Note: Descriptions are shown in the official language in which they were submitted.
CA 02639579 2008-10-02
r ~ e
Attorney Docket No. 7 1332.0060 1.PCT
P13 KINASE ANTAGONISTS
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent
Application No.
60/744,269, filed Apri14, 2006, and U.S. Provisional Patent Application No.
60/744,270,
filed Apri14, 2006, both of which are incorporated herein by reference in
their entirety for all
purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] The present invention was supported by a grant from the National
Institutes of
Health (AI44009). The Government has certain rights to the invention.
BACKGROUND OF THE INVENTION
[0003] Phosphoinositide 3-kinases (P13-Ks) catalyze the synthesis of the
phosphatidylinositol (PI) second messengers PI(3)P, PI(3,4)P2, and PI(3,4,5)P3
(PIP3)
(Fruman et al., 1998). In the appropriate cellular context, these three lipids
control diverse
physiological processes including cell growth, survival, differentiation and
chemotaxis
(Katso et al., 2001). The P13-K family comprises 15 kinases with distinct
substrate
specificities, expression patterns, and modes of regulation (Katso et al.,
2001). The class I
P13 -Ks (p 11 0a, p 1100, p 1108, and p 1107) are activated by tyrosine
kinases or G-protein
coupled receptors to generate PIP3, which engages downstream effectors such as
the
Akt/PDK1 pathway, the Tec family kinases, and the Rho family GTPases. The
class II and
III PI3-Ks play a key role in intracellular trafficking through the synthesis
of PI(3)P and
PI(3,4)P2. The PIKKs are protein kinases that control cell growth (mTORC 1) or
monitor
genomic integrity (ATM, ATR, DNA-PK, and hSmg-1).
[0004] The importance of these enzymes in diverse pathophysiology has made the
P13-K
family the focus of intense interest as a new class of drug targets (Ward et
al., 2003). This
interest has been fueled by the recent discovery that p110a is frequently
mutated in primary
tumors (Samuels et al., 2004) and evidence that the lipid phosphatase PTEN, an
inhibitor of
P13-K signaling, is a commonly inactivated tumor suppressor (Cantley and Neel,
1999).
Efforts are underway to develop small molecule P13-K inhibitors for the
treatment of
inflammation and autoimmune disease (pI108, pl 10y, and mTOR), thrombosis
(p110(3), viral
infection (the PIKKs) and cancer (p110a, mTOR, and others). Recently, the
first selective
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Attorney Docket No. 7 13 32.00601.PCT
inhibitors of these enzymes have been reported (Camps et al., 2005; Condliffe
et al., 2005;
Jackson et al., 2005; Knight et al., 2004; Lau et al., 2005; Sadhu et al.,
2003).
[0005] The present invention meets these and other needs in the art by
providing a new
class of P13-Kinase antagonists.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides certain qnovel compounds found to be
effective as
antagonists of P13-Kinases.
[0007] In one aspect, the present invention provides a PI3-Kinase affinity
pocket binding
antagonist (e.g. a P13-Kinase affinity pocket quinazolinone antagonist) or a
P13-kinase
antagonist as set forth in Formula (I), defined below.
[0008] In another aspect, the present invention provides methods of decreasing
the catalytic
activity of a P13-Kinase. The method includes the step of contacting the P13-
Kinase with an
activity decreasing amount of a P13-Kinase affinity pocket binding antagonist
(e.g. a P13-
Kinase affinity pocket quinazolinone antagonist) or a P13-Kinase antagonist of
Formula (I).
[0009] In another aspect, the present invention provides methods of treating
disease
mediated by treating a condition mediated by P13-Kinase activity in a subject
in need of such
treatment. The method includes administering to the subject a therapeutically
effective
amount of a P13-Kinase affinity pocket binding antagonist (e.g. a P13-Kinase
affinity pocket
quinazolinone antagonist) or a P13-Kinase antagonist of Formula (I).
[0010] In another aspect, the present invention provides methods of disrupting
the function
of a leukocyte or disrupting a function of an osteoclast. The method includes
contacting the
leukocyte or the osteoclast with a function disrupting amount of a PI3-Kinase
affinity pocket
binding antagonist (e.g. a P13-Kinase affinity pocket quinazolinone
antagonist) or a P13-
Kinase antagonist of Formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 illustrates structures of representative compounds from eleven
chemotypes
of P13-K inhibitors.
[0012] Figure 2 illstrates structures of isoform-selective P13-K inhibitors.
A. Structure of
ATP in the active site of p 110y, highlighting different regions of the ATP
binding pocket. B.
An alignment of all reported P13-K inhibitor co-crystal structures. Met 804
adopts an up
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Attorney Docket No. i i 332.00601.PCT
conformation in all structures except PIK-39. C. Structures or models of
isoform-selective
P13-K inhibitors bound to pl l0y. D. Structures or models of multi-targeted
P13-K inhibitors
bound to p 1107.
[0013] Figure 3 illustrates the probing of selectivity and an the P13-Kinase
affinity pocket.
A. The structure of PIK-39 bound to p 110y suggests a model for the binding of
IC87114.
PIK-293 and PIK-294 are pyrazolopyrimidine analogs of IC87114. PIK-294
projects a m-
phenol into the affinity pocket, and this compound is more potent against the
class I P13-Ks.
B. (Left) Ratio of IC50 values between mutant and wild-type for p110S
inhibitors and
pl l0a/multi-targeted inhibitors. (Center) Dose response curves for binding of
two p1108
inhibitors to wild-type, M7521, and M752V p110S (Right) Models suggesting the
impact of
the M7521 and M752V mutations in p1108 on the binding of the different classes
of
inhibitors.
[0014] Figure 4. Structures of additional P13-K inhibitors and inactive
analogs.
[0015] Figure 5. IC50 values ( M) for selected P13-K inhibitors against lipid
kinases.
[0016] Figure 6. Inhibition of protein kinases by PI3-K inhibitors. Values
represent %
activity remaining in the presence of 10 M inhibitor. Values are average of
triplicate
measurements. IC50 values are in parenthesis where appropriate ( M).
[0017] Figure 7 sets forth the sequence of a human p1106 kinase.
[0018] Figure 8 sets forth the sequence of a human pl l0y kinase.
[0019] Figure 9 sets forth the sequence of a human p l l0a kinase.
[00201 Figure 10 sets forth the sequence of a human p 110(3 kinase.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
10021] Abbreviations used herein have their conventional meaning within the
chemical and
biological arts.
[0022] Where substituent groups are specified by their conventional chemical
formulae,
written from left to right, they equally encompass the chemically identical
substituents that
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would result from writing the structure from right to left, e.g., -CH2O- is
equivalent to
-OCH2-.
[0023] The term "alkyl," by itself or as part of another substituent, means,
unless otherwise
stated, a straight (i.e. unbranched) or branched chain, or cyclic hydrocarbon
radical, or
combination thereof, which may be fully saturated, mono- or polyunsaturated
and can include
di- and multivalent radicals, having the number of carbon atoms designated
(i.e. Ci-Cio
means one to ten carbons). Examples of saturated hydrocarbon radicals include,
but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-
butyl, isobutyl, sec-
butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers
of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated
alkyl group is one
having one or more double bonds or triple bonds. Examples of unsaturated alkyl
groups
include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-
(butadienyl), 2,4-
pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and
the higher
homologs and isomers.
[0024] The term "alkylene" by itself or as part of another substituent means a
divalent
radical derived from an alkyl, as exemplified, but not limited, by -
CH2CH2CH2CH2-,
-CH2CH=CHCH2-, -CH2C=CCH2-, -CH2CH2CH(CH2CH2CH3)CH2-. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those groups having
10 or fewer
carbon atoms being preferred in the present invention. A "lower alkyl" or
"lower alkylene" is
a shorter chain alkyl or alkylene group, generally having eight or fewer
carbon atoms. An
"alkynylene" is a subset of an alkylene in which the alkylene includes at
least on triple bond
between adjacent carbon atoms.
[0025] The term "heteroalkyl," by itself or in combination with another term,
means, unless
otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon
radical, or
combinations thereof, consisting of at least one carbon atoms and at least one
heteroatom
selected from the group consisting of 0, N, P, Si and S, and wherein the
nitrogen,
phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen
heteroatom may
optionally be quaternized. The heteroatom(s) 0, N, P and S and Si may be
placed at any
interior position of the heteroalkyl group or at the position at which alkyl
group is attached to
the remainder of the molecule. Examples include, but are not limited to, -CHZ-
CH2-O-CH3,
-CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3,
-CHZ-CHZ-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-
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CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be
consecutive,
such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means a divalent
radical derived
from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -
CH2-S-
CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either
or both
of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,
alkylenediamino, and
the like). Still further, for alkylene and heteroalkylene linking groups, no
orientation of the
linking group is implied by the direction in which the formula of the linking
group is written.
For example, the formula -C(O)OR'- represents both -C(O)OR'- and -R'OC(O)-. As
described above, heteroalkyl groups, as used herein, include those groups that
are attached to
the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR',
-NR'R,-
OR', -SR, and/or -SO2R'. Where "heteroalkyl" is recited, followed by
recitations of specific
heteroalkyl groups, such as -NR'R or the like, it will be understood that the
terms heteroalkyl
and -NR'R" are not redundant or mutually exclusive. Rather, the specific
heteroalkyl groups
are recited to add clarity. Thus, the term "heteroalkyl" should not be
interpreted herein as
excluding specific heteroalkyl groups, such as -NR'R" or the like.
[0026] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in
combination
with other terms, represent, unless otherwise stated, cyclic versions of
"alkyl" and
"heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom
can occupy the
position at which the heterocycle is attached to the remainder of the
molecule. Examples of
cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-
cyclohexenyl, 3-
cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not
limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-
piperidinyl, 4-
morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like. The terms
"cycloalkylene"
and "heterocycloalkylene" refer to the divalent derivatives of cycloalkyl and
heterocycloalkyl, respectively.
[0027] The terms "halo" or "halogen," by themselves or as part of another
substituent,
mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
Additionally,
terms such as "haloalkyl," are meant to include monohaloalkyl and
polyhaloalkyl. For
example, the term "halo(C1-C4)alkyl" is mean to include, but not be limited
to,
trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
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[0028] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic,
hydrocarbon substituent which can be a single ring or multiple rings
(preferably from 1 to 3
rings) which are fused together or linked covalently. The term "heteroaryl"
refers to aryl
groups (or rings) that contain from one to four heteroatoms (in each separate
ring in the case
of multiple rings) selected from N, 0, and S, wherein the nitrogen and sulfur
atoms are
optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A
heteroaryl group
can be attached to the remainder of the molecule through a carbon or
heteroatom. Non-
limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-
naphthyl, 4-
biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-
imidazolyl,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-
isoxazolyl, 4-
isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-
furyl, 2-thienyl, 3-
thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-
benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-
quinoxalinyl, 3-
quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and
heteroaryl ring
systems are selected from the group of acceptable substituents described
below. The terms
"arylene" and "heteroarylene" refer to the divalent radicals of aryl and
heteroaryl,
respectively.
