Note: Descriptions are shown in the official language in which they were submitted.
CA 02761074 2011-11-04
Attorney Docket Number: VPU09-I 16 WO
Inventors: Juan-Miguel Jimenez, and Andrew Miller
4 SUBSTITUTED PYRAZOLOPIRIMIDINES USEFUL AS PKC-THETA
INHIBITORS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C. 119 to United
States
Provisional Application No. 61/175,908, filed May 6, 2009 and entitled "4
SUBSTITUTED
PYRAZOLOPYRIMIDINES", the entire contents of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 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, CA: 1995).
[0003] In general, protein kinases mediate intracellular signaling by
affecting 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 H202),
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.
[0004] 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] A serine/threonine kinase, protein kinase C-theta (PKC-theta), is a
member of the novel,
calcium independent PKC subfamily that is selectively expressed in T cells and
skeletal muscle.
Several lines of evidence indicate that PKC-theta has an essential role in T
cell activation. Upon
antigen stimulation of T cells, PKC-theta, but not other PKC isoforms, rapidly
translocates from the
cytoplasm to the site of cell contact between the T cell and antigen-
presenting cell (APC), where it
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localizes with the T cell receptor (TCR) in a region termed the central
supramolecular activation
cluster (cSMAC) (Monks et al., 1997, Nature, 385: 83-86; Monks et al., 1998,
Nature, 395: 82-86).
[0006] It has been reported that PKC-theta selectively activates the
transcription factors AP-1
and NF-KB and integrates TCR and CD28 co-stimulatory signals leading to the
activation of the
CD28 response element (CD28RE) in the IL-2 promotor (Baier-Bitterlich et al.,
1996, Mol. Cell.
Biol., 16: 1842-1850; Coudronniere et al., 2000, PNAS, 97: 3394-3399). The
specific role for PKC-
theta in CD3/CD28 co-stimulation of T cells is highlighted in a study where
expression of a kinase-
dead PKC-theta mutant, or anti-sense PKC-theta dose-dependently inhibited
CD3/CD28 co-
stimulated NF-KB activation, but not TNF-alpha-stimulated NF-KB activation.
This was not seen
with other PKC isoforms (Lin et al., 2000, Mol. Cell. Biol., 20: 2933-2940).
Recruitment of PKC-
theta to the SMAC is reported to be mediated by its N-terminal regulatory
domain and is necessary
for T cell activation, as an over-expressed PKC-theta catalytic fragment did
not translocate and was
unable to activate NF-KB, whereas a PKC-theta catalytic domain-Lck membrane-
binding domain
chimera was able to reconstitute signaling (Bi et al., 2001, Nat. Immunol.,
2:556-563).
[0007] Translocation of PKC-theta to the SMAC appears to be mediated by a
largely PLC-
gamma/DAG-independent mechanism, involving Vav and PI3-kinase (Villalba et
al., 2002, JCB
157: 253-263), whilst activation of PKC-theta requires input from several
signaling components
including Lck, ZAP-70, SLP-76, PLC-gamma, Vav and P13-kinase (Liu et al.,
2000, JBC, 275:
3606-3609; Herndon et al., 2001, J. Immunol., 166: 5654-5664; Dienz et al.,
2002, J. Immunol., 169:
365-372; Bauer et al., 2001 JBC., 276: 31627-31634). These biochemical studies
in human T cells
have gained credence from studies in PKC-theta knockout mice, which have
confirmed a crucial role
for this enzyme in T cell function. PKC-theta-/- mice are healthy and fertile,
have a normally
developed immune system, but exhibit profound defects in mature T cell
activation (Sun et al., 200,
Nature, 404:402-407). Proliferative responses to TCR and TCR/CD28 co-
stimulation were inhibited
(>90%) as were in vivo responses to antigen. In agreement with studies on
human T cells, activation
of the transcription factors AP-1 and NF-KB was abrogated, resulting in a
severe deficit in IL-2
production and IL-2 R upregulation (Baier-Bitterlich et al., 1996, MBC, 16,
1842; Lin et al., 2000,
MCB, 20, 2933; Courdonniere, 2000, 97, 3394). More recently, studies in PKC-
theta-deficient mice
have indicated a role for PKC-theta in the development of mouse models of
autoimmune diseases,
including multiple sclerosis (MS), rheumatoid arthritis (RA) and irritable
bowel disease (IBD)
(Salek-Ardakani et al., 2006; Tan et al., 2006; Healy et al., 2006; Anderson
et at., 2006). In these
models, PKC-theta-deficient mice exhibited a marked reduction in disease
severity that was
associated with a profound defect in the development and effector function of
autoreactive T cells.
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[0008] In addition to its role in T cell activation, PKC-theta is reported to
mediate the phorbol
ester-triggered survival signal that protects T cells from Fas- and UV-induced
apoptosis (Villalba et
al., 2001, J. Immunol. 166: 5955-5963; Berttolotto et al., 2000, 275: 37246-
37250). This pro-
survival role is of interest because the human PKC-theta gene has been mapped
to chromosome 10
(IOp15), a region associated with mutations leading to T cell leukaemias and
lymphomas (Erdel et
al., 1995, Genomics 25: 295-297; Verma et al., 1987, J. Cancer Res. Clin.
Oncol., 113: 192-196).
[0009] In vivo, the role for PKC-theta in immune responses to infection is
dependent on the type
of pathogen encountered. PKC-theta deficient mice elicit normal Thl and
cytotoxic T cell-mediated
responses to several viral infections and the protozoan parasite, Leishmania
major and effectively
clear these infections (Marsland et al., 2004; Berg-Brown et al., 2004;
Marsland et al., 2005;
Giannoni et al., 2005). However, PKC-theta deficient mice are unable to wage
normal Th2 T cell
responses against the parasite Nippostrongylus brasiliensis and certain
allergens (Marsland et al.,
2004; Salek-Ardakani et al., 2004) and are unable to clear Listeria
monocytogenes infection
(Sakowicz-Burkiewicz et al., 2008). Clearly in some circumstances, the
requirement for PKC-theta
in T cell activation can be bypassed and this is likely to involve the
provision of additional signals to
T cells, either from cells of the innate immune system, or directly from the
pathogen in the form of
pathogen associated molecular patterns (PAMPs) (Marsland et al., 2007).
[0010] More recently, studies in PKC-theta-deficient mice have indicated a
role for PKC-theta in
the development of mouse models of autoimmune diseases, including multiple
sclerosis, rheumatoid
arthritis and inflammatory bowel disease. In all cases where examined, PKC-
theta-deficient mice
exhibited a marked reduction in disease severity that was associated with a
profound defect in the
development of a newly discovered class of T cells, Th17 cells (Salek-Ardakani
et al., 2006; Tan et
al., 2006; Healy et al., 2006; Anderson et al., 2006; Nagahama et al., 2008).
PKC-theta therefore
appears to be essential for the development of pathogenic auto-reactive Th 17
cells in the context of
autoimmunity. These observations support the notion that targeting PKC-theta
will provide a way to
target autoimmune T cell responses, leaving many T cell responses (e.g., to
viral infections) intact.
[0011] In addition to its role in T cell activation, PKC-theta mediates the
phorbol ester-triggered
survival signal that protects T cells from Fas- and UV-induced apoptosis
(Villalba et al., 2001, J.
Immunol. 166: 5955-5963; Berttolotto et al., 2000, 275: 37246-37250). This pro-
survival role is of
interest because the human PKC-theta gene has been mapped to chromosome 10
(1Op15), a region
associated with mutations leading to T cell leukaemias and lymphomas (Erdel et
al., 1995, Genomics
25: 295-297; Verma et al., 1987, J. Cancer Res. Clin. Oncol., 113: 192-196).
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[0012 ] Together, these data indicate that PKC-theta is an attractive target
for therapeutic
intervention in inflammatory disorders, immune disorders, lymphomas and T cell
leukaemias.
[0013] Accordingly, there is a great need to develop compounds useful as
inhibitors of protein
kinases. In particular, it would be desirable to develop compounds that are
useful as inhibitors of
kinases such as PKC-theta, particularly given the inadequate treatments
currently available for the
majority of the disorders implicated in their activation.
SUMMARY OF THE INVENTION
[0014 ] This invention provides, in general, compounds that are useful as
kinase inhibitors.
In one embodiment the compounds of the present invention are represented by a
structural formula
a I or IA:
H N
H N
N I N
N CA N R5 R5
AA A'
K R2
R, )x `'1)x B
R3 R
R4 or y
IA
or a pharmaceutically acceptable salt thereof wherein
A and A' are independently -N- or -C(R+)-.
Ring B is five- or six-membered saturated carbocyclic or heterocyclic ring.
R1 is halogen, -CN, -NO2, or -T1-Q1.
TI is absent or a C1-10 aliphatic wherein one or more methylene units of TI
are optionally
and independently replaced by G wherein G is -0-, -S(O)p-, -N(R')-, or -C(O)-;
and TI is
optionally and independently substituted with one or more JTI.
Q1 is absent or a 3-8 membered saturated, partially saturated, or fully
unsaturated
monocyclic ring having 0-3 heteroatoms independently selected from the groups
consisting of 0,
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N, and S, or an 8-12 membered saturated, partially saturated, or fully
unsaturated bicyclic ring
having 0-5 heteroatoms independently selected from the group consisting of 0,
N, and S, wherein
Q1 is optionally and independently substituted with one or more JQI; wherein
when R, is TI-Ql,
then TI and Q I are not both absent.
R2 is -H, -(CR++2),,CN, -(CR++2)nC(O)N(R*)2, -(CR++2)n OR*, -(CR++2)n N(R*)2,
-(CR++2)õN(R*)C(O)R*, or C1-10 aliphatic optionally substituted with one or
more halogen, or
phenyl.
Each R3 and R4 independently are -H, halogen, C1-10 aliphatic, heterocyclyl,
heterocyclylalkyl, aryl, or aralkyl, wherein R3 and R4 are optionally and
independently substituted
with one or more selected from the group consisting of C1-10 alkyl, halogen, -
CN, -NO2, -N(R*)2,
-S(O)pR*, -S(O)pNR*, -C(O)N(R*)2, -NR*C(O), -OC(O)N(R*)2, -N(R*)C(O)OR*,
-N(R*)C(O)N(R*)2 and -OR*; or R3 and R4 taken together with the carbon to
which they are
attached form C=O, or a 3-8 membered saturated, partially saturated, or fully
unsaturated
monocyclic ring having 0-3 heteroatoms independently selected from the groups
consisting of 0,
N, and S, wherein the ring is optionally and independently substituted with
one or more selected
from the group consisting of =O, =S, =N-R*, C1-10 aliphatic, C1-10
haloaliphatic, halogen, -CN,
-N02, -N(R*)2, -S(O)pR*, -S(O)pNR*, -C(O)N(R*)2, -NR*C(O), -OC(O)N(R*)2, -
N(R*)C(O)OR*,
-N(R*)C(O)N(R*)2 and -OR*.
Each R5 is independently -H, halogen, C1-10 haloaliphatic, or C1-10 aliphatic.
Each R7 is independently C1-10 haloaliphatic, CI-10 aliphatic, halogen, -NO2,
-(CR++2)nCN, -(CR++2)õN(R**)2, -(CR++2)nOR**, or -(CR++2)nC(O)N(R**)2, or two
R7 groups
together with the carbon to which they are attached form C=O.
Each JTI is independently halogen, -ORA, -N(R^)2, or -CN.
Each JQI is independently halogen, C l -10 alkyl, C 1-10 haloalkyl, -OR", -
N(R")2, -CN, -
NO2, -S(O)pR -S(O)pNR", -C(O) N(R")2, -N(R")C(O)R", acyl, carbalkoxyalkyl, or
acetoxyalkyl.
Each R+ is independently -H, halogen, or C l -10 alkyl optionally and
independently
substituted with up to five halogen groups.
Each R++ is independently -H or halogen.
Each R' is independently -H or C1-10 alkyl optionally and independently
substituted with
up to five halogen groups.
Each R^ is independently -H, C1-10 alkyl, or aralkyl wherein each R^ is
optionally and
independently substituted with up to five halogen groups.
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Each R" is independently -H or C1-10 alkyl optionally and independently
substituted with
up to five halogen groups.
Each R* is independently -H or C-10 alkyl or aralkyl optionally and
independently
substituted with up to five halogen groups.
Each R** is independently -H or C1-10 alkyl optionally and independently
substituted with
up to five halogen groups.
xis0or1.
yis0, 1 or2.
Each n is independently 0, or 1-10.
Each p is independently 0, 1, or 2.
[0015 ] In one embodiment, the present invention is a method of treating or
preventing protein
kinase-mediated condition in a subject, comprising administering to the
subject an effective amount
of a compound or composition of the present invention.
[0016] In one embodiment the present invention is the manufacture of a
compound or
composition of the present invention for use in treating or preventing a
protein kinase-mediated
condition in a subject.
[0017 ] In another embodiment, the compounds and compositions of the present
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.
DETAILED DESCRIPTION OF THE INVENTION
[0018] This invention relates to compounds and compositions (such as,
pharmaceutical
compositions) useful as protein kinase inhibitors.
[0019] In one embodiment, the compounds and compositions of the present
invention are
effective as inhibitors of PKCtheta.
[0020] Compounds of this invention include those described generally 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`h Ed. Additionally, general principles
of organic chemistry
are described in "Organic Chemistry", Thomas Sorrell, University Science
Books, Sausalito: 1999,
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and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March,
J., John Wiley
& Sons, New York: 2001, the entire contents of which are hereby incorporated
by reference.
[0021] In one embodiment the compounds of the present invention are
represented by a
structural formula I and IA as described above.
[0022] In a first embodiment, the compounds of the present invention are
represented by
Formula I. In an alternative embodiment the compounds of the present invention
are represented by
Formula IA.
[0023] Ina second embodiment, for compounds of Formula I and IA, A is -N- or -
C(R+)-; and
A' is -C(R+)- and the remainder of the variables are as described above.
[0024] Ina third embodiment, for compounds of Formula I and IA R+ is -H, and
the remainder
of the variables are as described above for the second embodiment.
[0025] In a fourth embodiment for compounds of Formula I and IA R1 is halogen,
or -TI-QI and
the remainder of the variables are as described above for the third
embodiment.
[0026] In a fifth embodiment for compounds of Formula I and IA TI is absent or
a C1-10
aliphatic wherein up to three methylene units of T1 are optionally and
independently replaced by G
wherein G is -0-, -N(R')-, or -C(O)-; and TI is optionally and independently
substituted with one or
more JTI. QI is absent or a 3-8 membered saturated, partially saturated, or
fully unsaturated
monocyclic ring having 0-3 heteroatoms independently selected from the groups
consisting of 0, N,
and S, wherein Q1 is optionally and independently substituted with one or more
JQi and the
remainder of the variables are as described above for the fourth embodiment.
