U.S. patent application number 13/871174 was filed with the patent office on 2014-01-16 for compounds useful as protein kinase inhibitors.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. The applicant listed for this patent is Vertex Pharmaceuticals Incorporated. Invention is credited to Jean-Damien Charrier, Francoise Pierard.
Application Number | 20140018352 13/871174 |
Document ID | / |
Family ID | 40120082 |
Filed Date | 2014-01-16 |
United States Patent
Application |
20140018352 |
Kind Code |
A1 |
Pierard; Francoise ; et
al. |
January 16, 2014 |
COMPOUNDS USEFUL AS PROTEIN KINASE INHIBITORS
Abstract
The present invention relates to compounds useful as inhibitors
of protein kinase. The invention also provides pharmaceutically
acceptable compositions comprising said compounds and methods of
using the compositions in the treatment of various disease,
conditions, or disorders.
Inventors: |
Pierard; Francoise;
(Abingdon, GB) ; Charrier; Jean-Damien; (Abingdon,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertex Pharmaceuticals Incorporated |
Cambridge |
MA |
US |
|
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Cambridge
MA
|
Family ID: |
40120082 |
Appl. No.: |
13/871174 |
Filed: |
April 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12673287 |
May 25, 2011 |
8461149 |
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PCT/US2008/009786 |
Aug 15, 2008 |
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13871174 |
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60964825 |
Aug 15, 2007 |
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60980629 |
Oct 17, 2007 |
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Current U.S.
Class: |
514/220 ;
514/221; 514/249 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 475/00 20130101; A61P 37/02 20180101; A61P 25/28 20180101;
A61P 19/08 20180101; C07D 487/04 20130101; A61P 31/12 20180101;
A61P 7/00 20180101; A61K 31/519 20130101; A61K 31/551 20130101;
A61P 37/06 20180101; A61P 3/10 20180101; C07D 487/14 20130101; A61P
29/00 20180101; A61P 37/00 20180101; A61P 25/00 20180101; A61P
43/00 20180101; C07D 487/10 20130101; A61P 1/16 20180101; A61P 9/00
20180101; A61P 35/02 20180101; A61K 45/06 20130101 |
Class at
Publication: |
514/220 ;
514/221; 514/249 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 31/519 20060101 A61K031/519; A61K 31/551 20060101
A61K031/551; A61K 45/06 20060101 A61K045/06; C07D 487/10 20060101
C07D487/10 |
Claims
1-44. (canceled)
45. A method of inhibiting protein kinase activity in a patient or
a biological sample, comprising administering to the patient a
compound of formula I: ##STR00092## wherein R.sup.1 is ##STR00093##
R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic, and is
optionally substituted with 1 or 2 halogen atoms; X is O and
R.sup.2 is --CH.sub.3; or X is NR.sup.5 and, R.sup.2 and R.sup.5,
together with the atoms to which they are attached, form a
1,2,4-triazole; each of R.sup.3 and R.sup.4 is independently H,
methyl, or ethyl; or R.sup.3 and R.sup.4, together with the atoms
to which they are attached, form a cyclopropyl ring; and n is 0 or
1.
46. The method of claim 45, wherein X is O and R.sup.2 is
--CH.sub.3.
47. The method of claim 45, wherein X is NR.sup.5 and, R.sup.2 and
R.sup.5, together with the atoms to which they are attached, form a
1,2,4-triazole.
48. The method of claim 45, wherein R.sup.6 is C.sub.1-4 aliphatic
or C.sub.3-6 cycloaliphatic, and is optionally substituted with 1
or 2 halogen atoms.
49. The method of claim 45, wherein R.sup.3 and R.sup.4 are each
methyl.
50. The method of claim 45, wherein R.sup.3 is H and R.sup.4 is
ethyl.
51. The method of claim 45, wherein R.sup.3 and R.sup.4, together
with the atoms to which they are attached, form a cyclopropyl
ring.
52. The method of claim 45, wherein R.sup.6 is cyclopropyl
optionally substituted with 1 or 2 halogen atoms.
53. The method of claim 45, wherein R.sup.6 is cyclopentyl
optionally substituted with 1 or 2 halogen atoms.
54. The method of claim 45, wherein R.sup.6 is cyclohexyl
optionally substituted with 1 or 2 halogen atoms.
55. The method of claim 45, wherein R.sup.6 is C.sub.1-4 aliphatic
optionally substituted with 1 or 2 halogen atoms.
56. The method of claim 45, wherein the compound is
N-cyclopropyl-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,7,8,-
9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-methoxybenzamide-
;
4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,7,8,9-tetrahydro--
5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-N-(3-fluorocyclopentyl)-3-metho-
xybenzamide;
N-(3,3-difluorocyclopentyl)-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethy-
l-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-me-
thoxybenzamide;
N-cyclopropyl-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'-oxo-5',6',8',9-
'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diazepine]-2'-ylam-
ino)-3-methoxybenzamide;
N-(3,3-difluorocyclopentyl)-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'--
oxo-5',6',8',9'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diaz-
epine]-2'-ylamino)-3-methoxybenzamide;
4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[1,2,4]triazol-
o[4,3-f]pteridin-7-ylamino)-N-ethyl-3-methoxybenzamide;
N-cyclopropyl-4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro--
[1,2,4]triazolo[4,3-f]pteridin-7-ylamino)-3-methoxybenzamide;
4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-oxo-5,6,7,8-tet-
rahydropteridin-2-ylamino)-N-ethyl-3-methoxybenzamide; or
N-cyclopropyl-4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-o-
xo-5,6,7,8-tetrahydropteridin-2-ylamino)-3-methoxybenzamide.
57. The method of claim 45, wherein the protein kinase is a
PLK.
58. The method of claim 57, wherein the protein kinase is PLK1.
59. A method of treating a proliferative disorder, a
neurodegenerative disorder, an autoimmune disorder, an inflammatory
disorder, or an immunologically mediated disorder in a patient in
need thereof, comprising the step of administering to the patient a
compound of formula I: ##STR00094## wherein R.sup.1 is ##STR00095##
R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic, and is
optionally substituted with 1 or 2 halogen atoms; X is O and
R.sup.2 is --CH.sub.3; or X is NR.sup.5 and, R.sup.2 and R.sup.5,
together with the atoms to which they are attached, form a
1,2,4-triazole; each of R.sup.3 and R.sup.4 is independently H,
methyl, or ethyl; or R.sup.3 and R.sup.4, together with the atoms
to which they are attached, form a cyclopropyl ring; and n is 0 or
1.
60. The method according to claim 59, further comprising
administering to said patient an additional therapeutic agent
selected from a chemotherapeutic or anti-proliferative agent, an
anti-inflammatory agent, an immunomodulatory or immunosuppressive
agent, a neurotrophic factor, an agent for treating cardiovascular
disease, an agent for treating destructive bone disorders, an agent
for treating liver disease, an anti-viral agent, an agent for
treating blood disorders, an agent for treating diabetes, or an
agent for treating immunodeficiency disorders, wherein the
additional therapeutic agent is appropriate for the disease being
treated and is administered together with the compound of formula I
as a single dosage form or separately from the compound as part of
a multiple dosage form.
61. A method of treating melanoma, myeloma, leukemia, lymphoma,
neuroblastoma, or a cancer selected from colon, breast, gastric,
ovarian, cervical, lung, central nervous system (CNS), renal,
prostate, bladder, or pancreatic, in a patient in need thereof,
comprising administering to the patient a compound of formula I:
##STR00096## wherein R.sup.1 is ##STR00097## R.sup.6 is C.sub.1-4
aliphatic or C.sub.3-6 cycloaliphatic, and is optionally
substituted with 1 or 2 halogen atoms; X is O and R.sup.2 is
--CH.sub.3; or X is NR.sup.5 and, R.sup.2 and R.sup.5, together
with the atoms to which they are attached, form a 1,2,4-triazole;
each of R.sup.3 and R.sup.4 is independently H, methyl, or ethyl;
or R.sup.3 and R.sup.4, together with the atoms to which they are
attached, form a cyclopropyl ring; and n is 0 or 1.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Application No.
60/964,825, filed on Aug. 15, 2007, and U.S. Application No.
60/980,629, filed on Oct. 17, 2007. The entire contents of the
aforementioned applications are incorporated herein."
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to compounds useful as
inhibitors of protein kinases. The invention also provides
pharmaceutically acceptable compositions comprising the compounds
of the invention and methods of using the compositions in the
treatment of various disorders. The invention also provides
processes for preparing the compounds of the invention.
BACKGROUND OF THE INVENTION
[0003] The search for new therapeutic agents has been greatly aided
in recent years by a better understanding of the structure of
enzymes and other biomolecules associated with diseases. One
important class of enzymes that has been the subject of intensive
study is protein kinases.
[0004] Protein kinases constitute a large family of structurally
related enzymes that are responsible for the control of a variety
of signal transduction processes within the cell (see Hardie, G.
and Hanks, S. The Protein Kinase Facts Book, I and II, Academic
Press, San Diego, Calif.: 1995). Protein kinases are thought to
have evolved from a common ancestral gene due to the conservation
of their structure and catalytic function. Almost all kinases
contain a similar 250-300 amino acid catalytic domain. The kinases
may be categorized into families by the substrates they
phosphorylate (e.g., protein-tyrosine, protein-serine/threonine,
lipids etc). Sequence motifs have been identified that generally
correspond to each of these kinase families (See, for example,
Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576-596; Knighton et
al., Science 1991, 253, 407-414; Hiles et al, Cell 1992, 70,
419-429; Kunz et al, Cell 1993, 73, 585-596; Garcia-Bustos et al,
EMBO J. 1994, 13, 2352-2361).
[0005] In general, protein kinases mediate intracellular signaling
by effecting a phosphoryl transfer from a nucleoside triphosphate
to a protein acceptor that is involved in a signaling pathway.
These phosphorylation events act as molecular on/off switches that
can modulate or regulate the target protein biological function.
These phosphorylation events are ultimately triggered in response
to a variety of extracellular and other stimuli. Examples of such
stimuli include environmental and chemical stress signals (e.g.,
shock, heat shock, ultraviolet radiation, bacterial endotoxin, and
H.sub.2O.sub.2), cytokines (e.g., interleukin-1 (IL-1) and tumor
necrosis factor alpha (TNF-a), and growth factors (e.g.,
granulocyte macrophage-colony stimulating factor (GM-CSF), and
fibroblast growth factor (FGF)). An extracellular stimulus may
affect one or more cellular responses related to cell growth,
migration, differentiation, secretion of hormones, activation of
transcription factors, muscle contraction, glucose metabolism,
control of protein synthesis, survival and regulation of the cell
cycle.
[0006] Many diseases are associated with abnormal cellular
responses triggered by protein kinase-mediated events as described
above. These diseases include, but are not limited to, cancer,
autoimmune diseases, inflammatory diseases, bone diseases,
metabolic diseases, neurological and neurodegenerative diseases,
cardiovascular diseases, allergies and asthma, Alzheimer's disease
and hormone related diseases. Accordingly, there has been a
substantial effort in medicinal chemistry to find protein kinase
inhibitors that are effective as therapeutic agents.
[0007] The Polo-like kinases (PLK) belong to a family of
serine/threonine kinases that are highly conserved across the
species, ranging from yeast to man (reviewed in Lowery D M et al.,
Oncogene 2005, 24; 248-259). The PLK kinases have multiple roles in
cell cycle, including control of entry into and progression through
mitosis.
[0008] PLK1 is the best characterized of the PLK family members.
PLK1 is widely expressed and is most abundant in tissues with a
high mitotic index. Protein levels of PLK1 rise and peak in mitosis
(Hamanaka, R et al., J Biol Chem 1995, 270, 21086-21091). The
reported substrates of PLK1 are all molecules that are known to
regulate entry and progression through mitosis, and include CDC25C,
cyclin B, p53, APC, BRCA2 and the proteasome. PLK1 is upregulated
in multiple cancer types and the expression levels correlate with
severity of disease (Macmillan, J C et al., Ann Surg Oncol 2001, 8,
729-740). PLK1 is an oncogene and can transform NIH-3T3 cells
(Smith, M R et al., Biochem Biophys Res Commun 1997, 234, 397-405).
Depletion or inhibition of PLK1 by siRNA, antisense, microinjection
of antibodies, or transfection of a dominant negative construct of
PLK1 into cells, reduces proliferation and viability of tumour
cells in vitro (Guan, R et al., Cancer Res 2005, 65, 2698-2704;
Liu, X et al., Proc Natl Acad Sci USA 2003, 100, 5789-5794, Fan, Y
et al., World J Gastroenterol 2005, 11, 4596-4599; Lane, H A et
al., J Cell Biol 1996, 135, 1701-1713). Tumour cells that have been
depleted of PLK1 have activated spindle checkpoints and defects in
spindle formation, chromosome alignment and separation and
cytokinesis. Loss in viability has been reported to be the result
of an induction of apoptosis. In contrast, normal cells have been
reported to maintain viability on depletion of PLK1. In vivo knock
down of PLK 1 by siRNA or the use of dominant negative constructs
leads to growth inhibition or regression of tumours in xenograft
models.
[0009] PLK2 is mainly expressed during the G1 phase of the cell
cycle and is localized to the centrosome in interphase cells. PLK2
knockout mice develop normally, are fertile and have normal
survival rates, but are around 20% smaller than wild type mice.
Cells from knockout animals progress through the cell cycle more
slowly than in normal mice (Ma, S et al., Mol Cell Biol 2003, 23,
6936-6943). Depletion of PLK2 by siRNA or transfection of kinase
inactive mutants into cells blocks centriole duplication.
Downregulation of PLK2 also sensitizes tumour cells to taxol and
promotes mitotic catastrophe, in part by suppression of the p53
response (Burns T F et al., Mol Cell Biol 2003, 23, 5556-5571).
