R8124): Reaction buffer: 10 mM Tris-HCL pH 7.2 with 10 mM
MgCl2, 0.05% NaN3 and 0.01% Tween 20. Prior to use DTT was added to
1 mM DTT final concentration (complete reaction buffer).
[0297] Binding solution was prepared from buffer kit according to
the manufactures recommendations. Binding Reagent was diluted
1:1500 in 40% Binding buffer A and 60% Binding buffer B.
[0298] FLT3 enzyme used was recombinant human FLT3 from Upstate
(#14-500) 7.2 U/ml, N-terminal GST tagged, amino acids 564-end.
[0299] Substrate peptide used: FAM-CSKtide from Molecular Devices
(#R7269) 20 .mu.M, 5FAM-KKKKEEIYFFFG-NH2.
ATP stock solution 10 mM DTT stock solution 100 mM
[0300] Compound dilutions: 0.01% Tween20+1% DMSO in reaction
buffer. Reagents were diluted in complete reaction buffer to
working solutions.
Assay Conditions
Final Concentrations:
[0301] Flt3: 0.0125 U/ml (batch dependent) [0302] FAM-CSKtide: 100
nM [0303] ATP: 100 .mu.M
[0304] Compound dose response: eleven step dilution 1:3,
concentration range 25000-0.42 nM, 5000-0.085 nM, resp. 500-0.0085
nM depending of the potency of compound.
Protocol
[0305] I. Set up kinase reaction in 20 .mu.l volume for 1 h: [0306]
Pipette into 96-well black area plate: [0307] 5 .mu.l compound
dilution or vehicle [0308] 5 .mu.l substrate peptide (400 nM)
[0309] 5 .mu.l enzyme (0.05 U/ml) or complete reaction buffer for
non specific background (NSB) [0310] 5 .mu.l ATP (400 .mu.M) [0311]
Cover the plate and incubate at room temperature with gentle
agitation [0312] II. Binding incubation for 2 h (minimum time):
[0313] Add 60 .mu.l binding solution. [0314] Cover the plate and
incubate at room temperature with gentle agitation [0315] III.
Fluorescence Polarization analysis: [0316] Measure fluorescein
using a plate reader (Analyst AD) excitation wave length 485 and
emission wave length 530, reading with integration time of 0.1 sec.
(Alternatively Victor.sup.2 V Wallac 485/535 nm)
[0317] Stock concentrations of test compounds were made at 10 mM in
100% DMSO. In the assay, compounds were tested in single point at
10 and 1 micromolar, diluted in reaction buffer as described above.
Compounds with an inhibitory activity greater than 60% inhibition
at 1 micromolar were subsequently tested in dose-respons for
IC.sub.50 determinations, using an eleven point dilution range with
1:3 dilution steps (typically from 25000 nM to 0.42 nM, more potent
compounds were assayed from 500 nM to 0.0085 nM). IC.sub.50 values
were obtained by the equation (A+((B-A)/(1+((C/x) D)))) where A
equals min, B equals max, C equals IC.sub.50 and D equals Hill
slope.
[0318] The compounds in accordance with the invention can display
IC.sub.50 values between 1 nM and 2 .mu.M (e.g. between 1 nM and 1
.mu.M, between 1 nM and 500 nM, between 1 nM and 100 nM, between 1
nM and 25 nM, between 1 nM and 10 nM).
Cellular Assays
[0319] AML cell-line MV4-11 carries the FLT3-internal tandem
duplication. This cell-line has been widely used for evaluating the
effect of FLT3-kinase inhibitors on viability and
proliferation.
[0320] Briefly, cells are seeded at a low density into 96-well
plates. Serial dilution of compounds is added and the cells are
incubated for 72 hours. Total number of viable cells is measured
using flow cytometry at the end of treatment, and the effect of the
compounds is calculated as % inhibition compared to vehicle treated
cells.
Cells and Culture Conditions
[0321] All cells were cultured under standard cell culture
conditions, at 37.degree. Celsius in an atmosphere of 5% CO2 in 90%
humidity.
[0322] AML-cell line MV4-11 was cultured in DMEM Glutamax high
glucose (4500 g/l glucose) supplemented with 10% Fetal Bovine Serum
(FBS) from Invitrogen. Cells were subcultured twice weakly, growing
to a density of approx 2 million cells per ml prior to
subcultivation.
Viability and Proliferation Assay
[0323] For viability determination, 3000-5000 cells were seeded in
50 microliter culture medium into a 96-well plate. Serial dilutions
1:3 of compounds from 10 mM DMSO stock were made in serumfree
culture medium supplemented with penicillin and streptomycin. 50
microliter of the serial dilutions were added to the
cell-suspension. The final concentration of compounds was from 5
micromolar to 0.8 nM, or from 500 nM to 0.08 nM respectively. The
DMSO concentration was kept constant at 0.05%.
[0324] At the end of the treatment, 100 microliter viability
reagent (Guava ViaCount) was added to each well and number of cells
and viability was determined using flow cytometry (Guava 96-well
ViaCount assay). Typically the vehicle treated (0.05% DMSO)
cell-line cells had doubled three times during the experiment.
[0325] % Survival was calculated compared to the vehicle treated
cells at the end of experiment. EC50 values were determined using
the equation (A+((B-A)/(1+((C/x) D)))) where A equals min, B equals
max, C equals EC.sub.50 and D equals Hill slope.
Results
TABLE-US-00001 [0326] TABLE 1 Typical mean IC.sub.50 values (n =
4-8) determined in the FLT3 kinase assay. Example IC50 (nM) 15 60
28 159 32 560
TABLE-US-00002 TABLE 2 EC.sub.50 values determined in AML-cell
line. Example Cell data MV4-11 (nM) 15 184 28 178 32 373
In Vitro Assay for Combinations of FLT3-Inhibitor and
Chemotherapy
[0327] Sequence dependent synergisitic activities of compounds of
formula (I) and standard chemotherapy agents used in treating AML
is performed as described in Brown et al. (2006) Leukemia 20:
1368-1376, and the results analysed using Calcusyn Software
according to the principles of Chou and Talalay (1981) Eur J
Biochem.
* * * * *