[0027 ] In a sixth embodiment for compounds of Formula I and IA each JTI is
independently -
OR^, -N(R)2, or -CN. Each JQI is independently C 1-10 alkyl, -OR", -N(R")2, or
acyl and the
remainder of the variables are as described above for the fifth embodiment.
[0028) Ina seventh embodiment for compounds of Formula I R2 is -H, -
(CR++2)nCN,
-(CR++2),,C(O)N(R*)2, -(CR++2)õ OR*, -(CR++2)nN(R*)2, or C1-3 aliphatic
optionally substituted with
one or more halogen. Each R3 and R4 independently is -H, C1-10 aliphatic,
cycloalkylalkyl,
heterocyclyl, heterocyclylalkyl, aryl, or aralkyl wherein R3 and R4 are
optionally and independently
substituted with one or more selected from the group consisting of halogen, -
CN, -NO2, -N(R*)2, and
-OR*; or R3 and R4 taken together with the carbon to which they are attached
form C=O, or a 3-8
membered saturated, partially saturated, or fully unsaturated monocyclic ring
having 0-3
heteroatoms independently selected from the groups consisting of 0, N, and S,
wherein the ring is
optionally and independently substituted with one or more selected from the
group consisting of =O,
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=S, C1-10 aliphatic, C1-10 haloaliphatic, halogen, -CN, -N(R*)2, and -OR* and
the remainder of the
variables are as described above for the sixth embodiment.
[0029] In an eighth embodiment for compounds of Formula I each R3 and R4
independently is
-H, C1-10 aliphatic, cycloalkylalkyl, wherein R3 and R4 are optionally and
independently substituted
with one or more selected from the group consisting of halogen, -CN, -NO2, -
N(R*)2, and -OR*; or
[00301 R3 and R4 taken together with the carbon to which they are attached
form C=O, or a 3-8
membered saturated, partially saturated, or fully unsaturated monocyclic ring
having 0-3
heteroatoms independently selected from the groups consisting of 0, N, and S,
wherein the ring is
optionally and independently substituted with one or more selected from the
group consisting of Cl-
aliphatic, C 1-10 haloaliphatic, halogen, -CN, -N(R*)2, and -OR* and the
remainder of the
variables are as described above for the seventh embodiment.
[0031] Ina ninth embodiment for compounds of Formula I and IA A is -C(R+)-.
and the
remainder of the variables are as described above for the eighth embodiment.
[0032] Ina tenth embodiment for compounds of Formula I and IA JTI is -ORS. and
the remainder
of the variables are as described above for the ninth embodiment.
[0033] In an eleventh embodiment for compounds of Formula I R2 is -H, -
(CR++2)nCN, -
(CR++2)nOR*, -(CR++2)nN(R*)2, or C1-3 aliphatic optionally substituted with
one or more halogen
and the remainder of the variables are as described above for the tenth
embodiment.
[00341 Ina twelfth embodiment the compounds of the present invention are
represented by
structural formula IC:
H
H N
N\
N N
R5
R2
R,
X
R4 R3
IC
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[0035] or a pharmaceutically acceptable salt thereof and the remainder of the
variables are as
described above for the eleventh embodiment.
[0036] Ina thirteenth embodiment for compounds of Formula I and IC R2 is -H, -
(CR++2)nCN, -
(CR++2)nOR*, -(CR++2)nN(R*)2, or CI-3 aliphatic optionally substituted with
one or more halogen.
R3 and R4 taken together with the carbon to which they are attached form a 3-8
membered saturated,
or partially saturated monocyclic ring having 0-3 heteroatoms independently
selected from the
groups consisting of 0, N, and S, wherein the ring is optionally and
independently substituted with
one or more selected from the group consisting of =O, =S, CI-10 aliphatic, CI-
10 haloaliphatic,
halogen, -CN, -N(R*)2, and -OR*. and the remainder of the variables are as
described above for the
twelfth embodiment.
[0037] Ina fourteenth embodiment for compounds of Formula I and IC R2 is -H, -
(CR++2)nCN, -
(CR++2)nOR*, -(CR++2)nN(R*)2, or CI-3 aliphatic optionally substituted with
one or more halogen.
R3 and R4 taken together with the carbon to which they are attached form a
monocyclic ring selected
from the group consisting of cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl,
azetidinyl,
pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl,
tetrahydrofuranyl, tetrahydropyranyl,
oxetanyl, imidazolinyl, thiazolidinyl, or oxazolidinyl, wherein the ring is
optionally and
independently substituted with one or more selected from the group consisting
of =O, =S, C1-10
aliphatic, C1-10 haloaliphatic, halogen, -CN, -N(R*)2, and -OR* and the
remainder of the variables
are as described above for the thirteenth embodiment.
[0038] Ina fifteenth embodiment for compounds of Formula I and IC
[0039] R2 is -H, -(CR++2)nCN, -(CR++2)nOR*, -(CR++2)nN(R*)2, or C1-3 aliphatic
optionally
substituted with one or more halogen. R3 and R4 taken together with the carbon
to which they are
attached form a monocyclic ring selected from the group consisting of
azetidinyl, pyrrolidinyl,
piperidinyl, piperazinyl, azepanyl, diazepanyl, tetrahydrofuranyl,
tetrahydropyranyl, oxetanyl,
imidazolinyl, thiazolidinyl, or oxazolidinyl, wherein the ring is optionally
and independently
substituted with one or more selected from the group consisting of =O, =S, C1-
10 aliphatic, C1-10
haloaliphatic, halogen, -CN, -N(R*)2, and -OR* and the remainder of the
variables are as described
above for the fourteenth embodiment.
[0040] Ina sixteenth embodiment for compounds of Formula I and IC R2 is -H, -
(CR++2)nCN, -
(CR++2)nOR*, -(CR++2)nN(R*)2, or C1-3 aliphatic optionally substituted with
one or more halogen.
R3 and R4 taken together with the carbon to which they are attached form a
monocyclic ring selected
from the group consisting of cyclopropyl, cyclobutyl, cyclohexyl, or
cyclopentyl, wherein the ring is
optionally and independently substituted with one or more selected from the
group consisting of =O,
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=S, C 1-10 aliphatic, C 1-10 haloaliphatic, halogen, -CN, -N(R*)2, and -OR*
and the remainder of the
variables are as described above for the fourteenth embodiment.
[0041] Ina seventeenth embodiment for compounds of Formula I and IC
[0042] R2 is-H, -(CR++2)nCN, -(CR++2)nOR*, -(CR++2)nN(R*)2, or CI-3 aliphatic
optionally
substituted with one or more halogen. Each R3 and R4 independently is -H, C 1-
10 aliphatic,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, or aralkyl wherein R3
and R4 are optionally
and independently substituted with one or more selected from the group
consisting of halogen, -CN,
-NO2, -N(R*)2, and -OR* and the remainder of the variables are as described
above for the twelfth
embodiment.
[0043] In an eighteenth embodiment for compounds of Formula I, IA and IC R5 is-
H, Cl, CI-4
haloalkyl, or C 1-4 alkyl and the remainder of the variables are as described
above for the fifteenth,
sixteenth or seventeenth embodiments.
[0044] Ina nineteenth embodiment for compounds of Formula I, IA and IC R5 is -
H, Cl,
trifluoromethyl, methyl, ethyl, or cyclopropyl and the remainder of the
variables are as described
above for the eighteenth embodiment.
[0045] Ina twentieth embodiment for compounds of Formula I, IA and IC R5 is
trifluoromethyl
and the remainder of the variables are as described above for the nineteenth
embodiment.
[0046] In a twenty first embodiment for compounds represented by structural
formula IA, ring B
is five- or six-membered saturated carbocyclic ring and the remainder of the
variables are as
described above for the sixth embodiment.
[0047] In a twenty second embodiment for compounds represented by structural
formula IA,
each R7 is independently CI-10 aliphatic, C1-10 haloaliphatic, halogen, -CN, -
N(R**)2, or -OR**; or
two R7 groups together with the carbon to which they are attached form C=O and
the remainder of
the variables are as described above for the twenty fisrt embodiment.
[0048] Ina twenty thirdh embodiment for compounds represented by structural
formula IA,
[0049] A is -C(R+)-. JTI is -ORA. Each JQI is independently CI-10 alkyl, -OR",
-N(R")2, or
acyl. Ring B is five-membered saturated carbocyclic ring and the remainder of
the variables are as
described above for the twenty second embodiment.
[0050] As used herein "one or more" means, for example, that all substitutable
carbon atoms can
be substituted, for example, up to 6 carbons atoms, up to 5 carbon atoms, up
to 3 carbon atoms, up to
2 carbon atoms, or one carbon atom can be substituted.
[00511 As described herein, a specified number range of atoms includes any
integer therein. For
example, a group having from 1-4 atoms could have 1, 2, 3, or 4 atoms.
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[0052] As used here the terms "absent" and "a bond" can be used
interchangeably to mean the
variable does not exits in that embodiment, that is the variable does not
represent an atom or groups
of atoms.
[0053] 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, storage, 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 C or less, in the absence of moisture or other chemically reactive
conditions, for at least a
week.
[0054] The term "aliphatic" or "aliphatic group", as used herein, means a
straight-chain (i.e.,
unbranched), branched, or cyclic hydrocarbon chain that is completely
saturated or that contains one
or more units of unsaturation but is non-aromatic. 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. In certain
embodiments, aliphatic
groups may be linear or branched. Unless indicated aliphatic groups include,
but are not limited to,
alkyl, alkenyl, or alkynyl groups. Specific examples include, but are not
limited to, methyl, ethyl,
isopropyl, n-propyl, sec-butyl, vinyl, methenyl (=CH2), ethenyl, n-butenyl,
ethynyl, and tert-butyl.
Specifically examples, include but are not limited to, for example, a C1-10
aliphatic includes n-
butylene substituted with cyclohexyl.
[0055] The term "alkyl" as used herein means a saturated straight, branched or
cyclic
hydrocarbon. The term "alkenyl" as used herein means a straight or branched
chain hydrocarbon
comprising one or more double bonds. The term "alkynyl" as used herein means a
straight or
branched chain hydrocarbon comprising one or more triple bonds. Unless
otherwise specified, alkyl,
alkenyl and alkynyl groups contain 1-20 carbon atoms. In some embodiments,
alkyl, alkenyl and
alkynyl groups contain 1-10 carbon atoms. In other embodiments, alkyl, alkenyl
and alkynyl groups
contain 1-8 carbon atoms. In still other embodiments, alkyl, alkenyl and
alkynyl groups contain 1-6
carbon atoms, and in yet other embodiments alkyl, alkenyl and alkynyl groups
contain 1-4 carbon
atoms.
[0056] The term "cycloaliphatic" (or "carbocycle" or "carbocyclyl" or
"carbocyclic") refers to a
non-aromatic monocyclic or polycyclic carbon containing ring which can be
saturated or contain one
or more units of unsaturation, having three to fourteen ring carbon atoms. The
term includes
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polycyclic fused, Spiro or bridged carbocyclic ring systems wherein the
radical or point of
attachment is on the carbocyclic ring. The term also includes polycyclic ring
systems in which the
carbocyclic ring can be attached to one or more non-aromatic carbocyclic or
heterocyclic rings or
one or more aromatic rings or combination thereof, wherein the radical or
point of attachment is on
the carbocyclic ring. Fused bicyclic ring systems comprise two rings which
share two adjoining ring
atoms, bridged bicyclic group comprise two rings which share three or four
adjacent ring atoms,
spiro bicyclic ring systems share one ring atom. Examples of cycloaliphatic
groups include, but are
not limited to, cycloalkyl and cycloalkenyl groups. Specific examples include,
but are not limited to,
cyclohexyl, cyclopropentyl, and cyclobutyl.
[0057] The term "heterocycle" (or "heterocyclyl", or "heterocyclic") as used
herein means refers
to a non-aromatic monocyclic or polycyclic ring which can be saturated or
contain one or more units
of unsaturation, having three to fourteen ring atoms in which one or more ring
carbons is replaced by
a heteroatom such as, N, S, or 0. The term includes polycyclic fused, Spiro or
bridged heterocyclic
ring systems wherein the radical or point of attachment is on the heterocyclic
ring. The term also
includes polycyclic ring systems in which the heterocyclic ring can be
attached to one or more non-
aromatic carbocyclic or heterocyclic rings or one or more aromatic rings or
combination thereof,
wherein the radical or point of attachment is on the heterocyclic ring.
Examples of heterocycles
include, but are not limited to, piperidinyl, piperizinyl, pyrrolidinyl,
pyrazolidinyl, imidazolidinyl,
azepanyl, diazepanyl, triazepanyl, azetidinylazocanyl, diazocanyl,
triazocanyl, oxazolidinyl,
oxetenyl, isoxazolidinyl, thiazolidinyl, imidazolinyl, isothiazolidinyl,
oxazocanyl, oxazepanyl,
thiazepanyl, thiazocanyl, benzimidazolonyl, tetrahydrofuranyl,
tetrahydrofuranyl,
tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiophenyl, morpholino,
including, for example,
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,
benzothiolanyl,
benzodithianyl, 3-(1-alkyl)-benzimidazol-2-onyl, and 1,3-dihydro-imidazol-2-
onyl.
[0058] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen,
phosphorus, or
silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or
silicon; 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)).
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[0059] The term "unsaturated", as used herein, means that a moiety has one or
more units of
unsaturation.
[0060] The term "alkoxy", or "thioalkyl", as used herein, refers to an alkyl
group, as defined
herein, attached to the molecule through an oxygen ("alkoxy" e.g., -0-alkyl)
or sulfur ("thioalkyl"
e.g., -S-alkyl) atom.
[0061] The terms "haloalkyl", "haloalkenyl", "haloaliphatic", and "haloalkoxy"
(or
"aminoalkyl", "hydroxyalkyl" etc.,) mean alkyl, alkenyl, aliphatic, or alkoxy,
as the case may be,
substituted with one or more halogen atoms (or amino or hydroxy). The terms
haloalkyl etc.,
include, mono- di- and tri- halo substituted groups. In particular, these
terms include perfluorinated
alkyl groups, such as -CF3 and -CF2CF3.
[0062] The terms "halogen", "halo", and "hal" mean F, Cl, Br, or I.
(0063] The term "acyl group" means -C(O)R wherein R is an aliphatic groups as
defined herein,
or an aryl group as defined herein.
[0064] The term "aryl" used alone or as part of a larger moiety as in
"heteroaryl", "aralkyl",
"aralkoxy", or "aryloxyalkyl" refers to both carbocyclic and or heterocyclic
aromatic ring systems.
The term "aryl" may be used interchangeably with the term "aryl ring".