[0029] For brevity, the term "aryl" when used in combination with other terms
(e.g.,
aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroaryl rings as
defined above.
Thus, the term "arylalkyl" is meant to include those radicals in which an aryl
group is
attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the
like) including
those alkyl groups in which a carbon atom (e.g., a methylene group) has been
replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-
naphthyloxy)propyl, and the like). However, the term "haloaryl," as used
herein is meant to
cover only aryls substituted with one or more halogens.
[0030] Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a
specific number of
members (e.g. "3 to 7 membered"), the term "member" referrers to a carbon or
heteroatom.
[0031] The term "oxo" as used herein means an oxygen that is double bonded to
a carbon
atom.
[0032] Each of above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl, and
"heterocycloalkyl", "aryl," "heteroaryl" as well as their divalent radical
derivatives) are meant
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to include both substituted and unsubstituted forms of the indicated radical.
Preferred
substituents for each type of radical are provided below.
[0033] Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl
monovalent and
divalent derivative radicals (including those groups often referred to as
alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, and
heterocycloalkenyl) can be one or more of a variety of groups selected from,
but not limited
to: -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -
C(O)R',
-CO2R',-C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)OR',
-NR-C(NR'R")=NR"', -S(O)R', -S(0)2R', -S(0)2NR'R", -NRSOZR', -CN and NO2 in a
number ranging from zero to (2m'+1), where m' is the total number of carbon
atoms in such
radical. R', R", R"' and R"" each preferably independently refer to hydrogen,
substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted
with 1-3 halogens),
substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl
groups. As used
herein, an "alkoxy" group is an alkyl attached to the remainder of the
molecule through a
divalent oxygen radical. When a compound of the invention includes more than
one R group,
for example, each of the R groups is independently selected as are each R',
R", R"' and R""
groups when more than one of these groups is present. When R' and R" are
attached to the
same nitrogen atom, they can be combined with the nitrogen atom to form a 4-,
5-, 6-, or 7-
membered ring. For example, -NR'R" is meant to include, but not be limited to,
l.-
pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one
of skill in the
art will understand that the term "alkyl" is meant to include groups including
carbon atoms
bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -
CH2CF3) and
acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).
[0034] Similar to the substituents described for alkyl radicals above,
exemplary substituents
for aryl and heteroaryl groups ( as well as their divalent derivatives) are
varied and are
selected from, for example: halogen, -OR', -NR'R", -SR', -halogen, -SiR'R"R"',
-OC(O)R',
-C(O)R', -COzR', -C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"',
-NR"C(O)OR', -NR-C(NR'R"R"')=NR ", -NR-C(NR'R")=NR"', -S(O)R', -S(0)2R',
-S(O)ZNR'R", -NRSOZR', -CN and -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxo,
and
fluoro(CI-C4)alkyl, in a number ranging from zero to the total number of open
valences on
aromatic ring system; and where R', R", R"' and R"" are preferably
independently selected
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from hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
When a
compound of the invention includes more than one R group, for example, each of
the R
groups is independently selected as are each R', R", R"' and R"" groups when
more than one
of these groups is present.
[0035] Two of the substituents on adjacent atoms of aryl or heteroaryl ring
may optionally
form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are
independently -NR-,
-0-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
Alternatively, two of the
substituents on adjacent atoms of aryl or heteroaryl ring may optionally be
replaced with a
substituent of the formula -A-(CHZ)r B-, wherein A and B are independently -
CRR'-, -0-,
-NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer
of from 1 to 4.
One of the single bonds of the new ring so formed may optionally be replaced
with a double
bond. Alternatively, two of the substituents on adjacent atoms of aryl or
heteroaryl ring may
optionally be replaced with a substituent of the formula -(CRR')S-X'-(C"R"')d-
, where s and d
are independently integers of from 0 to 3, and X' is -0-, -NR'-, -S-, -S(O)-, -
S(O)2-, or
-S(O)2NR'-. The substituents R, R', R" and R"' are preferably independently
selected from
hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted
cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, and
substituted or unsubstituted heteroaryl.
[0036] As used herein, the term "heteroatom" or "ring heteroatom" is meant to
include
oxygen (0), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
[0037] An "aminoalkyl" as used herein refers to an amino group covalently
bound to an
alkylene linker. The amino group is -NR'R", wherein R' and R" are typically
selected from
hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0038] A "substituent group," as used herein, means a group selected from the
following
moieties:
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[0039] (A) -OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, and
[0040] (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and
heteroaryl,
substituted with at least one substituent selected from:
[0041] (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, and
[0042] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and
heteroaryl,
substituted with at least one substituent selected from:
[0043] (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -NOZ, halogen, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, and
[0044] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl, substituted
with at least one substituent selected from oxo, -OH, -NH2, -SH, -CN, -CF3, -
NO2, halogen,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,
unsubstituted
heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.
[0045] A "size-limited substituent" or " size-limited substituent group," as
used herein
means a group selected from all of the substituents described above for a
"substituent group,"
wherein each substituted or unsubstituted alkyl is a substituted or
unsubstituted Cl-C2o alkyl,
each substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or
unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted
heterocycloalkyl is a
substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
[0046] A "lower substituent" or " lower substituent group," as used herein
means a group
selected from all of the substituents described above for a "substituent
group," wherein each
substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C$
alkyl, each
substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2
to 8 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or
unsubstituted C5-
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C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a
substituted or
unsubstituted 5 to 7 membered heterocycloalkyl.
[0047] The compounds of the present invention may exist as salts. The present
invention
includes such salts. Examples of applicable salt forms include hydrochlorides,
hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates,
citrates, fumarates,
tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures thereof including
racemic mixtures,
succinates, benzoates and salts with amino acids such as glutamic acid. These
salts may be
prepared by methods known to those skilled in art. Also included are base
addition salts such
as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, oi-
a similar
salt. When compounds of the present invention contain relatively basic
functionalities, acid
addition salts can be obtained by contacting the neutral form of such
compounds with a
sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of
acceptable acid addition salts include those derived from inorganic acids like
hydrochloric,
hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric,
hydriodic, or phosphorous acids and the like, as well as the salts derived
organic acids like
acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic,
fumaric, lactic,
mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the
like. Also included are salts of amino acids such as arginate and the like,
and salts of organic
acids like glucuronic or galactunoric acids and the like. Certain specific
compounds of the
present invention contain both basic and acidic functionalities that allow the
compounds to be
converted into either base or acid addition salts.
[0048] The neutral forms of the compounds are preferably regenerated by
contacting the
salt with a base or acid and isolating the parent compound in the conventional
manner. The
parent form of the compound differs from the various salt forms in certain
physical
properties, such as solubility in polar solvents.
[0049] Certain compounds of the present invention can exist in unsolvated
forms as well as
solvated fornis, including hydrated forms. In general, the solvated forms are
equivalent to
unsolvated forms and are encompassed within the scope of the present
invention. Certain
compounds of the present invention may exist in multiple crystalline or
amorphous forms. In
general, all physical forms are equivalent for the uses contemplated by the
present invention
and are intended to be within the scope of the present invention.
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[0050] Certain compounds of the present invention possess asymmetric carbon
atoms
(optical or chiral centers) or double bonds; the enantiomers, racemates,
diastereomers,
tautomers, geometric isomers, stereoisometric forms that may be defined, in
terms of absolute
stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and
individual isomers are
encompassed within the scope of the present invention. The compounds of the
present
invention do not include those which are known in art to be too unstable to
synthesize and/or
isolate. The present invention is meant to include compounds in racemic and
optically pure
forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared
using chiral
synthons or chiral reagents, or resolved using conventional techniques. When
the compounds
described herein contain olefinic bonds or other centers of geometric
asymmetry, and unless
specified otherwise, it is intended that the compounds include both E and Z
geometric
isomers.
[0051] The term "tautomer," as used herein, refers to one of two or more
structural isomers
which exist in equilibrium and which are readily converted from one isomeric
form to
another.
[0052] It will be apparent to one skilled in the art that certain compounds of
this invention
may exist in tautomeric forms, all such tautomeric forms of the compounds
being within the
scope of the invention.
[0053] Unless otherwise stated, structures depicted herein are also meant to
include all
stereochemical forms of the structure; i.e., the R and S configurations for
each asymmetric
center. Therefore, single stereochemical isomers as well as enantiomeric and
diastereomeric'
mixtures of the present compounds are within the scope of the invention.
[0054] Unless otherwise stated, structures depicted herein are also meant to
include
compounds which differ only in the presence of one or more isotopically
enriched atoms. For
example, compounds having the present structures except for the replacement of
a hydrogen
by a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-
enriched carbon are
within the scope of this invention.
[0055] The compounds of the present invention may also contain unnatural
proportions of
atomic isotopes at one or more of atoms that constitute such compounds. For
example, the
compounds may be radiolabeled with radioactive isotopes, such as for example
tritium (3H),
iodine-125 (125I) or carbon-14 (14C) . All isotopic variations of the
compounds of the present
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invention, whether radioactive or not, are encompassed within the scope of the
present
invention.
[0056] The term "pharmaceutically acceptable salts" is meant to include salts
of active
compounds which are prepared with relatively nontoxic acids or bases,
depending on the
particular substituent moieties found on the compounds described herein. When
compounds
of the present invention contain relatively acidic functionalities, base
addition salts can be
obtained by contacting the neutral form of such compounds with a sufficient
amount of the
desired base, either neat or in a suitable inert solvent. Examples of
pharmaceutically
acceptable base addition salts include sodium, potassium, calcium, ammonium,
organic
amino, or magnesium salt, or a similar salt. When compounds of the present
invention
contain relatively basic functionalities, acid addition salts can be obtained
by contacting the
neutral form of such compounds with a sufficient amount of the desired acid,
either neat or in
a suitable inert solvent. Examples of pharmaceutically acceptable acid
addition salts include
those derived from inorganic acids like hydrochloric, hydrobromic, nitric,
carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric,
dihydrogenphosphoric,
sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the
salts derived from relatively nontoxic organic acids like acetic, propionic,
isobutyric, maleic,
malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-
tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included
are salts of amino
acids such as arginate and the like, and salts of organic acids like
glucuronic or galactunoric
acids and the like (see, for example, Berge et al., "Pharmaceutical Salts",
Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the
present
invention contain both basic and acidic functionalities that allow the
compounds to be
converted into either base or acid addition salts.
