Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02779184 2012-05-31
PROTEIN KINASE INHIBITORS
FIELD OF INVENTION
The present invention relates to a novel family of inhibitors of protein
kinases. In particular, the present invention relates to inhibitors of the
members of the Tec and Src protein kinase families, more particularly Btk.
BACKGROUND OF THE INVENTION
Protein kinases are a large group of intracellular and transmembrane
signaling proteins in eukaryotic cells. These enzymes are responsible for
transfer of the terminal (gamma) phosphate from ATP to specific amino acid
residues of target proteins. Phosphorylation of specific tyrosine, serine or
threonine amino acid residues in target proteins can modulate their activity
leading to profound changes in cellular signaling and metabolism. Protein
kinases can be found in the cell membrane, cytosol and organelles such as
the nucleus and are responsible for mediating multiple cellular functions
including metabolism, cellular growth and division, cellular signaling,
modulation of immune responses, and apoptosis. The receptor tyrosine
kinases are a large family of cell surface receptors with protein tyrosine
kinase activity that respond to extracellular cues and activate intracellular
signaling cascades (Plowman et al. (1994) DN&P, 7(6):334-339).
Aberrant activation or excessive expression of various protein kinases are
implicated in the mechanism of multiple diseases and disorders
characterized by benign and malignant proliferation, excess angiogenesis, as
well as diseases resulting from inappropriate activation of the immune
system. Thus, inhibitors of select kinases or kinase families are expected to
be useful in the treatment of cancer, autoimmune diseases, and
inflammatory conditions including, but not limited to: solid tumors,
hematological malignancies, arthritis, graft versus host disease, lupus
erythematosus, psoriasis, colitis, illeitis, multiple sclerosis, uveitis,
coronary
artery vasculopathy, systemic sclerosis, atherosclerosis, asthma, transplant
rejection, allergy, dermatomyositis, pemphigus and the like.
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CA 02779184 2012-05-31
Examples of kinases that can be targeted to modulate disease include
receptor tyrosine kinases such as members of the platelet-derived growth
factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)
families and intracellular proteins such as members of the Syk, SRC, and Tec
families of kinases.
Tec kinases are non-receptor tyrosine kinases predominantly, but not
exclusively, expressed in cells of hematopoietic origin (Bradshaw 3M. Cell
Signal. 2010,22:1175-84). The Tec family includes Tec, Bruton's tyrosine
kinase (Btk), inducible T-cell kinase (Itk), resting lymphocyte kinase
(RIk/Txk), and bone marrow-expressed kinase (Bmx/Etk). Btk is a Tec
family kinase which is important in B-cell receptor signaling. Btk is
activated
by Src-family kinases and phosphorylates PLC gamma leading to effects on
B-cell function and survival. Additionally, Btk is important in signal
transduction in response to immune complex recognition by macrophage,
mast cells and neutrophils. Btk inhibition is also important in survival of
lymphoma cells (Herman, SEM. Blood 2011, 117:6287-6289) suggesting that
inhibition of Btk may be useful in the treatment of lymphomas. As such,
inhibitors of Btk and related kinases are of great interest as anti-
inflammatory as well as anti-cancer agents.
cSRC is the prototypical member of the SRC family of tyrosine kinases which
includes Lyn, Fyn, Lck, Hck, Fgr, Blk, Syk, Yrk, and Yes. cSRC is critically
involved in signaling pathways involved in cancer and is often over-expressed
in human malignancies (Kim LC, Song L, Haura EB. Nat Rev Clin Oncol. 2009
6(10):587-9). The role of cSRC in cell adhesion, migration and bone
remodeling strongly implicate this kinase in the development and progression
of bone metastases. cSRC is also involved in signaling downstream of
growth factor receptor tyrosine kinases and regulates cell cycle progression
suggesting that cSRC inhibition would impact cancer cell proliferation.
Additionally, inhibition of SRC family members may be useful in treatments
designed to modulate immune function. SRC family members, including Lck,
regulate T-cell receptor signal transduction which leads to gene regulation
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CA 02779184 2012-05-31
events resulting in cytokine release, survival and proliferation. Thus,
inhibitors of Lck have been keenly sought as immunosuppressive agents with
potential application in graft rejection and T-cell mediated autoimmune
disease (Martin et al. Expert Opin Ther Pat. 2010, 20:1573-93).
Inhibition of kinases using small molecule inhibitors has successfully led to
several approved therapeutic agents used in the treatment of human
conditions. Herein, we disclose a novel family of kinase inhibitors. Further,
we demonstrate that modifications in compound substitution can influence
kinase selectivity and therefore the biological function of that agent.
SUMMARY OF THE INVENTION
The present invention relates to a novel family of kinase inhibitors.
Compounds of this class have been found to have inhibitory activity against
members of the Tec and Scr protein kinase families, more particularly Btk.
One aspect of the present invention is directed to a compound of Formula 1:
NH2 y_z_w
N N,
Formula 1
wherein
n is an integer from 0 to 2;
X is selected from the group consisting of:
1) hydrogen,
2) alkyl,
3) heteroalkyl,
4) carbocyclyl,
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CA 02779184 2012-05-31
5) heterocyclyl;
wherein the alkyl, heteroalkyl, carbocyclyl and heterocyclyl may be further
substituted by the groups consisting of:
1) hydroxy,
2) alkoxy,
3) alkyl,
4) -0C(0)R4,
5) -0C(0)NR5R6,
6) -C(0)R4,
7) -C(0)NR5R6,
8) -NR5R6,
9) -NR2C(0)R4,
10) -NR2S(0)nR4,
11) -NR2C(0)NR5R6;
Y is selected from:
1 o
-0-
(X2)n
Z is selected from:
X1 and X2 are independently selected from hydrogen, halogen or cyano;
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CA 02779184 2012-05-31
W is independently selected from:
1) alkyl,
2) aralkyl,
3) heteroaralkyl,
4) -0R3,
5) -0C(0)R4,
6) -0C(0)NR5R6,
7) -CH2O-R4,
8) -NR5R6,
9) -NR2C(0)R4,
10) -NR2S(0)R4,
11) -NR2C(0)NR5R6;
wherein the alkyl, aralkyl and heteraralkyl may be further substituted;
R2 is selected from hydrogen or alkyl;
R3 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl,
heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, aralkyl or
heteroaralkyl;
R4 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl,
heteroalkyl, carbocyclyl, heterocyclyl, aryl or heteroaryl;
R5 and R6 are independently selected from hydrogen, alkyl, alkenyl, alkynyl,
heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl or R5 and R6 can be
fused to form a 3 to 8 membered heterocyclyl ring system.
CA 02779184 2012-05-31
Preferred embodiment includes compounds of Formula 1 where W is selected
from -0R3 and R3 is selected from substituted or unsubstituted aralkyl, or
substituted or unsubstituted heteroaralkyl.