[0010] PLK3 is expressed throughout the cell cycle and increases
from G1 to mitosis. Expression is upregulated in highly
proliferating ovarian tumours and breast cancer and is associated
with a worse prognosis (Weichert, W et al., Br J Cancer 2004, 90,
815-821; Weichert, W et al., Virchows Arch 2005, 446, 442-450). In
addition to regulation of mitosis, PLK3 is believed to be involved
in Golgi fragmentation during the cell cycle and in the DNA-damage
response. Inhibition of PLK3 by dominant negative expression is
reported to promote p53-independent apoptosis after DNA damage and
suppresses colony formation by tumour cells (Li, Z et al., J Biol
Chem 2005, 280, 16843-16850).
[0011] PLK4 is structurally more diverse from the other PLK family
members. Depletion of this kinase causes apoptosis in cancer cells
(Li, J et al., Neoplasia 2005, 7, 312-323). PLK4 knockout mice
arrest at E7.5 with a high fraction of cells in mitosis and partly
segregated chromosomes (Hudson, J W et al., Current Biology 2001,
11, 441-446).
[0012] Molecules of the protein kinase family have been implicated
in tumour cell growth, proliferation and survival. Accordingly,
there is a great need to develop compounds useful as inhibitors of
protein kinases. The evidence implicating the PLK kinases as
essential for cell division is strong. Blockade of the cell cycle
is a clinically validated approach to inhibiting tumour cell
proliferation and viability. It would therefore be desirable to
develop compounds that are useful as inhibitors of the PLK family
of protein kinases (e.g., PLK1, PLK2, PLK3 and PLK4), that would
inhibit proliferation and reduce viability of tumour cells,
particularly as there is a strong medical need to develop new
treatments for cancer, including treatments that would be
administered orally.
SUMMARY OF THE INVENTION
[0013] Compounds of this invention, and pharmaceutically acceptable
compositions thereof, are useful as inhibitors of protein kinases.
In some embodiments, these compounds are useful as inhibitors of
PLK protein kinases; in some embodiments, as inhibitors of PLK1
protein kinases. These compounds have the formula I, as defined
herein or pharmaceutically acceptable salts thereof.
[0014] These compounds, and pharmaceutically acceptable salts
thereof, are useful for treating or preventing a variety of
diseases, disorders or conditions, including, but not limited to,
an autoimmune, inflammatory, proliferative, or hyperproliferative
disease, a neurodegenerative disease, or an
immunologically-mediated disease. The compounds provided by this
invention are also useful for the study of kinases in biological
and pathological phenomena; the study of intracellular signal
transduction pathways mediated by such kinases; and the comparative
evaluation of new kinase inhibitors.
[0015] In some instances, the compounds of this invention
demonstrate PLK1 inhibition at concentrations of less than 1 nM. In
other instances, the compounds of this invention demonstrate PLK1
inhibition at concentrations of between 1 nM and 10 nM. Further,
the compounds of the invention demonstrate advantageous
phrmaco-kinetic properties.
DETAILED DESCRIPTION OF THE INVENTION
[0016] In one aspect, the invention features a compound of formula
I:
##STR00001##
[0017] wherein:
[0018] R.sup.1 is
##STR00002##
[0019] R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic,
and is substituted with 1 to 2 halogen atoms (e.g., fluorine);
[0020] X is O and R.sup.2 is CH.sub.3; or X is NR.sup.5 and,
R.sup.2 and R.sup.5, together with the atoms to which they are
attached, form a 1,2,4-triazole;
[0021] each of R.sup.3 and R.sup.4 is independently H, methyl, or
ethyl; or R.sup.3 and R.sup.4, together with the atoms to which
they are attached, form a cyclopropyl ring; and
[0022] n is 0 or 1.
[0023] Embodiments of the compound of this invention may include
one or more of the following features: X is O and R.sup.2 is
--CH.sub.3; R.sup.6 is a C.sub.3-6 cycloaliphatic optionally
substituted with 1 to 2 halogen (e.g., fluorine) atoms; R.sup.3 is
methyl; R.sup.4 is methyl; R.sup.3 is H; R.sup.4 is ethyl; R.sup.6
is cyclopropyl optionally substituted with 1 to 2 fluorine atoms;
R.sup.6 is cyclopentyl optionally substituted with 1 to 2 halogen
(e.g., fluorine) atoms; R.sup.6 is cyclohexyl optionally
substituted with 1 to 2 halogen (e.g., fluorine) atoms; R.sup.6 is
C.sub.1-4 aliphatic optionally substituted with 1 to 2 halogen
(e.g., fluorine) atoms.
[0024] Specific examples of the compounds of this invention
include, but are mot limited to, [0025]
N-cyclopropyl-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,7,8,-
9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-methoxybenzamide-
; [0026]
4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,7,8,9-tetr-
ahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-N-(3-fluorocyclopentyl)--
3-methoxybenzamide; [0027]
N-(3,3-difluorocyclopentyl)-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethy-
l-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-me-
thoxybenzamide; [0028]
N-cyclopropyl-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'-oxo-5',6',8',9-
'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diazepine]-2'-ylam-
ino)-3-methoxybenzamide; [0029]
N-(3,3-difluorocyclopentyl)-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'--
oxo-5',6',8',9'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diaz-
epine]-2'-ylamino)-3-methoxybenzamide; [0030]
4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[1,2,4]triazol-
o[4,3-f]pteridin-7-ylamino)-N-ethyl-3-methoxybenzamide; [0031]
N-cyclopropyl-4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro--
[1,2,4]triazolo[4,3-f]pteridin-7-ylamino)-3-methoxybenzamide;
[0032]
4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-oxo-5,6,7,8-tet-
rahydropteridin-2-ylamino)-N-ethyl-3-methoxybenzamide; and [0033]
N-cyclopropyl-4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-o-
xo-5,6,7,8-tetrahydropteridin-2-ylamino)-3-methoxybenzamide.
[0034] In some embodiments, the compounds of formula I are of
formula I-A:
##STR00003##
[0035] In some other embodiments, the compounds of formula I are of
formula I-B:
##STR00004##
[0036] In still some other embodiments, the compounds of formula I
are of formula I-C:
##STR00005##
[0037] In some embodiments when the compounds of formula I are of
formula I-A, R.sup.3 and R.sup.4 are each methyl; or, one of
R.sup.3 and R.sup.4 is H and the other one is methyl; or, R.sup.3
and R.sup.4, together with the atoms to which they are attached,
form cyclopropyl.
[0038] In some other embodiments when the compounds of formula I
are of formula I-B, one of R.sup.3 and R.sup.4 is H and the other
one is ethyl; and the asymmetric carbon has an (R) configuration as
shown below.
##STR00006##
[0039] In some other embodiments when the compounds of formula are
of formula I-C, one of R.sup.3 and R.sup.4 is H and the other one
is ethyl; and the asymmetric carbon has an (R) configuration as
shown below.
##STR00007##
[0040] In some embodiments of the compounds of this invention,
R.sup.1 is
##STR00008##
[0041] In another aspect, the present invention provides a process
for preparing a compound of formula I-A:
##STR00009##
[0042] In formula I-A, R.sup.1 is
##STR00010##
R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic, and is
optionally substituted with 1 or 2 halogen atoms (e.g., 2 fluorine
atoms); and each of R.sup.3 and R.sup.4 is independently H, methyl,
or ethyl; or R.sub.3 and R.sub.4, together with the atoms to which
they are attached, form a cyclopropyl ring. This process includes
the step of reacting a compound of formula 5A:
##STR00011##
wherein LG.sub.2 is a leaving group, with H.sub.2NR.sup.1 to form
the compound of formula I-A.
[0043] In some embodiments, the process further includes the step
of reacting a compound of formula 4A:
##STR00012##
with Me-LG.sub.3, wherein LG.sub.3 is a leaving group capable of
being displaced by an NH amide under suitable conditions, to form
the compound of formula 5A.
[0044] In some other embodiments, the process further includes the
step of reductive cyclization of a compound of formula 3A:
##STR00013##
wherein R is C.sub.1-6 aliphatic or hydrogen, to form a compound of
formula 4A.
[0045] In still some other embodiments, the process further
includes cyclizing a compound of formula 3A-a:
##STR00014##
under cyclo-condensation conditions to form a compound of formula
4A.
[0046] In yet still some other embodiments, the process further
includes the step of reacting a compound of formula 3A:
##STR00015##
under reduction conditions to form a compound of formula 3A-a.
[0047] Examples of suitable reduction conditions are described in,
e.g., J. W. Bae et al., Chem. Commun., 2000, 1857-1858
(N-alkylaminobenzenes were prepared in a simple and efficient
one-pot synthesis by reduction of nitrobenzenes followed by
reductive amination with decaborane (B10H14) in the presence of 10%
Pd/C); R. J. Rahaim et al., Org. Lett., 2005, 7, 5087-5090
(palladium-catalyzed reduction of aromatic nitro groups to amines
can be accomplished in high yield, with wide functional group
tolerance and short reaction times at r.t. using aqueous potassium
fluoride and polymethylhydrosiloxane (PMHS) for aromatic nitro
groups; Aliphatic nitro compounds are reduced to the corresponding
hydroxylamines using triethylsilane instead of PMHS/KF); G. S.
Vanier, Synlett, 2007, 131-135 (a generally applicable method for
the introduction of gaseous hydrogen into a sealed reaction system
under microwave irradiation allows the hydrogenation of various
substrates in short reaction times with moderate temperatures
between 80.degree. C. and 100.degree. C. with 50 psi of hydrogen);
S. Chandrasekhar et al., J. Org. Chem., 2006, 71, 2196-2199
(poly(ethylene glycol) (PEG) (400) has been found to be a superior
solvent over ionic liquids by severalfold in promoting the
hydrogenation of various functional groups using Adams' catalyst;
both the catalyst and PEG were recycled efficiently over 10 runs
without loss of activity, and without substrate cross
contamination); H. Berthold et al., Synthesis, 2002, 1607-1610 (a
microwave-assisted, palladium-catalyzed catalytic transfer
hydrogenation of different homo- or heteronuclear organic compounds
using formate salts as a hydrogen source was performed in
([bmim][PF6]; essentially pure products could be isolated in
moderate to excellent yields by simple liquid-liquid extraction);
and C. Yu et al., J. Org. Chem., 2001, 66, 919-924 (a mild and
efficient electron-transfer method for the chemoselective reduction
of aromatic nitro groups using samarium(0) metal in the presence of
a catalytic amount of 1,1'-dioctyl-4,4'-bipyridinium dibromide
gives aromatic amines in good yield with selectivity over a number
of other functional and protecting groups).
[0048] In yet still some other embodiments, the process further
includes the steps of (a) reacting the compound of formula 3A-a
with an alkylating agent under suitable conditions to form a
compound of formula 3A-b;
##STR00016##
and (b) cyclizing the compound of formula 3A-b under suitable
cyclo-condensation conditions to form a compound of formula 5A.
Examples of alkylating agents include alkyl halide. See, e.g., U.S.
Pat. No. 4,783,554.
[0049] In still some other embodiments, the process further
includes reacting a compound of formula 2a:
##STR00017##
with a compound of formula 1:
##STR00018##
under suitable displacement conditions to form the compound of
formula 3A.
[0050] In some other embodiments, the process further includes
reacting a compound of formula 11:
##STR00019##
with a compound of formula 12:
##STR00020##
under suitable reductive amination conditions to form the compound
of formula 2a.
[0051] In yet still other embodiments, the process further includes
the steps of (a) reacting compound of formula 11, e.g., with
1,3,5-triazine or its derivatives, under suitable conditions to
form hexahydro-1,3,5-triazine of formula 13;
##STR00021##
and (b) reacting the compound of formula 13 with a ketene silyl
acetal of formula 14;
##STR00022##
under suitable conditions to form the compound of formula 2a.
[0052] Still another aspect of this invention provides a process
for preparing a compound of formula I-C:
##STR00023##
In formula I-C, R.sup.1 is
##STR00024##
R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic, and is
optionally substituted with 1 or 2 halogen atoms (e.g., 2 fluorine
atoms); and each of R.sup.3 and
[0053] R.sup.4 is independently H, methyl, or ethyl; or R.sup.3 and
R.sup.4, together with the atoms to which they are attached, form a
cyclopropyl ring. This process includes the step of reacting a
compound of formula 5C:
##STR00025##
wherein LG.sub.2 is a leaving group, with H.sub.2NR.sup.1 to form
the compound of formula I-C.
[0054] In some embodiments, this process further includes the step
of reacting a compound of formula 4C:
##STR00026##
with Me-LG.sub.3, wherein LG.sub.3 is a leaving group capable of
being displaced by an NH amide under suitable conditions, to form
the compound of formula 5C
[0055] In some other embodiments, the process of this invention
further includes the step of reductive cyclization of a compound of
formula 3C:
##STR00027##
under suitable conditions to form a compound of formula 4C.
[0056] In some other embodiments, the process further includes
cyclizing a compound of formula 3C-a:
##STR00028##
under cyclo-condensation conditions to form a compound of formula
4C.
[0057] In some other embodiments, the process further includes the
step of reacting a compound of formula 3C:
##STR00029##
under suitable reduction conditions to form a compound of formula
3C-a.
[0058] In some other embodiments, the process further includes the
steps of (a) reacting the compound of formula 3C-a with an
alkylating agent under suitable conditions to form a compound of
formula 3C-b;
##STR00030##
and (b) cyclizing the compound of formula 3C-b under suitable
cyclo-condensation conditions to form a compound of formula 5C.
Examples of suitable alkylating agents include alkyl halide.
[0059] In some other embodiments, the process further includes the
step of reacting a compound of formula 2b:
##STR00031##
with a compound of formula 1:
##STR00032##
under suitable displacement conditions to form the compound of
formula 3C.
[0060] In some other embodiments, the process further includes the
step of reacting a compound of formula 11:
##STR00033##
with a compound of formula 15:
##STR00034##
under suitable displacement conditions, to form the compound of
formula 2b.
[0061] In yet another aspect, the present include provides a
process for preparing a compound of formula I-B:
##STR00035##
In formula I-B, R.sup.1 is
##STR00036##
R.sup.6 is C.sub.1-4 aliphatic or C.sub.3-6 cycloaliphatic, and is
optionally substituted with 1 or 2 halogen atoms (e.g., 2 fluorine
atoms); and each of R3 and R4 is independently H, methyl, or ethyl;
or R3 and R4, together with the atoms to which they are attached,
form a cyclopropyl ring. This process includes the step of reacting
a compound of formula 10:
##STR00037##
wherein LG.sub.2 is a suitable leaving group, with H.sub.2NR.sup.1
under suitable conditions to form the compound of formula I-B.