[0057] In addition to salt forms, the present invention provides compounds,
which are in a
prodrug form. Prodrugs of the compounds described herein are those compounds
that readily
undergo chemical changes under physiological conditions to provide the
compounds of the
present invention. Additionally, prodrugs can be converted to the compounds of
the present
invention by chemical or biochemical methods in an ex vivo environment. For
example,
prodrugs can be slowly converted to the compounds of the present invention
when placed in a
transdermal patch reservoir with a suitable enzyme or chemical reagent.
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[0058] The terms "a," "an," or "a(n)", when used in reference to a group of
substituents
herein, mean at least one. For example, where a compound is substituted with
"an" alkyl or
aryl, the compound is optionally substituted with at least one alkyl and/or at
least one aryl.
Moreover, where a moiety is substituted with an R substituent, the group may
be referred to
as "R-substituted." Where a moiety is R-substituted, the moiety is substituted
with at least
one R substituent and each R substituent is optionally different.
[0059] Description of compounds of the present invention are limited by
principles of
chemical bonding known to those skilled in the art. Accordingly, where a group
may be
substituted by one or more of a number of substituents, such substitutions are
selected so as
to comply with principles of chemical bonding and to give compounds which are
not
inherently unstable and/or would be known to one of ordinary skill in the art
as likely to be
unstable under ambient conditions, such as aqueous, neutral, and several known
physiological
conditions. For example, a heterocycloalkyl or heteroaryl is attached to the
remainder of the
molecule via a ring heteroatom in compliance with principles of chemical
bonding known to
those skilled in the art thereby avoiding inherently unstable compounds.
[0060] The terms "treating" or "treatment" refers to any indicia of success in
the treatment
or amelioration of an injury, pathology or condition, including any objective
or subjective
parameter such as abatement; remission; diminishing of symptoms or making the
injury,
pathology or condition more tolerable to the patient; slowing in the rate of
degeneration or
decline; making the final point of degeneration less debilitating; improving a
patient's
physical or mental well-being. The treatment or amelioration of symptoms can
be based on
objective or subjective parameters; including the results of a physical
examination,
neuropsychiatric exams, and/or a psychiatric evaluation. For example, the
certain methods
presented herein successfully treat cancer by decreasing the incidence of
cancer and or
causing remission of cancer.
[0061] An "effective amount" is an amount sufficient to contribute to the
treatnient,
prevention, or reduction of a symptom or symptoms of a disease. An "effective
amount may
also be referred to as a "therapeutically effective amount." A "reduction" of
a symptom or
symptoms (and grammatical equivalents of this phrase) means decreasing of the
severity or
frequency of the symptom(s), or elimination of the symptom(s). A
"prophylactically
effective amount" of a drug is an amount of a drug that, when administered to
a subject, will
have the intended prophylactic effect, e.g., preventing or delaying the onset
(or reoccurrence)
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a disease, or reducing the likelihood of the onset (or reoccurrence) of a
disease or its
symptoms. The full prophylactic effect does not necessarily occur by
administration of one
dose, and may occur only after administration of a series of doses. Thus, a
prophylactically
effective amount may be administered in one or more administrations. An
"activity
decreasing amount," as used herein, refers to an amount of antagonist required
to decrease the
activity of an enzymne relateive to the absence of the antagonist. A "function
disrupting
amount," as used herein, refers to the amount of antagonist required to
disrupt the function of
an osteoclast or leukocyte relative to the absence of the antagonist.
[0062] As used herein, the "antagonist" or "the compound of the present
invention" refers
to a compound fo Formula (I), or a P13-Kinase affinity pocket binding
antagonist (e.g. a P13-
Kinase affinity pocket quinazolinone antagonist). A "compound of Formula (I)"
includes all
embodiments of Formula (I) as described below.
II. P13-Kinase Antagonists
[0063] In one aspect, the present invention provides novel P13-kinase
antagonists. In some
embodiments, the P13-kinase antagonist is a P13-Kinase affinity pocket binding
antagonist
(e.g. a P13-Kinase affinity pocket quinazolinone antagonist). The P13-Kinase
affinity pocket
binding antagonist of the present invention is a compound containing a P13-
Kinase affinity
pocket binding moiety. The P13-Kinase affinity pocket quinazolinone
antagonists of the
present invention are substituted quinazolinone compounds containing a P13-
Kinase affinity
pocket binding moiety. The P13-Kinase affinity pocket binding moiety is a
substituent
which, upon contacting a p l l 0a, p 110(3, p 110y, or p 1106 kinase, fills
space within the
corresponding P13-Kinase affinity pocket. In some embodiments, the P13-Kinase
affinity
pocket binding moiety displaces at least one water molecule within the P13-
Kinase affinity
pocket. The P13-Kinase affinity pocket binding moiety may also interact with
one or more
amino acids that from part of the P13-Kinase affinity pocket. A description of
the P13-Kinase
affinity pocket and methods of determining whether a substituent fills space
within the P13-
Kinase affinity pocket are set forth below.
[0064] In some embodiments, the P13-Kinase affinity pocket quinazolinone
antagonist
further include a pyrazolopyrimidine substituent or a pyrrolopyrimidine
substituent. In some
related embodiments, the pyrazolopyrimidine substituent or pyrrolopyrimidine
substituent is
covalently bonded to the quinazolinone core, and the P13-Kinase affinity
pocket binding
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moiety is covalently attached to the pyrazolopyrimidine substituent or
pyrrolopyrimidine
substituent.
[00651 In some embodiments, the P13-kinase antagonist of the present invention
has the
formula:
R2)
q
R3 O
N
\ N~ )
z
N N
X~ N
~ Ll R4
R~ (I).
[0066] In Formula (I) above, q is an integer from 0 to 5 (e.g. 1); z is an
integer from 0 to 10
(e.g. 1); and X is =CH- or =N-. Ll is a bond, substituted or unsubstituted
alkylene,
substituted or unsubstituted heteroalkylene, substituted or unsubstituted
cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted
arylene, or
substituted or unsubstituted heteroarylene.
[0067] R' and R2 are independently halogen, -CN, -OR5, -S(O)r,R6, -NR7 RB, -
C(O)R9,
substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an
integer from 0 to
2. R' may also be a P13-Kinase affinity pocket binding moiety. R3 and R4 are
independently
hydrogen, halogen, -CN, -OR5, -S(O)õR6, -NR'Rg, -C(O)R9, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted
or unsubstituted heteroaryl, where n is an integer from 0 to 2.
[0068] R5 is independently hydrogen, -C(O)R10, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R6 is independently hydrogen, -NR11R12, substituted
or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
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cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl. Where n is 1 or 2, R6 is other than
hydrogen.
[0069] R7 is independently hydrogen, substituted or unsubstituted alkyl,
substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
R8 is independently hydrogen, -S(O),,R13, -C(O)R14, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl.
[0070] R9 is independently -NR15R16, hydrogen, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R10 is independently hydrogen, -NR17R18, substituted
or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl.
[0071] R14 is independently hydrogen, -NR19R20, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl; and
15 Ri ,
12 R'3, R ,
[0072] R", R ,
6 R", R18, R'9, and R20 are independently hYdrogen
,
substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0073] In some embodiments, the P13-Kinase affinity pocket binding moiety is
capable of
forming a hydrogen bond with the side chain amine of K833, an amino acid
within the P13-
Kinase affinity pocket.
[0074] In some embodiments, R' is halogen, substituted or unsubstituted
halo(C1-C6)alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or
unsubstituted aryl(C1-C6)alkyl, or substituted or unsubstituted heteroaryl(CI-
C6)alkyl. R'
may also be halogen, substituted or unsubstituted phenyl, substituted or
unsubstituted furanyl,
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substituted or unsubstituted pyrrolyl, substituted or unsubstituted
thiophenyl, or substituted or
unsubstituted benzothiophenyl, substituted or unsubstituted indolyl,
substituted or
unsubstituted quinolinyl, substituted or unsubstituted pyridinyl, substituted
or unsubstituted
1H-pyrrolo[2,3-c]pyridinyl, substituted or unsubstituted 1H-pyrrolo[2,3-
b]pyridinyl,
substituted or unsubstituted thiazolyl, substituted or unsubstituted
imidazolyl, substituted or
unsubstituted oxazolyl, substituted or unsubstituted isoxazolyl, substituted
or unsubstituted
pyrazolyl, substituted or unsubstituted isothiazolyl, substituted or
unsubstituted cylcohexyl,
substituted or unsubstituted morpholino, substituted or unsubstituted
piperidinyl, or
substituted or unsubstituted tetrahydropyridinyl.
[0075] In other embodiments, R' is phenyl, furanyl, pyrrolyl, thiophenyl, or
benzothiophenyl, each of which are optionally substituted with one or more R21
substituent(s). R21 is independently (1) or (2) as defined in this paragraph.
Thus, R21 may be
(1) halogen, -CN, -ORZZ, -C(O)R23, -NR2aR 25, -S(O)WNR26R27, or -S(O)WR 28 .
The yrn
s bol w is
an integer from 0 to 2. RZZ, R23, R24, R25, R26, R27, and RZg are
independently hydrogen,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyl-
alkyl,
heterocycloalkyl-alkyl, arylalkyl, or heteroarylalkyl, optionally substituted
with unsubstituted
alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl,
unsubstituted
heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted
heteroarylalkyl. R21 may
also be (2) (CI-Clo)alkyl, 2 to 10 membered heteroalkyl, C3-C8 cycloalkyl, 3
to 8 membered
heterocycloalkyl, aryl or heteroaryl optionally substituted with halogen, -OH,
-CN, -NH2,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,
unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted
cycloalkyl-alkyl,
unsubstituted heterocycloalkyl-alkyl, unsubstituted arylalkyl, or
unsubstituted
heteroarylalkyl.
[0076] In some embodiments, R' is phenyl substituted at the meta and para
positions, or
substituted at the meta and meta positions. That is, R' is a 4,5-substituted
phenyl or a 3,5-
substituted phenyl. In some related embodiments, the 4,5-substituted phenyl or
3,5-
substituted phenyl is substituted, independently, with R21 (as defined in the
previous
paragraph). In some embodiments, R21 is halogen or -OR22. RZ' may also be
fluorine and R 22
may be hydrogen or unsubstituted CI-Ca alkyl (e.g. methyl). In other
embodiments, Rl is
phenyl substituted para position (i.e. a 4-substituted phenyl).