Preferred embodiment includes compounds of Formula 1 where W is selected
from the group consisting of:
N,
F3C
4
¨0 = ¨0 II 00 ¨0 ¨0
0 0
Ho
¨0 /
lit ¨0 N
rN rN
--0/ / \
s-
or
Preferred embodiment includes compounds of Formula 1 where X is selected
from the group consisting of:
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JVVV JVL/11 'NW
JVV,/
.Aftn/
VVIIV
NH
H, Me, acetyl, cis.? 0 0 , 0
ONV
JVW
0
A0
Ny0< N rN)
( ,
4VVV
./VVV
N)
, ,or
More preferred embodiment includes compounds of Formula 1 where W is
selected from the group consisting of:
or
More preferred embodiment includes compounds of Formula 1 where Z is
selected from the group consisting of:
j
Another aspect of the present invention provides a pharmaceutical
composition comprising an effective amount of a compound of Formula 1 and
a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect of the present invention, there is provided a use of the
compound of Formula 1 as an inhibitor of protein kinase, more particularly,
as an inhibitor of Btk.
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Another aspect of the present invention provides a method of modulating
kinase function, the method comprising contacting a cell with a compound of
the present invention in an amount sufficient to modulate the enzymatic
activity of a given kinase or kinases, such as Btk, thereby modulating the
kinase function.
Another aspect of the present invention provides a method of modulating the
target kinase function, the method comprising a) contacting a cell with a
compound of the present invention in an amount sufficient to modulate the
target kinase function, thereby b) modulating the target kinase activity and
signaling.
Another aspect of the present invention provides a probe, the probe
comprising a compound of Formula 1 labeled with a detectable label or an
affinity tag. In other words, the probe comprises a residue of a compound of
Formula 1 covalently conjugated to a detectable label. Such detectable
labels include, but are not limited to, a fluorescent moiety, a
chemiluminescent moiety, a paramagnetic contrast agent, a metal chelate, a
radioactive isotope-containing moiety, or biotin.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention relates to novel kinase inhibitors. These compounds
are found to have activity as inhibitors of protein kinases: including members
of the tyrosine kinases Aurora, SRC (more specifically Lck) and Tec (more
specifically Btk) kinase families.
Compounds of the present invention may be formulated into a
pharmaceutical composition which comprises an effective amount of a
compound of Formula 1 with a pharmaceutically acceptable diluent or carrier.
For example, the pharmaceutical compositions may be in a conventional
pharmaceutical form suitable for oral administration (e.g., tablets, capsules,
granules, powders and syrups), parenteral administration (e.g., injections
(intravenous, intramuscular, or subcutaneous)), drop infusion preparations,
inhalation, eye lotion, topical administration (e.g., ointment), or
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suppositories. Regardless of the route of administration selected the
compounds may be formulated into pharmaceutically acceptable dosage
forms by conventional methods known to those skilled in the art.
The phrase "pharmaceutically acceptable" is employed herein to refer to
those ligands, materials, cornpositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition, or vehicle, such as a
liquid or solid filler, diluent, excipient, solvent or encapsulating material.
Each carrier must be acceptable in the sense of being compatible with the
other ingredients of the formulation, including the active ingredient, and not
injurious or harmful to the patient. Some examples of materials which can
serve as pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose, and sucrose; (2) starches, such as corn starch, potato
starch, and substituted or unsubstituted (3-cyclodextrin; (3) cellulose, and
its
derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;
(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil,
and
soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as
ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl
alcohol; (20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations.
The term "pharmaceutically acceptable salt" refers to the relatively non-
toxic,
inorganic and organic acid addition salts of the compound(s). These salts
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can be prepared in situ during the final isolation and purification of the
compound(s), or by separately reacting a purified compound(s) in its free
base form with a suitable organic or inorganic acid, and isolating the salt
thus
formed. Representative salts include the hydrobromide, hydrochloride,
sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,
stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,
lactobionate, laurylsulphonate salts, and amino acid salts, and the like (See,
for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:
1-19).
In other cases, the compounds of the present invention may contain one or
more acidic functional groups and, thus, are capable of forming
pharmaceutically acceptable salts with pharmaceutically acceptable bases.
The term "pharmaceutically acceptable salts" in these instances refers to the
relatively non-toxic inorganic and organic base addition salts of a
compound(s). These salts can likewise be prepared in situ during the final
isolation and purification of the compound(s), or by separately reacting the
purified compound(s) in its free acid form with a suitable base, such as the
hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal
cation, with ammonia, or with a pharmaceutically acceptable organic
primary, secondary, or tertiary amine. Representative alkali or alkaline earth
salts include the lithium, sodium, potassium, calcium, magnesium, and
aluminum salts, and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like
(see, for example, Berge et al., supra).
As used herein, the term "affinity tag" means a ligand or group, linked either
to a compound of the present invention or to a protein kinase domain, that
allows the conjugate to be extracted from a solution.
The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon
groups, including straight-chain alkyl and branched-chain alkyl groups,
CA 02779184 2012-05-31
including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl,
etc. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, cyclopropylmethyl,
n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The terms "alkenyl" and
"alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls described above,
but that contain at least one double or triple bond respectively.
Representative alkenyl groups include vinyl, propen-2-yl, crotyl, isopenten-2-
yl, 1,3-butadien-2-y1), 2,4-pentadienyl, and 1,4-pentadien-3-yl.
Representative alkynyl groups include ethynyl, 1- and 3-propynyl, and 3-
butynyl. In certain preferred embodiments, alkyl substituents are lower alkyl
groups, e.g., having from 1 to 6 carbon atoms. Similarly, alkenyl and alkynyl
preferably refer to lower alkenyl and alkynyl groups, e.g., having from 2 to 6
carbon atoms. As used herein, "alkylene" refers to an alkyl group with two
open valencies (rather than a single valency), such as -(0-12)1-10- and
substituted variants thereof.
The term "alkoxy" refers to an alkyl group having an oxygen attached
thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,
tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by
an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an
ether is or resembles an alkoxy.
The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy
group, thereby forming an ether.
The terms "amide" and "amido" are art-recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the general
formula:
0
RI 9
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wherein R9, R1 are as defined above. Preferred embodiments of the amide
will not include imides, which may be unstable.
The terms "amine" and "amino" are art-recognized and refer to both
unsubstituted and substituted amines and salts thereof, e.g., a moiety that
can be represented by the general formulae:
R9 R9
1
or ¨N¨+
Rl
Rlo R1o.
wherein R9, Rim and Rim' each independently represent a hydrogen, an alkyl,
an alkenyl, -(CH2)m-R8, or R9 and R' taken together with the N atom to
which they are attached complete a heterocycle having from 4 to 8 atoms in
the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocyclyl or a polycyclyl; and m is zero or an integer from 1 to 8. In
preferred embodiments, only one of R9 or 111 can be a carbonyl, e.g., R9,
R10,
and the nitrogen together do not form an imide. In even more preferred
embodiments, R9 and R1 (and optionally R10') each independently represent
a hydrogen, an alkyl, an alkenyl, or -(CH2)m-R8. In certain embodiments, the
amino group is basic, meaning the protonated form has a pKa > 7.00.