[0062] In some other embodiments, the process further includes the
step of reacting a compound of formula 9:
##STR00038##
under suitable cyclization conditions known in the art for
converting hydrazides into 1,2,4-triazoles, to form a compound of
formula 10.
[0063] In some other embodiments, the process further includes the
step of reacting a compound of formula 8:
##STR00039##
wherein LG.sub.3 is a leaving group capable of being displaced by
an NH amide under suitable conditions, with hydrazine to form the
compound of formula 9.
[0064] In some other embodiments, the process further includes
reacting a compound of formula 4C:
##STR00040##
under suitable conditions known in the art for converting amides
into activated amides, to form a compound of formula 8.
[0065] In some other embodiments of the process, R.sup.2 and
R.sup.5, together with the atoms to which they are attached, form a
1,2,4-triazole.
[0066] Compounds of this invention include those described herein,
and are further illustrated by the classes, subclasses, and species
disclosed herein. As used herein, the following definitions shall
apply unless otherwise indicated. For purposes of this invention,
the chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 75.sup.th Ed. Additionally, general principles of
organic chemistry are described in "Organic Chemistry", Thomas
Sorrell, University Science Books, Sausalito: 1999, and "March's
Advanced Organic Chemistry", 5.sup.th Ed., Ed.: Smith, M. B. and
March, J., John Wiley & Sons, New York (2001), the entire
contents of which are hereby incorporated by reference.
[0067] As described herein, a specified number range of atoms
includes any integer therein, including the upper and lower limits
of the range. For example, a group having from 1 to 4 (or 1-4)
atoms could have 1, 2, 3, or 4 atoms.
[0068] As described herein, compounds of the invention may
optionally be substituted with one or more substituents, such as
are illustrated generally above, or as exemplified by particular
classes, subclasses, and species of the invention. It will be
appreciated that the phrase "optionally substituted" is used
interchangeably with the phrase "substituted or unsubstituted." In
general, the term "substituted", whether preceded by the term
"optionally" or not, refers to the replacement of hydrogen radicals
in a given structure with the radical of a specified substituent.
Unless otherwise indicated, an optionally substituted group may
have a substituent at each substitutable position of the group, and
when more than one position in any given structure may be
substituted with more than one substituent selected from a
specified group, the substituent may be either the same or
different at every position. Combinations of substituents
envisioned by this invention are preferably those that result in
the formation of stable or chemically feasible compounds.
[0069] The term "stable", as used herein, refers to compounds that
are not substantially altered when subjected to conditions to allow
for their production, detection, recovery, purification, and use
for one or more of the purposes disclosed herein. In some
embodiments, a stable compound or chemically feasible compound is
one that is not substantially altered when kept at a temperature of
40.degree. C. or less, in the absence of moisture or other
chemically reactive conditions, for at least a week.
[0070] The term "aliphatic" or "aliphatic group," as used herein,
means a straight-chain (i.e., unbranched), branched, or cyclic
substituted or unsubstituted hydrocarbon chain that is completely
saturated or that contains one or more units of unsaturation that
has a single point of attachment to the rest of the molecule.
[0071] Unless otherwise specified, aliphatic groups contain 1-20
aliphatic carbon atoms. In some embodiments, aliphatic groups
contain 1-10 aliphatic carbon atoms. In other embodiments,
aliphatic groups contain 1-8 aliphatic carbon atoms. In still other
embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms,
and in yet other embodiments aliphatic groups contain 1-4 aliphatic
carbon atoms. Suitable aliphatic groups include, but are not
limited to, linear or branched, substituted or unsubstituted alkyl,
alkenyl, or alkynyl groups. Specific examples include, but are not
limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl,
n-butenyl, ethynyl, and tert-butyl.
[0072] It should be understood that if the aliphatic is alkenyl or
alkynyl, then the aliphatic group has at least 2 carbon atoms.
[0073] The term "cycloaliphatic" refers to a monocyclic
C.sub.3-C.sub.8 hydrocarbon or bicyclic C.sub.7-C.sub.12
hydrocarbon that is completely saturated or that contains one or
more units of unsaturation, but which is not aromatic, that has a
single point of attachment to the rest of the molecule wherein any
individual ring in said bicyclic ring system has 3-7 members.
Suitable cycloaliphatic groups include, but are not limited to,
cycloalkyl and cycloalkenyl groups. Specific examples include, but
are not limited to, cyclohexyl, cyclopropenyl, and cyclobutyl.
[0074] The term "heteroaliphatic", as used herein, means aliphatic
groups wherein one or two carbon atoms are independently replaced
by one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon.
Heteroaliphatic groups may be substituted or unsubstituted,
branched or unbranched, cyclic or acyclic, and include
"heterocycle", "heterocyclyl", "heterocycloaliphatic", or
"heterocyclic" groups. The term "heterocycle", "heterocyclyl", or
"heterocyclic" as used herein means non-aromatic, monocyclic,
bicyclic, or tricyclic ring systems in which one or more ring
members are an independently selected heteroatom. In some
embodiments, the "heterocycle", "heterocyclyl", or "heterocyclic"
group has three to fourteen ring members in which one or more ring
members is a heteroatom independently selected from oxygen, sulfur,
nitrogen, or phosphorus, and each ring in the system contains 3 to
7 ring members.
[0075] Suitable heterocycles include, but are not limited to,
3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,
2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,
3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,
2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino,
1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl,
1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl,
3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl,
5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,
4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl,
1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,
5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and
1,3-dihydro-imidazol-2-one.
[0076] Cyclic groups, (e.g., cycloaliphatic and heterocycles), can
be linearly fused, bridged, or spirocyclic.
[0077] The term "heteroatom" means one or more of oxygen, sulfur,
nitrogen, or phosphorus, (including, any oxidized form of nitrogen,
sulfur, or phosphorus; the quaternized form of any basic nitrogen
or; a substitutable nitrogen of a heterocyclic ring, for example N
(as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR' (as
in N-substituted pyrrolidinyl)).
[0078] The term "unsaturated", as used herein, means that a moiety
has one or more units of unsaturation.
[0079] The term "nonaromatic", as used herein, describes rings that
are either saturated or partially unsaturated.
[0080] The term "aromatic", as used herein, describes rings that
are fully unsaturated.
[0081] The term "alkoxy", or "thioalkyl", as used herein, refers to
an alkyl group, as previously defined, attached to the principal
carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl")
atom.
[0082] The terms "haloalkyl", "haloalkenyl", "haloaliphatic", and
"haloalkoxy" mean alkyl, alkenyl or alkoxy, as the case may be,
substituted with one or more halogen atoms. The terms "halogen",
"halo", and "hal" mean F (fluorine), Cl (chlorine), Br (bromine),
or I (iodine).
[0083] The term "aryl" used alone or as part of a larger moiety as
in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic,
bicyclic, and tricyclic ring systems having a total of five to
fourteen ring members, wherein at least one ring in the system is
aromatic and wherein each ring in the system contains 3 to 7 ring
members. The term "aryl" may be used interchangeably with the term
"aryl ring". The term "aryl" also refers to heteroaryl ring systems
as defined hereinbelow.
[0084] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroaralkyl" or "heteroarylalkoxy", refers to
monocyclic, bicyclic, and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, at least one ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
Suitable heteroaryl rings include, but are not limited to,
2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl,
5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g.,
3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl
(e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and
5-triazolyl), 2-thienyl, 3-thienyl, benzofuryl, benzothiophenyl,
indolyl (e.g., 2-indolyl), pyrazolyl (e.g., 2-pyrazolyl),
isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,
1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,
1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,
4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,
3-isoquinolinyl, or 4-isoquinolinyl).
[0085] The term "protecting group" and "protective group" as used
herein, are interchangeable and refer to an agent used to
temporarily block one or more desired reactive sites in a
multifunctional compound. In certain embodiments, a protecting
group has one or more, or preferably all, of the following
characteristics: (a) is added selectively to a functional group in
good yield to give a protected substrate that is (b) stable to
reactions occurring at one or more of the other reactive sites; and
(c) is selectively removable in good yield by reagents that do not
attack the regenerated, deprotected functional group. Exemplary
protecting groups are detailed and provided by T. W. Greene et al.
in "Protective Groups in Organic Synthesis", Third Edition, John
Wiley & Sons, New York (1999) (and other editions of the book),
the entire contents of which are hereby incorporated by reference.
The term "nitrogen protecting group," as used herein, refers to an
agents used to temporarily block one or more desired nitrogen
reactive sites in a multifunctional compound. Certain exemplary
nitrogen protecting groups are also detailed and provided by T. W.
Greene et al., in Chapter 7 of "Protective Groups in Organic
Synthesis", Third Edition, John Wiley & Sons, New York (1999),
the entire contents of which are hereby incorporated by
reference.
[0086] In some embodiments, an alkyl or aliphatic chain can be
optionally interrupted with another atom or group. This means that
a methylene unit of the alkyl or aliphatic chain is optionally
replaced with said other atom or group. Examples of such atoms or
groups would include, but are not limited to, --NR--, --O--, --S--,
--CO.sub.2--, --OC(O)--, --C(O)CO--, --C(O)--, --C(O)NR--,
--C(.dbd.N--CN)--, --NRCO--, --NRC(O)O--, --SO.sub.2NR--,
--NRSO.sub.2--, --NRC(O)NR--, --OC(O)NR--, --NRSO.sub.2NR--,
--SO--, or --SO.sub.2--, wherein R is defined herein. Unless
otherwise specified, the optional replacements form a chemically
stable compound. Optional interruptions can occur both within the
chain and at either end of the chain; i.e., both at the point of
attachment and/or also at the terminal end. Two optional
replacements can also be adjacent to each other within a chain so
long as it results in a chemically stable compound. The optional
interruptions or replacements can also completely replace all of
the carbon atoms in a chain. For example, a C.sub.3 aliphatic can
be optionally interrupted or replaced by --NR--, --C(O)--, and
--NR-- to form --NRC(O)NR-- (a urea).
[0087] Unless otherwise specified, if the replacement or
interruption occurs at the terminal end, the replacement atom is
bound to an H on the terminal end. For example, if
--CH.sub.2CH.sub.2CH.sub.3 were optionally interrupted with --O--,
the resulting compound could be --OCH.sub.2CH.sub.3,
--CH.sub.2OCH.sub.3, or --CH.sub.2CH.sub.2OH.
[0088] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention.
[0089] Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the
invention.
[0090] Unless otherwise stated, a substituent can freely rotate
around any rotatable bonds. For example, a substituent drawn as
##STR00041##
also represents
##STR00042##
[0091] Additionally, unless otherwise stated, structures depicted
herein are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
compounds having the present structures except for the replacement
of hydrogen by deuterium or tritium, or the replacement of a carbon
by a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this invention. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
[0092] As used herein, the term "pharmaceutically acceptable salt"
refers to salts of a compound which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
humans and lower animals without undue toxicity, irritation,
allergic response and the like, and are commensurate with a
reasonable benefit/risk ratio.
[0093] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al., describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66,
1-19, incorporated herein by reference. Pharmaceutically acceptable
salts of the compounds of this invention include those derived from
suitable inorganic and organic acids and bases. These salts can be
prepared in situ during the final isolation and purification of the
compounds. Acid addition salts can be prepared by 1) reacting the
purified compound in its free-based form with a suitable organic or
inorganic acid and 2) isolating the salt thus formed.
[0094] Examples of pharmaceutically acceptable, nontoxic acid
addition salts are salts of an amino group formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, phosphoric acid,
sulfuric acid and perchloric acid or with organic acids such as
acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid or malonic acid or by using other methods used in the
art such as ion exchange. Other pharmaceutically acceptable salts
include adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, glycolate, gluconate, hemisulfate, heptanoate,
hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,
lactate, laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, palmoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
salicylate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C.sub.1-4alkyl).sub.4 salts. This
invention also envisions the quaternization of any basic
nitrogen-containing groups of the compounds disclosed herein. Water
or oil-soluble or dispersible products may be obtained by such
quaternization.
[0095] Base addition salts can be prepared by (1) reacting the
purified compound in its acid form with a suitable organic or
inorganic base, and (2) isolating the salt thus formed. Base
addition salts include alkali or alkaline earth metal salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate. Other
acids and bases, while not in themselves pharmaceutically
acceptable, may be employed in the preparation of salts useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid or base addition salts.
[0096] The following abbreviations are used: [0097] PG protecting
group [0098] LG leaving group [0099] DCM dichloromethane [0100] Ac
acetyl [0101] DMF dimethylformamide [0102] EtOAc ethyl acetate
[0103] DMSO dimethyl sulfoxide [0104] MeCN acetonitrile [0105] TCA
trichloroacetic acid [0106] ATP adenosine triphosphate [0107] EtOH
ethanol [0108] Ph phenyl [0109] Me methyl [0110] Et ethyl [0111] Bu
butyl [0112] DEAD diethylazodicarboxylate [0113] HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [0114] BSA
bovine serum albumin [0115] DTT dithiothreitol [0116] MOPS
4-morpholinepropanesulfonic acid [0117] NMR nuclear magnetic
resonance [0118] HPLC high performance liquid chromatography [0119]
LCMS liquid chromatography-mass spectrometry [0120] TLC thin layer
chromatography [0121] Rt retention time
[0122] In some embodiments, the compounds of this invention are
represented in Table 1.
TABLE-US-00001 TABLE 1 ##STR00043## I-1 ##STR00044## I-2
##STR00045## I-3 ##STR00046## I-4 ##STR00047## I-5 ##STR00048## I-6
##STR00049## I-7 ##STR00050## I-8 ##STR00051## I-9
General Synthetic Methodology
[0123] The compounds of this invention may be prepared in general
by methods such as those depicted in the general schemes below.
Unless otherwise indicated, all variables in the following schemes
are as defined herein.