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Attorney Docket No. 71332.00601.PCT
[0077] In some embodiments, Ll is substituted or unsubstituted alkylene (e.g.
a substituted
or unsubstituted alkynylene. In other embodiments, Ll is substituted or
unsubstituted
methylene, substituted or unsubstituted ethylene, substituted or unsubstituted
propylene,
substituted or unsubstituted butylenes, substituted or unsubstituted
ethynylene, or substituted
or unsubstituted prop-2-ynylene. In some related embodiments, R' is -CN, -ORS,
NR'R8,
R21-substituted or unsubstituted cycloalkyl, R21-substituted or unsubstituted
aryl, R21-
substituted or unsubstituted heteroaryl, R21-substituted or unsubstituted C1-
C4 alkyl. R21 may
be halo en -OR22 NR24Rz5 or unsubstituted C-C4 alk 1. RS R' R8 RZZ R 24 and
R25 ma
g , ~ -, i Y , , , ~ Y
independently be hydrogen or unsubstituted C1-C4 alkyl (e.g. methyl).
[0078] In some embodiments, R2 is halogen, -OH, -CN, -NH2, unsubstituted
alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl,
unsubstituted
heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted
heteroarylalkyl. R2 may also
be halogen or unsubstituted alkyl. In some embodiments, R2 is fluorine or
unsubstituted C1-
C4 alkyl (e.g. methyl).
[0079] R3 may be halogen, -OH, -CN, -NH2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
unsubstituted
heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl-
alkyl, unsubstituted
arylalkyl, or unsubstituted heteroarylalkyl. R3 may also be unsubstituted CI-
C4 alkyl (e.g.
methyl).
[0080] In some embodiments, R4 is halogen, -OH, -CN, -NHz, unsubstituted
allcyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl,
unsubstituted
heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted
heteroarylalkyl.
[0081] In some embodiments, R2 and R3 are independently unsubstituted C1-C4
alkyl, R4 is
NHZ, q is 1, and z is 1.
[0082] In some embodiments, each substituted group described above in the
compound of
Formula (I) is substituted with at least one substituent group. More
specifically, in some
embodiments, each substituted alkyl, substituted heteroalkyl, substituted
cycloalkyl,
substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted cycloalkyl-
alkyl, substituted heterocycloalkyl-alkyl, substituted arylalkyl, and/or
substituted
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heteroarylalkyl, described above in the compounds of Formula (I) is
substituted with at least
one substituent group. In other embodiments, at least one or all of these
groups are
substituted with at least one size-limited substituent group. Alternatively,
at least one or all
of these groups are substituted with at least one lower substituent group.
[0083] In other embodiments of the compounds of Formula (I), each substituted
or
unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, including
those alkyl groups
forming part of a cycloalkyl-alkyl (i.e. a cycloalkyl-(CI -C20)alkyl),
heterocycloalkyl-alkyl
(i.e. a heterocycloalkyl-(C1-CZO)alkyl), arylalkyl (i.e. an aryl-(C1-
CZO)alkyl), or substituted
heteroarylalkyl (i.e. a heteroaryl-(CI-CZO)alkyl). Each substituted or
unsubstituted heteroalkyl
is a substituted or unsubstituted 2 to 20 membered heteroalkyl. Each
substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl,
including those
cycloalkyl groups forming part of a cycloalkyl-alkyl (i.e. a C4-C8 cycloalkyl-
alkyl, or a C4-C8
cycloalkyl-(Cl-C20)alkyl). Each substituted or unsubstituted heterocycloalkyl
is a substituted
or unsubstituted 4 to 8 membered heterocycloalkyl, including those
heterocycloalkyl groups
forming part of a heterocycloalkyl-alkyl (i.e. a 4 to 8 membered
heterocycloalkyl-alkyl, or a 4
to 8 membered heterocycloalkyl-(CI-C20)alkyl).
[0084] Alternatively, each substituted or unsubstituted alkyl is a substituted
or
unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a
substituted or
unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted
cycloalkyl is a
substituted or unsubstituted C5-C7 cycloalkyl, each substituted or
unsubstituted
heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered
heterocycloalkyl,
including cycloalkyl-alkyl groups, heterocycloalkyl-alkyl groups,
heteroarylalkyl groups, and
arylalkyl groups, as described in the preceding paragraph.
[0085] In another embodiment, the compounds of the present invention include
the
compounds of any one or all of those listed in Table 1 below.
III. The P13-Kinase Affinity Pocket
[0086] The term "P13-Kinase affinity pocket," as used herein, refers to a
cavity within
p 110a, p 110(3, p 110y, and p 1108 corresponding to the lightly shaded region
shown in Figures
2A, 2C, and 2D labeled "Affinity Pocket." Figures 2A, 2C, and 2D illustrate a
computer
model of the p110y crystal structure. In p110y, the surface of the P13-Kinase
affinity pocket
is bound, at least in part, by the side chain of K833, D964, I879, and D841
(p110r
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Attorney Docket No. 71332.00601.PCT
numbering, see Figure 8). The surface of the corresponding cavity in p 1105 is
bound, at least
in part, by the side chain of K779, D911, 1825, and D787 (p110S numbering, see
Figure 7).
The corresponding cavity within p110a is bound, at least in part, by the side
chains of K802,
D933, 1848, and D810 (p110a numbering, see Figure 9). The corresponding cavity
within
pl 10(3 is bound, at least in part, by the side chains of K805, D937, I851,
and D813 (p110(3
numbering, see Figure 10). The P13-Kinase affinity pocket is not accessed by
ATP.
[0087] The P13-Kinase affinity pocket of p 110b may be referred to herein as
the p 1108
affinity pocket. Likewise, the P13-Kinase affinity pocket of p l 10y may be
referred to herein
as the p110y affinity pocket. The PI3-Kinase affinity pocket includes lysine
779, which,
according to computer models, forms a hydrogen bond with the pyridine nitrogen
of PIK-90
and the phenol oxygen of PI 103 (Figure 2D), both of which are inhibitors of
p1108. Based
on these computer modeling results, a novel antagonist was designed based on
the chemical
structure of PIK-39 and IC87114, as detailed below.
[0088] As shown in Figure 2C, PIK-39 does not contain a P13-Kinase binding
pocket
moiety. And as shown in Figure 3A, IC87114 maintains contacts to E880 and V882
in the
ATP binding region of p 1105, but is also missing a P13-Kinase binding pocket
moiety. By
inserting m-phenol (a P13-Kinase binding pocket moiety) at the C3 of the
pyrazolopyrimidine
of IC87114, the P13-Kinase affinity pocket is accessed (FIG. 3A) resulting in
a 60-fold
increase in p110S inhibition potency.
[0089] As described above, a P13-Kinase binding pocket moiety is a substituent
which,
upon contacting upon contacting p 110a, p 110[3, p 110y, or p 1108, fills
space within the
corresponding P13-Kinase binding pocket. For example, a P13-Kinase affinity
pocket binding
moiety is a substituent which, upon contacting upon contacting p110S, fills
space within the
p1108 affinity pocket. Likewise, a pl l0a affinity pocket binding moiety is a
substituent
which, upon contacting upon contacting p110a, fills space within the p110a
affinity pocket.
In some embodiments, the antagonist interact with or displaces the side chain
of rnethionine
804 of p110y, or the equivalent methionine present in pl IOa, p110(3, or p110S
(See Figures
7-10).
[0090] In some embodiments, the P13-Kinase binding pocket moiety additionally
interacts
(e.g. bonds) with an amino acid that forms part of the P13-Kinase binding
pocket. In some
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related embodiments, the interaction is a hydrogen bond, van der Waals
interaction, ionic
bond, covalent bond (e.g. disulfide bond) or hydrophobic interaction.
IV. Determining Space Filling Within the P13-Kinase Affinity Pocket
[0091] To determine whether the P13-Kinase affinity pocket binding moiety
fills space
within the P13-Kinase affinity pocket, computer modeling techniques are
employed. A query
P13-Kinase affinity pocket binding antagonist (i.e. a test compound) is fit
into a computer
image of p 1 l 0y. The p 1 l 0y computer image is derived from the solved co-
crystal structure of
human p 1 l0y bound to PIK-39. The PyMOL Molecular Graphics System may be
employed
to generate the image. An example is presented in Figure 3A, wherein IC87114
and PIK-294
are built into the computer image of p 110y kinase, derived from the p 11 Oy -
PIK-39 co-
crystal. See Knight, et al., Cell 125: 733-745 (2006).
[0092] The computer models are typically analyzed to prevent any gross steric
clashes and
to satisfy key hydrogen bonds between the query P13-Kinase affinity pocket
binding
antagonist and the p110y protein (e.g. V882 and M804). In some embodiments,
energy
minimization calculations are performed to optimize binding energy. Using
these techniques,
one skilled in the art can easily determine whether a query P13-Kinase
affinity pocket binding
antagonist includes a P13-Kinase affinity pocket binding moiety that fills
space within the
P13-Kinase affinity pocket.
[0093] In some embodiments, the query P13-Kinase affinity pocket binding
antagonist is
analyzed to determine whether at least one bond (e.g. a hydrogen bond) is
formed between
the query P13-Kinase affinity pocket binding antagonist and an amino acid that
form part of
the P13-Kinase affinity pocket. Using a computer modeling technique as
described above,
the distance between one or more amino acids that form part of the P13-Kinase
affinity
pocket and a potential contact point on the P13-Kinase affinity pocket binding
moiety is
determined. Based on this distance, one skilled in the art may determine
whether at least one
bond is formed between one or more amino acids that form part of the P13-
Kinase affinity
pocket and a P13-Kinase affinity pocket binding moiety.
V. General Syntheses
[0094] The compounds of the invention are synthesized by an appropriate
combination of
generally well known synthetic methods. Techniques useful in synthesizing the
compounds
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of the invention are both readily apparent and accessible to those of skill in
the relevant art.
The discussion below is offered to illustrate certain of the diverse methods
available for use
in assembling the compounds of the invention. However, the discussion is not
intended to
define the scope of reactions or reaction sequences that are useful in
preparing the
compounds of the present invention.