The term "aralkyl", as used herein, refers to an alkyl group substituted with
an aryl group, for example -(CH2)n-Ar.
The term "heteroaralkyl", as used herein, refers to an alkyl group substituted
with a heteroaryl group, for example -(CH2)õ-Het.
The term "aryl" as used herein includes 5-, 6-, and 7-membered substituted
or unsubstituted single-ring aromatic groups in which each atom of the ring
is carbon. The term "aryl" also includes polycyclic ring systems having two
or more cyclic rings in which two or more carbons are common to two
adjoining rings wherein at least one of the rings is aromatic, e.g., the other
cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,
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CA 02779184 2012-05-31
heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene,
phenanthrene, phenol, aniline, anthracene, and phenanthrene.
The terms "carbocycle" and "carbocyclyl", as used herein, refer to a non-
aromatic substituted or unsubstituted ring in which each atom of the ring is
carbon. The terms "carbocycle" and "carbocycly1" also include polycyclic ring
systems having two or more cyclic rings in which two or more carbons are
common to two adjoining rings wherein at least one of the rings is
carbocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Representative
carbocyclic groups include cyclopentyl, cyclohexyl, 1-cyclohexenyl, and 3-
cyclohexen-1-yl, cycloheptyl.
The term "carbonyl" is art-recognized and includes such moieties as can be
represented by the general formula:
0
x,R"
wherein X is a bond or represents an oxygen or a sulfur, and RH represents a
hydrogen, an alkyl, an alkenyl, -(CH2)m-R8 or a pharmaceutically acceptable
salt. Where X is an oxygen and RH is not hydrogen, the formula represents
an "ester". Where X is an oxygen, and Ril is a hydrogen, the formula
represents a "carboxylic acid".
The terms "heteroaryl" includes substituted or unsubstituted aromatic 5- to
7-membered ring structures, more preferably 5- to 6-membered rings,
whose ring structures include one to four heteroatoms. The term
"heteroaryl" also includes polycyclic ring systems having two or more cyclic
rings in which two or more carbons are common to two adjoining rings
wherein at least one of the rings is heteroaromatic, e.g., the other cyclic
rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls,
and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan,
thiophene, imidazole, isoxazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like.
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The term "heteroatom" as used herein means an atom of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and
sulfur.
The terms "heterocycly1" or "heterocyclic group" refer to substituted or
unsubstituted non-aromatic 3- to 10-membered ring structures, more
preferably 3- to 7-membered rings, whose ring structures include one to four
heteroatoms. The term terms "heterocycly1" or "heterocyclic group" also
include polycyclic ring systems having two or more cyclic rings in which two
or more carbons are common to two adjoining rings wherein at least one of
the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heterocyclyl groups include, for example, tetrahydrofuran, tetrahydropyran,
piperidine, piperazine, pyrrolidine, morpholine, lactones, and lactams.
The term "hydrocarbon", as used herein, refers to a group that is bonded
through a carbon atom that does not have a =0 or =S substituent, and
typically has at least one carbon-hydrogen bond and a primarily carbon
backbone, but may optionally include heteroatoms. Thus, groups like
methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be
hydrocarbyl for the purposes of this application, but substituents such as
acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which
is linked through oxygen, not carbon) are not. Hydrocarbyl groups include,
but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl,
alkenyl,
alkynyl, and combinations thereof.
The terms "polycycly1" or "polycyclic" refer to two or more rings (e.g.,
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or
heterocyclyls) in which two or more carbons are common to two adjoining
rings, e.g., the rings are "fused rings". Each of the rings of the polycycle
can
be substituted or unsubstituted.
As used herein, the term "probe" means a compound of the invention which
is labeled with either a detectable label or an affinity tag, and which is
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CA 02779184 2012-05-31
capable of binding, either covalently or non-covalently, to a protein kinase
domain. When, for example, the probe is non-covalently bound, it may be
displaced by a test compound. When, for example, the probe is bound
covalently, it may be used to form cross-linked adducts, which may be
quantified and inhibited by a test compound.
The term "substituted" refers to moieties having substituents replacing a
hydrogen on one or more carbons of the backbone. It will be understood
that "substitution" or "substituted with" includes the implicit proviso that
such substitution is in accordance with permitted valence of the substituted
atom and the substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation such
as by rearrangement, cyclization, elimination, etc. As used herein, the term
"substituted" is contemplated to include all permissible substituents of
organic compounds. In a broad aspect, the permissible substituents include
acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and non-aromatic substituents of organic compounds. The
permissible substituents can be one or more and the same or different for
appropriate organic compounds. For purposes of this invention, the
heteroatoms such as nitrogen may have hydrogen substituents and/or any
permissible substituents of organic compounds described herein which satisfy
the valences of the heteroatoms. Substituents can include, for example, a
halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a
phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an
azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a
sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in the art that
the moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate.
CA 02779184 2012-05-31
Compounds of the invention also include all isotopes of atoms present in the
intermediates and/or final compounds. Isotopes include those atoms having
the same atomic number but different mass numbers. For example, isotopes
of hydrogen include deuterium and tritium.
General Synthetic Methods
General Synthetic Method A:
W W W
0
Base, ligand, IW ir W catalyst
0 4:S: NaOH
) oxalyl chloride
0 ---b- 0
OH a Bo2c w Ho2c ir
cloc le
Br .'
1-i 1-iii 1-iv 1-v
1-ii
0
40w 0=w 0 =
1-v
malononitrile io TMS-diazomethane 0 hydrazine
. W
r- r
NC
i \
CN meo -,.. CN ,N
HO H2N N
CN CN H
1-vi 1-ii 1-viii
0 = 0 =
formamidine
________ 3. NH2 = W __________________ fa W
Ph3P, DIAD
1-viii . NH2
R1 OH
1,1 N N N 1-x
R,
1-ix
Scheme 1
Ulmann condensation of phenol 1-i with ester 1-ii provided intermediate 1-iii.
Saponification of intermediate 1-iii yielded intermediate 1-iv. Conversion of
intermediate 1-iv to its acid chloride, using for example oxalyl chloride and
DMF, provided intermediate 1-v. Condensation of intermediate 1-v with
malononitrile yielded intermediate 1-vi. Methylation of intermediate 1-vi with
TMS-diazomethane provided intermediate 1-vii. Condensation of 1-vii with
hydrazine yielded intermediate 1-viii. Condensation of intermediate 1-viii
with formamidine yielded intermediate 1-ix. Intermediate 1-ix was treated
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CA 02779184 2012-05-31
with alcohol R1OH, under Mitsunobu conditions, to provide the desired
compounds or intermediates of general formula 1-x.