[0124] In one method, the compounds of the invention wherein X is O
may be prepared as illustrated in Scheme 1.
##STR00052##
[0125] Referring to Scheme 1, the nitro pyrimidine 1, wherein
LG.sub.1 and LG.sub.2 are, for example, chlorine, reacts with
.alpha.- or .beta.-aminoesters 2 (in which n is 0 or 1) to provide
an adduct 3. Reduction of the nitro group under known conditions,
followed by cyclization, provides bicyclic compound 4. The amide
N--H may be functionalized by reaction with, for example, an alkyl
halide in the presence of a strong base such as, for example,
sodium hydride to provide compound 5. Reaction of compound 5 with
R.sup.1R.sup.8NH, optionally in the presence of a palladium
catalyst, provides a compound of formula I.
[0126] An alternate method for synthesizing the compounds of this
invention is illustrated in Scheme 2.
##STR00053##
[0127] Referring to Scheme 2, compound 3 wherein LG.sub.2 is, for
example, chlorine, reacts with R.sup.1R.sup.8NH, optionally in the
presence of a palladium catalyst, to provide compound 6. Reduction
of the nitro group in compound 6 as previously described, followed
by cyclization provides a bicyclic compound 7. The amide group in
compound 7 is functionalized to provide compounds of formula I.
[0128] Illustrated below in Scheme 3 is a method for preparing
compounds of this invention wherein X is --NR.sup.5 and R.sup.5 and
R.sup.2, together with the atoms to which they are attached, form a
triazole ring.
##STR00054##
[0129] Referring to Scheme 3, activation of the lactam functional
group in compound 4, provides the intermediate 8 wherein LG.sub.3
is, for example, chlorine. The displacement of the group LG.sub.3
in compound 8 with hydrazine provides the intermediate of formula
9. Reaction of compound 9 with an orthoformate ester (e.g., methyl
orthoformate) provides the triazole intermediate 10. Reaction of
compound 10 with R.sup.1R.sup.8NH as previously described provides
a compound of formula I-b.
[0130] Shown below in Scheme 4 is a method for preparing compounds
of formula 2 wherein R.sup.7 is 3,3-difluorocyclopentyl and n is 1,
which are shown in the Scheme as formula 2a.
##STR00055##
[0131] Referring to Scheme 4, 3,3-difluorocyclopentanamine 11
(described in WO 2007/062308 and WO 2007/062314) reacts with an
aldhyde 12 under known reductive amination conditions to provide
intermediates 2a. Suitable reductive amination conditions as used,
e.g., in Eschweiler-Clarke Reaction, and in addition are described
in literature. See, e.g., A. F. Abdel-Magid et al., J. Org. Chem.,
1996, 61, 3849-3862; J. W. Bae et al., J. Chem. Soc., Perkin Trans.
1, 2000, 145-146; B. T. Cho et al., Tetrahedron, 2005, 61,
5725-5734; M. McLaughlin et al., Org. Lett., 2006, 8, 3307-3310; T.
Mizuta et al., J. Org. Chem., 2005, 70, 2195-2199.
[0132] An alternative method for preparing intermediates of formula
2a is illustrated in Scheme 5.
##STR00056##
[0133] In Scheme 5, 3,3-difluorocyclopentanamine 11 reacts with
formaldehyde in the presence of sodium hydroxide to provide the
1,3,5-triazine 13. Reaction of compound 13 with a ketene silyl
acetal of formula 14 provides an intermediate of formula 2a.
[0134] Shown below in Scheme 6 is a method for preparing compound
of formula 2 in which n is 0 (shown in the Scheme as formula
2b).
##STR00057##
[0135] Referring to Scheme 6, a compound of formula 15 wherein LG
is, for example, bromine, reacts with 3,3-difluorocyclopentanamine
11 to give an aminoester of formula 2b.
[0136] Another aspect of this invention provides compounds that are
inhibitors of protein kinases, and thus are useful for the
treatment of the diseases, disorders, or conditions (collectively
"disorders") implicated by protein kinases, along with other uses
described herein. Examples of such conditions include proliferative
disorders, neurodegenerative disorders, autoimmune disorders,
inflammatory disorders, or immunologically mediated disorders that
are implicated or mediated by protein kinases (e.g., PLK1, pLK2,
PLK3, or PLK4). Specific examples of such conditions include, but
are not limited to, melanoma, myeloma, leukemia, lymphoma,
neuroblastoma, or a cancer selected from colon, breast, gastric,
ovarian, cervical, lung, central nervous system (CNS), renal,
prostate, bladder, or pancreatic.
[0137] In another aspect of the present invention, pharmaceutically
acceptable compositions are provided, wherein these compositions
each comprise any of the compounds described herein, and optionally
a pharmaceutically acceptable carrier, adjuvant or vehicle.
[0138] In certain embodiments of the compositions of this
invention, these compositions each further comprise one or more
additional therapeutic agents. Examples of such additional
therapeutic agents include, but are not limited to, a
chemotherapeutic or anti-proliferative agent, an anti-inflammatory
agent, an immunomodulatory or immunosuppressive agent, a
neurotrophic factor, an agent for treating cardiovascular disease,
an agent for treating destructive bone disorders, an agent for
treating liver disease, an anti-viral agent, an agent for treating
blood disorders, an agent for treating diabetes, or an agent for
treating immunodeficiency disorders. The additional therapeutic
agent can be administered together with the compound or the
pharmaceutical composition of as a single dosage form or separately
from the compound or pharmaceutical composition as part of a
multiple dosage form.
[0139] The present invention provides compounds and compositions
that are useful as inhibitors of protein kinases. In some
embodiments, the protein kinases are PLKs (e.g., PLK1, PLK2, PLK3,
or PLK4). In some embodiments, PLK1.
[0140] As inhibitors of protein kinases, the compounds and
compositions of this invention are particularly useful for treating
or lessening the severity of a disease, condition, or disorder
where a protein kinase is implicated in the disease, condition, or
disorder. In one aspect, the present invention provides a method
for treating or lessening the severity of a disease, condition, or
disorder where a protein kinase is implicated in the disease state.
In another aspect, the present invention provides a method for
treating or lessening the severity of a kinase disease, condition,
or disorder where inhibition of enzymatic activity is implicated in
the treatment of the disease. In another aspect, this invention
provides a method for treating or lessening the severity of a
disease, condition, or disorder with compounds that inhibit
enzymatic activity by binding to the protein kinase. Another aspect
provides a method for treating or lessening the severity of a
kinase disease, condition, or disorder by inhibiting enzymatic
activity of the kinase with a protein kinase inhibitor.
[0141] In some embodiments, said protein kinase inhibitor is a PLK
inhibitor.
[0142] One aspect of the invention relates to a method of
inhibiting protein kinase activity in a patient, which method
comprises administering to the patient a compound of formula I, or
a composition comprising said compound.
[0143] In some embodiments, said method is used to treat or prevent
a condition selected from autoimmune diseases, inflammatory
diseases, proliferative and hyperproliferative diseases,
immunologically-mediated diseases, bone diseases, metabolic
diseases, neurological and neurodegenerative diseases,
cardiovascular diseases, hormone related diseases, allergies,
asthma, and Alzheimer's disease. In some embodiments, said protein
kinase in PLK. In other embodiments, said condition is selected
from a proliferative disorder and a neurodegenerative disorder.
[0144] Depending upon the particular protein kinase-mediated
conditions to be treated or prevented, additional drugs, which are
normally administered to treat or prevent that condition, may be
administered together with the inhibitors of this invention. For
example, chemotherapeutic agents or other anti-proliferative agents
may be combined with the protein kinase inhibitors of this
invention to treat proliferative diseases.
[0145] Those additional agents may be administered separately, as
part of a multiple dosage regimen, from the protein kinase
inhibitor-containing compound or composition. Alternatively, those
agents may be part of a single dosage form, mixed together with the
protein kinase inhibitor in a single composition.
[0146] As inhibitors of protein kinases, the compounds and
compositions of this invention are also useful in biological
samples. One aspect of the invention relates to inhibiting protein
kinase activity in a biological sample, which method comprises
contacting said biological sample with a compound of formula I or a
composition comprising said compound. The term "biological sample",
as used herein, means an in vitro or an ex vivo sample, including,
without limitation, cell cultures or extracts thereof; biopsied
material obtained from a mammal or extracts thereof; and blood,
saliva, urine, feces, semen, tears, or other body fluids or
extracts thereof.
[0147] Inhibition of protein kinase activity in a biological sample
is useful for a variety of purposes that are known to one of skill
in the art. Examples of such purposes include, but are not limited
to, blood transfusion, organ-transplantation, and biological
specimen storage.
[0148] Another aspect of this invention relates to the study of
protein kinases in biological and pathological phenomena; the study
of intracellular signal transduction pathways mediated by such
protein kinases; and the comparative evaluation of new protein
kinase inhibitors. Examples of such uses include, but are not
limited to, biological assays such as enzyme assays and cell-based
assays.
[0149] The activity of the compounds as protein kinase inhibitors
may be assayed in vitro, in vivo or in a cell line. In vitro assays
include assays that determine inhibition of either the kinase
activity or ATPase activity of the activated kinase. Alternate in
vitro assays quantitate the ability of the inhibitor to bind to the
protein kinase and may be measured either by radiolabelling the
inhibitor prior to binding, isolating the inhibitor/kinase complex
and determining the amount of radiolabel bound, or by running a
competition experiment where new inhibitors are incubated with the
kinase bound to known radioligands. Detailed conditions for
assaying a compound utilized in this invention as an inhibitor of
PLK1, PLK2, PLK3, and PLK4 are set forth in the Examples below.
[0150] One aspect of this invention provides compounds that are
useful for the treatment of diseases, disorders, and conditions
characterized by excessive or abonormal cell proliferation. Such
diseases include, a proliferative or hyperproliferative disease,
and a neurodegenerative disease.
[0151] Examples of proliferative and hyperproliferative diseases
include, without limitation, cancer.
[0152] The term "cancer" includes, but is not limited to, the
following cancers: breast; ovary; cervix; prostate; testis,
genitourinary tract; esophagus; larynx, glioblastoma;
neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid
carcinoma, large cell carcinoma, small cell carcinoma, lung
adenocarcinoma; bone; colon; colorectal; adenoma; pancreas,
adenocarcinoma; thyroid, follicular carcinoma, undifferentiated
carcinoma, papillary carcinoma; seminoma; melanoma; sarcoma;
bladder carcinoma; liver carcinoma and biliary passages; kidney
carcinoma; myeloid disorders; lymphoid disorders, Hodgkin's, hairy
cells; buccal cavity and pharynx (oral), lip, tongue, mouth,
pharynx; small intestine; colon-rectum, large intestine, rectum;
brain and central nervous system; chronic myeloid leukemia (CML),
and leukemia. The term "cancer" includes, but is not limited to,
the following cancers: myeloma, lymphoma, or a cancer selected from
gastric, renal, or and the following cancers: head and neck,
oropharangeal, non-small cell lung cancer (NSCLC), endometrial,
hepatocarcinoma, Non-Hodgkins lymphoma, and pulmonary.
[0153] For the avoidance of doubt, the term "cancer" also includes,
but is not limited to, the following cancers: epidermoid Oral:
buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma
(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),
myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:
bronchogenic carcinoma (squamous cell or epidermoid,
undifferentiated small cell, undifferentiated large cell,
adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;
Gastrointestinal: esophagus (squamous cell carcinoma, larynx,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma,
lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,
insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma),
small bowel or small intestines (adenocarcinoma, lymphoma,
carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel or large intestines
(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,
leiomyoma), colon, colon-rectum, colorectal; rectum, Genitourinary
tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],
lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,
transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma,
biliary passages; Bone: osteogenic sarcoma (osteosarcoma),
fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma,
Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma),
multiple myeloma, malignant giant cell tumor chordoma,
osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell
tumors; Nervous system: skull (osteoma, hemangioma, granuloma,
xanthoma, osteitis deformans), meninges (meningioma,
meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,
glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,
oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),
spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological: uterus (endometrial carcinoma), cervix (cervical
carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian
carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma,
unclassified carcinoma], granulosa-thecal cell tumors,
Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (squamous cell carcinoma, intraepithelial carcinoma,
adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell
carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal
rhabdomyosarcoma), fallopian tubes (carcinoma), breast;
Hematologic: blood (myeloid leukemia [acute and chronic], acute
lymphoblastic leukemia, chronic lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant
lymphoma] hairy cell; lymphoid disorders; Skin: malignant melanoma,
basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma,
keratoacanthoma, moles dysplastic nevi, lipoma, angioma,
dermatofibroma, keloids, psoriasis, Thyroid gland: papillary
thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid
carcinoma, undifferentiated thyroid cancer, multiple endocrine
neoplasia type 2A, multiple endocrine neoplasia type 2B, familial
medullary thyroid cancer, pheochromocytoma, paraganglioma; and
Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as
provided herein, includes a cell afflicted by any one of the
above-identified conditions.
[0154] In some embodiments, the compounds of this invention are
useful for treating cancer, such as colorectal, thyroid, breast,
and lung cancer; and myeloproliferative disorders, such as
polycythemia vera, thrombocythemia, myeloid metaplasia with
myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic
leukemia, hypereosinophilic syndrome, juvenile myelomonocytic
leukemia, and systemic mast cell disease.
[0155] In some embodiments, the compounds of this invention are
useful for treating hematopoietic disorders, in particular,
acute-myelogenous leukemia (AML), chronic-myelogenous leukemia
(CML), acute-promyelocytic leukemia (APL), and acute lymphocytic
leukemia (ALL).
[0156] Examples of neurodegenerative diseases include, without
limitation, Alzheimer's disease.
[0157] Another aspect of this invention provides a method for the
treatment or lessening the severity of a disease selected from a
proliferative or hyperproliferative disease, or a neurodegenerative
disease, comprising administering an effective amount of a
compound, or a pharmaceutically acceptable composition comprising a
compound, to a subject in need thereof.
[0158] In certain embodiments, an "effective amount" of the
compound or pharmaceutically acceptable composition is that amount
effective in order to treat said disease. The compounds and
compositions, according to the method of the present invention, may
be administered using any amount and any route of administration
effective for treating or lessening the severity of said
disease.