[0065] Carbocyclic aromatic ring groups have only carbon ring atoms (typically
six to fourteen)
and include monocyclic aromatic rings such as phenyl and fused polycyclic
aromatic ring systems in
which one carbocyclic aromatic rings is fused to one or more aromatic rings in
which the radical or
point of attachment is on the carbocyclic aromatic ring. Examples include 1-
naphthyl, 2-naphthyl,
1-anthracyl and 2-anthracyl. Also included within the scope of the term
"carbocyclic aromatic ring",
as it is used herein, is a group in which an aromatic ring is fused to one or
more non-aromatic rings
(carbocyclic or heterocyclic), such as in an indanyl, phthalimidyl,
naphthimidyl, phenanthridinyl, or
tetrahydronaphthyl, where the radical or point of attachment is on the
carbocyclic aromatic ring.
[0066] The term "heteroaryl", "heteroaromatic", "heteroaryl ring", "heteroaryl
group" and
"heteroaromatic group", used alone or as part of a larger moiety as in
"heteroaralkyl" or
"heteroarylalkoxy", refers to heteroaromatic ring groups having five to
fourteen members, including
monocyclic heteroaromatic rings and polycyclic aromatic rings in which a
monocyclic heteroaryl
ring is fused to one or more other aromatic ring in which the radical or point
of attachment is on the
heteroaryl ring. Heteroaryl groups have one or more ring heteroatoms. Also
included within the
scope of the term "heteroaryl", as it is used herein, is a group in which an
aromatic ring is fused to
one or more non-aromatic rings (carbocyclic or heterocyclic), where the
radical or point of
attachment is on the heteroaryl ring. Bicyclic 6,5 heteroaromatic ring, as
used herein, for example,
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is a six membered heteroaromatic ring fused to a second five membered ring,
wherein the radical or
point of attachment is on the six membered ring. Examples of heteroaryl groups
include pyridyl,
pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrrolyl, pyrazolyl,
triazolyl, tetrazolyl, oxazolyl,
isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl or thiadiazolyl including,
for example, 2-furanyl, 3-
furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-
isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,
3-pyrazolyl, 4-
pyrazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-
pyridyl, 2-pyrimidinyl, 4-
pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-
thiazolyl, 2-triazolyl, 5-
triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl,
benzothienyl, benzofuranyl,
indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl,
isoquinolinyl, indolyl,
isoindolyl, acridinyl, benzisoxazolyl, 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., I -isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl).
[0067] The terms "aralkyl", "heteroaralkyl", "cycloaliphaticalkyl", and
"heterocyclylakyl", refer
to alkyl groups as defined herein substituted with aryl, heteroaryl,
cycloaliphatic, or heterocyclic
groups respectively.
[0068] 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 functional
groups in a compound
with multiple reactive sites. 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. As would be understood by one
skilled in the art, in
some cases, the reagents do not attack other reactive groups in the compound.
In other cases, the
reagents may also react with other reactive groups in the compound. Examples
of protecting groups
are detailed in Greene, T.W., Wuts, P. G 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 agent used to temporarily block one or more desired nitrogen reactive sites
in a multifunctional
compound. Preferred nitrogen protecting groups also possess the
characteristics exemplified for a
protecting group above, and certain exemplary nitrogen protecting groups are
also detailed in
Chapter 7 in Greene, T.W., Wuts, P. G in "Protective Groups in Organic
Synthesis", Third Edition,
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John Wiley & Sons, New York: 1999, the entire contents of which are hereby
incorporated by
reference.
[0069] In some embodiments, where indicated a methylene unit of an aliphatic
group or alkyl
group is optionally replaced with another atom or group. Examples of such
atoms or groups include,
but are not limited to, -N(R')-, -0-, -C(O)-, -C(=N-CN)-, -C(=N R')-, -C(=NO
R')-, -S-, -S(O)-, and
-S(0)2-. These atoms or groups can be combined to form larger groups. Examples
of such larger
groups include, but are not limited to, -OC(O)-, -C(O)CO-, -C02-, -C(O)N R'-, -
C(=N-CN),
-N(R')C(O)-, -N(R')C(O)O-, -S(O)2N(R')-, -N(R')S02-, -N(R')C(O)N(R')-, -
OC(O)N(R')-, and
-N(R')S02N(R')-, wherein R' is defined herein.
[0070] Only those replacement and combinations of groups that result in a
stable structure are
contemplated. Optional replacements can occur both within the chain and/or 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 replacements can also completely replace all of the
carbon atoms in a
chain. For example, a C3 aliphatic can be optionally replaced by -N(R')-, -
C(O)-, and -N(R')- to
form -N(R')C(O)N(R')- (a urea).
[00711 Unless otherwise indicated, if the replacement occurs at the terminal
end, the replacement
atom is bound to an H on the terminal end. For example, if a methylene unit in
-CH2CH2CH3 were
optionally replaced with -0-, the resulting compound could be -OCH2CH3, -
CH2OCH3, or
-CH2CH2OH.
[0072 ] Unless otherwise indicated, structures depicted herein are also meant
to include all
isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, and
rotational) 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 are included in this
invention. As would be
understood to one skilled in the art, a substituent can freely rotate around
any rotatable bonds. For
N N-
example, a substituent drawn as also represents
[0073] Therefore, single stereochemical isomers as well as enantiomeric,
diastereomeric,
geometric, conformational, and rotational mixtures of the present compounds
are within the scope of
the invention.
[00741 Unless otherwise indicated, all tautomeric forms of the compounds of
the invention are
within the scope of the invention.
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[0075] Additionally, unless otherwise indicated, 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 13C- or 14C-enriched
carbon are within the
scope of this invention. Such compounds are useful, for example, as analytical
tools or probes in
biological assays.
[0076] As described herein, where indicated compounds and groups of the
invention may
optionally be substituted with one or more substituents, such as are
illustrated generally herein, 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. Therefore, where it is not
indicated that a compound
or group is substituted, it is understood that the group is not substituted.
That is, if the terms
"optionally substituted" or "substituted" is not present in an instance of the
definition of a compound
or group it is understood that the compound or group is not substituted in
that instance. For
example, Ri is alkyl, Rii is optionally substituted alkyl, and Riii is alkyl
optionally substituted with
halo, means that Rii, and Riii are optionally substituted and Ri is not
substituted in this instance.
[0077 ] Only those choices and combinations of substituents that result in a
stable structure are
contemplated. Such choices and combinations will be apparent to those of
ordinary skill in the art
and may be determined without undue experimentation.
[0078] The term "ring atom" is an atom such as C, N, O or S that is in the
ring of an aromatic
group, cycloalkyl group or non-aromatic heterocyclic ring.
[0079] A "substitutable ring atom" in an aromatic group is a ring carbon or
nitrogen atom
bonded to a hydrogen atom. The hydrogen can be optionally replaced with a
suitable substituent
group. Thus, the term "substitutable ring atom" does not include ring nitrogen
or carbon atoms
which are shared when two rings are fused. In addition, "substitutable ring
atom" does not include
ring carbon or nitrogen atoms when the structure depicts that they are already
attached to a moiety
other than hydrogen, or when the structure depicts they are already bound by a
hydrogen.
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[0080] An optionally substituted aryl group as defined herein contains one or
more substitutable
ring atoms, which may be optionally bonded to one or more suitable
substituent. Examples of
suitable substituents on a substitutable ring carbon atom of an aryl group
includes Rk. Rk is -Ra, -
Br, -Cl, -1, -F, -ORa, -SRa, -O-CORa, -CORa, -CSRa, -CN, -NO2, -NCS, -SO3H, -
N(RaRb), -
COORa, -NRcNRcCORa, -NRcNRcCO2Ra, -CHO, -CON(RaRb), -OC(O)N(RaRb), -CSN(RaRb),
-NRcCORa, -NRcCOORa, -NRcCSRa, -NRcCON(RaRb), -NRcNRcC(O)N(RaRb),
-NRcCSN(RaRb), -C(=NRc)-N(RaRb), -C(=S)N(RaRb), -NRd-C(=NRc)-N(RaRb), -
NRcNRaRb,
-S(O)pNRaRb, -NRcSO2N(RaRb), -NRcS(O)pRa, -S(O)pRa, -OS(O)pNRaRb or -OS(O)pRa;
wherein
p is 1 or 2.
[0081] Ra-Rd are each independently -H, an aliphatic group, aromatic group,
non-aromatic
carbocyclic or heterocyclic group or -N(RaRb), taken together, form a non-
aromatic heterocyclic
group. The aliphatic, aromatic and non-aromatic heterocyclic group represented
by Ra-Rd and the
non-aromatic heterocyclic group represented by -N(RaRb) are each optionally
and independently
substituted with one or more groups represented by RI. Preferably Ra-Rd are
unsubstituted.
[0082] Rl is halogen, Rm, -ORm, -SR', -NO2, -CN, -N(Rm)2, -CORm, -COORm, -
NHCO2Rm,
-NHC(O)Rm, -NHNHC(O)Rm, -NHC(O)N(Rm)2, -NHNHC(O)N(Rm)2, -NHNHCO2Rm, -
C(O)N(Rm)2, -OC(O)Rm, -OC(O)N(Rm)2, -S(O)2Rm, -SO2N(Rm)2, -S(O)Rm, -
NHSO2N(Rm)2, -
NHSO2Rm, -C(=S)N(Rm+)2, or -C(=NH)-N(Rm)2.
[0083] Rm is-H, a C1-C4 alkyl group, a monocyclic aryl group, a non-aromatic
carbocyclic or
heterocyclic group each optionally substituted with unsaubsituted alkyl,
haloalkyl, alkoxy,
haloalkoxy, halogen, -CN, -NO2, amine, alkylamine or dialkylamine. Preferably
Rm is unsubstituted.
[0084] An optionally substituted aliphatic or a non-aromatic heterocyclic or
carbocyclic group as
defined herein contains one or more substitutable atoms which may optionally
be bonded to one or
more suitable substituents. Examples of suitable substituents for an aliphatic
group or a ring carbon
of a non-aromatic heterocyclic group is Rn. Rn include those substituents
listed above for Rk and
=O, =S, =NNHRo, =NN(Ro)2, =NNHC(O)Ro, =NNHCO2 (alkyl), =NNHSO2 (alkyl), =NRo,
Spiro
cycloalkyl group or fused cycloalkyl group. Each Ro is independently selected
from hydrogen, an
unsubstituted alkyl group or a substituted alkyl group. Examples of
substituents on the alkyl group
represented by Ro include amino, alkylamino, dialkylamino, aminocarbonyl,
halogen, alkyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy,
dialkylaminocarbonyloxy,
alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy,
haloalkoxy, or haloalkyl.
Preferably Ro is unsubstituted.
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[0085] When a heterocyclyl, heteroaryl, or heteroaralkyl group contains a
nitrogen atom, it may
be substituted or unsubstituted as indicated herein. When a nitrogen atom in
the aromatic ring of a
heteroaryl group has a substituent the nitrogen may be a quaternary nitrogen.
[0086] In certain embodiments non-aromatic nitrogen-containing heterocyclic
group or
heteroaryl groups are optionally substituted at the nitrogen ring atom.
Suitable substituents on the
nitrogen of a non-aromatic heterocyclic group or heteroaryl group include -Rq,
-N(Rq)2, -C(O)Rq,
CO2Rq, -C(O)C(O)Rq, -SO2Rq, SO2 N(Rq)2, -C(=S)N(Rq)2, -C(=NH)-N(Rq)2, and -
NRgSO2Rq;
wherein Rq is hydrogen, an aliphatic group, a substituted aliphatic group,
aryl, substituted aryl,
heterocyclic or carbocyclic ring or a substituted heterocyclic or carbocyclic
ring. Examples of
substituents on the group represented by R^ include alkyl, haloalkoxy,
haloalkyl, alkoxyalkyl,
sulfonyl, alkylsulfonyl, halogen, nitro, cyano, hydroxy, aryl, carbocyclic or
heterocyclic ring, oxo,
amino, alkylamino, dialkylamino, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyloxy,
alkoxy, carboxy, alkoxycarbonyl, or alkylcarbonyl. Preferably R^ is not
substituted.
[0087] Non-aromatic nitrogen containing heterocyclic rings and heteroaryl that
are substituted
on a ring nitrogen and attached to the remainder of the molecule at a ring
carbon atom are said to be
N substituted. For example, an N alkyl piperidinyl group is attached to the
remainder of the molecule
at the two, three or four position of the piperidinyl ring and substituted at
the ring nitrogen with an
alkyl group. Non-aromatic nitrogen containing heterocyclic rings such as
piperazinyl that are
substituted on a ring nitrogen and attached to the remainder of the molecule
at a second ring nitrogen
atom are said to be N' substituted-N-heterocycles. For example, an N' acyl N-
piperazinyl group is
attached to the remainder of the molecule at one ring nitrogen atom and
substituted at the second
ring nitrogen atom with an acyl group.
[0088] As used herein an optionally substituted aralkyl can be substituted on
both the alkyl and
the aryl portion. In certain embodiments, optionally substituted aralkyl is
optionally substituted on
the aryl portion.
[0089] The compounds of the invention are defined herein by their chemical
structures and/or
chemical names. Where a compound is referred to by both a chemical structure
and a chemical
name, and the chemical structure and chemical name conflict, the chemical
structure is determinative
of the compound's identity.
[0090] The compounds of this invention can exist in free form for treatment,
or where
appropriate, as a pharmaceutically acceptable salt.
[0091 ] 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
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humans and lower animals without undue side effects, such as, toxicity,
irritation, allergic response
and the like, and are commensurate with a reasonable benefit/risk ratio.
[0092] 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.
[00931 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, glycolate,
hemisulfate, heptanoate,
hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-
ethanesulfonate, lactobionate,
lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate,
nicotinate, nitrate, oleate, oxalate, palmoate, palmoate, pectinate,
persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate,
sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
[0094] 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. Salts derived from
appropriate bases include alkali metal (e.g., sodium, lithium, and potassium),
alkaline earth metal
(e.g., magnesium and calcium), ammonium and N+(C1_4alkyl)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] 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
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preparation of salts useful as intermediates in obtaining the compounds of the
invention and their
pharmaceutically acceptable acid or base addition salts.
[0096] It should be understood that this invention includes
mixtures/combinations of different
pharmaceutically acceptable salts and also mixtures/combinations of compounds
in free form and
pharmaceutically acceptable salts.
[0097] In addition to the compounds of this invention, pharmaceutically
acceptable solvates
(e.g., hydrates) and clathrates of the compounds of this invention may also be
employed in
compositions to treat or prevent the herein identified disorders.