General Synthetic Method B:
malononitrile TMS-diazomethane hydrazine
CN
,c) CN
NC
\
0 CI HO CN NH
CN
H2N
2-i 2-ii 2-ili 2-iv
X=I, Br
X 0*
X
2-iv
formamidine Ph3P, DIAD, NH2 Base, ligand,
= NH2
catalyst
NH2 N \ \
RICH L ,N 1 N IN
I
N". \N N, N N
10 w
, R1
N N
2-vii
2 OH-v 2-vi 2-viii
Scheme 2
Benzoyl chlorides of formula 2-i were condensed with malononitrile to
provide intermediate 2-ii. Methylation of intermediate 2-ii with TMS-
diazomethane provided intermediate 2-iii. Condensation of intermediate 2-iii
with hydrazine provided intermediate 2-iv. Further condensation of
intermediate 2-iv with formamidine provided intermediate 2-v. Intermediate
2-v was treated with alcohol WON, under Mitsunobu conditions, to provide
intermediate 2-vi. Ullmann condensation of intermediate 2-vi with phenolic
intermediates 2-vii provided the desired compounds or intermediates of
general formula 2-viii.
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Exemplification
The following synthetic methods are intended to be representative of the
chemistry used to prepare compounds of Formula 1 and are not intended to
be limiting.
Synthesis of Compound 1:
,L0
)LOH 0 OBn
OH K2CO3 Al OBn CuCI, Cs2CO3
________________ = ______________________ =
Benzyl bromide IP 0 0
OH OH 0 0
EtO2C
3-a Br 3-b
OBn
S I. OBn
NaOH oxaly1 chloride
3-b ________ .. o __________ = o
11110 10
Ho2c CIOC
3-c 3-d
0411
0 0 0 0
DIPEA TMS-diazomethane
3-d ________ 0.
malononitrile (1101 0 0
\ CN \ CN
HO Me0
CN CN
3-e 3-f
0 . 0 *
hydrazine 0 formamidine 0
3-f _______ =
NC NH2 *
N .
/ \ N
= \
H2N N N'' I ,
H N [NI
3-g Compound 1
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Scheme 3
Step 1: Intermediate 3-a
Benzyl bromide (27.0 ml, 227 mmol) was added drop wise to a stirred
suspension of resorcinol (25.0 g, 227 mmol) and potassium carbonate (31.4
g, 227 mmol) in acetone (150 ml) and the reaction was heated under reflux
overnight. Volatiles were removed under reduced pressure. Water and ethyl
acetate were added, the organic layer was separated, washed with brine,
dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by silica gel chromatography provided intermediate 3-a as a
beige oil.
Step 2: Intermediate 3-b
To a solution of 3-a (15.0 g, 74.9 mmol) in 1,4-dioxane (200 ml) were
sequentially added ethyl 4-bromobenzoate (20.59 g, 90 mmol), N,N-
dimethylglycine (4.25 g, 41.2 mmol), copper(I) chloride(3.71 g, 37.5 mmol)
and cesium carbonate (61.0 g, 187 mmol). The reaction mixture was stirred
at reflux overnight and then cooled to room temperature. Water and ethyl
acetate were added, the organic layer was separated, washed with saturated
aqueous NaHCO3, brine, dried over MgSO4, filtered and concentrated under
reduced pressure. Purification by silica gel chromatography provided
intermediate 3-b as a colorless oil.
Step 3: Intermediate 3-c
To a solution of 3-b (17.5 g, 50.2 mmol) in THF (200 ml) and Me0H (100 ml)
was added 2N sodium hydroxide (100 ml, 200 mmol) and the reaction was
stirred at room temperature overnight. Volatiles were removed under
reduced pressure. 10% Aqueous HCI and ethyl acetate were added to the
residue, the organic layer was separated, washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure to provide
intermediate 3-c as beige solid.
19
CA 02779184 2012-05-31
Step 4: Intermediate 3-d
To a suspension of 3-c (16.1 g, 50.3 mmol) in dichloromethane (100 ml)
were added DMF (0.1 ml, 1.29 mmol) and oxalyl chloride (4.4 ml, 50.3
mmol). The solution was stirred at room temperature for 2 hours. Volatiles
were removed under reduced pressure to provide intermediate 3-d as beige
solid.
Step 5: Intermediate 3-e
To a solution of intermediate 3-d (16.5 g, 48.9 mmol) in toluene (50 ml) and
THF (7 ml), cooled to -10 C, were added malononitrile (3.19 ml, 50.2 mmol)
and DIPEA (17.5 ml, 100 mmol) in toluene (50 mL), drop wise, over a period
of 30 minutes. After the addition was completed, the reaction was stirred for
1 hour at 0 C and room temperature overnight. Volatiles were removed
under reduced pressure. 1M aqueous HCI and ethyl acetate were added, the
organic layer was separated, washed with 1M HCI and brine, dried over
MgSO4, filtered and concentrated under reduced pressure to provide
intermediate 3-e as beige solid.
Step 6: Intermediate 3-f
To a solution of intermediate 3-e (18.1 g, 49.1 mmol) in acetonitrile (177 ml)
and methanol (19.0 ml), cooled to 0 C, were added DIPEA (10.3 ml, 59.0
mmol) and a 2M solution of (diazomethyl)trimethylsilane in hexanes (27.0
ml, 54.0 mmol). After the addition was completed, the reaction was stirred at
room temperature overnight. Acetic acid (0.56 ml, 9.83 mmol) was added,
the reaction was then stirred for 30 minutes and volatiles were removed
under reduced pressure. Saturated aqueous NaHCO3 and ethyl acetate were
added, the organic layer was separated, washed with brine, dried over
CA 02779184 2012-05-31
MgSO4, filtered and concentrated under reduced pressure. Purification by
silica gel chromatography provided intermediate 3-f as yellow solid.
Step 7: Intermediate 3-g
To a suspension of intermediate 3-f (8.05 g, 21.1 mmol) in ethanol (10.5 ml)
was added a solution of hydrazine nionohydrate (2.76 ml, 56.8 mmol). The
reaction was stirred at 100 C for 1 hour and then cooled to room
temperature. Water was added; a precipitate formed and was collected by
filtration, washed with diethyl ether and dried in vacuo to provide
intermediate 3-g as an off-white solid.
Step 8: Compound 1
Intermediate 3-g (8 g, 20.92 mmol) was added to a solution of formamidine
(58.4 ml, 1464 mmol) and the reaction was stirred at 180 C for 2 hours and
then cooled to room temperature. Water and ethyl acetate were added; the
organic layer was separated, washed with brine, dried over MgSO4, filtered
and concentrated under reduced pressure to provide compound 1 as beige
solid. MS (m/z) M+H= 410.2
Synthesis of Compound 2:
o * o *
NH2 * Ph3P, DIAD NH2 =
N \ OH N \
N
, 1_ I ,N
N N
Compound 1 Compound 2
Scheme 4
21
CA 02779184 2012-05-31
To a solution of cyclopentanol (316 mg, 3.66 mmol) in THF was added
triphenylphosphine (961 mg, 3.66 mmol) and DIAD (712 id, 3.66 mmol). The
yellow solution was stirred 5 minutes, compound 1 (1.0 g, 2.44 mmol) was
added and the reaction was then stirred at room temperature overnight.