[0159] In some embodiments, said disease is a protein-kinase
mediated condition. In some embodiments, said disease is a
PLK-mediated disease.
[0160] The term "protein kinase-mediated condition", as used
herein, means any disease or other deleterious condition in which a
protein kinase plays a role. Such conditions include, without
limitation, autoimmune diseases, inflammatory diseases,
proliferative and hyperproliferative diseases,
immunologically-mediated diseases, bone diseases, metabolic
diseases, neurological and neurodegenerative diseases,
cardiovascular diseases, hormone related diseases, allergies,
asthma, and Alzheimer's disease.
[0161] The term "PLK-mediated condition", as used herein means any
disease or other deleterious condition in which PLK plays a role.
Such conditions include, without limitation, a proliferative or
hyperproliferative disease, or a neurodegenerative disease.
[0162] In another aspect of the present invention, pharmaceutically
acceptable compositions are provided, wherein these compositions
comprise any of the compounds as described herein, and optionally
comprise a pharmaceutically acceptable carrier, adjuvant or
vehicle.
[0163] In certain embodiments, these compositions optionally
further comprise one or more additional therapeutic agents.
[0164] For example, chemotherapeutic agents or other
anti-proliferative agents may be combined with the compounds of
this invention to treat proliferative diseases and cancer.
[0165] Examples of known chemotherapeutic agents include, but are
not limited to, Gleevec.TM., adriamycin, dexamethasone,
vincristine, cyclophosphamide, fluorouracil, topotecan, taxol,
interferons, and platinum derivatives.
[0166] Other examples of agents the inhibitors of this invention
may also be combined with include, without limitation: treatments
for Alzheimer's Disease such as Aricept.RTM. and Excelon.RTM.;
treatments for Parkinson's Disease such as L-DOPA/carbidopa,
entacapone, ropinrole, pramipexole, bromocriptine, pergolide,
trihexephendyl, and amantadine; agents for treating Multiple
Sclerosis (MS) such as beta interferon (e.g., Avonex.RTM. and
Rebif.RTM.), Copaxone.RTM., and mitoxantrone; treatments for asthma
such as albuterol and Singulair.RTM.; agents for treating
schizophrenia such as zyprexa, risperdal, seroquel, and
haloperidol; anti-inflammatory agents such as corticosteroids, TNF
blockers, IL-1 RA, azathioprine, cyclophosphamide, and
sulfasalazine; immunomodulatory and immunosuppressive agents such
as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil,
interferons, corticosteroids, cyclophophamide, azathioprine, and
sulfasalazine; neurotrophic factors such as acetylcholinesterase
inhibitors, MAO inhibitors, interferons, anti-convulsants, ion
channel blockers, riluzole, and anti-Parkinsonian agents; agents
for treating cardiovascular disease such as beta-blockers, ACE
inhibitors, diuretics, nitrates, calcium channel blockers, and
statins; agents for treating liver disease such as corticosteroids,
cholestyramine, interferons, and anti-viral agents; agents for
treating blood disorders such as corticosteroids, anti-leukemic
agents, and growth factors; and agents for treating
immunodeficiency disorders such as gamma globulin.
[0167] As described herein, the pharmaceutically acceptable
compositions of the present invention additionally comprise a
pharmaceutically acceptable carrier, adjuvant, or vehicle, which,
as used herein, includes any and all solvents, diluents, or other
liquid vehicle, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's Pharmaceutical
Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co.,
Easton, Pa., 1980) discloses various carriers used in formulating
pharmaceutically acceptable compositions and known techniques for
the preparation thereof. Except insofar as any conventional carrier
medium is incompatible with the compounds of the invention, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutically acceptable composition, its use is
contemplated to be within the scope of this invention.
[0168] Some examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, or potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, wool fat, sugars such
as lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propylene glycol or polyethylene glycol;
esters such as ethyl oleate and ethyl laurate; agar; buffering
agents such as magnesium hydroxide and aluminum hydroxide; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
[0169] The protein kinase inhibitors or pharmaceutical salts
thereof may be formulated into pharmaceutical compositions for
administration to animals or humans. These pharmaceutical
compositions, which comprise an amount of the protein inhibitor
effective to treat or prevent a protein kinase-mediated condition
and a pharmaceutically acceptable carrier, are another embodiment
of the present invention. In some embodiments, said protein
kinase-mediated condition is a PLK-mediated condition.
[0170] The exact amount of compound required for treatment will
vary from subject to subject, depending on the species, age, and
general condition of the subject, the severity of the infection,
the particular agent, its mode of administration, and the like. The
compounds of the invention are preferably formulated in dosage unit
form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of agent appropriate for the patient to be treated.
It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided
by the attending physician within the scope of sound medical
judgment. The specific effective dose level for any particular
patient or organism will depend upon a variety of factors including
the disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed, and like
factors well known in the medical arts. The term "patient", as used
herein, means an animal, preferably a mammal, and most preferably a
human.
[0171] The pharmaceutically acceptable compositions of this
invention can be administered to humans and other animals orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, or drops),
bucally, as an oral or nasal spray, or the like, depending on the
severity of the infection being treated. In certain embodiments,
the compounds of the invention may be administered orally or
parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg
and preferably from about 1 mg/kg to about 25 mg/kg, of subject
body weight per day, one or more times a day, to obtain the desired
therapeutic effect. In a preferred embodiment, compounds of this
invention are administered orally.
[0172] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0173] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0174] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0175] In order to prolong the effect of a compound of the present
invention, it is often desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by
dissolving or suspending the compound in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the
compound in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of compound to
polymer and the nature of the particular polymer employed, the rate
of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0176] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0177] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0178] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0179] The active compounds can also be in microencapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0180] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, eardrops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0181] In addition to the compounds of this invention,
pharmaceutically acceptable derivatives or prodrugs of the
compounds of this invention may also be employed in compositions to
treat or prevent the above-identified disorders.
[0182] The compounds of this invention can also exist as
pharmaceutically acceptable derivatives.
[0183] A "pharmaceutically acceptable derivative" is an adduct or
derivative which, upon administration to a patient in need, is
capable of providing, directly or indirectly, a compound as
otherwise described herein, or a metabolite or residue thereof.
Examples of pharmaceutically acceptable derivatives include, but
are not limited to, esters and salts of such esters.
[0184] A "pharmaceutically acceptable derivative or prodrug" means
any pharmaceutically acceptable ester, salt of an ester or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing, either
directly or indirectly, a compound of this invention or an
inhibitorily active metabolite or residue thereof. Particularly
favoured derivatives or prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a patient (e.g., by allowing an
orally administered compound to be more readily absorbed into the
blood) or which enhance delivery of the parent compound to a
biological compartment (e.g., the brain or lymphatic system)
relative to the parent species.
[0185] Pharmaceutically acceptable prodrugs of the compounds of
this invention include, without limitation, esters, amino acid
esters, phosphate esters, metal salts and sulfonate esters.
[0186] Pharmaceutically acceptable carriers that may be used in
these pharmaceutical compositions include, but are not limited to,
ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat.
[0187] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes, but is
not limited to, subcutaneous, intravenous, intramuscular,
intra-articular, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional and intracranial injection or infusion
techniques. Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
[0188] Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as carboxymethyl
cellulose or similar dispersing agents which are commonly used in
the formulation of pharmaceutically acceptable dosage forms
including emulsions and suspensions. Other commonly used
surfactants, such as Tweens, Spans and other emulsifying agents or
bioavailability enhancers which are commonly used in the
manufacture of pharmaceutically acceptable solid, liquid, or other
dosage forms may also be used for the purposes of formulation.
[0189] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers commonly
used include, but are not limited to, lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried cornstarch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0190] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient that is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include, but
are not limited to, cocoa butter, beeswax and polyethylene
glycols.
[0191] The pharmaceutical compositions of this invention may also
be administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
including diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs.
[0192] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0193] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0194] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with or without a preservative such
as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
pharmaceutical compositions may be formulated in an ointment such
as petrolatum.
[0195] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0196] The amount of protein kinase inhibitor that may be combined
with the carrier materials to produce a single dosage form will
vary depending upon the host treated, the particular mode of
administration. Preferably, the compositions should be formulated
so that a dosage of between 0.01 and 100 mg/kg body weight/day of
the inhibitor can be administered to a patient receiving these
compositions.
[0197] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of inhibitor will also
depend upon the particular compound in the composition.
[0198] According to another embodiment, the invention provides
methods for treating or preventing a protein kinase-mediated
condition (in some embodiments, a PLK-mediated condition)
comprising the step of administering to a patient one of the
above-described pharmaceutical compositions. The term "patient", as
used herein, means an animal, preferably a human.
[0199] In some embodiments, said method is used to treat or prevent
a condition selected from a proliferative disorder, such as cancer,
a neurodegenerative disorder, an autoimmune disorder, an
inflammatory disorder, and an immunologically-mediated disorder. In
some embodiments, said method is used to treat or prevent a
condition selected from cancers such as cancers of the breast,
colon, prostate, skin, pancreas, brain, genitourinary tract,
lymphatic system, stomach, larynx and lung, including lung
adenocarcinoma and small cell lung cancer; stroke, diabetes,
myeloma, hepatomegaly, cardiomegaly, Alzheimer's disease, cystic
fibrosis, and viral disease, or any specific disease described
above.
[0200] The compounds of this invention may be prepared in general
by methods known to those skilled in the art. Those compounds may
be analyzed by known methods, including but not limited to LCMS
(liquid chromatography mass spectrometry) and NMR (nuclear magnetic
resonance). Compounds of this invention may be also tested
according to these examples. It should be understood that the
specific conditions shown below are only examples, and are not
meant to limit the scope of the conditions that can be used for
making, analyzing, or testing the compounds of this invention.
Instead, this invention also includes conditions known to those
skilled in that art for making, analyzing, and testing the
compounds of this invention.
EXAMPLES
[0201] As used herein, the term "Rt(min)" refers to the HPLC
retention time, in minutes, associated with the compound. Unless
otherwise indicated, the HPLC method utilized to obtain the
reported retention time is as follows: [0202] Column: ACE C8
column, 4.6.times.150 mm [0203] Gradient: 0-100%
acetonitrile+methanol 50:50 (20 mM Tris phosphate) [0204] Flow
rate: 1.5 mL/minute [0205] Detection: 225 nm.
[0206] Mass spec. samples were analyzed on a MicroMass Quattro
Micro mass spectrometer operated in single MS mode with
electrospray ionization. Samples were introduced into the mass
spectrometer using chromatography.
[0207] .sup.1H-NMR spectra were recorded at 400 MHz using a Bruker
DPX 400 instrument.
[0208] The following compounds of formula I were prepared and
analyzed as follows.
Example 1
(R)--N-cyclopropyl-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,-
7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-methoxybenza-
mide
##STR00058##
[0209] Step 1: tert-butyl (1R,3
S)-3-hydroxycyclopentylcarbamate
##STR00059##
[0211] Di-tert-butyl dicarbonate (432 mg, 1.98 mmol) was added to a
solution of (1S,3R)-3-aminocyclopentanol (200 mg, 1.98 mmol) and
triethylamine (0.662 ml, 4.75 mmol) in dichloromethane (20 mL) at
0.degree. C. After complete addition, the reaction mixture was
allowed to warm up to room temperature and was stirred for 18
hours. The reaction mixture was concentrated under vacuo and
purified on silica gel by flash column chromatography to give the
required compound (380 mg, 95% yield).
[0212] .sup.1H NMR (CDCl3, 400 MHz) .delta. 1.45 (9H, s), 1.64 (1H,
br d), 1.78-1.96 (4H, m), 1.96-2.11 (2H, m), 4.05 (1H, br s), 4.39
(1H, br s).
Step 2: (R)-tert-butyl 3-oxocyclopentylcarbamate
##STR00060##
[0214] Dess-Martin periodinane (961 mg, 2.27 mmol) was added
portionwise to a solution of tert-butyl
(1R,3S)-3-hydroxycyclopentylcarbamate (380 mg, 1.89 mmol) in
dichloromethane (10 mL) at 0.degree. C. After complete addition,
the reaction mixture was stirred for 1 hour at 0.degree. C., then,
allowed to warm up to room temperature and stirred for 18 hours.
The reaction mixture was quenched with a 50/50 of a saturated
aqueous solution of sodium bicarbonate and a saturated aqueous
solution of sodium thiosulfate. The aqueous layer was extracted 3
times with dichloromethane. The combined organic layers were washed
with brine, dried over magnesium sulfate and concentrated in vacuo.
The residue was purified on silica gel by flash column
chromatography to give the title compound (316 mg, 84% yield).
[0215] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.47 (9H, s),
1.01-1.13 (1H, m), 2.13 (1H, dd), 2.25 (1H, m), 2.31-2.44 (2H, m),
2.65 (1H, dd), 4.24 (1H, br s), 4.62 (1H, br s).
Step 3: (R)-tert-butyl 3,3-difluorocyclopentylcarbamate
##STR00061##
[0217] Deoxofluor.RTM. [bis(2-methoxyethyl)aminosulfur trifluoride,
0.574 ml, 3.12 mmol] was added dropwise to a solution of
(R)-tert-butyl 3-oxocyclopentylcarbamate (310 mg, 1.56 mmol) in
dichloromethane (8 mL) at 0.degree. C. After complete addition, the
reaction mixture was allowed to warm up to room temperature and
stirred for 18 hours. The reaction mixture was poured slowly into
an ice-cold saturated aqueous solution of sodium bicarbonate. The
aqueous layer was extracted 3 times with dichloromethane. The
combined organic layers were dried over magnesium sulfate and
concentrated under vacuo. The residue was purified on silica gel by
flash column chromatography to give the title compound (226 mg, 66%
yield).
[0218] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 1.46 (9H, s),
1.61-1.74 (1H, m), 1.90-2.32 (4H, m), 2.52 (1H, dq), 4.18 (1H, br
s), 4.65 (1H, br s).