[0098] As used herein, the term "pharmaceutically acceptable solvate," is a
solvate formed from
the association of one or more pharmaceutically acceptable solvent molecules
to one of the
compounds the invention. The term solvate includes hydrates (e.g.,
hemihydrate, monohydrate,
dihydrate, trihydrate, tetrahydrate, and the like).
[0099] As used herein, the term "hydrate" means a compound of the present
invention or a salt
thereof, that further includes a stoichiometric or non-stoichiometric amount
of water bound by non-
covalent intermolecular forces.
[00100] As used herein, he term "clathrate" means a compound of the present
invention or a
salt thereof in the form of a crystal lattice that contains spaces (e.g.,
channels) that have a guest
molecule (e.g., a solvent or water) trapped within.
[ 00101 ] 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 herein identified disorders.
[001021 As used herein and unless otherwise indicated, the term "prodrug"
means a
derivative of a compound that can hydrolyze, oxidize, or otherwise react under
biological conditions
(in vitro or in vivo) to provide a compound of this invention. Prodrugs may
become active upon
such reaction under biological conditions, or they may have activity in their
unreacted forms.
Examples of prodrugs contemplated in this invention include, but are not
limited to, analogs or
derivatives of compounds of the invention that comprise biohydrolyzable
moieties such as
biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates,
biohydrolyzable
carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
Other examples of
prodrugs include derivatives of compounds of the invention that comprise -NO, -
N02, -ONO, or -
ONO2 moieties. Prodrugs can typically be prepared using well-known methods,
such as those
described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995) 172-178,
949-982 (Manfred E. Wolff ed., 5th ed).
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[001031 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.
[00104] A "pharmaceutically acceptable derivative or prodrug" includes any
pharmaceutically acceptable ester, salt of an ester or other derivative or
salt thereof 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.
[001051 Pharmaceutically acceptable prodrugs of the compounds of this
invention include,
without limitation, esters, amino acid esters, phosphate esters, metal salts
and sulfonate esters.
[00106] As used herein, the phrase "side effects" encompasses unwanted and
adverse effects
of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are
always unwanted, but
unwanted effects are not necessarily adverse. An adverse effect from a therapy
(e.g., prophylactic or
therapeutic agent) might be harmful or uncomfortable or risky. Side effects
include, but are not
limited to fever, chills, lethargy, gastrointestinal toxicities (including
gastric and intestinal
ulcerations and erosions), nausea, vomiting, neurotoxicities,
nephrotoxicities, renal toxicities
(including such conditions as papillary necrosis and chronic interstitial
nephritis), hepatic toxicities
(including elevated serum liver enzyme levels), myelotoxicities (including
leukopenia,
myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste,
prolongation of
gestation, weakness, somnolence, pain (including muscle pain, bone pain and
headache), hair loss,
asthenia, dizziness, extra-pyramidal symptoms, akathisia, cardiovascular
disturbances and sexual
dysfunction.
[00107 ] In one embodiment the present invention is a pharmaceutical
composition
comprising a compound of the present invention and a pharmaceutically
acceptable carrier, diluent,
adjuvant or vehicle. In one embodiment the present invention is a
pharmaceutical composition
comprising an effective amount of compound of the present invention and a
pharmaceutically
acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable
carriers include, for
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example, pharmaceutical diluents, excipients or carriers suitably selected
with respect to the intended
form of administration, and consistent with conventional pharmaceutical
practices.
[00108] A pharmaceutically acceptable carrier may contain inert ingredients
which do not
unduly inhibit the biological activity of the compounds. The pharmaceutically
acceptable carriers
should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or
devoid of other
undesired reactions or side-effects upon the administration to a subject.
Standard pharmaceutical
formulation techniques can be employed.
[00109] The pharmaceutically acceptable carrier, adjuvant, or vehicle, 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.
[00110 ] 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
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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.
[00111] The protein kinase inhibitors or pharmaceutical salts thereof may be
formulated into
pharmaceutical compositions for administration to a subject as defined herein.
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.
[ 00112 ] In one embodiment the present invention is a method of treating or
preventing a protein
kinase-mediated disorder in a subject in need thereof, comprising
administering to the subject an
effective amount of a compound composition or a pharmaceutically acceptable
salt of the present
invention as described herein. In another embodiment, the present invention is
the use of an
effective amount of a compound, composition or a pharmaceutically acceptable
salt described herein
for treating or preventing a disease or disorder, described herein, in a
subject in need thereof. In
another embodiment, the present invention is the use of an effective amount of
a compound,
composition or a pharmaceutically acceptable salt described herein for
treating a disease or disorder,
described herein, in a subject in need thereof. In yet another embodiment, the
present invention is
the use of an effective amount of a compound, composition or a
pharmaceutically acceptable salt
described herein for the manufacture of a medicament method for the treatment
or prevention of a
disease or disorder, described herein, in a subject in need thereof. In yet
another embodiment, the
present invention is the use of an effective amount of a compound, composition
or a
pharmaceutically acceptable salt described herein for the manufacture of a
medicament method for
the treatment of a disease or disorder, described herein, in a subject in need
thereof. In one
embodiment the protein kinase mediated disease is a protein kinase C (PKC)
mediated disease. In
another embodiment the protein kinase mediated disease is a protein kinase C
theta (PKCtheta)-
mediated disease.
[00113] As used herein, the terms "subject", "patient" and "mammal" are used
interchangeably.
The terms "subject" and "patient" refer to an animal (e.g., a bird such as a
chicken, quail or turkey,
or a mammal), preferably a mammal including a non-primate (e.g., a cow, pig,
horse, sheep, rabbit,
guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey,
chimpanzee and a human), and
more preferably a human. In one embodiment, the subject is a non-human animal
such as a farm
animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea
pig or rabbit). In a
preferred embodiment, the subject is a human.
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[00114] As used herein, an "effective amount" refers to an amount sufficient
to elicit the desired
biological response. In the present invention the desired biological response
is to reduce or
ameliorate the severity, duration, progression, or onset of a protein kinase-
mediated condition,
prevent the advancement of a protein kinase-mediated condition, cause the
regression of a protein
kinase-mediated condition, prevent the recurrence, development, onset or
progression of a symptom
associated with a protein kinase-mediated condition, or enhance or improve the
prophylactic or
therapeutic effect(s) of another therapy. The precise amount of compound
administered to a subject
will depend on the mode of administration, the type and severity of the
disease or condition and on
the characteristics of the subject, such as general health, age, sex, body
weight and tolerance to
drugs. It will also depend on the degree, severity and type of protein kinase-
mediated condition, and
the mode of administration. The skilled artisan will be able to determine
appropriate dosages
depending on these and other factors. When co-administered with other agents,
e.g., when co-
administered with an protein kinase-mediated condition agent, an "effective
amount" of the second
agent will depend on the type of drug used. Suitable dosages are known for
approved agents and can
be adjusted by the skilled artisan according to the condition of the subject,
the type of condition(s)
being treated and the amount of a compound of the invention being used. In
cases where no amount
is expressly noted, an effective amount should be assumed.
[00115 ] As used herein, the terms "treat", "treatment" and "treating" refer
to the reduction or
amelioration of the progression, severity and/or duration of a protein kinase-
mediated condition, or
the amelioration of one or more symptoms (preferably, one or more discernible
symptoms) of a
protein kinase-mediated condition resulting from the administration of one or
more therapies (e.g.,
one or more therapeutic agents such as a compound of the invention). In
specific embodiments, the
terms "treat", "treatment" and "treating" refer to the amelioration of at
least one measurable physical
parameter of a protein kinase-mediated condition. In other embodiments the
terms "treat",
"treatment" and "treating" refer to the inhibition of the progression of a
protein kinase-mediated
condition, either physically by, e.g., stabilization of a discernible symptom,
physiologically by, e.g.,
stabilization of a physical parameter, or both. In other embodiments the terms
"treat", "treatment"
and "treating" refer to the reduction or stabilization of a protein kinase-
mediated condition.
[00116 ] As used herein, the terms "prevent", "prevention" and "preventing"
refer to the
reduction in the risk of acquiring or developing a given protein kinase-
mediated condition, or the
reduction or inhibition of the recurrence or a protein kinase-mediated
condition. In one embodiment,
a compound of the invention is administered as a preventative measure to a
patient, preferably a
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human, having a genetic predisposition to any of the conditions, diseases or
disorders described
herein.
[ 00117 ] As used herein, the terms, "disease", "disorder" and "condition" may
be used
interchangeably here to refer to a protein kinase-mediated condition.
[001181 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. In some embodiments, said protein kinase inhibitor
is a PKCtheta
inhibitor.
[00119] 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, immuno-deficiency disorders,
immunomodulatory or
immunosuppressive disorder, bone diseases, metabolic diseases, neurological
and neurodegenerative
diseases, cardiovascular diseases, hormone related diseases, diabetes,
allergies, asthma, and
Alzheimer's disease. In one embodiment, the protein-kinase mediated condition
is a PKC-mediated
condition.
[00120 ] The term "PKC-mediated condition", as used herein means any disease
or other
deleterious condition in which PKC plays a role. Such conditions include,
without limitation, those
listed above, and in particular, T-cell mediated diseases, including without
limitation autoimmune
diseases, chronic or acute inflammatory diseases, and proliferative and
hyperproliferative diseases.
In one embodiment, the PKC-mediated condition is a PKCtheta-mediated condition
[00121] The term "PKCtheta-mediated condition", as used herein means any
disease or other
deleterious condition in which PKCtheta plays a role. Such conditions include,
without limitation,
those listed above, and in particular, autoimmune diseases, chronic or acute
inflammatory diseases,
and proliferative and hyperproliferative diseases.
[00122 ] As used herein, the term "inflammatory disease" or "inflammatory
disorder" refers to
pathological states resulting in inflammation, typically caused by leukocyte
infiltration. Examples of
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such disorders include inflammatory skin diseases, including, without
limitation, psoriasis and atopic
dermatitis; systemic scleroderma and sclerosis; responses associated with
inflammatory bowel
disease (IBD) (such as Crohn's disease and ulcerative colitis); ischemic
reperfusion disorders
including surgical tissue reperfusion injury, myocardial ischemic conditions
such as myocardial
infarction, cardiac arrest, reperfusion after cardiac surgery and constriction
after percutaneous
transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms;
cerebral edema
secondary to stroke; cranial trauma, hypovolemic shock; asphyxia; adult
respiratory distress
syndrome; acute-lung injury; Behcet's Disease; dermatomyositis; polymyositis;
multiple sclerosis
(MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus
nephritis; autoimmune
diseases such as rheumatoid arthritis (RA), Sjorgen's syndrome, vasculitis;
diseases involving
leukocyte diapedesis; central nervous system (CNS) inflammatory disorder,
multiple organ injury
syndrome secondary to septicaemia or trauma; alcoholic hepatitis; bacterial
pneumonia; antigen-
antibody complex mediated diseases including glomerulonephritis; sepsis;
sarcoidosis;
immunopathologic responses to tissue or organ transplantation; inflammations
of the lung, including
pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,
bronchiectasis, diffuse
panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis
(IPF), and cystic
fibrosis; etc.
[00123] Proliferative or hyperproliferative diseases are characterized by
excessive or abnormal
cell proliferation. Such diseases include, without limitation, cancer and
myeloproliferative
disorders.
[00124] The term "cancers" includes, but is not limited to, the following
cancers: epidermoid
Oral: Cardiac: Lung: Gastrointestinal: Genitourinary tract: Liver: Bone:
Nervous system:
Gynecological: Hematologic: Thyroid gland: and Adrenal glands. Hematologic
cancers include:
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, and psoriasis.
Thus, the term "cancerous cell" as provided herein, includes a cell afflicted
by any one of the above-
identified conditions.
[00125] The term "myeloproliferative disorders", includes disorders such as
polycythemia vera,
thrombocythemia, myeloid metaplasia with myelofibrosis, hypereosinophilic
syndrome, juvenile
myelomonocytic leukaemia, systemic mast cell disease, and hematopoietic
disorders, in particular,
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acute-myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute-
promyelocytic
leukemia (APL), and acute lymphocytic leukemia (ALL).
[00126] Examples of neurodegenerative diseases include, without limitation,
Alzheimer's
disease Huntington's disease, Parkinson's disease, AIDS-associated dementia,
and bipolar disorder.
[00127] In one embodiment the PKCtheta mediated disease includes, without
limitation, chronic
inflammation, autoimmune diabetes, rheumatoid arthritis (RA), rheumatoid
spondylitis, gouty
arthritis and other arthritic conditions, multiple sclerosis (MS), asthma,
systemic lupus
erythrematosis, adult respiratory distress syndrome, Behcet's disease,
psoriasis, chronic pulmonary
inflammatory disease, graft versus host reaction, Crohn's Disease, ulcerative
colitis, inflammatory
bowel disease (IBD), which includes celiac disease and irritable bowel
syndrome; Alzheimer's
disease, T-cell leukaemia, lymphoma, transplant rejection, cancer and pyresis,
along with any
disease or disorder that relates to inflammation and related disorders.
[00128] In one embodiment the PKCtheta mediated disease includes, diseases
such as, but not
limited to, arthritis, rheumatoid arthritis, osteoarthritis, joint
inflammation, lupus, multiple sclerosis,
asthma, psoriasis, cancer, T-cell lymphomas, leukaemia,diabetes type I or 11,
and inflammatory
bowel diseases, transplant rejection, Crohn's disease and colitis.
[00129] Examples of autoimmune diseases include, without limitation, multiple
sclerosis,
rheumatoid arthritis and irritable bowel disease.
[001301 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.
[00131] 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.
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[00132] 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.
[001331 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.
[00134] 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.
[00135] 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.
[00136] 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,
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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.
[00137 ] 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.
[00138] 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.
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[001391 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.
[00140] 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.
[00141 ] 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
CA 02761074 2011-11-04
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in the manufacture of pharmaceutically acceptable solid, liquid, or other
dosage forms may also be
used for the purposes of formulation.
[00142 ] 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.
[00143] 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 which 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.
[00144 ] 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.
[00145] 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.
[00146] 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.
[00147] For ophthalmic use, the pharmaceutical compositions may be formulated
as
micronized suspensions in isotonic, pH adjusted sterile saline, or,
preferably, as solutions in isotonic,
31
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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.
[00148] 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.
[00149] The dosage regimen utilizing the compounds of Structural Formula I,
IA, and IC
can be selected in accordance with 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 renal and hepatic function of the subject; and the particular compound or
salt thereof 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 skilled artisan
can readily determine
and prescribe the effective amount of the compound of Structural Formula I,
IA, and IC required to
treat, for example, to prevent, inhibit (fully or partially) or arrest the
progress of the disease.