Volatiles were removed under reduced pressure. Purification by silica gel
chromatography provided compound 2 as an off-white solid. MS (m/z)
M+H=478.2
Synthesis of Compound 3:
0= 0=
0 0
Ph3P, DIAD
NH2 qk
410NH2
OH 410
\ NV' \
N
I N
N
N
Compound 1 0
0
Compound 3
Scheme 5
To a solution of (S)-tert-butyl 3-hydroxypiperidine-1-carboxylate (5.65 g,
28.1 mmol) in THF was added triphenylphosphine (7.37 g, 28.1 mmol) and
DIAD (5.46 ml, 28.1 mmol). The yellow solution was stirred 5 minutes,
compound 1 (10 g, 24.42 mmol) was added and the reaction was then
stirred at room temperature overnight. Volatiles were removed under
reduced pressure. Purification by silica gel chromatography provided
compound 3 as white foam. MS (m/z) M+H= 593.1
Synthesis of Compound 4:
22
CA 02779184 2012-05-31
0* 0*
0
NH2 HCI*
NH2 *
elkN "- N
N N
N N
a NH
0
Compound 4
Compound 3
Scheme 6
To a solution of compound 3 (1.88 g, 3.17 mmol) in dichloromethane was
added 4N HCI in 1,4-dioxane (19.82 ml, 79.0 mmol) and the reaction was
stirred at room temperature for 2 hours. Volatiles were removed under
reduced pressure. Purification by reverse phase chromatography eluting with
a 1% aqueous HCl/methanol gradient provided compound 4.2HCI as white
solid. MS (m/z) M+H= 493.1
Synthesis of compound 5
* *
0 0
NH2 TEA
0 NH2
N "N N
I' 1 N
N N
NH 2HCI N
L bN
Compound 4 Compound 5
Scheme 7
23
CA 02779184 2012-05-31
To a solution of compound 4.2HCI (100 mg, 0.17 mmol) in dichloromethane
(2 ml) cooled to 0 C were sequentially added TEA (99 pl, 0.70 mmol) and
acryloyl chloride (17.61 mg, 0.19 mmol). The reaction was stirred at 0 C for
1 hour. A saturated aqueous solution of ammonium chloride was added, the
organic layer was separated, washed with brine, dried over MgSO4, filtered
and concentrated under reduced pressure. Purification by silica gel
chromatography provided compound 5 as white solid. MS (m/z) M+H= 547.1
Synthesis of compound 6
0* 0 =
0 0
TEA
NH2 NH2
\NI 0
2HCI N \ N ,
N N N N
LNH
Compound 4 Compound 6
Scheme 8
To a solution of compound 42HCI (1.8 g, 3.18 mmol) in dichloromethane (32
ml) cooled to 0 C were sequentially added TEA (1.77 ml, 12.73 mmol) and
acetyl chloride (249 pl, 3.50 mmol). The reaction was stirred at 0 C for 1
hour and room temperature overnight. Saturated aqueous ammonium
chloride was added, the organic layer was separated, washed with brine,
dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by reverse phase chromatography eluting with 1% aqueous
HCl/methanol gradient provided compound 6=FICI as beige solid. MS (m/z)
M+H= 535.1
24
CA 02779184 2012-05-31
Compounds 7 and 8 were prepared in a similar manner to compounds 6 by
acylation of compound 4 with butanoyl chloride and iso-butanoyl chloride,
respectively.
Synthesis of intermediate 9-d
Br
Br
Br
DIPEA
malononitrile 1110 TMS-diazomethane
1011
CN
CN
HO 0
0 CI CN
CN
9-a 9-b
Br Br
hydrazine
formamidine
9-b __________________________________________ NH2
NC N
N
\
NH N
H2N
9-c 9-d
Scheme 9
Step 1: Intermediate 9-a
To a solution of 4-bromobenzoyl chloride (25 g, 114 mmol) in toluene (200
ml) and THF (30 ml), cooled to -10 C, were sequentially added malononitrile
(7.60 ml, 120.0 mmol) and DIPEA (39.8 ml, 228 mmol) in toluene (50 mL)
drop wise over a period of 1 hour. After the addition was completed, the
reaction was stirred for 1 hour at 0 C and room temperature overnight.
Volatiles were removed under reduced pressure. 1M HCI and ethyl acetate
were added to the residue, the organic layer was separated, washed twice
with 1M HCI and brine, dried over MgSO4, filtered and concentrated under
reduced pressure to provide intermediate 9-a as yellow solid.
CA 02779184 2012-05-31
Step 2: Intermediate 9-b
To a solution of intermediate 9-a (26.4 g, 106 mmol) in acetonitrile (300 ml)
and methanol (35.0 ml), cooled to 0 C, was added DIPEA (22.2 ml, 127
mmol) and (diazomethyl)trimethylsilane (58.3 ml, 117 mmol). After the
addition was completed, the reaction was stirred at room temperature
overnight. Acetic acid (1.21 ml, 21.2 mmol) was added, the reaction was
stirred for 30 minutes and volatiles were removed under reduced pressure.
Saturated aqueous NaHCO3 and ethyl acetate were added, the organic layer
was separated, washed with brine, dried over MgSO4, filtered and
concentrated under reduced pressure. Purification by silica gel
chromatography provided intermediate 9-b as a yellow solid.
Step 3: Intermediate 9-c
To a suspension of intermediate 9-b (4.49 g, 17.07 mmol) in ethanol (8.5
ml) was added a solution of hydrazine monohydrate (2.23 ml, 46.1 mmol)
and the reaction was stirred at 100 C for 1 hour and then cooled to room
temperature. Volatiles were removed under reduced pressure to provide
intermediate 9-c as a yellow solid.
Step 4: Intermediate 9-d
Intermediate 9-c (4.49 g, 17.07 mmol) was added to a solution of
formamidine (40.8 ml, 1024 mmol) and the reaction was stirred at 180 C
for 3 hours and then cooled to room temperature. Ethanol was added; a
precipitate formed and was collected by filtration, dried in vacuo to provide
intermediate 9-d as beige solid.
Synthesis of intermediate 10-a
26
CA 02779184 2012-05-31
Br
Ph3P, DIAD
9-d NH2
OH
IµV \ N
'
N N
10-a
Scheme 10
To a solution of intermediate 9-d (1.0 g, 3.45 mmol) in THF was added
triphenylphosphine (1.35 g, 5.17 mmol), cyclopentanol (0.47 ml, 5.17 mmol)
and DIAD (1.0 ml, 5.17 mmol) and the reaction was then stirred at room
temperature overnight. Volatiles were removed under reduced pressure.