Scheme 1
R3 O R3 O R3 O I
OH N
` H R2)q N \ R2)
NH2 NH2 q ` q
N
El E2 E3 CI
I
R3 O R3 0 / I
I/ N \ R2/q N R2)N N q
,N =
X\ N\\ X\ / N`
R1 _NI I _N>
H2N H2N
E5 E4
In Scheme 1, R~, R2, R3, X, and q are as defined above. The anthranilic acid
El may be
converted to the acid chloride using, for example, SOC12 and then directly
reacted with the
amino functionality of an aniline to yield the corresponding amide E2.
Subsequent
cyclization of E2 may be accomplished using chloroacetylchloride. Substitution
of the E3
chlorine with the iodo-pyrazolopyrimidine or iodo-pyrrolopyrimidine is
performed in the
presence of base to form E4. Finally, the iodine of E4 is substituted with R'
by a Suzuki-
Miyaura coupling with the appropriate boronic acid.
VI. Methods
[0095] In another aspect, the present invention provides methods of decreasing
the catalytic
activity of a P13 kinase, such as p1108 kinase or p110y kinase. The method
includes the step
of contacting the P13 kinase (e.g. p 1105 kinase) with an activity decreasing
amount of a P13-
Kinase antagonist (i.e. a P13-Kinase affinity pocket binding antagonist or a
P13-Kinase
22
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Attorney Docket No. 71332.00601.PCT
antagonist of Formula (I)). In some embodiments, the antagonist is a P13-
Kinase affinity
pocket quinazolinone antagonist. In some embodiments, the P13-Kinase
antagonist is
specific to p I 108 relative to the antagonist action against p I l 0a, p
110(3, and/or p 110y. In
some embodiments, the P13-Kinase antagonist is specific to p1108 relative to
the antagonist
action against p110a and/or p110(3. In some embodiments, the P13-Kinase
antagonist is
specific to p1108 relative to the antagonist action against p110a. In some
embodiments, the
P13-Kinase antagonist is specific to pl l0y relative to the antagonist action
against p110a
and/or p110(3. In some embodiments, the P13-Kinase antagonist is specific to
p110y relative
to the antagonist action against p11 0a.
[0096] In some embodiments, where the P13-Kinase antagonist is specific to
P1107 relative
to the antagonist action against p110(3, and/or p110a, the P13-Kinase
antagonist is the P13-
Kinase antagonist of Formula (I), where Rl is a 4,5-substituted phenyl. In
some related
embodiments, the 4,5-substituted phenyl is substituted, independently, with
R21. R21 may be
halogen or -ORZZ. R21 may also be fluorine and RZZ may be hydrogen or
unsubstituted C1-C4
alkyl (e.g. methyl).
[0097] In other embodiments, where the P13-Kinase antagonist is specific to pI
l0S relative
to the antagonist action against p 110a, p 110(3, and/or p 110y, the P13-
Kinase antagonist is the
P13-Kinase antagonist of Formula (I), where R' is a 3,5-substituted phenyl. In
so:me related
embodiments, the 3,5-substituted phenyl is substituted, independently, with
R21. Rzl may be
halogen or -ORZZ. R21 may also be fluorine and R22 may be hydrogen or
unsubstituted C1-C4
alkyl (e.g. methyl).
[0098] In some embodiments, the IC50 against the p1105 kinase and/or p110y is
at least
1.5, 2.0, 3.0, 4.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or
100 fold lower than
the IC50 against p 110a, and/or p 110(3. In other embodiments, the IC50 of the
antagonist
against p 1108 kinase and/or p 1107 is less than 100 M, 50 M, 40 M, 30 M,
20 pM, 10
M, 5 M, 1 M, 0.5 M, 0.1 lVl, 50 nM, 10 nM, 1 nM. 0.5 nM, 0.1 nM, 50 pM, 10
pM, or
1 pM.
[0099] In another aspect, the present invention provides methods of treating a
disease
mediated by P13-Kinase activity (e.g. p110S kinase activity or pl 107 kinase
activity) in a
subject in need of such treatment. The method includes administering to the
subject a
therapeutically effective amount of a P13-Kinase antagonist (i.e. a P13-Kinase
affinity pocket
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Attorney Docket No. 71332.00601.PCT
antagonist or P13-Kinase antagonist of Formula (I)). In some embodiments, the
antagonist is
a P13-Kinase affinity pocket quinazolinone antagonist.
[0100] In some embodiments, the disease is a hematologic malignancy,
inflammation,
autoimmune disease, or cardiovascular disease. In some embodiments, the
disease is a
hematologic malignancy or autoimmune disease. Examples of hematologic
malignancies
include acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),
mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and
myelodysplastic syndrome (MDS). Examples of inflammation disorders and
autoimmune
disease include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),
and asthma.
Other disorders include bone-resorption disorders and thromobsis.
[0101] The disorder may also be a type of cancer or cancer metastasis,
including, for
example, leukemia, carcinomas and sarcomas, such as cancer of the brain,
breast, cervix,
colon, head & neck, liver, kidney, lung, non-small cell lung, melanoma,
mesothelioma,
ovary, sarcoma, stomach, uterus and medulloblastoma. Additional examples
include,
Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastorna,
ovarian
cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,
primary
brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid,
urinary bladder
cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid
cancer,
neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant
hypercalcemia,
endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine and
exocrine
pancreas, and prostate cancer. A detailed description of conditions and
disorders mediated by
p1108 kinase activity is set forth in Sadu et al., WO 01/81346, which is
incorporated herein
by reference in its entirety for all purposes.
[0102] In another aspect, the present invention provides methods of disrupting
the function
of a leukocyte or disrupting a function of an osteoclast. The method includes
contacting the
leukocyte or the osteoclast with a function disrupting amount of a P13-Kinase
antagonist (i.e.
a P13-Kinase affinity pocket antagonist or P13-Kinase antagonist of Formula
(I)). In some
embodiments, the antagonist is a P13-Kinase affinity pocket quinazolinone
antagonist.
VII. Pharmaceutical Formulations
[0103] In another aspect, the present invention provides a pharmaceutical
composition
including a P13-Kinase affinity pocket binding antagonist or a compound of
Formula (I) in
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Attorney Docket No. 71332.00601.PCT
admixture with a pharmaceutically acceptable excipient. One of skill in the
art will recognize
that the pharmaceutical compositions include the pharmaceutically acceptable
salts of the
P13-Kinase antagonists of the present invention described above.
[0104] In therapeutic and/or diagnostic applications, the compounds of the
invention can be
formulated for a variety of modes of administration, including systemic and
topical or
localized administration. Techniques and formulations generally may be found
in
Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott,
Williams & Wilkins
(2000).
[0105] The compounds according to the invention are effective over a wide
dosage range.
For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg,
from 0.5 to
100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of
dosages that
may be used. A most preferable dosage is 10 to 30 mg per day. The exact dosage
will depend
upon the route of administration, the form in which the compound is
administered, the subject
to be treated, the body weight of the subject to be treated, and the
preference and experience
of the attending physician.
[0106] Pharmaceutically acceptable salts are generally well known to those of
ordinary
skill in the art, and may include, by way of example but not limitation,
acetate,
benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide,
calcium ecletate,
camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate,
fumarate, gluceptate,
gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,
hydrobi-omide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate,
malate, maleate,
mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate),
pantothenate,
phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate,
succinate, sulfate,
tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be
found in, for
example, Remington: The Science and Practice of Pharmacy (20`h ed.)
Lippincott, Williams
& Wilkins (2000). Preferred pharmaceutically acceptable salts include, for
example, acetate,
benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride,
maleate,
mesylate, napsylate, pamoate (embonate), phosphate, salicylate, succinate,
sulfate, or tartrate.
[0107] Depending on the specific conditions being treated, such agents may be
formulated
into liquid or solid dosage forms and administered systemically or locally.
The agents may
be delivered, for example, in a timed- or sustained- low release form as is
known to those
skilled in the art. Techniques for formulation and administration may be found
in Remington:
CA 02639579 2008-10-02
Attorney Docket No. 713 32.00601.PCT
The Science and Practice of Pharmacy (20`h ed.) Lippincott, Williams & Wilkins
(2000).
Suitable routes may include oral, buccal, by inhalation spray, sublingual,
rectal, transdermal,
vaginal, transmucosal, nasal or intestinal administration; parenteral
delivery, including
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct
intraventricular, intravenous, intra-articullar, intra -sternal, intra-
synovial, intra-hepatic,
intralesional, intracranial, intraperitoneal, intranasal, or intraocular
injections or other modes
of delivery.
[0108] For injection, the agents of the invention may be formulated and
diluted in aqueous
solutions, such as in physiologically compatible buffers such as Hank's
solution, Ringer's
solution, or physiological saline buffer. For such transmucosal
administration, penetrants
appropriate to the barrier to be permeated are used in the formulation. Such
penetrants are
generally known in the art.
[0109] Use of pharmaceutically acceptable inert carriers to formulate the
compounds herein
disclosed for the practice of the invention into dosages suitable for systemic
administration is
within the scope of the invention. With proper choice of carrier and suitable
manufacturing
practice, the compositions of the present invention, in particular, those
formulated as
solutions, may be administered parenterally, such as by intravenous injection.
The
compounds can be formulated readily using pharmaceutically acceptable carriers
well known
in the art into dosages suitable for oral administration. Such carriers enable
the compounds
of the invention to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries,
suspensions and the like, for oral ingestion by a subject (e.g. patient) to be
treated.
[0110] For nasal or inhalation delivery, the agents of the invention may also
be formulated
by methods known to those of skill in the art, and may include, for example,
but riot limited
to, examples of solubilizing, diluting, or dispersing substances such as,
saline, preservatives,
such as benzyl alcohol, absorption promoters, and fluorocarbons.
[0111] Pharmaceutical compositions suitable for use in the present invention
include
compositions wherein the active ingredients are contained in an effective
amount to achieve
its intended purpose. Determination of the effective amounts is well within
the capability of
those skilled in the art, especially in light of the detailed disclosure
provided herein.
[0112] In addition to the active ingredients, these pharmaceutical
compositions may contain
suitable pharmaceutically acceptable carriers comprising excipients and
auxiliaries which
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Attorney Docket No. 713 32.00601.PCT
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. The preparations formulated for oral administration may be
in the form of
tablets, dragees, capsules, or solutions.
[0113] Pharmaceutical preparations for oral use can be obtained by combining
the active
compounds with solid excipients, optionally grinding a resulting mixture, and
processing the
mixture of granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee
cores. Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations, for example, maize starch,
wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-
cellulose, sodium carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone
(PVP:
povidone). If desired, disintegrating agents may be added, such as the cross-
linked
polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0114] Dragee cores are provided with suitable coatings. For this purpose,
concentrated
sugar solutions may be used, which may optionally contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/or titanium
dioxide,
lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-
stuffs or pigments
may be added to the tablets or dragee coatings for identification or to
characterize different
combinations of active compound doses.