(00150] Dosages of the compounds of Structural Formula I, IA, and IC can range
from
between about 0.01 to about 100 mg/kg body weight/day, about 0.01 to about 50
mg/kg body
weight/day, about 0.1 to about 50 mg/kg body weight/day, or about Ito about 25
mg/kg body
weight/day. It is understood that the total amount per day can be administered
in a single dose or
can be administered in multiple dosings such as twice, three or four times per
day.
[001511 The compounds for use in the method of the invention can be formulated
in unit
dosage form. The term "unit dosage form" refers to physically discrete units
suitable as unitary
dosage for subjects undergoing treatment, with each unit containing a
predetermined quantity of
active material calculated to produce the desired therapeutic effect,
optionally in association with a
suitable pharmaceutical carrier. The unit dosage form can be for a single
daily dose or one of
multiple daily doses (e.g., about I to 4 or more times per day). When multiple
daily doses are used,
the unit dosage form can be the same or different for each dose.
[00152 ] An effective amount can be achieved in the method or pharmaceutical
composition
of the invention employing a compound of Structural Formula I, IA, and IC or a
pharmaceutically
acceptable salt or solvate (e.g., hydrate) thereof alone or in combination
with an additional suitable
32
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therapeutic agent, for example, a cancer-therapeutic agent. When combination
therapy is employed,
an effective amount can be achieved using a first amount of a compound of
Structural Formula I, IA,
and IC or a pharmaceutically acceptable salt or solvate (e.g., hydrate)
thereof and a second amount
of an additional suitable therapeutic agent.
[001531 In one embodiment, the compound of Structural Formula I, IA, and IC
and the
additional therapeutic agent, are each administered in an effective amount
(i.e., each in an amount
which would be therapeutically effective if administered alone). In another
embodiment, the
compound of Structural Formula I, IA, and IC and the additional therapeutic
agent, are each
administered in an amount which alone does not provide a therapeutic effect (a
sub-therapeutic
dose). In yet another embodiment, the compound of Structural Formula I, IA,
and IC can be
administered in an effective amount, while the additional therapeutic agent is
administered in a sub-
therapeutic dose. In still another embodiment, the compound of Structural
Formula I, IA, and IC can
be administered in a sub-therapeutic dose, while the additional therapeutic
agent, for example, a
suitable cancer-therapeutic agent is administered in an effective amount.
[00154 ] As used herein, the terms "in combination" or "coadministration" can
be used
interchangeably to refer to the use of more than one therapies (e.g., one or
more prophylactic and/or
therapeutic agents). The use of the terms does not restrict the order in which
therapies (e.g.,
prophylactic and/or therapeutic agents) are administered to a subject.
[001551 Coadministration encompasses administration of the first and second
amounts of
the compounds of the coadministration in an essentially simultaneous manner,
such as in a single
pharmaceutical composition, for example, capsule or tablet having a fixed
ratio of first and second
amounts, or in multiple, separate capsules or tablets for each. In addition,
such coadministration also
encompasses use of each compound in a sequential manner in either order.
[001561 When coadministration involves the separate administration of the
first amount of a
compound of Structural Formula I, IA, and IC and a second amount of an
additional therapeutic
agent, the compounds are administered sufficiently close in time to have the
desired therapeutic
effect. For example, the period of time between each administration which can
result in the desired
therapeutic effect, can range from minutes to hours and can be determined
taking into account the
properties of each compound such as potency, solubility, bioavailability,
plasma half-life and kinetic
profile. For example, a compound of Structural Formula I, IA, and IC and the
second therapeutic
agent can be administered in any order within about 24 hours of each other,
within about 16 hours of
each other, within about 8 hours of each other, within about 4 hours of each
other, within about I
hour of each other or within about 30 minutes of each other.
33
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[001571 More, specifically, a first therapy (e.g., a prophylactic or
therapeutic agent such as a
compound of the invention) can be administered prior to (e.g., 5 minutes, 15
minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72
hours, 96 hours, 1 week,
2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or
subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, I week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6
weeks, 8 weeks, or 12 weeks after) the administration of a second therapy
(e.g., a prophylactic or
therapeutic agent such as an anti-cancer agent) to a subject.
[00158 ] It is understood that the method of coadministration of a first
amount of a
compound of Structural Formula I, IA, and IC and a second amount of an
additional therapeutic
agent can result in an enhanced or synergistic therapeutic effect, wherein the
combined effect is
greater than the additive effect that would result from separate
administration of the first amount of
the compound of Structural Formula I, IA, and IC and the second amount of the
additional
therapeutic agent.
[00159] As used herein, the term "synergistic" refers to a combination of a
compound of the
invention and another therapy (e.g., a prophylactic or therapeutic agent),
which is more effective
than the additive effects of the therapies. A synergistic effect of a
combination of therapies (e.g., a
combination of prophylactic or therapeutic agents) permits the use of lower
dosages of one or more
of the therapies and/or less frequent administration of said therapies to a
subject. The ability to
utilize lower dosages of a therapy (e.g., a prophylactic or therapeutic agent)
and/or to administer said
therapy less frequently reduces the toxicity associated with the
administration of said therapy to a
subject without reducing the efficacy of said therapy in the prevention,
management or treatment of
a disorder. In addition, a synergistic effect can result in improved efficacy
of agents in the
prevention, management or treatment of a disorder. Finally, a synergistic
effect of a combination of
therapies (e.g., a combination of prophylactic or therapeutic agents) may
avoid or reduce adverse or
unwanted side effects associated with the use of either therapy alone.
[00160] The presence of a synergistic effect can be determined using suitable
methods for
assessing drug interaction. Suitable methods include, for example, the Sigmoid-
Emax equation
(Holford, N.H.G. and Scheiner, L.B., Clin. Pharmacokinet. 6: 429-453 (1981)),
the equation of
Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol.
114: 313-326
(1926)) and the median-effect equation (Chou, T.C. and Talalay, P., Adv.
Enzyme Regul. 22: 27-55
(1984)). Each equation referred to above can be applied with experimental data
to generate a
corresponding graph to aid in assessing the effects of the drug combination.
The corresponding
34
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VPI/09-1 I6 WO
graphs associated with the equations referred to above are the concentration-
effect curve,
isobologram curve and combination index curve, respectively.
[ 001611 In some embodiments, said additional therapeutic agent is selected
from a cancer-
therapeutic agent, such as, an anti-cancer agent, an anti-proliferative agent,
or a chemotherapeutic
agent.
[ 00162 ] In some embodiments, said additional therapeutic agent is selected
from
camptothecin, the MEK inhibitor: U0126, a KSP (kinesin spindle protein)
inhibitor, adriamycin,
interferons, and platinum derivatives, such as Cisplatin.
[001631 In other embodiments, said additional therapeutic agent is selected
from taxanes;
inhibitors of bcr-abl (such as Gleevec, dasatinib, and nilotinib); inhibitors
of EGFR (such as Tarceva
and Iressa); DNA damaging agents (such as cisplatin, oxaliplatin, carboplatin,
topoisomerase
inhibitors, and anthracyclines); and antimetabolites (such as AraC and 5-FU).
[00164 ] In yet other embodiments, said additional therapeutic agent is
selected from
camptothecin, doxorubicin, idarubicin, Cisplatin, taxol, taxotere,
vincristine, tarceva, the MEK
inhibitor, U0126, a KSP inhibitor, vorinostat, Gleevec, dasatinib, and
nilotinib.
[00165] In another embodiment, said additional therapeutic agent is selected
from Her-2
inhibitors (such as Herceptin); HDAC inhibitors (such as vorinostat), VEGFR
inhibitors (such as
Avastin), c-KIT and FLT-3 inhibitors (such as sunitinib), BRAF inhibitors
(such as Bayer's BAY
43-9006) MEK inhibitors (such as Pfizer's PD0325901); and spindle poisons
(such as Epothilones
and paclitaxel protein-bound particles (such as Abraxane )=
[00166] Other therapies or anticancer agents that may be used in combination
with the
inventive agents of the present invention include surgery, radiotherapy (in
but a few examples,
gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton
therapy,
brachytherapy, and systemic radioactive isotopes, to name a few), endocrine
therapy, biologic
response modifiers (interferons, interleukins, and tumor necrosis factor (TNF)
to name a few),
hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g.,
antiemetics), and other
approved chemotherapeutic drugs, including, but not limited to, alkylating
drugs (mechlorethamine,
chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites
(Methotrexate), purine
antagonists and pyrimidine antagonists (6-Mercaptopurine, 5-Fluorouracil,
Cytarabile,
Gemcitabine), spindle poisons (Vinblastine, Vincristine, Vinorelbine,
Paclitaxel), podophyllotoxins
(Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin,
Mitomycin), nitrosoureas
(Carmustine, Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes
(Asparaginase), and
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hormones (Tamoxifen, Leuprolide, Flutamide, and Megestrol), GleevecTM,
adriamycin,
dexamethasone, and cyclophosphamide.
[00167 ] A compound of the instant invention may also be useful for treating
cancer in
combination with any of the following therapeutic agents: abarelix (Plenaxis
depot ); aldesleukin
(Prokine ); Aldesleukin (Proleukin ); Alemtuzumabb (Campath ); alitretinoin
(Panretin );
allopurinol (Zyloprim ); altretamine (Hexalen ); amifostine (Ethyol );
anastrozole (Arimidex );
arsenic trioxide (Trisenox ); asparaginase (Elspar ); azacitidine (Vidaza );
bevacuzimab
(Avastin ); bexarotene capsules (Targretin ); bexarotene gel (Targretin );
bleomycin
(Blenoxane ); bortezomib (Velcade ); busulfan intravenous (Busulfex(&);
busulfan oral
(Myleran ); calusterone (Methosarb ); capecitabine (Xeloda(K); carboplatin
(Paraplatin );
carmustine (BCNU , BiCNU ); carmustine (Gliadel ); carmustine with
Polifeprosan 20 Implant
(Gliadel Wafer ); celecoxib (Celebrex ); cetuximab (Erbitux(K); chlorambucil
(Leukeran(K);
cisplatin (Platinol ); cladribine (Leustatin , 2-CdA ); clofarabine
(Clolar(K); cyclophosphamide
(Cytoxan , Neosar ); cyclophosphamide (Cytoxan Injection(g); cyclophosphamide
(Cytoxan
Tablet ); cytarabine (Cytosar-U ); cytarabine liposomal (DepoCyt );
dacarbazine (DTIC-Dome );
dactinomycin, actinomycin D (Cosmegen ); Darbepoetin alfa (Aranesp(R);
daunorubicin liposomal
(DanuoXome ); daunorubicin, daunomycin (Daunorubicin(V); daunorubicin,
daunomycin
(Cerubidine ); Denileukin diftitox (Ontak(&); dexrazoxane (Zinecard );
docetaxel (Taxotere(K);
doxorubicin (Adriamycin PFS ); doxorubicin (Adriamycin , Rubex ); doxorubicin
(Adriamycin
PFS Injection ); doxorubicin liposomal (Doxil ); dromostanolone propionate
(dromostanolone );
dromostanolone propionate (masterone injection ); Elliott's B Solution
(Elliott's B Solution );
epirubicin (Ellence ); Epoetin alfa (epogen ); erlotinib (Tarceva );
estramustine (Emcyt );
etoposide phosphate (Etopophos ); etoposide, VP-16 (Vepesid ); exemestane
(Aromasin );
Filgrastim (Neupogen ); floxuridine (intraarterial) (FUDR ); fludarabine
(Fludara(V); fluorouracil,
5-FU (Adrucil ); fulvestrant (Faslodex ); gefitinib (Iressa ); gemcitabine
(Gemzar(K);
gemtuzumab ozogamicin (Mylotarg(K); goserelin acetate (Zoladex Implant );
goserelin acetate
(Zoladex ); histrelin acetate (Histrelin implant ); hydroxyurea (Hydrea );
Ibritumomab Tiuxetan
(Zevalin ); idarubicin (Idamycin ); ifosfamide (IFEX ); imatinib mesylate
(Gleevec ); interferon
alfa 2a (Roferon A ); Interferon alfa-2b (Intron A ); irinotecan (Camptosar );
lenalidomide
(Revlimid(R); letrozole (Femara(R); leucovorin (Wellcovorin , Leucovorin );
Leuprolide Acetate
(Eligard ); levamisole (Ergamisol(K); lomustine, CCNU (CeeBU );
meclorethamine, nitrogen
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VPI/09-116 WO
mustard (Mustargen ); megestrol acetate (Megace ); melphalan, L-PAM (Alkeran
);
mercaptopurine, 6-MP (Purinethol ); mesna (Mesnex(R); mesna (Mesnex tabs );
methotrexate
(Methotrexate(&); methoxsalen (Uvadex ); mitomycin C (Mutamycin ); mitotane
(Lysodren );
mitoxantrone (Novantrone ); nandrolone phenpropionate (Durabolin-50 );
nelarabine (Arranon );
Nofetumomab (Verluma ); Oprelvekin (Neumega ); oxaliplatin (Eloxatin );
paclitaxel (Paxene );
paclitaxel (Taxol ); paclitaxel protein-bound particles (Abraxane(&);
palifermin (Kepivance(R);
pamidronate (Aredia ); pegademase (Adagen (Pegademase Bovine) ); pegaspargase
(Oncaspar );
Pegfilgrastim (Neulasta(R); pemetrexed disodium (Alimta(&); pentostatin
(Nipent ); pipobroman
(Vercyte ); plicamycin, mithramycin (Mithracin(V); porfimer sodium (Photofrin
); procarbazine
(Matulane ); quinacrine (Atabrine(R); Rasburicase (Elitek(R); Rituximab
(Rituxan ); sargramostim
(Leukine ); Sargramostim (Prokine ); sorafenib (Nexavar ); streptozocin
(Zanosar ); sunitinib
maleate (Sutent ); talc (Sclerosol ); tamoxifen (Nolvadex ); temozolomide
(Temodar );
teniposide, VM-26 (Vumon ); testolactone (Teslac ); thioguanine, 6-TG
(Thioguanine ); thiotepa
(Thioplex ); topotecan (Hycamtin ); toremifene (Fareston ); Tositumomab
(Bexxar );
Tositumomab/I-131 tositumomab (Bexxar ); Trastuzumab (Herceptin(&); tretinoin,
ATRA
(Vesanoid(&); Uracil Mustard (Uracil Mustard Capsules(V); valrubicin (Valstar
); vinblastine
(Velban(&); vincristine (Oncovin ); vinorelbine (Navelbine ); zoledronate
(Zometa ) and
vorinostat (Zolinza(&).
[001681 For a comprehensive discussion of updated cancer therapies see,
http://www.nci.nih.gov/, a list of the FDA approved oncology drugs at
http://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,
Seventeenth Ed. 1999,
the entire contents of which are hereby incorporated by reference.