Purification by silica gel chromatography provided intermediate 10-a as white
solid. MS (m/z) M+H= 359.6
=
27
CA 02779184 2012-05-31
Synthesis of Compound 9
CI
40 OH OTBS Ph3P, DIAD, roo 0 40
imidazole
TBSCI CI
OH OH HO OTBS
11-a 11-b
ci
401 0
TBAF
11-b
OH
11-c
0*
0
CuCI, CS2CO3,
1O-a+ 11-c ____________ = OH NH2 Wilk
\ CI
1 NjN
1St N
Compound 9
Scheme 11
Step 1: Intermediate 11-a
To a solution of resorcinol (15 g, 136 mmol) in DMF (100 ml), cooled to 0 C,
were added imidazole (19.48 g, 286 mmol) and tert-
butylchlorodimethylsilane (21.56 g, 143 mmol). The reaction was then stirred
at room temperature overnight. Saturated aqueous ammonium chloride and
ethyl acetate were added; the organic layer was separated, washed 3 times
with a saturated aqueous solution of ammonium chloride and brine, dried
over MgSO4, filtered and concentrated under reduced pressure. Purification
by silica gel chromatography provided intermediate 11-a as a colorless oil.
28
CA 02779184 2012-05-31
Step 2: Intermediate 11-b
To a solution of (4-chlorophenyl) methanol (1.52 g, 10.70 mmol) in THF (20
mL) were sequentially added intermediate 11-a (2.88 g, 12.84 mmol),
triphenylphosphine (3.37 g, 12.84 mmol) and DIAD (2.53 ml, 12.84 mmol)
drop wise at room temperature and the reaction was then stirred for 1 hour.
Saturated aqueous ammonium chloride and ethyl acetate were added, the
organic layer was separated, washed with brine, dried over MgSO4, filtered
and concentrated under reduced pressure. Purification by silica gel
chromatography provided intermediate 11-b as a colorless oil.
Step 3: Intermediate 11-c
Tetrabutylammonium fluoride trihydrate (3.93 g, 12.47 mmol) was added to
a solution of intermediate 11-b (2.9 g, 8.31 mmol) in THF (15 mL) and the
reaction was stirred at room temperature overnight. Saturated aqueous
ammonium chloride and ethyl acetate were added, the organic layer was
separated, washed with brine, dried over MgSO4, filtered and concentrated
under reduced pressure. Purification by silica gel chromatography provided
intermediate 11-c as a colorless oil.
Step 4: Compound 9
A solution of intermediate 10-a (200 mg, 0.56 mmol), intermediate 11-c
(229 mg, 0.977 mmol), quinolin-8-ol (16.21 mg, 0.112 mmol), copper (I)
chloride (11.05 mg, 0.11 mmol) and cesium carbonate (546 mg, 1.67
mmol), in dimethylacetamide (1 ml), was degassed with argon for 10
minutes, sealed and heated in a sealed tube at 140 C overnight. The
solution was cooled to room temperature, water and ethyl acetate were
added, the organic layer was separated, the aqueous layer was extracted
twice with ethyl acetate, the combined organic extracts were washed with
brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by reverse phase chromatography eluting with a 1%
29
CA 02779184 2012-05-31
HCl/methanol gradient provided compound 9.1-1CI as yellow solid. MS (m/z)
M+H= 512.2
Synthesis of intermediate 12-a
Br
NH2 4Ik
Ph3P, DIAD
9-d ________________________________ N
N
Me0H N
12-a
Scheme 12
To a solution of intermediate 9-d (500 mg, 1.72 mmol), in THF (8.6 mL),
were sequentially added methanol (105 pl, 2.59 mmol), triphenylphosphine
(678 mg, 2.59 mmol) and DIAD (503 pi, 2.59 mmol), drop wise, at room
temperature. The solution was then stirred at room temperature overnight.
A precipitate formed and was collected by filtration and dried in vacuo to
provide intermediate 12-a as a white solid.
Synthesis of compound 16
0*
1410i, 23,
12-a + cueCSC0NH2 44Ik
OH __
OH N1 ,N
3-a
Compound 16
Scheme 13
CA 02779184 2012-05-31
A solution of intermediate 12-a (235 mg, 0.77 mmol), intermediate 3-a (271
mg, 1.35 mmol), quinolin-8-ol (22.4 mg, 0.15 mmol), copper (I) chloride
(15.3 mg, 0.15 mmol) and cesium carbonate (755 mg, 2.31 mmol) in
dimethylacetamide (1 ml) was degassed with nitrogen for 10 minutes,
heated in a sealed tube at 140 C overnight and then cooled to room
temperature. Water and ethyl acetate were added, the organic layer was
separated, the aqueous layer was extracted twice with ethyl acetate, the
combined organic extracts were washed with brine, dried over MgSO4,
filtered and concentrated under reduced pressure. Purification by reverse
phase chromatography eluting with 1% HCl/methanol gradient provided
compound 16=HCI as a beige solid. MS (m/z) M+H= 424.2
Synthesis of compound 17
CHO CHO
DIPEA, TBSCI NaBH4 OH
OH OTBS OTBS
14-a 14-b
00
14-b ________
Ph3P, DIAD, 40 0 410 TBAF 0
101
CN CN
OTBS OH
HO
CN 14-c 14-d
0* CN
CuCI, CS2CO3, 0
12-a + 14-d
OH NH2
N
\ Compound 17
31
CA 02779184 2012-05-31
Scheme 14
Step 1: Intermediate 14-a
To a solution of 3-hydroxybenzaldehyde (14.73 g, 121 mmol) in
dichloromethane (100 mL) were sequentially added triethylamine (25.08 ml,
181 mmol), tert-butylchlorodimethylsilane (20.0 g, 133 mmol), portion wise,
and the reaction was stirred at room temperature overnight. 10% Citric acid
was added, the organic layer was separated, washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. Purification by
silica gel chromatography provided intermediate 14-a as a yellow oil.
Step 2: Intermediate 14-b
To a solution of intermediate 14-a (16.0 g, 67.7 mmol) in methanol (100 ml)
cooled to 0 C was added portion wise sodium borohydride (1.28 g, 33.8
mmol). After the addition was completed the reaction was stirred at room
temperature for 2 hours. Volatiles were removed under reduced pressure.
Water and ethyl acetate were added to the residue, the organic layer was
separated, washed with brine, dried over Mg504, filtered and concentrated
under reduced pressure to provide intermediate 14-b as a yellow oil.