[0115] Pharmaceutical preparations that can be used orally include push-fit
capsules made
of gelatin, as well as soft, sealed capsules made of gelatin, and a
plasticizer, such as glycerol
or sorbitol. The push-fit capsules can contain the active ingredients in
admixture with filler
such as lactose, binders such as starches, and/or lubricants such as talc or
magnesium stearate
and, optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols
(PEGs). In addition, stabilizers may be added.
[0116] Depending upon the particular condition, or disease state, to be
treated or prevented,
additional therapeutic agents, which are normally administered to treat or
prevent that
condition, may be administered together with the inhibitors of this invention.
For example,
chemotherapeutic agents or other anti-proliferative agents may be combined
with the
inhibitors of this invention to treat proliferative diseases and cancer.
Examples of known
chemotherapeutic agents include, but are not limited to, adriamycin,
dexamethasone,
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Attorney Docket No. 713 32.00601.PCT
vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons,
and platinum
derivatives.
[0117] Other examples of agents the inhibitors of this invention may also be
combined with
include, without limitation, anti-inflammatory agents such as corticosteroids,
TNF blockers,
IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory
and
immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,
mycophenolate
mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and
sulfasalazine;
neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors,
interferons,
anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonian
agents; agents for
treating cardiovascular disease such as beta-blockers, ACE inhibitors,
diuretics, nitrates,
calcium channel blockers, and statins; agents for treating liver disease such
as corticosteroids,
cholestyramine, interferons, and anti-viral agents; agents for treating blood
disorders such as
corticosteroids, anti-leukemic agents, and growth factors; agents for treating
diabetes such as
insulin, insulin analogues, alpha glucosidase inhibitors, biguanides, and
insulin sensitizers;
and agents for treating immunodeficiency disorders such as gamma globulin.
[0118] These additional agents may be administered separately, as part of a
multiple dosage
regimen, from the inhibitor-containing composition. Alternatively, these
agents niay be part
of a single dosage form, mixed together with the inhibitor in a single
composition.
[0119] The present invention is not to be limited in scope by the exemplified
embodiments,
which are intended as illustrations of single aspects of the invention.
Indeed, various
modifications of the invention in addition to those described herein will
become apparent to
those having skill in the art from the foregoing description. Such
modifications are intended
to fall within the scope of the invention. Moreover, any one or more features
of any
embodiment of the invention may be combined with any one or more other
features of any
other embodiment of the invention, without departing from the scope of the
invention. For
example, the P13-Kinase agonists of the present invention described above are
equally
applicable to the methods of treatment and methods of inhibiting kinases
described herein.
References cited throughout this application are examples of the level of
skill in the art and
are hereby incorporated by reference herein in their entirety for all
purposes, whether
previously specifically incorporated or not.
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VIII. Examples
The following examples are meant to illustrate certain embodiments of the
invention, and not
to limit the scope of the invention.
A. Exemplary Synthesis Scheme
Scheme 2
CH O CH O
C~ CFVO ~ CHO ~
~/N~ 0
~ I Cl~CI N
I OM $OCI_ \ CI
CH, C116
CI
~ "`+ roh
c i
o
~ HxNHr N ~ \ Oy".~O
( v N ~ ~
~N N II / N II /N
F(CI)
S19-S23
" + K_CO S25-S39
N S12, S13
cl " H~ GJ~L
\4roA P'
4a
CH~ O NHr CHa O
" \ ~- NII \ \ KiCO, I \ " "
CH, l / / /~ CH("/ \I N H "/ \I PJ(PPA,4 EIOH,NaiCO3 S3-J11
c' S14-S18
N\ /
I I
HiN
Scheme 2 illustrates synthetic routes to certain compounds listed in Table 1
below. Using the
information provided in Schemes 1 and 2, and the detailed synthesis
information of certain
compounds provided below, one skilled in the art would immediately recognize
the synthetic
routes to the compounds of the present invention.
B. Detailed Synthesis of Certain Compounds
1. Synthesis of 2-amino-6-methyl-N-o-tolylbenzamide
(0120] 2-amino-6-methylbenzoic acid (25 g, 165 mmol) was dissolved in benzene
(250
mL). Thionyl chloride (37.5 mL, 500 mmol) was added and the reaction heated to
reflux
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Attorney Docket No. 7 i332.00601.PCT
overnight. The following day the reaction was concentrated in vacuo, and then
taken up
twice in benzene (200 mL) and solvent removed in vacuo again to give a black
oil. The oil
was dissolved in CHC13 (400 mL), o-toluidine (44 mL, 412 mmol) was added ancl
the
reaction heated to reflux. Reaction was complete after two hours, and the
product was
purified by three silica gel chromatographies (15% EtOAc/Hexanes) to yield a
tan solid (29
g, 73.4% yield). LR-ESI MS (M+H)+ m/z calcd 241.1, found 240.9.
2. Synthesis of 2-(chloromethyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one
[0121] Chloroacetylchloride (29 mL, 363 mmol) was added to a solution of 2-
amino-6-
methyl-N-o-tolylbenzamide (29 g, 121 mmol) in acetic acid (600 mL) and the
reaction heated
to reflux. After two hours the reaction was cooled to RT, and concentrated in
vacuo. The
product was purified by three silica gel chromatographies (twice in 15%
EtOAc/Hexanes
followed by 10% diethylether/hexanes) to yield a white solid (8.3 g, 23%
yield). LR-ESI MS
(M+H)+ m/z calcd 299.1, found 298.8.
3. Synthesis of 2-((4-amino-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-
3-o-tolylquinazolin-4(3H)-one
[0122] 2-(chloromethyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one (0.15 g, 0.5
mmol) and
1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.101 g, 0.05 mmol) were added to DMF (10
mL)
and K2C03 (0.138 g, 1 mmol) and allowed to stir at RT in the dark for 24
hours. The product
was precipitated by addition of water (800 mL) and collected by filtration.
The precipitate
was further purified by RP-HPLC (MeCN:H20:0.1 % TFA). LR-ESI MS (M+H)+ m/z
calcd
398.2, found 398.1.
4. Synthesis of Compound S2
[0123] 2-(chloromethyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one (3 g, 10.0
mmol) and 3-
iodo-lH-pyrazolo[3,4-d]pyrimidin-4-amine (3.91 g, 15.05 mmol) were added to
DMF (50
mL) and K2CO3 (2.77 g, 20 mmol) and allowed to stir at RT in the dark for 24
hours. The
product was precipitated by addition of water (900 mL) and collected by
filtration. The
precipitate was further purified by silica gel chromatography (2%
MeOH/CH2C12). LR-ESI
MS (M+H)+ m/z calcd 524.1, found 523.9.
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Attorney Docket No. 713 32.00601.PCT
5. Synthesis of Compound S3
[0124] 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (50 mg, 0.096 mmol), m-phenol boronic acid (14.5 mg,
0.105
mmol) and tetrakis(triphenylphosphine)palladium (22 mg, 0.019 mmol) were
dissolved in a
solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous Na2CO3 (2.75 mL).
The
reaction was heated to reflux overnight under an argon atmosphere. The
following day the
reaction was poured into water, and the aqueous phase extracted three times
with CH2C12.
The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1%
TFA). LR-ESI MS (M+H)+ m/z calcd 490.2, found 490.1.
6. Synthesis of Compound S4
[0125] 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (50 mg, 0.096 mmol), 5-formylbenzo[b]thiophene-2-
boronic ester
(30.3 mg, 0.105 mmol) and tetrakis(triphenylphosphine)palladium (22 mg, 0.019
mmol) were
dissolved in a solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous
Na2CO3 (2.75
mL). The reaction was heated to reflux overnight under an argon atmosphere.
The following
day the reaction was poured into water, and the aqueous phase extracted three
times with
CH2C12. The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1 % TFA). LR-ESI MS (M+H)+ m/z calcd 558.2, found 558Ø
7. Synthesis of Compound S5
[0126] 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (50 mg, 0.096 mmol), 5-formyl-3-methylthiophene-2-
boronic acid
(18.9 mg, 0.105 mmol) and tetrakis(triphenylphosphine)palladium (22 mg, 0.019
mmol) were
dissolved in a solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous
Na2CO3 (2.75
mL). The reaction was heated to reflux ovenrnight under an argon atmosphere.
The following
day the reaction was poured into water, and the aqueous phase extracted three
times with
CH2C12. The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1 % TFA). LR-ESI MS (M+H)+ m/z calcd 522.2, found 522Ø
8. Synthesis of S6
[01271 2-((4-amino-3-iodo-1 H-pyrazolo [3,4-d]pyrimidin-1-yl)methyl)-5-methyl-
3-o-
tolylquinazolin-4(3H)-one (100 mg, 0.192 mmol), 3,4-dimethoxyphenyl boronic
ester (38.2
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Attorney Docket No. 713 32.00601.PCT
mg, 0.21 mmol) and tetrakis(triphenylphosphine)palladium (44 mg, 0.038 mmol)
were
dissolved in a solution of DME (20 mL), EtOH (3.2 mL) and saturated aqueous
NazCO3 (5.5
mL). The reaction was heated to reflux overnight under an argon atmosphere.
The following
day the reaction was poured into water, and the aqueous phase extracted three
times with
CHZCl2. The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1% TFA). LR-ESI MS (M+H)+ m/z calcd 534.2, found 534Ø
9. Synthesis of S7
2-((4-amino-3-iodo-1 H-pyrazolo [3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-
tolylquinazolin-
4(3H)-one (100 mg, 0.192 mmol), 4-phenoxyphenyl boronic acid (44.9 mg, 0.21
mmol) and
tetrakis(triphenylphosphine)palladium (44 mg, 0.038 mmol) were dissolved in a
solution of
DME (20 mL), EtOH (3.2 mL) and saturated aqueous Na2CO3 (5.5 mL). The reaction
was
heated to reflux overnight under an argon atmosphere. The following day the
reaction was
poured into water, and the aqueous phase extracted three times with CH2C12.
The organic
extract was concentrated in vacuo and purified by RP-HPLC (MeCN:H20:0.1% TFA).