[001691 Other examples of agents the compounds of this invention may also be
combined
with include, without limitation: treatments for Alzheimer's Disease such as
Aricept and Excelon ;
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 and Rebif ), Copaxone , and
mitoxantrone; treatments
for asthma such as albuterol and Singulair ; agents for treating schizophrenia
such as zyprexa,
risperdal, seroquel, and haloperidol; anti-inflammatory agents such as
corticosteroids, TNF blockers,
IL-I RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory
and
immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,
mycophenolate mofetil,
interferons, corticosteroids, cyclophophamide, azathioprine, and
sulfasalazine; neurotrophic factors
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VPI/09-116 WO
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.
[00170] 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, IA, and IC 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.
[00171 ] Inhibition of protein kinase activity in a biological sample is
useful fora 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.
[00172 ] 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.
[00173] 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 are set forth in the Examples below.
[00174 ] Another aspect of the invention provides a method for modulating
enzyme activity
by contacting a compound of Formula 1, IA, and IC with a protein kinase.
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Abbreviations
[00175] The following abbreviations are used:
DMSO dimethyl sulfoxide
TCA trichloroacetic acid
ATP adenosine triphosphate
BSA bovine serum albumin
DTT dithiothreitol
MOPS 4-morpholinepropanesulfonic acid
NMR nuclear magnetic resonance
HPLC high performance liquid chromatography
LCMS liquid chromatography-mass spectrometry
TLC thin layer chromatography
Rt retention time
[001761 In some embodiments, the compounds of this invention are represented
in Table 1.
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VPI/09-116 WO
Table 1
3
4 a= 6
It
It #
' ;=^.`' till JI
SI
In some embodiments the variables used herein, such as x, y, A, A', R2, R3,
R4, R5, R7, are as defined
in Table 1.
General synthetic methodology
[001771 The compounds of this invention may be prepared in light of the
specification using
steps generally known to those of ordinary skill in the art. Those compounds
may be analyzed by
CA 02761074 2011-11-04
VPI/09-116 WO
known methods, including but not limited to LCMS (liquid chromatography mass
spectrometry)
HPLC and NMR (nuclear magnetic resonance). 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 compounds of this invention. Instead, this invention also
includes conditions that
would be apparent to those skilled in that art in light of this specification
for making the compounds
of this invention. Unless otherwise indicated, all variables in the following
schemes are as defined
herein. General Schemes:
[00178] Scheme I
Br RXO ORX
N
N H
]No a
A \A I
RZ N
RZ
(Rt)x
R3 (Rl)x
1 R4 2 R3 CI
R4 3 Rx is -H or two RX
together form:
N
N H 7~C
ri N;
N
b
A A'
j I RZ
(R1)
R3
4 R4
Reagents and conditions: a) K2CO3, [B(OR7)2]2, Pd(dppf)2CI2.DCM, DME, 100 C;
b) Na2CO3, Pd[P(tBu)3]2, dioxane, 70 C.
[00179] Scheme I above describes another general synthetic route for preparing
compounds
of Formula I, IA, and IC of this invention where x, A, A', R1, R2, R3 and R4
are as described herein
(it is understood that R5 and R6 can also be present and that ring B
substituted with (RA)Y can
replace the CR2R3R4 group). Starting materials for 1 are either commercially
available or can be
prepared by reactions well known in the art (e.g. Knochel, Buchwald).
Boronation of derivative 1,
followed by Suzuki-Miyaura cross-coupling reaction with intermediate 3 leads
to compounds 4 of
this invention.
41
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VPI/09-116 WO
[00180] Scheme 2:
N N rN N
II
N N N
N
a
A A' A A'
I~ ( CN
j\ NH2
(Rt (Ri)x
R R3
R4 6 R4
Reagents and conditions: a) LiAlH4, THE or alane:dimethyethylamine 0.5 in
toluene, THE
[ 00181] Scheme 2 above shows a general synthetic route for preparing
compounds of
Formula I, IA, and IC of this invention where x, A, A', R1, R3 and R4 are as
described herein and
R2 is CH2NH2 (it is understood that R5 and R6 can also be present and that
ring B substituted with
(R7)y can replace the CR2R3R4 group).. Compounds 6 of this invention can be
prepared by
reduction of the cyano functionality using conditions well known in the art.
[001821 Scheme 3:
RXO\ ORX
Br
a N H
N
A) A' A ~A + (~ \ \N
RZ RZ N
Br /
R3 Br
7 R4 8 R3 CI RX is -H or two RX
R4 9 together form.
rN N N H
N ri N
b N N
C
A A' A A'
I Rz RZ
Br (R1)z
R3 Rs
R4 11 R4
42
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Reagents and conditions: a) B(OR7)2(OMe), `PrMgCI.LiCI, THF, -20 C; b)
Na2CO3, Pd[P(tBu)3]2, dioxane, 70 C; c) R'B(OH)2, Na2CO3, Pd(PPh3)4, DME,
mwave
irradiations, 150 C.
[00183] Scheme 3 above shows a general synthetic route for preparing compounds
of
Formula I, IA, and IC of this invention where x, A, A', R1, R2, R3 and R4 are
as described herein (it
is understood that R5 and R6 can also be present and that ring B substituted
with (R7)y can replace
the CR2R3R4 group).. Derivatives 8, obtained by boronation of 7, undergo
Suzuki-Miyaura cross-
coupling reactions to form compounds of formula 10. After introducing R'
substituent by reactions
well known in the art (e.g. Knochel or Suzuki-Miyaura), compounds 11 of this
invention were
obtained.
Scheme 4:
N
/ CI N CI N CI N N
N / a NI R4 b R5 R4 C NI N
p Rs
C112 CI 13 OH CI 14 p 15 CI
d
H R
N CI 15 R5
N N Ng RXis-HortwoRX
N rN
\ \ together form:
/ N R'O~ OR'
N N '7'L B
i
R5 f Rs
A~ A' e A A'
NHP
NHz NHP (R
(R1) (Rj Ry
R R3 R2 R~ 16 Rz
18 Z 17
43
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Reagents and conditions: a) LDA, THF, aldehyde, -78 C; b) Cr03, acetone, 0 C ;
c)
NH2NH2, THF, pressure tube, 90 C; d) Na2CO3, Pd(PPh3)4, dioxane, mwave
irradiation, 150 C; e)
deprotection conditions.
[00184] Scheme 4 above shows another general synthetic route for preparing
compounds of
Formula I, IA, and IC of this invention where x, A, A', R1, R2, R3, and R5 are
as described herein
(it is understood that R6 can also be present). Intermediate 12 was
transformed to the alcohol 13
and then oxidized to ketone 14. This was cyclised using hydrazine to give 15
which was coupled
with intermediate 16 using Suzuki-Miyaura cross-coupling reactions to yield
compounds 17.
Compounds 18 of this invention were finally obtained after deprotection.
Scheme 5:
O-Na' I
N a N b H2N N C N N d N
~ ~CF3 N N 0 \ N N N
0 0 0 0 \CF3 CF3 OH CF3
0 0 21
17 18 19 20
N H
N\ N r N
R'O. ORx ,N N - N
N~ N 6N ~
e 3
N / iN + A/-A' f~ A~ A CF3 F3 g A~ A CF
CF3 INHP . õx= L/ I NHz
(Rt)x/ R3 ~w urn (Rt)x/ R NHP 22 R R3
22 23 Rz Rz 3 z
24 25
Reagents and conditions: a) NaOEt, EtOOCCF3, RT; b) tBuNHNH2, TFA,
dimethylcarbonate
80 C; c) (CH3)2NC(OMe)2, MeCN, 50 C; d) NH3, MeOH, 100 C in a sealed tube; e)
S(O)CI2,
DMF, 77 C, overnight; f) Na2CO3, [P(tBu3)3]2, dioxane, 60 C; g) deprotection
conditions.
[00185] Scheme 5 above shows another general synthetic route for preparing
compounds of
Formula I, IA, and IC of this invention where x, A, A', R1, R2 and R3 are as
described herein (it is
understood that R6 can also be present). Intermediate 19 was prepared by two-
step process that
involves condensation reaction and cyclazation using hydrazine. The
intermediate 19 was cyclazed
to produce intermediate 21 by condensing 19 with NN, dimethylformamide
dimethylacetal and
then treated with ammonia under pressurize conditions. Intermediate 21 was
transformed in a
44
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chloro derivative using thionyl chloride. Compounds of formula 21 then
underwent a Suzuki-
Miyaura cross-coupling reaction with intermediate 23 to lead to compounds 24.
Compounds 25 of
this invention were finally obtained after deprotection.
[00186] Scheme 6:
~Br Br
O 0 A/ \\A A/ \ A
a / b I __j
O 0 OB NHP
26 27 28
p 29
0 O
R`O~ B /OR"
RX is -H or two RX
together form:
s7~ A ~A'
NHP
N N P9
N 30
N NrN N 0
II N
CF3 CF3 P9
A A f A / A' e rN N
/~ N ~ NHP N (Rl)x (RI).
I 31 CF3
33 32 0
Reagents and conditions: a) 2-triphenylphosphoranylideneacetate, DCM, 0 C -
RT; b) i)3-bromo-
phenyl boronic acid, [Rh(cod)CI]2, dioxane, KOH, ethyl 2-(oxetan-3-ylidene)
acetate, ii) NaOH,
MeOH, 0 C; c) DPPA, triethylamine, tBuOH, 80 C; d) B(OR7)2(OMe),
Pd[dppf)]C12.DCM,
dioxane, KOAc, 90 C; e) Na2CO3, [P(tBu3)3]2, dioxane, 60 C; f) deprotection
conditions.
[00187] Scheme 6 above shows another general synthetic route for preparing
compounds of
Formula I, IA, and IC of this invention where x, A, A', and R1 are as
described herein it is
understood that R6 can also be present). Intermediate 26 was converted to 27
under Wittig reaction
conditions and then coupled to form 28, using Rh as catalyst. A Curtius
reaction gave 29, which
was converted to the boronate 30 and then underwent a Suzuki-Miyaura cross-
coupling reaction
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with intermediate 31 to lead to compounds 32. Final compounds 33 were obtained
after
deprotection.
EXAMPLES
[00188] HPLC Methods
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. Mobile phase for all mass spec. analyses
consisted of 10mM
pH 7 ammonium acetate and a 1:1 acetonitrile-methanol mixture. Column gradient
conditions were
5%-100% acetonitrile-methanol over 3.5 mins gradient time and 4.8 mins run
time on an ACE5C8
3.0 x 75mm column. Flow rate was 1.2 ml/min.
As used herein, the term "Rt(min)" refers to the LCMS retention time, in
minutes, associated
with the compound. Unless otherwise indicated, the LCMS method utilized to
obtain the reported
retention time is as detailed above.
[00189 ] I H-NMR spectra were recorded at 400 MHz using a Bruker DPX 400
instrument.
[001901 The following compounds of Formula I, IA, and IC were prepared and
analyzed as
follows.
Example 1 : 1-(3-(3-cyclopropyl-lH-pyrazolo[3,4-dlpyrimidin-4-yl)phenyl)
cyclopropanecarbonitrile (Compound 1)
N H
H N
(N CI N\ CI Step 2 N CI Step 3 N Step 4 N
Step I N N
N N/ OH N/ 0 N N
CI CI CI CI =N
Compound I
Method A :
Step 1 : Cyclopropyl(4,6-dichloropyrimidin-5-yl)methanol
A solution of diisopropylamine (1.358 g, 1.881 mL, 13.42 mmol) in THE (15 mL)
was allowed to
stir at -78 C and treated with nBuLi (5.704 g, 8.388 mL of 1.6 M, 13.42 mmol).
The reaction
mixture was allowed to stir at this temp for 5 minutes before being allowed to
warm to 0 C for 30
minutes. The LDA solution was then cooled to -78 C before a previously
prepared solution of 4,6-
dichloropyrimidine (2 g, 13.42 mmol) in THE (12 mL) was added dropwise over 15
minutes. The
mixture was then allowed to stir at -78 C for 45 minutes before being treated
dropwise with a
46
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solution of cyclopropanecarbaldehyde (987.6 mg, 1.053 mL, 14.09 mmol) in THE
(10 mL). The
reaction was stirred at -78 C for 1 hour and then diluted with H20/EtOAc and
allowed to warm to
ambient temperature overnight. The organic layer was separated, washed with
saturated brine,
dried (MgSO4) and concentrated in vacuo. The resultant residue was purified by
column
chromatography (ISCO Companion TM, 80g column, EtOAc / Petroleum ether) to
give the required
product (1.092g, 37% Yield). 'H NMR (CDC13, 400 MHz) 6 0.43 (4H, m), 0.79 (1H,
m), 3.09 (1H,
m), 4.53 (1H, m), 8.68 (IH, s); MS (ES) 221.81
Step 2 : Cyclopropyl(4,6-dichloropyrimidin-5-yl)methanone
A solution of cyclopropyl-(4,6-dichloropyrimidin-5-yl)methanol (I g, 4.565
mmol) in anhydrous
acetone (20 mL) was allowed to stir at 0 C and treated with Cr03 (1.461 g,
541.1 L, 14.61
mmol). The reaction was allowed to stir at this temperature for 40 minutes and
then treated with
isopropanol (4mL) and stirred for a further 10 minutes before being diluted
with saturated
NaHCO3. The reaction was filtered through a celite pad and washed with EtOAc.
The organics
were separated, washed with saturated NaCl, dried (MgSO4), filtered and
concentrated in vacuo to
give an oil. The resultant residue was purified by column chromatography (ISCO
CompanionTM,
40g column, EtOAc / Petroleum ether) to give the required product (771 mg, 78%
Yield). ' H NMR
(CDC13, 400 MHz) 6 1.18 (2H, m), 1.37 (2H, m), 2.19 (1 H, m), 8.75 (1 H, s);
MS (ES) 217.94
Step 3 : 4-chloro-3-cyclopropyl-IH-pyrazolo[3,4-d]pyrimidine
A solution of cyclopropyl-(4,6-dichloropyrimidin-5-yl)methanone (771 mg, 3.552
mmol) in THE
(30 mL) was allowed to stir at RT and treated with anhydrous hydrazine (1M in
THF) (4.191 mL
of 1M, 4.191 mmol). The mixture was stirred at ambient temperature for 4 hours
and then diluted
with EtOAc/H20 and the organic layer washed with saturated brine, dried
(MgSO4), filtered and
concentrated in vacuo. The resultant residue was purified by column
chromatography (ISCO
CompanionTM, 40g column, MeOH / DCM) to give the required product (296mg, 43%
Yield).