Step 3: Intermediate 14-c
To a solution of intermediate 14-b (1.0 g, 2.09 mmol) in THF (42 mL) were
sequentially added 2-hydroxybenzonitrile (600 mg, 5.03 mmol),
triphenylphosphine (1.32 g, 5.03 mmol) and DIAD (991 pl, 5.03 mmol) drop
wise at room temperature; the reaction was then stirred at reflux for 2 hours
then cooled to room temperature. Saturated aqueous ammonium chloride
and ethyl acetate were added, the organic layer was separated, washed with
brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by silica gel chromatography provided intermediate 14-c as a
colorless oil.
32
CA 02779184 2012-05-31
Step 2: Intermediate 14-d
To a solution of intermediate 14-c (1.22 g, 3.62 mmol) in THF (36.0 ml) was
added tetrabutylammonium fluoride (946 mg, 3.62 mmol) and the reaction
was stirred at room temperature for 1 hour. Saturated aqueous ammonium
chloride and ethyl acetate were added, the organic layer was separated,
washed with brine, dried over MgSO4, filtered and concentrated under
reduced pressure. Purification by silica gel chromatography provided
intermediate 14-d as a white solid.
Step 2: Compound 17
A solution of intermediate 12-a (200 mg, 0.6 mmol), intermediate 14-cl (259
mg, 1.15 mmol), quinolin-8-ol (19.0 mg, 0.13 mmol), copper (I) chloride
(13.0 mg, 0.13 mmol) and cesium carbonate (643 mg, 1.97 mmol) in
dimethylacetamide (3.0 ml) was degassed with argon for 10 minutes, heated
in a sealed tube at 140 C overnight. After cooling to room temperature,
water and ethyl acetate were added, the organic layer was separated, the
aqueous layer was extracted twice with ethyl acetate, the combined organic
extracts were washed with brine, dried over MgSO4, filtered and concentrated
under reduced pressure. Purification by silica gel chromatography provided
compound 17 as a white solid. MS (m/z) M+H= 449.3
Synthesis of compound 18
33
CA 02779184 2012-05-31
rN
LIAIH4 Ph3P, DIAD,
HO
14-b
0 OTBS
15-a 15-b
Oc
TBAF
OH
15-c
0*
CuCI, CS2CO3
12-a +15-cOH NH2 sz,N
NH
, N
N N\ Compound 18
Scheme 15
Step 1: Intermediate 15-a
To a solution of ethyl 2-methylthiazole-5-carboxylate (5.82 g, 34.0 mmol) in
THF (170 ml), cooled to 0 C, was added a 1.0M solution of LiAIH4in THF (34
ml, 34.0 mmol) and the reaction was slowly warmed to room temperature
and stirred overnight. Water (1.3 ml) was slowly added, followed by 15%
NaOH (1.3 mL). The solution was stirred for 2 hours at room temperature
then filtered on celite. The filtrate was concentrated under reduced pressure
to provide intermediate 15-a as a yellow oil.
Step 2: Intermediate 15-b
To a solution of intermediate 15-a (7.75 g, 34.5 mmol) and intermediate 14-
b (4.25 g, 32.9 mmol), in THF (33 mL), were sequentially added
triphenylphosphine (10.35 g, 39.5 mmol) and DIAD (7.68 ml, 39.5 mmol),
drop wise, at room temperature. The reaction was then stirred for 18 hours.
Volatiles were removed in vacuo. Purification by silica gel chromatography
provided intermediate 15-b as a colorless oil.
34
CA 02779184 2012-05-31
Step 3: Intermediate 15-c
To a solution of intermediate 15-b (5.5 g, 16.39 mmol), in THF (82.0 ml),
was added a 1.0M solution of tetrabutylammonium fluoride in THF (16.4 ml,
16.4 mmol) and the reaction was stirred at room temperature for 30
minutes. Saturated aqueous ammonium chloride and ethyl acetate were
added, the organic layer was separated, washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. Purification by
silica gel chromatography provided intermediate 15-c as beige solid.
Step 4: Compound 18
A solution of intermediate 12-a (200 mg, 0.65 mmol), intermediate 15-c
(146 mg, 0.65 mmol), quinolin-8-ol (19.0 mg, 0.13 mmol), copper (I)
chloride (13.0 mg, 0.13 mmol) and cesium carbonate (643 mg, 1.97 mmol)
in dimethylacetamide (6.5 ml) was degassed with argon for 10 minutes,
ealed, heated in a sealed tube at 140 C for 2 hours and then cooled to room
temperature. Water and ethyl acetate were added, the organic layer was
separated, the aqueous layer was extracted twice with ethyl acetate, the
combined organic extracts were washed with brine, dried over MgSO4,
filtered and concentrated under reduced pressure. Purification by reverse
phase chromatography eluting with 1% HCl/methanol gradient provided
compound 15.1-1CI as beige solid. MS (m/z) M+H= 445.1
Table 1 summarizes representative compound of Formula 1.
CA 02779184 2012-05-31
Table 1: Example Compounds of Formula 1
Compound Structure MS (m/z)
0*
0
1 NH2 [M+H]=410.2
\N
I N'
0=
0
NH2 =
2
[M+Hr=478.2
,N
N
0=
0
NH2 th
\ [M+H]+=593.1
N L ,N
3
UN-.?
36
CA 02779184 2012-05-31
0=
0
NH2 4IW
4
410 [M+H]=493.1
\
I ,N
N N
L\NH
0=
0
NH2 fa
[M+H]=547.1
\
I ,N
N N
446
0
NH2 40
6
[M+H]=535.1
N "N
I ,
N N
37
CA 02779184 2012-05-31
0=
0
NH2
7 N' ,
I ,N [M+H]=563.1
N N
0*
0
NH2
8 N [M+H]=563.1
L ,N
N
L\N
*
0
9
NH2
[M+Hr=512.2
CI
N \N
L I =
N
38
CA 02779184 2012-05-31
0*
0
NH2 =
O [M+H]=508.1
N ' , \
L I ,N --0
14 N
d
0*
n.
NH2 = 0=
0, [M+H]=508.2
NV \
I'N
N N),....,
U
_
0*
CN
NH2 0 glis illit
12 [M+Hr=503.2
NI "
I ,NI
N N
d
0*
0
NH2 = =
13 [M+Hr=478.2
N' , "
I ,N
N N
d
39
CA 02779184 2012-05-31
0*
14 NH2 fi SN
[M+Hr=485.2
N' \N
I ,
N
0 0 CN
15 NH2 110
[M+H]=503.3
N' \
I'N
N N
0*
16
0
441k
NH2 41k [M+H]=424.2
N \N
I ,
N N
0
CN
0
17 NH2 [M+H]=449.3
N' \N
I ,
N N
CA 02779184 2012-05-31
0=
18 NH2 * [M+H]=445.1
\N
'
N N
4Ik
19
NH2 410
[M+H]=431.4
\
L ,N
N
4Ik
CF3
NH2 0
[M+1-1]=492.1
,
,N
N N
0 =
0
NH2
21 'IN[M+Hr=494.2
N
U0
41
CA 02779184 2012-05-31
0 .