LR-
ESI MS (M+H)+ m/z calcd 566.2, found 566Ø
10. Synthesis of S8
[0128] 2-((4-amino-3-iodo-IH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (100 mg, 0.192 mmol), 4-benzyloxyphenyl boronic acid
(47.9 mg,
0.21 mmol) and tetrakis(triphenylphosphine)palladium (44 mg, 0.038 mmol) were
dissolved
in a solution of DME (20 mL), EtOH (3.2 mL) and saturated aqueous Na2CO3 (5.5
mL). The
reaction was heated to reflux overnight under an argon atmosphere. The
following day the
reaction was poured into water, and the aqueous phase extracted three times
with CH2C12.
The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1%
TFA).
11. Synthesis of S33
[0129] 2-((4-amino-3-iodo-1 H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (50 mg, 0.096 mmol), 3-cyanophenyl boronic acid
(15.8 mg, 0.105
mmol) and tetrakis(triphenylphosphine)palladium (22 mg, 0.019 mmol) were
dissolved in a
solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous Na2CO3 (2.75
niL). The
reaction was heated to reflux overnight under an argon atmosphere. The
following day the
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reaction was poured into water, and the aqueous phase extracted three times
with CH2C12.
The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1 %
TFA). LR-ESI MS (M+H)+ m/z calcd 499.2, found 499Ø
12. Synthesis of 2-amino-N-(2-chlorophenyl)-6-methylbenzamide
[0130] 2-amino-6-methylbenzoic acid (2.5 g, 16.5 mmol) was dissolved in
benzene (75
mL). Thionyl chloride (3.0 mL, 41.1 mmol) was added and the reaction heated to
reflux
overnight. The following day the reaction was concentrated in vacuo, and then
taken up
twice in benzene (75 mL) and solvent removed in vacuo again to give a black
oil. The oil
was dissolved in CHC13 (75 mL), 2-chloroaniline (3.5 mL) was added and the
reaction heated
to reflux. Reaction was complete after four hours, at which point the reaction
was filtered,
the filtrate concentrated in vacuo, and the the product was purified by silica
gel
chromatography (25% EtOAc/Hexanes) to yield a brown oil (1.94 g, 45% yield).
HR-El MS
(M)+ m/z calcd 260.07, found 260.0715.
13. Synthesis of 2-(chloromethyl)-3-(2-chlorophenyl)-5-methylquinazolin-4(3H)-
one
[0131] Chloroacetylchloride (0.72 mL, 9 mmol) was added to a solution of 2-
arnino-N-(2-
chlorophenyl)-6-methylbenzamide (0.8 g, 3.06 mmol) in acetic acid (10 mL) and
the reaction
heated to reflux. After 2.5 hours the reaction was cooled to RT, and
concentrated. in vacuo.
The product was purified by silica gel chromatography (10% EtOAc/Hexanes) to
yield a
white solid (0.353 g, 36% yield). HR-El MS (M)+ m/z calcd 318.0327, found
318.0321.
14. Synthesis of 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-
(2-chlorophenyl)-5-methylquinazolin-4(3H)-one
[0132] 2-(chloromethyl)-3-(2-chlorophenyl)-5-methylquinazolin-4(3H)-one (0.112
g, 0.35
mmol) and 3-iodo-IH-pyrazolo[3,4-d]pyrimidin-4-amine (0.138 g, 0.053 mmol)
were added
to DMF (5 mL) and K2CO3 (0.096 g, 0.7 mmol) and allowed to stir at RT in the
dark for 72
hours. The product was precipitated by addition of water (50 mL) and collected
by filtration.
The precipitate was further purified by RP-HPLC (MeCN:H20: 0. 1% TFA).
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15. Synthesis of S1
[0133] 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-
chlorophenyl)-
5-methylquinazolin-4(3H)-one (60 mg, 0.11 mmol), m-phenol boronic acid (17
rng, 0.121
mmol) and tetrakis(triphenylphosphine)palladium (25 mg, 0.022 mmol) were
dissolved in a
solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous Na2CO3 (2.75 mL).
The
reaction was heated to reflux overnight under an argon atmosphere. The
following day the
reaction was poured into water, and the aqueous phase extracted three times
with CH2C12.
The organic extract was concentrated in vacuo and purified by RP-HPLC
(MeCN:H20:0.1 %
TFA). LR-ESI MS (M+H)+ m/z calcd 510.1, found 510Ø
16. Synthesis of S34
[0134] 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-
o-
tolylquinazolin-4(3H)-one (50 mg, 0.096 mmol), benzene 3-sulphonamide boronic
ester (29.7
mg, 0.105 mmol) and tetrakis(triphenylphosphine)palladium (22 mg, 0.019 mmol)
were
dissolved in a solution of DME (10 mL), EtOH (1.6 mL) and saturated aqueous
Na2CO3 (2.75
mL). The reaction was heated to reflux overnight under an argon atmosphere.
The following
day the reaction was poured into water, and the aqueous phase extracted three
times with
CHZC12. LR-ESI MS (M+H)+ m/z calcd 553.2, found 553Ø
C. P13-Kinase Structural Studies
[0135] Crystal structures of p 110y have been reported, alone and in complex
with ATP or
pan-specific inhibitors such as LY294002 and wortmannin (Walker et al., 2000;
Walker et
al., 1999). To explore how potent and selective inhibitors bind, the crystal
structures of P13-
K inhibitors from three chemotypes bound to human p 110y were determined at
2.5 - 2.6 A
resolution: the quinazoline purine PIK-39, the imidazopyridine PIK-90 and the
phenylthiazole PIK-93 (Figure 2).
[0136] Based on these co-crystal structures and a conserved arylmorpholine
pharmacophore model, structural models were generated for three additional
chemotypes
bound to pl l0y: the pyridinylfuranopyrimidine PI-103, the morpholinochromone
PIK-108,
and the morpholinopyranone KU-55933 (Figure 2). Model-building for these
inhibitors was
guided by the observation that each compound contains the key arylmorpholine
pharmacophore found in LY294002.
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[0137] PIK-39 is an isoquinoline purine that inhibits p1108 at mid-nanomolar
concentrations, p 11 Oy and p 110(3 at concentrations - 100-fold higher, and
shows no activity
against any other P13-K family member, including p110a, at concentrations up
to 100 M
(Figure 5). The biochemical selectivity of this compound is achieved through
an unusual
binding mode revealed in its co-crystal structure with p110y (Figure 2C). Only
the
mercaptopurine moiety of PIK-39 makes contacts within the interior of the ATP
binding
pocket, and this ring system is rotated -110 and twisted -35 out of the
plane relative to the
adenine of the ATP. In this orientation, it satisfies hydrogen bonds to the
backbone amides of
Val 882 and Glu 880 (thereby recapitulating the hydrogen bonds made by Nl and
N6 of
adenine).
[0138] In contrast to other P13-K inhibitor structures, PIK-39 does not access
the deeper
pocket in the active site interior (Figure 2C, lightly shaded area labeled as
"Affinity Pocket").
Instead, the aryl-isoquinoline moiety of PIK-39 extends out to the entrance of
the ATP
binding pocket (Figure 2B). In this region, the kinase accommodates the
inhibitor by
undergoing a conformational rearrangement in which Met 804 shifts from an "up"
position,
in which it forms the ceiling of the ATP binding pocket, to a "down" position
which it packs
against the isoquinoline moiety. The effect of this movement, which is unique
to the PIK-39
structure (Figure 2B), is to create a novel hydrophobic pocket between Met 804
and Trp 812
at the entrance to the ATP binding site. This induced-fit pocket buries -180
A2 of solvent
accessible inhibitor surface area, enabling PIK-39 to achieve nanomolar
affinity despite
limited contacts within the active site core.
[0139] Co-crystal structures of PIK-90 and PIK-93 compounds bound to p110y
were
determined. PIK-90 and PIK-93 both make a hydrogen bond to the backbone amide
nitrogen
of Val 882 (Figure 2D), an interaction conserved among all known P13-K
inhibitors (Walker
et al., 2000). In addition to this hydrogen bond, PIK-93 makes a second
hydrogen bond to
the backbone carbonyl of Val 882 and a third between its sulphonamide moiety
and the side
chain of Asp 964. PIK-93 is one of the most polar inhibitors in our panel
(clogP = 1.69) and
these extended polar interactions may compensate for its limited hydrophobic
surface area.
[0140] PIK-90 binds in a mode similar to PIK-93, although this larger compound
makes
more extensive hydrophobic interactions, burying 327 A 2 of solvent accessible
surface area.
To achieve this, PIK-90 projects its pyridine ring into a deeper cavity that
is partially
accessed by PIK-93 but not occupied by ATP (Figure 2D, lightly shaded circle).
In this
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region, the pyridine ring of PIK-90 is poised to make a hydrogen bond to Lys
833, and we
find that replacement of this pyridine nitrogen with carbon results in a 100-
fold loss in
affinity (PIK-95, Figure 4). PI-103, a third multi-targeted P13K inhibitor,
projects a phenol
into the same pocket based on an arylmorpholine pharmacophore model (Figure
2D).
[0141] Two structural features distinguish these potent, multi-targeted
inhibitors from the
more selective compounds in our panel. First, these compounds adopt a flat
conformation in
the ATP binding pocket, whereas highly selective inhibitors project out of the
plane occupied
by ATP (Figure 2). Second, the most potent inhibitors project into a deeper
binding pocket
that is not accessed by ATP (Figure 2A). Much of the surface of this affinity
pocket is
contributed by the side-chain of Ile 879.
[0142] The mercaptopurine in the PIK-39 structure was replaced with adenine to
yield a
model of IC87114 (Figure 3A). This substitution provided the adenine of IC871
l.4 in the
correct orientation to make the same hydrogen bonds as the mercaptopurine of
PIK-39, even
though these two ring systems are rotated by 110 with respect to each other.
[0143] Unlike other inhibitor chemotypes, PIK-39 does not exploit the P13-
kinase affinity
pocket (Figure 2C). The pyrazolopyrimidine analog of IC87114 (PIK-293) as well
as a novel
analog containing an m-phenol (PIK-294, Figure 3A) were then tested for
inhibition of the
class I P13-Ks. PIK-294 was up to 60-fold more potent than PIK-293 (Figure
3A).