'H NMR (DMSO, 400 MHz) 6 0.97 - 1.09 (5H, m), 8.75 (1H, s), 14.10 (1H, br s);
MS (ES)
195.00
Step 4 : 1-(3-(3-cyclopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)
cyclopropanecarbonitrile
A solution of 4-chloro-3-cyclopropyl-IH-pyrazolo[3,4-d]pyrimidine (290 mg,
1.490 mmol) in
dioxane (30 mL) was treated with 1-[3-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-
2-
47
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yl)phenyl]cyclopropanecarbonitrile (445.6 mg, 1.490 mmol) and sodium carbonate
(2.235 mL of 2
M, 4.470 mmol). The reaction mixture was degassed (vacuum/nitrogen cycles) and
then treated
with Pd[P(tBu)3]2 (114.2 mg, 0.2235 mmol) and the reaction heated at 70 C for
a total of 7 hours.
The mixture was allowed to cool to RT and diluted with EtOAc/H2O. The organics
were separated,
washed with saturated NaCl, dried (MgSO4), passed through a short SiO2 pad and
concentrated to
give an oil. This was purified by column chromatography (ISCO CompanionTM,
120g column, 0-
6% MeOH / DCM) to give the required product (54.2mg, 12% Yield).
' H NMR (DMSO, 400 MHz) 6 13.82 (brs, 1 H), 8.99 (s, I H), 7.88 (m, 2H), 7.65 -
7.54 (m, 2H),
1.89 - 1.77 (m, 3H), 1.66 - 1.57 (m, 2H) and 1.05 - 0.85 (m, 4H) ppm; MS (ES)
302.0
The following compounds were prepared in general by a similar route to that
described in Example
I.
Compound 2
Compound 4
Example 2 : 1-(3-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)cyclopropane
carbonitrile
(Compound 3) N N
H ii N
(NyCI Step I (i N\ N=N Step 2 N
N ro/H N
CI O CI - N
Compound -3
Method B
Step 1 : 4-chloro-1 H-pyrazolo[3,4-d]pyrimidine
A suspension of 4,6-dichloropyrimidine-5-carbaldehyde (500 mg, 2.825 mmol) in
THE (IOmL)
was allowed to stir at RT and treated with 3A sieves and hydrazine (3.461 g,
3.390 mL of I M in
THF, 3.390 mmol) followed by triethylamine (571.7 mg, 787.5 .tL, 5.650 mmol).
The reaction was
allowed to stir at RT for 10 minutes before being heated to 160 C for 20
minutes in the
microwave. The mixture was diluted with EtOAc/water and the organic layer
washed with
48
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saturated NaCl, dried (MgSO4), and concentrated in vacuo. This was purified by
column
chromatography (ISCO CompanionTM, 40g column, MeOH / DCM) to give the required
product
(117mg, 27% Yield).
I H NMR (DMSO, 400 MHz) 8 8.45 (1 H, s), 8.84 (1 H, s), 14.54 (1 H, br s) ppm;
MS (ES) 154.96
Step 2 : 1-(3-(1H-pyrazolo[3,4-d]pyrimidin-4-
yl)phenyl)cyclopropanecarbonitrile
A solution of 4-chloro-1 H-pyrazolo[3,4-d]pyrimidine (110 mg, 0.7117 mmol) in
dioxane (20 mL)
was treated with 1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl]cyclopropanecarbonitrile (230.7 mg, 0.7715 mmol) and then 2M sodium
carbonate
(1.068 mL of 2 M, 2.135 mmol). The reaction mixture was degassed
(vacuum/nitrogen cycles) and
then treated with Pd[P(tBu)3]2 (36.37 mg, 0.07117 mmol) and the reaction
heated at 70 C
overnight. The reaction mixture was allowed to cool to room temperature and
diluted with
EtOAc/H20. The organics were separated, washed with saturated NaCl, dried
(MgSO4), passed
through a short silica pad and concentrated to give an oil. This was purified
by column
chromatography (ISCO Companion TM, 80g column, 0-10% MeOH / DCM) to give the
required
product (2.8mg, 2% Yield).
'H NMR (DMSO, 400 MHz) 8 1.65 - 1.69 (m, 2H), 1.84 - 1.87 (m, 2H), 7.57 (d, J
= 8.2 Hz, 1 H),
7.67 (t, J = 8.0 Hz, I H), 8.24 - 8.26 (m, 2H), 8.69 (s, 1 H), 9.09 (s, I H)
and 14.27 (brs, 1 H) ppm;
MS (ES-) 261.0
Example 3 : (1-(3-(3-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)phenyl)cyclopropyl)
methanamine (Compound 5)
N CI Method A N H Method C N
Steps I-3 Step 1 \ / Step 2 - NN Step 3 NN
N / N N N
CI CI CI
I =N ~ I NHS
N\
r N
Step 4 N
I
NHp
Compound -5
Compound 5 was prepared using Method A, Steps 1-3 followed by Method C, Steps
1-4.
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Method C :
Step 1 : 4-chloro-3-ethyl-I-trityl-1H-pyrazolo[3,4-d]pyrimidine
4-chloro-3-ethyl-IH-pyrazolo[3,4-d]pyrimidine (1.96 g, 10.73 mmol) was
dissolved in dry DMF
(30 mL) and cooled in an ice-bath. Sodium hydride (472.0 mg, 11.80 mmol) was
added in one
portion and the resultant mixture was stirred at 0 C for 40 minutes. Trityl
chloride (3.142 g, 11.27
mmol) was added in one portion and the resultant solution was stirred at 0 C
for 60 minutes. The
reaction mixture was concentrated under reduced pressure and the residue was
partitioned between
DCM and brine. The aqueous layer was extracted with DCM and the combined
organics were
washed with brine, dried over MgSO4, filtered and concentrated in vacuo. This
was purified by
column chromatography (ISCO CompanionTM, 120g column, DCM) to give the
required product
(2.90g, 64% Yield). 'H NMR (DMSO, 400 MHz) 6 1.34 (3H, t), 3.11 (2H, q), 7.27
(15H, m) and
8.42 (1 H, s) ppm; MS (ES) 425.00
Step 2 : 1-(3 -(3 -ethyl- I -trityl- I H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)
cyclopropanecarbonitrile
A solution of 4-chloro-3 -ethyl- I -trityl-pyrazolo[3,4-d]pyrimidine (255.7
mg, 0.6017 mmol) in
dioxane (12 mL) was treated with 1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-
yl)phenyl]cyclopropanecarbonitrile (100 mg, 0.3344 mmol) and then 2M sodium
carbonate (978.0
L of 2 M, 1.956 mmol). The reaction mixture was degassed (vacuum/nitrogen
cycles) and then
treated with Pd[P(tBu)3]2 (50 mg, 0.09784 mmol) and heated at 70 C for 3
hours. The reaction
was allowed to cool to RT and diluted with EtOAc/H20. The organics were
separated, washed
with saturated NaCl, dried (MgS04), and concentrated in vacuo. The resultant
residue was purified
by column chromatography (ISCO CompanionTM, 40g column, EtOAc / Petroleum
ether) to give
the required product (209mg, 65% Yield). 'H NMR (DMSO, 400 MHz) 6 0.64 (3H,
t), 0.71 (4H,
m), 2.54 (2H, q), 7.03 - 7.09 (15H, m), 7.25 - 7.31 (4H, m) and 8.49 (1H, s)
ppm; MS (ES) 532.21
Step 3 : (1-(3-(3-ethyl-I-trityl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)phenyl)cyclopropyl)
methanamine
1-[3-(3-ethyl-I-trityl-pyrazolo[3,4-d]pyrimidin-4-
yl)phenyl]cyclopropanecarbonitrile (208 mg,
0.3912 mmol) was dissolved in dry THE (30 mL) and cooled in an ice-bath.
Alane:dimethylethylamine, 0.5M in PhMe (4.694 mL of 0.5 M, 2.347 mmol) was
added slowly
dropwise and the resultant mixture was stirred at 0 C and allowed to warm to
RT overnight. The
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reaction was allowed to cool to 0 C and carefully quenched with a THF:H20
mixture (1:1) to give
a suspension which was passed through a celite pad and washed with
EtOAc/saturated NaCl. The
organic layer was separated and the aqueous layer extraced with EtOAc. The
organics were
combined, dried (MgSO4), and concentrated in vacuo. The resultant residue was
purified by
column chromatography (ISCO CompanionTM, 40g column, DCM / MeOH) to give the
required
product (81mg, 39% Yield). 'H NMR (DMSO, 400 MHz) 8 0.89 (4H, m), 0.93 (3H,
m), 2.73 (2H,
m), 2.89 (2H, m), 3.36 (2H, br s), 7.30 (15H, m), 7.53 (3H, m), 7.67 (1H, m)
and 8.64 (IH, s) ppm;
MS (ES) 536.24
Step 4 : (1-(3-(3-ethyl-IH-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)cyclopropyl)
methanamine
A solution of [1-[3-(3-ethyl-l-trityl-pyrazolo[3,4-d]pyrimidin-4-
yl)phenyl]cyclopropy1]
methanamine (81 mg, 0.1512 mmol) in DCM (20mL) was allowed to stir at 0 C and
treated with
triethylsilane (105.5 mg, 144.9 L, 0.9072 mmol) followed by a solution of TFA
(689.6 mg, 465.9
L, 6.048 mmol) in DCM (IOmL). The mixture was allowed to stir at 0 C for 10
minutes and then
at RT for 30 minutes. The reaction was concentrated in vacuo and then
neutralized by washing
through PL-HCO3 MP-resin SPE cartidges, the fractions were concentrated to
give a solid which
was lyophilised from MeCN/H20 to give a solid (44mg, 77% Yield). 'H NMR (DMSO,
400 MHz)
8 8.88 (d, J = 3.6 Hz, IH), 7.68 - 7.43 (m, 4H), 3.50 - 3.20 (m, 4H), 2.79 (m,
2H) and 0.98 - 0.68
(m, 7H) ppm; MS (ES) 294.0
The following compound was prepared in general by a similar route to that
described in Example
3.
Compound 6
Example 4 : 1-(3-(3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)
cyclopropanecarboxamide (Compound 7)
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4- 4-
O"Na'
N4,,,~ S eth'd D N CFa Step 2 H2N N,N Step 3 NON N N Step 4 NYY N
-'-0 0 0 O CF3 CF3 OH \CF
0 0
//
N
N N
N i N N iN
Step 5 N 1 NN Step 6 CF3 Step 7 CF3
N I 1 0
CI CFa N NHz
Compound -7
Method D :
Step 1 : Sodium 3-cyano-4-ethoxy-1,1,1-trifluoro-4-oxobutan-2-olate
A solution of sodium ethoxide (previously prepared by dissolving sodium (2.4
g, 104.4 mmol) in
EtOH (40 mL, 685.1 mmol)) was treated dropwise with ethyl 2-cyanoacetate
(11.36 g, 10.71 mL,
100.4 mmol) at RT. The reaction was allowed to stir at RT for 45 minutes
before ethyl
trifluoroacetate (14.97 g, 105.4 mmol) was added dropwise with stirring. The
mixture was then
allowed to stir at RT for a further 3 hours and then concentrated in vacuo
(25.72g, quantitative
yield). 'H NMR (DMSO, 400 MHz) 6 1.16 (3H, m), 4.01 (3H, m), 4.37 (IH, m) ppm
Step 2 : Ethyl 5-amino- I -tert-butyl-3-(trifluoromethyl)- I H-pyrazole-4-
carboxylate
A mixture of ethyl 2-cyano-3-hydroxy-4,4,4-trifluoro-2-butenoate sodium salt
(15 g, 64.34 mmol),
tert-butylhydrazine (14.43 g, 115.8 mmol), TFA (15.04 g, 10.16 mL, 131.9 mmol)
and 3A
molecular sieves (28g) in dimethyl carbonate (140 mL) was heated at 80 C
overnight. The
reaction mixture was allowed to cool to RT, diluted with EtOAc and filtered
through celite. The
filtrate was concentrated in vacuo and the residue diluted with EtOAc and
washed with saturated
NaHCO3, 5% NaOH, H2O followed by saturated NaCI, dried (MgSO4), filtered and
concentrated
in vacuo to give an oil (1.781g, 10% Yield). 'H NMR (CDC13, 400 MHz) 6 1.35
(3H, m), 1.65
(9H, m), 4.29 (2H, m), 5.50 (2H, br s); MS (ES) 280.10
Step 3 : Ethyl I-tert-butyl-5-((dimethylamino)methyleneamino)-3-
(trifluoromethyl)-IH-pyrazole-
4-carboxylate
52
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A solution of ethyl 5-amino-l-tert-butyl-3-(trifluoromethyl)pyrazole-4-
carboxylate (1.78 g, 6.374
mmol) in MeCN (100.0 mL) was treated with N,N-dimethy I foram ide dimethyl
acetal (7.595 g,
8.467 mL, 63.74 mmol) and the mixture heated to 50 C for 5 hours. The reaction
was allowed to
cool, concentrated in vacuo and purified by column chromatography (ISCO
CompanionTM, 120g
column, EtOAc/ Petroleum ether) to give the product as an oil which solidified
on standing
(1.063g, 50% Yield). 'H NMR (CDCI3, 400 MHz) S 1.23 (3H, m), 1.56 (9H, m),
3.03 (6H, s), 4.14
(2H, m), 7.72 (1H, s); MS (ES) 335.13
Step 4 : 1-tert-butyl-3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol
A solution of ethyl 1-tert-butyl-5-(dimethylaminomethyleneamino)-3-
(trifluoromethyl) pyrazole-4-
carboxylate (1 g, 2.991 mmol) in MeOH (4mL) was dissolved in NH3 in MeOH
(22.43 mL of 2
M, 44.86 mmol) and then transferred to a bomb which was heated at 100 C
overnight. Analysis
showed starting material remaining so the reaction was charged with further 2M
NH3 (20mL)
added and heated to 165 C overnight. The reaction mixture was concentrated in
vacuo and the
residue taken up in formamide (20mL), treated with ammonium carbonate (344.9
mg, 3.589
mmol), 3A sieves and heated at 180 C for 48 hours. The reaction mixture was
allowed to cool to
RT, diluted with EtOAc/water, the two layers were separated, dried (MgS04),
filtered and
concentrated in vacuo to give the product as an oil. The residue was purified
by column
chromatography (ISCO Companion TM, 120g column, MeOH/DCM) to give the required
product
(628mg, 81% Yield). 'H NMR (CDCI3, 400 MHz) S 1.73 (9H, s), 7.98 (1 H, s),
11.76 (1 H, br s);
MS (ES) 261.05
Step 5 : 1-tert-butyl-4-chloro-3-(trifluoromethyl)-1H-pyrazolo[3,4-
d]pyrimidine
DMF (168.6 mg, 178.6 L, 2.306 mmol) was added dropwise to a mixture of I-tert-
butyl-3-
(trifluoromethyl)pyrazolo[3,4-d]pyrimidin-4-ol (300 mg, 1.153 mmol) and
thionyl chloride (4.938
g, 3.028 mL, 41.51 mmol) and the reaction mixture heated at 77 C for 16 hours.