Step 4: (R)-3,3-difluorocyclopentanamine hydrochloride
##STR00062##
[0220] Hydrochloric acid (4M in dioxane, 60 mL) was added to a
solution of (R)-tert-butyl 3,3-difluorocyclopentylcarbamate (4.61
g, 20.8 mmol) in dioxane (40 mL) at 0.degree. C. After complete
addition, the reaction mixture was allowed to warm to room
temperature and stirred for 2 hours. The solvent was removed in
vacuo and the residue was triturated with diethylether to afford
the title compound as an off-white solid (2.68 g, 82% yield).
[0221] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.76-1.88 (1H,
m), 2.10-2.40 (4H, m), 2.45-2.60 (1H, m), 3.68 (1H, quint), 8.33
(3H, s).
Step 5:
1,3,5-tris((R)-3,3-difluorocyclopentyl)-1,3,5-triazinane
##STR00063##
[0223] (R)-3,3-difluorocyclopentanamine hydrochloride (2.68 g, 17
mmol) in ethanol (17 mL) was cooled to 0.degree. C. Aqueous sodium
hydroxide (2 M, 8.5 ml, 17 mmol) was added followed by 37%
formaldehyde (1.38 ml, 17 mmol) were added dropwise. After complete
addition, the reaction mixture was stirred for 15 minutes at
0.degree. C. then allowed to warm to room temperature and stir for
a further hour. The title compound was isolated as an white solid
by filtration (1.75 g, 77% yield).
[0224] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.60-1.75 (3H,
m), 1.90-2.15 (9H, m), 2.18-2.43 (6H, m), 3.11 (3H, br s), 3.39
(6H, br s).
Step 6: (R)-methyl
3-(3,3-difluorocyclopentylamino)-2,2-dimethylpropanoate
##STR00064##
[0226] Triflic acid (58 .mu.l, 0.66 mmol) was added to a solution
of 1,3,5-tris((R)-3,3-difluorocyclopentyl)-1,3,5-triazinane (1.75
g, 4.38 mmol) and 1-methoxy-2-methyl-1-(trimethylsiloxy)propene
(2.29 g, 13.14 mmol) in dichloromethane (40 mL) at 0.degree. C.
After complete addition, the reaction mixture was allowed to warm
up to room temperature and stirred for 2 hours. The reaction
mixture was diluted with dichloromethane, washed with an aqueous
saturated solution of sodium bicarbonate and brine. The organic
layer was dried over magnesium sulfate and concentrated in vacuo.
The residue was purified on silica gel by flash column
chromatography to afford the title compound as oil (2.72 g, 88%
yield).
[0227] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.05 (6H, s),
1.30-1.56 (3H, m), 1.72 (1H, dq), 1.80-1.95 (1H, m), 2.00-2.28 (2H,
m), 2.49 (2H, dd), 3.09 (1H, quint), 3.54 (3H, s).
Step 7: (R)-methyl
3-((2-chloro-5-nitropyrimidin-4-yl)(3,3-difluorocyclopentyl)amino)-2,2-di-
methylpropanoate
##STR00065##
[0229] 2,4-Dichloro-5-nitropyrimidine (2.18 g, 11.25 mmol) was
added to a mixture of (R)-methyl
3-(3,3-difluorocyclopentylamino)-2,2-dimethylpropanoate (2.65 g,
11.25 mmol) and sodium bicarbonate (3.78 g, 44.98 mmol) in
dichloromethane (10 mL) and petroleum ether (40 mL). The reaction
mixture was stirred at room temperature for 18 hours. The reaction
mixture was diluted with dichloromethane and dried over magnesium
sulfate. The solids were filtered off, rinsing with more
dichloromethane. The mother liquors were adsorbed on silica gel and
concentrated in vacuo. The residue was purified by flash column
chromatography to afford the title compound as yellow solid (2.74
g, 62% yield).
[0230] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.23 (6H, s),
1.90-2.07 (2H, m), 2.11-2.40 (3H, m), 2.40-2.55 (1H, m), 3.70 (3H,
s), 3.72-3.84 (3H, m), 8.84 (1H, s); MS (ES+) 393.
Step 8:
(R)-2-chloro-9-(3,3-difluorocyclopentyl)-7,7-dimethyl-8,9-dihydro--
5H-pyrimido[4,5-b][1,4]diazepin-6(7H)-one
##STR00066##
[0232] A mixture of (R)-methyl
3-((2-chloro-5-nitropyrimidin-4-yl)(3,3-difluorocyclopentypamino)-2,2-dim-
ethylpropanoate (2.74 g, 6.98 mmol) and iron powder (0.799 g, 14.31
mmol) in glacial acetic acid (30 mL) was heated to 70.degree. C.
for 2 hours. The reaction mixture was filtered hot and the cake was
further washed with acetic acid. The mother liquors were
concentrated in vacuo. The residue was taken up in a 15% solution
of methanol in dichloromethane and filtered through a pad of silica
gel rinsing with more methanol-dichloromethane solution. The
filtrates were concentrated in vacuo. The residue was triturated
with methanol and the solid was filtered to afford the title
compound as an off-white solid (1.67 g, 72% yield).
[0233] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.30 (6H, s),
1.89 (1H, m), 2.03-2.23 (3H, m), 2.30-2.44 (1H, m), 2.48-2.64 (1H,
m), 3.36 (2H, s), 5.52 (1H, quint), 7.73 (1H, s), 7.86 (1H, s); MS
(ES+) 331, (ES-) 329.
Step 9:
(R)-2-chloro-9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-8,9-dihyd-
ro-5H-pyrimido[4,5-b][1,4]diazepin-6(7H)-one
##STR00067##
[0235] 60% Sodium hydride in mineral oil (0.210 g, 5.24 mmol) was
added to a mixture of
(R)-2-chloro-9-(3,3-difluorocyclopentyl)-7,7-dimethyl-8,9-dihydro-5H-pyri-
mido[4,5-b][1,4]diazepin-6(7H)-one (1.65 g, 4.99 mmol) and methyl
iodide (0.34 ml, 5.49 mmol) in dimethylacetamide (18 mL). The
reaction mixture was stirred at room temperature for 25 minutes.
Ice was added to the reaction mixture and the resultant precipitate
collected and rinsed with water. The solid was dried in a pistol in
vacuo for 3 hours. The title compound was obtained as a white solid
(1.68 g, 98% yield).
[0236] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 1.09 (6H, s),
1.87-2.22 (3H, m), 2.22-2.48 (3H, m), 3.19 (3H, s), 3.49 (2H, s),
5.26 (1H, quint), 8.12 (1H, s).
[0237] MS (ES+) 345.
Step 10:
(R)--N-cyclopropyl-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-
-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-met-
hoxybenzamide
##STR00068##
[0239] Concentrated hydrochloric acid (52 .mu.l) was added to a
mixture of
(R)-2-chloro-9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-8,9-dihydro-5H-p-
yrimido[4,5-b][1,4]diazepin-6(7H)-one (100 mg, 0.29 mmol) and
4-amino-N-cyclopropyl-3-methoxybenzamide (90 mg, 0.44 mmol) in
ethanol (1.4 mL) and water (5.2 mL). The reaction mixture was
heated to 85.degree. C. and stirred for 48 hours. The reaction
mixture was concentrated in vacuo. The residue was dissolved in
ethyl acetate, washed with an aqueous saturated solution of sodium
bicarbonate and brine. The organic layer was dried over magnesium
sulfate and concentrated in vacuo. The residue was purified on
silica gel by flash column chromatography to afford the title
compound as a white solid (83 mg, 56% yield).
Step 11: Mesylate salt formation
[0240]
(R)--N-cyclopropyl-4-(9-(3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-
-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-3-metho-
xybenzamide (83 mg, 0.16 mmol) was dissolved in hot (50.degree. C.)
methanol (4 mL) and treated with methane sulfonic acid (10.5 .mu.L,
0.16 mmol), the mixture was evaporated under reduced pressure and
azeotroped three times with diethyl ether. The residue was
triturated with ether and filtered to give the methane sulfonate
salt (69 mg, 71% yield).
[0241] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.54-0.61 (2H,
m), 0.68-0.75 (2H, m), 1.14 (6H, d), 1.98-2.18 (3H, m), 2.28-2.46
(3H, m), 2.31 (3H, s), 2.80-2.86 (1H, m), 3.18 (3H, s), 3.56 (2H,
s), 3.94 (3H, s), 5.34 (1H, dt), 7.49 (1H, d), 7.54 (1H, s), 8.03
(1H, d), 8.05 (1H, s), 8.41 (1H, d), 9.03 (1H, br s).
[0242] MS (ES+) 515, (ES-) 513.
[0243] Other compounds of formula I of this invention have been
prepared via a sequence similar to the one described in Example
1.
Example 2
4-(9-((R)-3,3-difluorocyclopentyl)-5,7,7-trimethyl-6-oxo-6,7,8,9-tetrahydr-
o-5H-pyrimido[4,5-b][1,4]diazepin-2-ylamino)-N-((1R,3R)-3-fluorocyclopenty-
l)-3-methoxybenzamide
##STR00069##
[0245] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 1.10 (6H, d),
1.55-2.40 (12H, m), 3.20 (3H, s), 3.45 (2H, s), 3.95 (3H, s), 4.45
(1H, sext), 5.20 (0.5H, s), 5.35 (0.5H, s), 5.40 (1H, quint), 7.45
(1H, d), 7.50 (1H, s), 7.80 (1H, s), 8.05 (1H, s), 8.25 (1H, d),
8.35 (1H, d).
[0246] MS (ES+) 561, (ES-) 559.
Example 3
N--((R)-3,3-difluorocyclopentyl)-4-(9-((R)-3,3-difluorocyclopentyl)-5,7,7--
trimethyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-ylami-
no)-3-methoxybenzamide
##STR00070##
[0248] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 1.10 (6H, d),
1.80-2.50 (12H, m), 3.20 (3H, s), 3.45 (2H, s), 3.95 (3H, s), 4.45
(1H, sextet), 5.40 (1H, quintet), 7.45 (1H, d), 7.50 (1H, s), 7.80
(1H, s), 8.05 (1H, s), 8.35 (1H, d), 8.45 (1H, d).
[0249] MS (ES+) 579, (ES-) 577.
Example 4
(R)--N-cyclopropyl-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'-oxo-5',6',-
8',9'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diazepine]-2'--
ylamino)-3-methoxybenzamide
##STR00071##
[0250] Step 1: methyl 1-formylcyclopropanecarboxylate
##STR00072##
[0252] Dimethyl cyclopropane-1,1-dicarboxylate (6.90 ml, 50 mmol)
was dissolved in dichloromethane (100 mL) and cooled to -78.degree.
C. DIBAL (1.0 M in DCM, 100 ml, 100 mmol) was added slowly over a
period of 30 minutes. The reaction mixture was stirred for 6.5
hours at -78.degree. C. then treated carefully with an aqueous
saturated solution of ammonium chloride (16 mL) followed by HCl
(1.0 M, 20 mL). The reaction was allowed to warm to room
temperature over the weekend. The solids were filtered off and
washed with DCM. The filtrate was washed with brine, dried over
magnesium sulfate and the solvent removed under reduced pressure
(200 mbar at rt). The crude product was purified on silica gel by
flash column chromatography to afford the desired compound as a 45%
w/w solution in EtOAc/DCM/ether as judged by .sup.1H NMR (3.295
g).
[0253] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.58-1.63 (2H,
m), 1.64-1.70 (2H, m), 3.81 (3H, s), 10.38 (1H, s).
Step 2: (R)-methyl
1-((3,3-difluorocyclopentylamino)methyl)cyclopropanecarboxylate
##STR00073##
[0255] Methyl 1-formylcyclopropanecarboxylate (45% w/w, 3.205 g,
11.26 mmol) and (R)-3,3-difluorocyclopentanamine hydrochloride
(1.774 g, 11.26 mmol) were dissolved in dichloromethane (20 mL) at
0.degree. C. under nitrogen. To this solution was added sodium
acetate (0.923 g, 11.26 mmol) followed by sodium
triacetoxyborohydride (3.46 g, 16.32 mmol). The reaction mixture
was allowed to warm up to room temperature overnight. The reaction
mixture was quenched with a saturated aqueous solution of sodium
bicarbonate and stirred at room temperature for a further 10
minutes. The aqueous layer was extracted 3 times with
dichloromethane. The combined organic layers were washed with
brine, dried over magnesium sulfate and concentrated under vacuo.
The residue was purified on silica gel by flash column
chromatography to give the required compound (2.631 g, quantitative
yield).
[0256] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.79-0.82 (2H,
m), 1.25-1.29 (2H, m), 1.55-1.61 (1H, m), 1.82-2.08 (4H, m),
2.12-2.39 (2H, m), 2.67 (2H, dd), 3.26 (1H, quint), 3.65 (3H,
s).
Step 3: (R)-methyl
1-(((2-chloro-5-nitropyrimidin-4-yl)(3,3-difluorocyclopentyl)amino)methyl-
)cyclopropane-carboxylate
##STR00074##
[0258] 2,4-Dichloro-5-nitropyrimidine (2.16 g, 11.15 mmol) was
added to a mixture of (R)-methyl
1-((3,3-difluorocyclopentylamino)methyl)cyclopropanecarboxylate
(2.6 g, 11.15 mmol) and sodium bicarbonate (3.75 g, 44.64 mmol) in
dichloromethane (15 mL) and petroleum ether (60 mL). The reaction
mixture was stirred at room temperature for 18 hours. The reaction
mixture was diluted with dichloromethane and dried over magnesium
sulfate. The solids were filtered off, rinsing with more
dichloromethane. The mother liquors were adsorbed on silica gel and
concentrated in vacuo. The residue was purified by flash column
chromatography to afford the desired product as a yellow solid
(3.974 g, 91% yield).
[0259] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.96-1.05 (2H,
m), 1.40-1.48 (2H, m), 2.06-2.25 (3H, m), 2.34-2.78 (3H, m), 3.53
(2H, dd), 3.59 (3H, s), 4.04 (1H, quint), 8.84 (1H, s).
[0260] MS (ES+) 391.