CN
0
22 NH2 ik
[M+Hr=519.2
N' , \
1 ,N
N N
o
0
0 .
u3
0
23 NH2 O Ilk
[M+H]=562.1
N' \N
I ,
N
U
0
0=
O_______\
24 NH2 Th
410 sN
[M+Hr=501.2
N' , \
I ,N
N N
a0
42
CA 02779184 2012-05-31
0* CN
0
25 NH2
N' \ [M+H]=560.2
I ,N
N
UN-{
0
0*
CF3
0
26 NH2 110 110
N \N [M+H]=603.1
I N'
0
0*
S,f`J
27 NH2
N' \N [M+H]=542.2
I ,
N
0
43
CA 02779184 2012-05-31
O CN
0
2 NH2
8 410
[M+Hr=560.2
N7 "N
'
N
UN(
0
0
CF3
0
29
NH2 410 [M+Hr=546.2
"N
= I
N
0 =
CN
0
NH2 [M+H]=449.4
\
= I ,N
N N
0 0 CF3
31
NH2 [M+H]+=492.1
N7 \
I ,N
N N
44
CA 02779184 2012-05-31
0
0 CF3
32 NH2 =
[M+H]=546.1
N
I ,N
N N
0=
o CF3
33
NH2*
N , [M+H]=603.1
L ,N
N
0
0=
34 NH2 sN
[M+H]=488.3
,
I ,N
N N
N--
CA 02779184 2012-05-31
0Q
0
35 NH2 4. NC 410
[M+H]=519.2
\N
I '
N
0 =
0
36 NH2 44, F3C
[M+Hr=562.2
"N
I '
N
0 =
37 NH2 40 [M-I-H]=499.1
"N
I'
N
46
CA 02779184 2012-05-31
0 =
38 NH2 410
[M+H]=515.1
'N
N N)Th
0
0 =
NH2
39 [M+H]=514.2
,N
N N\
N,.
0*
NH2 410
N'
40 \ ,N1 [M+H]=543.1
N N\
47
CA 02779184 2012-05-31
0*
0'\ _
NH2 41
N
41 N \ [M+Fir=522.1
IN
N N
\---6
N
0 =
NH2 40 N
42 N' \ [M+H]=521.2
L, I ,N
N N
111
0*
NH2 fa N
43 N' , \ [M+H]=500.2
L... 1 ,N
N Nym
C--.)
N
H
48
CA 02779184 2012-05-31
0*
0"---_
NH2 4*
N
44 N \ N [M+H]=530.1
I ,
N Nv
--A
(N---\
=--0/
0=
0
45 NH2 lli . [M+H]=452.1
NV \N
I ,
N N\
/=0
F
0*
0---A___
46 NH2 * N [M+H]=519.1
NV \N
I'
N r).Th
\-- )
0
49
CA 02779184 2012-05-31
0*
NH2,
47 INV \
I ,N [M+Hr=542.1
N N)Th
/10
0=
, NI/
NH2 410
48 [M+Hr=498.2
\N
'
N
0Q
NH2 4110
49 N \ [M +H] =600.1
L ,N
N
aN
CA 02779184 2012-05-31
0*
()Ars
50 NH2 * [M+I-1]+=503.2
N'
'N
N
0Il
=
0
NH2
51 [M+Hr=500.2
N' N
I '
N N
L\NH
Kinase Binding
Btk Kinase Inhibition Assay
Fluorescence polarization-based kinase assays were performed in 384 well-
plate format using histidine tagged recombinant human full-length Bruton
Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the
KinEASE TM FP Fluorescein Green Assay supplied from Millipore. Kinase
reaction were performed at room temperature for 60 minutes in presence of
250 11M substrate, 10 M ATP and variable test article concentrations. The
reaction was stopped with EDTA/kinease detection reagents and the
polarization measured on a Tecan 500 instrument. From the dose-response
curve obtained, the ICso was calculated using Graph Pad Prisms using a
non linear fit curve. The Km for ATP on each enzyme was experimentally
determined and the Ki values calculated using the Cheng-Prusoff equation
51
CA 02779184 2012-05-31
(see: Cheng Y, Prusoff WH. (1973) Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes 50 per cent
inhibition (I50) of an enzymatic reaction". Biochem Pharmacol 22 (23): 3099-
108).
k, values are reported in Tables 2:
Table 2: Inhibition of Btk
Compound ki (nM) Compound k, (nM) Compound k(nM)
1 a 16 a 31 a
2 a 17 a 32 a
3 a 18 a 33 a
4 a 19 a 34 a
a 20 a 35 a
6 a 21 a 36 a
7 a 22 a 37 a
8 a 23 a 38 a
9 a 24 a 39 a
a 25 a 40 a
11 a 26 a 41 a
12 a 27 a 42 a
13 a 28 a 43 a
14 a 29 a 44 a
a 30 a 45 a
a - Less than 100 nM; b - less than 1000 nM, c - more than 1000 nM
52
CA 02779184 2012-05-31
Splenic Cell Proliferation Assay
Splenocytes were obtained from 6 week old male CD1 mice (Charles River
Laboratories Inc.). Mouse spleens were manually disrupted in PBS and
filtered using a 70um cell strainer followed by ammonium chloride red blood
cell lysis. Cells were washed, resuspended in Splenocyte Medium (HyClone
RPMI supplemented with 10% heat-inactivated FBS, 0.5X non-essential
amino acids, 10mM HEPES, 50uM beta mercaptoethanol) and incubated at 37
C, 5% CO2 for 2h to remove adherent cells. Suspension cells were seeded in
96 well plates at 50,000 cells per well and incubated at 37 C, 5% CO2 for 1h.
Splenocytes were pre-treated in triplicate with 10,000 nM curves of Formula
1 compounds for 1h, followed by stimulation of B cell proliferation with
2.5ug/m1 anti-IgM F(a131)2 (Jackson ImmunoResearch) for 72h. Cell
proliferation was measured by Cell Titer-Glo Luminescent Assay (Promega).
EC50 values (50% proliferation in the presence of compound as compared to
vehicle treated controls) were calculated from dose response compound
curves using GraphPad Prism Software.
EC50 values are reported in Table 2:
Table 2: Inhibition of splenic cell proliferation
Compound ki (nM) Compound k, (nM) Compound k1(nM)
1 b 16 b 31 b
2 b 17 b 32 a
3 b 18 a 33 a
4 b 19 b 34 b
a 20 a 35 a
6 a 21 a 36 a
7 b 22 a 37 a
8 a 23 a 38 a
9 b 24 a 39 b
b 25 a 40 b
11 b 26 a 41
12 a 27 a 42 a
53
CA 02779184 2012-05-31
13 b 28 a 43 b
14 a 29 a 44 a
15 a 30 a 45
a - Less than 100 nM; b - less than 1000 nM, c ¨ more than 1000 nM
54