The reaction was
allowed to cool and concentrated in vacuo. The residue was diluted with
EtOAc/saturated NaHCO3
and the two layers separated. The organics were washed with water followed by
saturated NaCl,
dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by
column
chromatography (ISCO CompanionTM, 40g column, EtOAc/ Petroleum ether) to give
the required
product (239mg, 74% Yield). 'H NMR (CDCI3, 400 MHz) S 1.78 (9H, s), 8.75 (1 H,
s); MS (ES)
279.01
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Step 6 : 1-(3-(1-tent-butyl-3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)
phenyl)cyclopropanecarbonitrile
A solution of 1-tert-butyl-4-chloro-3-(trifluoromethyl)pyrazolo[3,4-
d]pyrimidine (238 mg, 0.8541
mmol) in dioxane (30.01 mL) was treated with 1-[3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-
yl)phenyl]cyclopropane-l-carbonitrile (255.4 mg, 0.8541 mmol) and then 2M
sodium carbonate
(1.281 mL of 2 M, 2.562 mmol). The reaction mixture was degassed
(nitrogen/vacuum cycles) and
then treated with Pd[P(tBu)3]2 (65.47 mg, 0.1281 mmol) and allowed to heat at
67 C for 5 hours.
The reaction was allowed to cool and diluted with EtOAc/water and the two
layers separated. The
organics were washed with saturated NaCl, dried (MgSO4), filtered and
concentrated in vacuo. The
residue was purified by column chromatography (ISCO CompanionTM, 120g column,
EtOAc/
Petroleum ether) to give the required product (226mg, 69% Yield). 'H NMR
(CDC13, 400 MHz) 6
1.54 (2H, m), 1.92 (9H, s), 7.30 (3H, m), 7.68 (1H, m), 9.14 (1H, s); MS (ES+)
388.96
Step 7 : 1-(3-(3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)
cyclopropanecarboxamide
1-[3-[ 1-tert-butyl-3-(trifluoromethyl)pyrazolo[3,4-d]pyrimidin-4-yl]phenyl]
cyclopropanecarbonitrile (225 mg, 0.5838 mmol) was treated with methane
sulfonic acid (7.406 g,
5.001 mL, 77.06 mmol) and the reaction allowed to heat at 60 C for 2 hours.
The mixture was
allowed to cool to RT, poured onto ice and made basic (pH 8) by addition of
solid NaHCO3. This
was treated with EtOAc/water and the two layers separated. The organics were
washed with water
followed by saturated NaCI, dried (MgSO4), filtered and concentrated in vacuo.
The residue was
purified by column chromatography (ISCO CompanionTM, 40g column, DCM/MeOH) to
give the
required product (72mg, 36% Yield). 'H NMR (DMSO, 400 MHz) 6 1.54 (2H, m),
1.92 (9H, s),
7.30 (3H, m), 7.68 (1H, m), 9.14 (1H, s); MS (ES) 388.96
Example 5 : (1-(3-(3-ethyl-1H-pyrazolo[3,4-dIpyrimidin-4-yl)-5-fluorophenyl)
cyclobutyl)methanamine (Compound 8)
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\ I / / N NN
N CI Method A N N Method E N. Step 2 N
Step 1 N
Steps I-3 N
Method C
CI Step I CI O F NH2
F NO
H Compound -8
Compound 1-8 was prepared using Method A, Steps 1-3 followed by Method C, Step
1 followed
by Method E, Steps 1-2.
Method E :
Step 1 : Tert-butyl (1-(3-(3-ethyl-l-trityl-IH-pyrazolo[3,4-d]pyrimidin-4-yl)-
5-
fluorophenyl)cyclobutyl)methylcarbamate
A solution of 4-chloro-3-ethyl-l-trityl-pyrazolo[3,4-d]pyrimidine (1 g, 2.353
mmol) in dioxane
(100 mL) was treated with tert-butyl N-[[I-[3-fluoro-5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-
yl)phenyl]cyclobutyl]methyl]carbamate (1.431 g, 3.530 mmol) followed by 2M
sodium carbonate
(3.530 mL of 2 M, 7.059 mmol). The reaction mixture was degassed
(nitrogen/vacuum cycles) and
then treated with Pd[P(tBu)3]2 (180.4 mg, 0.3530 mmol) and heated at 67 C for
24 hours. The
mixture was allowed to cool and diluted with EtOAc/water and the two layers
separated. The
organics were washed with saturated NaCl, dried (MgSO4), filtered and
concentrated in vacuo. The
residue was purified by column chromatography (ISCO Companion TM, 120g column,
EtOAc/
Petroleum ether) to give the required product (1.407g, 90% Yield). IH NMR
(CDC13, 400 MHz) S
1.54 (2H, m), 1.92 (9H, s), 7.30 (3H, m), 7.68 (1H, m), 9.14 (IH, s); MS (ES)
388.96
Step 2 :(1-(3-(3 -ethyl- I H-pyrazolo[3,4-d]pyrim idin-4-yl)-5-
fluorophenyl)cyclobutyl)
methanamine
A solution of tert-butyl N-[[1-[3-(3-ethyl-l-trityl-pyrazolo[3,4-d]pyrimidin-4-
yl)-5-fluoro-
phenyl]cyclobutyl]methyl]carbam ate (1.4 g, 2.096 mmol) in DCM (40 mL) was
cooled to 0 C.
and treated with triethylsilane (974.9 mg, 1.339 mL, 8.384 mmol) followed by a
solution of TFA
(2.390 g, 1.615 mL, 20.96 mmol) in DCM (IOmL). The reaction mixture was then
allowed to
warm to RT overnight, concentrated in vacuo and purified by column
chromatography (ISCO
CompanionTM, 80g column, 95:5:1 DCM/MeOH/NH4OH) to give the required product
(385mg,
53% Yield). 'H NMR (CDCI3, 400 MHz) S 8.97 (s, IH), 7.40 (d, J = 8.7 Hz, I H),
7.28 (s, 1H),
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7.13 (d, J = 9.8 Hz, I H), 3.70 (brs, 2H), 2.85 (brs, 2H), 2.78 (q, J = 7.5
Hz, 2H), 2.32 - 1.64 (m,
6H) and 0.98 (t, J = 7.5 Hz, 3H) ppm; MS (ES) 326Ø
[00191 ] Table 2 below depicts data for certain exemplary compounds made in
general by a
similar route to that outlined in the Examples above.
M +1 RT
# (obs) (min) 1 H-NMR
H NMR (400.0 MHz, DMSO) d 13.82 (brs, 1 H), 8.99 (s, 1 H), 7.88 (m, 2H), 7.65 -
1 302 2.87 7.54 (m, 2H), 1.89 - 1.77 (m, 3H), 1.66 - 1.57 (m, 2H) and 1.05 -
0.85 (m, 4H) m
H NMR (400.0 MHz, DMSO) d 13.90 (brs, 1 H), 8.99 (s, 1 H), 7.80 - 7.51 (m,
4H),
2 290 2.78 2.78 , 2H), 1.86 (m, 2H), 1.63 (m, 2H) and 1.00 (t, 3H) m
H NMR (400.0 MHz, DMSO) d 1.65 - 1.69 (m, 2H), 1.84 - 1.87 (m, 2H), 7.57 (d, J
= 8.2 Hz, 1 H), 7.67 (t, J = 8.0 Hz, 1 H), 8.24 - 8.26 (m, 2H), 8.69 (s, 1 H),
9.09 (s,
3 261 2.74 1 H and 14.27 (brs, 1 H m
H NMR (400.0 MHz, DMSO) d 1.60 (m, 2H), 1.85 (m, 2H), 2.36 (s, 3H), 7.52 (m,
4 276 2.61 2H), 7.78 (m, 2H), 8.99 (s, 1 H and 13.85 (brs, 1 H m
H NMR (400.0 MHz, DMSO) d 8.88 (d, J = 3.6 Hz, 1 H), 7.68 - 7.43 (m, 4H), 3.50
-
294 2.06 3.20 (m, 4H), 2.79 (m, 2H) and 0.98 - 0.68 (m, 7H) m
H NMR (400.0 MHz, DMSO) d 0.69 - 0.90 (m, 4H), 2.37 (s, 3H), 2.78 (s, 2H),
3.09
6 280 1.89 - 3.72 (brs, 2H), 7.43 - 7.73 (m, 4H) and 8.91 (s, 1 H) m
H NMR (400.0 MHz, DMSO) d 15.19 (brs, 1 H), 9.20 (s, 1 H), 7.67 - 7.54 (m,
4H),
7 348 2.66 7.16 (s, 1 H), 6.13 (s, 1 H), 1.39 - 1.36 (m, 2H) and 1.05 - 1.02
(m, 2H) m
H NMR (400.0 MHz, DMSO) d 8.97 (s, 1 H), 7.40 (d, J = 8.7 Hz, 1 H), 7.28 (s, 1
H),
7.13 (d, J = 9.8 Hz, 1 H), 3.70 (brs, 2H), 2.85 (brs, 2H), 2.78 (q, J = 7.5
Hz, 2H),
8 326 2.24 2.32 - 1.64 (m, 6H) and 0.98 (t, J = 7.5 Hz, 3H) m
[00192] Example 6:
[001931 PKC theta
[00194 ] An assay buffer solution was prepared which consisted of 100 mM HEPES
(pH
7.5), 10 mM MgC12, 25 mM NaCI, 0.1 mM EDTA and 0.01% Brij. An enzyme buffer
containing
reagents to final assay concentrations of 0.00001% Triton X-100, 200 g/mL
Phosphatidylserine, 20
pg/mL Diacylglycerol, 360 M NADH, 3 mM phosphoenolpyruvate, 70 g/mL pyruvate
kinase, 24
g/mL lactate dehydrogenase, 2 mM DT-17, 100 M substrate peptide
(ERMRPRKRQGSVRRRV
SEQ ID NO. 1) and 18 nM PKC theta kinase was prepared in assay buffer. To 60
L of this enzyme
buffer, in a 384 well plate, was added 2 L of VRT stock solution in DMSO. The
mixture was
allowed to equilibrate for 10 mins at 30 C. The enzyme reaction was initiated
by the addition of 5
L stock ATP solution prepared in assay buffer to a final assay concentration
of 240 M. Initial rate
data was determined from the rate of change of absorbance at 340 nM
(corresponding to
56
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stoichiometric consumption of NADH) using a Molecular Devices Spectramax plate
reader
(Sunnyvale, CA) over 15 mins at 30 C. For each Ki determination 12 data
points covering the VRT
concentration range of 0 - 20 M were obtained in duplicate (DMSO stocks were
prepared from an
initial 10 mM VRT stock with subsequent 1:2 serial dilutions). Ki values were
calculated from
initial rate data by non-linear regression using the Prism software package
(Prism 4.0a, Graphpad
Software, San Diego, CA). Ki values are represented as A < 0.05 .tM, B < 0.5
M, B* > 0.7 [IM, C
<2.8pM,C**. 1.25pM,D>2.8MM.
A compounds are: 2, 3, 4, 5, and 8.
B compounds are: 1 and 6.
B* compounds are: 7.
[001951 PKC Delta
[00196] An assay buffer solution was prepared which consisted of 100 mM HEPES
(pH
7.5), 10 mM MgCI2, 25 mM NaCl, 0.1 mM EDTA and 0.01% Brij. An enzyme buffer
containing
reagents to final assay concentrations of 0.002% Triton X-100, 200 g/mL
Phosphatidylserine, 20
g/mL Diacylglycerol, 360 pM NADH, 3 mM phosphoenolpyruvate, 70 g/mL pyruvate
kinase, 24
g/mL lactate dehydrogenase, 2 mM DTT, 150 M substrate peptide
(ERMRPRKRQGSVRRRV
SEQ ID NO. 2) and 46 nM PKC delta kinase was prepared in assay buffer. To 16
L of this enzyme
buffer, in a 384 well plate, was added 1 L of VRT stock solution in DMSO. The
mixture was
allowed to equilibrate for 10 mins at 30 C. The enzyme reaction was initiated
by the addition of 16
L stock ATP solution prepared in assay buffer to a final assay concentration
of 150 MM. Initial rate
data was determined from the rate of change of absorbance at 340 nM
(corresponding to
stoichiometric consumption of NADH) using a Molecular Devices Spectramax plate
reader
(Sunnyvale, CA) over 15 mins at 30 C. For each Ki determination 12 data
points covering the VRT
concentration range of 0 - 20 M were obtained in duplicate (DMSO stocks were
prepared from an
initial 10 mM VRT stock with subsequent 1:2 serial dilutions). Ki values were
calculated from
initial rate data by non-linear regression using the Prism software package
(Prism 4.0a, Graphpad
Software, San Diego, CA).
B compounds are: 5 and 8.
C compounds are 3 and 6.
C** compounds are 1, 4 and 7.
[00197] PKC Alpha
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[00198] An assay buffer solution was prepared which consisted of 100 mM HEPES
(pH
7.5), 10 mM MgC12, 25 mM NaCl, 0.1 mM EDTA, 100 M CaC12 and 0.01% Brij. An
enzyme
buffer containing reagents to final assay concentrations of 0.002% Triton X-
100, 100 g/mL
Phosphatidylserine, 20 g/mL Diacylglycerol, 360 pM NADH, 3 mM
phosphoenolpyruvate, 70
g/mL pyruvate kinase, 24 pg/mL lactate dehydrogenase, 2 mM DTT, 150 M
substrate peptide
(RRRRRKGSFKRKA SEQ ID NO. 3) and 4.5 nM PKC alpha kinase was prepared in assay
buffer.
To 16 L of this enzyme buffer, in a 384 well plate, was added 1 L of VRT
stock solution in
DMSO. The mixture was allowed to equilibrate for 10 mins at 30 C. The enzyme
reaction was
initiated by the addition of 16 L stock ATP solution prepared in assay buffer
to a final assay
concentration of 130 M. Initial rate data was determined from the rate of
change of absorbance at
340 nM (corresponding to stoichiometric consumption of NADH) using a Molecular
Devices
Spectramax plate reader (Sunnyvale, CA) over 15 mins at 30 C. For each Ki
determination 12 data
points covering the VRT concentration range of 0 - 20 pM were obtained in
duplicate (DMSO
stocks were prepared from an initial 10 mM VRT stock with subsequent 1:2
serial dilutions). Ki
values were calculated from initial rate data by non-linear regression using
the Prism software
package (Prism 4.0a, Graphpad Software, San Diego, CA).
B compounds are: 8.
C compounds are: 5.
C** compounds are: 1, 2, 3, 4, 6, and 7.
[00199] 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 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 rather than by the specific
embodiments that have
been represented by way of example herein.
58