Step 4:
(R)-2'-chloro-9'-(3,3-difluorocyclopentyl)-8',9'-dihydrospiro[cycl-
opropane-1,7'-pyrimido[4,5-b][1,4]diazepin]-6'(5'H)-one
##STR00075##
[0262] A mixture of (R)-methyl
1-(((2-chloro-5-nitropyrimidin-4-yl)(3,3-difluorocyclopentyl)amino)methyl-
)cyclopropane-carboxylate (3.90 g, 9.98 mmol) and iron powder
(1.143 g, 20.46 mmol) in glacial acetic acid (50 mL) was heated to
70.degree. C. for 2 hours. The reaction mixture was filtered hot
and the cake was further washed with acetic acid. The mother
liquors were concentrated in vacuo. The residue was taken up in a
15% solution of methanol in dichloromethane and filtered through a
path of silica gel rinsing with more methanol-dichloromethane
solution. The mother liquors were concentrated in vacuo. The
residue was triturated with methanol and the solid was filtered to
afford the desired compound as a light pink solid (1.953 g, 60%
yield).
[0263] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.87-0.95 (2H,
m), 1.14-1.20 (2H, m), 1.75-1.86 (1H, m), 1.92-2.40 (5H, m), 3.48
(2H, dd), 5.01 (1H, quint), 7.80 (1H, s), 9.92 (1H, s).
[0264] MS (ES+) 329, (ES-) 327.
Step 5:
(R)-2'-chloro-9'-(3,3-difluorocyclopentyl)-5'-methyl-8',9'-dihydro-
spiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diazepin]-6'(5'H)-one
##STR00076##
[0266] 60% sodium hydride in mineral oil (0.25 g, 6.23 mmol) was
added to a mixture of
(R)-2'-chloro-9'-(3,3-difluorocyclopentyl)-8',9'-dihydrospiro[cyclopropan-
e-1,7'-pyrimido[4,5-b][1,4]diazepin]-6'(5'H)-one (1.95 g, 5.94
mmol) and methyl iodide (0.41 ml, 6.53 mmol) in dimethylacetamide
(20 mL). The reaction mixture was stirred at room temperature for
25 minutes. Ice was added to the reaction mixture. A solid crashed
out and was filtered off, rinsing with water. The solid was dried
in a pistol under vacuo for 3 hours. The compound was obtained as a
light pink solid (1.863 g, 92% yield).
[0267] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.70-0.73 (2H,
m), 0.90-0.96 (2H, m), 1.77-1.92 (1H, m), 2.00-2.45 (5H, m), 3.17
(3H, s), 3.59 (2H, dd), 4.89 (1H, quint), 8.12 (1H, s).
[0268] MS (ES+) 343.
Step 6:
(R)--N-cyclopropyl-4-(9'-(3,3-difluorocyclopentyl)-5'-methyl-6'-ox-
o-5',6',8',9'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][1,4]diazep-
ine]-2'-ylamino)-3-methoxybenzamide
##STR00077##
[0270] Concentrated hydrochloric acid (45 .mu.l) was added to a
mixture of
(R)-2'-chloro-9'-(3,3-difluorocyclopentyl)-5'-methyl-8',9'-dihydrospiro[c-
yclopropane-1,7'-pyrimido[4,5-b][1,4]diazepin]-6'(5'H)-one (100 mg,
0.29 mmol) and 4-amino-N-cyclopropyl-3-methoxybenzamide (90 mg,
0.44 mmol) in ethanol (1.4 mL) and water (5.2 mL). The reaction
mixture was heated to 85.degree. C. and stirred for 18 hours. The
reaction mixture was concentrated in vacuo. The residue was taken
up with ethyl acetate, washed with an aqueous saturated solution of
sodium bicarbonate and brine. The organic layer was dried over
magnesium sulfate and concentrated in vacuo. The residue was
purified on silica gel by flash column chromatography to afford the
desired compound as a white solid (49 mg, 33% yield).
[0271] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.53-0.60 (2H,
m), 0.67-0.75 (4H, m), 0.82-0.95 (2H, m), 1.84-1.91 (1H, m),
2.04-2.31 (4H, m), 2.39-2.51 (1H, m), 2.81 (1H, m), 3.17 (3H, s),
3.51 (2H, dd), 3.93 (3H, s), 5.00 (1H, quint), 7.44 (1H, d), 7.47
(1H, s), 7.80 (1H, s), 8.04 (1H, s), 8.29-8.34 (2H, m).
[0272] MS (ES+) 513, (ES-) 511.
Example 5
N--((R)-3,3-difluorocyclopentyl)-4-(9'-((R)-3,3-difluorocyclopentyl)-5'-me-
thyl-6'-oxo-5',6',8',9'-tetrahydrospiro[cyclopropane-1,7'-pyrimido[4,5-b][-
1,4]diazepine]-2'-ylamino)-3-methoxybenzamide
##STR00078##
[0274] This compound was analyzed as a free base.
[0275] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.65-0.73 (2H,
m), 0.82-0.96 (2H, m), 1.80-1.93 (2H, m), 2.04-2.30 (8H, m),
2.40-2.56 (2H, m), 3.17 (3H, s), 3.52 (2H, dd), 3.95 (3H, s), 4.43
(1H, dt), 5.01 (1H, quint), 7.47 (1H, d), 7.50 (1H, s), 7.82 (1H,
s), 8.05 (1H, s), 8.34 (1H, d), 8.41 (1H, d).
[0276] MS (ES+) 577, (ES-) 575.
Example 6
4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[1,2,4]-triazol-
o[4,3-f]pteridin-7-ylamino)-N-ethyl-3-methoxybenzamide
##STR00079##
[0277] Step 1: (S)-tert-butyl
2-(trifluoromethylsulfonyloxy)butanoate
##STR00080##
[0279] A solution of tert-butyl (s)-2-hydroxybutyrate (1.0 g, 6.242
mmol) in dichloromethane (30 mL), cooled down to 0.degree. C., was
treated dropwise with 2,6-lutidine (2.0 mL). The resultant solution
was then treated dropwise with triflic anhydride (3.346 g, 1.995
ml, 11.86 mmol) added over 2-3 minutes. The reaction mixture was
stirred at 0.degree. C. for 40 minutes, then poured onto a mixture
of brine (70 mL) and 1 M HCl (35 mL), extracted further with
dichloromethane, dried over magnesium sulfate and concentrated
under reduced pressure at room temperature to give a pale brown oil
(2.6 g). The crude mixture was redissolved in dichloromethane and
washed further with a 2:1 solution of saturated aqueous brine and 1
M HCl (2.times.20 mL) then brine, dried over magnesium sulfate and
concentrated at room temperature to give pale brown oil (1.772 g,
97% yield).
[0280] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.08 (3H, t),
1.53 (9H, s), 2.00-2.09 (2H, m), 4.97 (1H, dd).
Step 2: (R)-tert-butyl 2-((R)-3,3-difluorocyclopentyl
amino)butanoate
##STR00081##
[0282] (R)-3,3-difluorocyclopentanamine hydrochloride (1.888 g,
11.98 mmol) in water (4 mL) was basified with potassium carbonate
and extracted with dichloromethane (15 times) (total 50 mL). The
solution was dried over magnesium sulfate, filtered, and added onto
(5)-tert-butyl 2-(trifluoromethylsulfonyloxy)butanoate (1.75 g,
5.988 mmol). Dimethylsulfoxide (2 mL) was added and the mixture was
concentrated at room temperature under 280 mbar (down to 8 mL
volume). The resultant solution was transferred into a glass
pressure tube and heated at 60.degree. C. overnight. The resultant
mixture was diluted with ethyl acetate and washed with an aqueous
saturated solution of bicarbonate, dilute brine and dried over
magnesium sulfate. The residue was purified on silica gel by flash
column chromatography to afford the desired compound as colourless
oil (1.17 g, 74% yield).
[0283] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.94 (3H, t),
1.49 (9H, s), 1.54-1.67 (3H, m), 1.82-2.07 (3H, m), 2.21-2.40 (2H,
m), 3.00 (1H, t), 3.22 (1H, quint); MS (ES+) 264.
Step 3: (R)-methyl 2-((R)-3,3-difluorocyclopentylamino)
butanoate
##STR00082##
[0285] (R)-tert-butyl 2-((R)-3,3-difluorocyclopentyl
amino)butanoate (1.0 g, 3.798 mmol) was dissolved in methanol (70
mL) and cooled down to 0.degree. C. The resultant mixture was
saturated with HCl gas, then stirred at room temperature for 3
hours. The reaction mixture was warmed to 40.degree. C. for 90
minutes then concentrated under reduced pressure. The residue was
partitioned between DCM and an aqueous solution of NaHCO.sub.3. The
aqueous phase was extracted with DCM three times, dried over
magnesium sulfate and concentrated under reduced pressure to afford
the desired product as colourless oil (645 mg, 78% yield).
[0286] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.95 (3H, t),
1.57-1.74 (3H, m), 1.83-2.09 (3H, m), 2.19-2.40 (2H, m), 3.15 (1H,
t), 3.21 (1H, quint).
[0287] MS (ES+) 222.
Step 4: (R)-methyl
2-((2-chloro-5-nitropyrimidin-4-yl)((R)-3,3-difluorocyclopentyl)amino)but-
anoate
##STR00083##
[0289] 2,4-Dichloro-5-nitropyrimidine (603.3 mg, 3.110 mmol) was
added to a mixture of (R)-methyl
2-((R)-3,3-difluorocyclopentylamino) butanoate (688 mg, 3.110 mmol)
and sodium bicarbonate (1.045 g, 12.44 mmol) in dichloroethane (5
mL) and petroleum ether (12 mL) in a sealed tube. The reaction
mixture was heated at 60.degree. C. for 4 days. The reaction
mixture was diluted with dichloroethane and washed with an aqueous
solution of sodium bicarbonate and brine, dried over magnesium
sulfate and concentrated in vacuo. The residue was purified by
flash column chromatography to afford the desired product as yellow
solid (698 mg, 59% yield).
[0290] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 1.05 (3H, t),
1.98-2.04 (3H, m), 2.26-2.66 (5H, m), 3.71-3.76 (1H, m), 3.80 (3H,
s), 3.90 (1H, quint), 8.76 (1H, s).
[0291] MS (ES+) 379.
Step 5:
(R)-2-chloro-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-7,8-dihydropt-
eridin-6(5H)-one
##STR00084##
[0293] A mixture of (R)-methyl
2-((2-chloro-5-nitropyrimidin-4-yl)((R)-3,3-difluorocyclopentyl)amino)but-
anoate (677 mg, 1.787 mmol) and iron powder (179.7 mg, 3.217 mmol)
in glacial acetic acid (8 mL) was heated to 70.degree. C. for 1
hours. The reaction mixture was filtered hot and the cake was
further washed with acetic acid. The mother liquors were
concentrated in vacuo. The residue was taken up in a 15% solution
of methanol in dichloromethane and filtered through a path of
silica gel rinsing with more methanol-dichloromethane solution. The
mother liquors were concentrated in vacuo. The residue was
triturated with ethanol and the solid was filtered to afford the
desired compound as white solid (348 mg, 61% yield).
[0294] .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 0.76 (3H, t),
1.66-1.86 (2H, m), 2.03-2.24 (3H, m), 2.33-2.50 (2H, m), 2.67-2.82
(1H, m), 4.22-4.31 (2H, m), 7.61 (1H, s), 10.89 (1H, s).
[0295] MS (ES+) 317, (ES-) 315.
Step 6:
(R)-7-chloro-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[-
1,2,4]triazolo[4,3-f]pteridine
##STR00085##
[0297] Potassium tert-butoxide (1M in THF, 697.7 .mu.L, 0.6977
mmol) was added at -20.degree. C. to a solution of
(R)-2-chloro-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-7,8-dihydropteridin--
6(5H)-one (170 mg, 0.5367 mmol) in THF (3 mL). The reaction mixture
was warmed up to 0.degree. C. for 25 minutes after complete
addition. The reaction mixture was cooled down at -40.degree. C.
and diethylchlorophosphate (120.4 mg, 100.8 .mu.L, 0.6977 mmol) was
added. After complete addition, the reaction mixture was warmed up
to room temperature for 45 minutes. The resulting mixture was added
dropwise to 1 M hydrazine in THF (8.050 ml, 8.050 mmol) and the
reaction mixture was stirred at room temperature for 18 hours. The
reaction mixture was concentrated in vacuo, partitioned between DCM
and an aqueous solution saturated in NaHCO.sub.3. The organic phase
was dried (MgSO.sub.4) and concentrated in vacuo to afford a pale
coloured oil (200 mg). The crude oil was dissolved in
trimethylorthoformate (2.847 g, 2.935 mL, 26.83 mmol) and heated to
110.degree. C. for 1 hour. The reaction mixture was concentrated in
vacuo and purified on silica gel by flash column chromatography to
afford the compound as colourless solid (0.143 gr, 78% yield).
[0298] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 0.85 (3H, t),
1.83-1.95 (1H, m), 2.04-2.39 (4H, m), 2.48-2.83 (3H, m), 4.43 (1H,
quint), 5.19 (1H, dd), 8.33 (1H, s), 8.68 (1H, s).
[0299] MS (ES+) 341.
Step 7:
4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[1,2,4]-
-triazolo[4,3-f]pteridin-7-ylamino)-N-ethyl-3-methoxybenzamide
##STR00086##
[0301] Concentrated hydrochloric acid (36 .quadrature.l) was added
to a mixture of
(R)-7-chloro-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[1,2,4]t-
riazolo[4,3-f]pteridine (70 mg, 0.21 mmol) and
4-amino-N-ethyl-3-methoxybenzamide (59.84 mg, 0.31 mmol) in ethanol
(0.9 mL) and water (3.6 mL). The reaction mixture was heated to
95.degree. C. and stirred for 18 hours. The reaction mixture was
concentrated in vacuo. The residue was taken up with ethyl acetate,
washed with an aqueous saturated solution of sodium bicarbonate and
brine. The organic layer was dried over magnesium sulfate and
concentrated in vacuo. The residue was purified on silica gel by
flash column chromatography to afford the desired compound as a
white solid (82 mg, 80% yield).