[0144] The structure of PIK-39 bound to p110y reveals a conformational
rearrangement of
Met 804 that creates an induced pocket, and we have hypothesized that this
conformational
rearrangement underlies the selectivity of PIK-39 for p1108. A prediction of
this model is
that mutation of Met 804 should perturb the binding of p1108-selective
inhibitors (which
access the induced pocket), but not affect other classes of inhibitors (which
do not access this
pocket). Modeling suggests that mutation of Met 804 to a(3-branched amino acid
(such as
valine or isoleucine) should restrict the pocket formed by rearrangement of
that residue
(Figure 3B, right). Therefore, we mutated the corresponding residue in p 1106
(Met 752) to
valine or isoleucine, expressed and purified these kinases, and tested them
for sensitivity to
P13-K inhibitors (Figure 3B). We find that M7521 and M752V p1108 are resistant
to the
p1106-selective inhibitors PIK-39 and IC87114, but retain sensitivity to the
p1l0a/multi-
targeted inhibitors PIK-90, PIK-93, and PI-103. This chemotype-specific
resistance supports
the unique role of Met 752 in gating an inducible selectivity pocket.
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[0145] Antagonist modeling was performed using the PyMOL Molecular Graphics
System.
All p 1 10y crystal structures (PDB codes in parentheses), including the Apo
(1E8Y), ATP
(1E8X), Wortmannin (1E7U), LY294002 (1E7V), Quercetin (1E8W), Myricetin
(1E90), and
Staurosporine (lE8Z), PIK-90, PIK-93, and PIK-39 bound forms were structurally
aligned
using PyMOL's align function. Models for the inhibitors PIK-108, KU-55933, and
PI-103
were built on top of the LY294002 arylmorpholine scaffold (lE7V) using
PyMO:L's fragment
building function. A model for the inhibitor IC87114 was similarly built on
top of the PIK-
39 aryl-isoquinoline scaffold.
[0146] The model for PI-103 was built into the protein structure of p110y
bound to PIK-90,
because the PIK-90 structure contains the enlarged affinity pocket that is
necessary to
accommodate PIK-103's phenolic moiety (the PIK-90 p110y structure otherwise
does not
exhibit any conformational differences in the arymorpholine-binding region in
comparison to
the LY294002-bound p110y structure). The models for PIK-108, KU-55933, and
IC87114
were built into the protein structure of p110y bound to PIK-39 because these
inhibitors
possess bulky groups that project out of the adenine plane and are likely to
exploit the unique
"Met 804 down" induced-fit pocket. In all inhibitor models, the choice of
protein structure
and inhibitor binding mode is based on extensive biochemical SAR as well as
inhibitor
geometry. The protein structures and inhibitor models have not been minimized
to optimize
binding energy, but care was taken to prevent any gross steric clashes and to
satisfy key
hydrogen bonds.
D. Expression and Assays of p110a/p85a, p1100/p85a, p1108/p85a, and
p110y
[0147] The class I P13-Ks were either purchased (p110a/p85a, p110(3/p85a,
p1108/p85(x from Upstate, and p110y from Sigma) or expressed as previously
described
(Knight et al., 2004). IC50 values were measured using either a standard TLC
assay for lipid
kinase activity (described below) or a high-throughput membrane capture assay.
Kinase
reactions were performed by preparing a reaction mixture containing kinase,
inhibitor (2%
DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgC12), and
freshly
sonicated phosphatidylinositol (100 g/ml). Reactions were initiated by the
addition of ATP
containing 10 Ci of y-32P-ATP to a final concentration 10 or 100 M, as
indicated in Figure
5, and allowed to proceed for 5 minutes at room temperature. For TLC analysis,
reactions
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were then terminated by the addition of 105 t 1N HC1 followed by 160 l
CHC13:MeOH
(1:1). The biphasic mixture was vortexed, briefly centrifuged, and the organic
phase
transferred to a new tube using a gel loading pipette tip precoated with
CHC13. This extract
was spotted on TLC plates and developed for 3 - 4 hours in a 65:35 solution of
n-
propanol:lM acetic acid. The TLC plates were then dried, exposed to a
phosphorimager
screen (Storm, Amersham), and quantitated. For each compound, kinase activity
was
measured at 10 - 12 inhibitor concentrations representing two-fold dilutions
from the highest
concentration tested (typically, 200 M). For compounds showing significant
activity, IC50
determinations were repeated two to four times, and the reported value is the
average of these
independent measurements.
[0148] Results are set forth in Table 1 below.
Table 1
Compound Structure IC50
MW 110a 1109 110 1103
Na 00
N
CI
N'I-, N N
Fp N
S 1 H2N
(509.9464) ~ + ++ +++ +++
CH3 0
~ I
N \
CH,
N
N
S2 N
(523.33) H2N
++ ++ +++ +++
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Compound Structure IC50
MW 110a 110/3 110y 1106
CH3 0
~ I
N
CH3
N~N N
HO N
S3 H2N
(489.53) + +++ +++ +++
CH3 O
N
CH3
N
N/ N
\ / \
N
s HzN
O
S4
H + + ++ +++
(557.63)
CH3 O
N
CH3
N
N
N~
N
s HZN
H3C
S5
(521.59) 0 H + ++ +++ +++
CH3 O ~ I
N \
N
~,) CH3
/N
N~
N~ / ~
/ N
H~CO \ / HzN
S6
(533.58) H3CO + ++ +++ +++
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Compound Structure IC50
MW pIlOa 110Q pIlOy 1103
~
CH3 0
\
CH3
/N N
N~ ~
/ N
QH2I
S7 ~-~ o
(565.62) + ++ ++ +++
CH3 0 N
1) ~ CH3
N
/ N
N
N~
N
\ HZN
S8
579.65 0 ++ ++ ++ +++
CH3 0 N
CHy
N N
N
HO \ H2N
S9
(507.52) F ++ +++ +++ +++
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Compound Structure IC50
MW plIOa 110Q 110y 110d
CH3 0 ~
N
CH3
N
N
N NN
N
F HZN
S10
(507.52) HO ++ +++ +++ +++
CH3 0 ~
N
CH3
N
N
N
HZN
S11
(503.55) H3CO ++ ++ +++ +++
CH3 0
~ I
N
CH3
N
N~ N
N
HO H2N
S12
523.97 c, ++ ++ +++ +++
CH3 0 ~ I
N
CH3
N~N N
N
HO HZN
S13
507.52 F ++ ++ +++ +++
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Compound Structure IC50
MW 110a 110 110y pl
CH3 0
N
CHy
N N
N
N
HN
S14 H2N
512.56 + ++ +++ +++
CH3 0
N
CH'
N~N N
\ / !
~N
HN HpN
S15 ~
512.56 + ++ ++ +++
CH3 0
N
CH3
N~N N
~ r
~~N
S16 H3C0 \ H2N
/
521.55 F + ++ ++ +++
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Compound Structure IC50
Mw pllOa 110fl 110y 110a
CH3 0
N
/ CH3
N
I'll N N
N
N
F \ / HZN
S17
(521.55) H3CO ++ ++ +++ +++
CH3 0 N
CH3
N
N N
N
FyC \ HZN
S18
(608.52) CF3 + + + ++
CH3 0
N
CH3
N
N~ N
/ N
HZN
S19
(461.52) + ++ ++ +++
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Compound Structure IC50
MW pllOa 110,Q pllOy 110d
CH3 0 N
/ CH3
N
N
NIZ N
N
// HZN
S20 HO
(513.55) ++ ++ +++ +++
CH3 O
N \
CH3
N
N N
N
HZN
H2N
S21
(512.56) ~ + ++ ++ ++
CH3 O ~
N \
~ CH3
N
Nz N N
N
H2N
S22 N
498.54 + ++ ++ +++
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Compound Structure IC50
MW pllOa pllOg pIlOy 110(5
CH3 0
N
CH3
NN N
N
HZN
N
S23
498.54 + ++ +++ +++
CH3 0
N
) CH3
N
N
N N
N
HzN
S24
(524.58) - N ++ ++ +++ +++
CH3 0
N
CH3
NN N
N
S25 HZN
(451.48) HO ++ +++ ++ +++
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Com ound Structure IC50
MW pIlOa 110,Q pllOy '110d
CH3 O
N
CH3
N
N N
N
HZN
S26 HN
(464.52) 1 +++
CH3 0 N
CH3
N
N N
N
HZN
S27 \N
(478.55) 1 ++
CH3 O ~
N \
) CH3
N
N
N N
N
HZN
S28 HOllm.
(465.51) CH, + ++ ++ +++
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Com ound Structure IC50
MW pllOa 110/3 pllOy 1109
CH3 O
N
CH3
N
N~ N
N
HZN
S29 HO
(465.51) cH, ++ +++ +++ +++
CH3O / I
N \
CH3
N
N
N
N
HZN
NH
S30
NHZ ++ + ++ +++
(493.52)
CH3 O /
\
CH3
N
N N
N
H2N
S31
(488.54) N= +++
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Com ound Structure IC50
MW pllOa pllOg pIlOy pl
CH3 0
~ I
N \
CH3
N /N N~
i N
O O HZN
S32
CH3 0
~ I
N \
N"~ CH3
/N
N N~
~
~N
S33 NC-C7 HZN
CH3 0
~ I
/ CH3
N
N N N~
~
O N
HZN-II HZN
S34
The symbol +++ represents an IC50 of less than 1 M; the symbol ++ represents
an IC50
value froml [M to 100 M; and + represents an IC50 value of more than 100 M.
IX. References
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Fukushima, Y., Yazaki, Y., Kikuchi, M., et al. (2000). p1 l0beta is up-
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[0152] Bi, L., Okabe, I., Bernard, D. J., Wynshaw-Boris, A., and Nussbaum, R.
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(2005). Phosphoinositide 3-kinase catalytic subunit deletion and regulatory
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T.,
Okkenhaug, K., Vanhaesebroeck, B., Turner, M., Webb, L., Wymann, M. P., et al.
(2005).
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[0159] Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998).
Phosphoinositide kinases.
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[0160] Harrington, L. S., Findlay, G. M., and Lamb, R. F. (2005). Restraining
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mTOR signalling goes back to the membrane. Trends Biochem Sci 30, 35-42.
[0161] Hickson, I., Zhao, Y., Richardson, C. J., Green, S. J., Martin, N. M.,
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Reaper, P. M., Jackson, S. P., Curtin, N. J., and Smith, G. C. (2004).
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[0162] Jackson, S. P., Schoenwaelder, S. M., Goncalves, I., Nesbitt, W. S.,
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Wright, C. E., Kenche, V., Anderson, K. E., Dopheide, S. M., Yuan, Y., et al.
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[0163] Katso, R., Okkenhaug, K., Ahmadi, K., White, S., Timms, J., and
Waterfield, M. D.
(2001). Cellular function of phosphoinositide 3-kinases: implications for
development,
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Coan, K.,
Abraham, R. T., and Shokat, K. M. (2004). Isoform-specific phosphoinositide 3-
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