[0302] The compound was analyzed as a mesylate salt.
[0303] .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 0.71 (3H, t), 1.11
(3H, t), 1.79-2.34 (6H, m), 2.35 (3H, s), 2.40-2.50 (1H, m),
2.64-2.82 (1H, m), 3.27-3.34 (2H, m), 3.91 (3H, s), 4.52 (1H,
quin), 5.35 (1H, t), 7.51 (1H, d), 7.56 (1H, s), 7.96 (1H, d), 8.44
(1H, t), 8.61 (1H, s), 8.89 (1H, br s), 9.30 (1H, s).
[0304] MS (ES+) 499, (ES-) 497.
Example 7
N-cyclopropyl-4-((R)-5-((R)-3,3-difluorocyclopentyl)-4-ethyl-4,5-dihydro-[-
1,2,4]triazolo[4,3-f]pteridin-7-ylamino)-3-methoxybenzamide
##STR00087##
[0306] This compound was analyzed a mesylate salt.
[0307] .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 0.55-0.60 (2H, m),
0.65-0.75 (5H, m), 1.76-2.30 (6H, m), 2.33 (3H, s), 2.44-2.51 (1H,
m), 2.65-2.80 (1H, m), 2.80-2.90 (1H, m), 3.90 (3H, s), 4.52 (1H,
quin), 5.34 (1H, q), 7.48 (1H, d), 7.50 (1H, s), 7.98 (1H, d), 8.40
(1H, d), 8.61 (1H, s), 8.77 (1H, br s), 9.30 (1H, s).
[0308] MS (ES+) 511, (ES-) 509.
Example 8
4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetr-
ahydropteridin-2-ylamino)-N-ethyl-3-methoxybenzamide
##STR00088##
[0309] Step 1:
(R)-2-chloro-8((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-7,8-dihydrop-
teridin-6(5H)-one
##STR00089##
[0311] 60% sodium hydride in mineral oil (22.41 mg, 0.56 mmol) was
added to a mixture of
(R)-2-chloro-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-7,8-dihydropteridin--
6(5H)-one (169 mg, 0.53 mmol) and methyl iodide (36.54 .mu.l, 0.59
mmol) in dimethylacetamide (1.7 mL). The reaction mixture was
stirred at room temperature for 1 hour. Ice was added to the
reaction mixture. A solid crashed out and was filtered off, rinsing
with water. The solid was dried in a pistol under vacuo at
70.degree. C. The compound was obtained as a white solid (165 mg,
94% yield).
[0312] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.69-0.76 (3H,
m), 1.65-1.87 (2H, m), 2.07-2.37 (3H, m), 2.38-2.82 (3H, m), 3.24
(3H, s), 4.31 (1H, m), 4.43 (1H, m), 7.92 (1H, s).
[0313] MS (ES+) 331.
Step 2:
4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-oxo-5,6,-
7,8-tetrahydropteridin-2-ylamino)-N-ethyl-3-methoxybenzamide
(I-8)
##STR00090##
[0315] Concentrated hydrochloric acid (42 .mu.l) was added to a
mixture of
(R)-2-chloro-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-7,8-dihydro-
pteridin-6(5H)-one (80 mg, 0.24 mmol) and
4-amino-N-ethyl-3-methoxybenzamide (70.47 mg, 0.36 mmol) in ethanol
(1.2 mL) and water (4.8 mL). The reaction mixture was heated to
95.degree. C. and stirred for 18 hours. The reaction mixture was
concentrated in vacuo. The residue was taken up with
dichloromethane, washed with an aqueous saturated solution of
sodium bicarbonate and brine. The organic layer was dried over
magnesium sulfate and concentrated in vacuo. The residue was
purified on silica gel by flash column chromatography to afford the
desired compound as a white solid (89 mg, 75% yield).
[0316] This compound was analyzed as a mesylate salt.
[0317] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.75 (3H, t),
1.13 (3H, t), 1.70-2.20 (6H, m), 2.30 (3H, s), 2.40-2.50 (2H, m),
3.23 (3H, s), 3.30 (2H, quint), 3.91 (3H, s), 4.39 (1H, m), 4.50
(1H, m), 7.51 (1H, d), 7.57 (1H, s), 7.76 (1H, d), 8.87 (1H, br s),
8.47 (1H, m), 9.11 (1H, br s).
[0318] MS (ES+) 489, (ES-) 487.
Example 9
N-cyclopropyl-4-((R)-8-((R)-3,3-difluorocyclopentyl)-7-ethyl-5-methyl-6-ox-
o-5,6,7,8-tetrahydropteridin-2-ylamino)-3-methoxybenzamide
##STR00091##
[0320] This compound was analyzed as a mesylate salt.
[0321] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta. 0.55-0.59 (2H,
m), 0.70-0.77 (5H, m), 1.75-2.48 (7H, m), 2.30 (3H, s), 2.50-2.70
(1H, m), 3.22 (3H, s), 3.90 (3H, d), 4.30-4.44 (1H, m), 4.53-4.57
(1H, m), 7.50 (1H, d), 7.57 (1H, s), 7.69-7.75 (2H, m), 8.47 (1H,
s), 9.57 (1H, br s).
[0322] MS (ES+) 501, (ES-) 499.
Example 10
PLK Assays
[0323] The compounds of the present invention are evaluated as
inhibitors of human PLK kinase using the following assays.
PLK1 Inhibition Assay:
[0324] Compounds were screened for their ability to inhibit PLK1
using a radioactive-phosphate incorporation assay. Assays were
carried out in a mixture of 25 mM HEPES (pH 7.5), 10 mM MgCl.sub.2,
0.1% BSA, and 2 mM DTT. Final substrate concentrations were 150
.mu.M (350 .mu.M for determining values of less than 1 nM)
[.gamma.-33P]ATP (115mCi 33P ATP/mmol ATP, Amersham Pharmacia
Biotech/Sigma Chemicals) and 300 .mu.M (450 .mu.M for determining
values of <1 nM) peptide (KKKISDELMDATFADQEAK) (SEQ ID NO:1).
Assays were carried out at 25.degree. C. in the presence of 4 nM (1
nM for determining values of less than 1 nM) PLK1. An assay stock
buffer solution was prepared containing all of the reagents listed
above, with the exception of ATP and the test compound of interest.
30 .mu.L of the stock solution was placed in a 96 well plate
followed by addition of 2 .mu.L of DMSO stock containing serial
dilutions of the test compound (typically starting from a final
concentration of 10 .mu.M with 2-fold serial dilutions) in
duplicate (final DMSO concentration 5%). The plate was
pre-incubated for 10 minutes at 25.degree. C. and the reaction
initiated by addition of 8 .mu.L [.gamma.-33P]ATP (final
concentration 150 .mu.M (350 .mu.M for determining values of less
than 1 nM))).
[0325] The reaction was stopped after 90 minutes (240 minutes for
determining values of less than 1 nM) by the addition of 100 .mu.L
0.14M phosphoric acid. A multiscreen phosphocellulose filter
96-well plate (Millipore, Cat. No. MAPHN0B50) was pretreated with
100 .mu.L 0.2M phosphoric acid prior to the addition of 125 .mu.L
of the stopped assay mixture. The plate was washed with 200 .mu.L
0.2M phosphoric acid for 4 times. After drying, 100 .mu.L Optiphase
`SuperMix` liquid scintillation cocktail (Perkin Elmer) was added
to the well prior to scintillation counting (1450 Microbeta Liquid
Scintillation Counter, Wallac).
[0326] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San
Diego Calif., USA).
[0327] In general, compounds of the invention are effective for the
inhibition of PLK1. The following compounds showed Ki less than 1
nM in the radioactive incorporation assay: I-2, I-3, I-6, I-7, I-8,
I-9. The following compounds showed Ki between 1 nM and 10 nM in
the radioactive incorporation assay: I-1, I-4, I-5.
PLK2 Inhibition Assay:
[0328] Compounds were screened for their ability to inhibit PLK2
using a radioactive-phosphate incorporation assay. Assays were
carried out in a mixture of 25 mM HEPES (pH 7.5), 10 mM MgCl.sub.2,
0.1% BSA, and 2 mM DTT. Final substrate concentrations were 200
.mu.M [.gamma.-33P]ATP (57 mCi 33P ATP/mmol ATP, Amersham Pharmacia
Biotech/Sigma Chemicals) and 300 .mu.M peptide
(KKKISDELMDATFADQEAK) (SEQ ID NO:1). Assays were carried out at
25.degree. C. in the presence of 25 nM PLK2. An assay stock buffer
solution was prepared containing all of the reagents listed above,
with the exception of ATP and the test compound of interest. 30
.mu.L of the stock solution was placed in a 96 well plate followed
by addition of 2 .mu.L of DMSO stock containing serial dilutions of
the test compound (typically starting from a final concentration of
1 .mu.M with 2-fold serial dilutions) in duplicate (final DMSO
concentration 5%). The plate was pre-incubated for 10 minutes at
25.degree. C. and the reaction initiated by addition of 8 .mu.L
[.gamma.-33P]ATP (final concentration 200 .mu.M).
[0329] The reaction was stopped after 90 minutes by the addition of
100 .mu.L 0.14M phosphoric acid. A multiscreen phosphocellulose
filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated
with 100 .mu.L 0.2M phosphoric acid prior to the addition of 125
.mu.L of the stopped assay mixture. The plate was washed with 200
.mu.L 0.2M phosphoric acid for 4 times. After drying, 100 .mu.L
Optiphase `SuperMix` liquid scintillation cocktail (Perkin Elmer)
was added to the well prior to scintillation counting (1450
Microbeta Liquid Scintillation Counter, Wallac).
[0330] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San
Diego Calif., USA).
PLK3 Inhibition Assay:
[0331] Compounds were screened for their ability to inhibit PLK3
using a radioactive-phosphate incorporation assay. Assays were
carried out in a mixture of 25 mM HEPES (pH 7.5), 10 mM MgCl.sub.2,
and 1 mM DTT. Final substrate concentrations were 75 .mu.M
[.gamma.-33P]ATP (60mCi 33P ATP/mmol ATP, Amersham Pharmacia
Biotech/Sigma Chemicals) and 10 .mu.M peptide (SAM68 protein
.DELTA.332-443). Assays were carried out at 25.degree. C. in the
presence of 5 nM PLK3 (S38-A340). An assay stock buffer solution
was prepared containing all of the reagents listed above, with the
exception of ATP and the test compound of interest. 30 .mu.L of the
stock solution was placed in a 96-well plate followed by addition
of 2 .mu.L of DMSO stock containing serial dilutions of the test
compound (typically starting from a final concentration of 10 .mu.M
with 2-fold serial dilutions) in duplicate (final DMSO
concentration 5%). The plate was pre-incubated for 10 minutes at
25.degree. C. and the reaction initiated by addition of 8 .mu.L
[.gamma.-33P]ATP (final concentration 75 .mu.M).
[0332] The reaction was stopped after 60 minutes by the addition of
100 .mu.L 0.14M phosphoric acid. A multiscreen phosphocellulose
filter 96-well plate (Millipore, Cat No. MAPHN0B50) was pretreated
with 100 .mu.L 0.2 M phosphoric acid prior to the addition of 125
.mu.L of the stopped assay mixture. The plate was washed with 200
.mu.L 0.2 M phosphoric acid for 4 times. After drying, 100 .mu.L
Optiphase `SuperMix` liquid scintillation cocktail (Perkin Elmer)
was added to the well prior to scintillation counting (1450
Microbeta Liquid Scintillation Counter, Wallac).
[0333] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San
Diego Calif., USA).
PLK4 Inhibition Assay:
[0334] Compounds were screened for their ability to inhibit PLK4
using a radioactive-phosphate incorporation assay. Assays were
carried out in a mixture of 8 mM MOPS (pH 7.5), 10 mM MgCl.sub.2,
0.1% BSA and 2 mM DTT. Final substrate concentrations were 15 .mu.M
[.gamma.-33P]ATP (227mCi 33P ATP/mmol ATP, Amersham Pharmacia
Biotech/Sigma Chemicals) and 300 .mu.M peptide (KKKMDATFADQ) (SEQ
ID NO: 2). Assays were carried out at 25.degree. C. in the presence
of 25 nM PLK4. An assay stock buffer solution was prepared
containing all of the reagents listed above, with the exception of
ATP and the test compound of interest. 30 .mu.L of the stock
solution was placed in a 96-well plate followed by addition of 2
.mu.L of DMSO stock containing serial dilutions of the test
compound (typically starting from a final concentration of 10 .mu.M
with 2-fold serial dilutions) in duplicate (final DMSO
concentration 5%). The plate was pre-incubated for 10 minutes at
25.degree. C. and the reaction initiated by addition of 8 .mu.L
[.gamma.-33P]ATP (final concentration 15 .mu.M).
[0335] The reaction was stopped after 180 minutes by the addition
of 100 .mu.L 0.14M phosphoric acid. A multiscreen phosphocellulose
filter 96-well plate (Millipore, Cat No. MAPHN0B50) was pretreated
with 100 .mu.L 0.2M phosphoric acid prior to the addition of 1254
of the stopped assay mixture. The plate was washed with 200 .mu.L
0.2M phosphoric acid for 4 times. After drying, 100 .mu.L Optiphase
`SuperMix` liquid scintillation cocktail (Perkin Elmer) was added
to the well prior to scintillation counting (1450 Microbeta Liquid
Scintillation Counter, Wallac).
[0336] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San
Diego Calif., USA).
[0337] While we have described a number of embodiments of this
invention, it is apparent that our basic examples may be altered to
provide other embodiments that utilize or encompass the compounds,
methods, and processes of this invention. Therefore, it will be
appreciated that the scope of this invention is to be defined by
the appended claims.
Sequence CWU 1
1
2119PRTArtificial SequenceSynthetically generated peptide 1Lys Lys
Lys Ile Ser Asp Glu Leu Met Asp Ala Thr Phe Ala Asp Gln 1 5 10 15
Glu Ala Lys 211PRTArtificial SequenceSynthetically generated
peptide 2Lys Lys Lys Met Asp Ala Thr Phe Ala Asp Gln 1 5 10
* * * * *